【摘要】 为制备鸽腺病毒Ⅰ型(PiAdV-1)六邻体蛋白(Hexon)和纤突蛋白(Fiber)-2的多克隆抗体，用蛋白结构分析软件对Hexon基因和Fiber-2基因序列进行分析，设计其相关抗原片段，克隆至载体pET-28a(+)中，获得重组质粒p ET-28a-Hexon和pET-28a-Fiber2。利用大肠杆菌BL-21(DE3),IPTG诱导表达，并通过Ni亲和层析树脂纯化。然后，用纯化的重组蛋白免疫新西兰大白兔，制备多克隆抗体并利用Protein A层析柱纯化。SDS-PAGE结果分别出现大小约27和54 k Da的目的条带，表明重组Hexon和Fiber蛋白成功表达。ELISA方法检测免疫后兔血清，抗Hexon和Fiber-2多抗血清效价均高达1∶10~8。Western blot检测表明制备的多克隆抗体具有目标蛋白的结合活性。本研究为建立PiAdV-1的检测方法奠定基础，也为PiAdV-1亚单位疫苗研制提供支持。更多还原

【Abstract】 In order to prepare polyclonal antibodies against Hexon protein and Fiber-2 protein of pigeon adenovirus 1( PiAdV-1),the sequences of the Hexon gene and the Fiber-2 gene were analyzed by protein structure analysis software,and the related antigen fragments of the two genes were cloned into the vector p ET-28a( +). The recombinant plasmid p ET-28a-Hexon and p ET-28a-Fiber2 were obtained and transformed into E. coli BL21( DE3) and induced with IPTG. The recombinant protein was purified by High-Affinity Ni Resin. Polyclonal antibodies were purified by protein A resin from the serum of New Zealand white rabbits which was immunized by the recombinant proteins. The target bands of about 27 and 54 k Da appeared in SDS-PAGE,indicating that the recombinant Hexon and Fiber protein were successfully expressed. The ELISA results showed that the titers of the anti-Hexon and Fiber-2 polyclonal antibodies were as high as 1 ∶ 10~8.The Western blot analysis indicated that the polyclonal antibodies had binding activity to the Hexon and Fiber-2 proteins,respectively. This study laid a foundation for establishment of PiAdV detection method and also provided support for development of PiAdV-1 subunit vaccine.更多还原