摘要
非洲猪瘟(ASF)是由非洲猪瘟病毒(ASFV)感染引起的一种高致病性病毒性传染病。为制备兔抗ASFV A137R蛋白多克隆抗体,本研究以ASFV Pig/HLJ/2018分离株基因组DNA为模板扩增ASFV A137R基因,将其克隆至原核表达载体pET-21a(+)中,构建重组表达质粒pET-21a-A137R,转化大肠杆菌BL21(DE3)感受态细胞后经IPTG诱导表达,SDS-PAGE检测结果显示,表达的A137R重组蛋白分子质量约15.0 ku,其主要以可溶形式表达。利用HisTrap亲和层析和凝胶过滤层析纯化获得高纯度的A137R重组蛋白,利用该纯化重组蛋白免疫新西兰大白兔,制备抗ASFV A137R蛋白多克隆抗体,经Protein G亲和层析介质纯化后经western blot、间接免疫荧光和免疫共沉淀试验检测结果显示,制备的兔抗ASFV A137R蛋白的多克隆抗体能特异性地识别HEK293T细胞中瞬时表达的ASFV Flag-A137R蛋白和ASFV感染猪肺泡巨噬细胞中表达的ASFV A137R蛋白。本研究为进一步探究ASFV A137R蛋白的功能、其在ASFV感染及致病性中的作用和诊断技术研究奠定了基础。
African swine fever(ASF)is a highly pathogenic viral infectious disease caused by African swine fever virus.To prepare rabbit polyclonal antibody against ASFV A137R protein,the genomic DNA isolated from ASFV Pig/HLJ/2018 strain was used as a template to amplify ASFV A137R gene.The target gene was then subcloned into the bacterial expression vector pET-21a to construct the recombinant plasmid pET-21a-A137R.The recombinant plasmid was transformed into E.coli BL21(DE3)and induced by the addition of IPTG to express A137R protein.SDS-PAGE result showed that the recombinant protein was expressed in a soluble form with a molecular mass of 15.0ku.The recombinant protein A137R was purified by HisTrap affinity chromatography and gel filtration chromatography.Two New Zeal white rabbits were immunized with the purified recombinant protein A137R to prepare anti-ASFV A137R polyclonal antibody,and the antibody was then purified by a protein G affinity chromatography column.The results of western blot,indirect immunofluorescence assay and immunoprecipitation showed that rabbit anti-ASFV A137R polyclonal antibody can specifically detect the ASFV Flag-A137R protein in HEK293 cells that were transfected with pCAGGS-Flag-A137R plasmid and the ASFV A137R proteinin ASFV-infected porcine alveolar macrophage.This study lays the foundation for further exploring the function of A137R protein,the role of A137R in ASFV infection-mediated pathogenicity,and diagnostic techniques based on A137R.
作者
向志达
张涛清
张元峰
黄丽
杨玉莹
翁长江
XIANG Zhi-da;ZHANG Tao-qing;ZHANG Yuan-feng;HUANG Li;YANG Yu-ying;WENG Chang-jiang(College of Animal Science,Yangtze University,Jingzhou 434025,China;Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,State Key Laboratory of Veterinary Biotechnology,Harbin 150069,China)
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2021年第1期60-64,共5页
Chinese Journal of Preventive Veterinary Medicine
基金
非洲猪瘟病毒感染与致病机制(31941002、CAAS-ZDXT2018007)。
