人tumstatin基因的克隆表达及其鉴定

Cloning of Human tumstatin Gene and Expression of Its Recombinant Protein

目的重组表达人tumstatin,为人tumstatin的肿瘤受体显像奠定基础。方法提取HEK293细胞总RNA,通过RT-PCR扩增人tumstatin基因,导入pMD19-T,测序,然后亚克隆至pET28a,IPTG诱导表达,产物进行SDS-PAGE分析和Western blot鉴定。结果提取的总RNA经电泳,有清晰的28S和18S两条带。RT-PCR扩增产物长度与理论值750bp相一致。测序证实tumstatin基因正确插入载体中。重组人tumstatin在大肠杆菌中高效表达,表达量约占菌体总蛋白量的30%。Western blot证实所表达蛋白为目的蛋白。结论重组人tumstatin蛋白在大肠杆菌中高效表达,为进一步的tumstatin的肿瘤受体显像奠定基础。

Objective To clone human tumstatin gene, express its recombinant protein for further research. Methods Total RNA was extracted from HEK293 cell. The tumstatin gene was amplified by RT-PCR, then cloned into pMD19-T and sequenced. Subsequently, the tumstatin gene was cloned into the pET28a and transformed into E. coli BL21 （DE3） where it was induced to express by IPTG. The products were identified by SDS-PAGE and Western blot. Results RT-PCR product was about 750bp, its sequence was the same as that of tumstatin reported. The expression vector pET28a-tumstatin was constructed successfully, and there was a new protein band about Mr 29 KD on SDS-PAGE. The ratio of the expressed product to total bacterial proteins was 30%. The result of Western blot demonstrated that the expressed product was the tumstatin protein. Conclusion Human tumstatin gene was cloned and its recombinant proteins were expressed successfully in this study.