共表达γ干扰素的乙型脑炎病毒与猪细小病毒核酸疫苗的构建及其免疫原性研究  

Research on Construction of DNA Vaccine of Recombinant Porcine Parvovirus Virus Coexpressing E10 Protein of Japanese Encephalitis Virus Fused with poIFN-γ gene and Its Immunogenicity in Mice

作　　者：叶茂[1] 郭万柱[1] 徐志文[1] 王小玉[1] 李云云[1] 

机构地区：[1]四川农业大学动物医学院动物生物技术中心,四川雅安625014

出　　处：《西南农业学报》2010年第3期890-896,共7页Southwest China Journal of Agricultural Sciences

基　　金：教育部长江学者和创新团队发展计划项目资助(IRT0848)

摘　　要：为了构建和筛选良好的猪乙型脑炎和猪细小病毒的核酸疫苗,并提高核酸疫苗的免疫效果,用重叠PCR法把PPV VP2和JEV E10连接,再将扩增的IFN-γ连入构建载体pcDNA.VEI使其共同表达。用脂质体将pcDNA.VEI介导入Vero细胞,通过间接免疫荧光法检测目的基因的体外表达。用pcDNA.VEI和不表达IFN-Κ的pcDNAZ.VE分别免疫Balb/c小鼠,同时设立PBS、猪细小病毒灭活疫苗、乙型脑炎弱毒疫苗和pcDNA3.1空白质粒免疫对照组。共免疫两次,间隔2周检测免疫指标。结果表明,通过鉴定核酸疫苗的构建达到了预期目标,并可在荧光显微镜下观察到了其转染的Vero细胞。该核酸疫苗能够诱导脾淋巴细胞增殖。ELISA结果显示,与pcDNA.VE和阴性对照组进行比较,pcDNA.VEI所诱导产生的两种抗体都较高。pcDNA.VEI组能较稳定的诱导产生T细胞亚群,并检测到CD4+/CD8+比例于首免2周后都高于pcDNA.VE。因此表明该核酸疫苗能产生特异诱导体液免疫和细胞免疫且是安全的,证明了IFNγ基因具有免疫增强效果,为乙脑和猪细小病毒核酸疫苗研究提供了实验依据。To construct the PPV and JEV DNA vaccine and then to screen out the highly effective one for its immunogenicity,PPV VP2 and JEV E10 were connected,which were cloned into pcDNA-3.1 and coexpressed with poIFNγ gene.The Balb/c mice were immunized with this DNA vaccine named pcDNA.VEI,pcDNA.VE within no IFN-γ expressed,Inactivated PPV Vaccine,JE live Attenuated Vaccine,and pcDNA3.1 as the blank control groups.The mice were immunized 2 times and immune indexes were tested per 2 weeks.The result showed that the DNA vaccine was successfully constructed and the vero cells transfected by it were observed by fluorescence microscope.The stimulation of spleen lymphocytes was proved on the DNA vaccine.The ELISA results showed that the level of two sorts of antibodies introduced by pcDNA.VEI was higher than the IFNγ-negative control group.The T Lymphocyte Subsets were steadily induced by group of pcDNA.VEI,and the rates of CD4＋/CD8＋ induced by which were all higher than group of pcDNA.VE after 2 weeks to the first immunizing.The results showed that the DNA vaccine enhanced the level of specific humoral and cellular inmmne response elicited in mice,and the safty could be proved.As the promising immune adjuvant gene,IFNγ cloned in this DNA vaccine had the effect of immunologic enhancement.

关 键 词：猪Γ干扰素 乙型脑炎病毒 猪细小病毒 核酸疫苗 免疫原性 增强免疫 

分 类 号：S828[农业科学—畜牧学]