pBV-IL-6重组质粒构建及其在E.coli中的高效表达

CONSTRUCTION OF RECOMBINANT INTERLEUKIN-6 PLASMID pBVIL-6 AND ITS OVEREXPRESSION IN E.coli

目的构建人ＩＬ－６重组质粒（ｒｈＩＬ－６），并使其在Ｅ．ｃｏｌｉ中高效表达。②方法经计算机分析设计，人工合成３条寡核苷酸引物，通过定点诱变并优化起始区，应用ＰＣＲ扩增及重组ＤＮＡ技术，构建重组质粒ｐＢＶ－ＩＬ－６，并转化至Ｅ．ｃｏｌｉ宿主菌ＤＨ５α中诱导表达，对产物进行纯化和复性，ＭＴＴ法检测生物活性。③结果获得２种ｒｈＩＬ－６高效表达的Ｅ．ｃｏｌｉＤＨ５α／ｐＢＶ－ＩＬ－６工程菌，一种表达２０．８ｋｕＩＬ－６全蛋白；另一种表达１６．８ｋｕ，Ｎ端缺失２５个氨基酸ＩＬ－６蛋白。薄层密度扫描分析表达产率，前者为２８．３％，后者为３３．４％，且均具有生物学活性。④结论ｒｈＩＬ－６构建成功。

Objective To construct recombinant interleukin-6 plasmid, which can high-level express rhIL-6 in E. coli.Methods Three sets of primers were designed using computer analysis PCR amplification, sitespecific mutagenesis, optimization of translation initiation and DNA recombinant in vitro were used for constructing the recombinant plasmid pBVIL-6 which was induced to express in E. coli. After isolation and purification, their biological activity was determined by means of MTT. ResultsTwo types of recombinant engineering bacteria, E.coli DH5α/pBVIL-6, were constructed, which overproduced human interleukin6(rhIL-6). One with molecular weight 20.8ku accounted for 28.3% of the E.coli proteins, another one with molecular weight 16.8ku accounted for 33. 4%, they both had biological activity. Conclusion The recombinant human interleukin-6 plasmid pBVIL-6 was constructed successfully, which could express rhIL-6 in E.coli at high level.