中文摘要：

目的 重组表达抗线粒体抗体二亚型（AMA-M2）的二个靶抗原丙酮酸脱氢酶复合体E2（PDC-E2）、2-氧戊二酸脱氢酶复合体E2（OGDC-E2）及其连接形成的二联体PO-E2融合蛋白,已用于人原发性胆汁性肝硬化（PBC）的早期发现和临床诊断.方法 针对PDC-E2,OGDC-E2的cDNA序列设计引物,从正常人的淋巴细胞中提取RNA,通过反转录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（＋）-PDC-E2,pET28a（＋）-OGDC-E2,pET28a（＋）-P0-E2,转化大肠埃希菌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PO-E2融合蛋白将有利于原发性胆汁性肝硬化的实验室诊断.

英文摘要：

Objective To express PO-E2 recombinant fusion protein in E. coli. in order that it can be used in early diagnosis of primary biliary cirrhosis （PBC）. Methods The cDNA encoding PDC-E2 and OGDC-E2 was obtained by RT-PCR （ reverse transcription 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 induced by IPTG exhibited the antigenicity of AMA-M2 by Western blotting. Conclusion The PO-E2 recombinant fusion protein could be applied to the diagnosis of PBC.