Novel triple expression hybrid clone from human sourcesfor the detection of M2 antibodies

Abstract

OBJECTIVE: Primary biliary cirrhosis (PBC) or, more accurately, chronic non-suppurative destructive cholangitis, is a chronic cholestatic liver disease that is considered to have an autoimmune basis. In most patients, PBC is characterized by the presence of M2 antibodies. The major autoantigens recognized by M2 antibodies are members of the 2-oxo-acid dehydro­genase complex, including pyruvate dehydrogenase complex E2 (PDC-E2), branched chain 2-oxo-acid dehydrogenase complex E2 (BCOADC-E2), 2-oxo-glutarate dehydrogenase complex E2 (OGDC-E2), E1 subunits of the pyruvate dehydrogenase complex and protein X of the PDC. The aim of this study was to express the immunodominant epitopes of BCOADC-E2, PDC-E2 and OGDC-E2 separately, to create a triple hybrid clone (designated as BPO) containing epitopes of three autoantigens, and to use BPO as a tool for the detection of M2 antibodies specific for primary biliary cirrhosis (PBC). METHODS: The cDNA fragments from human sources encoding the M2-reactive epitopes of BCOADC-E2, PDC-E2 and OGDC-E2 were amplified using reverse transcription−polymerase chain reaction with total RNA extracted from human peripheral mononuclear blood cells. The fragments were cloned into pQE-30 and then transferred into plasmid Escherichia coli M15. Transformants were induced by isopropylthio-β-d-galactoside and confirmed with sodium dodecyl sulfate−polyacrylamide gel electrophoresis and western blotting. RESULTS: Four specific proteins produced 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 tool for the detection of M2-specific autoantibodies in the diagnosis of PBC.