• 中文核心期刊
  • CSCD来源期刊
  • 中国科技核心期刊
  • CA、CABI、ZR收录期刊

一株鸭肝炎病毒的基因组序列及其VP1基因的多克隆抗体制备

Genomic Sequencing and VP1 Polyclonal Antibody Preparation for a Duck Hepatitis Virus

  • 摘要:
      目的  确定福建某鸭场雏鸭以出现角弓反张和肝脏出现肿大并伴有密集出血点为主要病征的高死亡率病害的病原,并制备可特异性检测的多克隆抗体,以期为福建省鸭流行病学研究提供素材。
      方法  取该鸭场病死鸭肝脏组织进行病毒的分离鉴定,并对病毒进行全基因测序。利用pGEX-4T-1载体构建该病毒优势抗原表位基因的高效表达系统,制备VP1多克隆抗体,并用Western-blotting检测其特异性。
      结果  应用非免疫的鸭胚从疑似鸭病毒性肝炎的鸭肝脏中分离到1株病毒。该分离毒在鸭胚连续传6代后死亡率约为70%,且死胚尿囊液分别在鸭、鸡、小鼠和兔血液中均未出现血凝现象。注射分离毒的尿囊液的1日龄雏鸭死亡率达100%。RT- PCR扩增结果、全基因组测序结果表明分离毒为I型鸭病毒性肝炎病毒,毒株DQ226541.1的基因序列同源性达99.4%,其VP1氨基酸序列与毒株DQ226541.1氨基酸序列一致。将毒株命名为Fujian2015。成功构建重组质粒pGEX-4T-1-VP1,并制备了可特异性检测VP1蛋白的VP1多抗血清。
      结论  成功分离到了1株I型鸭病毒性肝炎病毒,同时制备了该毒株的VP1多克隆抗体,为后续检测I型鸭病毒性肝炎病毒相关研究奠定了基础。

     

    Abstract:
      Objective  The pathogen that caused the high mortality disease showing symptoms of anterior arch reflexion and liver enlargement with dense bleeding points on ducklings at a duck farm in Fujian was identified. Specifically detectable polyclonal antibody was prepared for epidemiological study on the disease.
      Method  Liver tissues of the affected and died ducklings were collected for virus identification. Sequence of the virus was determined, and the pGEX-4T-1 vector used to construct a high-efficiency expression system for the dominant epitope gene. A VP1 polyclonal antibody was prepared, and specificity confirmed by western-blotting.
      Result  A virus was isolated from the liver of a duck suspected of viral hepatitis using non-immune duck embryos. The mortality rate by the isolated virus on the embryos after 6 generations of continuous transmission was approximately 70%. But the allantoic fluid of the dead embryo did not show hemagglutination in the blood of duck, chicken, mouse, or rabbit. In contrast, the mortality rate on 1-day-old ducklings injected with the allantoic fluid was 100%. The RT-PCR amplification and the entire genome sequence indicated the isolated virus to be positive for duck viral hepatitis virus 1. Furthermore, the gene homology between the virus and DQ226541.1 was 99.4%, and the sequence of VP1 amino acids consistent with that of DQ226541.1. The virus was code named Fujian 2015. Subsequently, the recombinant plasmid pGEX-4T-1-VP1 was successfully constructed, and the VP1 polyclonal antibody serum capable of specifically detecting VP1 protein prepared.
      Conclusion   A duck hepatitis virus type I was successfully isolated, and a VP1 polyclonal antibody of this strain prepared. Further study for the detection of duck hepatitis virus type I is in order.

     

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