人胰岛素原基因在大肠杆菌中的可溶性表达及分离纯化研究  

Soluble Expression and Purification of Human Proinsulin in Escherichia coli.

作　　者：沈加斌 徐攀[1] 李鑫雨[1] 张立[1] 郑海燕[1] 雍彬[1] 

机构地区：[1]四川师范大学生命科学学院,四川成都610101

出　　处：《食品与生物技术学报》2016年第7期721-727,共7页Journal of Food Science and Biotechnology

基　　金：国家自然科学基金项目(21472132)

摘　　要：根据NCBI中的人胰岛素原基因序列及大肠杆菌密码子偏爱性设计特异引物,PCR扩增得到人胰岛素原基因,构建该基因的原核表达质粒p ET32-PI,重组质粒转化大肠杆菌BL21(DE3)。对重组菌进行温度和IPTG浓度优化,发现重组菌在30℃和终浓度为0.6 mmol/L的IPTG条件下表达效果最好且没有包涵体,经SDS-PAGE和Westem blot检测,表达蛋白的相对分子质量与理论相对分子质量一致且具有胰岛素的免疫原性,证明胰岛素原基因得到了正确表达。对重组菌进行流加发酵,发酵液离心收集菌体,破菌后上清液过亲和层析柱和离子交换柱分离纯化目的蛋白,透析后的样品用肠激酶和羧肽酶酶切,利用亲和柱去除融合蛋白与His标签,最终分离胰岛素样品,利用免疫酶标法,l m L样品测得活性92μIU,证明实验所得样品具有人胰岛素活性。The human proinsulin gene(PI) was cloned by PCR using specific primers designed according to the PI gene sequcence in NCBI and codon in Escherichia coli. The expression plasmid p ET32-PI was constructed to express fusion protein of PI in E. coli BL21(DE3). The recombinant PI protein in E. coli exhibited the best expression at an optimized condition of 30 ℃ and 0.6 mmol/L Isopropyl β-D-Thiogalactoside(IPTG). The results of SDS-PAGE and Western blot analysis indicated that the PI protein was successfully expressed at the same molecular weight as the known PI protein. After the Fed-batch fermentation and centrifugation,this protein was purified to homogeneity using the Ni-NTA affinity chromatography and ion exchange chromatography,respectively. Proinsulin was converted to mature insulin through the digestion of enterokinase and carboxypeptidase. The digested product was purified by Ni-NTA affinity chromatography. The enzyme activity of recombinant insulin was 92 μl U/m L and the product was proved to possess the activity of human insulin.

关 键 词：人胰岛素原 大肠杆菌 发酵 分离纯化 

分 类 号：Q789[生物学—分子生物学]