摘要
猪萨佩罗病毒(PSV)是一种小RNA病毒,在临床上多与猪流行性腹泻病毒、捷申病毒等引起混合感染。近年来,我国多个省份报道在猪体内分离到该病毒,因而有必要加强对PSV的相关研究。参照PSV-HuN2株序列,将VP1基因插入pGEX-6p-1中进行重组蛋白表达。将蛋白质溶液与弗氏佐剂混合,按照标准程序免疫6~8周龄BALB/c小鼠,第3次免疫后,小鼠眼眶采血,IFA鉴定抗体水平,选取抗体水平高的小鼠脾细胞进行细胞融合。使用IFA法筛选阳性杂交瘤细胞,经过2次有限稀释法亚克隆,最终获得1株PSV VP1单克隆抗体,命名为33-2A。通过IFA、WB、IP试验,33-2A均可与PSV结合。同时对该单抗的B细胞抗原表位进行鉴定,其所识别的抗原表位在PSV VP1蛋白第40—46位氨基酸,多肽序列为^(40)PALTAAE^(46)。结果显示,该单抗与猪萨佩罗病毒VP1蛋白具有良好的反应特异性,并且将其抗原表位与NCBI中上传的PSV毒株序列进行分析,33-2A可识别大多数PSV毒株,具有一定的广谱性。这一研究为猪萨佩罗病毒病原学及其新型疫苗的研制奠定了基础。
As a member of the family Picornaviridae,porcine sapelovirus(PSV)is often infected with porcine epidemic diarrhea virus,teschovirus and so on.In recent years,PSV has been isolated from porcine in many provinces of China.It suggests that it is necessary to strengthen the research on PSV.In this study,according to the sequence of PSV HuN2 strain,VP1 gene was inserted into the pGEX-6P-1 vector,and expressed the recombinant protein.BALB/c mice aged 6-8 weeks were immunized according to the standard procedure.After the third immunization,the mouse orbital blood was collected to identify the antibody level.The highly positive mouse spleen cells were selected for cell fusion.The positive hybridoma cells and two subclones were screened by IFA method,and then a PSV VP1 monoclonal antibody was obtained,named as 33-2A.The results of IFA showed that PSV could be recognized by 33-2A MAb,and specific green fluorescence appeared in the cytoplasm;The results of WB and IP showed that PSV infected porcine cell could specifically bind to 33-2A,and there was a specific band at 32 ku.We also identified the B-cell antigen epitope of 33-2A,it was at amino acids 40-46 of PSV VP1 protein,and the polypeptide sequence was ^(40)PALTAAE^(46).The results showed that the monoclonal antibody can react with PSV VP1 protein.The epitope was analyzed with the PSV sequences uploaded in NCBI,33-2A antibody can react with most PSV strains and has a certain universal to PSV.This study laid a foundation for the study of the etiology and pathogenesis of PSV.
作者
王羽茜
牟春晓
陈振海
WANG Yuxi;MOU Chunxiao;CHEN Zhenhai(Jiangsu Co-Innovation Center for Important Animal Infectious Diseases and Zoonoses/College of Veterinary Medicine,Yangzhou University,Yangzhou 225009,China)
出处
《扬州大学学报（农业与生命科学版）》
CAS
北大核心
2021年第6期48-53,66,共7页
Journal of Yangzhou University：Agricultural and Life Science Edition
基金
江苏高校优势学科建设工程项目(PAPD)
江苏高校动物重要疫病与人兽共患病防控协同创新中心开放课题(201810)。
