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1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 I OI I i
(12) United States Patent
(10) Patent No.:
Prior et al.
(45) Date of Patent:
(56)
(54) IMMUNOGENIC SEQUENCES
Jun. 12, 2012
References Cited
U.S. PATENT DOCUMENTS
(75) Inventors: Joann Lisa Prior, Salisbury (GB);
Richard Geoffrey Prior, Salisbury
(GB); Paul Gareth Hitchen, Wiltshire
(GB); Anne Dell, London (GB);
Richard William Titball, Salisbury
(GB)
3,148,120 A
9/1964 Otto
5,066,596 A
11/1991 Manning et al.
5,951,987 A * 9/1999 Cherwonogrodzky
et al . .......................... 424/252.1
6,261,568 B1
6,268,171 B1
6,350,454 B1
6,406,705 B1
644,804 Al
6,444,210 B1
6,444,445 B2
6,444,804 B1
6,552,006 B2
7,399,756 B2
(73) Assignee: The Secretary of State for Defence
(GB)
(*) Notice:
US 8,198,430 B2
Subject to any disclaimer, the term of this
patent is extended or adjusted under 35
U.S.C. 154(b) by 366 days.
7/2001
7/2001
2/2002
6/2002
9/2002
9/2002
9/2002
9/2002
4/2003
7/2008
Gicquel et al.
Meyer et al.
Thune
Davis et al.
Lam et al.
Kournikakis et al.
Nikolich et al.
Lam et al.
Raz et al.
Jomaa et al.
(Continued)
(21)
Appl. No.: 12/277,077
(22)
Filed:
FOREIGN PATENT DOCUMENTS
Nov. 24, 2008
EP
0250614 Al
1/1988
(Continued)
(65)
Prior Publication Data
US 2010/0080828 Al
Apr. 1, 2010
Related U.S. Application Data
(63) Continuation of application No. 10/516,215, filed as
application No. PCT/GB03/02338 on May 30, 2003,
now abandoned.
(30)
Foreign Application Priority Data
May 31, 2002
(51)
(52)
(GB) ................................... 0212666.2
Int. Cl.
A61K39102
(2006.01)
CO 7H 21/00
(2006.01)
U.S. Cl . ................... 536/123.1; 536/23.2; 536/23.1;
536/24.33; 435/320.1; 435/41; 435/6.12;
435/6.1
Belanger, M et al, Microbiology, 1999, vol. 145, pp. 3505-3521,
Functional analysis of genes responsible for the synthesis of the
B-band 0 antigen of Pseudomonas aeruginosa serotype 06
lipopolysaccharide.*
(Continued)
Primary Examiner Albert Navarro
Assistant Examiner Ginny Portner
(74) Attorney, Agent, or Firm Kilpatrick Townsend &
Stockton LLP
(57)
ABSTRACT
The application relates to nucleic acids which encode
enzymes responsible for the production of the O-antigen of
Francisella tularensis, and their use as or in the production of
vaccines and in diagnosis.
(58) Field of Classification Search ........................ None
See application file for complete search history.
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US 8,198,430 B2
Page 2
U.S. PATENT DOCUMENTS
7,588,744 B1
7,592,326 B2
2 00 1/00 246 53 Al
2003/0022226 Al
2004/0087555 Al
2006/0280759 Al
2007/0066801 Al
2007/0128225 Al
2 00 7/02 18086 Al
2007/0264233 Al
2 00 7/029 23 86 A9
2008/0207568 Al
2009/0087456 Al
2009/0196887 Al
2010/0021501 Al
2010/0047283 Al
2010/0119524 Al
2010/0204184 Al
9/2009
9/2009
9/2001
1/2003
5/2004
12/2006
3/2007
6/2007
9/2007
11/2007
12/2007
8/2008
4/2009
8/2009
1/2010
2/2010
5/2010
8/2010
Sylvester
Karaolis
Gicquel et al.
Hooper et al.
Belmant et al.
Titball et al.
Engler et al.
Prior et al.
Tiollier
Michell et al.
Campbell et al.
Belmant
Eyles et al.
Morita et al.
Michell et al.
Michell et al.
Ulaeto et al.
Montero et al.
FOREIGN PATENT DOCUMENTS
EP
GB
GB
GB
GB
GB
RU
WO
WO
WO
WO
WO
WO
WO
WO
WO
WO
WO
WO
WO
WO
WO
WO
WO
WO
WO
WO
WO
WO
WO
WO
WO
WO
WO
WO
2123285
2321103 A
212666.2
0511722.1
0625587.1
2445028 A
2240822 C2
WO-88/08430
WO-92/13871
WO-93/11791
WO-9741234
WO-01/26683
WO-01/58485
WO-02/18600
WO-02/60935
WO-03/068151
WO-03/102191
WO-2004/004654
WO-2004/084935
WO-2004/098491
WO-2005/013918
WO-2005/021708
WO-2005/054258
WO-2005/063802
WO-2006/067635
WO-2006/103568
WO-2006/111019
WO-2006/131752
WO-2007/028985
WO-2007/034166
WO-2007/097789
WO-2008/012538
WO-2008/075075
WO-2010/086617
WO-2010/119245
11/2009
7/1998
7/2002
6/2005
12/2007
6/2008
4/2004
11/1988
1/1992
6/1993
11/1997
4/2001
8/2001
3/2002
8/2002
8/2003
12/2003
1/2004
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U.S. Patent
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Sheet 1 of 19
LL
US 8,198,430 B2
Fig.3.
WbtL?
WbtL
WbtJ
WbtE
WbtE
WbtM
WbtF
WbtF
WbtA
WbtN
WbtH
WbtH
WbtC
WbtK
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WbtD
Wbt6
U.S. Patent
Jun. 12, 2012
Sheet 3 of 19
US 8,198,430 B2
Fig.4.
SEQ ID NO 41
1
tcttttataa atgatgatag caaacaaaaa ataataggtt ctgtgcacaa aaaacttaaa
ttatattgaa aatagctaaa ccgctgttat ttaagatttg aaaagcaata aatatcaatg
61
121 gtttagcaaa tgaattatca tataaaagaa gtattctggt caattatttt atcattctta
181 aaatcacaaa aaggtataca taccaatgat gaagccaaat taagattgtt tattgaagct
241 gtattttatg tgttacgtac aggctgtcaa tggagaatgt taccatttta ttatggtaaa
301 tatagatcaa tacataagcg ttttaaagat tggtgtgata aagatatatt ttctagatta
361 tttaaatcag tacaaaaccc tgatttacaa gaagtcatgc ttgattcaac aatagcaaga
421 gcacatgctt gtgctacggg atatgataaa gatgataacc aagcaattgg tagatcagtt
481 ggtaggataa ccactaaaat ccatgctatg actgatgctt taggtaatcc aatagaaata
541 ttgttgtcag aggataaaac tcatgatagt aaagtagcta tagatttact aaaaaatgta
601 tataatacaa aagttatcgc tgatagagca tatcattcta atgaaatcag gcagcatatt
661 caaggtatat cctctgaagc tgttatccct tgtaaatcaa atactctaaa ccatatacct
721 tttgatagtc atgtatataa agaaagacat ttgatagaga atttcttttc taaaattaag
781 cattttagaa gagtattctc tagatttgat aaaaccattt tagcatatat aggaatgatt
841 aaattagctt gtacttttat ttggttacga tgaatattta tttttgtgca cagaacctaa
901
tttgcatttt tgtgcacaaa gaaaattttt ttgatataat agactttaat aggatatttt
961
ctaaaaatta acaaatgtct ttctacgata atagaacgct taatttcgtg gtaataatag
1021 ttttaactat tattactgtt aattggactt tctatatttt caagcaagat gttaatttac
1081 attttttact tgcattagtt ttgctgagat gcttgtcatc ttttttacta cttagagatt
1141 atatggctag ttggcgtaag tcgactcaaa aaactttttt acgtaaggct tttattaatt
1201 tgccagtatt tttcatagtg gcattatttt tttatggcaa agtcactttt tcgttgatat
1261 tctctgagtt tttattttat gtttttttga tcagtttaag tgtctacttt tattggtatt
1321 tgatgaacag aggatcagtg gataaaagta aaactgcggt tatttatggt gcaggtgctg
1381 caggaacaaa gattgctcaa gaacttgctt ctgctggtta tcgcatcaaa tgttttgttg
1441 atgacaatga aactttacaa aaaagaagta ttgatagtaa aaaggttcta tctaaagctg
1501 aattaacaaa actattgcta tctagtagat ttgacctttt ggttattgca ttgccaagaa
1561 atgcaaacca agtagtcaaa aatatatata aagaatttga aaaggatttt aatcagatta
1621 gaattatgcc gcctcttgag gaaattcttc aagatgagaa ttttatgtca cagttgaagc
1681 ctgtttcact ctatgatcta ttagcgcgtg atactaagag tttagataaa gaatctatct
1741 ctaattttat caaaaataag gtggtgctag tcacaggagc tggaggtagt ataggttctg
1801 aaatagtaca tcaatgtatc aagtatcagg caaaagagtt gatattggtt gatcatagtg
1861 agtttaactt atataaaatt actgaggagt gtagtcattt taatatcaat agtgtgctat
1921 gttctgtttg tgatagaaaa gcattggctg aggtttttca aaagtatact ccaaatatag
1981 tatttcatgc tgctgcctac aagcatgttc ccttagttga ggagaatatc tctagagcaa
2041 ttagaaataa tatcttaggt actaagaatg ctatagatct ggctatagaa gctggtgttg
2101 agtcatttat attgatttcc actgataaag cagtgcgacc aacgaatgtt atgggggcta
2161 ccaagagagt ttgtgagctg tatttacaga atgttgatcc caaaaatacc aagcttgctg
2221 cagtgcgttt tggtaatgtg cttggtagta gtggcagtgt gattccaaaa tttgaagagc
2281 aaataagaaa aggtggtcct gttacagtta ctcatcctga aattacacgt tattttatgt
2341 tgataccaga agcttgtgaa ctggtcctac aagctggtgc tattgcaaaa aattcagagg
2401 tctttgtctt agatatgggg caacctgtca agattattga tcttgctaaa caatttatta
2461 gactttctgg tagaggtgat attgatatta aaatagttgg tttgcgtcca ggagagaaac
2521 tttacgaaga gcttttgata gaggaagatg atgttagtac cgactataaa gatattttta
2581 ttggtagaag gactttttac gatattaata ctctaaacca agatattgaa tcgttgatca
2641 aggatgatgt tgatcagctt gtgatattaa agaaaattgt tccggaattt gaacatagat
2701 tgaatgggta gtggttttat gttttatgag gtttttaaaa gattgcttga tattttactt
2761 tcttttatgg ggttgttgtt attaagtcct attttcttaa ttattatttt tatgataaag
2821 aaagattcaa aaggacctat attttttaaa caaaagcgct atggtaaaga taagcaattt
2881 ttttacatat ataagtttag aactatgtat gttgatactc caaaagatat gccaacgcac
2941 atgttacagg atccatcgaa atgtataact aaggttggag gatttttaag gaaatcatct
3001 ttagatgagt tgccacaaat tataaatatt ctaaaaggtg aaatgagcat cgtgggtcca
3061 agaccagcat tatggaatca agatgactta atagcacaaa gagataagta tggggcaaat
U.S. Patent
Jun. 12, 2012
Sheet 4 of 19
US 8,198,430 B2
Fig.4 (Cont I).
3121 gctgtgcctg tgggactgac tggctgggca cagattaatg gtagggatga attaccaata
3181 cctgataaag ctaaacttga tggtgattat gtaaaaaata aaagtacatg gtttgattta
3241 aaatgtattt ttttgacagt attttctgtt tttgccaaaa agggcgtcgt tgagggtggt
3301 actggagctt taggtaacaa agaggattta aagtagtatg aaaaaaagaa tcttagttac
3361 aggtttgagt agctatattg gtaactcatt tgcggctaaa tataactcag attttagtat
3421 cgataaaata tctttgcgcg atgtttcgtg ggcaaatata gacttaagtg gttatgatgc
3481 tgtattgcat gtcgctggaa ttgcccatac ttcaaaggat cctaaactaa aagaaaaata
3541 ctataaaata aatacgcaat taacttatga tctggcaaaa caagctaaag atcaaggtgt
3601 tcgacagttt gtgtttttaa gtagtattat agtttatggt gatagtgcgc caataggtca
3661 acaaaaagtt ataactaaat ataccgaacc taaaccagat gatttttatg gagatagtaa
3721 gcttcaaact gaaattaagc taaatagcct ggctagtgat gactttaata ttgctataat
3781 cagaccacca atggtatatg gagaaggctc aaaaggcaac tatccaaagt tggttaaact
3841 tgcaaagtat acttttattt ttcctaatat taataaccaa agaagtgtta tatctataga
3901 taatttatct aaagagattg cagaaataat tttgcaaact aaacatggag tttttctact
3961 tcaagataat gaatattttt gcacttcaca gtttataaaa aactatagaa aagatgtttt
4021 aggtaagaga acttatctga caaaaatttt taatccaatt ataagattgc ttgctaaaaa
4081 agtagatttt attaataaag tttttgggaa tttgacttat gagaagtaag ttattattca
4141 tagctaatga ttttgatatt gtaatatatc gtttcagaag agaagtaatc gagtcttttg
4201 ctgctaaaga gtatgagata gtactagtaa caccatattc taagaaagca gaggtttttt
4261 gtaaaagtct tggtgttaag tatataaatg ttgatataga tagacgaggc aaaaatcctt
4321 ttaaggattt gcttctttta tttaactatt tcaaaataat aaaaaaagaa aaacctgatt
4381 acatttttag ctatacaatt aaaccaaatt tgtatgttgg gttagtgaat ttgtttttta
4441 ggaagaagtt ttatccaaat gtaacaggct taggaagtgt ttttgctaat catggtattg
4501 ttcagaagtt tataatatct ttatataagt tatcatttaa aagcaccaca aaagtattct
4561 ttcagaatga gcaaaataaa aagttattta tagctaagaa aataatcagt ggagaaaaat
4621 caatattatt accaggttct ggggtaaact tagatgaaaa taaatatgtt gactatccta
4681 aagaccaagg aatattaaaa ttcgtttttc ttggccgaat aatgaaagaa aaggggattt
4741 atgaattgtt agaagccttt gctatacttg agaaaaaata taaaaatatt agtcttgaca
4801 tttatggttt ttgtgatgaa aataaatcta attttatggg aaaggttaat acgataaaat
4861 cagtaaaatt ttatggtttt actgataata ctaaagaaaa aatagctagt gcacatgcag
4921 ttgttttgcc atcttaccat gaaggaatgt caaatgtgct gttagaagca gctgcgatag
4981 gtagacctgt aattgcgtca gatattcctg ggtgtagaga aatttttgat gatggtctct
5041 ctggcttatc atgtaaccct aatgatgtga gttctttacg taactcatta gagcagttta
5101 taaatatgtc gtatactgat aaaatagcta tgagctataa agctagagct aagatagaaa
5161 aagattttga tagaagtatt gttgtcaatg catacttaca gcaaaattaa taataagggt
5221 ttaaattatg agtttatatg aggatatagt cgctaaaaga gaaaaggttt cattggttgg
5281 cttgggttat gttggtttac caatagctat tgcatttgca aaaaaaatag atgtgttagg
5341 atttgatatt tgtgaaacaa aagttcaaca ttataaggat ggttttgatc caacaaaaga
5401 agtaggagat gaggctgtca gaaatacgac aatgaaattt agttgtgatg aaacaagtct
5461 taaagagtgt aaatttcata ttgttgcagt tcctacacca gttaaagcag ataaaactcc
5521 tgatttgacg ccgattatta aggcaagtga gacggttggt aggaatcttg tcaaaggcgc
5581 ttatgttgtg tttgaatcaa ctgtttatcc tggtgttaca gaagatgttt gcgtaccaat
5641 acttgaaaaa gagtctggct tgaggtctgg tgaagatttc aaagttggtt actctcctga
5701 gaggataaat cctggtgata aggttcatag gttagaaaca attatcaaag tagtatctgg
5761 tatggatgaa gagtctttag atactatagc aaaagtttat gagctagtag tagacgcagg
5821 agtttataga gctagtagta taaaagtggc tgaagctgct aaggttatag aaaactctca
5881 aagagatgtt aatatagctt ttgttaatga gttatcgata atatttaatc agatgggtat
5941 tgatactcta gaggttttag cagcagctgc aactaaatgg aatttcttaa actttaagcc
6001 tggtcttgtt ggtggacatt gtattggtgt tgacccatat tacctaacgt acaaggcagc
6061 tgagcttgga tatcattctc aggtaatatt atctggtcgt aggataaatg atagtatggg
6121 taaatttgta gttgagaatt tagtcaaaaa actgatatct gcagatatac ctgttaagcg
6181 agctagagta gcaattttcg gctttacttt taaagaagac tgtcctgaca ctaggaatac
6241 tcgagttata gatatggtaa aagagctcaa cgagtatggt atagagccat atattataga
6301 tccggtagct gataaagaag aggctaaaca tgagtatgga cttgagtttg atgatctaag
6361 taaaatggtc aatctagatg cgatcattat tgctgttagt cacgaacagt ttaaagatat
U.S. Patent
Jun. 12, 2012
Sheet 5 of 19
US 8,198,430 B2
Fig.4 (Cont
6421 aacaaagcaa cagtttgata ggctatatgc gcataattct agaaagatta tatttgacat
6481 caaaggtagt ttagataaat ctgagtttga aaaagattat atttattgga gattgtagtg
6541 gcttacgata atgttaaatt tcctcatggt tcgttttttt tggtgactgg aggtgcgggt
6601 tttattggct ctaatttatg tgaagtttta cttagtaagg gttatagagt taggtgttta
6661 gatgatctct caaatggtca ctatcacaat gttgagccgt ttttaactaa ttctaattat
6721 gagtttataa aaggtgatat tagagattta gatacttgca tgaaagcttg tgaaggtatt
6781 gattatgttc tacatcaagc tgcttgggga agcgtaccaa gaagtattga gatgccatta
6841 gtgtatgaag atataaatgt taaaggtgca ttaaatatgc ttgaagcggc tagacaaaat
6901 aacgttaaaa aatttgtcta tgcttctagt tcatcagtat atggtgatga gccaaattta
6961 cctaaaaaag aaggtagaga aggaaatgtt ttatcaccct atgcatttac aaagaaagct
7021 aatgaagagt gggcgagact atacacaaag ttatatggtc tagatactta tggtctaaga
7081 tattttaatg ttttcggtag aagacaagat cctaatggtg cgtatgcagc agttatacct
7141 aaatttatca aacagttatt aaatgatgaa gcgccaacta taaatggaga tggtaaacag
7201 tcgagagatt ttacatatat agagaatgtt attgaggcaa atcttaaagc atgtttagca
7261 gatagtaagt atgccggaga gtcttttaat atagcttatg gaggtagaga gtatcttata
7321 gatttgtact ataatctttg tgatgccttg ggtaaaaaaa tagagccaaa ttttggtcca
7381 gatagagcgg gtgatattaa gcatagtaat gctgatattt cgaaggctag gaatatgctc
7441 ggatataatc cggaatatga ttttgaatta ggcataaagc atgctgttga gtggtattta
7501 attaattaaa tggtatttta atcaagtgta cataaaaaaa gtgtctttta aaattttata
7561 tttatattta ctagcttttt gtattatttt tagtttagaa tttaaatttg ctatattgaa
7621 tattatagtt tatcttccgg cttgtatttt gggtttttta gctcttaaaa aactatttgt
7681 cggaaatatt gttaagaaac aattagcttt cctttttttc tttttctttt tatcaatgat
7741 ttatttaata atagtccaaa taatcttact tgatgcagca tcattgtttc ctcagttttt
7801 atttaacatt ttgatcgcga taggtttttg taactttatt tttgtttcat atgataataa
7861 tgaaaattat ttttttaata tgtctaaaat aatatttttt gttactttct tacaatctat
7921 ttttgtattt ctttcaaggt attatatatt tttaaatgat tggatattct tttttttagt
7981 gaaaaaaggg aatattgaga tttcgaatgt tattgaatat aagttaagag tattcggact
8041 tagtaacgct ggaggggatg gtttaggatt ttcaattact ataggattat gtttttctat
8101 attttatttt atcaaatata ttaaaggtaa atctatattt accaaactta tgctgtttgt
8161 acctttaatt cttattgtgt tttctaatat tttcatatct agaacatcac tcttaacttc
8221 ttcacttata ttgttaataa caatatttta tatatatatt aaaaaagaaa aattactgtt
8281 tattataata ttggcgctat tctttttatc aatatggata ttgttcaaat taaatttgaa
8341 tttgagttgg gcttttgaaa atatttactc gtacattcaa tctggcgatt tttcacatgg
8401 aagtctaagt gttttaatca ataaaatgct ttttgtgcca gataaccttt tgacttggat
8461 atttggttgt gaggatgtta gtaatactga tattggttat attaaatatt tatactatta
8521 tgggattata tttagtatgt ttttttatat tcttattatt ttcttgtact ttgaaatgag
8581 aaaatgtttt atattttcag agtatcgatc attatttcta ttgttgttaa tagtatgttt
8641 agtttttcaa gcaaaaataa tttttttgac agtaggatta tttactaaat taaccattat
8701 attatttatt ttttctctta aagaaaacag ctttacaact aggagtgtga tttgaaaagg
8761 tttgtacatt taataataaa ccttaaccaa ggtggtgctg aaacaatgct ttataaactt
8821 tgcaaatcta tggataagtc aatatatcat attacgatta tatcacttat gggtagggga
8881 gtatttgcaa ataagttaga agcttatggt gttaaagttt atacattaaa tttaaataaa
8941 tttaatgtac tatttgtatt gtttaaatat attaagatta tcagaagaat aaagcctgat
9001 gttattcatg cttggatgta tcatgcaaat gtaatttcta tattatgcaa gcctttttat
9061 agaaagacta aatatataaa tagtataaga atgggattgg agaattatga tggtcataag
9121 aatcttacaa agtttatgat aaagttgaat gcaaaatttt ctaagttctc agatttaaca
9181 ttaaataatt caaagaaatc attagaagat catcaaaata taggttttaa aaaccaatgc
9241 tttatagcaa atggttttga taaagatgtt tttaaaccga gctttttaaa gtatgaaaaa
9301 tttcgtttaa ataatgattt agatgataat gttaaaatta taggtatcat agcaagaaat
9361 catgctgata aaaatatttc tcgtttctta caaatagcta atttattgtt aaaaagtaat
9421 cctagtttac ggtttttaat tgctggaaga gagtgttcga aaatagatat aggtagttat
9481 ctagataaca aaagtaatgt aaataagttt tttgtatttg aatctgtgga ttctagtgaa
9541 tacttaccag tattagattt atatttgtct acatcaaaag ttgaaggttt tccaaatata
9601 cttgcagaag ccatgctatg tgaagttcct attgttgctt ctaatgttgg agattgtaaa
9661 gatatactta atggatacgg tgaagttttt gagcttagtc aaggtaataa agaaataata
U.S. Patent
Jun. 12, 2012
Sheet 6 of 19
US 8,198,430 B2
Fig.4 (Cont III).
9721 gaaaagatta tgaaagtttt agaaacaacg gtagtcatga aaaagcgcat gagagaatat
9781 ataataaata attttagtat agaagctatt ttggaaaaac"acgaaaaact ttatcatgag
9841 ggcagtgtct aatgtgtgga gtagtaggct tttactcatt taataaagaa gaaggttttg
9901 actcaataat taatcaatca ttgctttcta taaagcatag agggtcggat gatagtgggt
9961 attggtgcga caatcaagtt actctggggc atactagatt atcaatacac gatataacta
10021 atgcgggaca tcagccaatg ttatctaata gcggtaatac tgctattgtg tttaatggag
10081 aaatatataa ttacttatcc ataaaaaatc agctattaag tgaatattca aatcttaaat
10141 ttaaaagtaa cagtgatact gaggttttgg tcaatgctat tgaactttgg ggtatagata
10201 aaactttaga aaaatgcata ggaatgtttg cttttggagt ttacagtaga aaaactagtt
10261 gcttaatact agctagagat agatttggcg agaagccatt atattttggt atccaaaatg
10321 gtattttggg ttttgcatca gaattgaagg cacttaagcc attaaaggaa tgtggctgga
10381 ggtttgatat agatagagat gctttagcaa catatatgag gtatgcttat gtaccaacac
10441 catactctat ttataaaaat atatctaaac taaatgtagg tagttacata aaatttgatg
10501 ctaaaggtaa tagtaaagag tataaatatt gggattctaa aaaagtacta gattcagaaa
10561 aatataaaga ttcgtatgat caagcaatcc tagatttaga aattaagctt aaaagtacac
10621 tatcaataca aatgcagtca gatgttcctc taggagcatt tttatccgga ggaattgact
10681 caacaactgt agttgctctt atgcaaagta tgtctaaaga taagataaac acttttagta
10741 taggttttaa tcaaaaagaa tataatgaag ctgagcatgc aagagcagta gcaaaacata
10801 taggtacaaa ccacacagat atgtatgtta cagaaagaga tgctcttgat gtaataccaa
10861 aacttgctgg aatatatgac gagccctttg ctgattcatc acaaatacca acgtatcttg
10921 tgagtaaaat agctaagtcg aaagtaacag ttgcactatc aggtgacgct ggtgatgagc
10981 tctttggcgg ttataataga tactttttag caccaaatat tgctaaaaaa atcaaatttg
11041 ctaagttact taaatatgca ccagatgctt ggataaaaaa agctgagata ttaaattttg
11101 gtaagttcgc tttattagca gataaactac taaaactaaa aagagttctc gaaaaagcaa
11161 aaacaaataa agagctttat gtactacttt gttcacaaat aaatgatact agctttgtgt
11221 taggagcaaa agagtatgat atattaagag ataagaatat ttatgatatt ccacaattat
11281 ctttccaaga gtggatgatg tttgttgatt ctaatacata tatgatagat gatatattgg
11341 ttaaggttga tagagcagct atggctaact ctctagagac aagagtgcca tttttagatc
11401 ataatattta tgaatttgct tattccttac caattgacta taaaatacaa cgaggtaacg
11461 gaaaaagaat tttgaaagat ttgttatata aatatgtgcc agaaagtttg gtcaataggt
11521 ctaagatggg gtttggtatt ccgcttgcta aatggttaag agaagattta cgagagtggg
11581 cagataattt actggattat agtaaaatag acaagcaagg ttacttaagt cctgaggtgg
11641 tgcaaaaata ttggcaagag catttgagtg gtaaaagaaa ttggcaagca atattatgga
11701 atattctaat ttttcaggag tggttagata atgagtaaag taaatgtaac aaaaccatac
11761 ttaccagata taaataaata taaaagctat gtaaataaaa tatacaaaaa tggatggctt
11821 actaataatg gtccgttagt gcaagagcta gaaaaaagac ttgcaaagta tctaggtgtt
11881 aaaaatatag ttttagtatc aaatggtaca attgcattag aaatcgcgta tagagcgtta
11941 ggagtcaaag gaagtgcaat tactactcca ttttcatttg ttgctactac atcttcattg
12001 gtttctaaca atgtaaaacc agtgtttgtt gatattgatg agaatactct aagtatagac
12061 gtctctaaaa ttaagtatgc tattgaagag gatacttcag ctattgtgcc agttcatgtg
12121 tttggaaatg gttgtgaagt tgaaaaaata gacatgctgg ctaaaaaaca taacttaaaa
12181 gttatttatg atgcagcaca tgcttttgat gttaagtata agggtgagag tatattaaac
12241 tatggtgata tttcgacatt aagttttcat gcaacaaaga tttttcattc tattgaagga
12301 ggtgcgctta tcattaatga tgatagtctt gttgaaaaag ttcgttattt cattaatttt
12361 ggtatagaaa gctcagaatc aataccttac ttaggtacta atgctaaaat gaatgaattt
12421 gaggcggcta tgggactttg tgttctagat gatattatag aaattaagag caaaaggaaa
12481 gttattacag agatatatga ggctgggtta gatggattgg taaagtttca agaacagaat
12541 cagcattcta gtaggaatta tagctatttt ccagtaatat ttaggactga ggaggaactt
12 601 ctcagagtac agaaagcact aatacaaaat gatataatat cgcgtagata tttttatcca
12661 tcattagata gtcttagtta tatagagcca aagcagtata tgccaatctc aagagatata
12721 tctaaaagaa tattatgttt gccaatttat gcagagttag aagacgataa aattaataaa
12781 ataattaata atatcaaaga ggtttcctca tgaaaaaaat atttgttgtt acagataata
12841 gaactattct aagtgatttt aaaaatatca ttggtagtaa aaatgatgta caggttgatt
12901 atttttgtag tttcaagagt caaacttctt ttgccaaaga aatatataac agtgagatta
12961 agccaataga tatgaaaaaa aatggcaatg atcttattgg taagtatgat ttaggttttt
U.S. Patent
Jun. 12, 2012
Sheet 7 of 19
US 8,198,430 B2
Fig.4 (Cont IV).
13021 cttgtcattc gaaacaatta tttccagcaa aattagttaa ttcagtatta tgtataaata
13081 ttcatcctgg acttaatcca tataatagag ggtggtttcc acaggtcttc tctattataa
13141 ataaactacc tataggagca actattcatg tgatggatga agagatagat catggagata
13201 taatcattca ggaagaagtt gaagttaatt ctttcgaaaa ctcttttgat gtttatgcta
13261 aagttcaaaa aaaagaagtt gagttgttca ctaaagtcat agatgatatt ttgaataata
13321 agttcactcg aatcaaacct aactccgaag gcaactataa ttcaattcat gattataaaa
13381 acatgtgtga aattgattta gataaaatag taacaatgcg ggaagcaatt gactatctaa
13441 gggctatgac acaccctcca tataaaaata gttatttcat tgatgagcat ggaaataaag
13501 tatttgttgc tcttgaactt gaaaagataa gttagaaaaa tgagccttaa aaaaaataca
13561 atatcaaatt atataacaca actatatact agcttaattg gtattgttat acttcctttg
13621 tatttacaac atttaagtca tgatgcattt ggtctgattg gtttttttac agtttttcaa
13681 acgtggttac ggttgttgga tgttggtata acaccaactt tatcaagaga agtggctcat
13741 gttagaggta gtactgatga ctatcattac ttacgcaagt tggttagatc gttagagcta
13801 tttttcatta ttgttggtgt tctggtattt attgtaatta gtacacattc aaggtatata
13861 tccacctctt ggttacatat aggctcgcta gatgctgata gtgtaagtgt atgtattgca
13921 cttatgggtt taatgtttgc attaagatgg gtgtctgatc tatatggtgg tggtttgcgt
13981 ggctttgaaa gacaggttct ttataataat ttaagtatca tacaaacgac actacagttt
14041 attggtggat tattatttat ctgctatgtg tctactaata ttatgtatta ttttgtatat
14101 cagacaataa ttgcgatact atatctagta tgtattgcaa ttgcatttta taaaatacta
14161 ccatcatcat ttagcgtggg tttaaggttt gattttaaaa taattagaaa agtgcttcca
14221 tttgcactag gcattgcata ttctacaaca gtttggatta ttgtcactca atctgataaa
14281 ttagtgttct cacatgtatt accattatct gagtatggtt atttatcttt attgatagtg
14341 atatctagtg ctgttacgat attgtcctct ccgattagca tagctattca gcctagaatg
14401 acaatgctat tagcccaaca aaatgtaaaa ggaatggaaa gcttatattt aaaatcatcc
14461 ttgatctcaa ttactttttt atctgctgta gtaacatgtg ttttgatgta ttctcatcag
14521 ctgttgcagt catggacagg aagtatggaa attgctaatt ggggtagtaa tatcttaaat
14581 atatatgttt tatcagcatc tattatttgt ataatatcat ttcaatattt tttacagtat
14641 gcttatggta agttaaagct acataataca tataatacaa ttagtttagt attttttgct
14701 cctatagtta tatatactgc ttataattat ggagtgtata ctacagcact attatggctt
14761 ggatatgcta tagtggggct gataatctgg atgcctattg tacaccatgt atttgctaaa
14821 ggtatcaata ggtatttttt tataaattta gcagttatta ctatagtatg ttttttatta
14881 tcgttaatat ttaagggttg gtatatttat ccaagtaaaa ttgggttggt agaattaata
14941 ttgattgggt ttgcattttt atttatacaa atttgtatag agtatgtttt gtttcggtac
15001 aaggttttga ggtgtataga tgattaaagt ttcagtatgt gtgatgacat acaatcaaga
15061 aaagtatatt ggtcaatgtt tagagtcttt ggttactcaa gagactgatt ttgactttga
15121 gataatcgtt ggagatgatt tttctacaga tggtacaaga gatgttattc aagagtatca
15181 aaaaaagtat ccggatatca taaagccagt ttttagagat aagaatgtgg gaattactga
15241 aaatattaaa gaaatctatt ttgttgcaaa tggtgagtat atagctcata tggatggtga
15301 tgattatgca ttgcctggta aacttcaaat tcaggctgat tttttggata ataatccaag
15361 atgtacggga gtttttcata atataaatat actctatcca aatggtaata tacaacatag
15421 taggtttgct tgttcaaata agagtatatt caatttatca gacactttac gcggagttgc
15481 tgttggtgca aatagttcaa aaatgttcag aacatcggtt ttggatgatt tgattttacc
15541 ggatatagag cttctagatt attattttca tgttataaca gcagaaaaag gttatttaag
15601 ttttttaaat tctaatgaat cctatagtgt gtacagaaaa ggtattggta tcacatctaa
15661 gtctaaggaa aaaatctata atacttatgc tggattattt gaatattttt tggatagata
15721 tcctgaagag aaattaaata tttgtatccc.tgttgtgcaa atgataattt cggctattaa
15781 agggagatgt tttattagtg ctattcgtct attcaaaatt ttaattagat caagatgtat
15841 tccattagta agttggttta aatatagatt tgaaaaataa atatcattta gaggattatg
15901 tgaaatgaag ggaataattc tagctggtgg cagtggtaca aggctatatc cacttacctt
15961 gggtgttagc aaacagctgc tacctgttta tgacaagcca ttgttatact atccactatc
16021 tgtgcttatg cttgcaggta ttagggagat attaattatc tctacagtgc gtgatatctc
16081 acttatccaa gagcttcttg gtgatggttc acaatttggt atacagttga gttataaaat
16141 ccagccatca ccagatgggc ttgctcaagc atttattctt ggtgaggagt ttttggcggg
16201 tgactcagct tgtttgatat taggagataa tatctactat ggtcaaggta tgactacaat
16261 gctagagtct gcaagagcac agtgtggagg tccagctggt ggcgcttgtg tttttggtta
U.S. Patent
Jun. 12, 2012
Sheet 8 of 19
US 8,198,430 B2
Fig.4 (Cont V).
16321 ttatgttaat gatccgcata gatatggtat agtcgaattt gataagcaaa aaaatgtaat
16381 ttcggtagag gaaaagccac agaatcctaa gtcacactat gctatcacag gtttatattt
16441 ttatgataat aatgttgttg agtatgctaa acaagtcaaa ccatctgcac gtggtgagct
16501 agagattact tcacttaatg agttatatct aaaagaaaat aagctaaatg tcgaactctt
16561 agggcgtggc tttgcttggc ttgatgctgg tacgcatgat tcattgctag aggcaggtca
16621 atatgtcgca actattgaga aaagacaagg gcttaaaatt gcatgtttgg aagaaattgc
16681 atggcgtaaa ggctttatct caacacaaca agttctagct caagctgaaa aactttctaa
16741 gacagagtat ggtcagtatc tgaagaattt aattaaggat ggtttataaa ttaatccgtc
16801 atacccatga aggtgggtat ctcataaaag ttggatatgt tttggagatt ccaatctgcg
16861 cagtaatgac aggtttggta atatatagcg atgttttaca atgactaaaa atggttttat
16921 gtatattctt acaaataagg ataatactgt tctgtacata gttgtaacat ctaatttgat
16981 aaaaagaatg tatgagcata aacatagcct tgcagatggt tttactaaaa atataatgtt
17041 aataagttag tttattttga aatttatgaa gatataaaag cagcaattct gtgagaaaag
17101 cagttgaaaa aatgaaacag atcttggaaa gaacgaatta ttaatgagat gaatccgaat
17161 tggaatgatt tatatgaatt aatatgtgag taaaactttt gtcttactgg tgcagatagg
17221 tatctctaaa tatcagatgt gattgggaga ttaccgccta cgcggtaatg acaagtttat
17281 gcggtaatga tagtttagtg agagaatgac tagtcactat aggaatgatg atgtaatgag
17341 gaatgaaaaa atgaactaca aaccaaaaaa tatcctagta acaggtgcgg cgggatttat
17401 tggtagtaac tatgtgcgta tgatgttatc acgctatagt gatatcaaaa taatctcgta
17461 tgataagctt acttatgcgg gtagtttaga taatctaaaa gacttgaata atgaacataa
17521 ccatactttt ataaaaggtg atatttgtga tgaagtttta gtatatcaaa cactgaaaga
17581 atataaaatt gatacgatag tacattttgc tgcagaatcg catgttgata attcaattgc
17641 taatccaaag gtatttttag aaacgaatgt gataggtaca tttacacttt tagattgtgc
17701 taaaaggtat tggttagatg agctaggttt agaagaaact agttgtaggt ttcatcatgt
17761 atctactgat gaggtatatg gtaccttggc aaaagatgaa ccagccttta ctgagattaa
17821 ggcttatgag ccaaattcac cgtattcggc atctaaggcg ggatctgatc atatttctag
17881 agcatatcat catacctata aacttccggt aacaatttca aattgttcaa acaactatgg
17941 accataccaa catcgagaga aattaatccc tgtagtgata aatagttgta taaactacaa
18001 gcctattcct gtttacggag atggttcgaa tattcgagat tggctatatg tagaagatca
18061 ctgcgatgct atccagacaa ttgttgagaa aggagtggtt ggagaggttt ataatattgg
18121 tggtattaat gaagttgata atctaacctt ggtaaaaact atctgtaaac taatggatga
18181 atataaacca gaaaatgctc cacattctaa cttaatcaca tttgtggaag atagaaaagg
18241 acatgattgg cgttatgcta ttgataacag caagattcag aatgagttag gatggaagcc
18301 atcacaagat tttgataaga tgtttagaca aactattgag ttttatctat agcttaaata
18361 tttatcttat gagtatctct aaaaaatcaa tttaatttat ttttgtgtta aaaagtagtg
18421 ttcgcaagaa tatagttaat ccgaaagata tttgtagaaa aagatatttg tagaaatgtt
18481 ataatgtcta ataaaaatgc catcatatag ccaagatttt agagacatcg taattaataa
18541 acatgaagaa ggtatgacgg agttcgagct gagtaagttt tttaacatag ataagcgtac
18601 agttgtttca tggatagagt tttataaaag aaccggagat tatagttcaa agcaaggagt
18661 tggttgtggc agagtcgcta gctttaccga taaaacattg attgaacagt atttgataga
18721 tcatccagat gcaagtgcat tagatataaa agaagcatta gcccctgata ttccaagaag
18781 tacattttat gattgtctta atagacttgg ttttagtttt aaaaaaagac tccaaaatat
18841 aagcaaagaa aagaacatga aaggttggag tatatagaaa aactaaaaga aatagccaat
18901 aaatttgatg tacaaatatt atatctacct ccgtactctc cagatttaaa tcctattgaa
18961 aaggtttggg ctaactatta aaaaaatatt tagaaaagtg aataatagtt ttgaaaaatt
19021 ttgtgatgct atctcttatg tgtttaacaa aatactctcg gattaactat atcatgctgc
19081 taaaatattc ttggtattct ctggtcaaaa ctgacataat gatgctctac tttgtataag
19141 gtttgctaca aatattatct aaacaaacat acaaaggtaa tttttagaga tcctattata
19201 aacctactat ctaaatttag taagttaagt tatgacaata tttaatttgc tgatttattg
19261 ttgaatatat tagctttcta tataattaat caatatcaaa gttatttagg tttttataat
19321 gattactcct attatcttat ctggaggatt cggctcaagg ctatggccac tatcacgaga
19381 ggcatcgcca aagcagttta tcggcttggt tgatgaacat agtctattag aaaatacaat
19441 taagcgacta gataatgtca aggatataac ttcacctgta gttgtctgta atgaaagtca
19501 tagattccaa gttgctgaag tgttgcggaa aatcaataaa aaaggcgata tactcctaga
19561 gccattagcc agaaatactg ctccagcaat tgcacttgca gcactacatt tagctattaa
U.S. Patent
Jun. 12, 2012
Sheet 9 of 19
US 8,198,430 B2
Fig.4 (Cont VI).
19621 tgatccaaat acaattatgc tagttttagc tgctgaccat catattgaaa atctggagat
19681 ttttcatcaa gctatcgaaa aagcacagca aaaagttatt aaagatgatt ctttagttac
19741 ctttggcatt acaccaactt gtcctcatga aggctatggt tatattaaac aaggggtaca
19801 gactactgta aatggagttt ataaggtaga taaatttgtt gagaagccta gtgtggtcgt
19861 tgcacaagag tatttagata gtggcaaata ctattggaat agcggtatgt ttatgttcac
19921 agctagagtg tatttagagg ttttagagaa gttacagcca gagatttaca gaggatgtga
19981 aaaaacttat caaaagtcac agcaggattt agattttgtg cgttttgata aacaaagctt
20041 tgccctagtt caatcacagt caatagacta cgcagttatg gagaaagcaa ctaatgttgc
20101 tatagtgcct atgcaacaaa gtggctggtc tgatgttggc tcttgggact ctttgtatga
20161 tattgctgca aaagatagtt gtggtaatgt ggttattggc gatgtgatta ctagtaatgt
20221 caaaaatagt tatttacgct cgcatgatcg tttattggct gcagtcggag ttaatgattt
20281 aataattgtt gaaacagcag atgctatact tgtcgcggat aagaacaaaa ctcaagatgt
20341 caaaaaaata gtcgaagttt tgaaaattca gcagcgaagt gaattattac agcataagca
20401 aatttataaa ccttggggtt cagcgacaat attagaggat aagtctggtt ataagataca
20461 ggcgattcaa cttgaaccgg gcaagaagtt atcattacag caacattatc accgtagtga
20521 gcattggatt gtgatttctg gaactgctac ggtaactatt ggtactacta agtctattgt
20581 tagaccaaat gagtctgtat atataaaaat aggcgaatct cacagacttg aaaataatgg
20641 caagattcca gttattctta tagaagtaca agttggagaa tatataagtg aagacgatat
20701 tgttagacta gatacaagta gttaatataa aaacaattag atagaaaaaa atataatgag
20761 acaaactata ataaaagaaa taatcaaatc tagcggcgta aagtttggta ctagtggagt
20821 tagaggtctt gtttcagcta tgacagataa gatctgttgg ctttatacaa aagcttttat
20881 tcaattccta gagcaaaaat actctattgc taagggtact aaaattgcta tagctcatga
20941 tctacgtgag agtagcccta gaataacaac agttgttatt aaagctatca tagatagtgg
21001 tcatgagcca atatactgtg gtgagatacc atcaccagct gtaatgctat atggtatatc
21061 taatcagata ccgtcagtta tggttactgg tagtcatatt ccagaggata gaaatggtat
21121 taagtttaat actccatatg gtgaagttct caaagaagat gaagaaatga ttgttagcca
21181 aactatcagc attgatgaaa gtatttttga taaaaatggc atgtttttac aaaaactaga
21241 attaccagag cctagtaagc aagcatatac acagtatatt gacaggtatg tagatttttt
21301 ccctaacaac tgtctagcag gtaagactat agggctttat cagcactcat ctgtaggacg
21361 agagatagtc aaagagattc tagagaaact aggtgctaag gttatcttgc tagaattttc
21421 cgaaaaattt gtatctgtag ataccgaggc aattcgccag gaagatgtaa agcttgctaa
21481 gcagtgggca agcaagtata aagttgatag tatagtttca actgatggcg atgctgatag
21541 gccactagtt agtgatgagt atggcaattg gctaaaaggt gatattttag gtgtactgac
21601 agctaaatat ctccaagcca atgttatcgt gacaccagta agtagcaata ctgtggcaga
21661 aaagataggt tattttagta acgtgattag aactaaaata ggctcgccgt atgtaattgc
21721 tgcaatgaat gaattactct caaataatca aaatgctgtg gttggatatg aggcaaatgg
21781 aggatttcta ttggctagtg atatttgtaa agatgataaa actctaaaag cgctgcctac
21841 aagagatgct gttataccaa tgttggctgt aatgatgcta tctatcaact ctaataaaac
21901 cgtgtcagag cttttatttg atttgccatc tcgatataca gcaagtagta aaattgatga
21961 ttttgcttcc gagaaaagcc aagaaatctt gaagtcaata ttagcaggtg aatcagatct
22021 tttagataaa attatatcgg agcattttga tggtaaaaat agcattgaaa atatcgatac
22081 tacagatggt gttagagtaa ctttgacaaa tcaagatatt atccatctta gaccatctgg
22141 taatgctcca gagcttaggt gctatacaga ggcagctagt gatgagcagg caaaaagttt
22201 aaatcaatat tgtgtggatt tgattaacaa aaacatttga agatcagtca aaaatattcc
22261 ctaacttttc tcttcaccat tgaaccatta ctaaccttat ctatagctag ccacagataa
22321 aaatgtcatg ctggatttat ttcagcgttt cattataaat atcaatttta ttgagatcct
22381 gaaactagtt caggatgaca g
U.S. Patent
Jun. 12, 2012
Sheet 10 of 19
US 8,198,430 B2
Fig.5.
SEQ ID NO 2
MNYHIKEVFWSIILSFLKSQKGIHTNDEAKLRLFIEAVFYVLRT
GCQWRMLPFYYGKYRSIHKRFKDWCDKDIFSRLFKSVQNPDLQEVMLDSTIARAHACA
TGYDKDDNQAIGRSVGRITTKIHAMDALGNPIEILLSEDKTHDSKVAIDLLKNVYNT
KVIADRAYHSNEIRQHIQGISSEAVIPCKSNTLNHIPFDSHVYKERHLIENFFSKIKH
FRRVFSRFDKTILAYIGMIKLACTFIWLR
SEQ ID NO 3 MSFYDNRTLNFVVIIVLTIITVNWTFYIFKQDVNLHFLLALVLL
RCLSSFLLLRDYMASWRKSTQKTFLRKAFINLPVFFIVALFFYGKVTFSLI£SEFLFY
VFLISLSVYFYWYLMNRGSVDKSKTAVIYGPIGAAGTKIAQELASAGYRIKCFVDDNET
LQKRSIDSKKVLSKAELTKLLLSSRFDLLVIALPRNANQVVKNIYKEFEKDFNQIRIM
PPLEEILQDENFMSQLKPVSLYDLLARDTKSLDKESISNFIIQJKVVLVTGAGGSIGSE
IVHQCIKYQAKELILVDHSEFNLYKITEECSHFNINSVLCSVCDRKALAEVFQKYTPN
IVFHARAYKHVPLVEENISRAIRNNILGTKNAIDLAIEAGVESFILISTDKAVRPTNV
MGATKRVCELYLQNVDPKNTKLAAVRFGNVLGSSGSVIPKFEEQIRKGGPVTVTHPEI
TRYFMLIPEACELVLQPLGAIAKNSEVFVLDMGQPVKIIDLAKQFIRLSGRGDIDIKIV
GLRPGEKLYEELLIEEDDVSTDYKDIFIGRRTFYDINTLNQDIESLIKDDVDQLVILK
KIVPEFEHRLNG
SEQ ID NO 4 MFYEVFKRLLDILLSFMGLLLLSPIFLIIIFMIKKDSKGPIFFK
QKRYGKDKQFFYIYKFRTMYVDTPKDMPTHMLQDPSKCITKVGGFLRKSSLDELPQII
NILKGEMSIVGPRPALWNQDDLIAQRDKYGANAVPVGLTGWAQINGRDELPIPDKAKL
DGDYVKNKSTWFDLKCIFLTVFSVFAKKGVVEGGTGALGNKEDLK
SEQ ID NO 5 MKKRILVTGLSSYIGNSFAAKYNSDFSIDKISLRDVSWANIDLS
GYDAVLHVAGIAHTSKDPKLKEKYYKINTQLTYDLAKQAKDQGVRQFVFLSSIIVYGD
SAPIGQQKVITKYTEPKPDDFYGDSKLQTEIKLNSLRSDDFNIAIIRPPMVYGEGSKG
NYPKLV LAKYTFIFPNINNQRSVISIDNLSKEIAEIILQTKHGVFLLQDNEYFCTSQ
FIKNYRKDVLGKRTYLTKIFNPIIRLLAKKVDFINKVFGNLTYEK
SEQ ID NO 6
MR SKLLFIANDFDIVIYRFRREVIESFAAKEYEIVLVTPYSKKA
EVFCKSLGVKYINVDIDRRGKNPFKDLLLLFNYFKIIKKEKPDY I FSYTIKPNLYVGL
VNLFFRKKFYPNVTGLGSVFANHGIVQKFIISLYKLSFKSTTKVFFQNEQNKKLFIAK
KIISGEKSILLPGSGVNLDENKYVDYPKDQGILKFVFLGRIMKEKGIYELLEAFAILE
KKYKNISLDIYGFCDENKSNFMGKVNTIKSVKFYGFTDNTKEKIASAHAVVLPSYHEG
MSNVLLERAAIGRPVIASDIPGCREIFDDGLSGLSCNPNDVSSLRNSLEQFINMSYTD
KIAMSYKARAKIEKDFDRSIVVNAYLQQN
SEQ ID NO 7 MSLYEDIVAKREKVSLVGLGYVGLPIAIAF KKIDVLGFDICET
KVQHYKDGFDPTKEVGDEAVRNTTMKFSCDETSLKECKFHIVAVPTPVKADKTPDLTP
IIKASETVGRNLVKGAYVVFESTVYPGVTEDVCVPILEKESGLRSGEDFKVGYSPERI
NPGDKVHRLETIIKVVSGMDEESLDTIAKVYELVVDAGVYRASSIKVAEAAKVIENSQ
RDVNIAFVNELSIIFNQMGIDTLEVLAAAATKWNFLNFKPGLVGGHCIGVDPYYLTYK
AAELGYHSQVILSGRRINDSMGKFVVENLVKKLISADIPVKRARVAIFGFTFKEDCPD
TRNTRVIDMVKELNEYGIEPYIIDPVADKEEAKHEYGLEFDDLSKMVNLDAIIIAVSH
EQFKDITKQQFDRLYAHNSRKIIFDIKGSLDKSEFEKDYIYWRL
U.S. Patent
Jun. 12, 2012
Sheet 11 of 19
US 8,198,430 B2
Fig.5 (Cont I).
SEQ ID NO 8
SEQ ID NO 9
VAYDNVKFPHGS£FLVTGGAGFIGSNLCEVLLSKGYRVRCLDDL
SNGHYHNVEPFLTNSNYEFIKGDIRDLDTCMKACEGIDYVLHQAAWGSVPRSIEMPLV
YEDINVKGALNMLEAARQNNVKKFVYASSSSVYGDEPNLPKKEGREGNVLSPYAFTKK
ANEEWARLYTKLYGLDTYGLRYFNVFGRRQDPNGAYAAVIPKFIKQLLNDEAPTINGD
GKQSRDFTYIENVIEANLKACLADSKYAGESFNIAYGGREYLIDLYYNLCDALGKKIE
PNFGPDRAGDIKHSNADISKARN! GYNPEYDFELGIKHAVEWYLIN
VYIKKVSFKILYLYLLAFCIIFSLEFKFAILNIIVYLPACILGF
L7L•FVGNIVKKQLAFLFFFFFLSMIYLIIVQIILLDAASLFPQFLFNILIAIGFC
NFIFVSYDNNENYFFNMSKIIFFVTFLQSIFVFLSRYYIFLNDWIFFFLVKKGNIEIS
NVIEYKLRVFGLSNAGGDGLGFSITIGLCFSIFYFIKYIKGKSIFTKLMLFVPLILIV
FSNIFISRTSLLTSSLILLITIFYIYIKKEKLLFIIILALFFLSIWILFKLNLNLSWA
FENIYSYIQSGDFSHGSLSVLINKMLFVPDNLLTWIFGCEDVSNTDIGYIKYLYYYGI
IFSMFFYILII FLY FEMRKCFIFSEYRSLFLLLLIVCLVFQAKIIFLTVGLFTKLTII
LFIFSLKENSFTTRSVI
SEQ ID NO 10 LKRFVHLIINLNQGGAETMLYKLCKSMDKSIYHITIISLMGRGV
FANKLEAYGVKVYTLNLNKFNVLFVLFKYIKIIRRIKPDVIHAWMYHANVISILCKPF
YRKTKYINSIRMGLENYDGHKNLTKFMIKLNAKFSKFSDLTLNNSKKSLEDHQNIGFK
NQCFIANGFDKDVFKPSFLKYEKFRLNNDLDDNVKIIGIIARNHADKNISRFLQIANL
LLKSNPSLRFLIAGRECSKIDIGSYLDNKSNVNKFFVFESVDSSEYLPVLDLYLSTSK
VEGFPNILAEn .CEVPIVASNVGDCKDILNGYGEVFELSQGNKEIIEKIMKVLETTV
VMKKRIIREYIINNFSIEAILEKHEKLYHEGSV
SEQ ID NO 11 MCGVVGFYSFNKEEGFDSIINQSLLSIKHRGSDDSGYWCDNQVT
LGHTRLSIHDITNAGHQPMLSNSGNTAIVFNGEIYNYLSIKNQLLSEYSNLKFKSNSD
TEVLVNAIELWGIDKTLEKCIGMFAFGVYSRKTSCLILARDRFGEKPLYFGIQNGILG
FA SELKALKPLKECGWRFDIDRDALATYMRYAYVPTPYSIYKNISKLNVGSYIKFDAK
GNSKEYKYWDSKKVLDSEKYKDSYDQAILDLEIKLKSTLSIQMQSDVPLGAFLSGGID
STTVVALMQSMSKDKINTFSIGFNQKEYNEAEHARAVAKHIGTNHTDMYVTERDALDV
IPKLAGIYDEPFADSSQIPTYLVSKIAKSKVTVALSGDAGDELFGGYNRYFLAPNIAK
KIKFAKLLKYAPDAWIKKAEILNFGKFALLADKLLKLKRVLEKRKTNKELYVLLCSQI
NDTSFVLGAKEYDILRDKNIYDIPQLSFQEWMMFVDSNTYMIDDILVKVDRAAMANSL
ETRVPFLDHNIYEFAYSLPIDYKIQRGNGKRILKDLLYKYVPESLVNRSKMGFGIPLA
KWLREDLREWADNLLDYSKIDKQGYLSPEVVQKYWQEHLSGKRNWQAILWNILIFQEW
LDNE
SEQ ID NO 12 MSKVNVTKPYLPDINKYKSYVNKIYKNGWLTNNGPLVQELEKRL
AKYLGVEQJIVLVSNGTIALEIAYRALGVKGSAITTPFSFVATTSSLVSNNVKPVFVDI
DENTLSIDVSKIKYAIEEDTSAIVPVHVFGNGCEVEKIDDG AKKHNLKVIYDAAHAFD
VKYKGESILNYGDISTLSFHATKIFHSIEGGALIINDDSLVEKVRYFINFGIESSESI
PYLGTNAIQ~IIJEFEAAMGLCVLDDIIEIKSKRKVITEIYEAGLDGLVKFQEQNQHSSRN
YSYFPVIFRTEEELLRVQKALIQNDIISRRYFYPSLDSLSYIEPKQYMPISRDISKRI
LCLPIYAELEDDKINKIINNIKEVSS
SEQ ID NO 13 MK KIFVVTDNRTILSDFKNIIGSKNDVQVDYFCSFKSQTSFAKE
IYNSEIKPIDMKKNGNDLIGKYDLGFSCHSKQLFPAKLVNSVLCINIHPGLNPYNRGW
FPQVFSIINKLPIGATIHVMDEEIDHGDIIIQEEVEVNSFENSFDVYAKVQKKEVELF
TKVIDDILNNKFTRIKPNSEGNYNSIHDYKNMCEIDLDKIVTMREAIDYLRAMTHPPY
KNSYFIDEHGNKVFVALELEKIS
U.S. Patent
Jun. 12, 2012
Sheet 12 of 19
US 8,198,430 B2
Fig.5 (Cont II).
SEQ ID NO 14 MS LKKNTISNYITQLYTSLIGIVILPLYLQHLSHDAFGLIGFFT
VFQTWLRLLDVGITPTLSREVAHVRGSTDDYHYLRKLVRSLELFFIIVGVLVFIVIST
HSRYISTSWLHIGSLDADSVSVCIALMGLMFALRWVSDLYGGGLRGFERQVLYNNLSI
IQTTLQFIGGLLFICYVSTNIMYYFVYQTIIAILYLVCIAIAFYKILPSSFSVGLRFD
FKIIRKVLPFALGIAYSTTVWIIVTQSDKLVFSHVLPLSEYGYLSLLIVISSAVTILS
S PIS IAIQPRMTMLLAQQNVKGMESLYLKSSLISITFLSAVVTCVLMYSHQLLQSWTG
SME LANWGSNI LNI YVLSAS I ICI I S FQY FLQYAY GKLKLHNTYNTI S LV FFAPIV I Y
TAYNYGVYTTALLWLGYAIVGLIIWMPIVHHVFAKGINRYFFINLAVITIVCFLLSLI
FKGWYIYPSK.IGLVELILIGFAFLFIQICIEYVLFRYKVLRCIDD
SEQ ID NO 15
MIKVSVCVMTYNQEKYIGQCLESLVTQETDFDFEIIVGDDFSTD
GTRDVIQEYQKKYPDIIKPVFRDKNVGITENIKEIYFVANGEYIAHMDGDDYALPGKL
QIQADFLDNNPRCTGVFHNINILYPNGNIQHSRFACSNKSIFNLSDTLRGVAVGANSS
KMFRTSVLDDLILPDIELLDYYFHVITAEKGYLSFLNSNESYSVYRKGIGITSKSKEK
IYNTYAGLFEYFLDRYPEEKLNICI PWQMIISAIKGRCFI SAIRLFKILIRSRCI PL
VSWFKYRFEK
SEQ ID NO 16
MKGIILAGGSGTRLYPLTLGVSKQLLPVYDKPLLYYPLSVLMLA
GIREILIISTVRDISLIQELLGDGSQFGIQLSYKIQPSPDGLAQAFILGEEFLAGDSA
CLILGDNIYYGQGMTTMLESARAQCGGPAGGACVFGYYVNDPHRYGIVEFDKQKNVIS
VEEKPQNPKSITYAITGLYFYDNNVVEYAKQVKPSARGELEITSLNELYLKENKLNVEL
LGRGFAWLDAGTHDSLLEAGQYVATIEKRQGLKIACLEEIAWRKGFISTQQVLAQAEK
LSKTEYGQYLKNLIKDGL
SEQ ID NO 17
MTSITYRNDDVMRNEKNBIYKPKNILVTGAAGFIGSNYVR MLSRY
SDIKIISYDKLTYAGSLDNLKDLNNEHNHTFIKGDICDEVLVYQTLKEYKIDTIVHFA
AESHVDNSIANPKVFLETNVIGTFTLLDCAKRYWLDELGLEETSCRFHHVSTDEVYGT
LAKDEPAFTEIKAYEPNSPYSASKAGSDHISRAYHHTYKLPVTISNCSNNYGPYQHRE
KLIP VINSCINYKPIPVYGDGSNIRDWLY WDHCDAIQTIVEKGWGEVYNIGGINE
VDNLTLVKTICKLMDEYKPENAPHSNLITFVEDRKGHDWRYAIDNSKIQNELGWKPSQ
DFDKMFRQTIEFYL
SEQ ID NO 18
MPSYSQDFRDIVINKHEEGMTEFELSKFFNIDKRTVVSWIEFYK
RTGDYSSKQGVGCGRVASFTDKTLIEQYLIDHPDASALDIKEALAPDIPRSTFYDCLN
RLGFSFKKRLQNISKEKNMKGWSI
SEQ ID NO 19 MI TPI I LSGGFGSRLWPLSREAS PKQFIGLVDEHSLLENTIKRL
DNVKDITSPVWCNESHRFQVAEVLRKINKKGDILLEPLARNTAPAIALAALHLAIND
PNTIMLVLAADHHIENLEIFHQAIEKAQQKVIKDDSLVTFGITPTCPHEGYGYIKQGV
QTTVNGVYKVDKFVEKPSVVVAQEYLDSGKYYWNSGMFMFrARVYLEVLEKLQPEIYR
GCEKTYQKSQQDLDFVRFDKQSFALVQSQSIDYAVMEKATNVAIVPMQQSGWSDVGSW
DS LYDIAAKDSCGNWIGDVITSNVKNSYLRSHDRLLAAVGVNDLIIVETADAILVAD
KNKTQDVKKIVEVLKIQQRSELLQHKQIYKPWGSATILEDKSGYKIQAIQLEPGKKLS
LQQHYHRSEHWIVISGTATVTIGTTKSIVRPNESVYIKIGESHRLENNGKIPVILIEV
QVGEYISEDDIVRLDTSS
U.S. Patent
Jun. 12, 2012
Sheet 13 of 19
US 8,198,430 B2
Fig.5 (Cont III).
SEQ ID NO 20 MR QTIIKEIIKSSGVKFGTSGVRGLVSAMTDKICWLYTKAFIQF
LEQKYSIAKGTKIAIAHDLRESSPRITTVVIKAIIDSGHEPIYCGEIPSPAVMLYGIS
NQIPSVMVTGSHIPEDRNGIKFNTPYGEVLKEDEEMIVSQTISIDESIFDKNGMFLQK
LELPEPSKQAYTQYIDRYVDFFPNNCLAGKTIGLYQHSSVGREIVKEILEKLGAKVIL
LE FSEKFVSVDTEAIRQEDVKLAKQWASKYKVDSIVSTDGDADRPLVSDEYGNWLKGD
ILGVLTAKYLQANVIVTPVSSNTVAEKIGYFSNVIRTKIGSPYVIAAMNELLSNNQNA
VVGYEANGGFLLASDICKDDKTLKALPTRDAVIPMLAVMMLSINSNKTVSELLFDLPS
RYTASSKIDDFASEKSQEILKSILAGESDLLDKIISEHFDGKNSIENIDTTDGVRVTL
TNQDIIHLRPSGNAPELRCYTEAASDEQAKSLNQYCVDLINKNI
U.S. Patent
Jun. 12, 2012
Sheet 14 of 19
Fig.6.
US 8,198,430 B2
SEQ ID NO 1
atatttatttttgtgcacagaacctaatttgcatttttgtgcacaaagaaaatttttttg
atataatagactttaataggatattttctaaaaattaacaaatgtctttctacgataata
gaacgcttaatttcgtggtaataatagttttaactattattactgttaattggactttct
atattttcaagcaagatgttaatttacattttttacttgcattagttttgctgagatgct
tgtcatcttttttactacttagagattatatggctagttggcgtaagtcgactcaaaaaa
cttttttacgtaaggcttttattaatttgccagtatttttcatagtggcattattttttt
atggcaaagtcactttttcgttgatattctctgagtttttattttatgtttttttgatca
gtttaagtgtctacttttattggtatttgatgaacagaggatcagtggataaaagtaaaa
ctgcggttatttatggtgcaggtgctgcaggaacaaagattgctcaagaacttgcttctg
ctggttatcgcatcaaatgttttgttgatgacaatgaaactttacaaaaaagaagtattg
atagtaaaaaggttctatctaaagctgaattaacaaaactattgctatctagtagatttg
accttttggttattgcattgccaagaaatgcaaaccaagtagtcaaaaatatatataaag
aatttgaaaaggattttaatcagattagaattatgccgcctcttgaggaaattcttcaag
atgagaattttatgtcacagttgaagcctgtttcactctatgatctattagcgcgtgata
ctaagagtttagataaagaatctatctctaattttatcaaaaataaggtggtgctagtca
caggagctggaggtagtataggttctgaaatagtacatcaatgtatcaagtatcaggcaa
aagagttgatattggttgatcatagtgagtttaacttatataaaattactgaggagtgta
gtcattttaatatcaatagtgtgctatgttctgtttgtgatagaaaagcattggctgagg
tttttcaaaagtatactccaaatatagtatttcatgctgctgcctacaagcatgttccct
tagttgaggagaatatctctagagcaattagaaataatatcttaggtactaagaatgcta
tagatctggctatagaagctggtgttgagtcatttatattgatttccactgataaagcag
tgcgaccaacgaatgttatgggggctaccaagagagtttgtgagctgtatttacagaatg
ttgatcccaaaaataccaagcttgctgcagtgcgttttggtaatgtgcttggtagtagtg
gcagtgtgattccaaaatttgaagagcaaataagaaaaggtggtcctgttacagttactc
atcctgaaattacacgttattttatgttgataccagaagcttgtgaactggtcctacaag
ctggtgctattgcaaaaaattcagaggtctttgtcttagatatggggcaacctgtcaaga
ttattgatcttgctaaacaatttattagactttctggtagaggtgatattgatattaaaa
tagttggtttgcgtccaggagagaaactttacgaagagcttttgatagaggaagatgatg
ttagtaccgactataaagatatttttattggtagaaggactttttacgatattaatactc
taaaccaagatattgaatcgttgatcaaggatgatgttgatcagcttgtgatattaaaga
aaattgttccggaatttgaacatagattgaatgggtagtggttttatgttttatgaggtt
tttaaaagattgcttgatattttactttcttttatggggttgttgttattaagtcctatt
ttcttaattattatttttatgataaagaaagattcaaaaggacctatattttttaaacaa
aagcgctatggtaaagataagcaatttttttacatatataagtttagaactatgtatgtt
gatactccaaaagatatgccaacgcacatgttacaggatccatcgaaatgtataactaag
gttggaggatttttaaggaaatcatctttagatgagttgccacaaattataaatattcta
aaaggtgaaatgagcatcgtgggtccaagaccagcattatggaatcaagatgacttaata
gcacaaagagataagtatggggcaaatgctgtgcctgtgggactgactggctgggcacag
attaatggtagggatgaattaccaatacctgataaagctaaacttgatggtgattatgta
aaaaataaaagtacatggtttgatttaaaatgtatttttttgacagtattttctgttttt
gccaaaaagggcgtcgttgagggtggtactggagctttaggtaacaaagaggatttaaag
tagtatgaaaaaaagaatcttagttacaggtttgagtagctatattggtaactcatttgc
ggctaaatataactcagattttagtatcgataaaatatctttgcgcgatgtttcgtgggc
aaatatagacttaagtggttatgatgctgtattgcatgtcgctggaattgcccatacttc
aaaggatcctaaactaaaagaaaaatactataaaataaatacgcaattaacttatgatct
ggcaaaacaagctaaagatcaaggtgttcgacagtttgtgtttttaagtagtattatagt
ttatggtgatagtgcgccaataggtcaacaaaaagttataactaaatataccgaacctaa
accagatgatttttatggagatagtaagcttcaaactgaaattaagctaaatagcctggc
tagtgatgactttaatattgctataatcagaccaccaatggtatatggagaaggctcaaa
aggcaactatccaaagttggttaaacttgcaaagtatacttttatttttcctaatattaa
taaccaaagaagtgttatatctatagataatttatctaaagagattgcagaaataatttt
gcaaactaaacatggagtttttctacttcaagataatgaatatttttgcacttcacagtt
U.S. Patent
Jun. 12, 2012
Sheet 15 of 19
US 8,198,430 B2
Fig.6 (Cont I).
tataaaaaactatagaaaagatgttttaggtaagagaacttatctgacaaaaatttttaa
tccaattataagattgcttgctaaaaaagtagattttattaataaagtttttgggaattt
gacttatgagaagtaagttattattcatagctaatgattttgatattgtaatatatcgtt
tcagaagagaagtaatcgagtcttttgctgctaaagagtatgagatagtactagtaacac
ca tattctaagaaagcagaggttttttgtaaaagtcttggtgttaagtatataaatgttg
atatagatagacgaggcaaaaatccttttaaggatttgcttcttttatttaactatttca
aaataataaaaaaagaaaaacctgattacatttttagctatacaattaaaccaaatttgt
atgttgggttagtgaatttgttttttaggaagaagttttatccaaatgtaacaggcttag
gaagtgtttttgctaatcatggtattgttcagaagtttataatatctttatataagttat
catttaaaagcaccacaaaagtattctttcagaatgagcaaaataaaaagttatttatag
ctaagaaaataatcagtggagaaaaatcaatattattaccaggttctggggtaaacttag
atgaaaataaatatgttgactatcctaaagaccaaggaatattaaaattcgtttttcttg
gccgaataatgaaagaaaaggggatttatgaattgttagaagcctttgctatacttgaga
aaaaatataaaaatattagtcttgacatttatggtttttgtgatgaaaataaatctaatt
ttatgggaaaggttaatacgataaaatcagtaaaattttatggttttactgataatacta
aagaaaaaatagctagtgcacatgcagttgttttgccatcttaccatgaaggaatgtcaa
atgtgctgttagaagcagctgcgataggtagacctgtaattgcgtcagatattcctgggt
gtagagaaatttttgatgatggtctctctggcttatcatgtaaccctaatgatgtgagtt
ctttacgtaactcattagagcagtttataaatatgtcgtatactgataaaatagctatga
gctataaagctagagctaagatagaaaaagattttgatagaagtattgttgtcaatgcat
acttacagcaaaattaataataagggtttaaattatgagtttatatgaggatatagtcgc
taaaagagaaaaggtttcattggttggcttgggttatgttggtttaccaatagctattgc
atttgcaaaaaaaatagatgtgttaggatttgatatttgtgaaacaaaagttcaacatta
taaggatggttttgatccaacaaaagaagtaggagatgaggctgtcagaaatacgacaat
gaaatttagttgtgatgaaacaagtcttaaagagtgtaaatttcatattgttgcagttcc
tacaccagttaaagcagataaaactcctgatttgacgccgattattaaggcaagtgagac
ggttggtaggaatcttgtcaaaggcgcttatgttgtgtttgaatcaactgtttatcctgg
tgttacagaagatgtttgcgtaccaatacttgaaaaagagtctggcttgaggtctggtga
agatttcaaagttggttactctcctgagaggataaatcctggtgataaggttcataggtt
agaaacaattatcaaagtagtatctggtatggatgaagagtctttagatactatagcaaa
agtttatgagctagtagtagacgcaggagtttatagagctagtagtataaaagtggctga
agctgctaaggttatagaaaactctcaaagagatgttaatatagcttttgttaatgagtt
atcgataatatttaatcagatgggtattgatactctagaggttttagcagcagctgcaac
taaatggaatttcttaaactttaagcctggtcttgttggtggacattgtattggtgttga
cccatattacctaacgtacaaggcagctgagcttggatatcattctcaggtaatattatc
tggtcgtaggataaatgatagtatgggtaaatttgtagttgagaatttagtcaaaaaact
gatatctgcagatatacctgttaagcgagctagagtagcaattttcggctttacttttaa
agaagactgtcctgacactaggaatactcgagttatagatatggtaaaagagctcaacga
gtatggtatagagccatatattatagatccggtagctgataaagaagaggctaaacatga
gtatggacttgagtttgatgatctaagtaaaatggtcaatctagatgcgatcattattgc
tgttagtcacgaacagtttaaagatataacaaagcaacagtttgataggctatatgcgca
taattctagaaagattatatttgacatcaaaggtagtttagataaatctgagtttgaaaa
agattatatttattggagattgtagtggcttacgataatgttaaatttcctcatggttcg
ttttttttggtgactggaggtgcgggttttattggctctaatttatgtgaagttttactt
agtaagggttatagagttaggtgtttagatgatctctcaaatggtcactatcacaatgtt
gagccgtttttaactaattctaattatgagtttataaaaggtgatattagagatttagat
acttgcatgaaagcttgtgaaggtattgattatgttctacatcaagctgcttggggaagc
gtaccaagaagtattgagatgccattagtgtatgaagatataaatgttaaaggtgcatta
aatatgcttgaagcggctagacaaaataacgttaaaaaatttgtctatgcttctagttca
tcagtatatggtgatgagccaaatttacctaaaaaagaaggtagagaaggaaatgtttta
tcaccctatgcatttacaaagaaagctaatgaagagtgggcgagactatacacaaagtta
tatggtctagatacttatggtctaagatattttaatgttttcggtagaagacaagatcct
aatggtgcgtatgcagcagttatacctaaatttatcaaacagttattaaatgatgaagcg
ccaactataaatggagatggtaaacagtcgagagattttacatatatagagaatgttatt
gaggcaaatcttaaagcatgtttagcagatagtaagtatgccggagagtcttttaatata
U.S. Patent
Jun. 12, 2012
Sheet 16 of 19
US 8,198,430 B2
Fig.6 (Cont II).
gcttatggaggtagagagtatcttatagatttgtactataatctttgtgatgccttgggt
aaaaaaatagagccaaattttggtccagatagagcgggtgatattaagcatagtaatgct
gatatttcgaaggctaggaatatgctcggatataatccggaatatgattttgaattaggc
ataaagcatgctgttgagtggtatttaattaattaaatggtattttaatcaagtgtacat
aaaaaaagtgtcttttaaaattttatatttatatttactagctttttgtattatttttag
tttagaatttaaatttgctatattgaatattatagtttatcttccggcttgtattttggg
ttttttagctcttaaaaaactatttgtcggaaatattgttaagaaacaattagctttcct
ttttttctttttctttttatcaatgatttatttaataatagtccaaataatcttacttga
tgcagcatcattgtttcctcagtttttatttaacattttgatcgcgataggtttttgtaa
ctttatttttgtttcatatgataataatgaaaattatttttttaatatgtctaaaataat
attttttgttactttcttacaatctatttttgtatttctttcaaggtattatatattttt
aaatgattggatattcttttttttagtgaaaaaagggaatattgagatttcgaatgttat
tgaatataagttaagagtattcggacttagtaacgctggaggggatggtttaggattttc
aattactataggattatgtttttctatattttattttatcaaatatattaaaggtaaatc
tatatttaccaaacttatgctgtttgtacctttaattcttattgtgttttctaatatttt
catatctagaacatcactcttaacttcttcacttatattgttaataacaatattttatat
atatattaaaaaagaaaaattactgtttattataatattggcgctattctttttatcaat
atggatattgttcaaattaaatttgaatttgagttgggcttttgaaaatatttactcgta
cattcaatctggcgatttttcacatggaagtctaagtgttttaatcaataaaatgctttt
tgtgccagataaccttttgacttggatatttggttgtgaggatgttagtaatactgatat
tggttatattaaatatttatactattatgggattatatttagtatgtttttttatattct
tattattttcttgtactttgaaatgagaaaatgttttatattttcagagtatcgatcatt
atttctattgttgttaatagtatgtttagtttttcaagcaaaaataatttttttgacagt
aggattatttactaaattaaccattatattatttattttttctcttaaagaaaacagctt
tacaactaggagtgtgatttgaaaaggtttgtacatttaataataaaccttaaccaaggt
ggtgctgaaacaatgctttataaactttgcaaatctatggataagtcaatatatcatatt
acgattatatcacttatgggtaggggagtatttgcaaataagttagaagcttatggtgtt
aaagtttatacattaaatttaaataaatttaatgtactatttgtattgtttaaatatatt
aagattatcagaagaataaagcctgatgttattcatgcttggatgtatcatgcaaatgta
at ttctatattatgcaagcctttttatagaaagactaaatatataaatagtataagaatg
ggattggagaattatgatggtcataagaatcttacaaagtttatgataaagttgaatgca
aaattttctaagttctcagatttaacattaaataattcaaagaaatcattagaagatcat
caaaatataggttttaaaaaccaatgctttatagcaaatggttttgataaagatgttttt
aaaccgagctttttaaagtatgaaaaatttcgtttaaataatgatttagatgataatgtt
aaaattataggtatcatagcaagaaatcatgctgataaaaatatttctcgtttcttacaa
atagctaatttattgttaaaaagtaatcctagtttacggtttttaattgctggaagagag
tgttcgaaaatagatataggtagttatctagataacaaaagtaatgtaaataagtttttt
gtatttgaatctgtggattctagtgaatacttaccagtattagatttatatttgtctaca
tcaaaagttgaaggttttccaaatatacttgcagaagccatgctatgtgaagttcctatt
gttgcttctaatgttggagattgtaaagatatacttaatggatacggtgaagtttttgag
cttagtcaaggtaataaagaaataatagaaaagattatgaaagttttagaaacaacggta
gtcatgaaaaagcgcatgagagaatatataataaataattttagtatagaagctattttg
gaaaaacacgaaaaactttatcatgagggcagtgtctaatgtgtggagtagtaggctttt
actcatttaataaagaagaaggttttgactcaataattaatcaatcattgctttctataa
agcatagagggtcggatgatagtgggtattggtgcgacaatcaagttactctggggcata
ctagattatcaatacacgatataactaatgcgggacatcagccaatgttatctaatagcg
gtaatactgctattgtgtttaatggagaaatatataattacttatccataaaaaatcagc
tattaagtgaatattcaaatcttaaatttaaaagtaacagtgatactgaggttttggtca
atgctattgaactttggggtatagataaaactttagaaaaatgcataggaatgtttgctt
ttggagtttacagtagaaaaactagttgcttaatactagctagagatagatttggcgaga
agccattatattttggtatccaaaatggtattttgggttttgcatcagaattgaaggcac
ttaagccattaaaggaatgtggctggaggtttgatatagatagagatgctttagcaacat
atatgaggtatgcttatgtaccaacaccatactctatttataaaaatatatctaaactaa
atgtaggtagttacataaaatttgatgctaaaggtaatagtaaagagtataaatattggg
attctaaaaaagtactagattcagaaaaatataaagattcgtatgatcaagcaatcctag
U.S. Patent
Jun. 12, 2012
Sheet 17 of 19
US 8,198,430 B2
Fig.6 (Cont III).
atttagaaattaagcttaaaagtacactatcaatacaaatgcagtcagatgttcctctag
gagcatttttatccggaggaattgactcaacaactgtagttgctcttatgcaaagtatgt
ctaaagataagataaacacttttagtataggttttaatcaaaaagaatataatgaagctg
agcatgcaagagcagtagcaaaacatataggtacaaaccacacagatatgtatgttacag
aaagagatgctcttgatgtaataccaaaacttgctggaatatatgacgagccctttgctg
attcatcacaaataccaacgtatcttgtgagtaaaatagctaagtcgaaagtaacagttg
cactatcaggtgacgctggtgatgagctctttggcggttataatagatactttttagcac
caaatattgctaaaaaaatcaaatttgctaagttacttaaatatgcaccagatgcttgga
taaaaaaagctgagatattaaattttggtaagttcgctttattagcagataaactactaa
aactaaaaagagttctcgaaaaagcaaaaacaaataaagagctttatgtactactttgtt
cacaaataaatgatactagctttgtgttaggagcaaaagagtatgatatattaagagata
agaatatttatgatattccacaattatctttccaagagtggatgatgtttgttgattcta
atacatatatgatagatgatatattggttaaggttgatagagcagctatggctaactctc
tagagacaagagtgccatttttagatcataatatttatgaatttgcttattccttaccaa
ttgactataaaatacaacgaggtaacggaaaaagaattttgaaagatttgttatataaat
atgtgccagaaagtttggtcaataggtctaagatggggtttggtattccgcttgctaaat
ggttaagagaagatttacgagagtgggcagataatttactggattatagtaaaatagaca
agcaaggttacttaagtcctgaggtggtgcaaaaatattggcaagagcatttgagtggta
aaagaaattggcaagcaatattatggaatattctaatttttcaggagtggttagataatg
agtaaagtaaatgtaacaaaaccatacttaccagatataaataaatataaaagctatgta
aataaaatatacaaaaatggatggcttactaataatggtccgttagtgcaagagctagaa
aaaagacttgcaaagtatctaggtgttaaaaatatagttttagtatcaaatggtacaatt
gcattagaaatcgcgtatagagcgttaggagtcaaaggaagtgcaattactactccattt
tcatttgttgctactacatcttcattggtttctaacaatgtaaaaccagtgtttgttgat
attgatgagaatactctaagtatagacgtctctaaaattaagtatgctattgaagaggat
acttcagctattgtgccagttcatgtgtttggaaatggttgtgaagttgaaaaaatagac
atgctggctaaaaaacataacttaaaagttatttatgatgcagcacatgcttttgatgtt
aagtataagggtgagagtatattaaactatggtgatatttcgacattaagttttcatgca
acaaagatttttcattctattgaaggaggtgcgcttatcattaatgatgatagtcttgtt
gaaaaagttcgttatttcattaattttggtatagaaagctcagaatcaataccttactta
ggtactaatgctaaaatgaatgaatttgaggcggctatgggactttgtgttctagatgat
attatagaaattaagagcaaaaggaaagttattacagagatatatgaggctgggttagat
ggattggtaaagtttcaagaacagaatcagcattctagtaggaattatagctattttcca
gtaatatttaggactgaggaggaacttctcagagtacagaaagcactaatacaaaatgat
ataatatcgcgtagatatttttatccatcattagatagtcttagttatatagagccaaag
cagtatatgccaatctcaagagatatatctaaaagaatattatgtttgccaatttatgca
gagttagaagacgataaaattaataaaataattaataatatcaaagaggtttcctcatga
aaaaaatatttgttgttacagataatagaactattctaagtgattttaaaaatatcattg
gtagtaaaaatgatgtacaggttgattatttttgtagtttcaagagtcaaacttcttttg
ccaaagaaatatataacagtgagattaagccaatagatatgaaaaaaaatggcaatgatc
ttattggtaagtatgatttaggtttttcttgtcattcgaaacaattatttccagcaaaat
tagttaattcagtattatgtataaatattcatcctggacttaatccatataatagagggt
ggtttccacaggtcttctctattataaataaactacctataggagcaactattcatgtga
tggatgaagagatagatcatggagatataatcattcaggaagaagttgaagttaattctt
tcgaaaactcttttgatgtttatgctaaagttcaaaaaaaagaagttgagttgttcacta
aagtcatagatgatattttgaataataagttcactcgaatcaaacctaactccgaaggca
actataattcaattcatgattataaaaacatgtgtgaaattgatttagataaaatagtaa
caatgcgggaagcaattgactatctaagggctatgacacaccctccatataaaaatagtt
atttcattgatgagcatggaaataaagtatttgttgctcttgaacttgaaaagataagtt
agaaaaatgagccttaaaaaaaatacaatatcaaattatataacacaactatatactagc
ttaattggtattgttatacttcctttgtatttacaacatttaagtcatgatgcatttggt
ctgattggtttttttacagtttttcaaacgtggttacggttgttggatgttggtataaca
ccaactttatcaagagaagtggctcatgttagaggtagtactgatgactatcattactta
cgcaa gttggttagatc gttagagctatttttcattattgttggtgttctggtatttatt
gtaattagtacacattcaaggtatatatccacctcttggttacatataggctcgctagat
U.S. Patent
Jun. 12, 2012
Sheet 18 of 19
US 8,198,430 B2
Fig.6 (Cont IV).
gctgatagtgtaagtgtatgtattgcacttatgggtttaatgtttgcattaagatgggtg
tctgatctatatggtggtggtttgcgtggctttgaaagacaggttctttataataattta
agtatcatacaaacgacactacagtttattggtggattattatttatctgctatgtgtct
actaatattatgtattattttgtatatcagacaataattgcgatactatatctagtatgt
attgcaattgcattttataaaatactaccatcatcatttagcgtgggtttaaggtttgat
tttaaaataattagaaaagtgcttccatttgcactaggcattgcatattctacaacagtt
tggattattgtcactcaatctgataaattagtgttctcacatgtattaccattatctgag
tatggttatttatctttattgatagtgatatctagtgctgttacgatattgtcctctccg
attagcatagctattcagcctagaatgacaatgctattagcccaacaaaatgtaaaagga
atggaaagcttatatttaaaatcatccttgatctcaattacttttttatctgctgtagta
acatgtgttttgatgtattctcatcagctgttgcagtcatggacaggaagtatggaaatt
gctaattggggtagtaatatcttaaatatatatgttttatcagcatctattatttgtata
atatcatttcaatattttttacagtatgcttatggtaagttaaagctacataatacatat
aatacaattagtttagtattttttgctcctatagttatatatactgcttataattatgga
gtgtatactacagcactattatggcttggatatgctatagtggggctgataatctggatg
cctattgtacaccatgtatttgctaaaggtatcaataggtatttttttataaatttagca
gttattactatagtatgttttttattatcgttaatatttaagggttggtatatttatcca
agtaaaattgggttggtagaattaatattgattgggtttgcatttttatttatacaaatt
tgtatagagtatgttttgtttcggtacaaggttttgaggtgtatagatgattaaagtttc
agtatgtgtgatgacatacaatcaagaaaagtatattggtcaatgtttagagtctttggt
tactcaagagactgattttgactttgagataatcgttggagatgatttttctacagatgg
tacaagagatgttattcaagagtatcaaaaaaagtatccggatatcataaagccagtttt
tagagataagaatgtgggaattactgaaaatattaaagaaatctattttgttgcaaatgg
tgagtatatagctcatatggatggtgatgattatgcattgcctggtaaacttcaaattca
ggctgattttttggataataatccaagatgtacgggagtttttcataatataaatatact
ctatccaaatggtaatatacaacatagtaggtttgcttgttcaaataagagtatattcaa
tttatcagacactttacgcggagttgctgttggtgcaaatagttcaaaaatgttcagaac
atcggttttggatgatttgattttaccggatatagagcttctagattattattttcatgt
tataacagcagaaaaaggttatttaagttttttaaattctaatgaatcctatagtgtgta
cagaaaaggtattggtatcacatctaagtctaaggaaaaaatctataatacttatgctgg
attatttgaatattttttggatagatatcctgaagagaaattaaatatttgtatccctgt
tgtgcaaatgataatttcggctattaaagggagatgttttattagtgctattcgtctatt
caaaattttaattagatcaagatgtattccattagtaagttggtttaaatatagatttga
aaaataaatatcatttagaggattatgtgaaatgaagggaataattctagctggtggcag
tggtacaaggctatatccacttaccttgggtgttagcaaacagctgctacctgtttatga
caagccattgttatactatccactatctgtgcttatgcttgcaggtattagggagatatt
aattatctctacagtgcgtgatatctcacttatccaagagcttcttggtgatggttcaca
at ttggtatacagttgagttataaaatccagccatcaccagatgggcttgctcaagcatt
tattcttggtgaggagtttttggcgggtgactcagcttgtttgatattaggagataatat
ctactatggtcaaggtatgactacaatgctagagtctgcaagagcacagtgtggaggtcc
agctggtggcgcttgtgtttttggttattatgttaatgatccgcatagatatggtatagt
cgaatttgataagcaaaaaaatgtaatttcggtagaggaaaagccacagaatcctaagtc
acactatgctatcacaggtttatatttttatgataataatgttgttgagtatgctaaaca
agtcaaaccatctgcacgtggtgagctagagattacttcacttaatgagttatatctaaa
agaaaataagctaaatgtcgaactcttagggcgtggctttgcttggcttgatgctggtac
gcatgattcattgctagaggcaggtcaatatgtcgcaactattgagaaaagacaagggct
taaaattgcatgtttggaagaaattgcatggcgtaaaggctttatctcaacacaacaagt
tctagctcaagctgaaaaactttctaagacagagtatggtcagtatctgaagaatttaat
taaggatggtttataaattaatccgtcatacccatgaaggtgggtatctcataaaagttg
gatatgttttggagattccaatctgcgcagtaatgacaggtttggtaatatatagcgatg
ttttacaatgactaaaaatggttttatgtatattcttacaaataaggataatactgttct
gtacatagttgtaacatctaatttgataaaaagaatgtatgagcataaacatagccttgc
agatggttttactaaaaatataatgttaataagttagtttattttgaaatttatgaagat
at aaaagcagcaattctgtgagaaaagcagttgaaaaaatgaaacagatcttggaaagaa
cgaattattaatgagatgaatccgaattggaatgatttatatgaattaatatgtgagtaa
U.S. Patent
Jun. 12, 2012
Sheet 19 of 19
US 8,198,430 B2
Fig.6 (Cont V).
aacttttgtcttactggtgcagataggtatctctaaatatcagatgtgattgggagatta
ccgcctacgcggtaatgacaagtttatgcggtaatgatagtttagtgagagaatgactag
tcactataggaatgatgatgtaatgaggaatgaaaaaatgaactacaaaccaaaaaatat
cctagtaacaggtgcggcgggatttattggtagtaactatgtgcgtatgatgttatcacg
ctatagtgatatcaaaataatctcgtatgataagcttacttatgcgggtagtttagataa
tctaaaagacttgaataatgaacataaccatacttttataaaaggtgatatttgtgatga
agttttagtatatcaaacactgaaagaatataaaattgatacgatagtacattttgctgc
agaatcgcatgttgataattcaattgctaatccaaaggtatttttagaaacgaatgtgat
aggtacatttacacttttagattgtgctaaaaggtattggttagatgagctaggtttaga
agaaactagttgtaggtttcatcatgtatctactgatgaggtatatggtaccttggcaaa
agatgaaccagcctttactgagattaaggcttatgagccaaattcaccgtattcggcatc
taaggcgggatctgatcatatttctagagcatatcatcatacctataaacttccggtaac
aatttcaaattgttcaaacaactatggaccataccaacatcgagagaaattaatccctgt
agtgataaatagttgtataaactacaagcctattcctgtttacggagatggttcgaatat
tcgagattggctatatgtagaagatcactgcgatgctatccagacaattgttgagaaagg
agtggttggagaggtttataatattggtggtattaatgaagttgataatctaaccttggt
aaaaactatctgtaaactaatggatgaatataaaccagaaaatgctccacattctaactt
aatcacatttgtggaagatagaaaaggacatgattggcgttatgctattgataacagcaa
gattcagaatgagttaggatggaagccatcacaagattttgataagatgtttagacaaac
tattgagttttatctatagcttaaatatttatcttatgagtatctctaaaaaatcaattt
aatttatttttgtgttaaaaagtagtgttcgcaagaatatagttaatccgaaagatattt
gtagaaaaagatatttgtagaaatgttataatgtctaataaaa
US 8,198,430 B2
1
IMMUNOGENIC SEQUENCES
2
There are no reports of LPS isolation from the more virulent subspecies tularensis strains.
CROSS REFERENCE TO RELATED
When LPS structure is studied in other species, it is freAPPLICATIONS
quently observed that the only difference in structure between
5 strains is the composition of the 0-antigen. Therefore, it
This application is a continuation of U.S. patent applicawould be useful to elucidate the structure of the O-antigen
tion Ser. No. 10/516,215, filed Sep. 1, 2005, which is the U.S.
part of the LPS molecule in virulent subspecies in order to
national phase of International Application No. PCT/
provide the basis for diagnostic tests and also to allow it to be
GB2003/02338, filed May 30, 2003, and published in English
produced recombinantly, to avoid handling a pathogenic
as International Publication No. WO 03/102191 Al Dec. 11, io organism.
2003, which application claims priority to Great Britain
However, the genetic basis of 0-antigen expression is comApplication No. 0212666.2, filed May 31, 2002, the contents
plex; in most bacteria the genes required for production of a
of each of the foregoing are incorporated by reference herein.
complete 0-antigen are located in a cluster on the bacterial
The present invention relates to nucleic acid sequences, in
chromosome. Therefore identification and isolation of genes
particular genes that encode the enzymes which produce the 15 responsible for the 0-antigen is not straightforward. Further0-antigen of Francisella tularensis, and their use as or in the
more, the identification and isolation of LPS from virulent
production of vaccines and in diagnosis.
strains is further complicated because it is difficult to stain
Francisella tularensis is a small Gram-negative coccobausing conventional methods.
cillus, which causes the zoonotic disease Tularemia. AccordThe applicants have now determined the genetic basis of
ing to Bergey's manual of systematic bacteriology the genus 20 O-antigen production in F. tularensis subspecies tularensis.
Francisella contains two species: F. tularensis and FranFurthermore, they have established the efficacy of LPS from
cisclla novicida. However, recently several workers have sugvarious F. tularensis strains as a vaccine.
gested that F. novicida be considered a subspecies of F. tulaAccording to the present invention there is provided a
rensis (Hollis D G, et al., J. Clin. Micro. 27: 1601-1608). The
nucleic acid which encodes a series of enzymes or enzyme
closely related bacterium Yersinia philomiragia is now also 25 fragments which, when expressed together in a cell, are able
considered a member of the genus Francisella, due to its high
to produce an immunogenic moiety able to produce an
degree of relatedness at the DNA level. There are several
immune response in an animal to which it is administered,
proposed subspecies of F. tularensis other than novicida;
which response is protective against Francisella tularensis
these are: subspecies tularensis, subspecies holarctica and
infection, said nucleic acid encoding at least some of the
subspecies mediaasiatica. The subspecies tularensis and hol- 30 enzymes of SEQ ID NOS 3-17, or modifications thereof.
arctica can be identified on the basis of virulence, citrulline
The expression "modification" refers to sequences of
ureidase activity and acid production from glycerol (Olsufjev
amino acids, which differ from the base sequence from which
N G, et al. (1959) J. Hyg. Epidemiol. Xicrobiol. Immunol. 3:
they are derived in that one or more amino acids within the
138-149. Francisella tularensis subspecies mediaasiatica is
sequence are substituted for other amino acids. Amino acid
predominantly found in central asian republics of the former 35 substitutions may be regarded as "conservative" where an
USSR. Strains of this subspecies possess citrulline ureidase
amino acid is replaced with a different amino acid with
activity, and are able to ferment glycerol, but are less virulent
broadly similar properties. Non-conservative substitutions
than strains of F. tularensis subspecies tularensis in rabbit.
are where amino acids are replaced with amino acids of a
Tularemia is a disease occurring in the northern hemidifferent type. Broadly speaking, fewer non-conservative
sphere; with cases frequently found in Europe, N. America, 40 substitutions will be possible without altering the biological
Asia, N. Russia and Japan. Rodents are thought to be the main
activity of the polypeptide. Suitably modifications will be at
reservoir of the bacteria, with ticks as one of the main vectors.
least 60% identical, preferably at least 75% identical, and
The lipopolysaccharide (LPS) of Gram-negative bacteria is
more preferably at least 90% identical to the base sequence.
the major component of the outer membrane. The molecule is
Identity in this instance can be judged for example using
composed of 3 regions, lipid-A, which is embedded in the 45 the algorithm of Lipman-Pearson, with Ktuple:2, gap penouter membrane and has a conserved structure between spealty:4, Gap Length Penalty: 12, standard PAM scoring matrix
cies, and two polysaccharides, the core oligosaccharide
(Lipman, D. J. and Pearson, W. R., Rapid and Sensitive Prowhich can vary in complexity between species, and the 0-antein Similarity Searches, Science, 1985, vol. 227, 1435tigen which is a very polymorphic structure (Kenne L, et al.
1441).
(1983) Bacterial Polysaccharides The polysaccharides. Aca- 50
In particular, the invention comprises a nucleic acid which
demic Press, pp. 287-362). The LPS molecule is thought to be
encodes enzymes of SEQ ID NOS 3-17.
required by the bacteria for protection against serum killing
A preferred example of such a nucleic acid comprises SEQ
(Whitfield C, et al, (1997) Mol. Micro. 23: 629-638) and
ID NO 1 or a variant thereof. In particular the nucleic acid is
cationic antimicrobial peptides (Groisman E A. (1994).
of SEQ ID NO 1.
Trends. Microbiol. 2: 444-449).
55
The term "variant thereof' in relation to a nucleic acid
The structure and immunogenicity of LPS isolated from
sequences means any substitution of, variation of, modificathe less virulent F tularensis subspecies holarctica strains has
tion of, replacement of, deletion of, or the addition of one or
been studied to some degree (Dreisbach V C, et al. (2000)
more nucleic acid(s) from or to a polynucleotide sequence
Infect. Immun 68: 1988-1996). Animals immunised with this
providing the resultant protein sequence encoded by the polyLPS are protected against a subspecies holarctica strain chal- 6o nucleotide exhibits the similar properties as the protein
lenge (Fulop M J, et al. (1995). Vaccine 13: 1220-1225), but
encoded by the basic sequence. The term therefore includes
not a subspecies tularensis strain challenge (Fulop M J, et al.
alleleic variants, degenerate variants which encode similar
(2001). Vaccine 19: 4465-4472). However, the LPS from a
proteins but vary only as a result of the degeneracy of the
subspecies holarctica strain appears to be less toxic than other
genetic code. It also includes a polynucleotide which substanGram-negative LPS and its O-antigen contains rare sugars 65 tially hybridises to the polynucleotide sequence of the present
which are related in structure to those found in Pseudomonas
invention. Preferably, such hybridisation occurs at, or
aeruginosa 06 and Shigella dysenteriae type 7.
between low and high stringency conditions. In general
US 8,198,430 B2
3
4
terms, low stringency conditions can be defined as 3xSSC at
about ambient temperature to about 55° C. and high stringency condition as 0.1 xSSC at about 65° C. SSC is the name
of the buffer of 0.15M NaCl, 0.015M tri-sodium citrate.
3xSSC is three times as strong as SSC and so on.
Typically, variants have 65% or more of the nucleotides in
common with the polynucleotide sequence of the present
invention, more typically 70%, preferably 75%, even more
preferably 80% or 85% and, especially preferred are 90%,
95%, 98% or 99% or more identity.
Variants may comprise the basic sequence which has been
modified to ensure that the codon usage is enhanced or optimised, as would be understood in the art, for a particular
organism in which it is required that the sequence is expressed
in a desired organism, for example a prokaryotic cell such as
E. coli. This may involve modifying the content of particular
nucleotides, for instance changing the percentage of G and C
present in the sequence, to suit that usually found in genes
which are highly expressed in the target organism. In addition, particular variants of SEQ ID NO 1 are synthetic variants, engineered to remove codons rarely found in highly
expressed genes from common expression hosts such as E.
coli and, at the same time, avoid the introduction of codons
rarely found in genes coding for O-antigens. For example,
wherever possible the codons for the amino acids arg, leu, ile,
gly and pro are changed to CGT or CGC (arg), CTG, CTT or
CTC (leu), ATC or ATT (ile), GGT or GGC (gly), and CCG
CCA or CCT (pro), thus eliminating rare codons.
When comparing nucleic acid sequences for the purposes
of determining the degree of identity, programs such as
BESTFIT and GAP (both from Wisconsin Genetics Computer Group (GCG) software package). BESTFIT, for
example, compares two sequences and produces an optimal
alignment of the most similar segments. GAP enables
sequences to be aligned along their whole length and fins the
optimal alignment by inserting spaces in either sequence as
appropriate. Suitably, in the context of the present invention
when discussing identity of nucleic acid sequences, the comparison is made by alignment of the sequences along their
whole length.
SEQ ID NO 1 comprises a series of genes which encode a
number of enzymes which are shown hereinafter in FIG. 5
and SEQ ID NOs 3-17. Preferably any variants of SEQ ID NO
1 encode enzymes of SEQ ID NOS 3-17 or modifications of
these.
The expression "fragment" used in relation to amino acid
sequences refers to any portion of the given amino acid
sequence which has the same activity as the complete amino
acid sequence. Fragments will suitably comprise at least 20
and preferably at least 50 consecutive amino acids from the
basic sequence.
The term "fragments" is also used in relation to nucleic
acid sequences. Fragments of SEQ ID NO 1 may have applications in diagnostics, and these form a further aspect of the
invention. For diagnostic purposes, fragments may be quite
short, for example from 5-30 bases, which may be used as
primers or probes. Particular characteristic regions of SEQ ID
NO 1 from which suitable fragments for diagnostic purposes
may be identified are elucidated hereinafter. Fragments
which are useful in therapy would generally be expected to be
longer, for example from 600-17,000 bases long.
A region of genome of the F. tularensis strain Schu 24
(subspecies tularensis) which includes SEQ ID NO 1, and
which is responsible for expression of the set of enzymes
necessary for constructing the polysaccharide, has been identified. It is shown hereinafter in FIG. 4 as SEQ ID NO 41. This
sequence includes a number of genes including a series of
genes that encode the enzymes illustrated in FIG. 5 hereinafter as SEQ ID NOS 3-20. Putative functions were applied to
these genes by comparison with known sequences as illustrated in Table 1.
5
TABLE 1
SEQ ID F. tularensis
NO
protein
10
15
20
25
30
35
40
45
50
55
6o
65
Gene product
size (aa)
Putative function
2
Transposase
247
3
4
WbtA
WbtB
578
205
5
6
7
WbtC
WbtD
WbtE
263
363
436
8
9
WbtF
Wzy
323
409
10
11
12
WbtG
WbtH
WbtI
366
628
360
13
14
15
16
WbtJ
Wzx
WbtK
WbtL
241
495
286
294
17
WbtM
348
18
19
Transposase
ManC
126
468
20
ManB
494
Hypothetical protein
Transposase
Sugar epimerase
Galactosyl transferase
Glycosyl transferase
UDP-glucose 4-epimerase
Sugar transferase
LPS biosynthesis
Dehydrogenase
C 4-epimerase
Membrane protein/O-antigen
polymerase
Transferase
Asparagine synthetase
Sugar transaminase/
perosamine synthetase
Formyl transferase
o-antigen flippase
Glycosyl transferase
Glucose-l-phosphate
thymidylyltransferase
dTDP-D-glucose 4,6dehydratase
dTDP-D-glucose 4,6dehydratase
Transposase
Mannose-l-phosphate
guanylyltransferase
phosphomannomutase
In particular the proteins illustrated as SEQ ID NOS 3-17
are believed to be involved in O-antigen biosynthesis. The
O-antigen itself has applications both in diagnostics and as a
prophylactic or therapeutic vaccine.
When the nucleic acids of the invention are expressed
together in a host cell, they will result in the construction of an
antigen that produces an immune response in an animal
including a human, which is protective against infection by F.
tularensis. Thus they may be used in the production of prophylactic or therapeutic vaccines.
The nucleic acid may be included in a vector such as a live
viral vaccine, for instance, adenovirus vector or vaccinia, or
in a plasmid to form so-called "naked DNA" vaccines, or
preferably in a bacterial vector such as attenuated Salmonella
species. In this case, the nucleic acid will be under the control
of suitable control elements such as promoters, signal
sequences, enhancers and the like, as would be understood in
the art. In this case, the nucleic acid is expressed either within
the cells of the patient to whom the vaccine is administered, or
in the case of bacterial vectors, within the host cell itself. As
a result a series of enzymes are produced which are able to
construct the protective 0-antigen in situ.
The vector is suitably combined with a pharmaceutically
acceptable carrier in a vaccine formulation. The nature of the
carrier depends upon the type of vector being used, as would
be understood in the art. In particular, when the vaccine
comprises a recombinant Salmonella, it is suitably in the form
of a composition which is suitable for oral administration.
Alternatively, the nucleic acid may be included in an
expression vector which is used to transform a host cell.
Suitable host cells are prokaryotic or eukaryotic cells, but are
preferably prokaryotic cells such as E. coli. In particular, the
nucleic acid used is a synthetic variant of SEQ ID NO 1,
US 8,198,430 B2
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6
optimised for expression in the particular host cells. The
sequence and particularly SEQ ID NO 1. Primers and probes
of this kind form a further aspect of the invention.
protective O-antigen can then be recovered from these cells
By selection of particular primers and probes, it may also
after culture thereof.
be possible to allow differentiation between strains ofF tulaThus in a further aspect there is provided a method of
preparing a prophylactic or therapeutic vaccine, which 5 rensis infection. For instance, the applicants have found that
primers comprising SEQ ID NOS 21 and 22 and 35 and 36 set
method comprises transforming a host cell with a nucleic acid
out hereinafter will allow distinction between strains of F
of the invention, culturing said host cell, and recovering a
tularensis subspecies tularensis, andF tularensis subspecies
moiety which produces a protective immune response against
holarctica as described below. In the former case, this possiF tularensis therefrom.
io bility arises because of differences in the downstream
Expression vectors and host cells for use in this method,
sequence, and in the latter, because of differences in deletions
together with the product thereof form further aspects of the
in the flanking transposase sequence. Consequently, analysis
invention.
using primers or probes based upon these regions may be used
Vaccines of this type will suitably be in the form of a
to determine whether any particular strain is F tularensis
pharmaceutical composition, in which the antigen is com- 15 subspecies tularensis or otherwise.
bined with a pharmaceutically acceptable carrier, as would be
In order to discover whether the LPS from a subspecies
understood in the art.
tularensis strain has similar structure (and properties) to that
The compositions of the invention may be in a form suitfrom a subspecies holarctica strain, LPS from F tularensis
able for oral use, for administration by inhalation (for
strain Schu S4 (subspecies tularensis) was extracted.
example as a finely divided powder or a liquid aerosol), for 20
LPS extracted from F tularensis strain Schu S4 was shown
administration by insufflation (for example as a finely divided
to have a characteristic ladder pattern after gel electrophorepowder) or for parenteral administration (for example as a
sis. However, the LPS was difficult to stain and required
sterile aqueous or oily solution for intravenous, subcutaneadditional oxidation in order to visualise the O-antigen bands.
ous, intramuscular or intramuscular dosing or as a supposiThis may suggest that the sugars in the 0-antigen of F. tulatory for rectal dosin.
25 rensis strain Schu S4 are not oxidised in the same way as the
The compositions of the invention may be obtained by
O-antigen sugars found in most other bacteria.
conventional procedures using conventional pharmaceutical
The F. tularensis strain Schu S4 0-antigen gene cluster
excipients, well known in the art.
contained 15 genes, the putative functions of which was
For further information on Formulation the reader is
assigned (see Table 1 above) based on the BLAST results and
referred to Chapter 25.2 in Volume 5 of Comprehensive 30 structural information about the sugars contained in the 0-anMedicinal Chemistry (Corwin Hansch; Chairman of Editotigen. Genes within the cluster are likely to be responsible for
rial Board), Pergamon Press 1990.
the production of the 0-antigen molecule as well as the transThe amount of active ingredient that is combined with one
portation of the molecule out of the bacterial cell.
or more excipients to produce a single dosage form will
There are two main O-antigen synthesis modes, O-antigen
necessarily vary depending upon the host treated and the 35 polymerase (wzy)-dependent and wzy-independent. In the
particular route of administration. For example, a formulation
wzy-dependent system it is thought that the polymerase
intended for oral administration to humans will generally
(wzy), flippase (wzx) and chain length determinant (wzz) are
contain, for example, from 0.5 mg to 2 g of active agent
part of a complex in the cell wall which facilitates polymericompounded with an appropriate and convenient amount of
sation and export of the LPS molecule. In the wzy-indepenexcipients which may vary from about 5 to about 98 percent 4o dent system a different set of proteins are involved in the
by weight of the total composition. Dosage unit forms will
transportation and polymerisation of the LPS molecule. The
generally contain about 1 mg to about 500 mg of an active
transporter is ATP driven and composed of two proteins wzt
ingredient. For further information on Routes of Administraand wzm that belong to the ABC-transporter family.
tion and Dosage Regimes the reader is referred to Chapter
In the F. tularensis O-antigen gene cluster, proteins with
25.3 in Volume 5 of Comprehensive Medicinal Chemistry 45 high identity to wzy and wzx are present, suggesting that
(Corwin Hansch; Chairman of Editorial Board), Pergamon
transportation and polymerisation of the O-antigen is via a
Press 1990.
wzy-dependent pathway.
The size of the dose for therapeutic or prophylactic purThe TMHMM analyses of the putative O-antigen flippase
poses of moiety of the invention will naturally vary according
(Wzx) and polymerase (Wzy) proteins supported their
to the age and sex of the animal or patient and the route of 5o assigned functions based on sequence similarity. The preadministration, according to well known principles of medidicted numbers of trans-membrane helices for the F. tularencine.
sis proteins of 14 and 11 for Wzx and Wzy respectively are
Thus in a further aspect the invention provides recombisimilar to those predicted for other bacteria, in which these
nant O-antigen of F. tularensis which is obtainable from a
cytoplasmic membrane proteins have been predicted to have
host cell which expresses proteins of SEQ ID NO 3-17, or 55 around 10-12 trans-membrane helices. The prediction of 2
modifications thereof.
large periplasmic domains for the F. tularensis Wzy protein is
Furthermore, the applicants' realisation of the sequence of
consistent with the two large periplasmic domains of the
the O-antigen sequence provides the possibility that this
Shigella flexneri Wzy protein.
sequence can form the basis of diagnostic tests, to determine
No gene that could encode a Wzz homologue was identiwhether a patient has an F. tularensis infection. In such case, 60 fled, which may indicate that one is not present in the F.
samples such as blood or saliva samples may be taken from
tularensis genome.
the patient and the presence of SEQ ID NO 1 or variants
The close proximity or overlapping of the genes wbtA to
thereof detected.
wbtL suggests these are transcribed as one operon.
Specific detection methods are well known in the art, and
Approximately 0.5 Kb downstream is wbtM, which has a
may include amplification procedures such as the polymerase 65 putative promoter of its own. Downstream of the second
chain reaction (PCR) and/or other detection methods using
transposase are manC and manB, which also have their own
for example labelled probes that hybridise to the target
putative promoter and are probably not involved in biosyn-
US 8,198,430 B2
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8
thesis of the 0-antigen as mannose was not found to be part of
FIG. 1. SDS-PAGE analysis of LPS isolated from E. coli
the structure of the F. tularensis O-antigen, nor is it one of the
strain K325, 1.25 tg (track 1) and F. tularensis strain Schu S4,
intermediate products required for its synthesis.
50 tg (track 2).
The two genes manC and manB may once have been
FIG. 2. The genetic organisation of the 0-antigen gene
involved in biosynthesis of the 0-antigen in an ancestor of F 5 cluster in F tularensis strain Schu S4. The G+C content of the
tularensis. The presence of transposases flanking the 0-antiO-antigen cluster is shown in the upper panel.
gen biosynthetic gene cluster wbtA to wbtM suggests this
FIG. 3. Schematic structure of an O-antigen subunit of F.
tularensis strain Schu S4 and the assignment of putative funccluster may have been horizontally acquired, perhaps replactions to the 0-antigen gene cluster genes. A single O-unit is
ing an ancestral polysaccharide gene cluster.
10 shown with sugar residues and glycosidic linkages indicated.
The 0-antigen gene cluster appears to be present in all
FIG. 4. Shows the region of the genome the nucleic acid
subspecies tularensis and B strains screened. However, there
sequence of the F tularensis genome which encodes all the
is at least one difference between the clusters in subspecies
proteins shown in FIG. 5.
tularensis and B strains within a region containing a transFIG. 5. Shows the amino acid sequences of proteins
posase. BLAST analysis using the partially deleted trans15 encoded by SEQ ID NO 1, as well as a number of flanking
posase has revealed possibly over 50 copies of it in the F
gene sequences,
tularensis Schu S4 genome. It is possible that the insertion
FIG. 6. Shows the nucleic acid sequence (SEQ ID NO 1)
sequence originated in the F tularensis genome from S. pneuwhich encodes the enzymes necessary for 0-antigen producmonia and was copied randomly within the genome. The
tion.
open reading frames flanking the insertion sequence have no 20
significant homology within the F tularensis genome, sugEXAMPLE 1
gesting that these genes were not imported to this locus with
the insertion.
Methods
In subspecies tularensis strains, this insertion has become
Bacterial Strains and Growth Conditions
deleted to leave only fragments of the transposase and down- 25
Bacterial strains used in this study are shown in Table 2 and
stream sequence. The overall similarity between the subspewere cultured at 37° C. on BCGA agar for 48 hrs.
cies tularensis and subspecies holarctica clusters seems to
indicate that the insertions took place in F tularensis before
TABLE 2
division of the subspecies. Partial deletion of the subspecies
Species and Strain
Subspecies
tularensis transposase would have the effect of stabilising this 30
F. tularensis Schu4
tularensis
region of DNA, as this enzyme has been found to be necessary
F. tularensis 199
tularensis
for insertion events to take place.
F. tularensis 230
tularensis
It seems unlikely that this will affect expression of the
F. tularensis 041
tularensis
F. tularensis LVS
holarctica
cluster in either subspecies tularensis or B strains. It could be 35
F. tularensis 200
holarctica
that in subspecies tularensis strains part of the transposase has
F. tularensis 025
holarctica
been lost due to genome down sizing. However, the gross
F. tularensis 075
holarctica
difference in size of PCR products generated across this
F. tularensis HN63
holarctica
F. tularensis 147
mediaasiatica
region when amplifying DNA from different subspecies may
be utilised in diagnostic procedures.
40
The applicants have found that a similar O-antigen gene
LPS Purification
cluster to that found in F tularensis strain Schu S4 is present
LPS was purified from F tularensis strain Schu S4 using a
in other strains of F. tularensis. This includes subspecies
hot-phenol and water extraction method (Westphal 0, et al.
holarctica strains. Consequently, a vaccine which utilises the
(1965). Methods in Carbohydrate Chemistry 5: 83-91).
0-antigen to produce a protective immune response is likely 45 Gel Electrophoresis and Silver Staining
to provide protection against infection by several virulent
Glycine gel electrophoresis was performed according to
strains of F. tularensis.
the method of Laemmli (Laemmli U K. (1970). Nature 227:
The applicants have demonstrated that LPS from F. tula680-685.) using a 12.5% separating gel with a 4.5% stacking
rensis subspecies tularensis strains is protective. In particular,
gel. Ten ltl of each sample were electrophoresed for approx 2
it appears to be protective against challenge from strains other 5o hat 100 my in the Mini-protean II slab system (Biorad).
than F. tularensis subspecies tularensis, and in particular
Gels were silver stained according to the method of Chart
against challenge with F. tularensis subspecies holartica.
(Chart IT (1994) LPS: Isolation and Characterisation. In:
This finding is unexpected in view of the results reported
Raton B, Arbor A (eds.) Methods in Practical Laboratory
above which suggest that LPS from F. tularensis subspecies
Bacteriology. CRC Press, London, Tokyo, pp. 11-20). Howholartica is not protective against infection by other F. tula- 55 ever, the oxidation step was increased to 10 min.
rensis species. Thus recombinant vaccines as described above
Nucleotide Sequence Analysis
will be particularly useful.
The sequence encoding the 0-antigen biosynthetic cluster
Thus in a further aspect, the invention provides LPS obtainwas identified and extracted from the Known protein
able from F. tularensis subspecies tularensis for use as a
sequences (obtained from GenBank) involved in the biosynvaccine against infection by F. tularensis. Vaccine composi- 60 thesis of the 0-antigen of other bacteria were used to probe the
tions containing LPS from F tularensis subspecies tularensis
F. tularensis Schu S4 partial genome sequence (Prior RG, et
are also novel and form a further aspect of the invention.
al. (2001) Journal of applied microbiology 91: 614-620),
These will comprise pharmaceutically acceptable carriers as
using TBLASTN (Altschul SF. et al. (1997) Nucleic acids
described above.
research 25: 3389-3402). The contig containing the putative
The invention will now be particularly described by way of 65 O-antigen gene cluster was extracted and subsequently analyexample with reference to the accompanying diagrammatic
sed using the annotation tool Artemis (Wellcome Trust
drawings in which:
Sanger Institute, UK). This allowed visualization of
US 8,198,430 B2
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10
BLASTN, BLASTX and BLASTP searches, GC content and
other analyses performed on the sequence and the predicted
proteins.
The protein sequences encoded by the putative 0-antigen
flippase gene (wzx) and 0-antigen polymerase gene (wzy)
were analysed for trans-membrane helices using TMHMM
(Sonnhammer EL L, et al. (1998) In: Glasgow J, Littlejohn T,
et al. (eds.) The sixth international conference on intelligent
systems for molecular biology. AAAI Press, Menlo Park,
Calif., pp. 175-182).
PCR Analysis of the Putative O-Antigen Gene Cluster
DNA was prepared from the F tularensis strains shown in
table 1, by phenol extraction, as described by Karlsson et al
2000 (Microb. Comp. Genom. 5: 25-39). Ten pairs of overlapping PCR primers were designed to amplify the whole of
the putative 0-antigen gene cluster in approximate 2 kilobase
segments using the DNAstar program PrimerSelect. The
primers were designed with annealing temperatures ranging
from 42 to 59° C., although all were used successfully at 49°
C.
The structures of these primer pairs is summarised in Table
3.
TABLE 3
Primer Forward/
set
reverse Structure
SEQ ID
NO
1
Forward ATAATGAAATCAATCCACGAG
Reverse CCAGCCAGTCAGTCCCACAG
21
22
2
Forward TGTCTTAGATATGGGGCAACC
Reverse ACAAATATCAAATCCTAACACATC
23
24
3
Forward TAGAAGCAGCTGCGATAGGTAGAC
Reverse TTAAATAAAAACTGAGGAAACA
25
26
4
Forward ATGGTATTTTAATCAAGTGT
Reverse CTAGTATGCCCCAGAGT
27
28
5
Forward TGGTGCGACAATCAAGTTA
Reverse AGAAGTTCCTCCTCAGTC
29
30
6
Forward AGAAATTAAGAGCAAAAGGAAAGT
Reverse ATCTCAAAGTCAAAATCAGTCTCT
31
32
7
Forward TACGATATTGTCCTCTCCGATTAG
Reverse TAGTTGCGACATATTGACCTG
33
34
8
Forward AGGCAGGTCAATATGTCGCAACT
Reverse TTTCCGCAACACTTCAGCAACTT
35
36
9
Forward GCTATGGCCACTATCACGAGAGG
Reverse TATACTTGCTTGCCCACTGCTTAG
37
38
Forward ACCGTAGTGAGCATTGGATTGT
Reverse ACTAGGGCCTCTGACCGTTCTC
39
40
10
PCR amplification using each pair of primers with each
template DNA was carried out in the following mixture:
1 xPCR buffer (including 1.5 mM MgC12), 0.2 mM deoxynucleoside triphosphates (dNTPs), 2.5 mM forward primer,
2.5 mM reverse primer, 2.0 ld template DNA, 0.5U Tag polymerase and filtered sterile water to a final volume of 20 µl. The
reaction mixtures were incubated at 90° C. for 1 min and then
cycled at 90° C. for 1 min, 49° C. for 1 min and 72° C. for 2
min 25 s for 30 cycles, with a final incubation at 72°C. for 10
min. PCR products were visualised on 0.5% agarose gels,
with ethidium bromide staining. PCR buffer, dNTPs, and
polymerase were from Roche. PCR primers were synthesised
by MWG-Biotech.
Cloning of PCR Products
PCR products amplified from Schu S4, HN63 and LVS
DNA using primer pair 8 were cloned into pGEM-T easy
(Promega) for sequence analysis. Ligated DNA was trans5 formed in E. coli JM109 chemically competent cells
(Promega) and putative clones were screened using both
colony PCR and digestion with restriction endonucleases. All
DNA manipulations, including ligations, transformations,
colony PCR, restriction endonuclease digestions and agarose
io gel electrophoresis were carried out according to methods
described by Sambrook et al (1987) Molecular cloning: A
laboratory manual. Cold Spring Harbor, N.Y.).
Purification of PCR products from agarose gel was
achieved using the QIAquick Gel Extraction Kit (Qiagen)
15 according to the manufacturer's instructions.
The three constructs were sequenced at Oswel by the
dideoxynucleotide chain-termination method (Sanger F, et al.
Proc. Natl. Acad. Sci. U.S.A. 74: 5463-5467) using universal
primers. Each sequence was compared and the BLAST (Alts20 chul S F, et al. (1990) Basic local alignment search tool. J.
Mol. Biol. 215: 403-410) function of the ARTEMIS software
package was used for homology searches in the locally held
GenBank databases to identify the functions of the differential regions of DNA.
25 Mass Spectrometry Analysis of the 0-Antigen Molecule
Results
LPS Purification
The hot phenol-water extraction method was used to purify
LPS from 2.2 g of freeze dried F tularensis strain Schu S4.
3o This resulted in 7 mg of LPS, which is a yield of 0.3%. The
LPS was difficult to visualise after SDS-PAGE and silver
staining. The oxidation step was increased from 5 min to 10
min to visualise a ladder pattern (FIG. 1).
F tularensis 0-Antigen Biosynthetic Gene Cluster
35
The F tularensis 0-antigen biosynthetic gene cluster was
found to be 17 Kb in length and contain 15 genes putatively
identified as being involved in 0-antigen biosynthesis,
flanked by two transposases (FIG. 2). Possible promoter sites
were identified just upstream of the genes wbtA and wbtM.
4o Downstream of the second transposase are located the genes
manC and manB, with a possible promoter just upstream of
manC.
FIG. 2 also shows the G+C content plot of the cluster using
a window size of 500 bases. The overall G+C content of this
45 region of the genome at 31.27% is slightly lower than the
genome average of approximately 33%. The plot shows that
the central section of the cluster, from wzy to wbtK, generally
has an even lower G+C content.
Downstream from manC, on the opposite strand, are
50 located the genes for the transcription termination factor rho
and thioredoxin. In E. coli both of these genes are also found
flanking one end of a polysaccharide biosynthetic gene cluster that of the enterobacterial common antigen.
The 0-antigen repeat unit of F tularensis is shown in FIG.
55 3, together with the putative role of the genes involved in
0-antigen biosynthesis. Based on their homology to other
LPS and sugar biosynthetic genes, in particular P. aeruginosa
serotype 06 which expresses a similar 0-antigen repeat
structure (Knirel Y A, et al. (1985) Eur. J. Biochem. 150:
60 541-550), the putative role of the gene products have been
assigned.
It is proposed that the biosynthesis of 2-acetamido-2,6dideoxy-D-glucose (QuiNAc) involves WbtA, a dehydratase
and WbtC, which shows homology to UDP-Glc 4-epime65 rases. WbtA and WbtC share homology to WbpM and WbpV
of P. aeruginosa strain 06, both thought to be involved in
QuiNAc biosynthesis and shown to be essential for 06 0-an-
US 8,198,430 B2
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12
tigen synthesis. WbtE, WbtF and WbtH are proposed to be
PCR Analysis of the 0-Antigen Gene Cluster
Eight of the PCR products (primer sets 2, 3, 4, 5, 6, 7, 9 and
involved in 2-acetamido-2-deoxy-D-galactouronamide (Gal10) from each template DNA appeared to bethe same size
NAcAN) biosynthesis. WbtF shows homology to UDP-gluwhen viewed by agarose gel electrophoresis. Primer pair 1,
cose 4-epimerases, including WbpP and VipB, whilst WbtE
shows homology to WbpO and VipA, UDP-Ga1NAc dehy- 5 covering the start of the gene cluster, had to be designed to
amplify a 4.8 Kb region due to lack of suitable priming sites
drogenases involved in the formation of 2-acetamido-2upstream of the cluster because of the presence of an insertion
deoxy-D-galactouronic acid (Ga1NAcA) inE. aeruginosa and
element found many times in the F tularensis Schu S4
Salmonella enterica var typhi respectively. WbtH produces
genome. This primer pair 1 produced the relevant size product
significant alignments with glutamine amidotransferases,
io for F tularensis Schu S4, but when used on subspecies holincluding WbpS of P. aeruginosa serotype 06, which may
arctica, strain LVS, did not produce a product. Thus this
putatively be involved in the formation of the Ga1NAcAN
primer pair may have particular applications in diagnostics
amido group. Biosynthesis of the fourth sugar, 4N-formylwhere distinction betweenF tularensis subspecies tularensis
quinovosamine (Qui4NFm) most likely involves Wbtl, WbtJ,
and F tularensis subspecies holarctica is required. Where
WbtL and WbtM. Sequence homology suggests that WbtL i5 samples containing DNA from the former is present, a PCR
may be involved in the formation of the activated sugar
using primer pair 1 will generate a product, which would not
dTDP-D-Glucose with WbtM functioning as a dTDP-D-Glube present in the second case.
cose 4,6-dehydratase. Wbtl is proposed to be involved in
The PCR using primer pair 8 revealed a difference in size
Qui4NFm amination since it shows homology to RfbE, a
between subspecies tularensis strains and subspecies holarcperosamine synthetase. Finally, WbtJ is likely to be respon- 20 tica and subspecies mediaasiatica. Subspecies tularensis
sible for the addition of the N-formyl moiety, showing sigstrains show a deletion of 303 nucleotides when compared to
subspecies holarctica strains (including LVS) and subspecies
nificant homology to formyltransferases.
mediaasiatica. Cloning and sequence analysis of this region
Specific glycosyltransferases are required to form the olifrom the subspecies tularensis strain Schu S4, the subspecies
gosaccharide units of the O-antigen repeat. Four glycosyl25 holarctica strain HN63 and LVS has shown that the deletion
transferases would be necessary for the synthesis of each
in Schu S4 occurs at the beginning of a putative transposase
0-antigen unit in F tularensis. Based on homology, WbtB is
that is similar to IS630-spn 1 transposase ORF 1 of Streptoproposed to mediate the addition of QuiNAc to undecaprenyl
coccus pneumoniae.
phosphate (Und-P) to initiate O-antigen biosynthesis. WbtD
Thus primer pair 8 may also be particularly useful in disand WbtG are probable Ga1NAcAN transferases, possibly 30 tinguishing between strains of F tularenis. Following a PCR
involved in the addition of the two consecutive Ga1NAcAN
reaction on samples containing DNA using these primers, a
residues onto the O-antigen unit. WbtD shares homology to
separation of the products on the basis of size, for example on
WbpU of P. aeruginosa strain 06, proposed to transfer 2-fora gel, should reveal distinguishable differences therebetween.
mamido-2-deoxy-D-galactouronamide (Ga1NFmAN) onto
EXAMPLE 2
QuiNAc (Belanger M, et al. (1999). Microbiology 145: 3505- 35
3521). WbtG is homologous to WbpT of P. aeruginosa,
Protective Effects
thought to be involved in addition of Ga1NAcA to Ga1NFLPS Purification
mAN. WbtK is probably the fourth glycosyltransferase,
LPS was purified from F tularensis strain Schu S4 or from
which
adds
4,6-dideoxy-4-formamido-D-glucose
40 strain LVS using a hot-phenol and water extraction method
(QuiNA4Fm) to complete the tetrasaccharide 0 unit.
mentioned above in Example 1.
Wzx and Wzy
Immunization with LPS and Protection Studies
Once assembled, the O-antigen repeat units are transloThe ability of F. tularensis strain LVS or strain Schu S4
LPS to protect BALB/c mice from a F tularensis was detercated to the periplasmic face of the inner membrane via Wzx,
a transporter/flippase. Wzy then acts as an O-antigen repeat 45 mined by immunizing groups of six female BALB/c mice by
the i.p. route with the purified LPS obtained. On each dosing
unit polymerase. When analysed using TMHMM, the F tulaoccasion, mice were given 50 µg of LPS in phosphate buffrensis Wzx protein had a predicted 14 trans-membrane heliered saline (PBS). The mice received three immunizations,
ces, with both termini on the cytoplasmic side of the memeach 7 days apart.
brane. The F tularensis Wzy protein had a predicted 11
Mice were challenged i.p. with F tularensis LVS (1x105
trans-membrane helices, with the amino terminus predicted 50
CFU)
21 days after the last immunization. All control animals
to be on the cytoplasmic side of the membrane, and the
died after challenge. Mice which had been immunised LPS
carboxy terminus on the periplasmic side. Additionally, the
isolated from the LVS strain were protected from death. Mice
Wzy protein was predicted to have two large periplasmic
which had been immunised with LPS from either the SchuS4
domains from amino acids 142-178 and 268-327.
55 or LVS strain showed and extended time to death. At a chalA gene with homology to the O-antigen chain length deterlenge dose of 10 cfu animals Immunised with SchS4 LPS
minant (wzz) was not identified in the current F tularensis
survived for an average of 64 hours (with 99% confidence)
Schu S4 sequence dataset.
longer than the unimmunised controls.
SEQUENCE LISTING
<160> NUMBER OF SEQ ID NOS: 41
<210> SEQ ID NO 1
<211> LENGTH: 17623
o8ZZ 6PDPD666jD 66jDe6jDe6 66I6IDD616 jD6jPPPD66 666eeje 6e6eeeDPD6
OZZZ
PjPPjjDe6j e6PPDjee66 jPjjPD6PDD e6PPDD 666 16D DBPBI eee6j66eee
09TZ
PjDjjPj222 jPjjPPPDPD D6116PBIPB P111DIPDIP PP66PPIllI IP66266II6
OOTZ
6J22IDPPIPI 6633 3663
ObOZ
JIBIP161P 13 6Pjjj62 PjPjPj2D2j 1111112236 22j26222j6 6363622
086T
LLDLLLjJjj IIL IL j33 6 636 2226222 2Fi ~2~~~~~2~~ PlIpplIDII
OZ6T
0981
33~6
6
6
66363
66666
6II j2266 336
26223 633 2236666
OOST
6223 33 6 6366
or
3363
0881
662~62~66 366
6662~
3222~36
36663
3622633
633
66
633~2
66636
6D6 636363 62233
3336
6366 66662 233 36666 66~226 3 33 636
09Z1 6e36eee~e6 3e336
OZOT
6633 636
63366 662222622 222 66226
OZBT
0801
62233222~
6222~2~3 6336
6662 62~663
0951
0DTT
636
3662263
OZ9T
OOZT
6636
36666
6~26~26226
3336
636 ~2 Fi2222~~~
266~26
63
0DLT
089T
3361PIP6
6666
6626
008T
63363
36~226
6e6116166 36ee6
3663
366
66662~
33363 6223 336 3636~23
666366
36636
3336
62222 Fi
6366
66666
666
096
223663
006
23 62~36~6 6~6622~222 22~~2~~~~2 PIDIDIPIDI 226222 262 ~~~62622~~
08
08L
636
336
6~63636
62233
226663 336336
6333
662~6
009
66226
0b5
63363
663
622262233
6~266
6336
636
6663
6363666
36~2663
081
3366
09
6633
63663
23666
66363
6333
6222366~2
66636
36662~23
636636
o
OZT
636
633
O
00
66 22226~~~22
3366 22222~62~2
08
09E
62~662 6636663
6336ee6~~
OZL
099
6366
666ee
36
662~366
66
366
63
3
662
363
6~262236
6663
63
3636
662~22~~~~ 2Fi2~22~2~2
6336
6
36
63366
T :3DM QO S <00t>
sTsu9.z2Tnl 2TT9sTDu2zd :WSINFI02IO <£TZ>
FINQ :3dI,L <ZTZ>
p9nUT UOD1,j
Uj
ZU O17'861'8 S 1
O89 b
D 6e6j6PPD 66eejjejje 6DD6DP6jjj e6jDDjDPee eje6PD6eee Jj6PDD Dej
OZ9b
DD 62D6 6jJPj PDjjj 222j6j6262 22jjDj622D PPP6j P6j6j 16PIlIPPPB
095b
j22D2FDPjP 2262D 6jD6 626j26266P j6pp62222D PPDD 6111 I66I266PPI
oo
Pjj2D22Djj 6P2PPOPP26 1H111PTPFi1 HPBiBi2ABI 6jP62j2P22 PPP2D6jjj2
Obbti
D611PIDBPI PPDD66 1161211666 JID6611661 j2D
08E
D6D 6PjPjP 66P6jpj2jj 16PBIPIIPP P111666PPI P2jppjjppp PD6PDPjjDP
OZ£b
jPD6jPPDj6 11611el6ee 6PIP61111P 6eeeee6eje 6PPjD6e6ej D6PPPjPjD6
09ZDI
P61PIDBPIP 222j26jDPj P16DIBIPIP PPIPI116PD 626D D 22j6DPjjjD
oo
116PBlBIPB jPPjDDDeej 6jPDjPjjD6 6ID D D 66 je6je6jjjj jeee6e6ej6
OvTD,
1666IDD
66PP 22RP6i222Pj
6D 6D6 IPP16IDDPB P166PIPBDB jD62D62262 JI6ID6I6I2
080b PPDj6jP266 PP6jPDD2jj D1PDD6H11 6jJ62D6jPD 2D6j6PjD62 q2222P2Fi22
OZOb
PIDPIPPIP6 IDPI111661 ejjjjeeeej 6PDjPPPeje 6DPjPPjj66 eee666jejj
666
096E
D6DD6
006E
Ob8£
D6D ~62~~2~222 22~2~22222
6
666 6222262226
6eee~DD
6D6
OZL
DD6
099E
D666
6D66D
6~222DD
~6~~~222~~ 222~~22~2~
08bE
LDH1LIDLL 111LIJ11DQ D666
09££
DeDee~6e~D
D666D2 Fit ~2 Fi2 ~2~2
6
6
6222222~D 6D662
ppjjjjj222 22~26~~~2~ ~~2262622~ 666
6222262~2~ ~222222~2~
666~2D
1111221222 6D666
D6D
222DD6622 6666 6~22DDDD2 6D
6
666222DD
D
DeeD66
6
D22D662
D666
666
DD6D66
66DDe
666~e~~
63 66622 D~26222~D6 22D2222D66
DD~266222
OOL
D
D2j2DDD
666D6
D6D6
6D6~26
0852 D66616D 6~26D6D6
2~66
09bZ 6
00
D222D6
662 26222~~22~
OZ8Z ee~DDee6DD
O9
66D66
66D6
882 D66DD6
09L
6666~2
6D
000
O6
66226
D6DD 2222262222 222~22~222
OZT
090E
DD6
6e6eee~D 6~D6D 6e6DIeej6e e6e6ee6e~~
666
ObZ~
08TE
666
6D
00
D6
66D6
66
62222~~~2D
666
ObSV
OZb~
ee~eeee6~e
6 666D66 ~~2~~2~~2~ 22~~222226 266I62DI22 ~2222622~~
08L~ 6
009E
D66
6DD6
66
66622~~ ~262~2~222
D66
D62~66
66D62~ ~~~2262222 2226~2~62~
6D666 D66666 e66D6D6 66eeeeeDD6
6D6
ObEZ 2~6~2~~26~ 666D
66D6 2222 22222
D6222~26
6~2666 66~22~~2
p9nUT UOD-
91
91
ZU O17'861'8 S 1
080E
111112IPIP gq2q6622Dq 11DIIIP161 11112IDI22 DpgqDqqqDp 1161111112
OZOL
I22I2222ID 161PIPPIll jjjj2jj222 P61PP IPPIP 61PIPD
0969 ppqfiq11V6i 62~26D6D
DDDFi
66D~6~~~2~ ~222222~~~ lDf3pgqqqqq
6
08L9 666
D66DDD
6D6 p1OphIpi2 1112121111 2222gqqqDq Fi~Fi2222222
OZL9
0999
D66
0099
D662~~226
O9
D66
089
1666JIDD6
09
09E9
00E9
09
636
66
6666D66
6D6
62~2~2~2D
662 6D6D
66
66D
666666
DD6
DDD~26
DD66
6D~222~26
D662~26
26D22DD62 62222~66
ObES
22jjjjDPjj D66D
0825
jDPPPPPPDj 6
OZZS
DI2112I22~ 66DDD
66622~62
666D6 6666D6 66~~~~~~~~
62~662 22DD6~~
6D62~66 DD6
6DD2~2266
66
D22D6D6D
6D66
6D6
6~2666
D62~66
6D66
09TS p6 6166
66
D66D6
6666
DD66
666D
D666
666
DD6DD ~6~~262262
6DD2~2~26 2D6D6
666D66
66D6 26D6D662 2D6D22~D D
DDD
66DD622~~
662~DD
6D6
6666
6D
6222DDD2 22266 622~D6D62
086b 26D666
666D6D2 62~62~62~D 626~2~~~62
222D6D
6622666
662~2D 66
BBIDD
26D6D2
66~6
6D22D6222 D22~2~26
oo
o t
266D622
D 6D6~262~D
666D66
22622DD2~6
D6662~6 666~~2~
6D66D
26~2D222~D 666226
008b
6662
666
6DD66
D2D~66
09b5
098
6DDDD~
66
09L
O b0
6D66
62~D66D622 6D6~2~22
66DD6
6~22D2D
OZSS D6
D62226
D6D6
~2Fi2~~~2Fi2 6
2D6D6
D22DD
DD66~266
D666
09
D~222D666
66~2666
666
0885
06
6~2D6
666D6
62~62~6 6D666~22
D66D
2D6D
6
DD62 62 2222222
6662~6
D~626266DD
D6226666 D6D622D
DOTS
6DD66
D6
0b65
0855
662~D662 26D
DD~2622D2 62262~66D
0009
OOLS
66DD6
66
0Z19
OZ85
6666D 6~2D6222~2
66DD
0819
0909
OlDDIB611 PO BD6pD61
DD 6 2~22~22~~~ pM jp F3j PpO j211111DIZ 111DMIIIZ
0069
0b89
6 11111PI11D
62~6222D~2 ~~22~22262
666 DD~222~26 666DDD D666 222~~~~262
e6D66D
6D22D~226
6D666 ee6e~e~~6~
D6D66
p9nUT UOD-
81
Li
ZU O17'861'8 S 1
662~6666
O8b6
D D66226jj 2262D D6j 1116661111 P1661PPPPD D66
O6
266D66
09E6
IlD6gqq6q2 2662~2D6
00E6
2D16611116 626~~2~26~ 62~22~6222 2~~~222~~~ j222DjjPjP 26~622~~2~
D6D
PjPjjP DD62
D66D2 222262~6D
6D
6666D
6D62~22~D
6~22DD6 2D~2D666D 6D
D6666D
OZT6
D6D666
6DD
6666
D662~6
0006
66666
D6
6
66~2D6D62
666D6
112IDD 6
6eee~6
6D6
DD66
6622 662222D
D666D ~22Fi~~~2~Fi
66 eeeeDee~e6
098
0858 6662266 D6
66D6
6DD
0Z58
66
008
D6 66eIele6eI eeee6D 6~
DD622
6D6
D~222622 D62~2D6 62~2~~2222
6D
D66DD222
098
6~222D6
D6~22DD
62~2~2222D
D66 ~~2~~22262 22~~~22~22 2jj2D22jj~ 262~~~~~62 2~~~~~~222
0E8
eD6~ee66
66666
DD6eeD6~e~
0ZZ8
6~222D6
0918
D666
D6D
66DD6 222226226 2~~2~~2622
H2I2IP22t 11611PI61~ ~2~~2~Fi~22 ~~~222~222 fjJ222Jfl2D 2~2~~~Fi222
6~66
008
D6226pI jf3
626666
66D
086L
6622DD
0Z6L
D6D
D6
666DD
D6~22D 2226D666
6622226
6662 662~D22D2~
D62D2222 6222~~DD
098E
662
D622D~~~~
008E
0 DLL
6666
DD6226D6
00L8
0018
6DD222226
6222~22~6 622D6D
OZ88 6e6~~~~~6e
088
6~266D6 6662~2D62
D66 D6666~2D
08
09L8
D6D
D666 226226222
0906
O68
26D6~2
D6D2~22~6
0DZ6 D62D
0816
666~~
~~2~~2Fi~~2 ~Fi2 Fit ~~~~~ 2~2~~~~Fi~2 222Fi2 Fi~222 6D6~~
66
089L
66~26
OZ9L
66666
D2
66D
26DD
62266~2DD
095E
6DD
OOSL
6DD66~
6D66
D66662 Fi
666~2
0DDL
ILLDIL1111 IDI IL jDf3Df3 Fi~~2~22~2~ ~2~~~Fi~~2~ ~22222Fi222 222~~2~2~2
08EL
ILILIIJILI LLDLLj22j~ Fi~~2~2~~~2 ~~~~~~~22~ jDjDPDjPDP 2Fi2~~~2~2~
DD66
OZEL
09ZL
OOZE
0DTL
D6D222
666
D66
D66
DD2~~~2~2~
66~26666 266D6D
6~226D
6D66D~ ~2~Fi2Fi22~~ Fi22~2~22Fi~
666
666~222
p9nUT UOD-
OZ
61
ZU O17'861'8 S 1
O88TT 2D6T2IH2P OD6H1612I RTPP6PP22 TDIPIPRB2 6PPD D D D6RZPI62D
OZ8TT 6PPPDD6e6e IPIPTI6eA DZ6PIP6eM POZeODTeH AIPZP62Z6 DBDIPIPPIP
D D622 262D2I6P6P DIDIID 66 666D66 2111212216
09LT
OOLTT eDDVIIPID 6
66 PI6PIDUeD
6666
Ob9TT Ie6eII666I D66e6IPIPI e6P6PDP Ae II6eee66ee PPD6e6PPH PPP6PIPHP
08STT R6ZP6i2TDT 1616HZDP6i 66RZD66D6 626IIIPP6I PPHT2222IR 6D66
OZSTT e M DLTIDD IPPDIPe6PD ID6eee6PIP 166H 11PPI IPDI AP~T6 D66
09bTT A6TTD6 e61e6IPPII PDIPIID6D6 T66e66ee6I TPTRM2DU AZP6PPPDP
6D
6D H1ue6166 TPIDeeeIIe IPI6e6P6I6 666Pe
OOtiTI PD61P D
ObETT 1161261111 D6RR2D62R 6IP612I U2 II62PPPAD PPI2DP2P2P 2TD66TR6R
6666 6666 1H6i16i1PR1 T6~DD6I6M MD62DgDP
08ZTI
OZZTT I266P6226I 12ZR6R16P 2AP222IDZ DT6D~6~R~ 622DIR2IP 2626R6T~2
09111 Ie61161116 I6PDDeeeeI 6IPPDPPIDI 116611PD11 DIPDPIDPID 611611 1PD
OOTTT IIILDD D DPUPPD6I6 ee66PPDDI6 P66P116D6P 6PIPI6D6D Pu62IIPD6
OoOIT IIeeDPI66I PeeDIeI6eI 1116PIeIee PePII6I66P IDIPI6eeeD 6IIDP6eeee
0860T ee6e~D6e6e PD6l6en6D D66
OZ60T
ID
D66 66~eeeeeDe ~e~eeee~ee
6D6 2PRIPDPIP 22~2~262~~ PjjDPjPDDP 222~22~6~2 22~6222~62
09801 6I22I2BP11 66~62662D
OO8OI p16616e6
66
D6622D6
D622D6 6~~2226222
D666666
DD6D
0DLOT PDPf3eleeee l6~le hP66 IDPIIIeeIP 6eD666~6e6
0890T IeeeID611D 633 riel66l 1166661e6
D66
0901 ILLLILILII 666 IIII2P62P2 2266D
6622D62
6e6ee1166
D666
e6DD66~e
66 26D22D2~22 P2 F TD2f3II
0901 Le DLITDD 12RDf3llIP Pf312I112IP 2ZPDI2f32ll IIZPDD6l62 6D66
D6 6D62D62 62~2666
OOSOT DIDID
o or PID
6I262 j26 j2j2j2D2j2
6b6 11161e6Ie6 6~6e6eeDD~ ~~~~e~~ee~ PDD11eIPf3l PIIIPIpe6e
08E01 2 Rf3P62PZI 2Z2I2612I6 262222 626
Df32lD2I26l 2P2IPP2DPD
OZ£OT 11611ZD D el6le h lD6 ef3PePIPeeD
6DlDllf3e 6eeeeeIDee
6~D6
0901
Df3DMf3e P166VHP2 PUu26D6l D6epppppel
OOIOI e66D6~e6 eDDeD6lele ePIIDPll6e elb6llleee DTeePeeeeI bf3llelppeD
0DTOT DPDf3P h h I beIe6giIeeT PU66D66e ID D6e6le6 66~D6De6 66eDlelDpb
08001 f3llf3PDP2l6 222f3Dlf32PI D6PlPPPPl6 P616UbTP1 6D DD 82 2DPbIPDTR
OZOOT 6ID611TDDD 66D6
66~D6
DPPeeDDPIe el6le6UDl D666eee
662D2D 2DD222D2~6
0966
0066 6D6226
6DD6
0b86
D66
D6 622D6~2D62
66
62~6D22D
6222~D
6666DD
D66
08L6
6DDD6 6D6D6 ~222~2~22~ ~2~~2~2~62 222~~~622~ ~22262~~~2
OZL6
6DD~22D6 22D 66
6D6 22222~~~~2
62~22~66 D6~26
0996 666
0096 LLjDLLLfDj 2j2jPLLLLj UJMRZb2 IP DD2D DD 6D6
06
D22D6
D6~266
6666D66
p9nUT UoDe
lz
ZU O17'861'8 S 1
62~662~6~2
666~2~2
DD622~~
OSZbT 66Ie6PDPID I U IIe6IP6 P66H6D
6e6PPDID
oZZbT 16611ID16e 6eAI6IPPD 166HP~P16 eeee6PPDIP PDPIPDe6Ie BIBIBIe16e
666 166e611116 6PPDPI66D 116111161e I6P6PIPI6I
09TbT
oOTvT 1ILLLDLILI IIe UIIIPD 611166611e 611PIPP11e e6PI66II66 BIIPPPP16P
o ovr LODleV1U P166U666e eH1PIPPIl 6DIeAeIII IIFIPI6PI PIDeUPH6
086ET PDH2I MPP2 1211111112 1H6i2I22DI2 Z6622PID6I RPI61PDDP DPI6HPIDD
OZ6£T 6Ie66ID
Ie6ID66661 6elelD6
66D66
DD6D
D66
09SET Pf3BlPu2P1 PlIDBIDPIP IPZPAHPZP IDD D61111 A2I6PIII6 ~VPPDPIP2
008ET ILILDLILLI 2DPIDf3222I 1H22IF61PZ lDRl2lF3PDP 1111112122 DUIPDIPIP
0DL£T PZP16U 1eI IPIDIPD6PD IPV11fi1e1 eZe1Pee V D ZPIPPl6Pl6 666OPPID6
089E1 IIePP66K1 6ee66PDe66 IPDI6PD6II 6ID6PDIPDI D66 AIFZ6IPDP
OZ9ET Pl6~l6lD6l D~~ZUZZZD U1CCDZDIP BllDD1PDZP 2P2UZPRI ~~Fi222 FiFi~2
62N2ID6l2 PDP6lP2f3Pl DDfi2DII2ID 62 D6PA2
09SET
OOSET 6DD D DD 6 112IPHRPII 6ID6162IRZ 2I2Fi16i2I26i APRIRZZ 11PI1661PI
DDellPl6le D D D 6~6 elIPPeIefi1 DZePDIDPDI 666l
or
08EST IIFPD2eDM D A2Z2D6
OZ££T
D662ID D6
DD D6 Ifi 2e2P62H 22 RP22R I
666 V1666~6D6 eA 1PDIPDI eDDPIDPIPe a IPIIIZPD FIIPeD6IIP
09ZET IFiIP1Fi2IDI 21PID2I26b f3llP2IP2DP F3PRI2IPlFit IZ12I12lf3l 2A2IePID2
OOZ£T IDIBIBIPLD BIDIP H 1M IPMe66166 IIMAfPR2 IDeDe6beee DPIPDIPI6e
OTET 2 A R2Ze21 2lVD 66
D666D6
08oEY 6I6661e6Pe 11PD611161 PeT11666
666666
DD6
D6D~
66e~e6D6
OZO£T Ie6PID6D D 66eIeIPDeI Z66H D DD DDIPIPIe16 6ePDIIeDeD e16P AeeZ6
O96ZT ll2blet66 ID 616611 6hPlPD
D6e6
6Dl6ell 666eeD6D
006ZT L H DcZZLDZ eIDgi6Ie6ZD PI6eI66P6e II6IPDID66 T6Pe6P6PeD IeZIIDePDD
0DSZT PDPeIe1Ffi1 ~6~e66U6
08eZT
66D66 6DgiPPDIIII l6PDPIIIII U666~D
66D6 e6leDl6eeI IIeDeeDeII Le1611IDRL IDeIP11611 e166UePIt
OIUY DF2IDPI2I2 IDP2D2D22I 2IeII2ePDI PI2PDPIP22 PPP22IIDD6 2FI2222262
099ZT M6PPIe62P PP6ltDe26t IDID611611 lPlf3PP2I2P 266 D66~
OO9ZT ll62l22222 IPIPDDIDDD 2DPD2f3lPlD 6662eIDZ2I D26ll22D62 2666D6~22D
ObSZT 22l62l2P22 I262III26I 1222616161 2D22222IPI R6ZRRgiP DII22I2IDP
08bZT PD66ee6DD DPPIDDPeeb Tee6DIDPDI 16PeIeeIPP 61111PIe61 P6eIPDI6eP
OIliZT eIDPD11611 6e66ee6e ePeeegieDII 6ePPID6IeI V61e6H11 DIDPeee6Dl
09EZT IlDlIPeI16 Pe6H6egifiP PF6PDIIPDI PPIPIe6e66 IPDIe6eZe6 P6ee6Ie66I
OOEZT 26l6l2DiI2 QD D62662 IPIDDPID22 eIe2PI2II2 IRIDARZ66 PDPDD 66
OfiZZT X6662 Fit X22 IeI2DDZ22I IDe6iFiIDDZ2 DLI2IPPPT2 IFiI2IIPLFi2 DII2MI62Z
08TZT TP2eeD6eDD
6D
D6 HDZVI166 PIII2F3I2IFi PPM11ZZeII
OTT D~26~22D66 (222222226 IPIP62l22D D622~~2626 lF2D22Z2I2 IPP2FP2PDD
090ZT 6M ltDllD2 e2D 62622D 11166 16111 IIeRP6IZ6 6PDPI6I26I 2PPP2I62I6
OOOZY f3ll2DI2I22 222IlIl26l 622IDII2ID P262l22I26 2DPI161161 IIPI222222
Oli6TT P6lgiD(DD
6ee666
p9nUT UOD-
1,z
fz
ZU O17'861'8 S 1
0899T 2612616111 2I26I66222 PI2 H 1IDPI 2DD22IPDPP 6
611 D262222IDI
OZ99T PPIP6PHI6 e1666D6
616PIPID
09S9T 6DPDIPII6I 26IPI6D6I6 IPIDPPI6PI 66
OOS9T ILILLLLLLD D
Obb9T
6 66D66D6I66 2D
62IDD
DPIDPP 6Ieeeeee6I ee66e6IPPI 6Ie61e6Iee 66PIPID DI
666 6D6IPIII6e PDe6IPPI66 D6D DD6DD
6D66
08E91 PIIP6e666I Ie6I6Ie6PD IPIPPPIDID IPI66PIP6e D6I66ID
D 6IIIID
O£91 PPI6e6I6Ie IPPIIPe6Ie IPIIIP6Iee 66IIee6DD ee6Ie6e6Ie PIIPIIee6D
0991 ee6eee66II D 6PDeee6 Ieeeeee6II 6PD6eeee6e BIBID11PPD 6PD6PPPPIP
0091 R6P26IPIZ IPPPFIIHP AI6i2ZI6iP2 RPlIBIP2I PR2PPPIDP 1111661262
0 1 9T D6HDD6
DPPPIPD626
DI2D22I6 II6PIPDPI6
0809T IDIIHZD2Z2 PRFiFiPPZPP POPAOZZPI 2161PI1116 6iIP22PPID2 BIPPDPI111
0091 6Ie6D6eIeI eIPcI66H 1 66eDe6IPPI 6PD6D6ZDLe eDDIIe6e66 111161eIe6
DIDLeL666Z 66ee6~eDDD DLcDl6DDIe elIPPFIeH I661P66PPI
096ST 66
0061 IPPIII2P6P 26IDI2I62D L66R ~6P62 D26P2IDILI D2PPP26ID6 22D D62ID
081 l6gieDPPDPD PPDiDIP Q T D66DPPI6D6 6iPD6Aeee 6e66V 161 ze6IIPPPPI
08LST ID666PPDe6 PPPP6e6Ac ID D6D 6I PIPPDI66PD 66c6PID6A PDAe6~PD6
OZLST D 66ID6Ie 6D 66D6 lTlD66l6D6 66PN D D 6D 6D 622 222262
099ST LLLLDLULI l626RPLZD 2DVD2M26 26 D666 66DD6D DD222D62
0091 PDPPPZD6B 16261161116 TPPIPPZP6I D11111PIPI ZI662D D
ObSST D 622IDD 26DDD622 66666 D
08D,S1 1621216612 R6D6DD
6
66
6
ZD6IPID D
66D
66D6 D6666D62
OZbST DD 66666 16PDPD6e6e PD6IDI6e6e IDBIPPDPID e6IPI66PPD I66IeID D
09E1 IPI2PIPfi2fi 6ellPlP6h I6tlD62D D 26666D66
6666
66D~~2~
Oo£ST APD6ePDZD 6IID666Ie6 PDD DIPDD6 eDDIeePeIe V6P6LI6PD eIe166H11P
ObZST PD2DN66tP 61166VDZlD 6262PDDIPI ID2DIDIPIP 6~6~6~62~2 ZDID R lZ22
0811
6e666 c Ncl66eD6 LlD6leNDf3 I61DIeIDeD DieZDeIPII 6hPDD6PPD
D D6ID6PD PPPD6PA6L 666LLDD
OZTST
DPDDIPIPID 6666I
090S1 62D661661D 62LD1122ZP 2666226 22 26lf3lPllPf3 6P62lllPDI PR22LP222
OOOST Pf3lllPfiPl2 R22L11I661 lfiP2lFi2ll2 DD
6126 PPDIP6Pll2 2IIIIP222D
0b6bT HeLD 6D PlD6I6c he llIl6le6e6 66~ee AeID 66DMDPZe 6ZeeeD6I61
0881 IBIDDDI216 H1PLP2PZl 222fi26i22FII DDZPI262I2 661111112I 2P63III2II2
081 66ID6ILIID 2I22I2IDI2 222226622 D6
DD
6622226D
09Lb1 21Fi1631632I2 IDDIPPFi122 IDIIP22III IIIF3P2III2 II66PP2226 PDFi2D22I2I
OOLbT 161PD1V1e 11eAP6PtD ND6c6dPl P6f3RRcAII e6h~e6~P6 6H111166D
091 Dee6eDll6j PePePDIIf3e IePeD61661 36ZD6H6c6 6D6D D De6eDIPIII
081 DDDhdPIio
61LD6H16 6el6eleDee DPIPIPeI66 IPPPDD
6666
0 ZS bT
6~2622DD
66
D
6D66
091 eDIIPeeDII DeeeI66IDD 6VPD6ZcIZ P61c61661e 66IeIeDID6 eIPIeI6e6I
OObbT 66I222D6N 611112IDI2 PP6222II2Z P2226ID2II 2266616122 622~2662~
Ob£bT llM6 DD6e ePIPDIeIe6 6DDlel6eee eeecDlel6e 6D6 TP6e6eeDPI
p9rUT UOD-
9Z
SZ
ZU O17'861'8 S 1
US 8,198,430 B2
27
28
-continued
agttttagta tatcaaacac tgaaagaata taaaattgat acgatagtac attttgctgc 16740
agaatcgcat gttgataatt caattgctaa tccaaaggta tttttagaaa cgaatgtgat 16800
aggtacattt acacttttag attgtgctaa aaggtattgg ttagatgagc taggtttaga 16860
agaaactagt tgtaggtttc atcatgtatc tactgatgag gtatatggta ccttggcaaa 16920
agatgaacca gcctttactg agattaaggc ttatgagcca aattcaccgt attcggcatc 16980
taaggcggga tctgatcata tttctagagc atatcatcat acctataaac ttccggtaac 17040
aatttcaaat tgttcaaaca actatggacc ataccaacat cgagagaaat taatccctgt 17100
agtgataaat agttgtataa actacaagcc tattcctgtt tacggagatg gttcgaatat 17160
tcgagattgg ctatatgtag aagatcactg cgatgctatc cagacaattg ttgagaaagg 17220
agtggttgga gaggtttata atattggtgg tattaatgaa gttgataatc taaccttggt 17280
aaaaactatc tgtaaactaa tggatgaata taaaccagaa aatgctccac attctaactt 17340
aatcacattt gtggaagata gaaaaggaca tgattggcgt tatgctattg ataacagcaa 17400
gattcagaat gagttaggat ggaagccatc acaagatttt gataagatgt ttagacaaac 17460
tattgagttt tatctatagc ttaaatattt atcttatgag tatctctaaa aaatcaattt 17520
aatttatttt tgtgttaaaa agtagtgttc gcaagaatat agttaatccg aaagatattt 17580
gtagaaaaag atatttgtag aaatgttata atgtctaata aaa
<210>
<211>
<212>
<213>
SEQ ID NO 2
LENGTH: 247
TYPE: PRT
ORGANISM: Francisella tuiarensis
<400> SEQUENCE: 2
Met Asn Tyr His Ile Lys Glu Val Phe Trp Ser Ile Ile Leu Ser Phe
1
5
10
15
Leu Lys Ser Gin Lys Gly Ile His Thr Asn Asp Glu Ala Lys Leu Arg
20
25
30
Leu Phe Ile Glu Ala Val Phe Tyr Val Leu Arg Thr Gly Cys Gin Trp
35
40
45
Arg Met Leu Pro Phe Tyr Tyr Gly Lys Tyr Arg Ser Ile His Lys Arg
50
55
60
Phe Lys Asp Trp Cys Asp Lys Asp Ile Phe Ser Arg Leu Phe Lys Ser
65
70
75
80
Val Gin Asn Pro Asp Leu Gin Glu Val Met Leu Asp Ser Thr Ile Ala
85
90
95
Arg Ala His Ala Cys Ala Thr Gly Tyr Asp Lys Asp Asp Asn Gin Ala
100
105
110
Ile Gly Arg Ser Val Gly Arg Ile Thr Thr Lys Ile His Ala Met Thr
115
120
125
Asp Ala Leu Gly Asn Pro Ile Glu Ile Leu Leu Ser Glu Asp Lys Thr
130
135
140
His Asp Ser Lys Val Ala Ile Asp Leu Leu Lys Asn Val Tyr Asn Thr
145
150
155
160
Lys Val Ile Ala Asp Arg Ala Tyr His Ser Asn Glu Ile Arg Gin His
165
170
175
Ile Gin Gly Ile Ser Ser Glu Ala Val Ile Pro Cys Lys Ser Asn Thr
180
185
190
Leu Asn His Ile Pro Phe Asp Ser His Val Tyr Lys Glu Arg His Leu
195
200
205
17623
US 8,198,430 B2
29
30
-continued
Ile Glu Asn Phe Phe Ser Lys Ile Lys His Phe Arg Arg Val Phe Ser
210
215
220
Arg Phe Asp Lys Thr Ile Leu Ala Tyr Ile Gly Met Ile Lys Leu Ala
225
230
235
240
Cys Thr Phe Ile Trp Leu Arg
245
<210>
<211>
<212>
<213>
SEQ ID NO 3
LENGTH: 578
TYPE: PRT
ORGANISM: Franciseila tularensis
<400> SEQUENCE: 3
Met Ser Phe Tyr Asp Asn Arg Thr Leu Asn Phe Val Val Ile Ile Val
1
5
10
15
Leu Thr Ile Ile Thr Val Asn Trp Thr Phe Tyr Ile Phe Lys Gin Asp
20
25
30
Val Asn Leu His Phe Leu Leu Ala Leu Val Leu Leu Arg Cys Leu Ser
35
40
45
Ser Phe Leu Leu Leu Arg Asp Tyr Met Ala Ser Trp Arg Lys Ser Thr
50
55
60
Gin Lys Thr Phe Leu Arg Lys Ala Phe Ile Asn Leu Pro Val Phe Phe
65
70
75
80
Ile Val Ala Leu Phe Phe Tyr Gly Lys Val Thr Phe Ser Leu Ile Phe
85
90
95
Ser Glu Phe Leu Phe Tyr Val Phe Leu Ile Ser Leu Ser Val Tyr Phe
100
105
110
Tyr Trp Tyr Leu Met Asn Arg Gly Ser Val Asp Lys Ser Lys Thr Ala
115
120
125
Val Ile Tyr Gly Ala Gly Ala Ala Gly Thr Lys Ile Ala Gin Glu Leu
130
135
140
Ala Ser Ala Gly Tyr Arg Ile Lys Cys Phe Val Asp Asp Asn Glu Thr
145
150
155
160
Leu Gin Lys Arg Ser Ile Asp Ser Lys Lys Val Leu Ser Lys Ala Glu
165
170
175
Leu Thr Lys Leu Leu Leu Ser Ser Arg Phe Asp Leu Leu Val Ile Ala
180
185
190
Leu Pro Arg Asn Ala Asn Gin Val Val Lys Asn Ile Tyr Lys Glu Phe
195
200
205
Glu Lys Asp Phe Asn Gin Ile Arg Ile Met Pro Pro Leu Glu Glu Ile
210
215
220
Leu Gin Asp Glu Asn Phe Met Ser Gin Leu Lys Pro Val Ser Leu Tyr
225
230
235
240
Asp Leu Leu Ala Arg Asp Thr Lys Ser Leu Asp Lys Glu Ser Ile Ser
245
250
255
Asn Phe Ile Lys Asn Lys Val Val Leu Val Thr Gly Ala Gly Gly Ser
260
265
270
Ile Gly Ser Glu Ile Val His Gin Cys Ile Lys Tyr Gin Ala Lys Glu
275
280
285
Leu Ile Leu Val Asp His Ser Glu Phe Asn Leu Tyr Lys Ile Thr Glu
290
295
300
Glu Cys Ser His Phe Asn Ile Asn Ser Val Leu Cys Ser Val Cys Asp
305
310
315
320
Arg Lys Ala Leu Ala Glu Val Phe Gin Lys Tyr Thr Pro Asn Ile Val
325
330
335
US 8,198,430 B2
32
31
-continued
Phe His Ala Ala Ala Tyr Lys His Val Pro Leu Val Glu Glu Asn Ile
340
345
350
Ser Arg Ala Ile Arg Asn Asn Ile Leu Gly Thr Lys Asn Ala Ile Asp
355
360
365
Leu Ala Ile Glu Ala Gly Val Glu Ser Phe Ile Leu Ile Ser Thr Asp
370
375
380
Lys Ala Val Arg Pro Thr Asn Val Met Gly Ala Thr Lys Arg Val Cys
385
390
395
400
Glu Leu Tyr Leu Gln Asn Val Asp Pro Lys Asn Thr Lys Leu Ala Ala
405
410
415
Val Arg Phe Gly Asn Val Leu Gly Ser Ser Gly Ser Val Ile Pro Lys
420
425
430
Phe Glu Glu Gln Ile Arg Lys Gly Gly Pro Val Thr Val Thr His Pro
435
440
445
Glu Ile Thr Arg Tyr Phe Met Leu Ile Pro Glu Ala Cys Glu Leu Val
450
455
460
Leu Gln Ala Gly Ala Ile Ala Lys Asn Ser Glu Val Phe Val Leu Asp
465
470
475
480
Met Gly Gln Pro Val Lys Ile Ile Asp Leu Ala Lys Gln Phe Ile Arg
485
490
495
Leu Ser Gly Arg Gly Asp Ile Asp Ile Lys Ile Val Gly Leu Arg Pro
500
505
510
Gly Glu Lys Leu Tyr Glu Glu Leu Leu Ile Glu Glu Asp Asp Val Ser
515
520
525
Thr Asp Tyr Lys Asp Ile Phe Ile Gly Arg Arg Thr Phe Tyr Asp Ile
530
535
540
Asn Thr Leu Asn Gln Asp Ile Glu Ser Leu Ile Lys Asp Asp Val Asp
545
550
555
560
Gln Leu Val Ile Leu Lys Lys Ile Val Pro Glu Phe Glu His Arg Leu
565
570
575
Asn Gly
<210>
<211>
<212>
<213>
SEQ ID NO 4
LENGTH: 205
TYPE: PRT
ORGANISM: Francisella tularensis
<400>
SEQUENCE:
4
Met Phe Tyr Glu Val Phe Lys Arg Leu Leu Asp Ile Leu Leu Ser Phe
1
5
10
15
Met Gly Leu Leu Leu Leu Ser Pro Ile Phe Leu Ile Ile Ile Phe Met
20
25
30
Ile Lys Lys Asp Ser Lys Gly Pro Ile Phe Phe Lys Gln Lys Arg Tyr
35
40
45
Gly Lys Asp Lys Gln Phe Phe Tyr Ile Tyr Lys Phe Arg Thr Met Tyr
50
55
60
Val Asp Thr Pro Lys Asp Met Pro Thr His Met Leu Gln Asp Pro Ser
65
70
75
80
Lys Cys Ile Thr Lys Val Gly Gly Phe Leu Arg Lys Ser Ser Leu Asp
85
90
95
Glu Leu Pro Gln Ile Ile Asn Ile Leu Lys Gly Glu Met Ser Ile Val
100
105
110
Gly Pro Arg Pro Ala Leu Trp Asn Gln Asp Asp Leu Ile Ala Gln Arg
115
120
125
Asp Lys Tyr Gly Ala Asn Ala Val Pro Val Gly Leu Thr Gly Trp Ala
US 8,198,430 B2
34
33
-continued
130
135
140
Gin Ile Asn Gly Arg Asp Glu Leu Pro Ile Pro Asp Lys Ala Lys Leu
145
150
155
160
Asp Gly Asp Tyr Val Lys Asn Lys Ser Thr Trp Phe Asp Leu Lys Cys
165
170
175
Ile Phe Leu Thr Val Phe Ser Val Phe Ala Lys Lys Gly Val Val Glu
180
185
190
Gly Gly Thr Gly Ala Leu Gly Asn Lys Glu Asp Leu Lys
195
200
205
<210>
<211>
<212>
<213>
SEQ ID NO 5
LENGTH: 263
TYPE: PRT
ORGANISM: Francisella tularensis
<400> SEQUENCE: 5
Met Lys Lys Arg Ile Leu Val Thr Gly Leu Ser Ser Tyr Ile Gly Asn
1
5
10
15
Ser Phe Ala Ala Lys Tyr Asn Ser Asp Phe Ser Ile Asp Lys Ile Ser
20
25
30
Leu Arg Asp Val Ser Trp Ala Asn Ile Asp Leu Ser Gly Tyr Asp Ala
35
40
45
Val Leu His Val Ala Gly Ile Ala His Thr Ser Lys Asp Pro Lys Leu
50
55
60
Lys Glu Lys Tyr Tyr Lys Ile Asn Thr Gin Leu Thr Tyr Asp Leu Ala
65
70
75
80
Lys Gin Ala Lys Asp Gin Gly Val Arg Gin Phe Val Phe Leu Ser Ser
85
90
95
Ile I1e Val Tyr Gly Asp Ser Ala Pro Ile Gly Gin Gin Lys Val Ile
100
105
110
Thr Lys Tyr Thr Glu Pro Lys Pro Asp Asp Phe Tyr Gly Asp Ser Lys
115
120
125
Leu Gin Thr Glu Ile Lys Leu Asn Ser Leu Ala Ser Asp Asp Phe Asn
130
135
140
Ile Ala Ile Ile Arg Pro Pro Met Val Tyr Gly Glu Gly Ser Lys Gly
145
150
155
160
Asn Tyr Pro Lys Leu Val Lys Leu Ala Lys Tyr Thr Phe Ile Phe Pro
165
170
175
Asn Ile Asn Asn Gin Arg Ser Val Ile Ser Ile Asp Asn Leu Ser Lys
180
185
190
Glu Ile Ala Glu Ile Ile Leu Gin Thr Lys His Gly Val Phe Leu Leu
195
200
205
Gin Asp Asn Glu Tyr Phe Cys Thr Ser Gin Phe Ile Lys Asn Tyr Arg
210
215
220
Lys Asp Val Leu Gly Lys Arg Thr Tyr Leu Thr Lys Ile Phe Asn Pro
225
230
235
240
Ile Ile Arg Leu Leu Ala Lys Lys Val Asp Phe Ile Asn Lys Val Phe
245
250
255
Gly Asn Leu Thr Tyr Glu Lys
260
<210>
<211>
<212>
<213>
SEQ ID NO 6
LENGTH: 363
TYPE: PRT
ORGANISM: Francisella tularensis
<400> SEQUENCE: 6
US 8,198,430 B2
35
36
-continued
Met Arg Ser Lys Leu Leu Phe Ile Ala Asn Asp Phe Asp Ile Val Ile
1
5
10
15
Tyr Arg Phe Arg Arg Glu Val Ile Glu Ser Phe Ala Ala Lys Glu Tyr
20
25
30
Glu Ile Val Leu Val Thr Pro Tyr Ser Lys Lys Ala Glu Val Phe Cys
35
40
45
Lys Ser Leu Gly Val Lys Tyr Ile Asn Val Asp Ile Asp Arg Arg Gly
50
55
60
Lys Asn Pro Phe Lys Asp Leu Leu Leu Leu Phe Asn Tyr Phe Lys Ile
65
70
75
80
Ile Lys Lys Glu Lys Pro Asp Tyr Ile Phe Ser Tyr Thr Ile Lys Pro
85
90
95
Asn Leu Tyr Val Gly Leu Val Asn Leu Phe Phe Arg Lys Lys Phe Tyr
100
105
110
Pro Asn Val Thr Gly Leu Gly Ser Val Phe Ala Asn His Gly Ile Val
115
120
125
Gin Lys Phe Ile Ile Ser Leu Tyr Lys Leu Ser Phe Lys Ser Thr Thr
130
135
140
Lys Val Phe Phe Gin Asn Glu Gin Asn Lys Lys Leu Phe Ile Ala Lys
145
150
155
160
Lys Ile Ile Ser Gly Glu Lys Ser Ile Leu Leu Pro Gly Ser Gly Val
165
170
175
Asn Leu Asp Glu Asn Lys Tyr Val Asp Tyr Pro Lys Asp Gin Gly Ile
180
185
190
Leu Lys Phe Val Phe Leu Gly Arg Ile Met Lys Glu Lys Gly Ile Tyr
195
200
205
Glu Leu Leu Glu Ala Phe Ala Ile Leu Glu Lys Lys Tyr Lys Asn Ile
210
215
220
Ser Leu Asp Ile Tyr Gly Phe Cys Asp Glu Asn Lys Ser Asn Phe Met
225
230
235
240
Gly Lys Val Asn Thr Ile Lys Ser Val Lys Phe Tyr Gly Phe Thr Asp
245
250
255
Asn Thr Lys Glu Lys Ile Ala Ser Ala His Ala Val Val Leu Pro Ser
260
265
270
Tyr His Glu Gly Met Ser Asn Val Leu Leu Glu Ala Ala Ala Ile Gly
275
280
285
Arg Pro Val Ile Ala Ser Asp Ile Pro Gly Cys Arg Glu Ile Phe Asp
290
295
300
Asp Gly Leu Ser Gly Leu Ser Cys Asn Pro Asn Asp Val Ser Ser Leu
305
310
315
320
Arg Asn Ser Leu Glu Gin Phe Ile Asn Met Ser Tyr Thr Asp Lys Ile
325
330
335
Ala Met Ser Tyr Lys Ala Arg Ala Lys Ile Glu Lys Asp Phe Asp Arg
340
345
350
Ser Ile Val Val Asn Ala Tyr Leu Gin Gin Asn
355
360
<210>
<211>
<212>
<213>
SEQ ID NO 7
LENGTH: 436
TYPE: PRT
ORGANISM: Francisella tularensis
<400> SEQUENCE: 7
Met Ser Leu Tyr Glu Asp Ile Val Ala Lys Arg Glu Lys Val Ser Leu
1
5
10
15
US 8,198,430 B2
37
38
-continued
Val Gly Leu Gly Tyr Val Gly Leu Pro Ile Ala Ile Ala Phe Ala Lys
20
25
30
Lys Ile Asp Val Leu Gly Phe Asp Ile Cys Glu Thr Lys Val Gln His
35
40
45
Tyr Lys Asp Gly Phe Asp Pro Thr Lys Glu Val Gly Asp Glu Ala Val
50
55
60
Arg Asn Thr Thr Met Lys Phe Ser Cys Asp Glu Thr Ser Leu Lys Glu
65
70
75
80
Cys Lys Phe His Ile Val Ala Val Pro Thr Pro Val Lys Ala Asp Lys
85
90
95
Thr Pro Asp Leu Thr Pro Ile Ile Lys Ala Ser Glu Thr Val Gly Arg
100
105
110
Asn Leu Val Lys Gly Ala Tyr Val Val Phe Glu Ser Thr Val Tyr Pro
115
120
125
Gly Val Thr Glu Asp Val Cys Val Pro Ile Leu Glu Lys Glu Ser Gly
130
135
140
Leu Arg Ser Gly Glu Asp Phe Lys Val Gly Tyr Ser Pro Glu Arg Ile
145
150
155
160
Asn Pro Gly Asp Lys Val His Arg Leu Glu Thr Ile Ile Lys Val Val
165
170
175
Ser Gly Met Asp Glu Glu Ser Leu Asp Thr Ile Ala Lys Val Tyr Glu
180
185
190
Leu Val Val Asp Ala Gly Val Tyr Arg Ala Ser Ser Ile Lys Val Ala
195
200
205
Glu Ala Ala Lys Val Ile Glu Asn Ser Gln Arg Asp Val Asn Ile Ala
210
215
220
Phe Val Asn Glu Leu Ser Ile Ile Phe Asn Gln Met Gly Ile Asp Thr
225
230
235
240
Leu Glu Val Leu Ala Ala Ala Ala Thr Lys Trp Asn Phe Leu Asn Phe
245
250
255
Lys Pro Gly Leu Val Gly Gly His Cys Ile Gly Val Asp Pro Tyr Tyr
260
265
270
Leu Thr Tyr Lys Ala Ala Glu Leu Gly Tyr His Ser Gln Val Ile Leu
275
280
285
Ser Gly Arg Arg Ile Asn Asp Ser Met Gly Lys Phe Val Val Glu Asn
290
295
300
Leu Val Lys Lys Leu Ile Ser Ala Asp Ile Pro Val Lys Arg Ala Arg
305
310
315
320
Val Ala Ile Phe Gly Phe Thr Phe Lys Glu Asp Cys Pro Asp Thr Arg
325
330
335
Asn Thr Arg Val Ile Asp Met Val Lys Glu Leu Asn Glu Tyr Gly Ile
340
345
350
Glu Pro Tyr Ile Ile Asp Pro Val Ala Asp Lys Glu Glu Ala Lys His
355
360
365
Glu Tyr Gly Leu Glu Phe Asp Asp Leu Ser Lys Met Val Asn Leu Asp
370
375
380
Ala Ile Ile Ile Ala Val Ser His Glu Gln Phe Lys Asp Ile Thr Lys
385
390
395
400
Gln Gln Phe Asp Arg Leu Tyr Ala His Asn Ser Arg Lys Ile Ile Phe
405
410
415
Asp Ile Lys Gly Ser Leu Asp Lys Ser Glu Phe Glu Lys Asp Tyr Ile
420
425
430
Tyr Trp Arg Leu
US 8,198,430 B2
39
40
-continued
435
<210>
<211>
<212>
<213>
SEQ ID NO 8
LENGTH: 323
TYPE: PRT
ORGANISM: Francisella tularensis
<400> SEQUENCE: 8
Val Ala Tyr Asp Asn Val Lys Phe Pro His Gly Ser Phe Phe Leu Val
1
5
10
15
Thr Gly Gly Ala Gly Phe Ile Gly Ser Asn Leu Cys Glu Val Leu Leu
20
25
30
Ser Lys Gly Tyr Arg Val Arg Cys Leu Asp Asp Leu Ser Asn Gly His
35
40
45
Tyr His Asn Val Glu Pro Phe Leu Thr Asn Ser Asn Tyr Glu Phe Ile
50
55
60
Lys Gly Asp Ile Arg Asp Leu Asp Thr Cys Met Lys Ala Cys Glu Gly
65
70
75
80
Ile Asp Tyr Val Leu His Gln Ala Ala Trp Gly Ser Val Pro Arg Ser
85
90
95
Ile Glu Met Pro Leu Val Tyr Glu Asp Ile Asn Val Lys Gly Ala Leu
100
105
110
Asn Met Leu Glu Ala Ala Arg Gln Asn Asn Val Lys Lys Phe Val Tyr
115
120
125
Ala Ser Ser Ser Ser Val Tyr Gly Asp Glu Pro Asn Leu Pro Lys Lys
130
135
140
Glu Gly Arg Glu Gly Asn Val Leu Ser Pro Tyr Ala Phe Thr Lys Lys
145
150
155
160
Ala Asn Glu Glu Trp Ala Arg Leu Tyr Thr Lys Leu Tyr Gly Leu Asp
165
170
175
Thr Tyr Gly Leu Arg Tyr Phe Asn Val Phe Gly Arg Arg Gln Asp Pro
180
185
190
Asn Gly Ala Tyr Ala Ala Val Ile Pro Lys Phe Ile Lys Gln Leu Leu
195
200
205
Asn Asp Glu Ala Pro Thr Ile Asn Gly Asp Gly Lys Gln Ser Arg Asp
210
215
220
Phe Thr Tyr Ile Glu Asn Val Ile Glu Ala Asn Leu Lys Ala Cys Leu
225
230
235
240
Ala Asp Ser Lys Tyr Ala Gly Glu Ser Phe Asn Ile Ala Tyr Gly Gly
245
250
255
Arg Glu Tyr Leu Ile Asp Leu Tyr Tyr Asn Leu Cys Asp Ala Leu Gly
260
265
270
Lys Lys Ile Glu Pro Asn Phe Gly Pro Asp Arg Ala Gly Asp Ile Lys
275
280
285
His Ser Asn Ala Asp Ile Ser Lys Ala Arg Asn Met Leu Gly Tyr Asn
290
295
300
Pro Glu Tyr Asp Phe Glu Leu Gly Ile Lys His Ala Val Glu Trp Tyr
305
310
315
320
Leu Ile Asn
<210>
<211>
<212>
<213>
SEQ ID NO 9
LENGTH: 409
TYPE: PRT
ORGANISM: Francisella tularensis
<400> SEQUENCE: 9
US 8,198,430 B2
41
42
-continued
Val Tyr Ile Lys Lys Val Ser Phe Lys Ile Leu Tyr Leu Tyr Leu Leu
1
5
10
15
Ala Phe Cys Ile Ile Phe Ser Leu Glu Phe Lys Phe Ala Ile Leu Asn
20
25
30
Ile Ile Val Tyr Leu Pro Ala Cys Ile Leu Gly Phe Leu Ala Leu Lys
35
40
45
Lys Leu Phe Val Gly Asn Ile Val Lys Lys Gin Leu Ala Phe Leu Phe
50
55
60
Phe Phe Phe Phe Leu Ser Met Ile Tyr Leu Ile Ile Val Gin Ile Ile
65
70
75
80
Leu Leu Asp Ala Ala Ser Leu Phe Pro Gin Phe Leu Phe Asn Ile Leu
85
90
95
Ile Ala Ile Gly Phe Cys Asn Phe Ile Phe Val Ser Tyr Asp Asn Asn
100
105
110
Glu Asn Tyr Phe Phe Asn Met Ser Lys Ile Ile Phe Phe Val Thr Phe
115
120
125
Leu Gin Ser Ile Phe Val Phe Leu Ser Arg Tyr Tyr Ile Phe Leu Asn
130
135
140
Asp Trp Ile Phe Phe Phe Leu Val Lys Lys Gly Asn Ile Glu Ile Ser
145
150
155
160
Asn Val Ile Glu Tyr Lys Leu Arg Val Phe Gly Leu Ser Asn Ala Gly
165
170
175
Gly Asp Gly Leu Gly Phe Ser Ile Thr Ile Gly Leu Cys Phe Ser Ile
180
185
190
Phe Tyr Phe Ile Lys Tyr Ile Lys Gly Lys Ser Ile Phe Thr Lys Leu
195
200
205
Met Leu Phe Val Pro Leu Ile Leu Ile Val Phe Ser Asn Ile Phe Ile
210
215
220
Ser Arg Thr Ser Leu Leu Thr Ser Ser Leu Ile Leu Leu Ile Thr Ile
225
230
235
240
Phe Tyr Ile Tyr Ile Lys Lys Glu Lys Leu Leu Phe Ile Ile Ile Leu
245
250
255
Ala Leu Phe Phe Leu Ser Ile Trp Ile Leu Phe Lys Leu Asn Leu Asn
260
265
270
Leu Ser Trp Ala Phe Glu Asn Ile Tyr Ser Tyr Ile Gin Ser Gly Asp
275
280
285
Phe Ser His Gly Ser Leu Ser Val Leu Ile Asn Lys Met Leu Phe Val
290
295
300
Pro Asp Asn Leu Leu Thr Trp Ile Phe Gly Cys Glu Asp Val Ser Asn
305
310
315
320
Thr Asp Ile Gly Tyr Ile Lys Tyr Leu Tyr Tyr Tyr Gly Ile Ile Phe
325
330
335
Ser Met Phe Phe Tyr Ile Leu Ile Ile Phe Leu Tyr Phe Glu Met Arg
340
345
350
Lys Cys Phe Ile Phe Ser Glu Tyr Arg Ser Leu Phe Leu Leu Leu Leu
355
360
365
Ile Val Cys Leu Val Phe Gin Ala Lys Ile Ile Phe Leu Thr Val Gly
370
375
380
Leu Phe Thr Lys Leu Thr Ile Ile Leu Phe Ile Phe Ser Leu Lys Glu
385
390
395
400
Asn Ser Phe Thr Thr Arg Ser Val Ile
405
<210> SEQ ID NO 10
US 8,198,430 B2
-continued
<211> LENGTH: 366
<212> TYPE: PRT
<213> ORGANISM: Francisella tularensis
<400> SEQUENCE: 10
Leu Lys Arg Phe Val His Leu Ile Ile Asn Leu Asn Gin Gly Gly Ala
1
5
10
is
Glu Thr Met Leu Tyr Lys Leu Cys Lys Ser Met Asp Lys Ser Ile Tyr
20
25
30
His Ile Thr Ile Ile Ser Leu Met Gly Arg Gly Val Phe Ala Asn Lys
35
40
45
Leu Glu Ala Tyr Gly Val Lys Val Tyr Thr Leu Asn Leu Asn Lys Phe
50
55
60
Asn Val Leu Phe Val Leu Phe Lys Tyr Ile Lys Ile Ile Arg Arg Ile
65
70
75
80
Lys Pro Asp Val Ile His Ala Trp Met Tyr His Ala Asn Val Ile Ser
85
90
95
Ile Leu Cys Lys Pro Phe Tyr Arg Lys Thr Lys Tyr Ile Asn Ser Ile
100
105
110
Arg Met Gly Leu Glu Asn Tyr Asp Gly His Lys Asn Leu Thr Lys Phe
115
120
125
Met Ile Lys Leu Asn Ala Lys Phe Ser Lys Phe Ser Asp Leu Thr Leu
130
135
140
Asn Asn Ser Lys Lys Ser Leu Glu Asp His Gin Asn Ile Gly Phe Lys
145
150
155
160
Asn Gin Cys Phe Ile Ala Asn Gly Phe Asp Lys Asp Val Phe Lys Pro
165
170
175
Ser Phe Leu Lys Tyr Glu Lys Phe Arg Leu Asn Asn Asp Leu Asp Asp
180
185
190
Asn Val Lys Ile Ile Gly Ile Ile Ala Arg Asn His Ala Asp Lys Asn
195
200
205
Ile Ser Arg Phe Leu Gin Ile Ala Asn Leu Leu Leu Lys Ser Asn Pro
210
215
220
Ser Leu Arg Phe Leu Ile Ala Gly Arg Glu Cys Ser Lys Ile Asp Ile
225
230
235
240
Gly Ser Tyr Leu Asp Asn Lys Ser Asn Val Asn Lys Phe Phe Val Phe
245
250
255
Glu Ser Val Asp Ser Ser Glu Tyr Leu Pro Val Leu Asp Leu Tyr Leu
260
265
270
Ser Thr Ser Lys Val Glu Gly Phe Pro Asn Ile Leu Ala Glu Ala Met
275
280
285
Leu Cys Glu Val Pro Ile Val Ala Ser Asn Val Gly Asp Cys Lys Asp
290
295
300
Ile Leu Asn Gly Tyr Gly Glu Val Phe Glu Leu Ser Gin Gly Asn Lys
305
310
315
320
Glu Ile Ile Glu Lys Ile Met Lys Val Leu Glu Thr Thr Val Val Met
325
330
335
Lys Lys Arg Met Arg Glu Tyr Ile Ile Asn Asn Phe Ser Ile Glu Ala
340
345
350
Ile Leu Glu Lys His Glu Lys Leu Tyr His Glu Gly Ser Val
355
360
365
<210>
<211>
<212>
<213>
SEQ ID NO 11
LENGTH: 628
TYPE: PRT
ORGANISM: Francisella tularensis
US 8,198,430 B2
-continued
<400> SEQUENCE: 11
Met Cys Gly Val Val Gly Phe Tyr Ser Phe Asn Lys Glu Glu Gly Phe
1
5
10
15
Asp Ser Ile Ile Asn Gin Ser Leu Leu Ser Ile Lys His Arg Gly Ser
20
25
30
Asp Asp Ser Gly Tyr Trp Cys Asp Asn Gin Val Thr Leu Gly His Thr
35
40
45
Arg Leu Ser Ile His Asp Ile Thr Asn Ala Gly His Gin Pro Met Leu
50
55
60
Ser Asn Ser Gly Asn Thr Ala Ile Val Phe Asn Gly Glu Ile Tyr Asn
65
70
75
80
Tyr Leu Ser Ile Lys Asn Gin Leu Leu Ser Glu Tyr Ser Asn Leu Lys
85
90
95
Phe Lys Ser Asn Ser Asp Thr Glu Val Leu Val Asn Ala Ile Glu Leu
100
105
110
Trp Gly Ile Asp Lys Thr Leu Glu Lys Cys Ile Gly Met Phe Ala Phe
115
120
125
Gly Val Tyr Ser Arg Lys Thr Ser Cys Leu Ile Leu Ala Arg Asp Arg
130
135
140
Phe Gly Glu Lys Pro Leu Tyr Phe Gly Ile Gin Asn Gly Ile Leu Gly
145
150
155
160
Phe Ala Ser Glu Leu Lys Ala Leu Lys Pro Leu Lys Glu Cys Gly Trp
165
170
175
Arg Phe Asp Ile Asp Arg Asp Ala Leu Ala Thr Tyr Met Arg Tyr Ala
180
185
190
Tyr Val Pro Thr Pro Tyr Ser Ile Tyr Lys Asn Ile Ser Lys Leu Asn
195
200
205
Val Gly Ser Tyr Ile Lys Phe Asp Ala Lys Gly Asn Ser Lys Glu Tyr
210
215
220
Lys Tyr Trp Asp Ser Lys Lys Val Leu Asp Ser Glu Lys Tyr Lys Asp
225
230
235
240
Ser Tyr Asp Gin Ala Ile Leu Asp Leu Glu Ile Lys Leu Lys Ser Thr
245
250
255
Leu Ser Ile Gin Met Gin Ser Asp Val Pro Leu Gly Ala Phe Leu Ser
260
265
270
Gly Gly Ile Asp Ser Thr Thr Val Val Ala Leu Met Gin Ser Met Ser
275
280
285
Lys Asp Lys Ile Asn Thr Phe Ser Ile Gly Phe Asn Gin Lys Glu Tyr
290
295
300
Asn Glu Ala Glu His Ala Arg Ala Val Ala Lys His Ile Gly Thr Asn
305
310
315
320
His Thr Asp Met Tyr Val Thr Glu Arg Asp Ala Leu Asp Val Ile Pro
325
330
335
Lys Leu Ala Gly Ile Tyr Asp Glu Pro Phe Ala Asp Ser Ser Gin Ile
340
345
350
Pro Thr Tyr Leu Val Ser Lys Ile Ala Lys Ser Lys Val Thr Val Ala
355
360
365
Leu Ser Gly Asp Ala Gly Asp Glu Leu Phe Gly Gly Tyr Asn Arg Tyr
370
375
380
Phe Leu Ala Pro Asn Ile Ala Lys Lys Ile Lys Phe Ala Lys Leu Leu
385
390
395
400
Lys Tyr Ala Pro Asp Ala Trp Ile Lys Lys Ala Glu Ile Leu Asn Phe
405
410
415
US 8,198,430 B2
48
-continued
Gly Lys Phe Ala Leu Leu Ala Asp Lys Leu Leu Lys Leu Lys Arg Val
420
425
430
Leu Glu Lys Ala Lys Thr Asn Lys Glu Leu Tyr Val Leu Leu Cys Ser
435
440
445
Gln Ile Asn Asp Thr Ser Phe Val Leu Gly Ala Lys Glu Tyr Asp Ile
450
455
460
Leu Arg Asp Lys Asn Ile Tyr Asp Ile Pro Gln Leu Ser Phe Gln Glu
465
470
475
480
Trp Met Met Phe Val Asp Ser Asn Thr Tyr Met Ile Asp Asp Ile Leu
485
490
495
Val Lys Val Asp Arg Ala Ala Met Ala Asn Ser Leu Glu Thr Arg Val
500
505
510
Pro Phe Leu Asp His Asn Ile Tyr Glu Phe Ala Tyr Ser Leu Pro Ile
515
520
525
Asp Tyr Lys Ile Gln Arg Gly Asn Gly Lys Arg Ile Leu Lys Asp Leu
530
535
540
Leu Tyr Lys Tyr Val Pro Glu Ser Leu Val Asn Arg Ser Lys Met Gly
545
550
555
560
Phe Gly Ile Pro Leu Ala Lys Trp Leu Arg Glu Asp Leu Arg Glu Trp
565
570
575
Ala Asp Asn Leu Leu Asp Tyr Ser Lys Ile Asp Lys Gln Gly Tyr Leu
580
585
590
Ser Pro Glu Val Val Gln Lys Tyr Trp Gln Glu His Leu Ser Gly Lys
595
600
605
Arg Asn Trp Gln Ala Ile Leu Trp Asn Ile Leu Ile Phe Gln Glu Trp
610
615
620
Leu Asp Asn Glu
625
<210>
<211>
<212>
<213>
SEQ ID NO 12
LENGTH: 360
TYPE: PRT
ORGANISM: Francisella tularensis
<400>
SEQUENCE:
12
Met Ser Lys Val Asn Val Thr Lys Pro Tyr Leu Pro Asp Ile Asn Lys
1
5
10
15
Tyr Lys Ser Tyr Val Asn Lys Ile Tyr Lys Asn Gly Trp Leu Thr Asn
20
25
30
Asn Gly Pro Leu Val Gln Glu Leu Glu Lys Arg Leu Ala Lys Tyr Leu
35
40
45
Gly Val Lys Asn Ile Val Leu Val Ser Asn Gly Thr Ile Ala Leu Glu
50
55
60
Ile Ala Tyr Arg Ala Leu Gly Val Lys Gly Ser Ala Ile Thr Thr Pro
65
70
75
80
Phe Ser Phe Val Ala Thr Thr Ser Ser Leu Val Ser Asn Asn Val Lys
85
90
95
Pro Val Phe Val Asp Ile Asp Glu Asn Thr Leu Ser Ile Asp Val Ser
100
105
110
Lys Ile Lys Tyr Ala Ile Glu Glu Asp Thr Ser Ala Ile Val Pro Val
115
120
125
His Val Phe Gly Asn Gly Cys Glu Val Glu Lys Ile Asp Met Leu Ala
130
135
140
Lys Lys His Asn Leu Lys Val Ile Tyr Asp Ala Ala His Ala Phe Asp
145
150
155
160
US 8,198,430 B2
50
-continued
Val Lys Tyr Lys Gly Glu Ser Ile Leu Asn Tyr Gly Asp Ile Ser Thr
165
170
175
Leu Ser Phe His Ala Thr Lys Ile Phe His Ser Ile Glu Gly Gly Ala
180
185
190
Leu Ile Ile Asn Asp Asp Ser Leu Val Glu Lys Val Arg Tyr Phe Ile
195
200
205
Asn Phe Gly Ile Glu Ser Ser Glu Ser Ile Pro Tyr Leu Gly Thr Asn
210
215
220
Ala Lys Met Asn Glu Phe Glu Ala Ala Met Gly Leu Cys Val Leu Asp
225
230
235
240
Asp Ile Ile Glu Ile Lys Ser Lys Arg Lys Val Ile Thr Glu Ile Tyr
245
250
255
Glu Ala Gly Leu Asp Gly Leu Val Lys Phe Gin Glu Gin Asn Gin His
260
265
270
Ser Ser Arg Asn Tyr Ser Tyr Phe Pro Val Ile Phe Arg Thr Glu Glu
275
280
285
Glu Leu Leu Arg Val Gin Lys Ala Leu Ile Gin Asn Asp Ile Ile Ser
290
295
300
Arg Arg Tyr Phe Tyr Pro Ser Leu Asp Ser Leu Ser Tyr Ile Glu Pro
305
310
315
320
Lys Gin Tyr Met Pro Ile Ser Arg Asp Ile Ser Lys Arg Ile Leu Cys
325
330
335
Leu Pro Ile Tyr Ala Glu Leu Glu Asp Asp Lys Ile Asn Lys Ile Ile
340
345
350
Asn Asn Ile Lys Glu Val Ser Ser
355
360
<210>
<211>
<212>
<213>
SEQ ID NO 13
LENGTH: 241
TYPE: PRT
ORGANISM: Francisella tularensis
<400> SEQUENCE: 13
Met Lys Lys Ile Phe Val Val Thr Asp Asn Arg Thr Ile Leu Ser Asp
1
5
10
15
Phe Lys Asn Ile Ile Gly Ser Lys Asn Asp Val Gin Val Asp Tyr Phe
20
25
30
Cys Ser Phe Lys Ser Gin Thr Ser Phe Ala Lys Glu Ile Tyr Asn Ser
35
40
45
Glu Ile Lys Pro Ile Asp Met Lys Lys Asn Gly Asn Asp Leu Ile Gly
50
55
60
Lys Tyr Asp Leu Gly Phe Ser Cys His Ser Lys Gin Leu Phe Pro Ala
65
70
75
80
Lys Leu Val Asn Ser Val Leu Cys Ile Asn Ile His Pro Gly Leu Asn
85
90
95
Pro Tyr Asn Arg Gly Trp Phe Pro Gin Val Phe Ser Ile Ile Asn Lys
100
105
110
Leu Pro Ile Gly Ala Thr Ile His Val Met Asp Glu Glu Ile Asp His
115
120
125
Gly Asp Ile Ile Ile Gin Glu Glu Val Glu Val Asn Ser Phe Glu Asn
130
135
140
Ser Phe Asp Val Tyr Ala Lys Val Gin Lys Lys Glu Val Glu Leu Phe
145
150
155
160
Thr Lys Val Ile Asp Asp Ile Leu Asn Asn Lys Phe Thr Arg Ile Lys
165
170
175
US 8,198,430 B2
52
51
-continued
Pro Asn Ser Glu Gly Asn Tyr Asn Ser Ile His Asp Tyr Lys Asn Met
180
185
190
Cys Glu Ile Asp Leu Asp Lys Ile Val Thr Met Arg Glu Ala Ile Asp
195
200
205
Tyr Leu Arg Ala Met Thr His Pro Pro Tyr Lys Asn Ser Tyr Phe Ile
210
215
220
Asp Glu His Gly Asn Lys Val Phe Val Ala Leu Glu Leu Glu Lys Ile
225
230
235
240
Ser
<210>
<211>
<212>
<213>
SEQ ID NO 14
LENGTH: 495
TYPE: PRT
ORGANISM: Francisella tularensis
<400> SEQUENCE: 14
Met Ser Leu Lys Lys Asn Thr Ile Ser Asn Tyr Ile Thr Gin Leu Tyr
1
5
10
15
Thr Ser Leu Ile Gly Ile Val Ile Leu Pro Leu Tyr Leu Gin His Leu
20
25
30
Ser His Asp Ala Phe Gly Leu Ile Gly Phe Phe Thr Val Phe Gin Thr
35
40
45
Trp Leu Arg Leu Leu Asp Val Gly Ile Thr Pro Thr Leu Ser Arg Glu
50
55
60
Val Ala His Val Arg Gly Ser Thr Asp Asp Tyr His Tyr Leu Arg Lys
65
70
75
80
Leu Val Arg Ser Leu Glu Leu Phe Phe Ile Ile Val Gly Val Leu Val
85
90
95
Phe Ile Val Ile Ser Thr His Ser Arg Tyr Ile Ser Thr Ser Trp Leu
100
105
110
His Ile Gly Ser Leu Asp Ala Asp Ser Val Ser Val Cys Ile Ala Leu
115
120
125
Met Gly Leu Met Phe Ala Leu Arg Trp Val Ser Asp Leu Tyr Gly Gly
130
135
140
Gly Leu Arg Gly Phe Glu Arg Gin Val Leu Tyr Asn Asn Leu Ser Ile
145
150
155
160
Ile Gin Thr Thr Leu Gin Phe Ile Gly Gly Leu Leu Phe Ile Cys Tyr
165
170
175
Val Ser Thr Asn Ile Met Tyr Tyr Phe Val Tyr Gin Thr Ile Ile Ala
180
185
190
Ile Leu Tyr Leu Val Cys Ile Ala Ile Ala Phe Tyr Lys Ile Leu Pro
195
200
205
Ser Ser Phe Ser Val Gly Leu Arg Phe Asp Phe Lys Ile Ile Arg Lys
210
215
220
Val Leu Pro Phe Ala Leu Gly Ile Ala Tyr Ser Thr Thr Val Trp Ile
225
230
235
240
Ile Val Thr Gin Ser Asp Lys Leu Val Phe Ser His Val Leu Pro Leu
245
250
255
Ser Glu Tyr Gly Tyr Leu Ser Leu Leu Ile Val Ile Ser Ser Ala Val
260
265
270
Thr Ile Leu Ser Ser Pro Ile Ser Ile Ala Ile Gin Pro Arg Met Thr
275
280
285
Met Leu Leu Ala Gin Gin Asn Val Lys Gly Met Glu Ser Leu Tyr Leu
290
295
300
US 8,198,430 B2
54
53
-continued
Lys Ser Ser Leu Ile Ser Ile Thr Phe Leu Ser Ala Val Val Thr Cys
305
310
315
320
Val Leu Met Tyr Ser His Gin Leu Leu Gin Ser Trp Thr Gly Ser Met
325
330
335
Glu Ile Ala Asn Trp Gly Ser Asn Ile Leu Asn Ile Tyr Val Leu Ser
340
345
350
Ala Ser Ile Ile Cys Ile Ile Ser Phe Gin Tyr Phe Leu Gin Tyr Ala
355
360
365
Tyr Gly Lys Leu Lys Leu His Asn Thr Tyr Asn Thr Ile Ser Leu Val
370
375
380
Phe Phe Ala Pro Ile Val Ile Tyr Thr Ala Tyr Asn Tyr Gly Val Tyr
385
390
395
400
Thr Thr Ala Leu Leu Trp Leu Gly Tyr Ala Ile Val Gly Leu Ile Ile
405
410
415
Trp Met Pro Ile Val His His Val Phe Ala Lys Gly Ile Asn Arg Tyr
420
425
430
Phe Phe Ile Asn Leu Ala Val Ile Thr Ile Val Cys Phe Leu Leu Ser
435
440
445
Leu Ile Phe Lys Gly Trp Tyr Ile Tyr Pro Ser Lys Ile Gly Leu Val
450
455
460
Glu Leu Ile Leu Ile Gly Phe Ala Phe Leu Phe Ile Gin Ile Cys Ile
465
470
475
480
Glu Tyr Val Leu Phe Arg Tyr Lys Val Leu Arg Cys Ile Asp Asp
485
490
<210>
<211>
<212>
<213>
SEQ ID NO 15
LENGTH: 286
TYPE: PRT
ORGANISM: Francisella tularensis
<400>
SEQUENCE:
495
15
Met Ile Lys Val Ser Val Cys Val Met Thr Tyr Asn Gin Glu Lys Tyr
1
5
10
15
Ile Gly Gin Cys Leu Glu Ser Leu Val Thr Gin Glu Thr Asp Phe Asp
20
25
30
Phe Glu Ile Ile Val Gly Asp Asp Phe Ser Thr Asp Gly Thr Arg Asp
35
40
45
Val Ile Gin Glu Tyr Gin Lys Lys Tyr Pro Asp Ile Ile Lys Pro Val
50
55
60
Phe Arg Asp Lys Asn Val Gly Ile Thr Glu Asn Ile Lys Glu Ile Tyr
65
70
75
80
Phe Val Ala Asn Gly Glu Tyr Ile Ala His Met Asp Gly Asp Asp Tyr
85
90
95
Ala Leu Pro Gly Lys Leu Gin Ile Gin Ala Asp Phe Leu Asp Asn Asn
100
105
110
Pro Arg Cys Thr Gly Val Phe His Asn Ile Asn Ile Leu Tyr Pro Asn
115
120
125
Gly Asn Ile Gin His Ser Arg Phe Ala Cys Ser Asn Lys Ser Ile Phe
130
135
140
Asn Leu Ser Asp Thr Leu Arg Gly Val Ala Val Gly Ala Asn Ser Ser
145
150
155
160
Lys Met Phe Arg Thr Ser Val Leu Asp Asp Leu Ile Leu Pro Asp Ile
165
170
175
Glu Leu Leu Asp Tyr Tyr Phe His Val Ile Thr Ala Glu Lys Gly Tyr
180
185
190
US 8,198,430 B2
55
56
-continued
Leu Ser Phe Leu Asn Ser Asn Glu Ser Tyr Ser Val Tyr Arg Lys Gly
195
200
205
Ile Gly Ile Thr Ser Lys Ser Lys Glu Lys Ile Tyr Asn Thr Tyr Ala
210
215
220
Gly Leu Phe Glu Tyr Phe Leu Asp Arg Tyr Pro Glu Glu Lys Leu Asn
225
230
235
240
Ile Cys Ile Pro Val Val Gin Met Ile Ile Ser Ala Ile Lys Gly Arg
245
250
255
Cys Phe Ile Ser Ala Ile Arg Leu Phe Lys Ile Leu Ile Arg Ser Arg
260
265
270
Cys Ile Pro Leu Val Ser Trp Phe Lys Tyr Arg Phe Glu Lys
275
280
285
<210>
<211>
<212>
<213>
SEQ ID NO 16
LENGTH: 294
TYPE: PRT
ORGANISM: Francisetla tularensis
<400> SEQUENCE: 16
Met Lys Gly Ile Ile Leu Ala Gly Gly Ser Gly Thr Arg Leu Tyr Pro
1
5
10
15
Leu Thr Leu Gly Val Ser Lys Gin Leu Leu Pro Val Tyr Asp Lys Pro
20
25
30
Leu Leu Tyr Tyr Pro Leu Ser Val Leu Met Leu Ala Gly Ile Arg Glu
35
40
45
Ile Leu Ile Ile Ser Thr Val Arg Asp Ile Ser Leu Ile Gin Glu Leu
50
55
60
Leu Gly Asp Gly Ser Gin Phe Gly Ile Gin Leu Ser Tyr Lys Ile Gin
65
70
75
80
Pro Ser Pro Asp Gly Leu Ala Gin Ala Phe Ile Leu Gly Glu Glu Phe
85
90
95
Leu Ala Gly Asp Ser Ala Cys Leu Ile Leu Gly Asp Asn Ile Tyr Tyr
100
105
110
Gly Gin Gly Met Thr Thr Met Leu Glu Ser Ala Arg Ala Gin Cys Gly
115
120
125
Gly Pro Ala Gly Gly Ala Cys Val Phe Gly Tyr Tyr Val Asn Asp Pro
130
135
140
His Arg Tyr Gly Ile Val Glu Phe Asp Lys Gin Lys Asn Val Ile Ser
145
150
155
160
Val Glu Glu Lys Pro Gin Asn Pro Lys Ser His Tyr Ala Ile Thr Gly
165
170
175
Leu Tyr Phe Tyr Asp Asn Asn Val Val Glu Tyr Ala Lys Gin Val Lys
180
185
190
Pro Ser Ala Arg Gly Glu Leu Glu Ile Thr Ser Leu Asn Glu Leu Tyr
195
200
205
Leu Lys Glu Asn Lys Leu Asn Val Glu Leu Leu Gly Arg Gly Phe Ala
210
215
220
Trp Leu Asp Ala Gly Thr His Asp Ser Leu Leu Glu Ala Gly Gin Tyr
225
230
235
240
Val Ala Thr Ile Glu Lys Arg Gin Gly Leu Lys Ile Ala Cys Leu Glu
245
250
255
Glu Ile Ala Trp Arg Lys Gly Phe Ile Ser Thr Gin Gin Val Leu Ala
260
265
270
Gin Ala Glu Lys Leu Ser Lys Thr Glu Tyr Gly Gin Tyr Leu Lys Asn
275
280
285
US 8,198,430 B2
57
58
-continued
Leu Ile Lys Asp Gly Leu
290
<210>
<211>
<212>
<213>
SEQ ID NO 17
LENGTH: 348
TYPE: PRT
ORGANISM: Francisella tularensis
<400> SEQUENCE: 17
Met Thr Ser His Tyr Arg Asn Asp Asp Val Met Arg Asn Glu Lys Met
1
5
10
15
Asn Tyr Lys Pro Lys Asn Ile Leu Val Thr Gly Ala Ala Gly Phe Ile
20
25
30
Gly Ser Asn Tyr Val Arg Met Met Leu Ser Arg Tyr Ser Asp Ile Lys
35
40
45
Ile Ile Ser Tyr Asp Lys Leu Thr Tyr Ala Gly Ser Leu Asp Asn Leu
50
55
60
Lys Asp Leu Asn Asn Glu His Asn His Thr Phe Ile Lys Gly Asp Ile
65
70
75
80
Cys Asp Glu Val Leu Val Tyr Gin Thr Leu Lys Glu Tyr Lys Ile Asp
85
90
95
Thr Ile Val His Phe Ala Ala Glu Ser His Val Asp Asn Ser Ile Ala
100
105
110
Asn Pro Lys Val Phe Leu Glu Thr Asn Val Ile Gly Thr Phe Thr Leu
115
120
125
Leu Asp Cys Ala Lys Arg Tyr Trp Leu Asp Glu Leu Gly Leu Glu Glu
130
135
140
Thr Ser Cys Arg Phe His His Val Ser Thr Asp Glu Val Tyr Gly Thr
145
150
155
160
Leu Ala Lys Asp Glu Pro Ala Phe Thr Glu Ile Lys Ala Tyr Glu Pro
165
170
175
Asn Ser Pro Tyr Ser Ala Ser Lys Ala Gly Ser Asp His Ile Ser Arg
180
185
190
Ala Tyr His His Thr Tyr Lys Leu Pro Val Thr Ile Ser Asn Cys Ser
195
200
205
Asn Asn Tyr Gly Pro Tyr Gin His Arg Glu Lys Leu Ile Pro Val Val
210
215
220
Ile Asn Ser Cys Ile Asn Tyr Lys Pro Ile Pro Val Tyr Gly Asp Gly
225
230
235
240
Ser Asn Ile Arg Asp Trp Leu Tyr Val Glu Asp His Cys Asp Ala Ile
245
250
255
Gin Thr Ile Val Glu Lys Gly Val Val Gly Glu Val Tyr Asn Ile Gly
260
265
270
Gly Ile Asn Glu Val Asp Asn Leu Thr Leu Val Lys Thr Ile Cys Lys
275
280
285
Leu Met Asp Glu Tyr Lys Pro Glu Asn Ala Pro His Ser Asn Leu Ile
290
295
300
Thr Phe Val Glu Asp Arg Lys Gly His Asp Trp Arg Tyr Ala Ile Asp
305
310
315
320
Asn Ser Lys Ile Gin Asn Glu Leu Gly Trp Lys Pro Ser Gin Asp Phe
325
330
335
Asp Lys Met Phe Arg Gin Thr Ile Glu Phe Tyr Leu
340
345
<210> SEQ ID NO 18
<211> LENGTH: 126
US 8,198,430 B2
59
60
-continued
<212> TYPE: PRT
<213> ORGANISM: Francisel1a tu1arensis
<400> SEQUENCE: 18
Met Pro Ser Tyr Ser Gin Asp Phe Arg Asp Ile Val Ile Asn Lys His
1
5
10
15
Glu Glu Gly Met Thr Glu Phe Glu Leu Ser Lys Phe Phe Asn Ile Asp
20
25
30
Lys Arg Thr Val Val Ser Trp Ile Glu Phe Tyr Lys Arg Thr Gly Asp
35
40
45
Tyr Ser Ser Lys Gin Gly Val Gly Cys Gly Arg Val Ala Ser Phe Thr
50
55
60
Asp Lys Thr Leu Ile Glu Gin Tyr Leu Ile Asp His Pro Asp Ala Ser
65
70
75
80
Ala Leu Asp Ile Lys Glu Ala Leu Ala Pro Asp Ile Pro Arg Ser Thr
85
90
95
Phe Tyr Asp Cys Leu Asn Arg Leu Gly Phe Ser Phe Lys Lys Arg Leu
100
105
110
Gin Asn Ile Ser Lys Glu Lys Asn Met Lys Gly Trp Ser Ile
115
120
125
<210>
<211>
<212>
<213>
SEQ ID NO 19
LENGTH: 468
TYPE: PRT
ORGANISM: Francisella tularensis
<400> SEQUENCE: 19
Met I1e Thr Pro Ile Ile Leu Ser Gly Gly Phe Gly Ser Arg Leu Trp
1
5
10
15
Pro Leu Ser Arg Glu Ala Ser Pro Lys Gin Phe Ile Gly Leu Val Asp
20
25
30
Glu His Ser Leu Leu Glu Asn Thr Ile Lys Arg Leu Asp Asn Val Lys
35
40
45
Asp Ile Thr Ser Pro Val Val Val Cys Asn Glu Ser His Arg Phe Gin
50
55
60
Val Ala Glu Val Leu Arg Lys Ile Asn Lys Lys Gly Asp Ile Leu Leu
65
70
75
80
Glu Pro Leu Ala Arg Asn Thr Ala Pro Ala Ile Ala Leu Ala Ala Leu
85
90
95
His Leu Ala Ile Asn Asp Pro Asn Thr Ile Met Leu Val Leu Ala Ala
100
105
110
Asp His His Ile Glu Asn Leu Glu Ile Phe His Gin Ala Ile Glu Lys
115
120
125
Ala Gin Gin Lys Val Ile Lys Asp Asp Ser Leu Val Thr Phe Gly Ile
130
135
140
Thr Pro Thr Cys Pro His Glu Gly Tyr Gly Tyr Ile Lys Gin Gly Val
145
150
155
160
Gin Thr Thr Val Asn Gly Val Tyr Lys Val Asp Lys Phe Val Glu Lys
165
170
175
Pro Ser Val Val Val Ala Gin Glu Tyr Leu Asp Ser Gly Lys Tyr Tyr
180
185
190
Trp Asn Ser Gly Met Phe Met Phe Thr Ala Arg Val Tyr Leu Glu Val
195
200
205
Leu Glu Lys Leu Gin Pro Glu Ile Tyr Arg Gly Cys Glu Lys Thr Tyr
210
215
220
Gin Lys Ser Gin Gin Asp Leu Asp Phe Val Arg Phe Asp Lys Gin Ser
US 8,198,430 B2
62
61
-continued
225
230
235
240
Phe Ala Leu Val Gln Ser Gln Ser Ile Asp Tyr Ala Val Met Glu Lys
245
250
255
Ala Thr Asn Val Ala Ile Val Pro Met Gln Gln Ser Gly Trp Ser Asp
260
265
270
Val Gly Ser Trp Asp Ser Leu Tyr Asp Ile Ala Ala Lys Asp Ser Cys
275
280
285
Gly Asn Val Val Ile Gly Asp Val Ile Thr Ser Asn Val Lys Asn Ser
290
295
300
Tyr Leu Arg Ser His Asp Arg Leu Leu Ala Ala Val Gly Val Asn Asp
305
310
315
320
Leu Ile Ile Val Glu Thr Ala Asp Ala Ile Leu Val Ala Asp Lys Asn
325
330
335
Lys Thr Gln Asp Val Lys Lys Ile Val Glu Val Leu Lys Ile Gln Gln
340
345
350
Arg Ser Glu Leu Leu Gln His Lys Gln Ile Tyr Lys Pro Trp Gly Ser
355
360
365
Ala Thr Ile Leu Glu Asp Lys Ser Gly Tyr Lys Ile Gln Ala Ile Gln
370
375
380
Leu Glu Pro Gly Lys Lys Leu Ser Leu Gln Gln His Tyr His Arg Ser
385
390
395
400
Glu His Trp Ile Val Ile Ser Gly Thr Ala Thr Val Thr Ile Gly Thr
405
410
415
Thr Lys Ser Ile Val Arg Pro Asn Glu Ser Val Tyr Ile Lys Ile Gly
420
425
430
Glu Ser His Arg Leu Glu Asn Asn Gly Lys Ile Pro Val Ile Leu Ile
435
440
445
Glu Val Gln Val Gly Glu Tyr Ile Ser Glu Asp Asp Ile Val Arg Leu
450
455
460
Asp Thr Ser Ser
465
<210>
<211>
<212>
<213>
SEQ ID NO 20
LENGTH: 494
TYPE: PRT
ORGANISM: Francisella tularensis
<400> SEQUENCE: 20
Met Arg Gln Thr Ile Ile Lys Glu Ile Ile Lys Ser Ser Gly Val Lys
1
5
10
15
Phe Gly Thr Ser Gly Val Arg Gly Leu Val Ser Ala Met Thr Asp Lys
20
25
30
Ile Cys Trp Leu Tyr Thr Lys Ala Phe Ile Gln Phe Leu Glu Gln Lys
35
40
45
Tyr Ser Ile Ala Lys Gly Thr Lys Ile Ala Ile Ala His Asp Leu Arg
50
55
60
Glu Ser Ser Pro Arg Ile Thr Thr Val Val Ile Lys Ala Ile Ile Asp
65
70
75
80
Ser Gly His Glu Pro Ile Tyr Cys Gly Glu Ile Pro Ser Pro Ala Val
85
90
95
Met Leu Tyr Gly Ile Ser Asn Gln Ile Pro Ser Val Met Val Thr Gly
100
105
110
Ser His Ile Pro Glu Asp Arg Asn Gly Ile Lys Phe Asn Thr Pro Tyr
115
120
125
Gly Glu Val Leu Lys Glu Asp Glu Glu Met Ile Val Ser Gln Thr Ile
US 8,198,430 B2
64
63
-continued
130
135
140
Ser Ile Asp Glu Ser Ile Phe Asp Lys Asn Gly Met Phe Leu Gln Lys
145
150
155
160
Leu Glu Leu Pro Glu Pro Ser Lys Gln Ala Tyr Thr Gln Tyr Ile Asp
165
170
175
Arg Tyr Val Asp Phe Phe Pro Asn Asn Cys Leu Ala Gly Lys Thr Ile
180
185
190
Gly Leu Tyr Gln His Ser Ser Val Gly Arg Glu Ile Val Lys Glu Ile
195
200
205
Leu Glu Lys Leu Gly Ala Lys Val Ile Leu Leu Glu Phe Ser Glu Lys
210
215
220
Phe Val Ser Val Asp Thr Glu Ala Ile Arg Gln Glu Asp Val Lys Leu
225
230
235
240
Ala Lys Gln Trp Ala Ser Lys Tyr Lys Val Asp Ser Ile Val Ser Thr
245
250
255
Asp Gly Asp Ala Asp Arg Pro Leu Val Ser Asp Glu Tyr Gly Asn Trp
260
265
270
Leu Lys Gly Asp Ile Leu Gly Val Leu Thr Ala Lys Tyr Leu Gln Ala
275
280
285
Asn Val Ile Val Thr Pro Val Ser Ser Asn Thr Val Ala Glu Lys Ile
290
295
300
Gly Tyr Phe Ser Asn Val Ile Arg Thr Lys Ile Gly Ser Pro Tyr Val
305
310
315
320
Ile Ala Ala Met Asn Glu Leu Leu Ser Asn Asn Gln Asn Ala Val Val
325
330
335
Gly Tyr Glu Ala Asn Gly Gly Phe Leu Leu Ala Ser Asp Ile Cys Lys
340
345
350
Asp Asp Lys Thr Leu Lys Ala Leu Pro Thr Arg Asp Ala Val Ile Pro
355
360
365
Met Leu Ala Val Met Met Leu Ser Ile Asn Ser Asn Lys Thr Val Ser
370
375
380
Glu Leu Leu Phe Asp Leu Pro Ser Arg Tyr Thr Ala Ser Ser Lys Ile
385
390
395
400
Asp Asp Phe Ala Ser Glu Lys Ser Gln Glu Ile Leu Lys Ser Ile Leu
405
410
415
Ala Gly Glu Ser Asp Leu Leu Asp Lys Ile Ile Ser Glu His Phe Asp
420
425
430
Gly Lys Asn Ser Ile Glu Asn Ile Asp Thr Thr Asp Gly Val Arg Val
435
440
445
Thr Leu Thr Asn Gln Asp Ile Ile His Leu Arg Pro Ser Gly Asn Ala
450
455
460
Pro Glu Leu Arg Cys Tyr Thr Glu Ala Ala Ser Asp Glu Gln Ala Lys
465
470
475
480
Ser Leu Asn Gln Tyr Cys Val Asp Leu Ile Asn Lys Asn Ile
485
490
<210>
<211>
<212>
<213>
<220>
<223>
SEQ ID NO 21
LENGTH: 21
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 21
ataatgaaat caatccacga g
21
US 8,198,430 B2
65
66
-continued
<210>
<211>
<212>
<213>
<220>
<223>
SEQ ID NO 22
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 22
ccagccagtc agtcccacag
<210>
<211>
<212>
<213>
<220>
<223>
20
SEQ ID NO 23
LENGTH: 21
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 23
tgtcttagat atggggcaac c
<210>
<211>
<212>
<213>
<220>
<223>
21
SEQ ID NO 24
LENGTH: 24
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 24
acaaatatca aatcctaaca catc
<210>
<211>
<212>
<213>
<220>
<223>
24
SEQ ID NO 25
LENGTH: 24
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 25
tagaagcagc tgcgataggt agac
<210>
<211>
<212>
<213>
<220>
<223>
24
SEQ ID NO 26
LENGTH: 22
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 26
ttaaataaaa actgaggaaa ca
<210>
<211>
<212>
<213>
<220>
<223>
22
SEQ ID NO 27
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 27
atggtatttt aatcaagtgt
<210>
<211>
<212>
<213>
<220>
<223>
SEQ ID NO 28
LENGTH: 17
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic oligonucleotide
20
US 8,198,430 B2
67
68
-continued
<400> SEQUENCE: 28
ctagtatgcc ccagagt
<210>
<211>
<212>
<213>
<220>
<223>
17
SEQ ID NO 29
LENGTH: 19
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 29
tggtgcgaca atcaagtta
<210>
<211>
<212>
<213>
<220>
<223>
19
SEQ ID NO 30
LENGTH: 18
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 30
agaagttcct cctcagtc
<210>
<211>
<212>
<213>
<220>
<223>
18
SEQ ID NO 31
LENGTH: 24
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 31
agaaattaag agcaaaagga aagt
<210>
<211>
<212>
<213>
<220>
<223>
24
SEQ ID NO 32
LENGTH: 24
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 32
atctcaaagt caaaatcagt ctct
<210>
<211>
<212>
<213>
<220>
<223>
24
SEQ ID NO 33
LENGTH: 24
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 33
tacgatattg tcctctccga ttag
<210>
<211>
<212>
<213>
<220>
<223>
24
SEQ ID NO 34
LENGTH: 21
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 34
tagttgcgac atattgacct g
<210> SEQ ID NO 35
<211> LENGTH: 23
<212> TYPE: DNA
21
US 8,198,430 B2
69
70
-continued
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 35
aggcaggtca atatgtcgca act
<210>
<211>
<212>
<213>
<220>
<223>
23
SEQ ID NO 36
LENGTH: 23
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 36
tttccgcaac acttcagcaa ctt
<210>
<211>
<212>
<213>
<220>
<223>
23
SEQ ID NO 37
LENGTH: 23
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 37
gctatggcca ctatcacgag agg
<210>
<211>
<212>
<213>
<220>
<223>
23
SEQ ID NO 38
LENGTH: 24
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 38
tatacttgct tgcccactgc ttag
<210>
<211>
<212>
<213>
<220>
<223>
24
SEQ ID NO 39
LENGTH: 22
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 39
accgtagtga gcattggatt gt
<210>
<211>
<212>
<213>
<220>
<223>
22
SEQ ID NO 40
LENGTH: 22
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 40
actagggcct ctgaccgttc tc
<210>
<211>
<212>
<213>
22
SEQ ID NO 41
LENGTH: 22401
TYPE: DNA
ORGANISM: Francisella tularensis
<400> SEQUENCE: 41
tcttttataa atgatgatag caaacaaaaa ataataggtt ctgtgcacaa aaaacttaaa
60
ttatattgaa aatagctaaa ccgctgttat ttaagatttg aaaagcaata aatatcaatg
120
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D66
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09111 PPD622P2P6 D D 62622 PP2ID2222I D2IDP2PI26
D6D
66
626~D62 222222 266 hD6l262DD Pb612I222I IDPI16PPID
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ObOTT 6h IPPPDIP 22P22PID6l R12PPDDPb 62lllIlb
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6
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6262
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Obb9T U1e1e1 H6 6PDPDIPID6 IPID DPDI6 PPIDDIee6e D DD6eeee6 6e6PI66D
08£9T IPPlf3lPePe eeeD6ee~e6
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0 1 9T IP22Pl2llFi PHpf3POPIP lf3f3VlePOP D6666 6llDllD626 PPDDIPIID
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0091 DD DD IDP1P116H 2DD622D26 P1116IDD
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6lDf362DZ 1e2PDHOPP P166 DD6 1 PDFil2V2Fil
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DIPee6 PPPAPZePP
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091 RMPD2111 ZV2I22D11 IPDZ2RMe I6IIIPIIPi D D62DDI H161PIPZP
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6 22PDIPI22D R2IIPPI2l 2IDAIDFi2I 12RI2P6i11
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09Z6T
OOZ6T LILIILIDDI 26262lllll 22~66222~2 IPDPPPD222 IDI2112I22 PDPID61116
ObT6T Fi2PI216111 D D D6I26 IPPIPD 6ID 2222D 66ID IDIIPI66II DIIPIP222I
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ID6I261611
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09681 226112IDD P2PIU2f3PD DID D 6DD IDDPIDIPIP V2I222D2I 6l26lll2P2
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6D6
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08L8T 622622DD
2I26IDDDD6 PIIPD62262 PPPIPI262I IP D6I622D6 I262DD D
OZL8T P6PIe6UlP l6eDee6lle 6llPDPPee~ e6DDD6
09981 I6e66PPD6e PPDII6PIPI Ie6e66DD
D6D~6e6e D661611661
6PPPPIPIII I6e6PIP66I PD1116116e
00981 D 6D622I2 6PlPDPPIII pl6P2I626 lD626D 62 66D666 226226~2D
0581 PPIPPIRgI 6D~2D26262 hII26 DD 6glPRD D DBIP2222I2 PIDIBI22I2
0881 II6Ieee6PI 6IIIPIP6ee eee6PI6III PIP6eee6DD IPPII6PIPI ee6PPD6D
0Zb81 666PP22 21161 61111 I211122Ill 22DI222222 IDIDIPI6P6 I211DI2111
09E81 2I22P V D6P IPIDIPIIII 6~6V2LD22 gDg6g A I6L 26PPIPf3A L I2622D D
00E81 DD6ee66~e6 6PNf3e6IPe 6eDIIe6eeD 6eDeeIe6ll PID6Ie116D 666De
081 66eeee6
6ee6616111 PDPDIPPIID PPIDIIPDPD DlD6leeee6 PDDPPPIPIP
08181 261266I22I DPPPI6ID IDPPPP2I66 A DDPPIDIP PIP6H6PP6 L2PA2I66I
6626266 66~62662 226266
0Z181 66
09081 PDIe6ee6el BIPIPID66I le6e6D
6D 66
D 62DD
D66D6D
666D6
DDDD6
00081 PPDPID222I 2161162122 2I26162T61 DDDIPPVgP 262626DIPD PPDDPI2DD
06L1 66IPIDPPDP 22DII6II22 PDIII22DP2 If36DDIID22 2I2IDDPIPD IPDI2I2Df32
088LT
6PID111PIP DIP61DIP66 6D66PPID
OZ8LT 22Il2626lD 2IIIDD62DD
09LLT l6lPDIPD
l662l6llf3
D66DIIPI6D DPDIIPPPDD 6e6IPIID66
D66DD6
6666
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662~D62 6666~ ~2~Fi Fi2222~
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6 6PPPDDIPPI
09L1 D6heeD Ve eIe6ll6leD 63 6eD6 D6pIlPDPI 66D6 VeePPLPLe
08SL1 262226~D2D PPPDlPl2l6 2111162261 26161112I2 6~662222~2 IL U DPIPDD
OZSLT PPIPDee6Ie PIPP6IID6 PPPPIDIPPI e6PIII6PI6 66D6IPIID IID6PPIP6I
09bLT P16DIDIPPI PPPPDIeIe6 l6elelD6D DIP1161e61 e16DBIBIPI D666
OObLT 1eIlle666D 66D6I66PDe e16PIDD
PPPPPPDDPP PDPIDPP6Ie eeeee6Iee6
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08ZLT leluf3PPDP 666D6D PIDD6DD
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OZZLT 66e~e6eD6 66IDPIIDI6 LA IDPPPPI 6P6161PIPP Mee6LPLPL HP6IPP66I
09TLT Iee6DD 6 IP6P6lPPH eAPP6D 6 PPe66h D
OOTLT 6eeee6e6~6 LD
D6eD 6
6ll6eD
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0869T Ie6111PPID LPDPeI6Vf3 PIPDPI6IDI IBIDPIPPIP 66ee~eeeDe MDlIPIP16
p9nUT UOD-
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099TZ e6PD66I6ID PIPPD6PI6e PI6PDD Deb 16DIP1161e PDD6PPDDD IPIPPPID6e
009TZ D 6ID 6I6 6
6 I66PPPPID6 6IIPPD66Ie I6e6Ie6I6e II6PID DD6
ObSTZ 6PIPBID6Ie 6D66Ie6IDe PD1116PIPI 6PIP6II6ee PIPI6PPD6e PD666I6PD6
08bTZ PPID6IID6e PPI6Ie6ee6 6PDD6DIIPP D66e6DD
6PI6ID
6 IIIeeeee6D
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09ETZ 6D 666D IPDDD62D IPIIID6662 IPIDP622I6 62D62ID 6I DPPDPPIDDD
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00£T
ObZTZ 262ID22222 DPI1111612 D66I22222I 2611111216 2226I26II2 D6PDIPID
O8TTZ PDD6PII6II e6Ieee6ee6 Ie6ee6PPPD ID 6ee6I6 6IPIPDD D IPPI U6ee1
OZTTZ IPI66Ieee6 PIP66e6PDD IIPIPDI6PI 66D66 PII6PDI6DD PIP6PDIPPI
090TZ DIPIPI66Ie 1PID6Iee16 ID6PDD DIP DDPIP6e6I6 BIBIDPIPIP PDD6e6IPDI
O0OTZ 66I6PIP6PI PDIPID6eee 11PI16116e D DPPIPP6 PIDDD6PI6e 6e6I6D D
Ob60Z
6D
D6
PlD6ll2222 ID 66622I D6112ID D
D626 PIDDII22D
666D6 2PjP6PDP6j PjD62Djjj6 JjDj66P6Pj
0880
OZ80Z J6e66j6PjD e1661116ee ej6D66D6ej DjPPPDjeej eee6eeeeje PjPjDPPPDP
09LOZ 626I22IPIP 2222226212 6PMPPDP2P PPIPIP2I16 2I622D
6 2ID26PII6I
OOLOZ 1eIe6D6 616PPIPIPI ee6e666e PDel6ee6el PllDllell6 DD
6D262D2D D~226D66
Ob90Z 66
08502 16111 ID16D PIDPIDPI66 11PID
DD62~
66 D D6jD2D6 BIDIlIP616 11PBBIIPDB
OZSOZ 2662~6DD
622622D6 66DD226D 22D6D66
66D6
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6D
6626
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o o
J6j2622D D 2222D 622 j266D6D 6j IDPIPID6I2 62D6PDP226 1161122122
08ZOZ U126i1P2iI 6266D 62D6 ID6611P M l 6D 6J2D6D jD6DPjjjPj 16PIPPPPPD
OZ2OI I6IPP16MR 2AP6161PB D66HPI166 IBIPP16616 11 Fi2IPBiP22 PD6jD6jjPfi
09TOZ pf3lPlfilllD 1R2666NDl 366 6~26 D 66ID6616 222D22D6
DD662~2~
OOTOZ D6U6IPeIR eeD6ePP6e6 61e A6PD6D eIDP6PIeeD I6eDeDIePD A6e1DDD61
Obo0
D6222D
66D
0866T 266~2666
66D62 DD62222D ~2~~~22222
DD6D6
666
6662~D62
OZ66T ReDU6IelI I61eI66D6e IPP66IIPID PIPPPD66I6 PIP6PIIIPI 6P6PPDPD6I
0986T 6D6666 DD6ee6e6
666
6DD6
0086T PDPI6666ee DPPPIIPIPI 1661PID66e e6IPDIDD 6 IIDPPDDPDP IIPD66IIID
D62DD622 2226D D6 PPRI2DM11
ObL6T
0896T Ie6e661D
eee6llPlPD
DD 6D6 D6ellll6el D6lPllPPDe IPPPDD 6l
OZ96T PPIIPID62l IIPD D D6 2D6IIDD6I I22D62DDD 6IDPIPP262 DD6PIIPDD6
09S61 262IDD D
6D66222 222I22DI22 2266D66 6226~D66 22DD
62j
00561 eDl6eee6le PIBID 6116 el6lDD DII DPPIPIe66e PD161PPIP6 PID 6D6eeI
061 IPPDPIeeee 6PIIPIDI6e IPDee6Ie6I I66IID66D PIII6PD6ee PDD6D D66
08E61 2626DPDIPI D DD66l2lD 6622DD66D
66266~D I211RI21R IDDIDPIl26
p9nUT UOD-
88
L8
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US 8,198,430 B2
89
90
-continued
tgcaatgaat gaattactct caaataatca aaatgctgtg gttggatatg aggcaaatgg 21780
aggatttcta ttggctagtg atatttgtaa agatgataaa actctaaaag cgctgcctac 21840
aagagatgct gttataccaa tgttggctgt aatgatgcta tctatcaact ctaataaaac 21900
cgtgtcagag cttttatttg atttgccatc tcgatataca gcaagtagta aaattgatga 21960
ttttgcttcc gagaaaagcc aagaaatctt gaagtcaata ttagcaggtg aatcagatct 22020
tttagataaa attatatcgg agcattttga tggtaaaaat agcattgaaa atatcgatac 22080
tacagatggt gttagagtaa ctttgacaaa tcaagatatt atccatctta gaccatctgg 22140
taatgctcca gagcttaggt gctatacaga ggcagctagt gatgagcagg caaaaagttt 22200
aaatcaatat tgtgtggatt tgattaacaa aaacatttga agatcagtca aaaatattcc 22260
ctaacttttc tcttcaccat tgaaccatta ctaaccttat ctatagctag ccacagataa 22320
aaatgtcatg ctggatttat ttcagcgttt cattataaat atcaatttta ttgagatcct 22380
gaaactagtt caggatgaca g
22401
The invention claim is:
9. A method of diagnosing infection by F tularensis, which
1. An isolated nucleic acid which encodes fifteen enzymes 25 method comprises detecting SEQ ID NO: 1 in a sample taken
involved in O-antigen biosynthesis, wherein the enzymes,
from a patient suspected of having an infection.
when expressed together in a cell, are able to produce an
10. A vector comprising an isolated nucleic acid molecule
immunogenic moiety able to produce an immune response
that
encodes fifteen enzymes involved in O-antigen biosynagainst Francisella tularensis infection in an animal.
thesis, wherein the enzymes, when expressed in a cell, are
2. The isolated nucleic acid of claim 1, which encodes
enzymes of SEQ ID NOS 3-17.
30 able to produce an immunogenic moiety able to produce an
immune response against Francisella tularensis infection in
3. The isolated nucleic acid of claim 1, having the sequence
an animal, wherein the isolated nucleic acid molecule
of SEQ ID NO 1.
4. The isolated nucleic acid of claim 1, wherein the nucleic
encodes enzymes of SEQ ID NOS 3-17.
acid comprises codons that have been optimised for expres11. The vector of claim 10, wherein the vector is a bacterial
sion in a bacterial cell.
35 vector.
5. The isolated nucleic acid of claim 4, wherein the bacte12. The vector of claim 11, wherein the bacterial vector is
rial cell is E. co/i.
a Salmonella vector.
6. An expression vector comprising the isolated nucleic
13. A composition comprising the vector of claim 10 in
acid of claim 1.
combination with a pharmaceutically acceptable carrier.
7. An isolated host cell transformed with the vector of 40
14. A host cell transformed with the vector of claim 6,
claim 6.
wherein the host cell is not a cell of a human patient to whom
8. A method of preparing an immunogenic composition,
the isolated nucleic acid is administered.
which method comprises:
transforming a host cell with the isolated nucleic acid of
claim 1; and,
culturing said host cell.
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