ABCE1基因siRNA表达质粒的构建及在95-D肺腺癌细胞中的表达

Construction and expression of a plasmid expressing SiRNAs in lung carcinoma cells(95-D)aimed at ABCE1 gene

目的构建针对人ABCE1基因siRNA表达质粒,为进一步研究该基因功能奠定基础。方法合成含靶向ABCE1基因siRNA转录模板的茎环结构,与两端分别有BamHⅠ、HindⅢ酶切位点的RNAi-ReadypSIREN-DNR-DsRed-Express质粒连接,大肠杆菌中扩增,测序鉴定,转染到95-D肺腺癌细胞中,观察其表达。结果转化大肠杆菌涂布平板长出阳性菌落,重组质粒电泳初步说明质粒构建成功,测序结果表明RNAi-ReadypSIREN-DNR-DsRed-Express的酶切位点BamHⅠ、HindⅢ之间有71bp的插入片段,其序列与所设计、合成的ABCE1的siRNA转录模板序列一致,该质粒转染到95-D细胞中表达为红色荧光蛋白。结论 siRNA转录模板完整正确地插入RNAi-Ready pSIREN-DNR-DsRed-Express质粒中。

[Objective] To construct siRNAs expressing plasmid aimed at ABCE1 gene and study the function of ABCE1 with RNA interference technology. [Methods] First, a target sequence in the middle of ABCE1 gene was selected, then according to the sequence, two complementary 71 met oligonucleotides were synthesized with 5 single-stranded over-hangs according to the kit manual, which was ligated with the linearized RNAi-Ready pSIREN-DNR-DsRed-Express. The plasmid was transformed into DHJot bacteria to amplify and then purified. The purified plasmid was identified by gel electrophoresis and sequencing. [Results] The results of gel eleetrophoresis and sequencing showed that the plasmid was identical with the positive con- trol, and the sequence was identical with what we have inserted in, and there was no aberrations such as mutation, deletion, or insertion. [Conclusion] The plasmid which can express siRNAs aimed at ABCE1 gene has been constructed successfully.