禽腺病毒4型fiber-2与8b型fiber截短融合蛋白的原核表达及其免疫原性分析

doi:10.11843/j.issn.0366-6964.2023.12.026

摘要/Abstract

摘要： 利用原核表达系统表达Ⅰ群禽腺病毒血清4型(FAdV-4)fiber2和8b型(FAdV-8b)fiber全长蛋白、截短蛋白及二价融合蛋白,并评价其免疫原性。基于FAdV-4 fiber2和FAdV-8b fiber抗原性好的截短片段(FP和BP),利用SOE-PCR构建二价融合片段(BP-FP),分别对截短蛋白、融合蛋白及全长(FL和BL)在大肠杆菌中进行表达,采用SDS-PAGE和Western blot对重组蛋白进行鉴定。纯化后的重组蛋白免疫SPF鸡并攻毒,计算各组存活率、间接ELISA检测免疫鸡血清抗体水平、qPCR检测泄殖腔拭子排毒量和肝脏中细胞因子IL-4、IFN-γ和TNF-α mRNA表达水平以及进行组织病理观察。结果显示:构建的重组质粒均在大肠杆菌中成功可溶表达;BP-FP4组存活率为58.3%,其他组存活率均为100%;全长组(FL组和BL组)抗体水平均高于各自片段组(FP组和BP组),但差异不显著(P＞0.05);攻毒后3 和7 d C8b组排毒量均显著高于免疫组(P＜0.05),攻毒后7 d BP-FP8b组排毒量显著高于BP和BL组(P＜0.05),BL组和BP组差异不显著(P＞0.05);免疫组IL-4、IFN-γ和TNF-α mRNA表达均显著高于阴性对照组(P＜0.05),FL组IFN-γ和TNF-α mRNA表达均显著低于BP-FP4组(P＜0.05),FL组和FP组差异不显著(P＞0.05),BL组IL-4和IFN-γ mRNA表达均显著低于BP-FP8b组(P＜0.05)。组织病理观察发现,攻毒组肝脏病变最严重,FP组与BP组次之,FL组与BL组无明显病变。本研究表明FAdV-4 fiber2和FAdV-8b fiber的全长蛋白和截短蛋白免疫保护效果优于融合蛋白,截短蛋白可替代全长蛋白用于制备禽腺病毒亚单位疫苗。

关键词: 禽腺病毒, 纤突蛋白, 原核表达, 亚单位疫苗

Abstract: The purpose of this study was to express the full-length, truncated and bivalent fusion proteins of group Ⅰ fowl adenovirus serotype 4 (FAdV-4) fiber2 and serotype 8b(FAdV-8b) fiber by prokaryotic expression system, and evaluate its immunogenicity. Based on good antigenic truncated fragments (FP and BP) of FAdV-4 fiber2 and FAdV-8b fiber, a bivalent fusion fragment was constructed by SOE-PCR. The truncated protein, full-length and fusion protein were individually expressed in E. coli and identified by SDS-PAGE and Western blot. The SPF chickens were immunized using purified recombinant proteins and challenged with FAdV-4 and FAdV-8b. Survival rate of each group was calculated, and serum antibody levels of immunized chickens were detected by indirect ELISA. Viral loads of cloacal swab and the mRNA expression levels of cytokines IL-4, IFN-γ and TNF-α in liver were detected by qPCR post-challenge and histopathological observation. The constructed recombinant plasmids were successfully soluble expression in E. coli. The survival rate of BP-FP4 group was 58.3%, and that of other groups was 100%. The antibody level of full-length group (FL group and BL group) was higher than that of its corresponding fragment group (FP group and BP group), but the difference was not significant (P>0.05). At 3 days and 7 days post-challenge, the viral load in C8b group was significantly higher than that of immunized group (P<0.05). At 7 days post-challenge, the viral load in BP-FP8b group was significantly higher than that in BP and BL groups (P<0.05), but there was no significant difference between BL and BP group (P>0.05). The mRNA expression of three cytokines (IL-4,IFN-γ and TNF-α) in immunized groups were significantly higher compared to the negative control group (P<0.05). The mRNA expression of IFN-γ and TNF-α in FL group were significantly lower than those in BP-FP4 group (P<0.05), and there was no significant difference between FL group and FP group (P>0.05). The mRNA expression of IL-4 and IFN-γ in BL group were significantly lower than those in BP-FP8b group (P<0.05). Histopathological observation revealed that the liver lesions in the challenge groups were the most severe, followed by the FP and BP groups, and no significant pathological changes were observed in the FL and BL groups. This study showed that the immunoprotection of the truncated protein and the full-length protein of FAdV-4 fiber2 and FAdV-8b was better than that of the fusion protein. Therefore, the truncated protein could be used to replace the full-length protein in the preparation of fowl adenovirus subunit vaccine.

Key words: fowl adenovirus, fiber, prokaryotic expression, subunit vaccine

中图分类号:

S852.659.1

迟丽丽, 王君娜, 张宇名, 刘健, 陈志远, 李树凡, 尹燕博, 徐守振. 禽腺病毒4型fiber-2与8b型fiber截短融合蛋白的原核表达及其免疫原性分析[J]. 畜牧兽医学报, 2023, 54(12): 5162-5170.

CHI Lili, WANG Junna, ZHANG Yuming, LIU Jian, CHEN Zhiyuan, LI Shufan, YIN Yanbo, XU Shouzhen. Prokaryotic Expression and Immunogenicity Analysis of Truncated Fusion Protein of FAdV-4 Fiber2 and FAdV-8b Fiber[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(12): 5162-5170.

参考文献

[1] EL-SHALL N A, EL-HAMID H S A, ELKADY M F, et al. Epidemiology, pathology, prevention, and control strategies of inclusion body hepatitis and hepatitis-hydropericardium syndrome in poultry:a comprehensive review[J]. Front Vet Sci, 2022, 9:963199.
[2] LIU A J, ZHANG Y, CUI H Y, et al. Advances in vaccine development of the emerging novel genotype fowl adenovirus 4[J]. Front Immunol, 2022, 13:916290.
[3] WANG Z, ZHAO J. Pathogenesis of hypervirulent fowl adenovirus serotype 4:the contributions of viral and host factors[J]. Viruses, 2019, 11(8):741.
[4] 王白玉, 黄庆, 乔麒龙, 等. 表达FAdV-8b Fiber蛋白重组FAdV-4的构建与鉴定[J]. 中国兽医学报, 2022, 42(2):230-236.WANG B Y, HUANG Q, QIAO Q L, et al. Construction and identification of recombinant fowl adenovirus 4 expressing the fiber gene of fowl adenovirus 8b[J]. Chinese Journal of Veterinary Science, 2022, 42(2):230-236. (in Chinese)
[5] SOHAIMI N M, HAIR-BEJO M. A recent perspective on fiber and hexon genes proteins analyses of fowl adenovirus toward virus infectivity-A review[J]. Open Vet J, 2021, 11(4):569-580.
[6] CHEN L, YIN L J, ZHOU Q F, et al. Epidemiological investigation of fowl adenovirus infections in poultry in China during 2015-2018[J]. BMC Vet Res, 2019, 15(1):271.
[7] HARAKUNI T, ANDOH K, SAKAMOTO R I, et al. Fiber knob domain lacking the shaft sequence but fused to a coiled coil is a candidate subunit vaccine against egg-drop syndrome[J]. Vaccine, 2016, 34(27):3184-3190.
[8] GUARDADO-CALVO P, LLAMAS-SAIZ A L, FOX G C, et al. Structure of the C-terminal head domain of the fowl adenovirus type 1 long fiber[J]. J Gen Virol, 2007, 88(9):2407-2416.
[9] DE LUCA C, SCHACHNER A, MITRA T, et al. Fowl adenovirus (FAdV) fiber-based vaccine against inclusion body hepatitis (IBH) provides type-specific protection guided by humoral immunity and regulation of B and T cell response[J]. Vet Res, 2020, 51(1):143.
[10] 郭瑞珍, 苏冰倩, 王棋, 等. 禽腺病毒血清4型Fiber2蛋白单克隆抗体制备及其抗原表位鉴定[J]. 畜牧兽医学报, 2021, 52(7): 2000-2012.GUO R Z, SU B Q, WANG Q, et al. Preparation of the monoclonal antibody against the serotype 4 fowl adenovirus fiber2 protein and identification of the antigenic epitope[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(7): 2000-2012. (in Chinese)
[11] GUPTA A, AHMED K A, AYALEW L E, et al. Immunogenicity and protective efficacy of virus-like particles and recombinant fiber proteins in broiler-breeder vaccination against fowl adenovirus (FAdV)-8b[J]. Vaccine, 2017, 35(20):2716-2722.
[12] SCHACHNER A, MAREK A, JASKULSKA B, et al. Recombinant FAdV-4 fiber-2 protein protects chickens against hepatitis-hydropericardium syndrome (HHS)[J]. Vaccine, 2014, 32(9):1086-1092.
[13] LI P H, ZHENG P P, ZHANG T F, et al. Fowl adenovirus serotype 4:epidemiology, pathogenesis, diagnostic detection, and vaccine strategies[J]. Poult Sci, 2017, 96(8):2630-2640.
[14] JIA Z P, PAN X H, ZHI W J, et al. Probiotics surface-delivering fiber2 protein of fowl adenovirus 4 stimulate protective immunity against hepatitis-hydropericardium syndrome in chickens[J]. Front Immunol, 2022, 13:919100.
[15] 邹开宇, 高倩文, 江之瑶, 等. 我国Ⅰ群禽腺病毒主要流行血清型间交叉中和作用的初步研究[J]. 黑龙江畜牧兽医, 2018(11):150-153.ZOU K Y, GAO Q W, JIANG Z Y, et al. Study on the cross-neutralization between the main serotypes of group Ⅰ fowl adenovirus in China[J]. Heilongjiang Animal Science and Veterinary Medicine, 2018(11):150-153. (in Chinese)
[16] 吴志金. 鸡传染性支气管炎病毒广西分离株的致病性研究和细胞因子实时荧光RT-PCR相对定量检测方法的建立[D]. 南宁:广西大学, 2013.WU Z J. Pathogenicity of infectious bronchitis virus Guangxi isolates and development of real-time RT-PCR assays for chicken cytokines[D]. Nanning:Guangxi University, 2013. (in Chinese)
[17] MENG K, YUAN X Y, YU J, et al. Identification, Pathogenicity of novel fowl adenovirus serotype 4 SDJN0105 in Shandong, China and immunoprotective evaluation of the newly developed inactivated oil-emulsion FAdV-4 vaccine[J]. Viruses, 2019, 11(7):627.
[18] YIN L J, CHEN L, LUO Y Y, et al. Recombinant fiber-2 protein protects Muscovy ducks against duck adenovirus 3(DAdV-3)[J]. Virology, 2019, 526:99-104.
[19] ZHANG X H, ZHONG Y J, ZHOU Z H, et al. Molecular characterization, phylogeny analysis and pathogenicity of a Muscovy duck adenovirus strain isolated in China in 2014[J]. Virology, 2016, 493:12-21.
[20] TIAN K Y, GUO H F, LI N, et al. Protection of chickens against hepatitis-hydropericardium syndrome and newcastle disease with a recombinant Newcastle disease virus vaccine expressing the fowl adenovirus serotype 4 fiber-2 protein[J]. Vaccine, 2020, 38(8):1989-1997.
[21] SONG Y P, WEI Q, LIU Y C, et al. Development of novel subunit vaccine based on truncated fiber protein of egg drop syndrome virus and its immunogenicity in chickens[J]. Virus Res, 2019, 272:197728.
[22] 程增青, 范根成, 窦小龙, 等. 1群4型禽腺病毒的分离鉴定及Fiber-2蛋白的免疫效果分析[J]. 畜牧兽医学报, 2020, 51(10):2463-2471.CHENG Z Q, FAN G C, DOU X L, et al. Isolation and identification of group I type 4 avian adenovirus and analysis in immune effect of fiber-2 protein[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(10):2463-2471. (in Chinese)
[23] GUO X, CHANG J, LU S Y, et al. Multiantigen epitope fusion recombinant proteins from capsids of serotype 4 fowl adenovirus induce chicken immunity against avian Angara disease[J]. Vet Microbiol, 2023, 278:109661.
[24] GRAFL B, PROKOFIEVA I, WERNSDORF P, et al. Infection with an apathogenic fowl adenovirus serotype-1 strain (CELO) prevents adenoviral gizzard erosion in broilers[J]. Vet Microbiol, 2014, 172(1-2):177-185.
[25] FINGERUT E, GUTTER B, GALLILI G, et al. A subunit vaccine against the adenovirus egg-drop syndrome using part of its fiber protein[J]. Vaccine, 2003, 21(21-22):2761-2766.
[26] SHAH M S, ASHRAF A, KHAN M I, et al. Fowl adenovirus:history, emergence, biology and development of a vaccine against hydropericardium syndrome[J]. Arch Virol, 2017, 162(7):1833-1843.
[27] UGWU C C, HAIR-BEJO M, NURULFIZA M I, et al. Efficacy, humoral, and cell-mediated immune response of inactivated fowl adenovirus 8b propagated in chicken embryo liver cells using bioreactor in broiler chickens[J]. Vet World, 2022, 15(11):2681-2692.
[28] PAN Q, YANG Y C, GAO Y L, et al. An inactivated novel genotype fowl adenovirus 4 protects chickens against the hydropericardium syndrome that recently emerged in China[J]. Viruses, 2017, 9(8):216.
[29] 刘玲, 焦鹏涛, 王萌, 等. 鸡干扰素γ与白介素2对外周血中Th1细胞分化相关细胞因子的影响[J]. 生物工程学报, 2022, 38(9):3329-3343.LIU L, JIAO P T, WANG M, et al. Effects of chicken interferon γ and interleukin-2 on cytokines related to Th1 cell differentiation in peripheral blood[J]. Chinese Journal of Biotechnology, 2012, 38(9):3329-3343. (in Chinese)
[30] SARFRAZ M, SULEMAN M, TIKOO S K, et al. Immune responses to in ovo vaccine formulations containing inactivated fowl adenovirus 8b with poly[di(sodium carboxylatoethylphenoxy)] phosphazene (PCEP) and avian beta defensin as adjuvants in chickens[J]. Vaccine, 2017, 35(6):981-986.