RESUMO
The class II major histocompatibility complex molecules play a major role in presentation of exogenous antigenic peptides to the CD4 positive helper T cell. These are heterodimeric cell surface glycoproteins consisting of alpha- and beta-chains. In the present study, we cloned and expressed the alpha- and beta-chain of HLA-DR4 lacking the transmembrane and cytoplasmic domain separately in Escherichia coli using the pET-5a expression vector system. The expressed alpha- and beta-chains were purified in a denaturing condition by an ion exchange chromatography on Q-Sepharose and a gel filtration chromatography on Sephacryl S-200, respectively. The recombinant proteins were refolded and reassembled by removing the denaturing agent and concomitant reoxidation of the disulfide bond. The refolded heterodimeric rDR4 molecule was resolved by 12.5% SDS-PAGE in a nonreducing condition and confirmed by Western blot using polyclonal antibody against DR-alpha and the monoclonal antibody (L243) for the conformationally correct DR molecule. The rDR4 molecules were reconstituted with a 50-fold molar excess biot-HA (307-319), and the bound peptides to the heterodimer complex were determined by a microplate assay coated with L243 antibody using Extravidin-HRP conjugate.
Assuntos
Antígeno HLA-DR4/química , Sequência de Aminoácidos , Sequência de Bases , Primers do DNA/genética , Escherichia coli/genética , Expressão Gênica , Antígeno HLA-DR4/genética , Antígeno HLA-DR4/isolamento & purificação , Humanos , Conformação Proteica , Dobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
The antigens of HLA-DR locus were determined in 127 patients with rheumatoid arthritis and in 175 healthy persons. The frequency of HLA-DR1 antigen was discovered in 45% of the patients and in 22.3% of the examinees from the control group (p less than 0.001). The relative risk is 2.84. 69% of the patients were seropositive. The HLA-DR4 antigen was found in 34% of the patients and in 21% of the healthy examinees (p less than 0.05). The relative risk is 1.91. The rheumatoid factor was determined in 70% of the patients with this antigen.