枯草芽孢杆菌启动子的克隆及功能验证
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摘要
以pHT01穿梭质粒为骨架,构建以卡那霉素抗性基因为报告基因的启动子探针载体pHT-kan.利用该探针载体在大肠杆菌中克隆枯草芽孢杆菌168的启动子活性片段,挑取得到100个重组子.通过卡那霉素浓度梯度筛选出2个抗性最强的片段进行序列测定和分析,将启动子片段命名为BSP25、BSP31.将抗性最高的两个载体转入枯草芽孢杆菌168菌株,结果表明,它们可以在枯草芽孢杆菌中启动卡那霉素抗性基因的表达,重组菌株表现出卡那霉素抗性.
A shuttle promoter-probe vector pHT-kan was constructed with pHT01,it contains kanamycin resistance gene as repoter gene.The promoter fragments of Bacillus subtilis 168 were cloned in E.coli by pHT-kan,we get 100 recombinants.Then we increase the concentration of kanamycin to select the promoters with the highest kanamycin resistance,which was named BSP25 and BSP31.The two vectors with most efficient promoter fragments were transformed into B.subtilis 168 by electroporation,the kanamycin resistant gene was expressed in B.subtilis 168 with kanamycin resistance.
A shuttle promoter-probe vector pHT-kan was constructed with pHT01,it contains kanamycin resistance gene as repoter gene.The promoter fragments of Bacillus subtilis 168 were cloned in E.coli by pHT-kan,we get 100 recombinants.Then we increase the concentration of kanamycin to select the promoters with the highest kanamycin resistance,which was named BSP25 and BSP31.The two vectors with most efficient promoter fragments were transformed into B.subtilis 168 by electroporation,the kanamycin resistant gene was expressed in B.subtilis 168 with kanamycin resistance.
引文
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[13]Contreras J A R,Pedraza-Reyes M,Ordoez L G,et al.Replicative and integrative plasmids for production of human inter-feron gamma in Bacillus subtilis[J].Plasmid,2010,64:170-176.
[14]Kim J H,Hwang B Y,Roh J,et al.Comparison of PaprE,PamyE,and PP43 promoter strength forβ-galactosidase and staphy-lokinase expression in Bacillus subtilis[J].Biotechnology and Bioprocess Engineering,2008,3:313-318.
[2]Terpe K.Overview of bacterial expression systems for heterologous protein production:from molecular and biochemical funda-mentals to commercial systems[J].Appl Microbiol Biotechnol,2006,72:211-222.
[3]Park Y C,Jun S Y,Seo J H.Construction and characterization of recombinant Bacillus subtilis JY123 able to transport xylose ef-ficiently[J].Journal of Biotechnology,2012,161:402-406.
[4]夏雨.枯草芽孢杆菌食品级表达系统的构建和分泌表达研究[D].无锡:江南大学,2007.
[5]Vavrova L,Muchova K,Barak I.Comparison of different Bacillus subtilis expression systems[J].Research in Microbiology,2010,161:791-797.
[6]Simonen M,Palva I.Protein secretion in Bacillus species[J].Microbiological Reviews,1993,57(1):109-137.
[7]周冰,张惟材.枯草芽孢杆菌蛋白质分泌机制研究进展[J].生物技术通讯,2004,15(3):281-284.
[8]Zhang Ai-ling,Liu Hui,Yang Ming-ming,et al.Assay and characterization of a strong promoter element from B.subtilis[J].Biochemical and Biophysical Research Communications,2007,354:90-95.
[9]佟小雪,牛丹丹,陈献忠,等.地衣芽孢杆菌高温α-淀粉酶异源表达研究[J].生物技术,2009,19(5):11-13.
[10]Phan T T P,Nguyen H D,Schumann W.Novel plasmid-based expression vectors for intra-and extracellular production ofrecombinant proteins in Bacillus subtilis[J].Protein Expression and Purification,2006,46:189-195.
[11]Xue Gang-ping,Johnson J S,Dalrymple B P.High osmolarity improves the electro-transformation efficiency of the gram-positive bacteria Bacillus subtilis and Bacillus licheniformis[J].Journal of Microbiological Methods,1999,34:183-191.
[12]Liu Sen-lin,Du Kun.Enhanced expression of an endoglucanase in Bacillus subtilis by using the sucrose-inducible sacB pro-moter and improved properties of the recombinant enzyme[J].Protein Expression and Purification,2012,83:164-168.
[13]Contreras J A R,Pedraza-Reyes M,Ordoez L G,et al.Replicative and integrative plasmids for production of human inter-feron gamma in Bacillus subtilis[J].Plasmid,2010,64:170-176.
[14]Kim J H,Hwang B Y,Roh J,et al.Comparison of PaprE,PamyE,and PP43 promoter strength forβ-galactosidase and staphy-lokinase expression in Bacillus subtilis[J].Biotechnology and Bioprocess Engineering,2008,3:313-318.