【摘要】 将编码柯萨奇Ｂ３ 病毒（ＣＶＢ３） 衣壳蛋白ＶＰ１ 和ＶＰ２ 的基因，分别克隆到具有７．５ｋ 启动子的痘苗病毒表达载体ｐＧＪＰ５ 上；将ＣＶＢ３ 衣壳蛋白全基因克隆到具有Ｔ７ 启动子的痘苗表达载体ｐＴＭ１ 上，并筛选到相应的重组痘苗病毒ＶＶＰ１ 、ＶＶＰ２ 和ＶＶＰ／４／２／３／１ 。ＶＶＰ１ 和ＶＶＰ２ 稳定表达产物为ＣＶＢ３ 衣壳蛋白ＶＰ１ 和ＶＰ２ ，而ＶＶＰ／４／２／３／１ 的产物ＶＰ４／２／３／１ 为一无分泌性的多聚蛋白，且这三种表达产物均属无分泌性蛋白。为了表达成熟的ＣＶＢ３ 衣壳蛋白，将去除５′端非编码区的ＣＶＢ３ 全基因克隆到具有Ｔ７ 启动子的痘苗表达载体ｐＴＭ１ 上，利用重组痘苗病毒ＶＴ７ 所提供的Ｔ７ＲＮＡ 聚合酶，在哺乳动物细胞中进行了暂时表达研究。Ｗｅｓｔｅｒｎ ｂｌｏｔ 分析提示，ＶＰ１、ＶＰ２ 和ＶＰ３ 蛋白得到成熟表达，且发现表达产物具有分泌性。本研究将为今后研究ＣＶＢ免疫性打下基础。更多还原

【Abstract】 The DNA fragments coding CVB3 capsid protein VP1 and VP2 were put under the control of 7.5k promoter in a vaccinia virus plasmid pGJP5, which were used to screen the recombinant vaccinia viruses VVP1 carrying CVB3 VP1 gene and the VVP2 carrying CVB3 VP2 gene through homology recombination respectively. The VP1 or VP2 protein was able to be observed in Vero cells infected with VVP1 or VVP2 by Western blot, though they were not secreted into the media. Then CVB3 capsid protein VP4/2/3/1 gene was inserted into the vaccinia virus expression plasmid pTM1 on the downstream side of the promoter T7, and the recombinant vaccinia virus VVP4/2/3/1 was constructed through homology recombination. The CVB3 VP4/2/3/1 ploypeptide in Vero cells was found on Western blot using the T7 RNA polymerase encoded by vT7. The results suggest that the mature expression of CVB3 capsid proteins has a strict requirement of the nonstructural region downstream. Thus, the entire coding region of CVB3 genome was introduced into a vaccinia virus expression plasmid pTM1 with the 5’ untranslated region totally excluded, under the control of a T7 promoter. The transient expression in mammalian cells was studied using the T7 RNA polymerase encoded by vT7. The CVB3 proteins were able to be detected by ELISA both in the media and in the cells, and the proteins CVB3 VP1, VP2, VP3 in mature forms were all observed on Western blot using antisera against CVB3, CVB3 VP1 protein and CVB3 VP4/2/3 protein respectively. The work will provide basis for future study on the purification and immunological analysis of CVB proteins.更多还原