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The Application Of Agarase And Serine Protease From Vibrio Sp In The Immunoprotect Of The Japenese Flounder

Posted on:2010-08-17Degree:DoctorType:Dissertation
Country:ChinaCandidate:W W ZhangFull Text:PDF
GTID:1103360275969344Subject:Marine biology
Abstract/Summary:PDF Full Text Request
Vibrio sp V134 was isolated from a fish farm in Huangdao Shandong Province. agaV gene was cloned and expressed in BL21(DE3), the recombinant protein AgaV was purified with nichel-nitrilotriacetic acid agarose under native conditions. The purified protein was analyzed using agarose as substrate to establish the optimum temperature and pH of the agarase, which was around 40℃and pH 7.0, with a narrow agarolytic range of pH respectively. AgaV was demonstrated in two aspects: first, as an agarolytic enzyme, the purified recombinant AgaV could be employed in the recovery of DNA from agarose gels; second, as a secretion protein, AgaV was explored at genetic level and used as a reporter in the construction of a secretion signal trap which proved to be a simple and efficient molecular tool for selection of genes encoding secretion proteins from both G+ and G- bacteria.Of the six signal sequences selected by pBU from fish pathogen T4, a Vibrio harveyi strain, one characterized to be a serine protease and named degQVh, was tested to be an immunoprotective protein agaist T4. The protease exhibited highly RPS (Relative Percentage Survival)≈65% aganist the disease caused by T4. To enhance RPS, AgaV-DegQVh fusion protein was expressed in DH5αto form the antigen delivery system, which was proved to increase the RPS about 30%. degQVh was cloned into PET258 and expressed in BL21 (DE3), the recombinant DegQvh was purified and characterized, which showed the highest protelytic activity around 50℃ and pH 8.0. Five mutations concluding three site mutations and two PDZ deletion mutations demonstrated that the protease activity of DegQVh need His(83), Asp (113) and Ser (188), the three typical catalytic amino acid and both of the PDZ domains. Analysis indicated that the expression of degQVh was regulated by temperature and cell density. The sequence alignment of up stream of degQVh indicated that it contained oneσE–dependent promoter. Further study indicated that degQVh could complement the function of DegP in E. coli.
Keywords/Search Tags:agarase, serine protease, secretion sequence, Japanese Flounder
PDF Full Text Request
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