| Primary biliary cirrhosis(PBC) is a chronic progressive cholestatic liver disease with autoimmune basis. Patients are typically females aged 35-65 years.According to some research , there is a wide spread impression that the number of patients with PBC is increasing in recent years.Many patients with PBC have no specific symptoms, rather,they present with unexplained liver function abnormalities. Progression occurs over years or decades. The end stage is an established biliary cirrhosis.A hall mark features of PBC is the presence of high titer antimitochondrial antibodies (AMA) in patient sera. AMA are devided into nine subgroups termed M1-M9 according to the biochemical knowledge of the antigens they recognized. It is established that only those antibodies known as M2 were sepecific for PBC. M2 antibodies are detectable years or decades before the clinical and histological features of PBC. The major autoantigens recognized by M2 antibodies are members of the 2-oxo-acid dehydrogenase complex, including the pyruvate dehydrogenase complix E2 (PDC-E2),the branched chain 2-oxo-acid dehydrogenase complex E2 (BCOADC-E2), the 2-oxo-glutarate dehydrogenase complex E2 (OGDC-E2). The immunodominant epitopes of PDC-E2, BCOADC-E2 and OGDC-E2 have been mapped within the lipoyl domains. Antibodies to PDC-E2 can be detected in the serum of more than 95% of patients with PBC. 53%-55% and 39%-88% of PBC sera reactive with BCOADC-E2 and OGDC-E2 respectively . Using three , 92%-100% of patients with PBC can be verified. In contrast,there was no report that M2 were found in other disease other thanPBC.In the research, we have designed a triple hybrid clone, designated as BPO , that coexpresses the three immunodominant lipoyl domains of PDC-E2, BCOADC-E2 and OGDC-E2 from human souces. We established and then used clinically the specific immunogical methods with purified BPO to detect the M2 antibodies which were sepecific for PBC. We used these methods clinically and evaluate the value of measurement of M2 autoantibodies in diagnosis PBC .The research work comprise following three parts:Part 1.Cloning , expression and identification of M2 autoantigen and its trimerTotal RNA were extracted from human peripheral mononuclear blood cell, and cDNA were synthesizwd by reverse transcriptase.Using this as a template,the inner lipoyl domain and part of the outer lipoyl domain PDC-E2,the lipoyl domain of BCOADC-E2 and the lipoyl domain of OGDC-E2 were amplified by the polymerase chain reaction.The amplified polymerase chaim reaction products were digested with restriction endonuclease and puried. The puried cDNA fragments were inserted into expression vector pQE-30. Sequence analysis confirmed that the DNA sequence of the insert was completely consistent with the published data.The recombinant plasmids pQE-30/BCOADC-E2, pQE-30/PDC-E2, pQE-30/OGDC-E2 and pQE-30/BPO were transformed into plasmid E. Coli. Ml5(pREP4). Its products were induced by isopropylthio-β-D-galactoside and confirmed with SDS-PAGE,Western-blot and Immunoabsorption test . The molecular mass of the 4 recombinant proteins was examined by SDS-PAGE in 15% polycrylamide gel.,14kD (BCOADC-E2),18kD (PDC-E2),10kD (OGDC-E2),42kD (BPO) protein bands were visualized.Seven M2-positive sera were used to confirm the antigenic reactivity of the recombinant proteins. The presence of M2 antibodies was established by theEuroimmun Reseach Center (Germany) using beef heart mitochondrial preparation in immunoblotting. Of the 7 M2-positive sera, 3 reacted BCOADC-E2 PDC-E2 and OGDC-E2 respectively.3 reacted with BCOADC-E2 and PDC-E2, 1 reacted with PDC-E2 and OGDC-E2. These sera all reacted with BPO.Sera were absorbed with E.coli lysates from BPO overnight. M2-positive sera became M2-negative in Western-blot after absorped by the BPO, but there were not any changes in control sera. These verified that BPO contain the immunodominant epitopes of PDC-E2, BCOADC-E2 and OGDC-E2 .Part 2.Establishment of the sepecific immunogical methods for the diagno...
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