抗非洲猪瘟病毒药物筛选Cell-ELISA法的建立及应用  

Establishment and application of Cell-ELISA assay for anti-African swine fever virus drug screening

作　　者：钱炳旭 胡永新 郑东霞[2] 李金明[2] 苗雨润 戈胜强[2] 巩明霞[2] 韩秀琚[2] 曾德新 汤芳[1] 廖凯 彭宛清 吴晓东[2] 戴建君[1] 王志亮[2] 薛峰[1,3] QIAN Bing-xu;HU Yong-xin;ZHENG Dong-xia;LI Jin-ming;MIAO Yu-run;GE Sheng-qiang;GONG Ming-xia;HAN Xiu-ju;ZENG De-xin;TANG Fang;LIAO Kai;PENG Wan-qing;WU Xiao-dong;DAI Jian-jun;WANG Zhi-liang;XUE Feng(College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China;China Animal Health and Epidemiology Center,Qingdao 266032,China;Sanya Institute of Nanjing Agricultural University,Sanya 572000,China;Hefei Customs Technology Center,Hefei 230022,China;Technology Center of Hefei Customs,and Anhui Province Key Laboratory of Analysis and Detection for Food Safety,Hefei 230022,China)

机构地区：[1]南京农业大学动物医学院,江苏南京210095 [2]中国动物卫生与流行病学中心,山东青岛266032 [3]南京农业大学三亚研究院,海南三亚572000 [4]合肥海关技术中心,安徽合肥230022 [5]合肥海关技术中心,食品安全分析与检测安徽省重点实验室,安徽合肥230022

出　　处：《中国兽医科学》2023年第11期1348-1354,共7页Chinese Veterinary Science

基　　金：国家重点研发计划项目(2021YFD1800500,2022YFD1800500);海南省科技专项资金(ZDYF2022XDNY248);江苏省农业科技自主创新基金项目[CX(21)20381];江苏省重点研发计划(现代农业)重点项目(BE2020398);南京农业大学三亚研究院指导基金项目(NAUSY-ZDO8)。

摘　　要：为了建立一种抗非洲猪瘟病毒(ASFV)药物高通量筛选(HTS)方法,采用ASFV感染细胞作为包被抗原,以p30单克隆抗体作为检测抗体,通过优化抗体稀释度、孵育时间等试验条件,成功建立了Cell-ELISA方法,并使用该方法对Selleck天然产物库中的1036个化合物进行了筛选。结果显示,该Cell-ELISA法筛选得到5个具有抗ASFV活性的化合物;当5个化合物浓度为10μmol/L时,PAM细胞存活率均大于80%;进一步分析ASFV p72的转录和翻译水平,结果显示,ASFV在经化合物处理的PAM上被显著抑制,确保了Cell-ELISA筛选结果的准确性。上述结果表明,该Cell-ELISA法可作为一种HTS方法,用于筛选抗ASFV药物,并促进ASFV抗病毒药物的开发,对ASF疫情防控具有重要意义。To establish a high throughput screening(HTS)method for anti-African swine fever virus(ASFV)drugs.Using ASFV-infected cells as the coated antigen and p30 monoclonal antibody as the detection antibody,the Cell-ELISA was successfully established by optimizing experimental conditions such as antibody dilution and incubation time.The method was applied to screen 1036 compounds from the Selleck natural product library.The results revealed that Cell-ELISA identified five compounds with anti-ASFV activity.When the concentration of these five compounds was 10μmol/L,the cell viability of PAM cells exceeded 80%.Further analysis of the transcription and translation levels of ASFV p72 revealed significant inhibition of ASFV in PAM cells treated with the compounds,ensuring the accuracy of the Cell-ELISA screening results.These findings indicate that the Cell-ELISA can serve as a HTS method for identifying anti-ASFV drugs and facilitating the development of ASFV antiviral drugs,which is of great significance for the prevention and control of ASF.

关 键 词：非洲猪瘟病毒 Cell-ELISA 抗病毒药物 高通量筛选 小分子化合物 

分 类 号：S852.659.1[农业科学—基础兽医学]