Prokaryotic expression of recombinant mouse histidyl-tRNA synthetase-maltose binding protein fusion gene and identification of its antigen specificity / 中华风湿病学杂志
Chinese Journal of Rheumatology
;
(12): 182-185,封3, 2013.
Article
in Chinese
| WPRIM
| ID: wpr-598221
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.
Full text:
Available
Index:
WPRIM (Western Pacific)
Type of study:
Diagnostic study
/
Prognostic study
Language:
Chinese
Journal:
Chinese Journal of Rheumatology
Year:
2013
Type:
Article
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