CN116425888A - 一种多肽tat-v2r1c及其应用 - Google Patents
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Abstract
本发明公开了一种多肽TAT‑V2R1C及其应用,该多肽的氨基酸序列如SEQ ID NO.1所示;其中,1‑11位为穿膜肽TAT,12‑54位为V2R1C。本发明提供的多肽TAT‑V2R1C来源于V2R,该多肽TAT‑V2R1C能够和V2R羧基末端竞争性的与β‑arrestin 2相结合,干扰β‑arrestin 2所介导信号通路,从而抑制β‑arrestin 2介导的肿瘤细胞增殖、迁移和侵袭。本发明的多肽TAT‑V2R1C在制备治疗肾癌等有V2R表达的癌症的药物中具有应用潜力。
Description
技术领域
本发明属于医药技术领域,具体涉及一种多肽TAT-V2R1C及其应用。
背景技术
β-arrestin 2在肾癌组织中表达显著增加,β-arrestin 2的高表达和不良预后密切相关。β-arrestin 2通过其介导的信号通路影响癌细胞侵袭、转移和增殖等过程,干预其信号通路可能成为临床治疗的有效策略。
β-arrestin 2在肾癌组织中过表达,β-arrestin 2高表达和肿瘤进展以及细胞整体生存率相关,说明β-arrestin 2是不良预后的一个有效地生物标志物,基因沉默β-arrestin 2能够明显抑制癌细胞增殖、迁移和侵袭。证实β-arrestin 2是癌症发生、发展的关键分子,靶向β-arrestin 2的药物研究将蕴含巨大的临床治疗潜力。
β-arrestin 2作为GPCR(G protein coupled receptor)调控的关键蛋白,其作用也可能受到GPCR的影响。肾癌组织中高表达的后叶加压素2型受体(vasopressin type-2receptor,V2R)作为一种G蛋白偶联受体,其功能受到β-arrestin 2的调控,两个蛋白的作用是相互的,V2R通过胞内结构域和β-arrestin2发生相互作用。β-arrestin 2竞争性地与V2R细胞质区域结合,一方面将其从G蛋白偶联中释放出来,另一方面由于β-arrestin 2覆盖了V2R的细胞质功能区域,将阻止更多G蛋白的结合。随后β-arrestin 2介导的信号通路开始在肿瘤发生、侵袭和转移等过程中发挥作用。
V2R是一种典型的七次跨膜(trans-membrane,TM)受体,7个α螺旋结构通过3个胞外loop(extracellular loops,ECLs 1-3)和3个胞内loop(intracellular loops,ICLs 1-3)连接。根据以往GPCR研究显示:V2R和β-arrestin 2发生相互作用的部位主要为羧基末端。
而选择性干扰β-arrestin 2和V2R的相互作用,能够阻止β-arrestin 2到V2R的聚集,可能阻止β-arrestin 2介导的信号通路。因此,研究和开发阻断β-arrestin2的多肽药物,将具有很好的治疗癌症的作用。但现有技术中目前仍缺少相关研究。
发明内容
针对现有技术的不足,本发明提供一种多肽TAT-V2R1C及其应用,该多肽在制备治疗肾癌等癌症的药物中具有应用潜力。
本发明是通过以下技术方案实现的:
一种多肽TAT-V2R1C,所述多肽TAT-V2R1C的氨基酸序列如SEQ ID NO.1所示;其中,1-11位为穿膜肽TAT,12-54位为V2R1C。
编码上述多肽TAT-V2R1C的基因,所述基因的核苷酸序列如SEQ ID NO.2所示;其中,自5’末端起第1-33位为编码穿膜肽TAT的核苷酸序列,第34-162位为编码V2R1C的核苷酸序列。
含有上述基因的表达盒、重组载体或重组菌。
上述多肽TAT-V2R1C、上述基因或上述表达盒、重组载体或重组菌在制备抗肿瘤药物中的应用。
优选地,所述肿瘤为有V2R表达的肿瘤组织或细胞。
优选地,所述肿瘤为肾癌。
优选地,所述药物为任何药物治疗学上可接受的剂型。
优选地,所述药物还包括药学上可接受的递药载体。
本发明的有益效果如下:
本发明提供的多肽TAT-V2R1C来源于V2R,该多肽TAT-V2R1C能够和V2R羧基末端竞争性的与β-arrestin 2相结合,干扰β-arrestin 2所介导信号通路,从而抑制β-arrestin2介导的肿瘤细胞增殖、迁移和侵袭。本发明的多肽TAT-V2R1C在制备治疗肾癌等有V2R表达的癌症的药物中具有应用潜力。
附图说明
图1为实施例2中多肽TAT-V2R1C对肾癌细胞增殖的影响;
图2为实施例2中多肽TAT-V2R1C对肾癌细胞迁移的影响;
图3为实施例2中多肽TAT-V2R1C肾癌细胞侵袭能力的影响。
具体实施方式
下面结合附图与具体实施例对本发明做进一步详细说明,给出的实施例仅为了阐明本发明,而不是为了限制本发明的范围。
以下实施例中的定量实验,如无特殊说明,均设置三次重复,结果取平均值。
以下实施例中的实验方法,如无特殊说明,均为常规的分子生物学方法。
以下实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
以下实施例中肾癌细胞A498、786-O、ACHN均来源于中国科学院上海生命科学研究院细胞资源中心。
实施例1多肽TAT-V2R1C的获取
本实施例阐述了具有穿模活性的多肽TAT-V2R1C的获取过程,具体步骤如下:
(1)提取SKOV3细胞总RNA,反转录得到cDNA,以cDNA为模板通过PCR获得V2R羧基末端多肽序列,其上游引物如SEQ ID NO.3所示,下游引物如SEQ ID NO.4所示,在上游引物内插入TAT编码序列(如SEQ ID NO.5所示),在上、下游引物分别加入XhoI和BamHI酶切位点,通过酶切链接的方法,构建pWaldo-TAT-V2R1C大肠杆菌表达载体,并转化BL21(DE3)表达菌株,获得重组子。
其中,pWaldo是pET28(a+)基础上插入了GFP-8His的载体,其合成可参考文献:Drew,D.E.,von Heijne,G.,Nordlund,P.&de Gier,J.W.Green fluorescent protein asan indicator to monitor membrane protein overexpression in Escherichiacoli.FEBS Lett 507,220-4(2001).
(2)接种重组子菌株于100mL LB培养基中,37℃过夜培养。转种至2L LB培养基中,37℃培养至OD600约0.5~0.6之间,加入终浓度为0.5mM的IPTG,20℃培养24h,8000g离心10min收集细胞。
(3)将细胞悬浮于100mL裂解缓冲液中(20mM Tris-HCl,pH 8.5,300mM NaCl和10%甘油),加入50μg/mL溶菌酶,200U DnaseI和1片蛋白酶抑制剂(cocktail),高压均质机1200bar,4℃破碎细胞,18,000rpm,4℃离心30min去除细胞碎片;将含有目的蛋白质的上清样品自然通过柱子一次;冲洗缓冲液(20mM Tris-HCL,pH 8.5,300mM NaCl,10%甘油和30mM咪唑)洗120mL,洗脱(20mM Tris-HCL,pH 8.5,300mM NaCl,10%甘油和300mM咪唑)并收集蛋白质样品,通过分子筛柱子(缓冲液:20mM Tris-HCL,pH 8.5,300mM NaCl,10%甘油)进行纯化,此步骤能够获得较高纯度的多肽TAT-V2R1C融合蛋白,可用于后续实施例的相关研究。
多肽TAT-V2R1C的氨基酸序列如SEQ ID NO.1所示,由54个氨基酸残基组成;其中,1-11位氨基酸残基组成具有穿透细胞膜作用的区域,即穿膜肽TAT,该区域和12-54位氨基酸前后位置可以互换,并可以被具有相同作用的其他序列替换;12-54位氨基酸残基构成能够干扰β-arrestin 2蛋白质功能的区域,即V2R1C。
多肽TAT-V2R1C编码基因的核苷酸序列如SEQ ID NO.2所示,长度为162bp;其中,自5’末端起第1-33位为编码穿膜肽TAT的核苷酸序列,第34-162位为编码V2R1C的核苷酸序列。
实施例2多肽TAT-V2R1C的应用研究
1、MTT检测
A498、786-O、ACHN细胞分别接种于96孔板,贴壁12h后加入0.2mg/mL实施例1获取的多肽TAT-V2R1C,2h后再次加入0.2mg/mL多肽TAT-V2R1C,加入PBS的细胞作对照。给药处理48h后细胞孔中加入5mg/mL MTT溶液10μL,低速摇床震荡温育3~4h,随后移除MTT和培养基的混合液,加入100μL DMSO溶液。由于MTT的存在,活细胞将形成晶体,而结晶物随后溶解于DMSO溶液,酶联免疫检测仪检测490nm波长处的吸光值。
2、细胞迁移和侵袭
(1)A498、786-O、ACHN细胞迁移检测使用24孔Transwell细胞培养小室,小室内放置有8μm孔的聚碳酸酯滤膜。将培养好的细胞用胰蛋白酶消化(消化前加入0.2mg/mL实施例1获取的多肽TAT-V2R1C处理2h)并重新悬浮于RPMI-1640培养基,使其终浓度为2×105个/mL。500μL 10% FBS/RPMI-1640培养基或者500μL SDF-1溶液加入到小室底层,100μL细胞悬液加入到滤膜上层。
(2)侵袭实验则先在Transwell小室中铺无血清的RPMI-1640培养基1:1稀释的Matrigel胶50μL,37℃孵育2h后接种细胞悬液400μL。恒温培养箱中静置培养24h后,用湿棉签轻轻擦去Matrigel凝胶和聚碳酸酯膜表面的细胞,以无水乙醇固定滤膜,0.1%结晶紫染色15min,PBS清洗,倒置显微镜20倍放大视野下随机取5个不重复视野,计数每个视野穿过Transwell小室底部膜下部的细胞数。
3、实验结果
给予多肽后细胞形态未发现明显异常。如图1所示,多肽TAT-V2R1C(图中简写为V2R1C,下同)能够明显抑制肾癌细胞的增殖。如图2、图3所示,多肽TAT-V2R1C能够明显抑制肾癌细胞的迁移和细胞侵袭。
由本实施例的实验结果可知,本发明的多肽TAT-V2R1C能够竞争性地与β-arrestin 2结合,进而抑制β-arrestin 2所介导的肾癌细胞迁移、侵袭和增殖等过程。
以上显示和描述了本发明的具体实施方式,本行业的技术人员应该了解,本发明不受上述实施例的限制,在不脱离本发明精神和范围的前提下本发明还会有各种变化和改进,这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等同物界定。
Claims (8)
1.一种多肽TAT-V2R1C,其特征在于,所述多肽TAT-V2R1C的氨基酸序列如SEQ ID NO.1所示;其中,1-11位为穿膜肽TAT,12-54位为V2R1C。
2.编码权利要求1所述多肽TAT-V2R1C的基因,其特征在于,所述基因的核苷酸序列如SEQ ID NO.2所示;其中,自5’末端起第1-33位为编码穿膜肽TAT的核苷酸序列,第34-162位为编码V2R1C的核苷酸序列。
3.含有如权利要求2所述基因的表达盒、重组载体或重组菌。
4.权利要求1所述多肽TAT-V2R1C、权利要求2所述基因或权利要求3所述表达盒、重组载体或重组菌在制备抗肿瘤药物中的应用。
5.根据权利要求4所述的应用,其特征在于,所述肿瘤为有V2R表达的肿瘤组织或细胞。
6.根据权利要求5所述的应用,其特征在于,所述肿瘤为肾癌。
7.根据权利要求4所述的应用,其特征在于,所述药物为任何药物治疗学上可接受的剂型。
8.根据权利要求4所述的应用,其特征在于,所述药物还包括药学上可接受的递药载体。
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