天然产物库中体外颉颃A型塞内卡病毒化合物的筛选及作用途径研究  

Screening and Action Pathway of Compounds Antagonistic Senacavirus A in vitro from Natural Product Library

作　　者：王西彤 沙洲 迟田英[2] 崔进 郑辉[2] 陈峰 曹振山 张慧[2,3] 戈胜强 魏荣[2] 尼博 黄保续[1,2] WANG Xitong;SHA Zhou;CHI Tianying;CUI Jin;ZHENG Hui;CHEN Feng;CAO Zhenshan;ZHANG Hui;GE Shengqiang;WEI Rong;NI Bo;HUANG Baoxu(College of Veterinary Medicine,Northeast Agricultural University,Harbin 150006,China;China Animal Health and Epidemiology Center,Qingdao 266033,China;Qingdao Key Laboratory of Modern Bioengineering and Animal Disease Research,Qingdao 266032,China;Key Laboratory of Animal Biosafety Risk Prevention and Control(South),Ministry of Agriculture and Rural Affairs,Qingdao 266032,China;Shandong Animal Disease Prevention and Control Center,Jinan 250100,China;Key Laboratory of Animal Biosafety,Qingdao,Qingdao 266032,China)

机构地区：[1]东北农业大学动物医学学院,哈尔滨150006 [2]中国动物卫生与流行病学中心,青岛266033 [3]青岛市现代生物工程及动物疫病研究重点实验室,青岛266032 [4]农业农村部动物生物安全风险预警及防控重点实验室(南方),青岛266032 [5]山东省动物疫病预防与控制中心,济南250100 [6]青岛市动物生物安全重点实验室,青岛266032

出　　处：《中国畜牧兽医》2024年第7期3155-3165,共11页China Animal Husbandry & Veterinary Medicine

基　　金：“十四五”国家重点研发计划“口蹄疫病毒的分子流行病学与传播机制”(2021YFD1800300);中国动物卫生与流行病学中心创新基金(DW2021009)。

摘　　要：【目的】寻找对A型塞内卡病毒(SVA)具有抑制活性的天然产物,并深入研究其颉颃途径。【方法】将天然产物浓度统一稀释至10μmol/L,与荧光素酶重组塞内卡病毒(rSVA-NLuc)相结合,在天然产物小分子库中筛选出具有荧光素酶抑制活性的化合物,采用实时荧光定量RT-PCR方法验证其活性,通过乳酸脱氢酶(LDH)法进行细胞毒性试验确定最大无毒浓度,结合病毒滴度测定、实时荧光定量RT-PCR、Western blotting、间接免疫荧光试验(IFA)等技术,在病毒感染周期的不同阶段,对其颉颃SVA的效果及途径进行研究。【结果】通过初筛,从136种天然产物中筛选出莫能霉素钠盐、孕酮、叶下珠次素、4-羟基德里辛和2-甲基雌酮5种能体外颉颃SVA的化合物。进一步通过实时荧光定量RT-PCR复核验证和细胞毒性检测,筛选出一种能体外颉颃SVA的天然产物——孕酮。与空白对照组相比,其在体外可导致SVA mRNA表达水平极显著下降(P<0.01),最大无毒浓度达50μmol/L。进一步研究表明,孕酮能在感染后8、12、24、36 h持续抑制SVA增殖,与对照组相比,在感染后24和36 h病毒滴度显著下降(P<0.05)。与对照组相比,不同浓度(10、20和50μmol/L)孕酮在吸附阶段导致SVA吸附水平极显著下降(P<0.01);在复制阶段使SVA VP3水平显著下降且使病毒滴度极显著下降(P<0.01);在释放阶段使释放胞外的病毒比例极显著下降(P<0.01);但对其他途径无显著影响。说明孕酮能在体外抑制SVA吸附、复制和释放,但对其入胞、组装途径无明显作用。【结论】本研究从天然产物小分子库中筛选出一种具有低细胞毒性且对SVA具有较强颉颃作用的天然产物——孕酮。该产物对SVA吸附、复制和释放阶段具有抑制作用,从而颉颃SVA的感染。本研究为促进抗SVA药物的开发和研究SVA的感染偏好性提供了科学参考。【Objective】The experiment was aimed to search for natural products with inhibitory activity against Senecavirus A(SVA)and further investigate its antagonistic pathway.【Method】The concentration of the natural products were uniformly diluted to 10μmol/L.Combined with the recombinant Senecavirus A with luciferase(rSVA-NLuc),compounds with luciferase inhibitory activity were screened from the natural products library,and their activity was further verified using Real-time quantitative RT-PCR.The maximum non-toxic concentration was determined by cytotoxicity testing using lactate dehydrogenase(LDH)method.Virus titer determination,Real-time quantitative RT-PCR,Western blotting,IFA and other techniques were used to study the effect and pathway of antagonism against SVA.【Result】Five compounds,monensin sodium salt,progesterone,hypophyllanthin,4-hydroxyderricin and 2-methoxyestrone,which could antagonize SVA in vitro,were screened from 136 natural products.Progesterone,a natural product that could antagonize SVA in vitro,was screened by Real-time quantitative RT-PCR and cytotoxicity detection.Compared with blank control group,the mRNA expression level of SVA was extremely significantly decreased in vitro(P<0.01),and the maximum non-toxic concentration reached 50μmol/L.Further study showed that progesterone could continuously inhibit the proliferation of SVA at 8,12,24 and 36 h after infection,and the virus titer was significantly decreased at 24 and 36 h after infection compared with control group(P<0.05).Compared with control group,different concentrations of progesterone(10,20 and 50μmol/L)extremely significantly decreased the adsorption level of SVA at the adsorption stage(P<0.01).In the replication stage,the level of SVA VP3 and virus titer were extremely significantly decreased(P<0.01).The proportion of released extracellular virus extremely significantly decreased(P<0.01).But there was no significant effect on other approaches.These results indicated that progesterone could inhibit the adsorption,repli

关 键 词：孕酮 A型塞内卡病毒(SVA) 天然产物 颉颃途径 

分 类 号：S859.3[农业科学—临床兽医学]