表达鸡传染性法氏囊病病毒VP2基因的重组火鸡疱疹病毒的构建及生物学特性分析

doi:10.11843/j.issn.0366-6964.2023.02.022

畜牧兽医学报 ›› 2023, Vol. 54 ›› Issue (2): 656-662.doi: 10.11843/j.issn.0366-6964.2023.02.022

表达鸡传染性法氏囊病病毒VP2基因的重组火鸡疱疹病毒的构建及生物学特性分析

李晓涵, 杨伏春, 刘芮, 高立, 崔红玉, 张艳萍, 刘长军, 祁小乐, 王笑梅*, 高玉龙*, 李凯*

中国农业科学院哈尔滨兽医研究所, 兽医生物技术国家重点实验室禽免疫抑制病创新团队, 哈尔滨 150069

收稿日期:2022-08-01 出版日期:2023-02-23 发布日期:2023-02-21
通讯作者: 李凯,主要从事动物病毒学和免疫学研究,E-mail:likai01@caas.cn;高玉龙,主要从事动物病毒学和免疫学研究,E-mail:gaoyulong@caas.cn;王笑梅,主要从事动物病毒学和免疫学研究,E-mail:wangxiaomei@caas.cn
作者简介:李晓涵(1998-),女,山东泰安人,硕士生,主要从事动物病毒学研究
基金资助:
国家自然科学基金联合基金项目（U20A2061）

Construction of Recombinant Herpesvirus of Turkey Expressing Infectious Bursal Disease Virus VP2 Gene and Analysis of Its Biological Characteristics

LI Xiaohan, YANG Fuchun, LIU Rui, GAO Li, CUI Hongyu, ZHANG Yanping, LIU Changjun, QI Xiaole, WANG Xiaomei*, GAO Yulong*, LI Kai*

Avian Immunosuppressive Diseases Division, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China

Received:2022-08-01 Online:2023-02-23 Published:2023-02-21

摘要/Abstract

摘要： 鸡传染性法氏囊病（IBD）是一种严重危害养禽业的高度致死性和免疫抑制性传染病。为研制IBD重组火鸡疱疹病毒（HVT）活载体疫苗，本研究构建了表达鸡传染性法氏囊病病毒（IBDV）保护性抗原VP2基因的重组HVT并对其体外生物学特性进行了分析。通过RT-PCR扩增IBDV超强毒株VP2基因并克隆入pCI载体，获得重组真核表达质粒pCI-VP2。用限制性内切酶将携带CMV启动子的VP2基因表达框架切下，连接于入门质粒pENTR，构建获得重组入门质粒pENTR-VP2。将pENTR-VP2与HVT重组黏粒H3-Kan/ccdB进行LR重组反应，构建重组表达黏粒H3-VP2。用H3-VP2与其他4个相互重叠并覆盖HVT全基因组的黏粒共同转染鸡胚成纤维细胞（CEF），拯救获得重组病毒rHVT-VP2。将重组病毒在CEF中连续传至20代后用PCR、间接免疫荧光试验和免疫印迹试验进行检测，并绘制重组病毒体外生长曲线，分析其体外复制特性。结果表明，重组病毒rHVT-VP2能够稳定表达VP2蛋白，rHVT-VP2在CEF中的复制能力与亲本病毒无明显差异。重组病毒rHVT-VP2免疫鸡后能够诱导产生IBDV中和抗体，并对IBDV强毒株攻击引起的死亡提供90%免疫保护。重组病毒rHVT-VP2的构建为研制IBD重组HVT活载体疫苗奠定了基础，对IBD的防控具有重要意义。

关键词: 鸡传染性法氏囊病, 火鸡疱疹病毒, VP2基因, 生物学特性

Abstract: Infectious bursal disease (IBD) is a highly lethal and immunosuppressive infectious disease that seriously harms the poultry industry. In order to develop a recombinant herpesvirus of turkey (HVT) vector vaccine against IBD, a recombinant HVT expressing the protective antigen VP2 gene of infectious bursal disease virus (IBDV) was constructed and its biological characteristics in vitro were analyzed. The VP2 gene of very virulent IBDV (vvIBDV) was amplified by RT-PCR and cloned into pCI vector to obtain the recombinant eukaryotic expression plasmid pCI-VP2. The expression frame of VP2 gene carrying CMV promoter was cut off by restriction endonuclease and ligated into entry plasmid pENTR to construct a recombinant entry plasmid pENTR-VP2. The recombinant expression fosmid H3-VP2 were constructed by LR recombination reaction between pENTR-VP2 entry plasmid and HVT recombinant fosmid H3-Kan/ccdB. The recombinant virus rHVT-VP2 was obtained by co-transfection of H3-VP2 with four other fosmids that overlap each other and cover the whole genome of HVT into chicken embryo fibroblasts (CEF). After continuously passaged in CEF for 20 generations, the recombinant virus was detected by PCR, indirect immunofluorescence, and Western blot. The growth curve of recombinant virus in vitro was determined and its replication characteristics in vitro were analyzed. The results showed that the recombinant virus rHVT-VP2 stably expressed VP2 protein, and the replication ability of rHVT-VP2 in CEF was not significantly different from that of the parent virus. The recombinant virus rHVT-VP2 induced virus neutralization antibodies against IBDV and provided 90% protection against vvIBDV-induced death in chickens. The construction of recombinant virus rHVT-VP2 lays a foundation for the development of recombinant HVT live vector vaccine against IBD and is of great significance for the prevention and control of this disease.

Key words: infectious bursal disease, herpesvirus of turkey, VP2 gene, biological characteristics

中图分类号:

S852.659.4

李晓涵, 杨伏春, 刘芮, 高立, 崔红玉, 张艳萍, 刘长军, 祁小乐, 王笑梅, 高玉龙, 李凯. 表达鸡传染性法氏囊病病毒VP2基因的重组火鸡疱疹病毒的构建及生物学特性分析[J]. 畜牧兽医学报, 2023, 54(2): 656-662.

LI Xiaohan, YANG Fuchun, LIU Rui, GAO Li, CUI Hongyu, ZHANG Yanping, LIU Changjun, QI Xiaole, WANG Xiaomei, GAO Yulong, LI Kai. Construction of Recombinant Herpesvirus of Turkey Expressing Infectious Bursal Disease Virus VP2 Gene and Analysis of Its Biological Characteristics[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(2): 656-662.

参考文献 21

[1]	VAN DEN BERG T P. Acute infectious bursal disease in poultry:a review[J]. Avian Pathol, 2000, 29(3):175-194.
[2]	NOORUZZAMAN M, HOSSAIN I, RAHMAN M M, et al. Comparative pathogenicity of infectious bursal disease viruses of three different genotypes[J]. Microb Pathog, 2022, 169:105641.
[3]	CHEN Z X, LIAN J M, LIANG Z S, et al. Characterization and pathogenicity of infectious bursal disease virus in Southern China[J]. Poult Sci, 2022, 101(10):102018.
[4]	ALIYU H B, HAMISU T M, BEJO M H, et al. Comparative pathogenicity of Malaysian variant and very virulent infectious bursal disease viruses in chickens[J]. Avian Pathol, 2022, 51(1):76-86.
[5]	SNYDER D B, LANA D P, SAVAGE P K, et al. Differentiation of infectious bursal disease viruses directly from infected tissues with neutralizing monoclonal antibodies:evidence of a major antigenic shift in recent field isolates[J]. Avian Dis, 1988, 32(3):535-539.
[6]	JIANG N, WANG Y L, ZHANG W Y, et al. Genotyping and molecular characterization of infectious bursal disease virus identified in important poultry-raising areas of China during 2019 and 2020[J]. Front Vet Sci, 2021, 8:759861.
[7]	VAN DEN BERG T P, GONZE M, MEULEMANS G. Acute infectious bursal disease in poultry:isolation and characterisation of a highly virulent strain[J]. Avian Pathol, 1991, 20(1):133-143.
[8]	WANG Y S, WANG Z C, TANG Y D, et al. Comparison of four infectious bursal disease viruses isolated from different bird species[J]. Arch Virol, 2007, 152(10):1787-1797.
[9]	VAN DEN BERG T P, MORALES D, ETERRADOSSI N, et al. Assessment of genetic, antigenic and pathotypic criteria for the characterization of IBDV strains[J]. Avian Pathol, 2004, 33(5):470-476.
[10]	LI G P, KUANG H Y, GUO H X, et al. Development of a recombinant VP2 vaccine for the prevention of novel variant strains of infectious bursal disease virus[J]. Avian Pathol, 2020, 49(6):557-571.
[11]	LI K, GAO H L, GAO L, et al. Adjuvant effects of interleukin-18 in DNA vaccination against infectious bursal disease virus in chickens[J]. Vaccine, 2013, 31(14):1799-1805.
[12]	TENG M, ZHENG L P, LI H Z, et al. Pathogenicity and pathotype analysis of henan isolates of Marek's disease virus reveal long-term circulation of highly virulent MDV variant in China[J]. Viruses, 2022, 14(8):1651.
[13]	ZELNÍK V. Marek's disease virus research in the post-sequencing era:new tools for the study of gene functions and virus-host interactions[J]. Avian Pathol, 2003, 32(4):323-333.
[14]	BIGGS P M, NAIR V. The long view:40 years of Marek's disease research and Avian pathology[J]. Avian Pathol, 2012, 41(1):3-9.
[15]	REDDY S M, IZUMIYA Y, LUPIANI B. Marek's disease vaccines:Current status, and strategies for improvement and development of vector vaccines[J]. Vet Microbiol, 2017, 206:113-120.
[16]	ROMANUTTI C, KELLER L, ZANETTI F A. Current status of virus-vectored vaccines against pathogens that affect poultry[J]. Vaccine, 2020, 38(45):6990-7001.
[17]	REEMERS S, VERSTEGEN I, BASTEN S, et al. A broad spectrum HVT-H5 avian influenza vector vaccine which induces a rapid onset of immunity[J]. Vaccine, 2021, 39(7):1072-1079.
[18]	JIA W F, ZHANG X H, WANG H R, et al. Construction and immune efficacy of a recombinant turkey herpesvirus vaccine strain expressing fusion protein of genotype VII Newcastle disease virus[J]. Vet Microbiol, 2022, 268:109429.
[19]	SHARMA J M, KIM I J, RAUTENSCHLEIN S, et al. Infectious bursal disease virus of chickens:pathogenesis and immunosuppression[J]. Dev Comp Immunol, 2000, 24(2-3):223-235.
[20]	MVLLER H, ISLAM M R, RAUE R. Research on infectious bursal disease-the past, the present and the future[J]. Vet Microbiol, 2003, 97(1-2):153-165.
[21]	LIAO Y L, BAJWA K, REDDY S M, et al. Methods for the manipulation of herpesvirus genome and the application to Marek's disease virus research[J]. Microorganisms, 2021, 9(6):1260.

表达鸡传染性法氏囊病病毒VP2基因的重组火鸡疱疹病毒的构建及生物学特性分析

Construction of Recombinant Herpesvirus of Turkey Expressing Infectious Bursal Disease Virus VP2 Gene and Analysis of Its Biological Characteristics

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献 21

相关文章 15

编辑推荐

Metrics

本文评价

[1]	李昭燕, 高江, 郭时惠, 赵茹茜, 马文强. 猫过敏原检测方法与控制措施的研究进展[J]. 畜牧兽医学报, 2023, 54(6): 2272-2279.
[2]	刘青, 吴少鹏, 石彬, 邵红霞, 钱琨, 叶建强, 秦爱建. 鸡CTLA-4蛋白在昆虫细胞中的表达及其单克隆抗体的制备[J]. 畜牧兽医学报, 2023, 54(6): 2596-2604.
[3]	李德豪, 李宜穗, 马静云, 孙媛, 张祥斌. 一株沙门菌温和噬菌体PEA2-3生物学特性及基因组分析[J]. 畜牧兽医学报, 2023, 54(4): 1632-1640.
[4]	韩生义, 李玲霞, 李淑萍, 胡国元, 李生庆. 沙门菌噬菌体SP3的生物学特性及基因组分析[J]. 畜牧兽医学报, 2023, 54(12): 5228-5239.
[5]	程宝钰, 李子荷, 崔燕蕾, 李嘉慧, 杨鑫玮, 于永乐, 杨海燕, 单虎, 张传美. 猫细小病毒的遗传演化及分离毒株的致病性分析[J]. 畜牧兽医学报, 2022, 53(9): 3121-3131.
[6]	岑鑫, 阳亭亭, 赵尊福, 文永平, 张焕容. 沙门菌烈性噬菌体的分离鉴定、生物学特性及基因组分析[J]. 畜牧兽医学报, 2022, 53(8): 2677-2688.
[7]	曹莉, 程如楠, 武周慧, 王家伟, 王瑜, 张永红, 吴清民, 王真. 鼠伤寒沙门菌sapC基因缺失株的构建及生物学特性分析[J]. 畜牧兽医学报, 2022, 53(7): 2282-2289.
[8]	周群, 杨苑, 宋鑫, 何欣怡, 曹慧, 张斌. 2017—2021年成都市猫细小病毒的检测及其VP2基因的变异分析[J]. 畜牧兽医学报, 2022, 53(4): 1303-1309.
[9]	刘畅, 刘教, 陈进, 王勉之, 熊文广, 曾振灵. 1株可裂解bla_NDM-5及mcr-1阳性大肠杆菌噬菌体的分离与生物学特性[J]. 畜牧兽医学报, 2022, 53(3): 857-866.
[10]	孙培皓, 赵鑫哲, 吴涵潇, 吕策, 杨利国, 梁爱心. 奶牛乳腺上皮细胞的原代培养及其生物学特性分析[J]. 畜牧兽医学报, 2022, 53(2): 447-458.
[11]	吴居业, 卢宝春, 祝宇凡, 贾坤. 血小板凝血酶蛋白1对犬乳腺肿瘤细胞体外生物学特性的影响分析[J]. 畜牧兽医学报, 2022, 53(12): 4470-4481.
[12]	吴立婷, 刘半红, 田源, 王娟, 包红朵, 周艳, 庞茂达, 王冉, 张辉. 产气荚膜梭菌噬菌体鉴定及其环境消减效果评价[J]. 畜牧兽医学报, 2022, 53(10): 3550-3560.
[13]	邵颖, 傅丹丹, 吴晓妍, 谷一, 宋祥军, 涂健, 祁克宗. ETT2结构基因epaPQR对禽致病性大肠杆菌生物学特性及致病性的影响[J]. 畜牧兽医学报, 2022, 53(10): 3631-3641.
[14]	巩倩雯, 李一昊, 曾颃, 于沛欣, 钱新杰, 王瑜欣, 戴建君, 汤芳. 特异性识别K1荚膜大肠杆菌的噬菌体PNJ1809-36生物学特性及全基因组分析[J]. 畜牧兽医学报, 2021, 52(6): 1677-1688.
[15]	李淑雅, 黄赞, 朱伟云, 林焱. 1株C3样奇异变形杆菌烈性噬菌体的生物学特性及基因组分析[J]. 畜牧兽医学报, 2021, 52(4): 1069-1078.