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新冠病毒N蛋白纳米抗体制备及检测卡组装

吴方晖 阴忆烽 刘艳丽 雷美玲 宋云扬

吴方晖, 阴忆烽, 刘艳丽, 雷美玲, 宋云扬. 新冠病毒N蛋白纳米抗体制备及检测卡组装[J]. 中国公共卫生. doi: 10.11847/zgggws1140589
引用本文: 吴方晖, 阴忆烽, 刘艳丽, 雷美玲, 宋云扬. 新冠病毒N蛋白纳米抗体制备及检测卡组装[J]. 中国公共卫生. doi: 10.11847/zgggws1140589
WU Fang-hui, YIN Yi-feng, LIU Yan-li, . Preparation of nanobody against N protein of SARS-CoV-2 and assembly of colloidal gold immunochromatography card[J]. Chinese Journal of Public Health. doi: 10.11847/zgggws1140589
Citation: WU Fang-hui, YIN Yi-feng, LIU Yan-li, . Preparation of nanobody against N protein of SARS-CoV-2 and assembly of colloidal gold immunochromatography card[J]. Chinese Journal of Public Health. doi: 10.11847/zgggws1140589

新冠病毒N蛋白纳米抗体制备及检测卡组装

doi: 10.11847/zgggws1140589
基金项目: 国民核生化灾害防护国家重点实验室应急科研基金(SKLNBC2020-5)
详细信息
    作者简介:

    吴方晖(1970 – ),男,湖北武汉人,副研究员,博士,研究方向:药理学与分子生物学

    通讯作者:

    宋云扬,E-mail:syyfeng@126.com

  • 中图分类号: R 373

Preparation of nanobody against N protein of SARS-CoV-2 and assembly of colloidal gold immunochromatography card

  • 摘要:   目的  通过制备新冠病毒N蛋白纳米抗体,将其应用于新冠病毒N蛋白抗原检测。  方法  将新冠病毒N蛋白免疫羊驼,构建N蛋白纳米抗体基因文库,通过噬菌体展示技术淘筛对新冠病毒N蛋白具有亲和力的纳米抗体,将纳米抗体基因克隆到酵母载体,挑取阳性克隆菌株诱导表达,亲和层析纯化分泌蛋白,透析脱盐和浓缩后,采用ELISA法筛选对新冠病毒N蛋白高亲和性纳米抗体,交叉配对筛选出1对纳米抗体制备成胶体金免疫层析检测卡,滴加N蛋白溶液到样品孔观察检测线和质控线。  结果  共筛选出46株不同序列的纳米抗体,优选其中6株表达、纯化和浓缩得到纳米抗体,抗体浓度 > 1.0 mg/mL。ELISA筛选结果显示,6株纳米抗体均对N蛋白有高亲和力,抗体配对实验筛选到1对抗体可以夹心法检测新冠病毒N蛋白,组装成胶体金免疫层析检测卡,对新冠病毒N蛋白抗原检测可见明显检测线和质控线条带,检测限 ≥ 10 μg/mL。  结论  制备的纳米抗体可以用于胶体金纳米抗体免疫层析检测卡,实现对新冠病毒N蛋白的检测。
  • 图  1  新冠病毒N蛋白SDS-PAGE电泳图

    注:1:蛋白Marker;2:亲和层析纯化N蛋白;3:超滤浓缩N蛋白;4:诱导表达阳性菌株破壁上清液。

    图  2  菌落PCR琼脂糖凝胶DNA电泳结果

    注:M :DNA Marker;第1~50泳道 阳性克隆PCR扩增产物;第51泳道 克隆PCR扩增产物无目标DNA。

    图  3  胶体金纳米抗体免疫层系检测卡组装示意图

    图  4  纳米抗体免疫检测版对N蛋白的检测结果

    注:1~4 分别是浓度为 100、30、10、3 μg/mL的N蛋白溶液;5无N蛋白阴性对照溶液。

    表  1  纳米抗体噬菌体展示库筛选结果

    筛选轮数输入效价
    (pfu)
    筛选效价
    (pfu)
    对照效价
    (pfu)
    富集程度相差倍数
    第1轮 1 × 10124.4 × 1082.4 × 1064.4 × 10–4183
    第2轮5.2 × 10112.4 × 1081.6 × 1064.1 × 10–4150
    第3轮8.4 × 10111.6 × 1092.4 × 1061.9 × 10–3667
    下载: 导出CSV

    表  2  ELISA检测纳米抗体对抗原N蛋白和阴性对照BSA的吸光值

    编号123456
    N蛋白1.2271.4361.2980.6010.9850.455
    BSA0.1230.1450.0730.0810.0840.076
    下载: 导出CSV
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出版历程
  • 收稿日期:  2022-10-13
  • 网络出版日期:  2022-12-02

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