鲤疱疹病毒3型ORF148 DNA疫苗对建鲤鱼苗的免疫保护

鲤疱疹病毒3型(Cyprinid herpesvirus 3, CyHV-3)又称为锦鲤疱疹病毒(Koi herpesvirus, KHV),是一种高传染性、致死性病毒。为开发新型CyHV-3 DNA疫苗,前期研究中将CyHV-3 ORF148基因插入pEGFP-N1构建重组质粒,在此基础上,本研究通过转染试验、间接免疫荧光试验证实pORF148-EGFP融合蛋白可以在CCB-J细胞系和建鲤体内表达;将重组质粒作为DNA疫苗,肌肉注射免疫建鲤鱼苗,ELISA检测表明免疫pEGFP-ORF148重组质粒可以显著提高建鲤血清特异性抗体水平; RT-qPCR检测显示,免疫pEGFP-ORF148重组质粒后,建鲤脾脏和头肾中的免疫相关基因如IFN-a1、Mx-1、CXCa、CXCR1、TNF-α、IL-1β及IgM基因表达量均显著提高。攻毒实验显示, CyHV-3攻毒21 d后, PBS组、pEGFP-N1组和pEGFP-ORF148组的建鲤存活率分别为30%、35%和85%,免疫pEGFP-ORF148可以显著提高建鲤的存活率(P0.01)。本研究旨在为CyHV-3 DNA疫苗的应用提供理论依据。

Immunization of Cyprinus carpio var. jian with Cyprinid herpesvirus 3 ORF148 DNA vaccine against koi herpesvirus disease

Cyprinid herpesvirus 3 (CyHV-3), also known as koi herpesvirus (KHV), is a highly contagious and lethal virus. At present, a kind of commercial attenuated vaccine has been used in Israel against this virus. However, this vaccine, as well as other reported gene-modified attenuated vaccines against CyHV-3 infection, cannot exclude the risk of reversion to virulence. In contrast to live vaccines, DNA vaccines are easy to produce and safe to apply. CyHV-3 has a genome of 295 kb, which is predicted to encode 164 different open reading frames (ORFs). Among them, ORF148 encodes a glycosylated type I membrane protein and bioinformatics analysis has shown that this protein has good immunogenicity. During our previous study, CyHV-3 ORF148 was inserted into pEGFP-N1, generating a pEGFP-ORF148 recombinant plasmid. In the present study, it was confirmed that a pORF148-EGFP fusion protein could be successfully expressed by transfection assay and immunofluorescence analysis, respectively. The carps, n=45), were intramuscularly injected with the pEGFP-ORF148 recombinant plasmid, pEGFP-N1 and PBS three times at two-week intervals. The results of ELISA showed that immunization with the pEGFP-ORF148 recombinant plasmid could significantly (<0.01) improve the tire of specific antibodies against CyHV-3. The RT-qPCR revealed that injection with the pEGFP-ORF148 recombinant plasmid caused 145.30-, 131-, 52.27-, 12.16-, 4.19-, 3.05-, 4.03-fold increases of , gene expression in the head kidney, respectively, when the PBS immunized carps served as a control group. In the spleen, the expression of from pEGFP-ORF148 recombinant plasmid injected carps were 22.44-, 4.63-, 6.14-, and 4.23 times higher than those of control group. Post-infection with CyHV-3, the survival rates of carps immunized with PBS, pEGFP-N1, and the pEGFP-ORF148 recombinant plasmid were 30%, 35% and 85%, respectively, suggesting that immunization with the pEGFP-ORF148 recombinant plasmid significantly protected the carps against the CyHV-3 infection (<0.01). In conclusion, DNA vaccination based on the pEGFP-ORF148 recombinant plasmid could elicit the activation of an immune response and production of a specific antibody to produce effective protection against CyHV-3 infection. Thus, this study highlights a potential DNA vaccine candidate against CyHV-3.