CN114315784A - 一种组氨酸标签荧光探针及其制备方法和应用 - Google Patents
一种组氨酸标签荧光探针及其制备方法和应用 Download PDFInfo
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- CN114315784A CN114315784A CN202111440062.0A CN202111440062A CN114315784A CN 114315784 A CN114315784 A CN 114315784A CN 202111440062 A CN202111440062 A CN 202111440062A CN 114315784 A CN114315784 A CN 114315784A
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- histidine
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Abstract
一种组氨酸标签荧光探针及其制备方法和应用。本发明公开了一种基于罗丹明B衍生物的组氨酸标签荧光化合物及其制备方法和应用。所述荧光化合物Rho‑IDA能螯合不同的金属离子，其中螯合二价钴离子得到的荧光探针衍生物Rho‑IDA‑CoII具有特异性标记组氨酸标签融合蛋白的功能。本发明的荧光探针具有合成方法简便，分子量小，激发和发射波长位于近红外光区的优点。在应用于生物成像时，可以降低背景环境荧光杂质的干扰；同时能够对凝胶中的组氨酸标签融合蛋白进行快速、专一的标记染色，为相关生物化学研究提供了可靠的研究工具。
Description
技术领域
本发明涉及化学和生物技术领域，具体涉及一种基于罗丹明B衍生物的组氨酸标签荧光化合物及其制备方法和应用。
背景技术
蛋白质的生化标记可以阐明蛋白质的功能、定位和动态。基于小分子的重组蛋白荧光标记作为荧光蛋白融合技术的替代方案，具有特殊的前景，且不会对蛋白质功能造成有害干扰。典型的技术是基于小分子标记的荧光成像，该技术是设计合成一个能与特定短肽结合的荧光探针，通过将特定短肽融合到目标蛋白上，实现小分子荧光探针的蛋白质标记。近几十年来，基于小分子探针标记蛋白质的研究取得了巨大的进展。其中，蛋白质的金属螯合标记技术由于其操作简单和高特异性而备受关注。由于His6-Ni2+-NTA体系被广泛应用于亲和层析蛋白纯化，因此该系统通过偶联荧光基团能对组氨酸标签（His-tag）融合蛋白进行特异性标记。目前常见的是基于单个或多个NTA（次氮基三乙酸酯）衍生物偶联的荧光探针，虽然这些探针克服了组氨酸标签与Ni2+结合力弱的缺点。但由于这类荧光探针的分子量较大，存在应用成本高、合成方法复杂、蛋白标记缓慢等缺点，使得这些荧光探针不能应用于现有的大部分组氨酸标签融合蛋白库。因此，设计灵敏度高、特异性好、应用范围广的组氨酸标签融合蛋白荧光探针具有重要意义。
发明内容
本发明的目的是提供一种基于罗丹明B衍生物的组氨酸标签荧光探针及其制备方法和应用。
本发明采用的技术方案是：
一种基于罗丹明B衍生物的荧光化合物Rho-IDA，其结构式如（I）所示：
本发明还涉及制备所述荧光化合物Rho-IDA的方法，所述方法为：将罗丹明B和N-羟基丁二酰亚胺反应生成罗丹明B-NHS酯，罗丹明B-NHS酯再与亚氨基二乙酸反应生成Rho-IDA。
具体的，所述方法包括如下步骤：
（1）将罗丹明B和N-羟基丁二酰亚胺混合，室温下反应过夜，充分活化罗丹明B的羧基，过滤去除杂质；
（2）向步骤（1）得到的反应液中加入亚氨基二乙酸和三乙胺，在室温下反应过夜；
（3）HCl溶液调节反应液pH至2.0，室温搅拌至沉淀析出；
（4）过滤收集沉淀并用HCl溶液漂洗，蒸干溶剂，得到所述荧光化合物Rho-IDA。
本发明还涉及荧光化合物Rho-IDA的金属螯合物Rho-IDA-M，其结构式如（II）所示：
式（II）中，M为能被荧光探针Rho-IDA螯合的金属元素，例如过渡金属元素。
所述金属螯合物Rho-IDA-M由荧光化合物Rho-IDA等摩尔的M2+混匀进行螯合反应制得。
优选的，所述M为下列之一：Cu2+，Co2+，Ni2+。
本发明还涉及荧光化合物Rho-IDA及其金属螯合物Rho-IDA-M在制备荧光探针中的应用。
具体的，所述荧光探针用于特异性荧光标记组氨酸标签融合蛋白，或者用于定量检测痕量组氨酸标签融合蛋白。
本发明的有益效果主要体现在：本发明设计并合成了一种基于IDA（亚氨基二乙酸）偶联罗丹明B的新型组氨酸标签的荧光化合物，该荧光化合物作为荧光探针的优势在于其激发波长和发射波长位于近红外光区，在应用于荧光成像时可以降低样本自身荧光背景的干扰，且灵敏度高、特异性强，能特异性检测1 pmol的组氨酸标签蛋白；该荧光探针能在常温常压条件下通过两步法快速制备，并能结合不同的二价过渡金属离子，在荧光标记金属离子相互作用的研究中提供了可靠的研究工具。
附图说明
图1为本发明中探针Rho-IDA的核磁氢谱图。
图2为本发明中探针Rho-IDA的ESI-MS谱图。
图3为本发明中探针Rho-IDA-CoII的荧光光谱图。
具体实施方式
下面结合具体实施例对本发明进行进一步描述，但本发明的保护范围并不仅限于此：
试验材料和试剂：
1、本发明所使用的分析纯化学试剂均采购自国药集团化学试剂有限公司。
2、SDS-PAGE实验仪器与试剂： SurePAGETM梯度浓度（4~20%）预制胶购买自南京金斯瑞生物科技有限公司；恒压电泳使用伯乐Mini-PROTEAN® Tetra电泳槽；凝胶成像使用伯乐ChemiDoc XRS+系统。
3、固定剂：将去离子水、乙醇、乙酸按照10:5:1的比例配制并充分混匀。
4、荧光染液：Rho-IDA-CoII 1 μM，Tris-HCl缓冲液（pH 8.0） 10 mM和NaCl 300mM。
5、洗涤液：咪唑 50 mM，Tris-HCl缓冲液（pH 8.0） 10 mM和NaCl 300 mM。
说明：以下实施例中未作具体说明的分子生物学实验方法，均参照《分子克隆实验指南》(第三版) J.萨姆布鲁克一书中所列的具体方法进行，或者按照试剂盒和产品说明书进行
实施例1：荧光探针Rho-IDA的化学合成
1) 在装有磁力搅拌子的50 mL反应瓶中，加入原料罗丹明B 0.1 mmol，二氯甲烷10 mL，充分溶解。
2)缓慢加入0.15 mmol N-羟基丁二酰亚胺和0.15 mmol N, N'-二环己基碳二亚胺，室温反应过夜，过滤去除沉淀。
3) 缓慢加入三乙胺0.2 mmol，混匀。
4) 加入亚氨基二乙酸0.15 mmol，室温反应过夜。
5)减压蒸干溶剂，剩余物用30 mL二氯甲烷溶解，继续加入30 mL饱和食盐水，萃取，分取二氯甲烷层。
6) 过滤去除杂质，使用硅胶柱层析，洗脱剂为石油醚:乙酸乙酯=5:1，得到玫瑰红粉末，总收率为64%。
使用Bruker Avance III光谱仪和Therm LCQ TM Deca XP plus质谱仪对上述获得的合成产物进行鉴定，结果参见图1和图2。电喷雾电离质谱（ESI-MS）的鉴定结果为：m/z：558.46，与Rho-IDA的计算值[M]+：558相符；其核磁1H-NMR鉴定氢原子数与荧光探针结构一致。结果说明根据上述方法合成得到的玫瑰红粉末即为荧光探针Rho-IDA。
实施例2：荧光探针Rho-IDA-CoII的荧光性能测定
1) 将相同摩尔浓度的Rho-IDA和CoCl2等量混匀，得到荧光探针Rho-IDA-CoII；
2) 取0.1 mM的荧光探针Rho-IDA-CoII水溶液200 μL至微量石英比色皿中，使用紫外-可见光分光光度仪对Rho-IDA-CoII样品进行全波长扫描，波长范围是200 nm~850nm，以超纯水作为空白对照。全波长扫描结果表明，荧光探针Rho-IDA-CoII在550 nm和575nm两个波长处存在最大吸收峰。
3) 将步骤2中的荧光探针Rho-IDA-CoII溶液样品转移至微量荧光比色皿中，设定激发波长为550 nm，扫描荧光探针Rho-IDA-CoII在400~750 nm波长范围内的荧光强度；
4)检测结果表明（图3），荧光探针Rho-IDA-CoII在550 nm波长的可见光激发下，能发射580 nm的强荧光。
实施例3：基于荧光探针Rho-IDA-CoII的组氨酸标签蛋白体外染色
1) 待检测的组氨酸标签融合蛋白（SEQ ID NO. 1）与电泳上样缓冲液以3:1的比例混匀后，沸水浴5 min；
2) 使用4~20%梯度凝胶（金斯瑞，中国）对蛋白样品进行SDS-PAGE分离，电泳条件为200 V恒压，40 min；
3) 电泳结束后，将凝胶小心置于固定液中浸泡1 h；
4) 使用去离子水漂洗凝胶2次，每次10 min，充分去除固定剂；
5) 将凝胶转移至荧光染液中避光染色1 h；
6) 使用洗涤液漂洗凝胶2次，每次10 min；
7) 使用伯乐CCD相机捕捉荧光凝胶图像；
8)将步骤7中记录完荧光染色结果的凝胶使用考马斯亮蓝染色，比较分析荧光染色的效果；
9) 结果表明，荧光探针Rho-IDA-CoII能特异性识别标记组氨酸标签融合蛋白。对比考马斯亮蓝法染色结果，荧光探针Rho-IDA-CoII对组氨酸标签融合蛋白的标记染色基本不受非标签的蛋白的干扰，并且能清晰标记微量的组氨酸标签融合蛋白。
实施例4：荧光探针Rho-IDA-CoII检测灵敏度测定
1) 精确配制浓度分别为1，2，3，5，10，50，100，150，200 ng/μL的组氨酸标签融合蛋白（SEQ ID NO. 1）标准溶液；
2) 按照实施例3的方法对BSA样品进行SDS-PAGE分析，标准蛋白样品的上样量为10 μL；
3) 使用荧光探针Rho-IDA-CoII标记后，检测荧光染色效果；
4)凝胶成像结果表明，当蛋白电泳上样量在30 ng（约1 pmol）以上时能发出仪器可清晰成像的条带。荧光探针Rho-IDA-CoII的检测灵敏度为1 pmol的组氨酸标签融合蛋白。
尽管已经示出和描述了本发明的实施例，对于本领域的普通技术人员而言，可以理解在不脱离本发明的原理和精神的情况下可以对这些实例进行多种变化、修改、替换和变型，本发明的范围由所附权利要求及其等同物限定。
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405 410 415
Glu Glu Glu Leu Ala Lys Ile Val Thr Arg Asp Ser Met Ile Gly Val
420 425 430
Ala Lys Val Gln Pro Ser Ser Val Thr Val Arg
435 440
Claims (8)
1.一种基于罗丹明B衍生物的荧光化合物Rho-IDA，其结构式如（I）所示：
2.制备权利要求1所述荧光化合物Rho-IDA的方法，其特征在于，所述方法为：将罗丹明B和N-羟基丁二酰亚胺反应生成罗丹明B-NHS酯，罗丹明B-NHS酯再与亚氨基二乙酸反应生成Rho-IDA。
3.如权利要求2所述的方法，其特征在于所述方法包括：
（1）将罗丹明B和N-羟基丁二酰亚胺混合，室温下反应过夜，过滤去除杂质；
（2）向步骤（1）得到的反应液中加入亚氨基二乙酸和三乙胺，在室温下反应过夜；
（3）HCl溶液调节反应液pH至2.0，室温搅拌至沉淀析出；
过滤收集沉淀并用HCl溶液漂洗，蒸干溶剂，得到所述荧光化合物Rho-IDA。
4.荧光化合物Rho-IDA的金属螯合物Rho-IDA-M，其结构式如（II）所示： （II） 式
（II）中，M为能被荧光化合物Rho-IDA螯合的金属元素。
5.如权利要求3所述的金属螯合物Rho-IDA-M，其特征在于所述M为下列之一：Cu2+，Co2+，Ni2+。
6.荧光化合物Rho-IDA及其金属螯合物Rho-IDA-M在制备荧光探针中的应用。
7.如权利要求5所述的应用，其特征在于所述荧光探针用于特异性荧光标记组氨酸标签融合蛋白。
8.如权利要求5所述的应用，其特征在于所述荧光探针用于定量检测痕量组氨酸标签融合蛋白。
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