Construction And Expression Of Recombinant Plasmid PET-OmpU And Its Imraunoprotective Effect To Koi Carp

The ascites disease in aquatic animals is an important disease caused by Vibriomimicus. But there isn’t an effective vaccine for the prevention of the disease atpresent. The outer membrane protein U (OmpU) is relatively conserved antigenamong pathogenic vibrio spp, and has been proven to be a major protective antigen ofVibrio cholerae and Vibrio vulnificus. However, it is not clear whether OmpU ofVibrio mimicus possess immune protective effect. In this study, The OmpU protein ofVibrio mimicus was expressed successfully by prokaryotic expression system. Theimmunogenicity, immunoreactivity and immune protective effect to koi carp of thesoluble recombinant OmpU protein were detected. The results laid the foundation forthe further development of the OmpU protein vaccine of Vibrio mimicus.In order to prepare special antibody against Vibrio mimicus，rabbit wasimmunized with inactivated suspension of04-13strain emulsified in Oil adjuvant A5fully three times. The result showed that the titer of rabbit anti-Vm antisera was1:225detected by agglutination test after the third immunization, which could fully meet therequirements of the following Western blot experiment.For the purpose of obtaining highly efficient expression of the solublerecombinant OmpU protein. First of all, signal peptide of OmpU protein was analyzedby SignalP on-line software based on OmpU gene sequence of V.mimicus strain04-13isolated from Anhui Province(GenBank: DQ846743), it was found that there was asignal peptide composed with22amino acids encoded by66bp base pairs from5’end.A pair of specific primers were designed and OmpU gene without signalpeptide(1004bp base pairs)was amplified by PCR and was cloned into pET-32a（+）vector to constract recombinand plasmid pET-32a-OmpU. Based on the orthogonalexperimental design L（934）and the analysis of SDS-PAGE, The four factors includingmedia induced temperature, induced time and IPTG concentration were optimized.The result showed that the gene engineering E.coil strain pET-32a-OmpU/Rossetagrowing at37℃for3hours in LB medium was incduced for4hours by0.5mmol/LIPTG, the expression amounts of soluble recombinant OmpU protein reached496mg/L.In order to analyze the immunogenicity, immunoreactivity and immuneprotective effect to the koi carp(锦鲤) of the soluble recombinant OmpU protein,OmpU protein was expressed in the optimized condition and was purified with Niaffinity chromatography colum.The purified protein with98%purity and3.2mg/mL concentration could react specifically with rabbit anti-Vm antibody by Western blot.60healthy experimental koi carps, which were divided into four groups on average,were immunized with the purified recombinant protein, tagged protein His,inactivated suspension of04-13strain and sterile saline (control) respectively. on the7th day after the fourth immunization, the IgM levels in different sera were detectedby commercial ELISA kit, at the same time The Experimental fish were challengedwith3×109cfu/mL suspension of04-13strain.The result showed that the levels of IgMin antisera increased in varying degrees after four immunization compared with thelevel of control serum,the IgM amounts were3.32mg/mL、2.95mg/mL and4.51mg/mL respectively in recombinant OmpU protein antisera, tagged proteinantisera and Vm antisera.Survival rate of immunized fish with recombinant OmpUprotein and inactivated bacteria after challenge were73%and87%, while100%mortality was found in fish immunized with His tagged protein and control fish.These results suggest recombinant protein OmpU possess a good immunogenicityand reactogenicity and immune protective effect to koi carp in some certain degree.