人U_6启动子表达载体的构建及在胃癌细胞中的活性鉴定

目的构建人U6(hU6)snRNA启动子驱动的小RNA分子的表达载体,并在人胃低分化腺癌SGC-7901细胞中鉴定其表达外源性小RNA分子的活性。方法以人基因组DNA为模板,用PCR方法扩增hU6snRNA启动子序列,并将其克隆入PUC19载体中,命名为PUC-hU6-extra,经测序证明其序列的正确性;用脂质体法转染SGC-7901细胞;用RT-PCR检测U6启动子的转录活性,并测定培养细胞的生长曲线。结果hU6snRNA基因启动子和紧接转录起始点hU6snRNA的前27个核苷酸被成功地克隆入PUC19质粒载体,重组载体在SGC-7901细胞中能高表达小分子RNA,并且未观察到后者对细胞体外增殖的影响。结论成功构建了以hU6snRNA启动子驱动的、能在人胃低分化腺癌SGC-7901细胞内高效转录小分子RNA的表达质粒PUC-hU6-extra。

Construction of an expression vector directed by human U6 small nuclear RNA promoter and identification of expression in gastric carcinoma cell

Objective To construct an expression vector directed by hU6 snRNA promoter for synthesizing small RNA and to identify its functional activity in the gastric carcinoma cells SGC-7901. Methods Using human genomic DNA as template, U6 snRNA promoter was obtained by PCR method, and then cloned into PUC19 vector to produce the recombinant plasmid PUC-hU6-extra, which was sequenced and then transfected into gastric carcinoma cell SGC-7901 with liposome. The effect of expression directed by U6 promoter was detected by RTPCR method, and the cell proliferation curve analysis was performed by stained dye. Results The hU6 snRNA promoter with the first 27 nucleotides followed were successfully cloned into PUC19 plasmid. The recombinant vector could efficiently transcribe small RNA molecules and exerted no effect on cell proliferation in SGC-7901 cells in vitro. Conclusion We have successfully constructed the recombinant PUC-hU6-extra plasmid vector that can efficiently transcribe small RNA molecules directed by hU6 snRNA promoter in the gastric carcinoma cells SGC- 7901.