The Preliminary Research On The Interaction Of Complemet C3 And Its Deletion Mutants With CLIC1

Objective This experiment for studying on the interaction of C3 and its deletion mutants C3(1-840),C3(824-1663) with CLIC1 protein,we constructed respectively eukaryotic expression vectors of C3 gene and its deletion mutants C3(1-840),C3(824-1663),tagged with FLAG. The plasmids of C3 and its deletion mutants C3(1-840),C3(824-1663) were transiently cotransfected with CLIC1 into COS7cells respectively to observe whether two expressed proteins were colocalied in intracellular. For yeast two-hybid experiment,We builded yeast expression vectors of its deletion mutants.Finally, The plasmids of C3 and its deletion mutants C3(1-840),C3(824-1663) were transiently cotransfected with CLIC1 into HEK293 T cells respectively,we confirmed the interaction of C3 and its deletion mutants C3(1-840),C3(824-1663) with CLIC1 by co-immunoprecipitation assay.Methods Firstly, The sequence of wild-type C3 and its deletion mutants C3(1-840)、C3(824-1663) were amplified and digested by restriction enzymes, and then amplified sequences inserted the p CDNA3.1 vector tagged with FLAG. In this way,we constructed p GADT7- C3(1-840), p GBKT7- C3(1-840), p GADT7- C3(824-1663) and p GBKT7- C3(824-1663).All plasmids were confirmed by Enzyme identification and DNA sequencing.Secondly, yeast expression vectors were into groups and co-transformed into AH109 separately with CLIC1,this was performed on SD/-Leu/-Trp/-His/3AT/X-α-gal solid medium.Clones exhibiting blue were transformants with active α-gal activity.Thirdly,plasmid was transiently transfected into COS7 cells to show the localization of expressed proteins.C3 and its deletion mutants C3(1-840),C3(824-1663) were cotransfected with CLIC1 into COS7 cells and observed by fluorescence microscopy.Finally,the interaction of C3 and its deletion mutants C3(1-840),C3(824-1663) were performed in HEK293 T cells with CLIC1 by co-immunoprecipitation assayResults According to the enzyme identification and sangon sequencing results proved that all kinds of plasmid build is successful.Yeast two-hybrid assay shows C3(1-840) not have blue with CLIC1 in yeast cells. But C3(824-1663) and CLIC1 shows have blue and had α-galactosidase activity. In the immunefluorescence microscope,C3 protein and its deletion mutants C3(1-840) 、 C3(824-1663) mainly distributed in cytoplasm,CLIC1 mainly distributed in nucleus especially concentrated in nuclear membrane,and less in cytoplasm. But the deletion mutant protein of C3(824-1663) inaddition to the distribution in the cytoplasm, had a bright spot in the nuclear membrane concentration distribution.Colocalized experiment observed C3 and C3(1-840) have no obvious colocalization,but C3(824-1663) had. Co-immunoprecipitation assay shows that both C3 or C3(1-840) have no interaction with CLIC1.Conclusion By yeast two-hybrid,immunofluorescence and Co-immunoprecipitation show that the C3 and C3(1-840) protein canot interacts with CLIC1 protein in cells.However, C3(824-1663) and CLIC1 protein maybe have interaction exists in a cell.