人GST-AWP1融合蛋白的原核表达及其抗体制备

Expression of Human GST-AWP1 Fusion Protein in E. coli and Preparation of Polyclonal Antibody Against This Protein

为进一步研究人的一新蛋白———蛋白激酶C相关激酶 1相关蛋白 (AWP1)的结构、功能及与其相互作用的蛋白而进行GST AWP融合蛋白表达载体的构建、原核表达、纯化及其抗体的制备 .采用逆转录PCR(RT PCR)法从人ECV30 4内皮细胞中扩增AWP1cDNA编码区 ,并将其重组于谷胱甘肽硫转移酶 (GST)融合蛋白表达质粒pGEX KG中 .经酶切、序列鉴定分析后 ,用该重组质粒转化大肠杆菌BL2 1,并经异丙基 β D 硫代半乳糖苷 (IPTG)诱导产生GST AWP1融合蛋白 ,继而纯化获得了分子量约 5 6kD的融合蛋白 .将此融合蛋白免疫新西兰兔 ,经ELISA和Western印迹检测获得了效价高、免疫活性强的兔抗人多克隆抗体 .结果表明成功构建了GST AWP1融合蛋白表达载体 ,在大肠杆菌高效表达了GST AWP1融合蛋白 ,并获得高效多抗 。

In order to further study the structure and biological function of a human novel protein that associates with serine/theronine kinase PRK1 (AWP1) and to investigate the proteins interacted with AWP1, GST-AWP1 fusion protein vector was constructed. GST-AWP1 fusion protein was expressed and purified in prokaryotic system, and its polyclonal antibody was prepared. AWP1 cDNA codon domain was amplified from human EC304 cell line by RT-PCR method and recombined into pGEX-KG plasmid expressing glutathione S-transferase (GST) fusion protein. After identified by the restriction enzyme digestion and sequencing, the recombinant clone was transformed into the competent expressive cells of E. coli BL21. GST-AWP1 fusion protein was induced by IPTG and further purified by GST agarose to obtain a fusion protein with molecular weight of 56 kD. Then New Zealand rabbits were injected with purified GST-AWP1 fusion protein to induce immunoreaction, and a highly reactive and specific antiserum was prepared by ELISA and Western blotting. The results showed that GST-AWP1 fusion protein was successfully highly expressed and its polyclonal antibody was prepared.