CN115260289B - 一种炎症结肠靶向肽及其筛选方法 - Google Patents
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Abstract
本发明公开了一种炎症结肠靶向肽及其筛选方法。靶向肽的氨基酸序列选自No.1~No.7之一,且在制备炎症性肠病药物中的应用,并衍射出生物活性片段、多聚核苷酸序列、复合材料。本发明筛选得到的噬菌体对炎症结肠具有很强的靶向性,为IBD靶向治疗药物的开发与疾病诊断奠定了基础,应用前景广阔。
Description
技术领域
本发明属于生物技术领域的一种靶向肽及其筛选方法,具体是涉及了一种炎症结肠靶向肽及其筛选方法。
背景技术
炎症性肠病(IBD)是一种病因不明的肠道慢性炎症性疾病,包括克罗恩病和溃疡性结肠炎。腹痛、腹泻、粘液血便、体重减轻等疾病症状极大地降低了IBD患者的生活质量,给他们造成了巨大的经济负担。IBD的传统治疗药物包括氨基水杨酸制剂、糖皮质激素和免疫抑制剂等,它们的疗效有限且副作用明显。对IBD免疫相关信号通路的深入了解导致了生物制剂的开发,例如肿瘤坏死因子TNFα抑制剂、整合素拮抗剂和白细胞介素(IL)抗体。然而,这些生物制剂的全身性分布容易导致机会性感染、恶性肿瘤等不良事件的发生,进一步增加了疾病的复杂性。因此,开发新的靶向、高效且生物安全的IBD治疗途径已成为当务之急。
噬菌体展示技术是一种分子生物学技术,通过基因工程将外源肽或蛋白质基因插入到噬菌体蛋白质基因中,由外源基因编码的肽或蛋白质以融合蛋白的形式存在于噬菌体表面。展示的肽或蛋白质可以保持相对的空间结构和生物学活性,能够结合并识别靶分子。基于噬菌体展示技术,研究人员开发了一个随机的噬菌体多肽文库。该多肽文库含有上亿种噬菌体,每种噬菌体展示有不同随机外源多肽,构成了一个高通量的随机多肽展示库。利用随机多肽噬菌体文库,能够筛选出可靶向肿瘤或特定器官的肽。
目前只有对离体发炎肠道进行筛选的方法,还没有报道过用体内生物淘选技术进行炎症结肠靶向肽筛选的研究,而采用体内生物淘选技术筛选得到的靶向肽更符合体内复杂的生理环境。
发明内容
相较于已有的方法,本发明提出了一种炎症结肠靶向肽的体内筛选方法,利用商业化的Ph.D.-12TM噬菌体多肽文库IBD造模小鼠进行靶向筛选,筛选得到的噬菌体对炎症结肠具有很强的靶向性,为IBD靶向治疗药物的开发与疾病诊断提供了新的思路。
本发明的技术方案是:
一、一种炎症结肠靶向肽,所述靶向肽的氨基酸序列选自No.1~No.7之一。如下表:
编号 | 多肽序列 |
No、1 | VVGRAMAYSTIP |
No、2 | QMGFMTSPKHSV |
No、3 | ESYSAKHRIMLT |
No、4 | VSVPGIITGTLR |
No、5 | CFAGTPSILMLA |
No、6 | GVLNSSPSTRFV |
No、7 | ATMRGDQSVRIF |
所述的炎症结肠靶向肽在制备炎症性肠病药物中应用。
二、一种生物活性物质,包含靶向肽,包括但不限于化学偶联的化合物,改性的药物等。
该生物活性物质能够发挥所述靶向肽相同的功能。
三、一种多聚核苷酸序列,能够编码所述的靶向肽,或者能够编码所述的生物活性物质。
四、一种复合材料,包含所述靶向肽。该复合材料能够发挥所述靶向肽相同的功能。
所述亲和多肽、所述生物活性片段、所述多聚核苷酸序列或者所述复合材料在制备用于炎症的生物医学材料和药物中的应用。具体是在制备用于炎症结肠的生物医学材料和药物中的应用。
本发明是先构建IBD疾病模型,再进行体内筛选,通过心脏灌流去除不能靶向炎症结肠组织的噬菌体,解剖分离出炎症结肠组织,通过研磨得到含有靶向噬菌体的组织匀浆。然后对靶向炎症结肠的噬菌体进行DNA测序,获得噬菌体外壳蛋白展示的多肽序列。
与现有方法相比,本发明采用体内生物淘选技术筛选得到的靶向肽更符合体内复杂的生理环境,对炎症结肠具有更强的靶向性,为IBD靶向治疗药物的开发与疾病诊断奠定了基础。
附图说明
图1为实施例1中利用Ph.D.-12TM噬菌体多肽文库对IBD造模小鼠进行4轮筛选的过程中,每一轮筛选输出与投入比的折线图。
图2为实施例2中对第三轮筛选得到的炎症结肠靶向噬菌体进行DNA测序分析后,得到的一系列频次较高的序列。
图3为实施例2中对第四轮筛选得到的炎症结肠靶向噬菌体进行DNA测序分析后,得到的一系列频次较高以及第三轮和第四轮同时出现的序列。
图4为实施例3中对7种高频次噬菌体进行体内炎症结肠靶向能力验证实验的结果。
具体实施方式
下面结合实施例和附图对本发明作进一步的详细说明,以下实施例是本发明的优选实施例而本发明并不局限于以下实施例。凡在本发明的精神和原则之内,所作的的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
本发明的实施例如下:
实施例1:构建C57B6/J小鼠IBD模型
1)环境适应:选择6-8周龄的雄性C57B6/J小鼠,先在无特定病原体(SPF)环境下适应七天;
2)化学诱导:给予小鼠含3%DSS的饮用水连续七天,其中每两天更换一次新鲜的含有3%DSS的饮用水,得到IBD模型小鼠。
实施例2:炎症结肠靶向肽的筛选
1)噬菌体多肽文库注射:向造模小鼠尾静脉注射Ph.D.-12TM噬菌体多肽文库,在小鼠体内血液循环1小时;
2)心脏灌流:在气体麻醉的条件下,用无菌生理盐水缓慢灌注心脏,以洗脱不能特异性靶向炎症结肠的噬菌体;
3)组织称重:解剖取出整个炎症结肠组织,用无菌PBS洗涤,在滤纸上干燥后称重;
4)组织研磨:将炎症结肠组织转移到含有组织细胞裂解液和PMSF的研磨容器中,在冰上彻底研磨;
5)离心:将得到的组织匀浆离心,得到组织匀浆上清液;
6)涂板:提前将配制好的LB/IPTG/Xgal平板于37℃恒温箱预热。将步骤4)收集到的组织匀浆上清液取一部分稀释至合适倍数,取10μL稀释后的溶液与200μL活化的E.coil、ER2738菌液孵育15分钟。将孵育后的噬菌体/细菌混合液转移至LB/IPTG/Xgal平板上,用一次性无菌涂布棒旋涂均匀后,在37℃培养箱中培养过夜,次日计数噬菌体蓝斑数目;
7)噬菌体扩增:将剩下的组织匀浆上清液加入到活化的ER2738菌液中,在37℃温度的摇床中以220rpm培养4-6h,得到噬菌体和ER2738菌的混合液;
8)噬菌体纯化:将混合液进行离心除菌,再加入PEG/NaCl混合溶液进行沉降过夜,次日离心,用无菌PBS重悬沉淀,得到纯化后的噬菌体,重复纯化2次,纯化后的噬菌体液作为下一轮筛选的子库;
9)将步骤1-8经过3-5次重复循环,保证每一轮的噬菌体投入量一致,得到特异性靶向炎症结肠的噬菌体;
测试1:计算投入产出比
步骤6)中计数的噬菌体蓝斑数目除以投入的噬菌体数目,即可得到每一轮筛选的投入产出比。图1为炎症结肠靶向肽筛选的投入产出比结果图。根据筛选结果,每轮的投入产出比逐轮递增,说明展示有炎症结肠靶向肽的噬菌体产生了明显的富集。
DNA测序:保留筛选得到的特异性靶向炎症结肠的噬菌体样本,并对其进行基因测序,通过基因序列推导出对应的氨基酸序列,第三轮筛选的得到的频次较高的序列如图2所示,第四轮筛选得到的频次较高的序列以及第三轮第四轮同时出现的序列结果如图3所示。
测试2:通过体内实验验证筛选所得噬菌体的炎症结肠靶向能力。
1)特异性靶向炎症结肠的噬菌体的扩增与纯化:将保存的7种噬菌体样品、展示有随机序列的噬菌体和野生型噬菌体样品在室温下融化后,取200μL加入到20mL处于对数生长期的E.coil、ER2738菌液中,孵育20分钟后,置于37℃的摇床中在220rpm的转速下振摇4.5小时。将噬菌体/细菌混合液离心除菌后,在上清中加入五分之一体积的PEG/NaCl溶液,在4℃条件下过夜沉降。次日,离心后保留沉淀,用无菌PBS重悬沉淀,即完成一次噬菌体的纯化。重复两次纯化过程,得到的噬菌体溶液用于炎症结肠靶向能力验证;
7种亲和噬菌体具体分别为包含有VVGRAMAYSTIP序列、QMGFMTSPKHSV序列、ESYSAKHRIMLT序列、VSVPGIITGTLR序列、CFAGTPSILMLA序列、GVLNSSPSTRFV序列ATMRGDQSVRIF序列的噬菌体。
2)构建C57B6/J小鼠IBD模型:选择6-8周龄的雄性C57B6/J小鼠,先在无特定病原体(SPF)环境下适应七天。再给予小鼠含3%DSS的饮用水连续七天,其中每两天更换一次新鲜的含有3%DSS的饮用水,得到IBD模型小鼠。
3)噬菌体注射:对造模后的小鼠分别尾静脉注射相同浓度和体积的上述九种噬菌体,在小鼠体内血液循环1小时;
4)心脏灌流:在气体麻醉的条件下,用无菌生理盐水缓慢灌注心脏,以洗脱没有结合再炎症结肠组织部位的噬菌体;
5)组织称重:解剖取出整个炎症结肠组织,用无菌PBS洗涤,在滤纸上干燥后称重;
6)组织研磨:将炎症结肠组织转移到含有组织细胞裂解液和PMSF的研磨容器中,在冰上彻底研磨;
7)离心:将得到的组织匀浆离心,得到组织匀浆上清液;
8)涂板计数:提前将配制好的LB/IPTG/Xgal平板于37℃恒温箱预热。将步骤7)收集到的组织匀浆上清液取一部分稀释至合适倍数,取10μL稀释后的溶液与200μL活化的E.coil、ER2738菌液孵育15分钟。将孵育后的噬菌体/细菌混合液转移至LB/IPTG/Xgal平板上,用一次性无菌涂布棒旋涂均匀后,在37℃培养箱中培养过夜,次日计数噬菌体蓝斑数目;
9)靶向能力量化:步骤8)中计数的噬菌体蓝斑数目除以炎症结肠组织的质量,即可得到单位质量炎症结肠组织中结合的靶向噬菌体数目。图4为单位质量炎症结肠组织中9种噬菌体结合数目的柱状图,其中,展示有VVGRAMAYSTIP多肽的噬菌体显示出最强的炎症结肠靶向能力。
本发明涉及的氨基酸序列如下:
SEQ ID No.1;
名称:靶向肽1的氨基酸序列
来源:人工序列(Artificial Sequence)
VVGRAMAYSTIP
SEQ ID No.2;
名称:靶向肽2的氨基酸序列
来源:人工序列(Artificial Sequence)
QMGFMTSPKHSV
SEQ ID No.3;
名称:靶向肽3的氨基酸序列
来源:人工序列(Artificial Sequence)
ESYSAKHRIMLT
SEQ ID No.4;
名称:靶向肽4的氨基酸序列
来源:人工序列(Artificial Sequence)
VSVPGIITGTLR
SEQ ID No.5;
名称:靶向肽5的氨基酸序列
来源:人工序列(Artificial Sequence)
CFAGTPSILMLA
SEQ ID No.6;
名称:靶向肽6的氨基酸序列
来源:人工序列(Artificial Sequence)
GVLNSSPSTRFV
SEQ ID No.7;
名称:靶向肽7的氨基酸序列
来源:人工序列(Artificial Sequence)
ATMRGDQSVRIF。
序列表
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Claims (4)
1.一种炎症结肠靶向肽,其特征在于,所述靶向肽的氨基酸序列为SEQ ID No.1。
2.一种多聚核苷酸,其特征在于,能够编码权利要求1所述的靶向肽。
3.权利要求1所述炎症结肠靶向肽、权利要求2所述多聚核苷酸的应用,其特征在于:在制备用于靶向炎症结肠的生物医学材料中的应用。
4.权利要求1所述炎症结肠靶向肽、权利要求2所述多聚核苷酸的应用,其特征在于:在制备用于靶向炎症结肠的药物中的应用。
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WO2012096411A1 (ja) * | 2011-01-12 | 2012-07-19 | 国立大学法人宮崎大学 | 難治性炎症性腸疾患の予防又は治療方法 |
CN113262212A (zh) * | 2021-04-26 | 2021-08-17 | 北京大学口腔医学院 | 一种靶向炎症区域的细胞膜微囊泡及其应用 |
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WO2012096411A1 (ja) * | 2011-01-12 | 2012-07-19 | 国立大学法人宮崎大学 | 難治性炎症性腸疾患の予防又は治療方法 |
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