Gene cloning and fusion expression of human autoantigen Jo-1 in E.coli
- VernacularTitle:自身抗原组氨酰转移核糖核酸合成酶的基因克隆和原核表达研究
- Author:
Xiangyue YANG
;
Xiaopeng LAN
;
Jian LIAO
;
Zhiqiang ZHONG
;
Zhongyong ZHU
- Publication Type:Journal Article
- Keywords:
Autoantigens;
Cloning,molecular;
Gene fusion
- From:
Chinese Journal of Laboratory Medicine
2003;0(11):-
- CountryChina
- Language:Chinese
-
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.
