ObjectiveTo investigate the significant effect of costimulatory pathway B7CD28/CTLA4 on the islets of Langerhans transplantation. MethodsThe literatures were reviewed to summarize the molecular structure and functions of the pathway and the related animal experiments.ResultsThe costimulatory pathway B7CD28/CTLA4 was one of the signaling pathways of T cells activation and proliferation. If the costimulatory signals were absent, Tlymphocyte would be induced to the clonalanegy. Through blocking the costimulatory pathway mediated by CD28, CTLA4Ig prolonged to the islets of Langerhans survival in recipients. ConclusionBy the studies of the costimulatory pathway, it is helpful to understand the immune mechanism of the survival of islet grafts.
ObjectiveA mammalian expression vector encoding TAG72specific single chain variable region(scFv) fused to the transmembrane and intracellular domains of the signal transducing chain of CD28 was constructed, to generate for targeting of genetically modified T cells to gastrointestinal cancer, and to investigate the cytotoxicity against TAG72 positive target cells.MethodsThe transmembrane and intracellular domains of CD28 cDNA was amplified from human T lymphocytes using RTPCR to clone into a mammalin expression vecter, and CD28 fragments were ligated downstream of the antiTAG72 scFv cDNA and sequence verified.ResultsA 729base pair of antiTAG72 scFv was in accordance with sequence concerned; a 240base pair of cDNA of the transmembrane and intracellular domains of CD28 was confirmed as sequence concerning of Genebank.ConclusionWe constructed a mammalian expression vector encoding fusing gene to activate tumorassociated antigenspecific T lymphocyte, for generation of modified T lymphocytes to gastrointestinal tumors.
To verify the role of mAbCD28 in allograft transplantation. The biological action of mAbCD28 had been tested in mixed-lymphocyte-reaction and parathyroid gland allotranplantation in rats. Results: mAbCD28 could significantly suppress the proliferation of T cells in vitro and prolong the survival time of allograft in vivo. The results showed that mAbCD28 could block the costimulatory signals transmitted by CD28 molecules, and played an immunosuppressive role in parathyroid gland transplantation in rats.
Objective To construct a green fluorescent protein expression plasmid pEGFP-C3-anti-TAG72 scFv-CD28, containing anti-TAG72 single chain variable fragment (scFv) fused into the transmembrane and intracellular domain of the signal-transducing chain of CD28 gene, and to transfect it into peripheral blood mononuclear cells． Methods Recombinant transmembrane and intracellular domain of CD28 cDNA and anti-TAG72 scFv cDNA fragment was subcloned into pEGFP-C3 vector. Recombinant clones were selected by Kanamyein， and then identified by PCR， enzyme digestion analysis and DNA sequencing. The recombinant plasmid was transfected into peripheral blood mononuclear cells by means of lipofection. The recombinant protein expression was confirmed by immunocytochemistry, laser scanning confocal microscope, PCR and Western blot analysis. Results The fused gene fragment of anti-TAG72 scFv-CD28 was successfully inserted into pEGFP-C3 plasmid, and it was confirmed by enzyme digestion and DNA sequencing. The fused anti-TAG72 scFv-CD28 gene and its protein was identified in peripheral blood mononuclear cells． Conclusion The eukaryotic expression plasmid pEGFP-C3-anti-TAG72 scFv-CD28 was successfully constructed and transiently expressed in peripheral blood mononuclear cells， which would lay a foundation for further studies on the role of it to activate tumor-associated antigen-specific T lymphocyte, for generating of modified T lymphocytes targeting gastrointestinal tumors.