人源胸腺素α原及白介素2融合蛋白基因克隆及原核表达.docVIP

人源胸腺素α原及白介素2融合蛋白基因克隆及原核表达 　【摘要】; 目的 克隆人胸腺素α原/白介素2 融合基因，构建其原核表达载体并在大肠杆菌系统中有效表达。方法 采用RTPCR方法，从人白血病细胞和人T淋巴细胞中分别扩增得到胸腺素α原/白介素2 基因，利用基因重组技术将融合基因片段重组于pET42a原核表达载体上，构建成pET42aPI。经酶切、测序鉴定后，将重组质粒转化大肠杆菌Rosseta，IPTG诱导蛋白表达。表达产物经离子交换和分子筛纯化后进行Western blot分析，通过人外周血单核细胞玫瑰花结实验对融合蛋白进行了初步的活性测定。结果 凝胶电泳显示PCR产物的长度约750 bp，测序结果表明，扩增片段与预期序列一致。重组菌在IPTG诱导下表达相对分子质量为28 kDa的蛋白，纯化后的融合蛋白能提高人外周血单核细胞玫瑰花结形成率，表明具有一定活性。结论 成功克隆和构建了人胸腺素α原/白介素2融合基因的原核表达载体，并在原核表达系统中得到有效表达。 【关键词】; 胸腺素α原；白介素2；融合蛋白；表达 　　Abstract:Objective To clone human prothymosin α and interleukin2 fusion gene into the prokaryotic expression vector and to express the fusion protein effectively in E.coli system.Methods The human prothymosin α and interleukin2 gene were amplified by RTPCR from human leukemia cells and T lymphocytes respectively. The two genes were fused with a linker and the fusion gene fragment was used to construct a prokaryotic expression vector pET42aPI by DNA recombinant technique. After identification by restriction analysis and DNA sequencing, the recombinant plasmid was transformed into E.coli Rosetta. The overexpressed protein induced by IPTG was purified by anion exchange chromatography and Sephacryl S200 chromatography. The biological activity of the fusion protein was assayed using T cell Erosette formation test of human blood. Results The PCR product was about 750 bp in length, which was consistent with the expected size of the fusion gene. Plasmid pET42aPI was transformed into Rosetta and a new protein with a relative molecular weight of 28 kDa was expressed. The purified fusion protein could increase the Erosette formation rate of human peripheral mononuclear cells.Conclusion The encoding sequence of human prothymosin α and interleukin2 fusion gene was successfully cloned into the prokaryotical expression vector pET42a and can be expressed effectively in E.coli system. 　　Key words:prothymosin α; interleukin 2; fusion protein; expression 　　人源胸腺素α原(prothymosinα，proTα)是由109个氨基酸组成的一种小分子酸性蛋白。它具有多种不同的生物学活性