牛抗菌肽Bac7-Bac5-β defense串联基因在昆虫杆状病毒系统中的表达及其产物的活性分析  

Expression of the Fusion Genes Encoding Bovine Antimicrobial Peptides Bac7-Bac5-βdefense in Insect Rod-shaped Virus Systemand Analysis of the Fusion Protein Antimicrobial Activity

作　　者：赵昆[1] 刘思国[1] 王春来[1] 宫强[1] 迟磊[1] 刘建东[1] 王勇[1] 云孟克[1] 刘慧芳[1] 孙延鸣[2] 

机构地区：[1]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,哈尔滨150001 [2]石河子大学动物科技学院,石河子832003

出　　处：《农业生物技术学报》2008年第3期380-384,共5页Journal of Agricultural Biotechnology

摘　　要：根据GenBank中公布的牛抗菌肽bac7、bac5和βdefense(LAP基因)成熟肽基因序列,人工合成了抗菌肽融合基因Bac7-Bac5-βdefense,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-Bac7-Bac5-βdefense,转化DH10Bac大肠杆菌(Escherichia coli)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-Bac7-Bac5-βdefense,转染昆虫草地夜蛾(Bombyx mandarina)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,表达量约占细胞总蛋白的10.6%。体外抑菌实验表明重组的r Bac7-Bac5-βdefense蛋白对大肠杆菌,具有抑菌活性。Based on the gene sequences encoding mature bovine antimicrobial peptides bac7, bac5 and β- defense （LAP） registered in GenBank, the fragment of the fusion gene Bac7-Bac5-β defense 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-Bac7-Bac5-β defense was then transformed into DH10BAC^TM competent cell. High molecular weight DNA, the recombinant baemid, was prepared fi＇om the ovemight cultures of selected Escherichia coil DH10BAC^TM colonies. By transfecting spodoptera fragiperda 9 （Sf9） cells with the recombinant bacmid, the recombinant baculovirus fi＇om the supematant was obtained, which could further infect Sf9 cells and express the target protein. The expression of rBac7-Bac5-β defense was confirmed by 12% SDS-PAGE and accounted for 10.6% of total bacterial protein, its molecular weight of rBac7-Bac5-β defense was 20 kD.In vitro antimierobial test showed that rBac7-Bac5-β defense antimicrobial had activity in E. coli.

关 键 词：牛抗菌肽 Bac7 Bac5和β defiense融合基因 昆虫杆状病毒表达系统 SPODOPTERA fragiperda 9(Sf9)细胞 重组表达 抑菌活性 

分 类 号：S188[农业科学—农业基础科学]