CN110467667A - 一种抗肿瘤活性肽及其应用 - Google Patents
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Abstract
本发明公开一种抗肿瘤活性肽及其应用。本发明所述活性肽的氨基酸序列如SEQ ID NO:1‑SEQ ID NO:5所示。本发明提供的活性肽具有较好的抗肿瘤活性,尤其可以显著抑制卵巢癌细胞的增殖,可用于抗肿瘤药物和保健品的开发。
Description
技术领域
本发明涉及活性肽技术领域。更具体地,涉及一种抗肿瘤活性肽及其应用。
背景技术
胶原蛋白属于结构蛋白,广泛存在于真皮、骨、鳞、鳔、肌肉、软骨等结缔组织中。同时也是哺乳动物体内含量最多,分布最广的功能性蛋白。胶原蛋白种类繁多,以Ⅰ型、Ⅱ型、Ⅲ型、Ⅳ型、Ⅺ型较为常见。胶原蛋白具有良好的生物相容性,可生物降解性,并具有多种生物活性,比如抗癌、抗氧化、增强免疫、调节骨代谢等,因此广泛应用于食品、医药、组织工程等领域。
生物活性肽是指对生物机体的生命活动有益或具有生理作用的肽类化合物。活性肽的结构可以从较小的二肽到较大分子的多肽。而且生物活性肽来源非常广泛,包括动物、植物、微生物等。生物活性肽制备方法包括酸法、碱法和酶法,其中以酶法制备生物活性肽较为普遍,目前,市面上很多骨胶原蛋白来源的活性肽多为骨胶原蛋白的酶解产物,而没有单一纯化后的活性肽产品,产品成分不明确,在后期药物和保健品开发过程会遇到各种问题。
发明内容
本发明的第一个目的在于提供牛骨胶原蛋白来源的活性肽。
本发明的第二个目的在于提供上述活性肽在抗肿瘤方面的应用。
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,本发明提供一种活性肽,所述活性肽的氨基酸序列包括如下序列中的一种或多种:
DAGPVG,如SEQ ID NO:1所示;
ERGEEGPAG,如SEQ ID NO:2所示;
HGDRGELGPAG,如SEQ ID NO:3所示;
ADGDRGEAGPAGPAGPAGPR,如SEQ ID NO:4所示;
GPAGPEGPRGDKGETGEEGDR,如SEQ ID NO:5所示。
可选的,所述活性肽还可以是与上述任一活性肽的氨基酸序列具有80%及以上同源性的活性肽,该活性肽和上述活性肽的功能相同或相似。例如,所述活性肽的氨基酸序列可以是与上述任一活性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活性肽的多核苷酸。
在已知活性肽的氨基酸序列的情况下,本领域技术人员可以根据对于活性肽表达的需要,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活性肽的编码基因。
在本发明中,示例性的,上述活性肽的核苷酸序列如序列表SEQ ID NO:6-SEQ IDNO:10所示。
本发明的活性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,本发明提供所述活性肽在制备抗肿瘤药物或保健品中的应用。优选的,用于抗女性卵巢癌。从而本发明进一步涉及一种药物或一种保健品,其中含有本发明的活性肽,例如DAGPVG,ERGEEGPAG,HGDRGELGPAG,ADGDRGEAGPAGPAGPAGPR,和GPAGPEGPRGDKGETGEEGDR中的一种或多种。
本发明的药物和保健品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。
本发明的药物和保健品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MTT细胞毒活性为追踪指标,将牛骨胶原蛋白酶解产物依次进行反相硅胶柱层析、凝胶柱层析以及高效液相色谱,并对活性最强的组分进行LC-MS-MS分析,最终鉴定到5种具有较强抗肿瘤细胞活性的活性肽,尤其可以显著抑制卵巢癌细胞的增殖。
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的快速反相硅胶柱层析色谱图。
图2示出超滤组分CP1经过快速反相硅胶柱层析得到的6个组分的MTT细胞毒活性测试结果。
图3示出快速反相硅胶柱层析组分CP1-1凝胶色谱图。
图4示出快速反相硅胶柱层析组分CP1-1经过凝胶柱层析得到的3个组分的MTT细胞毒活性测试结果。
图5示出凝胶柱层析组分CP1-1-1反相高效液相色谱图。
图6示出凝胶柱层析组分CP1-1-1经过反相高效液相色谱纯化得到的3个组分的MTT细胞毒活性测试结果。
图7示出采用MTT法验证本发明5种活性肽的细胞毒活性测试结果。
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活性肽制备
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:使用ODS-AQ反相硅胶柱(50μm,mm×100mm)对生物活性肽进行分离纯化。采用无水乙醇-超纯水为流动相以流速5mL/min对超滤截留的CP1进行梯度洗脱。洗脱条件为:100%的超纯水洗脱10min;96%的超纯水洗脱20min;90%的超纯水洗脱20min;87%的超纯水冲洗20min;83%的超纯水洗脱5min;78%的超纯水洗脱10min;100%的无水乙醇洗脱12min。220nm为检测波长,分别收集到6个洗脱峰并冷冻干燥放-80℃低温冰箱保存待用,结果见图1。对6个组分进行MTT细胞毒活性测试,结果如图2所示。CP1-1具有最强的细胞毒活性,遂对此组分进行进一步分离。
凝胶柱层析:对反相硅胶柱层析分离得到的最强活性的组分CP1-1采用Superdex75 10/300GL凝胶柱层析进行进一步纯化。凝胶柱首先用约50mL 20mM磷酸缓冲液(PH=4.2)进行平衡,之后上样,以0.3mL/min的流速,对样品进行洗脱,所用的流动相为20mM磷酸缓冲液(PH=4.2)。220nm为检测波长,收集到3个洗脱峰并冷冻干燥放-80℃低温冰箱备用,结果见图3。对3个组分进行MTT细胞毒活性测试,结果如图4所示。CP1-1-1具有最强的细胞毒活性,遂对此组分进行下一步分离。
高效液相色谱:对凝胶柱分离得到的活性最强的组分CP1-1-1采用高效液相色谱进行进一步纯化。ODS-AQ反相柱(20μm,2.5mm×150mm)首先用100%的超纯水平衡至基线呈一条直线,之后上样,以流速1mL/min对样品进行梯度洗脱。色谱条件为:100%的超纯水洗脱3min;100%的色谱乙腈洗脱12min。收集3个主要色谱峰并冷冻干燥放-80℃备用,结果见图5。对3个组分进行MTT细胞毒活性测试,结果如图6所示。CP1-1-1-2具有最强的细胞毒活性,遂对此组分进行LC-MS-MS分析。
利用液相色谱质谱联用仪对活性最强的组分CP1-1-1-2进行氨基酸序列分析,共鉴定到5条活性肽。结果见表1。
表1 从牛骨胶原蛋白酶解液中鉴定到的活性肽序列
实施例2
基于实施例1鉴定得到的5种活性肽的氨基酸序列,通过固相合成方法获得实验样品,进行抗肿瘤活性验证。结果表明活性肽具有较强的细胞毒活性,结果如图7所示。
固相合成肽公司:吉尔生化(上海)有限公司。
细胞毒活性的检测:
首先以每孔3000个人卵巢癌细胞系SKOV3接种于96孔板中,24h待细胞贴壁后,加入不同浓度活性肽(1mg/ml;0.25mg/ml;0.0625mg/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5d,然后每孔加入20μL5mg/ml MTT继续在培养箱中培养4h。吸取培养液,加入150μL DMSO二甲基亚砜。最后在490nm波长下,在酶标仪上进行吸光度A的测定,并计算其细胞相对生存率%=As/Ac×100。
公式中:As:活性肽样品吸光度的平均值Ac:空白对照吸光度的平均值;
活性以IC50表示,即细胞生存率达到50%时的浓度。
结果表明本发明合成的5条活性肽具有较强的抗癌活性,IC50分别为1mg/ml。
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。
序列表
<110> 中国科学院理化技术研究所
<120> 一种抗肿瘤活性肽及其应用
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Asp Ala Gly Pro Val Gly
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ggccccgccg gccccgaggg ccccaggggc gacaagggcg agaccggcga ggagggcgac 60
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Claims (6)
1.一种活性肽,其特征在于,所述活性肽的氨基酸序列包括如下序列中的一种或多种:
DAGPVG;
ERGEEGPAG;
HGDRGELGPAG;
ADGDRGEAGPAGPAGPAGPR;
GPAGPEGPRGDKGETGEEGDR。
2.一种多核苷酸,其特征在于,编码权利要求1所述的任一活性肽。
3.权利要求1所述的活性肽在制备抗肿瘤药物或保健品中的应用。
4.根据权利要求3所述的应用,其特征在于,所述肿瘤为卵巢癌。
5.一种药物,其特征在于,包含权利要求1所述的活性肽。
6.一种保健品,其特征在于,包含权利要求1所述的活性肽。
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