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
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.
