人源M2三联体靶抗原BPO-E2融合蛋白的克隆表达与鉴定
肝硬化,胆汁性；自身抗原；重组融合蛋白质类,,],肝硬化，胆汁性；自身抗原；重组融合蛋白质类,1资料与方法,2结果,3讨论,[参考文献]
[摘要] 目的 用基因工程的方法克隆表达抗线粒体抗体二亚型(AMA-M2)的3个靶抗原侧链二氧酸脱氢酶复合体E2(BCOADC-E2)、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)及其连接形成的三联体BPO-E2融合蛋白，以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BCOADC-E2、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(+)-BCOADC-E2、pET28a(+)-PDC-E2、pET28a(+)-OGDC-E2、pET28a(+)-BPO-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4种重组质粒。IPTG诱导表达后，获得4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 重组表达的融合蛋白将有利于原发性胆汁性肝硬化(PBC)的实验室诊断。[关键词] 肝硬化，胆汁性；自身抗原；重组融合蛋白质类
cDNA cloning and expression of autoantigen BPO-E2
ZHOU Ye,YAO Dingkang,CHEN Yan,et al.
Labortary Diagnostics,Changzheng Hospital,Second Military Medical University,and Clinical Immunology Center of PLA,Shanghai 200003,China
[Abstract] Objective To express BPOE2 recombinant fusion protein in E.coli.in order to be used in early diagnosis of PBC.Methods The cDNA encoding BCOADC-E2,PDC-E2 and OGDC-E2 were obtained by RT-PCR,confirmed by DNA sequencing,subcloned unidirectionally into the bacterial expression plasmid pET28a(+) and then transformed into E.coli.,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 BPO-E2 recombinant fusion protein could be used for diagnosis of PBC. ......
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