CN108640986B - 一种鸡蛋清过敏原分离方法 - Google Patents

一种鸡蛋清过敏原分离方法 Download PDF

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CN108640986B
CN108640986B CN201810501671.4A CN201810501671A CN108640986B CN 108640986 B CN108640986 B CN 108640986B CN 201810501671 A CN201810501671 A CN 201810501671A CN 108640986 B CN108640986 B CN 108640986B
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麻小娟
梁汭
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Abstract

本发明公开了一种鸡蛋清过敏原分离方法，属于蛋白分离技术领域，其步骤包含：1）鸡蛋清处理液调pH和离心配制处理，2）层析柱制备，3）层析分离，4）蛋白纯度检测，5）、蛋白质的浓缩。本发明应用阳离子交换层析法，将鸡蛋清处理后上样，通过洗脱液pH值和盐浓度调整，使四种蛋白质依次洗脱下来，经蛋白纯度检测验证，BCA法检测后计算卵白蛋白、卵转铁蛋白、卵类粘蛋白和溶菌酶的提取率分别为60.0%、52.1%、29.6%和90.2%。

Description

一种鸡蛋清过敏原分离方法
技术领域
本发明涉及蛋白质分离技术领域，具体为一种鸡蛋清过敏原分离方法。
背景技术
鸡蛋中的过敏原主要存在于蛋清中，目前公认蛋清中的主要过敏原有4种，分别为卵白蛋白（45kDa），卵类粘蛋白(28kDa)，卵转铁蛋白(76 kDa)和溶菌酶(14kDa)。过敏原标准品使用方便，然而已经证实很多标准品受到细菌内毒素(LPS)污染。LPS能够导致Th1/Th2类细胞因子失衡，因此在建立致敏动物模型或细胞实验时要严格控制过敏原中LPS的含量。分离纯化的过敏原可以不受LPS污染，是用于致敏模型的好选择。
然而目前报道的分离纯化方法一次只能分离一种过敏原，不能同时对鸡蛋清中主要的四种过敏原进行分离。
发明内容
本发明的目的在于克服上述背景技术困难，提供一种将鸡蛋清中四种主要过敏原进行分离的方法。
为达到上述目的，采用的技术方案为：一种鸡蛋清过敏原分离方法，其步骤包含：
1）、鸡蛋清处理液配制：新鲜鸡蛋去壳并分离出蛋清，冰浴中用玻璃棒缓慢搅拌至没有块状物质后，加入等体积蒸馏水并用1M盐酸调pH至6.0，4℃搅拌3h后，3000g离心10分钟；收集上清液，用1M盐酸调pH到3.8，4℃搅拌3h后再次离心，收集上清液于-20℃冻存备用；
2）、层析柱制备：取GE公司的CM-sepharose FF柱材，蒸馏水置换5-6次除去酒精，按常规装柱1.6cm×20cm，最终柱体积为30ml；
3）、层析分离：用20 mmol/L 醋酸-醋酸钠 pH4.0平衡柱子，至紫外监测仪读数稳定；上样5ml鸡蛋清处理液，控制流速在1.5ml/min，待穿过峰洗出后换上含0～0.2mol/LNaCl 醋酸-醋酸钠 pH4.3 洗脱，200min后换成用含0.1～0.2mol/L NaCl 醋酸-醋酸钠pH5.5 洗脱；待315 min时第4峰出峰结束，换上含0～0.4 mol/L NaCl PBS缓冲液 pH7.4洗脱，直至出完最后两峰，分离结束；
4）、蛋白纯度检测：将分离所得蛋白收集，取样10μl与10μl上样缓冲液混合后，取15μl加入聚丙烯酰胺凝胶 SDS-PAGE 胶孔内，电泳后，凝胶经考马斯亮蓝显色进行检测；
5）、蛋白质的浓缩：将收集到的四种蛋白质纯品用PEG浓缩，透析后冻干，于-20℃冻存。
所述聚丙烯酰胺凝胶 SDS-PAGE为12%分离胶，5%浓缩胶。
采用上述方案的有益效果为：这种鸡蛋清过敏原分离方法应用阳离子交换层析法，将鸡蛋清处理后上样，通过洗脱液pH值和盐浓度调整，使四种蛋白质依次洗脱下来，经蛋白纯度检测验证，BCA法检测后计算卵白蛋白、卵转铁蛋白、卵类粘蛋白和溶菌酶的提取率分别为60.0%、52.1%、29.6%和90.2%。
附图说明
图1为本发明实施例中离子交换层析曲线示意图。
图2为本发明实施例中过敏原蛋白SDS-PAGE的分离图。
具体实施方式
下面结合实施例进一步介绍本发明，但本发明不仅限于下述实施例，可以预见本领域技术人员在结合现有技术的情况下，实施情况可能产生种种变化。
一种鸡蛋清过敏原分离方法，其步骤包含：
1.鸡蛋清处理液的制备：新鲜鸡蛋去壳并分离出蛋清，冰浴中用玻璃棒缓慢搅拌至没有块状物质后，加入等体积蒸馏水并用1M盐酸调pH至6.0。4℃搅拌3h后，3000g离心10分钟。上清收集后用1M盐酸调pH到3.8，4℃搅拌3h后离心。上清液于-20℃冻存，即为鸡蛋清处理液。
2.层析柱的制备：将GE公司的CM-sepharose FF柱材取出适量，蒸馏水置换5-6次以除去酒精。按常规装柱(1.6cm×20cm)，最终柱体积约30ml。
3.层析分离：用20mmol/L 醋酸-醋酸钠(pH4.0)平衡柱子，至紫外监测仪读数稳定。上样5ml鸡蛋清处理液。控制流速在1.5ml/min。 待穿过峰洗出后换上含0～0.2mol/LNaCl 醋酸-醋酸钠(pH4.3)洗脱， 200min后换成用含0.1～0.2mol/L NaCl 醋酸-醋酸钠(pH5.5)洗脱。待315min时第4峰出峰结束，换上含0～0.4mol/L NaCl PBS缓冲液(pH7.4)洗脱，直至出完最后两峰，分离纯化结束。
4.蛋白纯度检测：将分离所得蛋白收集，取样10μl与10μl上样缓冲液混合后，取15μl加入聚丙烯酰胺凝胶(SDS-PAGE)胶孔内(15%分离胶，5%浓缩胶)，电泳后，凝胶经考马斯亮蓝显色。
5.蛋白质的浓缩：将收集到的四种蛋白质纯品（卵转铁蛋白的前一个峰纯度不高，只收集第二个峰）用PEG浓缩，透析后冻干。于-20℃冻存。
6.提取率计算：用BCA法测定蛋清处理液中以及四种分离纯化蛋白的蛋白质含量，查阅文献，蛋清中卵白蛋白、卵转铁蛋白、卵类粘蛋白和溶菌酶的质量分数分别为54%、12%、11%和3.4%，再结合上样体积和收集的蛋白液体积，计算出四种主要过敏原的提取率分别为60.0%、52.1%、29.6%和90.2%。
如图1，为实施例中离子交换层析曲线示意图。
如图2为实施例中过敏原蛋白SDS-PAGE的分离图，右图为浓缩后的溶菌酶和卵转铁蛋白。由于卵类黏蛋白含20%-25%的糖基成分，电泳时分子量显示偏大，条带弥散属正常现象。

Claims (2)

1.一种鸡蛋清过敏原分离方法，其特征步骤为：
1）、鸡蛋清处理液配制：新鲜鸡蛋去壳并分离出蛋清，冰浴中用玻璃棒缓慢搅拌至没有块状物质后，加入等体积蒸馏水并用1M盐酸调pH至6.0，4℃搅拌3h后，3000g离心10分钟；收集上清液，用1M盐酸调pH到3.8，4℃搅拌3h后再次离心，收集上清液于-20℃冻存备用；
2）、层析柱制备：取GE公司的CM-sepharose FF柱材，蒸馏水置换5-6次除去酒精，按常规装柱1.6cm×20cm，最终柱体积为30ml；
3）、层析分离：用20mmol/L 醋酸-醋酸钠pH4.0平衡柱子，至紫外监测仪读数稳定；上样5ml鸡蛋清处理液，控制流速在1.5ml/min，待穿过峰洗出后换上含0～0.2mol/L NaCl 醋酸-醋酸钠pH4.3梯度洗脱，200min后换成用含0.1～0.2mol/L NaCl 醋酸-醋酸钠pH5.5 梯度洗脱；待315min时第4峰出峰结束，换上含0～0.4mol/L NaCl PBS缓冲液pH7.4 梯度洗脱，直至出完最后两峰，分离结束；
4）、蛋白纯度检测：将分离所得蛋白收集，取样10μl与10μl上样缓冲液混合后，取15μl加入聚丙烯酰胺凝胶SDS-PAGE胶孔内，电泳后，凝胶经考马斯亮蓝显色进行检测；
5）、蛋白质的浓缩：将收集到的四种蛋白质纯品用PEG浓缩，透析后冻干，于-20℃冻存。
2.根据权利要求1所述的鸡蛋清过敏原分离方法，其特征是：所述步骤4）中，聚丙烯酰胺凝胶SDS-PAGE为12%分离胶，5%浓缩胶。
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