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。
【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-4 T-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 was100%. The RT-PCR amplification and the entire genome sequence indicated the isolated virus to be positive for duck viralhepatitis 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-4 T-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.
作者
杨世丽
常巍
袁梦
黄雅贞
陈仕龙
马燕梅
YANG Shili;CHANG Wei;YUAN Meng;HUANG Yazhen;CHEN Shilong;MA Yanmei(College of Animal Science(College of Bee Science),Fujian Agriculture and Forestry University,Fuzhou,Fujian 350002,China;Institute of Animal Husbandry and Veterinary Medicine,Fujian Academy of Agricultural Sciences,Fuzhou,Fujian 350013,China)
出处
《福建农业学报》
CAS
CSCD
北大核心
2021年第10期1145-1152,共8页
Fujian Journal of Agricultural Sciences
基金
福建省自然科学基金项目(2020J06016)
福建农林大学科技创新专项基金(CXZX2018017)
福建农林大学动物科学学院教师科研扶持基金项目(2018DK003)。
关键词
鸭病毒性肝炎病毒
分离鉴定
VP1基因
原核表达
多克隆抗体
duck hepatitis virus
isolation and identification
VP1 gene
prokaryotic expression
polyclonal antibody
