Immune Effect Of Carp Interleukin-10 And Typeâ… Interferon And Koi Herpesvirus Nucleic Acid Vaccine Combined Immunization

Interleukin 10(IL-10) is a multi-effect cytokines with dual immunomodulatory activity of immune stimulation and immunosuppression. IL-10 can promote differentiation and proliferation of B cells and natural killer cells.IL-10 can promote colony of monocytes, macrophages and mast cell. IL-10 is homologous genes which is closely related to viral genome. In the immune response, IL-10 can balance the relationship between the tissue damage and prevent pathogen invasion. In the process of against pathogens, it can reduce inflammation, and protect tissues from excessive inflammation injury.Interferon(Interferon, IFN) is a kind of glycoprotein. Cell and organism often require the stimulation such as virus, nucleic acid and bacterial endotoxin, to produce interferon. It has the prominent effect on virus infection rapidly and has antiviral effect. Type Ⅰ interferon(Ⅰ-IFN) is usually an important component to participate in innate immune response. Ⅰ-IFNcan activate the transcription of the IFN stimulate factor(ISGs), and code different antiviral proteins. It can release antiviral substances, and interfere the replication and proliferation of virus. The growth and differentiation of the body’s cells can be regulated by Ⅰ-IFN.Koi Herpesvirus(KHV) is the pathogen of Koi Herpesvirus disease(KHVD), which has a highly contagious and fatal rate between Koi and common carp. With the rapid development of global trade of aquatic products, the disease in the world spread wantonly and it is difficult to control, which will be resulting in serious damage to the economy of carp aquaculture. Nucleic acid vaccine can be used for the prevention and treatment of brocade KHVD, but the effect has a certain gap with target. The carp IL-10 and Ⅰ-IFNwere selected to build pEGFP-N1-IL-10 and pEGFP-N1- Ⅰ-IFN eukaryotic expression plasmid. The immune effect of Mixed plasmid pEGFP-N1-IL-10(pEGFP-N1-Ⅰ-IFN) and nucleic acid vaccine PIRES-ORF81 was studied. New way for prevention and treatment of KHVD was provided.On the basis of IL-10 mRNA and Ⅰ-IFNmRNA gene sequence Genbank login, two pairs of specific primers were designed. The extraction of RNA from carp tissues, then through RT-PCR, The cDNA was amplified by PCR and gain the two target genes, they are 540 bp and 552 bp, respectively. Recombinant plasmid pMD18-T-IL-10 and pMD18-T- Ⅰ-IFNwere constructed, confirmed by bioinformatics, IL-10 homology of 99%, Ⅰ-IFN homology of 100%. The eukaryotic recombinant plasmid pEGFP-N1-IL-10 and pEGFP-N1-Ⅰ-IFNwere constructed. After a large number of extraction and purification of eukaryotic recombinant plasmid were transfected into the Kio fin cell, to obverse whether there is a green fluorescent protein expression.Recombinant plasmid expressed successfully to test immune fish. The fish was injected by different doses of pEGFP-N1-IL-10(pEGFP-N1- Ⅰ-IFN) and nucleic acid vaccine PIRES-ORF81 mixed plasmid with the blank group, the PBS group and single PIRES-ORF81 group. Three immune were 14 days interval, and blood sampling was before and after immunization. And then the indirect ELISA antibody level in the serum was evaluated. ELISA results showed that antibody levels will increase gradually with the increase of number and dose of immune, The highest antibody levels was on the first week after three immune. Then the antibody levels gradually decline. Compared with single nucleic acid vaccine pIRES-ORF81, antibody levels combined by the immune of pEGFP-N1-IL-10(pEGFP-N1-Ⅰ-IFN) and nucleic acid vaccine pIRES-ORF81 increase obviously, but it was not significant(p> 0.05).