Study Of Anti-complement Activity Of Mutant Human CD59 Before And After Glycation

Objective: To establish a eukaryotic expression vector and a expression cell line of mutant human CD59(HMCD59).To investigate the effect of HMCD59 on diabetic vascular complications.To laid a foundation for explaining the mechanism of diabetic vascular complication. Methods: Two recombinant palter plasmid containing whole CDNA sequences of HMCD59 transfected CHO cells by lipofectamine with PCDNA respectively. Then G418 was used to select positive clone. A series of methods such as fluorescent antibody technique, immunoenzymatic histochemistry, western blot and flow cytometrey were used to make sure that HMCD59 protein expressed on cell membrane ;CHO cells was harvested to immunize rabbit for muticlonol antibody; it's activity of anti-complement were studied through the method of BCECF release. Results:1.Two stable cell lines expressing HMCD59 were successfully established, using lipofectamine mediating method. Two recombinant mutant CD59-palter transfected into CHO cells with PCDNA respectively. After two weeks cultivation with RPMI- 1640 medium containing 400ug/ml G418, positive clones was identified. G418-resistant clones was confirmed that HMCD59 molecules expressed on the surface of CHO cells by the method of fluorescent antibody technique and immunoenzymatic histochemistry .The rate of expression of HM5-CHO is 44. 06%;HM6-CHO,64.67%. The cell lysates were identified by western blot that there are a protein band at the position of the relative molecule mass of 20 000 which molecular weight is the same to CD59 molecule. 2, Muticlonol antibody to CHO cells was successfully acquired , and its titer was determined beyond 1 :160, 000. 3, Two expression product possesthe character of resisting complement, and the character weakened if the expression product was glycated. Conclusion: Successfully established two cells expressing HMCD59, acquired high-titered muticlonal antibody to CHO cells. Made a preliminary study of the resisting-complement character of HMCD59, and found that both of the two HMCD59s bore the character of resisting complement, and the character of them weakened after glycation.