摘要
猪伪狂犬病(pseudorabies,PR)是由伪狂犬病病毒(pseudorabies virus,PRV)引起的多种家畜和野生动物共患的传染病,猪是病毒唯一的自然宿主,严重危害养猪业的安全,目前主要通过接种灭活疫苗或基因缺失弱毒疫苗预防。为了开发安全有效的新型基因工程疫苗,本研究以PRV疫苗株Bartha-K61为参考毒株,利用杆状病毒-昆虫细胞系统表达了gB蛋白,非变性蛋白电泳证实gB蛋白能够形成三聚体结构,将纯化的gB蛋白免疫小鼠,通过攻毒保护试验评价免疫效果。结果显示,gB抗原能够诱导小鼠产生较强的免疫反应,血清中和抗体效价达到1:70;淋巴细胞刺激指数大于1.29,干扰素γ(interferon gamma,IFN-γ)释放水平高于100 pg/mL,免疫后攻毒[200μL/,10~4半数培养感染剂量(median tissue culture infectious dose,TCID50)/mL]临床的保护率达100%。免疫组化、组织病理切片及组织病毒载量检测显示,gB抗原免疫组的病理损伤及病毒载量相对PBS组显著降低。本研究获得的gB蛋白免疫可诱导小鼠产生强烈的体液免疫和细胞免疫应答,为研制重组gB蛋白亚单位疫苗奠定了基础。
Pseudorabies(PR)is an infectious disease caused by the pseudorabies virus(PRV),affecting various domesticated and wild animals.Since pigs are the only natural hosts of PRV,PR poses a serious threat to the pig farming industry.Currently,PR is primarily prevented through vaccination with inactivated vaccines or genetically modified attenuated live vaccines.Developing safe and effective genetically engineered vaccines would facilitate the eradication and control of PR.In this study,the PRV vaccine strain Bartha-K61 was used as the reference strain.The gB protein was expressed via the baculovirus-insect cell expression system.Non-denaturing gel electrophoresis confirmed that the gB protein could form a trimeric structure.The purified gB protein was used to immunize mice,and the immune effect was evaluated by a challenge test.The results showed that the gB antigen induced a strong immune response in mice,with the serum-neutralizing antibody titer above 1:70.The lymphocyte stimulation index reached more than 1.29,and the level of(interferon gamma,IFN-γ)release was higher than 100 pg/mL.After immunization,mice were challenged with the virus at a dose of 104 TCID₅₀/mL,200μL per mouse,and the clinical protection rate was 100%.Immunohistochemistry,histopathological section,and tissue viral load results showed that the pathological damage and viral load in the gB-immunized group were significantly lower than those in the PBS group.In summary,the gB protein obtained in this study induced strong humoral and cellular immune responses in mice,laying a foundation for developing a recombinant gB protein subunit vaccine.
作者
王金
王锴
张莹
谭书桢
孙世琪
郭慧琛
尹双辉
牛家强
WANG Jin;WANG Kai;ZHANG Ying;TAN Shuzhen;SUN Shiqi;GUO Huichen;YIN Shuanghui;NIU Jiaqiang(Key Laboratory of Hydatid Disease Control in Xizang,Ministry of Agriculture and Rural Affairs/Key Laboratory of Universities of Tibet Plateau Animal Disease Research,College of Animal Science,Xizang Agricultural and Animal Husbandry University,Nyingchi 860000,Xizang,China;State Key Laboratory for Animal Disease Control and Prevention,College of Veterinary Medicine and Biosafety,Lanzhou University,Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730000,Gansu,China)
出处
《生物工程学报》
北大核心
2025年第7期2694-2706,共13页
Chinese Journal of Biotechnology
基金
甘肃省重点研发计划(25YFNA014)
甘肃省科技重大专项(24ZDWA004)
成都农业科技中心地方财政专项(NASC2024KR06)
2023兰州市人才创业创新项目(2023-RC-3)。
关键词
昆虫细胞表达
伪狂犬病病毒
gB蛋白
小鼠
免疫评价
insect cell expression
pseudorabies virus
gB protein
mice
immunogenic evaluation
