Mda7/IL-24基因的克隆及其融合蛋白在E.coli中的表达

Cloning of human interleukin 24(mda7/IL-24) gene and its fusion protein expression in E.coli

目的 :克隆人黑素瘤分化相关基因 (melanomadifferentiationassociatedgene ,mda 7/IL 2 4 ) ,并在大肠杆菌中表达。方法 :从PHA刺激培养的人外周血淋巴细胞提取总RNA ,根据mda 7/IL 2 4基因序列设计引物 ,用PCR法扩增mda 7/IL 2 4基因。将mda 7/IL 2 4基因插入表达载体pET 2 8a(+)中 ,构建表达载体pET 2 8a mda 7/IL 2 4 ,用IPTG诱导目的蛋白在E .coli中表达。通过Western印迹分析对表达产物进行鉴定。结果 :经RT PCR从人外周血分离的淋巴细胞中扩增到mda 7/IL 2 4cDNA ,序列分析与文献报道一致 ;SDS PAGE分析表明 ,经 0 .5mmol/LIPTG 37℃诱导 4h后在E .coli中有大量mda 7/IL 2 4融合蛋白表达 ,相对分子质量约为 2 8× 10 3 ,融合蛋白约占诱导菌总蛋白的30 % ;Western印迹分析提示 ,诱导表达产物能与His单抗发生特异性反应。结论 :从人外周血淋巴细胞中获得了mda 7/IL 2 4的全长cDNA ,融合蛋白在E .coli中获得高效表达。

Objective: To clone and express the mda-7/IL-24 gene in E.coli. Methods: The mda-7/IL-24 cDNA was amplified by RT-PCR from human peripheral blood lymphocytes induced by PHA for 70 h at final concentration of 5 μg/ml and cloned into pGEM-T vector. The mda-7/IL-24 cDNA was subcloned into expression vector pET-28a(+) and expressed in E.coli. The fusion protein was identified by Western-blot using anti-His monoclonal antibody. Results: The mda-7/IL-24 cDNA was cloned from human peripheral blood lymphocytes. Sequence analysis revealed identity to the GenBank report. The recombinant His-mda-7/IL-24 could express in E.coli as a fusion protein of 28×10 3. The recombinant protein was mostly expressed in the inclusion bodies (about 30% in total protein) on the induction by 0.5 mmol/L IPTG at 37℃ for 4 hours. Western-blot analysis showed the recombinant protein can be recognized by His monoclonal antibody. Conclusion: The mda-7/IL-24 cDNA is cloned from human peripheral blood lymphocytes successfully. The fusion protein His-mda-7/IL24 can express in E.coli highly.