人IL-17F重组逆转录病毒载体的构建及其在293T细胞中的稳定表达

Construction of Human IL-17F-recombinant Retrovirus Vector and Its Stable Expression in 293T Cells

目的构建hIL-17F重组逆转录病毒载体并研究它在293T细胞中的表达。方法以测序验证的pUCm-T/hIL-17F为模板,采用聚合酶链式反应(PCR)技术扩增目的基因片段(包括20aa信号肽序列,而且hIL-17F基因内EcoR I酶切位点GAA T TC→GAA C TC同义点突变,即AA T→AA C,均为Asn),并正向插入逆转录病毒载体pSIV-1后经PCR、双酶切和测序鉴定。将构建正确的pSIV-1/hIL-17F与辅助病毒载体pHIT456和pHIT60混合后,采用脂质体法共转染293T包装细胞,获得具有感染能力的成熟重组逆转录病毒感染293T细胞,经G418筛选获得阳性细胞株,并应用PCR、RT-PCR和dot-ELISA法检测hIL-17F目的基因在293T细胞中的整合、转录和表达。结果成功构建了hIL-17F基因内EcoRI酶切位点GAA T TC→GAA C TC同义点突变的重组逆转录病毒载体pSIV-1/hIL-17F,并在293T细胞中稳定表达。结论以逆转录病毒为载体感染的稳定表达rhIL-17F的293T转基因细胞株的首次获得,为进一步研究hIL-17F的生物学功能及其机制奠定了良好的基础。

Objective To construct human IL-17F-recombinant stably in 293T cells. Methods The recombinant retrovirus vector retrovlruS vector pSIV-1/hIL-17F and express it （ including the 20aa signal peptide sequence, moreover, hlL-17F gene inside GAATTC→GAACTC synonymous mutation）was constructed by PCR according to pUCm-T/hIL-17F and identified by PCR, double en-onuclease digestion and DNA sequencing. The pSIV-1/hIL-17F together with its two helper virus vectors pHIT456 and pHIT60 was cotransfected into the package cell 293T by lipofectin to produce mature recombinant retrovirus, then which was used to infect 293T cell. The transgenic 293T cell line stably expressing rhIL-17F protein was selected in the presence of G418 and the integration, transcription, expression of hIL-17F target gene in 293T cell were identified by PCR, RT-PCR and dot-ELISA, respectively. Results The recombinant retrovirus vector pSIV-1/hIL-17F and its transgenic 293T cell expressing stably rhIL-17F protein were obtained successfully. Conclusion This is the first time for us to obtain the transgenic 293T cells expressing stably rhIL-17F protein in China, which paves the way for future study on biological functions and mechanism of hIL-17F.