Construction of mammalian cell expression vector of human leukocyte antigen-C gene and expression in JAR cells
- VernacularTitle:HLA-C基因真核细胞表达载体的构建、鉴定及表达
- Author:
Weidang LI
;
Lian FAN
;
- Publication Type:Journal Article
- Keywords:
Human leukocyte antigen RT PCR Gene cloning Gene expression
- From:
Chinese Journal of Immunology
2000;0(11):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To construct mammalian cell expression vector of human leukocyte antigen C gene and express it in JAR cell and study its functions.Methods:Total cell RNA was extracted from peripheral blood mononuclear cells and the cDNA was amplified by RT PCR;the cDNA fragments were directly inserted into Pbluescript KS(+/-) and the recombinant PBS HLA C was identified by restriction endonuclease digestion and sequencing;then the plasmid of pcDNA HLA C was transfected into JAR cells.Lactate Dehydrogenase(LDH) release assay was employed to detect the cytotoxicities of NK cells proliferated in vitro against HLA C transfectants JAR cells.Results:The recombinant mammalian cell expression vectors of pcDNA3 HLA C was constructed,the sequences of the insert was identical to the published sequences encoding HLA Cw *0602 gene.Several transfected JAR clones were obtained,which expressed HLA Cw *0602 in high level and constitutively.The specific lysis was detected for NK cells stimulated by IL 2 against JAR cells and HLA C transfectants JAR cells.But both cells were resistant to freshly isolated PBL cells.Conclusion:The recombinant mammalian cell expression vectors of pcDNA3 HLA Cw *0602 was successfully constructed,JAR cells resistance to NK lysis could involve an HLA C independent mechanism.
