牛抗菌肽Bac7-Bac5融合基因在大肠杆菌中的过表达,纯化及抑菌活性  

Overexpression of the Fusion Gene Encoding Bovine Antimicrobial Peptides Bac7-Bac5 in Escherichia coli,and Purification and Antimicrobial Activity of the Fusion Protein

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] 常月红[1] 刘慧芳[1] 周媛媛[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动物细菌病研究室,哈尔滨150001 [2]石河子大学,石河子832003

出　　处：《中国生物工程杂志》2007年第3期65-70,共6页China Biotechnology

摘　　要：牛抗菌肽Bac7和Bac5是一种线性阳离子小分子多肽,在机体天然免疫和获得性免疫中都发挥着重要作用。根据Gen bank中公布的牛抗菌肽bac7和bac5成熟肽基因序列,人工合成了融合基因Bac7-Bac5片段,克隆于原核表达载体pET32(a+)中构建了重组表达载体pET-B7-B5,将其转化于E.coli BL21(DE3)中实现了重组蛋白B7-B5(rB7-B5)的过表达,表达的rB7-B5以包涵体形式存在,rB7-B5表达量约占细菌总蛋白的36.6%,分子量大小为33kDa,与预测大小相符。以8mol/L尿素变性包含体后用Ni亲和层析柱纯化目的蛋白,经多步透析法复性的rB7-B5对猪胸膜肺炎放线杆菌和耐药性大肠杆菌具有很好的抑菌活性,为新型抗菌制剂的研制和开发奠定了基础。Bovine antimicrobial peptides Bac7 and Bac5 are linear cation micromolecular polypeptides, which play an important role in both innate and acquired immunity. Based on the gene sequences encoding mature bovine antimicrobial peptides bac7 and bac5 as registered in Genbank, the fragment of the fusion gene Bac7-Bac5 was synthesized and cloned into the prokaryotic expression vector pET32 （a ＋ ） to construct a recombinant expression vector, pET-BT-B5. The recombinant construct was then transformed into E. coli BL21 （DE3） to over-express the recombinant protein B7-B5 （rB7-B5）. The expressed rB7-B5 was localized in inclusion bodies and accounted for 36.6% of total bacterial protein. The molecular weight of rB7-B5 was 33kDa, a figure consistent with the predicted value. Following purification by Ni affinity chromatography and stepwise renaturation by dialysis, rBT-B5 showed sound antimicrobial activity in porcine actinobacillus pleuropneumoniae and antibiotics-resistant E. coli. So a basis for the research and development of novel antibacterial preparations were provided.

关 键 词：牛抗菌肽Bac7和Bac5 融合基因 重组表达 抑菌活性 

分 类 号：Q786[生物学—分子生物学]