[A6-Ala,A11-Ala]-人胰岛素突变体的构建及其生物活性  

Construction and Activity of [A6-Ala,A11-Ala]-Insulin

作　　者：陈新浦[1] 尹东 唐建国[1] 

机构地区：[1]北京大学生命科学学院蛋白质工程及植物基因工程国家重点实验室,北京100871

出　　处：《中国生物化学与分子生物学报》2000年第5期612-615,共4页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金!( 3 95 0 0 0 3 2 );杰出青年科学基金!( 3 95 2 5 0 0 8)资助

摘　　要：利用 PCR技术 ,将胰岛素原基因中 A6和 A1 1的 Cys序列突变成 Ala序列 ,以去除 A链的链内二硫键 .突变基因连接到质粒 p BV2 2 0 ,构建了表达质粒 p JG40 3,并且在大肠肝菌中得到表达 .经过 Sephadex G- 50层析可得到纯化的突变人胰岛素原 ( Mut- HPI- Ala) .纯化产物用胰蛋白酶和羧肽酶处理 ,并经 Resource Q阴离子交换柱层析可进一步获得纯化的突变人胰岛素 ( Mut- HI- Ala) .Native- PAGE分析表明 Mut- HI- Ala的结构松散 .Mut- HPI- Ala的放射免疫活性和胰岛素受体结合活性是非突变人胰岛素原 ( Met- HPI)的 4.6%和 2 .4% .Mut- HI- Ala的放射免疫活性和胰岛素受体结合活性是非突变人胰岛素 ( Met- HI)的 4.3%和 4.6% .实验结果表明 ,胰岛素The mutant proinsulin gene was constructed with the codons for A6 and A11 Cys changed to Ala to modify the intra A chain disulfide bond by the PCR mutagenesis.After expression,processing and purification,(A6,A11 Ala) human proinsulin(Mut HPI Ala)was gained.It showed 4.6% radioimmuno activity(RIA)and 2.4% receptor binding activity(RBA)as compared with Met human proinsulin(Met HPI)which was prepared in the same way.The(A6,A11 Ala) human insulin(Mut HI Ala)was obtained after trypsin and carboxypeptidase B(CPB)cleavage and FPLC Resource Q anion exchanged column seperation.Mut HI kept 4.3% radioimmuno activity and 4.6% receptor binding activity with Met HI as control.The mobility of Mut HI was lower than Met HI and HI on native PAGE.These results indicate that modification of intra A chain disulfide bond may cause change on the conformation which is needed to display its immuno activity and receptor binding activity.

关 键 词：胰岛素突变体 二硫键 生物活性 免疫活性 

分 类 号：Q578[生物学—生物化学] Q78