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) أنموذج ( أ ) الخاص برسائل الماجستير و اطاريح الدكتوراة ( اخر شهادة University of Baghdad College Name College of science Department Department of biology Full Name as written inPassport e-mail Dr Bahaa Abdullah Laftaah Al-Rubai [email protected] Career Assistant Lecturer Master Thesis Title Year Lecturer may Bahaa Abdullah Laftaah AlRubai AssistantProfessor Professor phD Bahaa Abdullah Laftaah Al-Rubai Role of Proteus mirabilis metalloprotease in degradation of different animal proteins and cloning of responsible gene in E .coli BL21 2009 ) أنموذج ( أ ) الخاص برسائل الماجستير و اطاريح الدكتوراة ( اخر شهادة I Summary Abstract 1. The clinical strain of Proteus mirabilis 49118-3 causes Urinary Tract Infection was obtained from China Gene Bank; this strain was ability to hydrolyze casein of skimed milk agar (clear zone around the colonies). Different liquid media containing various pertinacious compounds included peptone, tryptone, yeast extract and tryptic soy were used, Luria-Bertani (L.B) broth which contain (tryptone and yeast extract) gave the highest protease specific activity 145 unit/ mg protein when it was inoculated with P. mirabilis and incubated for 48hr at 37°C in shaking incubator with 180 rpm. The metalloprotease P. mirabilis was purified by precipitation with 60% saturation of ammonium sulfate, dialysis and gel filtration with Sephadex G-100 column, three peaks of protein appeared in last step, the protease activity was observed in third peak. The molecular weight of P. mirabilis metalloprotease was Daltons. 44755determined by SDS-PAGE it was approximately P. mirabilis metalloprotease was treated with different therapeutic agents included: anti-swarming agent and antibiotics to determine their effect on the activity of protease. The anti-swarming agent pNitrophenylglycerol (PNPG) inhibited the protease activity at concentration 10-500 µg. whereas the antibiotics varied in their effect, some of them decreased protease activity such as kanamycin , streptomycin, penicillin-G, erythromycin and tetracycline, the remaining activity of the enzyme treated with 75µg/ml of these antibiotics was 73 , 62 , 55, 35, and 22 % respectively, while the ampicillin revealed ) أنموذج ( أ ) الخاص برسائل الماجستير و اطاريح الدكتوراة ( اخر شهادة different effect depending on its concentrations since, the activity of enzyme increased to 107 , 125 % at 25 and 50 µg/ml respectively, and decreased to 72% at 75µg/ml of ampicillin. The effect of P. mirabilis metalloprotease on the human ß-defencen 1 (hBD-1) was studied; the bactericidal activity of this proteins was inhibited after treatment with P. mirabilis metalloprotease against Escherichia coli 0138 for 2 hours. In this study the metalloprotease of P. mirabilis revealed cytotoxic activity on monolayer of Vero cells which completely damaged after 7296 h and causing death of the cell culture, the enzyme was able to degrade different animal cell matrix compounds including collagen IV, elastin, laminin and fibronectin, also the metalloproteases able to hydrolyze other anmals proteins such as hemoglobin, gelatine, and bovin serum albumin (BSA) as well as casein. Zap A gene encoded P. mirabilis metalloprotease was amplified by polymerase chain reaction technique (PCR) and cloned in pGEX-KG plasmid to construct new recombinant pGEX-KG (ZapA) expression vector and transformed into E.coli BL21 DE3 to overexpression the recombinant metalloprotease , E.coli failed in expression of the cloned gene.