鸭肠炎病毒UL41基因缺失株的构建及其体外增殖能力分析

doi:10.11843/j.issn.0366-6964.2022.08.026

畜牧兽医学报 ›› 2022, Vol. 53 ›› Issue (8): 2689-2696.doi: 10.11843/j.issn.0366-6964.2022.08.026

鸭肠炎病毒UL41基因缺失株的构建及其体外增殖能力分析

杨伏春, 刘芮, 李晓涵, 高立, 刘长军, 祁小乐, 崔红玉, 王笑梅, 高玉龙*, 李凯*

中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室禽免疫抑制病创新团队, 哈尔滨 150069

收稿日期:2021-11-29 出版日期:2022-08-23 发布日期:2022-08-23
通讯作者: 李凯,主要从事动物病毒学和免疫学研究,E-mail:likai01@caas.cn;高玉龙,主要从事动物病毒学和免疫学研究,E-mail:gaoyulong@caas.cn
作者简介:杨伏春(1995-),男,江苏淮安人,硕士生,主要从事动物病毒学研究,E-mail:18852310149@163.com
基金资助:
黑龙江省优秀青年科学基金（YQ2020C024）

Construction of UL41 Gene Deletion Strain of Duck Enteritis Virus and Analysis of Its Replication Ability in vitro

YANG Fuchun, LIU Rui, LI Xiaohan, GAO Li, LIU Changjun, QI Xiaole, CUI Hongyu, WANG Xiaomei, GAO Yulong*, LI Kai*

Avian Immunosuppressive Diseases Division, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China

Received:2021-11-29 Online:2022-08-23 Published:2022-08-23

摘要/Abstract

摘要： 旨在构建鸭肠炎病毒（DEV）UL41基因缺失毒株，并对其生物学特性进行分析，本研究以克隆有DEV UL41基因的重组黏粒D1为骨架，利用Red/ET重组技术构建缺失UL41基因的重组黏粒D1 dUL41；将UL41基因缺失重组黏粒D1 dUL41与其他4个克隆有DEV基因组片段的亲本黏粒共转染鸭胚成纤维细胞（DEF），拯救获得UL41基因缺失毒株rDEV-SD19/dUL41。将该基因缺失病毒感染DEF后，提取病毒基因组进行PCR鉴定及测序，并利用间接免疫荧光试验检测该病毒感染细胞中UL41基因表达情况；绘制拯救的基因缺失病毒的生长曲线，分析其体外复制特性。PCR及测序结果显示，本研究成功构建了缺失UL41基因的重组黏粒D1 dUL41。将该基因缺失黏粒与其他含有DEV基因组的亲本黏粒共转染DEF后能够产生典型的蚀斑病变。PCR及测序结果显示，UL41基因成功从DEV基因组中缺失；间接免疫荧光试验发现，基因缺失病毒rDEV-SD19/dUL41感染DEF后，未见UL41蛋白表达。综上表明，本研究成功构建了DEV UL41基因缺失病毒。体外生长曲线显示，rDEV-SD19/dUL41在DEF中的复制能力明显低于亲本病毒，提示UL41蛋白在DEV复制中发挥重要作用。UL41基因缺失DEV的构建为进一步研究UL41基因在DEV感染和致病中的作用机制奠定了基础。

关键词: 鸭肠炎病毒, UL41基因, 基因缺失, 复制能力

Abstract: In order to construct the UL41 gene deletion strain of duck enteritis virus (DEV), the recombinant fosmid D1 dUL41 with UL41 gene deletion was constructed by using Red/ET recombinant technique with the recombinant fosmid D1 containing UL41 gene as the backbone. The UL41 gene deletion fosmid D1 dUL41 was co-transfected into duck embryo fibroblasts (DEF) with four other parent fosmids containing DEV genomic fragments, and the UL41 gene deletion strain rDEV-SD19/dUL41 was obtained. After the UL41 deletion virus was inoculated on DEF, the virus genome was extracted for PCR identification and sequencing, and the expression of UL41 gene in the infected cells was detected by indirect immunofluorescence assay. The growth curve of the rescued gene deletion virus was tested and its replication characteristics in vitro were analyzed. The PCR and sequencing results showed that the recombinant fosmid D1 dUL41 with UL41 deletion was obtained in this study. Typical plaque lesions can be produced after co-transfection of the UL41-deletion fosmid with other parent fosmids containing DEV genomic fragments. The results of PCR and sequencing showed that UL41 gene was successfully deleted from the DEV genome, and indirect immunofluorescence assay showed that there was no expression of UL41 protein after the inoculation of rDEV-SD19/dUL41 in DEFs. The growth curve in vitro showed that the replication ability of rDEV-SD19/dUL41 in DEF was significantly lower than that of the parent virus, indicating that UL41 gene plays important roles in DEV replication. The construction of DEV UL41 gene deletion strain lays a foundation for the study of the function of UL41 gene in DEV infection and pathogenesis.

Key words: duck enteritis virus, UL41 gene, gene deletion, replication ability

中图分类号:

S852.659.1

杨伏春, 刘芮, 李晓涵, 高立, 刘长军, 祁小乐, 崔红玉, 王笑梅, 高玉龙, 李凯. 鸭肠炎病毒UL41基因缺失株的构建及其体外增殖能力分析[J]. 畜牧兽医学报, 2022, 53(8): 2689-2696.

YANG Fuchun, LIU Rui, LI Xiaohan, GAO Li, LIU Changjun, QI Xiaole, CUI Hongyu, WANG Xiaomei, GAO Yulong, LI Kai. Construction of UL41 Gene Deletion Strain of Duck Enteritis Virus and Analysis of Its Replication Ability in vitro[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(8): 2689-2696.

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鸭肠炎病毒UL41基因缺失株的构建及其体外增殖能力分析

Construction of UL41 Gene Deletion Strain of Duck Enteritis Virus and Analysis of Its Replication Ability in vitro

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