生物学杂志

生物学杂志 ›› 2022, Vol. 39 ›› Issue (6): 20-.doi: 10.3969/j.issn.2095-1736.2022.06.020

• 研究报告 • 上一篇    下一篇

一种人B淋巴细胞表面抗原的重组表达和纯化

  

  1. 1. 清华大学 生命科学学院 蛋白质研究技术中心， 北京 100084； 2. 清华大学 
    结构生物学高精尖创新中心， 北京 100084; 3. 北京市第四中学国际校区， 北京 100031
  • 出版日期:2022-12-18 发布日期:2022-12-12
  • 作者简介:常卿，博士，高级工程师，研究方向为蛋白质制备与鉴定、生物分子相互作用分析，E-mail: changqing@mail.tsinghua.edu.cn
  • 基金资助:
    国家自然科学基金面上项目（81971469）

Recombinant expression and purification of the extracellular domain of human B lymphocyte surface antigen

  1. 1. Technology Center for Protein Research, School of Life Sciences, Tsinghua University, Beijing 100084, China;
    2. Beijing Advanced Innovation Center for Structural Biology, Beijing 100084, China;
    3. Beijing No.4 High School International Campus, Beijing 100031, China
  • Online:2022-12-18 Published:2022-12-12

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摘要： 人源CD20作为B淋巴细胞表面特异性表达的膜蛋白，可以作为B淋巴细胞瘤免疫治疗的有效靶点。通过分子克隆引物设计，将CD20的胞外区以重复串联的方式克隆到pET28a载体上，两者之间通过Linker连接区（-GSSGGSSG-）连接，构建表达载体pET28a-Bi20。测序正确后转化至大肠杆菌Transetta(DE3)中重组表达，优化表达条件为当菌液OD600为0.6~0.8时，以IPTG终浓度1 mmol/L，37 ℃诱导5 h。表达产物带有C端6个组氨酸的亲和标签，以包涵体形式表达。通过包涵体洗涤条件优化，变性条件下的镍亲和层析纯化，透析复性条件的摸索和优化，最终获得纯度大于95%的可溶CD20胞外区同源二聚体Bi20，建立在大肠杆菌表达系统中规模化量产CD20胞外区多肽的方法。经Western Blot和ELISA检测证实纯化复性后CD20胞外区多肽Bi20具有良好的免疫原性。

关键词: 人源B淋巴细胞表面抗原, 细胞治疗, 重组表达, 连接区, 包涵体变复性

Abstract: Human CD20 is a B lymphocyte surface antigen abnormally high-expressed in B-cell lymphoma, chosen as a cell-therapy target of B-cell malignancies. By molecular cloning, the extracellular domain of human CD20 was successfully constructed into pET28a vector as twins connected with a linker (-GSSGGSSG-）, and with a C-terminal 6His-tag. After confirming by DNA sequencing, the expression vector named pET28a-Bi20 was transformed into E. coli Transetta(DE3). The expression of the recombinant product Bi20 was induced by adding 1 mmol/L IPTG. When the optical density at 600 nm (OD600) of the bacterial suspension reached 0.6-0.8, at 37 ℃ for 5 h. The recombinant Bi20 was expressed as inclusion body. Through washing of inclusion body, purifying by Ni-affinity column under denatured condition, and screening and optimizing the refolding experimental conditions of inclusion body, the target peptide was finally purified as a soluble form of high purity. Western blotting and ELISA demonstrated that the purified Bi20 protein had a desirable immunogenicity.

Key words: human B lymphocyte surface antigen, cell therapy, recombinant expression, linker, inclusion body refolding

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