CN106831944A - 一种肿瘤坏死因子alpha的高亲和性肽及其应用 - Google Patents
一种肿瘤坏死因子alpha的高亲和性肽及其应用 Download PDFInfo
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Abstract
本发明属于生物技术领域，具体涉及一种肿瘤坏死因子alpha的高亲和性肽及其应用。本发明肽的氨基酸序列为SEQ.ID.NO.1和SEQ.ID.NO.3所示，或者为SEQ.ID.NO.1和SEQ.ID.NO.3单次重复或多次重复的串联或分支的肽分子序列，其氨基酸序列为SEQ.ID.NO.4所示；能和肿瘤坏死因子alpha高度亲力结合并可拮抗其功能。使用该肽直接注射动物，可显著降低动物体炎症的发生程度，提高动物体对炎症损伤的抵抗性。本发明的肽可用作开发肿瘤坏死因子alpha拮抗药物，治疗因各种急慢性炎症所造成的机体损伤，如各种物理、化学和生物因子所引起的炎性的、自身免疫和应激损伤，具有极其广泛的应用前景，且分子量小、免疫原性低、易于合成，避免了以往单克隆抗体药物拮抗剂的副作用和不足。
Description
技术领域
本发明属于生物技术领域，具体涉及一种肿瘤坏死因子alpha（TNFα）的高亲和性肽（TNFα拮抗肽）及其应用。
背景技术
肿瘤坏死因子开始是在1975年由内毒素注射而诱导产生，被鉴定为可诱导移植到小鼠体内的肿瘤凋亡的血清活性因子，因此才被命名为肿瘤坏死因子。随着研究的深入，发现TNFα是一种十分重要的炎症因子。在免疫反应诱发的炎症性疾病中，TNFα在组织中的表达上升，同时诱导一系列的病原性反应和相关炎症因子的表达，最终导致组织的损伤，例如骨骼破坏、软骨降解、成纤维细胞增殖、角质细胞增殖等等，使得TNFα成为治疗多种炎症性疾病的重要靶点。
TNFα已有的拮抗剂主要集中于单克隆抗体以及单克隆抗体衍生分子上，但是单克隆抗体药物的免疫原性强，在机体内可诱发抗药物抗体的产生，即抗抗体的产生，毫无疑问会影响药物疗效，另一方面还可能诱导抗体依赖的细胞介导的细胞毒作用和抗体介导的补体依赖的细胞毒作用，易于引起对机体的伤害和副作用，由于抗抗体产生也会使药效下降。不同的分子结构决定了分子的免疫原性，肽与抗体相比其免疫原性低，而且分子小使得肽类物质的组织渗透性更强，因此肽是一种更有优势的TNFα拮抗分子。采用人工合成的方法得到序列特异性的肽，使用ELISA方法检测拮抗肽与TNFα的亲和性，然后使用该拮抗肽注射炎症动物，可以很方便地观察到该肽是否具有对炎症发生的抑制作用。
发明内容
本发明的目的是提供一种肿瘤坏死因子alpha的高亲和性肽以及该亲和肽在拮抗肿瘤坏死因子alpha功能上的应用。
本发明提供的TNFα高亲和性肽，其氨基酸序列如SEQ.ID.NO.1所示，记为632肽或TNFα拮抗肽，具有如下功能特征：①可与TNFα高度亲和，测得解离常数Kd达到138nM（Kd值表示一半受体被配体结合时配体的浓度，Kd值越小代表受体与配体的亲和性越高）；②注射到炎症动物模型体内后可有效抑制炎症的发生，起到对炎症损伤的抵抗作用。
本发明还提供编码所述TNFα高亲和性肽的亲和肽的基因（由相应氨基酸各简并密码子构成），其核苷酸序列如SEQ.ID.NO.2所示。
通过生物技术改造，本发明还获得了一种具有与所述TNFα高亲和性肽功能相同的肽序列，其氨基酸序列如SEQ.ID.NO.3所示，记为636肽。
本发明还提供一种同时含有TNFα高亲和性肽氨基酸序列的SEQ.ID.NO.1和氨基酸序列为SEQ.ID.NO.3的肽序列，为单次重复或多次重复的串联或分支的肽分子序列，及含有这些核心序列特征（同源性在70%以上）的分子，其肽氨基酸序列为SEQ.ID.NO.4所示。
本发明还提供一种经过生物和化学基团修饰的肽，其含有TNFα高亲和性肽SEQ.ID.NO.1和SEQ.ID.NO.3作为核心序列的C端（或N端，或侧链）基团上加抗原或药物或PEG修饰或其他分子基团共价修饰的结构特征分子。
本发明还提供一种经过修饰的肽，包括上述各种TNFα高亲和性肽，其TNFα高亲和性肽的加FITC等荧光基团修饰、同位素标记、化学发光基团或酶标试剂修饰的分子，用于检测TNFα。
本发明还提供上述各种TNFα高亲和性肽，在制备TNFα拮抗剂中的应用。
本发明还提供上述各种TNFα高亲和性肽，在黏TNFα表达检测和临床检测中的应用。
本发明还提供如核苷酸序列为SEQ.ID.NO.2的肽基因在制备表达TNFα拮抗剂中的应用和在示踪检测中的应用。
本发明的氨基酸序列为SEQ.ID.NO.1的肽可作为TNFα拮抗药物。该肽与TNFα具有很强的亲和性，可抑制TNFα的生物学活性，抵抗TNFα诱导的炎症损伤。本发明的氨基酸序列为SEQ.ID.NO.3的肽同样可以作为潜在的TNFα拮抗药物，该肽同样与TNFα具有较强的亲和性，并可抑制TNFα的生物学活性。
该肽与TNFα亲和性实验：使用2μg/ml的TNFα溶液4℃过夜包被96孔酶标板，加入不同浓度的带有FITC标记的632肽后孵育2h，再加入带有HRP标记的抗FITC的单克隆抗体孵育1h，然后加入ABTS显色液显色1h，通过酶标仪读取OD410数值，GraphPad Prism5作图并分析。结果证实632肽与TNFα蛋白具有极强的亲和力，解离常数Kd为138nM。
该肽对炎症动物模型炎症发生程度的影响：将实验动物分为空白组、无关肽对照组和拮抗肽组（632肽）。以小鼠为例，使用6周大小的雄性昆明鼠，右侧腹部皮下注射，连续3天。最后一次注射后使用对二甲苯涂抹小鼠右耳耳廓两侧，1h后处死小鼠，剪下左右耳，使用耳肿打耳器打下耳片，同一只小鼠的右耳重量减去左耳重量为鼠耳肿胀度。比较不同组之间的差异，发现该肽可以显著抑制小鼠耳部肿胀，即该肽可以抵抗炎症诱导的损伤。
由此可见，该肽对TNFα蛋白具有极强的亲和性，而且该肽可以抵抗TNFα诱导的炎症损伤，因此可以用以抑制TNFα生物活性的发挥，从而在TNFα强烈相关的炎症性疾病中起到治疗作用。
附图说明
图1. 632肽对TNFα蛋白的亲和性。
图2. 636肽对TNFα蛋白的亲和性。
图3. 632肽抑制昆明小鼠炎症模型炎症发生的程度。
具体实施方式
一、肽序列的获得与改造
通过化学的方法人工合成所需的肽，肽具有与TNFα蛋白亲和的作用。
二、肽的合成与纯化
Lys（Dde）-WangResin用DCM浸泡10min后抽干DCM，然后加入树脂体积三倍的25%哌啶（哌啶/DMF)，用氮气鼓吹20min后抽干哌啶。加入DMF鼓吹1min，循环6次后抽干，取树脂茚三酮检测呈蓝色。产物为：H-Lys（Dde）-WangResin。加入树脂3倍当量的Fmoc-Val-OH，HATU，DIEA在DMF中。用氮气鼓吹20min后抽干DMF反应液，加入DMF用氮气鼓吹1min后抽干，循环3次，茚三酮检测树脂呈透明。产品为：Fmoc-Val-Lys（Dde）-WangResin，依次类推，得到粗产品。用乙腈、Milli-Q水通过汉邦YCM C18柱子进行纯化。从而获得高特异性、高活性的目的肽。
三、632肽与TNFα的亲和试验及对动物模型炎症发生程度的影响
1、该肽与TNFα蛋白亲和性实验结果：将2μg/ml的TNFα蛋白4℃过夜包被在96孔酶标板上，BSA封闭后加入不同浓度的带有FITC标记的632肽，共孵育2h，加入带HRP标记的抗FITC的单克隆抗体孵育1h，然后加入ABTS显色液，显色1h后通过酶标仪读取OD410数值，GraphPad Prism 5作图并分析。结果证实632肽与TNFα蛋白具有极强的亲和力，解离常数Kd为138nM。
2、该肽对炎症动物模型炎症发生程度的影响：将实验动物分为空白组、无关肽对照组和拮抗肽组（632肽）。使用6周大小的雄性昆明鼠，右侧腹部皮下注射肽，连续3天。末次注射后30min，各组动物用对二甲苯0.03ml/只涂于小鼠右耳廓两面致炎，左耳不涂作为正常耳。致炎1h后处死动物，完整剪下小鼠耳朵并做好标记，区分同一只小鼠的左右耳。使用同一张纸包裹住鼠耳两面，用直径8mm的耳肿打孔器打下双耳相同部位耳片，连同白纸一起置于天平上称重，记录数值。鼠耳肿胀度= 致炎侧耳片重一非致炎侧耳片重。
结果发现，632肽与TNFα蛋白具有极强的亲和作用。动物实验结果表明，注射632肽的实验组动物的鼠耳肿胀度显著小于对照组。以上结果说明632肽可以通过拮抗TNFα活性的发挥，从而达到抵抗炎症损伤的效果。如图1、图2所示。
四、具体应用
肿瘤坏死因子alpha作为一种重要的炎症因子，在炎症发生发展的过程中起到了十分重要的作用，而在炎症诱导的免疫性疾病中，TNFα表达上升的同时诱导一系列的病原性反应和相关细胞因子的表达，造成组织损伤。多种炎症性疾病，例如风湿性关节炎、银屑病等中对TNFα的拮抗治疗已被证实为有效的治疗方式。但是目前的拮抗物集中在单克隆抗体及其衍生物上，副作用中强，尤其是免疫原性，限制了药物使用。因此本发明提供的TNFα高亲和性肽除了与TNFα亲和力强以外，还可以抑制TNFα生物活性的发挥，从而抵抗相关的炎症损伤，可作为潜在的TNFα拮抗药物。另外经过修饰的632肽，可以作为检测试剂检测TNFα蛋白的表达。
序列表
SEQ.ID.NO.1（N端→C端）：HYIDFRW
SEQ.ID.NO.2：CATTATATTGATTTTAGGTGG
SEQ.ID.NO.3（N端→C端）：KASGSPSGFWPS
SEQ.ID.NO.4（N端→C端）：HYIDFRWDMKASGSPSGFWPS 。
SEQUENCE LISTING
<110> 复旦大学
<120> 一种肿瘤坏死因子alpha的高亲和性肽及其应用
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Claims (7)
1.一种肿瘤坏死因子alpha的高亲和性肽，其特征在于，氨基酸序列为SEQ.ID.NO.1和SEQ.ID.NO.3之一；或者为SEQ.ID.NO.1和SEQ.ID.NO.3单次重复或多次重复的串联或分支的肽分子序列，其氨基酸序列为SEQ.ID.NO.4。
2.一种编码氨基酸序列为SEQ.ID.NO.1的肿瘤坏死因子alpha的高亲和性肽的基因，其特征在于，核苷酸序列为SEQ.ID.NO.2所示。
3.一种经过生物和化学基团修饰的肽，其特征在于，含有如权利要求1所述的肿瘤坏死因子alpha高亲和性肽作为核心序列的C端，或N端，或侧链基团上加抗原或药物或PEG修饰或其他分子基团共价修饰的结构特征分子。
4.一种经过修饰的肽，其特征在于，如权利要求1所述的肿瘤坏死因子alpha的高亲和性肽，其肿瘤坏死因子alpha高亲和性肽的加FITC荧光基团修饰、同位素标记、化学发光基团或酶标试剂修饰的分子，用于检测肿瘤坏死因子alpha。
5.如权利要求1所述的肿瘤坏死因子alpha高亲和性肽在制备肿瘤坏死因子alpha拮抗剂中的应用。
6.如权利要求1所述的肿瘤坏死因子alpha高亲和性肽在制备肿瘤坏死因子alpha表达检测物和临床检测物中的应用。
7.如权利要求2所述的肿瘤坏死因子alpha的高亲和性肽的基因在制备表达肿瘤坏死因子alpha拮抗剂中的应用和在示踪检测中的应用。
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