Establishment of a eukaryotic expressing system for human decay accelerating factor / 中国免疫学杂志
Chinese Journal of Immunology
; (12)1999.
Artigo
em Chinês
| WPRIM (Pacífico Ocidental)
| ID: wpr-547050
Biblioteca responsável:
WPRO
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.
Texto completo:
Disponível
Base de dados:
WPRIM (Pacífico Ocidental)
Tipo de estudo:
Estudo prognóstico
Idioma:
Chinês
Revista:
Chinese Journal of Immunology
Ano de publicação:
1999
Tipo de documento:
Artigo