RESUMO
Objective:To set up a eukaryotic system for high expressing human CD137 and to investigate the role of CD137 and CD137L on signals transduction of cells.Methods:The pCDNA3 plasmid containing full length of human CD137 cDNA sequence(CMV ILA SEN,CIS) and pSV2 dhfr plasmid were cotransfected into dhfr CHO cells by lipoid mediating method. The positive clone was selected with G418. Expression of CD137 on dhfr CHO cells were induced by MTX and detected by RT PCR, immunocytochemistry and flow cytometry.It's activity study was done by method of incorporating 3H TdR.Results:CD137 expressed on the surface of dhfr CHO, expression rate was 96.07%, it's activity study indicated that CD137 increase the proliferation of PBMC stimulated by anti CD3 monoclonal antibody.Conclusion:dhfr CHO cells that highly express CD137 were established. CD137 can increase the proliferation of PBMC stimulated by anti CD3 monoclonal antibody.
RESUMO
Objective: To amplify two human mutant CD59 eukaryotic expressing systems and investigate mutant CD59 functional activity. Methods: Mammalian expression vector PLATER of mutant CD59 cDNAs was transfected into CHO together with the pcDNA by lipofectamine,which confered resistance to G418(400 ?g/ml). The positive clones were tested by FIH. Activity of both mutants CD59 was determined by BCECF release assay. Results: Mutant CD59 cDNAs subcloned into the mammalian expression vector PLATER and transfected CHO together with the pcDNA,which confered resistance to G418. The positive clones were tested by FIH.Activity of both mutants CD59 before and after glycation was determined by BCECF release assay,both of them could restrict MAC formation ,and glycation could inhibit CD59. Conclusion: A eukaryotic system that expressing mutant CD59 cDNA was successfully set up.It was found that mutant CD59 could restrict MAC formation,and glycation could inhibit mutant CD59. These would be helpful for the furthur study of link mutant CD59 and the vascular proliferative of diabetes.