SA11株轮状病毒VP7基因的克隆及表达

Cloning and Expression of SA11 Rotavirus VP7 Gene

采用RT PCR方法 ,从提取的SA1 1株轮状病毒总RNA中扩增出VP7基因片段 ,进行鉴定后 ,将该片段克隆于真核表达载体pEF1 HisC dhfr,构建成重组质粒pEF1 HisC dhfr VP7,然后用质脂体法转染COS 7细胞 ,进行真核系统的表达 .获得了长 981bp的PCR片段 ,序列分析结果与已知VP7序列相同 .表达后经细胞超声破碎 ,Westernblot检测表达产物 ,在相对分子质量 38× 1 0 3 处有表达条带 ,表达蛋白主要存在于上清中 .因此 ,获得了VP7基因 ,并在COS 7细胞中获得了表达 ,表达蛋白质免疫Balb c小鼠 。

VP7 gene fragment was amplified from total RNA of SRV strain(SA11) by RT-PCR method.The fragment was identified and cloned into eukaryotic expression vector pEF1/HisC-dhfr and constructed recombinant plasmid pEF1/HisC-dhfr/VP7.Then liposome method was employed to transfect the recombinant vector into COS-7 cell and then the gene was expressed.The results showed that an about 980?bp length PCR product was obtained and the sequence was the same as known VP7 gene.The expression product was detected by Western blot after ultrasonic crash,and an expression band about 38×10 3 MW was found,and most of the aim protein was in supernatant.Thus,VP7 gene of SA11 rotavirus was obtained and can be expressed in COS-7 cell.The expressed protein was used to immune Balb/c mouse,and the antibody which had the specific binding activity was obtained.