Establishment of a eukaryotic expressing system for human decay accelerating factor
- VernacularTitle:人衰变加速因子DAF基因真核表达系统的构建
- Author:
Li XU
;
Zhouzhou ZHAO
;
Hui LIU
;
Wenxin LI
- Publication Type:Journal Article
- Keywords:
DAF;
NIH3T3 cell;
Stable transfection;
Eukaryotic expression
- From:
Chinese Journal of Immunology
1999;0(12):-
- CountryChina
- Language:Chinese
-
Abstract:
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.
