牛副流感3型病毒样颗粒的制备及免疫原性评价  

Preparation and immune efficacy evaluation of bovine parainfluenza type 3 virus like particles

作　　者：朱晨曦 黄向月 朱庆 丁露 邓梏男 阿克阿加 贺春赛 马元珍 吴锦波 张朝辉 张斌[1,4] ZHU Chenxi;HUANG Xiangyue;ZHU Qing;DING Lu;DENG Gunan;AKE Ajia;HE Chunsai;MA Yuanzhen;WU Jinbo;ZHANG Chaohui;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,Sichuan 624400,China;Center for Animal Disease Control and Prevention,Ganzi Tibetan Autonomous Prefecture,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,四川马尔康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404-411,442,共9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)。

摘　　要：针对牛副流感病毒3型(bovine parainfluenza virus type 3,BPIV3)的基质蛋白(M)和血凝素-神经氨酸酶(HN)基因进行密码子优化并构建重组穿梭质粒Dual-M+HN;使用杆状病毒表达系统制备BPIV3病毒样颗粒(virus-like particles,VLPs),并采用Western blot、间接免疫荧光和电镜对其进行验证;将验证成功的VLPs与MF59佐剂和CpG-ODN免疫增强剂混合通过肌注方式免疫小鼠。通过检测小鼠血清特异性抗体、中和抗体和血凝抑制抗体来评估BPIV3 VLPs的免疫效果。结果显示,优化后的M和HN蛋白基因密码子适应指数(codon adaptation index,CAI)分别为0.96和0.95,CG含量分别达到54.1%和53.1%;构建的重组质粒转化至DH10Bac进行蓝白斑筛选,将验证正确的重组杆粒转染至Sf9细胞,获得杆状病毒pFastBac-M+HN,电镜下观察可见直径约180 nm的BPIV3 VLPs;IFA和Western blot试验均证明目的蛋白的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=5时蛋白表达量最高;将50μg VLPs与MF59佐剂和CpG-ODN混合后肌注方式免疫小鼠,结果显示VLPs免疫组在首免2周时抗体开始上升,在二免后21 d时达到最高,平均IgG抗体滴度为1∶40228,中和抗体滴度平均为1∶298,血凝抑制抗体滴度为1∶549,达到灭活苗水平(P≥0.05),表明本试验制备的VLPs能诱导机体产生体液免疫反应。结果表明,本试验成功制备了能自组装表达BPIV3 HN和M蛋白的VLPs,并能诱导小鼠机体产生体液免疫反应,为后续BPIV3 VLPs疫苗研究奠定了基础。Codon optimization was performed for the M and HN genes of bovine parainfluenza virus type 3(BPIV3),and the recombinant shuttle plasmid Dual-M+HN was constructed.BPIV3 VLPs was prepared using the baculovirus expression system,and verified by Western blot,IFA and electron microscopy.The successfully verified virus-like particle(VLPs)were mixed with MF59adjuvant and CpG-ODN immunoenhancer to immunize mice by intramuscular-injection,and BPIV3inactivated vaccine group and adjuvant control group were set up.The immune effect of BPIV3VLPs was evaluated by monitoring mouse serum specific antibodies,neutralizing antibodies and hemagglutination inhibition antibodies.The results showed that the optimized codon adaptation index(CAI)of the M and HN protein genes were 0.96 and 0.95,respectively,and the CG content reached 54.1%and 53.1%,respectively.The constructed recombinant plasmid was transformed into DH10Bac for blue and white spot screening.The validated recombinant rod particles were transfected into Sf9 cells to obtain the rod-shaped virus pFastBac-M+HN.Under electron microscopy,BPIV3 VLPs with a diameter of approximately 180 nm were observed.IFA and Western blot experiments confirmed the successful expression and biological activity of the target protein.Through protein optimization,it was found that the protein expression was highest at an infection dose of MOI=5.After mixing 50μg VLPs with MF59 adjuvant and CpG-ODN,mice were immunized by intramuscular injection.The results showed that the antibodies in the VLPs immunized group began to rise at 2 weeks of the first immunization and reached their peak at 21 days of the second immunization,with an average IgG antibody titer of 1:40228;The average titer of neutralizing antibodies is 1:298;The titer of hemagglutination inhibition antibody is 1:549,reaching the level of inactivated vaccine(P≥0.05),indicating that the VLPs prepared in this experiment can induce humoral immune response in the body.In summary,this study successfully prepared VLPs capable of self-assembly

关 键 词：牛副流感病毒3型 基质蛋白(M) 血凝素-神经氨酸酶(HN) 病毒样颗粒 免疫原性 

分 类 号：S852.65[农业科学—基础兽医学]