牛病毒性腹泻病毒E0-E2基因融合腺病毒的构建及小鼠免疫效果评价

doi:10.16431/j.cnki.1671-7236.2022.06.005

摘要/Abstract

摘要： 【目的】研究牛病毒性腹泻病毒（Bovine viral diarrhea virus，BVDV）E0和E2串联基因重组腺病毒作为基因工程疫苗的应用潜力。【方法】采用PCR扩增、E0-E2基因融合并构建重组穿梭质粒pDC316-E0-E2，将其与AdMax腺病毒系统的骨架质粒共转染HEK293T细胞包装成重组腺病毒，通过Western blotting进行验证，并通过Reed-Muench法测定病毒滴度，通过肌内、皮下免疫接种小鼠后用ELISA方法及流式细胞检测进行免疫效果试验。【结果】成功扩增到E0、E2基因目的片段，大小分别为681和1 122 bp，得到了完整的腺病毒Ad5-E0-E2；测定其滴度为1.1×10¹⁰ PFU/mL；Western blotting检测结果显示，Ad5-E0-E2外源基因在HEK293T细胞中表达，得到了与预期相符的目的条带（65 ku）；ELISA检测结果表明，通过肌内和皮下注射Ad5-E0-E2均能产生较高的抗体水平；流式细胞检测显示首免、二免后肌内和皮下注射Ad5-E0-E2组CD4^＋、CD4^＋/CD8^＋比值均极显著高于PBS对照组（P<0.01）。【结论】本试验成功构建重组腺病毒Ad5-E0-E2，且具有较好的反应原性和免疫原性，能诱导机体产生针对BVDV的特异性抗体。

关键词: 牛病毒性腹泻病毒(BVDV); 基因融合; 腺病毒

Abstract: 【Objective】 The experiment was aimed to study the application potential of recombinant Adenovirus with E0 and E2 tandem genes of Bovine viral diarrhea virus (BVDV) as genetic engineering vaccine.【Method】 The recombinant Adenovirus was amplified by PCR, E0-E2 gene fusion was performed by overlapping extended PCR and the recombinant shuttle plasmid pDC316-E0-E2 was constructed, which was co-transfected into HEK293T cells with AdMax skeleton plasmid for packaging, and verified by Western blotting.The titer of the virus was determined by Reed-Muench method.The immune effect was studied by ELISA and flow cytometry after the mice were injected by muscle and subcutaneous injection.【Result】 The target fragments of E0 and E2 genes were successfully amplified, which were 681 and 1 122 bp, and the complete Adenovirus AD5-E0-E2 with titer of 1.1×10¹⁰ PFU/mL was obtained.Western blotting was used to detect the expression of Ad5-E0-E2 exogenous gene in HEK293T cells, and the target band (65 ku) was obtained, which was consistent with expectations.ELISA results showed that high antibody levels could be generated by both muscle and subcutaneous injection.Flow cytometry results showed that the ratios of CD4⁺ and CD4⁺/CD8⁺ in groups of intramuscular and subcutaneous injection Ad5-E0-E2 were both extremely significantly higher than that in PBS group (P<0.01).【Conclusion】 Adenovirus Ad5-E0-E2 was successfully constructed in this experiment, which had good reactivity and immunogenicity, and could induce the body to produce specific antibodies against BVDV.

Key words: Bovine viral diarrhea virus (BVDV); gene fusion; Adenovirus

中图分类号:

S852.65⁺3

任杰, 林泠, 汤德元, 曾智勇, 王彬, 禹光美, 郑如雯, 吴道义, 黄涛. 牛病毒性腹泻病毒E0-E2基因融合腺病毒的构建及小鼠免疫效果评价[J]. 中国畜牧兽医, 2022, 49(6): 2056-2063.

REN Jie, LIN Ling, TANG Deyuan, ZENG Zhiyong, WANG Bin, YU Guangmei, ZHENG Ruwen, WU Daoyi, HUANG Tao. Construction of Bovine Viral Diarrhea Virus E0-E2 Gene Fusion Adenovirus and Evaluation of Immune Effect in Mice[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(6): 2056-2063.

参考文献

[1] TAUTZ N, THIELH J, DUBOVI E J, et al. Pathogenesis of mucosal disease:A cytopathic Pestivirus generated by an internal deletion[J].Virology, 1994, 68:3289-3297.
[2] DEMIL E, FENTIE T, VIDAL G, et al.Prevalence of Bovine viral diarrhea virus antibodies and risk factors in dairy cattle in Gondar city, Northwest Ethiopia[J].Preventive Veterinary Medicine, 2021, 191:105363.
[3] BENAVIDES B, CASSL J, DIEGUEZ J, et al.Quantitative risk assessment of introduction of BVDV and BoHV-1 through indirect contacts based on implemented biosecurity measures in dairy farms of Spain[J].Preventive Veterinary Medicine, 2021, 188:105263.
[4] DE OLIVEIRA P S B, SILVA JÚNIOR J V J, WEIBLEN R, et al.Subtyping Bovine viral diarrhea virus (BVDV):Which viral gene to choose?[J].Infection, Genetics and Evolution, 2021, 92:104891.
[5] NGUYE T N T, SHA S, HONG M S, et al.Mechanistic model for production of recombinant adeno-associated virus via triple transfection of HEK293 cells[J].Molecular Therapy. Methods & Clinical Development, 2021, 21:642-655.
[6] MATTHEWS Q, SIBLEYD A, UW H, et al.Genetic incorporation of a Herpes simplex virus type 1 thymidine kinase and firefly luciferase fusion into the Adenovirus protein Ⅸ for functional display on the virion[J].Molecular Imaging, 2006, 5(4):510-519.
[7] WU J, CHEN G, ZHUANG F C, et al.Long-term toxicity, pharmacokinetics and immune effects of a recombinant Adenovirus vaccine expressing Human papillomavirus 16 E6 and E7 proteins (HPV16 E6E7-Ad5 Vac) in primates[J].American Journal of Translational Research, 2018, 10(5):1539-1551.
[8] ELAHI S M, SHEN S H, TALBOT B G, et al.Induction of humoral and cellular immune responses against the nucleocapsid of Bovine viral diarrhea virus by an Adenovirus vector with an inducible promoter[J].Virology, 1999, 261(1):1-7.
[9] CHEN X, DING X Y, ZHU L Q, et al.The identification of a B-cell epitope in Bovine viral diarrhea virus (BVDV) core protein based on a mimotope obtained from a phage-displayed peptide library[J].International Journal of Biological Macromolecules, 2021, 183:2376-2386.
[10] 陈文龙.抗牛病毒性腹泻病毒Erns蛋白单克隆抗体制备及鉴定[D].兰州:甘肃农业大学, 2020. CHEN W L.Preparation and identification of monoclonal antibody against Bovine viral diarrhea virus Erns protein[D].Lanzhou:Gansu Agricultural University, 2020.(in Chinese)
[11] 李智勇, 石顺利, 王艳杰, 等.内蒙古地区奶牛病毒性腹泻/黏膜病血清流行病学调查[J].畜牧与饲料科学, 2014, 35(3):106-108. LI Z Y, SHI S L, WANG Y J, et al.Seroepidemiological investigation of viral diarrhea/mucosal disease in dairy cows in Inner Mongolia[J].Animal Husbandry and Feed Science, 2014, 35(3):106-108.(in Chinese)
[12] 曲萍, 赵柏林, 胡冬梅, 等.我国西部地区牛病毒性腹泻流行情况调查[J].黑龙江畜牧兽医, 2016, 6:111-113. QU P, ZHAO B L, HU D M, et al.Investigation on epidemic situation of bovine viral diarrhea in Western China[J].Heilongjiang Animal Science and Veterinary Medicine, 2016, 6:111-113.(in Chinese)
[13] 陈新诺, 张斌.牛病毒性腹泻病毒的分子生物学研究进展[J].中国畜牧兽医, 2017, 44(11):3137-3142. CHEN X N, ZHANG B.Advances in molecular biology of Bovine viral diarrhea virus[J].China Animal Husbandry & Veterinary Medicine, 2017, 44(11):3137-3142.(in Chinese)
[14] 姚志兰, 傅宏庆, 崔平福, 等.江浙部分地区牛病毒性腹泻病毒分子流行病学调查及BVDV-2型毒株分离鉴定[J].扬州大学学报(农业与生命科学版), 2019, 40(1):40-46. YAO Z L, FU H Q, CUI P F, et al.Molecular epidemiological investigation of Bovine viral diarrhea virus and isolation and identification of BVDV-2 strain in Jiangsu and Zhejiang provinces[J].Journal of Yangzhou University (Agriculture and Life Science Edition), 2019, 40(1):40-46.(in Chinese)
[15] 石柯, 刘巧玲, 张森, 等.贵州省部分牛场BVDV、BHV-1、BCoV、MB的感染情况调查及BVDV分子特征分析[J].畜牧与兽医, 2021, 53(9):83-88. SHI K, LIU Q L, ZHANG S, et al.Investigation of BVDV, BHV-1, BCoV and MB infection and molecular characteristics analysis of BVDV in some cattle farms in Guizhou province[J].Animal Husbandry & Veterinary Medicine, 2021, 53(9):83-88.(in Chinese)
[16] 吴桐忠, 努尔赛力克·努素甫, 黄新, 等.表达牛病毒性腹泻病毒E2蛋白的重组乳酸菌的构建及蛋白免疫原性分析[J].中国畜牧兽医, 2021, 48(8):2975-2981. WU T Z, NURSELIK N, HUANG X, et al.Construction and immunogenicity analysis of recombinant Lactobacillus expressing E2 protein of Bovine viral diarrhea virus[J].China Animal Husbandry & Veterinary Medicine, 201, 48(8):2975-2981.(in Chinese)
[17] 陈为宏, 周玉龙, 尹辉, 等.牛病毒性腹泻病毒E0和E2蛋白的融合表达及纯化[J].中国生物制品学杂志, 2014, 27(10):1268-1271. CHEN W H, ZHOU Y L, YIN H, et al.Fusion expression and purification of E0 and E2 proteins of Bovine viral diarrhea virus[J].Chinese Journal of Biologicals, 2014, 27(10):1268-1271.(in Chinese)
[18] 孙凡婷, 张云, 张妍, 等.牛病毒性腹泻病毒E₀-E₂融合基因重组卡介苗的构建[J].畜牧与兽医, 2015, 47(10):89-92. SUN F T, ZHANG Y, ZHANG Y, et al.Construction of recombinant BCG vaccine of Bovine viral diarrhea virus E₀-E₂ fusion gene[J].Animal Husbandry & Veterinary Medicine, 2015, 47(10):89-92.(in Chinese)
[19] 马小静, 李梦莹, 张淮瑜, 等.共表达牛病毒性腹泻病毒E0基因和牛传染性鼻气管炎病毒gD基因的重组质粒DNA构建及其对小鼠的免疫效应[J].畜牧兽医学报, 2021, 52(1):154-165. MA X J, LI M Y, ZHANG H Y, et al.Construction of recombinant plasmid DNA co-expressing Bovine viral diarrhea virus E0 gene and Bovine infectious rhinotracheitis virus gD gene and its immune effect on mice[J].Chinese Journal of Animal Science and Veterinary Medicine, 2021, 52(1):154-165.(in Chinese)
[20] 梁霄勇.牛病毒性腹泻病毒的分离鉴定及其E0核酸疫苗与细胞因子佐剂联合免疫研究[D].乌鲁木齐:新疆农业大学, 2016. LIANG X Y.Isolation and identification of Bovine viral diarrhea virus and study on E0 nucleic acid vaccine and cytokine adjuvant combined immunization[D].Urumqi:Xinjiang Agricultural University, 2016.(in Chinese)