禽腺病毒Hexon蛋白多克隆抗体制备及特异性鉴定

Preparation and Specificity Identification of Polyclonal Antibodies Against Fowl Adenovirus Hexon Protein

本研究旨在制备禽腺病毒(FAd V)Hexon蛋白多克隆抗体并对其特异性进行分析鉴定。通过构建重组的原核表达载体p ET-28a-Hexon,转入表达菌E. coli BL21 (DE3),IPTG诱导,并运用SDS-PAGE进行重组蛋白的鉴定,最后纯化蛋白。利用纯化的融合蛋白进行常规新西兰大白兔免疫,以制备Hexon蛋白的多克隆抗体。采用间接免疫荧光、免疫组织化学和Western blot的方法鉴定多克隆抗体的特异性。结果表明,Hexon蛋白的原核表达量较高,并且是以包涵体的形式存在,分子质量大小约为43 k Da;免疫兔后获得能与FAd V发生特异性反应且高效价的多克隆抗体。因此,本试验制备的FAd V Hexon蛋白的多克隆抗体证明特异性良好,为后期禽腺病毒病的诊断、致病机制研究及亚单位疫苗的研制提供了良好的物质基础。

The purpose of this study is to prepare polyclonal antibodies against fowl adenovirus(FAdV)Hexon protein and to identify its specificity.The recombinant prokaryotic expression plasmid pET-28a-Hexon was constructed and transformed into E.coli BL21(DE3),IPTG induction,the recombinant protein was identified by SDS-PAGE,and the protein was purified.The polyclonal antibodies of Hexon protein was prepared by routine immunity in New Zealand white rabbits with purified fusion protein.The specificity of polyclonal antibodies was identified by indirect immunofluorescence,immunohistochemistry and Western blot.The results showed the prokaryotic expression of Hexon protein was high and existed in the form of inclusion body,and its molecular weight was about 43 kDa;Polyclonal antibodies having specific reaction with FAdV and high potency could be obtained in the immunized rabbits.The above results indicated the polyclonal antibody of FAdV Hexon protein prepared in this study proved to be of good specificity and provided a good material basis for the diagnosis,pathogenesis study and subunit vaccine development of avian adenovirus diseases.