CN114437175B - 一种肽探针及其应用和包含该肽探针的试剂盒 - Google Patents
一种肽探针及其应用和包含该肽探针的试剂盒 Download PDFInfo
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Abstract
本发明涉及肽探针技术领域，具体涉及一种肽探针及其应用和包含该肽探针的试剂盒。肽探针为Y形结构，Y形结构的一条分支为Suc‑Val‑Pro‑PheP‑(OPh)2，另一支分支为Asp‑Ala‑His‑Lys‑孟加拉红，所述肽探针用于制备血清Cu(Ⅱ)及组织蛋白酶G的检测试剂盒。本发明提供的肽探针包含一条蛋白酶抑制剂分支和一条金属离子螯合剂分支，能够同时检测血液中组织蛋白酶G和Cu(Ⅱ)水平；该肽探针的化学结构非常简单，与抗体和酶相比，制造成本低廉，且与临床样本中各种生物分子的相互作用模式也相对清晰，不易受到样品中非特异性干扰蛋白的影响。
Description
技术领域
本发明涉及肽探针技术领域，具体涉及一种肽探针及其应用和包含该肽探针的试剂盒。
背景技术
近年来，随着人们生活水平发展及其饮食结构改变，急性心肌梗死患病率逐年增加，已成为危害人类健康的首要疾病。再灌注治疗是急性ST段抬高心肌梗死最主要的治疗措施，再灌注损伤是其常见并发症，因其发病过程易于一般的损伤出血相混淆，最终延误治疗时间，因此，如何提高缺血再灌注损伤的诊断显得尤为关键。由于心肌在再灌注过程中会引发过度的氧化应激和炎症反应，细胞还原/氧化平衡被破坏，生成大量活性氧，释放组织蛋白酶G并通过中性粒细胞进入血液。组织蛋白酶与活性化学物质会发生协同作用诱导缺血/再灌注后的心肌重塑和功能障碍，因此有必要对这些酶与活性化学物质进行持续的检测，以提供预后信息。
然而，由于传统的蛋白质检测是基于抗体和酶的生物分析，这些生物大分子相对昂贵，因此对大多数患者而言，每天频繁进行检测是难以负担的。基于此，有必要提供一种成本更低的检测试剂。
发明内容
针对传统生物分析技术检测成本高的技术问题，本发明提供一种肽探针及其应用和包含该肽探针的试剂盒，该肽探针包含一条蛋白酶抑制剂分支Suc-Val-Pro-PheP-(OPh)2和一条金属离子螯合剂分支Asp-Ala-His-Lys-孟加拉红，能够同时检测血液中组织蛋白酶G和Cu(Ⅱ)水平；该肽探针的化学结构非常简单，与抗体和酶相比，制造成本低廉，且与临床样本中各种生物分子的相互作用模式也相对清晰，不易受到样品中非特异性干扰蛋白的影响。
第一方面，本发明提供一种肽探针，所述肽探针为Y形结构，Y形结构的一条分支为Suc-Val-Pro-PheP-(OPh)2，另一支分支为Asp-Ala-His-Lys-孟加拉红。
进一步的，所述肽探针的结构式如下：
第二方面，本发明提供一种上述肽探针的应用，所述肽探针用于制备血清Cu(Ⅱ)及组织蛋白酶G的检测试剂盒。
第三方面，本发明提供一种包含上述肽探针的试剂盒。
进一步的，所述肽探针的剂型为冻干粉。
进一步的，所述肽探针的剂型为溶液。
进一步的，肽探针溶液的浓度为20μM，肽探针溶液由PBS缓冲液溶解肽探针冻干粉制备。
进一步的，PBS缓冲液的浓度为100mM，pH值为7.4。
进一步的，肽探针冻干粉的纯度为>95％。
本发明的有益效果在于：
本发明提供的Y形肽探针的其中一条分支Suc-Val-Pro-PheP-(OPh)2中包含一个特殊的P原子，当该序列被酶识别后，该P原子能够不可逆地攻击酶的丝氨酸部分，并形成共价键；另一条分支是Asp-Ala-His-Lys-孟加拉红，其中孟加拉红能起到光敏剂作用，该分支不仅具有捕获Cu(Ⅱ)的作用，更重要的是，其能够利用血液样品中的干扰蛋白实现检测信号的有效放大，具体原理如下：
本发明提供的肽探针与样品一起孵育，Cu(Ⅱ)和组织蛋白酶G能够被对应的分支捕获，同时借助电化学扫描和白光(可见光)照射，Cu(Ⅱ)产生的电流峰值能够作为信号读出，白光(可见光)则可激发孟加拉红产生活性氧；另一方面，电化学扫描还能够将Cu(Ⅱ)还原至Cu(Ⅰ)，Cu(Ⅰ)能够像过氧化物酶一样利用活性氧，通过过度的氧化应激破坏离它最近的化学键，如金属离子螯合剂本身的肽键，这部分被释放的螯合剂可以充当人工酶，利用光和电化学能量生成活性氧，并氧化修饰血清样本中的干扰蛋白，由于干扰蛋白与组织蛋白酶G都含有络氨酸，它们会交联形成双络氨酸产物，使干扰蛋白转化为2D纳米蛋白质网络，实现组织蛋白酶G检测信号的放大。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，对于本领域普通技术人员而言，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1是验证例1的ITC实验结果图。
图2是验证例2的SPR实验结果图。
图3是验证例3无组织蛋白酶G存在时的实验结果图。
图4是验证例3有组织蛋白酶G存在时的实验结果图。
图5是设计肽探针的工作原理图。
图6是电化学扫描前后的原子力显微镜照片。
图7是应用例1血清样品与患者样品的检测结果对照图。
具体实施方式
为了使本技术领域的人员更好地理解本发明中的技术方案，下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都应当属于本发明保护的范围。
实施例1
一种Y形结构的肽探针，该Y形结构的一条分支为Suc-Val-Pro-PheP-(OPh)2，另一支分支为Asp-Ala-His-Lys-孟加拉红，具体结构式如下：
该肽探针委托上海科肽生物科技有限公司合成，合成方法为本领域技术人员都知道的普通方法，该肽探针的剂型为冻干粉，纯度>95％。
该肽探针可用于制备血清Cu(Ⅱ)及组织蛋白酶G的检测试剂盒。
为验证该肽探针对血清组织蛋白酶G的特异性等特性，用PBS缓冲液(100mM，pH值为7.4，下同)溶解该肽探针及人重组组织蛋白酶G(购自OriGene，CAT#：TP761143)，得到浓度为20μM的肽探针溶液和浓度为100nM的靶蛋白溶液。将肽探针溶液、靶蛋白溶液分装成小瓶，以确保每个小瓶仅够一次性使用，从而避免冻融循环。
样品共包括三组，第一组为标准样品，使用PBS缓冲液稀释人重组组织蛋白酶G；第二组为血清样品，使用随机抽取的10例健康志愿者血清稀释人重组组织蛋白酶G，健康志愿者心肌肌钙蛋白和CK-MB值正常，运动心电图阴性，定量冠状动脉造影显示直径狭窄<20％；第三组为患者样品，由随机抽取的10例ST段抬高的急性心肌梗死患者提供，患者在胸痛发作后12小时内成功经皮冠状动脉介入治疗，并于再灌注48小时后采集血样、分离血清。下述验证例未特别说明的，所使用的均为标准样品。
验证例所使用的水为MiliQ水纯化系统制备的双蒸水(电阻率为18MΩ·cm)，其他试剂均为分析级。
验证例1ITC实验
使用等温滴定量热仪(MicroCal iTC200，GE Healthcare Life Sciences)验证肽探针与组织蛋白酶G之间的非共价氢键识别，由于实施例1肽探针(以下称“设计肽探针”)其中一条分支中Phe的特殊P原子会干扰非共价过程，因此本实验使用没有活性P原子、其他结构不变的对照肽探针进行。本实验在25℃下进行，探针溶液为含有0.01mM对照肽探针的PBS缓冲液，蛋白质溶液为含有1μM组织蛋白酶G的PBS缓冲液，共连续向蛋白质溶液中滴加38次探针溶液，每次探针溶液的体积为1μL，相邻两次滴定间隔至少120s，滴定前所有溶液都去除气泡。
图1上图表示用探针溶液滴定蛋白质溶液时的热峰值，下图为根据上图每个热峰值进行积分计算得出的ITC曲线，可以看出，该ITC曲线为典型的饱和曲线，表明肽探针可以与组织蛋白酶G特异性相互作用。
验证例2SPR实验
使用Autolab ESPRIT系统(Echo Chemie B.V.)检测肽探针与组织蛋白酶G的共价反应，该系统配备670nm单色p偏振光光源。分别将修饰后的设计肽探针与对照肽探针滴加在传感表面上，并记录两种肽探针与组织蛋白酶G相互作用的表面等离子体共振情况。
其中，传感表面按照如下方法制备：
将氧化铟锡(ITO)导电玻璃切成小块，并依次用双蒸水、乙醇和甲醇超声清洗，以去除ITO导电玻璃表面吸附的有机杂质，最后用双蒸水再次冲洗ITO导电玻璃表面。
用16-磷酸十六烷酸残基(Sigma-Aldrich)对设计肽探针及对照肽探针进行修饰，修饰方法参考文献Biointerfaces on indium-tin oxide prepared fromorganophosphonic acid self-assembled monolayers(Chockalingam et al.，2014)。设计肽探针经修饰后，结构式如下：
将含有修饰后的肽探针的PBS缓冲液分别滴在处理后的ITO导电玻璃表面，并用记号笔标出液滴覆盖区域，然后将ITO导电玻璃在4℃下避光放置16h，进行自组装。最后用大量双蒸水冲洗ITO导电玻璃，在高纯氮气流下干燥。
将标准样品分别滴加在载有两种不同肽探针的传感表面上，记录两种肽探针的SPR响应情况，结果如图2中0-1段所示。由缺少特殊活性P原子的对照肽探针的曲线可以看出，组织蛋白酶G流过传感表面，肽探针可以首先以非共价的形式捕获它，这与验证例一结果无异。对比设计肽探针的曲线可以看出，设计肽探针曲线略高于对照肽探针曲线。
为进一步研究该现象，首先使用温和的表面活性剂处理载有两探针的传感表面，结果如图2中1-2段所示，发现对照肽探针的SPR信号变化明显，而设计肽探针的信号丢失程度要小得多，这表明至少有一些表面捕获的组织蛋白酶G与设计肽探针形成了稳定的连接；然后再加入变性洗涤剂洗涤传感表面，结果如图2中2-3段所示，对照肽探针的SPR信号几乎锐减至添加组织蛋白酶G前的基线水平，而设计肽探针仍保留强烈的残余信号，进一步证实设计肽探针可以与部分表面捕获的组织蛋白酶分子形成共价键。
验证例3
按照验证例2相同方法，制备载有修饰后的设计肽探针的传感表面，将传感表面置于含有不同Cu(Ⅱ)浓度(0.33、0.1、0.033、0.01、0.0033、0.001nM)的PBS缓冲液中各孵育5min，然后取出置于PBS缓冲液中使用CHI660D电化学工作站在-0.2～0.8V范围内进行电化学扫描，电化学测试采用三电极体系，传感表面为工作电极，饱和甘汞电极(SCE)为参比电极，铂丝为对电极，扫描时间为5s。测试结果如图3a所示，在没有组织蛋白酶G存在的情况下，几种不同量的Cu(Ⅱ)导致传感表面的电化学信号响应成比例。
将传感表面置于含有0.33nM Cu(Ⅱ)的PBS缓冲液中孵育5min，然后分别设置扫描时间为5s、1min、3min、5min、7min、10min，其余条件同上，测试结果如图3b所示。将扫描后的传感表面取出，并向PBS缓冲液中放入新的载有修饰后的设计肽探针的传感表面，并再次进行电化学测试，记录PBS缓冲液中残余的Cu信号，结果如图3c所示。对比图3b、c可以看出，随电化学扫描时间的延长，Cu信号逐渐从传感表面转移至缓冲液中。在组织蛋白酶G存在下重复上述实验，结果基本相似，图4a、b分别对应图3b、c条件，说明设计肽探针与组织蛋白酶的相互作用可以发生在探针-离子相互作用一侧，几乎没有可见的干扰。
在白光照明下，向缺少组织蛋白酶G的含Cu(Ⅱ)PBS缓冲液中加入1μM的荧光红染料(amplex red，波长570nm)，将载有修饰后的设计肽探针的传感表面置于该缓冲液中孵育不同时间，使用配备氙灯的QM-4/2005荧光光谱仪测量传感表面的荧光发射光谱，以检测铜离子结合设计肽探针生成活性氧的能力，结果如图3d所示。设计肽探针上的孟加拉红(光敏剂)产生的活性氧可以被设计肽探针所捕获的Cu(Ⅱ)利用，在使用活性氧氧化荧光红染料后，Cu(Ⅱ)被还原，此时需要进行电化学扫描以将离子循环回Cu(Ⅱ)状态。作为对照，在没有电化学扫描的情况下，重复该实验，仅能观察到可忽略的荧光信号。综上，验证例1-3证实了设计肽探针可以与组织蛋白酶G、Cu(Ⅱ)相互作用，且Cu(Ⅱ)能够以光电化学的形式激发设计肽探针生产、消耗活性氧。
如图5所示，本发明的设计肽探针工作时，血清中的微量Cu(Ⅱ)能够消耗设计肽探针上光敏剂产生的活性氧以氧化白蛋白等血清蛋白，并与组织蛋白酶G一起共价锚定在传感表面上，达到放大信号的效果。由图6的原子力显微镜照片可以发现，Cu(Ⅱ)和组织蛋白酶G被捕获在传感表面后，未进行电化学扫描时，传感表面的形貌相对平坦，经过电化学扫描后，被氧化的血清蛋白与组织蛋白酶G发生交联，传感表面出现网状形貌。
应用例1
分别应用设计肽探针检测血清样品和患者样品，检测方法如下：
在白光照明下，将样品分别滴加在载有修饰后的设计肽探针的传感表面上，孵育5min后，以传感表面为工作电极、饱和甘汞电极(SCE)为参比电极、铂丝为对电极，使用CHI660D电化学工作站进行测试，首先以0.1V/s的电压扫描速率在0.5～1V范围内进行电化学扫描10min；然后在10μM urazol交联剂存在下，对传感表面进行第二步电化学扫描(扫描范围-0.3～0.9V)，分析组织蛋白酶G水平；同时对传感表面的Cu(Ⅱ)进行荧光强度测试。
结果如图7所示，患者的Cu(Ⅱ)和组织蛋白酶G水平均有统计学意义上的升高，提示随后发生心肌重塑和功能障碍的风险高。
尽管通过参考附图并结合优选实施例的方式对本发明进行了详细描述，但本发明并不限于此。在不脱离本发明的精神和实质的前提下，本领域普通技术人员可以对本发明的实施例进行各种等效的修改或替换，而这些修改或替换都应在本发明的涵盖范围内/任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到变化或替换，都应涵盖在本发明的保护范围之内。
Claims (7)
1.一种肽探针，其特征在于，所述肽探针的结构式如下：
2.一种如权利要求1所述的肽探针的应用，其特征在于，所述肽探针用于制备血清Cu(Ⅱ)及组织蛋白酶G的检测试剂盒。
3.一种包含如权利要求1所述的肽探针的试剂盒。
4.如权利要求3所述的试剂盒，其特征在于，所述肽探针的剂型为冻干粉。
5.如权利要求4所述的试剂盒，其特征在于，肽探针冻干粉的纯度为>95％。
6.如权利要求3所述的试剂盒，其特征在于，所述肽探针的剂型为溶液。
7.如权利要求6所述的试剂盒，其特征在于，肽探针溶液的浓度为20μM。
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