人源M2二联体靶抗原BP-E2融合蛋白的克隆表达与鉴定

cDNA Cloning and Expression of Autoantigen BP-E2

目的克隆表达抗线粒体抗体二亚型(AMA-M2)的二个靶抗原侧链2-氧酸脱氢酶复合体E2(BCOADC-E2)、丙酮酸脱氢酶复合体E2(PDC-E2)及其连接形成的二联体BP-E2融合蛋白,用于人原发性胆汁性肝硬化(PBC)的早期临床诊断。方法根据BCOADC-E2,PDC-E2的cDNA序列设计引物,从正常人的淋巴细胞中提取RNA,通过RT-PCR方法扩增得到相应的基因片段,经测定序列验证后插入表达载体pET28a(+),构建重组表达载体pET28a(+)-BCOADC-E2,pET28a(+)-PDC-E2,pET28a(+)-BP-E2,转化大肠杆菌BL21(DE3)后诱导表达蛋白质。表达蛋白经SDS-PAGE,Western blot等鉴定。结果经核苷酸序列测定和酶切鉴定结果表明,成功地构建了三种重组质粒。IPTG诱导表达后,获得三种融合蛋白。经免疫学鉴定,重组抗原片段具有AMA-M2的免疫原性。结论重组表达的BP-E2融合蛋白将有利于原发性胆汁性肝硬化(PBC)的实验室诊断。

Objective To express BP-E2 recombinant fusion protein in E. coll. in order to be used in early diagnosis of primary biliary cirrhosis （PBC）. Methods The cDNA encoding BCOADC-E2 and PDC-E2 were obtained by RT-PCR,confirmed by DNA sequencing,subcloned unidirectionally into the bacterial expression plasmid pET28a （＋） and then transformed into E. coil. BL21（DE3） to express the recombinant fusion protein induced by IPTG. Results The recombinant fusion protein exhibited the antigenicity of AMA-M2 by western blotting. Conclusion The BP-E2 recombinant fusion protein could be used for diagnosis of PBC.