A链21位Asn缺失胰岛素的分离纯化及性质研究

THE STUDY OF (△N)A21 INSULIN

将A2 1位缺失胰岛素原基因直接克隆到温度诱导型表达载体pBV2 2 0上 ,在E .coli系统中得到高效表达 ,表达量为 8% - 15 %。表达产物经过超声波破碎或高压匀浆 ,包涵体裂解 ,等电点沉淀、二硫键还原及A ,B链重组等后加工过程后 ,得到高纯度的突变胰岛素原。用酶切的方法将其活化为A2 1位缺失胰岛素 ,并进行性质和活性测定 ,证明其免疫活性和受体结合活性均大幅下降。因此认为A2 1Asn在维持胰岛素发挥其生物功能所必须的特定空间结构方面有重要作用 ,A2

In this paper, (△N)A21Proinsulin was directly inserted into the multiple cloning site-EcoRI and BamHI, and the resulting plasmid was used to transform E.coil strain DH 5α (△N) A21Proinsulin have been expressed and the expressing rate is 8%～15%. The target proinsulin was obtained with highly purity by series of processing, which consists of disintegration cell membrane,the fission of inclusion body and other steps. After cheavaged by trypsin and carboxypeptidase B, (△N) A21Proinsulin was transformed into corresponding mutant insulin. (△N) A21insulin was analyzed by Native-PAGE, Radioimmuno Assay (RIA), recptor binging assay (RBA). According to the result, the activity of (△N) A21proinsulin is muchlower than natural insulin. This result implies that A21 is very important for insulin to sustain certain spatial conformation. A21Asn isn't an important amino acid which is bound with insulin receptor, but the absention of A21Asn will the affect total activity of insulin. Therefore, A21Asn can be replaced by other amino acids but it can't be absent.