Co-expression of human complement regulatory proteins DAF and MCP with an IRES-mediated dicistronic mammalian vector enhances their cell protective effects.
C3 convertase regulatory proteins, decay accelerating factor (DAF, CD55) and membrane cofactor protein (MCP, CD46), have complementary function and transfected into non-human cells might confer protection against human complement. This may be an effective strategy to alleviate C-mediated cell damage by combining the two activities. In this study, we constructed a dicistronic mammalian expression vector pcDNA3-MCPIRESDAF using the internal ribosomal entry sites (IRES) of the encephalomyocarditis virus (EMCV), and stable cell lines were obtained by G418 screening. Integration of extraneous genes was identified by PCR. RT-PCR and Western blotting analysis demonstrated that the EMCV IRES allowed for efficient co-expression of hMCP and hDAF in NIH3T3 cells stably transfected with pcDNA3-MCPIRESDAF. Human complement-mediated cytolysis assays showed that co-expressed DAF and MCP proteins could provide more significant protection against complement-mediated cytolysis than either hMCP or hDAF alone. These results suggest that DAF and MCP synergize the actions of each other, and the IRES-mediated polycistronic vector should improve the efficiency and effectiveness of multi-gene delivery. The pcDNA3-MCPIRESDAF vector has potential therapeutic value for effectively controlling complement activation, thereby increasing the possibility of inter-species transplantation.
Xu
Li
L
State Key Laboratory of Virology, College of Life Science, Wuhan University, Wuhan, 430072, PR China.
Zhao
Zhouzhou
Z
Sheng
Jiqun
J
Zhu
Chengang
C
Liu
Hui
H
Jiang
Dahe
D
Mao
Xin
X
Guo
Mingxiong
M
Li
Wenxin
W
D000818:Animals; D018958:CD55 Antigens / Q000378:metabolism*; D002470:Cell Survival; D002478:Cells, Cultured; D050577:Complement C3-C5 Convertases / Q000378:metabolism; D015938:Complement Membrane Attack Complex / Q000378:metabolism; D003165:Complement System Proteins / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D051916:Membrane Cofactor Protein / Q000378:metabolism*; D051379:Mice; D041681:NIH 3T3 Cells; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012270:Ribosomes / Q000378:metabolism; D014162:Transfection
D018958:CD55 Antigens; D015938:Complement Membrane Attack Complex; D051916:Membrane Cofactor Protein; D003165:Complement System Proteins; D050577:Complement C3-C5 Convertases