人类错配修复基因hMLH1和hMSH2错义突变真核表达载体的构建及表达

The Construction and Expression of Eukaryotic Expression Vectors of Missense Variants in the Human Mismatch Repair Genes,hMLH1 and hMSH2

目的构建人类错配修复基因hMLH1和hMSH2错义突变真核表达载体并检测它们在瞬时转染细胞中的蛋白表达。方法重组pcDNA3.1-hMLH1-wt和pcDNA3.1-hMSH2-wt质粒为模板,用定点诱变的方法分别构建hMLH1基因和hMSH2基因错义突变真核表达载体,将诱变前后的重组质粒瞬时转染至HCT-116细胞(hMLH1基因缺陷型)或LoVo细胞(hMSH2基因缺陷型)中,免疫印迹法和免疫荧光法分别检测这两种基因在各自的转染细胞中的蛋白表达。结果测序证实,定点突变成功,构建出hMLH1基因错义突变pcDNA3.1-hMLH1-T117M和pcDNA3.1-hMLH1-V384D真核表达载体以及hMSH2基因错义突变pcDNA3.1-hMSH2-L390F、pcDNA3.1-hMSH2-Q419K和pcDNA3.1-hMSH2-Q629R真核表达载体。结果表明转染后,除hMLH1-T117M突变体蛋白表达缺失外,其余的野生型及突变型真核表达载体在转染细胞中均能表达相应的蛋白。结论成功构建了在人的肿瘤细胞系中能有效表达的两种hMLH1基因错义突变和三种hMSH2基因错义突变的真核表达载体,为进一步研究这些错义突变的病因学作用和功能意义奠定了实验基础。

Objective To create eukaryotic expression vectors carrying either hMLH1 and hMSH2 missense variants and study the protein expression of these mutations in transient transfected cells. Methods Plasmids pcDNA3, 1- hMLH1-wt and pcDNA3.1-hMSH2-wt contained the wild type hMLH1 and hMSH2 respectively. Wild-type plasmids were used as template for site-directed mutagenesis to create the hMLH1 and hMSH2 missense mutation eukaryotic expression vectors. The wild-type plasmids and recombinant plasmids were then transiently transfected into HCT 116 （hMLH1-defective cell lines） and LoVo （hMSH2-defective cell lines） using Fugene 6 and protein expression of these two genes were determined using western blot analysis and immunofluorescence staining. Results Two hMLH1 missense mutation and three hMSH2 missense mutation recombinant plasmids were generated in which the mutation in these recombinant plasmids had been verified by DNA sequencing. Both willd-type and mutant proteins were found expressed in their transfected ceils. Conclusion These recombinant plasmids can be used as the foundation for future experiments in determining the etiological role and functional significance of these missense mutations.