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GenBank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51 000,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 clone ,express and identify the nuclear antigen Sm B' in E. coli to establish a new assay for detecting autoantibody to Sm B'. Methods A full length cDNA of Sm B' was cloned from cell line HL-60 by RT-PCR. The PCR product was TA clonedand sequenced and inserted into the vector pGEX-ST. The recombinant plasmid was transformed into E. coil BL21. The positive cloneswere identified by restricted enzymes and induced by IPTG. The expression product was analyzed by SDS-PAGE and Western blot. Resuits The PCR product was about 700 bp in size which was in accordance with predicted 657 bp and sequencing result showed consistent with the sequence in GenBank. The pGEX-ST-Sm B' positive clone produced a 51 000 kD of fusion protein which was immunoreacrive with anti-Sin B' confirmed by SDS-PAGE and Western blot. Conclusion The successful cloning and expression of nuclear antigenSm B' laid a foundation for further research work.
出处
《临床检验杂志》
CAS
CSCD
北大核心
2006年第3期166-168,共3页
Chinese Journal of Clinical Laboratory Science
基金
福建省青年人才科技创新基金资助项目(2002J060)。
关键词
核抗原
克隆
分子
融合基因
nuclear antigen
cloning
molecular
gene fusion