细胞核抗原Sm B′的基因克隆和原核表达  

GENE CLONING AND FUSION EXPRESSION OF NUCLEAR ANTIGEN SM B'IN E.COLI

作　　者：杨湘越[1] 兰小鹏[1] 廖剑[1] 钟智强[1] 朱忠勇[1] 

机构地区：[1]福州总医院全军检验医学研究所

出　　处：《福州总医院学报》2007年第3期151-152,共2页Journal of Fuzhou General Hospital

摘　　要：目的:为建立抗Sm自身抗体检测方法,自行克隆、表达和鉴定细胞核抗原Sm B′。方法:应用逆转录聚合酶链反应(RT-PCR)技术,从HL-60细胞株中克隆Sm B′全长基因,将PCR产物直接进行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,阳性克隆鉴定后在异丙基-β-D-硫代半乳糖苷(IPTG)诱导下表达,产物行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质印迹法(Western blot)鉴定。结果:PCR产物约为700 bp,与预期657 bp接近,测序结果与GenBard核酸数据库报道完全一致。pGEX-5T-Sm B′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51000,具有与抗Sm B′抗体的反应性。结论:成功克隆表达核抗原Sm B′,为建立抗Sm自身抗体检测方法奠定了基础。Objective： To establish a new assay for detecting autoantibody Sm B＇, cloning and expressing nucleaR antigen Sm B＇ in E. coli. Method： A full length cDNA of Sm B＇ waS cloned from cell line HL - 60 by RT - PCR. 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 700 bp in size which waS in accordance with predicted 657 bp and seqencing result showed the same with GenBank＇ s report. The pGEX - 5T - Sm B＇ positive clone produced a 51 000 fusion protein which had immunoreactivity of anti - Sm B＇ by SDS - PAGE and Western blot. Conclusion： Successfully cloning and expression of nuclear antigen Sm B＇ laid a foundation for further research work.

关 键 词：核抗原 克隆 分子 融合基因 

分 类 号：R454.2[医药卫生—治疗学]