[1]梁宇佳,缪殿南,闫国和,等.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(08):789-792.
 Liang Yujia,Miao Diannan,Yan Guohe,et al.Construction, prokaryotic expression and purification of human sTrail fusion protein activated by uPA[J].J Third Mil Med Univ,2011,33(08):789-792.
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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(/HTML )
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《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
33卷
期数:
2011年第08期
页码:
789-792
栏目:
论著
出版日期:
2011-04-30

文章信息/Info

Title:
Construction, prokaryotic expression and purification of human sTrail fusion protein activated by uPA
作者:
梁宇佳缪殿南闫国和戴晓天熊玮
第三军医大学西南医院呼吸内科
Author(s):
Liang Yujia Miao Diannan Yan Guohe Dai Xiaotian Xiong Wei
Department of Respiratory Diseases,Southwest Hospital,Third Military Medical University,Chongqing, 400038,China
关键词:
克隆融合基因融合蛋白抗原性
Keywords:
cloning fusion gene uPA-sTrail antigenicity
分类号:
R394-33;R394.3
文献标志码:
A
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。
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 plasmid pET-32a/his-uPA-sTrail. After transforming Escherichia coli BL21(DE3) with pET-32a/his-uPA-sTrail, fusion protein Trx-his-uPA-sTrail was expressed under induction, and then purified and digested by enterokinase (EK) to obtain target fusion protein sTrail-uPA. The target fusion protein was purified, and its antigenicity was detected through Western blotting.     Results    Plasmid pET-32a/his-uPA-sTrail could express fusion protein Trx-his-uPA-sTrail (38×103). Fusion protein sTrail-uPA (19.5×103), obtained through digesting Trx-his-uPA-sTrail with EK, possessed antigenicity of human sTrail.     Conclusion    Fusion protein sTrail-uPA (19.5×103) with human sTrail antigenicity and uPA recognition site as well as its prokaryotic expression vector are successfully constructed.

参考文献/References:

梁宇佳, 缪殿南, 闫国和, 等. 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):789-792.

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更新日期/Last Update: 2011-04-13