耐热选育对新城疫LaSota疫苗株热稳定性的影响  

Effect of Heat-Resistant Breeding on Thermal Stability of LaSota Vaccine Strain of Newcastle Disease Virus

作　　者：赵庆庆 曾哲[2] 肖倩妮 李丽 冯贺龙 姚伦 罗青平[2,3] 温国元 商雨[2] ZHAO Qing-qing;ZENG Zhe;XIAO Qian-ni;LI Li;FENG He-long;YAO Lun;LUO Qing-ping;WEN Guo-yuan;SHANG Yu(School of Animal Science and Nutritional Engineering,Wuhan Polytechnic University,Wuhan,Hubei,430048,China;Key Laboratory of Prevention and Control Agents for Animal Bacteriosis(Ministry of Agriculture and Rural Affairs),Hubei Provincial Key Laboratory of Animal Pathogenic Microbiology,Institute of Animal Husbandry and Veterinary Medicine,Hubei Academy of Agricultural Sciences,Wuhan,Hubei,430064,China;Hubei Hongshan Laboratory,Wuhan,Hubei,430070,China)

机构地区：[1]武汉轻工大学动物科学与营养工程学院,湖北武汉430048 [2]湖北省农业科学院畜牧兽医研究所,农业农村部畜禽细菌病防治制剂创制重点实验室&畜禽病原微生物学湖北重点实验室,湖北武汉430064 [3]湖北洪山实验室,湖北武汉430070

出　　处：《动物医学进展》2025年第2期35-41,共7页Progress In Veterinary Medicine

基　　金：湖北省重点研发计划项目(2023BBB034);国家自然科学基金项目(U23A20239,32302869);国家现代农业产业技术体系资助项目(CARS-41);武汉市创新专项(2022020801010337)。

摘　　要：为了探究新城疫病毒(Newcastle disease virus,NDV)LaSota疫苗株热稳定性相关的潜在氨基酸位点,将其在SPF鸡胚中自然传代、间隔耐热选育和连续耐热选育至20代,监测血凝活性和细胞感染力的热稳定性;对不同代次的病毒进行全基因组测序,筛选传代过程中的突变位点,并利用蛋白结构生物学方法分析突变位点在病毒蛋白结构热稳定性中的作用。结果显示,经过间隔耐热选育和连续耐热选育的病毒血凝活性和细胞感染力热稳定性均有所提升。测序结果分析表明,与自然传代毒株相比,间隔耐热选育和连续耐热选育病毒分别共计有11和12个碱基突变,其中5个为错义突变(间隔耐热选育病毒为4个),NP蛋白Q169K(仅发生在连续耐热选育病毒)、HN蛋白G171S和V191I、M蛋白E170D以及L蛋白E1612K。蛋白结构分析结果显示,L蛋白E1612K的突变导蛋白二级结构改变;HN蛋白G171S的突变导致蛋白三级结构改变。通过耐热选育筛选出了5个可能与NDV LaSota疫苗株热稳定性相关的关键氨基酸位点,为NDV的耐热机理研究及ND耐热疫苗的研发提供理论支撑。To investigate potential amino acid sites related to the thermal stability of the Newcastle disease virus LaSota vaccine strain,it was passaged in SPF chicken embryos through natural propagation,interval heat resistance breeding,and continuous heat resistance breeding up to the 20th generation.Hemagglutination activity and cell infectivity thermal stability were monitored.Whole-genome sequencing of viruses from different passages was conducted to identify mutation sites,and the role of these mutations in the thermal stability of viral protein structures was analyzed using protein structural biology methods.The results indicated that the viruses subjected to interval and continuous heat resistance breedings showed improved thermal stability in hemagglutination activity and cell infectivity.Sequence analysis revealed that compared to the naturally passaged strain,interval and continuous heat resistance breedings resulted in 11 and 12 base mutations,respectively,with five being nonsynonymous mutations:NP protein Q169K(only in continuously bred virus),HN protein G171S and V191I,M protein E170D,and L protein E1612K.Structural analysis demonstrated that the E1612K mutation in the L protein altered its secondary structure,while the G171S mutation in the HN protein led to changes in the tertiary structure.In summary,this study identified five key amino acid sites potentially associated with the thermal stability of the NDV LaSota vaccine strain through heat resistance breeding.This provides methodological and theoretical support for research on the thermal resistance mechanism of NDV and the development of heat-resistant NDV vaccines.

关 键 词：新城疫病毒 LaSota疫苗株 耐热选育 鸡胚传代 热稳定性 

分 类 号：S852.659.95[农业科学—基础兽医学]