CVB3VP1/pcDNA3真核表达质粒的构建及免疫效果观察

用ＲＴ ＰＣＲ从ＣＶＢ３ 感染细胞中扩增出ＶＰ１ 片段，重组入真核表达质粒ｐｃＤＮＡ３ 中，转化大肠杆菌Ｃ６００，扩增后的质粒经ＣｓＣｌ 密度梯度离心纯化，体外转染ＣＯＳ ７ 细胞，用ＳＤＳ ＰＡＧＥ和Ｗｅｓｔｅｒｎ ｂｌｏｔｔｉｎｇ 检测表达产物；并经胫骨前肌注射小鼠，每鼠１００μｇ／ 次，隔４ 周加强一次，共３ 次。免疫后不同时间检测抗体、淋巴细胞增殖反应等免疫指标。结果显示重组质粒体外转染真核细胞表达ＶＰ１ 蛋白，免疫小鼠后产生了特异性抗体和淋巴细胞增殖反应。表明ＣＶＢ３ ＶＰ１ ＤＮＡ疫苗获得初步有效结果。

Construction of Eukaryotic Expression Plasmid CVB3VP1/pcDNA3 and Observation of Its Immune Effects on Mice

To observe the immune effects of DNA vaccine,CVB3VP1/pcDNA3,VP1 segment was amplified from CVB3 infected cells with RT PCR and recombinated into eukaryotic expression plasmid,pcDNA3 The COS 7 cells were transfected with the recombinant plasmid CVB3VP1/pcDNA3 in vitro Mice were immunized with this DNA vaccine by injecting into the tibialis anterior muscle,100μg per mice,and boosting twice at 4 weeks interval In the different times after immunization antibody and lymphocyte proliferation were detected The recombinant plasmid CVB3VP1/pcDNA3 expressed VP1 in the transfected eukaryotic cells in vitro The specific antibody and lymphocyte proliferation response could be detected in the immunized mice compared with the control mice This results proved that CVB3VP1 DNA vaccine can induce immune responses