重组人胰岛素类似物蛋白在大肠杆菌中克隆、表达与纯化

目的:设计合成编码Des30、B26 H、B28D和B26 H-B28D基因,用大肠杆菌BL21(DE3)表达上述4种速效人胰岛素原蛋白,为探知B26 H-B28D功能和用植物系统表达速效胰岛素原类似物研究做必要的准备。方法:基于人胰岛素氨基酸和小C肽(TYPGDVPK)序列,按植物(油菜)偏爱密码子设计合成了198 bp编码人速效胰岛素原基因Des30。随后以Des30为模板,用PCR突变技术扩增并创造了基因B26 H、B28D和B26 H-B28D,并构建了4个原核表达载体,转化大肠杆菌BL21(DE3)后,经IPTG诱导、用Ni-NTA亲和柱纯化、复性、胰岛素体外成熟(酶解)获得重组人胰岛素突变体蛋白。结果:4种人速效胰岛素原类似物在宿主菌中均以包涵体形式存在,IPTG诱导时间以8 h为佳。Western blot结果表明,带his-tag的速效人胰岛素原蛋白已成功在宿主菌中表达,用Ni-NTA亲和柱纯化获得了较纯的速效人胰岛素原蛋白。纯化的包涵体蛋白通过低温透析复性,然后用胰蛋白酶和羧肽酶B酶切,Western blot结果显示,释放出的单体胰岛素与阳性对照一样具有免疫活性,RP-HPLC和MALDI-TOF质谱检测表明,酶切产物分子量峰值分别与预测的人胰岛素类似物分子量一致。结论:该研究为B26 H-B28D功能研究和用植物系统表达人类胰岛素的研究奠定基础。

Cloning, Expression, and Purification of Recombinant Human-Insulin Analogue Gene in E.coli

Objective: To explore the functions of B26H-B28D gene and to develop new kinds of and novel production ways for short-acting insulin analogues.Methods: Based on the amino acid sequences of human insulin and mini C peptide(TYPGDVPK),the 198 bp gene Des30 of coded short-acting human proinsulin analogue were designed and synthesized according to codon usage bias of rape(Brassica napus L).Subsequently,genes of the B26H,B28D and B26H-B28D were created by PCR mutational technique,the DNA sequences of the Des30 was used as template,and four prokaryotic expression vectors were constructed.The plasmids of the expression vectors were transformed into BL21(DE3)of the Escherichia coli.Target proteins were successfully induced by isopropyl β-D-1-thiogalactopyranoside(IPTG),isolated and purified by Ni-NTA bind resin,and target proteins were refolded and digested,and then the bioactivity were assayed for four insulin analogues.Results: The tricine-SDS-PAGE assay and expression of target proteins showed that all the four short-acting human proinsulin analogues were expressed at inclusion body form,and the optimized induction time was 8 hours by IPTG.Western blot result implied that the short-acting human proinsulin analogues with his-tag were expressed by E.coli system,and target proteins were successfully isolated and purified by Ni-NTA-his bind resin.MALDI/TOF mass spectrometry showed that the mass of the cleaved proinsulin analogues were identical with the presumed human insulin.Conclusion: This research laid foundation to study functions of the B26H-B28D and to produce short-acting human proinsulin analogues by plant expression systems.