Construction Of Cyprinid Herpesvirus 2 Envelope Protein ORF25n Expression Vector And Its Immune Effect

Carassiusauratus gibelio has become an important freshwater product in Chinese aquaculture due to its delicious taste.Recently,the outbreak of herpesviral haematopoietic necrosis characterized by gill hemorrhage,the main pathogen was carp herpesvirus type 2(Cy HV-2)has caused huge economic losses to C.gibelio.Cy HV-2 is a linear double stranded DNA virus.Its genome size was 290 kb,the wirus was 120 nm.It was known that there were 151 open reading frames.The nucleocapsid of the virus was icosahedral symmetry.The main function of virus envelope protein was to help the virus enter the host cell.The ORF25 n gene of Cy HV-2 belongs to the ORF25 multi-gene family.The membrane protein encoded by the virus contains immunoglobulin like domain.Our previously research showed that ORF25 n protein has good conservatism and immunogenicity and can be used as an ideal antigen.In the present study,ORF25 were cloned and connected to the prokaryotic expression vector p ET-32 a,and transformed into Escherichia coli BL21.E.coli was induced to express overnight by IPTG.A rapid detection kit for Cy HV-2 was also developed.The eukaryotic expression plasmid of Cy HV-2 ORF25 n was also constructed.ORF25 n gene was amplified by template Cy HV-2 DNA.The PCR product was recovered and purified by enzyme digestion and connected to the eukaryotic expression vector pc DNA3.C.gibelio were immunized with different concentrations of plasmids.After 28 days of immunization,immunized fish were challenged with Cy HV-2 virus to determine the immune protection rate.Our result showed that ORF25 n gene was successfully amplified with a length of 585 bp,and the eukaryotic expression plasmid pc DNA-ORF25 n of ORF25 n was successfully constructed.The results of immune challenge showed that the recombinant plasmid pc DNA-ORF25 n groups showed a greater immune protection than control group and empty carrier group.After the first immunization,the protection rate of the 2.5μg immunization group could reached 50%,and after the second immunization,the protection rate of the 2.5μg and 5μg immunization groups could reached up to 75%.In addition,the presence of pc DNA-ORF25 n plasmid can be detected in the muscle tissue and liver,spleen,kidney,and brain of the injection site from 12 h to 7 days after immunization.It was shown that after injection,the eukaryotic expression plasmid pc DNA-ORF25 n can be replicated in fish in a short period of time,and the fish can be immunized by in vivo antigen expression,so that the immune fish can obtain resistance to CyHV-2.The results from q PCR demonstrated that the expression level of IFN-γ,C3,and Ig M in the immunized group showed no difference with control group(p> 0.05).In the spleen tissue,the expression of IL-1β in 5 μg immunized group were increased first and then decreased,and were significantly higher than the control group(p <0.05),it reached a peak on day 5th,and the expression of MHC I in the spleen was also significantly higher than that in the control group(p <0.05).)And reached its peak at 5th d.The results showed that the immune cells of fish were activated after being immunized with the nucleic acid vaccine,and the mutual recognition ability of the immune cells was enhanced which indicated that the pc DNA-ORF25 n plasmid immunization not only improve the fish’s ability to resist Cy HV-2,but also enhance the immune response.