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目的构建HBVDNAPTP1基因的原核表达载体,诱导其在大肠埃希菌中表达,并对融合蛋白进行纯化。方法利用逆转录-PCR获得乙型肝炎病毒(HBV)DNA聚合酶(Polymerase)反式调节人类新基因HBVD-NAPTP1,测序正确后插入至原核表达载体pET-32a(+)中,转化BL21(DE3)宿主菌进行诱导,并利用组氨酸亲和层析方法对融合蛋白进行纯化。结果 HBVDNAPTP1原核表达载体转化宿主菌后,经0.5 mmol/L IPTG、30℃诱导5 h获得了分子量约为31 kD的HBVDNAPTP1融合蛋白的优化表达,Western blotting证实融合蛋白的特异性。亲和层析纯化后得到较纯的HBVDNAPTP1融合蛋白,每升培养菌液中可获得2.24 mg的纯化蛋白。结论成功获得纯化的HBVDNAPTP1融合蛋白,为今后开展HBVDNAPTP1的生物学功能研究奠定了物质基础。
Objective To construct prokaryotic expression vector of HBVDNAPTP1 gene and induce its expression in Escherichia coli. The fusion protein was purified. Methods HBVD-NAPTP1 gene was trans-regulated by polymerase chain reaction (RT-PCR) and inserted into prokaryotic expression vector pET-32a (+). The recombinant plasmid was transformed into BL21 ) Host bacteria were induced, and the use of histidine affinity chromatography purification of the fusion protein. Results HBVDNAPTP1 prokaryotic expression vector was transformed into host strain. After induced by 0.5 mmol / L IPTG for 30 min at 5 ℃, the recombinant protein of HBVDNAPTP1 with molecular weight of about 31 kD was obtained. The specificity of the fusion protein was confirmed by Western blotting. The pure HBVDNAPTP1 fusion protein was purified by affinity chromatography, and 2.24 mg of purified protein was obtained per liter of culture broth. Conclusion The successful purification of HBVDNAPTP1 fusion protein has laid the material foundation for the further study on the biological function of HBVDNAPTP1.