人胰岛素突变体GIRR的分离纯化及性质研究  

Purification and Characterization of the Mutant of Recombinant Human Insulin GIRR

作　　者：解军[1] 于保锋[1] 陈显久[1] 张悦红[1] 常冰梅[1] 胡晓年[2] 牛勃[1] 程牛亮[1] 

机构地区：[1]山西医科大学生物化学与分子生物学教研室,太原030001 [2]协和医科大学基础医学研究所,北京100730

出　　处：《中国生物工程杂志》2004年第11期47-50,共4页China Biotechnology

基　　金：山西省青年科技基金资助项目 ( 2 0 0 3 10 61;0 2 0 0 110 2 8)

摘　　要：构建人胰岛素原突变体GIRR(A2 1Asn→Gly,B1 7Leu→Ile ,B31Arg ,B32Arg)基因的原核融合表达载体并进行表达和纯化 ,进一步对其性质进行研究。将人胰岛素原突变体基因重组到pTXB1融合表达载体上 ,在E .coliBL2 1 (DE3)中得到高效表达。表达产物经几丁质亲和柱层析分离以及胰蛋白酶和羧肽酶B的酶促转化等步骤 ,得到纯的人胰岛素突变体GIRR ,其纯度经HPLC鉴定达到 95 % ,氨基酸组成和设计相符 ,其受体结合活性是猪胰岛素的 1 0 5 % ,生物活性为 2 6 8IU mg。其血浆半衰期为 2 0分钟 ,比标准猪胰岛素提高 3倍多。结果提示本突变体具有更长的半衰期 ,为长效胰岛素的制备提供了新的思路。The gene of human proinsulin mutant (A21Asn→Gly,B17Leu→Ile,B31Arg,B32Arg,GIRR ) was synthesized and recombinated to the fusion protein expression vector pTXB1 and expressed in E.coli BL21(DE3) with high protein level. The expressed fusion protein was purified by chitin affinity filtration and converted to the mutant of human insulin by trypsin and carboxypeptidase B. The insulin mutant, purified by DEAE-Sephadex A-25, with a purity of 95% determined by HPLC, has an expected amino acid composition. The receptor binding activity of this mutant is 105% of that of standard porcine insulin and its biological activity is 26.8IU/mg. The half-life of the mutant insulin in rabbit plasma is 20 minutes, that is more than three times longer than the half-life of standard porcine insulin. This indicates that the mutant insulin has a longer plasma half-life, it may provide a new way for preparation of long-acting insulin.

关 键 词：人胰岛素 融合表达 栽体 血浆 原核 HPLC 突变体 高效表达 性质研究 受体结合活性 

分 类 号：Q78[生物学—分子生物学] R587[医药卫生—内分泌]