人胰高血糖素样肽-1突变体基因的构建和表达  

Construction of prokaryotic recombinant with complete human GLP-1 mutant cDNA

作　　者：刘莉[1] 胡红琳[1] 丁振兴[1] 王佑民[1] 

机构地区：[1]安徽医科大学内分泌代谢病研究所,第一附属医院内分泌科,合肥230022

出　　处：《安徽医科大学学报》2006年第6期632-635,共4页Acta Universitatis Medicinalis Anhui

基　　金：安徽省科技厅攻关项目(编号:06023064);安徽医科大学校级基金(2005Kj58);2006年度第一附属医院博士经费资助项目

摘　　要：目的构建人胰高血糖素样肽-1突变体(2Gly-hGLP-1)基因,并表达GST-2Gly-hGLP-1。方法用限制性内切酶双酶切载有hGLPcDNA的重组克隆质粒PMD18Tsimple和原核表达质粒pGEX-4T-1,将GLP-1定向插入原核表达载体pGEX-4T-1+中,筛选出阳性克隆。在获得重组hGLP-1基因工程菌的基础上,利用定向突变的技术改造其第2位丙氨酸为甘氨酸,经酶切克隆于pGEX-4T-1+中,构建pGEX-4T-1+/2Gly-hGLP-1表达载体。转化大肠杆菌BL21并进行诱导表达。结果经测序分析,证明2Gly-hGLP-1已经克隆到pGEX-4T-1+中。Westernblot证实,该蛋白可被特异性hGLP-1识别。结论成功构建和表达了人胰高血糖素样肽-1突变体原核表达质粒。Objective To clone the gene of hGLP- 1 mutant （ ^2Gly - hGLP - 1 ） and express the GST - ^2Gly - hGLP- 1.Methods The recombinant plasmid PMDI8T simple/GLP-I and prokaryotic expression vector pGEX-4T-1 were digested by two restrictive endonucleases. The complete hGLP cDNA were orientaly inserted into pGEX-4T-1^＊ and its prokaryotic recombinant was obtained. Using recombinant hGLP-1 expression system, the 2 nd alanine was changed to be glycine by means of site-directed mutagenesis. The mutant gene was cloned to the plasmid vected pGEX-4T-1^＊ after cleavage with restrictive endonuclease and then inserted into the MCS of the expression vector pGEX-4T-1. The recombinant plasmid was expressed in E. coli. BL21 and induced by IPTG. Results The recombinant hGLP-1 gene mutation was identified by DNA sequencing. Western blot analysis showed that it could be specially recogonized by the antibody of hGLP-1. Conclusion The prokaryotic expressing recombinant with complete GLP-1 mutant cDNA is constructed and expressed.

关 键 词：胰高血糖素/遗传学 基因表达 

分 类 号：R349.83[医药卫生—基础医学]