非洲猪瘟病毒pE248R蛋白的原核表达及其多克隆抗体制备
Prokaryotic Expression of African Swine Fever Virus pE248R Protein and Preparation of Polyclonal Antibodies
杜楠楠 1陈金霞 1曹云雷 1张宽 1童武 2李丽薇 2赵冉 3童光志 2高飞2
作者信息
- 1. 中国农业科学院上海兽医研究所,上海 200241
- 2. 中国农业科学院上海兽医研究所,上海 200241;扬州大学江苏省动物重要疫病与人兽共患病防控协同创新中心,扬州 225009
- 3. 厦门市动物疫病预防控制中心,厦门 361009
- 折叠
摘要
本研究利用PCR方法从非洲猪瘟病毒的灭活样品中扩增出747 bp的E248R基因全长序列,利用同源重组法构建原核表达质粒pCold Ⅰ-pE248R,经1 mmol/L的IPTG诱导1h后,利用SDS-PAGE对重组蛋白进行表达鉴定和反应原性分析,表达蛋白的分子量约为32 kDa,利用纯化后得到pE248R重组蛋白作为免疫原经过4次免疫后制备鼠源抗pE248R多克隆抗体.利用该多克隆抗体检测实验室构建并拯救的已证明能够稳定表达ASFV pE248R蛋白的重组病毒rPRRSV-E248R,结果显示制备的抗pE248R的多克隆抗体能够与rPRRSV-E248R发生特异性结合反应,证明利用本试验中原核表达的pE248R蛋白制备的多克隆抗体具有抗pE248R蛋白的特异性,为进一步针对ASFV E248R基因建立快速,特异性的血清学检测方法奠定了基础,也为针对pE248R蛋白的功能性研究提供了研究基础.
Abstract
In this study,the 747 bp E248R gene was amplified by PCR from inactivated samples of African swine fever virus(ASFV),and the prokaryotic expression plasmid pCold Ⅰ-pE248R was constructed by homologous recombination method.After induced with 1 mmol/L IPTG for 16 h,the recombinant protein was identified with a molecular mass of 32 kDa in SDS-PAGE.The purified pE248R recombinant protein was used to immunize mice 4 times to prepare anti-pE248R polyclonal antibodies.Then the polyclonal antibodies were used to detect the recombinant viruses rPRRSV-E248R,which was constructed and rescued by our laboratory and had been proved to stably express ASFV pE248R protein.The results showed that the prepared polyclonal antibodies specifically bound to rPRRSV-E248R.The availability of the polyclonal antibodies against the prokaryotic pE248R protein laid a foundation for developing a rapid and specific serological detection method for ASFV E248R gene,and also provided a research foundation for the functional study of pE248R protein.
关键词
非洲猪瘟病毒/E248R基因/pE248R蛋白/多克隆抗体Key words
African swine fever virus/E248R gene/pE248R protein/polyclonal antibody引用本文复制引用
基金项目
国家自然科学基金ASFV专项(31941017)
中央级公益性科研院所基本科研业务费专项(Y2020YJ15)
广东省重点领域研发计划(2019B020211003)
出版年
2024
国家科技期刊平台
NSTL
维普
万方数据