CN113092427A - 异硫氰酸荧光素在标记β内啡肽中的应用 - Google Patents
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Abstract
本发明公开了异硫氰酸荧光素在标记β内啡肽中的应用，其具有灵敏度高、选择性好、响应时间短、使用方便、精准检测、可视化检测、保持生物样本完整性的优点，解决了β内啡肽在神经传递过程中无法观察荧光信号的问题，为神经科学的发展提供了一种有效的手段。
Description
技术领域
本发明属于生物技术领域，具体涉及异硫氰酸荧光素(FITC)在标记β内啡肽中的应用。
背景技术
某些物质在光的照射下，吸收光能进入激发态，从激发态回到基态时，可以电磁辐射形式放出所吸收的光能，此现象称为光致发光。在此现象中，如用一短波长的光照射该物质，它在极短的时间内能发射出波长比照射光长的光，即为荧光。荧光素是一些具有这种特性的染料。FITC是生物学应用最广泛的一种绿色荧光素衍生物，其异硫氰酸基团能与蛋白上的氨基末端和首胺反应，从而将荧光素标记到蛋白上，包括抗体和凝集素。FITC的生物应用包括蛋白的荧光标记，蛋白的荧光示踪，以及荧光抗体的病原体快速鉴定技术。FITC的最大吸收波长为495nm，呈黄-橙色；最大发射波长为525nm，呈黄-绿色。
现有技术中，还没有对脑内β内啡肽进行标记的技术，因此，本发明通过脑立体定位注射技术，用FITC对β内啡肽进行标记，以观察β内啡肽不同核团之间的转运，为神经科学的发展提供了一种有效的手段。
发明内容
发明目的：本发明所要解决的技术问题是针对现有技术的不足，提供异硫氰酸荧光素在标记β内啡肽中的应用。
本发明还提供了一种异硫氰酸荧光素标记的β内啡肽及其应用。
本发明还提供了一种小鼠模型及其应用。
为了解决上述第一个技术问题，本发明公开了异硫氰酸荧光素在标记β内啡肽中的应用。
为了解决上述第二个技术问题，本发明公开了一种异硫氰酸荧光素标记的β内啡肽。
其中，所述异硫氰酸荧光素标记的β内啡肽为异硫氰酸荧光素和β内啡肽串联形成的新肽链FITC-YGGFMTSEKSQTPLVTLFKNAIIKNVHKKGQ。
其中，上述异硫氰酸荧光素标记的β内啡肽在研究神经递质科学中的应用也在本发明的保护范围内。
优选地，所述异硫氰酸荧光素标记的β内啡肽在脑中研究神经递质科学。
进一步优选地，所述异硫氰酸荧光素标记的β内啡肽在中脑中研究神经递质科学。
其中，所述在研究神经递质科学中的应用为通过对β内啡肽进行异硫氰酸荧光素标记，β内啡肽在神经传递的过程中，可以从异硫氰酸荧光素对β内啡肽标记的荧光图中观察到不同脑区的荧光信号，从而看出β内啡肽不同核团之间的转运。
其中，所述研究神经递质科学为通过脑立体定位注射技术，向脑中注射异硫氰酸荧光素标记的β内啡肽。
其中，所述异硫氰酸荧光素标记的β内啡肽的用量为0.5-2μL/20g。
优选地，所述异硫氰酸荧光素标记的β内啡肽的用量为1-2μL/20g。
进一步优选地，所述异硫氰酸荧光素标记的β内啡肽的用量为1μL/20g。
其中，所述注射速度为0.01-1μL/min。
优选地，所述注射速度为0.05μL/min。
为了解决上述第三个技术问题，本发明公开了一种小鼠模型。
其中，所述小鼠模型含有异硫氰酸荧光素标记的β内啡肽。
优选地，所述小鼠脑中含有异硫氰酸荧光素标记的β内啡肽。
进一步优选地，所述小鼠中脑中含有异硫氰酸荧光素标记的β内啡肽。
其中，上述小鼠模型在研究神经递质科学中的应用也在本发明的保护范围内。
有益效果：与现有技术相比，本发明具有如下优势：
(1)本发明首次在β内啡肽内进行FITC荧光标记，其具有灵敏度高、选择性好、响应时间短、使用方便、精准检测、可视化检测、保持生物样本完整性的优点。
(2)在β内啡肽进行FITC荧光标记，β内啡肽在神经传递的过程中，可以观察到不同脑区的荧光信号，解决了β内啡肽在神经传递过程中无法观察荧光信号的问题，为神经科学的发展提供了一种有效的手段。
附图说明
下面结合附图和具体实施方式对本发明做更进一步的具体说明，本发明的上述和/或其他方面的优点将会变得更加清楚。
图1为FITC标记β内啡肽的荧光图。
图2为β内啡肽的分析报告。
图3为β内啡肽的MASS图。
图4为β内啡肽的HPLC图。
具体实施方式
下述实施例中所述实验方法，如无特殊说明，均为常规方法；所述试剂和材料，如无特殊说明，均可从商业途径获得。
荧光探针是建立在光谱化学和化学波导与量测技术基础上的将分析对象的化学信息以荧光信号表达的传感装置。信号表达包括荧光的增强或减弱、光谱的移动、荧光寿命的变化等。
在医学研究中，脑立体定位技术被广泛运用于脑部给药、脑部损毁、刺激和脑电记录的精确定位中，成为研究脑结构和功能必不可少的工具。
实施例1：
一、实验前准备：
脑立体定位仪、常规手术器械、颅骨钻、微量注射器、干棉球、1％的戊巴比妥钠、生理盐水、1mL注射器、C57小鼠。
用1％的戊巴比妥钠麻醉小鼠，注射量为50mg/100g。从饲养笼中取出C57小鼠，通过腹腔注射的方式麻醉小鼠。5-10min后，麻醉剂起效。待小鼠麻醉后，用剃毛器将小鼠头部毛发剔除干净。
二、固定小鼠
将麻醉剔毛后的小鼠固定到立体定位仪上。固定时，先将小鼠门齿卡在适配器门齿夹上，轻轻压上门齿夹横杆，调整适配器高度前后，使耳杆可以方便进入小鼠外耳道。左手托起小鼠头部，将左侧耳杆插入小鼠耳道，调节左右侧耳杆使动物头部保持在U型开口的中心位置，先锁紧固定一侧耳杆，使动物头部不能晃动，同时旋紧门齿夹螺丝。检查是否固定成功：鼻对正中，头部不动，提尾不掉，目测大脑放置水平。用手术刀划开小鼠头部皮肤，去除颅骨表面结缔组织，干棉球擦除血迹，暴露前后囟。分别将颅骨移到前后囟位置，看矢状缝是否正中，颅骨是否水平。位置调整无误后以Bregma(前囟)为0点按照预先确定好的坐标移动颅骨钻，钻孔。钻好孔后可用细针轻轻试探颅骨是否钻透。
三、注射FITC标记的β内啡肽
(1)委托丹港生物科技有限公司按照固相合成法合成FITC标记的β内啡肽FITC-YGGFMTSEKSQTPLVTLFKNAIIKNVHKKGQ。
(2)将步骤(1)所得FITC标记的β内啡肽注射到步骤二处理小鼠(20g)的中脑中脑导水管周围灰质(PAG)区。注射体积1μL，速度0.05μL/min。注射完成后缝合小鼠头皮。取下小鼠放入笼中继续饲养。
(3)饲养4-5周取小鼠中脑PAG区、切片、观察FITC标记β内啡肽的荧光信号，结果如图1所示，可以看出在中脑PAG区，FITC标记的β内啡肽的流动。
步骤(1)中，FITC标记的β内啡肽合成报告如图2-4所示，可以看荧光显色物质确实是β内啡肽；具体地，图2中，从Sequence可以看出，这个物质是β内啡肽，β内啡肽纯度的标准是＞95％，从检测报告的Results纯度＞95.97％。另外，图3是MASS图，所得到的峰说明了本物质是β内啡肽。图4是HPLC图，为了说明β内啡肽的纯度，最高峰的conc(浓度)是95.9769，＞95％，说明β内啡肽的纯度是处于标准范围的。
本发明提供了异硫氰酸荧光素在标记β内啡肽中的应用的思路及方法，具体实现该技术方案的方法和途径很多，以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。本实施例中未明确的各组成部分均可用现有技术加以实现。
Claims (9)
1.异硫氰酸荧光素在标记β内啡肽中的应用。
2.一种异硫氰酸荧光素标记的β内啡肽。
3.权利要求2所述异硫氰酸荧光素标记的β内啡肽在研究神经递质科学中的应用。
4.根据权利要求3所述的应用，其特征在于，所述异硫氰酸荧光素标记的β内啡肽在脑中研究神经递质科学。
5.根据权利要求3所述的应用，其特征在于，所述异硫氰酸荧光素标记的β内啡肽在中脑中研究神经递质科学。
6.一种小鼠模型，其特征在于，所述小鼠模型含有异硫氰酸荧光素标记的β内啡肽。
7.根据权利要求6所述的模型，其特征在于，所述小鼠脑中含有异硫氰酸荧光素标记的β内啡肽。
8.根据权利要求6所述的模型，其特征在于，所述小鼠中脑中含有异硫氰酸荧光素标记的β内啡肽。
9.权利要求6-8中任意一项所述小鼠模型在研究神经递质科学中的应用。
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