Expression Of Human Mutant CD59 In Eukaryotic Expression System And Its Activity Study

OBJECTIVE To set up a eukaryotic expression system for human mutantCD59.To get CHO cell lines that highly expression of human mutantCD59,and to detect the function of glycating and nonglycating human mutant CD59. Discuss the important function of complement regulatory membrane protein CD59 restricting complement membrane attack complex formation in diabetes' pathology. Help to explain the pathological mechanisms of humans to develop vascular proliferative complications of diabetes.METHODS To set up a eukaryotic system that highly expressing humanmutantCD59 : The recombinant PALTER-MAX plasmid containing human mutantCD59 cDNA and PCDNA plasmid were co-transfected into CHO cell by cation lipoid mediating method .The positive clones were selected by G418,and highly expressing clones were more selected by flow cytometry ,and get stable highly expressing CHO cell lines . Stable highly expressing CHO cell lines were more detected by immunocytochemistry and immunofluorescence technology . CD59 protein were obtained by spalling CHO cells . Stable highly expressing cells were validated by SDS-PAGE immunoblot analysis and solid phase enzyme immunoassay . Stable highly expressing cells were cultivated in high glucose ,and the function of CD59 restricting complement membrane attack complex formation were investigated by BCECF releasing test.RESULTS A eukaryotic expression system for high expression humanmutantCD59 were successfully set up : The recombinant PALTER-MAX plasmid containing human mutantCD59 cDNA and PCDNA plasmid were co-transfected into CHO cell by cation lipoid mediating method ;and the cells were grown in F12 medium containing 400ug/ml G418 for 14 days, positive clones were grown in RPMI1640 medium to get stable expressing cell lines . Highly expressing clones were selected by flow cytometry ,and were named PALTER-CD59-CHO1PALTER-CD59-CHO2 . Flow cytometry indicated that expression rates of PALTER-CD59-CHO1 and PALTER-CD59-CHO2 were 53.7%and 54.5% . Further more, Stable highly expressing CHO cell lines were more detected by immunocytochemistry and immunofluorescence technology . PALTER-CD59 -CHO1 and PALTER-CD59-CHO2 were grown in RPMI1640 to get a large of cells . CD59 protein were obtained by spalling PALTER- CD59- CHO1 and PALTER - CD59 - CHO2 cells . Stable highly expressing cells were further validated by SDS-PAGE, immunoblot analysis and solid enzyme immunoassay . PALTER - CD59 - CHO1 and PALTER - CD59 - CHO2 were glycated in RPMI1640 of 50mM ribose for 72 hours .BCECF releasing test indicted that the releasing rate of PALTER-CD59-CHO1 or PALTER-CD59-CHO2 was less high than PALTER-CHO ,and the releasing rate of glycated PALTER-CD59 -CHOI or PALTER-CD59-CHO2 was higher than unglycated ones . PALTER -CD59-CHO1 and PALTER -CD59 -CHO2 were glycated in RPMI1640 of 50mM ribose for 72 hours .BCECF releasing test indicted that the releasing rate of PALTER - CD59 - CHOI or PALTER - CD59 - CHO2 was less high than PALTER-CHO ,and the releasing rate of glycated PALTER-CD59-CHO1 or PALTER - CD5 9-CHO2 was higher than unglycated ones .CONCLUSIONS1. To constrcture a eukaryotic expression system of human mutant CD59 HM1 HM2 and get CHO cell lines that highly expression of human mutant CD59 .2.Conform the complement regulating function of human mutant CD59 and the function decreasing of glycated CD59 .Conform the important function of complement membrane regulatory protein CD59 restricting complement membrane attack complex formation ,and the decreasing funtion of glycated CD59 possibly result in or accelerate to develop human vascular proliferative complications of diabetes.