可溶性HLA-A~*0203-BSP原核表达载体的构建及其在大肠杆菌中的高效表达  

Construction of Prokaryotic Expression Vector for Soluble HLA-A~*0203-BSP and Its High Yield Expression in Escherichia coli

作　　者：贾仟涛[1] 徐丽慧[1] 迟晓云[1] 查庆兵[1] 李丰耀[1] 何贤辉[1] 

机构地区：[1]暨南大学生命科学技术学院组织移植与免疫实验中心

出　　处：《中国生物工程杂志》2006年第9期5-10,共6页China Biotechnology

基　　金：国家自然科学基金资助项目(30230350;30371651;30572199)

摘　　要：目的：构建HLA—A＊0203重链胞外域羧基端融合生物素化酶BirA底物肽（BSP）的融合蛋白CHLA-A＊0203一BSP的原核表达载体，并在大肠杆菌中进行表达。方法：以RT-PCR方法从HLA—A28供者外周血单个核细胞（PBMC）中克隆HLA-A＊0203重链基因的cDNA，并测序鉴定，然后以PCR方法构建HLA-A＊0203一BSP的原核表达载体，在大肠杆菌BL21（DE3）菌株中诱导表达并以免疫印迹鉴定。结果：DNA测序显示，从3名HLA-A2＊供者PBMC中克隆的cDNA中，只有从供者2获得编码HLA-A＊0203重链基因的cDNA。将编码重链胞外域1-276的序列和编码BSP的序列融合，构建HLA-A＊0203-BSP融合蛋白的原核表达载体并经测序验证。该融合蛋白在BL21（ED3）中获得高效表达，约占菌体总蛋白的30％；产物相对分子质量约为34kDa，与理论大小一致。Western印迹分析显示融合蛋白完全存在于包涵体中。结论：成功克隆HLA-A＊0203重链基因的cDNA，构建HLA-A＊0203-BSP融合蛋白的原核表达载体，并在大肠杆菌中获得高效表达，为制备HLA-A＊0203四聚体打下基础。Objective： To clone the cDNA of HLA-A＊ 0203 heavy chain and to construct the prokaryotic expression vector for the ectodomain of HLA-A＊0203 fused with a BirA substrate peptide （BSP） at its carboxyl terminus （ HLA-A ＊ 0203-BSP ） and express the recombinant protein in Escherichia coli. Methods： The cDNA for HLA-A ＊ 0203 heavy chain was cloned by RT-PCR from the PBMC of three HLA-A2 ^＋ donors and confirmed by DNA sequencing. DNA fragment encoding HLA-A ＊ 0203-BSP was amplified by PCR with the sequence-verified cDNA as a template. It was then cloned into pET-3d vector. After confirmed by DNA sequencing once again, the prokaryotic expression vector was transformed into BL21 （ DE3 ） strain. The recombinant protein was expressed in E. coli after IPTG induction. SDS-PAGE and Western blot analyses were employed to detect the expressed fusion protein. Results ： DNA sequence analysis showed that the eDNA encoding the heavy chain of HLA -A ＊0203 was cloned from donor 2, which had been confirmed to be HLA-A2 ^＋ by flow cytometry. The expression vector for the recombinant HLA-A ＊ 0203-BSP fusion protein was then constructed and verified by DNA sequencing. SDS-PAGE analysis revealed that the fusion protein was highly expressed in E. coli and accounted for 30% of total bacterial proteins. Furthermore, Western blotting showed that all the recombinant protein existed in the inclusion bodies. The fusion protein had a,molecular weight of about 34 kDa, which is in accordance with the theoretical value. Conclusion： The eDNA of HLA-A＊ 0203 heavy chain was cloned and the prokaryotic expression vector for the fusion protein of HLA-A＊ 0203-BSP was constructed. The recombinant protein was highly expressed as the form of inclusion bodies in E. coli, which would facilitate the preparation of soluble HLA-A ＊ 0203 tetramer.

关 键 词：人类白细胞抗原 胞外域 融合蛋白 原核表达 包涵体 

分 类 号：Q78[生物学—分子生物学]