中国兽医科学2024，Vol.54Issue(9)：1224-1231,8.DOI:10.16656/j.issn.1673-4696.2024.0148
贵州蛋鸡源高致病性FAdV-4分离株Hexon蛋白多克隆抗体的制备
Preparation of polyclonal antibodies against Hexon protein of pathogenic isolates of FAdV-4 from layers in Guizhou Province,China
摘要
Abstract
In order to prepare a serological diagnostic antigen of fowl adenovirus serotype 4(FAdV-4)with good reactogenicity,the Hexon gene was amplified from chicken liver cancer cells(LMH)infected with highly pathogenic isolates of FAdV-4 from layers,and it was ligated into the pET-30a vec-tor using homologous recombination technology to obtain the recombinant plasmid pET-30a-Hexon.The re-combinant plasmid was then transferred into E.coli BL21(DE3),and Hexon protein expression was induced by isopropyl β-D-thiogalac toside(IPTG).The purified recombinant protein was further used to immu-nize New Zealand White rabbits to produce rabbit polyclonal antibodies against the Hexon protein.The results showed that the recombinant prokaryotic expression plasmid pET-30a-Hexon was confirmed to be correctly constructed by electrophoresis and sequencing.The recombinant Hexon protein was success-fully expressed in the form of inclusion bodies with a molecular mass of approximately 37 ku.The ex-pression of Hexon protein in LMH was detected by Western-blot and indirect immunofluorescence assay(IFA)using the prepared polyclonal antibody as the primary antibody,the result indicated that the pre-pared polyclonal antibody could specifically react with the Hexon protein,and proved that the recombi-nant protein had good immunogenicity.The antibody titer was 1:2 048 000 as determined by indirect ELISA.This study establishes the groundwork for developing a serologic diagnostic technique for FAdV-4,preventing and controlling the disease,mapping the role of the Hexon proteins in the pathogenic process,and creating a subunit vaccine in Guizhou Province,China.关键词
血清4型禽腺病毒/Hexon蛋白/多克隆抗体Key words
fowl adenovirus serotype 4/Hexon protein/polyclonal antibody分类
农业科技引用本文复制引用
罗东还,温贵兰,邓乔木,朱国强,徐飞,赵静,杨洪兴..贵州蛋鸡源高致病性FAdV-4分离株Hexon蛋白多克隆抗体的制备[J].中国兽医科学,2024,54(9):1224-1231,8.基金项目
2021年度贵州省科技支撑计划项目(黔科合支撑[2021]一般164) （黔科合支撑[2021]一般164）
贵州省农业科技支撑计划项目(黔科合支撑[2019]2286号) （黔科合支撑[2019]2286号）
2020年贵州省高等学校教学内容和课程体系改革项目(GZJG20200035) （GZJG20200035）
