ABSTRACT
Recombinant expression vector pcDNA3-DAFMCP-DP containing human membrane complement regulatory proteins (hCRPs) decay accelerating factor (DAF) and membrane cofactor protein (MCP) cDNA was constructed by using two independent promoters. After transfected into NIH3T3 cells by calcium phosphate-DNA precipitate method, NIH3T3 pcDNA3-DAFMCP-DP transfectants were obtained by G418 selection. Extraneous genes integration was identified by PCR. The co-expression of human DAF and MCP at both mRNA and protein levels was confirmed by using RT-PCR and Western blot analysis. Human DAF and MCP cDNA were integrated into NIH3T3 pcDNA3-DAFMCP-DP genomic DNA after continuous 30 times passages, indicating that NIH3T3 pcDNA3-DAFMCP-DP were stable cell lines. Human C-mediated cytolysis assays showed that NIH3T3 cells transfected stably with pcDNA3-DAF, pcDNA3-MCP, and pcDNA3-DAFMCP-DP were protected from C-mediated damage and co-expressed human DAF and MCP provided more excellent protection against C-mediated attack, which was compared with either DAF or MCP alone. These results suggest that the dicistronic vector could improve the efficiency of multi-gene delivery and benefit the synergic effect of human membrane complement regulatory proteins DAF and MCP.
Subject(s)
Animals , Humans , Mice , 3T3 Cells , CD55 Antigens , Genetics , Pharmacology , DNA, Complementary , Genetics , Drug Synergism , Graft Rejection , Membrane Cofactor Protein , Genetics , Pharmacology , Recombinant Fusion Proteins , Genetics , Pharmacology , TransfectionABSTRACT
Objective:To construct recombinant expressing vector pcDNA3-DAF and to develop the NIH3T3 cell model expess human complement regulatory protein decay accelerating factor(DAF,CD55)stably after transfected.Methods:Human membrane complement regulatory protein(hCRP) DAF cDNA containing the full-length of encoding region was cloned into expressing vector pcDNA3.After identification by restriction enzyme digestion,PCR and sequencing,the recombinant plasmid was transfected into NIH3T3 cells with calcium phosphate-DNA precipitate method.A stably-transfected cell line was established by G418 selection.Extraneous gene integration was identified by PCR.Expression of DAF at both mRNA and protein levels was analyzed by RT-PCR,Western blot and indirect immunofluorescence microscopy.Results:The eukaryotic expression vector pcDNA3-DAF was successfully constructed and the DAF gene was transfected stably into NIH3T3 cells,a stably-transfected cell line was established and DAF was efficiently expressed on the surface of transfected NIH3T3 cells.Human DAF cDNA was integrated into NIH3T3 pcDNA3-DAF genomic DNA after continuous 30 times passages,indicating that NIH3T3 pcDNA3-DAF was stable cell line.Conclusion:The establishment of the stably-transfected cell line and the expression of the target gene provide a base for further studies on the function of the DAF and the cooperative fashion among different human complement regulatory proteins in alleviating the complement-mediated cytolysis.