CN109627344A

CN109627344A - CAMP fluorescence probe and its application

Info

Publication number: CN109627344A
Application number: CN201811620932.0A
Authority: CN
Inventors: 储军; 王亮
Original assignee: Shenzhen Institute of Advanced Technology of CAS
Current assignee: Shenzhen Institute of Advanced Technology of CAS
Priority date: 2018-12-28
Filing date: 2018-12-28
Publication date: 2019-04-16
Anticipated expiration: 2038-12-28
Also published as: CN109627344B

Abstract

The present invention relates to field of biological detection, in particular to cAMP fluorescence probe and its application.Compared to current existing green fluorescence probe, the probe of the invention or with bigger dynamic range (compared with cADDis/cAMPr), or with good fluorescent brightness (compared with Flamindo2), with cAMP affinity also in moderate concentration.Although should not be directly compared with red cAMP probe, compared to current existing red fluorescence probe Pink-Flamindo2 and R-FlincA, the fluorescent brightness that the probe of the invention has had.To sum up, #252 probe provided by the invention has taken into account the performance of fluorescent brightness, dynamic range, affinity etc., keeps its application range wider.

Description

CAMP fluorescence probe and its application

Technical field

The present invention relates to field of biological detection, in particular to cAMP fluorescence probe and its application.

Background technique

Cyclic adenosine monophosphate (cAMP) is the downstream courier point of current maximum drug targets g protein coupled receptor (GPCR) family Son, cAMP fluorescence probe and micro-imaging research are the basic research of GPCR signal path and the important research side of drug development To.CAMP fluorescence probe is broadly divided into fluorescence resonance energy transfer probe based on fluorescin and based on single fluorescin Probe, the latter's dynamic range are big compared with the former and using simple.

CAMP fluorescence imaging refers to the expression of cAMP fluorescence probe in cell in living cells, then utilizes fluorescence microscope The Strength Changes of detection probe fluorescence.Fluorescence probe is the key that cAMP fluorescence imaging analysis.It is existing to be based on single fluorescence egg White cAMP probe and its critical nature are as shown in the table, and wherein #252 is the probe involved in the present invention arrived.From the table it is found that Probe based on single fluorescin is divided into green and red 2 groups.The Flamindo2 of its Green and Pink of red The dynamic ranges such as Flamindo2/R-FlincA are larger, but fluorescent brightness is extremely low in 37 DEG C of culture cells；Because it is to ring phosphorus Sour guanosine (cGMP) also has response, not too much high to cAMP selectivity, especially when cGMP concentration is higher (> 10 μM).Other Probe dynamic range is smaller.

Table 1

It is divided into green and red 2 groups currently based on the cAMP probe of single fluorescin, the former mainly has Flamindo2, cADDis and cAMPr, the latter mainly have Pink Flamindo, Red cADDis and R-FlincA.Practical application In, fluorescent brightness, dynamic range, affinity and specificity are 4 critically important parameters.And the sensitivity of probe is (with dynamic model Enclose and the correlations such as affinity) and signal-to-noise ratio (related to fluorescent brightness) be imaging in obtain accurately cAMP concentration variation and space-time The key of distributed intelligence.But the dynamic range of current cADDis, cAMPr and Red cADDis is smaller, and signal intensity amplitude does not surpass Cross 60% (i.e. fluorescent brightness knots modification Δ F/F₀, F₀、F₁Respectively be free of the probe of cAMP and in conjunction with saturated concentration cAMP The fluorescent brightness of probe, Δ F=(F₁-F₀)/F₀).Flamindo2, Pink Flamindo and R-FlincA are in 37 DEG C of physiology Brightness is very low in temperature culture cell, and also has very big response to 10 μM and higher concentration cGMP, and specificity is not enough.To sum up, right It is required to improve in the performance of existing green and the red cAMP probe based on single fluorescin, above-mentioned 4 aspect, with Meets the needs of highly sensitive and high s/n ratio fluorescence imaging in 37 DEG C of culture cells.

Summary of the invention

In view of this, the present invention provides a kind of cAMP fluorescence probe and its application.The present invention is for cAMP imaging technique Probe portion optimizes, and obtains a green probe, makes the performances such as its signal intensity amplitude, fluorescent brightness, affinity It gets a promotion.

In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:

The present invention provides a kind of cAMP fluorescence probes, comprising: the polypeptide sensitive to cAMP and fluorescin.

In some specific embodiments of the invention, the polypeptide sensitive to cAMP is with cAMP binding characteristic MlotiK1 CNBD structural domain or its function fragment, analog, derivative or mutant.

In some specific embodiments of the invention, the MlotiK1 CNBD structural domain is included

(I), the amino acid sequence as shown in SEQ ID NO:1；

(II), such as (I) described amino acid sequence is substituted, lacks or adds the amino acid that one or more amino acid obtain Sequence, and with (I) described amino acid sequence amino acid sequence functionally identical or similar；

(III), with the amino acid sequence of (I) or (II) described amino acid sequence at least 80% homology.

In some specific embodiments of the invention, the fluorescin is the green fluorescent protein that cyclisation is reset cpEGFP。

In some specific embodiments of the invention, the fluorescin is included

(IV), the amino acid sequence as shown in SEQ ID NO:2；

(V), such as (IV) described amino acid sequence is substituted, lacks or adds the amino acid that one or more amino acid obtain Sequence, and with (IV) described amino acid sequence amino acid sequence functionally identical or similar；

(VI), with the amino acid sequence of (IV) or (V) described amino acid sequence at least 80% homology.

In some specific embodiments of the invention, the fluorescin is inserted into the MlotiK1 CNBD structural domain Site is P/N (74/75), i.e., the described fluorescin be inserted into the MlotiK1 CNBD structural domain site be P74 and N75 it Between.

In some specific embodiments of the invention, the cAMP fluorescence probe has structure shown in formula I:

MlotiK1 CNBD-N-linker1-cpEGFP-linker2-MlotiK1 CNBD-C

Formulas I

Wherein, MlotiK1 CNBD-N is the N-terminal of MlotiK1 CNBD, comprising:

(VII), the amino acid sequence as shown in SEQ ID NO:3；

(VIII), such as (VII) described amino acid sequence is substituted, lacks or adds the ammonia that one or more amino acid obtain Base acid sequence, and with (VII) described amino acid sequence amino acid sequence functionally identical or similar；

(IX), with the amino acid sequence of (VII) or (VIII) described amino acid sequence at least 80% homology；

The MlotiK1 CNBD-C is the C-terminal of MlotiK1 CNBD, comprising:

(X), the amino acid sequence as shown in SEQ ID NO:4；

(XI), such as (X) described amino acid sequence is substituted, lacks or adds the amino acid that one or more amino acid obtain Sequence, and with the amino acid sequence functionally identical or similar of amino acid sequence shown in (X)；

(XII), with the amino acid sequence of (X) or (XI) described sequence at least 80% homology；

Linker1 is the first link peptide, comprising:

(XIII), the amino acid sequence as shown in SEQ ID NO:5；

(XIV), such as (XIII) described amino acid sequence is substituted, lacks or adds the ammonia that one or more amino acid obtain Base acid sequence, and with (XIII) described amino acid sequence amino acid sequence functionally identical or similar；

(XV), with the amino acid sequence of (XIII) or (XIV) described amino acid sequence at least 80% homology；

Linker2 is the second link peptide, comprising:

(XVI), the amino acid sequence as shown in SEQ ID NO:6；

(XVII), such as (XVI) described amino acid sequence is substituted, lacks or adds the ammonia that one or more amino acid obtain Base acid sequence, and with (XVI) described amino acid sequence amino acid sequence functionally identical or similar；

(XVIII), with the amino acid sequence of (XVI) or (XVII) described amino acid sequence at least 80% homology.

In some specific embodiments of the invention, the cAMP fluorescence probe is included

(XIX), the amino acid sequence as shown in SEQ ID NO:7；

(XX), such as (XIX) described amino acid sequence is substituted, lacks or adds the amino that one or more amino acid obtain Acid sequence, and with the amino acid sequence functionally identical or similar of amino acid sequence shown in (XIX)；

(XXI), with the amino acid sequence of (XIX) or (XX) described amino acid sequence at least 80% homology.

On this basis, the present invention also provides the nucleotide for encoding the cAMP fluorescence probe.

In some specific embodiments of the invention, the nucleotide has:

(XXII), there is nucleotide sequence shown in SEQ ID NO:8；

(XXIII), such as (XXII) described nucleotide sequence is modified, replaces, misses or adds one or more bases and obtain The nucleotide sequence obtained；

(XXIV), there is the sequence of at least 80% homology with (XXII) or (XXIII) nucleotide sequence；

(XXV), with the complementary series of (XXII), (XXIII) or (XXIV) described nucleotide sequence.

The present invention also provides expression vectors, the nucleotide including encoding the cAMP fluorescence probe.

The present invention also provides the host cells of conversion or the transfection expression vector.

In addition, the present invention also provides the preparation methods of the cAMP fluorescence probe, comprising: it is thin to cultivate the host The expression of born of the same parents, the induction cAMP fluorescence probe.

On the basis of the studies above, the present invention also provides application of the cAMP fluorescence probe in detection cAMP.

In addition, the present invention also provides the applications of cAMP fluorescence probe variation of cAMP in detection living cells.

The present invention also provides the detection methods of cAMP, include the following steps:

Step 1, construction of expression vector, the expression vector include coding cAMP as described in any one of claim 1~8 glimmering The nucleotide of light probe；

Step 2, the host cell for being converted or being transfected the expression vector；

The expression of step 3, culture host cell as claimed in claim 11, the induction cAMP fluorescence probe；

Step 4, by the cAMP fluorescence probe to the corresponding of cAMP described in sample to be tested, obtain the dense of the cAMP Degree.

On this basis, the present invention also provides kits, including the cAMP fluorescence probe.

In present specification, the CNBD is MlotiK1 CNBD.

Core of the invention is that screen mutation has gone out a kind of new, based on single protein fluorescence albumen cAMP probe # 252.Currently, comprehensively considering the parameters such as dynamic range, fluorescent brightness and cAMP affinity, #252 in the probe of green-series It is best one.In actual use, in being expressed in mammalian cell, common fluorescence microscope, Ji Kejian are utilized Survey cell after by particular stimulation cAMP concentration whether change.

Because the practical application performance of probe is related with various performance parameters, it is difficult to assert which is visited at present Needle is best.In green probe series, compared with available comprehensive information, optimal Flamindo2, #252 of the invention exists (Δ F/F0) is more preferable (315%) in signal intensity amplitude, and fluorescent brightness, which improves a lot, (about improves 60 in HEK293 cell Times), it can work in 37 DEG C of culture cells.#252 is compared to red probe Pink-Flamindo and R-FlincA, in brightness On be significantly improved.

Compared to current existing green fluorescence probe, the probe of the invention or with bigger dynamic range (compared with cADDis/cAMPr), or with good fluorescent brightness (compared with Flamindo2), with cAMP affinity also suitable In concentration.Although should not be directly compared with red cAMP probe, compared to current existing red fluorescence probe Pink- Flamindo2 and R-FlincA, the fluorescent brightness that the probe of the invention has had.To sum up, #252 provided by the invention is visited Needle has taken into account the performance of fluorescent brightness, dynamic range, affinity etc., keeps its application range wider.

Detailed description of the invention

In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below There is attached drawing needed in technical description to be briefly described.

Fig. 1 shows the design of #252 and its compared with Flamindo2 brightness；Wherein, Fig. 1 (A), which shows, is inserted into cpEGFP In cAMP is affine structural domain, the link peptide on the left side and the right is respectively WG and RV, obtains #252 probe；Fig. 1 (B) show by Flmindo2 and #252 converts bacterium, after 34 DEG C are cultivated 15 hours, carries out fluorescence imaging to plate；

Fig. 2 shows the amino acid sequence of #252；Underscore amino acid is respectively the component of front and back link peptide；It is between WG and RV It is cyclized the green fluorescent protein sequence reset；It is CNBD-N sequence before WG, is CNBD-C sequence after RV；

Fig. 3 shows the dynamic range measurement of #252 probe；Fig. 3 (A) shows the #252 probe dilution purified from bacterium in pH In 7.3 HEPES solution, final concentration of 2 μM；Fig. 3 (B) shows that concentration and probe concentration is glimmering in HEPES solution and saturated concentration cAMP Luminescence exitation spectrum；The ratio of red curve and black curve in Fig. 3 (A)；It can be seen that it is excited in wave band in 440-500nm, dynamic Range is between 3.5-4.15 times；

Fig. 4 shows Flamindo2 and #252 to the response curve of cAMP or cGMP；Fluorescence is by 500nm light source activation herein；

Fig. 5 shows brightness and response of the #252 probe in HEK293T cell；Fig. 5 (A) is transfected using Lipofectamine HEK cell blank plasmid, plasmid containing Flamindo2, plasmid containing #252 and contain mEGFP (monomer green fluorescent protein) plasmid, mistake After night culture, with without phenol red and serum DMEM cell culture fluid it is 6 hours hungry after be imaged with fluorescence microscope；It is visited in cell The fluorescent brightness average value of needle is marked out, it is seen that #252 brightness is much higher than Flamindo2；Fig. 5 (B) is by 60 μM Culture solution is added in Forskolin and 100 μM of IBMX, and fluorescence imaging is carried out before and after dosing, before dosing stimulation is illustrated in figure The fluorescent brightness of (such as 1500 seconds) after (0 second) and dosing for a period of time；Fig. 5 (C) is 1/2/3/4 cell selected in (B) The change curve of middle fluorescent brightness；

Fig. 6 show the 285-ins and #252 of purifying in conjunction with cAMP after excitation spectrum variation；Dotted line is that cAMP is not added to swash Luminous spectrum (Fig. 6 A), solid line are the excitation spectrum (Fig. 6 B) being added after final concentration of 1mM cAMP；As it can be seen that #252 probe have compared with Big positive variation；

Fig. 7 shows brightness testing result；Show that 252 brightness are (87-40)/(40-26)=3.35 times of 285-ins.

Specific embodiment

The invention discloses a kind of cAMP fluorescence probe and its application, those skilled in the art can use for reference present disclosure, It is suitably modified realization of process parameters.In particular, it should be pointed out that all similar substitutions and modifications carry out those skilled in the art Say it is it will be apparent that they are considered as being included in the present invention.Method and application of the invention has passed through preferred embodiment It is described, related personnel can obviously not depart from the content of present invention, in spirit and scope to method described herein and answer With being modified or appropriate changes and combinations, carry out implementation and application the technology of the present invention.

Term used herein " fluorescence probe " refers to the polypeptide sensitive to environment cAMP blended with fluorescin, institute Stating the polypeptide sensitive to cAMP in environment specifically can be Epac albumen, using cAMP binding structural domain specific in Epac with The conformation change that cAMP is generated after combining causes the conformation change of fluorescin, changes so as to cause the fluorescence of generation, leads to The fluorescence of the fluorescin measured under difference cAMP concentration is crossed to draw standard curve, and then intracellular for detecting and analyzing The presence of cAMP is horizontal.

Terms used herein " fusion protein " is identical as " fluorescent fusion protein " and " recombination fluorescent fusion protein " meaning, refers to Amino acid sequence comprising the polypeptide of specific binding domain or its segment, derivative or the like.

Terms used herein " function fragment ", " analog ", " derivative " refer to substantially with CNBD structure in the present invention The albumen of domain biological activity having the same.Function fragment, analog or the derivative of CNBD structural domain of the invention can be with It is:

(1) there are one or more conservative or non-conservative amino acid residues (preferably conservative amino acid) substituted Albumen, and amino acid residue substituted in this way can be and may not be by genetic coding, or

(2) in one or more amino acid residues with the albumen of substituent group, or

(3) additional amino acid sequence merges the albumen to be formed with this protein sequence, or

(4) maturation protein merges the albumen to be formed with another compound.

These function fragments, analogs and derivatives belong to scope known to those skilled in the art.

In the present invention, term " fluorogen " used is synonymous with " fluorescin ", refers to that itself issues fluorescence or under irradiation The protein of fluorescence is issued, fluorescin is often used as detection means.

" the phase same sex " of two or more polypeptides or sequence of nucleic acid molecules of the present invention or " phase same sex percentage " be On specified region or comparison window, using sequence comparison algorithm known in the art, two are analyzed by manually estimating comparison Or multiple sequences maximum correspondence when, part of sequence has the amino acid residue or core of certain percentage in specified region Thuja acid is identical (such as 35%, 50%, 70%, 85%, 90%, 95% or 100% are identical), is suitable for measuring sequence similarity and phase The optimization algorithm of same sex percentage is BLAST algorithm, and for details, reference can be made to ALtschul etc. (1977) Nucleic Acids Res.25:3389。

In the present invention, " sequence with 80% homology " is preferred embodiment, and those skilled in the art being capable of basis Actual needs is obtained with sequence provided by the invention with the sequence of the 70% homology, sequence of 50% homology, 40% homologous Property sequence, the sequence of the sequence of 35% homology or 30% homology, and then obtain fluorescence probe similar in function, the present invention This will not be repeated here, and above-mentioned technical proposal is within the scope of the present invention.

When mentioning certain more peptide or protein, term used herein " variant " includes having the identical function of more peptide or proteins Can but the different variant of sequence.These variants include but is not limited to: lacking, are inserted into the polypeptide or protein sequence And/or replace one or more (usually 1-30, preferably 1-5) amino acid, and recurrence N-terminal and/or C-terminal addition one The sequence that a or multiple (usually 20 within) amino acid obtains.In the art, with performance is similar or similar amino acid When being replaced, the function of more peptide or proteins is not usually changed.In the art, the similar amino acid of performance often refers to have The amino acid residues of similar side chain.It is well known to those skilled in the art, in gene cloning experimental implementation, it usually needs design is suitable Restriction enzyme site, this certainly will introduce one or more incoherent residues in expressed more peptide or protein ends, but this is simultaneously The activity of desired polypeptides or albumen is not influenced.

Term used herein " nucleic acid " can be DNA form or rna form, DNA include cDNA, genomic DNA or Artificial synthesized DNA.DNA can be coding strand or noncoding strand.The coding region sequence of encoding mature albumen is also possible to degeneracy Variant.

When mentioning nucleic acid, terms used herein " variant " can be the allelic variant that natural or non-natural occurs.This A little nucleotide variants include substitution variants, insertion variant and Deletion variants.Allelic variant is a kind of alternative forms, It can be the substitution, insertion or missing of one or more nucleotide, but not from the function of substantially changing encoded albumen Characteristic.

The full length sequence of fluorescence probe or fusion protein of the present invention or its segment can usually use PCR amplification method or artificial conjunction It is obtained at method.For PCR amplification, design primer can be carried out by disclosed related nucleotide sequence according to the present invention, and with commercially available The library cDNA prepares the library cDNA as template by method known to those skilled in the art, and amplification obtains related sequence.Once obtaining Obtain related sequence, so that it may in high volume obtain related sequence with recombination method, be usually to be cloned into carrier, then be transferred to cell Separation and purifying obtain related fusion protein from host cell by conventional method.

Currently, can obtain encoding by chemical synthesis process completely albumen of the present invention or its segment or its analog, The DNA sequence dna of derivative, variant, and then be introduced into various existing carriers or cell known to this research field.It is another Mutation can be introduced into protein sequence of the present invention by aspect by the methods of chemical synthesis or mutation PCR.

Expression vector of the invention can be used for expressing fluorescence probe or fusion egg of the invention in protokaryon or eukaryotic It is white, thus the present invention relates to the host cell for having imported expression vector of the present invention, host cell can be thin for any protokaryon or eukaryon Born of the same parents, the preferably various cells for being conducive to gene product expression or fermenting and producing, and known to this research field and commonly use, such as respectively Kind Bacillus coli cells core yeast cells.In one embodiment, Escherichia coli building has been selected to express fusion of the present invention The host cell of albumen.This hair can be expressed with being suitble to the conventional method culture of the host cell expression to obtain transformed cells Bright fluorescence probe albumen.According to host cell used, culture medium used in culture is various conventional mediums, then suitable It is cultivated under conditions of host cell growth.It, can be with suitably but after host cell growth to cell density appropriate Method such as chemical agent induction induce the promoter of selection, and cell is continued culture a period of time later.

Recombination fusion protein in the above-mentioned methods can be expressed or be secreted into the cell or on cell membrane carefully It is extracellular.As needed, its physics, chemistry and other characteristics be can use, restructuring destination protein is carried out by different separation methods Isolated or purified.These methods include but is not limited to: conventional centrifugation, ultrasonication processing or osmometry processing, renaturation The technologies such as processing, protein precipitant facture, affinity chromatography, ion-exchange chromatography, sieve chromatography, high performance liquid chroma- tography and The combination of these methods.

In one embodiment, this hair is produced by the inclusion of the Escherichia coli fermentation of fusion protein coded sequence of the present invention Bright fluorescence probe or fusion protein, and the fluorescence probe of the present invention of pure form has been obtained by affinity chromatography and gel chromatography Or fusion protein.The purposes of fluorescence probe of the present invention includes but is not limited to: the level of cAMP, screening medicine under detection physiological status Object, diagnosis disease relevant to cAMP level etc..

Raw materials used and reagent is available on the market in cAMP fluorescence probe provided by the invention and its application.

Below with reference to embodiment, the present invention is further explained:

Embodiment 1

CNBD-N-linker1-cpEGFP-linker2-CNBD-C (Cyclic nucleotide- is constructed first Binding domain, CNBD, cyclic nucleotide binding structural domain；The N-terminal of CNBD-N, CNBD；The C-terminal of CNBD-C, CNBD； CpEGFP is cyclized the green fluorescent protein of rearrangement；Linker, link peptide).Linker1 and linker2 are screened, # is obtained 252 probes, linker1 and linker2 are respectively WG and RV (Figure 1A).Flamindo2 and #252 are expressed respectively in bacterium In, after 34 DEG C are cultivated 15 hours, it is seen that the brightness of #252 fluorescence probe is obviously than 29 times of Flamindo2 high (Figure 1B).The ammonia of #252 Base acid sequence, which has also been given, comes out (Fig. 2).

Mesorhizobium loti MAFF303099DNA, complete genome RC (ORF sequece) is (such as Shown in SEQ ID No.11)

ATGTCGGTACTGCCTTTCTTAAGAATTTACGCGCCGCTCAACGCGGTGCTGGCTGCGCCTGGGTTGCTG GCGGTGGCTGCGCTCACGATACCGGACATGTCCGGACGAAGCAGACTGGCTCTGGCTGCCCTGCTCGCTGTCATCTG GGGCGCCTATCTCCTGCAACTGGCCGCGACGCTGCTCAAGCGCCGGGCGGGAGTCGTACGGGACAGGACGCCCAAAA TCGCCATCGATGTGCTCGCAGTCTTGGTTCCACTCGCCGCATTTCTGCTCGACGGCTCGCCTGACTGGAGCCTCTAC TGTGCTGTCTGGCTGCTGAAACCGCTGCGCGACTCGACTTTCTTCCCGGTCCTGGGCAGGGTCCTGGCCAACGAAGC ACGCAATCTGATCGGCGTCACCACGCTCTTCGGCGTCGTTCTGTTCGCAGTGGCGCTCGCAGCCTATGTCATCGAGC GCGATATCCAACCGGAAAAGTTCGGCAGCATTCCCCAGGCAATGTGGTGGGCGGTGGTCACGCTGTCCACCACCGGC TATGGGGACACTATCCCGCAAAGCTTCGCCGGCCGCGTCCTTGCCGGGGCGGTCATGATGAGTGGCATCGGCATCTT CGGACTCTGGGCCGGCATTCTTGCCACAGGCTTCTATCAAGAAGTCCGTCGCGGGGATTTCGTCCGCAATTGGCAAT TGGTCGCCGCCGTGCCGTTGTTTCAGAAGCTCGGCCCGGCCGTGCTGGTCGAGATCGTGCGCGCCTTAAGAGCCCGC ACGGTGCCGGCGGGCGCCGTGATCTGCCGCATTGGCGAGCCCGGCGATCGGATGTTCTTCGTCGTGGAGGGGAGCGT CAGCGTCGCGACGCCGAATCCGGTGGAGCTTGGCCCTGGCGCCTTCTTCGGCGAGATGGCGCTGATCAGCGGCGAAC CGCGTTCGGCGACCGTCAGCGCCGCAACGACGGTCTCACTCCTGTCGCTGCATTCGGCGGATTTCCAGATGTTGTGC AGCAGCAGCCCGGAGATCGCGGAAATCTTCCGCAAGACCGCGCTCGAGCGTCGCGGCGCTGCGGCGAGCGCT

MICNBD protein:(is as shown in SEQ ID No.12)

MSVLPFLRIYAPLNAVLAAPGLLAVAALTIPDMSGRSRLALAALLAVIWGAYLLQLAATLLKRRAGVVR DRTPKIAIDVLAVLVPLAAFLLDGSPDWSLYCAVWLLKPLRDSTFFPVLGRVLANEARNLIGVTTLFGVVLFAVALA AYVIERDIQPEKFGSIPQAMWWAVVTLSTTGYGDTIPQSFAGRVLAGAVMMSGIGIFGLWAGILATGFYQEVRRGDF VRNWQLVAAVPLFQKLGPAVLVEIVRALRARTVPAGAVICRIGEPGDRMFFVVEGSVSVATPNPVELGPGAFFGEMA LISGEPRSATVSAATTVSLLSLHSADFQMLCSSSPEIAEIFRKTALERRGAAASA

Embodiment 2

By the expression of #252 probe in bacterium, 2 days collection thallus of incubated at room temperature (contain in the HEPES buffer solution of pH=7.3 150mM KCl and 50mM HEPES) in ultrasonication, utilize HisPur CobaltResin (be purchased from Pierce Corporation) purifying to visit Needle, and probe is dissolved in by the HEPES in pH=7.3 by Econo-Pac10DG desalting column (being purchased from U.S. Bio-Rad company) In buffer, with BCA kit (being purchased from U.S. Thermo scientific company) measurement concentration and probe concentration.Take 2mM probe molten Liquid, the response using multi-function microplate reader Infinite M1000PRO detection probe to various concentration cAMP and cGMP, Flamindo2 is as control.It can be seen that #252 after saturated concentration cAMP (~500 μM) are added, is excited in 440-500nm wave band Fluorescent brightness be increased to 3.5-4.15 times (Fig. 3).Under 50 μM of cAMP concentration, the fluorescent brightness excited at 500nm changes (ΔF/F₀) it is~2.5 times (Fig. 4, red curve)；Under 50 μM of cGMP concentration, the fluorescent brightness excited at 500nm changes (ΔF/F₀) it is -16% (Fig. 4, green curve).When being greater than 5 μM of cGMP, #252's because of signal intensity width caused by cGMP Degree is significantly less than Flamindo2.When less than 5 μM of cGMP, Flamindo2 is compared with #252 because of signal intensity width caused by cGMP Spend small, but the signal intensity is smaller (Fig. 4, green curve and black dotted lines, data are as shown in table 2) at this time.

Table 2

Embodiment 3

#252, Flamindo2, mEGFP (monomer green fluorescent protein) are building up on carrier for expression of eukaryon (CAG respectively Promoter), pass through HEK293T cell of 2000 kit of the Lipofectamine transfection culture in the culture dish of glass bottom (purchase from GE Healthcare Dharmacon company), after being incubated overnight, with it is without serum, without phenol red culture medium (being purchased from GIBCO company) starved cells 6 hours.The IX83 fluorescence microscope detection probe voluntarily built using this laboratory it is bright Degree, it is seen that #252 brightness under cell quiescent condition about improves 60 times of [(3000-225)/(225- far beyond Flamindo2 high 180)=61.6] (Fig. 5 A).Cell is by 60 μM of Forskolin and 100 μM of IBMX (being purchased from green skies biotech company) thorn CAMP concentration increases after swashing, and the signal intensity amplitude of #252 probe is 100%-250% (Fig. 5 B-C).So far it is dynamic to complete lactation The fluorescence imaging step of object intracellular cAMP concentration variation.

The mutation of 4 fluorescence probe of embodiment

285-ins is probe before optimizing, and 252 be the probe after optimization.Two kinds are compared, different amino acid overstriking font Be marked out: left side Linker the former be LE, the latter WG.The right linker the former be LP, the latter RV.

The change made on fluorescin is respectively as follows: K153T, I171V, S30R, Y39N, S72A, F99V from left to right, N105T.These serial numbers carry out autofluorescence albumen, rather than serial number of the amino acid on probe.

Mutation method: first selecting the site to be mutated, then the amino acid comprising mutation on the primer of design, segmentation PCR amplification respectively comprising the segment being mutated, is finally 1 overlap PCR and integrates these mutation, then do library and select out.

252-ORF (as shown in SEQ ID No.8):

ATGCGGGGTTCTCATCATCATCATCATCATGGTATGGCTAGCATGACTGGTGGACAGCAAATGGGTCGG GATCTGTACGACGATGACGATAAGGATCCGATGGGCTTCTATCAAGAAGTCCGTCGCGGGGATTTCGTCCGCAATTG GCAATTGGTCGCCGCCGTGCCGTTGTTTCAGAAGCTCGGCCCGGCCGTGCTGGTCGAGATCGTGCGCGCCTTAAGAG CCCGCACGGTGCCGGCGGGCGCCGTGATCTGCCGCATTGGCGAGCCCGGCGATCGGATGTTCTTCGTCGTGGAGGGG AGCGTCAGCGTCGCGACGCCGTGGGGGAACGTCTATATCACAGCCGACAAGCAGAAGAACGGCATCAAGGCGAACTT CAAGATCCGCCACAACGTTGAGGACGGCGGCGTGCAGCTCGCCTACCACTACCAGCAGAACACCCCCATCGGCGACG GCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCGTGCAGTCCAAACTTTCGAAAGACCCCAACGAGAAGCGCGAT CACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGGGCGGTACCGG AGGGAGCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAA ACGGCCACAAGTTCAGCGTGCGTGGCGAGGGTGAGGGCGATGCCACCAATGGCAAGCTGACCCTGAAGTTCATCTGC ACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCGCACGCTA CCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACATCCAGGAGCGCACCATCGTTT TCAAGGACGACGGCACCTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTG AAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACCGCGTGAATCCGGTGGAGCT TGGCCCTGGCGCCTTCTTCGGCGAGATGGCGCTGATCAGCGGCGAACCGCGTTCGGCGACCGTCAGCGCCGCAACGA CGGTCTCACTCCTGTCGCTGCATTCGGCGGATTTCCAGATGTTGTGCAGCAGCAGCCCGGAGATCGCGGAAATCTTC CGCAAGACCGCGCTCGAGCGTCGCGGCGCTGCGGCGAGCGCT

252protein (as shown in SEQ ID No.7):

MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDPMGFYQEVRRGDFVRNWQLVAAVPLFQKLGPAVLVEI VRALRARTVPAGAVICRIGEPGDRMFFVVEGSVSVATPWGNVYITADKQKNGIKANFKIRHNVEDGGVQLAYHYQQN TPIGDGPVLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFTGVVPILVE LDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYIQ ERTIVFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNRVNPVELGPGAFFGEMALISGEPRSAT VSAATTVSLLSLHSADFQMLCSSSPEIAEIFRKTALERRGAAASA

285-ins-ORF (as shown in SEQ ID No.9):

ATGCGGGGTTCTCATCATCATCATCATCATGGTATGGCTAGCATGACTGGTGGACAGCAAATGGGTCGG GATCTGTACGACGATGACGATAAGGATCCGATGGGCTTCTATCAAGAAGTCCGTCGCGGGGATTTCGTCCGCAATTG GCAATTGGTCGCCGCCGTGCCGTTGTTTCAGAAGCTCGGCCCGGCCGTGCTGGTCGAGATCGTGCGCGCCTTAAGAG CCCGCACGGTGCCGGCGGGCGCCGTGATCTGCCGCATTGGCGAGCCCGGCGATCGGATGTTCTTCGTCGTGGAGGGG AGCGTCAGCGTCGCGACGCCGCTCGAGAACGTCTATATCAAGGCCGACAAGCAGAAGAACGGCATCAAGGCGAACTT CAAGATCCGCCACAACATCGAGGACGGCGGCGTGCAGCTCGCCTACCACTACCAGCAGAACACCCCCATCGGCGACG GCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCGTGCAGTCCAAACTTTCGAAAGACCCCAACGAGAAGCGCGAT CACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGGGCGGTACCGG AGGGAGCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAA ACGGCCACAAGTTCAGCGTGTCCGGCGAGGGTGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGC ACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTA CCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACATCCAGGAGCGCACCATCTTCT TCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTG AAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACCTGCCGAATCCGGTGGAGCT TGGCCCTGGCGCCTTCTTCGGCGAGATGGCGCTGATCAGCGGCGAACCGCGTTCGGCGACCGTCAGCGCCGCAACGA CGGTCTCACTCCTGTCGCTGCATTCGGCGGATTTCCAGATGTTGTGCAGCAGCAGCCCGGAGATCGCGGAAATCTTC CGCAAGACCGCGCTCGAGCGTCGCGGCGCTGCGGCGAGCGCT

285-ins protein (as shown in SEQ ID No.10):

MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDPMGFYQEVRRGDFVRNWQLVAAVPLFQKLGPAVLVEI VRALRARTVPAGAVICRIGEPGDRMFFVVEGSVSVATPLENVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQN TPIGDGPVLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFTGVVPILVE LDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYIQ ERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNLPNPVELGPGAFFGEMALISGEPRSAT VSAATTVSLLSLHSADFQMLCSSSPEIAEIFRKTALERRGAAASA

Mutant probe bring effect:

1, dynamic range becomes larger.

Fig. 6 show the 285-ins and #252 of purifying in conjunction with cAMP after excitation spectrum variation.Dotted line is that cAMP is not added to swash Luminous spectrum (A), solid line are the excitation spectrum (B) being added after final concentration of 1mM cAMP.As it can be seen that #252 probe have it is biggish just Variation.

2. brightness improves.

Fig. 7 shows that 252 brightness are (87-40)/(40-26)=3.35 times of 285-ins.

The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered It is considered as protection scope of the present invention.

Sequence table

<110>Shenzhen Xianjin Technology Academe

<120>cAMP fluorescence probe and its application

<130> MP1829175

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

<211> 355

<212> PRT

<213> CNBD

<400> 1

Met Ser Val Leu Pro Phe Leu Arg Ile Tyr Ala Pro Leu Asn Ala Val

1 5 10 15

Leu Ala Ala Pro Gly Leu Leu Ala Val Ala Ala Leu Thr Ile Pro Asp

20 25 30

Met Ser Gly Arg Ser Arg Leu Ala Leu Ala Ala Leu Leu Ala Val Ile

35 40 45

Trp Gly Ala Tyr Leu Leu Gln Leu Ala Ala Thr Leu Leu Lys Arg Arg

50 55 60

Ala Gly Val Val Arg Asp Arg Thr Pro Lys Ile Ala Ile Asp Val Leu

65 70 75 80

Ala Val Leu Val Pro Leu Ala Ala Phe Leu Leu Asp Gly Ser Pro Asp

85 90 95

Trp Ser Leu Tyr Cys Ala Val Trp Leu Leu Lys Pro Leu Arg Asp Ser

100 105 110

Thr Phe Phe Pro Val Leu Gly Arg Val Leu Ala Asn Glu Ala Arg Asn

115 120 125

Leu Ile Gly Val Thr Thr Leu Phe Gly Val Val Leu Phe Ala Val Ala

130 135 140

Leu Ala Ala Tyr Val Ile Glu Arg Asp Ile Gln Pro Glu Lys Phe Gly

145 150 155 160

Ser Ile Pro Gln Ala Met Trp Trp Ala Val Val Thr Leu Ser Thr Thr

165 170 175

Gly Tyr Gly Asp Thr Ile Pro Gln Ser Phe Ala Gly Arg Val Leu Ala

180 185 190

Gly Ala Val Met Met Ser Gly Ile Gly Ile Phe Gly Leu Trp Ala Gly

195 200 205

Ile Leu Ala Thr Gly Phe Tyr Gln Glu Val Arg Arg Gly Asp Phe Val

210 215 220

Arg Asn Trp Gln Leu Val Ala Ala Val Pro Leu Phe Gln Lys Leu Gly

225 230 235 240

Pro Ala Val Leu Val Glu Ile Val Arg Ala Leu Arg Ala Arg Thr Val

245 250 255

Pro Ala Gly Ala Val Ile Cys Arg Ile Gly Glu Pro Gly Asp Arg Met

260 265 270

Phe Phe Val Val Glu Gly Ser Val Ser Val Ala Thr Pro Asn Pro Val

275 280 285

Glu Leu Gly Pro Gly Ala Phe Phe Gly Glu Met Ala Leu Ile Ser Gly

290 295 300

Glu Pro Arg Ser Ala Thr Val Ser Ala Ala Thr Thr Val Ser Leu Leu

305 310 315 320

Ser Leu His Ser Ala Asp Phe Gln Met Leu Cys Ser Ser Ser Pro Glu

325 330 335

Ile Ala Glu Ile Phe Arg Lys Thr Ala Leu Glu Arg Arg Gly Ala Ala

340 345 350

Ala Ser Ala

355

<210> 2

<211> 241

<212> PRT

<213> cpEGFP

<400> 2

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 Asp 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> 3

<211> 285

<212> PRT

<213> CNBD-N

<400> 3

Met Ser Val Leu Pro Phe Leu Arg Ile Tyr Ala Pro Leu Asn Ala Val

1 5 10 15

Leu Ala Ala Pro Gly Leu Leu Ala Val Ala Ala Leu Thr Ile Pro Asp

20 25 30

Met Ser Gly Arg Ser Arg Leu Ala Leu Ala Ala Leu Leu Ala Val Ile

35 40 45

Trp Gly Ala Tyr Leu Leu Gln Leu Ala Ala Thr Leu Leu Lys Arg Arg

50 55 60

Ala Gly Val Val Arg Asp Arg Thr Pro Lys Ile Ala Ile Asp Val Leu

65 70 75 80

Ala Val Leu Val Pro Leu Ala Ala Phe Leu Leu Asp Gly Ser Pro Asp

85 90 95

Trp Ser Leu Tyr Cys Ala Val Trp Leu Leu Lys Pro Leu Arg Asp Ser

100 105 110

Thr Phe Phe Pro Val Leu Gly Arg Val Leu Ala Asn Glu Ala Arg Asn

115 120 125

Leu Ile Gly Val Thr Thr Leu Phe Gly Val Val Leu Phe Ala Val Ala

130 135 140

Leu Ala Ala Tyr Val Ile Glu Arg Asp Ile Gln Pro Glu Lys Phe Gly

145 150 155 160

Ser Ile Pro Gln Ala Met Trp Trp Ala Val Val Thr Leu Ser Thr Thr

165 170 175

Gly Tyr Gly Asp Thr Ile Pro Gln Ser Phe Ala Gly Arg Val Leu Ala

180 185 190

Gly Ala Val Met Met Ser Gly Ile Gly Ile Phe Gly Leu Trp Ala Gly

195 200 205

Ile Leu Ala Thr Gly Phe Tyr Gln Glu Val Arg Arg Gly Asp Phe Val

210 215 220

Arg Asn Trp Gln Leu Val Ala Ala Val Pro Leu Phe Gln Lys Leu Gly

225 230 235 240

Pro Ala Val Leu Val Glu Ile Val Arg Ala Leu Arg Ala Arg Thr Val

245 250 255

Pro Ala Gly Ala Val Ile Cys Arg Ile Gly Glu Pro Gly Asp Arg Met

260 265 270

Phe Phe Val Val Glu Gly Ser Val Ser Val Ala Thr Pro

275 280 285

<210> 4

<211> 70

<212> PRT

<213> CNBD-C

<400> 4

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 Ser 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

<210> 5

<211> 2

<212> PRT

<213> linker1

<400> 5

Trp Gly

1

<210> 6

<211> 2

<212> PRT

<213> linker2

<400> 6

Arg Val

1

<210> 7

<211> 422

<212> PRT

<213>cAMP fluorescence probe (cAMP fluorescent probe)

<400> 7

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 Pro 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 Asp 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 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> 8

<211> 1266

<212> DNA

<213>cAMP fluorescence probe (cAMP fluorescent probe)

<400> 8

atgcggggtt ctcatcatca tcatcatcat ggtatggcta gcatgactgg tggacagcaa 60

atgggtcggg atctgtacga cgatgacgat aaggatccga tgggcttcta tcaagaagtc 120

cgtcgcgggg atttcgtccg caattggcaa ttggtcgccg ccgtgccgtt gtttcagaag 180

ctcggcccgg ccgtgctggt cgagatcgtg cgcgccttaa gagcccgcac ggtgccggcg 240

ggcgccgtga tctgccgcat tggcgagccc ggcgatcgga tgttcttcgt cgtggagggg 300

agcgtcagcg tcgcgacgcc gtgggggaac gtctatatca cagccgacaa gcagaagaac 360

ggcatcaagg cgaacttcaa gatccgccac aacgttgagg acggcggcgt gcagctcgcc 420

taccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 480

tacctgagcg tgcagtccaa actttcgaaa gaccccaacg agaagcgcga tcacatggtc 540

ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagggc 600

ggtaccggag ggagcatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc 660

ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgcgtgg cgagggtgag 720

ggcgatgcca ccaatggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc 780

gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cgcacgctac 840

cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacatccag 900

gagcgcacca tcgttttcaa ggacgacggc acctacaaga cccgcgccga ggtgaagttc 960

gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc 1020

aacatcctgg ggcacaagct ggagtacaac cgcgtgaatc cggtggagct tggccctggc 1080

gccttcttcg gcgagatggc gctgatcagc ggcgaaccgc gttcggcgac cgtcagcgcc 1140

gcaacgacgg tctcactcct gtcgctgcat tcggcggatt tccagatgtt gtgcagcagc 1200

agcccggaga tcgcggaaat cttccgcaag accgcgctcg agcgtcgcgg cgctgcggcg 1260

agcgct 1266

<210> 9

<211> 1266

<212> DNA

<213> 285-ins-ORF

<400> 9

atgcggggtt ctcatcatca tcatcatcat ggtatggcta gcatgactgg tggacagcaa 60

atgggtcggg atctgtacga cgatgacgat aaggatccga tgggcttcta tcaagaagtc 120

cgtcgcgggg atttcgtccg caattggcaa ttggtcgccg ccgtgccgtt gtttcagaag 180

ctcggcccgg ccgtgctggt cgagatcgtg cgcgccttaa gagcccgcac ggtgccggcg 240

ggcgccgtga tctgccgcat tggcgagccc ggcgatcgga tgttcttcgt cgtggagggg 300

agcgtcagcg tcgcgacgcc gtgggggaac gtctatatca cagccgacaa gcagaagaac 360

ggcatcaagg cgaacttcaa gatccgccac aacgttgagg acggcggcgt gcagctcgcc 420

taccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 480

tacctgagcg tgcagtccaa actttcgaaa gaccccaacg agaagcgcga tcacatggtc 540

ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagggc 600

ggtaccggag ggagcatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc 660

ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgcgtgg cgagggtgag 720

ggcgatgcca ccaatggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc 780

gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cgcacgctac 840

cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacatccag 900

gagcgcacca tcgttttcaa ggacgacggc acctacaaga cccgcgccga ggtgaagttc 960

gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc 1020

aacatcctgg ggcacaagct ggagtacaac cgcgtgaatc cggtggagct tggccctggc 1080

gccttcttcg gcgagatggc gctgatcagc ggcgaaccgc gttcggcgac cgtcagcgcc 1140

gcaacgacgg tctcactcct gtcgctgcat tcggcggatt tccagatgtt gtgcagcagc 1200

agcccggaga tcgcggaaat cttccgcaag accgcgctcg agcgtcgcgg cgctgcggcg 1260

agcgct 1266

<210> 10

<211> 422

<212> PRT

<213> 285-ins-ORF

<400> 10

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 Pro Leu Glu Asn Val Tyr

100 105 110

Ile Lys Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile

115 120 125

Arg His Asn Ile Glu Asp 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 Ser Gly Glu Gly Glu

225 230 235 240

Gly Asp Ala Thr Tyr 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 Ser 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

Phe Phe Lys Asp Asp Gly Asn 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 Leu Pro

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> 11

<211> 1065

<212> DNA

<213> mICNBD

<400> 11

atgtcggtac tgcctttctt aagaatttac gcgccgctca acgcggtgct ggctgcgcct 60

gggttgctgg cggtggctgc gctcacgata ccggacatgt ccggacgaag cagactggct 120

ctggctgccc tgctcgctgt catctggggc gcctatctcc tgcaactggc cgcgacgctg 180

ctcaagcgcc gggcgggagt cgtacgggac aggacgccca aaatcgccat cgatgtgctc 240

gcagtcttgg ttccactcgc cgcatttctg ctcgacggct cgcctgactg gagcctctac 300

tgtgctgtct ggctgctgaa accgctgcgc gactcgactt tcttcccggt cctgggcagg 360

gtcctggcca acgaagcacg caatctgatc ggcgtcacca cgctcttcgg cgtcgttctg 420

ttcgcagtgg cgctcgcagc ctatgtcatc gagcgcgata tccaaccgga aaagttcggc 480

agcattcccc aggcaatgtg gtgggcggtg gtcacgctgt ccaccaccgg ctatggggac 540

actatcccgc aaagcttcgc cggccgcgtc cttgccgggg cggtcatgat gagtggcatc 600

ggcatcttcg gactctgggc cggcattctt gccacaggct tctatcaaga agtccgtcgc 660

ggggatttcg tccgcaattg gcaattggtc gccgccgtgc cgttgtttca gaagctcggc 720

ccggccgtgc tggtcgagat cgtgcgcgcc ttaagagccc gcacggtgcc ggcgggcgcc 780

gtgatctgcc gcattggcga gcccggcgat cggatgttct tcgtcgtgga ggggagcgtc 840

agcgtcgcga cgccgaatcc ggtggagctt ggccctggcg ccttcttcgg cgagatggcg 900

ctgatcagcg gcgaaccgcg ttcggcgacc gtcagcgccg caacgacggt ctcactcctg 960

tcgctgcatt cggcggattt ccagatgttg tgcagcagca gcccggagat cgcggaaatc 1020

ttccgcaaga ccgcgctcga gcgtcgcggc gctgcggcga gcgct 1065

<210> 12

<211> 355

<212> PRT

<213> mICNBD

<400> 12

Met Ser Val Leu Pro Phe Leu Arg Ile Tyr Ala Pro Leu Asn Ala Val

1 5 10 15

Leu Ala Ala Pro Gly Leu Leu Ala Val Ala Ala Leu Thr Ile Pro Asp

20 25 30

Met Ser Gly Arg Ser Arg Leu Ala Leu Ala Ala Leu Leu Ala Val Ile

35 40 45

Trp Gly Ala Tyr Leu Leu Gln Leu Ala Ala Thr Leu Leu Lys Arg Arg

50 55 60

Ala Gly Val Val Arg Asp Arg Thr Pro Lys Ile Ala Ile Asp Val Leu

65 70 75 80

Ala Val Leu Val Pro Leu Ala Ala Phe Leu Leu Asp Gly Ser Pro Asp

85 90 95

Trp Ser Leu Tyr Cys Ala Val Trp Leu Leu Lys Pro Leu Arg Asp Ser

100 105 110

Thr Phe Phe Pro Val Leu Gly Arg Val Leu Ala Asn Glu Ala Arg Asn

115 120 125

Leu Ile Gly Val Thr Thr Leu Phe Gly Val Val Leu Phe Ala Val Ala

130 135 140

Leu Ala Ala Tyr Val Ile Glu Arg Asp Ile Gln Pro Glu Lys Phe Gly

145 150 155 160

Ser Ile Pro Gln Ala Met Trp Trp Ala Val Val Thr Leu Ser Thr Thr

165 170 175

Gly Tyr Gly Asp Thr Ile Pro Gln Ser Phe Ala Gly Arg Val Leu Ala

180 185 190

Gly Ala Val Met Met Ser Gly Ile Gly Ile Phe Gly Leu Trp Ala Gly

195 200 205

Ile Leu Ala Thr Gly Phe Tyr Gln Glu Val Arg Arg Gly Asp Phe Val

210 215 220

Arg Asn Trp Gln Leu Val Ala Ala Val Pro Leu Phe Gln Lys Leu Gly

225 230 235 240

Pro Ala Val Leu Val Glu Ile Val Arg Ala Leu Arg Ala Arg Thr Val

245 250 255

Pro Ala Gly Ala Val Ile Cys Arg Ile Gly Glu Pro Gly Asp Arg Met

260 265 270

Phe Phe Val Val Glu Gly Ser Val Ser Val Ala Thr Pro Asn Pro Val

275 280 285

Glu Leu Gly Pro Gly Ala Phe Phe Gly Glu Met Ala Leu Ile Ser Gly

290 295 300

Glu Pro Arg Ser Ala Thr Val Ser Ala Ala Thr Thr Val Ser Leu Leu

305 310 315 320

Ser Leu His Ser Ala Asp Phe Gln Met Leu Cys Ser Ser Ser Pro Glu

325 330 335

Ile Ala Glu Ile Phe Arg Lys Thr Ala Leu Glu Arg Arg Gly Ala Ala

340 345 350

Ala Ser Ala

355

Claims

1. a kind of cAMP fluorescence probe characterized by comprising the polypeptide sensitive to cAMP and fluorescin.

2. cAMP fluorescence probe as described in claim 1, which is characterized in that the polypeptide sensitive to cAMP is with cAMP The MlotiK1 CNBD structural domain of binding characteristic.

3. cAMP fluorescence probe as claimed in claim 2, which is characterized in that the MlotiK1 CNBD structural domain includes

(I), the amino acid sequence as shown in SEQ ID NO:1；

(II), such as (I) described amino acid sequence is substituted, lacks or adds the amino acid sequence that one or more amino acid obtain Column, and with (I) described amino acid sequence amino acid sequence functionally identical or similar；

4. cAMP fluorescence probe as described in any one of claims 1 to 3, which is characterized in that the fluorescin is cyclisation weight The green fluorescent protein cpEGFP of row.

5. such as the described in any item cAMP fluorescence probes of Claims 1-4, which is characterized in that the fluorescin includes

(IV), the amino acid sequence as shown in SEQ ID NO:2；

(V), such as (IV) described amino acid sequence is substituted, lacks or adds the amino acid sequence that one or more amino acid obtain Column, and with (IV) described amino acid sequence amino acid sequence functionally identical or similar；

6. such as the described in any item cAMP fluorescence probes of claim 2 to 5, which is characterized in that described in the fluorescin insertion The site of MlotiK1 CNBD structural domain is A/T (283/284), T/P (284/285), P/N (285/286), N/P (286/ 287)。

7. cAMP fluorescence probe as claimed in claim 6, which is characterized in that the cAMP fluorescence probe has shown in formula I Structure:

MlotiK1 CNBD-N-linker1-cpEGFP-linker2-MlotiK1 CNBD-C

Formulas I

Wherein, MlotiK1 CNBD-N is the N-terminal of MlotiK1 CNBD, comprising:

(VII), the amino acid sequence as shown in SEQ ID NO:3；

(VIII), such as (VII) described amino acid sequence is substituted, lacks or adds the amino acid that one or more amino acid obtain Sequence, and with (VII) described amino acid sequence amino acid sequence functionally identical or similar；

The MlotiK1 CNBD-C is the C-terminal of MlotiK1 CNBD, comprising:

(X), the amino acid sequence as shown in SEQ ID NO:4；

(XI), such as (X) described amino acid sequence is substituted, lacks or adds the amino acid sequence that one or more amino acid obtain Column, and with the amino acid sequence functionally identical or similar of amino acid sequence shown in (X)；

Linker1 is the first link peptide, comprising:

(XIII), the amino acid sequence as shown in SEQ ID NO:5；

(XIV), such as (XIII) described amino acid sequence is substituted, lacks or adds the amino acid that one or more amino acid obtain Sequence, and with (XIII) described amino acid sequence amino acid sequence functionally identical or similar；

Linker2 is the second link peptide, comprising:

(XVI), the amino acid sequence as shown in SEQ ID NO:6；

(XVII), such as (XVI) described amino acid sequence is substituted, lacks or adds the amino acid that one or more amino acid obtain Sequence, and with (XVI) described amino acid sequence amino acid sequence functionally identical or similar；

8. cAMP fluorescence probe as described in any one of claim 1 to 7, which is characterized in that it is included

(XIX), the amino acid sequence as shown in SEQ ID NO:7；

(XX), such as (XIX) described amino acid sequence is substituted, lacks or adds the amino acid sequence that one or more amino acid obtain Column, and with the amino acid sequence functionally identical or similar of amino acid sequence shown in (XIX)；

9. encoding the nucleotide of the cAMP fluorescence probe as described in any one of claim 1 to 8.

10. nucleotide as claimed in claim 9 comprising:

(XXII), there is nucleotide sequence shown in SEQ ID NO:8；

(XXIII), as (XXII) described nucleotide sequence is modified, replaces, misses or adds one or more bases acquisitions Nucleotide sequence；

11. expression vector, the nucleotide including encoding the cAMP fluorescence probe as described in any one of claim 1~8.

12. conversion or the host cell for transfecting expression vector as claimed in claim 11.

13. the preparation method of cAMP fluorescence probe as described in any one of claim 1~8, comprising: culture is such as claim 11 institute The expression of the host cell, the induction cAMP fluorescence probe stated.

14. application of the cAMP fluorescence probe in detection cAMP as described in any one of claim 1~8.

15. the application of cAMP fluorescence probe variation of cAMP in detection living cells as described in any one of claim 1~8.

The detection method of 16.cAMP, which comprises the steps of:

Step 1, construction of expression vector, the expression vector include that coding cAMP fluorescence as described in any one of claim 1~8 is visited The nucleotide of needle；

The expression of step 3, culture host cell as claimed in claim 12, the induction cAMP fluorescence probe；

Step 4, by the cAMP fluorescence probe to the corresponding of cAMP described in sample to be tested, obtain the concentration of the cAMP.

17. kit, which is characterized in that including the cAMP fluorescence probe as described in any one of claim 1~8.