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
罗东还 1温贵兰 1邓乔木 1朱国强 2徐飞 1赵静 1杨洪兴1
作者信息
- 1. 贵州大学动物科学学院,贵州 贵阳 550025;贵州省动物生物制品工程技术研究中心,贵州贵阳 550016
- 2. 扬州大学兽医学院,江苏扬州 225009
- 折叠
摘要
为制备反应原性良好的血清4型禽腺病毒(fowl adenovirus serotype 4,FAdV-4)血清学诊断抗原,本试验从在鸡肝癌细胞(LMH)上接种培养的蛋鸡源高致病性FAdV-4分离株中扩增到Hexon基因,利用同源重组技术将它连接至载体pET-30a,获得重组质粒pET-30a-Hexon,又将重组质粒转入大肠杆菌(E.coli)BL21(DE3)感受态细胞,经异丙基硫代半乳糖苷(isopropyl β-D-thiogalac toside,IPTG)诱导表达后纯化Hexon蛋白,进一步采用纯化后重组蛋白免疫新西兰大白兔,制备兔抗Hexon蛋白的多克隆抗体.结果显示,重组原核表达质粒pET-30a-Hexon经电泳和测序鉴定证明构建正确;重组蛋白Hexon以包涵体的形式被成功表达,分子质量约37 ku;以制备的多克隆抗体为一抗,通过Western-blot和间接免疫荧光(indirected immunofluorescence assay,IFA)检测到LMH中的Hexon蛋白表达,表明制备的多克隆抗体能与Hexon蛋白发生特异性反应,证明该重组蛋白具有良好的免疫原性;经间接ELISA测定,抗体效价为1:2 048 000.本研究为建立FAdV-4血清学诊断技术、防控该疾病、摸索Hexon蛋白在致病过程中的作用及研制亚单位疫苗等奠定了基础.
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引用本文复制引用
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)
贵州省农业科技支撑计划项目(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)
出版年
2024
国家科技期刊平台
NSTL
万方数据