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

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 degQ_Vh, 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-DegQ_Vh fusion protein was expressed in DH5αto form the antigen delivery system, which was proved to increase the RPS about 30%. degQ_Vh 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 DegQ_Vh 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 degQ_Vh was regulated by temperature and cell density. The sequence alignment of up stream of degQ_Vh indicated that it contained oneσE–dependent promoter. Further study indicated that degQ_Vh could complement the function of DegP in E. coli.