Pro370Leu突变型MYOC基因真核表达质粒的构建及其表达特点

Construction and Expression Property of Eukaryotic Expression Plasmid Inserted by Pro370Leu Mutation of the MYOC Gene

目的:构建Pro370Leu突变型MYOC基因真核表达质粒,并在Hela细胞中表达以研究蛋白的定位和分泌特点。方法:以pGEM-T-MYOC质粒中MYOC为模板,用PCR介导的定点突变技术,得到Pro370Leu突变型MYOC基因(mMYOC),并定向亚克隆到真核表达质粒pDsRed2-N1上,得到pDsRed2-N1-mMYOC重组表达质粒,再用限制性内切酶消化和DNA测序鉴定,最后用脂质体包埋转染法转染Hela细胞,进行荧光显微镜观察,并用WesternBlot分析蛋白分泌情况。结果:经酶切和DNA序列测定,证实重组质粒构建成功,mMYOC基因能在Hela细胞中表达,并且蛋白只定位在细胞质中,经WesternBlot分析,mMYOC蛋白不能分泌到培养液中。结论:成功构建Pro370Leu突变型MYOC基因真核表达质粒pDsRed2-N1-mMYOC,并能有效表达,其表达的蛋白定位在细胞质,并且不能分泌。

Objective： To construct eukaryotic expression plasmid inserted by Pro370Leu mutation of the MYOC gene and express it in Hela cells for studying location and secretory property of mutant protein. Methods： Pro370Leu mutation MYOC gene was obtained by PCR site-directed mutagenesis with pGEM-T-MYOC plasmid as plate, which was subcloned into eukaryotic expression plasmid pDsRed2-N1 to construct recombinant plasmid pDsRed2-N1-mMYOC which was transfected into Hela cells by lipofectin method. The accuracy of pDsRed2-N1-mMYOC was confirmed by restricting enzyme digestion analysis and DNA sequencing, and the expression of mMYOC in Hela cells were observed under fluorescence microscope. The secretory property was evaluated by Western blot analysis. Results： With restricting enzyme digestion analysis and DNA sequencing, recombinant plasmid pDsRed2-N1-mMYOC was constructed correctly and mMYOC gene was expressed in transfected Hela cells. The protein distributed in cell plasma.mMYOC could not be secreted to media confirmed by western Blot analysis. Conclusion： Eukaryotic expression plasmid pDsRed2-N1-mMYOC was constructed and expressed successfully. The mMYOC protein only distributed in cell plasma and failed to secrete.