CN106518970B - 可与甲胎蛋白特异结合的多肽序列及其应用 - Google Patents
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Abstract
本发明涉及一条可与甲胎蛋白特异结合的多肽序列及其应用，属于多肽设计及病毒抗原检测领域。本发明借助于分子对接及虚拟筛选技术，在甲胎蛋白晶体结构的基础上，通过分子对接技术，搜寻虚拟多肽库中与靶标蛋白结合模式与亲和力最佳的多肽配体，最终得到可与甲胎蛋白特异结合的多肽序列，其多肽序列为WYRVGY，即P217。人工合成P217，并与甲胎蛋白进行亲和力鉴定，P217序列与人工表达的甲胎蛋白之间相互作用的平衡解离常数KD为1.47×10‑9M，即1.47nM，说明亲和力较好。本发明设计而成的P217序列与人工表达蛋白、人工纯化蛋白均可以结合，除与甲胎蛋白反应性较高外，与其它蛋白均不发生交叉性反应，特异性较好。
Description
技术领域
本发明涉及一条可与甲胎蛋白特异结合的多肽序列及其应用，属于多肽设计及病毒抗原检测领域。
背景技术
基于分子对接的虚拟筛选技术是一种新兴的研究多肽与蛋白质之间相互作用的技术手段。这一技术主要是运用计算机快速运算实现多肽与相应靶标蛋白在空间构象上的对接，将虚拟多肽数据库中的分子逐一与靶标蛋白晶体结构的特定活性位点进行对接，通过计算机快速运算并不断调整多肽与靶标蛋白结合的位置、构象、分子内部可旋转键的二面角和靶标蛋白的氨基酸残基侧链和骨架，寻找多肽分子与靶标蛋白在空间结构上的最佳构象，并预测两者之间的结合模式与亲和力，通过评分值挑选出接近天然构象的与靶标蛋白亲和力最佳的多肽配体的一种理论模拟分子间相互作用的方法。
甲胎蛋白(AFP)是一种糖蛋白，正常情况下，这种蛋白主要来自胚胎的肝细胞，胎儿出生后约两周甲胎蛋白从血液中消失，因此正常人血清中甲胎蛋白的含量不到20μg/L。甲胎蛋白在产妇羊水或母体血浆中可用于胎儿产前监测。如在神经管缺损、脊柱裂、无脑儿等时，AFP可由开放的神经管进入羊水而导致其在羊水中含量显著升高。胎儿在宫腔内死亡、畸胎瘤等先天缺陷亦可有羊水中AFP增高。AFP可经羊水部分进入母体血循环。在85％脊柱裂及无脑儿的母体，血浆AFP在妊娠16-18周可见升高而有诊断价值。在成人，AFP可以在大约80％的肝癌患者血清中升高，在生殖细胞肿瘤出现AFP阳性率为50％。在其它肠胃管肿瘤，如胰腺癌或肺癌及肝硬化等患者体内也会出现不同程度的升高。因此，甲胎蛋白的检测结果可以作为多种病症的判定依据之一。
发明内容
本发明借助于分子对接及虚拟筛选技术，在甲胎蛋白晶体结构的基础上，通过分子对接技术，搜寻虚拟多肽库中与靶标蛋白结合模式与亲和力最佳的多肽配体，其多肽序列为WYRVGY，即P217。人工合成P217，利用表达纯化的甲胎蛋白进行表面等离子体共振(Surface Plasmon Resonance，SPR)与ELISA结合试验，结果表明，人工合成的P217与甲胎蛋白有良好的结合能力，由此证明，借助本发明设计而成的多肽，可用于血清中甲胎蛋白进行定量和定性的快速检测。
为了实现上述目的，本发明所采用的技术方案是：
可与甲胎蛋白特异结合的多肽序列，所述的多肽序列为WYRVGY。
所述的可与甲胎蛋白特异结合的多肽序列，包括以所述的多肽序列为核心，任何对该多肽序列所进行的相应调整或修饰；修饰材料包括但不限制在纳米材料、荧光材料、酶类、生物素及特定的蛋白质。
将所述的多肽序列用于血清中甲胎蛋白的检测，其中包括但不局限于酶联免疫吸附试验(ELISA)检测。
一种所述的多肽序列在甲胎蛋白的快速检测中的应用，其中包括但不限于哺乳动物血清中、人工重组表达或人工纯化手段得到的甲胎蛋白。
一种所述的多肽序列在甲胎蛋白定量和定性的快速检测中的应用。
本发明的有益效果：
1、本发明借助于分子对接及虚拟筛选技术，在甲胎蛋白晶体结构的基础上，通过分子对接技术，搜寻虚拟多肽库中与靶标蛋白结合模式与亲和力最佳的多肽配体，最终得到可与甲胎蛋白特异结合的多肽序列，其多肽序列为WYRVGY，即P217。人工合成P217，并与甲胎蛋白进行亲和力鉴定，P217序列与人工表达的甲胎蛋白之间相互作用的平衡解离常数KD为1.47×10-9M，即1.47nM，说明亲和力较好。
2、由于人工表达甲胎蛋白的局限性，针对甲胎蛋白的抗体的特异性比较差，本发明设计得到的P217序列很好地避免了这一难题，实现快速人工合成与低廉检测成本。
3、本发明设计而成的P217序列与人工表达蛋白、人工纯化蛋白均可以结合，除与甲胎蛋白反应性较高外，与其它蛋白均不发生交叉性反应，特异性较好。
4、本发明与传统噬菌体多肽库筛选相比，具有简单，快速及成本低的特点；通过计算机辅助实施分子对接，可为实现甲胎蛋白结构功能解析提供较好的理论指导。
5、本发明与人工表达纯化的蛋白进行免疫来获得针对蛋白抗体的过程相比，具有操作简单，省时省力，费用低等优点；通过对筛选P217序列进行标记，可实现对甲胎蛋白进行定性和定量的快速检测。
附图说明
图1为P217序列与甲胎蛋白的对接结果展示。
图2为P217序列与人工表达甲胎蛋白的SPR亲和力鉴定结果。其中，纵坐标代表传感器所检测到的信号值；横坐标代表样品在传感器中相互作用的时间。图中，从上到下各曲线对应的P217溶液浓度逐渐降低。
图3为P217序列与甲胎蛋白阳性血清的ELISA鉴定结果。
具体实施方式
以下结合实施例对本发明的具体实施方式作进一步详细说明。
实施例1分子对接及虚拟肽库的筛选
1、甲胎蛋白的准备
从PDB数据库中搜寻哺乳动物甲胎蛋白的晶体结构(3MRK)，借助计算机程序对晶体结构进行分析，选定其第100到200氨基酸残基之间的特定氨基酸残基作为对接设定区域，以进行分子对接。
2、虚拟多肽库的设计
建立不同氨基酸残基的空间结构，借助计算机程序，实现对输入目标多肽进行批量生成，以满足分子对接计算机程序的自动调用和处理。虚拟多肽库均以直链形式生成，不对任何侧链、首尾氨基和羧基进行修饰。单一虚拟多肽库的生成以不超过四位氨基酸残基为宜。
3、对接结果的评断
分别计算多肽与蛋白结合的自由能，氢链，范得华力等力学参数进行综合评定，以此来判定筛选结果，并筛选得到P217，其多肽序列为色氨酸-酪氨酸-精氨酸-缬氨酸-甘氨酸-酪氨酸(Trp-Tyr-Arg-Val-Gly-Tyr，简写WYRVGY)(SEQ ID NO.1)，其与甲胎蛋白对接的相互作用位置结果见图1。
实施例2 P217序列与人工表达甲胎蛋白的亲和力鉴定
1、将人工表达纯化的甲胎蛋白采用PBS缓冲液(pH 7.4)稀释为1μg/ml(蛋白量计)，采用活泼酯法，分别将1-(3-二甲氨基丙基)-3-乙基碳二亚胺/N-羟基琥珀酰亚胺(EDC/NHS，1:1)、甲胎蛋白注入安装有氨基芯片的SPR检测仪中，保证EDC/NHS和甲胎蛋白分别与氨基芯片相互作用5min，实施甲胎蛋白与氨基芯片的偶联。偶联完成后，该传感器就可以用于测量甲胎蛋白与P217序列之间的相互作用。
2、向传感器中注入250μl PBS缓冲液(pH 7.4)，以最大流速(150μl/min)运行缓冲液，达到信号基线，将缓冲液的流速降至20μl/min，以获得较为稳定的基线。
3、将人工合成并在氨基端生物素化修饰的P217干粉使用PBS缓冲液(pH 7.4)分别稀成浓度为303.5μM、75.87μM、37.94μM、18.97μM，4.72μM、2.37μM的P217溶液，从低浓度开始依次向传感器中注入250μl的P217溶液，每次注入样品均采用20μl/min的流速并且与传感器相互作用5min，最后用PBS缓冲液(pH 7.4)冲洗5min。以得到的不同浓度P217溶液与甲胎蛋白相互作用的结合与解离曲线为依据，进行P217序列与甲胎蛋白相结合的亲和力分析(见图2)。
结果表明，P217序列与人工表达的甲胎蛋白具有较好的亲和性结合，两者之间相互作用的平衡解离常数KD为1.47×10-9M，即1.47nM。
实施例3 P217序列与血清的甲胎蛋白的ELISA鉴定
1、取哺乳动物血液(阳性)进行离心，去除血细胞，取血清进行酶标板包被；以同样方式将不同样品，即正常人血清、兔血清、牛血清进行酶标板包被，并和同体积PBS缓冲液同时作为对照。其中，包被抗原均采用碳酸盐(CBS)缓冲液进行稀释，每孔50μl加入96孔酶标板中，置于4℃条件下过夜，用PBST缓冲液洗涤5次后再用质量分数2％的BSA液进行封闭。
2、将人工合成并在氨基端生物素化修饰的P217干粉使用PBS缓冲液(pH 7.4)稀释成500ng/ml的浓度，以每孔50μl的体积加入到上述酶标板中，混匀后，置于37℃条件下，避光温育30min。
3、用PBST缓冲液洗涤5次，并甩干酶标板孔中的液体；使用PBST缓冲液稀释偶联辣根过氧化物酶的链霉亲和素(1:1000)，以每孔50μl的体积加入甩干的酶标板中，混匀后，置于37℃条件下，避光温育30min。
4、根据试验所需量，将TMB显色液以每孔100μl的体积加入到上述酶标板中，充分混匀30s之后，室温条件下，显色10min。
5、将2M的硫酸终止液以每孔50μl的体积加入上述酶标板中，充分混匀30s之后，在酶标仪器上，读取各孔在450nm处的吸光值，判定结果。
结果表明，P217序列与哺乳动物血清中的甲胎蛋白具有较好的亲和性与特异性结合，与其它阴性血清均不发生反应(见图3)。
SEQUENCE LISTING
<110> 郑州大学第一附属医院
<120> 可与甲胎蛋白特异结合的多肽序列及其应用
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 6
<212> PRT
<213> 人工序列
<400> 1
Trp Tyr Arg Val Gly Tyr
1 5
Claims (2)
1.可与甲胎蛋白特异结合的多肽序列，其特征在于，所述的多肽序列为WYRVGY。
2.一种如权利要求1所述的多肽序列在制备甲胎蛋白的快速检测试剂或者试剂盒中的应用，其中包括但不限于哺乳动物血清中、人工重组表达或人工纯化手段得到的甲胎蛋白。
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