新城疫载体系统表达天蚕素AD抗菌肽及其抑菌效果  

Expression of cecropin AD in Newcastle disease virus vector system and its bacteriostatic effect

作　　者：任项霏 冯贺龙 徐英英 李丽 杨宏春 王鑫 商雨[2,3,4,5] 阳柳君 周祖涛 李自力 刘梅[1,6] 罗青平 胡思顺[1,6] 温国元 REN Xiangfei;FENG Helong;XU Yingying;LI Li;YANG Hongchun;WANG Xin;SHANG Yu;YANG Liujun;ZHOU Zutao;LI Zili;LIU Mei;LUO Qingping;HU Sishun;WEN Guoyuan(College of Veterinary Medicine/College of Animal Science and Technology,Huazhong Agricultural University,Wuhan 430070,China;Institute of Animal Husbandry and Veterinary Medicine,Hubei Academy of Agricultural Sciences,Wuhan430064,China;Key Laboratory of Prevention and Control Agents for Animal Bacteriosis,Ministry of Agriculture and Rural Affairs,Wuhan 430064,China;Hubei Provincial Key Laboratory of Livestock and Poultry Pathogen Microbiology,Wuhan 430064,China;Hubei Hongshan Laboratory,Wuhan 430070,China;Hubei Provincial Key Laboratory of Preventive Veterinary Medicine,Wuhan 430070,China)

机构地区：[1]华中农业大学动物科学技术学院/动物医学院,湖北武汉430070 [2]湖北省农业科学院畜牧兽医研究所,湖北武汉430064 [3]农业农村部畜禽细菌病防治制剂创制重点实验室,湖北武汉430064 [4]畜禽病原微生物学湖北省重点实验室,湖北武汉430064 [5]湖北洪山实验室,湖北武汉430070 [6]湖北省预防兽医学重点实验室,湖北武汉430070

出　　处：《中国兽医学报》2023年第9期1820-1827,共8页Chinese Journal of Veterinary Science

基　　金：国家现代农业产业技术体系专项资助项目(CARS-41-G13);湖北省农业科技创新中心资助项目(2019-620-000-001-17);湖北省科技创新项目重大专项资助项目(2020ABA016);湖北省农业科学院青年科学基金资助项目(2022NKYJJ10);鸭源支原体分离鉴定及其灭活疫苗和亚单位疫苗研发横向课题资助项目(707121067)。

摘　　要：为探究天蚕素AD抗菌肽是否能在新城疫病毒(NDV)载体系统中表达,且通过该种表达系统得到的CAD是否有生物活性。采用同源重组的方式将CAD插入至NDV载体pTS09-C中,并利用反向遗传操作系统成功拯救出重组病毒rTS-CAD,该重组病毒能在SPF鸡胚中稳定增殖。通过生物学特性分析,在NDV TS09-C株的P和M基因之间插入CAD基因并未改变其的弱毒和增殖特性。间接免疫荧光试验证实重组病毒能在BHK-21细胞中稳定表达CAD抗菌肽。抑菌试验结果显示,NDV载体系统表达的CAD对金黄色葡萄球菌(G+)和大肠杆菌(G-)均有一定的抑制作用:在微孔板法抑菌试验中,重组病毒表达的CAD抗菌肽对金黄色葡萄球菌的抑制率为79.6%~91.7%,对大肠杆菌的抑制率为87.5%~89.8%;在与DF-1细胞共培养试验中,重组病毒表达的CAD抗菌肽对金黄色葡萄球菌的抑制率可以达到74.5%~77.7%,对大肠杆菌的抑制率为35.0%~45.0%。本研究表明,NDV载体系统能够成功表达出有活性的CAD抗菌肽,且有良好的抑菌效果。这一研究为探索NDV载体表达抗菌肽和进一步研究CAD的局部效应奠定了一定的理论基础。This study aims to study whether the cecropin AD(CAD)antimicrobial peptide can be expressed in the Newcastle disease virus vector system,and whether the CAD obtained through this expression system had biological activity.CAD was inserted into the NDV pTS09-C by homologous recombination,and the recombinant virus was rescued by reverse genetic operating system,the recombinant virus can stably proliferate in SPF chicken embryo.Indirect immunofluorescence assay confirmed that the recombinant virus could stably express CAD antimicrobial peptide in BHK-21cells.Through biological characterization,the insertion of CAD genes between P and M genes of NDV TS09-C strain did not alter its virulence and proliferative characteristics.The results of bacteriostatic test showed that CAD expressed by NDV vector system had a certain inhibitory effect on Staphylococcus aureus(G+)and Escherichia coli(G-).In the microplate bacteriostatic test,the inhibition rate of CAD antimicrobial peptides expressed by recombinant virus against Staphylococcus aureus was 79.6%-91.7%.The inhibition rate against Escherichia coli is 87.5%-89.8%.In the experiment of co-culture with DF-1cells,the inhibition rate of CAD antimicrobial peptides expressed by recombinant viruses against Staphylococcus aureus can reach 74.46%-77.68%.The inhibition rate against Escherichia coli is 35%-45%,This study showed that NDV vector system can successfully express the active CAD antimicrobial peptide and had antibacterial effect.This study laid the foundation for exploring the NDV vector expression of antimicrobial peptides and further study of CAD local effects.

关 键 词：天蚕素AD 新城疫病毒载体 反向遗传操作系统 抗菌肽 

分 类 号：S852.65[农业科学—基础兽医学]