胰高血糖素样多肽.doc

胰高血糖素样多肽-2的重组表达及鉴定 陈渝 赵云 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、纯化获得高纯度的胰高血糖素样多肽-2并鉴定胰高血糖素样多肽-2的抗原性，为胰高血糖素样多肽-2的作用机制的探究奠定基础。方法 人工合成胰高血糖素样多肽-2序列并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、Western blot 鉴定重组蛋白。结果 成功合成胰高血糖素样多肽-2序列，与Genebank收录序列一致；成功构建原核表达质粒GLP-2/pET31b(+)，优化表达条件，获取较纯的GLP-2重组蛋白，溴化氰裂解获得了蛋白的单体。该蛋白具有生物活性。 Construction of Glucagons like peptide 2 prokaryotic expression vector and expression of Glucagons like peptide 2 gene Abstract: Objective To construct prokaryotic expression vector of GLP-2, express and purify the recombinant GLP-2 in E.coli BLR(DE3), the research establish the base for further study physiological and pharmaceutical function of GLP-2. Methods GLP-2 DNA fragment was synthesis useing chemistry method , and the gene was cloned into prokaryotic expression vector pET31b(+), gained the recombinant expression vector GLP-2/pET31b(+).After identified by DNA sequencing,The recombinant plasmid GLP-2/pET31b(+) was transformed into E.coli BLR(DE3). After IPTG induction, the expressed protein products were verified by SDS - PAGE and Western blotting, purified by Ni2 +- NTA agarose affinity chromatography and desalinization by molecular sieve . the final GLP-2 monomer was gained by clearaged of bromide hydrogen .Result gene sequencing proved that the fragment amplified was inserted into prokaryotic expression vector pET31b(+)correctly. Tricine SDSanalysis and Western blotting showed that the proteins have good antigenicity and specificity. Conclusion Successfully constructed GLP-2/pET31b(+) prokaryotic expression vector, expressed recombinant GLP-2 in E.coli BLR(DE3), and achieved high purity recombinant GLP-2 protein monomer 【关键词】：胰高血糖素样多肽-2；原核表达；肠道保护；烧伤；缺氧；细胞增殖 严重烧伤后造成肠粘膜血流量下降，肠粘膜组织损伤及通透性增加，使肠道屏障功能障碍, 造成肠道细菌移位，引发多器官功能障碍,是导致伤后高代谢的重要原因。因此促进严重烧伤后肠道修复，能减轻脏器炎症反应，防止肠源性感染，改善代谢障碍，加快创面愈合，是临床救治重要措施之一。肠道作为人体最大的内分泌器官，其自身分泌许多肠道激素对肠道结构功能起着重要的保护作用，随着近年来对胰高血糖素样多肽-2(Glucagons like peptide 2,GLP-2)认识的不断深入