上海交通大学学报（医学版）

上海交通大学学报（医学版）

• 论著（基础研究） • 上一篇    下一篇

重组人源抑制型受体TIGIT在大肠埃希菌中的制备及其作用特性研究

陈孝云 1，周爱武 2，叶玮 1   

  1. 上海交通大学 医学院1.附属第九人民医院口腔预防科,上海市口腔医学重点实验室，上海 200011；2.细胞分化与凋亡教育部重点实验室，上海 200025
  • 出版日期:2017-02-28 发布日期:2017-02-28
  • 作者简介:陈孝云（1990—），女，硕士生；电子信箱：63021038@163. com。
  • 基金资助:

    上海交通大学医学院附属第九人民医院院级基金（院2012-04）

Expression, purification and characterization of human inhibitory receptor TIGIT in E.coli

CHEN Xiao-yun1, ZHOU Ai-wu2, YE Wei1   

  1. 1. Department of Preventive and Pediatric Dentistry, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China; 2. Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China

  • Online:2017-02-28 Published:2017-02-28
  • Supported by:

    Foundation of Shanghai Ninth Peoples’s Hospital, Shanghai Jiao Tong University School of Medicine, 2012-04

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摘要：

目的 ·在大肠埃希菌中制备人源 GST-TIGIT融合蛋白并探究其与具核梭杆菌（Fn）互相作用特性。方法 ·对人源免疫调节蛋白TIGIT的基因进行扩增，重组到pGEX4T2表达载体中并转入大肠埃希菌BL21（DE3）中表达融合蛋白；利用GST亲和层析方法纯化 GST-TIGIT融合蛋白；采用特异性黏附试验检测GST-TIGIT融合蛋白与Fn的相互作用。结果 · GST-TIGIT融合蛋白在大肠埃希菌中成功表达并利用GST亲和层析柱得到纯化蛋白；GST-TIGIT融合蛋白可特异性地结合于Fn表面，但与嗜酸乳杆菌结合较弱。结论 ·通过原核表达系统得到有活性的人源GST-TIGIT融合蛋白，并初步证明了其与Fn的黏附作用，为后续研究TIGIT蛋白与Fn特异性相互作用的机制奠定了基础。

关键词: TIGIT, 克隆, 表达, 纯化, 具核梭杆菌, 黏附性

Abstract:

Objective · To prepare recombinant human TIGIT protein in E.coli and characterize its ability in binding Fusobacterium nucleatum (Fn). Methods · The gene of immunomodulatory protein of human TIGIT was amplified and cloned into pGEX4T2, and recombinant plasmid was transformed into E.coli BL21 (DE3) for GST-TIGIT fusion proteins were purified by the GST affinity chromatography and the interaction between GST-TIGIT fusion protein and Fn was tested by a pulldown assay. Results · Recombinant GST-TIGIT fusion protein expressed successfully in E.coli and was purified to homogeneity by GST affinity column. This protein could specifically bind to Fn, but not Lactobacillus acidophilus. Conclusion · High purify and activity of human GST-TIGIT fusion protein can be achieved by the prokaryotic expression system, and the adhesion between this protein and Fn has been preliminarily explored, which provides basis for further characterize interaction between them.

Key words: TIGIT, clone, expression, purification, Fusobacterium nucleatum, adhesion