hGH-双精氨酸C肽人胰岛素原基因的克隆表达及纯化研究  

Study on Cloning,Expression and Purification of hGH-double Arg C Peptide Human Proinsulin Gene

作　　者：胡兵[1,2] 黄超[1,2] 李文静[1,2] 邓柳红[2] 肖苏生[2] 张春发[2] 

机构地区：[1]海南大学农学院,海口570228 [2]中国热带农业科学院热带生物技术研究所,海口571101

出　　处：《生物技术通报》2009年第12期96-101,共6页Biotechnology Bulletin

摘　　要：旨在提高基因重组人胰岛素在大肠杆菌中表达的稳定性及表达包涵体蛋白的复性水平。在人胰岛素原N端前融合人生长素N端的一段序列来充当前导肽,同时将C肽设计为两个精氨酸,分10段合成长链寡核苷酸链,利用重叠延伸PCR技术(SOE PCR)扩增得到该基因片段。与表达载体PET-30a连接,转化E.coliBL21(DE3),IPTG诱导表达。表达的融合蛋白采用Ni-NTA亲和层析纯化,纯化后的蛋白经复性、冻干等步骤后用胰蛋白酶,羧肽酶B双酶切再过DEAE Sepharose FastFlow阴离子交换柱,收集洗脱峰。对制备所得的胰岛素用SDS-PAGE,Western blot进行性质鉴定,及皮下注射小鼠测定生物活性。结果显示,目的蛋白在大肠杆菌BL21(DE3)中得到了表达,表达产物以不溶性包涵体形式纯在,约占大肠杆菌总蛋白的30%。经Ni-NTA亲和层析得到的重组蛋白纯度为85%,DEAE Sepharose Fast Flow阴离子交换纯化得到单组分胰岛素。Western Blot显示制备所得的胰岛素具有胰岛素免疫原性,皮下给药注射小鼠活性测定表明具有明显的降血糖活性。获得了一种高效生产基因重组人胰岛素的方法,为研究胰岛素类似物奠定了前期基础同时也为今后探索胰岛素的非注射给药途径提供了原料。It was to enhance the stability of recombinant human insulin expressed in E. coli and improve renaturation efficiency of inclusion body protein of human insulin. The sequence which is similar to the N-terminal of human growth hormone（hGH） was integrated onto human insulin as as precursor peptide. At the same time,the C-peptide is replaced with two arginines,to construct hGH-double Arg C peptide human proinsulin gene. The high efficiency expression vector expressed in E. coli BL21 （ DE3 ） successfully induced by IPTG.. Then the expressed fusion protein was purified by Ni-NTA Affinity chromatography. After renaturation, and freeze-dried procedures, the purified protein was digested by trypsinase and carboxypeptidase B. At last, the digestion products were under DEAE Sepha- rose fast flow purification procedure to get purified human insulin,and biological activity was carried out by injecting mice directly. Re- suits demonstrated that the recombinant protein was expressed in E. coli BL21 （ DE3 ） successfully in the form of insoluble inclusion bodies, accounting for about 30% of total proteins in E. coli. The purity was up to nearly 85% by using Ni-NTA affinity chromatography. Western Blot showed that the recombiant protein possessed insulin antigenicity, and the subcutaneous injection of mice showed the insulin have a significant ability to lower blood glucose. Thus,we achieved a approach to produce recombinant human insulin efficiently, which lay a theoretical foundation for the study of all kinds of insulin analogues as well as provide raw materials for the future exploration of Non-injecting route.

关 键 词：hGH-双精氨酸C肽人胰岛素原 重叠延伸PCR 融合蛋白表达 纯化 活性测定 

分 类 号：Q78[生物学—分子生物学] TQ467.32[化学工程—制药化工]