Prokaryotic Expression And Immunogenicity Of ORF30and ORF131Genes Of Koi Herpesvirus

Koi herpes virus (KHV) infection causes Koi herpes virus disease (KHVD).Koi herpesvirus (KHV) infection with occurs in carp and koi (Cyprinus carpio) only. It will causeinterstitial nephritis and gill necrosis and other symptoms, which has a highly contagious andfatal rate, KHV was first detected in Magan Micha1998in Israel. So far, the virus has beenreported in around the world. A large koi Death by KHV. And there is no effective vaccine forits prevention and treatment. It has been listed in aquatic animal disease directory by the OfficeInternational Des Epizooties (OIE), China’s Ministry of agriculture listed as animal epidemic ofclass II.The virus has dodecahedron three-dimensional spherical structure. mature virus havediolame or envelope. KHV has linear structure of double strands of DNA, genome includes156Open reading frame(ORF),the sum of the Guanine and Cytosine content is59.2%.The currentunderstanding of the virus and is not well understood, only a small part of structure and functionto be understood, but the study of genomics, immunology, molecular biology still exist manyunknown and controversial. Vaccine research KHVD just started, in terms of inactivatedvaccines, live attenuated vaccines, gene vaccine has been some progress, but efficient, safe andwidely recognized vaccine has not yet appeared.Hence, by genetic engineering unique, Selected Gene segments ORF30and ORF131ofKoi herpes virus which Sequence conservation And immunogenicity prominent，Koi immunizedwith the recombinant protein ORF30and ORF131，Koi for immunization to stimulate the bodyto produce high titers of specific neutralizing antibodies to protect free from Koi herpes virusthreats.Amplified ORF30and ORF131from brocade carp herpes virus genom and clone them intoPMD-18T vector. Amplify ORF30and ORF131again when the results of the identification ispositive and then cloned them into pET-32a constructing the recombinant expression plasmidpET-32a-ORF30and pET-32a-ORF131which were then transformed into （E.coil）BL21(DE3),and induced by IPTG. Purified the expressed recombinant fusion protein through anionexchange chromatography DEAE Sepharose4FF、 cation-exchange chromatography SPSepharose Fast Flow、hydrophobic chromatography Phenyl Sepharose6FF and metal chelatechromatography Chelating Sepharose Fast Flow. To obtain recombinant protein ORF30andORF131and analyze basic physical and chemical properties, then immune health carp withthem, which were determined reasonable immunization programs, while to establish ELISA monitoring methods, in order to monitor antibody titer levels and antibody ebb and flowrules by immune. When the antitoxic and protective experiment by using laboratory Koi finishes,the research assess the effect and result of it. Explore and develop an immunity to resist Koiherpes virus vaccine.The results prove that: After the PCR amplification and sequencing the recombinantplasmid was proved that the plasmid ORF30and ORF131was cloned into the vectorconstructed correctly. The relative molecular mass of fusion protein was approximately29.4KDa and55KDa. mainly existed in a soluble form and contained more than22.8%and33%of total somatic protein. The purified ORF30and ORF131recombination protein wasabove90%.In summary, the build obtained efficient fusion protein ORF30and ORF131arehighly immunogenic, stimulating carp to produce high titers of neutralizing antibodies andplaying a very good therapeutic effect on resistance to koi herpes virus infections. Which laid afoundation of development of immune agents for the cyprinid herpes virus (KHV).