CN109608525B - Large stokes orange fluorescent protein LSSmKO1 and application thereof - Google Patents
Large stokes orange fluorescent protein LSSmKO1 and application thereof Download PDFInfo
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Abstract
The invention provides a large Stokes orange fluorescent protein LSSmKO1 and application thereof. The large stokes orange fluorescent protein is a mutant orange fluorescent protein mKO kappa, and compared with the amino acid sequence of mKO kappa, the large stokes orange fluorescent protein has mutation sites: V160D.
Description
Technical Field
The invention relates to the technical field of biological optics and molecular imaging, in particular to a large Stokes orange fluorescent protein LSSmKO1 and application of the large Stokes orange fluorescent protein LSSmKO 1.
Background
When the fluorescent protein is used for live cell or living body imaging, multicolor simultaneous imaging is often difficult to realize, and if the limitation is broken through, the detection of a plurality of biological rapid reaction events can be realized, so that the development of neurobiology is greatly promoted.
In single photon imaging, multiple fluorescent proteins can be excited by light with different excitation wavelengths to realize simultaneous multicolor imaging. However, in multiphoton imaging, the cost of adding a secondary titanium sapphire laser or an optical parametric oscillator is high, and it is difficult to regulate them together, so that simultaneous imaging of three or more colors of fluorescent proteins is difficult to achieve. At present, the microscope method for simultaneously imaging a plurality of biomolecular events mainly depends on dynamic modulation of laser, if multicolor fluorescence simultaneous imaging is required, a plurality of sets of repeated optical devices and more accurate synchronous control are required, however, a plurality of technical difficulties in the aspect are not overcome, and therefore, the realization of the three-color or multicolor simultaneous imaging of fluorescent protein is very difficult. Therefore, it is necessary to develop a fluorescent protein for multi-channel simultaneous imaging of living body without changing the original microscopic system.
However, the development of large stokes fluorescent proteins could effectively solve this problem. The protein can realize that the same exciting light simultaneously excites three or more fluorescent proteins, thereby obtaining the fluorescence in different wavelength regions and achieving the purpose of simultaneously imaging the three or more fluorescent proteins. However, the excitation peak of the large stokes orange fluorescent protein (LSSOFP) is very close to that of the Green Fluorescent Protein (GFP) and the large stokes red fluorescent protein (LSSRFP), so that a beam of light with a specific wavelength can be found to simultaneously excite three proteins, the emission peaks of the three proteins are far apart, the emission spectra overlap less, and fluorescent signals can be respectively received, thereby realizing three-color simultaneous imaging of living cells.
However, there are very few types of existing LSSOFPs, and the emission spectrum of the existing LSSOFPs overlaps with the emission spectrum of the LSSRFP greatly, so that the imaging crosstalk is serious, and therefore, it is necessary to develop a new LSSOFP with blue-shifted emission spectrum.
Disclosure of Invention
According to some embodiments of the inventive concept, there is provided a large stokes orange fluorescent protein which is a mutated orange fluorescent protein mKO κ having a mutation site compared to the amino acid sequence of mKO κ: V160D.
According to some embodiments of the present inventive concept, there is provided the use of large stokes orange fluorescent proteins in single photon, multi-photon and super-resolution microscopy imaging.
According to some embodiments of the inventive concept, there is provided the use of a large stokes orange fluorescent protein in a bioluminescence resonance energy transfer system.
According to some embodiments of the inventive concept, there is provided the use of large stokes orange fluorescent proteins in super-resolution technology imaging based on random single molecule localization.
The large Stokes fluorescent protein LSSmKO1 provided by the invention has the following advantages:
(1) has excellent monomer property;
(2) compared with other orange fluorescent proteins with large Stokes shift, the fluorescent protein has the advantages of high brightness and short maturation time;
(3) the large Stokes displacement is realized, and the method can be used for simultaneous imaging of single photon, multi-photon and super-resolution microscopy multicolor, so that the green fluorescent protein mTFP2 and single-photon light can be simultaneously excited in a two-photon mode, the resolution of two-photon imaging is improved, and the method is applied to photo-plate nonlinear optical microscopy imaging;
(4) the BRET can be applied to a new bioluminescence resonance energy transfer system BRET, is used as a donor to be combined with receptor luciferase NanoLuc, can form a new bioluminescence resonance energy transfer system BRET with high sensitivity in an organism, and improves the depth and sensitivity of in-vivo imaging.
Drawings
The above and other objects and features of the present invention will become more apparent from the following description of the embodiments of the present invention taken in conjunction with the accompanying drawings, in which:
FIG. 1 is a graph comparing the amino acid sequence of the large Stokes orange fluorescent protein LSSmKO1 with the amino acid sequence of the orange fluorescent protein mKO κ;
FIG. 2 is the DNA sequence of the large Stokes orange fluorescent protein LSSmKO1 gene;
FIG. 3 is an excitation spectrum and an emission spectrum of the large Stokes orange fluorescent protein LSSmKO 1;
FIG. 4 is a high performance liquid chromatography peak of the large Stokes orange fluorescent protein LSSmKO 1.
Detailed Description
Hereinafter, the inventive concept will be described in more detail with reference to the accompanying drawings. The inventive concept may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided as examples so that this disclosure will be thorough and complete, and will fully convey the concept of the invention to those skilled in the art. Accordingly, experimental steps, conditions, instruments, etc. that are not necessary for a complete understanding of the various aspects and features of the inventive concepts may not be described by one of ordinary skill in the art. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and the present specification and will not be interpreted in an idealized and/or overly formal sense unless expressly so defined herein.
The stokes shift refers to the difference between the maximum emission wavelength and the maximum excitation wavelength of a fluorescent substance. If the difference is more than 100nm, the fluorescent protein is considered to have the characteristic of large Stokes shift. The large Stokes fluorescent protein can ensure the simultaneous fluorescence imaging of two or more fluorescent proteins with different emission spectra in a multicolor simultaneous imaging experiment, and effectively reduce the spectral crosstalk between different fluorescent proteins.
The first fluorescent protein with large Stokes shift reported is mKeima, which is a red fluorescent protein, and the emission peak and absorption peak are located at 440nm and 620nm, respectively, and the excitation of Cyan Fluorescent Protein (CFP) and mKeima with 458nm light can achieve the purpose of simultaneously exciting two fluorescent molecules, thereby realizing multicolor imaging with a single light source (Kogure T, Karasawa S, Araki T, Saito K, Kinjo M, Miyawaki A.A fluorescent variant of a protein from the same storage cladding fluorescent proteins, both single particle defects and products-color single-laser-fluorescence cross-correlation spectroscopy, Nat Biotechnol,2006,24: 577-. However, mKeima has the disadvantage that it has two excitation peaks, which in addition to being excited at 440nm, can also be excited at 584nm, which causes difficulties in multicolor imaging. To overcome this shortcoming of mKeima, Piatkevich k.d. et al, 2010, obtained fluorescent protein mutants LSSmKate1 and LSSmKate2, which had the same excitation wavelength but different emission wavelengths, so they could be better used to perform biochemical reactions such as two-photon in vivo observation of tumor cell metastasis (Piatkevich KD, Hulit J, Subach OM, Wu B, abdula a, Segall JE, Verkhusha vv, monomer red fluorescence samples shift. proc natl acad Sci USA,2010,107: 5369-. Jie Yang et al obtained a fluorescent protein mutant mBeRFP in 2013, which is a red fluorescent protein, and the emission peak and the absorption peak of the red fluorescent protein are respectively located at 446nm and 611nm, and the emission peak and the absorption peak can be used for exciting CFP and mBeRFP by adopting 458nm light. Compared with LSS-mKate2 and mKeima, mBeRFP has the advantages of high light intensity, good light stability, rapid maturation and the like (Yang J, Wang L, Yang F, Luo H, Xu L, Lu J, Zeng S, Zhang Z. mBeRFP, an improved large stocks shifted fluorescent protein. PLoS One,2013,8(6): e 64849.). Guany et al, 2015, obtained a monomeric fluorescent protein hmKeima8.5 with large Stokes shift, which constitutes a highly efficient fluorescent protein pair with the blue-green fluorescent protein mTFP1 for multiphoton and multicolor imaging, where the peak emission wavelengths of the two fluorescent proteins differ by 120nm, thus effectively avoiding crosstalk, and hmKeima8.5 was also significantly superior in brightness to its previous generation mKeima (guanY, Meurer M, Raghavan S, Rebane A, Lindquist JR, Santos, Kats I, Davidson MW, Maxitschek R, Hughes TE, Drobichev M, Knop M, Shah JV.Living-Cell multiphotofluorescence resonance imaging with fluorescence excitation of same wavelength, Cell 20511, Cell 19, Biofluorescence emission, Cell 19, Cell III, Sabdc. 3, Sa.
The existing large stokes orange fluorescent protein is only one of LSSmorange, and the protein is obtained from Shcherbakova DM and HinkMA research in 2012. The excitation peak and the emission peak of the protein are 437nm and 572nm respectively. The excitation peak of the protein is very close to that of Green Fluorescent Protein (GFP) and large Stokes red fluorescent protein (LSSRFP), so that a beam of light with a specific wavelength can be found to simultaneously excite three proteins, and the protein can be used for experiments of three-color simultaneous imaging of living cells. However, the emission peak of lssmoorange is relatively close to the emission peak wavelength of LSSRFP, and there is a certain spectral overlap, which causes a large crosstalk, so it is necessary to develop a large Stokes orange fluorescent protein with blue-shifted emission spectrum (shcherbakkova DM, Hink MA, Joosen L, gadela TW, Verkhusha vv. an orange fluorescent protein with a large storage shift for single-excitation multicolor FCCS and FRET imaging. jam Chem Soc,2012,134(18): 7913-23.).
In order to solve the existing technical problems, the invention provides a large Stokes orange fluorescent protein which is named as LSSmKO 1. The fluorescent protein is obtained by site-directed mutagenesis screening of orange fluorescent protein mKO kappa. Figure 1 shows a comparison of the amino acid sequence of the large stokes orange fluorescent protein LSSmKO1 with the amino acid sequence of orange fluorescent protein mKO κ, where the mutated amino acid positions are marked in grey, according to the inventive concept.
As shown in figure 1, the large stokes shift fluorescent protein LSSmKO1 has the following mutation sites compared to the amino acid sequence of mKO κ: V160D. The mutation site embodies the large stokes shift characteristic of LSSmKO1, and specifically, the valine at position 160 of the mKO kappa amino acid sequence is replaced by aspartic acid.
In one exemplary embodiment, as shown in figure 1, the amino acid sequence of LSSmKO1 also has the following mutation sites compared to the amino acid sequence of orange fluorescent protein mKO κ: M11T, E40A, T42K, R44K, a56S, L59I, a63S, H68N, V70A, E76A, L96M, S105K, Y118I, I134V, N137K, E167V, C174V, F178I, G196S, V200E, K202I, V211Y, H217S, S218Y, 219-: SNLGMDELYK are provided. These mutation sites characterize the maturation rate, brightness and monosomy of LSSmKO 1.
In addition, the mutation sites described above may specifically be: the methionine at position 11 of the amino acid sequence of mKO κ is substituted by threonine, the glutamic acid at position 40 is substituted by glycine, the threonine at position 42 is substituted by lysine, the arginine at position 44 is substituted by lysine, the alanine at position 56 is substituted by serine, the leucine at position 59 is substituted by isoleucine, the alanine at position 63 is substituted by serine, the histidine at position 68 is substituted by asparagine, the valine at position 70 is substituted by alanine, the glutamic acid at position 76 is substituted by alanine, the leucine at position 96 is substituted by methionine, the serine at position 105 is substituted by lysine, the tyrosine at position 118 is substituted by isoleucine, the isoleucine at position 134 is substituted by valine, the asparagine at position 137 is substituted by lysine, the glutamic acid at position 167 is substituted by valine, the cysteine at position 174 is substituted by valine, phenylalanine at position 178 was replaced by isoleucine, glycine at position 196 was replaced by serine, valine at position 200 was replaced by glutamic acid, lysine at position 202 was replaced by isoleucine, valine at position 211 was replaced by tyrosine, histidine at position 217 was replaced by serine, serine at position 218 was replaced by tyrosine, and serine, asparagine, leucine, glycine, methionine, aspartic acid, glutamic acid, leucine, tyrosine, lysine were added at position 219-228.
In another aspect, the present invention also provides a gene encoding the large stokes fluorescent protein LSSmKO 1. In terms of the degeneracy of codons, under the condition that the amino acid sequence of the large Stokes fluorescent protein LSSmKO1 is determined, the DNA base sequence of the gene has a plurality of cases, and the DNA base sequence belongs to the protection scope of the invention. Preferably, the DNA base sequence of the gene is shown in FIG. 2, and the base sequence can be obtained by means of gene synthesis.
In another aspect, the invention also provides the application of the large Stokes fluorescent protein LSSmKO1 in single-photon and multi-photon microscopic multicolor imaging. For example, the fluorescent protein can be effectively and simultaneously excited by single color light to carry out multicolor imaging in single-photon and multi-photon microscopic imaging together with green fluorescent protein, and can be applied to optical sheet type nonlinear optical microscopic imaging.
In another aspect, the invention also provides the application of the large stokes fluorescent protein LSSmKO1 in a novel bioluminescence resonance energy transfer system BRET. For example, the acceptor in the system may be luciferase NanoLuc, and the donor may be the above-mentioned fluorescent protein LSSmKO 1. The emission spectrum of the luciferase NanoLuc largely overlaps with the absorption spectrum of LSSmKO1, so if the two proteins are close enough, the energy generated by the oxidation reaction intermediate of the luciferase NanoLuc will be transferred to LSSmKO1 by the principle of bioluminescence resonance energy transfer, thus exciting the latter, causing the system to emit the emission spectrum of LSSmKO 1.
On the other hand, the invention also provides the application of the large stokes fluorescent protein LSSmKO1 in super-resolution technology imaging based on random single molecule positioning. For example, after the fluorescent protein is used for marking structures and organelles in cells, the excitation light is used for irradiating samples, and the obtained points on the images are not lighted at the same time when two close points are not lighted, so that super resolution can be realized in a positioning mode. Although only a few molecules can be obtained by one positioning, a high-resolution image can be obtained by positioning hundreds of thousands of single molecules by thousands of pictures.
Hereinafter, the features, properties, and implementation methods of the present invention will be described in detail.
1. Site-directed mutagenesis of orange fluorescent protein mKO kappa
The mKO kappa gene is amplified through polymerase chain reaction, then mutants are expressed and screened on a constitutive expression vector, the used expression strain is Stellar (purchased from Agilent technologies), 10 clones are arranged for each mutant in order to ensure the integrity of the library, and finally the fluorescence property of the mutants is detected through naked eye resolution and blue light LED exciting light penetrating through an orange acrylic filter, so that a monoclonal antibody expressing the fluorescent protein with large Stokes shift is screened, namely the fluorescent protein LSSmKO1 with large Stokes shift.
2. Excitation and emission spectrum detection experiments for LSSmKO1
The LSSmKO 1-expressing strain was first lysed with B-PER II (purchased from Pierce), followed by protein purification with HisPur Cobalt Resin (purchased from Pierce), followed by desalting through an Econo-Pac 10DG gravity flow chromatography column (purchased from Bio-Rad, USA). After completion of the above protein purification steps, the excitation spectrum and emission spectrum of LSSmKO1 were detected using an Infinite M1000 multifunctional microplate reader, the results of which are shown in fig. 3.
3. Monomeric detection assay for LSSmKO1
The LSSmKO 1-expressing strain was first lysed using B-PER II (purchased from Pierce), and the protein was then purified using HisPur Cobalt Resin (purchased from Pierce), followed by desalting through an Econo-Pac 10DG gravity flow chromatography column (purchased from Bio-Rad, USA). After the above protein purification steps were completed, the concentration of the fluorescent protein was concentrated to 10mg/ml, and the concentrated solution was placed in a sample bottle, and the monomer property of the fluorescent protein was detected by Shimadzu high performance liquid chromatography and size exclusion column, and the results are shown in FIG. 4.
4. Spectral properties and monosomy of LSSmKO1
Referring to fig. 3, the excitation spectrum of LSSmKO1 is represented by a dashed line and the emission spectrum thereof is represented by a solid line. The excitation and emission peaks of LSSmKO1 were at approximately 445nm and 568nm, respectively. In addition, the maturation rate of LSSmKO1 is significantly faster than that of currently used LSSmOrange, and LSSmKO1 will brighten before LSSmOrange for the same culture time and temperature.
Referring to fig. 4, after the LSSmKO1 protein was extracted and purified, and detected by high performance liquid chromatography and size exclusion column, it was confirmed that LSSmKO1 peak was very close to fused, which is clearly later than diploid LSSmKate 2. Fusion is a fluorescent protein with good monomer property at present, and the peak time is very close to that of the fusion, which indicates that LSSmKO1 is also a fluorescent protein with good monomer property. Since LSSmKO1 is a monomeric fluorescent protein, it does not cause mutual aggregation of fluorescent protein molecules under in vitro conditions.
Therefore, the invention provides a large stokes orange fluorescent protein LSSmKO1, a gene for coding the large stokes orange fluorescent protein LSSmKO1 and application of the large stokes orange fluorescent protein LSSmKO 1.
The large Stokes fluorescent protein LSSmKO1 provided by the invention has the following advantages:
(3) has excellent monomer property;
(4) compared with other orange fluorescent proteins with large Stokes shift, the fluorescent protein has the advantages of high brightness and short maturation time;
(3) the large Stokes displacement is realized, and the method can be used for simultaneous imaging of single photon, multi-photon and multi-color ultramolecular microscopy, so that the green fluorescent protein mTFP2 and single-color light can be simultaneously excited in a two-photon mode, the resolution of two-photon imaging is improved, and the method is applied to photo-plate type nonlinear optical microscopic imaging;
(4) the BRET can be applied to a new bioluminescence resonance energy transfer system BRET, is used as a donor to be combined with receptor luciferase NanoLuc, can form a new bioluminescence resonance energy transfer system BRET with high sensitivity in an organism, and improves the depth and sensitivity of in-vivo imaging.
<110> Shenzhen advanced technology research institute
<120> Dastokes orange fluorescent protein LSSmKO1 and application thereof
<160> 2
<210> 1
<211> 228
<212> PRT
<213> Artificial sequence
<400> 1
Met Val Ser Val Ile Lys Pro Glu Met Lys Thr Arg Tyr Tyr Met Asp
1 5 10 15
Gly Ser Val Asn Gly His Glu Phe Thr Ile Glu Gly Glu Gly Thr Gly
20 25 30
Arg Pro Tyr Glu Gly His Gln Ala Met Lys Leu Lys Val Thr Met Ala
35 40 45
Glu Gly Gly Pro Met Pro Phe Ser Phe Asp Ile Val Ser His Ser Phe
50 55 60
Cys Tyr Gly Asn Arg Ala Phe Thr Lys Tyr Pro Ala Glu Ile Pro Asp
65 70 75 80
Tyr Phe Lys Gln Ala Phe Pro Glu Gly Leu Ser Trp Glu Arg Ser Met
85 90 95
Glu Phe Glu Asp Gly Gly Ser Ala Lys Val Ser Ala His Ile Ser Leu
100 105 110
Arg Gly Asn Thr Phe Ile His Lys Ser Lys Phe Thr Gly Val Asn Phe
115 120 125
Pro Ala Asp Gly Pro Val Met Gln Lys Gln Ser Val Asp Trp Glu Pro
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Ser Thr Glu Lys Ile Thr Ala Ser Asp Gly Val Leu Lys Gly Asp Asp
145 150 155 160
Thr Met Tyr Leu Lys Leu Val Gly Gly Gly Asn His Lys Val Gln Ile
165 170 175
Lys Thr Thr Tyr Lys Ala Ala Lys Glu Ile Leu Glu Met Pro Gly Asp
180 185 190
His Tyr Ile Ser His Arg Leu Glu Arg Ile Thr Glu Gly Asn Ile Thr
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Glu Gln Tyr Glu Asp Ala Val Ala Ser Tyr Ser Asn Leu Gly Met Asp
210 215 220
Glu Leu Tyr Lys
225
<210> 2
<211> 684
<212> DNA
<213> Artificial sequence
<400> 2
ATGGTGAGTG TCATTAAACC AGAGATGAAG ACCAGGTACT ACATGGACGG CTCCGTCAAT 60
GGGCATGAGT TCACAATTGA AGGTGAAGGC ACAGGCAGAC CTTACGAGGG ACACCAAGCA 120
ATGAAACTAA AAGTCACAAT GGCCGAGGGC GGGCCAATGC CTTTCTCCTT CGACATCGTG 180
AGCCACTCCT TCTGTTACGG CAACAGAGCC TTTACTAAAT ATCCAGCAGA GATACCAGAC 240
TATTTCAAAC AAGCCTTTCC TGAAGGCCTC TCATGGGAAA GGTCGATGGA GTTCGAAGAT 300
GGTGGGTCCG CTAAAGTCAG TGCGCATATC AGCCTTAGAG GAAACACCTT CATCCACAAA 360
TCCAAATTTA CTGGGGTTAA CTTTCCTGCC GATGGTCCTG TCATGCAAAA GCAGAGCGTG 420
GACTGGGAGC CATCAACCGA GAAAATTACT GCCAGCGACG GAGTTCTGAA GGGTGATGAC 480
ACGATGTACC TAAAGCTTGT GGGAGGCGGC AATCACAAAG TCCAAATTAA GACTACTTAC 540
AAGGCGGCAA AAGAGATTCT TGAAATGCCA GGAGACCATT ACATCAGCCA TCGCCTCGAG 600
AGGATCACCG AAGGCAACAT TACTGAGCAG TACGAAGACG CAGTAGCTAG CTACAGCAAC 660
Claims (7)
1. A large Stokes orange fluorescent protein, characterized in that: the large Stokes orange fluorescent protein is a mutant orange fluorescent protein mKO kappa, and the amino acid sequence of the large Stokes orange fluorescent protein is shown as SEQ ID NO: 1 is shown.
2. The large stokes orange fluorescent protein of claim 1, wherein the DNA base sequence of the gene of the large stokes orange fluorescent protein is shown in SEQ ID NO: 2, respectively.
3. Use of the large stokes orange fluorescent protein of claim 1 in single photon, multiphoton, and super-resolution microscopy imaging.
4. Use of the large stokes orange fluorescent protein according to claim 1 in a bioluminescence resonance energy transfer system.
5. The use according to claim 4, wherein in the bioluminescent resonance energy transfer system, the acceptor is luciferase NanoLuc and the donor is the large Stokes orange fluorescent protein.
6. Use of the large stokes orange fluorescent protein according to claim 1 in super resolution imaging based on random single molecule localization.
7. Use according to claim 6, wherein the sample is irradiated with excitation light after labeling structures and organelles in the cell with the large Stokes orange fluorescent protein.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105461787A (en) * | 2015-12-29 | 2016-04-06 | 深圳先进技术研究院 | Large stokes displacement fluorescent protein CyOFP and application thereof |
| CN108503701A (en) * | 2017-02-24 | 2018-09-07 | 中国科学院深圳先进技术研究院 | A kind of fluorescin, fusion protein, the nucleic acid of separation, carrier and application |
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| US20140058065A1 (en) * | 2012-08-21 | 2014-02-27 | The Board Of Trustees Of The Leland Stanford Junior University | Far-red fluorescent proteins with improved detectability by red excitation light |
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| CN106290988A (en) * | 2015-05-25 | 2017-01-04 | 中国科学院上海应用物理研究所 | saturated resonance energy transfer super-resolution probe and its preparation method and application |
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| CN106831971B (en) * | 2017-02-24 | 2020-10-27 | 中国科学院深圳先进技术研究院 | Far-red fluorescent protein, fusion protein, isolated nucleic acid, vector and application |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105461787A (en) * | 2015-12-29 | 2016-04-06 | 深圳先进技术研究院 | Large stokes displacement fluorescent protein CyOFP and application thereof |
| CN108503701A (en) * | 2017-02-24 | 2018-09-07 | 中国科学院深圳先进技术研究院 | A kind of fluorescin, fusion protein, the nucleic acid of separation, carrier and application |
Non-Patent Citations (2)
| Title |
|---|
| A bright cyan-excitable orange fluorescent protein facilitates dual-emission microscopy and enhances bioluminescence imaging in vivo;Jun Chu 等;《Nature Biotechnol》;20161130;第34卷(第7期);第760-767页 * |
| 红色荧光蛋白的研究进展;王飞 等;《生物技术通报》;20170620;第33卷(第9期);第32-47页 * |
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| WO2020125545A1 (en) | 2020-06-25 |
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