CLONING,EXPRESSION,IMMUNOGENICITY OF EXTRACELLULAR PROTEINASE FROM VIBRIO HARVEYI

Vibrio harveyi is a causative agent of vibriosis that infects large yellow croaker(Pseudosciaena crocea) as well as some other marine fish and brings severe loss to their culture.The extracellular proteases of V.harveyi have a role in the virulence of the organism and are potential candidates for vaccine development.In this study,the specific primers of extracellular protease(ΔProA) gene excluding the region coding for signal peptide were designed according to the sequence of V.harveyi strain GYC1108-1 ΔProA gene(GeneBank No.:EF126129),and a piece of DNA sequence about 1552 bp was amplified by PCR from genomic DNA of GYC1108-1 strain.The ΔProA gene was cloned into pUC57-T easy vector and sequenced.Then the ΔProA gene fragment were subcloned into pET-28a(+) to construct expression plasmid pET28a-ΔProA.It expressed a 55 kD fusion protein HIS-ΔProA in E.coli TG1 when induced by isopropyl thiogalactoside(IPTG).SDS-PAGE analysis showed that the recombinant protein was expressed in the form of inclusion body and accumulated up to 21% of the total bacteria protein.It revealed that,by western blot analysis,the fusion proteins ΔProA had immunoreactivity.The purified fusion protein ΔProA used as antigen to immunize large yellow croaker(Pseudosciaena crocea) and mice,the relative percentage survival(RPS) were 75% and 91.6% respectively.This result indicates that the ΔProA may be one of the important protective antigens of V.harveyi,and may play a role in protecting fish from infection of V.harveyi.