共表达γ干扰素的猪细小病毒与猪乙型脑炎病毒核酸疫苗的构建及其免疫原性研究
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摘要
本实验为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连接起来,得到重组质粒pcDNA.VE,再设计—对特异性引物扩增IFN-γ并插入载体pcDNA.VE,构建含有PPV VP2基因、JEV E10基因和IFN-γ基因的重组质粒pcDNA.VEI,并使其共同表达。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,观察细胞绿色荧光。用pcDNA.VEI和不表达IFN-γ的pcDNA.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。观测脾淋巴细胞增殖、通过ELISA观测免疫小鼠的抗体水平,并进行T细胞亚群动态监测。
结果显示,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。在首免后2周时还不明显,但随后抗体水平逐渐增高,第4周时抗体水平显示出明显增高。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。结果表明：共表达γ干扰素的乙型脑炎病毒与猪细小病毒核酸疫苗构建成功,并能产生特异诱导体液免疫和细胞免疫,在小鼠免疫试验中IFNγ基因在核酸疫苗中发挥了免疫增强效果,为乙脑和猪细小病毒核酸疫苗研究提供了实验依据。
In this experiment, 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 the recombinant plasmid pcDNA.VE was obtained. The poIFN-y gene was amplified by a pairs of specific primers and inserted into plasmid pcDNA.VE. The plasmid pcDNA.VEI was constructed and coexpressed. Transfection to the vero cells was mediated by liposome, for its expression was observed by indirect immuno-fluorescence assay. The Balb/c mice were immunized with this DNA vaccine named pcDNA.VEI, pcDNA.VE within no IFN-y 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 stimulation of spleen lymphocytes, the level of two sorts of antibodies introduced by pcDNA.VEI and the number of T Lymphocyte Subsetsweres were measured.
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 IFNy-negative control group, which was not obvious in the first 2 weeks, but the level steadily increased and was much higher at the 4th week. 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 was successfully constructed and 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, IFNy cloned in this DNA vaccine had the effect of immunologic enhancement.
结果显示,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。在首免后2周时还不明显,但随后抗体水平逐渐增高,第4周时抗体水平显示出明显增高。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。结果表明：共表达γ干扰素的乙型脑炎病毒与猪细小病毒核酸疫苗构建成功,并能产生特异诱导体液免疫和细胞免疫,在小鼠免疫试验中IFNγ基因在核酸疫苗中发挥了免疫增强效果,为乙脑和猪细小病毒核酸疫苗研究提供了实验依据。
In this experiment, 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 the recombinant plasmid pcDNA.VE was obtained. The poIFN-y gene was amplified by a pairs of specific primers and inserted into plasmid pcDNA.VE. The plasmid pcDNA.VEI was constructed and coexpressed. Transfection to the vero cells was mediated by liposome, for its expression was observed by indirect immuno-fluorescence assay. The Balb/c mice were immunized with this DNA vaccine named pcDNA.VEI, pcDNA.VE within no IFN-y 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 stimulation of spleen lymphocytes, the level of two sorts of antibodies introduced by pcDNA.VEI and the number of T Lymphocyte Subsetsweres were measured.
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 IFNy-negative control group, which was not obvious in the first 2 weeks, but the level steadily increased and was much higher at the 4th week. 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 was successfully constructed and 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, IFNy cloned in this DNA vaccine had the effect of immunologic enhancement.
引文
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