猪CD205分子CysR-FN Ⅱ蛋白特异性纳米抗体的筛选与鉴定研究  

Research of screening and identification of nanobodies against porcine CD205 CysR-FN Ⅱ protein

作　　者：孙鑫 杨利[1,3] 郭东辉 马旭东 杜露平[1,3] 侯立婷[1,3] 陈瑾 冯秀丽[2] 郑其升 程海卫[1,3] SUN Xin;YANG Li;GUO Donghui;MA Xudong;DU Luping;HOU Liting;CHEN Jin;FENG Xiuli;ZHENG Qisheng;CHENG Haiwei(Institute of Veterinary Immunology&Engineering/National Research Center of Engineering and Technology for Veterinary Biologicals/Jiangsu Key Laboratory for Food Quality and Safety,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China;College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China;GuoTai(Taizhou)Center of Technology Innovation for Veterinary Biologicals,Taizhou 225300,China;Henan Qixiang Biotechnology Co.,Ltd.,Jiaozuo 454950,China;Guangzhou Experimental Station,Chinese Academy of Tropical Agricultural Sciences,Guangzhou 510140,China)

机构地区：[1]江苏省农业科学院动物免疫工程研究所/国家兽用生物制品工程技术研究中心/江苏省食品质量与安全重点实验室,江苏南京210014 [2]南京农业大学动物医学院,江苏南京210095 [3]兽用生物制品国泰(泰州)技术创新中心,江苏泰州225300 [4]河南祺祥生物科技有限公司,河南焦作454950 [5]中国热带农业科学院广州实验站,广东广州510140

出　　处：《南京农业大学学报》2024年第5期941-947,共7页Journal of Nanjing Agricultural University

基　　金：国家自然科学基金项目(32102690);“十四五”重点研发计划项目(2022YFD1800800)。

摘　　要：[目的]猪CD205分子作为猪树突状细胞(DC)特异性的抗原提呈受体,在抗原提呈过程中起关键作用。本研究通过噬菌体展示技术针对猪CD205分子CysR-FNⅡ蛋白进行筛选以获得其特异性的纳米抗体,为猪CD205靶向抗原提呈研究奠定基础。[方法]利用前期制备的猪CD205分子CysR-FNⅡ蛋白进行羊驼免疫,经6次皮下免疫后采集羊驼外周血淋巴细胞,提取总RNA后反转录获得cDNA,经PCR扩增编码纳米抗体的基因片段,构建猪CD205分子CysR-FNⅡ蛋白噬菌体展示纳米抗体基因文库,再运用噬菌体展示技术针对猪CD205分子CysR-FNⅡ蛋白进行亲和筛选,随机挑选单克隆菌落并使用Phage-ELISA方法鉴定出针对猪CD205分子CysR-FNⅡ蛋白的特异性纳米抗体。[结果]4轮亲和筛选后,通过Phage-ELISA方法鉴定和序列比对分析,共成功获得12个猪CD205分子CysR-FNⅡ蛋白特异性纳米抗体,其相对分子质量约为17×10^(3)。[结论]本研究成功筛选并获得猪CD205分子CysR-FNⅡ蛋白特异性的纳米抗体。[Objectives]Porcine CD205,as a receptor specific to porcine dendritic cells(DC),played a key role in the antigen presentation.In this study,nanobodies against porcine CD205 CysR-FNⅡprotein were screened by phage display technology,which laid a foundation for the research of porcine CD205-targeted antigen presentation.[Methods]CysR-FNⅡprotein of porcine CD205 molecule prepared in the previous research was used for the immunization of alpacas.After subcutaneous immunization for 6 times,peripheral blood lymphocytes of alpaca were collected,their total RNA was extracted and cDNA was obtained by reverse transcription.The gene fragments encoding nanobodies were obtained by PCR amplification,and the porcine CD205 phage display nanobody library was constructed.Bio-spannings were performed by phage display technology and monoclonal colonies were randomly selected to screen specific nanobodies against porcine CD205 CysR-FNⅡprotein by Phage-ELISA.[Results]After 4 rounds of bio-spanning,a total of 12 porcine CD205 CysR-FNⅡprotein specific nanobodies were selected by Phage-ELISA and sequence analysis with their molecular weight about 17×10^(3).[Conclusions]This study successfully selected and obtained porcine CD205 CysR-FNⅡprotein specific nanobodies.

关 键 词：猪 CD205 CysR-FNⅡ 纳米抗体 噬菌体展示 

分 类 号：S852.4[农业科学—基础兽医学]