复制缺陷型人泡沫病毒载体辅助质粒pΔGP的构建及转染小肠癌细胞的研究

Study on the Transfection of Constrction Replication-DefectiveVirus Vector Helper Plasmid pΔGP to HIC Cell

在人泡沫病毒原病毒全长克隆pHRSV13的基础上,缺失突变gag和pol基因,并且用SV40polyA加尾信号替代人泡沫病毒的3′LTR,构建辅助质粒pΔGP.将复制缺陷型人泡沫病毒载体质粒pGPSNI EGFP和辅助质粒pΔGP分别转染和共转染小肠癌HIC细胞系,荧光显微镜检测发现共转染pGPSNI EGFP和pΔGP的HIC细胞能够强烈表达绿色荧光蛋白,转染有复制缺陷型人泡沫病毒载体质粒pGPSNI EGFP的HIC细胞能够表达少量的绿色荧光蛋白,而转染有辅助载体pΔGP的HIC细胞不表达绿色荧光.结果证明复制缺陷型人泡沫病毒载体的构建成功,表明人泡沫病毒env基因3′端的内部启动子IP具有弱启动子的活性,并且bel基因产生的调控蛋白能够反式激活人泡沫病毒内部启动子IP和5′LTR的启动子.

Based on an infectious molecular clone of HFV(pHSRV1), helper vector pΔGP was successfully constructed by deletion of the gag and pol genes, substitution SV40 polyA signal for the 3′LTR of human foamy virus.Replication-defective vector pGPSNI-EGFP and helper vector pΔGP were cotransfected into HIC cell line. Moreover, replication-defective vector pGPSNI-EGFP and helper vector pΔGP were transfected into HIC cell line as a control, respectively. Using fluorescence microscopy, the cotransfected HIC cell with pGPSNI-EGFP and pΔGP vectors strongly expressed green fluorescence protein(GFP) and the transfected HIC cell with replication-defective vector pGPSNI-EGFP weakly expressed green fluorescence protein, while the transfected HIC cell with helper vector pΔGP did not completely express green fluorescence protein. The result showed not only that replication-defective vector pGPSNI-EGFP and helper vector pΔGP from human foamy virus were successfully constructed, but also the 3′ end sequence of human foamy virus′ env gene(internal promoter, IP) had weak promoter activity and Bel protein promoted by IP transactivated both IP and the 5′ LTR promoter of human foamy virus strongly.