[B10Glu,B24-Asp-B25]人胰岛素类似物的制备、鉴定与性质研究

Preparation,Identification and Properties of [B10Glu,B24-Asp-B25] Human Insulin Analogues

采用 PCR方法 ,将人胰岛素分子 B链 B1 0位 His突变为 Glu,在 B2 4和 B2 5位之间插入 Asp,构建了[B1 0 Glu,B2 4-Asp-B2 5 ]胰岛素原基因 .利用通用型质粒 p BV2 2 0构建表达载体 ,在大肠杆菌 DH5 α中表达 ,表达蛋白为包含体形式 ,约占菌体总蛋白的 2 0 %～ 3 0 % .经过复性和凝胶过滤得到胰岛素原融合蛋白 .用胰蛋白酶和羧肽酶 B酶切 ,经 DEAE离子交换和 RP-HPLC纯化得胰岛素突变体类似物 .用凝胶过滤法测定了蛋白质分子自身的缔合性质 ,用圆二色谱测定了构象变化 .放射性免疫活性及受体结合活性测定结果表明 ,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人胰岛素的 73 .6%和 1 46% .

Diabetes mellitus is a kind of serious endocrinopathy and insulin is the most effective medicine for the treatment of diabetes. The effective form of insulin when used as medicine is monomer. However,normal insulin usually forms polymers because of its high self-association,which makes it lag in the regulation of glucose level in blood. So it is necessary to develop monomeric insulin analogues. Research on the structure and function of insulin indicates that the interaction between the two insulin molecules of dimer insulin exists mainly between the two \%β\%-sheets of C terminus of B chains. There is a strong hydrophobic interaction between aromatic rings of B24,B25,B26 residues. The hydrophobic interaction is crucial for the formation of insulin dimer. B10 His residue is very important for the formation of hexamer. Based on these points,mutations were introduced to the interaction surface,expecting to get monomeric insulin with a low self-association. A mutant gene was constructed through asymmetric PCR. B10His residue was replaced with Glu and Asp was inserted between B24 and B25 residues. The gene was cloned into pBV220,which is a heat-inducing expression vector. The gene was expressed in \%E.Coli\% DH5\%α\% with a high level. Protein was purified through Sephacyl S-100 after denature and refolding. The proinsulin analogue was digested by trypsin and carboxypeptidase B,the product of which was purified through DEAE Sepharose and RP-HPLC. Protein identities were confirmed by MALDI-TOF mass spectrometry. Purified insulin analogue was obtained. The self-association of the insulin analogue was studied through analytical size exclusive chromatography with Superdex 75 column. The result showed an lower self-association than human insulin obviously. Conformation changes were studied through circular dichoism,results of which also showed the insulin analogue had a low self-association. These results indicated this insulin analogue had a strong monomeric property. B10His was replaced with Glu,and hexamer could not form. The acidic residue Asp was inserted between B24 and B25.It could destroy the hydrophobic interaction between molecules. These two factors could weaken the self-association of insulin greatly. The biological activity of the human insulin analogue was also studied \%in vitro\%. The relative activity of RIA was \{73.7%\}. The relative activity of RBA was 146%. The analogue retained high activities \%in vitro\%. It suggested that \[B10Glu,B24-Asp-B25\] human insulin could be a potential drug for the treatment of diabetes.