摘要目的 在大肠杆菌中表达小鼠组氨酰tRNA合成酶(HARS)与麦芽糖结合蛋白(MBP)融合基因,通过亲和层析纯化以获得具有抗原特异性的重组蛋白.方法 提取C57BL/6小鼠肌肉组织总RNA,反转录为cDNA,利用软件设计一对特异性引物,扩增HARS基因上游591对碱基序列.对扩增产物和载体质粒pMALc-5e分别进行双酶切,再用T4连接酶连接得到重组质粒.将其转化大肠杆菌感受态细胞,挑取单克隆培养并鉴定质粒序列.异丙基-β-D硫化吡喃半乳糖苷(IPTG)诱导融合蛋白表达后亲和层析纯化,聚丙烯酰胺凝胶电泳对融合蛋白进行相对分子质量粗鉴定,蛋白印迹法鉴定抗原特异性.结果 重组HARS-MBP融合蛋白基因在大肠杆菌胞质中高效表达,亲和层析纯化后的融合蛋白与预测相对分子质量66 000相符,与抗体具有良好的特异结合能力.结论 小鼠HARS-MBP融合基因能够在大肠杆菌中稳定高效地表达,表达的蛋白具有良好的抗原特异性,为后续炎性肌病的研究提供了基础.

abstractsObjective To express the recombinant mouse histidyl-tRNA synthetase (HARS) and maltose binding protein (MBP) gene in Escherichia coli and obtain the purified protein which possesses antigen specificity.Methods Total RNA was extracted from the myocytes of C57BL/6 mouse and reversely transcripted to cDNA.The gene of N-terminal origin of 591 base pairs was amplified,then cloned into pMALc-5e vector.The recombinant plasmid was transformed into Rosetta-gami B,then IPTG was used to induce the expression of HARS-MBP.Fusion protein was purified by affinity chromatograph.The molecular weight (MW) of HARS-MBP was roughly determined by SDS-PAGE.The antigen specificity was identified by Western blotting using anti-Jo-1 serum from patients,commercial anti-HARS and anti-MBP antibodies.Results The recombinant HARS-MBP protein gene was efficiently expressed in Escherichia coli,and the MW was consistent with predicted MW of 66 000.The fusion protein was specifically combined with its antibody.Conclusion The HARS-MBP fusion protein could be efficiently and steadily synthesized in Escherichia coli,which shows satisfactory antigen specificity and provides the key requirement for making a deep study of HARS in the pathogenesis of idiopathic inflammatory myopathy(IIM) and animal modeling of IIM.