人β2-微球蛋白基因克隆及原核中表达.docVIP

人β2-微球蛋白基因克隆及原核中表达 　 作者：孙万军，徐东刚，杜建芳，邹民吉，王金凤，蔡欣，王嘉玺，艾辉胜 【摘要】 本研究获取主要组织相容性复合体（MHC）Ⅰ类分子的轻链部分，即β2-微球蛋白（β2m）以制备MHCⅠ类分子-肽四聚体。根据已报道的序列设计特异引物，利用逆转录聚合酶链反应（RT-PCR）方法从人白细胞中克隆β2m的基因，并构建β2m的原核表达载体，在大肠杆菌中进行表达。结果显示： 构建的编码成熟β2m的pET-β2m可在大肠杆菌BL21(DE3)中高效表达β2m，表达量占菌体总蛋白的32%，β2m以包涵体的形式表达，经洗涤、变性后，以Sephacryl S-200 HR（S-200）柱层析纯化，纯度可达95%以上，纯化产物采用稀释法复性。Western blot分析表明，该蛋白具有与抗人天然β2m抗体反应的特性。结论：获得了高效表达人β2m的大肠杆菌工程菌株，建立了简便有效的β2m包涵体纯化、复性方法，为制备MHCⅠ类分子-肽四聚体奠定了基础。 【关键词】 β2-微球蛋白 　　Cloning of Human β2-microglobulin Gene and Efficient Expression in Escherichia Coli 　　Abstract Human β2-microglobulin (β2m) 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 class I tetramer. The present study was aimed to obtain recombinant human β2m expressed in Escherichia coli (E.coli) for preparing MHC class I tetramers. For cloning of human β2m gene，a pair of specific primers was designed based on the published sequence of this gene. A 300 bp specific DNA fragment corresponding to the encoding region of β2m lack of the signal peptide sequence was obtained by RT-PCR from the total RNA of human leukocytes. The amplified cDNA was inserted into the IPTG-inducible expression plasmid pET-17b by NdeⅠ and Bam HⅠ sites and its sequence was confirmed by DNA sequence analysis. The recombinant plasmid pET-β2m was transformed to the competent cells of E.coli BL21(DE3). The results showed that β2m was expressed in the form of inclusion body and amounted to over 32% of total cell proteins after IPTG induction. After washing with triton X-100 and urea，the inclusion body was dissolved with 4 mol/L urea and then purified with Sephacryl S-200 HR，and the final purity reached above 95%. The denatured protein was renatured by dilution method. Western blot assay indicated that the monoclonal antibody against human native β2m could react specifically with the recombinant protein. In conclusion，the human β2m gene was cloned succe