牛乳主要过敏原<i>α</i><sub>S1</sub>-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

doi:10.13386/j.issn1002-0306.2021110034

牛乳主要过敏原α_S1-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

何圣发^{1, 2,},
龙彩云³,
王娇¹,
熊梦¹,
赵江强¹,
燕燕¹,
董亚萍¹,
李欣^{2, 4},
陈红兵^{2, 5, ,}

1.
赣南医学院公共卫生与健康管理学院，江西赣州 341000
2.
南昌大学食品科学与技术国家重点实验室，江西南昌 330047
3.
赣州市疾病预防控制中心，江西赣州 341000
4.
南昌大学食品学院，江西南昌 330047
5.
南昌大学中德联合研究院，江西南昌 330047

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
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出版历程
- 收稿日期: 2021-11-05
- 网络出版日期: 2022-06-22
- 刊出日期: 2022-08-03

Purification, Identification and Polyclonal Antibody Development for Cow’s Milk Major Allergen α_S1-Casein

HE Shengfa^{1, 2
,},
LONG Caiyun³,
WANG Jiao¹,
XIONG Meng¹,
ZHAO Jiangqiang¹,
YAN Yan¹,
DONG Yaping¹,
LI Xin^{2, 4},
CHEN Hongbing^{2, 5
, ,}

1.
School of Public Health and Health Management, Gannan Medical University, Ganzhou 341000, China
2.
State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China
3.
Ganzhou Center for Disease Control and Prevention, Ganzhou 341000, China
4.
School of Food Science & Technology, Nanchang University, Nanchang 330047, China
5.
Jiangxi-OAI Joint Research Institute, Nanchang University, Nanchang 330047, China

摘要

摘要: 目的：从牛乳酪蛋白中纯化获得α_S1-酪蛋白并对其进行综合鉴定，以及制备特异性识别α_S1-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α_S1-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α_S1-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α_S1-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，α_S1-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α_S1-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α_S1-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α_S1-酪蛋白免疫学检测方法的建立提供了物质基础。
- 牛乳过敏原 /
- α_S1-酪蛋白 /
- 纯化 /
- 鉴定 /
- 多克隆抗体
Abstract: Objective: To purify and comprehensive identify α_S1-casein from bovine caseins and prepare rabbit polyclonal antibody against α_S1-casein. Method: DEAE Sepharose Fast Flow anion exchange chromatography was used to separate and purify α_S1-casein. The physicochemical property (isoelectric point and protein content), immunological technique and mass spectrometry were used to comprehensively identify α_S1-casein. Then the α_S1-casein was obtained by dialysis and lyophilization, polyclonal antibody was prepared by immunizing New Zealand white rabbit with the purified α_S1-casein, and the specificity of polyclonal antibody was analyzed. Result: Among the four kinds of caseins, α_S1-casein had the latest peak time, the largest peak area in anion exchange chromatography and the highest position in the electrophoretic diagram. Finally, the purity of α_S1-casein was 94.26%, and the yield was 27.19%. The titers of sera from two rabbits inoculated with purified α_S1-casein for five times reached 1280000 and 320000, respectively. In addition to the slight cross reaction with soybean protein (<0.25%), the antiserum showed no cross reaction with α-Lactalbumin, β-Lactoglobulin, egg white protein and peanut protein, indicating high specificity. Conclusion: This study prepared high purity α_S1-casein and high specificity polyclonal antibody against α_S1-casein, providing a new train of thought for α_S1-casein purification and comprehensive identification, and also providing material basis for the development of immunologic method for detection of α_S1-casein.
- cow’s milk allergen /
- α_S1-casein /
- purification /
- identification /
- polyclonal antibody

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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。

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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。

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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。

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图 4 泳道9（图2A）的二级质谱图及其在α_S1-酪蛋白氨基酸序列中的覆盖区域

Figure 4. Mass spectra and amino acid sequences in α_S1-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）。

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图 5 α_S1-酪蛋白透析前后的SDS-PAGE图

Figure 5. SDS-PAGE profile of α_S1-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α_S1-酪蛋白。

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图 6 A兔和B兔的血清效价

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

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图 7 pAb-α_S1-CN与食物过敏原蛋白的交叉反应（n=3）

Figure 7. Cross-reactivity of pAb-α_S1-CN with food allergen proteins (n=3)

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表 1 α_S1-酪蛋白不同分离纯化方法的对比

Table 1. Comparison of different methods for purifying α_S1-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%（α_S1-酪蛋白）	本研究
DEAE Sepharose Fast Flow 阴离子交换层析	第二高（低于α_S2-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	α_S1-酪蛋白高于α_S2-酪蛋白，位于20~31 kDa之间	理化性质	－	[8]
Q-Sepharose阴离子交换层析	α_S1-酪蛋白高于α_S2-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% （α_S-酪蛋白）	[12]
尿素沉淀+Q-Sepharose FF 阴离子交换层析	－	标准品	75.4% （α_S-酪蛋白）	[12]
膜分离+选择性沉淀法	－	－	61% （α_S-酪蛋白）	[32]
注：“－”表示无相关数据。

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表 2 间接ELISA确定α_S1-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 α_S1-casein and dilution multiple of HRP-IgG by indirect ELISA

一抗稀释倍数		5000	1万	2万	4万	8万	16万	5000	1万	2万	4万	8万	16万
α_S1-CN包被浓度（μg/mL）	血清类型	二抗稀释5000倍						二抗稀释1万倍
4	P	2.744	2.164	1.441	0.793	0.403	0.205	1.794	1.293	0.753	0.452	0.232	0.148
4	N	0.075	0.059	0.054	0.048	0.049	0.049	0.056	0.052	0.049	0.051	0.059	0.074
2	P	2.759	2.085	1.331	0.722	0.397	0.203	1.743	1.276	0.773	0.445	0.245	0.165
2	N	0.072	0.060	0.053	0.050	0.047	0.048	0.057	0.056	0.065	0.062	0.067	0.078
1	P	2.609	1.885	1.194	0.682	0.312	0.180	1.627	1.168	0.729	0.416	0.243	0.136
1	N	0.069	0.060	0.054	0.051	0.049	0.051	0.061	0.064	0.064	0.069	0.070	0.075
0.5	P	2.285	1.476	1.003	0.468	0.277	0.154	1.367	0.894	0.527	0.332	0.177	0.127
0.5	N	0.072	0.068	0.058	0.053	0.052	0.061	0.070	0.073	0.063	0.071	0.064	0.079
注：P为A兔的二免阳性血清，N为A兔的阴性血清。

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