Study Of The Relationship Between CD59 Mutagenisis And Signal Conduction Of The Cytoapoptosis

Objective To construct a eukaryotic express vector and a expression cell line of mutant human CD59 (HMCD59) . To investigate the effect of HMCD59 on the signal conduction of the cytogenisis furtherly, and try to illustrate the mechanism of all kinds of tumorous diseases.Methods Site-directed mutagenesis was performed by recombinant PCR and was confirmed by sequence analysis . In order to set up a eukaryotic system that highly expressing HMCD59, the recombinant PALTER-MAX plasmid containing HMCD59 CD and pcDNA plasmid were co-transfected into Chinese Hamster Ovary(CHO) cell by cation lipoid mediating method. The cell clones which were transfected steadily were selected by G418. The cell clones expressing CD59 mutagenisis highly were detected by immunocytochemistry, immunofluorescence technology, flow cytometry and Weston blot. To detect the consistency of Fas, Bcl-2 and NF-KB expressed by CHO cell before and after transfection.Result A eukaryotic expressing system for high expression HMCD59 were set up successfully. The recombinant PALTER-MAX plasmid containing HMCD59 cDNA and pcDNA plasmid were co-transfected into CHO cell by catin lipoid mediating method. And the cells were grown in RPMI1640 medium containing 400 ug/ml G418 for 14 days. The positive cell clones were selected to furtherly enlarge to get the stable expressing cells. Through detected, there were HMCD59 expressing on membrance of selected positive cell. The cell lysates were identified by Western blot when there were a protein band at the position of the relative molecule mass of 20000 which molecular weight is same to CD59 molecule. The difference of expressing of Fas, Bcl-2 and NF-KB in CHO cells after transfection was detected by immunocytochemistry, and initially, it was proved that CD59had the function of anti-apoptosis. Then, we sorted a good many of positive cell clones in order to search the function of CD59 furtherly.Conclusion The eukaryotic expressing system which based on HMCD59 and PALTER-MAX plasmid was constructed successfully. The recombinant plasmid was conducted into the eukaryotic cell by transfection, and there were HMCD59 expressed on the membrance of the eukaryotic cell after transfection. HMCD59 had the function of antiapoptosis, which set foundation for searching the mechanism of tumor.