Cloning and expression of histidyl-tRNA synthetase autoanfigen gene and its clinical application / 中华检验医学杂志

ABSTRACT

Objective To clone and construct the recombinant plasmid containing Jo-1 of HepG2 cells,then purify the protein and identify the immunoreactivity of the recombinant protein.and establish the enzyme linked immunosorbent assay(ELSA)to detect Jo-1 autoantigen correlative antibodies in diagnosis of polymyositis/dermatomyositis.Methods The constructed plasmid was transformed into E.coli.DH5α and BL21(DE3).This fusion protein was purified by Ni-NTA chromatography and its immunnoreactivity was identified by SDS-PAGE and Western blot.ELISA with the fusion protein was established to detect the Jo-1 autoantigen correlative antibodies in sernm samples of 75 patient with PM/DM,30 patients with SLE.30 patients with RA,10 patients with SS and 30 normal controls.Results The sequence of Jo-1 autoantigen gene Was the same as the sequence reported on the literatures.SDS-PAGE gel analysis showed the molecular weisat of fusion protein was approximately 55 000 Da. Western blotting analysis showed that the fusion protein had the same immunoreactivity as human Jo-1 autoantigen.The results of ELISA indicated that the positive rate of anti-Jo-1 antibody was 28%.but the antibody was negative in other controls.There was significant difierence of positivity of the autoantibody between PM/DM and disease controls or normal controls (x2=31.84,P＜0.01).Conclusions The plasmid containing Jo-1 is successfully cloned into E.coli.DH5α and BL21 (DE3).EUSA analysis shows its good antigenicity and specificity.