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

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(isopropylβ-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∶2048000。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。

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 vector using homologous recombination technology to obtain the recombinant plasmid pET-30a-Hexon.The recombinant plasmid was then transferred into E.coli BL21(DE3),and Hexon protein expression was induced by isopropylβ-D-thiogalac toside(IPTG).The purified recombinant protein was further used to immunize 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 successfully expressed in the form of inclusion bodies with a molecular mass of approximately 37 ku.The expression 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 prepared polyclonal antibody could specifically react with the Hexon protein,and proved that the recombinant protein had good immunogenicity.The antibody titer was 1∶2048000 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.