DNA错配修复酶hMSH2缺陷细胞株的建立

Construction of hMSH2-deficient cell strain of DNA mismatch repair enzyme

目的 建立并鉴定DNA错配修复酶hMSH2缺陷细胞株 ,用于研究hMSH2基因的作用机制及其缺陷与肿瘤发生的关系。方法 运用基因工程的方法获得hMSH2cDNA片段 ,并反向克隆到绿色荧光蛋白真核表达载体pEGFP-C1上 ,随后将构建好的hMSH2反义RNA绿色荧光蛋白真核表达载体转染入人胚肺成纤维细胞 (HLF) ,使之在HLF中表达 ,用蛋白免疫印迹法鉴定转染细胞中hMSH2基因的表达水平。结果 构建了hMSH2反义RNA绿色荧光蛋白真核表达载体 ,并在真核细胞成功表达 ;hMSH2酶缺陷细胞株hMSH2基因的蛋白表达水平下降了 4 4 %。结论 hMSH2缺陷细胞株的成功建立和鉴定为hMSH2基因功能研究提供了一种有效手段。

Objective To construct hMSH2-deficient cell strain of DNA mismatch repair enzyme which can be used in studying the functions and action mechanisms of hMSH2 gene. Methods The conservative region of hMSH2 gene cDNA was obtained by molecular biology technique.The eukaryotic expression plasmids of hMSH2 gene antisense RNA were constructed through inserting the conservative region fragments into pEGFP-C1 vectors reversedly.HLFs were transfected with the eukaryotic expression plasmids of hMSH2 gene antisense RNA to construct hMSH2-deficient cells(named as 'HLFS').The protein expression levels of hMSH2 gene in HLF and HLFS were detected by the western blotting to estimate the effects of antisense inhibition. Results DNA sequencing results was accorded with genbank at the rate of 99.5%,suggesting that the fragment was a part of hMSH2 cDNA.The construction of DNA mismatch repair enzyme hMSH2-deficient cells train was successful and the protein expression level of hMSH2 gene in HLFS was decreased 44% as compared with that in HLF. Conclusion The successes in constructing a hMSH2-deficient strain offered an effective cell strain for studying the function of hMSH2 gene.