CN110615831B - 一种环肽及其应用 - Google Patents
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- CN110615831B CN110615831B CN201910923710.4A CN201910923710A CN110615831B CN 110615831 B CN110615831 B CN 110615831B CN 201910923710 A CN201910923710 A CN 201910923710A CN 110615831 B CN110615831 B CN 110615831B
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Abstract
本发明设计了一种环肽，其特征在于，包括两部分，a.表面偶联半胱氨酸，b.天然结合蛋白模拟肽链功能序列——Arg，Phe，Ser，Tyr和Asp。本发明的优点是：本发明主要是设计了一种制备简便、绿色环保、操作简单，效果明显、价格低廉的用于黄曲霉毒素的新型探针。本发明主要是环肽序列的设计，比较新颖独特，环肽的性质比较稳定，找出了本发明设计的环肽结构中用于检测黄曲霉毒素灵敏度最高的环肽。
Description
技术领域
本发明涉及的是一种能够检测黄曲霉毒素的环肽，属于食品安全检测领域。
背景技术
酶是由活细胞产生的、对其底物具有高度特异性和高度催化效能的蛋白质或RNA，有高效催化功能的生物催化剂，自然界的一切生命现象都与酶的参与有关。酶催化反应因其具有高的催化效率、高的反应专一性和温和的反应条件等特点，使其在医药、化工、食品和农业等领域发挥着重要作用。然而天然酶在实际应用上存在明显缺陷，比如来源有限、提纯困难、易失活、价格昂贵、使用条件苛刻等等。这些都使得天然酶的应用受到了极大的限制。因而，开发具有酶功能的模拟酶的体系便应运而生。诺贝尔奖获得者Cram、Pederson与Lehn根据酶催化反应机理，提出了主-客体化学和超分子化学，奠定了模拟酶的重要理论基础。酶,在生命体中发挥着重要的作用,也在制药学、生命分析化学等实际领域中得到广泛的应用。但因为在实际使用中天然酶存在容易失活、降解,以及制备和纯化的操作繁琐等缺陷,寻找各种天然酶的替代物,即模拟酶,是解决这些问题的关键。
环肽是具有环状结构的多肽链。主要有自然界提取和人工合成，环的结构可以通过连接肽的一端与另一端通过酰胺键形成，或其他稳定的化学键如酯键、醚、硫醚、硫等。N-C(或头部到尾部)环化反应大多是氨基酸和羧基之间的酰胺键的形成，许多生物活性环肽都是由这种方式形成。
自然界中发现的几种环状肽在临床中被广泛应用，例如短杆菌肽和短杆菌酪的杀菌活性，环孢素A的免疫抑制活性和万古霉素的抗菌活性等等。虽然多肽一般被认为在药物开发方面一直被诟病，但肽药物也表现出一系列的优势。与天然分子相比，肽模拟物具有毒性小，不易蓄积的特点。即使肽可以被迅速降解，但这也会是一件好事。多肽类药物可减少对靶分子的有害作用，他们会迅速消失的，第二，肽模拟物被迅速代谢，由蛋白水解酶降解，成功被吸收。降解产物只是氨基酸，不会有毒性。肽可以非常有选择性地作用于它们的靶点。考虑到这些优势，市场上有许多肽类药物也就不足为奇了。这些肽类药物包括受体激动剂和拮抗剂、肽类激素和类似物、HIV蛋白酶抑制剂等。除了多肽作为药物分子的优点外，环状肽还可以制成更好的肽类药物。
黄曲霉毒素是黄曲霉和寄生曲霉等某些菌株产生的双呋喃环类毒素。其中以黄曲霉毒素B1的毒性最大，致癌性最强。黄曲霉毒素主要污染粮油及其制品，各种植物性与动物性食品也能被污染，常常存在于土壤、动植物、各种坚果特别是花生和核桃中，在我国华中、华南、华北地区产毒株多，产毒量也大，东北、西北地区较少。目前检测黄曲霉毒素的方法有薄层层析法为主，发展到高效液相色谱法、微柱法、酶联免疫吸附法等方法，这些方法的样品前处理过程较为繁琐，而且都涉及到一些专业仪器的操作，所以发展能实时、快速、灵敏的黄曲霉毒素检测方法十分必要。
发明内容
本发明的目的在于设计一种环肽可以特异性的结合黄曲霉毒素，并且可以利用这一特性提供一种检测黄曲霉毒素的新方法。
本发明的技术方案是：
一种环肽，包括两部分，a.表面偶联半胱氨酸，b.天然结合蛋白模拟肽链功能序列——Arg，Phe，Ser，Tyr和Asp。
优选的，包括如下五种：
①CP-1：cyclic[-RFSCDYY-]
②CP-2：cyclic[-SFRFCYDYY-]
③CP-3：cyclic[-FRFSCYDYY-]
④CP-4：cyclic[-RFSCDDY-]
⑤CP-5：cyclic[-SRFCDYY-]
其中C为半胱氨酸，S为丝氨酸，F为苯丙氨酸，R为精氨酸，Y为酪氨酸，D为天冬氨酸。
所述环肽作为黄曲霉毒素结合模拟蛋白用于黄曲霉毒素的检测。
环肽作为黄曲霉毒素结合模拟蛋白用于黄曲霉毒素的检测，包括以下步骤：
(1)将环肽冻干粉溶于含有15％甲醇10mM,PH为7.4的PBS溶液中制成0.3mM的环肽溶液；
(2)将黄曲霉毒素溶于15％甲醇10mM,PH为7.4的PBS溶液中制成0.025mM的黄曲霉毒素溶液；
(3)将0.3mM的环肽溶液逐滴滴入0.025mM的黄曲霉毒素溶液中，测量每滴一滴产生的热量，从而可以得出每条肽对黄曲霉毒素的结合能力的强弱。
由于实验使用的仪器是等温滴定量热仪，若多肽结合黄曲霉的能力强，则每一滴放出的热量就多，若多肽结合黄曲霉的能力弱，则每一滴放出的热量少。这个强弱可以说明检测的准确度。
优选的，将0.3mM的环肽溶液逐滴滴入0.025mM的黄曲霉毒素溶液中最佳吸附条件为：温度25℃，pH＝7.4，时间为40分钟。
乙酰胆碱酯酶中天然结合蛋白活性位点氨基酸包括5种：Ser(丝氨酸)、Phe(苯丙氨酸)、Arg(精氨酸)、Tyr(酪氨酸)、Asp(天门氨酸)。根据位点排布以及位点氨基酸与黄曲霉毒素之间的氢键作用，设计出多种环肽链，根据这些环肽链中氨基酸种类、个数以及位置排布的不同，设计出一系列环肽。按照本发明的方法测定出环肽CP-2的结合黄曲霉毒素的能力最好。用于检测黄曲霉毒素灵敏度最高，准确度最高。
本发明的优点是：本发明主要是设计了一种制备简便、绿色环保、操作简单，效果明显、价格低廉的用于黄曲霉毒素的新型探针。本发明主要是环肽序列的设计，比较新颖独特，环肽的性质比较稳定。找到了结合黄曲霉毒素的能力最好环肽CP-2。
附图说明
构成本发明创造的一部分的附图用来提供对本发明创造的进一步理解，本发明创造的示意性实施例及其说明用于解释本发明创造，并不构成对本发明创造的不当限定。在附图中：
图1为本发明创造实施例所述的利用MALDI-TOF-MS(基质辅助激光解吸电离飞行时间质谱)对CP-1～CP-5五种环肽结合黄曲霉毒素强弱关系图。
图2为本发明创造实施例所述的利用等温滴定量热仪对最优环肽CP-2选择性和敏感性的检测。图a～图d依次是环肽滴定空白溶液ITC图、环肽滴定黄曲霉毒素ITC图、空白滴定黄曲霉毒素ITC图、环肽滴定黄曲霉毒素扣除空白后的ITC拟合曲线。
图3为本发明创造实施例所述的最优肽CP-2自组装到金片电极后又与黄曲霉毒素结合的CV(循环伏安法)图和EIS(电化学阻抗)图。图a是CV(循环伏安法)图，图b是EIS(电化学阻抗)图
图4为本发明创造实施例所述的最优环肽CP-2吸附黄曲霉毒素前后的粒径大小关系图。图a是环肽吸附黄曲霉毒素前的粒径分布图，图b是环肽吸附黄曲霉毒素后的粒径分布图。
图5为本发明创造实施例所述的最优环肽CP-2与黄曲霉毒素浓度关系图。1#为纳米金溶液，2#为纳米金加入黄曲霉毒素溶液，3#—8#浓度为30μM、3μM、300nM、30nM、10nM、3nM，9#为纳米金加入环肽CP-2溶液。
图6 ITC实验原理图
具体实施方式
为了使本发明上述特征和优点更加清楚和容易理解，下面将结合附图对本发明的实施方式作进一步详细描述。
设计一种环肽，包括两部分，a.表面偶联半胱氨酸，b.天然结合蛋白模拟肽链功能序列——Arg，Phe，Ser，Tyr和Asp。
优选的，包括如下五种：
①CP-1：cyclic[-RFSCDYY-]
②CP-2：cyclic[-SFRFCYDYY-]
③CP-3：cyclic[-FRFSCYDYY-]
④CP-4：cyclic[-RFSCDDY-]
⑤CP-5：cyclic[-SRFCDYY-]
其中C为半胱氨酸，S为丝氨酸，F为苯丙氨酸，R为精氨酸，Y为酪氨酸，D为天冬氨酸。
按照设计的环肽结构由生物公司制备五种环肽。
实施例1：五种环肽对黄曲霉毒素的结合能力的测定。
将5μL 10μM的黄曲霉毒素与5μL 10μM的环肽溶液均匀混合，通过MALDI-TOF-MS(基质辅助激光解吸电离飞行时间质谱)测其质荷比。图1为五条环肽分别结合黄曲霉毒素的能力，通过Icp+afb/Icp的大小可以很清楚的看出是环肽CP-2的结合黄曲霉毒素的能力最好。
实施例2：利用等温滴定量热仪对环肽CP-2结合黄曲霉毒素的表征：
将多肽冻干粉溶于含有15％甲醇10mM,PH为7.4的PBS溶液中制成0.3mM的多肽溶液，同时将黄曲霉毒素溶于15％甲醇10mM,PH为7.4的PBS溶液中制成0.025mM的黄曲霉毒素溶液，保证两种的有机溶剂的含量，PH值一样。温度为25℃，pH＝7.4，时间为40分钟，转速为1000r/min,滴定时间间隔为2min。图2a与图2c是滴定实验的空白组，图2b是最优环肽滴定黄曲霉毒素的ITC图。图2d为整合之后ITC曲线，经过分析环肽CP-2结合黄曲霉毒素的K值达到2.59(0.35)M-1，ΔH值达到9.9(0.3)Kcal/mol，可以看出最优肽CP-2对黄曲霉毒素有非常强的结合作用。
实施例3：电化学方法对环肽CP-2结合黄曲霉毒素的表征：
所有的电化学实验采用三电极体系，包括工作电极(金电极)，参比电极(银/氯化银),对电极(铂)。电解质溶液是1mL浓度为0.1mol/L的高氯酸钠溶液，其中包含浓度为1mmol/L铁氰化钾、亚铁氰化钾(1:1)，循环伏安法的扫描电位-0.2-0.6V,扫描速度50mV/s，电化学阻抗采用的振幅是5mV，频率范围0.1Hz到100kHz。图3a表示的是环肽CP-2吸附到金电极表面后再次结合黄曲霉毒素的CV图，在图3a中最外侧的CV曲线是干净的金电极，当吸附了环肽后，就变成了其内侧的CV曲线，最后结合了黄曲霉毒素后就是最内侧的CV曲线。图3b表示的是环肽吸附黄曲霉毒素的EIS图，没有吸附黄曲霉毒素之前阻抗约为9KΩ，吸附黄曲霉毒素之后阻抗约为33KΩ。从两图中可以看出随着结合黄曲霉毒素，电位和电阻也是有相应的变化。
实施例4：利用粒径分析仪对环肽CP-2结合黄曲霉毒素的表征：
取1ml 0.1mM的环肽CP-2溶液，利用粒径分析仪测量其粒径尺寸，将1ml 0.1mM的环肽CP-2与1ml 0.01mM的黄曲霉毒素溶液混合，然后取1ml的混合溶液，利用粒径分析仪测量其粒径尺寸。图4a是环肽CP-2的粒径分布，大概分布在80-100nm，图4b是环肽结合黄曲霉毒素后的粒径分布，大概分布在450-600nm。从两图中可以看出环肽CP-2结合黄曲霉毒素前后粒径尺寸大小的相应变化。
实施例5：最优环肽CP-2与不同浓度黄曲霉毒素的关系：
取九支离心管分别加入300μL纳米金溶液，在3#—8#离心管中加入黄曲霉毒素溶液，使其含量分别为30μM、3μM、300nM、30nM、10nM、3nM。在2#离心管中加入黄曲霉毒素溶液。1#离心管不做任何处理，最后在3#--9#离心管中加入环肽CP-2,五分钟后，观察颜色变化。图5可以看出，与1#颜色相比2#离心管颜色没有任何变化，其他离心管颜色都发生变化，9#离心管中颜色发生变化，颜色最深，在3#—8#离心管中，随着黄曲霉毒素含量越少，纳米金颜色越来越深，越接近9#离心管的颜色。8#离心管中含有3nM的黄曲霉毒素，颜色和9#离心管接近，但也有一定的差异。这说明黄曲霉毒素不能使纳米金颜色发生变化，环肽可以使纳米金发生颜色变化，当同时间入黄曲霉毒素和环肽的时候，由于环肽能和黄曲霉毒素结合，所以黄曲霉毒素越多结合的环肽越多，能使纳米金显色的环肽越少，离心管的颜色就越浅，从图中可以看出环肽CP-2可以检测到3nM的黄曲霉毒素。
综上所述，环肽CP-2在设计的五条环肽中，是结合黄曲霉毒素能力最强的环肽，并且通过一系列的表征方法，也得出了环肽CP-2是五条肽中结合黄曲霉毒素能力最强的一条环肽。
以上所述仅为本发明创造的较佳实施例而已，并不用以限制本发明创造，凡在本发明创造的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明创造的保护范围之内。
Claims (1)
1.一种能够特异性结合黄曲霉毒素的环肽，其特征在于，包括两部分， a. 表面偶联半胱氨酸， b. 天然结合蛋白模拟肽链功能序列——Arg，Phe，Ser，Tyr和Asp,具体结构为：cyclic[-SFRFCYDYY-]，
其中C为半胱氨酸，S为丝氨酸，F为苯丙氨酸，R为精氨酸，Y为酪氨酸，D为天冬氨酸。
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