Co-expression of the complement regulatory proteins human DAF and CD59 with an IRES-mediated dicistronic mammalian vector enhances their cell protective effects.
The human complement regulatory proteins (hCRPs) decay accelerating factor (DAF/CD55) and protectin CD59 transfected into non-human cells could confer protection against human complement. The combination of DAF and CD59 would be an effective strategy to help overcome host complement-induced hyperacute rejection in xenotransplantation. We constructed a dicistronic mammalian expression vector pcDNA3-CD59IRESDAF by using the internal ribosomal entry sites (IRES) of the encephalomyocarditis virus (EMCV). RT-PCR, Western blotting and immunofluorescence microscopic analysis demonstrated that the EMCV IRES allowed for efficient co-expression of hCD59 and hDAF on the surface of NIH/3T3 cells transfected stably with pcDNA3-CD59IRESDAF. Human complement-mediated cytolysis assays showed that co-expressed DAF and CD59 proteins could provide more significant protection against complement-mediated cytolysis than either hCD59 or hDAF alone. These results suggest that IRES containing polycistronic vector should improve the efficiency and effectiveness of multi-gene delivery and that the construct pcDNA3-CD59IRESDAF vector has potential therapeutic value for effectively controlling complement activation and for preventing hyperacute rejection in clinical gene therapy.
Xu
Li
L
State Key Laboratory of Virology, College of Life Science, Wuhan University, P.R. China.
Shao
Huanjie
H
Wu
Wenlan
W
Cao
Zhijian
Z
Zhao
Zhouzhou
Z
Liu
Hui
H
Jiang
Dahe
D
Mao
Xin
X
Li
Wenxin
W
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D018958:CD55 Antigens / Q000235:genetics / Q000276:immunology*; D018957:CD59 Antigens / Q000235:genetics / Q000276:immunology*; D002470:Cell Survival / Q000235:genetics / Q000276:immunology; D003165:Complement System Proteins / Q000276:immunology*; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D051379:Mice; D008856:Microscopy, Fluorescence; D041681:NIH 3T3 Cells; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012270:Ribosomes / Q000378:metabolism; D014162:Transfection
D018958:CD55 Antigens; D018957:CD59 Antigens; D012333:RNA, Messenger; D003165:Complement System Proteins