摘要
登革病毒(DENV)是一种经伊蚊传播的人兽共患病毒,目前没有特异性抗DENV药物和安全且高效的疫苗。为制备可中和DENV的纳米抗体,本研究构建血清Ⅱ型DENV(DENV-2)特异性噬菌体展示文库,并筛选DENV E蛋白特异性纳米抗体。首先将灭活并浓缩的DENV-2免疫羊驼,采血分离外周血单核细胞(PBMC)并提取总RNA,通过RT-PCR扩增羊驼重链抗体可变区(VHH)并克隆入pCANTAB-5E质粒,构建成DENV特异性纳米抗体噬菌体展示文库,然后,以DENV-2 E蛋白为抗原淘筛纳米抗体库,将筛选到的2个VHH片段克隆入pPICZαA载体后在毕赤酵母(GS115株)中表达,并纯化抗体,最后,鉴定纯化的纳米抗体与E蛋白的反应性。结果显示:成功构建库容为9.1×10^(8)个的噬菌体展示文库;经3轮淘筛获得1株与DENV-2 E蛋白特异结合的纳米抗体DV2-Nb2。本研究为DENV诊断和抗体药物研发提供实验技术基础。
Dengue virus(DENV)is an important zoonotic arbovirus transmitted by Aedes mosquitoes.In order to construct and screen nanobodies that specifically bound to DENV E protein,an alpaca was immunized with inactivated DENV serotype 2(DENV-2)concentrates,from which peripheral blood mononuclear cells(PBMC)were collected for total RNA extraction.The variable region of heavy chain(VHH)of alpaca antibody was amplified by RT-PCR and cloned into pCANTAB-5E plasmid to generate DENV-specific nanobody library.DENV-2 E protein was used to screen the nanobody library using phage display technology.The two VHH fragments were expressed in Pichia pastoris GS115 by the pPICZαA vector followed by antibody purification and determination of reactivity.The results showed that the phage display library with a capacity of 9.1×108 was constructed and nanoparticle DV2-Nb2 antibodies specifically bound to the DENV-2 E protein were obtained by three rounds of screening.This study provided the experimental technology for DENV diagnosis and antibody drug development.
作者
陈玲玲
范瑞文
何雷
贾琼
秦蓉芬
陈玉红
李雷斌
黄袁慧
刘玉良
李义平
王传彬
CHEN Lingling;FAN Ruiwen;HE Lei;JIA Qiong;QIN Rongfen;CHEN Yuhong;LI Leibin;HUANG Yuanhui;LIU Yuliang;LI Yiping;WANG Chuanbin(College of Animal Science and Technology,Guangxi University,Nanning 530004,China;China Animal Disease Control Center,Beijing 102600,China;College of Veterinary Medicine,Shanxi Agricultural University,Taigu 030801,China;Key Laboratory of Tropical Disease Control and Research,Ministry of Education,Sun Yat-sen University Zhongshan School of Medicine,Guangzhou 510080,China)
出处
《中国动物传染病学报》
北大核心
2025年第4期170-176,共7页
Chinese Journal of Animal Infectious Diseases
基金
中山大学热带病防治研究教育部重点实验室开放基金。
