pBV220/hIL-4优化表达及其包涵体复性研究  

Optimizing Expression and Inclusion Body Renature Research of pBV220/hIL-4 in E.coli

作　　者：孙卫国[1] 李邦印[1] 张灵霞[1] 李国利[1] 熊志红[1] 程小星[1] 

机构地区：[1]解放军第309医院结核病研究所,北京100091

出　　处：《生物技术通报》2011年第5期205-208,222,共5页Biotechnology Bulletin

基　　金：国家传染病重大专项(2008ZX-10003-012)

摘　　要：旨在对人白细胞介素-4(hIL-4)上、下游序列进行优化设计,提高在原核系统中的表达量,并对表达的包涵体进行复性研究提高复性率。利用B ioSun的RNA二级结构预测模块辅助设计hIL-4起始密码子(AUG)上、下游的序列,使局部二级结构的自由能满足高表达要求;根据pBV220载体和原核系统的特点优化引物序列提高hIL-4原核表达量。优化hIL-4包涵体复性条件和方法,提高复性率和生物活性。结果显示,成功构建了pBV220/hIL-4高效表达载体,原核表达的重组人IL-4占细菌总蛋白的35%以上,明显高于优化前表达量;经过复性研究hIL-4包涵体复性率可达到15%以上,生物活性鉴定发现,纯化的hhIL-4蛋白的比活等同或高于国外同类产品。影响原核表达的条件较多,对表达序列的优化设计可以大大提高蛋白的表达量。针对人IL-4基因序列的优化设计提高了人IL-4基因的表达,复性方法的改良提高了复性率和生物活性。The study was to improve the expression quantity of human interleukin-4 gene in E.coli by optimizing sequence design and increase the renature ratio of inclusion body.Design up and downstream sequence of interleukin-4 start codon（AUG）region by ancillary BioSun soft to make free energy of RNA second structure content the demand of high expression levels;reform primer according to characteristic of pBV220 vector and prokaryon expression system to evaluate expression level;upgrade the condition and procedure of inclusion body renature to improve renature ratio and biological activity.Results showed that after optimizing design.The expressed hIL-4 was up to 35% of total bacterial protein with pBV220/hIL-4 vector expressed in E.coli.The renature ratio of recombinant hIL-4 inclusion body has also exceeded 15% and biological activity of purified recombinant hIL-4 was better than foreign congener when amending renature condition.It is important to optimize design expressed sequence of gene.The data showed that the optimized methods adopted in this study evaluate rhIL-4 expression level evidently and improve its renature ratio and biological activity efficiently.

关 键 词：人白细胞介素-4 优化表达 包涵体 生物活性 

分 类 号：Q78[生物学—分子生物学]