| 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 |
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| 引用本文： | 梁宇佳,缪殿南,闫国和,戴晓天,熊玮.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[J].第三军医大学学报,2011,33(8). |
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| 作者姓名： | 梁宇佳 缪殿南 闫国和 戴晓天 熊玮 |
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| 作者单位： | 第三军医大学西南医院呼吸内科,重庆,400038 |
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| 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.
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| 关 键 词： | 克隆 融合基因 融合蛋白 抗原性 |
Construction,prokaryotic expression and purification of human sTrail fusion protein activated by uPA |
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| Liang Yujia,Miao Diannan,Yan Guohe,Dai Xiaotian,Xiong Wei.Construction,prokaryotic expression and purification of human sTrail fusion protein activated by uPA[J].Acta Academiae Medicinae Militaris Tertiae,2011,33(8). |
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| Authors: | Liang Yujia Miao Diannan Yan Guohe Dai Xiaotian Xiong Wei |
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| Institution: | Liang Yujia,Miao Diannan,Yan Guohe,Dai Xiaotian,Xiong Wei(Department of Respiratory Diseases,Southwest Hospital,Third Military Medical University,Chongqing,400038,China) |
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| Abstract: | Objective To construct a prokaryotic expression vector carrying the gene of fusion protein sTrail-uPA that comprises human soluble tumor necrosis factor-related apoptosis inducing ligand(sTrail) and a peptide including urokinase plasminogen activator(uPA) recognition site,and to express and purify the fusion protein.Methods The gene of sTrail was cloned through RT-PCR.Chimeric gene his-uPA-sTrail was constructed through primer extension,and inserted into vector pET-32a to obtain prokaryotic expression plasm... |
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| Keywords: | cloning fusion gene uPA-sTrail antigenicity |
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