| Objective: CD28and CD137are critical co-stimulators for T cells. This projectaimed to construct eukaryotic expression vectors of human CD137/CD28-Ig fusionproteins and to prepare monoclonal antibodies specific to both molecules.Contents:1.Construction of CD137/CD28eukaryotic expression vectors;2.Expression and identification of CD137/CD28fusion proteins;3. Preparation ofmonoclonal antibody against CD137/CD28.Methods: CD137and CD28cDNA were cloned from the activated PBMC andcloned into pCDH vector or inserted into pD18Y vector. The recombinant plasmidspCDH-CD137/CD28and pD18Y-CD137/CD28were separately transfected intoCOS-7or CHO/dhfr-cells by Lipofectamine. The expression culture supernatant werecollected. The fusion proteins were purified by affinity chromatography and analyzedvia ELISA and Western-blot. pD18Y-CD137/CD28-Ig fusion protein was used forimmunizing Balb/c mice by the routine method. The spleen cells from immunizedmice were fused with SP2/0cells using selectively culture with HAT medium. ELISAand Western-blot were used to screen and identify the monoclonal antibodies.Results: The CD137/CD28cDNA were successfully amplified and cloned. Thefragments of CD137/CD28extracellular domains were subcloned into the expressionvectors with correct sequences. ELISA and Western-blot showed that the proteinspurified from the transfected cells culture supernatants that confirmed withCD137/CD28-Ig fusion proteins expression.At least five stable hybridomas specificfor CD137were obtained and also a group of CD28specific hybridomas that hadbeen obtained for further screening the stable clones.Conclusion: We successfully expressed human CD137/CD28-Ig fusion proteinsand prepared at least five specific McAbs(1D4,2C4,2C6,4C7,4E5) against CD137,and primarily got a group of human CD28specific monoclonal antibodies, which laid the foundation for the studies and applications for human T-cell activation. |
