Prokaryotic expression of recombinant mouse histidyl-tRNA synthetase-maltose binding protein fusion gene and identification of its antigen specificity
10.3760/cma.j.issn.1007-7480.2013.03.010
- VernacularTitle:小鼠组氨酰tRNA合成酶与麦芽糖结合蛋白融合基因在大肠杆菌中的表达与鉴定
- Author:
Yinli ZHANG
;
Xiaoming SHU
;
Xin LU
;
Changjun SHAO
;
Mingling GU
;
Guochun WANG
- Publication Type:Journal Article
- Keywords:
Histidine-tRNA ligase;
Recombinant fusion proteins;
HARS-MBP fusion protein;
Prokaryotic expression
- From:
Chinese Journal of Rheumatology
2013;(3):182-185,封3
- CountryChina
- Language:Chinese
-
Abstract:
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.
