非洲猪瘟病毒p30二聚体鉴定及胶体金免疫层析方法建立  

Identification of African swine fever virus p30 dimer and development of a colloidal gold immunochromatographic method for detecting it

作　　者：张胜楠 周雅维 张子羿 单玉平[4] 夏晓莉[1,2] 张鑫宇 ZHANG Shengnan;ZHOU Yawei;ZHANG Ziyi;SHAN Yuping;XIA Xiaoli;ZHANG Xinyu(College of Veterinary Medicine,Yangzhou University,Yangzhou 225009,China;Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseasesand Zoonoses,Yangzhou 225009,China;Shanghai Rohui Biotechnology Limited Company,Shanghai 201108,China;Animal Husbandry and Veterinary Station of Lianyungang,Lianyungang 222023,China)

机构地区：[1]扬州大学兽医学院,江苏扬州225009 [2]江苏省动物重要疫病防控协同创新中心,江苏扬州225009 [3]上海容晖生物科技有限公司,上海201108 [4]连云港市畜牧兽医站,江苏连云港222023

出　　处：《扬州大学学报（农业与生命科学版）》2023年第6期77-83,共7页Journal of Yangzhou University：Agricultural and Life Science Edition

基　　金：国家“十四五”重点研发项目(2021YFD1801202);上海市“科技创新行动计划”农业科技领域项目(20392002700);连云港市第六期“521高层次人才培养工程”科研项目(LYG06521202204)。

摘　　要：为建立一种制备简单、灵敏度高的非洲猪瘟病毒(ASFV)抗原快速检测方法,利用双表达载体pIRES在293A细胞上表达带有His标签和Flag标签的p30重组蛋白,裂解细胞后,通过免疫共沉淀发现p30重组蛋白以聚合体形式存在,进一步用质谱方法对原核系统表达纯化的p30重组蛋白进行分子量测定,结果证实,ASFV p30能自发形成二聚体。在此基础上,用抗p30的单克隆抗体6H9A10建立了单一单克隆抗体夹心检测p30二聚体的胶体金免疫层析方法,该方法对p30重组抗原最低检测浓度为2.1 ng·mL^(-1),对PRV、CSFV、PRRSV、PCV-2、PEDV、PPV、SVA等抗原检测结果均为阴性;用该胶体金免疫层析方法检测已知的100份临床样品,其中包含20份ASFV阴性血清、20份ASFV阴性组织、10份ASFV阴性血浆、21份ASFV阳性血清、21份ASFV阳性组织、8份ASFV阳性血浆,结果显示,检测出的48份阴性样品和49份阳性样品与已知结果一致。综上,建立的单个单克隆抗体夹心检测p30的胶体金免疫层析方法具有良好的特异性和敏感性,有望用于ASFV抗原的临床快速检测。To establish a simple preparation and sensitive method for the rapid detection of African swine fever virus(ASFV) antigen, the recombinant protein p30 with His tag and Flag tag was co-expressed in 293A cells using the expression vector pIRES. After lysing the cells, p30 was found to be a polymer by immunoprecipitation. The relative molecular mass of purified p30 was further determined by mass spectrometry, and the results confirmed that ASFV p30 could form dimers spontaneously. With that, a colloidal gold immunochromatographic method was established for the detection of p30 dimers by a single monoclonal antibody 6H9A10 against p30, and the ASFV p30 recombinant antigen at the concentration of 2.1 ng·mL^(-1) could be detected instead of the viral antigens, such as antigens of PRV, CSFV, PRRSV,PCV-2,PEDV,PPV and SVA.100 clinical samples including 20 ASFV negative sera,20 ASFV negative tissues,10 ASFV negative plasma,21 ASFV positive sera,21 ASFV positive tissues,and 8 ASFV positive plasma were tested by the immunochromatographic method,the results showed that the 48 negative samples and 49 positive samples detected were consistent with known samples.This study indicates that the established colloidal gold immuno-chromatographic method for the detection of p30 by single monoclonal antibody sandwich has good specificity and sensitivity,and is expected to be used for the rapid clinical detection of ASFV antigen.

关 键 词：非洲猪瘟病毒 P30 二聚体 胶体金免疫层析方法 

分 类 号：S852.65[农业科学—基础兽医学]