CN115322251A - 一种促进成骨细胞矿化的牛骨胶原肽及其应用 - Google Patents
一种促进成骨细胞矿化的牛骨胶原肽及其应用 Download PDFInfo
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Abstract
本发明公开了一种促进成骨细胞矿化的牛骨胶原肽及其应用。本发明所述牛骨胶原肽的氨基酸序列如SEQ ID NO:1‑SEQ ID NO:6所示。本发明提供的牛骨胶原肽可以有效促进成骨细胞矿化，预防和/或治疗骨质疏松，减轻骨质疏松患者的痛苦，且这些多肽具有高效低毒的特点，可以开发成药物或食品。
Description
本申请是本申请人于2021年03月23日提交的，申请号为202110308414.0，发明名称为“一种促进成骨细胞矿化的牛骨胶原肽及其应用”的发明申请的分案申请。
技术领域
本发明涉及活性肽技术领域。更具体地，涉及一种促进成骨细胞矿化的牛骨胶原肽及其应用。
背景技术
骨重塑一直贯穿人的一生。骨重塑主要包括骨形成和骨吸收两个过程。一旦骨形成的速度小于骨吸收的速度，就可能会导致骨质疏松的发生。骨质疏松是一种以骨密度减少为特征的一种全身性疾病。骨的矿化过程可以对骨密度的多少产生直接的影响。因此，寻找高效的有利于骨矿化过程的多肽对于抗骨质疏松药物或者食品的开发有着非常重要的意义。
发明内容
本发明的第一个目的在于提供活性高、毒性小的促进成骨细胞矿化的牛骨胶原肽。
本发明的第二个目的在于提供上述牛骨胶原肽在促进成骨细胞矿化，预防和/治疗骨质疏松方面的应用。
为达到上述目的，本发明采用下述技术方案：
根据本发明的第一个目的，本发明提供一种牛骨胶原肽，所述牛骨胶原肽的氨基酸序列包括如下序列中的一种或多种：
LPGIDGRPGPIGPAG，其中，2位脯氨酸被羟基化修饰形成羟脯氨酸，如SEQ ID NO:1所示；
GLTGPIGPPGPAG，其中，9位脯氨酸被羟基化修饰形成羟脯氨酸，如SEQ ID NO:2所示；
LPGIDGRPGPIGPAG，其中，2位和8位的脯氨酸被羟基化修饰形成羟脯氨酸，如SEQID NO:3所示；
APGPVGPVG，其中，2位脯氨酸被羟基化修饰形成羟脯氨酸，如SEQ ID NO:4所示；
GPAGPSGPAG，如SEQ ID NO:5所示；
GAPGSPGPAG，如SEQ ID NO:6所示。
可选的，所述牛骨胶原肽还可以是与上述任一牛骨胶原肽的氨基酸序列具有80％及以上同源性的活性肽，该牛骨胶原肽和上述牛骨胶原肽的功能相同或相似。例如，所述牛骨胶原肽的氨基酸序列可以是与上述任一牛骨胶原肽的氨基酸序列具有85％，90％，95％或97％同源性的序列。
本发明还提供了编码上述牛骨胶原肽的多核苷酸。
在已知牛骨胶原肽的氨基酸序列的情况下，本领域技术人员可以根据对于牛骨胶原肽表达的需要，基于密码子的简并性原则和不同物种对于密码子的使用偏好性，设计具有不同核苷酸序列的牛骨胶原肽的编码基因。
在本发明中，示例性的，上述牛骨胶原肽的核苷酸序列如序列表SEQ ID NO:7-SEQID NO:12所示。
进一步的，本发明提供一种融合多肽，所述融合多肽包含上述的牛骨胶原肽中的一种。所述的融合多肽可以是通过化学结合、酶切或者物理断裂方法获得。例如将多肽与卵清白蛋白或者牛血清白蛋白形成的蛋白复合体。
本发明的牛骨胶原肽可以从牛骨胶原蛋白酶解产物中经过分离纯化获得，也可以通过本领域已知的其他方法制备得到，例如固相合成的方法、生物工程的方法等等。
根据本发明的第二个目的，本发明提供所述牛骨胶原肽在促进成骨细胞矿化的药物或食品中的应用。进而提供所述牛骨胶原肽在制备预防和/或治疗骨质疏松的药物或食品中的应用。
本发明进一步涉及一种药物或一种食品，其中含有本发明的牛骨胶原肽，例如LPGIDGRPGPIGPAG(2位脯氨酸被羟基化修饰形成羟脯氨酸)，GLTGPIGPPGPAG(9位脯氨酸被羟基化修饰形成羟脯氨酸)，LPGIDGRPGPIGPAG(2位和8位的脯氨酸被羟基化修饰形成羟脯氨酸)，APGPVGPVG(2位脯氨酸被羟基化修饰形成羟脯氨酸)，GPAGPSGPAG，GAPGSPGPAG中的一种或多种。
本发明的药物可以制成本领域常见的各种形式，包括但不限于，适于口服的散剂、片剂(包括各种包衣片剂、缓释或控释片剂)、锭剂、胶囊剂(包括软胶囊和硬胶囊)、颗粒剂、丸剂、可分散粉末、水性或油性混悬剂、水性或油性溶液剂、乳剂、酏剂、糖浆剂等等，适于局部使用的霜剂、软膏剂、凝胶、水性或油性溶液剂、水性或油性混悬剂等等；适于吸入使用的粉末或液体气雾剂；适于经胃肠外给药的静脉内、皮下或肌内注射用无菌水性或油性的注射剂或冻干粉针剂、栓剂等等。
本发明的药物中可以进一步含有常规的各种辅料和/或其他活性成分。合适的辅料包括但不限于赋形剂、润滑剂、粘合剂、崩解剂、水溶性聚合物、无机盐、溶剂、溶解助剂、悬浮剂、等渗剂、缓冲液、防腐剂、抗氧剂、着色剂、甜味剂、酸味剂、起泡剂和调味剂等等。
本发明的有益效果如下：
本发明以促进成骨细胞矿化为活性筛选指标，依次采用反相硅胶柱层析、离子交换柱层析、凝胶柱层析，最终筛选到活性最强的组分，并采用高效液相色谱质谱联用仪对活性最强的组分进行氨基酸序列的鉴定。本发明获得牛骨胶原肽可以有效促进成骨细胞矿化，预防和/或治疗骨质疏松，减轻骨质疏松患者的痛苦，这些多肽具有高效低毒的特点。
附图说明
下面结合附图对本发明的具体实施方式作进一步详细的说明。
图1示出超滤组分CP1的快速反相柱层析色谱图。
图2示出超滤组分经过快速反相硅胶柱层析得到的6个组分的成骨细胞矿化活性测试结果。
图3示出快速反相硅胶柱层析组分CP1-5离子交换色谱图
图4示出离子交换柱层析组分CP1-5-1凝胶柱层析色谱图
图5示出离子交换柱层析组分CP1-5-2凝胶柱层析色谱图
图6示出CP1-5-1和CP1-5-2组分经过凝胶柱层析得到的4个组分的成骨细胞矿化活性测试结果。
图7示出本发明6种牛骨胶原肽的成骨细胞矿化沉积测试结果。
图8示出本发明6种牛骨胶原肽的成骨细胞上清骨钙素含量测试结果。
具体实施方式
为了更清楚地说明本发明，下面结合优选实施例和附图对本发明做进一步的说明。本领域技术人员应当理解，下面所具体描述的内容是说明性的而非限制性的，不应以此限制本发明的保护范围。
实施例1牛骨胶原肽制备
依据中国专利申请CN 105586379 A“一种具有抑制癌细胞增殖作用的胶原蛋白活性肽的制备方法”公开的内容，获得超滤组分CP1，即完成该专利申请中所公开的制备方法的步骤(1)-(8)。
反相硅胶柱层析：将超滤组分CP1样品溶解于1mL超纯水中，配制成10mg/mL溶液。离心并用0.22μm的一次性滤膜进行过滤。使用ODS-AQ反相硅胶柱(50μm,2.5×100mm)对生物活性肽进行分离纯化，其中，上样量为1mL，洗脱液为：超纯水(A液)和色谱乙醇(B液)。流速为：5mL/min。洗脱梯度为：0-10min，100％A；10-30min，96％A；30-50min，90％A；50-70min，87％A；70-75min，83％A；75-85min，78％A；85-97min，0％A。检测波长为220nm,共分离得到6个组分(CP1-1-CP1-6)并进行冷冻干燥，放-80℃低温冰箱保存待用，结果如图1所示。对6个组分进行成骨细胞矿化活性测试，结果如图2所示CP1-5可以显著的促进成骨细胞矿化，遂选择该组分进行进一步纯化。
离子交换柱柱层析：将反相柱层析得到的活性最强的组分(CP1-5)5mg溶解于100μL超纯水中，离心并用0.22μm的一次性滤膜进行过滤。使用离子交换柱对活性肽进行分离纯化，上样量为100μL，洗脱液为：超纯水(A液)和1M氯化钠溶液(B液)。流速为：1mL/min。洗脱梯度为：0-100％B液(0-1M)，60min。检测波长为220nm，共分离得到2个组分(CP1-5-1和CP1-5-2)并进行冷冻干燥放-80℃低温冰箱保存待用，结果如图3所示。
凝胶柱层析：将离子交换柱层析得到的组分CP1-5-1和CP1-5-2约2mg分别溶解于100μL超纯水中，离心并用0.22μm的一次性滤膜进行过滤，使用Sephadex G-15凝胶柱对活性肽进行分离纯化。上样量为100μL。洗脱液为：超纯水。流速为：0.8mLl/min。色谱条件为：100％超纯水洗脱50min。检测波长为220nm，共分离得到4个组分(CP1-5-1-A和CP1-5-1-B；CP1-5-2-A和CP1-5-2-B)并进行冷冻干燥，放-80℃低温冰箱保存待用，结果如图4和图5所示。对4个组分进行成骨细胞矿化活性测试，结果如图6所示CP1-5-2-B可以显著的促进成骨细胞矿化。
生物活性氨基酸序列的鉴定
组分CP1-5-2-B具有最强的生物学活性，遂对该组分进行LC-MS-MS液质联用分析，并用蛋白质组软件Peaks Studio进行数据分析。共鉴定到6条活性肽(牛骨胶原肽)，结果见表1。
表1从牛骨胶原蛋白酶解液中鉴定到的牛骨胶原肽序列
| 编号 | 牛股胶原肽序列 | 质荷比 | 理论分子量 | 长度(氨基酸) | 母体蛋白 |
| 1 | L<u>P</u>GIDGRPGPIGPAG | 695.3779 | 1388.7411 | 15 | Collagen alpha-2(I)chain |
| 2 | GLTGPIGP<u>P</u>GPAG | 553.796 | 1105.5768 | 13 | Collagen alpha-1(I)chain |
| 3 | L<u>P</u>GIDGR<u>P</u>GPIGPAG | 703.3752 | 1404.7361 | 15 | Collagen alpha-2(I)chain |
| 4 | A<u>P</u>GPVGPVG | 738.3782 | 737.3708 | 9 | Collagen alpha-1(I)chain |
| 5 | GPAGPSGPAG | 767.3674 | 766.3607 | 10 | Collagen alpha-2(I)chain |
| 6 | GAPGSPGPAG | 767.3674 | 766.3607 | 10 | Collagen alpha-1(Ill)chain |
P:表示羟基化修饰
实施例2
基于实施例1鉴定得到的6种牛骨胶原肽的氨基酸序列，通过固相合成方法获得实验样品，进行成骨细胞矿化活性验证。如图7、图8所示，6种牛骨胶原肽(编号1-6)均可以促进成骨细胞矿化沉积形成并可以提高成骨细胞骨钙素的含量，说明6种胶原蛋白肽均可以促进成骨细胞矿化。固相合成肽公司：吉尔生化(上海)有限公司。
细胞矿化沉积的检测：
首先以每孔50-100个小鼠MT3T3-E1接种24孔板中，24小时待细胞贴壁后，吸去培养液，加入6种多肽(浓度为0,05mg/mL)和三种成骨诱导剂(地塞米松10nmol，β-甘油磷酸钠10mmol,抗坏血酸5μg/ml)，并设置空白对照组,每组设置三个复孔，之后放入37℃、5％CO2浓度的恒温恒湿的细胞培养箱中培养21天。21天之后，弃去培养基，并用PBS清洗两遍。之后加入多聚甲醛固定液固定30min，用去离子水冲洗一次，加入1mL 0.2％茜素红S染色液，染色3min。之后用去离子水冲洗2次，以终止染色，用4x、10x显微镜进行拍照，拍照完毕之后，用image pro plus 6.0进行统计学分析。
细胞上清液骨钙素的检测：
将小鼠胚胎成骨细胞前体细胞MC3T3-E1接种于24孔细胞培养板中，过夜，然后弃去培养基，加入浓度为0.05mg/mL的多肽溶液培养21天，每三天换一次液。培养结束后，收集培养基，其骨钙素含量由放射免疫试剂盒进行检测。在4℃，样品中的骨钙素或者标准品和碘同位素标记的骨钙素和同一抗体一起孵育。之后分离试剂被加入以分离游离态和结合态的骨钙素。尔后测定结合态的放射性并计算结合率，用标准品制作标准曲线，根据标准曲线，以得到样品中骨钙素含量。
显然，本发明的上述实施例仅仅是为清楚地说明本发明所作的举例，而并非是对本发明的实施方式的限定，对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动，这里无法对所有的实施方式予以穷举，凡是属于本发明的技术方案所引伸出的显而易见的变化或变动仍处于本发明的保护范围之列。
SEQUENCE LISTING
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Claims (9)
1.一种牛骨胶原肽，其特征在于，所述牛骨胶原肽的氨基酸序列为APGPVGPVG；其中，2位脯氨酸被羟基化修饰形成羟脯氨酸。
2.一种多核苷酸，其特征在于，编码权利要求1所述的任一牛骨胶原肽。
3.一种融合多肽，其特征在于，其包含权利要求1所述的牛骨胶原肽。
4.根据权利要求3所述的融合多肽，其特征在于，所述的融合多肽是将权利要求1所述的牛骨胶原肽与载体蛋白连接形成的蛋白复合体。
5.根据权利要求4所述的融合多肽，其特征在于，所述载体蛋白为卵清白蛋白或牛血清白蛋白。
6.权利要求1所述的牛骨胶原肽在制备促进成骨细胞矿化的药物或食品中的应用。
7.权利要求1所述的牛骨胶原肽在制备预防和/或治疗骨质疏松的药物或食品中的应用。
8.一种药物，其特征在于，包含权利要求1所述的牛骨胶原肽。
9.一种食品，其特征在于，包含权利要求1所述的牛骨胶原肽。
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