My027融合蛋白表达载体构建及其在大肠杆菌的表达

Construction of recombination vector harboring My027 fusion protein and its expression in E. coli

目的探索My027蛋白的体外表达,通过原核表达获得大量可溶性My027融合蛋白。方法逆转录-聚合酶链反应(RT-PCR)法扩增出My027片段,插入原核表达载体pGEX-4T-2,构建质粒pGEX-My027。转化BL-21菌,IPTG低温诱导表达融合蛋白,亲和层析法获得融合蛋白,采用十二烷基硫酸钠(SDS)-聚丙烯酰胺凝胶(PAGE)、Western印迹法及质谱进行鉴定。结果融合蛋白以水溶形式分泌于大肠杆菌BL-21胞质中,纯化后的目的蛋白经SDS-PAGE、Western印迹法及质谱证实高效表达,氨基酸序列正确。结论人脂肪细胞新蛋白My027在pGEX-4T-2原核表达载体可获高效表达,为进一步研究其功能奠定基础。

Objective To investigate the expression of fusion protein My027 in vitro and to obtain abundant soluble fusion protein My027 by eukaryotic expression.Methods The full length My027 cDNA was amplified by reverse transcriptase polymerase chain reaction (RT-PCR) from the human adipocytes; and the fragment was inserted into vector pGEX-4T-2 to construct recombinant plasmid pGEX-My027. The latter was then used to transform E. coli BL-21 and the fusion protein was induced by IPTG. After purification, the protein M...