Identification and expression of human CD81 gene on murine NIH/3T3 cell membrane.
The human CD81 (hCD81) molecule has been identified as a putative receptor for hepatitis C virus (HCV). In this study, eukaryotic expression vector pCDM8-hCD81 containing hCD81 cDNA and pSV2neo helper plasmid was used to cotransfect with lipofectamine into murine fibroblast cell line NIH/3T3 to establish an hCD81-expressing cell line. Resistant cell clones were obtained 20 days after the selection with neomycin (600 micro/ml) and then cultured as monoclones. The expression of the transfected hCD81 gene in the cells was verified by RT-PCR and flow cytometry analyses. One of the selected cell clones showed obvious expression of hCD81 and was named NIH/3T3-hCD81. Competitive inhibition tests indicated that the binding of monoclonal anti-hCD81 (JS-81) to NIH/3T3-hCD81 cells was inhibited by recombinant HCV E2 protein, suggesting that the expressed hCD81 molecules on NIH/3T3-hCD81 cells maintain natural conformation of binding to HCV E2. The transfected NIH/3T3-hCD81 cells should be of great potential value in studies on HCV attachment and onset of infection.
Department of Microbiology, Second Military Medical University, Shanghai 200433, China.
J
J
Cao
P
P
Zhao
L J
LJ
Zhao
S M
SM
Wu
S Y
SY
Zhu
Z T
ZT
Qi
D016475:3T3 Cells; D000818:Animals; D015703:Antigens, CD / Q000235:genetics* / Q000378:metabolism*; D001667:Binding, Competitive; D002462:Cell Membrane / Q000235:genetics* / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism*; D051379:Mice; D060225:Tetraspanin 28; D014162:Transfection; D014759:Viral Envelope Proteins / Q000378:metabolism
D015703:Antigens, CD; C554798:CD81 protein, human; C554799:Cd81 protein, mouse; D008565:Membrane Proteins; D060225:Tetraspanin 28; D014759:Viral Envelope Proteins; C091015:glycoprotein E2, Hepatitis C virus