猪XCR1/FMDV双特异纳米抗体融合蛋白的制备与鉴定  

Preparation and identification of porcine XCR1/FMDV bispecific nanobody fusion protein

作　　者：杨利[1,2] 杜露平 侯立婷[1,2] 张浩明 于晓明[1,2] 李兰 乔绪稳[1,2] 张元鹏 秦竹[1,2] 王义伟 郑其升[1,2] 陈瑾 程海卫[1,2] YANG Li;DU Luping;HOU Liting;ZHANG Haoming;YU Xiaoming;LI Lan;QIAO Xuwen;ZHANG Yuanpeng;QIN Zhu;WANG Yiwei;ZHENG Qisheng;CHEN Jin;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;GuoTai(Taizhou)Center of Technology Innovation for Veterinary Biologicals,Taizhou 225300,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,南京210014 [2]兽用生物制品(泰州)国泰技术创新中心,江苏泰州225300

出　　处：《中国农业大学学报》2025年第1期131-141,共11页Journal of China Agricultural University

基　　金：江苏省自然科学基金项目(BK20231389);国家自然科学基金项目(32102690);“十四五”重点研发专项(2022YFD1800800)。

摘　　要：为通过将FMDV抗原靶向提呈至猪DC细胞进而提高疫苗免疫效力,本研究利用原核系统表达了猪XCR1目的蛋白,以该蛋白为免疫原免疫羊驼,提取其外周血淋巴细胞的总mRNA并反转录为cDNA,构建噬菌体展示纳米抗体文库,经过3轮亲和筛选获得猪XCR1特异性纳米抗体;利用SOE-PCR技术将该纳米抗体和猪O型FMDV特异性纳米抗体Nb205连接,构建XCR1/FMDV双特异纳米抗体融合蛋白。结果表明:1)成功原核表达猪XCR1目的蛋白,SDS-PAGE和Western blot结果显示,其相对分子量大小约35 ku,符合预期。2)羊驼血清效价为1∶64000,满足建库要求;构建的噬菌体展示文库库容量约为1.91×10^(8)CFU/mL,插入率为91.3%,多样性好;经3轮亲和筛选成功获得了纳米抗体Nb75,间接ELISA结果显示该纳米抗体能特异性与XCR1结合。3)利用SOE-PCR技术成功构建XCR1/FMDV双特异纳米抗体融合蛋白Nb75-205;SDS-PAGE和Western blot显示,该蛋白在大肠杆菌中可溶性表达,且呈现二聚体结构,间接ELISA结果显示Nb75-205能同时与XCR1目的蛋白和FMDV抗原结合。综上,本研究采用噬菌体展示技术成功获得猪XCR1特异性纳米抗体,并构建XCR1/FMDV双特异纳米抗体融合蛋白Nb75-205,且该蛋白为天然二聚体结构,与XCR1目的蛋白和FMDV抗原均具有较好的结合活性,为进一步开展FMDV抗原的猪XCR1靶向提呈研究奠定基础。In order to enhance the immune efficacy of FMDV vaccine by targeting FMDV to porcine dendritic cells,the target protein XCR1 was expressed using prokaryotic expression technology.Subsequently,alpacas were immunized with XCR1 as an immunogen.The total mRNA of peripheral blood lymphocytes of alpacas was extracted and reverse-transcribed into cDNA.A phage display nanobody library was constructed and XCR1-specific nanobodies were obtained after three rounds of affinity screening.Following this,a bispecific nanobody Nb75-205 targeting both XCR1 and FMDV was constructed by connecting Nb75 with Nb205 anti-FMDV nanobody using SOE-PCR technique.The results showed that:1)The target protein XCR1 was successfully obtained,SDS-PAGE and Western blot analysis showed that the molecular weight of this protein was about 35 ku,as expected.2)Antibody titers of alpacas were 1∶64000,meeting the requirement for database construction.The phage display library size was approximately 1.91×10^(8) CFU/mL,with an insertion rate of 91.3%and good diversity.After three rounds of affinity screening,the nanobody Nb75 was successfully obtained which could specifically bind to XCR1 as detected by Indirect ELISA.3)The XCR1/FMDV bispecific nanobody Nb75-205 was constructed through SOE-PCR technique.SDS-PAGE and Western blot results demonstrated that this fusion protein could be expressed in a soluble form in Escherichia coli,with a dimeric structure.Indirect ELISA results indicated that Nb75-205 was capable of binding to both XCR1 target protein and FMDV antigen.In summary,anti-porcine XCR1 nanobody was successfully obtained by phage screening technology,and XCR1/FMDV bispecific nanobody Nb75-205 was successfully constructed through SOE-PCR technology in this study.Nb75-205 was a dimer and could react with both XCR1 and FMDV,which laid a foundation for the further study on the presentation of FMDV targeted by porcine XCR1.

关 键 词：趋化因子受体1 双特异纳米抗体 二聚体 FMDV 

分 类 号：S852.4[农业科学—基础兽医学]