CN116693699A - 一种多肽tat-trpv1-c及其应用 - Google Patents
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Abstract
本发明公开了一种多肽TAT‑TRPV1‑C及其应用，该多肽TAT‑TRPV1‑C的氨基酸序列如SEQ ID NO.1所示；其中，1‑11位为穿膜肽TAT，12‑170位为TRPV1‑C。本发明的多肽TAT‑TRPV1‑C来源于TRPV1，该多肽能够与TRPV1胞内结构域竞争与H1R的结合，干扰H1R‑TRPV1轴所介导信号通路，从而抑制H1R‑TRPV1介导的炎症反应。本发明的多肽TAT‑TRPV1‑C在制备治疗TRPV1所介导的瘙痒等症状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-TRPV1-C及其应用。
背景技术
瘙痒是一种由多种原因引起的，能够刺激机体产生强烈抓挠欲望的不愉快的主观感觉，长时间持续瘙痒会严重影响患者生活。瘙痒是皮炎、湿疹等皮肤疾病的一种主要症状，同时瘙痒症状还常见于胆汁淤积、肿瘤、血液病、慢性肾衰竭以及艾滋病等疾病患者。瘙痒发生是人体习惯性的抓挠能有效缓解瘙痒的感觉，但是。长时间持续抓挠有可能导致皮肤损伤或者感染。然而，目前临床上面对多种类型的瘙痒却没有快速有效的治疗手段和治疗药物。
研究显示：瞬时感受器电位离子通道(transient receptor potential ionchannels，TRP)是许多瘙痒介质诱发的痒的关键离子通道。TRP通道是非选择性的阳离子通道，主要分布于细胞膜上，在哺乳动物神经与非神经系统中均有分布。外周的研究显示，瘙痒主要有组胺依赖和非组胺依赖的痒信号通路。其中组胺依赖的瘙痒信号通路主要通过对组胺刺激敏感，而对机械热刺激不敏感的无髓鞘C-类神经纤维传导。组胺一般通过组胺H1受体(histamine receptor type 1，H1R)激活磷脂酶C和磷脂酶A2，随后激活瞬时受体电位TRPV1(transient receptor potential vanilloid 1)通道，诱发背根神经节感觉神经元钙离子内流，最终产生瘙痒的感觉。组胺非依赖的瘙痒信号则是通过对机械热刺激敏感的C-类神经纤维传导。与野生型小鼠相比，组胺诱发的抓挠行为在TRPV1敲低的小鼠中显著减少。
TRPV1是一种6次跨膜蛋白，胞内羧基末端区域含有159个氨基酸，可能在胞内信号传导中发挥了关键作用。研究显示：组胺诱发的瘙痒主要通过H1R介导，而组胺H1受体则是通过激活TRPV1展示痒的表型，这一过程主要是通过H1R和TRPV1通道的羧基末端相互作用来实现的。
以TRPV1羧基末端氨基酸序列为基础设计多肽，该多肽将能够与TRPV1竞争性地与H1R结合，很可能能够阻断H1R-TRPV1信号轴所介导的信号通路，抑制其介导的炎症相关反应。因此，研究和开发靶向阻断H1R-TRPV1通路的多肽药物，可能具有临床应用前景，为瘙痒症状的治疗提供新的思路。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-TRPV1-C及其应用，该多肽在制备治疗TRPV1所介导的瘙痒等症状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-TRPV1-C，所述多肽TAT-TRPV1-C的氨基酸序列如SEQ ID NO.1所示；其中，1-11位为穿膜肽TAT，12-170位为TRPV1-C。
编码上述多肽TAT-TRPV1-C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34-510位为编码TRPV1-C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-TRPV1-C、上述基因或上述表达盒、重组载体或重组菌在制备治疗TRPV1介导的炎症性疾病症状的药物中的应用。
优选地，所述TRPV1介导的炎症性疾病症状为瘙痒。
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：
本发明的多肽TAT-TRPV1-C来源于TRPV1，该多肽能够与TRPV1胞内结构域竞争与H1R的结合，干扰H1R-TRPV1轴所介导信号通路，从而抑制H1R-TRPV1介导的炎症反应。本发明的多肽TAT-TRPV1-C在制备治疗TRPV1所介导的瘙痒等症状的药物中具有应用潜力。
附图说明
图1为实施例1中多肽TAT-TRPA1-C的纯化；
图2为实施例2中多肽TAT-TRPA1-C对炎症因子IL-4(A)和IL-5(B)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。
以下实施例中的小鼠均为实验用8周龄C57BL/6小鼠，来源于徐州医科大学实验动物中心。
实施例1多肽TAT-TRPV1-C的获取
本实施例阐述了具有穿模活性的多肽TAT-TRPV1-C的获取过程，具体步骤如下：
(1)以人源细胞cDNA为模板通过PCR获得TRPV1羧基末端多肽序列，上游引物如SEQID 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 IDNO.5所示)，在上、下游引物分别插入XhoI和BamHI酶切位点，通过酶切连接方法，构建pWaldo-TAT-TRPV1-C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+)插入了GFP-8His改造所得的载体，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0.15mM的IPTG，24℃培养22h，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 8.0，300mM NaCl和10％甘油)，加入200U DnaseI，50μg/mL溶菌酶和蛋白酶抑制剂，高压均质机1200bar，4℃破碎细胞，12,000rpm，4℃离心30min去除细胞碎片；将含有目的蛋白的上清自然流过镍柱；冲洗缓冲液(20mM Tris-HCL，pH 8.0，300mM NaCl，10％甘油和30mM咪唑)洗100mL，洗脱(20mMTris-HCL，pH 8.0，300mM NaCl，10％甘油和300mM咪唑)并收集蛋白质，随后通过分子筛柱子进一步纯化，此步骤能够获得纯度超过85％的TAT-TRPV1-C融合蛋白，如图1所示，可用于后续实施例的相关研究。
多肽TAT-TRPV1-C的氨基酸序列如SEQ ID NO.1所示，由170个氨基酸残基组成；其中，1-11位氨基酸残基组成具有穿过细胞膜功能的区域，即穿膜肽TAT，该区域和12-170位氨基酸前后位置可以互换，并且可以被具有相似作用的其他序列替换；12-170位氨基酸残基构成能够干扰H1R-TRPV1信号通路的区域，即TRPV1-C。
多肽TAT-TRPV1-C编码基因的核苷酸序列如SEQ ID NO.2所示，长度为510bp；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34-510位为编码TRPV1-C的核苷酸序列。
实施例2多肽TAT-TRPV1-C对瘙痒的影响
1、构建小鼠瘙痒模型并分组给药，具体如下：
(1)多肽给药组：实验前2d剃除小鼠颈背部的毛发(2cm×3cm)，实验开始第1、2、3天，于剃毛处涂抹1mg/mL实施例1获取的多肽TAT-TRPV1-C溶液。实验第4天涂完多肽TAT-TRPV1-C溶液后1h，皮下注射500μg组胺(50μL)，随后记录30min内小鼠行为学抓挠次数。
(2)瘙痒模型组：同多肽给药组操作，同样时间节点等剂量给与PBS替代多肽TAT-TRPV1-C溶液，第4天皮下注射500μg组胺(50μL)，记录30min内小鼠行为学抓挠次数。
(3)对照组：同多肽给药组操作，同样时间节点等剂量给与PBS替代多肽TAT-TRPV1-C溶液，记录30min内小鼠行为学抓挠次数。
2、小鼠肩背部皮下注射组胺后，立刻将小鼠置于透明观察盒，记录小鼠行为学抓痒次数。小鼠后爪抬起抓挠剃毛部位1次或连续抓挠多次，记录为1次抓痒行为，小鼠后爪落地或缩爪停顿表示结束本次抓痒。
表1多肽TAT-TRPV1-C对瘙痒的影响
实验结果如表1所示，多肽TAT-TRPV1-C能够显著抑制组胺诱发的小鼠瘙痒抓挠次数，差异具有统计学意义(P＜0.05)。
3、实验小鼠行为学检测结束后，眼球取血法取全血，离心提取血浆，随后采用ELISA试剂盒检测炎症因子IL-4和IL-5的含量。
实验结果如图2所示，多肽TAT-TRPV1-C能够有效降低炎症因子IL-4和IL-5含量，说明其对于缓解炎性瘙痒是有效的。多肽TAT-TRPV1-C缓解瘙痒的机制是抑制TRPV1激活所诱发的炎症因子增加。
由本实施例的实验结果可知，本发明的多肽TAT-TRPV1-C能够特异性抑制TRPV1激活所诱发的炎症因子释放，缓解瘙痒症状，从而达到治疗的目的。
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多种变化和改进，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 (7)
1.一种多肽TAT-TRPV1-C，其特征在于，所述多肽TAT-TRPV1-C的氨基酸序列如SEQ IDNO.1所示；其中，1-11位为穿膜肽TAT，12-170位为TRPV1-C。
2.编码权利要求1所述多肽TAT-TRPV1-C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34-510位为编码TRPV1-C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-TRPV1-C、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治疗TRPV1介导的炎症性疾病症状的药物中的应用。
5.根据权利要求4所述的应用，其特征在于，所述TRPV1介导的炎症性疾病症状为瘙痒。
6.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。
7.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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