人His-AWP1融合蛋白表达载体的构建及其在原核生物的表达

Construction of His-taged human AWP1 fusion protein vector and its expression in prokaryotic cells

目的 获取人His-AWP1融合蛋白,为下阶段深入研究AWP1的结构、功能及筛取与其相互作用的蛋白打下基础 方法 应用逆转录聚合酶链反府(RT-PCR)法从人ECV304内皮细胞系中克隆AWP1 cDNA,并将其重组于能表达6个组氨酸残基的原核表达质粒DET-14b中。经酶节、序列鉴定,选择正确重组克隆,将其质粒转化大肠杆菌BL21(DE3),IPTG诱导表达,用Ni2+-NTA His柱纯化和SDS-PAGE分离蛋门。结果克隆到一个627 bp的AWP1 cDNA片段.重组质粒目的DNA测序正确.纯化出了一个分子量约为38 kD的融合蛋白。结论用基因工程方法在原核细胞表达并成功纯化出His-AWP融合蛋白。

Objective To obtain the human His-AWPl fusion protein for further studying the structure and biological function of a human novel AWP1 protein and investigating and selecting on the proteins which interact with AWP1. Methods AWP1 cDNA was amplified by RT-PCR from human EC304 cell line and recombined into pET-14b vector expressing six consecutive His residues (6xHis tag). Then accuracy of DNA sequence was identifled by the assay of restrictional enzyme and sequencing, the recombinant clone transformed into the competent expressive cells of E.coli BL21 (DE3), the protein-expression induced by TPTG, the fusion protein purified by Ni2--NTA His agarose, and the protein separated by SDS-PAGE. Results A 627 bp AWP1 cDNA was cloned, the recombinant clone showed correct by DNA sequencing, and a 38 kD His fusion protein was obtained. Conclusion His-AWPl fusion protein can be expressed in prokaryotic cells by way of gene engineering and successfully purified.