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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 human melanoma differentiation related gene (mda 7 / IL 2 4) and express in E. coli. Methods: Total RNA was extracted from human peripheral blood lymphocytes stimulated by PHA. Primers were designed according to mda 7 / IL 2 4 gene sequence, and mda 7 / IL 2 4 gene was amplified by PCR. The mda 7 / IL 2 4 gene was inserted into the expression vector pET 2 8a (+) to construct the expression vector pET 2 8a mda 7 / IL 2 4, which was induced by IPTG in E.coli. Expression products were identified by Western blot analysis. Results: The mda 7 / IL 2 4 cDNA was amplified by RT PCR from lymphocytes isolated from human peripheral blood. The sequence analysis was consistent with that reported in the literature. SDS PAGE analysis showed that after induced by 0 .5 mmol / L IPTG at 37 ℃ for 4 h, A large number of mda 7 / IL 2 4 fusion protein was expressed in E. coli with a relative molecular mass of about 28 × 10 3. The fusion protein accounted for about 30% of the total bacterial induced protein. Western blot analysis showed that the expressed product could react with His monoclonal antibody Specific reaction occurred. Conclusion: The full-length cDNA of mda 7 / IL 2 4 was obtained from human peripheral blood lymphocytes and the fusion protein was highly expressed in E.coli.