| Human defensins is one kind of cations antibacterial peptide and rich in Arginine,it can mainly divided into two classes:human alpha-defensins and human beta-defensins,all of them have broad spectrum antimicrobial activities,cytotoxicity,anti-tumor,immunologic and other functions.And human defensins is one of an important micromolecule polypeptide and component of natural immune system.Because of it special antibacterial mechanism,the pathogenic microorganisms hardly to generate resistance to human defensins.So,in recently years,more and more researchers put their attentions on the study of human defensins.Through the study of the antimicrobial activities of human defensins to pathogenic bacteria,it may provide a direction for us to overcome the problem of bacterial resistance.Objective To established a cell line that expresses hBDl stablly, and detected the antimicrobial activity of the hBDl to the multidrug resistant bacterial strains.Methods Recombinant plasmid was introduced into COS-7cells by lipofectamine, COS-7cells were selected in culture medium containing G418to acquired the monoclonal cell lines,total RNA were extracted from the cultured cells,expression levels of hBDl mRNA was identified by RT-PCR,collected the supernatant solution of the cultured cell,expression levels of protein was identified by Western blotting.Put the expression products and resistant organisms mixed together,after incubation in different times in37℃,coating the mixtures in LB flat,then obtained the ratios between colonies number of experimental groups and colonies number of control groups,put those ratios as the survival rate of the drug resistance bacterias.Results1.The monoclonal cell lines had obtained after screened with G418,the hBDl gene could be detected both at transcriptional and protein levels;2.When the bacteria liquid concentration is2×104个/ml,under the influence of expression product hBDl,survival rate of multidrug-resistant Acinetobacter baumannii,multidrug-resistant Escherichia coli and multidrug-resistant Klebsiella pneumoniae could reduced to9%,22%and50%;3.When the bacteria liquid concentration is1×104个/ml,under the influence of expression product hBDl,survival rate of multidrug-resistant Acinetobacter baumannii,multidrug-resistant Escherichia coli and multidrug-resistant Klebsiella pneumoniae could reduced to12%,19%and46%;4.Survival rate of multidrug-resistant Stenotrophomonas maltophilia is not have apparente difference with the control group.Conclusion The stablly-transfected cell line of hBDl was successfully constructed,and the expression products of hBDl showed the antimicrobial activity toward multidrug resistant bacterial strains. Objective:To explore O-CHCS’s feasibility of being a gene vector for transfecting hBD2, using O-CHCS with the recombinant plasmid pCMV-hBD2transfeced L929cells.Methods:We constructed plasmid pCMV-hBD2, and the plasmid was introduced into L929cells by O-CHCS.Total RNA were extracted from the cultured cells, RT-PCR were performed with specific primers for hBD2and RT-PCR amplification products were done with agarose gel electrophoresis;the expression of hBD2protein was verified by Western blot;collecting the mediums from the cultured cells and detecting the antibacterial activity of purpose protein by Kirby-Bauer disk diffusion method, at the same time put the Lipofect as the positive control.Results:RT-PCR amplification products by agarose gel electrophoresis were the same size as the target gene by observation;hBD2gene expression at the protein level was detected by Western blot;mediums from the cultured cells transfecting with the pCMV-hBD2could form antibacterial circle against the Staphylococcus aureus, and these results were as same as the Lipofect.Conclusion:The eukaryotic expression vector pCMV-hBD2was successfully constructed and was introduced to the L929cells, by doing this we verified O-CHCS could be a gene vector for hBD2, which provided a economic and convenient way to artificial synthesis of hBD2. |
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