融合蛋白表达载体的构建及在大肠杆菌中的表达

Reconstruction and Expression of Regucalcin Fusion Protein in E. coli

目的构建人类regucalc in重组原核表达载体。方法用RT-PCR的方法从人类肝癌细胞系HepG2中扩增出regucalc in基因cDNA序列,应用T克隆载体及pET-32 a(+)表达载体后,将质粒转化进入原核表达菌株进行表达。结果表达产物经SDS-PAGE电泳分析后,在45 000处有一明显表达条带,与理论结果相符。结论成功构建融合表达载体Pet-regucalc in,并在原核细胞中表达出人类regucalc in-H is融合蛋白。

Objective To construct the prokaryotic expression vector of the human regucalcin gene. Methods To get the human regucalcin cDNA from human live cancer HepG： cells with RT-PCR, and then inserted the fragment of regucalcin into pMD-18 simple T vector. The amplified products were cloned into the prokaryotic expression vector pET-32a（ ＋ ）. Results There was one obvious band at the position of relative molecular weight 45 000 after SDS-PAGE analysis, and it was equivalent to the expected value. Conclusion To construct the recombinant fusion expression vector and to express the fusion protein in E. coli successfully.