逆转录病毒载体介导的T7RNA聚合酶在猪源细胞PK15及SK6中的稳定表达

Stable Expression of T7 RNA Polymerase Gene Mediated by Retroviral Vector in PK15 and SK6 Cells

本研究旨在利用逆转录病毒载体系统将T7RNA聚合酶基因导入猪源细胞,并对该基因的遗传稳定性进行分析。作者克隆并构建含T7RNA聚合酶基因的逆转录病毒重组载体pBABEpuro/T7。将pBABEpuro/T7与水泡性口炎病毒载体pVSV-G共转染GP2-293细胞,收获重组病毒并用该病毒在Polybrene的介导下分别感染PK15和SK6细胞,用嘌呤霉素筛选阳性细胞克隆。对此阳性细胞进行传代,并对不同代次细胞中的T7RNA聚合酶基因进行扩增,确定其遗传稳定性。用直接荧光法和聚丙烯酰胺凝胶电泳检测细胞中T7RNA聚合酶基因的表达及其活性。结果表明T7RNA聚合酶能够被稳定地整合进靶细胞基因组内,细胞系内的T7RNA聚合酶具有较好的转录活性,其活性传代不减弱。免疫荧光检测发现所表达的T7RNAP蛋白具有良好的反应原性。本研究表明T7RNA聚合酶基因稳定整合进猪源细胞,该基因的稳定整合为建立高效体内病毒拯救系统提供了良好的工具。

The experiment was conducted to insert T7 RNA polymerase（RNAP） gene into cells of swine by retroviral vector system,then to analyze its hereditary stability.The bacteriophage T7 RNAP gene was amplified via PCR from lysogen DE3,and the gene was cloned into pBABEpuro retrovial vector to construct a recombinant plasmid that named as pBABEpuro/T7.Then the pT7BABEpuro and pVSV-G plasmids were cotransfected into GP2-293 packaging cells by Lipfection 2000,some pseudotype viruses were ingathered and transfected into PK15 and SK6 cells under polybrene,respectively.The T7-PK15 and T7-SK6 cells were propagated in DMEM with puromycin respectively.The genome extraction from the cells transfected different times,and the T7 RNAP gene was amplified from the genome by PCR.Expression and activity of T7 RNAP gene in T7-PK15 and T7-SK6 cells were analyzed by immediate fluorescence and SDS-PAGE assay.Results showed that the T7 RNAP gene had been integrated into the chromosome of T7-PK15 and T7-SK6 cells,and expressed stably at high level.After 40 times passage,transcriptional activity of T7-PK15 and T7-SK6 cell lines were not weaked.T7 RNAP gene had been stably integrated into the chromosome of T7-PK15 and T7-SK6 cells,construction of cell lines provided favorable tools for virus rescue in vivo.