人干燥综合征B抗原的基因克隆及融合表达

目的：建立新的检测方法，克隆并表达人干燥综合征B抗原（SS-B）。方法：应用逆转录PCR（RT-PCR）技术，从HL-60细胞株中克隆SS-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-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，产物行十二烷基硫酸钠-多丙烯酰胺冻胶电泳（SDS-PAGE）和蛋白质印迹法（Western blot）试验。结果：PCR产物约为1200bp，与预期1224bp接近，测序结果与Gen-Bank报道的完全一致。pGEX-ST-SS-B重组阳性克隆酶切鉴定正确，SDS-PAGE和Western blot结果显示融合蛋白相对分子质量为71000，具有天然SS-B的免疫原性。结论：成功克隆表达SS-B，为下一步研究奠定了基础。

Gene cloning and fusion expression of human Sjogren syndrome antigen B in E.coli

Objective: To clone and express human Sjogren syndrome antigen B(SS-B)in E.coli.for establishing a new detecting method. Methods: A full length cDNA of SS-B was cloned from cell line HL-60 by RTPCR.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 blotting. Results: The PCR product was about 1 200 bp in size which was in accordance with predicted 1 224 bp and sequencing result showed the same with GenBank's report.The pGEX-5T-SS-B positive clone produced a molecular weight of 71 000 fusion protein which had natural immunogenicity of SS-B by SDS-PAGE and Western blotting. Conclusion: Successfully cloning and expression of human autoimmune antigen SS-B laid a foundation for further research work.