Construction, prokaryotic expression and purification of human sTrail fusion protein activated by uPA

Liang Yujia; Miao Diannan; Yan Guohe; Dai Xiaotian; Xiong Wei

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Construction, prokaryotic expression and purification of human sTrail fusion protein activated by uPA

更新时间：2025-01-15

- Construction, prokaryotic expression and purification of human sTrail fusion protein activated by uPA
- Journal of Army Medical University Vol. 33, Issue 8, Pages: 789-792(2011)
- 作者机构：
  
  第三军医大学西南医院呼吸内科
- 作者简介：
- 基金信息：
- DOI：
  CLC： R394.3;R394-33
- Published：2011，
  
  Published Online：30 April 2011，
- 稿件说明：
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LIANG YUJIA, MIAO DIANNAN, YAN GUOHE, et al. Construction, prokaryotic expression and purification of human sTrail fusion protein activated by uPA. [J]. Journal of army medical university, 2011, 33(8): 789-792.
DOI：

LIANG YUJIA, MIAO DIANNAN, YAN GUOHE, et al. Construction, prokaryotic expression and purification of human sTrail fusion protein activated by uPA. [J]. Journal of army medical university, 2011, 33(8): 789-792. DOI：

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38×10

含载体表达标签（Trx）的融合蛋白，EK酶切该蛋白获约19.5 ×10

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19.5×10

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 (38×10

). Fusion protein sTrail-uPA (19.5×10

)

obtained through digesting Trx-his-uPA-sTrail with EK

possessed antigenicity of human sTrail. Conclusion Fusion protein sTrail-uPA (19.5×10

) with human sTrail antigenicity and uPA recognition site as well as its prokaryotic expression vector are successfully constructed.

关键词

克隆融合基因融合蛋白抗原性

Keywords

cloningfusion geneuPA-sTrailantigenicity

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