应用非变性聚丙烯凝胶电泳筛选siRNA质粒的实验研究

Screening of siRNA expression plasmid by nondenaturing polyacrylamide gel electrophoresis

目的探讨利用非变性聚丙烯凝胶电泳筛选TGF-β1特异性siRNA表达质粒。方法以高表达TGF—β1人肺腺癌细胞株A549为肿瘤细胞模型。应用Angela原则选择siRNA序列，12％非变性聚丙烯凝胶电泳筛选siRNA表达质粒并经测序后转染A549细胞，间接免疫荧光和免疫印迹法检测转染前后A549细胞中TGF-β1表达水平。结果经典分子克隆方法成功构建pOligo1210、pOligo1216、pOligo1363。12％非变性聚丙烯凝胶可以清晰地分辨出被双酶切的插入片段。转染A549细胞后均产生特异性的表达抑制效应，抑制率为50％～83．2％。结论采用非变性聚丙烯凝胶电泳成功筛选出TGF—β1为靶点的siRNA表达质粒，并可广泛应用于筛选siRNA质粒。

Objective To screen TGF-β1 specific siRNA expression plasmid by nondenaturing polyacrylamide gel electrophoresis. Methods Human lung adenocarcinoma A549 as tumor cell model, siRNA sequences were selected by Angela principle and siR NA plasmids were screened by nondenaturing polyacrylamide gel electrophoresis. Then A549 cells were transfected with the re formed plasmids. The expression level of TGF-β1 were detected by indirect immunofluorescence assay（IFA） and Western blotting. Results POligo 1210,pOligo 1216,pOligo 1363 TGF-β1 were constructed successfully. The insert sequence can be distinguished in 12% nondenaturing polyacrylamide gel electrophoresis. SiRNA plasmids were screened by nondenaturing polyacrylamide gel clectrophoresis successfully. TGF-β1 expression can be inhibited with three plasmids ranged from 50% to 83.2 %. Conclusion TGF-β1 specific siRNA plasmids were screened by nondenaturing polyacrylamide gel electrophoresis successfully, It will be helpful to the study of TGF-β1 in tumor cells.