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

doi:10.11843/j.issn.0366-6964.2020.10.014

畜牧兽医学报 ›› 2020, Vol. 51 ›› Issue (10): 2463-2471.doi: 10.11843/j.issn.0366-6964.2020.10.014

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

程增青^1,2, 范根成², 窦小龙², 刘红祥², 申茂欣², 徐全刚³, 杨汉春^1*

1. 中国农业大学动物医学院, 北京 100193;
2. 青岛易邦生物工程有限公司/动物基因工程疫苗国家重点实验室, 青岛 266114;
3. 中国动物卫生与流行病学中心, 青岛 266032

收稿日期:2020-03-30 出版日期:2020-10-25 发布日期:2020-10-26
通讯作者: 杨汉春,主要从事动物分子病毒学与免疫学研究,E-mail:yanghanchun1@cau.edu.cn
作者简介:程增青(1983-),男,河南西峡人,兽医师,博士,主要从事动物用生物制品研发,E-mail:52191071@qq.com
基金资助:
青岛市动物保健品行业智库联合基金二期18-6-3-1-jch科技服务计划（科技金融专项-智库基金）

Isolation and Identification of Group I Type 4 Avian Adenovirus and Analysis in Immune Effect of Fiber-2 Protein

CHENG Zengqing^1,2, FAN Gencheng², DOU Xiaolong², LIU Hongxiang², SHEN Maoxin², XU Quangang³, YANG Hanchun^1*

1. College of Veterinary Medicine, China Agricultural University, Beijing 100193, China;
2. State Key Laboratory of Animal Genetic Engineering Vaccine/YEBIO Bioengineering Co., Ltd of Qingdao, Qingdao 266114, China;
3. China Animal Health and Epidemiology Center, Qingdao 266032, China

Received:2020-03-30 Online:2020-10-25 Published:2020-10-26

摘要/Abstract

摘要： 旨在鉴定河南某蛋鸡场以心包积液、肝肿大出血为特征的病原，并对病原的免疫原性蛋白进行表达和效果分析。本研究采用病毒分离、血清学试验、PCR检测及测序分析、动物回归试验、大肠杆菌表达、蛋白纯化、攻毒保护等方法进行研究。结果显示，所采集样品经卵黄囊途径接种7日龄SPF鸡胚，盲传2代后获得病毒；PCR可扩增出约900 bp的Hexon基因片段，经测序，其Hexon基因与血清C4型毒株的相似性为100%；血清中和试验结果表明分离的禽腺病毒为1群血清4型禽腺病毒，命名为HN-ZK株。该毒株可在鸡胚原代肝细胞上产生CPE，病毒含量可达10^7.5TCID₅₀·0.1 mL^-1。动物回归试验表明，该分离毒株可使35日龄SPF鸡100%（10/10）表现出发病症状。以分离株的DNA为模板，利用大肠杆菌原核表达系统，获得相对分子质量约为33 ku的Fiber-2蛋白，离心浓缩、纯化后蛋白含量为300 mg·mL^-1。将表达的Fiber-2蛋白用不同的剂量免疫SPF鸡，结果表明20 μg·只^-1的剂量能使鸡完全抵抗强毒株的攻击，说明Fiber-2蛋白具有较好的免疫原性。本研究可为禽心包积水-肝炎综合征的诊断和基因工程亚单位疫苗的研制提供数据参考。

关键词: I群禽腺病毒, 鉴定, Fiber-2蛋白, 亚单位疫苗

Abstract: The aim of this study was to identify pathogens, which caused pericardial effusion, hepatomegaly and bleeding at a chicken farm in Henan province, and furthermore to analyze effectiveness of immunogenic proteins of the pathogen. This study employed virus isolation, serological assays, PCR and sequencing analysis, animal experimentation, E. coli expression and protein purification, immunogenicity and challenge test and other methods. The results showed that virus was isolated in 7-day-old SPF chicken embryonated eggs, inoculated via the yolk sac route by two blind passages. Viral confirmation was carried out using PCR techniques, and showed a 900-bp-long fragment which shared a 100% homology with the Hexon gene of the serotype C4 strain. Serum neutralization results indicated that this isolate avian adenovirus was the group I type 4 avian adenovirus, named HN-ZK strain. The virus could induce CPE on primary hepatocytes of chicken embryo, and its titer was 10^7.5 TCID₅₀·0.1 mL^-1. Animal experimentation illustrated that the isolated virus caused 100% (10/10) typical symptoms and pathogenicity in 35-day-old SPF chickens. Furthermore, the DNA of the isolate virus was used as a template to express Fiber-2 fragment, with a molecular weight of 33 kDa by using the E. coli expression system, and the protein was concentrated and purified to 300 mg·mL^-1 after centrifugation and purification. SPF chickens were immunized with different doses of the purified Fiber-2 protein, and showed that a dose of 20 μg per chick was completely resistant to challenge of the virulent virus, suggesting the purified Fiber-2 protein has better immunogenicity. This study can provide data for diagnosing the hydropericardium hepatitis syndrome, and developing a genetic engineering subunit vaccine.

Key words: group I avian adenovirus, identification, fiber-2 protein, subunit vaccine

中图分类号:

S852.659.1

程增青, 范根成, 窦小龙, 刘红祥, 申茂欣, 徐全刚, 杨汉春. 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 Zengqing, FAN Gencheng, DOU Xiaolong, LIU Hongxiang, SHEN Maoxin, XU Quangang, YANG Hanchun. 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.

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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

Isolation and Identification of Group I Type 4 Avian Adenovirus and Analysis in Immune Effect of Fiber-2 Protein

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