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2025年4月5日 星期六
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牛抗菌肽Bac7-Bac5-β串联基因在昆虫杆状病毒系统中的表达及表达产物的研究
刘慧芳 赵昆
中国农业科学院哈尔滨兽医研究所 兽医生物技术国家重点实验室 动物细菌病研究室
Expression of the fusion gene encoding bovine antimicrobial peptides Bac7-Bac5-β defense in insect rod-shaped virus system, and antimicrobial activity of the fusion protein
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摘要 根据Gen bank中公布的牛抗菌肽bac7、bac5和防御素(LAP)成熟肽基因序列,人工合成了抗菌肽融合基因Bac7-Bac5-β,克隆到BAC-TO-BACTM重组杆状病毒表达系统的pFAST HTb载体中,构建了重组转座载体pFASTBac-B7-B5-β,转化DH10Bac大肠杆菌感受态细胞,PCR方法筛选阳性菌落,抽提大分子质粒DNA,获得重组杆状病毒穿梭载体Bacmid-B7-B5-B,转染昆虫草地夜蛾Sf9细胞出现病变后,收集含有重组病毒颗粒的培养上清,重新感染草地夜蛾Sf9单层细胞,收集Sf9细胞超声破碎,12%SDS-聚丙烯酰胺凝胶电泳可见表达的融合蛋白带,分子量大小为20kD,与预测大小相符,表达量约占细胞总蛋白的10.6%。体外抑菌实验表明重组的rB7-B5-B蛋白对大肠杆菌具有抑菌活性,本研究为新型抗菌制剂的研制和开发奠定了基础。
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刘慧芳赵昆
Abstract:In the present study, based on the gene sequences encoding mature bovine antimicrobial peptides bac7、bac5 and - defense(LAP) as registered in Genbank, the fragment of the fusion gene Bac7-Bac5-βdefense was synthesized and cloned into the pFASTHTb vector of BAC-TO-BACTM recombinant baculovirus expression system to construct a recombinant expression vector, The plasmid pFASTBac-B7-B5- was then transformed into DH10BACTM competent cell. High molecular weight DNA, the recombinant bacmid, was prepared from the overnight cultures of selected E. coli DH10BACTM colonies. By transfecting Sf9 cells with the recombinant bacmid the recombinant baculovirus from the superenatant was obtained, which could further infect Sf9 cells obtained and express the target protein. The expression of rB7-B5- was confirmed by 12% SDS-PAGE and accounted for 10.6% of total bacterial protein,the molecular weight of rB7-B5- was 20kD, a figure consistent with the predicted value. rB7-B5- showed antimicrobial activity in E. coli. The present study provides basis for the research and development of novel antibacterial preparations.
收稿日期: 2007-04-10     
通讯作者: 赵昆   
引用本文:   
刘慧芳 赵昆. 牛抗菌肽Bac7-Bac5-β串联基因在昆虫杆状病毒系统中的表达及表达产物的研究[J]. , 2008, 16(3): 0-.
. Expression of the fusion gene encoding bovine antimicrobial peptides Bac7-Bac5-β defense in insect rod-shaped virus system, and antimicrobial activity of the fusion protein. , 2008, 16(3): 0-.
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http://journal05.magtech.org.cn/Jwk_ny/CN/      或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2008/V16/I3/0
 
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