摘要
为探讨鹅β-防御素7（Av BD7）的生物学特性,将鹅Av BD7基因亚克隆到大肠杆菌原核表达载体p Pro EX HTa的EcoRⅠ和XhoⅠ双酶切位点上,构建重组表达质粒p Pro EX-Av BD7,将重组质粒转化到大肠杆菌Rosetta感受态细胞中,用异丙基-β-D-硫代半乳糖苷（IPTG）对其菌液进行诱导表达。经N-三（羟甲基）甲基甘氨酸十二烷基硫酸钠聚丙烯酰胺凝胶电泳（Tricine-SDS-PAGE）分析,该重组蛋白大小为10-15 ku,与预期大小结果一致。重组鹅Av BD7蛋白纯化后,通过菌落计数的方法测定其体外抗大肠杆菌、鸡白痢沙门氏菌、金黄色葡萄球菌、四联球菌、枯草芽孢杆菌等抑菌活性,盐离子浓度对其抗菌活性的影响及其对鸡红细胞的溶血活性。结果显示,重组鹅Av BD7蛋白对所测定的5种细菌均有显著抗菌活性（P〈0.05）,且其抑菌活性随蛋白浓度的增加而增强。高浓度盐离子（150 mmol/L）显著抑制重组鹅Av BD7蛋白的抗菌活性（P〈0.05）。重组鹅Av BD7蛋白对鸡红细胞没有溶血活性（P〉0.05）。由此可见,重组鹅Av BD7蛋白具有广谱抗菌活性,高浓度盐离子显著降低其抗菌活性,且该重组蛋白不具有溶血活性。
The objective of the study was to investigate the biological property of goose avian β-defensin（AvBD） 7.The gene of AvBD7 was cloned into EcoR I and Xho I sites of pProEX HTa vector to construct recombinant plasmid pProEX-AvBD7.The recombinant plasmid was transformed into Escherichia coli Rosetta and the bacteria was induced with isopropy-β-D-thiogalactoside（IPTG）.It was analyzed by tricine sodium dodecyl sulfate polyacrylamide gel electrophoresis（Tricine-SDS-PAGE） that the recombinant protein size was 10 to 15 ku,which was the same as the expected size.Antimicrobial activity of the recombinant fusion protein a—gainst Escherichia coli,Salmonella pullorum,Staphylococcus aureus,Micrococcus tetragenus and Bacillus subtilis was measured in vitro.In addition,the effect of ionic strength on the antibacterial activity and hemolytic activity of the recombinant fusion protein were investigated.The results showed that the recombinant goose AvBD7 protein exhibited significant antimicrobial activity against the five bacteria investigated（P〈0.05）,and antibacterial activity enhanced with the increasing of protein concentration.The antibacterial activity of recombinant goose AvBD7 protein was significantly inhibited by high salt concentration（150 mmol/L）（P〈0.05）.In addition,recombinant goose AvBD7 protein showed little hemolytic activity against chicken erythrocytes（P〉0.05）.In conclusion,recombinant goose AvBD7 protein exhibits extensive antimicrobial activity.High salt concentration significantly decrease its antibacterial activity.In addition,the recombinant goose AvBD7 protein shows little hemolytic activity.
出处
《动物营养学报》
CAS
CSCD
北大核心
2016年第4期1114-1121,共8页
CHINESE JOURNAL OF ANIMAL NUTRITION
基金
教育部博士点基金项目(20122325110016)
哈尔滨市科技创新人才研究专项资金项目(2013RFXXJ019)
黑龙江省应用技术研究与开发计划项目(PC13S02)
关键词
鹅β-防御素7
重组蛋白
抗菌活性
goose β-defensin 7
recombinant protein
antimicrobial activity
