Construction of eukaryotic expression vector of hMTH1 gene antisense RNA / 中华劳动卫生职业病杂志

ABSTRACT

<p><b>OBJECTIVE</b>To construct pEGFP-C1-T vector, an eukaryotic expression plasmid of hMTH1 gene antisense RNA.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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%.</p><p><b>CONCLUSION</b>The efficient expression vector of hMTH1 gene antisense RNA, pEGFP-C1-T has been constructed successfully.</p>