一种人B淋巴细胞表面抗原的重组表达和纯化  

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

作　　者：常卿 俞敏达 李文奇 CHANG Qing;YU Minda;LI Wenqi(Technology Center for Protein Research,School of Life Sciences,Tsinghua University,Beijing 100084,China;Beijing Advanced Innovation Center for Structural Biology,Beijing 100084,China;Beijing No.4 High School International Campus,Beijing 100031,China)

机构地区：[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　　处：《生物学杂志》2022年第6期20-24,共5页Journal of Biology

基　　金：国家自然科学基金面上项目(81971469)。

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OD_(600)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。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(OD_(600))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.

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 

分 类 号：Q78[生物学—分子生物学]