禽腺病毒4型Penton和Fiber1蛋白原核表达及多克隆抗体的制备  

Prokaryotic expression of FAdV-4 Penton and Fiber1 protein and preparation of their polyclonal antibodies

作　　者：方天 李乔斌 冯孝傲 朱二鹏 陈常秀 程振涛 FANG Tian;LI Qiaobin;FENG Xiao'ao;ZHU Erpeng;CHEN Changxiu;CHENG Zhentao(College of Animal Sciences,Guizhou University,Guiyang 550025,China;Guizhou Province Key Laboratory of Animal Disease and Veterinary Public Health,Guiyang 550025,China;Guizhou Center for Animal Disease Control and Prevention,Guiyang 550008,China)

机构地区：[1]贵州大学动物科学学院,贵州贵阳550025 [2]贵州省动物疫病与兽医公共卫生重点实验室,贵州贵阳550025 [3]贵州省动物疾病预防控制中心,贵州贵阳550008

出　　处：《畜牧与兽医》2024年第3期54-61,共8页Animal Husbandry & Veterinary Medicine

基　　金：贵州省科技计划项目(黔科合基础[2020]1Y409号);贵州省科技支撑计划项目(黔科合支撑[2021]一般161);贵州省优秀青年科技人才项目(黔科合平台人才[2021]5646)。

摘　　要：为开展禽腺病毒4型(FAdV-4)五邻体蛋白(Penton)和纤丝蛋白(Fiber1)多克隆抗体的制备及评价,研究通过对Penton和Fiber1基因优势抗原表位基因片段进行生物信息学分析,设计特异性引物PCR扩增获得Penton和Fiber1优势抗原表位基因片段,将其克隆至原核表达载体pColdⅠ,转入表达宿主菌BL21(DE3)进行异丙基硫代半乳糖苷(IPTG)诱导表达,将纯化、鉴定后的目的蛋白免疫BALB/c小鼠制备多克隆抗体,对抗体进行Western blot鉴定及间接ELISA抗体效价测定。结果可见,FAdV-4 Penton基因序列共编码525个氨基酸,Fiber1基因序列共编码432个氨基酸,其优势抗原表位点分别有7、6个区域,且均集中于N端;成功构建pColdⅠ-P和pColdⅠ-F1重组原核表达质粒,其诱导表达的重组蛋白Penton和Fiber1均与His标签单抗发生特异性反应,动物免疫获得的Penton、Fiber1多克隆抗体均能与目的蛋白反应形成特异性条带,且Penton多克隆抗体效价高于Fiber1多克隆抗体。综上,研究获得的纯化重组蛋白Penton较Fiber1蛋白表达量更高,免疫后血清抗体效价更高,更具有制备多克隆抗体的优势。In order to prepare and evaluate the polyclonal antibodies against fowl adenovirus serotype 4(FAdV-4)Penton and Fiber1 pro-tein,the dominant epitope gene fragments of the Penton and Fiber1 genes were screened by bioinformatics analysis and were obtained by spe-cific PCR amplification,and then they were cloned into the pColdⅠvector.Polyclonal antibodies were prepared by immunization of purified proteins of interest in BALB/c mice,and indirect ELISA and Western blot were used to detect the titers of the antibodies.The results showed that the FAdV-4 Penton gene sequence encoded a total of 525 amino acids,the Fiber1 gene sequence encoded a total of 432 amino acids,and they had 7 and 6 dominant antigen epitopes,respectively,which were located at the N-terminal.The recombinant prokaryotic expression plasmids pColdⅠ-P and pColdⅠ-F1 were successfully constructed,and the expressed recombinant protein Penton and the Fiber1 protein specifically reacted with the anti-His monoclonal antibodies.Both Penton-and Fiber1-generated polyclonal antibodies reacted with the pro-tein of interest to form specific bands,and the penton polyclonal antibodies had a higher titer than the Fiber1 polyclonal antibodies did.Taken together,the purified recombinant protein Penton obtained in this study had higher expression than the Fiber1 protein,exhibited stronger an-tibody binding ability,presented higher serum antibody titer after immunization,and possessed more advantages in preparing polyclonal anti-bodies.

关 键 词：禽腺病毒4型 Penton蛋白 Fiber1蛋白 生物信息学分析 多克隆抗体 

分 类 号：S855[农业科学—临床兽医学]