CN109265523A - Novel fluorescence marker and its fusion protein from BDFP near infrared light fluorescin - Google Patents
Novel fluorescence marker and its fusion protein from BDFP near infrared light fluorescin Download PDFInfo
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Abstract
The invention discloses a kind of far-red light fluorescins, the far-red light fluorescin includes BDFP near infrared light fluorescin, and including the mutation at the 113rd amino acids, and it does not mutate at the 120th amino acids, wherein, the amino acid sequence of the BDFP near infrared light fluorescin is as shown in SED ID NO:2.The present invention further discloses a kind of fusion fluorescins including the far-red light fluorescin.The invention also discloses a kind of nucleic acid for encoding the far-red light fluorescin or merging fluorescin, and the carrier including the nucleic acid.
Description
Technical field
The invention belongs to fluorescent marker fields.In particular it relates to a kind of far-red light fluorescin and its fusion
Fluorescin encodes the far-red light fluorescin or its nucleic acid and carrier for merging fluorescin.
Background technique
Far-red light (FR) or near infrared light (NIR) light absorption and light scattering in animal tissue are lower, there is higher penetrate
Property, it is the maximum SPECTRAL REGION of ability for penetrating the most tissues such as skin.Fluorescin with this kind of luminescent pigment group
It is more suitable for the imaging deep of animal live soma, is living imaging more preferably fluorescent marker.
At present there are mainly two types of such fluorescent markers, molecular size range is in 35kD or so.One kind being originated from green fluorescence
Albumen (GFP), energy self-catalysis forms chromophore, but spectral region has certain limitation, and maximum emission wavelength generally exists
670nm or so, such as marker TagRFP675.Another kind is originated from the receptor protein being present in bacterium, bacterial typing techniques
Albumen (BphP).BphP is mainly using the biliverdin (BV) of linear tetrapyrrol(e) structure as chromophore；Biliverdin BV is extensive simultaneously
It is present in eucaryote body, it means that the fluorescence marked object of BphP can be applied to zooblast and tissue living, and be not necessarily to
Any enzyme or external source confactor.The representative of BphP class marker has iFP series and iRFP series, and fluorescence emission wavelengths range is
670nm~720nm, such as IFP2.0 maximum emission wavelength 714nm.
But the molecular weight of the existing fluorescin for inspiring far-red light or near infrared light is larger, it is easily poly- when In vivo detection
Collection precipitating, and extreme environment is not tolerated, applicable situation is limited.
There are the fluorescent emission of far-red light range, mechanism and bacterial typing techniques for phycobniliprotein (phycobiliprotein)
Albumen (BphP) is similar, is derived mainly from the phycocyanobilin (PCB) in Non-covalent binding.Typical phycobniliprotein is fluorescence marked
Object such as ApcA, smURFP, ApcF2, their maximum emission wavelength are 698nm.
The sequence of core subunit ApcF2 of the Ding W L et al. based on phycobilisome obtains after carrying out genetic modification
Several new fluorescence phycobniliprotein are simultaneously named as BDFP, these BDFP albumen can covalent bond biliverdin BV, performance ratio
ApcF2 stablizes (Ding W L, Miao D, Hou Y N, et al.Small monomeric and highly stable
near-infrared fluorescent markers derived from the thermophilic
Phycobiliprotein, ApcF2 [J] .Biochimica et Biophysica Acta (BBA)-Molecular Cell
Research, 2017,1864 (10): 1877-1886).In addition, the molecular weight of these BDFP albumen is smaller, about 15kD is maximum
Fluorescence emission wavelengths are 710nm or so.
Although the BDFP albumen that Ding W L et al. is obtained compensates for the glimmering of existing far-red light or near infrared light well
The disadvantages mentioned above of photoprotein, but these protein fluorescence launch wavelengths are relatively simple (in 710nm or so), therefore cannot be effective
Ground is applied in combination.Therefore by genetic engineering transformation, obtain that brightness is higher, spectral property is more various and excellent fluorescence egg
It is white, it has very important significance.
Summary of the invention
It is an object of the present invention to comparing by homologous sequence, conservative functional site is selected, and determine targeted mutagenesis
Site.
Another object of the present invention is to provide a kind of phycobniliprotein through genetic modification for launching far-red light and its melt
Close fluorescin.
Another object of the present invention is to provide a kind of to encode the phycobniliprotein of the invention through genetic modification and its fusion
The nucleic acid of fluorescin, and encode the carrier of the nucleic acid.
In one aspect of the invention, a kind of far-red light fluorescin is provided, the far-red light fluorescin can be with
Amino acid sequence including near infrared light BDFP albumen, and including the mutation at the 113rd amino acids, and at the 120th
It does not mutate at amino acid.The amino acid sequence of the near infrared light BDFP albumen is as shown in SED ID NO:2.
Above-mentioned near infrared light BDFP albumen source is in truncated phycobniliprotein ApcF2, and the including ApcF2 the 20th~169
Amino acids are made of the 20th~169 amino acids of ApcF2, and including mutation S46T, I51V, N72C, Y82C,
Y92M, N136K, V160I and V161A.
Unless otherwise specified, the amino acid position in the present invention is carried out based on the protein sequence of phycobniliprotein ApcF2
Coding.
The phycobniliprotein ApcF2 of this paper refers to from Chroococcidiopsis thermalisPCC's 7203
ApcF2.Preferably, the amino acid sequence of phycobniliprotein ApcF2 of the invention is as shown in SEQ ID NO:1.
Further, in far-red light fluorescin of the invention, the 113rd leucine is sported phenylalanine or first
Methyllanthionine.
Further, far-red light fluorescin of the invention may include the mutation in the 125th amino acids.Preferably,
125th glycine can be sported cysteine.
Further, far-red light fluorescin of the invention may include the 30th, the 39th, the 47th, the 81st,
Mutation at 101st, the 143rd and/or the 151st amino acid.
Specifically, in far-red light fluorescin of the invention, the 30th phenylalanine is sported leucine or silk ammonia
Acid；39th glutamine is sported arginine；47th asparagine is sported serine；81st methionine
Sported threonine；101st aspartic acid is sported glycine；143rd valine is sported alanine；Or,
151st threonine is sported alanine.
Further, the amino acid sequence of far-red light fluorescin of the invention is as shown in SEQ ID NO.5.Alternatively, this
The far-red light fluorescin of invention is as shown in SEQ ID NO.7.Alternatively, far-red light fluorescin of the invention such as SEQ ID
Shown in NO.8.Alternatively, far-red light fluorescin of the invention is as shown in SEQ ID NO.9.
In still yet another aspect of the present, a kind of fusion fluorescin is provided, the fluorescent fusion protein includes this hair
Bright above-mentioned far-red light fluorescin.
Further, the fusion fluorescin may further include another far-red light fluorescin.Preferably, described
Another far-red light fluorescin is BDFP far-red light fluorescin.It is highly preferred that another far-red light fluorescin is this hair
Bright above-mentioned far-red light fluorescin.
Alternatively, the fusion fluorescin may further include non-far-red light fluorescin, the non-far-red light fluorescence
Albumen is near infrared light fluorescin, green fluorescent protein, blue fluorescent protein, cyan fluorescent protein, yellow fluorescence protein
And/or orange fluorescent protein.For example, the non-far-red light fluorescin can for EBFP, ECFP, mCerulean, TFP, GFP,
EGFP, EYFP, FRP, TagRFP or BDFP near infrared light fluorescin.The amino acid sequence of the nearly feux rouges fluorescin of the BDFP can
As shown in SED ID NO:2.
Further, it in fusion fluorescin of the invention, is connected between each fluorescin by connexon.It is preferred that
Ground, the connexon by 5~80 Amino acid profiles, such as by 5,6,7,8,9,10,11,12,13,14,15,16,17,18,
19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,40,45,50,55,60,65,70,75 or
80 Amino acid profiles.It is highly preferred that the connexon is by 5~70, preferably 5~60,5~55,5~50,5~45,5~
40,5~35 or 10~35 Amino acid profiles.
In still yet another aspect of the present, a kind of above-mentioned far-red light fluorescin of coding or above-mentioned fusion fluorescence egg are provided
White nucleic acid.
In still yet another aspect of the present, a kind of carrier including above-mentioned nucleic acid is provided.
As described above, modified BDFP far-red light fluorescin of the invention has lesser molecular weight, energy and pigment
Covalent bond, performance are stablized, and extreme environment is resistant to.
The maximum fluorescence emission peak of existing near infrared light BDFP albumen (near infrared light) near 710nm.The present invention is logical
Site mutation is crossed, blue shift occurs for the fluorescence emission peak of obtained BDFP albumen, and maximum emission wavelength is (remote near 670nm
Feux rouges).Therefore, the BDFP far-red light fluorescin that the present invention is obtained by site mutation can be with existing non-far-red light fluorescence
Albumen (such as BDFP near infrared light fluorescin) is applied in combination, and dual or multi-biological label is carried out to cell, tissue etc..
In addition, BDFP far-red light fluorescin of the invention can also be with another far-red light or non-far-red light fluorescin one
Building fusion fluorescin is played, to promote effective brightness when active somatic cell detection, or is combined fluorescent marker.Further
The fused in tandem albumen on ground, far-red light BDFP fluorescin of the invention and existing BDFP near infrared light fluorescin not only has
The effective brightness being significantly increased, and it is also equipped with the features such as large-scale Stokes is displaced, it can be used for fluorescence resonance energy and turn
The repercussion study etc. of shifting.
Detailed description of the invention
Fig. 1 shows the homologous sequence comparison diagram of ApcB, ApcF2, BDFP1.1, BDFP1.2, BDFP1.6.
Fig. 2 shows mutant in HEK 293T intracellular fluorescence microscope image (a) and effective brightness contrast
Scheme (b).Each mutant is co-expressed with eGFP (FPs:IRES:eGFP) in HEK293T into the cell respectively.Fig. 2 a is respectively illustrated
The green fluorescence of eGFP and the red fluorescence (channel FR or NIR) of each mutant are observed under fluorescence microscope.In figure 2b, respectively
The average far-red light fluorescent brightness of mutant first takes ratio with its average eGFP fluorescent brightness, corrects expression；Then again with
The value of BDFP1.6 (v23) compares, and obtains effective brightness final value.Cell imaging parameter: green channel (λex=470/40 nm,
λem=510/40nm), far-red light and near infrared light channel (λex=630/20nm, λem=690/50nm).BDFP1.1, v1,
The data acquisition time of v2, v3, v4, v7, v8, v14 are 30s, and the data acquisition time of other mutant is 5s.
Fig. 3 shows mutant v13~v32 in HEK 293T intracellular fluorescence microscope image (a) and effectively
Brightness contrast figure (b).Each mutant is co-expressed with eGFP in HEK 293T into the cell respectively.Fig. 3 a respectively illustrates aobvious in fluorescence
The red fluorescence (channel FR) of the green fluorescence of the eGFP observed under micro mirror and each mutant.In Fig. 3 b figure, mutant
Average far-red light fluorescent brightness first takes ratio with its average eGFP fluorescent brightness, corrects expression；Then again with BDFP1.6
(v23) value (it is set to 1) compares, and obtains effective brightness final value.Cell imaging parameter, green channel (λex=470/
40nm, λem=510/40nm), far-red light and near infrared light channel (λex=630/20nm, λem=690/50 nm).Data acquisition
Time, v17 30s, other mutant are 5s.
Fig. 4 show BDFP1.2 (v6), BDFP1.6 (v23) albumen and known far-red light fluorescin iRFP670,
The curve graph of the stability contrast of miRFP670 and smURFP in vitro.Fig. 4 a shows the fluorescence intensity of each fluorescin in pH
Change in fluorescence in 9.5~2 ranges, wherein the medium of pH2~5 is 0.1M sodium acetate and 0.3M sodium chloride, Jie of pH5.5~10
Matter is 0.1M NaH2PO4With 0.3M sodium chloride.Fig. 4 b shows mixing guanidine hydrochloride of the fluorescence intensity in various concentration of fluorescin
(pH7.2) variation in environment.Fig. 4 c shows variation of the fluorescence intensity of fluorescin after 80 DEG C of warm bath of solution.Fig. 4 d shows
Out photobleaching experiment as a result, medium be kaliumphosphate buffer (pH7.2), 103 W/2 light source of 100W HBO irradiation, far-red light
Filter setting (λEx/ bleaching=630/20, λem=690/50nm), light amasthenic lens is 100 × C-Apochromat oil immersion objective, hole
Diameter 1.2, microscope are Zeiss Axioscope A1, and image camera is cool-snap HQ2 CCD.
Fig. 5 shows the fusion protein that source of people albumen and monochromatic and double-colored BDFP albumen are expressed in living mammalian cells
Microphoto.(a) wide area microscope (wf) and structured lighting microscope (SIM, super-resolution microscope) image: HEK
293T expresses the fusion protein of each mutant and histone (H2B)；HeLa cell expresses each mutant and mitochondria imports sequence
(MTS), keratin (keratin), calcium connect the fusion protein of albumen (calnexin) or tubulin (α-tubulin)； U-
2OS cell expresses the fusion protein of each mutant and actin (β-actin)；Hamster CHO-K1 cell expresses mutant and flesh
The fusion protein of filamentous actin label (F-actin marker) lifeact.(b) far-red light (BDFP1.6,1.2 or 1.3) and close red
The dual colour imaging figure of outer light (BDFP1.1) fluorescin.(λ is arranged in far-red light image (left column) optical filterex=630/20 nm,
λem=667/30nm), (λ is arranged near infrared light imaging figure (middle column) optical filterex=685/20, λem=740/40nm)；It is double-colored
It synthesizes coverage diagram (right column).Scale bar: 10 μm.
Fig. 6 shows the effective of BDFP1.2, BDFP1.6 and their same sequence fusion protein and existing fluorescin
Brightness contrast figure.It (a) is fluorescent microscopy images.Wherein, by each BDFP fluorescin and existing fluorescin (iRFP670,
MiRFP670 and smURFP) with eGFP building in identical carrier (BDFPs:IRES:eGFP), it transiently transfects to HEK 293T
In cell.The fluorescent brightness of each BDFP fluorescin can be corrected based on the fluorescent brightness of eGFP.Observation channel setting be respectively
Green light (eGFP, λex=470/40nm, λem=510/40nm) and far-red light (λex=630/20nm, λem=690/50 nm).Than
Example ruler is set as 50 μm.(b) to the quantized result of the fluorescent brightness of (a).Data are handled using 8.0 software of Origin, single-factor
The variance analysis level of signifiance is α=0.05 (n=30).
Fig. 7 shows the same sequence of BDFP series albumen or the fluorescent brightness of different sequence series connection construct.(a) series connection building
The structural schematic diagram of body；(b) abosrption spectrogram for construct of connecting；(c) fluorescence spectra for construct of connecting；(d) row is on
The wide area dual colour imaging figure of Hela cell body internal labeling, lower row are SIM super-resolution imaging figure；(e) histogram of effective brightness,
Dark column indicates 630nm channel data (λex=630/20nm, λem=690/50nm), Bai Zhu indicates 660nm channel data (λex
=660/45nm, λem=710/50nm).Using 8.0 software data processing of Origin, the one-factor analysis of variance level of signifiance is α
=0.05 (n=30).
Specific embodiment
The following is a preferred embodiment of the present invention, it is noted that for those skilled in the art of the present technique,
Under the premise of not departing from general principles, several improvement can be made, these improvement are also contemplated as being within the scope of the present invention.
Divide that the present invention is described in detail by specific embodiment below.It is understood, however, that the present invention is not limited to following
Specific embodiment.Protection scope of the present invention is defined by claims, within its scope, can be following to the present invention
Embodiment carries out any change and combination.
Below in conjunction with specific embodiment, the present invention is explained further.
Material and method
1. carrier
PET28 (or pET30) and pACYCDuet (Novagen) is T7 promoter expression vector.PACYCDuet through designing,
It can be used in double objective gene sequences in the interior cotransformation expression of Escherichia coli (E.coli).Expression vector pcDNA3.1
It (Invitrogen) is the mammal expression vector with CMV promoter.
When HEK 293T is screened into the cell, using expression vector pcDNA3.1, amalgamation and expression sequence is BDFP:
IRES:eGFP.When making brightness contrast, the fluorescent brightness of BDFP albumen can be modified based on the fluorescent brightness of eGFP.
2. mutation
Mutation initiation template be BDFP1.1 (as shown in SEQ ID NO.2, i.e. ApcF2 (20-169)-S46T/I51V/
N72C/Y82C/Y92M/N136K/V160I/V161A)。
Locus specificity and site saturation mutagenesis (20 amino acid all use primer mixture to encode) use Zheng
Et al. (L.Zheng, U.Baumann, J.L.Reymond, An efficient one-step site-directed and
Site-saturation mutagenesis protocol, Nucleic Acid Res.32 (2004) e115) an effective step
Formula scheme carries out.Random mutagenesis is carried out by fallibility PCR, and used condition causes every 1000 base-pairs (bp) to generate
Up to 16 mutation frequency (O. Griesbeck, G.S.Baird, R.E.Campbell, D.A.Zacharias,
R.Y.Tsien, Reducing theenvironmental sensitivity of yellow fluorescent
Protein.Mechanism and applications, J.Biol.Chem.276 (2001) 29188-29194).
The gene library obtained by random mutagenesis is cloned on pET28 vector plasmid.Then the plasmid will be with generation
The plasmid pACYC-ho1 of BILE PIGMENTS (BV), conversion enters e. coli bl21 (DE3) together.First fine jade under the conditions of 37 DEG C of cell
Rouge plate culture 10h, then cultivates 48h under the conditions of 17 DEG C.Inducer uses isopropyl-β-D-thiogalactoside
(IPTG), concentration 0.05mM.Hyperfluorescence clone on plate, is the bacterium colony selection system using model Qpix 420
628/65nm/ is arranged in (Molecular Devices), feux rouges excitation channel, and 692/65nm is arranged in far-red light fluorescence channel.Each
The clone with high brightness fluorescent that library is selected, it will the fallibility pcr template as next round.
3. the expression of the recombinant protein in Escherichia coli
PET expression vector with encoding fluorescent protein is transformed into coli strain BL21 (DE3) (Novagen)
Afterwards, then by carrier pACYC-ho1 it is transformed into same bacterial strain.By transformed BL21 cell at 18 DEG C, culture is being supplemented with card
In that mycin (20 μ g/ml) and chloramphenicol (17 μ g/ml) LB culture medium.When O.D value reaches 0.4~0.6,1mM isopropyl is used
Base-β-D- thiogalactoside (IPTG) inducing expression 5~16 hours, then at 4 DEG C, 12,000 × g is centrifuged 3min, collects thin
Born of the same parents rinse 2 times through water, and short-term preservation is at 4 DEG C or is placed in -20 DEG C of long-term preservations.
4. the purifying of albumen and quantitative
Wet thallus is suspended in start buffer [kaliumphosphate buffer (KPB, 20mM, pH 7.2), the sodium chloride of ice pre-cooling
(NaCl, 0.5M)] in.It is broken through 50W power ultrasound (JY92-II, NingBo XinZhi Biology Science Co., Ltd, China), 5
Minute.At 4 DEG C, 12000 × g is centrifuged 60 minutes suspension.Obtained supernatant is through Ni2+Affinity column (Amersham
Biosciences it) purifies, wherein using start buffer [potassium phosphate (KPB, 20mM, pH 7.2) loading, using additionally containing
The buffer of 0.5M imidazoles is eluted.The sample being collected into, at least twice with start buffer (pH 7.2) dialysis.Pass through
Bradford method measures protein concentration, and bovine serum albumin(BSA) is used to be corrected as standard items.
5. the culture of mammalian cell
By HEK 293T, HeLa or U-2OS cell culture in the DMEM culture medium for containing 10% fetal calf serum
(Invitrogen) in, by CHO-K1 cell culture in the F-12K culture medium containing 10% fetal calf serum.It uses2000 (Invitrogen) are transfected.When transfection,2000 and DNA is with 3: 1 (μ l
: μ g) ratio in serum free medium Opti-Middle mixing after ten minutes, is added in the cell to be transfected immediately.5
After~6h, fresh DMEM culture medium is replaced.
6. (in-vivo imaging) is imaged in active somatic cell
It completes to transiently transfect in 20mm glass Tissue Culture Dish (Nest), starts to carry out active somatic cell imaging for 24 hours afterwards.At
It is first rinsed twice with 1mL PBS as before, is then rinsed once with DEME culture medium is (no phenol red).Imaging device is inverted microscope
Nikon Ti is furnished with 20 0.45-DIC L-WD object of cool-snap HQ2 CCD camera and Nikon Plan Fluor ELWD
Mirror.Excitation and transmitting are provided that eGFP is green channel, λex=470/40, λem=510/40nm；Far-red light fluorescin
For far-red light channel, λex=630/20, λem=690/50nm.Picture uses ImageJ software (National Institutes
Of Health) it is analyzed and is handled.
7. wide area and super-resolution microscope inspection
In room temperature, by stand-alone mode, Nikon is inverted using the ECLIPSE Ti-E equipped with 100 × 1.49NA oil immersion objective
Nikon structured lighting system on microscope obtains wide area and structured lighting microscope (SIM) photo.Use 640nm's
Semiconductor laser (100mW, CUBE 640-100C, COHERENT) excites FR fluorescence.Using soft by NIS-Elements AR
The electron multiplication CCD camera (Andor iXon3 DU897) of part (Nikon) control carries out data acquisition.Use NIS-
Elements AR handles image.
8. the building of fused in tandem albumen
The fluorescin of BDFP series can pass through following connexon (linker) construction of fusion protein:
The connexon of 11 amino acid residues: GHGTGSTGSGS；
The connexon of 23 amino acid residues: GHGTGSTGSGSSGTASSEDNNMA；
The connexon of 31 amino acid residues: GHGTGSTGSGSHGTGSTGSSGTASSEDNNMA.
Experiment discovery, the connexon of above-mentioned 3 kinds of different lengths have not significant impact that (result is not shown to the fluorescence of fusion protein
Out).
9. homologous sequence compares
BDFP fluorescin structure alignment, is completed on SWISS-MODEL remote server.The template sequence used is blunt
The phycobniliprotein ApcB (pdb code:1ALL) of top spirulina (Spirulina platensis), comparison software Swiss-
PDBViewer, edition 4 .1.Protein structure figure is created using PyMOL (http://www.pymol.org/).Using Clustal
Protein sequence comparison chart is completed in (http://www.clustal.org/) creation.
10. spectrum analysis
The ultraviolet-ray visible absorbing light of chromoprotein is detected by spectrophotometer (DU800, Beckman-Coulter)
Spectrum.The extinction coefficient of fluorescin is absorption coefficient ε=39,900M according to BILE PIGMENTS BV at 390nm-1cm-1, joined
Than conversion.
Fluorescence spectrum is detected by sepectrophotofluorometer (F320, Tianjin Gangdong Technology Development Co., Ltd.).
With reference to known iRFP670 fluorescin (ΦF=0.122) the sub- yield Φ of amount of fluorescence detectedF, sample detection environment is potassium phosphate
Salting liquid (20mM, pH 7.2, KPB).
11. the Detection of Stability of fluorescin
Detect the stability experiment under condition of different pH.The BDFP fluorescin solution of detection after purification, it is slow in different pH
Fluorescence intensity change under fliud flushing environment.It is 2~10 that range, which is arranged, in pH, and wherein pH2~5 uses 0.1M sodium acetate, 0.3M chlorination
The buffer system of sodium；PH5.5~10 uses 0.1M NaH2PO4, the buffer system of 0.3M sodium chloride.
Detect the stability experiment in strong denaturant environment.The BDFP fluorescin solution of detection after purification, different dense
Fluorescence intensity change under the guanidine hydrochloride solution environment of degree, pH7.2.
Detect stability experiment in a high temperauture environment.The BDFP fluorescin solution of detection after purification, in 80 DEG C of water-baths
In pot, fluorescence intensity changes with time.
Photobleaching experiment.Fluorescin solution (pH7.2,20mM KPB) is instilled in mineral oil, with 100W HBO 103
100% power illumination of W/2 light source, far-red light, which filters, is arranged (λex=630/20, λem=690/50nm), light amasthenic lens is
100 × oil mirror (C-Apochromat oil-immersion lens), aperture 1.2, using microscope Zeiss Axioscope
A1, image camera are cool-snap HQ2 CCD.With software I mageJ (National Institutes of Health)
Analyze data.
It will be understood by those skilled in the art that for the experimental methods of molecular biology for not making to illustrate herein, it can
Referring to specific method listed in " Molecular Cloning:A Laboratory guide " (fourth edition, M.R. Green and J. Pehanorm Brooker write) book into
Row, or carried out according to kit and product description.Used kit biomaterial unless otherwise specified can be from business
Approach obtains.
Embodiment
1. homologous sequence of embodiment compares analysis
Fig. 1 shows the comparison of several homologous sequences, and wherein ApcF2 and its derived sequence BDFP1.1 are all from
Chroococcidiopsis thermalisPCC 7203；ApcB comes from blunt top spirulina Spirulina platensis, should
Algae is not suitable with far-red light and near infrared light growing environment.It will be seen from figure 1 that L113 ApcB, ApcF2 and BDFP1.1 it
Between be highly conserved.
On the basis of BDFP1.1 sequence (SEQ ID NO.:2), fixed point saturation mutagenesis is carried out to L113；Screening obtains prominent
Variant is reacted by fallibility PCR, is carried out lasting random mutation, is established mutant library.Using external spectral property detection and
The method of active somatic cell imaging screens the mutant in mutant library according to following screening criteria: with BDFP1.1
It compares, obvious blue shift occurs for targeted mutagenesis body maximum emission wavelength, but is still greater than 660nm；Simultaneously with known fluorescin
Mark iRFP670 (ε Φfl=10.2mM-1cm-1) compare, molecule brightness is suitable.
By screening, 7 mutant are obtained first.This 7 mutant are sequenced, sequencing result finds that they distinguish
With amino acid mutation as shown in Table 1 below.
In this 7 mutant of expression in escherichia coli, then pass through Ni2+Each mutant of affinitive layer purification expression is gone forward side by side
Row protein quantification.The mutant protein of equivalent is placed in detection solution kaliumphosphate buffer (KPB, 20mM, pH 7.0) and 0.5M
In NaCl.The a length of 620nm of the excitation light wave of fluorescence emission spectrum；Molecule brightness contrast object of reference is iRFP670 (ε Φfl=
10.2 mM-1cm-1).The spectral property of 7 mutant is detected, as a result as shown in table 2 below.
The mutational site of each mutant and the spectral results of table 2 listed in conjunction with table 1 can be seen that BDFP1.1 most
Big launch wavelength is about 707nm；Mutant v1 and v2 mutate at L113, their maximum emission wavelength is blue shifted to about
700nm；Mutant v4 mutates at L113 and G125, and maximum emission wavelength is further blue shifted to about 670nm；Mutant
V6, v7 and v8 also have the mutation in other sites, but their emission maximum other than mutating at L113 and G125
Wavelength is in 670nm or so.
The above results show the mutation at L113 so that the blue spectral shift of fluorescin (is blue shifted to about from about 707nm
700nm), and L113 and G125 combinatorial mutagenesis the fluorescin blue spectral shift effect that is are more significant (being blue shifted to about 670nm).
Other sites, the mutation at F30, Q39, N47, M81, D101, V143 or T151 not will cause the further of fluorescin spectrum
Blue shift.
In addition, compared with BDFP1.1, it can be seen that L113, which is mutated, improves the molecule brightness of fluorescin, and L113 with
G125 combinatorial mutagenesis further improves the molecule brightness of fluorescin.Specifically, the molecule brightness of BDFP1.1 is 39, mutation
The molecule brightness of body v1 and v2 (being mutated with L113) are increased to 70 and 57 respectively；Mutant v4 (is combined with L113 and G125
Mutation) molecule brightness be further increased to 157；Point of other mutant v6~v8 with L113 and G125 combinatorial mutagenesis
Sub- brightness is above 150.
Embodiment 2. detects the effective brightness intracellular in HEK 293T of each BDFP fluorescin
The nucleic acid (FPs:IRES:eGFP) for expressing each mutant is constructed in expression vector pcDNA3.1, then instantaneously
It is intracellular to be transfected into HEK 293T, observes fluorescent brightness using inverted fluorescence microscope.As a result shown in Figure 2.
Fig. 2 a shows the fluorescent image observed under green channel and the channel FR/NIR respectively.Because BDFP1.1,
V1, v2, v3, v4, v7, v8, v14 are under the channel FR/NIR, and the 5s time for exposure does not collect the image of effective brightness, therefore
Take the 30s time for exposure.Other mutant take the 5s time for exposure.Mutant v6 (BDFP1.2), v7 are shown in Fig. 2 b
With the quantized result of the effective brightness of v8.
Can be seen that mutant v1, v2, v4, v6 (BDFP1.2), v7 and V8 from result above can lead in FR/NIR
Fluorescence is observed under road.In addition, though the molecule brightness of mutant v4, v7 and V8 are significantly improved, but thin in HEK 293T
Effective brightness intracellular is not high.In contrast, effective brightness of mutant v6 (BDFP1.2) is higher.
The screening of the further mutant of embodiment 3.
In order to further find the mutant of excellent, inventor has carried out another wheel mutation.With same as Example 1
Screening criteria, filter out 20 mutant v13~v32, as shown in table 3 below.
Meanwhile the spectral property of mutant v13~v32 is had detected, as a result shown in table 4 as above.
It can be seen that the maximum emission wavelength of v13~v32 is similar with v6 (BDFP1.2) from the result in table 4, exist
670nm or so, and molecule brightness is also higher.
Embodiment 4. detects mutant v13~v32 effective brightness intracellular in HEK 293T
In method same as Example 2, the expression vector for co-expressing each mutant and eGFP is transiently transfected to HEK
293T is intracellular, observes fluorescent brightness using inverted fluorescence microscope.As a result it is shown in Fig. 2 (v13~v15) and Fig. 3.
Fig. 2 a and 3a show the fluorescent image observed under green channel and the channel FR respectively.Wherein, due to mutation
Body v17 does not collect effective luminance picture in the 5s time for exposure, thus take the 30s time for exposure to acquire image (v14 exists
Effective luminance picture is not collected when the 30s time for exposure, therefore is being not shown in the figure) yet.Other mutant take 5s to expose
Time.Fig. 2 a and Fig. 3 b respectively quantify fluorescent brightness.
It can be seen from the figure that other mutant are equal in the intracellular effective brightness of HEK293T other than v14 and v17
It is very high.
Embodiment 5. detects the stability of each fluorescin under in vitro conditions
The present embodiment has detected fluorescin to conditions in vitro, such as the tolerance of soda acid, Denaturing, high temperature, photobleaching
Property.As a result shown in Figure 4.
Fig. 4 a shows fluorescence intensity of each fluorescin at different pH.It can be seen from the figure that BDFP1.2 (v6) and
The fluorescence intensity of BDFP1.6 (v23) fluorescin fluorescence in pH 2 also retains 50% or more (compared with when pH7), and
IRFP670 and miRFP670 fluorescence in pH3.5 is quenched completely.It is relatively stable that this, which illustrates BDFP1.2 at a low ph,
's.
Fig. 4 b shows fluorescence intensity of each fluorescin in the guanidine hydrochloride solution of various concentration.It can from figure
Out, BDFP1.2 (v6) still remains with 70% or more fluorescence under the conditions of 3M guanidine hydrochloride (pH7.2).BDFP1.6 (v23) is glimmering
The tolerance of photoprotein is higher, under the conditions of 3.5M guanidine hydrochloride, moreover it is possible to retain about 60% fluorescence.
Fig. 4 c shows each fluorescin time that fluorescence is kept under high temperature (80 DEG C).It can be seen from the figure that
BDFP1.2 (v6) and BDFP1.6 (v23) still remain with 40% or more fluorescence after 80 DEG C of warm bath 2h.And iRFP670's is glimmering
Light is quenched completely after 80 DEG C of warm bath 2h, and the fluorescence of smURFP is also reduced to 25% or less.
Fig. 4 d shows the retention time of each fluorescin fluorescence in photobleaching processing.It can be seen from the figure that
BDFP1.2 (v6) and BDFP1.6 (v23) are similar with iRFP670, miRFP670 and smURFP in photobleaching.
Based on the above results, it can be deduced that BDFP1.2 (v6) and BDFP1.6 (v23) is in low pH, concentrated hydrochloric acid guanidine solution
Or it in the environment of high temperature, all has excellent stability, and be resistant to photobleaching.
Embodiment 6. detects effective brightness and its polymerization state of each fluorescin in cell
Sample needs to pass through Ni before detecting spectrum2+Affinitive layer purification, detection solution environmental are KPB (20mM, pH
And 0.5M NaCl 7.0)；The a length of 660nm of the excitation light wave of fluorescence emission spectrum.The comparison object of reference of molecule brightness is
iRFP670 (ε·Φfl=10.2mM-1cm-1).In intracellular effective fluorescence (far-red light) brightness of HEK 293T, transfecting
Detection obtains pcDNA3.1-FPs:IRES:eGFP plasmid afterwards for 24 hours, without adding external source BILE PIGMENTS BV or coexpression ferroheme
Oxidizing ferment HO-1.As a result it is shown in the following table 5.
It is similar with result above, the maximum emission wavelength of BDFP1.1 be about 707nm, BDFP1.2 and BDFP1.6 most
Obvious blue shift, about 670nm occur for big launch wavelength.Therefore, BDFP1.2 and BDFP1.6 can be applied in combination with BDFP1.1,
Carry out double fluorescent label.
In addition, BDFP1.2 effective brightness in HEK 293T, every BV is 10.5, and every kDa is 21.8；BDFP1.6 is in HEK
Effective brightness in 293T, every BV are 61.6, and every kDa is 128.This shows that effective brightness of BDFP1.2 and BDFP1.6 is aobvious
It writes and is higher than BDFP1.1, and effective brightness of BDFP1.6 has been got back compared with BDFP1.2 and further increased.
Embodiment 7. detects the single expression of fluorescin in mammalian cells and its combinational expression with BDFP1.1
In the present embodiment, has detected BDFP1.2 and individually import sequence (MTS), keratin with histone (H2B), mitochondria
(keratin) or calcium connects the expression of the fusion protein of albumen (calnexin) in mammalian cells, has detected simultaneously
BDFP1.2, BDFP1.6 effect with BDFP1.1 composite marking respectively.As a result shown in Figure 5.
Fig. 5 a shows the result of BDFP1.2 separate marking.BDFP1.2 and histone are expressed in HEK 293T cell
(H2B) fusion protein；BDFP1.2 is expressed in HeLab cell individually imports sequence (MTS), keratin with mitochondria
(keratin) or calcium connects the fusion protein of albumen (calnexin).It can be seen from the figure that using the micro- (Wide of wide area
Field, wf) and structured lighting micro- (SIM, super-resolution microscope) the glimmering of fusion protein can be clearly observed
Light.
Figure 5b shows that the results that BDFP1.1 and BDFP1.2 or BDFP1.6 are combined label.By fusion protein
BDFP1.2-MTS is with BDFP1.1-NLS cotransfection with combinational expression in HeLa cell.Then it is detected under fluorescence microscope.
The optical filter of left column is set as λex=630/20nm, λem=667/30nm；The optical filter of middle column is set as λex=685/
20, λem=740/40nm；Right column is overlapping image.Similarly, fusion protein BDFP1.6-H2B and BDFP1.1-MTS is total to
Transfection is in HeLa cell with combinational expression.It can be seen from the figure that the fusion protein of BDFP1.2 and BDFP1.6 was issued
Far-red light fluorescence can be distinguished with the near infrared light fluorescence of BDFP1.1, so as to carry out double labeling to cell simultaneously.
Embodiment 8. detects the Absorption and emission spectra property of BDFP series albumen series connection construct
The present embodiment use 23 amino acid connexon, further construct series connection construct BDFP1.1:1.2,
BDFP1.1:1.6 (as shown in Figure 7a).
Series connection construct BDFP1.1:1.2, BDFP1.1:1.6, BDFP1.2:1.2 and BDFP1.6:1.6 of building are turned
Change into Escherichia coli, and inducing expression.Then the fusion fluorescent protein B DFP1.1 expressed by affinitive layer purification:
1.2, BDFP1.1:1.6, BDFP1.2:1.2 and BDFP1.6:1.6.Using ultraviolet-visible absorption spectroscopy instrument and Fluorescence Spectrometer
The spectral property for detecting these fusion fluorescins, is as a result shown in the following table 6.
It can be seen that the transmitting of same sequence series connection construct BDFP1.2:1.2 and BDFP1.6:1.6 from table 6 and Fig. 6 b, c
Spike length is each about 670nm, and significant change does not occur.And different sequence series connection construct BDFP1.1:1.2 and BDFP1.1:1.6,
Absorption peak is blue shifted near 640nm, and near fluorescence peak red shift to 710nm, this shows to increase Stokes displacement.
In addition, fluorescin becomes free state after fusion, it is not susceptible to aggregate and precipitate phenomenon (data are not shown).
Effective brightness of the detection series connection building of embodiment 9. and its polymerization state
In the present embodiment, by connect construct BDFP1.1:1.2, BDFP1.1:1.6, BDFP1.2:1.2 and BDFP1.6:
1.6 transiently transfect into HEK 293T cell, in fluorescence microscopy its effective brightness under the microscope, are as a result shown in table 6, Fig. 6 and figure
In 7e.
It can see from table 6 and Fig. 6 b, with the effective bright of sequence series connection construct BDFP1.2:1.2 and BDFP1.6:1.6
Degree is significantly improved, and is higher than the summation of effective brightness when two BDFP fluorescins are used alone.Fig. 7 e is also obtained
Similar results.This result shows that, connect construct in two BDFP fluorescin cooperating types improve series connection construct
Effective brightness.
It is arrived in addition, fusion protein BDFP1.2:1.2- histone and BDFP1.1:1.6- mitochondria are imported sequence cotransfection
Hela is intracellular, is observed under fluorescence microscope, is as a result shown in Fig. 7 d.Wherein, BDFP1.1:1.6- mitochondria imports
Sequence is observed using near infrared channels, and BDFP1.2:1.2- histone is observed using far infrared channel.It can from Fig. 7 d
The fluorescence of BDFP1.2:1.2 and BDFP1.1:1.6 can distinguish out, so as to carry out double labeling to cell.
SEQUENCE LISTING
<110>Guangzhou Tianbao Songyuan Biology Science & Technology Development Co., Ltd.
<120>the novel fluorescence marker and its fusion protein of BDFP near infrared light fluorescin are originated from
<130> P18GZ1NN03250CN
<160> 29
<170> PatentIn version 3.5
<210> 1
<211> 169
<212> PRT
<213> Chroococcidiopsis thermalis
<400> 1
Met Gln Asp Lys Leu Thr Ser Val Ala Lys Asn Cys Asp Leu Thr Gly
1 5 10 15
Ser Ser Leu Asn Arg Glu Val Val Glu Thr Leu Lys Glu Phe Leu Ala
20 25 30
Asp Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Ser Asn Ala
35 40 45
Ala Glu Ile Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro
50 55 60
Glu Leu Val Ser Pro Gly Gly Asn Ala Tyr Thr Thr Arg Arg Tyr Asn
65 70 75 80
Met Tyr Val Arg Asp Met Asn Tyr Phe Leu Arg Tyr Cys Ser Tyr Ala
85 90 95
Ile Val Ala Gly Asp Ala Ser Val Leu Asp Glu Arg Leu Leu Ala Gly
100 105 110
Leu Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Gly Pro Thr Ala
115 120 125
Arg Ser Ile Gln Leu Met Lys Asn Ile Val Lys Glu Lys Leu Val Thr
130 135 140
Ala Gly Met Thr Asn Ile Thr Phe Val Asp Glu Pro Phe Asp Tyr Val
145 150 155 160
Val Arg Glu Ile Ser Glu Thr Glu Ile
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<223> BDFP1.1
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Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Glu Phe Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asn Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Asp Ala Ser Val Leu Asp Glu Arg Leu Leu Ala Gly Leu
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Gly Pro Thr Ala Arg
100 105 110
Ser Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Val Thr Ala
115 120 125
Gly Met Thr Asn Ile Thr Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Glu Ile Ser Glu Thr Glu Ile
145 150
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Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Glu Phe Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asn Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Asp Ala Ser Val Leu Asp Glu Arg Leu Leu Ala Gly Phe
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Gly Pro Thr Ala Arg
100 105 110
Ser Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Val Thr Ala
115 120 125
Gly Met Thr Asn Ile Thr Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Glu Ile Ser Glu Thr Glu Ile
145 150
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Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Glu Phe Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asn Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Asp Ala Ser Val Leu Asp Glu Arg Leu Leu Ala Gly Met
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Gly Pro Thr Ala Arg
100 105 110
Ser Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Val Thr Ala
115 120 125
Gly Met Thr Asn Ile Thr Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Glu Ile Ser Glu Thr Glu Ile
145 150
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<223> v4
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Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Glu Phe Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asn Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Asp Ala Ser Val Leu Asp Glu Arg Leu Leu Ala Gly Phe
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Cys Pro Thr Ala Arg
100 105 110
Ser Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Val Thr Ala
115 120 125
Gly Met Thr Asn Ile Thr Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Glu Ile Ser Glu Thr Glu Ile
145 150
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<223> v5
<400> 6
Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Glu Phe Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asp Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Asp Ala Ser Val Leu Asp Glu Arg Leu Leu Ala Gly Phe
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Gly Pro Thr Ala Arg
100 105 110
Ser Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Val Thr Ala
115 120 125
Gly Met Thr Asn Ile Thr Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Glu Ile Ser Glu Thr Glu Ile
145 150
<210> 7
<211> 152
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<213> Artificial Sequence
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<223> v6
<400> 7
Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Glu Leu Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asn Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Gly Ala Ser Val Leu Asp Glu Arg Leu Leu Ala Gly Phe
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Cys Pro Thr Ala Arg
100 105 110
Ser Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Ala Thr Ala
115 120 125
Gly Met Thr Asn Ile Ala Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Glu Ile Ser Glu Thr Glu Ile
145 150
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<213> Artificial Sequence
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<223> v7
<400> 8
Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Glu Ser Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Ser Ala Ala
20 25 30
Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asn Thr
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Asp Ala Ser Val Leu Asp Glu Arg Leu Leu Ala Gly Phe
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Cys Pro Thr Ala Arg
100 105 110
Ser Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Val Thr Ala
115 120 125
Gly Met Thr Asn Ile Thr Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Glu Ile Ser Glu Thr Glu Ile
145 150
<210> 9
<211> 152
<212> PRT
<213> Artificial Sequence
<220>
<223> v8
<400> 9
Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Glu Phe Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Arg Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asn Thr
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Asp Ala Ser Val Leu Asp Glu Arg Leu Leu Ala Gly Phe
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Cys Pro Thr Ala Arg
100 105 110
Ser Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Val Thr Ala
115 120 125
Gly Met Thr Asn Ile Thr Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Glu Ile Ser Glu Thr Glu Ile
145 150
<210> 10
<211> 152
<212> PRT
<213> Artificial Sequence
<220>
<223> v13
<400> 10
Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Glu Leu Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asn Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Gly Ala Ser Val Leu Asp Gly Arg Met Leu Ala Gly Phe
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Cys Pro Thr Ala Arg
100 105 110
Gly Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Ala Thr Ala
115 120 125
Gly Met Thr Asn Ile Ala Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Val Ile Ser Glu Thr Glu Ile
145 150
<210> 11
<211> 152
<212> PRT
<213> Artificial Sequence
<220>
<223> v14
<400> 11
Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Glu Leu Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asn Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Gly Ala Ser Val Leu Asp Gly Arg Met Leu Ala Gly Phe
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Cys Pro Leu Cys Pro Thr Ala Arg
100 105 110
Gly Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Ala Thr Ala
115 120 125
Gly Met Thr Asn Ile Ala Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Val Ile Ser Glu Thr Glu Ile
145 150
<210> 12
<211> 152
<212> PRT
<213> Artificial Sequence
<220>
<223> v15
<400> 12
Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Glu Leu Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asn Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Gly Ala Ser Val Leu Asp Gly Arg Met Leu Ala Gly Phe
85 90 95
Arg Asp Thr Phe Asn Ser Cys Gly Ile Pro Leu Cys Pro Thr Ala Arg
100 105 110
Gly Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Ala Thr Ala
115 120 125
Gly Met Thr Asn Ile Ala Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Val Ile Ser Glu Thr Glu Ile
145 150
<210> 13
<211> 152
<212> PRT
<213> Artificial Sequence
<220>
<223> v16
<400> 13
Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Gly Leu Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asn Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Gly Ala Ser Val Leu Asp Gly Arg Met Leu Ala Gly Phe
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Cys Pro Thr Ala Arg
100 105 110
Gly Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Ala Thr Ala
115 120 125
Gly Met Thr Asn Ile Ala Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Val Ile Ser Glu Thr Glu Ile
145 150
<210> 14
<211> 152
<212> PRT
<213> Artificial Sequence
<220>
<223> v17
<400> 14
Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Glu Leu Leu Ala Asp
1 5 10 15
Gly Gly Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asn Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Gly Ala Ser Val Leu Asp Gly Arg Met Leu Ala Gly Phe
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Cys Pro Thr Ala Arg
100 105 110
Gly Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Ala Thr Ala
115 120 125
Gly Met Thr Asn Ile Ala Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Val Ile Ser Glu Thr Glu Ile
145 150
<210> 15
<211> 152
<212> PRT
<213> Artificial Sequence
<220>
<223> v18
<400> 15
Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Glu Leu Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Ala Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asn Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Gly Ala Ser Val Leu Asp Gly Arg Met Leu Ala Gly Phe
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Cys Pro Thr Ala Arg
100 105 110
Gly Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Ala Thr Ala
115 120 125
Gly Met Thr Asn Ile Ala Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Val Ile Ser Glu Thr Glu Ile
145 150
<210> 16
<211> 152
<212> PRT
<213> Artificial Sequence
<220>
<223> v19
<400> 16
Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Glu Leu Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Thr Ala Val Gly Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asn Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Gly Ala Ser Val Leu Asp Gly Arg Met Leu Ala Gly Phe
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Cys Pro Thr Ala Arg
100 105 110
Gly Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Ala Thr Ala
115 120 125
Gly Met Thr Asn Ile Ala Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Val Ile Ser Glu Thr Glu Ile
145 150
<210> 17
<211> 152
<212> PRT
<213> Artificial Sequence
<220>
<223> v20
<400> 17
Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Glu Leu Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Gly Thr Arg Arg Tyr Asn Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Gly Ala Ser Val Leu Asp Gly Arg Met Leu Ala Gly Phe
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Cys Pro Thr Ala Arg
100 105 110
Gly Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Ala Thr Ala
115 120 125
Gly Met Thr Asn Ile Ala Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Val Ile Ser Glu Thr Glu Ile
145 150
<210> 18
<211> 152
<212> PRT
<213> Artificial Sequence
<220>
<223> v21
<400> 18
Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Glu Leu Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Ala Arg Arg Tyr Asn Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Gly Ala Ser Val Leu Asp Gly Arg Met Leu Ala Gly Phe
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Cys Pro Thr Ala Arg
100 105 110
Gly Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Ala Thr Ala
115 120 125
Gly Met Thr Asn Ile Ala Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Val Ile Ser Glu Thr Glu Ile
145 150
<210> 19
<211> 152
<212> PRT
<213> Artificial Sequence
<220>
<223> v22
<400> 19
Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Glu Leu Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asn Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Gly Ala Ser Val Leu Ala Gly Arg Met Leu Ala Gly Phe
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Cys Pro Thr Ala Arg
100 105 110
Gly Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Ala Thr Ala
115 120 125
Gly Met Thr Asn Ile Ala Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Val Ile Ser Glu Thr Glu Ile
145 150
<210> 20
<211> 152
<212> PRT
<213> Artificial Sequence
<220>
<223>v23(EDFP1.6)
<400> 20
Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Glu Leu Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asn Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Gly Ala Ser Val Leu Asp Gly Arg Met Leu Ala Gly Phe
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Cys Pro Ala Ala Arg
100 105 110
Gly Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Ala Thr Ala
115 120 125
Gly Met Thr Asn Ile Ala Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Val Ile Ser Glu Thr Glu Ile
145 150
<210> 21
<211> 152
<212> PRT
<213> Artificial Sequence
<220>
<223> v24
<400> 21
Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Glu Leu Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asn Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Gly Ala Ser Val Leu Asp Gly Arg Met Leu Ala Gly Phe
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Cys Pro Thr Ala Arg
100 105 110
Gly Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Ala Gly Ala
115 120 125
Gly Met Thr Asn Ile Ala Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Val Ile Ser Glu Thr Glu Ile
145 150
<210> 22
<211> 152
<212> PRT
<213> Artificial Sequence
<220>
<223> v25
<400> 22
Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Glu Leu Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asn Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Gly Ala Ser Val Leu Asp Gly Arg Met Leu Ala Gly Phe
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Cys Pro Thr Ala Arg
100 105 110
Gly Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Ala Thr Ala
115 120 125
Gly Met Ala Asn Ile Ala Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Val Ile Ser Glu Thr Glu Ile
145 150
<210> 23
<211> 152
<212> PRT
<213> Artificial Sequence
<220>
<223> v26
<400> 23
Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Glu Leu Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asn Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Gly Ala Ser Val Leu Asp Gly Arg Met Leu Ala Gly Phe
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Cys Pro Ala Ala Arg
100 105 110
Gly Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Ala Gly Ala
115 120 125
Gly Met Thr Asn Ile Ala Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Val Ile Ser Glu Thr Glu Ile
145 150
<210> 24
<211> 152
<212> PRT
<213> Artificial Sequence
<220>
<223> v27
<400> 24
Met Ala Asn Arg Glu Val Phe Glu Thr Leu Lys Glu Leu Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Gly Thr Arg Arg Tyr Asn Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Gly Ala Ser Val Leu Ala Gly Arg Met Leu Ala Gly Phe
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Cys Pro Ala Ala Arg
100 105 110
Gly Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Ala Thr Ala
115 120 125
Gly Met Thr Asn Ile Ala Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Val Ile Ser Glu Thr Glu Ile
145 150
<210> 25
<211> 152
<212> PRT
<213> Artificial Sequence
<220>
<223> v28
<400> 25
Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Gly Leu Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Gly Thr Arg Arg Tyr Asn Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Gly Ala Ser Val Leu Asp Gly Arg Met Leu Ala Gly Phe
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Cys Pro Ala Ala Arg
100 105 110
Gly Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Ala Gly Ala
115 120 125
Gly Met Thr Asn Ile Ala Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Val Ile Ser Glu Thr Glu Ile
145 150
<210> 26
<211> 152
<212> PRT
<213> Artificial Sequence
<220>
<223> v29
<400> 26
Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Gly Leu Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Ala Arg Arg Tyr Asn Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Gly Ala Ser Val Leu Asp Gly Arg Met Leu Ala Gly Phe
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Cys Pro Ala Ala Arg
100 105 110
Gly Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Ala Gly Ala
115 120 125
Gly Met Thr Asn Ile Ala Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Val Ile Ser Glu Thr Glu Ile
145 150
<210> 27
<211> 152
<212> PRT
<213> Artificial Sequence
<220>
<223> v30
<400> 27
Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Gly Leu Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Gly Ala Arg Arg Tyr Asn Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Gly Ala Ser Val Leu Asp Gly Arg Met Leu Ala Gly Phe
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Cys Pro Ala Ala Arg
100 105 110
Gly Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Ala Gly Ala
115 120 125
Gly Met Thr Asn Ile Ala Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Val Ile Ser Glu Thr Glu Ile
145 150
<210> 28
<211> 152
<212> PRT
<213> Artificial Sequence
<220>
<223> v31
<400> 28
Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Gly Leu Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Ala Ala Val Gly Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Gly Thr Arg Arg Tyr Asn Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Gly Ala Ser Val Leu Asp Gly Arg Met Leu Ala Gly Phe
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Cys Pro Ala Ala Arg
100 105 110
Gly Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Ala Gly Ala
115 120 125
Gly Met Thr Asn Ile Ala Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Val Ile Ser Glu Thr Glu Ile
145 150
<210> 29
<211> 152
<212> PRT
<213> Artificial Sequence
<220>
<223> v32
<400> 29
Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Gly Leu Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Ala Ala Val Gly Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Ala Arg Arg Tyr Asn Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Gly Ala Ser Val Leu Asp Gly Arg Met Leu Ala Gly Phe
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Cys Pro Ala Ala Arg
100 105 110
Gly Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Ala Gly Ala
115 120 125
Gly Met Thr Asn Ile Ala Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Val Ile Ser Glu Thr Glu Ile
145 150
Claims (10)
1. a kind of far-red light fluorescin, which is characterized in that the far-red light fluorescin includes BDFP near infrared light fluorescence egg
It is white, and do not mutate including the mutation at the 113rd amino acids, and at the 120th amino acids,
Wherein, the amino acid sequence of the BDFP near infrared light fluorescin is as shown in SED ID NO:2.
2. far-red light fluorescin according to claim 1, which is characterized in that the far-red light fluorescin further wraps
Include the mutation at the 125th amino acids；
Preferably, the far-red light fluorescin further comprise the 30th, the 39th, the 47th, the 81st, the 101st,
Mutation at 143rd and/or the 151st amino acids.
3. far-red light fluorescin according to claim 1 or 2, which is characterized in that the 113rd leucine is sported benzene
Alanine or methionine；125th glycine is sported cysteine；
Preferably, the 30th phenylalanine is sported leucine or serine；39th glutamine is sported arginine；
47th asparagine is sported serine；81st methionine is sported threonine；101st aspartic acid is dashed forward
Become glycine；143rd valine is sported alanine；Or, the 151st threonine is sported alanine.
4. far-red light fluorescin according to claim 1 or 2, which is characterized in that the ammonia of the far-red light fluorescin
Base acid sequence is as shown in SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9, it is preferable that described remote
The amino acid sequence of feux rouges fluorescin is as shown in SEQ ID NO:7.
5. a kind of fusion fluorescin, which is characterized in that the fusion fluorescin includes such as any one of Claims 1 to 4
The far-red light fluorescin.
6. fusion fluorescin according to claim 5, which is characterized in that the fusion fluorescin further comprises another
One far-red light fluorescin；
Preferably, another far-red light fluorescin is BDFP far-red light fluorescin；
It is highly preferred that another far-red light fluorescin is far-red light fluorescence egg as described in any one of claims 1 to 4
It is white.
7. fusion fluorescin according to claim 5, which is characterized in that the fusion fluorescin further comprises non-
Far-red light fluorescin, the non-far-red light fluorescin are near infrared light fluorescin, green fluorescent protein, blue-fluorescence egg
White, cyan fluorescent protein, yellow fluorescence protein and/or orange fluorescent protein.
8. the fusion fluorescin according to any one of claim 5~7, which is characterized in that lead between each fluorescin
Cross connexon connection, it is preferable that the connexon is by 5~80 Amino acid profiles, more preferably by 5~70 Amino acid profiles.
9. a kind of encode far-red light fluorescin as described in any one of claims 1 to 4 or as in claim 5~8
The nucleic acid of described in any item fusion fluorescins.
10. a kind of carrier including nucleic acid as claimed in claim 9.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110386977A (en) * | 2019-07-01 | 2019-10-29 | 广州天宝颂原生物科技开发有限公司 | A kind of near infrared light fluorescin and its fusion protein |
| CN110577593A (en) * | 2019-07-01 | 2019-12-17 | 华中农业大学 | Small-molecule near-infrared light fluorescent protein and fusion protein thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1180312A (en) * | 1995-01-31 | 1998-04-29 | 伊莱利利公司 | Anti-obesity proteins |
-
2018
- 2018-08-30 CN CN201811009851.7A patent/CN109265523B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1180312A (en) * | 1995-01-31 | 1998-04-29 | 伊莱利利公司 | Anti-obesity proteins |
Non-Patent Citations (2)
| Title |
|---|
| DING,W 等: "far-red fluorescent protein BDFP1.2, partial [synthetic construct]，GenBank: ASN64561.1", 《NCBI DATABASE》 * |
| DING,W 等: "Synthetic construct far-red fluorescent protein BDFP1.2 gene, partial cds，GenBank: KY465660.1", 《NCBI DATABASE》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110386977A (en) * | 2019-07-01 | 2019-10-29 | 广州天宝颂原生物科技开发有限公司 | A kind of near infrared light fluorescin and its fusion protein |
| CN110577593A (en) * | 2019-07-01 | 2019-12-17 | 华中农业大学 | Small-molecule near-infrared light fluorescent protein and fusion protein thereof |
| CN110577593B (en) * | 2019-07-01 | 2021-09-14 | 华中农业大学 | Small-molecule near-infrared light fluorescent protein and fusion protein thereof |
| CN110386977B (en) * | 2019-07-01 | 2022-12-13 | 广州天宝颂原生物科技开发有限公司 | Near-infrared light fluorescent protein and fusion protein thereof |
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| CN109265523B (en) | 2021-11-09 |
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