hMSH2反义核糖核酸表达质粒的构建

Construction of hMSH2 antisense RNA expression plasmid1

利用逆转录－聚合酶链反应（ＲＴ－ＰＣＲ），特异地扩增ＨｅＬａ细胞中ｈＭＳＨ２ｃＤＮＡ的最保守区域，并将其克隆到ＴＡ载体，从重组质粒两端进行序列分析，证实为目的片段．将该目的片段反向克隆到哺乳动物表达载体ｐＲＥＰ９的ＢａｍＨⅠ和ＫｐｎⅠ位点之间，筛选后得ｐＲＥＰ９－ｈＭＳＨ２反义表达重组质粒．用磷酸钙－ＤＮＡ共沉淀法将重组质粒ＤＮＡ及载体ＤＮＡ分别导入ＨｅＬａ细胞，经Ｇ４１８筛选，扩大培养．凝胶阻滞实验证实转染有ｐＲＥＰ９－ｈＭＳＨ２重组质粒的ＨｅＬａ－ＭＳＨ２－细胞抽提物中Ｇ·Ｔ和Ａ·Ｃ错配结合蛋白质表达明显降低。

The most conserved region of hMSH2 cDNA in HeLa cells was amplified specifically by reverse transcription polymerase chain reaction (RTPCR). The PCR product was cloned into TA cloning vector. Sequencing from two ends of recombinant plasmid verified that the inserts in the recombinant plasmid was exactly the indicated fragment. The cDNA fragment was cloned into the BamHⅠand KpnⅠ site of mammalian expression vector pREP9 in antisense orientation by digestion and ligation. pREP9 vector DNA and antisense plasmid DNA were transfected into HeLa cells by calcium phosphateDNA coprecipitate respectively, selected by G418. It was found that G·T and A·C mismatch binding protein expression was inhibited significantly in the cell extracts of HeLaMSH2 cell which transfected with antisense hMSH2 expression recombinant plasmid and it provided an effective cell line to further study the roles of this gene.