抗非洲猪瘟病毒NP419L蛋白纳米抗体的筛选鉴定及其在抗体检测中的初步应用  

Screening and Identification of Nanobodies against NP419L Protein of African Swine Fever Virus and Its Preliminary Application of Antibody Detection

作　　者：王映 朱家宏 赵加凯 纪品品 陈旭 张路 刘宝元[1] 孙亚妮[1] 赵钦[1] WANG Ying;ZHU Jiahong;ZHAO Jiakai;JI Pinpin;CHEN Xu;ZHANG Lu;LIU Baoyuan;SUN Yani;ZHAO Qin(Yangling Observing and Experimental Station of National Data Center of Animal Health,College of Veterinary Medicine,Northwest A&F University,Yangling 712100,China)

机构地区：[1]西北农林科技大学动物医学院,农业农村部国家动物疫病数据中心杨凌观测实验点,杨凌712100

出　　处：《畜牧兽医学报》2023年第6期2509-2520,共12页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金面上项目(32273041);中央级公益性科研院所基本科研业务费院级统筹项目。

摘　　要：非洲猪瘟(African swine fever,ASF)是猪的一种高度传染性疾病,严重威胁全球养猪业的发展。本研究旨在筛选抗非洲猪瘟病毒(ASF virus,ASFV)NP419L蛋白的纳米抗体并对其在ASF抗体检测中的应用进行初步评价。大肠杆菌表达和Ni-NTA亲和层析纯化等技术表达纯化重组NP419L蛋白;将纯化的重组蛋白免疫双峰驼,5次免疫采集外周血淋巴细胞提取总RNA,经反转录、巢式PCR、酶切、连接和电转化等构建抗NP419L蛋白的噬菌体展示文库;利用噬菌体展示筛淘技术,经三轮筛淘获得抗NP419L蛋白的纳米抗体;构建纳米抗体与辣根过氧化物酶(HRP)融合蛋白的真核表达载体,转染HEK293T细胞,ELISA和间接免疫荧光(IFA)等鉴定融合蛋白的分泌表达及其与NP419L蛋白的结合;竞争ELISA初步评价获得的纳米抗体与HRP融合蛋白在ASF抗体检测中的应用。结果显示,成功制备了纯度较高的预期大小为48 ku的重组NP419L蛋白;构建获得了阳性率为89.5%,库容量为6×108的噬菌体展示文库;筛淘获得了6株抗NP419L蛋白的纳米抗体;分泌表达了该6株纳米抗体与HRP的融合蛋白,ELISA检测发现该6株融合蛋白可特异性结合重组NP419L蛋白,IFA检测发现1株融合蛋白与真核表达的NP419L蛋白结合,该融合蛋白可作为探针应用于ASF抗体的检测。本研究成功筛选出6株抗ASFV NP419L蛋白的特异性纳米抗体,并对其进行了结合蛋白特性的鉴定和抗体检测初步应用的评价,为后续ASF诊断技术的研发和NP419L蛋白功能研究奠定了基础。African swine fever(ASF)is a highly infectious disease of pigs,which seriously threatens the development of the global pig industry.The purpose of this study is to screen specific nanobodies against the NP419L protein of ASF virus(ASFV)and evaluate its application of anti-ASFV antibody detection.We prepared the recombinant NP419L protein by E.coli expression system and Ni-NTA affinity chromatography purification.The purified recombinant protein was used to immunize the Bactrian camels.After the fifth immunization,peripheral blood lymphocytes were collected and total RNA was extracted.A phage display library against NP419L protein was constructed by nested PCR,enzyme digestion,ligation and electroporation.Nanobodies against NP419L were obtained through three rounds of screening using phage display screening technology.The eukaryotic expression vectors of nanobodies fused with horseradish peroxidase(HRP)proteins were constructed and transfected into HEK293T cells.The secretory expression of the fusion protein and its binding to NP419L protein were identified by ELISA and indirect immunofluorescence(IFA),and preliminary evaluation of the obtained nanobody fused with HRP proteins in ASF detection using competitive ELISA.The results showed that the NP419L recombinant protein with the expected size of 48 ku was successfully prepared with high purity;a phage display library with a positive rate of 89.5%and a library capacity of 6×108 was constructed;six strains of anti-NP419L protein nanobodies were obtained by screening;the fusion proteins of these six nanobodies with HRP were secretively expressed,the results of ELISA showed that the six fusion proteins specifically bind to the recombinant NP419L protein,one fusion protein was found to bind to NP419L protein expressed with eukaryotic system by IFA,and this fusion protein can be used as a probe for the detection of ASFV antibodies.In the study,six nanobodies against ASFV NP419L protein were successfully screened.The preliminary characterization of its binding protein

关 键 词：非洲猪瘟病毒 NP419L蛋白 纳米抗体 

分 类 号：S852.659.1[农业科学—基础兽医学]