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
张家祺 1李格格 1朱庆 2杨洋 3喻琦胜 1陈涛云 1任玉鹏 1张斌 4张朝辉2
作者信息
- 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. 西南民族大学畜牧兽医学院,四川成都 610041;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
- 折叠
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∶102 400.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.
Abstract
To construct the Ad5-BVDV-E2 using the AdMax adenovirus packaging system and to e-valuate 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 adenovi-rus 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,in-tranasal,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 recom-binant adenovirus confirmed by IFA and Western blot analysis,exhibiting a good biological activity in vitro.In addition,high antibody levels were produced through intramuscular injection,in-tranasal,and oral administration.The antibody titer of the Ad5-BVDV-E2 group reached up to 1∶120 400 after one week of enhanced immunization.The successful preparation of Ad5-BVDV-E2 in-duced 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Key words
bovine viral diarrhea virus/E2 protein/recombinant adenovirus vector vaccine/immuno-genicity引用本文复制引用
基金项目
国家重点研发计划(十四五)(2021YFD1600203)
现代农业产业技术体系建设项目四川肉牛创新团队专项(SCCXTD-2020-13)
四川省转移支付科技计划(210015)
西南民族大学"双一流"项目(XM2023006)
出版年
2024
NETL
NSTL
万方数据