M2自身抗原及其三联体的克隆表达和初步鉴定

Cloning, expression and identification of M2 autoantigen and its trimer

目的 克隆人原发性胆汁性肝硬化 (PBC)特异性抗体M2的三个靶抗原 :支链 2 氧酸脱氢酶 (BCOADC)、丙酮酸脱氢酶 (PDC)及 2 氧戊二酸脱氢酶 (OGDC)的部分基因片段。表达BCOADC、PDC、OGDC的抗原多肽片段 ,并将其连接形成三联体 (BPO) ,用于PBC的早期发现和临床诊断。方法 针对三个靶抗原表位设计引物 ,通过反转录PCR方法 ,从正常人的淋巴细胞中扩增相应的基因片段 ,克隆入表达载体pQE 30 ,构建重组表达载体 pQE 30 /BCOADC、pQE 30 /PDC、pQE 30 /OGDC、pQE 30 /BPO ,测定序列 ,转化大肠杆菌M 15 (prep4)后诱导表达。表达蛋白经SDS PAGE、Western blot鉴定。结果 酶切鉴定和核苷酸序列测定结果表明 ,成功地构建了四种重组工程菌诱导表达后 ,经SDS PAGE、Western blot等证实四种抗原多肽片段获得表达 ,能与PBC病人起特异性反应。结论 获得PBC的特异性靶抗原多肽片段 ,并成功构建三联体 。

Objective To express the immunodominant epitopes of the branched chain 2 oxo acid dehydrogenase complex (BCOADC), the pyruvate dehydrogenase complex (PDC), the 2 oxo glutarate dehydrogenase complex (OGDC) and a triple hybrid clone (designated as BPO), and use BPO as a tool for the detection of M2 specific for primary biliary cirrhosis (PBC). Methods The cDNA fragments encoding the M2 reactive epitopes of BCOADC, PDC and OGDC were amplified using PCR with total RNA extracted from human peripheral mononuclear blood cell. The fragments were cloned into pQE 30 and then transformed into plasmid E.coli M15. Its products were induced by isopropylthio β D galactoside and confirmed with SDS PAGE and Western blot. Results Four specific proteins induced from the transformants containing the fused plasmid were shown to have antigenic reactivity with PBC sera, but not with normal control sera. Conclusions We succeeded in expressing three immunodominant epitopes and a hybrid clone which can be used as a powerful and specific method for the diagnosis of PBC.