增强型绿色荧光蛋白基因与轮状病毒VP6基因融合表达载体的构建及表达  

Construction of fusion expression vector of EGFP/rotavirus VP6 gene and expression in mammalian cells

作　　者：潘小霞[1] 张顺[1] 袁静[1] 文喻玲[1] 陈元鼎[1] 

机构地区：[1]中国医学科学院北京协和医学院医学生物学研究所,昆明650118

出　　处：《中国医药生物技术》2011年第4期241-245,共5页Chinese Medicinal Biotechnology

基　　金：中国医学科学院医学生物学研究所基金项目(IMB2009YB01)

摘　　要：目的构建增强型绿色荧光蛋白(EGFP)与轮状病毒(RV)VP6基因融合表达载体,研究融合蛋白表达及在细胞中的分布。方法提取A组人RVTB-Chen株VP6基因,用PCR扩增VP6编码cDNA片段。将此基因片段与真核表达载体pEGFP-C1携带的EGFP融合;运用脂质体的方法 ,将获得的重组质粒转染到MA104细胞中,在荧光显微镜下观察蛋白的表达及其在细胞中的分布情况。结果成功构建了融合表达质粒;转染细胞后,融合蛋白可在MA104细胞中表达且主要分布在胞质中。结论 RVVP6可与EGFP融合表达,融合蛋白在细胞质中呈均匀的分布,与RV感染细胞中合成的VP6蛋白的分布有较大的差别。Objective Construction of fusion expression vector of EGFP/rotavirus VP6 gene and expression in mammalian cells. The expression level and distribution of fusion protein was examined by fluorescence microscopy. Methods VP6 gene was cloned by PCR, digested with Sac I/BamH I, then inserted into the corresponding sites of the expression vector pEGFP-C1. The resulting plasmid pEGFP-VP6 was transfected into MA104 cells. The expression level of fusion protein was examined by fluorescence microscopy. Results The fusion expression vector was successfully constructed and expressed in MA104 cells and the fusion protein was distributed mainly in the cytoplasm. Conclusion The fusion protein EGFP-VP6 was successfully expressed in MA104 cells and distributed mainly in the cytoplasm and no viroplasms could be seen, which was differ widely from the VP6 protein synthesized in the rotavirus-infected cells.

关 键 词：胃肠炎 轮状病毒属 基因融合 绿色荧光蛋白质类 生物医学研究 

分 类 号：R373.1[医药卫生—病原生物学]