Gene cloning and fusion expression of human autoantigen Jo-1 in E.coli / 中华检验医学杂志

ABSTRACT

Objective To establish a new assay for detecting autoantibody Jo-1, cloning and expressing human autoantigen Jo-1 in E.coli.Methods A full length cDNA of human autoantigen Jo-1 was cloned from cell line HL-60 by RT-PCR. The PCR product was TA cloned, sequenced and inserted into the carrier pGEX-5T.The recombinant plasmid was transformed into E.coli BL-21. The positive clones were identified by restricted enzymes and induced by IPTG. The expression product was analyzed by SDS-PAGE and Western blot.Results The PCR product was about 1 500 bp in size which was in accordance with predicted 1 526 bp and sequencing result showed the same with GenBank′s report.The pGEX-5T- Jo-1 positive clone produced a 75 000 fusion protein which had natural immunogenicity of human autoantigen Jo-1 by SDS-PAGE and Western blot.Conclusion Successfully cloning and expression of human autoantigen Jo-1 laid a foundation for further research work.