高级检索

留言板

尊敬的读者、作者、审稿人, 关于本刊的投稿、审稿、编辑和出版的任何问题, 您可以本页添加留言。我们将尽快给您答复。谢谢您的支持!

姓名
邮箱
手机号码
标题
留言内容
验证码

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, 2023, 39(3): 389-393. 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, 2023, 39(3): 389-393. 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, 2023, 39(3): 389-393. 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, 2023, 39(3): 389-393. 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

    通信作者:

    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
  • [1] Jin YF, Yang HY, Ji WQ, et al. Virology, epidemiology, patho-genesis, and control of COVID-19[J]. Viruses, 2020, 12(4): 372. doi: 10.3390/v12040372
    [2] 中华人民共和国国家卫生健康委员会. 新型冠状病毒肺炎诊疗方案(第九版)[EB/OL]. 中华人民共和国国家卫生健康委员会. (2022-06-27). https://china.huanqiu.com/article/47CmXrga99W.
    [3] Mertens P, De Vos N, Martiny D, et al. Development and potential usefulness of the COVID-19 Ag respi-strip diagnostic assay in a pandemic context[J]. Frontiers in Medicine, 2020, 7: 225. doi: 10.3389/fmed.2020.00225
    [4] Muyldermans S. Nanobodies: natural single-domain antibodies[J]. Annual Review of Biochemistry, 2013, 82: 775 – 797. doi: 10.1146/annurev-biochem-063011-092449
    [5] 何晓婷, 董洁娴, 沈兴, 等. 纳米抗体的稳定性及其结构基础研究进展[J]. 生物化学与生物物理进展, 2022, 49(6): 1004 – 1017. doi: 10.16476/j.pibb.2021.0171
    [6] Vincke C, Muyldermans S. Introduction to heavy chain antibodies and derived nanobodies[J]. Methods in Molecular Biology, 2012, 911: 15 – 26.
    [7] Hamers-Casterman C, Atarhouch T, Muyldermans S, et al. Naturally occurring antibodies devoid of light chains[J]. Nature, 1993, 363(6428): 446 – 448. doi: 10.1038/363446a0
    [8] Salvador JP, Vilaplana L, Marco MP. Nanobody: outstanding features for diagnostic and therapeutic applications[J]. Analytical and Bioanalytical Chemistry, 2019, 411(9): 1703 – 1713. doi: 10.1007/s00216-019-01633-4
    [9] Cai HM, Yao HB, Li TT, et al. An improved fluorescent tag and its nanobodies for membrane protein expression, stability assay, and purification[J]. Communications Biology, 2020, 3(1): 753. doi: 10.1038/s42003-020-01478-z
    [10] Akazawa-Ogawa Y, Takashima M, Lee YH, et al. Heat-induced irreversible denaturation of the camelid single domain VHH antibody is governed by chemical modifications[J]. The Journal of Biological Chemistry, 2014, 289(22): 15666 – 15679. doi: 10.1074/jbc.M113.534222
    [11] Goldman ER, Liu JL, Zabetakis D, et al. Enhancing stability of camelid and shark single domain antibodies: an overview[J]. Frontiers in Immunology, 2017, 8: 865. doi: 10.3389/fimmu.2017.00865
    [12] Bever CRS, Majkova Z, Radhakrishnan R, et al. Development and utilization of camelid VHH antibodies from alpaca for 2, 2′, 4, 4′-tetrabrominated diphenyl ether detection[J]. Analytical Chemistry, 2014, 86(15): 7875 – 7882. doi: 10.1021/ac501807j
    [13] Vattekatte AM, Shinada NK, Narwani TJ, et al. Discrete analysis of camelid variable domains: sequences, structures, and in-silico structure prediction[J]. PeerJ, 2020, 8: e8408. doi: 10.7717/peerj.8408
    [14] Wesolowski J, Alzogaray V, Reyelt J, et al. Single domain antibodies: promising experimental and therapeutic tools in infection and immunity[J]. Medical Microbiology and Immun-ology, 2009, 198(3): 157 – 174. doi: 10.1007/s00430-009-0116-7
    [15] Deffar K, Shi HL, Li L, et al. Nanobodies – the new concept in antibody engineering[J]. African Journal of Biotechnology, 2009, 8(12): 2645 – 2652.
    [16] Shriver-Lake LC, Goldman ER, Dean SN, et al. Lipid-tagged single domain antibodies for improved enzyme-linked immuno-sorbent assays[J]. Journal of Immunological Methods, 2020, 481 – 482: 112790.
    [17] Sheng YM, Wang K, Lu QZ, et al. Nanobody-horseradish peroxidase fusion protein as an ultrasensitive probe to detect antibodies against Newcastle disease virus in the immunoassay[J]. Journal of Nanobiotechnology, 2019, 17(1): 35. doi: 10.1186/s12951-019-0468-0
    [18] Sun ZC, Lv JW, Liu X, et al. Development of a nanobody-AviTag fusion protein and its application in a streptavidin-biotin-amplified enzyme-linked immunosorbent assay for ochratoxin A in cereal[J]. Analytical Chemistry, 2018, 90(17): 10628 – 10634. doi: 10.1021/acs.analchem.8b03085
  • 加载中
图(4) / 表(2)
计量
  • 文章访问数:  431
  • HTML全文浏览量:  294
  • PDF下载量:  103
  • 被引次数: 0
出版历程
  • 接收日期:  2022-10-13
  • 网络出版日期:  2022-12-02
  • 刊出日期:  2023-03-10

目录

    /

    返回文章
    返回