人源M2二联体靶抗原BO-E2融合蛋白的克隆表达与鉴定  

CDNA cloning and expression of autoantigen B0-E2

作　　者：周晔[1] 姚定康[1] 陈燕[1] 蒋天舒[1] 陈波[1] 仲人前[1] 邓安梅[1] 

机构地区：[1]上海长征医院实验诊断科,上海市200003

出　　处：《世界华人消化杂志》2007年第22期2465-2467,共3页World Chinese Journal of Digestology

基　　金：国家自然科学基金项目;No.30471616~~

摘　　要：目的:表达二联体BO-E2重组融合蛋白,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:通过逆转录聚合酶链式反应(RT- PCR)方法扩增得到抗线粒体抗体二亚型(AMA-M2)的二个靶抗原侧链:二氧酸脱氢酶复合体E2(BCOADC-E2)、2-氧戊二酸脱氢酶复合体E2(OGDC-E2)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(+)-OGDC-E2,pET28a(+)-BO-E2,转化大肠杆菌BL21(DE3)后诱导表达蛋白质.表达蛋白经SDS-PAGE、westem 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3种重组质粒.异丙基-B-D-硫代半乳糖苷(IPTG)诱导表达后,获得3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BO-E2融合蛋白将有利于PBC的实验室诊断.AIM： To express BO-E2 recombinant fusion protein in Escherichia coli for use in early diagnosis of primary biliary cirrhosis （PBC）. METHODS： CDNA encoding BCOADC-E2 and OGDC-E2 were obtained by reverse transcription pdymerase chain reaction （RT-PCR）, confirmed by DNA sequencing, subcloned nondirectionally into the bacterial expression plasmid pET28a（＋）, and then transformed into E. coli BL21（DE3） to express the recombinant fusion protein, which was identified by SDS-PAGE and western blotting. RESULTS： Results of nucleotide sequencing and restriction analysis showed that the recombinant plasmid successfully constructed three species. Three types of fusion protein were expressed by isopropyl- Beta-d-thiogalactopyranoside （IPTG） inductiorL The recombinant fusion protein exhibited the antigenicity of AMA-M2 by western blotting. CONCLUSION： BO-E2 recombinant fusion protein can be used for in vitro diagnosis of PBC.

关 键 词：原发性胆汁性肝硬化 二氧酸脱氢酶复合体E2 2-氧戊二酸脱氢酶复合体E2 BO-E2融合蛋白 蛋白印迹 

分 类 号：R575.2[医药卫生—消化系统]