1.Cloning and expressing the E2 subunit of pyruvate dehydrogenase complex.
Jun ZHAO ; Cui-li SHU ; Li LEI ; Jing LI ; Rong GAO ; Yun CHENG
Chinese Journal of Hepatology 2003;11(10):602-604
OBJECTIVESTo construct the expression vector of the pyruvate dehydrogenase complex E2 subunit gene (PDC-E2).
METHODSThe 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.
RESULTSThe 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.
CONCLUSIONHigh efficient expression vector of PDC-E2 lays the foundation for serum assay of primary biliary cirrhosis patients with prokaryotic expressing PDC-E2.
Cloning, Molecular ; Dihydrolipoyllysine-Residue Acetyltransferase ; Enzyme-Linked Immunosorbent Assay ; Humans ; Liver Cirrhosis, Biliary ; blood ; diagnosis ; immunology ; Lymphocytes ; enzymology ; Polymerase Chain Reaction ; Pyruvate Dehydrogenase Complex ; analysis ; biosynthesis ; genetics
2.Prediction and identification of autoepitopes of PDC-E2 specific CD8+ CTL in primary biliary cirrhosis patients.
Hai-ying LIU ; Ding-kang YAO ; Xiao-qing TU ; Ye ZHOU ; Ye ZHU ; Yan CHEN ; Lie-ying FAN ; Ren-qian ZHONG
Acta Academiae Medicinae Sinicae 2004;26(5):500-504
OBJECTIVETo identify autoepitopes of E2 subunit of pyruvate dehydrogenase complex (PDC-E2) specific CD8+ CTL in primary biliary cirrhosis (PBC) patients.
METHODSAn 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.
RESULTSFive 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.
CONCLUSIONPeptides 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.
Antibody-Producing Cells ; cytology ; Autoantigens ; immunology ; Autoimmunity ; CD8-Positive T-Lymphocytes ; cytology ; immunology ; metabolism ; Cell Line ; Dihydrolipoyllysine-Residue Acetyltransferase ; Epitope Mapping ; Epitopes, T-Lymphocyte ; immunology ; HLA-A Antigens ; immunology ; HLA-A2 Antigen ; Humans ; Liver Cirrhosis, Biliary ; enzymology ; genetics ; immunology ; Phenotype ; Protein Binding ; Pyruvate Dehydrogenase Complex ; genetics ; immunology ; metabolism ; T-Lymphocytes, Cytotoxic ; immunology