Tat47-57-HBcAg融合基因表达载体的构建及融合蛋白的表达鉴定

目的：探讨融合蛋白Tat47-57-HBcAg原核表达的可行性,并分析其表达形式,为研究其功能提供理论基础。方法：合成Tat47-57编码序列,PCR技术扩增HBcAg基因,通过重叠延伸PCR片段拼接法,将Tat47-57编码序列和HBcAg基因拼接,将融合基因克隆到pET28原核表达载体中。挑选测序正确的构建质粒转化大肠埃希菌Rosetta-gami^TM2（DE3）,异丙基-β-D-硫代半乳糖苷诱导融合蛋白表达,表达产物超声破壁,十二烷基磺酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）分析融合蛋白的表达形式,Western印迹鉴定。结果：Tat47-57-HBcAg融合蛋白在大肠埃希菌中主要以可溶性形式过度表达,Western印迹分析表明Tat47-57-HBcAg融合蛋白可与HBcAg单克隆抗体反应。结论：融合蛋白Tat47-57-HBcAg以可溶性形式在原核表达系统中高效表达,并能特异性的被HBcAg单克隆抗体所识别。

Construction,expression and identification of fusion protein Tat_(47-57)-HBcAg in E. coli

Objective：To construct recombinant plasmid with fusion gene Tat47-57-HBcAg,express fusion protein Tat47-57-HBcAg in E.coli and identification the Tat47-57-HBcAg by western blot analysis.Methods：To synthesize the sequence of Tat47-57 and amplify HBcAg gene by PCR,splice the two sequences with splicing by overlap extension PCR,link fusion gene into pET28a,the sequences correct vector be transformed into E.coli Rosetta-gami^TM 2（DE3）,then the transformed E.coli is induced by isopropyl β-D-1-Thiogalactopyranoside and the expression product is analyzed by SDS-PAGE,furthermore,identification the expression product by Western blot with HBcAg monoclonal antibody.Results：Fusion protein Tat47-57-HBcAg is highly effective expressed in E.coli Rosetta-gamiTM 2（DE3）.The solubility analysis of expression product indicated that the fusion protein are mostly expressed in supernatant and expression product could react with HBcAg monoclonal antibody by western blot analysis.Conclusions：The Tat47-57-HBcAg fusion protein can be expressed in E.coli Rossetta-gami 2 correctly as soluble protein and be recognized by HBcAg monoclonal antibody.