CN105461787A - Large stokes displacement fluorescent protein CyOFP and application thereof - Google Patents
Large stokes displacement fluorescent protein CyOFP and application thereof Download PDFInfo
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0045—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent agent being a peptide or protein used for imaging or diagnosis in vivo
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/61—Fusion polypeptide containing an enzyme fusion for detection (lacZ, luciferase)
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Abstract
The invention provides large stokes displacement fluorescent protein CyOFP and application of the fluorescent protein to microimaging and highly-sensitive living creature light-emitting imaging. The fluorescent protein is obtained through mNeptune site directed mutagenesis, the 1-230 positions of the amino acid sequence of the fluorescent protein sequentially correspond to 4-234 positions shown by the SEQ ID No: 2, or the fluorescent protein can be further obtained by conducting gene synthesizing on the fluorescent protein DNA segment and expression. The invention further provides a novel BRET system and fusion protein Antares obtained through system optimization. The CyOFP can be stimulated by blue light, and a quite high quantum yield is achived; the CyOFP and the enhanced green fluorescent protein (EGFP) are jointly stimulated by monochromatic light at the same time in a two-photon mode, and the resolution ratio of two-photon imaging is raised; a novel BRET system formed together with fluorescent protease NanoLuc greatly improves the depth and sensitivity of living creature imaging.
Description
Technical field
The invention belongs to biomedical optical and molecular imaging technical field, be specifically related to a kind of large Stokes shift fluorescin CyOFP and application thereof.
Background technology
In biomedical optical and molecular imaging field, fluorescin has important application in the intragentic expression level of research active somatic cell, protein-interacting, protein conformation and protein-active.But, when using fluorescin to carry out living imaging, being often difficult to realize hyperchannel imaging simultaneously, if this restriction can be broken through, then can realizing, to the detection of multiple biological rapid reaction event, greatly promoting the development of neuroscience.In single photon image, can adopt by the multiple fluorescin of the optical excitation of different excitation wavelength to realize hyperchannel imaging simultaneously.But, in multi-photon imaging, add a secondary titanium sapphire laser or optical parametric oscillator costly, and be difficult to by they regulation and control to together, therefore, the more difficult realization of multiple fluorescin imaging simultaneously.The fluorescin developing large Stokes shift can effectively address this problem, and the light realizing same excitation wavelength excites two kinds of fluorescins simultaneously, then launches the light of different wavelength regions, reaches the object of two kinds of fluorescin multi-photons imaging simultaneously.The method has the advantages such as simple to operate, not high to equipment requirements, imaging effect is good.
The while of current, the microscopy of the multiple biomolecular events of imaging comprises, detect the super-resolution Structured Illumination of thin sample, the adaptive optics of resolving power is promoted in scattering tissue, improve the random scanning two-photon technology of image taking speed, and the mating plate formula that can improve again image taking speed while of can reducing phototoxicity and photobleaching is thrown light on, it can be realized by bessel beam or the mode such as Optical Lattices, incoherent focusing.These microscopies all depend on the dynamic modulation to laser, therefore, if (dual wavelength, the multi-wavelength) fluorescence imaging wanting to do polychrome just requires the optics that many covers repeat, and more accurate synchronization control, which results in some technical barriers.If develop a kind of fluorescin of large Stokes shift, and can and conventional green fluorescent protein to combine use, by making two kinds of different biological respinse incident visualizations, greatly reduce the high request to optics.
Noclilucence does not need optical excitation, the low feature of background, one of important means becoming chemiluminescence assay due to it.But brighter luciferase (producing photon by the biochemical reaction with substrate) not only quantum yield is low, and launch the wavelength shorter (excitation peak <600nm) of photon, therefore limit its application in animal body.Bioluminescence Resonance Energy transfer (BRET) based on luciferase and fluorescin then well solves the problems referred to above.But, at present based on the quantum yield of the red fluorescent protein in the bioluminescent probe of BRET relatively low (<0.4), and the emmission spectrum of luciferase and the absorption spectrum of fluorescin overlapping less, therefore BRET efficiency is lower.Therefore, need can spectrally there be high overlapping fluorescin with the brightest existing luciferase (Nluc, emission peak is at 460nm), and its emission peak is at 600nm place.
Stokes shift refers to the maximum emission wavelength of fluorescent substance and the difference of maximum excitation wavelength.If this difference is greater than 100nm, then think that this fluorescin has the characteristic of large Stokes shift.The fluorescin of large Stokes shift when the imaging of polychrome two-photon, can ensure two different spectrum fluorescin can by while fluorescence imaging, and effectively reduce the crosstalk of fluorescence spectrum.In addition, its (acceptor) can carry out radiationless energy trasfer in vivo together with luciferase (donor), namely Bioluminescence Resonance Energy transfer (BRET).Because BRET does not need external source optical excitation and background is low, therefore biodiversity resources sensitivity is very high, to detect and the bioanalysis association area such as high flux screening is widely used at protein-interacting, live body image, foranalysis of nucleic acids, proteolytic enzyme.
The fluorescin of the large Stokes shift reported the earliest is mKeima, it is a kind of red fluorescent protein, it is launched and absorption peak lays respectively at 440 and 620nm, adopt optical excitation CFP and mKeima of 458nm, the object simultaneously exciting two fluorescence molecules can be reached, thus realize the polychrome imaging (KogureT of single light source, KarasawaS, ArakiT, SaitoK, KinjoM, MiyawakiA.Afluorescentvariantofaproteinfromthestonycoral Montiporafacilitatesdual-colorsingle-laserfluorescencecr oss-correlationspectroscopy.NatBiotechnol, 2006, 24:577-581).But the shortcoming of mKeima is, it has two excitation peaks, except being excited at 440nm place, can also be excited at 584nm place, and this causes some difficulties to polychrome imaging.In order to overcome this shortcoming of mKeima, PiatkevichK.D. people is waited to obtain fluorescin mutant LSS-mKate1 and LSS-mKate2 in 2010, these two mutant have same excitation wavelength, but emission wavelength is different, therefore, them can be utilized preferably to carry out the biochemical reactions (PiatkevichKD such as the transfer of two-photon somatoscopy tumour cell, HulitJ, SubachOM, WuB, AbdullaA, SegallJE, VerkhushaVV.MonomericredfluorescentproteinswithalargeSto kesshift.ProcNatlAcadSciUSA, 2010, 107:5369-5374).The people such as Cui Boyu constructed a kind of BRET system based on bacterial luciferase LuxAB in 2014, using LuxAB as donor, enhancement type yellow fluorescence protein eYFP is as acceptor, this system effectively can accept the blue light that bacterial luciferase LuxAB launches, and inspire yellow fluorescence, produce obvious energy transfer signal, further confirm to find this system can also dynamic protein interaction in bacterial detection born of the same parents (Cui Boyu. based on BRET protein-interacting detection technique research [D] of bacterial luciferase. Xibei Univ. of Agricultural & Forest Science & Technology, 2014).
But in living organisms, the BRET system effect of two-photon synchronous imaging is unsatisfactory.Classical BRET technology uses acceptor mostly to be modified enhanced green fluorescent protein, and system utilizing emitted light is shorter, and the living imaging degree of depth is more shallow; Existing large Stokes shift fluorescin can not be excited efficiently together with green fluorescent protein, and launch the photon being different from EGFP, therefore two-photon image sensitivity is relatively low.
Summary of the invention
In order to solve the technical problem of above-mentioned existence, the object of the present invention is to provide a kind of large Stokes shift fluorescin CyOFP, this fluorescin is screened by far infrared fluorescin mNeptune site-directed mutagenesis and obtains.
Another object of the present invention is also to provide the application of above-mentioned large Stokes shift fluorescin CyOFP in micro-imaging and highly sensitive living body biological luminescence imaging.
Object of the present invention is achieved by the following technical programs:
A kind of large Stokes shift fluorescin, this fluorescin is a kind of far infrared fluorescin mNeptune of sudden change, compared with the aminoacid sequence of far infrared fluorescin mNeptune, large Stokes shift fluorescin CyOFP has following mutational site: M160K.This mutational site embodies the large Stokes shift characteristic of this large Stokes shift fluorescin CyOFP, the Met being specially the 160th of mNeptune aminoacid sequence replace by Lys; Wherein the 1-240 position of the aminoacid sequence of mNeptune corresponds to the 4-244 position as shown in SEQIDNo:1.
The aminoacid sequence of above-mentioned large Stokes shift fluorescin, compared with the aminoacid sequence of far infrared fluorescin mNeptune, has following mutational site: M11S, M15L, S28H, G41N, C61H, T94M, Y96F, C172V, L174F.These mutational sites embody the light characteristic of this large Stokes shift fluorescin CyOFP, the Met being specially the 11st of the aminoacid sequence of mNeptune replace by Ser, the Met of the 15th replace by Leu, the Ser of the 28th replace by His, the Gly of the 41st replace by Asn, the Cys of the 61st replace by His, the Thr of the 94th replace by Met, the Tyr of the 96th replace by Phe, the Cys of the 172nd replace by Val, the Leu of the 174th replace by Phe.
The aminoacid sequence of above-mentioned large Stokes shift fluorescin, compared with the aminoacid sequence of far infrared fluorescin mNeptune, has following mutational site: H10R, T18S, N21G, H23Q, T38K, T68V, N71K, H72Y, T73P, Q74A, G75D, I76L, F79Y, C114E, I121V, S128A, A142P, R179K, L187V, Y193H, H214Y, V216H.These mutational sites embody the maturing rate characteristic of this large Stokes shift fluorescin CyOFP, the His being specially the 10th of the aminoacid sequence of mNeptune replace by Arg, the Thr of the 18th replace by Ser, the Asn of the 21st replace by Gly, the His of the 23rd replace by Gln, the Thr of the 38th replace by Lys, the Thr of the 68th replace by Val, the Asn of the 71st replace by Lys, the His of the 72nd replace by Tyr, the Thr of the 73rd replace by Pro, the Gln of the 74th replace by Ala, the Gly of the 75th replace by Asp, the Ile of the 76th replace by Leu, the Phe of the 79th replace by Tyr, the Cys of the 114th replace by Glu, the Ile of the 121st replace by Val, the Ser of the 128th replace by Ala, the Ala of the 142nd replace by Pro, the Arg of the 179th replace by Lys, the Leu of the 187th replace by Val, the Tyr of the 193rd replace by His, the His of the 214th replace by Tyr, the Val of the 216th replace by His.
The aminoacid sequence of above-mentioned large Stokes shift fluorescin, compared with the aminoacid sequence of far infrared fluorescin mNeptune, has following mutational site: T95V, I171H, F194Y, C222S, D223N, P225G, S226G, K227G, Δ 228-234.These mutational sites embody the monosomy of this large Stokes shift fluorescin CyOFP, the Thr being specially the 95th of the aminoacid sequence of mNeptune replace by Val, the Ile of the 171st replace by His, the Phe of the 194th replace by Tyr, the Cys of the 222nd replace by Ser, the Asp of the 223rd replace by Asn, the Pro of the 225th replace by Gly, the Ser of the 226th replace by Gly, the Lys of the 227th replace by Gly, the aminoacid deletion of 228-234 position.
The aminoacid sequence of above-mentioned large Stokes shift fluorescin, compared with the aminoacid sequence of far infrared fluorescin mNeptune, has following mutational site: L147M, Δ 184-186.These mutational sites embody the light stability of this large Stokes shift fluorescin CyOFP.The Leu being specially the 147th of the aminoacid sequence of mNeptune replace by Met, the aminoacid deletion of 184-186 position.
The aminoacid sequence 1-230 position of above-mentioned large Stokes shift fluorescin CyOFP is corresponding in turn in the 4-234 position such as shown in SEQIDNo:2.
Above-mentioned large Stokes shift fluorescin procurement process:
Carry out site-directed mutagenesis to far infrared fluorescin mNeptune, wherein the 1-240 position of the aminoacid sequence of mNeptune is corresponding in turn in the 4-244 position such as shown in SEQIDNo:1; Then on constitutive expression carrier pNCS, mutant expressed and screen, expression strain used is XL-10Gold (buying from Agilent Technologies), for guaranteeing the integrity in library, each mutant arranges 10 clones, finally by discernable by eye and the blue-ray LED exciting light photoluminescent property through orange acrylic acid filter detection mutant, thus filter out the mono-clonal of the fluorescin of expressing large Stokes shift, name this fluorescin to be CyOFP, be above-mentioned large Stokes shift fluorescin CyOFP.
Shown by sequencing result: the aminoacid sequence of this fluorescin CyOFP is 41 amino acid sites and lacked two place's amino acid sequence segments, as shown in Fig. 1 and following table 1 of having suddenlyd change on the basis of far infrared fluorescin mNeptune aminoacid sequence:
Table 1
(Met that note: M160K represents the 160th of mNeptune aminoacid sequence replace by Lys; Δ 184-186 represents the disappearance of the amino acid sequence segments of the 184-186 position of the aminoacid sequence of mNeptune.)
Present invention also offers the gene of the above-mentioned large Stokes shift fluorescin CyOFP that encodes, preferably, the DNA base sequence of described gene is as shown in SEQIDNo:3, the amino acid encoded in this base sequence 13-702 position after transcription and translation corresponds to the 1-230 position of the aminoacid sequence of CyOFP, also correspond to the 4-234 position as shown in SEQIDNo:2, this base sequence can be obtained by the mode of gene chemical synthesis.
Present invention also offers the application of above-mentioned large Stokes shift fluorescin CyOFP in single photon and the micro-polychrome imaging of multi-photon.This large Stokes shift fluorescin CyOFP effectively can be excited by monochromatic ray simultaneously carry out polychrome imaging together with enhanced green fluorescence protein EGFP in single photon and multi-photon micro-imaging, is applied to mating plate formula nonlinear optics micro-imaging.
Present invention also offers a kind of new Bioluminescence Resonance Energy transfer system BRET, in this system, acceptor is luciferase NanoLuc, and donor is above-mentioned fluorescin CyOFP.The emmission spectrum of luciferase NanoLuc is substantially overlapping with the absorption spectrum of CyOFP, therefore, if the distance between these two kinds of albumen is enough near, the principle being shifted (BRET) by Bioluminescence Resonance Energy is transferred to CyOFP by the energy that the oxidizing reaction intermediate of NanoLuc produces, thus excite the latter, cause this systems radiate to go out the emmission spectrum of CyOFP.
The advantage of this new BRET system is, its emmission spectrum is the emmission spectrum of CyOFP, and its quantum yield is very high, can reach 0.76, and, wherein there is the emmission spectrum of half to be positioned at more than 600nm, improve the degree of depth of living body fluorescent imaging.
Present invention also offers a kind of fusion rotein Antares, adopt above-mentioned new Bioluminescence Resonance Energy transfer system BRET to react and merge link and formed.
The construction step of above-mentioned fusion rotein Antares is as follows:
As shown in Figure 2, an above-mentioned fluorescin CyOFP is adopted first to deliver a child into CyOFP (-4 with nitrogen end with luciferase NanoLuc fusion chain at carbon teminal respectively, 224)-NanoLuc (3,171) and NanoLuc-RH-CyOFP (3,230), then combined, finally, a fluorescin CyOFP merges to link with two luciferase NanoLuc and forms CyOFP (-4,224)-NanoLuc (3,171)-RH-CyOFP (3,230), is this fusion rotein Antares.Wherein, the numeral in bracket refers to and is included in first amino acid and last amino acid in NanoLuc and CyOFP fusion rotein fragment, and CyOFP coding is with consistent shown in Fig. 1, and link aminoacid sequence then uses single amino acids coded representation.
Beneficial effect of the present invention:
Large Stokes shift fluorescin CyOFP provided by the invention has following beneficial effect:
(1) this fluorescin has large Stokes shift, can be excited by cyan light, and has very high quantum yield (QY=0.76);
(2) this fluorescin and other oroteins have good amalgamation, can not interfere with studied albumen in intracellular location;
(3) this fluorescin has large Stokes shift, therefore can together with enhanced green fluorescence protein EGFP simultaneously by monochromatic ray with the mode excitation of two-photon, improve the resolving power of two-photon imaging;
(4) this fluorescin can be combined with acceptor fluorescent protein enzyme NanoLuc as donor, form a kind of high-sensitive Bioluminescence Resonance Energy transfer system BRET newly in vivo, the photon that this system produces is 18 times of luciferase in light of firefly polypide, substantially increase the degree of depth and the sensitivity of living imaging, the fusion rotein Antares that this system optimizes formation further has stronger fluorescent signal and sensitivity, substantially increases new BRET system to the penetration power of biological tissue.
Accompanying drawing explanation
Fig. 1 is the Amino acid sequences alignment figure of CyOFP and mNeptune;
Fig. 2 is fusion rotein Antares building process figure;
Fig. 3 is fluorescin " mNeptune+M160K " and mNeptune excitation and emission spectra comparison diagram in embodiment 2;
Fig. 4 is the fluorescin comparison diagram that fluorescin in embodiment 2 " mNeptune+M160K+ maturation and jump in brightness site " and " mNeptune+M160K " express in bacterium;
Fig. 5 is the native-PAGE protein electrophoresis comparison diagram of fluorescin in embodiment 2 " mNeptune+ monomer mutational site " and mNeptune;
Fig. 6 is fluorescin in embodiment 2 " mNeptune+ light stability mutational site " and mNeptune fluorescence intensity comparison diagram;
Fig. 7 is excitation spectrum and the utilizing emitted light spectrogram of CyOFP in embodiment 3;
Fig. 8 is the fluorogram (I: Actin muscle of the different subcellular organelle of HeLaCCL2 of expressing CyOFP in embodiment 4, II: Intercellular Bridge, III: endoplasmic reticulum, IV: endosome, V: cytoplasmic membrane, VI: actin cytoskeleton, VII: gorky, VIII: plastosome, IX: tubulin);
The fluorescence imaging figure (I: interval, II: in earlier stage, III: mid-term, IV: later stage, scale: 10 μm) of histone mitotic division different steps in HeLaS3 cell that Fig. 9 merges for CyOFP in embodiment 4;
Figure 10 is that (in Figure 10, a is the 3D model configuration sectional view of CyOFP and LSSmKate1 to CyOFP Structural Identification figure in embodiment 5, and main chain cartoon form represents, the amino acid group of chromophore and formation hydrogen bond represents with rod shape form; In Figure 10, b forms Excited state proton trans fer ESPT model in CyOFP; In Figure 10, c is the mode comparison diagram that CyOFP, DsRed and LSSmKate1 proximity thereto hydrogen bond is formed);
Figure 11 is the Two-photon excitation spectra figure of CyOFP, GCaMP6s, EGFP and fluorescein in embodiment 6;
Figure 12 is that in embodiment 6, tractin-CyOFP and cytosol EGFP carries out two-photon bessel beam mating plate formula micro-imaging figure in MV3 melanoma cell;
The monochromatic light sectional elevation that Figure 13 is formed by two-photon excitation imaging at 2/3 cone neurone place of mouse brain VI cortex for CyOFP and GCaMP6s in embodiment 7;
Figure 14 is the response value figure (grey collection of illustrative plates be single neurone, black collection of illustrative plates be average 5 neurones, above-listed arrow represent 8 directions that grating move) of GCaMP6s three co expression in the mouse of CyOFP in embodiment 7;
Figure 15 is luciferase NanoLuc emmission spectrum and CyOFP Absorption and emission spectra comparison diagram in embodiment 8;
Figure 16 is fusion rotein Antares and intermediate normalization method fluorescence spectrum figure in embodiment 8;
Figure 17 is Antares and other bioluminescent probe biodiversity resources comparison diagram in intravital mouse and mouse model in embodiment 9.
Embodiment
In order to there be understanding clearly to technical characteristic of the present invention, object and beneficial effect, existing following detailed description is carried out to technical scheme of the present invention, but can not be interpreted as to of the present invention can the restriction of practical range.In embodiment, the commercially available acquisition of each Starting reagents material, the experimental technique of unreceipted actual conditions is ordinary method and the normal condition of affiliated known, or according to the condition that apparatus manufacturer is advised.
Embodiment 1
Present embodiments provide a kind of large Stokes shift fluorescin CyOFP, this fluorescin on the basis of far infrared fluorescin mNeptune, carries out site-directed mutagenesis screening obtain, and its procurement process is as follows:
Carry out site-directed mutagenesis to far infrared fluorescin mNeptune, wherein the 1-240 position of the aminoacid sequence of mNeptune is corresponding in turn in the 4-244 position such as shown in SEQIDNo:1, then on constitutive expression carrier pNCS, mutant expressed and screen, expression strain used is XL-10Gold (buying from Agilent Technologies), for guaranteeing the integrity in library, each mutant arranges 10 clones, finally by discernable by eye and the blue-ray LED exciting light photoluminescent property through orange acrylic acid filter detection mutant, filter out the mono-clonal that a kind of expression has the fluorescin of large Stokes shift, the fluorescin with large Stokes shift in the present invention described in name is CyOFP, be above-mentioned large Stokes shift fluorescin CyOFP.
Shown by sequencing result: the aminoacid sequence of fluorescin CyOFP of the present invention is 41 amino acid sites and lacked two place's amino acid sequence segments, as shown in Fig. 1 and following table 2 of having suddenlyd change on the basis of far infrared fluorescin mNeptune aminoacid sequence:
Table 2
(Met that note: M160K represents the 160th of mNeptune aminoacid sequence replace by Lys; Δ 184-186 represents the disappearance of the amino acid sequence segments of the 184-186 position of the aminoacid sequence of mNeptune.)
The functional experiment that embodiment 2 mutational site is corresponding
(1) experiment of Stokes shift is embodied
Screened by the method random mutation mNeptune of fallibility PCR, select color and present orange bacterial colony, by order-checking, filter out the fluorescin only having M160K mutational site, be mNeptune+M160K.
Get 150 micro liter purified albumen to be placed in black 96 orifice plate, utilize the multi-functional microplate reader measurement of the SafireII of Tecan company to excite and emission spectrum.As shown in Figure 3, Fig. 3 is the present embodiment fluorescin " mNeptune+M160K " and mNeptune excitation and emission spectra comparison diagram, and Fig. 3 curve upper right corner picture is the white light figure of bacterial expression mNeptune and mNeptune-M160K.
As shown in Figure 3, M160K can make exciting of mNeptune and launch blue shift to occur simultaneously, but excites blue shift more, thus " mNeptune+M160K " can embody large Stokes shift.
(2) experiment of maturing rate and brightness is embodied
By method random mutation " mNeptune+M160K " the construction expression library of fallibility PCR, flat board is placed on incubated overnight in 30 DEG C of incubators, then within second day, the brightest bacterial colony is picked out, by order-checking, filter out in table 2 the mutant fluorescin with brightness and maturing rate mutational site, maturing rate and brightness screening are mainly screened by expressing the brightness of mutant bacteria, and that is maturing rate and the final performance of brightness are all the fluorescence grows of bacterium colony.Be the fluorescin in " mNeptune+M160K+ maturation and jump in brightness site ".
PNCS carrier (AlleleBiotechnology) is adopted to carry out the double digestion of BamHI and EcoRI, reclaim large fragment, then the large fragment after library and double digestion is connected, be transformed into DH5alpha bacterium, incubated overnight, choose the mono-clonal line of incubated overnight, then in incubated overnight.Then on DNA taps rubber instrument (BlueViewTransilluminator, Vernier company of the U.S.), fluorescence imaging is carried out.Contrast fluorescin is " mNeptune+M160K ", and as shown in Figure 4, Fig. 4 is the fluorescin comparison diagram that fluorescin " mNeptune+M160K+ maturation and jump in brightness site " and " mNeptune+M160K " express in bacterium to result.
As shown in Figure 4, in the fluorogram of bacterial expression fluorescin, " mNeptune+M160K+ maturation and jump in brightness site " is faster than the maturation of " mNeptune+M160K " and mutant is brighter.
(3) experiment of monosomy is embodied
Screened by the method random mutation mNeptune of fallibility PCR, the clone that random choose is bright, then purifying protein run non denatured glue to confirm which mutant is monomer, by order-checking, filter out the mutant fluorescin only in table 2 with monosomy mutational site, be the fluorescin in " mNeptune+ monomer mutational site ".
Get the purifying protein point sample of 20 microlitre 5uM in the Bis-Tris non denatured glue (Invitrogen company) of 4-16%, 120V voltage 90min.Conventional coomassie brilliant blue staining and decolouring is carried out after electrophoresis.Glue after decolouring carries out the imaging of white light figure by digital camera, and contrast fluorescin is mNeptune, and result as shown in Figure 5.Fig. 5 is the native-PAGE protein electrophoresis comparison diagram of fluorescin in the present embodiment " mNeptune+ monomer mutational site " and mNeptune.
As shown in Figure 5, " mNeptune+ monomer mutational site " strengthens than mNeptune monosomy.
(4) experiment of light stability is embodied
Screened by the method random mutation mNeptune of fallibility PCR, by random mutation technique construction expression library, all flat boards are placed in the homemade LED display sending out light orange, Continuous irradiation 30 minutes, then pick out the brightest clone, namely light stability is best.By order-checking, filter out the mutant fluorescin only in table 2 with light stability mutational site, be the fluorescin in " mNeptune+ light stability mutational site ".
Get 20 micro liter purified albumen and drop in bottom with in the 35mm capsule (MatTek company) of Cover Glass, protein liquid mineral oil (Sigma company) is closed.Under ware is positioned over inverted fluorescence microscope, light source intensity is set to 100%, and Continuous irradiation albumen drop, record fluorescence intensity, contrast fluorescin is mNeptune, and result is as shown in Figure 6 simultaneously.Fig. 6 is fluorescin in the present embodiment " mNeptune+ light stability mutational site " and mNeptune fluorescence intensity comparison diagram.
As shown in Figure 6, " mNeptune+ light stability mutational site " is more stable than mNeptune fluorescence.
Embodiment 3CyOFP excitation spectrum and emmission spectrum test experiments
B-PERII (buying from Pierre Si company) is adopted to carry out cracking to the bacterial strain of expressing CyOFP in above-described embodiment 1, then adopt HisPurCobaltResin (buying from Pierre Si company) purifying protein, desalt followed by Econo-Pac10DG run by gravity chromatographic column (buying from Bio-Rad company of the U.S.).After completing above protein purification steps, Lambda35UV/VISandLS-55 fluorescence spectrophotometer (buying from PerkinElmer company) is used to detect excitation spectrum and the emmission spectrum of CyOFP, as shown in Figure 7; Compare CyOFP and the contrast of some red fluorescent proteins on quantum yield at present in addition, as shown in table 3.
Table 3
Experimental result shows: the excitation spectrum scope of CyOFP is comparatively wide, between 488-526nm, still can keep more than 95% of the highest launching efficiency.The emission peak of CyOFP is positioned at 589nm place, and average emitted peak is positioned at 595nm place.Be different from the existing fluorescent red-orange albumen being applied to two-photon micro-imaging, CyOFP has the Stokes shift of about 90nm, and therefore, it can be excited by the more cyan light being positioned at about 440nm; From table 3 experimental data, the quantum yield of CyOFP is 0.76, and the fluorescin tdTomato quantum yield of the sub-productive rate of maximum amount of current all red fluorescent proteins is also only 0.7, therefore this large Stokes shift fluorescin CyOFP has very high quantum yield.
Embodiment 4CyOFP tests at intracellular targeting
In the present invention, aminoacid sequence according to above-mentioned large Stokes shift fluorescin CyOFP devises CyOFP gene, by CyOFP gene, (conventionally method is synthesized voluntarily, and correct through checking order) clone structure pCyOFP-N1 and pCyOFP-C1 on expression vector pEGFP-C1 and pEGFP-N1, (this base sequence 13-702 position amino acid of encoding after transcription and translation corresponds to the 1-230 position of the aminoacid sequence of CyOFP as shown in SEQIDNo:3 for the DNA base sequence of this CyOFP gene, also the 4-234 position as shown in SEQIDNo:2 is corresponded to), by the transfection of Lipofectamine2000 test kit, above-mentioned plasmid is proceeded to HEK293T, HeLaCCL2, HeLaS3 cell (buying from GEHealthcareDharmacon company), Eclipse80i fluorescent microscope (Nikon) is utilized to observe the location of CyOFP SW in cell.Result as shown in Figure 8 and Figure 9.
Fig. 8 is the fluorogram of the different subcellular organelle of HeLaCCL2 of expressing CyOFP, and as shown in Figure 8, CyOFP still has good amalgamation with other albumen at different subcellular locations; The fluorescence imaging figure of histone mitotic division different steps in HeLaS3 cell that Fig. 9 merges for CyOFP, as shown in Figure 9, merges and does not have cell cycle to produce interference at the histone of CyOFP.Illustrate thus, CyOFP does not disturb studied albumen in intracellular location.
There is the research of large Stokes shift molecular mechanism in embodiment 5CyOFP
There is the molecular mechanism of large Stokes shift in order to better understand CyOFP, its albumin crystal structure is resolved.First, by the CyOFP containing six Histidines by exclusion chromatography depolymerization, then, be concentrated into 13mg/mL and carry out crystallization by sitting the method for dripping vapor diffusion at 12 DEG C.The diffraction data of crystallization is obtained by AdvancedlightsourceonSIBYLSBeamline12.3.1.Crystalline structure then utilizes Phaser software to carry out molecule replacement method to obtain.Experimental result as shown in Figure 10.
By the protein structure (terminal of barrel-like structure points to below) of the CyOFP in Figure 10 shown in a, Lys
160be positioned at the below of chromophore, its amino (hydrogen bond receptor) and the phenolic hydroxyl group (hydrogen bond donor) of chromophore define a hydrogen bond, when 500nm excites, this hydrogen bond can occur ' Excited state proton trans fer ' phenomenon (ESPT) (as shown in b in Figure 10), and this ESPT is then the reason that CyOFP has large Stokes shift.With on the amino acid range of its formation hydrogen bond near the phenolic hydroxyl group of chromophore, CyOFP and DsRed crystalline structure has surprising similar, and the amino acid particularly coming 143 and 160 is Serine and Methionin (as shown in c in Figure 10) respectively.In addition, the existence of this hydrogen bond stabilizes luminophore, decreases its degree of freedom, therefore improves its quantum yield.The Interaction of substituents of CyOFP proximity thereto and LSSmKate1 are similar, the latter also includes Excited state proton trans fer acceptor, namely the glutamate side chain of 160 is positioned at, but their quantum yield still there are differences, this is perhaps because CyOFP also has the main difference in three places (in Figure 10 shown in c) compared to LSSmKate1.
The application of embodiment 6CyOFP on single photon and two-photon imaging
CyOFP-tractin and EGFP is entered cell MV3 by lentiviral vectors pLVX transfection, first one-photon excitation dual colour imaging is tested, then start to detect its two-photon imaging.The optical excitation of two-photon is the IX81 commercial microscopes (buying from Olympus Corp) of secondary titanium sapphire laser laser (buying from coherent company) of being coupled based on.
As shown in Figure 11, CyOFP can by the two-photon excitation of 900 to 1060nm.The light of 920-940nm wavelength is usually used to excite EGFP fluorescent probe, but under the exciting of this wavelength region, and the brightness of CyOFP is even than EGFP or to combine the conductor Calcium ion sensor GCaMP6s of EGFP taller.Therefore, CyOFP and EGFP fluorescent probe can simultaneously by monochromatic ray with the mode excitation of two-photon, then by light filter disc separately both light of launching.Find when using 3D mating plate formula imaging technique to observe, compare with one-photon excitation, two-photon imaging improves resolving power and can observe dynamic Actin muscle imaging (Figure 12).
Embodiment 7CyOFP and GCaMP6s is imaging contrast's experiment in mouse brain
Use AAV1 vector expression CyOFP and GCaMP6s gene, be then injected into the optic cortex of mouse, after 3 to 4 week of virus injection, carry out the implant surgery of cranium window and carry out living imaging.Figure 13 is the 2/3 basivertebral nerve unit place of CyOFP and GCaMP6s in mouse brain VI cortex, the monochromatic light sectional elevation formed by two-photon excitation imaging; Figure 14 is the response value of GCaMP6s at three coexpressions in the mouse of CyOFP.Single neurone (grey) and average 5 neurones (black); The most above-listed arrow represents 8 directions that grating moves.
Be can be observed by Figure 13, under the two-photon excitation of 940nm, the fluorescence of CyOFP is filled with whole pericaryon and dendron and does not have obvious ubcellular to be polymerized.Known in conjunction with Figure 14, fluorescent probe GCaMP6s now can the change of synchronous recording calcium ion in neurone.This further demonstrates that, CyOFP and GCaMP6s can under the exciting of monochromatic two-photon, carries out living imaging record to the neuron morphology of mouse brain and calcium transient.
The structure of the BRET system that embodiment 8 one kinds is new and fusion rotein Antares
Adopt LipofectamineLTX test kit transfectional cell HEK293A to make it to express mTurquoise2 and bioluminescent probe simultaneously.As shown in Figure 15, the emmission spectrum of luciferase NanoLuc is substantially overlapping with the absorption spectrum of CyOFP, therefore, if the distance between these two kinds of albumen is enough near, the principle being shifted (BRET) by Bioluminescence Resonance Energy is transferred to CyOFP by the energy that the oxidizing reaction intermediate of NanoLuc produces, thus excite the latter, cause this systems radiate to go out the emmission spectrum of CyOFP.The advantage of this new BRET system is, its emmission spectrum is the emmission spectrum of CyOFP, and its quantum yield is very high, can reach 0.76, and, wherein there is the emmission spectrum of half to be positioned at more than 600nm, improve the degree of depth of living body fluorescent imaging.
Fig. 2 illustrates fusion rotein Antares and builds and the flow process optimizing this BRET system, a fluorescin CyOFP is adopted first to deliver a child into CyOFP (-4 with nitrogen end with luciferase NanoLuc fusion chain at carbon teminal respectively, 224)-NanoLuc (3,171) and NanoLuc-RH-CyOFP (3,230), then combined, finally, a fluorescin CyOFP merges to link with two luciferase NanoLuc and forms CyOFP (-4,224)-NanoLuc (3,171)-RH-CyOFP (3,230), is this fusion rotein Antares.Wherein, the numeral in bracket refers to and is included in first amino acid and last amino acid in NanoLuc and CyOFP fusion rotein fragment, and CyOFP coding is with consistent shown in Fig. 1, and link aminoacid sequence then uses single amino acids coded representation.
Figure 16 is fusion rotein Antares and intermediate fluorescence spectrum figure, and from the result of Figure 16, Antares further increases the efficiency of NanoLuc and CyOFP.
Embodiment 9Antares and other bioluminescent probe biodiversity resources contrast experiment in intravital mouse and mouse model
In order to compare Antares and other bioluminescent probe performance in vivo, have employed " pseudo-mouse " model, it has same optical transport and scattering of light with real mouse.This method can monitor the effect that different luciferase acts on different bioluminescent probe, to avoid disturb by complex environment in active somatic cell.As shown in figure 17, FLuc, Nano-lantern, RLuc8.6-535 and BRET6 refer to different bioluminescent probes to result; Luciferin, sCTZ, ViviRen and FRZ refer to different luciferase substrate.
Result is as shown in a in Figure 17, Antares is sensitiveer than other fluorescent probes, when it is using FRZ as substrate, the brightness of its combination (Antares+FRZ) is 4.8 times of the second brightness luciferase and substrate combination (BRET6+sCTZ).And, as shown in b in Figure 17, when NanoLuc is independent and FRZ combines, weak 84 times of the combination of its signal intensity ratio Antares and FRZ, this further demonstrates and be transferred to will be greatly improved the penetration power of this system to biological tissue by Bioluminescence Resonance Energy transfer system by the utilizing emitted light of NanoLuc.
Then in real intravital mouse, be repeated above experiment, result is as shown in d in c and Figure 17 in Figure 17, and the signal that Antares produces is still the strongest.
In sum, large Stokes shift fluorescin CyOFP provided by the invention has large Stokes shift characteristic, can be excited by cyan light, and has very high quantum yield (QY=0.76); This CyOFP and other oroteins have good amalgamation, can not interfere with studied albumen in intracellular location; This CyOFP can together with enhanced green fluorescence protein EGFP simultaneously by monochromatic ray with the mode excitation of two-photon, improve the resolving power of two-photon imaging; This CyOFP can be combined with acceptor fluorescent protein enzyme NanoLuc as donor, form a kind of high-sensitive Bioluminescence Resonance Energy transfer system BRET newly in vivo, the photon that this system produces is 18 times of luciferase in light of firefly polypide, substantially increase the degree of depth and the sensitivity of living imaging, the fusion rotein Antares that this system optimizes formation further has stronger fluorescent signal and sensitivity, substantially increases new BRET system to the penetration power of biological tissue.
Sequence table
Claims (10)
1. a large Stokes shift fluorescin CyOFP, it is characterized in that: this fluorescin is a kind of far infrared fluorescin mNeptune of sudden change, compared with the aminoacid sequence of far infrared fluorescin mNeptune, large Stokes shift fluorescin CyOFP has following mutational site: M160K.
2. large Stokes shift fluorescin CyOFP according to claim 1, it is characterized in that, the aminoacid sequence of this fluorescin, compared with the aminoacid sequence of far infrared fluorescin mNeptune, has following mutational site: M11S, M15L, S28H, G41N, C61H, T94M, Y96F, C172V, L174F.
3. large Stokes shift fluorescin CyOFP according to claim 1, it is characterized in that, the aminoacid sequence of this fluorescin, compared with the aminoacid sequence of far infrared fluorescin mNeptune, has following mutational site: H10R, T18S, N21G, H23Q, T38K, T68V, N71K, H72Y, T73P, Q74A, G75D, I76L, F79Y, C114E, I121V, S128A, A142P, R179K, L187V, Y193H, H214Y, V216H.
4. large Stokes shift fluorescin CyOFP according to claim 1, it is characterized in that, the aminoacid sequence of this fluorescin, compared with the aminoacid sequence of far infrared fluorescin mNeptune, has following mutational site: T95V, I171H, F194Y, C222S, D223N, P225G, S226G, K227G, Δ 228-234.
5. large Stokes shift fluorescin CyOFP according to claim 1, it is characterized in that, the aminoacid sequence of this fluorescin, compared with the aminoacid sequence of far infrared fluorescin mNeptune, has following mutational site: L147M, Δ 184-186.
6. the large Stokes shift fluorescin CyOFP according to claim 1-5 any one, is characterized in that: the aminoacid sequence 1-230 position of this fluorescin is corresponding in turn in the 4-234 position such as shown in SEQIDNo:2.
7. the gene of the large Stokes shift fluorescin CyOFP described in any one of coding claim 1-6, preferably, the DNA base sequence of described gene is as shown in SEQIDNo:3.
8. the application of the large Stokes shift fluorescin CyOFP described in claim 1-6 any one in single photon and the micro-polychrome imaging of multi-photon.
9. a new Bioluminescence Resonance Energy transfer system BRET, is characterized in that, in this system, acceptor is luciferase NanoLuc, and donor is the fluorescin CyOFP described in claim 1-6 any one.
10. a fusion rotein Antares, is characterized in that, adopts new Bioluminescence Resonance Energy transfer system BRET according to claim 9 to react and merges link and formed.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108841846A (en) * | 2018-06-12 | 2018-11-20 | 中国科学院海洋研究所 | A kind of recombination fluorescence protein and preparation method thereof with big Stokes shift |
| WO2019068152A1 (en) * | 2017-10-04 | 2019-04-11 | Universidade Estadual De Campinas - Unicamp | Vector for expressing fluorescent proteins in haploid cells, expression cassettes and method for obtaining haploids of saccharomyces cerevisiae on a large scale |
| CN109608525A (en) * | 2018-12-17 | 2019-04-12 | 深圳先进技术研究院 | Big Stokes orange fluorescent protein LSSmKO1 and its application |
| CN109932347A (en) * | 2019-02-27 | 2019-06-25 | 天津大学 | A kind of near-infrared fluorescent albumen smURFP and the method for directly detecting biliverdin |
| CN111378022A (en) * | 2018-12-28 | 2020-07-07 | 中国科学院深圳先进技术研究院 | Fluorescent protein and preparation method and application thereof |
| CN117405636A (en) * | 2023-07-25 | 2024-01-16 | 中国科学院生物物理研究所 | Super-resolution light sheet fluorescence microscopic imaging system and method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1438239A (en) * | 2002-02-11 | 2003-08-27 | 厦门大学 | Novel fluorescent protein polynucleotide series coded thereof and use |
| WO2013151511A1 (en) * | 2012-04-05 | 2013-10-10 | Agency For Science, Technology And Research | Modified Dual-Colour Protein |
-
2015
- 2015-12-29 CN CN201511019266.1A patent/CN105461787A/en not_active Withdrawn
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1438239A (en) * | 2002-02-11 | 2003-08-27 | 厦门大学 | Novel fluorescent protein polynucleotide series coded thereof and use |
| WO2013151511A1 (en) * | 2012-04-05 | 2013-10-10 | Agency For Science, Technology And Research | Modified Dual-Colour Protein |
Non-Patent Citations (3)
| Title |
|---|
| MICHAEL Z. LIN1,ET AL: "Autofluorescent proteins with excitation in the optical window for intravital imaging in mammals", 《CHEM BIOL》 * |
| 杨杰等: "荧光蛋白研究进展", 《生物物理学报》 * |
| 杨松等: "可用于双色双光子显微成像的新的荧光蛋白对(英文)", 《生物化学与生物物理进展》 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019068152A1 (en) * | 2017-10-04 | 2019-04-11 | Universidade Estadual De Campinas - Unicamp | Vector for expressing fluorescent proteins in haploid cells, expression cassettes and method for obtaining haploids of saccharomyces cerevisiae on a large scale |
| CN108841846A (en) * | 2018-06-12 | 2018-11-20 | 中国科学院海洋研究所 | A kind of recombination fluorescence protein and preparation method thereof with big Stokes shift |
| CN108841846B (en) * | 2018-06-12 | 2021-08-06 | 中国科学院海洋研究所 | Recombinant fluorescent protein with large Stokes shift and preparation method thereof |
| CN109608525A (en) * | 2018-12-17 | 2019-04-12 | 深圳先进技术研究院 | Big Stokes orange fluorescent protein LSSmKO1 and its application |
| CN109608525B (en) * | 2018-12-17 | 2022-07-05 | 深圳先进技术研究院 | Large stokes orange fluorescent protein LSSmKO1 and application thereof |
| CN111378022A (en) * | 2018-12-28 | 2020-07-07 | 中国科学院深圳先进技术研究院 | Fluorescent protein and preparation method and application thereof |
| CN111378022B (en) * | 2018-12-28 | 2022-04-26 | 中国科学院深圳先进技术研究院 | Fluorescent protein and preparation method and application thereof |
| CN109932347A (en) * | 2019-02-27 | 2019-06-25 | 天津大学 | A kind of near-infrared fluorescent albumen smURFP and the method for directly detecting biliverdin |
| CN117405636A (en) * | 2023-07-25 | 2024-01-16 | 中国科学院生物物理研究所 | Super-resolution light sheet fluorescence microscopic imaging system and method |
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Inventor after: Chu Jun Inventor after: Ring Michael Inventor after: Zhang Chuqiu Inventor after: Guo Yuqi Inventor after: Wang Huina Inventor before: Chu Jun Inventor before: Zhang Chuqiu Inventor before: Wang Huina Inventor before: Guo Yuqi |
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