中文摘要：

目的构建钠泵α2亚基第4跨膜区与第5跨膜区之间的蛋白结构域M4～M5的原核表达载体,并在大肠杆菌中进行表达和纯化。方法通过PCR反应和酶切技术,构建表达载体pET28b（＋）-M4-5,并于大肠杆菌E.coliRosetta2进行表达,用6mol/L盐酸胍对表达形成的不溶包涵体进行变性,梯度稀释复性后,用Ni-NTA亲和柱进行纯化,得到目的蛋白,用Westernblot分析表达产物,并采用无机磷法测定其ATP酶活性。结果重组表达载体pET28b（＋）-M4-5经酶切与测序鉴定证实构建成功;导入大肠杆菌Rosetta2进行表达,表达产物相对分子质量为52×103左右,与预期值相符,表达量占细胞全蛋白的30%左右;SDS-PAGE分析表明,表达产物主要以包涵体形式存在,亲和柱纯化后,目的蛋白的纯度在95%以上;Westernblot分析表明表达产物与抗His抗体特异性结合;重组蛋白的ATP酶活性为（15.3±1.8）mmol·（g·h）-1。结论构建了原核表达载体pET28b（＋）-M4-5,并在大肠杆菌Rosetta2中成功表达,亲和纯化获得较高纯度的M4～M5融合蛋白。

英文摘要：

Objective To construct the prokaryotic expression vector of M4-M5 between the fourth and fifth transmembrane domains of Na ＋,K ＋-pumpα2 isoform,and express and purify it in E.coli.Methods PCR and enzyme digestion technology were used to construct a recombinant plasmid pET28b（＋）-M4-5,which was transformed into E.coli Rosetta 2.The expression plasmid in soluble inclusion bodies was degenerated with 6 mol/L GuHCl,refolded through gradient dilution in a buffer system.The target protein was purified by Ni2 ＋-NTA affinity chromatography,and verified by Western blotting,and its ATPase activity was detected by inorganic phosphonium colourimetry.Results Restriction endonuclease digestion and recombinant plasmid sequencing demonstrated that the expression vector pET28b（＋）-M4-5 was successfully constructed.After the target protein was induced into E.coli Rosetta 2,its relative molecular weight was about 52 × 103 as expected,accounting for about 30% of the total bacterial protein.SDS-PAGE analysis showed that the protein existed mostly in the form of inclusion bodies.After purification by affinity chromatography,the fusion protein had a purity of above 95%.Western blotting analysis showed that the expressed protein could specifically bind to anti-His antibody with an ATPase activity of 15.3 ± 1.8） mmol ·g-1 ·h-1.Conclusion The prokaryotic expression vector pET28b（＋）-M4-5 can be correctly constructed.The M4-M5 fusion protein can be expressed in E.coli Rosetta 2 and purified by affinity chromatography with a high purity.