目的:在毕赤酵母中表达新疆家蚕抗菌肽基因(Cecropin-XJ)并检测其活性。方法:根据作者实验室已克隆获得的新疆家蚕抗菌肽Cecropin-XJ基因设计引物,通过PCR方法扩增Cecropin-XJ,将PCR产物和表达载体pPIC9K用EcoRⅠ及NotⅠ双酶切,构建重...目的:在毕赤酵母中表达新疆家蚕抗菌肽基因(Cecropin-XJ)并检测其活性。方法:根据作者实验室已克隆获得的新疆家蚕抗菌肽Cecropin-XJ基因设计引物,通过PCR方法扩增Cecropin-XJ,将PCR产物和表达载体pPIC9K用EcoRⅠ及NotⅠ双酶切,构建重组表达质粒pPIC9K-Cecropin-XJ,酶切及测序正确后,电转化到毕赤酵母GS115,对分泌表达的重组蛋白进行活性检测。结果:PCR扩增获得192 bp Cecropin-XJ,成功构建pPIC9K-Cecropin-XJ,优化诱导条件证明在pH 6的BMMY培养液中,0.5%甲醇诱导约48h后,获得的表达产物活性较强,对多种革兰氏阴性菌和阳性菌具有抗菌活性,在100℃条件下,其活性可维持100min以上。结论:新疆家蚕抗菌肽在毕赤酵母中分泌表达,为大规模发酵生产奠定了基础。展开更多
Background CecropinXJ belongs to cecropinB, which is the most potent antibacterial peptide found naturally The aim of this study was to investigate the effects of cecropinXJ on growth and adherence of oral cariogenic ...Background CecropinXJ belongs to cecropinB, which is the most potent antibacterial peptide found naturally The aim of this study was to investigate the effects of cecropinXJ on growth and adherence of oral cariogenic bacteriaMethods Four oral cariogenic bacteria (Streptococcus mutans, Lactobacillus acidophilus, Actinomyces viscosus and Actinomyces naeslundii) were chosen for this experiment The minimal inhibitory concentrations (MICs) and reductive percent of bacterial growth were used to assay the antibacterial activity of cecropinXJ Mammalian cytotoxicity of cecropinXJ was tested with human periodontal membrane fibroblasts by tetrazolium (MTT) colorimetric assay The bacterial morphological changes induced by cecropinXJ were examined on scanning electron microscope (SEM) The influence of cecropinXJ on bacterial adhesion to salivacoated hydroxyapatite (SHA) was measured by scintillation countingResults The MICs of cecropinXJ for inhibition of the growth of four bacteria ranged from 40 to 428 μmol/L with the highest susceptible to A naeslundii and the lowest susceptible to L acidophilus At pH 68, 55 and 82, 1/2 MIC of cecropinXJ reduced the number of viable bacteria by 409%, 678% and 328% for S mutans and by 281%, 572% and 379% for L acidophilus The activities against Smutans and L acidophilus increased at pH 55 compared with pH 68 (P<001, respectively) In present of 50% saliva, 1/2 MIC of the peptide decreased the direct count of viable cells by 292% and 144% for S mutans and L acidophilus, respectively (P<001 and P>005, respectively), whereas almost no reduction counts were detected in the presence of 20% serum for both bacteria (P>005, respectively) Mammalian cytotoxicity of cecropinXJ from 10 to 100 μmol/L exhibited no cytotoxicity against human periodontal membrane fibroblasts (P>005) Bacterial morphological changes induced by MIC of cecropinXJ examined on SEM showed cell surface disruption Furthermore, the ability of A naeslundii adhesion to SHA decreased significantly with MIC of cecropinXJ for 10 and 20 minutes (P=0001 and 0000, respectively), and S mutans, A viscosus to SHA decreased significantly with MIC of cecropinXJ for 20 minutes( P=0000, respectively) Conclusions CecropinXJ exhibited bactericidal action against cariogenic pathogens, and the antibacterial activity enhanced in the acid environment The results also demonstrate that cecropinXJ prevents S mutans and actinomyces adsorption to SHA These findings suggest that CecropinXJ may have potential to prevent caries展开更多
文摘目的:在毕赤酵母中表达新疆家蚕抗菌肽基因(Cecropin-XJ)并检测其活性。方法:根据作者实验室已克隆获得的新疆家蚕抗菌肽Cecropin-XJ基因设计引物,通过PCR方法扩增Cecropin-XJ,将PCR产物和表达载体pPIC9K用EcoRⅠ及NotⅠ双酶切,构建重组表达质粒pPIC9K-Cecropin-XJ,酶切及测序正确后,电转化到毕赤酵母GS115,对分泌表达的重组蛋白进行活性检测。结果:PCR扩增获得192 bp Cecropin-XJ,成功构建pPIC9K-Cecropin-XJ,优化诱导条件证明在pH 6的BMMY培养液中,0.5%甲醇诱导约48h后,获得的表达产物活性较强,对多种革兰氏阴性菌和阳性菌具有抗菌活性,在100℃条件下,其活性可维持100min以上。结论:新疆家蚕抗菌肽在毕赤酵母中分泌表达,为大规模发酵生产奠定了基础。
文摘Background CecropinXJ belongs to cecropinB, which is the most potent antibacterial peptide found naturally The aim of this study was to investigate the effects of cecropinXJ on growth and adherence of oral cariogenic bacteriaMethods Four oral cariogenic bacteria (Streptococcus mutans, Lactobacillus acidophilus, Actinomyces viscosus and Actinomyces naeslundii) were chosen for this experiment The minimal inhibitory concentrations (MICs) and reductive percent of bacterial growth were used to assay the antibacterial activity of cecropinXJ Mammalian cytotoxicity of cecropinXJ was tested with human periodontal membrane fibroblasts by tetrazolium (MTT) colorimetric assay The bacterial morphological changes induced by cecropinXJ were examined on scanning electron microscope (SEM) The influence of cecropinXJ on bacterial adhesion to salivacoated hydroxyapatite (SHA) was measured by scintillation countingResults The MICs of cecropinXJ for inhibition of the growth of four bacteria ranged from 40 to 428 μmol/L with the highest susceptible to A naeslundii and the lowest susceptible to L acidophilus At pH 68, 55 and 82, 1/2 MIC of cecropinXJ reduced the number of viable bacteria by 409%, 678% and 328% for S mutans and by 281%, 572% and 379% for L acidophilus The activities against Smutans and L acidophilus increased at pH 55 compared with pH 68 (P<001, respectively) In present of 50% saliva, 1/2 MIC of the peptide decreased the direct count of viable cells by 292% and 144% for S mutans and L acidophilus, respectively (P<001 and P>005, respectively), whereas almost no reduction counts were detected in the presence of 20% serum for both bacteria (P>005, respectively) Mammalian cytotoxicity of cecropinXJ from 10 to 100 μmol/L exhibited no cytotoxicity against human periodontal membrane fibroblasts (P>005) Bacterial morphological changes induced by MIC of cecropinXJ examined on SEM showed cell surface disruption Furthermore, the ability of A naeslundii adhesion to SHA decreased significantly with MIC of cecropinXJ for 10 and 20 minutes (P=0001 and 0000, respectively), and S mutans, A viscosus to SHA decreased significantly with MIC of cecropinXJ for 20 minutes( P=0000, respectively) Conclusions CecropinXJ exhibited bactericidal action against cariogenic pathogens, and the antibacterial activity enhanced in the acid environment The results also demonstrate that cecropinXJ prevents S mutans and actinomyces adsorption to SHA These findings suggest that CecropinXJ may have potential to prevent caries
