基于E2蛋白BVDV腺病毒载体疫苗的制备及对小鼠的免疫原性分析  

Construction and immunogenicity evaluation of BVDV recombinant adenovirus based on E2 protein in mice

作　　者：张家祺 李格格 朱庆 杨洋 喻琦胜 陈涛云 任玉鹏 张斌[1,4] 张朝辉[2] ZHANG Jiaqi;LI Gege;ZHU Qing;YANG Yang;YU Qisheng;CHEN Taoyun;REN Yupeng;ZHANG Bin;ZHANG Chaohui(College of Animal Husbandry and Veterinary Medicine,Southwest Minzu University,Chengdu 610041,China;Center for Animal Disease Control and Prevention,Ganzi Tibetan Autonomous Prefecture,Kangding,Sichuan 626000,China;Ganzi County Agriculture,Animal Husbandry,Rural and Technology Bureau,Ganzi,Sichuan 626700,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]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 [3]甘孜县农牧农村和科技局,四川甘孜626700 [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》2024年第2期276-282,共7页Chinese Journal of Veterinary Science

基　　金：“十四五”国家重点研发计划资助项目(2021YFD1600203);国家农业产业技术体系四川肉牛创新团队专项基金资助项目(SCCXTD-2020-13);四川省转移支付科技计划资助项目(210015);西南民族大学"双一流"基金资助项目(XM2023006)。

摘　　要：选用牛病毒性腹泻病毒(bovine viral diarrhea virus,BVDV)SWU-Z6株的E2基因序列为模板,针对HEK293细胞密码子偏嗜性优化合成全长E2基因,利用AdMax腺病毒包装系统制备Ad5-BVDV-E2。采用PCR、间接免疫荧光(IFA)和Western blot验证了E2蛋白在HEK293细胞中成功表达,并通过肌注、滴鼻和口服免疫途径来免疫小鼠,同时设立BVDV商业灭活苗组和PBS空白对照组。结果显示,经酶切鉴定和测序验证表明,优化后的E2基因已正确克隆至腺病毒穿梭载体pDC316中,并获得重组质粒pDC316-E2,将pDC316-E2和腺病毒骨架质粒共转染至HEK293细胞,获得重组腺病毒Ad5-BVDV-E2。PCR验证了E2基因成功插入到腺病毒载体基因组中,IFA和Western blot证实了重组腺病毒能够正确表达E2蛋白,并具有良好的生物学活性。通过肌注、滴鼻和口服均能产生较高的抗体水平,其中Ad5-BVDV-E2肌注组小鼠加强免疫1周后抗体滴度最高达到1∶102400。结果表明,成功制备了Ad5-BVDV-E2,能诱导机体产生较强的体液免疫反应,表明Ad5-BVDV-E2具有临床应用的潜力。To construct the Ad5-BVDV-E2 using the AdMax adenovirus packaging system and to evaluate its immune efficacy in mice through intramuscular,intranasal,and oral immunization routes.The full length E2 gene sequence of BVDV-1 SWU-Z6 strain was optimized and synthesized based on the codon bias of HEK293 cells.Ad5-BVDV-E2 was prepared using the AdMax adenovirus packaging system.The expression of E2 protein in HEK293 was validated by PCR,the indirect immunofluorescence(IFA)and Western blot.Mice were immunized through intramuscular,intranasal,and oral routes,as well as BVDV commercial inactivated vaccine group and PBS blank were used as control groups.The recombinant plasmid pDC316-E2 was constructed by inserting the optimized E2 gene and verified through restriction endonuclease analysis and sequencing.Recombinant adenovirus Ad5-BVDV-E2 was obtained through co-transfection of pDC316-E2 and the adenovirus skeleton plasmid into HEK293 cells.The successful insertion of the E2 gene into the adenovirus vector genome was confirmed through PCR.E2 protein could be correctly expressed by the recombinant adenovirus confirmed by IF A and Western blot analysis,exhibiting a good biological activity in vitro.In addition,high antibody levels were produced through intramuscular injection,intranasal,and oral administration.The antibody titer of the Ad5-BVDV-E2 group reached up to 1:120400 after one week of enhanced immunization.The successful preparation of Ad5-BVDV-E2 induced efficient humoral immune responses in mice,indicating its potential for clinical application.

关 键 词：牛病毒性腹泻病毒 E2蛋白 重组腺病毒载体疫苗 免疫原性 

分 类 号：S852.65[农业科学—基础兽医学]