胰高血糖素样多肽-2的重组表达及鉴定  

Expression and identification of recombinant glucagons like peptide 2 prokaryotic

作　　者：陈渝[1] 赵云[1] 

机构地区：[1]第三军医大学西南医院烧伤研究所,重庆400038

出　　处：《西南国防医药》2009年第9期875-878,共4页Medical Journal of National Defending Forces in Southwest China

摘　　要：目的:构建胰高血糖素样多肽-2(GLP-2)原核表达载体,在大肠杆菌BLR(DE3)中表达,为胰GLP-2的作用机制的探究奠定基础。方法:人工合成GLP-2序列,磷酸化后连接到质粒pET31b(+),构建成功的质粒GLP-2/pET31b(+)转化至BLR(DE3)进行诱导表达,优化条件获得最大表达产量;纯化重组蛋白,溴化氰裂解蛋白获得单体蛋白,SDS-PAGE、W estern b lotting鉴定重组蛋白。结果:成功合成GLP-2序列,与Genebank收录序列一致;构建原核表达质粒GLP-2/pET31b(+),测序成功;37℃,OD600 nm为0.8时,加入1 mmol/L的IPTG诱导表达获取较纯的GLP-2重组蛋白产量较高,溴化氰裂解获得了蛋白的单体。结论:成功构建GLP-2原核表达载体,该蛋白具有生物活性。确定了优化表达条件,获得了具有生物活性的GLP-2单体蛋白,为进一步研究奠定基础。Objective： To construct prokaryotic expression vector of glucagons like peptide 2 （ GLP - 2 ）, and to express the recombinant GLP - 2 in E. coil BLR（ DE3 ） in order to establish the base for further study of physiological and pharmaceutical function of GLP - 2. Methods： GLP - 2 DNA sequence was synthesized using chemistry method, and the phosphorylated gene was cloned into prokaryotie expression vector pET31 b （ ＋ ）. Having been successfully constructed, the recombinant plasmid GLP - 2/pET31 b （ ＋ ） was transformed into E. coil BLR （ DE3 ） to induce the protein expression and the conditions were optimized to obtain maximum yield. The recombinant protein was purified and split into the monomer by cyanogen bromide. SDS - PAGE and Western blotting were applied to identify this recombination protein. Results： GLP - 2 DNA sequence was successfully synthesized and proved to be consistent with that recorded in Genebank. The sequencing results showed that prokaryutie expression vector pET31b（ ＋ ） was constructed eorrectly. When the temperature was 37℃ and OD600 ,m value was 0.8 ,the yield of relatively pure recombinant GLP - 2 was higher after 1 romoL/L of IPTG was added. Moreover, it was split into the monomer by cyanogen bromide eventually. Conclusion： GLP - 2/pET31 b（ ＋ ） prokaryotic expression vector with biological activity is successfully constructed. Optimized conditions of the recombinant GLP - 2 expression are determined and high purity monomer of recombinant GLP - 2 is achieved.

关 键 词：胰高血糖素样多肽-2 原核表达 重组蛋白 

分 类 号：R587.1[医药卫生—内分泌]