CAJ | 학술논문

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.
목적 재대장간균중표체소서조안선tRNA합성매(HARS)여맥아당결합단백(MBP)융합기인,통과친화층석순화이획득구유항원특이성적중조단백.방법 제취C57BL/6소서기육조직총RNA,반전록위cDNA,이용연건설계일대특이성인물,확증HARS기인상유591대감기서렬.대확증산물화재체질립pMALc-5e분별진행쌍매절,재용T4련접매련접득도중조질립.장기전화대장간균감수태세포,도취단극륭배양병감정질립서렬.이병기-β-D류화필남반유당감(IPTG)유도융합단백표체후친화층석순화,취병희선알응효전영대융합단백진행상대분자질량조감정,단백인적법감정항원특이성.결과 중조HARS-MBP융합단백기인재대장간균포질중고효표체,친화층석순화후적융합단백여예측상대분자질량66 000상부,여항체구유량호적특이결합능력.결론 소서HARS-MBP융합기인능구재대장간균중은정고효지표체,표체적단백구유량호적항원특이성,위후속염성기병적연구제공료기출.
Objective 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.