伪狂犬病病毒荧光标签毒株构建及其在抗病毒药物筛选中的初步应用  

Construction of Fluorescently Labelled Pseudorabies Viruses and Their Preliminary Application in Antiviral Drug Screening

作　　者：呙会会 张浩 杨丹 旷燕 李亚菲 刘绍蒙 刘青芸 王湘如[1,2] GUO Huihui;ZHAGN Hao;YANG Dan;KUANG Yan;LI Yafei;LIU Shaomeng;LIU Qingyun;WANG Xiangru(The Cooperative Innovation Center for Sustainable Pig Production,College of Veterinary Medicine,Huazhong Agricultural University,Wuhan 430070,China;National Key Laboratory of Agricultural Microbiology,Wuhan 430070,China)

机构地区：[1]华中农业大学动物医学院,生猪健康养殖协同创新中心,武汉430070 [2]农业微生物资源发掘与利用全国重点实验室,武汉430070

出　　处：《畜牧兽医学报》2024年第8期3600-3611,共12页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金(32122086)。

摘　　要：为筛选针对伪狂犬病病毒(pseudorabies virus,PRV)的有效抗病毒药物,本研究以PRV人源分离毒株hSD-1/2019和经典猪源流行毒株Ea为亲本病毒,采用同源重组技术将mCherry报告基因插入到PRV的UL 35基因终止子之前位点,构建了表达红色荧光蛋白mCherry的荧光标签毒株PRV-mCherry。基于构建的荧光标签病毒,以荧光强度为指标,建立高通量药物筛选平台,对天然产物库中1621种化合物进行抗病毒药物筛选,并初步探索药物特性。结果表明,mCherry标签插入位置正确且稳定遗传,荧光标签毒株PRV-mCherry与亲本毒株在PK-15细胞上的增殖曲线趋势一致,且荧光强度可反映病毒增殖情况。使用PRV-mCherry从天然产物库中成功筛选出3种可在体外有效抑制PRV增殖的药物,根据测定的50%细胞毒性浓度和50%抑制浓度,计算出3种药物的选择指数范围为203.63~564.58μmol·L^(-1),表明其具有良好的成药性。本研究为临床防治PRV感染的抗病毒药物发掘提供了新的策略和思路。In order to screen effective antiviral drugs against pseudorabies virus(PRV),we here constructed the recombinant strains PRV-mCherry expressing red fluorescent protein mCherry based on the human-originated isolate hSD-1/2019 and classical strain Ea.Specifically,mCherry reporter gene was inserted into the site before the terminator of PRV UL35 through homologous recombination technique.Based on the constructed recombinant fluorescent viruses,a high-throughput drug screening platform indicated by fluorescence intensity was established and applied for testing 1621 drugs in the natural product library for screening effective antiviral drugs.Properties of the drugs were preliminarily explored.The results showed that mCherry was inserted in the correct position and was stably inherited.The fluorescence intensity of the recombinant strain PRV-mCherry was representative of the viral proliferation.The strains PRV-mCherry showed similar proliferation curves as the parental strains,supporting the usability of the PRV-mCherry strains in drug screening.Three drugs that could effectively inhibit PRV proliferation in vitro were screened out from the natural product library by using PRV-mCherry.Based on the determined 50%cytotoxic concentration and 50%inhibitory concentration,the selection indices of the three drugs were calculated to be 203.63-564.58μmol·L^(-1),indicating that they had good drug-forming properties.The research provided new ideas in antiviral drugs for the treatment of PRV infection.

关 键 词：伪狂犬病病毒 人源分离株 荧光标签毒株 抗病毒药物 

分 类 号：S852.659.1[农业科学—基础兽医学]