非洲猪瘟病毒F334L基因编码蛋白的原核表达及多克隆抗体制备
Prokaryotic Expression of African Swine Fever F334L Gene Expressing Protein and Preparation of Its Polyclonal Antibody
刘佳晨 1李丽薇 2郭子强 1陈金霞 1曹云雷 1乔思娜 1刘长龙 1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
本研究通过将非洲猪瘟病毒(African swine fever virus,ASFV)的一种非结构蛋白编码基因F334L克隆至原核表达载体pCold-Ⅰ,完成重组表达质粒pCold-Ⅰ-ASFV-F334L的构建.将pCold-Ⅰ-ASFV-F334L转化受体菌感受态细胞,经1 mmol/L的IPTG诱导原核表达.SDS-PAGE和Western blot试验检测结果表明,F334L基因得到了良好的表达,将所得的F334L基因编码蛋白纯化后免疫小鼠,获得含多克隆抗体的血清.利用该抗体检测实验室构建并拯救的表达ASFV F334L基因编码蛋白的重组猪繁殖与呼吸综合征病毒rPRRSV-F334L,试验结果显示该多克隆抗体具有良好的反应原性和特异性,证明了ASFV F334L基因可在PRRSV活载体中稳定表达,为ASFV活载体疫苗的研发提供了材料.
Abstract
The recombinant expression plasmid pCold-Ⅰ-ASFV-F334L was constructed by cloning the F334L gene encoding one of non-structural protein of African swine fever virus(ASFV)into the prokaryotic expression vector pCold-Ⅰ.The pCold-Ⅰ-ASFV-F334L was transformed into competent cells,and the prokaryotic expression was induced by 1 mmol/L IPTG.Through SDS-PAGE and Western blot experiments,the results indicated that the F334L gene was well expressed.The purified F334L gene-encoded protein was used to immunize mice,resulting in the production of polyclonal antibodies in the serum.The generated antibodies were used to detect the recombinant porcine reproductive and respiratory syndrome virus expressing ASFV F334L gene-encoded protein(rPRRSV-F334L)constructed and rescued by the research team.The experimental results demonstrated that the polyclonal antibodies exhibited good reactivity and specificity,proving that the ASFV F334L gene can be stably expressed in the PRRSV live vector,thereby providing material for the development of ASFV live vector vaccines.
关键词
非洲猪瘟病毒/病毒活载体/F334L基因/重组病毒Key words
African Swine Fever Virus/viral live vector/F334L gene/recombinant virus引用本文复制引用
基金项目
国家重点研发计划课题(2021YFD1801401)
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)
上海市自然科学基金项目(21ZR1476900)
上海市自然科学基金项目(21ZR1477200)
出版年
2024
国家科技期刊平台
NSTL
万方数据