人β_2-微球蛋白基因克隆及其在大肠杆菌中的高效表达

Cloning of Human β_2-microglobulin Gene and Its High Expression in Escherichia coli

β2 微球蛋白 (β2 m)是主要组织相容性复合体 (MHC)Ⅰ类分子的轻链部分 ,为制备MHCⅠ类分子四聚体的必要成分。根据已报道的序列设计特异引物 ,利用RT PCR方法从人白细胞中克隆了 β2 m基因 ,并构建了成熟β2 m的原核表达载体 ,在大肠杆菌中得到高效表达。表达的β2 m大部分在包涵体中 ,经洗涤、变性和复性 ,并以强阴离子交换柱层析纯化 ,获得SDS PAGE纯的人重组 β2 m ,Western印迹法分析表明该蛋白具有与抗人天然 β2 m抗体反应的特性。此工作为制备MHCⅠ类分子四聚体奠定基础。

Human β 2-microglobulin (β 2m) is the light chain of major histocompatibility complex (MHC) class Ⅰ molecule. High-yield production of this protein is a prerequisite to the preparation of MHC Ⅰ tetramer. The present study aims to obtain recombinant human β 2m expressed in Escherichia coli (E. coli), for the purpose of preparing MHC class Ⅰ tetramers. For cloning of human β 2m gene, a pair of specific primers was designed based on the published sequence of this gene and the cDNA of full coding region for β 2m precursor was obtained by RT-PCR from the total RNA of human leukocytes. The amplified cDNA was subsequently cloned and its sequence was confirmed by DNA sequencing analysis (the sequence has been deposited in GenBank with accession number of AY187687). The prokaryotic expression vector containing a gene encoding mature β 2m was constructed by inserting the DNA fragment, which was generated by PCR reaction with the cloned β 2m gene as template, into an IPTG-inducible expression vector pET-3c plasmid. The first eight codons for N terminal amino acid residues of β 2m were optimized for its expression in E. coli. The complete sequence of β 2m gene in the expression vector was verified by DNA sequencing analysis. High-yield expression of β 2m was achieved in E. coli transformed with the expression vector, and most of the recombinant β 2m existed in the inclusion body after IPTG induction. The inclusion body was washed extensively and β 2m in the inclusion body was solublized with 8 mol/L urea. The β 2m was refolded by dialysis and purified by ion-exchange chromatography (Q-Sepharose). Western blotting assay indicated that the polyclonal antibody against human native β 2m could react specifically with the recombinant protein. The purified protein appeared as a single band on both SDS-PAGE and Western blotting, indicating that it was chemical and antigenic pure. This work establishes a convenient approach for renaturation and purification of large quantity of recombinant β 2m which is identical to the native protein without any tags fused except for a methionine residue at the amino terminus. This provides the basis for the preparation of MHC tetramers.