Expression and characterization of Mac-1-FP fusion protein in CHO cells  

作　　者：刁飞 严鸣 朱晓燕 杨勇骥 刘辉 徐仁宝 

机构地区：[1]Department of Pathophysiology, College of Basic Medical Sciences,Second Military Medical University,Shanghai 200433,China [2]Department of Pathophysiology,College of Basic Medical Sciences,Second Military Medical University,Shanghai 200433,China [3]Department of Pathophysiology, College of Basic Medical Sciences , Second Military Medical University, Shanghai 200433, China [4]Department of Cell Biology,College of Basic Medical Sciences,Second Military Medical University,Shanghai 200433,China [5]International Cooperation Laboratory on Signal Transduction,Eastern Hepatobiliary Surgery Institute,Shanghai,200438

出　　处：《Journal of Medical Colleges of PLA(China)》2004年第6期321-324,共4页中国人民解放军军医大学学报（英文版）

基　　金：Supported by National Natural Science Foundation of China (No.30000068, 39730210)

摘　　要：Objective: To construct mammalian cell expression vectors for Mac-1 with CFP and YFP and apply FRET to study the dimerization and function of CD11 b( Mac-1 α subunit) and CD18(Mac-1 β subunit). Methods: The mammalian cell expression vector for CD11b fused with CFP at the carboxyl terminal was constructed to create recombinant plasmid of pCD11b-CFP. Then pCD11b-CFP was co-transfected with pYFP-CD18 into CHO cell, a fibroblast like cell line, as a target cell within which there are some signal pathways involved in inflammatory stimulation but without endogenous Mac-1. Then CHO cells stably expressing both CD11b-CFP and YFP-CD18 fusion proteins were selected by Western blot and laser scanning confocal microscope. Results: The cyan and yellow fluorescence in co-transfected positive CHO cells were observed under a fluorescence microscope. CHO-Mac-1-FP cells stably expressing both CD11b-CFP and YFP-CD18 fusion proteins were obtained as demonstrated by Western blot successfully. The adhesive activity of CHO-Mac-1-FP cells with CHO-1CAM-1 cells was increased markedly by treatment with PMA, suggesting the translocation of GD11b-CFP and YFP-CD18 to the plasma membrane in CHO-Mac-1-FP cells and dimerization of CD11b-CFP and YFP-CD18 just as the function of the wild type Mac-1. Conclusion: CHO-Mac-1-FP cells with adhesive activity are established successfully, thus CHO-Mac-1-FP cells may be useful for the study of Mac-1 by FRET and for other purposes.To construct mammalian cell expression vectors for Mac-1 with CFP and YFP and apply FRETto study the dimerization and function of CD1-1b(Mac-1α subunit) and CD18(Mac-1 β subunit). Methods: The mammaliancell expression vectorfor CD1 1b fused with CFP at the carboxyl terminal was constructed to create recombinant plasmid ofpCD1 1b-CFP.Then pCD11b-CFP was co-transfected with pYFP-CD18 into CHO cell, a fibroblast like cell line, as a targetcell within which there are some signal pathways involved in inflammatory stimulation but without endogenous Mac-1. ThenCHO cells stably expressing both CD11b-CFP and YFP-CD18 fusion proteins were selected by Western blot and laser scan-ning confocal microscope. Results: The cyan and yellow fluorescence in co-transfected positive CHO cells were observed un-der a fluorescence microscope. CHO-Mac-1-FP cells stably expressing both CD11 b-CFP and YFP-CD18 fusion proteins wereobtained as demonstrated by Western blot successfully. The adhesive activity of CHO-Mac-1-FP cells with CHO-ICAM-1cells was increased markedly by treatment with PMA, suggesting the translocation of CD11b-CFP and YFP-CD18 to the plas-ma membrane in CHO-Mac-1-FP cells and dimerization of CD11b-CFP and YFP-CD18 just as the function of the wild typeMac-1. Conclusion: CHO-Mac-1-FP cells with adhesive activity are established successfully, thus CHO-Mac-1-FP cellsmay be useful for the study of Mac-1 by FRET and for other purposes.

关 键 词：MAC-1 cyan fluorescent protein yellow fluorescent protein fusion protein 

分 类 号：Q26[生物学—细胞生物学]