[Expression and Characterization of Single-chain Fv-Fc Fusion Protein against Human P185(erbB2)].
In order to increase therapeutic effects and decrease immunogenicity of mouse McAb, the single-chain Fv (scFv) created by fusing the light and heavy chain variable region genes of anti-human P185(erbB2) McAb was conjugated to the Fc gene of human IgG1 to construct a scFv-Fc fusion gene. The scFv-Fc fusion gene was cloned into the expression vector pCIDN. The scFv-Fc fusion protein was synthesized as secreted two-chain molecule in CHO cells, and purified by affinity chromatography on recombinant protein A. A special 185 kD P185(erbB2) protein was immunoprecipitated by the scFv-Fc fusion protein. The fluorescence-activated cell sorting (FACS) using SK-BR-3 cells as the target indicated that the fusion protein could bind to the extracellular domain of P185(erbB2). The affinity of the scFv-Fc fusion protein, determined by ELISA, was K=7.5x10(-10) (mol/L)(-1). This work laid basis for further studies on the anti-P185(erbB2) scFv-Fc fusion protein.
Xie
Zhi-Gang
ZG
Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing 100850, China. ningguo@nic.bmi.ac.cn
Guo
Ning
N
Shi
Ming
M
Feng
Jian-Nan
JN
Yu
Ming
M
Sun
Ying-Xun
YX
Shen
Bei-Fen
BF
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D016466:CHO Cells; D006224:Cricetinae; D005434:Flow Cytometry; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000235:genetics*; D007128:Immunoglobulin Fragments / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000235:genetics; D051379:Mice; D011233:Precipitin Tests; D011485:Protein Binding; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000276:immunology* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014407:Tumor Cells, Cultured
D007141:Immunoglobulin Fc Fragments; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D018719:Erb-b2 Receptor Tyrosine Kinases; D011993:Recombinant Fusion Proteins