稳定表达T_7RNA聚合酶的猪睾丸细胞系的建立

根据GenBank中登录的T7RNA聚合酶基因参考序列,设计合成了1对特异性引物扩增对T7RNA聚合酶基因进行扩增,将测序正确的T7RNA聚合酶基因和真核表达载体pIRES2-EGFP双酶切后进行连接构建pIRES2-EGFP-T7RNA RNA质粒。再将构建正确的pIRES2-EGFP-T7RNA质粒经用脂质体法转染猪睾丸细胞,通过G418筛选和单细胞克隆化,同时构建pET-32a-RED原核表达质粒载体,用其检测T7启动子控制下的红色荧光蛋白的表达。结果表明,建立的ST/T7RNA细胞系经20次传代仍然能稳定表达T7RNA聚合酶。结果显示,成功建立能稳定表达T7RNA聚合酶的猪睾丸细胞系,为猪瘟病毒反向遗传操作平台奠定了基础。

Establishment of a ST cell line stably expressing T7 RNA polymerase

Based on the published gene sequence of T7 RNA polymerase(T7 RNAP) in GenBank,a pair of specific primers was designed for amplifying the complete gene of T7 RNAP from E.coli BL21(DE3) cells,the sequencing of the T7 RNA polymerase gene and the eukaryotic expression vector pIRES2-EGFP by Bgl Ⅰ and EcoR Ⅰ restriction enzyme digestions and inserted into the eukaryotic expression vector pIRES2-EGFP to construct the recombinant plasmid pIRES2-EGFP-T7 RNAP.ST cell line stably expressing T7 RNA polymerase was established by using liposome method,G418 selection and cell cloning,at the same time construction of prokaryotic expression vector pET-32a-RED for detection of the red fluorescent protein expression under the control of T7 promoter.The results showed that the ST line of stably expressing T7 RNAP was successfully established.The ST line of stably expressing the T7 RNAP gene could provide a good platform for reverse genetic manipuLation of porcine RNA viruses in vitro.