CN116789753A - 血栓靶向多肽及其应用 - Google Patents
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Abstract
本发明提供了血栓靶向多肽及其应用，所述血栓靶向多肽的氨基酸序列分别如SEQ ID NO.1～3所示。本发明通过筛选得到的靶向血栓的多肽，能够特异性靶向小鼠体内的血栓组织，而不在其他主要器官富集，和治疗药物联用可以实现血栓部位的富集，提高治疗效果并降低全身给药的副作用。本发明靶向血栓组织的多肽的优势在于：(1)序列短小，合成生产成本低；(2)多肽片段结构稳定，降低了运输保存的成本及难度；(3)可通过多种方式进行给药。在血栓靶向治疗领域，为靶向药物的研发开拓了新的思路。
Description
技术领域
本发明涉及生物医学技术领域，尤其涉及血栓靶向多肽及其应用。
背景技术
噬菌体展示技术是一种通过基因工程将外源多肽或蛋白质的DNA序列插入到噬菌体基因中，从而在噬菌体衣壳蛋白表面呈现多肽或蛋白质的技术。通过构建多个具有不同随机多肽的噬菌体库，可以实现对特定靶标的筛选，以探究多肽与靶标之间的相互作用。该技术在生物学领域被广泛应用于研究多肽与蛋白质、DNA等分子相互作用，以及研究多肽与特定组织或器官的靶向性。
血管栓塞造成的动脉粥样硬化等血栓性疾病造成每年人类死亡的主要原因之一。体内异常血栓块的形成会堵塞血管，造成血流速度减慢甚至停滞，进而影响缺血区域的供血和供氧，导致组织缺血坏死，从而威胁人类生命。
当前，临床治疗血栓性疾病的首选方式是静脉注射纤溶药物，利用药物激活纤溶酶活性，分解由致密血纤维蛋白网状结构与血小板交织而成的血栓，促进血凝块溶解，恢复血管通畅。但因溶栓药物缺乏靶向性、半衰期短，且高剂量给药会影响凝血系统，易引起出血的风险。因此，寻找新的靶向途径已成为当务之急。
发明内容
为了解决背景技术中存在的问题，本发明提供一种与血栓组织具有高度亲和力并能够特异性靶向结合血栓组织中大量存在的纤维蛋白的多肽序列，以及该多肽在血栓靶向治疗领域中的用途。
具体技术方案如下：
本发明提供了三个血栓靶向多肽，其氨基酸序列分别如SEQ ID NO.1～3所示。
具体的，SEQ ID NO.1为：GPRPVTSEIHLK；SEQ ID NO.2为：GPRPTHPIYDTV；SEQ IDNO.3为：GPRPNPNTDAHA。
本发明利用Ph.D.-12TM噬菌体多肽文库对血栓主要成分纤维蛋白进行筛选，获得能够与纤维蛋白特异性亲和的噬菌体；再对纤维蛋白亲和噬菌体进行DNA测序，根据相应的DNA序列推导出亲和多肽的氨基酸序列；最后，验证多肽对纤维蛋白以及血栓的亲和能力。
本发明还提供了包含所述血栓靶向多肽的生物活性物质，为以下几种之一：
1)共价键连接所述血栓靶向多肽的化合物；
2)与血栓靶向多肽连接的工程化噬菌体；
3)连接血栓靶向多肽的多聚体混合物。
本发明提供了一种能够编码所述血栓靶向多肽的多聚核苷酸序列。
进一步地，所述的多聚核苷酸序列，其碱基序列如SEQ ID NO.6～8所示。
本发明还提供了包含所述的血栓靶向多肽的药物组合物。
本发明还提供了所述的血栓靶向多肽，所述的生物活性物质，所述的多聚核苷酸序列，或者所述的药物组合物在制备用于靶向结合血纤维蛋白的生物检测产品和/或药物中的应用。
本发明还提供了所述的血栓靶向多肽，所述的生物活性物质，所述的多聚核苷酸序列，或者所述的药物组合物在制备用于血栓靶向治疗的生物医学材料和/或药物中的应用。
与现有技术相比，本发明具有以下有益效果：
1)本发明通过筛选得到的靶向血栓的多肽，能够特异性靶向小鼠体内的血栓组织，而不在其他主要器官富集，和治疗药物联用可以实现血栓部位的富集，提高治疗效果并降低全身给药的副作用。
2)本发明靶向血栓组织的多肽的优势在于：(1)序列短小，合成生产成本低；(2)多肽片段结构稳定，降低了运输保存的成本及难度；(3)可通过多种方式进行给药。在血栓靶向治疗领域，为靶向药物的研发开拓了新的思路。
附图说明
图1为实施例1中在随机测序60个噬菌斑后各序列的个数分布图。
图2为实施例2中投入相同数量的不同噬菌体之后，每个种类噬菌体的输出数量图；其中，Wild作为阴性对照。
图3为实施例2中不同多肽搭载荧光后与血栓进行亲和，洗涤后的荧光强度结果图。
图4为实施例3中GK，GV和GA三种多肽对体内血栓靶向性能力对比验证。
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阐述，以下实施例仅为本发明的优选实施例，并不限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
实施例1
1.噬菌体12肽库的构建
利用通过噬菌体展示技术所构建的噬菌体12肽库，针对大量存在于血管栓塞处的血栓主要成分血纤维蛋白特异性结合阳性多肽进行四轮体外筛选。
(1)血纤维蛋白底物的制备：将3mg/μL的血纤维蛋白原溶液与10U凝血酶混合于1.5mL离心管底部。孵育1h后，血纤维蛋白原转化为血纤维蛋白，用TBST洗涤血纤维蛋白三次。
(2)噬菌体库去背景：将10μL的噬菌体(第一轮筛选使用ph.D.-12噬菌体库，后面几轮筛选使用前一轮扩增得到的字库稀释到500μL的TBST溶液中，加入到一个全新的1.5mLep管内，4℃孵育1h。
(3)将步骤(2)的液体全部吸出加入到步骤(1)的血纤维蛋白中，200rpm振荡环境中室温孵育1h。
(4)将步骤(3)的上清液吸出，并用PBST洗涤血纤维蛋白10次(5000rpm，10min)，洗去未与血纤维蛋白结合的噬菌体。
(5)噬菌体洗脱：向步骤(3)中加入400μL Gly-HCl缓冲液(pH＝2.2)，室温孵育10min，并加入70μL 1M Tris-HCl(pH＝9.1)中和液进行中和，5000rpm的转速离心10min后取出上清液保存在4℃冰箱中。
(6)滴度计数：取(5)洗脱后的噬菌体10μL，用TBS按10倍梯度稀释，再加入到90μL的EER2738大肠杆菌溶液中，侵染5min；再将侵染后的大肠杆菌溶液涂板于含有IPTG和X-gal的LB琼脂平板上。将平板避光倒置于37℃恒温培养箱中12h。
利用以下公式计算噬菌体的滴度：滴度＝1000×N×M pfu/mL，N为稀释倍数，M为蓝色噬菌斑个数。
(7)噬菌体扩增：将步骤(5)的剩余噬菌体溶液加入100mL活化的ER2738菌液中，室温静置孵育15min后放入200rpm，37℃的摇床培养8h。
(8)噬菌体纯化：将(7)中扩增所得的噬菌体8000rpm离心20min，上清液转入250mL锥形瓶中，加入9mL16.7％的PEG/NaCl，振荡混匀后4℃过夜沉降；第二天将溶液12000g离心30min，除去上清液，沉淀用1mL PBS重悬，测定浓度后作为下一轮筛选的输入库。
(9)重复上述步骤进行下一轮筛选，通过4轮筛选后，得到血纤维蛋白靶向结合的噬菌体。
(10)取10μL第2-4轮获得的噬菌体按步骤(6)做噬菌体滴度实验，获得蓝色噬菌斑。
2.噬菌体测序
随机挑选60个蓝色噬菌斑置入5mL含有四环素的LB培养基摇菌管中，150rpm，37℃过夜培养。将培养液用测序引物5’-CCCTCATAGTTAGCGTAACG-3’进行DNA测序，获得外源多肽序列及其个数分布，外源多肽序列具体如下：
GK的氨基酸序列为：GPRPVTSEIHLK(核苷酸序列ggtcctcgtcctgtgacttcggagattcatctgaaa)；GV的氨基酸序列为：GPRPTHPIYDTV(核苷酸序列gggccgcggcctactcatccgatttatgatactgtt)；GA的氨基酸序列为：GPRPNPNTDAHA(核苷酸序列gggcctcggcctaatccgaatacggatgcgcatgct)；HG的氨基酸序列为：HLFPSKPVMDAG；WR的氨基酸序列为：WVDVLELHTPVR。
结果如图1所示，图1为在噬菌体侵染大肠杆菌后挑选噬菌斑，对比每一种噬菌体的数量，在随机测序了60个噬菌斑后各序列的个数分布。由图1可知，五种序列的噬菌体个数频率不一，GK的个数最多，GV，GA，HG以及WR次之。表明在筛选过程中各个序列噬菌体的富集程度不一，GK的富集能力最强。
实施例2
通过构建体外血纤维蛋白模型对靶向肽进行亲和性验证
(1)将实施例1筛选所得的较高频次噬菌体克隆和野生M13噬菌体分别进行扩增、纯化。
(2)取100μL噬菌体溶液(1010pfu/mL)加入含血纤维蛋白的试管中，孵育1小时。
(3)用PBST洗涤血纤维蛋白10次(5000rpm，10min)，洗去未与血纤维蛋白结合的噬菌体。
(4)加入400μL Gly-HCl缓冲液(pH＝2.2)，室温孵育10min，并加入70μL 1M Tris-HCl(pH＝9.1)中和液进行中和，5000rpm的转速离心10min。
(5)取(4)洗脱后的噬菌体10μL，用TBS按10倍梯度稀释，再加入到90μL的EER2738大肠杆菌溶液中，侵染5min。再将侵染后的大肠杆菌溶液涂板于含有IPTG和X-gal的LB琼脂平板上。将平板避光倒置于37℃恒温培养箱中12h。
利用以下公式计算噬菌体的滴度：滴度＝1000×N×M pfu/mL，N为稀释倍数，M为蓝色噬菌斑个数。
(6)对噬菌体的氨基酸序列进行测序，得出亲和性最高的序列。
(7)将不同多肽(GK/GV/GA)与罗丹明B连接，通过荧光成像验证其亲和力。在96孔板中加入100μL血纤维蛋白原和10u凝血酶形成血纤维蛋白。
(8)PBS洗涤3次后加入经罗丹明修饰的多肽与血纤维蛋白孵育1h,孵育结束后用PBS洗涤5次，通过荧光显像仪测试其荧光强度。
结果如图2和3所示，图2为投入相同数量的不同噬菌体之后，每个种类的噬菌体输出数量。Wild作为阴性对照。由图2可知，GK的输出量最高，表现出最佳的亲和力。图3为不同的多肽搭载荧光后与血栓进行亲和，洗涤后表现出不同的荧光强度，荧光强度越强表示对血栓的亲和能力越强。由图2可知，GK最强。(RB为纯荧光Rhodamine B，作为阴性对照)。结果表明，GK，GV和GA在体外对血纤维蛋白有亲和能力，并且GK的亲和能力最强。
实施例3
通过构建小鼠体内血栓模型对靶向肽进行亲和性验证，具体步骤为：
(1)将筛选所得的较高频次噬菌体克隆和野生M13噬菌体分别进行扩增、纯化。
(2)取100μL噬菌体溶液(1010pfu/mL)加入含血纤维蛋白的试管中，孵育1小时
(3)用PBST洗涤血纤维蛋白10次(5000rpm，10min)，洗去未与血纤维蛋白结合的噬菌体
(4)加入400μL Gly-HCl缓冲液(pH＝2.2)，室温孵育10min，并加入70μL 1M Tris-HCl(pH＝9.1)中和液进行中和，5000rpm的转速离心10min。
(5)取(4)洗脱后的噬菌体10μL，用TBS按10倍梯度稀释，再加入到90μL的EER2738大肠杆菌溶液中，侵染5min。再将侵染后的大肠杆菌溶液涂板于含有IPTG和X-gal的LB琼脂平板上。将平板避光倒置于37℃恒温培养箱中12h。利用以下公式计算噬菌体的滴度：滴度＝1000×N×M pfu/mL，N为稀释倍数，M为蓝色噬菌斑个数。
(6)对噬菌体的氨基酸序列进行测序，得出亲和性最高的序列。
(7)将不同多肽(GK/GV/GA)与罗丹明B连接，通过尾静脉注射入颈动脉血栓模型的小鼠体内，1h后麻醉小鼠，对小鼠进行心脏灌流取下颈动脉进行荧光强度的观测。
(8)麻醉小鼠，切开小鼠胸口皮肤，分离心脏上方肌肉，剪断心脏上方的肋骨，利用手术器械撑开胸腔从而暴露心脏。将注射器从心尖插入心脏内，用手术剪剪开右心耳，同时打开生理盐水阀门使生理盐水通过心脏泵入体内，血液从右心耳置换流出直至小鼠四肢变白。然后将生理盐水切换成4％多聚甲醛以同样的方法再进行灌注。灌注结束后分理出颈动脉，剪下血栓部分浸泡于4％多聚甲醛溶液中。
(9)将颈动脉置于荧光显像仪下通过荧光强弱测试其荧光强度。
结果如图4所示，说明GK，GV和GA在体内对血栓有靶向亲和能力，且GK的亲和能力最强。
Claims (7)
1.血栓靶向多肽，其特征在于，氨基酸序列如SEQ ID NO.1～3所示。
2.包含如权利要求1所述血栓靶向多肽的生物活性物质，其特征在于，为以下几种之一：
1)共价键连接所述血栓靶向多肽的化合物；
2)与血栓靶向多肽连接的工程化噬菌体；
3)连接血栓靶向多肽的多聚体混合物。
3.一种能够编码权利要求1所述血栓靶向多肽的多聚核苷酸序列。
4.如权利按要求3所述的多聚核苷酸序列，其特征在于，其碱基序列如SEQ ID NO.6～8所示。
5.包含权利要求1所述的血栓靶向多肽的药物组合物。
6.如权利要求1所述的血栓靶向多肽，权利要求2所述的生物活性物质，权利要求3所述的多聚核苷酸序列，或者权利要求5所述的药物组合物在制备用于靶向结合血纤维蛋白的生物检测产品和/或药物中的应用。
7.如权利要求1所述的血栓靶向多肽，权利要求2所述的生物活性物质，权利要求3所述的多聚核苷酸序列，或者权利要求5所述的药物组合物在制备用于血栓靶向治疗的生物医学材料和/或药物中的应用。
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