重组TGF β1在大肠杆菌中的表达、纯化  

Expression,purification and preliminary identification TGF β1 in E.coli

作　　者：陈阳[1] 刘雪婷[1] 肖刚[1] 周智优[1] 汪炬[1] 

机构地区：[1]暨南大学生命科学技术学院,广东广州510632

出　　处：《沈阳药科大学学报》2011年第6期486-489,共4页Journal of Shenyang Pharmaceutical University

基　　金：广东省科技攻关资助项目(2009B030801237)

摘　　要：目的通过pET 43.1原核表达载体,表达重组人转化生长因子β1(human transform growth fac-tor,rhTGFβ1)。方法将人工整合了肠激酶作用位点的TGFβ1,插入到含有NusA蛋白的融合表达载体pET 43.1中。重组质粒转化大肠杆菌(E.coli)BL21(+)并用IPTG诱导表达。通过镍柱纯化重组蛋白,用Western blot杂交检测重组蛋白的免疫原性,用肠激酶酶切得到TGFβ1单体。结果 SDS-PAGE结果显示,相对分子质量约为70 000的融合蛋白大部分在上清中表达,目的融合蛋白量约占菌体总蛋白量的80%。取超声上清,通过His亲和色谱系统纯化目的融合蛋白,纯度质量分数为95%。肠激酶切割得到TGFβ1单体,经Western blot检测重组蛋白的免疫原性。结论人转化生长因子β1在大肠杆菌中实现了高效表达,获得了具有免疫原性的重组TGFβ1。Objective To was express TGF β1 in prokaryotic vector pET 43.1 for production.Methods TGF β1 gene synthesized with enterokinase（EK）digested sequence,the gene was cloned into the NusA protein fusion expression vector pET 43.1.Then the recombinant vector was introduced into E.coli BL21（＋） for expression which was induced by IPTG.Fusion protein was digested by EK and purified by Ni-affinity chromatography.Results SDS-PAGE result showed that the expression level of recombinant protein was up to about 80%,and most of them were soluble.The purity of target protein was about 95% after Ni-affinity chromatography.TGF β1 was harvested with a recovery of 80% by EK digestion then.The result showed that TGF β1 has strong immunogenicity against TGF β1 antibody.Conclusions Recombinant TGF β1 is produced with high yield in NusA-pET 43.1 system by E.coli BL21（＋）.

关 键 词：转化生长因子Β1 大肠杆菌 表达 纯化 

分 类 号：Q55[生物学—生物化学]