牛纽布病毒病毒样颗粒的制备及对小鼠的免疫效果评价  

Construction and immune efficacy evaluation of BNeV VLPs based on VP1 protein in mice

作　　者：丁露 黄向月 吴锦波 张朝辉 朱庆 朱晨曦 邓梏男 阿克阿加 贺春赛 马元珍 张斌[1,4] DING Lu;HUANG Xiangyue;WU Jinbo;ZHANG Chaohui;ZHU Qing;ZHU Chenxi;DENG Gunan;AKE Ajia;HE Chunsai;MA Yuanzhen;ZHANG Bin(College of Animal Husbandry and Veterinary Medicine,Southwest Minzu University,Chengdu 610041,China;Aba Tibetan and Qiang Autonomous Prefecture Animal Husbandry Science and Technology Research Institute,Aba,Sichuan 624400,China;Center for Animal Disease Control and Prevention,Ganzi Tibetan Autonomous Prefecture,Kangding,Sichuan 626000,China;Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization of Ministry of Education/Sichuan Province,Chengdu 610041,China)

机构地区：[1]西南民族大学畜牧兽医学院,四川成都610041 [2]阿坝藏族羌族自治州畜牧科学技术研究所,四川阿坝624400 [3]四川省甘孜藏族自治州动物疫病预防控制中心,四川康定626000 [4]青藏高原动物遗传资源保护与利用教育部/四川省重点实验室,四川成都610041

出　　处：《中国兽医学报》2025年第3期412-419,共8页Chinese Journal of Veterinary Science

基　　金：四川省中央引导地方科技发展专项资助项目(2024ZYD0072);国家农业产业技术体系四川兽药创新团队专项基金资助项目(SCCXTD-2020-18);四川省阿坝州应用技术研究与开发资金资助项目(R23YYJSYJ0001);西南民族大学2024年中央高校优秀学生培养工程资助项目(ZYN2024169)。

摘　　要：针对牛纽布病毒(bovine nebovirus,BNeV)VP1基因进行密码子优化并构建重组质粒pFastBac-Dual-VP1,使用杆状病毒表达系统制备BNeV-VP1病毒样颗粒(virus-like particles,VLPs)。采用Western blot、间接免疫荧光和电镜对其进行鉴定。将验证正确的VLPs与MF59佐剂、CpG-ODG免疫增强剂混合后免疫小鼠并评估其免疫效果。结果显示,优化后的VP1基因密码子适应指数(codon adaptation index,CAI)为0.93,GC含量为60.4%;将构建的重组质粒转化至DH10Bac进行蓝白斑筛选,验证成功后转染至SF9细胞,获得重组杆状病毒Baculo-BNeV-VP1;电镜下可观察到直径在35~40 nm的BNeV VLPs;IFA和Western blot试验均证明VP1蛋白可以成功表达并具有生物学活性;通过蛋白优化发现感染剂量MOI=0.5时蛋白表达量最高;将50μg VLPs与MF59佐剂和CpG-ODN混合后,以肌肉注射方式免疫小鼠,结果显示VLPs免疫组在首免后7 d产生IgG抗体,且抗体滴度逐步增加,于28 d达到峰值(1∶102400),35 d时有所下降,但仍保持较高水平;阻断效价BT50最高为640,可诱导小鼠产生BNeV VP1特异性阻断抗体。结果表明,利用杆状病毒表达系统表达了BNeV的VP1蛋白,并成功构建了BNeV VLPs,其能诱导小鼠机体产生体液免疫反应,为后续BNeV疫苗研究提供了参考。The codon was optimized for the bovine nebovirus(BNeV)VP1 gene and the recombinant plasmid pFastBac-Dual-VP1 was constructed,and BNeV-VP1 virus-like particles(VLPs)were prepared using a baculovirus expression system,and identified by Western blot,indirect immunofluorescence and electron microscopy.Successfully validated VLPs were mixed with MF59adjuvant and CpG-ODG,and mice were immunised by intramuscular injection and evaluated for immunity effects.The results showed that the optimized CAI(codon adaptation index)of VP1gene was 0.93 and the GC content was 60.4%.The constructed recombinant plasmid was transformed into DH10Bac for blue-white spot screening,and after successful verification,it was transrected into SF9 cells to obtain recombinant baculovirus Baculo-BNeV-VPl.BNeV virus-like particles with diameters ranging from 35 to 40 nm were observed under the electron microscope,and both IF A and Western blot experiments proved that the target proteins were successfully expressed and biologically active,and protein optimisation revealed that the highest protein expression was found at the infectious dose MOI=0.5.Mice were immunized by intramuscular injection after 50μg of VLPs were mixed with MF59 adjuvant and CpG-ODN.The results showed that the VLPs immunization group produced IgG antibodies 7 days after the first dose,and the antibody titer increased gradually,reaching a maximum of 1:102400,and declined at 35 d,but still maintained a high level;The blocking titer BT50 is up to 640,which can induce the production of BNeV VP1-specific blocking antibody in mice.In this study,the baculovirus expression system was used to express the VP1 protein of BNeV,and BNeV VLPs were successfully constructed,which could induce humoral immune response in mice,which provided a reference for the follow-up research of BNeV vaccine.

关 键 词：牛纽布病毒 VP1蛋白 病毒样颗粒 免疫原性 小鼠 

分 类 号：S852.65[农业科学—基础兽医学]