基于E2蛋白BVDV-1病毒样颗粒的构建及对小鼠的免疫效力评估

doi:10.11843/j.issn.0366-6964.2023.12.024

摘要/Abstract

摘要： 本研究利用昆虫细胞-杆状病毒表达系统构建牛病毒性腹泻病毒1型(BVDV-1) E2 病毒样颗粒(VLPs),并测定其对小鼠的免疫效力。选用BVDV-1 SWU-Z6株的E2基因序列为模板,针对昆虫细胞密码子偏嗜性优化合成E2基因,利用昆虫细胞-杆状病毒表达系统制备BVDV-1 E2 VLPs。采用间接免疫荧光试验(IFA)和Western blot验证E2蛋白在SF9细胞中的表达,通过电镜鉴定BVDV-1 E2 VLPs的组装。对获得的VLPs进行超离纯化,使用不同剂量的VLPs添加MF59佐剂和CpG-ODN免疫增强剂肌注免疫小鼠,同时设立BVDV商业灭活苗组和PBS空白对照组。二免4周后选用BVDV-1 SWU-Z6毒株以灌胃途径对小鼠进行攻毒,通过小鼠体重变化以及粪便中的病毒载量来评估BVDV-1 E2 VLPs的免疫保护效果。经酶切鉴定和测序验证表明,优化后的E2基因已正确克隆至杆状病毒穿梭载体pFast Dual中,并获得重组质粒pFast Dual BVDV-1 E2,将重组质粒转化到DH10.Bac感受态细胞经过蓝白斑筛选获得重组杆粒Bacmid-BVDV-1 E2,将重组杆粒转染至SF9细胞,获得重组杆状病毒Baculo-BVDV-1 E2。电镜下观察可见大量直径在80~100 nm的BVDV病毒样颗粒,IFA和Western blot结果证实重组杆状病毒能够正确表达E2蛋白,并具有良好的生物学活性。BVDV-1 E2 VLPs 50 μg组小鼠首免2周时ELSIA抗体效价约为1∶10 000,加强免疫2周后抗体效价达到1∶100 000。攻毒保护试验结果表明,免疫组小鼠在攻毒后第7天表现出体重下降,而后开始回升;在攻毒后第10天采集粪便没有检测到病毒拷贝。与非免疫组小鼠相对比,BVDV-1 E2 VLPs 50 μg剂量两次免疫后阻止了因病毒感染而导致体重下降以及消化道内病毒的复制。研究中成功制备了BVDV-1 E2 VLPs,能诱导机体产生较强的体液免疫反应,可使小鼠获得抵抗BVDV-1 SWU-Z6毒株感染的免疫保护力。

关键词: 牛病毒性腹泻病毒, E2蛋白, 杆状病毒, 病毒样颗粒, 免疫原性

Abstract: This study aimed to construct the BVDV-1(bovine viral diarrhea virus 1) E2 virus-like particles (VLP) by the insect cell-baculovirus expression system and to evaluate its immune efficacy in mice. The E2 gene sequence of BVDV-1 SWU-Z6 strain was optimized and synthesized according to the codon bias of insect cells, and used to generate the construct of the BVDV-1 E2 VLPs for insect cell-baculovirus expression system. The expression of E2 protein in recombinant baculovirus was validated by the indirect immunofluorescence (IFA) and Western blot, and the assembly of VLPs was evaluated through electron microscope. The VLPs were then purified using sucrose density centrifugation. Mice were immunized with different doses of VLPs plus MF59 adjuvant and CpG-ODN immune enhancer by intramuscular injection, with BVDV commercial inactivated vaccine group and PBS blank as control group. Four weeks after booster immunization, the mice were challenged using BVDV-1 SWU-Z6 strain via gavage. The immune protection effect of BVDV-1 E2 VLPs was evaluated based on weight changes and viral load in feces of mice. The recombinant plasmid pFast Dual BVDV-1 E2 was constructed by inserting the optimized E2 gene and verified by restriction endonuclease analysis and sequencing. After transformed into DH10.Bac competent cells, the recombinant baculovirus Baculo-BVDV-1 E2 were extracted and transfected into SF9 insect cells. The BVDV-1 E2 VLPs with diameter about 80-100 nm were observed by electron microscope. The E2 protein expressed by the recombinant baculovirus was further confirmed by IFA and Western blot analysis, exhibiting a good biological activity in vitro. The antibody titer in group injected with 50 μg of VLPs was about 1∶10 000 2 weeks after first immunization, and reached 1∶100 000 2 weeks after enhanced immunization by ELSIA. After the challenge with SWU-Z6 strain, the immunized mice showed weight loss 7 days after infection before beginning to recover. No virus was detected in feces collected on day 10 after infection. While the mice group injected 50 μg of BVDV-1 E2 VLPs twice showed no weight loss and virus replication in the digestive tract in comparison with the non-immunized mice. The BVDV-1 E2 VLPs expressed by the insect cell-baculovirus expression system induce an efficient BVDV-specific humoral immune responses in mice, and prevent the BVDV-1 SWU-Z6 strain infection.

Key words: bovine viral diarrhea virus, E2 protein, baculovirus, virus-like particles, immunogenicity

中图分类号:

S852.659.6

张家祺, 贾浠宁, 周群, 宋鑫, 朱晨曦, 李格格, 张斌. 基于E2蛋白BVDV-1病毒样颗粒的构建及对小鼠的免疫效力评估[J]. 畜牧兽医学报, 2023, 54(12): 5143-5153.

ZHANG Jiaqi, JIA Xining, ZHOU Qun, SONG Xin, ZHU Chenxi, LI Gege, ZHANG Bin. Construction of BVDV-1 Virus-like Particles based on E2 Protein and Evaluation of Its Immune Efficacy in Mice[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(12): 5143-5153.

参考文献

[1] WANG Z H, LIU M Y, ZHAO H R, et al. Induction of robust and specific humoral and cellular immune responses by bovine viral diarrhea virus virus-like particles (BVDV-VLPs) Engineered with baculovirus expression vector system[J]. Vaccines (Basel), 2021, 9(4):350.
[2] ZHOU Y, SHAO Z, DAI G, et al. Pathogenic infection characteristics and risk factors for bovine respiratory disease complex based on the detection of lung pathogens in dead cattle in Northeast China[J]. J Dairy Sci, 2023, 106(1):589-606.
[3] 王炜. 牛主要呼吸道病毒病血清学调查、牛病毒性腹泻病毒分离株鉴定及疫苗研究[D]. 北京:中国农业科学院, 2014.WANG W. Serosurvey of major bovine resporitary viruses and identification of BVDV isolates and vaccine development[D]. Beijing: Chinese Academy of Agricultural Sciences, 2014. (in Chinese)
[4] NEWCOMER B W, CHAMORRO M F, WALZ P H. Vaccination of cattle against bovine viral diarrhea virus[J]. Vet Microbiol, 2017, 206:78-83.
[5] SMITH D B, MEYERS G, BUKH J, et al. Proposed revision to the taxonomy of the genus Pestivirus, family Flaviviridae[J]. J General Virol, 2017, 98(8):2106-2112.
[6] DENG M L, CHEN N, GUIDARINI C, et al. Prevalence and genetic diversity of bovine viral diarrhea virus in dairy herds of China[J]. Vet Microbiol, 2020, 242:108565.
[7] CHANG L L, QI Y P, LIU D, et al. Molecular detection and genotyping of bovine viral diarrhea virus in Western China[J]. BMC Vet Res, 2021, 17(1):66.
[8] 任杰, 林泠, 汤德元, 等. 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 J, LIN L, TANG D Y, et al. Construction of bovine viral diarrhea virus E0-E2 gene fusion adenovirus and evaluation of immune effect in mice[J]. China Animal Husbandry & Veterinary Medicine, 2022, 49(6):2056-2063. (in Chinese)
[9] JIA S, HUANG X, LI H, et al. Immunogenicity evaluation of recombinant Lactobacillus casei W56 expressing bovine viral diarrhea virus E2 protein in conjunction with cholera toxin B subunit as an adjuvant[J]. Microb Cell Fact, 2020, 19(1):186. doi: 10.1186/s12934-020-01449-3.
[10] YANG N N, ZHANG J W, XU M G, et al. Virus-like particles vaccines based on glycoprotein E0 and E2 of bovine viral diarrhea virus induce humoral responses[J]. Front Microbiol, 2022, 13:1047001.
[11] 杨霞, 李雅茹, 丛佳南, 等. BVDV-1 VLPs的构建及免疫原性研究[J]. 中国病原生物学杂志, 2022, 17(1):43-48. YANG X, LI Y R, CONG J N, et al. Construction, identification, and determination of the immunogenicity of BVDV-1 VLPs[J]. Journal of Pathogen Biology, 2022, 17(1):43-48. (in Chinese)
[12] 高闪电, 张忠辉, 田占成, 等. 牛病毒性腹泻病毒1型病毒样颗粒的制备及其对豚鼠的免疫原性分析[J]. 生物工程学报, 2022, 38(1):130-138. GAO S D, ZHANG Z H, TIAN Z C, et al. Preparation of bovine viral diarrhea disease virus 1 virus-like particles and evaluation of its immunogenicity in a guinea pig model[J]. Chinese Journal of Biotechnology, 2022, 38(1):130-138. (in Chinese)
[13] QI S H, WO L J, SUN C, et al. Host cell receptors implicated in the cellular tropism of BVDV[J]. Viruses, 2022, 14(10):2302.
[14] LIU C Y, GUO H, ZHAO H Z, et al. Recombinant bovine herpesvirus type I expressing the bovine viral diarrhea virus E2 protein could effectively prevent infection by two viruses[J]. Viruses, 2022, 14(8):1618.
[15] POSSEE R D, CHAMBERS A C, GRAVES L P, et al. Recent developments in the use of baculovirus expression vectors[J]. Curr Issues Mol Biol, 2020, 34:215-230.
[16] IRONS S L, CHAMBERS A C, LISSINA O, et al. Protein production using the baculovirus expression system[J]. Curr Protocols Prot Sci, 2018, 91:5. 5. 1-5. 5. 22.
[17] CONTRERAS-GÓMEZ A, SÁNCHEZ-MIRÓN A, GARCÍA-CAMACHO F, et al. Protein production using the baculovirus-insect cell expression system[J]. Biotechnol Progr, 2014, 30(1):1-18.
[18] HANSOONGNERN P, PHECHARAT N, WASANASUK K, et al. Encapsidated-CpG ODN enhances immunogenicity of porcine circovirus type 2 virus-like particles[J]. Vet Microbiol, 2022, 275:109583.
[19] ALZUA G P, PIHL A F, OFFERSGAARD A, et al. Inactivated genotype 1a, 2a and 3a HCV vaccine candidates induced broadly neutralising antibodies in mice[J]. Gut, 2023, 72(3):560-572.
[20] KRIEG A M. CpG motifs in bacterial DNA and their immune effects[J]. Ann Rev Immunol, 2002, 20:709-760.
[21] U-TAYNAPUN K, CHIRAPONGSATONKUL N, ITAMI T, et al. CpG ODN mimicking CpG rich region of myxosporean Myxobolus supamattayai stimulates innate immunity in Asian sea bass (Lates calcarifer) and defense against Streptococcus iniae[J]. Fish Shellfish Immunol, 2016, 58:116-124.
[22] 阮文强, 陈新诺, 任玉鹏, 等. 不同来源的3株牛病毒性腹泻病毒对小鼠的致病性分析[J]. 畜牧兽医学报, 2018, 49(10):2232-2239. (in Chinese)RUAN W Q, CHEN X N, REN Y P, et al. Pathogenicity analysis of three bovine viral diarrhea viruses from different sources in mice[J]. Acta Veterinaria et Zootechnica Sinica, 2018, 49(10):2232-2239.
[23] LETELLIER C, KERKHOFS P. Real-time PCR for simultaneous detection and genotyping of bovine viral diarrhea virus[J]. J Virol Methods, 2003, 114(1):21-27.
[24] 陈新诺, 肖敏, 阮文强, 等. 川藏地区牦牛牛病毒性腹泻病毒分子流行病学调查及分离鉴定[J]. 畜牧兽医学报, 2018, 49(3):606-613.CHEN X N, XIAO M, RUAN W Q, et al. Molecular epidemiological investigation and isolation of bovine viral diarrhea virus in yak in Sichuan-Tibet plateau region[J]. Acta Veterinaria et Zootechnica Sinica, 2018, 49(3):606-613. (in Chinese)
[25] DEPTA P N, DOSTA M, WENZEL W, et al. Hierarchical coarse-grained strategy for macromolecular self-assembly:application to Hepatitis B virus-like particles[J]. Int J Mol Sci, 2022, 23(23):14699.
[26] 李亚军, 茹毅, 郝荣增, 等. CHO细胞表达牛病毒性腹泻病毒E2蛋白及免疫原性分析[J]. 畜牧兽医学报, 2022, 53(12): 4315-4324.LI Y J, RU Y, HAO R Z, et al. Expression of bovine viral diarrhea virus E2 protein in CHO cells and immunogenicity analysis[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(12): 4315-4324. (in Chinese)
[27] EL OMARI K, IOURIN O, HARLOS K, et al. Structure of a pestivirus envelope glycoprotein E2 clarifies its role in cell entry[J]. Cell Rep, 2013, 3(1):30-35.
[28] PANDE A, CARR B V, WONG S Y C, et al. The glycosylation pattern of baculovirus expressed envelope protein E2 affects its ability to prevent infection with bovine viral diarrhoea virus[J]. Virus Res, 2005, 114(1-2):54-62.
[29] WANG Y X, FENG B H, NIU C, et al. Dendritic cell targeting of bovine viral diarrhea virus E2 protein expressed by Lactobacillus casei effectively induces antigen-specific immune responses via oral vaccination[J]. Viruses, 2019, 11(6):575.
[30] SANGEWAR N, HASSAN W, LOKHANDWALA S, et al. Mosaic bovine viral diarrhea virus antigens elicit cross-protective immunity in calves[J]. Front Immunol, 2020, 11:589537.
[31] 曹慧. BVDV-1型E2蛋白亚单位疫苗的制备及免疫效果评价[D]. 成都:西南民族大学, 2023.CAO H. Preparation and immune effect evaluation of BVDV-1 E2 protein subunit vaccine[D]. Chengdu:Southwest Minzu University, 2023. (in Chinese)
[32] SHI S T, ZHU H R, XIA X Y, et al. Vaccine adjuvants:Understanding the structure and mechanism of adjuvanticity[J]. Vaccine, 2019, 37(24):3167-3178.
[33] WACK A, BAUDNER B C, HILBERT A K, et al. Combination adjuvants for the induction of potent, long-lasting antibody and T-cell responses to influenza vaccine in mice[J]. Vaccine, 2008, 26(4):552-561.
[34] SINGH M, KAZZAZ J, UGOZZOLI M, et al. MF59 oil-in-water emulsion in combination with a synthetic TLR4 agonist (E6020) is a potent adjuvant for a combination Meningococcus vaccine[J]. Human Vacc Immunotherapeut, 2012, 8(4):486-490.
[35] WANG S H, YANG G H, NIE J W, et al. Recombinant E^rns-E2 protein vaccine formulated with MF59 and CPG-ODN promotes T cell immunity against bovine viral diarrhea virus infection[J]. Vaccine, 2020, 38(22):3881-3891.