血清4型禽腺病毒Fiber-1蛋白截短表达及多克隆抗体制备  

Shortened expression and polyclonal antibody preparation of Fiber-1 protein of fowl adenovirus serotype 4

作　　者：罗昕雨 赵磊[1,2] 陈玉晴 缪欣怡 石家鑫 顾有方 李文超[1,2] 刘欣超 LUO Xinyu;ZHAO Lei;CHEN Yuqing;MIAO Xinyi;SHI Jiaxin;GU Youfang;LI Wenchao;LIU Xinchao(College of Animal Science,Anhui Science and Technology University,Fengyang 233100,China;Anhui Province Key Laboratory of Animal Nutrition Regulation and Health,Fengyang 233100,China)

机构地区：[1]安徽科技学院动物科学学院,安徽凤阳233100 [2]动物营养调控与健康安徽省重点实验室,安徽凤阳233100

出　　处：《安徽科技学院学报》2024年第2期25-30,共6页Journal of Anhui Science and Technology University

基　　金：农业农村部兽用药物与诊断技术广东科学观测实验站/广东省畜禽疫病防治研究重点实验室开放课题(YDWS202104);安徽省高校自然科学研究项目(2022AH051641);安徽省自然科学基金(1908085QC116);安徽科技学院人才引进项目(DKYJ201902)。

摘　　要：目的:制备血清4型禽腺病毒(FAdV-4)Fiber-1基因截短蛋白多克隆抗体,为FAdV-4病的诊断、检测及致病机制研究奠定基础。方法:对FAdV-4的CH/AHMC/2015分离株Fiber-1基因序列(MG148335.1:30459-31754)进行信号肽、疏水性和抗原决定簇分析,截取具有较高免疫原性的片段,设计合成特异性引物,以CH/AHMC/2015分离株基因组DNA为模板进行PCR扩增,获得截短Fiber-1(sFiber-1)基因片段,将其连接至原核表达载体pET-32a,验证后转化至大肠杆菌BL21感受态中,诱导表达sFiber-1蛋白。纯化后的蛋白与佐剂乳化后免疫SD大鼠,制备多克隆抗体,并测定多克隆抗体效价。用Western-blot检测抗体免疫原性。结果:成功构建了sFiber-1的原核表达载体,获得FAdV-4的sFiber-1重组蛋白,经SDS-PAGE鉴定,重组sFiber-1蛋白大小约为52 kDa,主要以包涵体形式表达;间接ELISA法测得sFiber-1多克隆抗体效价为1∶2^(13);Western blot结果显示制备的多克隆抗体能特异性识别出重组sFiber-1蛋白。结论:本研究成功表达了FAdV-4的重组sFiber-1蛋白,制备了具有较高免疫活性的sFiber-1多克隆抗体,可为FAdV-4的检测及诊断奠定基础。Objective:To prepare the polyclonal antibody against shortened Fiber-1(sFiber-1)protein of fowl adenovirus serotype 4(FAdV-4),and to provide a tool for the detection and diagnosis of FAdV-4.Methods:The signal peptide,hydrophobicity and antigen determination of the Fibre-1 gene sequence of CH/AHMC/2015 isolate of FAdV-4(MG148335.1:30459-31754)were analyzed,and the fragments with high immunogenicity were intercepted and the specific primers were designed and synthesized.The DNA of CH/AHMC/2015 isolate was amplified to obtain the sFiber-1 fragment,which was cloned into pET-32a,and transformed into the COMPETENT cell BL21 to express sFiber-1 protein.The purified sFiber-1 protein emulsified with adjuvant was used to immunize rats.Polyclond antibody was prepared,the titer of polyclonal antibody was analyzed by ELISA,and the immunogenicity of sFiber-1 was analyzed by Western blot.Results:The sFiber-1 prokaryotic expression vector was constructed to obtain recombinant sFiber-1 protein of FAdV-4.SDS-PAGE showed that sFiber-1 protein was mainly expressed in inclusion bodies with a molecular weight of 52 kDa.The titer of sFiber-1 antibody was 1∶2^(13).Western-blot results displayed that recombinant sFiber-1 protein can be recognized by the obtained antibody.Conclusion:In this study,recombinant sFiber-1 protein of FAdV-4 was successfully expressed and sFiber-1 polyclonal antibody with high immune activity was prepared,which could lay a foundation for the diagnosis and detection of FAdV-4.

关 键 词：血清4型禽腺病毒 Fiber-1基因 重组蛋白 多克隆抗体 

分 类 号：S855.3[农业科学—临床兽医学]