CN114591421B - 一种鉴别驯鹿源的特征多肽及其应用 - Google Patents
一种鉴别驯鹿源的特征多肽及其应用 Download PDFInfo
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Abstract
本发明属于生物技术领域，具体涉及一种鉴别驯鹿的特征多肽及其应用。本发明提供的特征多肽具体为：肽段1如SEQ ID No.1所示：MLTSEEK。本发明提供的特征多肽针对驯鹿具有优异的专属性和稳定性，特异性强，可用于驯鹿及其衍生产品鹿茸、鹿角药材及饮片、鹿皮、鹿血、鹿鞭、鹿筋的掺伪驯鹿或伪品鉴别，具有良好的应用前景。
Description
技术领域
本发明属于生物技术领域，具体涉及一种鉴别驯鹿源的特征多肽及其应用。
背景技术
鹿科动物具有很高的经济价值，其衍生产品为鹿茸、鹿角、鹿皮、鹿鞭、鹿肉、鹿血、鹿筋等。驯鹿广泛分布于欧亚及北美大陆的苔原、山地和林区，现有9个亚种，总数大约在400万头，数量上远远超过稀有的马鹿和梅花鹿。市场上常有使用驯鹿产品伪造梅花鹿或马鹿进行售卖。尤其因驯鹿的公鹿及母鹿均可长角，产量大易取得，相比于资源紧张的梅花鹿茸及马鹿茸，成本低廉。市场上有使用驯鹿茸切片泡血后冒充马鹿茸、梅花鹿茸进行售卖。而鹿皮、鹿鞭、鹿肉、鹿血、鹿筋等由于外观相似，主要成分也几乎相同，现有的鉴别方法无法分辨种属。
发明内容
针对现有技术中存在的技术空白，本发明提供了一种鉴别驯鹿源的特征多肽。
本发明还提供了上述特征多肽在鉴别驯鹿种属鉴别中的应用。
本发明为了实现上述目的所采用的技术方案为：
本发明提供了一种鉴别驯鹿鹿源的特征多肽，所述特征多肽具体为：肽段1如SEQID No.1所示：MLTSEEK。
本发明所发现的特征多肽肽段1存在于驯鹿血β珠蛋白中。
进一步的，所述SEQ ID No.1肽段1的质荷比为419.20（z=2）。
本发明还提供了一种用于鉴别驯鹿源的试剂盒，所述试剂盒中包含权利要求1所述的特征多肽。
本发明还提供了一种利用上述特征多肽鉴别驯鹿源的方法，包括以下步骤：
（1）对待测样品进行前处理，得待检测溶液；
（2）将待检测溶液通过液相-三重四级杆质谱法进行检测，分析对比待检测溶液中的质谱结果同肽段1的质谱图，当质谱图中出现肽段1的质谱图时，可判断待测样品为驯鹿或掺伪驯鹿。
进一步的，所述待测样品的前处理具体过程为：样品粉碎，称取样品50mg，加入10ml变性缓冲液，和1ml DTT溶液，摇匀，置80℃处理过夜，取出放冷至室温，离心，量取上清液500μL，加入100μL IAA溶液，避光反应30min，混匀，离心；对样品溶液脱盐后，量取上清液100μL，加入900μL碳酸氢铵溶液（1%）和10μL牛胰蛋白酶溶液，37℃酶解4h，取出放冷至室温，离心，取上清液，即得。
上述变性缓冲液具体制备过程为：称取573.1g 盐酸胍，121.1g 三羟甲基氨基甲烷，0.734g乙二胺四乙酸，加水溶解，加浓盐酸调pH至8.0，加水稀释至1L，摇匀，即得；所述DTT溶液的浓度为2.5M；所述IAA溶液的浓度为2M。
进一步的，所述牛胰蛋白酶溶液的具体制备过程为：液称取牛胰蛋白酶适量，用乙酸溶液溶解，制成浓度为10mg/ml的溶液，临用现配；所述乙酸溶液的浓度为2%。
本发明所使用的液相-三重四级杆质谱法中，液相条件为：色谱柱为ACQUITY UPLC® BEH C18 (2.1×50mm，1.7μm)，柱温43℃，流速0.3mL/min，流动相A为0.1%甲酸溶液，B为0.1%甲酸乙腈溶液，进行梯度洗脱。
上述梯度洗脱的条件为：0～9min,3%B→7.5%B；9～13min, 7.5%B→25%B；13～14min,25%B→90%B；14～17min, 90%B；17～17.5min, 10%B -97%B；17.5～21min,97%B，进样量为5μL。
本发明所使用的液相-三重四级杆质谱法中，质谱条件：采用质谱检测器，电喷雾离子化（ESI），正离子模式下，进行多反应监测；鞘气流速46L/hr；辅助气流速850 L/hr；喷雾电压3.5KV；离子源温度150℃；辅助气温度400℃；锥孔电压30V，碰撞电压35V)；溶剂延迟（solvent delay）为0～8min和14～20min；选择m/z（二电荷）419.20→258.1、419.20→217.1作为检测离子对。
本发明的有益效果为：
（1）本发明提供的特征多肽及检测方法，为掺伪驯鹿或使用驯鹿代替梅花鹿、马鹿产品的检查提供了解决方案。
（2）本发明提供的特征多肽针对鹿茸、鹿角、鹿皮、鹿鞭、鹿肉、鹿血、鹿筋等具有优异的专属性和稳定性，特异性强，具有良好的应用前景。
附图说明
图1为m/z（二电荷）419.20的二级质谱图及y离子、b离子归属。
图2 为 m/z（二电荷）419.20的专属性实验结果。
图3 为 MLTSEEK的BLAST(Basic Local Alignment Search Tool)结果。
具体实施方式
下面通过具体的实施方式对本发明的技术方案作进一步的解释和说明。
（一）仪器和试剂：Thermo EASY-nLC 1000纳升液相，Thermo ScientificOrbitrap-Fusion高分辨质谱，AB Triple Quad 6500+高效液相色谱质谱联用仪，赛多利斯XSE205电子天平，胰蛋白酶（Sigma公司生产，批号：SLBG6452V）盐酸胍、三羟甲基氨基甲烷（Tris）、乙二胺四乙酸(EDTA)、二硫苏糖醇（DTT）、碘代乙酰胺（IAA）、碳酸氢铵、乙酸均为分析纯。甲酸、乙腈均为色谱纯。
用于进行实验的为鹿茸、鹿角样品，由企业提供，经山东省农业科学院PCR实验检定鹿源。
实施例1
1、测定条件
1.1 液相-三重四极杆质谱测定条件
液相条件：色谱柱为ACQUITY UPLC® BEH C18 (2.1×50mm，1.7μm)，柱温43℃，流速0.3mL/min，流动相A为0.1%甲酸溶液，B为0.1%甲酸乙腈溶液，进行梯度洗脱。（0～9min,3%B→7.5%B；9～13min, 7.5%B→25%B；13～14min,25%B→90%B；14～17min, 90%B；17～17.5min, 10%B -97%B,17.5～21min,97%B）进样量为5 。质谱条件：采用质谱检测器，电喷雾离子化（ESI），正离子模式下，进行多反应监测；鞘气流速46L/hr；辅助气流速850 L/hr；喷雾电压3.5KV；离子源温度150℃；辅助气温度400℃。锥孔电压30V，碰撞电压35V)。溶剂延迟（solvent delay）为0～8min和14～20min。选择m/z（二电荷）419.20→258.1、419.20→217.1作为检测离子对。
、供试品溶液的制备
样品：使用鹿茸样品进行测定。首先样品粉碎，称取样品50mg，加入10ml变性缓冲液（称取573.1g 盐酸胍，121.1g 三羟甲基氨基甲烷，0.734g乙二胺四乙酸，加水溶解，加浓盐酸调pH至8.0，加水稀释至1L，摇匀，即得）。和1ml DTT溶液，摇匀，置80℃处理过夜，取出放冷至室温，离心（12000rpm，10min）。量取上清液500μL，加入100μL IAA溶液，避光反应30min，混匀，离心（12000rpm，10min）；对样品溶液脱盐后，量取上清液100μL，加入900μL碳酸氢铵溶液（1%）和10μL牛胰蛋白酶溶（液称取牛胰蛋白酶适量，用乙酸溶液溶解，制成浓度为10mg/ml的溶液，临用现配），37℃酶解4h，取出放冷至室温，离心（12000rpm，10min），取上清液，即得。实验结果见表1，结果图片见图1。
表1 专属性实验结果
由于找到的特征肽段归属于鹿血中β珠蛋白，故该特征肽段可以应用于鉴别驯鹿茸、鹿角、鹿皮、鹿鞭、鹿肉、鹿血、鹿筋等含血量丰富的鹿产品中。
对比例1
使用课题组前期探索的离子对(m/z)553.29（二电荷）→285.16、(m/z)553.29（二电荷）→329.19对鹿茸样品进行测定，发现除了驼鹿出现吸收峰外，马鹿于梅花鹿中也出现吸收峰。
<110>山东省食品药品检验研究院
<120>一种鉴别驯鹿源的特征多肽及其应用
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<210> 1
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Claims (8)
1.一种鉴别驯鹿的特征多肽，其特征在于，所述特征多肽具体为：如SEQ ID No.1所示的肽段1：MLTSEEK。
2.一种用于鉴别驯鹿的试剂盒，其特征在于，所述试剂盒中包含权利要求1所述的特征多肽。
3.一种利用权利要求1所述的特征多肽鉴别驯鹿的方法，其特征在于，包括以下步骤：
（1）样品粉碎，称取样品50mg，加入10ml变性缓冲液，和1ml DTT溶液，摇匀，置80℃处理过夜，取出放冷至室温，离心，量取上清液500μL，加入100μL IAA溶液，避光反应30min，混匀，离心；对样品溶液脱盐后，量取上清液100μL，加入900μL，浓度为1%的碳酸氢铵溶液和10μL牛胰蛋白酶溶液，37℃酶解4h，取出放冷至室温，离心，取上清液，即得；
（2）将待检测溶液通过液相-三重四级杆质谱法进行检测，分析对比待检测溶液中的质谱结果同肽段1的质谱图，当质谱图中出现肽段1的质谱图时，可判断待测样品中含有驯鹿源成分。
4.根据权利要求3所述的方法，其特征在于，所述变性缓冲液具体制备过程为：称取573.1g 盐酸胍，121.1g 三羟甲基氨基甲烷，0.734g乙二胺四乙酸，加水溶解，加浓盐酸调pH至8.0，加水稀释至1L，摇匀，即得；所述DTT溶液的浓度为2.5M；所述IAA溶液的浓度为2M。
5.根据权利要求4所述的方法，其特征在于，所述牛胰蛋白酶溶液的具体制备过程为：称取牛胰蛋白酶适量，用乙酸溶液溶解，制成浓度为10mg/ml的溶液，临用现配；所述乙酸溶液的浓度为2%。
6.根据权利要求3-5任一项所述的方法，其特征在于，所述液相-三重四级杆质谱法中，液相条件为：色谱柱为2.1×50mm，1.7μm 的ACQUITY UPLC® BEH C18，柱温43℃，流速0.3mL/min，流动相A为0.1%甲酸溶液，B为0.1%甲酸乙腈溶液，进行梯度洗脱。
7.根据权利要求6所述的方法，所述梯度洗脱的条件为：0~9min,3%B→7.5%B；9~13min,7.5%B→25%B；13~14min,25%B→90%B；14~17min, 90%B；17~17.5min,90%B -97%B；17.5~21min,97%B，进样量为5 μl。
8.根据权利要求7所述的方法，其特征在于，所述液相-三重四级杆质谱法中，质谱条件：采用质谱检测器，电喷雾离子化ESI，正离子模式下，进行多反应监测；鞘气流速46L/hr；辅助气流速850 L/hr；喷雾电压3.5KV；离子源温度150℃；辅助气温度400℃；锥孔电压30V，碰撞电压35V；溶剂延迟为0～8min和14～20min；选择m/z（二电荷）419.20→258.1、419.20→217.1作为检测离子对。
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