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M. P. Chumakov Institute of Poliomyelitis
and Viral Encephalitides
A NEW SEROLOGICAL SYSTEM FOR PREDICTING HCV TREATMENT RESPONSE,
PRELIMINARY RESULTS
1
Kuznetsova ,
1
Tallo ,
2
Brjalin ,
1
Reshetnjak ,
Tatiana
Tatjana
Vadim
Irina
3
3
1
Maria Smirnova , Alexei Shevelev , Valentina Tefanova
1Department
of Virology, NIHD, Tallinn, Estonia
2West-Tallinn Central Hospital, Tallinn, Estonia
3Federal State Budgetary Institution «M. P. Chumakov Institute of Poliomyelitis and Viral Encephalitides» of RAMS, Moscow, Russia
Background
Previously we analyzed complete NS5A sequences from Estonian patients with chronic HCV-1b infection who had received combination therapy with
PegIFNα-2a plus ribavirin. Phylogenetic analysis revealed four subclades tending to confer a different treatment outcome. These subclades were
characterized by several steady inherited amino acid substitutions. However, detection of amino acid substitutions in HCV is precluded by
irreproducible PCR of relevant NS5A region. Serological methods, e. g. solid-phase immune-assay (ELISA), provides an efficient and inexpensive
alternative to PCR for routine assay. However, there are no commercial kits available for assay of antibody response towards NS5A regions.
Additionally, NS5A protein is not easily available as an antigen for serological tests.
Objectives
Our work was focused on designing short derivatives of NS5A gene adapted for efficient production in E. coli and allowing NS5A putative subclades
discrimination within HCV1b genotype by serological testing.
The first stage required establishing an innovative screening method for designing truncated antigens. This approach included in vitro constructing
libraries of NS5A fragments and their screening for expression efficiency in bacteria.
The second stage included selection of an appropriate clones by serological methods (Western-blot).
The third stage involved engineering high-efficient producers of designed NS5A mini-derivatives fused with green fluorescent protein (GFP).
Results
Methods
1. Nicking PCR product with DNAse. NS5A PCR products were
treated with DNAse in several dilutions to produce nicks in DNA.
Probes with mild extent of degradation were used for the next
stage.
1. Selection of DNA probes
with mild extent of degradation
2. Generation of deletion derivatives. DNA probes purified by
phenol-chloroform extraction were treated with DNA
polymerase I for extending the gaps in DNA. Then DNA probes
were mixed with random primers bearing constant adapter
sequence at 5’-end and ligated. The library of deletion
derivatives was eventually produced by PCR with a single
adapter primer.
3. Cloning library of gene fragments into specially designed
LacZ-based vector was performed. Phenotypical selection for
brightness of colonies was carried out. Level of protein
expression was evaluated by beta-galactosidase activity test.
2. Results of PCR with a single adapter primer
4. PCR with standard primers for determination of insertion size
in the selected clones was performed. Obtained PCR products
were sequenced.
4. Insertion size range: from 50 bp to 700 bp
5. LacZ-based protein production and Western-blot with HCVpositive sera from patients were performed.
5. One out of 40 clones was immune-positive
6. Engineering GFP-based producers of selected NS5A
derivatives was carried out.
6. Primary purification of a fused protein with GFP
Delution of DNAse
104 105 106 conc. 1 kb Ladder
Probes: a
s
n
r 100 bp Ladder
3. Quantification of beta-galactosidase activity in soluble cell fractions
beta-galactosidase activity
Precipitate
Supernatant
PAGE electrophoresis under
denaturing and quasi-native conditions
kDa
Conclusions and plans
Extraction by: Tris SDS Ac.acid
Tris SDS Ac.acid
 The scheme of producing short derivatives library was designed.
 One water-soluble immune-positive protein was obtained. Its purification and further serological testing in ELISA with
panels of serum samples is in a progress.
 Testing alternative enzymes for extending the gaps in DNA, e.g. exonuclease III and lambda phage nuclease, is in course.
 Obtaining and testing new NS5A-derived proteins is considered.