CN113024674A - 荧光亮度宽幅变化的环磷酸腺苷荧光探针 - Google Patents

荧光亮度宽幅变化的环磷酸腺苷荧光探针 Download PDF

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CN113024674A
CN113024674A CN201911251920.XA CN201911251920A CN113024674A CN 113024674 A CN113024674 A CN 113024674A CN 201911251920 A CN201911251920 A CN 201911251920A CN 113024674 A CN113024674 A CN 113024674A
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储军
王亮
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Abstract

本发明涉及一种荧光亮度宽幅变化的环磷酸腺苷荧光探针，其具有如式I所示结构：MlotiK1 CNBD‑N‑linker1‑cpEGFP‑linker2‑MlotiK1 CNBD‑C 式I，其中，MlotiK1 CNBD‑N为MlotiK1 CNBD的N端，具有如SEQ ID NO:3所示的氨基酸序列；所述MlotiK1 CNBD‑C为MlotiK1 CNBD的C端，具有如SEQ ID NO:4所示的氨基酸序列；cpEGFP具有如SEQ ID NO:5所示的氨基酸序列。linker1为WG，linker2为RV。本发明的荧光探针荧光亮度宽幅变化大，对于提高探测灵敏度具有重要意义。

Description

荧光亮度宽幅变化的环磷酸腺苷荧光探针
技术领域
本发明属于生物检测领域，特别涉及cAMP荧光探针及其应用。
背景技术
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。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[1]、cADDis[2]及cAMPr[3]，后者主要有PinkFlamindo[4]、 Red cADDis[5]及R-FlincA[6]。实际应用中，动态范围(荧光亮度变化幅度,ΔF/F0)是很重要的参数，与检测灵敏度直接相关。在37℃生理温度培养细胞中，上述探针动态范围均较小。综上，提高探针在实际应用中的动态范围，对于提高探测灵敏度具有重要意义。
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探针及动态范围如下表所示，#252是本发明人2018申请专利设计到的探针。从该表可知，37℃生理温度培养细胞中。
Figure RE-GDA0002407334310000011
参考文献：
1.Odaka H,Arai S,Inoue T,Kitaguchi T(2014)Genetically-encoded yellowfluorescent cAMP indicator with an expanded dynamic range for dual-colorimaging.PLoS One 9: e100252.
2.Tewson PH,Martinka S,Shaner NC,Hughes TE,Quinn AM(2016)New DAG andcAMP Sensors Optimized for Live-Cell Assays in Automated Laboratories.JBiomol Screen 21: 298-305.
3.Hackley CR,Mazzoni EO,Blau J(2018)cAMPr:A single-wavelengthfluorescent sensor for cyclic AMP.Sci Signal 11.
4.Harada K,Ito M,Wang X,Tanaka M,Wongso D,et al.(2017)Red fluorescentprotein-based cAMP indicator applicable to optogenetics and in vivoimaging.Sci Rep 7: 7351.
5.https://montanamolecular.com/live-cell-camp-assay-caddis/red-caddis-camp-protocol/
6.Ohta Y,Furuta T,Nagai T,Horikawa K(2018)Red fluorescent cAMPindicator with increased affinity and expanded dynamic range.Sci Rep 8:1866.
7.CN109627344A
发明内容
为了解决cAMP探针荧光亮度变化幅度小的问题，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，其在37℃生理温度培养细胞中具目前最大的动态范围。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。
相较于目前已有的荧光探针，本发明创造的探针，在37℃培养细胞中具有更大的动态范围(ΔF/F0)，即具有更高的检测灵敏度。
本发明一个方面提供了一种cAMP荧光探针，所述cAMP荧光探针具有如式I所示结构：
MlotiK1 CNBD-N-linker1-cpEGFP-linker2-MlotiK1 CNBD-C 式I
其中，MlotiK1 CNBD-N为MlotiK1 CNBD的N端，具有如SEQ ID NO:3所示的氨基酸序列；
MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDPMGFYQEVRRGDFVRNWQLV AAVPLFQKLGPAVLVEIVRALRARTVPAGAVICRIGEPGDRMFFVVEGSVSVATN SEQ ID No.3
所述MlotiK1 CNBD-C为MlotiK1 CNBD的C端，具有如SEQ ID NO:4所示的氨基酸序列；
NVYITADKQKNGIKANFKIRHNVEGGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQS KLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFTGVVPILVELDG DVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMK QHDFFKSAMPEGYIQERTIVFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHK LEYN SEQ ID No.4
cpEGFP具有如SEQ ID NO:5所示的氨基酸序列
NPVELGPGAFFGEMALISGEPRVATVSAATTVSLLSLHSADFQMLCSSSPEIAEIFRKTALERRGAAASA SEQ ID No.5；
linker1为WG，linker2为RV。
在本发明的技术方案中，所述的cAMP荧光探针具有如SEQ ID No.2的序列。
本发明另一个方面提供了编码如上所述cAMP荧光探针的核苷酸。
本发明另一个方面提供了表达载体，其包括编码如上所述cAMP荧光探针的核苷酸。
本发明再一个方面提供了转化或转染如上所述的表达载体的宿主细胞。
本发明再一个方面提供了如上所述cAMP荧光探针的制备方法，包括：培养如上所述的宿主细胞、诱导所述cAMP荧光探针的表达。
本发明再一个方面提供了如上所述cAMP荧光探针在检测cAMP中的应用。
本发明再一个方面提供了如上所述cAMP荧光探针在37℃下活细胞内检测cAMP中的应用。
本发明再一个方面提供了一种包含上述cAMP荧光探针的试剂盒。
以下将结合附图对本发明进行详细说明。
(1)首先构建mICNBD-N-linker1-cpEGFP-linker2-mICNBD–C(Cyclicnucleotide-binding domain,CNBD,环核苷酸结合结构域；mICNBD-N，mICNBD的N端；mICNBD-C,mICNBD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(附图1)。#252的氨基酸序列也给了出来(附图1)。
(2)对#252的若干氨基酸进行突变，即得到G-Flamp1探针，序列如SEQ ID No.2所示。
MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDPMGFYQEVRRGDFVRNWQLV AAVPLFQKLGPAVLVEIVRALRARTVPAGAVICRIGEPGDRMFFVVEGSVSVATNWGNVYI TADKQKNGIKANFKIRHNVEGGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSKLSKDPN EKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFTGVVPILVELDGDVNGHKF SVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYIQERTIVFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNRVN PVELGPGAFFGEMALISGEPRVATVSAATTVSLLSLHSADFQMLCSSSPEIAEIFRKTALE RRGAAASA SEQ ID No.2
(3)将G-Flamp1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检测探针对饱和浓度cAMP(～500μM)的响应，可见其信号增加～8倍(附图2)。
(4)将cAMPr、Flamindo2、G-Flamp1、Pink-Flamindo及R-FlincA等探针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IX83荧光显微镜检测探针的亮度，可见细胞受60μM Forskolin(购自碧云天生物技术公司)刺激后，G-Flamp1具最大的信号变化幅度(ΔF/F0)，附图3。至此完成了哺乳动物细胞内cAMP浓度变化的荧光成像步骤。
附图说明
图1为#252探针及其本发明G-Flamp1探针的设计。将突变的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，WG之前为mlCNBD-N序列，RV 之后为mlCNBD-C序列，得到G-Flamp1探针探针。RSET为质粒载体上的先导序列，可以用于纯化蛋白。
图2为纯化G-Flamp1探针的动态范围测定。从细菌中纯化的G-Flamp1探针稀释在pH 7.3的HEPES溶液中，终浓度为2μM。图示探针浓度在HEPES溶液及饱和浓度cAMP中的荧光激发光谱。底部线为未加入cAMP探针的光谱，顶部线为加cAMP探针的光谱。虚线为激发谱，实线为发射谱。
图3为探针在HEK293T细胞中的亮度及响应。(A)利用Lipofectamine转染HEK细胞含cAMPr、Flamindo2、G-Flamp1、Pink-Flamindo探针的质粒，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60μM Forskolin刺激后荧光亮度变化。(B) R-FlincA探针的响应。
具体实施方式
为了使本发明的上述目的、特征和优点能够更加明显易懂，下面结合附图对本发明的具体实施方式做详细的说明，但不能理解为对本发明的可实施范围的限定。
实施例1
首先构建mICNBD-N-linker1-cpEGFP-linker2-mICNBD–C(Cyclic nucleotide-binding domain,CNBD,环核苷酸结合结构域；mICNBD-N，mICNBD的N端；mICNBD-C,mICNBD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(附图1)。#252的氨基酸序列如SEQID No.1所示。
252探针:SEQ ID No.1
MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDPMGFYQEVRRGDFVRNWQLV AAVPLFQKLGPAVLVEIVRALRARTVPAGAVICRIGEPGDRMFFVVEGSVSVATPWGNVYI TADKQKNGIKANFKIRHNVEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSKLSKDPN EKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFTGVVPILVELDGDVNGHKF SVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYIQERTIVFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNRVN PVELGPGAFFGEMALISGEPRSATVSAATTVSLLSLHSADFQMLCSSSPEIAEIFRKTALE RRGAAASA
对#252探针的若干氨基酸进行突变，见SEQ ID No.2加粗部分，即得到G-Flamp1探针，其序列见SEQ ID No.2。
G-Flamp1探针:SEQ ID No.2
MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDPMGFYQEVRRGDFVRNWQLV AAVPLFQKLGPAVLVEIVRALRARTVPAGAVICRIGEPGDRMFFVVEGSVSVATNWGNVYI TADKQKNGIKANFKIRHNVEGGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSKLSKDPN EKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFTGVVPILVELDGDVNGHKF SVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYIQERTIVFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNRVN PVELGPGAFFGEMALISGEPRVATVSAATTVSLLSLHSADFQMLCSSSPEIAEIFRKTALE RRGAAASA。
WG与RV之间是环化重排的绿色荧光蛋白序列。WG之前为mICNBD-N序列，RV之后为mICNBD-C序列。WG和RV为接头
实施例2
将G-Flamp1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检测探针对饱和浓度cAMP(～500μM)的响应，可见其荧光信号增加～8倍(附图2)。
实施例3
将cAMPr、Flamindo2、G-Flamp1、Pink-Flamindo及R-FlincA等探针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，可见细胞受60μM Forskolin(购自碧云天生物技术公司)刺激后， G-Flamp1具最大的信号变化幅度(ΔF/F0)达到2.2倍，见附图3。至此完成了哺乳动物细胞内cAMP浓度变化的荧光成像步骤。
SEQUENCE LISTING
<110> 中国科学院深圳先进技术研究院
<120> 荧光亮度宽幅变化的环磷酸腺苷荧光探针
<130> CP119011239C
<160> 5
<170> PatentIn version 3.3
<210> 1
<211> 422
<212> PRT
<213> 252探针
<400> 1
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Gly Asp Ala Thr Asn Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr
245 250 255
Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr
260 265 270
Gly Val Gln Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His Asp
275 280 285
Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Ile Gln Glu Arg Thr Ile
290 295 300
Val Phe Lys Asp Asp Gly Thr Tyr Lys Thr Arg Ala Glu Val Lys Phe
305 310 315 320
Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe
325 330 335
Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Arg Val
340 345 350
Asn Pro Val Glu Leu Gly Pro Gly Ala Phe Phe Gly Glu Met Ala Leu
355 360 365
Ile Ser Gly Glu Pro Arg Ser Ala Thr Val Ser Ala Ala Thr Thr Val
370 375 380
Ser Leu Leu Ser Leu His Ser Ala Asp Phe Gln Met Leu Cys Ser Ser
385 390 395 400
Ser Pro Glu Ile Ala Glu Ile Phe Arg Lys Thr Ala Leu Glu Arg Arg
405 410 415
Gly Ala Ala Ala Ser Ala
420
<210> 2
<211> 422
<212> PRT
<213> G-Flamp1探针
<400> 2
Met Arg Gly Ser His His His His His His Gly Met Ala Ser Met Thr
1 5 10 15
Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp
20 25 30
Pro Met Gly Phe Tyr Gln Glu Val Arg Arg Gly Asp Phe Val Arg Asn
35 40 45
Trp Gln Leu Val Ala Ala Val Pro Leu Phe Gln Lys Leu Gly Pro Ala
50 55 60
Val Leu Val Glu Ile Val Arg Ala Leu Arg Ala Arg Thr Val Pro Ala
65 70 75 80
Gly Ala Val Ile Cys Arg Ile Gly Glu Pro Gly Asp Arg Met Phe Phe
85 90 95
Val Val Glu Gly Ser Val Ser Val Ala Thr Asn Trp Gly Asn Val Tyr
100 105 110
Ile Thr Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile
115 120 125
Arg His Asn Val Glu Gly Gly Gly Val Gln Leu Ala Tyr His Tyr Gln
130 135 140
Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His
145 150 155 160
Tyr Leu Ser Val Gln Ser Lys Leu Ser Lys Asp Pro Asn Glu Lys Arg
165 170 175
Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu
180 185 190
Gly Met Asp Glu Leu Tyr Lys Gly Gly Thr Gly Gly Ser Met Val Ser
195 200 205
Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu
210 215 220
Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Arg Gly Glu Gly Glu
225 230 235 240
Gly Asp Ala Thr Asn Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr
245 250 255
Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr
260 265 270
Gly Val Gln Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His Asp
275 280 285
Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Ile Gln Glu Arg Thr Ile
290 295 300
Val Phe Lys Asp Asp Gly Thr Tyr Lys Thr Arg Ala Glu Val Lys Phe
305 310 315 320
Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe
325 330 335
Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Arg Val
340 345 350
Asn Pro Val Glu Leu Gly Pro Gly Ala Phe Phe Gly Glu Met Ala Leu
355 360 365
Ile Ser Gly Glu Pro Arg Val Ala Thr Val Ser Ala Ala Thr Thr Val
370 375 380
Ser Leu Leu Ser Leu His Ser Ala Asp Phe Gln Met Leu Cys Ser Ser
385 390 395 400
Ser Pro Glu Ile Ala Glu Ile Phe Arg Lys Thr Ala Leu Glu Arg Arg
405 410 415
Gly Ala Ala Ala Ser Ala
420
<210> 3
<211> 107
<212> PRT
<213> MlotiK1 CNBD-N
<400> 3
Met Arg Gly Ser His His His His His His Gly Met Ala Ser Met Thr
1 5 10 15
Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp
20 25 30
Pro Met Gly Phe Tyr Gln Glu Val Arg Arg Gly Asp Phe Val Arg Asn
35 40 45
Trp Gln Leu Val Ala Ala Val Pro Leu Phe Gln Lys Leu Gly Pro Ala
50 55 60
Val Leu Val Glu Ile Val Arg Ala Leu Arg Ala Arg Thr Val Pro Ala
65 70 75 80
Gly Ala Val Ile Cys Arg Ile Gly Glu Pro Gly Asp Arg Met Phe Phe
85 90 95
Val Val Glu Gly Ser Val Ser Val Ala Thr Asn
100 105
<210> 4
<211> 241
<212> PRT
<213> CNBD-C
<400> 4
Asn Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn
1 5 10 15
Phe Lys Ile Arg His Asn Val Glu Gly Gly Gly Val Gln Leu Ala Tyr
20 25 30
His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro
35 40 45
Asp Asn His Tyr Leu Ser Val Gln Ser Lys Leu Ser Lys Asp Pro Asn
50 55 60
Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly
65 70 75 80
Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Gly Gly Thr Gly Gly Ser
85 90 95
Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu
100 105 110
Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Arg Gly
115 120 125
Glu Gly Glu Gly Asp Ala Thr Asn Gly Lys Leu Thr Leu Lys Phe Ile
130 135 140
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
145 150 155 160
Leu Thr Tyr Gly Val Gln Cys Phe Ala Arg Tyr Pro Asp His Met Lys
165 170 175
Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Ile Gln Glu
180 185 190
Arg Thr Ile Val Phe Lys Asp Asp Gly Thr Tyr Lys Thr Arg Ala Glu
195 200 205
Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly
210 215 220
Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr
225 230 235 240
Asn
<210> 5
<211> 70
<212> PRT
<213> cpEGFP
<400> 5
Asn Pro Val Glu Leu Gly Pro Gly Ala Phe Phe Gly Glu Met Ala Leu
1 5 10 15
Ile Ser Gly Glu Pro Arg Val Ala Thr Val Ser Ala Ala Thr Thr Val
20 25 30
Ser Leu Leu Ser Leu His Ser Ala Asp Phe Gln Met Leu Cys Ser Ser
35 40 45
Ser Pro Glu Ile Ala Glu Ile Phe Arg Lys Thr Ala Leu Glu Arg Arg
50 55 60
Gly Ala Ala Ala Ser Ala
65 70

Claims (8)

1.一种cAMP荧光探针，所述cAMP荧光探针具有如式I所示结构：
MlotiK1 CNBD-N-linker1-cpEGFP-linker2-MlotiK1 CNBD-C式I
其中，MlotiK1 CNBD-N为MlotiK1 CNBD的N端，具有如SEQ ID NO:3所示的氨基酸序列；
MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDPMGFYQEVRRGDFVRNWQLVAAVPLFQKLGPAVLVEIVRALRARTVPAGAVICRIGEPGDRMFFVVEGSVSVATN SEQ ID No.3
所述MlotiK1 CNBD-C为MlotiK1 CNBD的C端，具有如SEQ ID NO:4所示的氨基酸序列；
NVYITADKQKNGIKANFKIRHNVEGGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYIQERTIVFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYN SEQ ID No.4
cpEGFP具有如SEQ ID NO:5所示的氨基酸序列
NPVELGPGAFFGEMALISGEPRVATVSAATTVSLLSLHSADFQMLCSSSPEIAEIFRKTALERRGAAASASEQ ID No.5；
linker1为WG，linker2为RV。
2.一种核酸，其编码如权利要求1所述cAMP荧光探针的核苷酸序列。
3.一种表达载体，其包括编码如权利要求1所述cAMP荧光探针的核酸。
4.转化或转染如权利要求3所述的表达载体的宿主细胞。
5.如权利要求1所述cAMP荧光探针的制备方法，包括：培养如权利要求4所述的宿主细胞、诱导所述cAMP荧光探针的表达。
6.如权利要求1所述cAMP荧光探针在检测cAMP中的应用。
7.如权利要求1所述cAMP荧光探针在37℃下活细胞内检测cAMP中的应用。
8.一种包含如权利要求1所述cAMP荧光探针的试剂盒。
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