摘要
为进一步研究鸭腺病毒3型(DAdV-3)Fiber-2蛋白,研究通过PCR扩增fiber-2基因,构建p ET32a-DAdV3-fiber2原核质粒,表达并纯化蛋白,以纯化的蛋白为免疫原,免疫6周龄雌性BALB/c小鼠制备抗Fiber-2单克隆抗体杂交瘤细胞,采用间接免疫荧光试验和免疫印迹试验进行鉴定,并用于DAdV-3 Fiber-2杆状病毒表达产物的鉴定。结果显示:原核表达获得1.77 mg/mL Fiber-2蛋白,筛选获得2株阳性单克隆细胞株2E10E4和4F7F10,两株单克隆抗体均为IgG 1型,轻链类型为κ;DAdV-3感染的LMH细胞中存在特异性荧光,免疫印迹试验确认在约59 ku处出现特异性条带;采用制备的单克隆抗体确认了表达Fiber-2蛋白的重组杆状病毒。研究获得了针对DAdV-3 Fiber-2蛋白的单克隆抗体,为深入探究Fiber-2在DAdV-3感染机制中的作用以及为建立DAdV-3抗原免疫测定技术奠定了基础。
To futher study Fiber-2 protein,fiber-2 gene was amplified by PCR,and pET32a-DAdV3-fiber2 prokaryotic plasmid was constructed,the protein was expressed and purified and used to immunize 6-week-age female BALB/c mice to prepare the anti-Fiber-2 monoclonal antibody hybridoma cells and identified by indirect immunofluorescence assay and Western blot,and the recombinant baculovirus expression product.The results showed that 1.77 mg/mL of Fiber-2 protein was obtained by prokaryotic expression and 2 positive cell lines named 2E10E4 and 4F7F10 were screened.All the strains′heavy chain type was IgG 1 and light chain type wasκ.The specific fluorescence was found in DAdV-3-infected LMH cells by IFA and Western blot identification revealed that a specific band appeared at about 59 ku.The recombinant baculovirus expression product of DAdV-3 Fiber-2 was identified by the monoclonal antibody.The results suggested that the prepared monoclonal antibodies against DAdV-3 Fiber-2 protein laid the foundation for an in-depth understanding of the role of Fiber-2 in DAdV-3 infection mechanism and the establishment of DAdV-3 antigen immunoassay technology.
作者
魏常青
史馨瑾
吕璐
刘英楠
谢振华
陈宗艳
陈鸿军
WEI Changqing;SHI Xinjin;LU Lu;LIU Yingnan;XIE Zhenhua;CHEN Zongyan;CHEN Hongjun(Shanghai Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Shanghai 200241)
出处
《中国家禽》
北大核心
2023年第2期46-51,共6页
China Poultry
基金
上海市自然科学基金项目(19ZR146880)。
