人自身抗原Sm B′的基因克隆和原核表达

Gene cloning and fusion expression of human autoimmune antigen Sm B’in E.coli

目的克隆并表达人自身抗原Sm B′。方法应用RT-PCR技术,从HL-60细胞株中克隆人自身抗原Sm B′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-ST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分析鉴定。结果PCR产物约为0.7kb,与预期657 bp接近,测序结果与GenBank报道的完全一致。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53KD,具有天然人自身抗原Sm B′的免疫原性。结论成功克隆表达人自身抗原Sm B′。

Objective To clone and express human autoimmune antigen Sm B’in E.coli.Methods A full length cDNA of human autoimmune antigen Sm B’was cloned from cell line HL-60 by RT-PCR and then the PCR product was TA cloned,sequenced wand in- serted into the carrier pGEX-5T.The recombinant plasmid was transformed into E.coli BL-21.The positive clones were identified by re- stricted enzymes and induced by IPTG.The expression product was analyzed by SDS-PAGE and Western blot.Results The PCR prod- uct was about 0.7kb in size which was in accordance with predicted 654bp and seqencing result showed the same with GenBank’s re- port.The pGEX-5T-Sm B’positive clone produced a 53KD fusion protein which had natrurai immunogenicity of human autoimmune an- tigen Sm B’by SDS-PAGE and Western blot,Conclusion Successfully cloning and expressing human autoimmune antigen Sm B’laid a foundation for further research work.