含uPA 裂解位点的人sTrail 融合基因的构建及其原核蛋白表达与纯化研究.docVIP

含uPA裂解位点的人sTrail融合基因的构建及其原核蛋白表达与纯化研究 梁宇佳, 缪殿南，闫国和, 戴晓天, 熊 玮 （400038 重庆，第三军医大学西南医院呼吸内科） [摘要] 目的 构建含尿激酶纤溶酶原激活剂（urokinase plasminogen activator，uPA）裂解位点的人可溶性凋亡诱导配体（soluble related apoptosis inducing ligand, sTrail)基因的原核载体，表达并纯化其融合蛋白sTrail-uPA。方法 RT-PCR法克隆sTrail基因后，经引物延伸法构建his-uPA-sTrail融合基因并亚克隆至原核表达载体pET-32a中，诱导其表达蛋白并将其纯化，肠肽酶（enterokinase，EK）切与再次纯化回收该蛋白，Western blot检测其抗原性。结果 表达载体经诱导成功获约38×103含载体表达标签（Trx）的融合蛋白，EK酶切该蛋白获约19.5 ×103的目的蛋白uPA-sTrail。检测表明，该目的蛋白具人Trail抗原性。结论 成功克隆uPA-sTrail融合基因并构建至原核表达载体，并获约19.5×103的融合蛋白，且该蛋白具人Trail抗原性。 [关键词] 克隆；融合基因；目融合蛋白；抗原性 [中图法分类号] [文献标志码] A The investigation on the construction of prokaryotic expression vector of human sTrail gene activited by uPA, and the expresson and purificaton of its recombination protein Liang Yujia, Miao Diannan, Yan Guohe,Dai Xiaotian,Xiong Wei (Department of Respiratory Diseases,Southwest Hospital,Third Military Medical University,Chongqing 400038,China) [Abstract] Objective To constructe the prokaryotic expression vector of human sTrail gene activited by Urokinase plasminogen activator（uPA）, induce the expression of its recombination protein in bacterium coli commune, get the interest protein by cutting the recombination protein with enterokinase（EK）, and identify the immunogenicity of the purified interest protein. Methods Cloning the fusion gene of his-uPA and sTrail by reverse transcriptase polymerase chain reaction (RT-PCR), and constructing its prokaryotic expression vctor pET-32a/his-uPA-sTrail. The vector was induced for getting its recombination protein Trx-his-uPA-sTrail,and the recombination protein was cut by EK in order to get interest protein uPA-sTrail. The uPA-sTrail was reclaimed and purified, and its antigenicity was tested with western blot. Result recombinant plasmid pET-32a/his-uPA-sTrail may expresse the protein Trx-his-uPA-sTrail, whose molecular weight is about 38 kD. Interest protein u-PA-sTrail, which is about 19.5 kD, was got by cutting