CN114933645A - 一种Rad51重组酶的抑制肽及其在提升化疗药物癌症杀伤效力中的应用 - Google Patents
一种Rad51重组酶的抑制肽及其在提升化疗药物癌症杀伤效力中的应用 Download PDFInfo
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Abstract
本发明涉及生物医学技术领域，具体涉及一种Rad51重组酶的抑制肽及其在提升化疗药物癌症杀伤效力中的应用本发明提供的抑制肽是一种新型Rad51重组酶抑制剂，由62个氨基酸组成，命名为CD2。采用本发明所提供的抑制肽CD2，能够降低癌症患者在化疗时，产生的Rad51重组酶的表达量，降低癌症病人体内DNA损伤修复能力，显著提升化疗药物对癌细胞杀伤力、提升放化疗治疗效果。
Description
技术领域
本发明涉及生物医学技术领域，具体涉及一种Rad51重组酶的抑制肽及其在提升化疗药物癌症杀伤效力中的应用。
背景技术
放化疗是恶性肿瘤的主要治疗手段，这些治疗主要是通过在癌症细胞内部产生DNA损伤来导致癌症细胞的死亡。然而，很多癌症病人体内Rad51重组酶呈现高表达。由于Rad51是介导同源重组(HR)无差错修复DNA损伤信号通路的关键因子，因此Rad51重组酶的高表达提高了对放化疗所造成的DNA损伤的修复能力，从而抑制癌症细胞的死亡，这些病人呈现明显的放化疗不敏感性，治疗效果差。因此，开发针对Rad51的抑制剂对于提高癌症治疗的有效性具有重要意义。
目前市面上的Rad51的抑制剂主要是合成类小分子化合物，包括B02、DIDS、CSB、IBR2、RI-1、Halenaquinone、RS-1等。对现有技术中，抑制Rad51的化合物进行鉴定，但尚未发现，现有技术中Rad51的抑制剂在提升人类恶性肿瘤放化疗治疗效果中的应用。
发明内容
本发明的目的是提高癌症患者对化疗药物的敏感性，提高化疗药物对肿瘤的杀伤力。
第一方面，本发明提供一种Rad51重组酶的抑制肽，所述抑制肽的氨基酸序列如SEQ ID NO.1所示。
本发明提供的抑制肽命名为CD2，由62个氨基酸组成：NAAPLHNFGEDFLQPYVQLQQNFSASDLEVNLEATRESHAHFSTPQALELFLNYSVTPPGEI。
本发明请求保护上述的抑制肽在干扰Rad51重组酶所介导的DNA双链损伤修复中的应用。
本发明提供的CD2与Rad51蛋白可直接结合，更具体地，CD2与Rad51蛋白的结合机制为：CD2通过干扰Rad51在DNA损伤部位的富集从而抑制了其对于DNA损伤的修复。
第二方面，本发明提供一种Rad51重组酶的抑制剂，所述抑制剂含有如SEQ IDNO.1所示的短肽。
本发明提供的抑制剂的剂型包括：片剂、胶囊、颗粒剂、口服液、缓释制剂、控释制剂、纳米制剂或注射剂。
根据本领域技术人员的理解，本发明还请求保护上述的抑制肽或上述的抑制剂在降低Rad51重组酶表达量中的应用。
以及，上述的抑制肽或上述的抑制剂在提高癌症患者对化疗药物敏感性中的应用。
以及，上述的抑制肽或上述的抑制剂在制备癌症化疗药物或构建癌症化疗药物递送系统中的应用。
在本发明的上述应用中，所述癌症包括：黑色素瘤、胃癌、乳腺癌、肾癌、肝癌、宫颈癌、卵巢癌、胰腺癌、前列腺癌、结肠癌。
第三方面，本发明请求保护一种癌症化疗药物，所述癌症化疗药物中含有如SEQID NO.1所示的短肽。
第四方面，本发明还请求保护一种癌症化疗药物递送系统，使用如SEQ ID NO.1所示的短肽介导癌症化疗药物递送到患者体内。
本发明的有益效果在于：
(1)本发明所提供的Rad51重组酶的抑制肽为62个氨基酸组成的短肽，具备良好的成药潜力。
(2)本发明提供的新型抑制肽分子CD2对Rad51重组酶的抑制效果好。
(3)本发明提供的新型抑制肽分子CD2显著提升了化疗药物对癌细胞的杀伤力。
附图说明
图1为本发明CD2与Rad51重组酶外源相互作用电泳图。
图2为本发明U2OS细胞中共转染I-SceI和CD2及转染空载体后，GFP阳性细胞数。
图3为本发明U2OS细胞中转染CD2及不含CD2，化疗药物喜树碱对肿瘤细胞的杀伤力对比图。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。在不背离本发明精神和实质的情况下，对本发明方法、步骤或条件所作的修改或替换，均属于本发明的保护范围。
若未特别指明，本发实施例中所用的实验材料、试剂、仪器等均可市售获得；若未具体指明，本发明实施例中所有的技术手段均为本领域技术人员所熟知的常规手段。
本实施例所用Rad51的抑制肽，命名为CD2，CD2的氨基酸序列(SEQ ID NO.1)为：NAAPLHNFGEDFLQPYVQLQQNFSASDLEVNLEATRESHAHFSTPQALELFLNYSVTPPGEI。
实施例1 CD2与Rad51直接相互作用
本实施例采用实验证实了CD2与Rad51重组酶是直接进行相互作用的，验证步骤如下：
(1)在原核表达载体上分别构建了CD2(GST标签)以及Rad51重组酶(His标签)的克隆：
将Rad51重组酶和CD2的编码序列分别构建到PET30a和pGEX6p-1表达载体中，得到的质粒转入BL21感受态中，体外诱导纯化Rad51重组酶和CD2。
(2)GST pull down实验：
将10μg融合有GST标签的CD2和Ctrl(GST)分别和10μg的融合有His标签的Rad51重组酶蛋白混合，反应体系补齐至500μl，取1％反应体系混合液作为Input，留样检测，低温摇床孵育过夜。
实验结果显示，融合有GST的CD2可共沉淀Rad51重组酶，表明CD2可与Rad51重组酶蛋白存在直接相互作用(图1)。
实施例2 CD2能够抑制Rad51重组酶介导的DNA双链损伤修复
本实施采用实验证实了，CD2能够抑制Rad51重组酶介导的DNA双链损伤修复。
已知Rad51重组酶介导同源重组的DNA损伤修复，为阐明CD2抑制Rad51重组酶活性，本实施例采用DR-GFP报告基因检测法研究了CD2对同源重组修复的影响，步骤如下。
在含有DR-GFP构建体的U2OS细胞中共转染I-SceI和CD2为实验组，同时共转染I-SceI和CD2对应空载作为对照组，转染48小时后，用流式细胞仪检测GFP阳性细胞数。不少于3次独立重复实验(双尾T检验，*P<0.05)。
实验结果：与对照组相比，表达CD2组GFP阳性细胞的数量显著减少。表明CD2可以抑制Rad51重组酶介导的同源重组DNA损伤修复(图2)。
实施例3 CD2有效提高了化疗药物对恶性肿瘤细胞的杀伤效果
为了探究CD2对化疗药物杀伤恶性肿瘤细胞的促进效果，本实施例采用人骨肉瘤U2OS细胞，通过过表达技术，检测CD2对化疗药物喜树碱(CPT)的肿瘤杀伤情况，步骤如下。
本实施例在人骨肉瘤U2OS细胞中分别表达GFP标记的CD2和空载的GFP载体，其中，空载的GFP载体转染作为对照组细胞。转染48小时后用2μM CPT处理4小时，然后进行细胞计数，计算人骨肉瘤U2OS细胞的相对生存能力，见图3。
实验结果：在对照组中，化疗药物喜树碱的添加在两天之后并未造成骨肉瘤细胞的显著死亡；但是，当在骨肉瘤细胞中通过外源表达CD2肽时，在短短的2天之内，化疗药物喜树碱处理导致了骨肉瘤细胞生存能力的显著下降，证明CD2显著提升了该药物的肿瘤杀伤效果。值得注意的是，由于单纯过表达CD2肽并不能导致骨肉瘤细胞的死亡，它抑制肿瘤生长的效应依赖于化疗药物喜树碱，这也支持了CD2通过抑制喜树碱等化疗药物造成的致死性双链损伤修复进而增强了肿瘤细胞对化疗药物的敏感性。综上，CD2肽能够促进化疗药物喜树碱对恶性骨肉瘤细胞的杀伤效力。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
序列表
<110> 首都师范大学
<120> 一种Rad51重组酶的抑制肽及其在提升化疗药物癌症杀伤效力中的应用
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Glu Ala Thr Arg Glu Ser His Ala His Phe Ser Thr Pro Gln Ala Leu
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Claims (10)
1.一种Rad51重组酶的抑制肽，其特征在于，所述抑制肽的氨基酸序列如SEQ ID NO.1所示。
2.权利要求1所述的抑制肽在干扰Rad51重组酶所介导的DNA双链损伤修复中的应用。
3.一种Rad51重组酶的抑制剂，其特征在于，所述抑制剂含有如SEQ ID NO.1所示的短肽。
4.根据权利要求3所述的抑制剂，其特征在于，所述抑制剂的剂型包括：片剂、胶囊、颗粒剂、口服液、缓释制剂、控释制剂、纳米制剂或注射剂。
5.权利要求1所述的抑制肽或权利要求3-4任一项所述的抑制剂在降低Rad51重组酶表达量中的应用。
6.权利要求1所述的抑制肽或权利要求3-4任一项所述的抑制剂在提高癌症患者对化疗药物敏感性中的应用。
7.权利要求1所述的抑制肽或权利要求3-4任一项所述的抑制剂在制备癌症化疗药物或构建癌症化疗药物递送系统中的应用。
8.根据权利要求6-7任一项所述的应用，其特征在于，所述癌症包括：黑色素瘤、胃癌、乳腺癌、肾癌、肝癌、宫颈癌、卵巢癌、胰腺癌、前列腺癌、结肠癌。
9.一种癌症化疗药物，其特征在于，所述癌症化疗药物中含有如SEQ ID NO.1所示的短肽。
10.一种癌症化疗药物递送系统，其特征在于，使用如SEQ ID NO.1所示的短肽介导癌症化疗药物递送到患者体内。
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