Preparation,Identification and Activities Research of [B10Glu,B25-Glu-B26] Human Insulin

采用PCR方法,将人胰岛素分子B链B10位His突变为Glu,在B25和B26位之间插入Glu,构建了[B10 Glu,B25-Glu-B26]胰岛素原融合蛋白基因.利用通用型质粒pBV220构建表达载体,在大肠杆菌DH5α中表达,表达蛋白为包含体形式,约占菌体总蛋白的20%～30%.经过复性、凝胶过滤等步骤得到胰岛素原融合蛋白.用胰蛋白酶和羧肽酶B酶切,经过DEAE离子交换和RP-HPLC纯化得到胰岛素突变体类似物,并经过质谱测定鉴定.凝胶过滤法测定了蛋白质分子自身的缔合性质,圆二色谱(CD)测定了构象的变化.并分别测定了放免活性、受体结合活性及小白鼠低血糖惊厥实验.结果表明,突变体分子缔合性明显下降.放免活性和受体结合活性分别约为标准胰岛素的63.5%和114.4%, 整体活力略高于天然胰岛素.[B10Glu, B25-Glu-B26] human proinsulin gene was obtained by asymmetry PCR and cloned into the expression vector pBV220 and expressed in Escherichia coli DH5alpha in the form of inclusion body. The target protein was refolded in vitro and purified. After lysis with trypsin and carboxypeptidase B, an insulin analogue was purified by DEAE ion exchange and RP-HPLC. The product was characterized by MALDI-TOF MS. Circular dichroism spectrum was studied. The results implied the changes of the secondary structure and the association. The association of the analogue was further studied by size exclusive chromatography with a strong monomeric property. The biology activities including RIA, RBA were assayed, exhibiting 63.5% and 114.4% those of native insulin respectively. The result of mouse convulsion test indicated the insulin analogue had in vivo activity nearly the same as native insulin.http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcApp=PARTNER_APP&SrcAuth=LinksAMR&KeyUT=WOS:000188119400008&DestLinkType=FullRecord&DestApp=ALL_WOS&UsrCustomerID=8e1609b174ce4e31116a60747a720701SCI(E)中文核心期刊要目总览(PKU)中国科技核心期刊(ISTIC)中国科学引文数据库(CSCD)1159-643