期刊文献+

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 被引量:6

Isolation and Identification of Group I Type 4 Avian Adenovirus and Analysis in Immune Effect of Fiber-2 Protein
下载PDF
导出
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^(7.5)TCID50·0.1 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20μg·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。 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 107.5 TCID 50·0.1 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 20μg 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.
作者 程增青 范根成 窦小龙 刘红祥 申茂欣 徐全刚 杨汉春 CHENG Zengqing;FAN Gencheng;DOU Xiaolong;LIU Hongxiang;SHEN Maoxin;XU Quangang;YANG Hanchun(College of Veterinary Medicine,China Agricultural University,Beijing 100193,China;State Key Laboratory of Animal Genetic Engineering Vaccine/YEBIO Bioengineering Co.,Ltd of Qingdao,Qingdao 266114,China;China Animal Health and Epidemiology Center,Qingdao 266032,China)
出处 《畜牧兽医学报》 CAS CSCD 北大核心 2020年第10期2463-2471,共9页 ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金 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)。
关键词 I群禽腺病毒 鉴定 Fiber-2蛋白 亚单位疫苗 group I avian adenovirus identification fiber-2 protein subunit vaccine
  • 相关文献

参考文献2

二级参考文献24

  • 1周斌,刘华雷,曹瑞兵,陈溥言.污染疫苗的禽腺病毒分离和鉴定[J].南京农业大学学报,2004,27(3):78-80. 被引量:21
  • 2戴志红,谢磊,赵耘.腺病毒的生物学特性[J].中国兽药杂志,2007,41(2):36-39. 被引量:6
  • 3Zhou Caiqin, Zhang Caiqiao. Protective effects of antioxidant vitamins on Aroclor 1254-induced toxicity in cultured chicken embryo hepatoeytes [J]. Toxieol in Vitro, 2005, 19:665-673. 被引量:1
  • 4Kennedy S W, Lorenzen A, James C A, et al. Ethoxyresorufin-O-deethylase and porphyrin analysis in chicken embryo hepatocyte cultures th a fluorescent multiwell plate reader [ J]. Analytical Biochemistry, 1993, 211 ( 1 ) : 102 - 112. 被引量:1
  • 5Fujii M, Yoshino I, Suzuki M, et al. Primary culture of chicken hepatocytes in serum-free medium( pH 7.8) secreted albumin and transferrin for a long period in free gas exchange with the atmosphere [ J ]. Iut J Biochem Cell B iol, 1996, 28 (12) : 1381 -1391. 被引量:1
  • 6Yellaturu C R, Deng X, Cagen L M, et al. Posttranslational processing of SREBP-1 in rat hepatoeytes is regulated by insulin and cAMP [J]. Biochemical and Biophysical Research Communications, 2005, 332(1) : 174- 180. 被引量:1
  • 7Sathiyaa Pt, Vijayan M M. Autoregulation of glucocorticoid receptor by cortisol in rainbow trout hepatocytes[J]. Am J Physiol Cell Physiol, 2003, 284:1508-1515. 被引量:1
  • 8Yamamoto N, Wu J, Zhang Y, et al. An optimal culture condition maintains human hepatocyte phenotype after long-term culture [J]. Hepatology Research, 2006, 35: (3) 169-177. 被引量:1
  • 9Li W C, Ralphs K L, Slack J M W, et al. Keratinocyte serum-free medium maintains long-term liver gene expression and function in cultured rat hepatocytes by preventing the loss of liver-enriched transcription factors [ J]. J Biochem Cell Biol, 2007, 39:541-554. 被引量:1
  • 10Dinesh Mittal,Naresh Jindal,Ashok Kumar Tiwari,Raj Singh Khokhar.Characterization of fowl adenoviruses associated with hydropericardium syndrome and inclusion body hepatitis in broiler chickens[J]. Indian Journal of Virology . 2014 (1) 被引量:1

共引文献25

同被引文献44

引证文献6

二级引证文献16

相关作者

内容加载中请稍等...

相关机构

内容加载中请稍等...

相关主题

内容加载中请稍等...

浏览历史

内容加载中请稍等...
;
使用帮助 返回顶部