Objective: To express the recombinant human bone morphogenetic protein-7(rhBMP-7) in Chinese hamster ovary(CHO) cells, and to establish the in vitro biological activity assay of rhBMP-7.Methods: Human BMP-7 cDNA was s...Objective: To express the recombinant human bone morphogenetic protein-7(rhBMP-7) in Chinese hamster ovary(CHO) cells, and to establish the in vitro biological activity assay of rhBMP-7.Methods: Human BMP-7 cDNA was subcloned into p114 mammalian expression vector and transfected to CHO cells by using the Lipofectamine 2000 transfection method. CHO cell supernatants were harvested and analyzed to identify the molecule mass of secreted rhBMP-7 and examine its biological activity in vitro to stimulate the synthesis of alkaline phophatase(ALP), a characteristic of osteoblast phenotypes. Results: rhBMP-7 was produced stably in CHO cells, as a processed mature disulfide-linked homodimer, with an apparent molecular mass of 36 000. Examination of the rhBMP-7 biological activity showed that rhBMP-7 specifically stimulated the production of ALP(4-fold increase at 100 ng of rhBMP-7/ml). Conclusion: The rhBMP-7 from CHO expression system has significant biological activity in induction of osteoblast phenotype, which demonstrates rhBMP-7 has the potential bone regeneration activity.展开更多
Objective: To prepare secretary recombinant human neutrophil peptidel (HNP1)and test its antimicrobial activity. Methods: The eukaryotic expression vector pcDNA3. 1/V5-His-TOPO-HNP1 was cotransfected with plasmid pDCH...Objective: To prepare secretary recombinant human neutrophil peptidel (HNP1)and test its antimicrobial activity. Methods: The eukaryotic expression vector pcDNA3. 1/V5-His-TOPO-HNP1 was cotransfected with plasmid pDCH1P11 carrying dhfr gene into dhfr- negative CHO (CHO-dhfr- ) cells and recombinant protein was verified by ELISA; G418 selective medium was used to screen the stably expressing cell clones followed by serial passages in 5×10-8 mol/L and 5×10 mol/L methotrexate (MTX) for gene amplification. Finally 4 cell clones with high expression level were obtained and confirmed by ELISA, RT-PCR and IFA. The bacteriastatic activity of concentrated supernatants was tested in vitro as well. Results: The expression level of recombinant HNPl ranged from 18.85 mg/L·48 h to 47.46 mg/L·48 h per 106 cells that was almost 200-fold increase than that in G418 selective medium. 303 bp segments were amplified from 4 stably tranfec tant clones which matched the length of HNPl cDNA by RT-PCR. Strong fluorescence was visible in cell plasma in the sta blly transfectant cells by IFA. K-B disc agar diffusion test showed obvious bacteriastatic diffusion on MH plate of E. coli. Conclusion: HNP1cDNA can be strongly expressed in CHO-dhfr- cells, which supernatants exhibited high inhibitive effect against bacteria.展开更多
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 mammalia...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.展开更多
文摘Objective: To express the recombinant human bone morphogenetic protein-7(rhBMP-7) in Chinese hamster ovary(CHO) cells, and to establish the in vitro biological activity assay of rhBMP-7.Methods: Human BMP-7 cDNA was subcloned into p114 mammalian expression vector and transfected to CHO cells by using the Lipofectamine 2000 transfection method. CHO cell supernatants were harvested and analyzed to identify the molecule mass of secreted rhBMP-7 and examine its biological activity in vitro to stimulate the synthesis of alkaline phophatase(ALP), a characteristic of osteoblast phenotypes. Results: rhBMP-7 was produced stably in CHO cells, as a processed mature disulfide-linked homodimer, with an apparent molecular mass of 36 000. Examination of the rhBMP-7 biological activity showed that rhBMP-7 specifically stimulated the production of ALP(4-fold increase at 100 ng of rhBMP-7/ml). Conclusion: The rhBMP-7 from CHO expression system has significant biological activity in induction of osteoblast phenotype, which demonstrates rhBMP-7 has the potential bone regeneration activity.
文摘Objective: To prepare secretary recombinant human neutrophil peptidel (HNP1)and test its antimicrobial activity. Methods: The eukaryotic expression vector pcDNA3. 1/V5-His-TOPO-HNP1 was cotransfected with plasmid pDCH1P11 carrying dhfr gene into dhfr- negative CHO (CHO-dhfr- ) cells and recombinant protein was verified by ELISA; G418 selective medium was used to screen the stably expressing cell clones followed by serial passages in 5×10-8 mol/L and 5×10 mol/L methotrexate (MTX) for gene amplification. Finally 4 cell clones with high expression level were obtained and confirmed by ELISA, RT-PCR and IFA. The bacteriastatic activity of concentrated supernatants was tested in vitro as well. Results: The expression level of recombinant HNPl ranged from 18.85 mg/L·48 h to 47.46 mg/L·48 h per 106 cells that was almost 200-fold increase than that in G418 selective medium. 303 bp segments were amplified from 4 stably tranfec tant clones which matched the length of HNPl cDNA by RT-PCR. Strong fluorescence was visible in cell plasma in the sta blly transfectant cells by IFA. K-B disc agar diffusion test showed obvious bacteriastatic diffusion on MH plate of E. coli. Conclusion: HNP1cDNA can be strongly expressed in CHO-dhfr- cells, which supernatants exhibited high inhibitive effect against bacteria.
基金Supported by National Natural Science Foundation of China (No.30000068, 39730210)
文摘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.
