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牛乳主要过敏原αS1-酪蛋白的纯化鉴定及其多克隆抗体的制备

何圣发 龙彩云 王娇 熊梦 赵江强 燕燕 董亚萍 李欣 陈红兵

何圣发,龙彩云,王娇,等. 牛乳主要过敏原αS1-酪蛋白的纯化鉴定及其多克隆抗体的制备[J]. 食品工业科技,2022,43(15):106−114. doi:  10.13386/j.issn1002-0306.2021110034
引用本文: 何圣发,龙彩云,王娇,等. 牛乳主要过敏原αS1-酪蛋白的纯化鉴定及其多克隆抗体的制备[J]. 食品工业科技,2022,43(15):106−114. doi:  10.13386/j.issn1002-0306.2021110034
HE Shengfa, LONG Caiyun, WANG Jiao, et al. Purification, Identification and Polyclonal Antibody Development for Cow’s Milk Major Allergen αS1-Casein[J]. Science and Technology of Food Industry, 2022, 43(15): 106−114. (in Chinese with English abstract). doi:  10.13386/j.issn1002-0306.2021110034
Citation: HE Shengfa, LONG Caiyun, WANG Jiao, et al. Purification, Identification and Polyclonal Antibody Development for Cow’s Milk Major Allergen αS1-Casein[J]. Science and Technology of Food Industry, 2022, 43(15): 106−114. (in Chinese with English abstract). doi:  10.13386/j.issn1002-0306.2021110034

牛乳主要过敏原αS1-酪蛋白的纯化鉴定及其多克隆抗体的制备

doi: 10.13386/j.issn1002-0306.2021110034
基金项目: 赣南医学院博士启动课题(QD201809);赣南医学院重点科研课题(ZD201816);南昌大学食品科学与技术国家重点实验室开放课题(SKLF-KF-202003);江西省自然科学基金项目(20202BAB215013);国家自然科学基金项目(32160606)。
详细信息
    作者简介:

    何圣发(1988−),男,博士,讲师,研究方向:食物过敏,E-mail:heshengfa11@hotmail.com

    通讯作者:

    陈红兵(1967–),男,博士,教授,研究方向:食物过敏,E-mail:chenhongbing@ncu.edu.cn

  • 中图分类号: TS201.6

Purification, Identification and Polyclonal Antibody Development for Cow’s Milk Major Allergen αS1-Casein

  • 摘要: 目的:从牛乳酪蛋白中纯化获得αS1-酪蛋白并对其进行综合鉴定,以及制备特异性识别αS1-酪蛋白的兔多克隆抗体。方法:采用DEAE Sepharose Fast Flow阴离子交换层析色谱对αS1-酪蛋白进行分离纯化,利用酪蛋白的理化性质(等电点和含量)、免疫学技术和质谱技术对纯化的αS1-酪蛋白进行综合鉴定,然后通过透析和冷冻干燥获得高纯度的αS1-酪蛋白,最后免疫新西兰大白兔制备多克隆抗体,并分析其特异性。结果:在4种酪蛋白中,αS1-酪蛋白在阴离子交换层析色谱中的出峰时间最晚、峰面积最大,在电泳图中的位置最高,最终获得纯度高达94.26%的αS1-酪蛋白,得率为27.19%。免疫5次后,两只兔子的抗血清效价分别为128万和32万,抗血清除了与大豆蛋白存在轻微交叉反应(<0.25%)外,与α-乳白蛋白、β-乳球蛋白、蛋清蛋白、花生蛋白均无交叉反应,表明特异性高。结论:本研究制备了高纯度的αS1-酪蛋白及其高特异性的多克隆抗体,为过敏原蛋白的纯化与综合鉴定提供了思路,为αS1-酪蛋白免疫学检测方法的建立提供了物质基础。
  • 图  1  牛乳酪蛋白的DEAE Sepharose Fast Flow阴离子交换层析图谱

    Figure  1.  Chromatogram of bovine casein using DEAE Sepharose Fast Flow anion-exchange chromatography

    注:A~D代表4个峰;1~9分别为第21、23、42、48、50、65、67、76、78管样品的位置,用于后续SDS-PAGE分析。

    图  2  DEAE Sepharose Fast Flow阴离子交换层析分离牛乳酪蛋白和牛乳蛋白的SDS-PAGE图

    Figure  2.  SDS-PAGE profile of bovine casein purified by DEAE Sepharose Fast Flow and milk proteins

    注:A:泳道1~9与阴离子交换层析图谱(图1)中所标注的“1~9”相对应;B:泳道1~3分别为脱脂牛乳、牛乳酪蛋白和牛乳α-酪蛋白,a~f分别指示牛乳中不同蛋白质的条带位置。

    图  3  间接ELISA分析阴离子交换层析样品的免疫反应性(n=3)

    Figure  3.  Analysis of immunoreactivity of bovine casein purified by DEAE Sepharose Fast Flow by indirect ELISA(n=3)

    注:样品组与对照组之间的差异显著性以*P<0.05和**P<0.01表示。

    图  4  泳道9(图2A)的二级质谱图及其在αS1-酪蛋白氨基酸序列中的覆盖区域

    Figure  4.  Mass spectra and amino acid sequences in αS1-casein of lane 9 (Fig.2A

    注:A~G为蛋白二级质谱图;H为氨基酸序列覆盖区域(红色)。

    图  5  αS1-酪蛋白透析前后的SDS-PAGE图

    Figure  5.  SDS-PAGE profile of αS1-casein before and after dialysis

    注:A:泳道1~9依次是第75~83管样品;B:泳道1是第78~81管样品冻干后的αS1-酪蛋白。

    图  6  A兔和B兔的血清效价

    Figure  6.  The serum titre of rabbit A and rabbit B

    图  7  pAb-αS1-CN与食物过敏原蛋白的交叉反应(n=3)

    Figure  7.  Cross-reactivity of pAb-αS1-CN with food allergen proteins (n=3)

    表  1  αS1-酪蛋白不同分离纯化方法的对比

    Table  1.   Comparison of different methods for purifying αS1-casein

    纯化方法在酪蛋白SDS-PAGE图中的位置鉴定依据/方法纯度参考文献
    DEAE Sepharose Fast Flow
    阴离子交换层析
    最高,位于35 kDa左右理化性质、免疫学技术、
    质谱技术
    94.26%(αS1-酪蛋白)本研究
    DEAE Sepharose Fast Flow
    阴离子交换层析
    第二高(低于αS2-酪蛋白),
    略低于35 kDa
    SDS-PAGE(分子量)[9]
    DEAE Sepharose Fast Flow
    阴离子交换层析
    第二高(低于β-酪蛋白),
    略低于26 kDa
    SDS-PAGE(分子量)[10]
    等电点沉淀法+HiPrep 16/10
    DEAE-FF阴离子交换层析
    αS1-酪蛋白高于αS2-酪蛋白,
    位于20~31 kDa之间
    理化性质[8]
    Q-Sepharose阴离子交换层析αS1-酪蛋白高于αS2-酪蛋白质谱技术、分子排阻色谱法、远紫外光谱[17]
    TSK-Gel Ether-5PW疏水作用层析有证标准物质[18]
    Q-Sepharose FF阴离子交换层析标准品66.84%
    αS-酪蛋白)
    [12]
    尿素沉淀+Q-Sepharose FF
    阴离子交换层析
    标准品75.4%
    αS-酪蛋白)
    [12]
    膜分离+选择性沉淀法61%
    αS-酪蛋白)
    [32]
    注:“-”表示无相关数据。
    下载: 导出CSV

    表  2  间接ELISA确定αS1-酪蛋白最佳抗原包被浓度和二抗稀释倍数

    Table  2.   Determination of the best antigen coating concentration of αS1-casein and dilution multiple of HRP-IgG by indirect ELISA

    一抗稀释倍数50001万2万4万8万16万50001万2万4万8万16万
    αS1-CN包被浓度(μg/mL)血清类型二抗稀释5000倍二抗稀释1万倍
    4P2.7442.1641.4410.7930.4030.2051.7941.2930.7530.4520.2320.148
    4N0.0750.0590.0540.0480.0490.0490.0560.0520.0490.0510.0590.074
    2P2.7592.0851.3310.7220.3970.2031.7431.2760.7730.4450.2450.165
    2N0.0720.0600.0530.0500.0470.0480.0570.0560.0650.0620.0670.078
    1P2.6091.8851.1940.6820.3120.1801.6271.1680.7290.4160.2430.136
    1N0.0690.0600.0540.0510.0490.0510.0610.0640.0640.0690.0700.075
    0.5P2.2851.4761.0030.4680.2770.1541.3670.8940.5270.3320.1770.127
    0.5N0.0720.0680.0580.0530.0520.0610.0700.0730.0630.0710.0640.079
    注:P为A兔的二免阳性血清,N为A兔的阴性血清。
    下载: 导出CSV
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  • 收稿日期:  2021-11-05
  • 网络出版日期:  2022-06-22
  • 刊出日期:  2022-08-03

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