[Eukaryotic expression of P-selectin functional segment on the membrane of CHO].
Cell adhesive molecular P-selectin was cloned, expressed and anchored on CHO cell membrane through GPI for selection specific antibodies. Total human platelet RNA was extracted and the functional segment of P-selectin gene was cloned by RT-PCR. The P-selectin functional segment gene was cloned into a eukaryotic expression vector pMCEw2-GPI containing an attenuated neo gene together with a downstream GPI, which was synthesized by overlapping PCR. The recombinant plasmid pMCEw2-GPI-P-selectin was then transfected to CHO(dhfr-) cells and screened with G418. ELISA, western-blot and immunofluorescence were carried out to detect the stability of P-selection expression on cell membrane. These results provided a necessary basis for the following study of selection the antibodies targeting P-selectin.
Lou
Xiaofang
X
Institute of Biotechnology, Academy of Military Medical Science, Beijing 100071, China.
Wang
Shuang
S
Du
Weishi
W
Yu
Rui
R
Yu
Weiyuan
W
Sun
Zhiwei
Z
D000818:Animals; D000906:Antibodies / Q000378:metabolism; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D004815:Epidermal Growth Factor / Q000378:metabolism; D017261:Glycosylphosphatidylinositols / Q000378:metabolism; D037102:Lectins / Q000378:metabolism; D019007:P-Selectin / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D012313:RNA / Q000378:metabolism; D014162:Transfection
D000906:Antibodies; D017261:Glycosylphosphatidylinositols; D037102:Lectins; D019007:P-Selectin; D004815:Epidermal Growth Factor; D012313:RNA