人TRPC1基因真核表达载体的构建及肝细胞表达

目的 构建人TRPC1基因真核表达载体,并观察其在人肝细胞株(HL-7702)中的表达.方法 提取细胞总RNA,逆转录-聚合酶链反应(RT-PCR)得到TRPC1基因的编码序列,经纯化回收后克隆入表达载体pGM-T中,酶切及琼脂糖凝胶电泳分析鉴定,最后转染HL-7702细胞,通过电泳和噻唑蓝(MTY)比色法检测基因表达与细胞生长.结果 扩增片段长度为455 bp,重组质粒pGM-T-TRPC1经酶切鉴定条带位置正确,证明表达载体成功构建.电泳结果显示转染重组质粒后的HL-7702细胞表达TRPC1增强.MTF检测发现转染前后肝细胞生长未受到明显影响(t值分别为0.43、-0.40、-2.01、-1.60、-0.41和-0.72,P值均0.05).结论 成功构建人TRPC1基因的真核表达载体pGM-T-TRPC1,并在人肝细胞株HL-7702中稳定表达.

Construction of human TRPC1 eukaryotic expression vector and its expression in HL-7702 cells

Objective To construct eukaryotic expression vector of human transient receptor potential canonical 1 (TRPC1) gene and observe its expression in human hepatocyte cell line (HL-7702).Methods Total RNA was extracted and the coding sequence of TRPC1 gene was amplified by RT-PCR.After purification, the fragment and eukaryotic expression vector pGM-T plasmid were ligated.The recombinant plasmid was verified by enzyme digestion and agarose gel electrophoresis.Expression of TRPC1 gene and growth of transfected HL-7702 cells were evaluated by electrophoresis and MTT method respectively.Results The length of fragment was 455 bp, and the plasmid showed two correct bands by digestion.It was suggested that TRPC1 gene had been cloned into pGM-T vector as expected.Electrophoresis results revealed that with the transfection, there were some changes in the expression of TRPC1.Furthermore, the growth of HL-7702 cells was not obviously affected by the transfection (t values were 0.43, - 0.40, -2.01, -1.60, -0.41 and -0.72 respectively,P 0.05).Conclusion The recombinant eukaryotic expression vector pGM-T-TRPC1 was successfully constructed and stably expressed in HL-7702 cells.