ABSTRACT
<p><b>OBJECTIVE</b>To identify autoepitopes of E2 subunit of pyruvate dehydrogenase complex (PDC-E2) specific CD8+ CTL in primary biliary cirrhosis (PBC) patients.</p><p><b>METHODS</b>An online database SYFPEITHI was applied to predict HLA-A*0201 restricted epitopes which located in PDC-E2 30-50 aa and 150-190 aa where B-cell epitopes clustered with CD4+ T-cell epitopes. T2 cell line reconstitution and stabilization assay, induction of specific CTL lines from peripheral blood mononuclear cells (PBMCs) of patients with PBC and cytotoxicity of peptides-induced CTL were performed to screen the epitopes from those candidates.</p><p><b>RESULTS</b>Five potential epitopes were predicted by database. Of the 5 candidates, two peptides 159-167 aa and 165-174 aa, with highly binding activity to HLA-A*0201 molecules, could stimulate PBMCs from most HLA-A*0201 positive PBC patients to proliferate and peptide-induced CTL lines showed specific cytotoxicity.</p><p><b>CONCLUSION</b>Peptides of KLSEGDLLA (159-167 aa) and LLAEIETDKA (165-174 aa) in the inner lipoyl domain of PDC-E2 are HLA-A*0201 restricted CD8+ CTL immunodominant epitopes in PBC.</p>
Subject(s)
Humans , Antibody-Producing Cells , Cell Biology , Autoantigens , Allergy and Immunology , Autoimmunity , CD8-Positive T-Lymphocytes , Cell Biology , Allergy and Immunology , Metabolism , Cell Line , Dihydrolipoyllysine-Residue Acetyltransferase , Epitope Mapping , Epitopes, T-Lymphocyte , Allergy and Immunology , HLA-A Antigens , Allergy and Immunology , HLA-A2 Antigen , Liver Cirrhosis, Biliary , Genetics , Allergy and Immunology , Phenotype , Protein Binding , Pyruvate Dehydrogenase Complex , Genetics , Allergy and Immunology , Metabolism , T-Lymphocytes, Cytotoxic , Allergy and ImmunologyABSTRACT
<p><b>OBJECTIVES</b>To construct the expression vector of the pyruvate dehydrogenase complex E2 subunit gene (PDC-E2).</p><p><b>METHODS</b>The PDC-E2 gene was amplified from human lymphocytes with RT-PCR, and was cloned into pExSecI vector to induce the PDC-E2 expression. The products were identified with western blot and ELISA.</p><p><b>RESULTS</b>The expression vector pExSecI/PDC-E2 was successfully constructed. The products could be identified by the specific self-antibodies in the sera from the primary biliary cirrhosis patients.</p><p><b>CONCLUSION</b>High efficient expression vector of PDC-E2 lays the foundation for serum assay of primary biliary cirrhosis patients with prokaryotic expressing PDC-E2.</p>