[The high level expression of CTLA-4/Ig fusion protein in CHO cell].
OBJECTIVE
To establish the basis in the function study and clinical application of the fusion protein of cytotoxic T lymphocyte antigen 4 CTLA-4 and IgG Crl (CTLA-4/Ig), by studying the expression of CTLA-4/Ig fusion protein in eukaryotic CHO cells and purifying CTLA-4/Ig fusion protein expressing in CHO cells.


METHODS
The CHO/dhfr- cells were transfected with linearized plasmids of pMM-CTLA-4-IgG4/WG and pMMGR. The clones of CHO/pCTLA-4+/pMMGR+ were in suspension cultured in serum-free culture media. The expression level of CTLA-4/Ig fusion protein in cell culture media was detected through Western blot. The biological identification of relative molecular mass, purity and antigen specificity of the expressing CTLA-4/Ig fusion protein that have been purified with protein A affinity chromatography was studied by SDS-PAGE or Western blot respectively.


RESULTS
The CHO cells that could steadily express at high level the CTLA-4/Ig fusion protein was selected for cloning. By SDS-PAGE for the CTLA-4/Ig fusion protein that have been purified with protein A affinity chromatography, a protein band was found to match well with the predicted relative molecular mass. This protein could specifically bind with human CTLA-4 monoclonal antibody.


CONCLUSIONS
The CHO cells that can express steadily CTLA-4/Ig fusion protein that is of the bioactivity at high level are successfully obtained.
Zheng
Li-heng
LH
Department of Cytobiology, College of Life Science, Sichuan University, Chengdu 610041, China.
Liu
Quan-sheng
QS
D000818:Animals; D000918:Antibody Specificity; D015703:Antigens, CD / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D015153:Blotting, Western; D016466:CHO Cells; D060908:CTLA-4 Antigen; D002846:Chromatography, Affinity; D006224:Cricetinae; D003412:Cricetulus; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D006801:Humans; D007136:Immunoglobulins / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D014162:Transfection
D015703:Antigens, CD; D060908:CTLA-4 Antigen; C556706:CTLA4 protein, human; D007136:Immunoglobulins; D011993:Recombinant Fusion Proteins