中国生物工程杂志

Recombinant expression vector pcDNA3-MCPCD59-DP containing human membrane complement regulatory proteins (hCRPs) MCP and CD59 cDNA was constructed successfully by using two independent promoters. After transfected into NIH3T3 cells with calcium phosphate-DNA precipitate method, NIH3T3 pcDNA3-MCPCD59-DP transfectants were obtained by G418 selection. Extraneous genes integration was identified by PCR. The co-expression of human CD59 and MCP at both mRNA and protein levels was confirmed by using RT-PCR and Western blot analysis. Human MCP and CD59 cDNA were integrated in NIH3T3 pcDNA3-MCPCD59-DP genomic DNA after continuous 30 times passages, indicating that NIH3T3 pcDNA3-MCPCD59-DP were stable cell lines. Human complement-mediated cytolysis assays showed that NIH3T3 cells transfected stably with pcDNA3-CD59, pcDNA3-MCP, and pcDNA3-MCPCD59-DP were protected from C-mediated damage and co-expressed human MCP and CD59 provided more excellent protection against C-mediated attack as compared with either CD59 or MCP expressed alone. These results suggest that the dicistronic vector represents an effective and efficacy strategy to overcome C-mediated damage and has potential therapeutic value for effectively controlling complement activation and finally for preventing hyperacute rejection in clinical gene therapy.