新型冠状病毒结构蛋白单克隆抗体的制备与鉴定  

Preparation and identification of monoclonal antibodies against structural proteins of SARS-CoV-2

作　　者：王玉玲 金伯泉 李娜 田莹 董芸 李琦 马樱 庄然 WANG Yuling;JIN Boquan;LI Na;TIAN Ying;DONG Yun;LI Qi;MA Ying;ZHUANG Ran(Department of Immunology,School of Basic Medical Sciences,Air Force Medical University,Xi'an 710032,China)

机构地区：[1]空军军医大学基础医学院免疫学教研室,陕西西安710032

出　　处：《空军军医大学学报》2023年第7期595-601,共7页Journal of Air Force Medical University

基　　金：国家自然科学基金(81871258);空军军医大学军事医学提升计划项目(2020SWAQ02)。

摘　　要：目的 制备针对新型冠状病毒(SARS-CoV-2)刺突蛋白(S蛋白)S1亚基、核衣壳蛋白(N蛋白)的小鼠源性单克隆抗体(mAbs),进行功能和应用的初步鉴定。制备具备中和活性的高亲和力人鼠嵌合抗体。方法 用重组表达的SARS-CoV-2 S蛋白S1亚基和N蛋白分别免疫BALB/c小鼠,利用常规B细胞杂交瘤技术制备抗S蛋白S1亚基mAbs和抗N蛋白mAbs,并鉴定其在Western blotting、免疫组织化学染色中的应用价值;建立检测可溶性S蛋白和N蛋白的夹心ELISA方法;基于假病毒-荧光素酶检测系统,检测特异性抗体的中和活性。将分泌具备中和活性抗S蛋白S1亚基mAbs的杂交瘤细胞株测序,制备人鼠嵌合抗体。结果 获得53株分泌小鼠抗SARS-CoV-2 S蛋白S1亚基mAbs的杂交瘤细胞株,克隆号分别命名XA326.1~XA326.53;获得42株分泌小鼠抗SARS-CoV-2 N蛋白mAbs的杂交瘤细胞株,克隆号分别命名XA327.1~XA327.42。初步鉴定出9株可用于Western blotting检测可溶性SARS-CoV-2 S蛋白S1亚基的mAbs、8株可用于Western blotting检测可溶性SARS-CoV-2 N蛋白的mAbs;9株可用于免疫组织化学染色的抗SARS-CoV-2 S蛋白S1亚基mAbs、4株可用于免疫组织化学染色的抗SARS-CoV-2 N蛋白mAbs。构建以mAb XA326.7为包被抗体、生物素标记的mAb XA326.19为检测抗体的检测可溶性SARS-CoV-2 S蛋白S1亚基的夹心ELISA试剂盒;采用mAb XA327.37作为包被抗体、生物素标记的mAb XA327.25为检测抗体,可以建立检测可溶性SARS-CoV-2 N蛋白的夹心ELISA试剂盒。XA326.4、XA326.7、XA326.8、XA326.49 mAbs具有中和活性,可以在假病毒感染实验中阻断病毒对人ACE2阳性细胞的感染。结论 成功制备出一套小鼠抗SARS-CoV-2 S蛋白S1亚基、N蛋白的mAbs,可用于多种免疫学检测技术,XA326.4、XA326.7、XA326.8、XA326.49中和活性抗体具有潜在的临床应用价值。Objective To prepare murine-derived monoclonal antibodies(mAbs)against the S1 subunit of spike protein(S protein)and nucleoprotein(N protein)of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)for preliminary identification of their functions and applications,and to prepare human-mouse chimeric antibodies with high affinity and neutralizing activity.Methods BALB/c mice were immunized with the S1 subunit of S protein and N protein of the recombinant SARS-CoV-2,respectively.MAbs against the S1 subunit of S protein and N protein were prepared by conventional B-cell hybridoma technique,and their application values were evaluated by Western blotting and immunohistochemical staining.A sandwich ELISA method for detecting the soluble S protein and N protein was established.The neutralizing activity of specific antibodies was detected based on pseudovirus-luciferase detection system.Hybridoma cell lines secreting neutralizing mAbs against the S1 subunit of S protein were sequenced to prepare human-mouse chimeric antibodies.Results A total of 53 hybridoma cell lines secreting murine mAbs against the S1 subunit of S protein of SARS-CoV-2 were obtained,with clone numbers named XA326.1-XA326.53,and 42 hybridoma cell lines secreting murine mAbs against SARS-CoV-2 N protein were obtained,with clone numbers named XA327.1-XA327.42.Preliminary identification showed that 9 strains of mAbs against the soluble S1 subunit of S protein and 8 strains of mAbs against soluble N protein of SARS-CoV-2 could be used for Western blotting,while 9 strains of mAbs against the S1 subunit of S protein and 4 strains of mAbs against N protein of SARS-CoV-2 could be applied to immunohistochemical staining.A sandwich ELISA kit with mAb XA326.7 as coating antibody and biotin-labeled mAb XA326.19 as detection antibody was established to detect the soluble S1 subunit of S protein of SARS-CoV-2 in serum,and a sandwich ELISA kit with mAb XA327.37 as coating antibody and biotin-labeled mAb XA327.25 as detection antibody was established to detect t

关 键 词：新型冠状病毒 刺突蛋白 核衣壳蛋白 单克隆抗体 中和抗体 免疫学检测方法 

分 类 号：R392[医药卫生—免疫学]