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鹅β-防御素3基因的分离、鉴定及其表达产物的生物学特性
引用本文:张名岳,周财源,韩宗玺,邵昙昊,刘胜旺,马得莹.鹅β-防御素3基因的分离、鉴定及其表达产物的生物学特性[J].生物工程学报,2011,27(12):1711-1721.
作者姓名:张名岳  周财源  韩宗玺  邵昙昊  刘胜旺  马得莹
作者单位:1. 东北农业大学动物营养研究所,哈尔滨150030;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/禽传染病研究室,哈尔滨150001
2. 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/禽传染病研究室,哈尔滨,150001
3. 东北农业大学动物营养研究所,哈尔滨,150030
摘    要:为了克隆鹅β-防御素(AvBD)3基因,并在原核表达重组鹅AvBD3蛋白,进一步研究鹅AvBD3蛋白的生物学特性,利用RT-PCR方法从鹅脾脏和法氏囊组织中扩增到鹅AvBD3基因片段,其cDNA片段大小为182 bp,编码60个氨基酸残基.经同源性分析发现鹅AvBD3氨基酸序列与鸡AvBD3氨基酸序列同源性最高,为100%.将该基因亚克隆到原核表达载体pGEX-6p-1的BamH Ⅰ和SalⅠ双酶切位点上,构建重组表达质粒pGEX-goose AvBD3.将重组质粒转化大肠杆菌BL21,于37℃用IPTG诱导表达,SDS-PAGE电泳表明,重组鹅AvBD3蛋白在原核高效表达(分子量约31 kDa).该重组蛋白经纯化后测定其体外抗菌活性与理化特性,结果显示,重组鹅AvBD3蛋白具有广谱的抗菌活性,对12种细菌,包括革兰氏阳性菌和革兰氏阴性菌均具有抑菌作用.高盐离子浓度显著降低重组鹅AvBD3蛋白的抗菌活性.此外,该重组蛋白的溶血活性极低,并对酸碱度具有较高的稳定性.

关 键 词:鹅AvBD3  融合蛋白  抗菌活性  盐浓度  溶血活性  酸碱度
收稿时间:2011/4/27 0:00:00

Isolation, identification and bioactivity characterization of goose avian beta-defensin 3
Mingyue Zhang,Caiyuan Zhou,Zongxi Han,Tanhao Shao,Shengwang Liu and Deying Ma.Isolation, identification and bioactivity characterization of goose avian beta-defensin 3[J].Chinese Journal of Biotechnology,2011,27(12):1711-1721.
Authors:Mingyue Zhang  Caiyuan Zhou  Zongxi Han  Tanhao Shao  Shengwang Liu and Deying Ma
Institution:Institute of Animal Nutrition, Northeast Agricultural University, Harbin 150030, China; Division of Avian Infectious Disease, National Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Scienc;Institute of Animal Nutrition, Northeast Agricultural University, Harbin 150030, China; Division of Avian Infectious Disease, National Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Scienc;Division of Avian Infectious Disease, National Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, China;Division of Avian Infectious Disease, National Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, China;Division of Avian Infectious Disease, National Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, China;Institute of Animal Nutrition, Northeast Agricultural University, Harbin 150030, China
Abstract:The objective of the study was to clone avian beta-defensin (AvBD) 3 gene from goose tissues, express the recombinant AvBD3 protein in Escherichia coli, and determine its antimicrobial activity. The mRNA of goose AvBD3 was cloned from spleen and bursa of Fabricius of the gooses by RT-PCR. The sequence analysis showed that the genefragment of AvBD3 contained 182 bp, and encoded 60 amino acids. Homology analysis showed that goose AvBD3 shared the highest percentage of amino acid homology (100%) with chicken AvBD3. The cDNA of goose AvBD3 was sub-cloned into BamH I and Sal I sites of pGEX-6p-1 vector to construct recombinant plasmid pGEX-goose AvBD3. The recombinant plasmid was transformed into E. coli BL21 and the bacteria was induced with IPTG It was demonstrated by SDS-PAGE that a 31 kDa protein which was equal to goose AvBD3 protein in molecular weight was highly expressed. The purified recombinant goose AvBD3 exhibited extensive antimicrobial activity against twelve bacteria strains, including Gram-positive and Gram-negative investigated. At high salt ions conditions, antimicrobial activity of recombinant goose AvBD3 protein against both Staphylococcus aureus and Pasteurella multocida decreased significantly. In addition, hemolysis activity of the recombinant protein was extremely low, and the recombinant protein remained antimicrobial activity under different pH values.
Keywords:goose AvBD3  fusion protein  antibacterial activity  salt concentration  hemolysis activity  pH values
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