【摘要】 目的克隆hIL-22成熟链基因并在大肠杆菌表达体系中进行有效表达。方法采用反转录PCR以及巢式PCR的方法克隆得到hIL-22基因。将hIL-22基因装入PQE3.0载体构建重组载体hIL-22/PQE3.0,继而转化宿主工程菌株M15。经异丙基B-D硫代半乳糖苷诱导表达产生hIL-22/His重组蛋白并纯化。重组蛋白经电泳分析和Westernblot验证。结果成功地克隆和构建了hIL-22/PQE3.0载体,转化的工程菌经过诱导表达18kd的hIL-22成熟链,利用亲和层析得到了纯化的重组蛋白。结论成功获得hIL-22/His重组蛋白,为下一步研究人IL-22在肿瘤细胞中的作用奠定了物质基础。更多还原

【Abstract】 Objective To clone the mature chain of hIL-22 and express the protein in E.coli efficiently.Methods The gene region of human IL-22 was cloned by RT-PCR and Nest PCR.After sequence identification,the hIL-22 gene encoding mature chain was inserted into expression plasmid PQE3.0 and transfected into E.coli M15.By the induction of Isoproplyl β-D-1-thiogalactopyranoside(IPTG),the recombinant hIL-22/His protein was effectively expressed in E.coli M15.The recombinant protein was purified by affinity chromatography.SDS-PAGE and Western blot were used to identified the recombinant hIL-22/His protein.Results The expression vector hIL-22/PQE3.0 was constructed successfully.The objective protein hIL-22 mature chain was expressed by bacterial cells as inclusion body.The molecular mass of the protein was confirmed by SDS-PAGE as an 18kD protein.Conclusion The pruified hIL-22/His recombinant protein can be used to make further study of cancer research.更多还原