Cloning And Eukaryotic Expression Of Surface Antigen Of Vibrio Harveyi

Vibrio harveyi is a marine Gram-negative luminous organism with a requirement for sodium chloride。It is a serious pathogen of marine fish and invertebrates, particularly large yellow croaker, resulting in severe economic losses every year. In fish, the diseases include vasculitis, gastro-enteritis and eye lesions.According to OmpU gene sequence published in GenBank,1primers were designed and the DNA fragment of about 1000 bp was amplified by PCR from genomic DNA of Vibrio Harveyi zj2008, isolated from disease Pseudosciaena crocea. The gene was cloned into pMD18-T vector and sequenced, then submitted to GenBank with the accession number FJ919231. BlastN result showed that the sequence shared identity of 99.0% with OmpU of Vibrio harveyi. The sequence was subcloned into pET30(a) resulted with the recombinant plasmid pET30(a)-OmpU. pET30(a)-OmpU was transferred into the host bacteria E.coli BL21(DE3) with IPTG induction for expression. SDS-PAGE profiles showed the expected MW of the recombinant OmpU,41 kDa. The protein was over expressed in insolvable fusion bodies and was preliminary purified by urea method. Forty pieces of large yellow croaker were individually immunized with 50μg of the recombinant protein by intraperitoneal injection. Four weeks after vaccination, ten pieces were artificially challenged by intraperitoneal injection with live bacteria and the mortality was recorded in the following 14 d. Relative percent survival rate of the immunized group reached 85%. The present study demonstrated strong immunogenicity of the recombinant OmpU. OmpU of V.harveyi should be given enough attention for their potential application in vaccine development.The OmpU genes was cloned from genomic DNA of Vhareyi zj2007, then submitted to GenBank with the accession number FJ919232. The strain was isolated from diseased Lateolabrax japonicus during the outbreak in 2007. The sequence was subcloned into pET30(a) resulted with the recombinant plasmid pET30(a)-OmpU. The pET30(a)-OmpU was transferred into the host bacteria E.coli BL21(DE3) with IPTG induction for expression. Recombinant OmpU showed a MW of 48.49kDa in SDS-PAGE,it moved more slowly than the calculated value (40.49kDa).Several outer membrane proteins of Vibrios such as OmpK, OmpW and OmpU are protective antigens to guard against vibriosis. The OmpW genes was cloned from genomic DNA of V.hareyi zj2008, then submitted to GenBank with the accession number FJ998268.The strain was isolated from diseased Janpnese seabass(Lateolabrax japonicus) during the outbreak in 2008. The sequence was subcloned into pET30(a) resulted with the recombinant plasmid pET30(a)-OmpU. The pET30(a)-OmpU was transferred into the host bacteria E.coli BL21(DE3) with IPTG induction for expression. Bandsan analysis showed that the molecular weight of recombinant OmpW was 28kDa, similar to the calculated value (26.69kDa). The present paper facilitated the further study on immunogenicity and vaccine development of this protein.According to OmpW gene sequence published in GenBank, primers were designed with restriction site,EcoRⅠand NoteⅠ,and the DNA fragment of about 600 bp was amplified by PCR from genomic DNA of Vibrio Harveyi zj2008, isolated from disease Pseudosciaena crocea.. Double Digests the PCR products and the plasmid pPIC9K,then,the OmpW gene was cloned into Pichia pastoris expression vector pPIC9K to construct the recombinant plasmid pPIC9K-OmpW,which was linearized with SacⅠ,and transformed into GS115 cells by electroporation. The positive clones were selected by MD/MM plates and confirmed by PCR. SDS-PAGE analysis showed that expression product could secreted into the supernatant.