条件性敲除D1133L基因的重组非洲猪瘟病毒的构建及增殖特性

doi:10.11843/j.issn.0366-6964.2023.02.027

畜牧兽医学报 ›› 2023, Vol. 54 ›› Issue (2): 706-714.doi: 10.11843/j.issn.0366-6964.2023.02.027

条件性敲除D1133L基因的重组非洲猪瘟病毒的构建及增殖特性

张婷, 冯涛, 杨金柯, 郝雨, 杨行, 张大俊, 史喜绢, 闫文倩, 陈玲玲, 刘湘涛, 郑海学*, 张克山*

中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室农业部畜禽病毒学重点开放实验室国家口蹄疫参考实验室, 兰州 730046

收稿日期:2022-04-06 出版日期:2023-02-23 发布日期:2023-02-21
通讯作者: 郑海学,主要从事动物传染病与流行病学研究,E-mail:zhenghaixue@caas.cn;张克山,主要从事兽医微生物及其分子生物学研究,E-mail:zks009@126.com
作者简介:张婷(1996-),女,甘肃古浪人,硕士生,主要从事兽医微生物及其分子生物学,E-mail1835631389@qq.com;冯涛(1989-),男,河南获嘉人,博士生,主要从事兽医微生物及其分子生物学研究。
基金资助:
国家重点研发计划（2021YFD1801300）；甘肃省重大专项（20ZD7NA006）；甘肃省科技重大专项（21ZD3NA001）；中国农业科学院重大任务（CAAS-ZDRW202006-03）

Construction and Growth Characteristics of Recombinant African Swine Fever Virus with Conditional Deletion of D1133L Gene

ZHANG Ting, FENG Tao, YANG Jinke, HAO Yu, YANG Xing, ZHANG Dajun, SHI Xijuan, YAN Wenqian, CHEN Lingling, LIU Xiangtao, ZHENG Haixue*, ZHANG Keshan*

State Key Laboratory of Veterinary Etiological Biology/National Foot-and-Mouth Disease Reference Laboratory/Key Laboratory of Animal Virology, Ministry of Agriculture/Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China

Received:2022-04-06 Online:2023-02-23 Published:2023-02-21

摘要/Abstract

摘要： 前期研究初步表明非洲猪瘟病毒（ASFV）编码的D1133L基因对ASFV复制至关重要，本研究拟进一步探究D1133L在ASFV复制中的作用。利用同源重组技术结合大肠杆菌lac阻遏操作系统实现条件性敲除D1133L基因，以pUC118为骨架重组转移载体ASFVΔi130，将重组转移载体转染骨髓源巨噬细胞（BMDMs），以ASFV CN/GS/2018为亲本毒株感染BMDM，在β-D-硫代半乳糖苷（IPTG）存在的条件下，经绿色荧光和PCR鉴定，获得条件性敲除D1133L重组毒株vD1133Li。利用荧光显微镜观察该重组毒株与亲本毒株在猪肺泡巨噬细胞（PAMs）中的复制差异，利用qPCR技术比较重组病毒与亲本毒株的复制差异，分析vD1133Li在无IPTG情况下回补D1133L蛋白后与在IPTG诱导情况下的复制差异。结果显示：本研究成功构建了条件性敲除D1133L的ASFV重组病毒vD1133Li，重组毒株不表达D1133L，在IPTG诱导下复制能力显著低于亲本毒株；在稳定表达D1133L的MA-104细胞系中，vD1133Li复制能力恢复。综上所述，D1133L基因对于ASFV复制至关重要，试验结果为进一步研究D1133L在ASFV致病中的机制及针对D1133L靶向药物研发提供了研究基础。

关键词: 非洲猪瘟病毒, 条件性D1133L基因缺失, 重组病毒, 增殖特性

Abstract: Previous studies have shown that the D1133L gene encoded by African swine fever virus (ASFV) is crucial to ASFV replication. In order to further explore the role of D1133L in ASFV replication, in this study, we used homologous recombination technology and Escherichia coli lac repression operating system to knockout D1133L gene conditionally, constructed recombinant transfer vector ASFV Δi130 using pUC118 as vector, transfected BMDM cells with recombinant transfer vector, and infected BMDM cells with ASFV CN/GS/2018 wild type strain. In the presence of isopropyl-β-D-thiogalactoside (IPTG), conditional knockout D1133L recombinant strain vD1133Li was identified by green fluorescence and qPCR. The replication of the recombinant virus strain and the parent virus strain in PAM cells was observed with fluorescence microscope. Differences between vD1133Li and the parent virus strain were analyzed by qPCR. The replication difference between conditionally knockout D1133L recombinant strain with or without IPTG was also further investigated. The vD1133Li with conditional deletion of D1133L was constructed successfully in this study. Replication level of vD1133Li was significantly lower than that of the wild type virus strain. In MA-104 cell lines that overexpressing stably D1133L, the replication ability of vD1133Li was recovered. In summary, D1133L is crucial for ASFV replication. These results provide a basis for further research on the function of D1133L in ASFV replication, the pathogenic mechanism and the development of D1133L targeted drugs.

Key words: African swine fever virus, conditional D1133L gene knockout recombinant virus, proliferation characterizations

中图分类号:

S852.659.1

张婷, 冯涛, 杨金柯, 郝雨, 杨行, 张大俊, 史喜绢, 闫文倩, 陈玲玲, 刘湘涛, 郑海学, 张克山. 条件性敲除D1133L基因的重组非洲猪瘟病毒的构建及增殖特性[J]. 畜牧兽医学报, 2023, 54(2): 706-714.

ZHANG Ting, FENG Tao, YANG Jinke, HAO Yu, YANG Xing, ZHANG Dajun, SHI Xijuan, YAN Wenqian, CHEN Lingling, LIU Xiangtao, ZHENG Haixue, ZHANG Keshan. Construction and Growth Characteristics of Recombinant African Swine Fever Virus with Conditional Deletion of D1133L Gene[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(2): 706-714.

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条件性敲除D1133L基因的重组非洲猪瘟病毒的构建及增殖特性

Construction and Growth Characteristics of Recombinant African Swine Fever Virus with Conditional Deletion of D1133L Gene

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