修复基因hMTH1反义RNA真核表达载体的构建

目的 构建人修复基因hMTH1反义RNA真核表达载体pEGFP-C1-T.方法 提取人胚肺成纤维细胞(HLF)总RNA，反转录-聚合酶链反应(RT-PCR)扩增hMTHI基因cDNA保守序列，经pGEMT载体克隆后双酶切，将cDNA保守序列反向插入绿色荧光蛋白表达载体pEGFP-Cl，构建hMTH1基因反义RNA真核表达载体pEGFP-C1-T，并转染细胞。用Western-blot法检验载体抑制hMTH1蛋白表达的效率。结果 经RT-PCR获得423bp产物，T载体克隆后，经DNA测序，确定该片段为hMTH1基因cDNA，进而构建反义RNA真核表达载体pEGFP-C1-T，测序后确证。该载体转染细胞后，可使hMTH1蛋白水平下降约46％。结论 成功构建hMTH1基因反义RNA真核表达载体pEGFP-C1-T.

Construction of eukaryotic expression vector of hMTH1 gene antisense RNA

Objective To construct pEGFP-C1-T vector,an eukaryotic expression plasmid of hMTH1 gene antisense RNA. Methods The conservative region of hMTH1 gene was amplified by RT-PCR after total RNA being extracted from human embryo lung fibroblast(HLF) and then cloned into pGEM-T vector.After the recombinant plasmid was certified by DNA sequencing,the conservative region of hMTH1 gene was inserted into pEGFP-C1 vector reversedly and pEGFP-C1-T vector was constructed.The efficiency of antisense inhibition was verified by Western blotting after cell transfection. Results 423 bp fragment including conservative region of hMTH1 gene was obtained by RT-PCR.After cloned by pGEM-T vector and certified by DNA sequencing,pEGFP-C1-T vector was successfully constructed by means of recombinant DNA technology.Additionally pEGFP-C1-T vector could efficiently decrease hMTH1 protein level by 46%. Conclusion The efficient expression vector of hMTH1 gene antisense RNA,pEGFP-C1-T has been constructed successfully.