[1]吴凤瑶,卢凤英,章丽娇,等.基于真核CMV启动子的3型鸭甲型肝炎病毒体内转录系统建立[J].江苏农业学报,2025,(12):2403-2408.[doi:doi:10.3969/j.issn.1000-4440.2025.11.012]
　WU Fengyao,LU Fengying,ZHANG Lijiao,et al.Establishment of an in vivo transcription system for duck hepatitis A virus type 3 based on the eukaryotic CMV promoter[J].,2025,(12):2403-2408.[doi:doi:10.3969/j.issn.1000-4440.2025.11.012]
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基于真核CMV启动子的3型鸭甲型肝炎病毒体内转录系统建立()

江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2025年12期
页码:
2403-2408
栏目:
畜牧兽医·水产养殖·益虫饲养
出版日期:
2025-12-31

文章信息/Info

Title:
Establishment of an in vivo transcription system for duck hepatitis A virus type 3 based on the eukaryotic CMV promoter
作者:
吴凤瑶; 卢凤英; 章丽娇; 韩凯凯; 杨婧; 赵冬敏; 黄欣梅; 刘宇卓; 尹馨; 苏丹; 张小飞; 刘青涛
(江苏省农业科学院兽医研究所/农业农村部兽用生物制品工程技术重点实验室,江苏南京210014)
Author(s):
WU Fengyao; LU Fengying; ZHANG Lijiao; HAN Kaikai; YANG Jing; ZHAO Dongmin; HUANG Xinmei; LIU Yuzhuo; YIN Xin; SU Dan; ZHANG Xiaofei; LIU Qingtao
(Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences/Key Laboratory of Veterinary Biological Engineering and Technology, Ministry of Agriculture and Rural Affairs, Nanjing 210014, China)
关键词:
3型鸭甲型肝炎病毒; 体内转录; CMV启动子; 反向遗传操作系统
Keywords:
duck hepatitis A virus type 3; in vivo transcription; CMV promoter; reverse genetics system
分类号:
S834
DOI:
doi:10.3969/j.issn.1000-4440.2025.11.012
文献标志码:
A
摘要:
本研究以pCI质粒为载体,将3型鸭甲型肝炎病毒(DHAV-3)强毒株的全基因组序列插入其真核启动子CMV之后,并在病毒全基因序列3′端添加丁型肝炎病毒核酶(HdvRz)序列获得重组质粒pCI-SD。将pCI-SD转染乳仓鼠肾(BHK-21)细胞后接种鸭胚,获得拯救病毒rSD。通过PCR扩增和序列测定，证明rSD为包含遗传标记(EcoR Ⅰ酶切位点)的拯救病毒。对rSD的半数致死量及其在感染雏鸭体内的组织分布进行测定和病理切片检测,结果显示,rSD与亲本病毒SD的毒价相近,且在感染雏鸭体内各组织的病毒拷贝数与亲本病毒相似,对各主要脏器的损伤也与亲本病毒相同,表明本研究成功构建了采用体内转录的DHAV-3反向遗传操作系统。该系统避免了体外转录核糖核酸(RNA)的弊端,简化了操作步骤,提高了拯救效率,为鸭甲型肝炎病毒的致病机制和新型疫苗研究提供了工具。
Abstract:
This study constructed a recombinant plasmid, pCI-SD, by inserting the full-length genome of a highly virulent duck hepatitis A virus type 3 （DHAV-3) strain downstream of the eukaryotic CMV promoter in the pCI vector, fo-llowed by the addition of a hepatitis delta virus ribozyme (HdvRz) sequence at the 3′ end. The pCI-SD plasmid was transfected into baby hamster kidney (BHK-21) cells, and the rescued virus (rSD) was subsequently obtained by inoculating duck embryos. PCR amplification and sequencing confirmed that rSD contained the genetic marker (an EcoRⅠ restriction site). Biological characterization showed that the median lethal dose (LD50) of rSD was similar to that of the parental virus SD. Tissue distribution analysis and histopathological examination in infected ducklings revealed that rSD exhibited comparable organ tropism and induced similar pathological damage as the parental virus, confirming the successful establishment of a DHAV-3 reverse genetics system based on in vivo transcription. This system avoids the drawbacks of in vitro RNA transcription, simplifies operational procedures, and improves rescue efficiency, providing a valuable tool for studying the pathogenesis of DHAV-3 and developing novel vaccines.

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备注/Memo

备注/Memo:
收稿日期:2025-08-12基金项目:国家自然科学基金项目(32202845)作者简介:吴凤瑶(1985-),女,四川成都人,博士,助理研究员,主要从事水禽疫病病原致病分子机制研究。(E-mail)cdwfyao@126.com通讯作者:张小飞,(E-mail)xiaofei0804@sina.com；刘青涛,(E-mail)taoqingliu2013@163.com
更新日期/Last Update: 2026-01-20