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

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（HLA-A*0203-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-A2+ 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-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34 kD，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。

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

Objective: To clone the cDNA of HLA-A0203 heavy chain and to construct the prokaryotic expression vector for the ectodomain of HLA-A0203 fused with a BirA substrate peptide (BSP) at its carboxyl terminus ( HLA-A0203-BSP ) and express the recombinant protein in Escherichia coli. Methods: The cDNA for HLA-A0203 heavy chain was cloned by RT-PCR from the PBMC of three HLA-A2~ donors and confirmed by DNA sequencing. DNA fragment encoding HLA-A0203-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 cDNA encoding the heavy chain of HLA -A0203 was cloned from donor 2, which had been confirmed to be HLA-A2~ by flow cytometry. The expression vector for the recombinant HLA-A0203-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 cDNA of HLA-A0203 heavy chain was cloned and the prokaryotic expression vector for the fusion protein of HLA-A0203-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-A0203 tetramer.