CN109627344B - cAMP fluorescent probe and application thereof - Google Patents
cAMP fluorescent probe and application thereof Download PDFInfo
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- CN109627344B CN109627344B CN201811620932.0A CN201811620932A CN109627344B CN 109627344 B CN109627344 B CN 109627344B CN 201811620932 A CN201811620932 A CN 201811620932A CN 109627344 B CN109627344 B CN 109627344B
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Abstract
本发明涉及生物检测领域，特别涉及cAMP荧光探针及其应用。相较于目前已有的绿色荧光探针，本发明创造的探针或者具有更大的动态范围(与cADDis/cAMPr比较)，或者具有好的荧光亮度(与Flamindo2比较)，与cAMP亲和力也在适中的浓度。虽然不宜直接与红色的cAMP探针比较，但相较于目前已有的红色荧光探针Pink‑Flamindo2及R‑FlincA，本发明创造的探针具有好的荧光亮度。综上，本发明提供的#252探针兼顾了荧光亮度、动态范围、亲和力等方面的性能，使其应用范围更广。The invention relates to the field of biological detection, in particular to a cAMP fluorescent probe and its application. Compared with the existing green fluorescent probes, the probes created by the present invention either have a larger dynamic range (compared with cADDis/cAMPr), or have good fluorescence brightness (compared with Flamindo2), and the affinity with cAMP is also high. Moderate concentration. Although it is not suitable to directly compare with the red cAMP probe, compared with the existing red fluorescent probes Pink-Flamindo2 and R-FlincA, the probe created by the present invention has good fluorescence brightness. To sum up, the #252 probe provided by the present invention takes into account the properties of fluorescence brightness, dynamic range, affinity, etc., so that its application range is wider.
Description
技术领域technical field
本发明涉及生物检测领域，特别涉及cAMP荧光探针及其应用。The invention relates to the field of biological detection, in particular to a cAMP fluorescent probe and its application.
背景技术Background technique
环磷酸腺苷(cAMP)是目前最大药物靶标G蛋白偶联受体(GPCR)家族的下游信使分子，cAMP荧光探针及显微成像研究是GPCR信号通路的基础研究和药物开发的重要研究方向。cAMP荧光探针主要分为基于荧光蛋白的荧光共振能量转移探针及基于单个荧光蛋白的探针，后者动态范围较前者大且使用简单。Cyclic adenosine monophosphate (cAMP) is the downstream messenger molecule of the largest drug target G protein-coupled receptor (GPCR) family at present. cAMP fluorescent probe and microscopic imaging research are the basic research of GPCR signaling pathway and an important research direction of drug development. . cAMP fluorescent probes are mainly divided into fluorescent resonance energy transfer probes based on fluorescent proteins and probes based on a single fluorescent protein. The latter has a larger dynamic range and is easier to use than the former.
活细胞中cAMP荧光成像是指将cAMP荧光探针表达在细胞中，然后利用荧光显微镜检测探针荧光的强度变化。荧光探针是cAMP荧光成像分析的关键。现有的基于单个荧光蛋白的cAMP探针及其重要性质如下表所示，其中#252是本发明所涉及到的探针。从该表可知，基于单个荧光蛋白的探针分为绿色和红色2小类。其中绿色的Flamindo2及红色的PinkFlamindo2/R-FlincA等动态范围较大，但是在37℃培养细胞中荧光亮度极低；因其对环磷酸鸟苷(cGMP)也有响应，其对cAMP选择性并不太高，尤其在cGMP浓度较高时(>10μM)。其他探针动态范围较小。Fluorescence imaging of cAMP in living cells refers to expressing cAMP fluorescent probes in cells, and then using a fluorescence microscope to detect changes in the fluorescence intensity of the probes. Fluorescent probes are the key to cAMP fluorescence imaging analysis. The existing cAMP probes based on a single fluorescent protein and their important properties are shown in the following table, among which #252 is the probe involved in the present invention. As can be seen from this table, probes based on a single fluorescent protein are divided into two subclasses, green and red. Among them, green Flamindo2 and red PinkFlamindo2/R-FlincA have large dynamic ranges, but the fluorescence brightness is extremely low in cells cultured at 37 °C; because they also respond to cyclic guanosine monophosphate (cGMP), their selectivity to cAMP is not Too high, especially at high cGMP concentrations (>10 μM). Other probes have smaller dynamic ranges.
表1Table 1
目前基于单个荧光蛋白的cAMP探针分为绿色和红色2小类，前者主要有Flamindo2、cADDis及cAMPr，后者主要有Pink Flamindo、Red cADDis及R-FlincA。实际应用中，荧光亮度、动态范围、亲和力及特异性是4个很重要的参数。而探针的灵敏度(与动态范围及亲和力等相关)和信噪比(与荧光亮度相关)是成像中获取精准的cAMP浓度变化及时空分布信息的关键。但目前cADDis、cAMPr及Red cADDis的动态范围较小，信号变化幅度不超过60％(即荧光亮度改变量ΔF/F0，F0、F1分别为不含cAMP的探针及与饱和浓度cAMP结合的探针的荧光亮度，ΔF＝(F1-F0)/F0)。Flamindo2、Pink Flamindo及R-FlincA均在37℃生理温度培养细胞中亮度很低，且对10μM及更高浓度cGMP也有很大响应，特异性不够。综上，对于现有的绿色和红色的基于单个荧光蛋白的cAMP探针，其上述4方面的性能均需要提高，以满足37℃培养细胞中高灵敏及高信噪比荧光成像的需求。At present, cAMP probes based on a single fluorescent protein are divided into two categories: green and red. The former mainly includes Flamindo2, cADDis and cAMPr, and the latter mainly includes Pink Flamindo, Red cADDis and R-FlincA. In practical applications, fluorescence brightness, dynamic range, affinity and specificity are four important parameters. The sensitivity of the probe (related to dynamic range and affinity) and signal-to-noise ratio (related to fluorescence brightness) are the keys to obtaining accurate cAMP concentration changes and spatial distribution information in imaging. However, the current dynamic range of cADDis, cAMPr and Red cADDis is small, and the signal change amplitude does not exceed 60% (that is, the change in fluorescence brightness ΔF/F 0 , F 0 and F 1 are the probe without cAMP and the cAMP with the saturated concentration, respectively). Fluorescence brightness of bound probe, ΔF=(F 1 -F 0 )/F 0 ). Flamindo2, Pink Flamindo and R-FlincA all showed very low brightness in cells cultured at 37°C physiological temperature, and also responded greatly to 10μM and higher concentrations of cGMP, with insufficient specificity. In summary, for the existing green and red cAMP probes based on a single fluorescent protein, the performance of the above four aspects needs to be improved to meet the needs of high sensitivity and high signal-to-noise ratio fluorescence imaging in cells cultured at 37°C.
发明内容SUMMARY OF THE INVENTION
有鉴于此，本发明提供一种cAMP荧光探针及其应用。本发明针对cAMP成像技术的探针部分进行优化，获得了一个绿色的探针，使其信号变化幅度、荧光亮度、亲和力等性能得到提升。In view of this, the present invention provides a cAMP fluorescent probe and its application. The invention optimizes the probe part of the cAMP imaging technology, and obtains a green probe, which improves the performance of signal change range, fluorescence brightness, affinity and the like.
为了实现上述发明目的，本发明提供以下技术方案：In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:
本发明提供了一种cAMP荧光探针，包括：对cAMP敏感的多肽和荧光蛋白。The present invention provides a cAMP fluorescent probe, comprising: a cAMP-sensitive polypeptide and a fluorescent protein.
在本发明的一些具体实施方案中，所述对cAMP敏感的多肽为具有cAMP结合特性的MlotiK1 CNBD结构域或其功能片段、类似物、衍生物或突变体。In some specific embodiments of the present invention, the cAMP-sensitive polypeptide is a MlotiK1 CNBD domain with cAMP-binding properties or a functional fragment, analog, derivative or mutant thereof.
在本发明的一些具体实施方案中，所述MlotiK1 CNBD结构域具有：In some specific embodiments of the invention, the MlotiK1 CNBD domain has:
(I)、如SEQ ID NO:1所示的氨基酸序列；(1), amino acid sequence as shown in SEQ ID NO:1;
(II)、如(I)所述氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的氨基酸序列，且与(I)所述氨基酸序列功能相同或相似的氨基酸序列；(II), an amino acid sequence obtained by substitution, deletion or addition of one or more amino acids to the amino acid sequence of (I), and an amino acid sequence that is functionally identical or similar to the amino acid sequence of (I);
(III)、与(I)或(II)所述氨基酸序列至少有80％同源性的氨基酸序列。(III), an amino acid sequence having at least 80% homology with the amino acid sequence described in (I) or (II).
在本发明的一些具体实施方案中，所述荧光蛋白为环化重排的绿色荧光蛋白cpEGFP。In some specific embodiments of the present invention, the fluorescent protein is a circularly rearranged green fluorescent protein cpEGFP.
在本发明的一些具体实施方案中，所述荧光蛋白具有：In some specific embodiments of the present invention, the fluorescent protein has:
(IV)、如SEQ ID NO:2所示的氨基酸序列；(IV), amino acid sequence as shown in SEQ ID NO:2;
(V)、如(IV)所述氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的氨基酸序列，且与(IV)所述氨基酸序列功能相同或相似的氨基酸序列；(V), the amino acid sequence obtained by replacing, deleting or adding one or more amino acids to the amino acid sequence described in (IV), and having the same or similar function as the amino acid sequence described in (IV);
(VI)、与(IV)或(V)所述氨基酸序列至少有80％同源性的氨基酸序列。(VI), an amino acid sequence having at least 80% homology with the amino acid sequence described in (IV) or (V).
在本发明的一些具体实施方案中，所述荧光蛋白插入所述MlotiK1 CNBD结构域的位点为P/N(74/75)，即所述荧光蛋白插入所述MlotiK1 CNBD结构域的位点为P74与N75之间。In some specific embodiments of the present invention, the site where the fluorescent protein is inserted into the MlotiK1 CNBD domain is P/N (74/75), that is, the site where the fluorescent protein is inserted into the MlotiK1 CNBD domain is Between P74 and N75.
在本发明的一些具体实施方案中，所述cAMP荧光探针具有如式I所示结构：In some specific embodiments of the present invention, the cAMP fluorescent probe has the structure shown in formula I:
MlotiK1 CNBD-N-linker1-cpEGFP-linker2-MlotiK1 CNBD-CMlotiK1 CNBD-N-linker1-cpEGFP-linker2-MlotiK1 CNBD-C
式IFormula I
其中，MlotiK1 CNBD-N为MlotiK1 CNBD的N端，具有：Among them, MlotiK1 CNBD-N is the N-terminus of MlotiK1 CNBD, with:
(VII)、如SEQ ID NO:3所示的氨基酸序列；(VII), the amino acid sequence shown in SEQ ID NO:3;
(VIII)、如(VII)所述氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的氨基酸序列，且与(VII)所述氨基酸序列功能相同或相似的氨基酸序列；(VIII), an amino acid sequence obtained by substitution, deletion or addition of one or more amino acids to the amino acid sequence described in (VII), and an amino acid sequence with the same or similar functions as the amino acid sequence described in (VII);
(IX)、与(VII)或(VIII)所述氨基酸序列至少有80％同源性的氨基酸序列；(IX), an amino acid sequence having at least 80% homology with the amino acid sequence described in (VII) or (VIII);
所述MlotiK1 CNBD-C为MlotiK1 CNBD的C端，具有：Described MlotiK1 CNBD-C is the C end of MlotiK1 CNBD, has:
(X)、如SEQ ID NO:4所示的氨基酸序列；(X), amino acid sequence as shown in SEQ ID NO:4;
(XI)、如(X)所述氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的氨基酸序列，且与(X)所示的氨基酸序列功能相同或相似的氨基酸序列；(XI), an amino acid sequence obtained by substitution, deletion or addition of one or more amino acids to the amino acid sequence described in (X), and an amino acid sequence with the same or similar function as the amino acid sequence shown in (X);
(XII)、与(X)或(XI)所述序列至少有80％同源性的氨基酸序列；(XII), an amino acid sequence with at least 80% homology to the sequence described in (X) or (XI);
linker1为第一连接肽，具有：linker1 is the first linker peptide with:
(XIII)、如SEQ ID NO:5所示的氨基酸序列；(XIII), the amino acid sequence shown in SEQ ID NO:5;
(XIV)、如(XIII)所述氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的氨基酸序列，且与(XIII)所述氨基酸序列功能相同或相似的氨基酸序列；(XIV), an amino acid sequence obtained by substitution, deletion or addition of one or more amino acids to the amino acid sequence described in (XIII), and an amino acid sequence with the same or similar functions as the amino acid sequence described in (XIII);
(XV)、与(XIII)或(XIV)所述氨基酸序列至少有80％同源性的氨基酸序列；(XV), an amino acid sequence having at least 80% homology with the amino acid sequence described in (XIII) or (XIV);
linker2为第二连接肽，具有：linker2 is the second linker peptide with:
(XVI)、如SEQ ID NO:6所示的氨基酸序列；(XVI), the amino acid sequence shown in SEQ ID NO:6;
(XVII)、如(XVI)所述氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的氨基酸序列，且与(XVI)所述氨基酸序列功能相同或相似的氨基酸序列；(XVII), an amino acid sequence obtained by substitution, deletion or addition of one or more amino acids to the amino acid sequence described in (XVI), and an amino acid sequence that has the same or similar functions as the amino acid sequence described in (XVI);
(XVIII)、与(XVI)或(XVII)所述氨基酸序列至少有80％同源性的氨基酸序列。(XVIII), an amino acid sequence having at least 80% homology with the amino acid sequence described in (XVI) or (XVII).
在本发明的一些具体实施方案中，所述cAMP荧光探针具有：In some specific embodiments of the present invention, the cAMP fluorescent probe has:
(XIX)、如SEQ ID NO:7所示的氨基酸序列；(XIX), the amino acid sequence shown in SEQ ID NO: 7;
(XX)、如(XIX)所述氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的氨基酸序列，且与(XIX)所示的氨基酸序列功能相同或相似的氨基酸序列；(XX), the amino acid sequence obtained by substitution, deletion or addition of one or more amino acids to the amino acid sequence described in (XIX), and an amino acid sequence with the same or similar function as the amino acid sequence shown in (XIX);
(XXI)、与(XIX)或(XX)所述氨基酸序列至少有80％同源性的氨基酸序列。(XXI), an amino acid sequence having at least 80% homology to the amino acid sequence of (XIX) or (XX).
在此基础上，本发明还提供了编码所述cAMP荧光探针的核苷酸。On this basis, the present invention also provides nucleotides encoding the cAMP fluorescent probe.
在本发明的一些具体实施方案中，所述核苷酸有：In some specific embodiments of the invention, the nucleotides are:
(XXII)、具有SEQ ID NO:8所示的核苷酸序列；(XXII), have the nucleotide sequence shown in SEQ ID NO:8;
(XXIII)、如(XXII)所述核苷酸序列经修饰、取代、缺失或添加一个或多个碱基获得的核苷酸序列；(XXIII), a nucleotide sequence obtained by modifying, replacing, deleting or adding one or more bases to the nucleotide sequence of (XXII);
(XXIV)、与(XXII)或(XXIII)所述核苷酸序列具有至少80％同源性的序列；(XXIV), a sequence having at least 80% homology to the nucleotide sequence of (XXII) or (XXIII);
(XXV)、与(XXII)、(XXIII)或(XXIV)所述核苷酸序列的互补序列。(XXV), a complementary sequence to the nucleotide sequence of (XXII), (XXIII) or (XXIV).
本发明还提供了表达载体，包括编码所述cAMP荧光探针的核苷酸。The present invention also provides an expression vector comprising nucleotides encoding the cAMP fluorescent probe.
本发明还提供了转化或转染所述的表达载体的宿主细胞。The present invention also provides host cells transformed or transfected with the expression vector.
此外，本发明还提供了所述cAMP荧光探针的制备方法，包括：培养所述的宿主细胞、诱导所述cAMP荧光探针的表达。In addition, the present invention also provides a preparation method of the cAMP fluorescent probe, comprising: culturing the host cell and inducing the expression of the cAMP fluorescent probe.
在上述研究的基础上，本发明还提供了所述cAMP荧光探针在检测cAMP中的应用。On the basis of the above research, the present invention also provides the application of the cAMP fluorescent probe in detecting cAMP.
此外，本发明还提供了所述cAMP荧光探针在检测活细胞内cAMP的变化的应用。In addition, the present invention also provides the application of the cAMP fluorescent probe in detecting the change of cAMP in living cells.
本发明还提供了cAMP的检测方法，包括如下步骤：The present invention also provides a method for detecting cAMP, comprising the steps of:
步骤1、构建表达载体，所述表达载体包括编码如权利要求1～8任一项所述cAMP荧光探针的核苷酸；
步骤2、获得转化或转染所述表达载体的宿主细胞；
步骤3、培养如权利要求11所述的宿主细胞、诱导所述cAMP荧光探针的表达；
步骤4、通过所述cAMP荧光探针对待测样品中所述cAMP的相应，获得所述cAMP的浓度。
在此基础上，本发明还提供了试剂盒，包括所述cAMP荧光探针。On this basis, the present invention also provides a kit including the cAMP fluorescent probe.
在本申请文件中，所述CNBD为MlotiK1 CNBD。In this application document, the CNBD is MlotiK1 CNBD.
本发明的核心在于突变筛选出了一种新的、基于单个蛋白荧光蛋白的cAMP探针The core of the present invention is that a new cAMP probe based on a single protein fluorescent protein has been screened out by mutation.
#252。目前，在绿色系列的探针中，综合考虑动态范围、荧光亮度、与cAMP亲和力等参数，#252是最好的一个。实际使用时，将其表达中哺乳动物细胞中，利用普通的荧光显微镜，即可检测细胞受特定刺激后cAMP浓度是否发生改变。#252. At present, among the green series of probes, considering parameters such as dynamic range, fluorescence brightness, and cAMP affinity, #252 is the best one. In actual use, it can be expressed in mammalian cells, and whether the cAMP concentration changes after specific stimulation of cells can be detected by ordinary fluorescence microscope.
因为探针的实际应用表现与多方面的参数性能有关，所以很难认定目前哪一个探针最好。在绿色探针系列中，与可获得全面信息的、最优的Flamindo2相比，本发明的#252在信号变化幅度上(ΔF/F0)是更好(315％)，荧光亮度有很大提高(HEK293细胞中约提高60倍)，可以在37℃培养细胞中工作。#252相较于红色探针Pink-Flamindo与R-FlincA，在亮度上有明显提高。Because the practical performance of probes is related to a variety of parameters, it is difficult to determine which probe is currently the best. In the green probe series, compared with the optimal Flamindo2 which can obtain comprehensive information, the #252 of the present invention is better (315%) in the signal change amplitude (ΔF/F0), and the fluorescence brightness is greatly improved (approximately 60-fold increase in HEK293 cells), can work in cells cultured at 37°C. Compared with the red probes Pink-Flamindo and R-FlincA, #252 has a significant improvement in brightness.
相较于目前已有的绿色荧光探针，本发明创造的探针或者具有更大的动态范围(与cADDis/cAMPr比较)，或者具有好的荧光亮度(与Flamindo2比较)，与cAMP亲和力也在适中的浓度。虽然不宜直接与红色的cAMP探针比较，但相较于目前已有的红色荧光探针Pink-Flamindo2及R-FlincA，本发明创造的探针具有好的荧光亮度。综上，本发明提供的#252探针兼顾了荧光亮度、动态范围、亲和力等方面的性能，使其应用范围更广。Compared with the existing green fluorescent probes, the probes created by the present invention either have a larger dynamic range (compared with cADDis/cAMPr), or have good fluorescence brightness (compared with Flamindo2), and the affinity with cAMP is also high. Moderate concentration. Although it is not suitable to directly compare with the red cAMP probe, compared with the existing red fluorescent probes Pink-Flamindo2 and R-FlincA, the probe created by the present invention has good fluorescence brightness. To sum up, the #252 probe provided by the present invention takes into account the properties of fluorescence brightness, dynamic range, affinity, etc., so that its application range is wider.
附图说明Description of drawings
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。In order to illustrate the embodiments of the present invention or the technical solutions in the prior art more clearly, the following briefly introduces the accompanying drawings that are required in the description of the embodiments or the prior art.
图1示#252的设计及其与Flamindo2亮度比较；其中，图1(A)示将cpEGFP插入到cAMP亲和结构域中，左边和右边的连接肽分别为WG与RV，得到#252探针；图1(B)示将Flmindo2与#252转化细菌，34℃培养15小时后，对平板进行荧光成像；Figure 1 shows the design of #252 and its brightness comparison with Flamindo2; wherein, Figure 1(A) shows the insertion of cpEGFP into the cAMP affinity domain, and the connecting peptides on the left and right are WG and RV, respectively, to obtain the #252 probe ; Figure 1(B) shows that Flmindo2 and #252 were transformed into bacteria, and after culturing at 34°C for 15 hours, the plate was subjected to fluorescence imaging;
图2示#252的氨基酸序列；下划线氨基酸分别为前后连接肽的组分；WG与RV之间是环化重排的绿色荧光蛋白序列；WG之前为CNBD-N序列，RV之后为CNBD-C序列；Figure 2 shows the amino acid sequence of #252; the underlined amino acids are the components of the connecting peptide before and after respectively; between WG and RV is the green fluorescent protein sequence of circular rearrangement; before WG is CNBD-N sequence, after RV is CNBD-C sequence;
图3示#252探针的动态范围测定；图3(A)示从细菌中纯化的#252探针稀释在pH7.3的HEPES溶液中，终浓度为2μM；图3(B)示探针浓度在HEPES溶液及饱和浓度cAMP中的荧光激发光谱；图3(A)中红色曲线与黑色曲线的比值；可见其在440-500nm激发波段内，动态范围在3.5-4.15倍之间；Figure 3 shows the dynamic range measurement of the #252 probe; Figure 3(A) shows the #252 probe purified from bacteria diluted in HEPES solution pH 7.3 at a final concentration of 2 μM; Figure 3(B) shows the probe Fluorescence excitation spectra in HEPES solution and saturated concentration of cAMP; the ratio of the red curve to the black curve in Figure 3(A); it can be seen that it is in the excitation band of 440-500nm, and the dynamic range is between 3.5-4.15 times;
图4示Flamindo2及#252对cAMP或cGMP的响应曲线；此处荧光由500nm光源激发；Figure 4 shows the response curves of Flamindo2 and #252 to cAMP or cGMP; here the fluorescence is excited by a 500nm light source;
图5示#252探针在HEK293T细胞中的亮度及响应；图5(A)利用Lipofectamine转染HEK细胞空白质粒、含Flamindo2质粒、含#252质粒及含mEGFP(单体绿色荧光蛋白)质粒，过夜培养后，用不含酚红及血清的DMEM细胞培养液饥饿6小时后用荧光显微镜成像；细胞中探针的荧光亮度平均值标记了出来，可见#252亮度远高于Flamindo2；图5(B)将60μMForskolin及100μM IBMX加入培养液，并在加药前后进行荧光成像，图中示意了加药刺激前(0秒)和加药一段时间后(比如1500秒)的荧光亮度；图5(C)为(B)中选定的1/2/3/4个细胞中荧光亮度的变化曲线；Figure 5 shows the brightness and response of #252 probe in HEK293T cells; Figure 5(A) HEK cells were transfected with blank plasmid, Flamindo2-containing plasmid, #252-containing plasmid and mEGFP (monomeric green fluorescent protein) plasmid using Lipofectamine, After overnight incubation, the cells were starved with DMEM cell culture medium without phenol red and serum for 6 hours and then imaged with a fluorescence microscope; the average value of the fluorescence brightness of the probes in the cells was marked, and the brightness of #252 was much higher than that of Flamindo2; Figure 5( B) 60 μM Forskolin and 100 μM IBMX were added to the culture medium, and fluorescence imaging was performed before and after dosing. The figure shows the fluorescence brightness before dosing stimulation (0 seconds) and after dosing for a period of time (such as 1500 seconds); Figure 5 ( C) is the change curve of fluorescence brightness in the selected 1/2/3/4 cells in (B);
图6示纯化的285-ins及#252与cAMP结合后的激发光谱变化；虚线为未加入cAMP激发光谱(图6A)，实线为加入终浓度为1mM cAMP后的激发光谱(图6B)；可见，#252探针有了较大的正变化；Figure 6 shows the change of the excitation spectrum of purified 285-ins and #252 combined with cAMP; the dotted line is the excitation spectrum without cAMP (Figure 6A), and the solid line is the excitation spectrum after adding a final concentration of 1 mM cAMP (Figure 6B); It can be seen that the #252 probe has a large positive change;
图7示亮度检测结果；表明252亮度为285-ins的(87-40)/(40-26)＝3.35倍。Fig. 7 shows the brightness detection results; it is shown that the brightness of 252 is (87-40)/(40-26)=3.35 times that of 285-ins.
具体实施方式Detailed ways
本发明公开了一种cAMP荧光探针及其应用，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。The invention discloses a cAMP fluorescent probe and its application, and those skilled in the art can learn from the content of this article and appropriately improve the process parameters to achieve. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are deemed to be included in the present invention. The method and application of the present invention have been described through the preferred embodiments, and it is obvious that relevant persons can make changes or appropriate changes and combinations of the methods and applications described herein without departing from the content, spirit and scope of the present invention to achieve and Apply the technology of the present invention.
本发明所用术语“荧光探针”是指与荧光蛋白相融合的对环境cAMP敏感的多肽，所述对环境中cAMP敏感的多肽具体可以是Epac蛋白，利用Epac中特异性的cAMP结合结构域与cAMP结合后产生的构象变化引起荧光蛋白的构象变化，从而导致产生的荧光发生改变，通过不同cAMP浓度下测定的荧光蛋白的荧光来绘制标准曲线，进而用于检测并分析细胞内cAMP的存在水平。The term "fluorescent probe" used in the present invention refers to a polypeptide sensitive to cAMP in the environment fused with a fluorescent protein, and the polypeptide sensitive to cAMP in the environment may specifically be an Epac protein, which utilizes the specific cAMP binding domain in Epac to interact with The conformational change generated after cAMP binding causes the conformational change of the fluorescent protein, which leads to the change of the generated fluorescence. The standard curve is drawn by the fluorescence of the fluorescent protein measured at different cAMP concentrations, which is then used to detect and analyze the presence level of intracellular cAMP. .
本文所用术语“融合蛋白”与“荧光融合蛋白”和“重组荧光融合蛋白”意义相同，指包含特异性结合结构域的多肽或其片段、衍生物或类似物的氨基酸序列。The term "fusion protein" as used herein has the same meaning as "fluorescent fusion protein" and "recombinant fluorescent fusion protein" and refers to the amino acid sequence of a polypeptide comprising a specific binding domain or a fragment, derivative or analog thereof.
本文所用术语“功能片段”、“类似物”、“衍生物”是指基本上与本发明中CNBD结构域具有相同的生物学活性的蛋白。本发明的CNBD结构域的功能片段、类似物或衍生物可以是：The terms "functional fragment", "analog", "derivative" as used herein refer to a protein having substantially the same biological activity as the CNBD domain of the present invention. Functional fragments, analogs or derivatives of CNBD domains of the present invention may be:
(1)有一个或多个保守或非保守的氨基酸残基(优选保守性氨基酸残基)被取代的蛋白，而这样被取代的氨基酸残基可以是也可以不是由遗传编码的，或(1) A protein in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues may or may not be genetically encoded, or
(2)在一个或多个氨基酸残基中具有取代基团的蛋白，或(2) A protein having a substituent group in one or more amino acid residues, or
(3)附加氨基酸序列与此蛋白序列融合形成的蛋白，或(3) A protein formed by fusion of an additional amino acid sequence with this protein sequence, or
(4)成熟蛋白与另一个化合物融合形成的蛋白。(4) A protein formed by fusion of a mature protein with another compound.
这些功能片段、类似物和衍生物属于本领域熟练技术人员公知的范围。These functional fragments, analogs and derivatives are well known to those skilled in the art.
在本发明中，所用术语“荧光团”与“荧光蛋白”同义，指自身发出荧光或在照射下发出荧光的蛋白质，荧光蛋白经常被用作检测手段。In the present invention, the term "fluorophore" is used synonymously with "fluorescent protein" and refers to a protein that fluoresces by itself or fluoresces under illumination, and fluorescent proteins are often used as detection means.
本发明所述两种或多种多肽或核酸分子序列的“相同性”或“相同性百分数”是在指定区域或比较窗口上，利用本领域已知的序列比较算法，通过人工目测比对来分析两个或多个序列的最大对应性时，其中部分序列在指定区域内有一定百分比的氨基酸残基或核苷酸相同(如35％、50％、70％、85％、90％、95％或100％相同)，适合于测定序列相似性和相同性百分数的优选算法是BLAST算法，具体可参见ALtschul等(1977)Nucleic AcidsRes.25:3389。The "identity" or "percent identity" of the sequences of two or more polypeptides or nucleic acid molecules of the present invention is determined by manual visual alignment over a specified region or comparison window using sequence comparison algorithms known in the art. When analyzing the maximum correspondence of two or more sequences, some of the sequences have a certain percentage of amino acid residues or nucleotides identical in the specified region (such as 35%, 50%, 70%, 85%, 90%, 95%) % or 100% identical), a preferred algorithm suitable for determining percent sequence similarity and identity is the BLAST algorithm, see ALtschul et al. (1977) Nucleic Acids Res. 25:3389.
在本发明中，“具有80％同源性的序列”为优选方案，本领域的技术人员能够根据实际需要获得与本发明提供的序列具有70％同源性的序列、50％同源性的序列、40％同源性的序列、35％同源性的序列或30％同源性的序列，进而获得功能相近的荧光探针，本发明在此不做赘述，上述技术方案均在本发明的保护范围之内。In the present invention, "a sequence with 80% homology" is a preferred solution, and those skilled in the art can obtain a sequence with 70% homology and a 50% homology with the sequence provided by the present invention according to actual needs. sequence, 40% homologous sequence, 35% homologous sequence or 30% homologous sequence to obtain fluorescent probes with similar functions, which are not repeated in the present invention, and the above technical solutions are all included in the present invention within the scope of protection.
提到某多肽或蛋白时，本发明所用术语“变异体”包括具有所述多肽或蛋白相同功能但序列不同的变异体。这些变异体包括但不限于：在所述多肽或蛋白序列中缺失、插入和/或取代一个或多个(通常为1-30个，较佳为1-5个)氨基酸，以及再起N端和/或C端添加一个或多个(通常为20个之内)氨基酸获得的序列。在本领域内，用性能相似或相近的氨基酸进行取代时，通常不会改变多肽或蛋白的功能。在本领域内，性能相似的氨基酸往往指具有相似侧链的氨基酸家族。本领域技术人员公知，在基因克隆实验操作时，通常需要设计合适的酶切位点，这势必在所表达的多肽或蛋白末端引入了一个或多个不相干的残基，但这并不影响目的多肽或蛋白的活性。When referring to a polypeptide or protein, the term "variant" as used in the present invention includes variants having the same function but different sequences of the polypeptide or protein. These variants include, but are not limited to, deletions, insertions and/or substitutions of one or more (usually 1-30, preferably 1-5) amino acids in the polypeptide or protein sequence, as well as reinvention of the N-terminus and A sequence obtained by adding one or more (usually within 20) amino acids to the C-terminus. In the art, substitution with amino acids with similar or similar properties usually does not change the function of the polypeptide or protein. In the art, amino acids with similar properties often refer to families of amino acids with similar side chains. It is well known to those skilled in the art that during gene cloning experiments, it is usually necessary to design suitable restriction sites, which inevitably introduces one or more irrelevant residues at the end of the expressed polypeptide or protein, but this does not affect the The activity of the polypeptide or protein of interest.
本发明所用术语“核酸”可以是DNA形式或是RNA形式，DNA包括cDNA、基因组DNA或人工合成的DNA。DNA可以是编码链或非编码链。编码成熟蛋白的编码区序列也可以是简并变体。The term "nucleic acid" as used in the present invention may be in the form of DNA or RNA, and DNA includes cDNA, genomic DNA or artificially synthesized DNA. DNA can be the coding or non-coding strand. The coding region sequence encoding the mature protein may also be a degenerate variant.
提到核酸时，本文所用术语“变异体”可以是天然或非天然发生的等位变异体。这些核苷酸变异体包括取代变异体、插入变异体和缺失变异体。等位变异体是一种替换形式，可以是一个或多个核苷酸的取代、插入或缺失，但不会从实质上改变所编码的蛋白的功能特性。The term "variant" as used herein in reference to a nucleic acid can be a naturally occurring or non-naturally occurring allelic variant. These nucleotide variants include substitution variants, insertion variants and deletion variants. An allelic variant is a form of substitution, which can be a substitution, insertion or deletion of one or more nucleotides that does not substantially alter the functional properties of the encoded protein.
本发明荧光探针或融合蛋白的全长序列或其片段通常可以用PCR扩增法或人工合成法来获得。对于PCR扩增，可根据本发明公开的有关核苷酸序列来设计引物，并用市售的cDNA库或按本领域技术人员已知的方法制备cDNA库作为模板，扩增得到有关序列。一旦获得有关序列，就可以用重组法来大批量获得有关序列，通常是将其克隆至载体，再转入细胞通过常规方法从宿主细胞中分离和纯化得到有关融合蛋白。The full-length sequences or fragments thereof of the fluorescent probes or fusion proteins of the present invention can usually be obtained by PCR amplification method or artificial synthesis method. For PCR amplification, primers can be designed according to the relevant nucleotide sequences disclosed in the present invention, and a commercially available cDNA library or a cDNA library prepared by methods known to those skilled in the art is used as a template to amplify the relevant sequences. Once the relevant sequences are obtained, the relevant sequences can be obtained in large quantities by recombinant methods, usually by cloning them into a vector, and then transferring them into cells. The relevant fusion proteins are isolated and purified from host cells by conventional methods.
目前，可以完全通过化学合成方法来得到编码本发明蛋白或其片段或其类似物、衍生物、变异体的DNA序列，进而将其引入本研究领域已知的各种现有载体或细胞中。另一方面，可以通过化学合成或突变PCR等方法将突变引入本发明蛋白序列中。At present, the DNA sequence encoding the protein of the present invention or its fragment or its analogs, derivatives, and variants can be obtained by chemical synthesis, and then introduced into various existing vectors or cells known in the research field. Alternatively, mutations can be introduced into the protein sequences of the present invention by methods such as chemical synthesis or mutational PCR.
本发明的表达载体可用于在原核或真核细胞中表达本发明的荧光探针或融合蛋白，因而本发明涉及已导入本发明表达载体的宿主细胞，宿主细胞可为任何原核或真核细胞，优选各种利于基因产物表达或发酵生产的细胞，且已被本研究领域熟知并常用，例如各种大肠杆菌细胞核酵母细胞。在一个实施方式中，选用了大肠杆菌构建表达了本发明融合蛋白的宿主细胞。可以用适合所述宿主细胞表达的常规方法培养获得转化细胞，表达本发明荧光探针蛋白。根据所用的宿主细胞，培养中所用的培养基为各种常规培养基，然后在适于宿主细胞生长的条件下进行培养。但宿主细胞生长到适当的细胞密度后，可以用合适的方法如化学剂诱导进行诱导选择的启动子，之后将细胞继续培养一段时间。The expression vector of the present invention can be used to express the fluorescent probe or fusion protein of the present invention in prokaryotic or eukaryotic cells, so the present invention relates to a host cell into which the expression vector of the present invention has been introduced, and the host cell can be any prokaryotic or eukaryotic cell, Various cells are preferred that facilitate expression or fermentative production of the gene product and are well known and commonly used in the art, such as various E. coli nucleolar yeast cells. In one embodiment, Escherichia coli is selected to construct the host cell expressing the fusion protein of the present invention. Transformed cells can be obtained by culturing and expressing the fluorescent probe protein of the present invention by conventional methods suitable for the expression of the host cells. The medium used in the culture is various conventional mediums according to the host cells used, and then the culture is carried out under conditions suitable for the growth of the host cells. However, after the host cell has grown to an appropriate cell density, the inducibly selected promoter can be induced by a suitable method such as a chemical agent, after which the cells can be cultured for a further period of time.
在上述方法中的重组融合蛋白可以在细胞内、或在细胞膜上表达、或者分泌到细胞外。根据需要，可以利用其物理、化学和其他特性通过不同分离方法对重组目的蛋白进行分离或纯化。这些方法包括但不局限于：常规的离心、超声破碎处理或渗透压法处理、复性处理、蛋白沉淀剂处理法、亲和层析、离子交换层析、分子筛层析、高效液相层析等技术以及这些方法的结合。The recombinant fusion protein in the above method can be expressed in the cell, or on the cell membrane, or secreted outside the cell. According to needs, the recombinant target protein can be separated or purified by different separation methods by utilizing its physical, chemical and other properties. These methods include, but are not limited to: conventional centrifugation, sonication or osmotic pressure treatment, renaturation treatment, protein precipitant treatment, affinity chromatography, ion exchange chromatography, molecular sieve chromatography, high performance liquid chromatography and other techniques and combinations of these methods.
在一个实施方式中，通过包含本发明融合蛋白编码序列的大肠杆菌发酵生产本发明荧光探针或融合蛋白，并通过亲和层析和凝胶层析纯化得到了纯形式的本发明荧光探针或融合蛋白。本发明荧光探针的用途包括但不局限于：检测生理状态下cAMP的水平、筛选药物、诊断与cAMP水平相关的疾病等。In one embodiment, the fluorescent probe or fusion protein of the present invention is produced by fermentation of Escherichia coli comprising the coding sequence of the fusion protein of the present invention, and purified by affinity chromatography and gel chromatography to obtain the fluorescent probe of the present invention in pure form or fusion protein. The uses of the fluorescent probe of the present invention include, but are not limited to: detecting the level of cAMP under physiological conditions, screening drugs, diagnosing diseases related to the level of cAMP, and the like.
本发明提供的cAMP荧光探针及其应用中所用原料及试剂均可由市场购得。The cAMP fluorescent probe provided by the present invention and the raw materials and reagents used in its application can be purchased from the market.
下面结合实施例，进一步阐述本发明：Below in conjunction with embodiment, the present invention is further elaborated:
实施例1Example 1
首先构建CNBD-N-linker1-cpEGFP-linker2-CNBD–C(Cyclic nucleotide-binding domain,CNBD,环核苷酸结合结构域；CNBD-N，CNBD的N端；CNBD-C,CNBD的C端；cpEGFP，环化重排的绿色荧光蛋白；linker,连接肽)。对linker1及linker2进行筛选，得到#252探针,其linker1及linker2分别为WG与RV(图1A)。将Flamindo2与#252分别表达在细菌中，34℃培养15小时后，可见#252探针荧光亮度明显比Flamindo2高29倍(图1B)。#252的氨基酸序列也给了出来(图2)。Firstly, CNBD-N-linker1-cpEGFP-linker2-CNBD-C (Cyclic nucleotide-binding domain, CNBD, cyclic nucleotide binding domain; CNBD-N, N-terminal of CNBD; CNBD-C, C-terminal of CNBD; cpEGFP, circularly rearranged green fluorescent protein; linker, linker peptide). The linker1 and linker2 were screened to obtain
Mesorhizobium loti MAFF303099DNA,complete genome RC(ORF sequece)(如SEQ ID No.11所示)Mesorhizobium loti MAFF303099DNA, complete genome RC (ORF sequece) (as shown in SEQ ID No. 11)
ATGTCGGTACTGCCTTTCTTAAGAATTTACGCGCCGCTCAACGCGGTGCTGGCTGCGCCTGGGTTGCTGGCGGTGGCTGCGCTCACGATACCGGACATGTCCGGACGAAGCAGACTGGCTCTGGCTGCCCTGCTCGCTGTCATCTGGGGCGCCTATCTCCTGCAACTGGCCGCGACGCTGCTCAAGCGCCGGGCGGGAGTCGTACGGGACAGGACGCCCAAAATCGCCATCGATGTGCTCGCAGTCTTGGTTCCACTCGCCGCATTTCTGCTCGACGGCTCGCCTGACTGGAGCCTCTACTGTGCTGTCTGGCTGCTGAAACCGCTGCGCGACTCGACTTTCTTCCCGGTCCTGGGCAGGGTCCTGGCCAACGAAGCACGCAATCTGATCGGCGTCACCACGCTCTTCGGCGTCGTTCTGTTCGCAGTGGCGCTCGCAGCCTATGTCATCGAGCGCGATATCCAACCGGAAAAGTTCGGCAGCATTCCCCAGGCAATGTGGTGGGCGGTGGTCACGCTGTCCACCACCGGCTATGGGGACACTATCCCGCAAAGCTTCGCCGGCCGCGTCCTTGCCGGGGCGGTCATGATGAGTGGCATCGGCATCTTCGGACTCTGGGCCGGCATTCTTGCCACAGGCTTCTATCAAGAAGTCCGTCGCGGGGATTTCGTCCGCAATTGGCAATTGGTCGCCGCCGTGCCGTTGTTTCAGAAGCTCGGCCCGGCCGTGCTGGTCGAGATCGTGCGCGCCTTAAGAGCCCGCACGGTGCCGGCGGGCGCCGTGATCTGCCGCATTGGCGAGCCCGGCGATCGGATGTTCTTCGTCGTGGAGGGGAGCGTCAGCGTCGCGACGCCGAATCCGGTGGAGCTTGGCCCTGGCGCCTTCTTCGGCGAGATGGCGCTGATCAGCGGCGAACCGCGTTCGGCGACCGTCAGCGCCGCAACGACGGTCTCACTCCTGTCGCTGCATTCGGCGGATTTCCAGATGTTGTGCAGCAGCAGCCCGGAGATCGCGGAAATCTTCCGCAAGACCGCGCTCGAGCGTCGCGGCGCTGCGGCGAGCGCTATGTCGGTACTGCCTTTCTTAAGAATTTACGCGCCGCTCAACGCGGTGCTGGCTGCGCCTGGGTTGCTGGCGGTGGCTGCGCTCACGATACCGGACATGTCCGGACGAAGCAGACTGGCTCTGGCTGCCCTGCTCGCTGTCATCTGGGGCGCCTATCTCCTGCAACTGGCCGCGACGCTGCTCAAGCGCCGGGCGGGAGTCGTACGGGACAGGACGCCCAAAATCGCCATCGATGTGCTCGCAGTCTTGGTTCCACTCGCCGCATTTCTGCTCGACGGCTCGCCTGACTGGAGCCTCTACTGTGCTGTCTGGCTGCTGAAACCGCTGCGCGACTCGACTTTCTTCCCGGTCCTGGGCAGGGTCCTGGCCAACGAAGCACGCAATCTGATCGGCGTCACCACGCTCTTCGGCGTCGTTCTGTTCGCAGTGGCGCTCGCAGCCTATGTCATCGAGCGCGATATCCAACCGGAAAAGTTCGGCAGCATTCCCCAGGCAATGTGGTGGGCGGTGGTCACGCTGTCCACCACCGGCTATGGGGACACTATCCCGCAAAGCTTCGCCGGCCGCGTCCTTGCCGGGGCGGTCATGATGAGTGGCATCGGCATCTTCGGACTCTGGGCCGGCATTCTTGCCACAGGCTTCTATCAAGAAGTCCGTCGCGGGGATTTCGTCCGCAATTGGCAATTGGTCGCCGCCGTGCCGTTGTTTCAGAAGCTCGGCCCGGCCGTGCTGGTCGAGATCGTGCGCGCCTTAAGAGCCCGCACGGTGCCGGCGGGCGCCGTGATCTGCCGCATTGGCGAGCCCGGCGATCGGATGTTCTTCGTCGTGGAGGGGAGCGTCAGCGTCGCGACGCCGAATCCGGTGGAGCTTGGCCCTGGCGCCTTCTTCGGCGAGATGGCGCTGATCAGCGGCGAACCGCGTTCGGCGACCGTCAGCGCCGCAACGACGGTCTCACTCCTGTCGCTGCATTCGGCGGATTTCCAGATGTTGTGCAGCAGCA GCCCGGAGATCGCGGAAATCTTCCGCAAGACCGCGCTCGAGCGTCGCGGCGCTGCGGCGAGCGCT
mICNBD protein:(如SEQ ID No.12所示)mICNBD protein: (as shown in SEQ ID No. 12)
MSVLPFLRIYAPLNAVLAAPGLLAVAALTIPDMSGRSRLALAALLAVIWGAYLLQLAATLLKRRAGVVRDRTPKIAIDVLAVLVPLAAFLLDGSPDWSLYCAVWLLKPLRDSTFFPVLGRVLANEARNLIGVTTLFGVVLFAVALAAYVIERDIQPEKFGSIPQAMWWAVVTLSTTGYGDTIPQSFAGRVLAGAVMMSGIGIFGLWAGILATGFYQEVRRGDFVRNWQLVAAVPLFQKLGPAVLVEIVRALRARTVPAGAVICRIGEPGDRMFFVVEGSVSVATPNPVELGPGAFFGEMALISGEPRSATVSAATTVSLLSLHSADFQMLCSSSPEIAEIFRKTALERRGAAASAMSVLPFLRIYAPLNAVLAAPGLLAVAALTIPDMSGRSRLALAALLAVIWGAYLLQLAATLLKRRAGVVRDRTPKIAIDVLAVLVPLAAFLLDGSPDWSLYCAVWLLKPLRDSTFFPVLGRVLANEARNLIGVTTLFGVVLFAVALAAYVIERDIQPEKFGSIPQAMWWAVVTLSTTGYGDTIPQSFAGRVLAGAVMMSGIGIFGLWAGILATGFYQEVRRGDFVRNWQLVAAVPLFQKLGPAVLVEIVRALRARTVPAGAVICRIGEPGDRMFFVVEGSVSVATPNPVELGPGAFFGEMALISGEPRSATVSAATTVSLLSLHSADFQMLCSSSPEIAEIFRKTALERRGAAASA
实施例2Example 2
将#252探针表达在细菌中，室温培养2天收集菌体，在pH＝7.3的HEPES缓冲液(含150mM KCl及50mM HEPES)中超声破碎，利用HisPur Cobalt Resin(购自皮尔斯公司)纯化探针，并通过Econo-Pac 10DG脱盐柱(购自美国Bio-Rad公司)将探针溶解于在pH＝7.3的HEPES缓冲液中，用BCA试剂盒(购自美国Thermo scientific公司)测定探针浓度。取2mM探针溶液，利用多功能酶标仪Infinite M1000 PRO检测探针对不同浓度cAMP及cGMP的响应，Flamindo2作为对照。可见#252在加入饱和浓度cAMP(～500μM)后，其在440-500nm波段激发的荧光亮度升高到3.5-4.15倍(图3)。在50μM cAMP浓度下，在500nm处激发的荧光亮度变化(ΔF/F0)为～2.5倍(图4，矩形示例的曲线)；在50μM cGMP浓度下，在500nm处激发的荧光亮度变化(ΔF/F0)为-16％(图4，×示例的曲线)。在大于5μM cGMP时，#252的因为cGMP引起的信号变化幅度明显小于Flamindo2。在小于5μM cGMP时,Flamindo2较#252因为cGMP引起的信号变化幅度小，但此时该信号变化均较小(图4，×示例的曲线与三角形示例的曲线，数据如表2所示)。The #252 probe was expressed in bacteria, cultured at room temperature for 2 days to collect bacterial cells, sonicated in pH=7.3 HEPES buffer (containing 150 mM KCl and 50 mM HEPES), and purified using HisPur Cobalt Resin (purchased from Pierce Corporation). The probe was dissolved in HEPES buffer at pH=7.3 through an Econo-Pac 10DG desalting column (purchased from Bio-Rad, USA), and the concentration of the probe was determined with BCA kit (purchased from Thermo scientific company, USA). . The 2mM probe solution was taken, and the multifunctional microplate reader Infinite M1000 PRO was used to detect the response of the probe to different concentrations of cAMP and cGMP, and Flamindo2 was used as a control. It can be seen that the fluorescence brightness of #252 in the 440-500 nm wavelength band increased to 3.5-4.15 times after adding a saturated concentration of cAMP (~500 μM) (Fig. 3). At 50 μM cAMP concentration, the change in fluorescence intensity excited at 500 nm (ΔF/F 0 ) was ~2.5-fold (FIG. 4, curve exemplified by rectangles); at 50 μM cGMP concentration, the change in fluorescence intensity at 500 nm excitation (ΔF /F 0 ) was -16% ( FIG. 4 , × example curve). When greater than 5 μM cGMP, the signal change amplitude of #252 due to cGMP was significantly smaller than that of Flamindo2. When less than 5μM cGMP, Flamindo2 has a smaller signal change amplitude than #252 due to cGMP, but the signal changes are smaller at this time (Fig. 4, the curve of the × example and the curve of the triangle example, the data are shown in Table 2).
表2Table 2
实施例3Example 3
将#252、Flamindo2、mEGFP(单体绿色荧光蛋白)分别构建到真核表达载体上(CAG启动子)，通过Lipofectamine 2000试剂盒转染培养在玻璃底的培养皿中的HEK293T细胞(购买自GE Healthcare Dharmacon公司)，过夜培养后，用不含血清的、不含酚红的培养基(购自GIBCO公司)饥饿细胞6小时。利用本实验室自行搭建的IX83荧光显微镜检测探针的亮度，可见#252在细胞静息状态下亮度远较Flamindo2高，约提高60倍[(3000-225)/(225-180)＝61.6](图5A)。细胞受60μM Forskolin及100μM IBMX(购自碧云天生物技术公司)刺激后cAMP浓度升高，#252探针的信号变化幅度为100％-250％(图5B-C)。至此完成了哺乳动物细胞内cAMP浓度变化的荧光成像步骤。#252, Flamindo2, and mEGFP (monomeric green fluorescent protein) were constructed into eukaryotic expression vectors (CAG promoter) respectively, and HEK293T cells (purchased from GE) cultured in glass-bottom dishes were transfected by
实施例4荧光探针的突变Example 4 Mutation of fluorescent probes
285-ins为优化前探针，252为优化后的探针。两种比较，不同的氨基酸用加粗字体标记了出来：左边Linker前者为LE，后者为WG。右边linker前者为LP，后者为RV。285-ins is the probe before optimization, and 252 is the probe after optimization. For the two comparisons, different amino acids are marked with bold fonts: the former is LE for the Linker on the left, and the latter is WG. The former linker on the right is LP and the latter is RV.
荧光蛋白上做的改变从左边到右边分别为：K153T,I171V,S30R,Y39N,S72A,F99V,N105T。这些序号来自荧光蛋白，而不是氨基酸在探针上的序号。The changes made on the fluorescent protein from left to right are: K153T, I171V, S30R, Y39N, S72A, F99V, N105T. These numbers are from the fluorescent protein, not the amino acid number on the probe.
突变方法：先选定要突变的位点，然后在设计的引物上包含突变的氨基酸，分段PCR扩增出各包含突变的片段，最后做1次overlap PCR将这些突变整合，然后做文库挑选。Mutation method: first select the site to be mutated, then include the mutated amino acid on the designed primer, amplify each fragment containing the mutation by segmental PCR, and finally do one overlap PCR to integrate these mutations, and then do the library selection .
252-ORF(如SEQ ID No.8所示):252-ORF (as shown in SEQ ID No. 8):
ATGCGGGGTTCTCATCATCATCATCATCATGGTATGGCTAGCATGACTGGTGGACAGCAAATGGGTCGGGATCTGTACGACGATGACGATAAGGATCCGATGGGCTTCTATCAAGAAGTCCGTCGCGGGGATTTCGTCCGCAATTGGCAATTGGTCGCCGCCGTGCCGTTGTTTCAGAAGCTCGGCCCGGCCGTGCTGGTCGAGATCGTGCGCGCCTTAAGAGCCCGCACGGTGCCGGCGGGCGCCGTGATCTGCCGCATTGGCGAGCCCGGCGATCGGATGTTCTTCGTCGTGGAGGGGAGCGTCAGCGTCGCGACGCCGTGGGGGAACGTCTATATCACAGCCGACAAGCAGAAGAACGGCATCAAGGCGAACTTCAAGATCCGCCACAACGTTGAGGACGGCGGCGTGCAGCTCGCCTACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCGTGCAGTCCAAACTTTCGAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGGGCGGTACCGGAGGGAGCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGCGTGGCGAGGGTGAGGGCGATGCCACCAATGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCGCACGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACATCCAGGAGCGCACCATCGTTTTCAAGGACGACGGCACCTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACCGCGTGAATCCGGTGGAGCTTGGCCCTGGCGCCTTCTTCGGCGAGATGGCGCTGATCAGCGGCGAACCGCGTTCGGCGACCGTCAGCGCCGCAACGACGGTCTCACTCCTGTCGCTGCATTCGGCGGATTTCCAGATGTTGTGCAGCAGCAGCCCGGAGATCGCGGAAATCTTCCGCAAGACCGCGCTCGAGCGTCGCGGCGCTGCGGCGAGCGCTATGCGGGGTTCTCATCATCATCATCATCATGGTATGGCTAGCATGACTGGTGGACAGCAAATGGGTCGGGATCTGTACGACGATGACGATAAGGATCCGATGGGCTTCTATCAAGAAGTCCGTCGCGGGGATTTCGTCCGCAATTGGCAATTGGTCGCCGCCGTGCCGTTGTTTCAGAAGCTCGGCCCGGCCGTGCTGGTCGAGATCGTGCGCGCCTTAAGAGCCCGCACGGTGCCGGCGGGCGCCGTGATCTGCCGCATTGGCGAGCCCGGCGATCGGATGTTCTTCGTCGTGGAGGGGAGCGTCAGCGTCGCGACGCCGTGGGGGAACGTCTATATCACAGCCGACAAGCAGAAGAACGGCATCAAGGCGAACTTCAAGATCCGCCACAACGTTGAGGACGGCGGCGTGCAGCTCGCCTACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCGTGCAGTCCAAACTTTCGAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGGGCGGTACCGGAGGGAGCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGCGTGGCGAGGGTGAGGGCGATGCCACCAATGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCGCACGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACATCCAGGAGCGCACCATCGTTTTCAAGGACGACGGCACCTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCA TCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACCGCGTGAATCCGGTGGAGCTTGGCCCTGGCGCCTTCTTCGGCGAGATGGCGCTGATCAGCGGCGAACCGCGTTCGGCGACCGTCAGCGCCGCAACGACGGTCTCACTCCTGTCGCTGCATTCGGCGGATTTCCAGATGTTGTGCAGCAGCAGCCCGGAGATCGCGGAAATCTTCCGCAAGACCGCGCGCGCGCGGCT
252 protein(如SEQ ID No.7所示):252 protein (as shown in SEQ ID No. 7):
285-ins-ORF(如SEQ ID No.9所示):285-ins-ORF (as shown in SEQ ID No. 9):
ATGCGGGGTTCTCATCATCATCATCATCATGGTATGGCTAGCATGACTGGTGGACAGCAAATGGGTCGGGATCTGTACGACGATGACGATAAGGATCCGATGGGCTTCTATCAAGAAGTCCGTCGCGGGGATTTCGTCCGCAATTGGCAATTGGTCGCCGCCGTGCCGTTGTTTCAGAAGCTCGGCCCGGCCGTGCTGGTCGAGATCGTGCGCGCCTTAAGAGCCCGCACGGTGCCGGCGGGCGCCGTGATCTGCCGCATTGGCGAGCCCGGCGATCGGATGTTCTTCGTCGTGGAGGGGAGCGTCAGCGTCGCGACGCCGCTCGAGAACGTCTATATCAAGGCCGACAAGCAGAAGAACGGCATCAAGGCGAACTTCAAGATCCGCCACAACATCGAGGACGGCGGCGTGCAGCTCGCCTACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCGTGCAGTCCAAACTTTCGAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGGGCGGTACCGGAGGGAGCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGTGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACATCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACCTGCCGAATCCGGTGGAGCTTGGCCCTGGCGCCTTCTTCGGCGAGATGGCGCTGATCAGCGGCGAACCGCGTTCGGCGACCGTCAGCGCCGCAACGACGGTCTCACTCCTGTCGCTGCATTCGGCGGATTTCCAGATGTTGTGCAGCAGCAGCCCGGAGATCGCGGAAATCTTCCGCAAGACCGCGCTCGAGCGTCGCGGCGCTGCGGCGAGCGCTATGCGGGGTTCTCATCATCATCATCATCATGGTATGGCTAGCATGACTGGTGGACAGCAAATGGGTCGGGATCTGTACGACGATGACGATAAGGATCCGATGGGCTTCTATCAAGAAGTCCGTCGCGGGGATTTCGTCCGCAATTGGCAATTGGTCGCCGCCGTGCCGTTGTTTCAGAAGCTCGGCCCGGCCGTGCTGGTCGAGATCGTGCGCGCCTTAAGAGCCCGCACGGTGCCGGCGGGCGCCGTGATCTGCCGCATTGGCGAGCCCGGCGATCGGATGTTCTTCGTCGTGGAGGGGAGCGTCAGCGTCGCGACGCCGCTCGAGAACGTCTATATCAAGGCCGACAAGCAGAAGAACGGCATCAAGGCGAACTTCAAGATCCGCCACAACATCGAGGACGGCGGCGTGCAGCTCGCCTACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCGTGCAGTCCAAACTTTCGAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGGGCGGTACCGGAGGGAGCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGTGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACATCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCA TCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACCTGCCGAATCCGGTGGAGCTTGGCCCTGGCGCCTTCTTCGGCGAGATGGCGCTGATCAGCGGCGAACCGCGTTCGGCGACCGTCAGCGCCGCAACGACGGTCTCACTCCTGTCGCTGCATTCGGCGGATTTCCAGATGTTGTGCAGCAGCAGCCCGGAGATCGCGGAAATCTTCCGCAAGACCGCGCGCGCGCGGCT
285-ins protein(如SEQ ID No.10所示):285-ins protein (shown as SEQ ID No. 10):
突变探针带来的效果：The effect brought by the mutation probe:
1,动态范围变大。1, the dynamic range becomes larger.
图6示纯化的285-ins及#252与cAMP结合后的激发光谱变化。虚线为未加入cAMP激发光谱(A)，实线为加入终浓度为1mM cAMP后的激发光谱(B)。可见，#252探针有了较大的正变化。Figure 6 shows the change in excitation spectra of purified 285-ins and #252 after binding to cAMP. The dotted line is the excitation spectrum (A) without cAMP added, and the solid line is the excitation spectrum (B) after adding a final concentration of 1 mM cAMP. It can be seen that the #252 probe has a large positive change.
2.亮度提高。2. Brightness is improved.
图7示252亮度为285-ins的(87-40)/(40-26)＝3.35倍。Figure 7 shows that 252 luminance is (87-40)/(40-26)=3.35 times of 285-ins.
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。The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, several improvements and modifications can be made. It should be regarded as the protection scope of the present invention.
序列表sequence listing
<110> 深圳先进技术研究院<110> Shenzhen Advanced Technology Research Institute
<120> cAMP荧光探针及其应用<120> cAMP fluorescent probe and its application
<130> MP1829175<130> MP1829175
<160> 12<160> 12
<170> SIPOSequenceListing 1.0<170> SIPOSequenceListing 1.0
<210> 1<210> 1
<211> 355<211> 355
<212> PRT<212> PRT
<213> CNBD<213> CNBD
<400> 1<400> 1
Met Ser Val Leu Pro Phe Leu Arg Ile Tyr Ala Pro Leu Asn Ala ValMet Ser Val Leu Pro Phe Leu Arg Ile Tyr Ala Pro Leu Asn Ala Val
1 5 10 151 5 10 15
Leu Ala Ala Pro Gly Leu Leu Ala Val Ala Ala Leu Thr Ile Pro AspLeu Ala Ala Pro Gly Leu Leu Ala Val Ala Ala Leu Thr Ile Pro Asp
20 25 30 20 25 30
Met Ser Gly Arg Ser Arg Leu Ala Leu Ala Ala Leu Leu Ala Val IleMet Ser Gly Arg Ser Arg Leu Ala Leu Ala Ala Leu Leu Ala Val Ile
35 40 45 35 40 45
Trp Gly Ala Tyr Leu Leu Gln Leu Ala Ala Thr Leu Leu Lys Arg ArgTrp Gly Ala Tyr Leu Leu Gln Leu Ala Ala Thr Leu Leu Lys Arg Arg
50 55 60 50 55 60
Ala Gly Val Val Arg Asp Arg Thr Pro Lys Ile Ala Ile Asp Val LeuAla Gly Val Val Arg Asp Arg Thr Pro Lys Ile Ala Ile Asp Val Leu
65 70 75 8065 70 75 80
Ala Val Leu Val Pro Leu Ala Ala Phe Leu Leu Asp Gly Ser Pro AspAla Val Leu Val Pro Leu Ala Ala Phe Leu Leu Asp Gly Ser Pro Asp
85 90 95 85 90 95
Trp Ser Leu Tyr Cys Ala Val Trp Leu Leu Lys Pro Leu Arg Asp SerTrp Ser Leu Tyr Cys Ala Val Trp Leu Leu Lys Pro Leu Arg Asp Ser
100 105 110 100 105 110
Thr Phe Phe Pro Val Leu Gly Arg Val Leu Ala Asn Glu Ala Arg AsnThr Phe Phe Pro Val Leu Gly Arg Val Leu Ala Asn Glu Ala Arg Asn
115 120 125 115 120 125
Leu Ile Gly Val Thr Thr Leu Phe Gly Val Val Leu Phe Ala Val AlaLeu Ile Gly Val Thr Thr Leu Phe Gly Val Val Leu Phe Ala Val Ala
130 135 140 130 135 140
Leu Ala Ala Tyr Val Ile Glu Arg Asp Ile Gln Pro Glu Lys Phe GlyLeu Ala Ala Tyr Val Ile Glu Arg Asp Ile Gln Pro Glu Lys Phe Gly
145 150 155 160145 150 155 160
Ser Ile Pro Gln Ala Met Trp Trp Ala Val Val Thr Leu Ser Thr ThrSer Ile Pro Gln Ala Met Trp Trp Ala Val Val Thr Leu Ser Thr Thr
165 170 175 165 170 175
Gly Tyr Gly Asp Thr Ile Pro Gln Ser Phe Ala Gly Arg Val Leu AlaGly Tyr Gly Asp Thr Ile Pro Gln Ser Phe Ala Gly Arg Val Leu Ala
180 185 190 180 185 190
Gly Ala Val Met Met Ser Gly Ile Gly Ile Phe Gly Leu Trp Ala GlyGly Ala Val Met Met Ser Gly Ile Gly Ile Phe Gly Leu Trp Ala Gly
195 200 205 195 200 205
Ile Leu Ala Thr Gly Phe Tyr Gln Glu Val Arg Arg Gly Asp Phe ValIle Leu Ala Thr Gly Phe Tyr Gln Glu Val Arg Arg Gly Asp Phe Val
210 215 220 210 215 220
Arg Asn Trp Gln Leu Val Ala Ala Val Pro Leu Phe Gln Lys Leu GlyArg Asn Trp Gln Leu Val Ala Ala Val Pro Leu Phe Gln Lys Leu Gly
225 230 235 240225 230 235 240
Pro Ala Val Leu Val Glu Ile Val Arg Ala Leu Arg Ala Arg Thr ValPro Ala Val Leu Val Glu Ile Val Arg Ala Leu Arg Ala Arg Thr Val
245 250 255 245 250 255
Pro Ala Gly Ala Val Ile Cys Arg Ile Gly Glu Pro Gly Asp Arg MetPro Ala Gly Ala Val Ile Cys Arg Ile Gly Glu Pro Gly Asp Arg Met
260 265 270 260 265 270
Phe Phe Val Val Glu Gly Ser Val Ser Val Ala Thr Pro Asn Pro ValPhe Phe Val Val Glu Gly Ser Val Ser Val Ala Thr Pro Asn Pro Val
275 280 285 275 280 285
Glu Leu Gly Pro Gly Ala Phe Phe Gly Glu Met Ala Leu Ile Ser GlyGlu Leu Gly Pro Gly Ala Phe Phe Gly Glu Met Ala Leu Ile Ser Gly
290 295 300 290 295 300
Glu Pro Arg Ser Ala Thr Val Ser Ala Ala Thr Thr Val Ser Leu LeuGlu Pro Arg Ser Ala Thr Val Ser Ala Ala Thr Thr Val Ser Leu Leu
305 310 315 320305 310 315 320
Ser Leu His Ser Ala Asp Phe Gln Met Leu Cys Ser Ser Ser Pro GluSer Leu His Ser Ala Asp Phe Gln Met Leu Cys Ser Ser Ser Pro Glu
325 330 335 325 330 335
Ile Ala Glu Ile Phe Arg Lys Thr Ala Leu Glu Arg Arg Gly Ala AlaIle Ala Glu Ile Phe Arg Lys Thr Ala Leu Glu Arg Arg Gly Ala Ala
340 345 350 340 345 350
Ala Ser AlaAla Ser Ala
355 355
<210> 2<210> 2
<211> 241<211> 241
<212> PRT<212> PRT
<213> cpEGFP<213> cpEGFP
<400> 2<400> 2
Asn Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala AsnAsn Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn
1 5 10 151 5 10 15
Phe Lys Ile Arg His Asn Val Glu Asp Gly Gly Val Gln Leu Ala TyrPhe Lys Ile Arg His Asn Val Glu Asp Gly Gly Val Gln Leu Ala Tyr
20 25 30 20 25 30
His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu ProHis Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro
35 40 45 35 40 45
Asp Asn His Tyr Leu Ser Val Gln Ser Lys Leu Ser Lys Asp Pro AsnAsp Asn His Tyr Leu Ser Val Gln Ser Lys Leu Ser Lys Asp Pro Asn
50 55 60 50 55 60
Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala GlyGlu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly
65 70 75 8065 70 75 80
Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Gly Gly Thr Gly Gly SerIle Thr Leu Gly Met Asp Glu Leu Tyr Lys Gly Gly Thr Gly Gly Ser
85 90 95 85 90 95
Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile LeuMet Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu
100 105 110 100 105 110
Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Arg GlyVal Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Arg Gly
115 120 125 115 120 125
Glu Gly Glu Gly Asp Ala Thr Asn Gly Lys Leu Thr Leu Lys Phe IleGlu Gly Glu Gly Asp Ala Thr Asn Gly Lys Leu Thr Leu Lys Phe Ile
130 135 140 130 135 140
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr ThrCys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
145 150 155 160145 150 155 160
Leu Thr Tyr Gly Val Gln Cys Phe Ala Arg Tyr Pro Asp His Met LysLeu Thr Tyr Gly Val Gln Cys Phe Ala Arg Tyr Pro Asp His Met Lys
165 170 175 165 170 175
Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Ile Gln GluGln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Ile Gln Glu
180 185 190 180 185 190
Arg Thr Ile Val Phe Lys Asp Asp Gly Thr Tyr Lys Thr Arg Ala GluArg Thr Ile Val Phe Lys Asp Asp Gly Thr Tyr Lys Thr Arg Ala Glu
195 200 205 195 200 205
Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys GlyVal Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly
210 215 220 210 215 220
Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu TyrIle Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr
225 230 235 240225 230 235 240
AsnAsn
<210> 3<210> 3
<211> 285<211> 285
<212> PRT<212> PRT
<213> CNBD-N<213> CNBD-N
<400> 3<400> 3
Met Ser Val Leu Pro Phe Leu Arg Ile Tyr Ala Pro Leu Asn Ala ValMet Ser Val Leu Pro Phe Leu Arg Ile Tyr Ala Pro Leu Asn Ala Val
1 5 10 151 5 10 15
Leu Ala Ala Pro Gly Leu Leu Ala Val Ala Ala Leu Thr Ile Pro AspLeu Ala Ala Pro Gly Leu Leu Ala Val Ala Ala Leu Thr Ile Pro Asp
20 25 30 20 25 30
Met Ser Gly Arg Ser Arg Leu Ala Leu Ala Ala Leu Leu Ala Val IleMet Ser Gly Arg Ser Arg Leu Ala Leu Ala Ala Leu Leu Ala Val Ile
35 40 45 35 40 45
Trp Gly Ala Tyr Leu Leu Gln Leu Ala Ala Thr Leu Leu Lys Arg ArgTrp Gly Ala Tyr Leu Leu Gln Leu Ala Ala Thr Leu Leu Lys Arg Arg
50 55 60 50 55 60
Ala Gly Val Val Arg Asp Arg Thr Pro Lys Ile Ala Ile Asp Val LeuAla Gly Val Val Arg Asp Arg Thr Pro Lys Ile Ala Ile Asp Val Leu
65 70 75 8065 70 75 80
Ala Val Leu Val Pro Leu Ala Ala Phe Leu Leu Asp Gly Ser Pro AspAla Val Leu Val Pro Leu Ala Ala Phe Leu Leu Asp Gly Ser Pro Asp
85 90 95 85 90 95
Trp Ser Leu Tyr Cys Ala Val Trp Leu Leu Lys Pro Leu Arg Asp SerTrp Ser Leu Tyr Cys Ala Val Trp Leu Leu Lys Pro Leu Arg Asp Ser
100 105 110 100 105 110
Thr Phe Phe Pro Val Leu Gly Arg Val Leu Ala Asn Glu Ala Arg AsnThr Phe Phe Pro Val Leu Gly Arg Val Leu Ala Asn Glu Ala Arg Asn
115 120 125 115 120 125
Leu Ile Gly Val Thr Thr Leu Phe Gly Val Val Leu Phe Ala Val AlaLeu Ile Gly Val Thr Thr Leu Phe Gly Val Val Leu Phe Ala Val Ala
130 135 140 130 135 140
Leu Ala Ala Tyr Val Ile Glu Arg Asp Ile Gln Pro Glu Lys Phe GlyLeu Ala Ala Tyr Val Ile Glu Arg Asp Ile Gln Pro Glu Lys Phe Gly
145 150 155 160145 150 155 160
Ser Ile Pro Gln Ala Met Trp Trp Ala Val Val Thr Leu Ser Thr ThrSer Ile Pro Gln Ala Met Trp Trp Ala Val Val Thr Leu Ser Thr Thr
165 170 175 165 170 175
Gly Tyr Gly Asp Thr Ile Pro Gln Ser Phe Ala Gly Arg Val Leu AlaGly Tyr Gly Asp Thr Ile Pro Gln Ser Phe Ala Gly Arg Val Leu Ala
180 185 190 180 185 190
Gly Ala Val Met Met Ser Gly Ile Gly Ile Phe Gly Leu Trp Ala GlyGly Ala Val Met Met Ser Gly Ile Gly Ile Phe Gly Leu Trp Ala Gly
195 200 205 195 200 205
Ile Leu Ala Thr Gly Phe Tyr Gln Glu Val Arg Arg Gly Asp Phe ValIle Leu Ala Thr Gly Phe Tyr Gln Glu Val Arg Arg Gly Asp Phe Val
210 215 220 210 215 220
Arg Asn Trp Gln Leu Val Ala Ala Val Pro Leu Phe Gln Lys Leu GlyArg Asn Trp Gln Leu Val Ala Ala Val Pro Leu Phe Gln Lys Leu Gly
225 230 235 240225 230 235 240
Pro Ala Val Leu Val Glu Ile Val Arg Ala Leu Arg Ala Arg Thr ValPro Ala Val Leu Val Glu Ile Val Arg Ala Leu Arg Ala Arg Thr Val
245 250 255 245 250 255
Pro Ala Gly Ala Val Ile Cys Arg Ile Gly Glu Pro Gly Asp Arg MetPro Ala Gly Ala Val Ile Cys Arg Ile Gly Glu Pro Gly Asp Arg Met
260 265 270 260 265 270
Phe Phe Val Val Glu Gly Ser Val Ser Val Ala Thr ProPhe Phe Val Val Glu Gly Ser Val Ser Val Ala Thr Pro
275 280 285 275 280 285
<210> 4<210> 4
<211> 70<211> 70
<212> PRT<212> PRT
<213> CNBD-C<213> CNBD-C
<400> 4<400> 4
Asn Pro Val Glu Leu Gly Pro Gly Ala Phe Phe Gly Glu Met Ala LeuAsn Pro Val Glu Leu Gly Pro Gly Ala Phe Phe Gly Glu Met Ala Leu
1 5 10 151 5 10 15
Ile Ser Gly Glu Pro Arg Ser Ala Thr Val Ser Ala Ala Thr Thr ValIle Ser Gly Glu Pro Arg Ser Ala Thr Val Ser Ala Ala Thr Thr Val
20 25 30 20 25 30
Ser Leu Leu Ser Leu His Ser Ala Asp Phe Gln Met Leu Cys Ser SerSer Leu Leu Ser Leu His Ser Ala Asp Phe Gln Met Leu Cys Ser Ser
35 40 45 35 40 45
Ser Pro Glu Ile Ala Glu Ile Phe Arg Lys Thr Ala Leu Glu Arg ArgSer Pro Glu Ile Ala Glu Ile Phe Arg Lys Thr Ala Leu Glu Arg Arg
50 55 60 50 55 60
Gly Ala Ala Ala Ser AlaGly Ala Ala Ala Ser Ala
65 7065 70
<210> 5<210> 5
<211> 2<211> 2
<212> PRT<212> PRT
<213> linker1<213> linker1
<400> 5<400> 5
Trp GlyTrp Gly
11
<210> 6<210> 6
<211> 2<211> 2
<212> PRT<212> PRT
<213> linker2<213> linker2
<400> 6<400> 6
Arg ValArg Val
11
<210> 7<210> 7
<211> 422<211> 422
<212> PRT<212> PRT
<213> cAMP荧光探针(cAMP fluorescent probe)<213> cAMP fluorescent probe
<400> 7<400> 7
Met Arg Gly Ser His His His His His His Gly Met Ala Ser Met ThrMet Arg Gly Ser His His His His His His Gly Met Ala Ser Met Thr
1 5 10 151 5 10 15
Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys AspGly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp
20 25 30 20 25 30
Pro Met Gly Phe Tyr Gln Glu Val Arg Arg Gly Asp Phe Val Arg AsnPro Met Gly Phe Tyr Gln Glu Val Arg Arg Gly Asp Phe Val Arg Asn
35 40 45 35 40 45
Trp Gln Leu Val Ala Ala Val Pro Leu Phe Gln Lys Leu Gly Pro AlaTrp Gln Leu Val Ala Ala Val Pro Leu Phe Gln Lys Leu Gly Pro Ala
50 55 60 50 55 60
Val Leu Val Glu Ile Val Arg Ala Leu Arg Ala Arg Thr Val Pro AlaVal Leu Val Glu Ile Val Arg Ala Leu Arg Ala Arg Thr Val Pro Ala
65 70 75 8065 70 75 80
Gly Ala Val Ile Cys Arg Ile Gly Glu Pro Gly Asp Arg Met Phe PheGly Ala Val Ile Cys Arg Ile Gly Glu Pro Gly Asp Arg Met Phe Phe
85 90 95 85 90 95
Val Val Glu Gly Ser Val Ser Val Ala Thr Pro Trp Gly Asn Val TyrVal Val Glu Gly Ser Val Ser Val Ala Thr Pro Trp Gly Asn Val Tyr
100 105 110 100 105 110
Ile Thr Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys IleIle Thr Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile
115 120 125 115 120 125
Arg His Asn Val Glu Asp Gly Gly Val Gln Leu Ala Tyr His Tyr GlnArg His Asn Val Glu Asp Gly Gly Val Gln Leu Ala Tyr His Tyr Gln
130 135 140 130 135 140
Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn HisGln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His
145 150 155 160145 150 155 160
Tyr Leu Ser Val Gln Ser Lys Leu Ser Lys Asp Pro Asn Glu Lys ArgTyr Leu Ser Val Gln Ser Lys Leu Ser Lys Asp Pro Asn Glu Lys Arg
165 170 175 165 170 175
Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr LeuAsp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu
180 185 190 180 185 190
Gly Met Asp Glu Leu Tyr Lys Gly Gly Thr Gly Gly Ser Met Val SerGly Met Asp Glu Leu Tyr Lys Gly Gly Thr Gly Gly Ser Met Val Ser
195 200 205 195 200 205
Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu LeuLys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu
210 215 220 210 215 220
Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Arg Gly Glu Gly GluAsp Gly Asp Val Asn Gly His Lys Phe Ser Val Arg Gly Glu Gly Glu
225 230 235 240225 230 235 240
Gly Asp Ala Thr Asn Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr ThrGly Asp Ala Thr Asn Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr
245 250 255 245 250 255
Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr TyrGly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr
260 265 270 260 265 270
Gly Val Gln Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His AspGly Val Gln Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His Asp
275 280 285 275 280 285
Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Ile Gln Glu Arg Thr IlePhe Phe Lys Ser Ala Met Pro Glu Gly Tyr Ile Gln Glu Arg Thr Ile
290 295 300 290 295 300
Val Phe Lys Asp Asp Gly Thr Tyr Lys Thr Arg Ala Glu Val Lys PheVal Phe Lys Asp Asp Gly Thr Tyr Lys Thr Arg Ala Glu Val Lys Phe
305 310 315 320305 310 315 320
Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp PheGlu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe
325 330 335 325 330 335
Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Arg ValLys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Arg Val
340 345 350 340 345 350
Asn Pro Val Glu Leu Gly Pro Gly Ala Phe Phe Gly Glu Met Ala LeuAsn Pro Val Glu Leu Gly Pro Gly Ala Phe Phe Gly Glu Met Ala Leu
355 360 365 355 360 365
Ile Ser Gly Glu Pro Arg Ser Ala Thr Val Ser Ala Ala Thr Thr ValIle Ser Gly Glu Pro Arg Ser Ala Thr Val Ser Ala Ala Thr Thr Val
370 375 380 370 375 380
Ser Leu Leu Ser Leu His Ser Ala Asp Phe Gln Met Leu Cys Ser SerSer Leu Leu Ser Leu His Ser Ala Asp Phe Gln Met Leu Cys Ser Ser
385 390 395 400385 390 395 400
Ser Pro Glu Ile Ala Glu Ile Phe Arg Lys Thr Ala Leu Glu Arg ArgSer Pro Glu Ile Ala Glu Ile Phe Arg Lys Thr Ala Leu Glu Arg Arg
405 410 415 405 410 415
Gly Ala Ala Ala Ser AlaGly Ala Ala Ala Ser Ala
420 420
<210> 8<210> 8
<211> 1266<211> 1266
<212> DNA<212> DNA
<213> cAMP荧光探针(cAMP fluorescent probe)<213> cAMP fluorescent probe
<400> 8<400> 8
atgcggggtt ctcatcatca tcatcatcat ggtatggcta gcatgactgg tggacagcaa 60atgcggggtt ctcatcatca tcatcatcat ggtatggcta gcatgactgg tggacagcaa 60
atgggtcggg atctgtacga cgatgacgat aaggatccga tgggcttcta tcaagaagtc 120atgggtcggg atctgtacga cgatgacgat aaggatccga tgggcttcta tcaagaagtc 120
cgtcgcgggg atttcgtccg caattggcaa ttggtcgccg ccgtgccgtt gtttcagaag 180cgtcgcgggg atttcgtccg caattggcaa ttggtcgccg ccgtgccgtt gtttcagaag 180
ctcggcccgg ccgtgctggt cgagatcgtg cgcgccttaa gagcccgcac ggtgccggcg 240ctcggcccgg ccgtgctggt cgagatcgtg cgcgccttaa gagcccgcac ggtgccggcg 240
ggcgccgtga tctgccgcat tggcgagccc ggcgatcgga tgttcttcgt cgtggagggg 300ggcgccgtga tctgccgcat tggcgagccc ggcgatcgga tgttcttcgt cgtggagggg 300
agcgtcagcg tcgcgacgcc gtgggggaac gtctatatca cagccgacaa gcagaagaac 360agcgtcagcg tcgcgacgcc gtgggggaac gtctatatca cagccgacaa gcagaagaac 360
ggcatcaagg cgaacttcaa gatccgccac aacgttgagg acggcggcgt gcagctcgcc 420ggcatcaagg cgaacttcaa gatccgccac aacgttgagg acggcggcgt gcagctcgcc 420
taccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 480taccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 480
tacctgagcg tgcagtccaa actttcgaaa gaccccaacg agaagcgcga tcacatggtc 540tacctgagcg tgcagtccaa actttcgaaa gaccccaacg agaagcgcga tcacatggtc 540
ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagggc 600ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagggc 600
ggtaccggag ggagcatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc 660ggtaccggag ggagcatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc 660
ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgcgtgg cgagggtgag 720ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgcgtgg cgagggtgag 720
ggcgatgcca ccaatggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc 780ggcgatgcca ccaatggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc 780
gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cgcacgctac 840gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cgcacgctac 840
cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacatccag 900cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacatccag 900
gagcgcacca tcgttttcaa ggacgacggc acctacaaga cccgcgccga ggtgaagttc 960gagcgcacca tcgttttcaa ggacgacggc acctacaaga cccgcgccga ggtgaagttc 960
gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc 1020gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc 1020
aacatcctgg ggcacaagct ggagtacaac cgcgtgaatc cggtggagct tggccctggc 1080aacatcctgg ggcacaagct ggagtacaac cgcgtgaatc cggtggagct tggccctggc 1080
gccttcttcg gcgagatggc gctgatcagc ggcgaaccgc gttcggcgac cgtcagcgcc 1140gccttcttcg gcgagatggc gctgatcagc ggcgaaccgc gttcggcgac cgtcagcgcc 1140
gcaacgacgg tctcactcct gtcgctgcat tcggcggatt tccagatgtt gtgcagcagc 1200gcaacgacgg tctcactcct gtcgctgcat tcggcggatt tccagatgtt gtgcagcagc 1200
agcccggaga tcgcggaaat cttccgcaag accgcgctcg agcgtcgcgg cgctgcggcg 1260agcccggaga tcgcggaaat cttccgcaag accgcgctcg agcgtcgcgg cgctgcggcg 1260
agcgct 1266agcgct 1266
<210> 9<210> 9
<211> 1266<211> 1266
<212> DNA<212> DNA
<213> 285-ins-ORF<213> 285-ins-ORF
<400> 9<400> 9
atgcggggtt ctcatcatca tcatcatcat ggtatggcta gcatgactgg tggacagcaa 60atgcggggtt ctcatcatca tcatcatcat ggtatggcta gcatgactgg tggacagcaa 60
atgggtcggg atctgtacga cgatgacgat aaggatccga tgggcttcta tcaagaagtc 120atgggtcggg atctgtacga cgatgacgat aaggatccga tgggcttcta tcaagaagtc 120
cgtcgcgggg atttcgtccg caattggcaa ttggtcgccg ccgtgccgtt gtttcagaag 180cgtcgcgggg atttcgtccg caattggcaa ttggtcgccg ccgtgccgtt gtttcagaag 180
ctcggcccgg ccgtgctggt cgagatcgtg cgcgccttaa gagcccgcac ggtgccggcg 240ctcggcccgg ccgtgctggt cgagatcgtg cgcgccttaa gagcccgcac ggtgccggcg 240
ggcgccgtga tctgccgcat tggcgagccc ggcgatcgga tgttcttcgt cgtggagggg 300ggcgccgtga tctgccgcat tggcgagccc ggcgatcgga tgttcttcgt cgtggagggg 300
agcgtcagcg tcgcgacgcc gtgggggaac gtctatatca cagccgacaa gcagaagaac 360agcgtcagcg tcgcgacgcc gtgggggaac gtctatatca cagccgacaa gcagaagaac 360
ggcatcaagg cgaacttcaa gatccgccac aacgttgagg acggcggcgt gcagctcgcc 420ggcatcaagg cgaacttcaa gatccgccac aacgttgagg acggcggcgt gcagctcgcc 420
taccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 480taccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 480
tacctgagcg tgcagtccaa actttcgaaa gaccccaacg agaagcgcga tcacatggtc 540tacctgagcg tgcagtccaa actttcgaaa gaccccaacg agaagcgcga tcacatggtc 540
ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagggc 600ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagggc 600
ggtaccggag ggagcatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc 660ggtaccggag ggagcatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc 660
ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgcgtgg cgagggtgag 720ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgcgtgg cgagggtgag 720
ggcgatgcca ccaatggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc 780ggcgatgcca ccaatggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc 780
gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cgcacgctac 840gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cgcacgctac 840
cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacatccag 900cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacatccag 900
gagcgcacca tcgttttcaa ggacgacggc acctacaaga cccgcgccga ggtgaagttc 960gagcgcacca tcgttttcaa ggacgacggc acctacaaga cccgcgccga ggtgaagttc 960
gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc 1020gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc 1020
aacatcctgg ggcacaagct ggagtacaac cgcgtgaatc cggtggagct tggccctggc 1080aacatcctgg ggcacaagct ggagtacaac cgcgtgaatc cggtggagct tggccctggc 1080
gccttcttcg gcgagatggc gctgatcagc ggcgaaccgc gttcggcgac cgtcagcgcc 1140gccttcttcg gcgagatggc gctgatcagc ggcgaaccgc gttcggcgac cgtcagcgcc 1140
gcaacgacgg tctcactcct gtcgctgcat tcggcggatt tccagatgtt gtgcagcagc 1200gcaacgacgg tctcactcct gtcgctgcat tcggcggatt tccagatgtt gtgcagcagc 1200
agcccggaga tcgcggaaat cttccgcaag accgcgctcg agcgtcgcgg cgctgcggcg 1260agcccggaga tcgcggaaat cttccgcaag accgcgctcg agcgtcgcgg cgctgcggcg 1260
agcgct 1266agcgct 1266
<210> 10<210> 10
<211> 422<211> 422
<212> PRT<212> PRT
<213> 285-ins-ORF<213> 285-ins-ORF
<400> 10<400> 10
Met Arg Gly Ser His His His His His His Gly Met Ala Ser Met ThrMet Arg Gly Ser His His His His His His Gly Met Ala Ser Met Thr
1 5 10 151 5 10 15
Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys AspGly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp
20 25 30 20 25 30
Pro Met Gly Phe Tyr Gln Glu Val Arg Arg Gly Asp Phe Val Arg AsnPro Met Gly Phe Tyr Gln Glu Val Arg Arg Gly Asp Phe Val Arg Asn
35 40 45 35 40 45
Trp Gln Leu Val Ala Ala Val Pro Leu Phe Gln Lys Leu Gly Pro AlaTrp Gln Leu Val Ala Ala Val Pro Leu Phe Gln Lys Leu Gly Pro Ala
50 55 60 50 55 60
Val Leu Val Glu Ile Val Arg Ala Leu Arg Ala Arg Thr Val Pro AlaVal Leu Val Glu Ile Val Arg Ala Leu Arg Ala Arg Thr Val Pro Ala
65 70 75 8065 70 75 80
Gly Ala Val Ile Cys Arg Ile Gly Glu Pro Gly Asp Arg Met Phe PheGly Ala Val Ile Cys Arg Ile Gly Glu Pro Gly Asp Arg Met Phe Phe
85 90 95 85 90 95
Val Val Glu Gly Ser Val Ser Val Ala Thr Pro Leu Glu Asn Val TyrVal Val Glu Gly Ser Val Ser Val Ala Thr Pro Leu Glu Asn Val Tyr
100 105 110 100 105 110
Ile Lys Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys IleIle Lys Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile
115 120 125 115 120 125
Arg His Asn Ile Glu Asp Gly Gly Val Gln Leu Ala Tyr His Tyr GlnArg His Asn Ile Glu Asp Gly Gly Val Gln Leu Ala Tyr His Tyr Gln
130 135 140 130 135 140
Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn HisGln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His
145 150 155 160145 150 155 160
Tyr Leu Ser Val Gln Ser Lys Leu Ser Lys Asp Pro Asn Glu Lys ArgTyr Leu Ser Val Gln Ser Lys Leu Ser Lys Asp Pro Asn Glu Lys Arg
165 170 175 165 170 175
Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr LeuAsp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu
180 185 190 180 185 190
Gly Met Asp Glu Leu Tyr Lys Gly Gly Thr Gly Gly Ser Met Val SerGly Met Asp Glu Leu Tyr Lys Gly Gly Thr Gly Gly Ser Met Val Ser
195 200 205 195 200 205
Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu LeuLys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu
210 215 220 210 215 220
Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly GluAsp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu
225 230 235 240225 230 235 240
Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr ThrGly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr
245 250 255 245 250 255
Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr TyrGly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr
260 265 270 260 265 270
Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln His AspGly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln His Asp
275 280 285 275 280 285
Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Ile Gln Glu Arg Thr IlePhe Phe Lys Ser Ala Met Pro Glu Gly Tyr Ile Gln Glu Arg Thr Ile
290 295 300 290 295 300
Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys PhePhe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe
305 310 315 320305 310 315 320
Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp PheGlu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe
325 330 335 325 330 335
Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Leu ProLys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Leu Pro
340 345 350 340 345 350
Asn Pro Val Glu Leu Gly Pro Gly Ala Phe Phe Gly Glu Met Ala LeuAsn Pro Val Glu Leu Gly Pro Gly Ala Phe Phe Gly Glu Met Ala Leu
355 360 365 355 360 365
Ile Ser Gly Glu Pro Arg Ser Ala Thr Val Ser Ala Ala Thr Thr ValIle Ser Gly Glu Pro Arg Ser Ala Thr Val Ser Ala Ala Thr Thr Val
370 375 380 370 375 380
Ser Leu Leu Ser Leu His Ser Ala Asp Phe Gln Met Leu Cys Ser SerSer Leu Leu Ser Leu His Ser Ala Asp Phe Gln Met Leu Cys Ser Ser
385 390 395 400385 390 395 400
Ser Pro Glu Ile Ala Glu Ile Phe Arg Lys Thr Ala Leu Glu Arg ArgSer Pro Glu Ile Ala Glu Ile Phe Arg Lys Thr Ala Leu Glu Arg Arg
405 410 415 405 410 415
Gly Ala Ala Ala Ser AlaGly Ala Ala Ala Ser Ala
420 420
<210> 11<210> 11
<211> 1065<211> 1065
<212> DNA<212> DNA
<213> mICNBD<213> mICNBD
<400> 11<400> 11
atgtcggtac tgcctttctt aagaatttac gcgccgctca acgcggtgct ggctgcgcct 60atgtcggtac tgcctttctt aagaatttac gcgccgctca acgcggtgct ggctgcgcct 60
gggttgctgg cggtggctgc gctcacgata ccggacatgt ccggacgaag cagactggct 120gggttgctgg cggtggctgc gctcacgata ccggacatgt ccggacgaag cagactggct 120
ctggctgccc tgctcgctgt catctggggc gcctatctcc tgcaactggc cgcgacgctg 180ctggctgccc tgctcgctgt catctggggc gcctatctcc tgcaactggc cgcgacgctg 180
ctcaagcgcc gggcgggagt cgtacgggac aggacgccca aaatcgccat cgatgtgctc 240ctcaagcgcc gggcgggagt cgtacgggac aggacgccca aaatcgccat cgatgtgctc 240
gcagtcttgg ttccactcgc cgcatttctg ctcgacggct cgcctgactg gagcctctac 300gcagtcttgg ttccactcgc cgcatttctg ctcgacggct cgcctgactg gagcctctac 300
tgtgctgtct ggctgctgaa accgctgcgc gactcgactt tcttcccggt cctgggcagg 360tgtgctgtct ggctgctgaa accgctgcgc gactcgactt tcttcccggt cctgggcagg 360
gtcctggcca acgaagcacg caatctgatc ggcgtcacca cgctcttcgg cgtcgttctg 420gtcctggcca acgaagcacg caatctgatc ggcgtcacca cgctcttcgg cgtcgttctg 420
ttcgcagtgg cgctcgcagc ctatgtcatc gagcgcgata tccaaccgga aaagttcggc 480ttcgcagtgg cgctcgcagc ctatgtcatc gagcgcgata tccaaccgga aaagttcggc 480
agcattcccc aggcaatgtg gtgggcggtg gtcacgctgt ccaccaccgg ctatggggac 540agcattcccc aggcaatgtg gtgggcggtg gtcacgctgt ccaccaccgg ctatggggac 540
actatcccgc aaagcttcgc cggccgcgtc cttgccgggg cggtcatgat gagtggcatc 600actatcccgc aaagcttcgc cggccgcgtc cttgccgggg cggtcatgat gagtggcatc 600
ggcatcttcg gactctgggc cggcattctt gccacaggct tctatcaaga agtccgtcgc 660ggcatcttcg gactctgggc cggcattctt gccacaggct tctatcaaga agtccgtcgc 660
ggggatttcg tccgcaattg gcaattggtc gccgccgtgc cgttgtttca gaagctcggc 720ggggatttcg tccgcaattg gcaattggtc gccgccgtgc cgttgtttca gaagctcggc 720
ccggccgtgc tggtcgagat cgtgcgcgcc ttaagagccc gcacggtgcc ggcgggcgcc 780ccggccgtgc tggtcgagat cgtgcgcgcc ttaagagccc gcacggtgcc ggcgggcgcc 780
gtgatctgcc gcattggcga gcccggcgat cggatgttct tcgtcgtgga ggggagcgtc 840gtgatctgcc gcattggcga gcccggcgat cggatgttct tcgtcgtgga ggggagcgtc 840
agcgtcgcga cgccgaatcc ggtggagctt ggccctggcg ccttcttcgg cgagatggcg 900agcgtcgcga cgccgaatcc ggtggagctt ggccctggcg ccttcttcgg cgagatggcg 900
ctgatcagcg gcgaaccgcg ttcggcgacc gtcagcgccg caacgacggt ctcactcctg 960ctgatcagcg gcgaaccgcg ttcggcgacc gtcagcgccg caacgacggt ctcactcctg 960
tcgctgcatt cggcggattt ccagatgttg tgcagcagca gcccggagat cgcggaaatc 1020tcgctgcatt cggcggattt ccagatgttg tgcagcagca gcccggagat cgcggaaatc 1020
ttccgcaaga ccgcgctcga gcgtcgcggc gctgcggcga gcgct 1065ttccgcaaga ccgcgctcga gcgtcgcggc gctgcggcga gcgct 1065
<210> 12<210> 12
<211> 355<211> 355
<212> PRT<212> PRT
<213> mICNBD<213> mICNBD
<400> 12<400> 12
Met Ser Val Leu Pro Phe Leu Arg Ile Tyr Ala Pro Leu Asn Ala ValMet Ser Val Leu Pro Phe Leu Arg Ile Tyr Ala Pro Leu Asn Ala Val
1 5 10 151 5 10 15
Leu Ala Ala Pro Gly Leu Leu Ala Val Ala Ala Leu Thr Ile Pro AspLeu Ala Ala Pro Gly Leu Leu Ala Val Ala Ala Leu Thr Ile Pro Asp
20 25 30 20 25 30
Met Ser Gly Arg Ser Arg Leu Ala Leu Ala Ala Leu Leu Ala Val IleMet Ser Gly Arg Ser Arg Leu Ala Leu Ala Ala Leu Leu Ala Val Ile
35 40 45 35 40 45
Trp Gly Ala Tyr Leu Leu Gln Leu Ala Ala Thr Leu Leu Lys Arg ArgTrp Gly Ala Tyr Leu Leu Gln Leu Ala Ala Thr Leu Leu Lys Arg Arg
50 55 60 50 55 60
Ala Gly Val Val Arg Asp Arg Thr Pro Lys Ile Ala Ile Asp Val LeuAla Gly Val Val Arg Asp Arg Thr Pro Lys Ile Ala Ile Asp Val Leu
65 70 75 8065 70 75 80
Ala Val Leu Val Pro Leu Ala Ala Phe Leu Leu Asp Gly Ser Pro AspAla Val Leu Val Pro Leu Ala Ala Phe Leu Leu Asp Gly Ser Pro Asp
85 90 95 85 90 95
Trp Ser Leu Tyr Cys Ala Val Trp Leu Leu Lys Pro Leu Arg Asp SerTrp Ser Leu Tyr Cys Ala Val Trp Leu Leu Lys Pro Leu Arg Asp Ser
100 105 110 100 105 110
Thr Phe Phe Pro Val Leu Gly Arg Val Leu Ala Asn Glu Ala Arg AsnThr Phe Phe Pro Val Leu Gly Arg Val Leu Ala Asn Glu Ala Arg Asn
115 120 125 115 120 125
Leu Ile Gly Val Thr Thr Leu Phe Gly Val Val Leu Phe Ala Val AlaLeu Ile Gly Val Thr Thr Leu Phe Gly Val Val Leu Phe Ala Val Ala
130 135 140 130 135 140
Leu Ala Ala Tyr Val Ile Glu Arg Asp Ile Gln Pro Glu Lys Phe GlyLeu Ala Ala Tyr Val Ile Glu Arg Asp Ile Gln Pro Glu Lys Phe Gly
145 150 155 160145 150 155 160
Ser Ile Pro Gln Ala Met Trp Trp Ala Val Val Thr Leu Ser Thr ThrSer Ile Pro Gln Ala Met Trp Trp Ala Val Val Thr Leu Ser Thr Thr
165 170 175 165 170 175
Gly Tyr Gly Asp Thr Ile Pro Gln Ser Phe Ala Gly Arg Val Leu AlaGly Tyr Gly Asp Thr Ile Pro Gln Ser Phe Ala Gly Arg Val Leu Ala
180 185 190 180 185 190
Gly Ala Val Met Met Ser Gly Ile Gly Ile Phe Gly Leu Trp Ala GlyGly Ala Val Met Met Ser Gly Ile Gly Ile Phe Gly Leu Trp Ala Gly
195 200 205 195 200 205
Ile Leu Ala Thr Gly Phe Tyr Gln Glu Val Arg Arg Gly Asp Phe ValIle Leu Ala Thr Gly Phe Tyr Gln Glu Val Arg Arg Gly Asp Phe Val
210 215 220 210 215 220
Arg Asn Trp Gln Leu Val Ala Ala Val Pro Leu Phe Gln Lys Leu GlyArg Asn Trp Gln Leu Val Ala Ala Val Pro Leu Phe Gln Lys Leu Gly
225 230 235 240225 230 235 240
Pro Ala Val Leu Val Glu Ile Val Arg Ala Leu Arg Ala Arg Thr ValPro Ala Val Leu Val Glu Ile Val Arg Ala Leu Arg Ala Arg Thr Val
245 250 255 245 250 255
Pro Ala Gly Ala Val Ile Cys Arg Ile Gly Glu Pro Gly Asp Arg MetPro Ala Gly Ala Val Ile Cys Arg Ile Gly Glu Pro Gly Asp Arg Met
260 265 270 260 265 270
Phe Phe Val Val Glu Gly Ser Val Ser Val Ala Thr Pro Asn Pro ValPhe Phe Val Val Glu Gly Ser Val Ser Val Ala Thr Pro Asn Pro Val
275 280 285 275 280 285
Glu Leu Gly Pro Gly Ala Phe Phe Gly Glu Met Ala Leu Ile Ser GlyGlu Leu Gly Pro Gly Ala Phe Phe Gly Glu Met Ala Leu Ile Ser Gly
290 295 300 290 295 300
Glu Pro Arg Ser Ala Thr Val Ser Ala Ala Thr Thr Val Ser Leu LeuGlu Pro Arg Ser Ala Thr Val Ser Ala Ala Thr Thr Val Ser Leu Leu
305 310 315 320305 310 315 320
Ser Leu His Ser Ala Asp Phe Gln Met Leu Cys Ser Ser Ser Pro GluSer Leu His Ser Ala Asp Phe Gln Met Leu Cys Ser Ser Ser Pro Glu
325 330 335 325 330 335
Ile Ala Glu Ile Phe Arg Lys Thr Ala Leu Glu Arg Arg Gly Ala AlaIle Ala Glu Ile Phe Arg Lys Thr Ala Leu Glu Arg Arg Gly Ala Ala
340 345 350 340 345 350
Ala Ser AlaAla Ser Ala
355 355
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| CN113024674B (en) * | 2019-12-09 | 2023-03-28 | 中国科学院深圳先进技术研究院 | Cyclic adenosine monophosphate fluorescent probe with wide-range change of fluorescence brightness |
| CN113567402B (en) * | 2020-04-29 | 2023-06-06 | 中国科学院深圳先进技术研究院 | Application of cAMP fluorescent probe G-Flamp1 |
| WO2021217479A1 (en) * | 2020-04-29 | 2021-11-04 | 中国科学院深圳先进技术研究院 | Application of camp fluorescent probe g-flamp1 |
| CN112480271B (en) * | 2020-12-15 | 2022-11-01 | 中国科学院深圳先进技术研究院 | High-performance red cAMP fluorescent probe and its application |
| CN113336859B (en) * | 2021-05-25 | 2022-11-18 | 大连理工大学 | Biological probe for identifying CD105 |
| CN117866067A (en) * | 2022-10-12 | 2024-04-12 | 深圳先进技术研究院 | cAMP fluorescent probes G-Flamp2 and G-Flamp2b, application and kit thereof |
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