非洲猪瘟病毒K205R蛋白特异性纳米抗体的筛选与鉴定  

Screening and identification of African swine fever virus K205R protein-specific nanobodies

作　　者：吴书雅 赵慧君[1,2] 任佳慧 董昊鑫 张铭芳 李嘉骥 杜永坤 万博[1,2] 张昂克 张改平[1,2,3] WU Shuya;ZHAO Huijun;REN Jiahui;DONG Haoxin;ZHANG Mingfang;LI Jiaji;DU Yongkun;WAN Bo;ZHANG Angke;ZHANG Gaiping(College of Veterinary Medicine,Henan Agricultural University,Zhengzhou 450046,China;International Joint Research Center of National Animal Immunology,Henan Agricultural University,Zhengzhou 450046,China;Key Laboratory of Animal Immunology,Ministry of Agriculture and Rural Affairs,Henan Academy of Agricultural Sciences,Zhengzhou 450002,China)

机构地区：[1]河南农业大学动物医学院,河南郑州450046 [2]河南农业大学动物医学院国家动物免疫学国际联合研究中心,河南郑州450046 [3]河南省农业科学院农业农村部动物免疫学重点实验室,河南郑州450002

出　　处：《河南农业大学学报》2023年第2期307-315,共9页Journal of Henan Agricultural University

基　　金：国家自然科学基金项目(31941001)。

摘　　要：【目的】利用噬菌体展示技术筛选非洲猪瘟病毒(African swine fever virus,ASFV)K205R蛋白的特异性纳米抗体。【方法】将原核表达、纯化的ASFV K205R蛋白免疫双峰驼,采集全血,分离外周血淋巴细胞,提取总RNA并反转录,巢式PCR扩增VHH基因,构建VHH噬菌体文库,利用噬菌体展示技术筛选K205R特异性纳米抗体。【结果】成功构建库容量为5×10^(8)的VHH噬菌体文库,然后利用噬菌体展示技术经过3轮淘选,特异性VHH噬菌体得到明显富集,氨基酸序列比对显示共筛选出5株K205R蛋白的特异性纳米抗体,分别为ASFV-K205R-Nb1、ASFV-K205R-Nb14、ASFV-K205R-Nb35、ASFV-K205R-Nb64和ASFV-K205R-Nb82。【结论】筛选的5株纳米抗体均具有良好的特异性,其中ASFV-K205R-Nb1、ASFV-K205R-Nb35和ASFV-K205R-Nb82具有更好的亲和力,为以纳米抗体作为灵敏探针建立的特异性强、灵敏度高、成本低的新型ASFV血清学诊断方法提供依据。【Objective】 Using phage display technology aims to screen specific nanobodies against African swine fever virus(ASFV) K205R protein.【Method】ASFV K205R protein was obtained by prokaryotic expression and purification,and immunized with Bactrian camels.Blood was collected on the seventh day after 4 immunizations,then isolated peripheral blood lymphocytes,extracted total RNA,and reversed transcribed into cDNA.The VHH genes were amplified using the cDNA as a template with nest PCR to construct a VHH phage library.Finally,ASFV K205R-specific nanobodies were screened by phage display technology.【Result】 The VHH phage library was successfully constructed with a library capacity of 5×10^(8).Then,after three rounds of panning by phage display technology,the specific VHH phages were significantly enriched.The amino acid sequence alignment found that five ASFV K205R protein-specific nanobodies were screened,named ASFV-K205R-Nb1,ASFV-K205R-Nb14,ASFV-K205R-Nb35,ASFV-K205R-Nb64 and ASFV-K205R-Nb82,respectively.【Conclusion】 The five screened nanobodies have good specificity,among which ASFV-K205R-Nb1,ASFV-K205R-Nb35 and ASFV-K205R-Nb82 have better affinity,they provide a material basis for the establishment of a new type of ASFV serological diagnosis method with strong specificity,high sensitivity and low cost using nanobody as a sensitive probe.

关 键 词：非洲猪瘟病毒 K205R蛋白 VHH噬菌体文库 噬菌体展示 纳米抗体 

分 类 号：S852.65[农业科学—基础兽医学]