[Co-expression and synergic effect of human complement regulatory proteins DAF and MCP].
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.
Xu
Li
L
State Key Lab of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, China.
Zhao
Zhouzhou
Z
Liu
Hui
H
Jiang
Dahe
D
Li
Wenxin
W
D016475:3T3 Cells; D000818:Animals; D018958:CD55 Antigens / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D018076:DNA, Complementary / Q000235:genetics; D004357:Drug Synergism; D006084:Graft Rejection / Q000517:prevention & control*; D006801:Humans; D051916:Membrane Cofactor Protein / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D051379:Mice; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D014162:Transfection
D018958:CD55 Antigens; D018076:DNA, Complementary; D051916:Membrane Cofactor Protein; D011993:Recombinant Fusion Proteins