| Koi herpesvirus (KHV) infection koi, carp, and its common variants cause the koi herpesvirus disease (KHVD), the disease infection of the gills and kidneys mainly, has a highly contagious and fatal rate. Once infected, the mortality rate as high as80%to100%.In1998, the disease first discovered in Israel, then spread and popular in Sweden, Britain, Germany, the United States, Indonesia, Japan and China Taiwan more than a dozen countries and regions, into China in2003;It is difficult to control that esulting in ahuge economic losses, has caused great concern in the world.Choose KHV’s two major protective antigen gene (ORF25and ORF81) as the target gene, the eukaryotic expression vector pIRES-neo as vector, build two eukaryotic expression recombinant plasmid pIRES-ORF25and pIRES-ORF81, after in vitro validation can get a good expression, carry out immune test and challenged experiment in carp that it has a good protective immunity.In this study, according to Genbank logged KHV ORF25and ORF81gene sequences, designed and synthesized two pairs of specific primers, to KHV’s nucleic acid as a template for PCR amplification;Two target genes were cloned into pMD-18T vector build pMD18T-25and pMD18T-81recombinant plasmid, identified the DNA sequencing then carry on simple bioinformatics analysis.The results showed that:the ORF25gene size of849bp, encoding283amino acids, the molecular weight of32kDa; ORF81gene size of771bp, encoding256amino acids, the molecular weight of28.2kDa. Bioinformatics analysis showed that: the two genes compared to GenBank logged three KHV, homology can be more than99%, and the two genes with good antigenicity.Two recombinant plasmids pIRES-ORF25and pIRES-ORF81were transiently transfected with a framed mirror carp caudal fin cells (MFC), and Western-blot method of testing proved that the two target proteins to get a good expression.The recombinant plasmid immunitied carp, set up to1μg,10μg and50ug three immunizing dose gradient for the plasmid pIRES-ORF25, pIRES-ORF81and pIRES-ORF25+pIRES-ORF81mixtures, and set the PBS, empty plasmid and blank three controlgroup; take intramuscular immunization three times successively, at intervals of two weeks:blood for the carp immunitied weekly, detectied of antibodies with ELISA method, analysis of the the carp humoral immune response; and carry out neutralization test detectied antibody levels; do inoculation experiments in the last, calculation of mortality, production and observation of the biopsy, verify that the protective immunity of the recombinant plasmid.ELISA results showed that:the KHV-specific antibodies could be detecte in carp’s blood, anti-body levels increased significantly after the boost immunization, the antibody levels were sig-nificantly different (p <0.05) Compare immune recombinant plasmid with the control group.The analysis of experimental results can be found:three dose gradient the recombinant plasmid,50μg dose induced antibody levels is best, but the difference was not significant (p>0.05); The two plasmids for mixed compared with single plasmid immunization induced antibody levels increased slightly, but the difference was not significant (p>0.05).Neutralization test results show:the injection of PBS, empty vector and blank control group carp did not produce neutralizing antibodies, the recombinant plasmid immunohistochemistry carp produced higher levels of neutralizing antibodies.Challenged experimental results show that:after inoculation, the injection of plasmid pIRES-ORF25、pIRES-ORF81and pIRES-ORF25+pIRES-ORF81mixture experimentsgroup protection rates were85%,82.5%and87.5%;The biopsy results showed that the organization of the control group there is a clear pathological changes, while the injection of eukaryotic expression recombinant plasmid group organization did not change significantly;explain that the two DNA vaccine plRES-ORF25and pIRES-ORF81has better KHV protective effect. |
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