CN109627344A - CAMP fluorescence probe and its application - Google Patents
CAMP fluorescence probe and its application Download PDFInfo
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- CN109627344A CN109627344A CN201811620932.0A CN201811620932A CN109627344A CN 109627344 A CN109627344 A CN 109627344A CN 201811620932 A CN201811620932 A CN 201811620932A CN 109627344 A CN109627344 A CN 109627344A
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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
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 (17)
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;
(III), with the amino acid sequence of (I) or (II) described amino acid sequence at least 80% homology.
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;
(VI), with the amino acid sequence of (IV) or (V) described amino acid sequence at least 80% homology.
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;
(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 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);
(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 amino acid that one or more amino acid obtain
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 amino acid that one or more amino acid obtain
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.
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);
(XXI), with the amino acid sequence of (XIX) or (XX) described amino acid sequence at least 80% homology.
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;
(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.
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;
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 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.
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CN112480271A (en) * | 2020-12-15 | 2021-03-12 | 中国科学院深圳先进技术研究院 | High-performance red cAMP fluorescent probe and application thereof |
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 application thereof |
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