人自身抗原SSA/Ro60的基因克隆和原核表达

Gene Cloning and Fusion Expression of Human Autoimmune Antigen SSA/Ro60 in E.coli

目的:克隆并表达人自身抗原SSA/Ro60。方法:应用RT-PCR技术,从HL-60细胞株中克隆人自身抗原SSA/Ro60全长基因,将PCR产物直接TA克隆、鉴定及测序,再定向克隆至pGEX-5T载体中,转入大肠杆菌BL-21,阳性克隆鉴定后在IPTG诱导下表达,产物行SDS-PAGE和Western blot。结果:PCR产物约为1.6 kb,与预期1614 bp接近,测序结果与Genebank报道的完全一致。pGEX-5T-SSA/Ro60重组阳性克隆酶切鉴定正确,SDS-PAGE和Western blot结果显示融合蛋白分子量为86 KD,具有天然人自身抗原SSA/Ro60的免疫原性。结论:成功克隆表达人自身抗原SSA/Ro60,为下一步工作奠定了基础。

Objective To clone and express human autoimmune antigen SSA/Ro60 in E. coll. Methods A full length cDNA of human autoimmune antigen SSA/Ro60 was cloned from cell line HL-60 by RT-PCR. Then the PCR product was TA cloned and 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 1.6 kb in size which was in accordance with predicted 1614 bp and seqencing result showed the same with Genebank＇s report. The pGEX-5T- SSA/Ro60 positive clone produce a 86KD fusion protein which had natrural immunogenicity of human autoimmune antigen SSA/Ro60 by SDS-PAGE and Western blot. Conclusion Successfully cloning and expression of human autoimmune antigenSSA/Ro60 laid a foundation for further reserth work.