趋化因子融合蛋白SDFKDEL慢病毒载体pLenti6/V5SK的构建和表达

Construction and expression of lentiviral vector pLenti6/V5SK on SDFKDEL

摘要:

目的构建含趋化因子融合蛋白SDFKDEL的慢病毒载体pLenti6/V5S K，为研究I型人免疫缺陷病毒（HIV1）感染的基因治疗奠定基础。方法应用聚合酶链反应（PCR）技术扩增目的基因片段SDFKDEL，纯化后的PCR产物定向连接入pLenti6/V5D TOPO载体，构建慢病毒载体pLenti6/V5S K，并在293FT细胞中建立慢病毒株，最后转染人宫颈癌HeLa细胞观察SDF1蛋白的表达。结果成功构建了慢病毒载体pLenti6/V5S K，并证实SDF1蛋白可在HeLa细胞系内表达。结论慢病毒载体可介导CXC细胞内趋化因子（CXCintrakine，SDFK）基因高效、稳定转染HeLa细胞，可用于抗HIV1感染的基因治疗。

Abstract:

ObjectiveTo study the HIVbased lentiviral vector, pLenti6/V5SK, which contains the gene of interest , CXCintrakine (SDFKDEL), on the gene therapy of HIV1 infection. MethodsA pLentibased expression vector, pLenti6/V5DTOPO, was used to produce the lentiviral vector, which was cotransfected with the ViraPowerTM Packaging Mix (pLP1, pLP2, and pLP/VSVG) into 293FT cells to produce a replicationincompetent lentivius stock. After lentiviral stock had been titrated the by  HeLa cells, the expression of the  interest gene of  of SDF1  could be assayed by  indirect immunofluorescence in transduced HeLa cells.ResultsThe lentiviral expression vector, pLenti6/V5SK, was confirmed by restriction enzyme digestion and sequencing. The lentivirus stock was constructed in 293 FT cell line. The fluorescent protein was mainly scattered in cytoplasm and perinucleus in transduced HeLa cells. ConclusionThese findings demonstrated the ability of the lentiviral vector to transduce multiple genes into HeLa cells, and the potential therapeutic effect on the treatment of HIV1 infection.