Near-infrared fluorescent proteins and their applications

Abstract

The present invention relates to a near-infrared fluorescent protein and its application. The near-infrared fluorescent protein comprises an amino acid sequence selected from SEQ ID No.: 21-40. The present invention also relates to a fusion protein comprising the near-infrared fluorescent protein. The near-infrared fluorescent protein of the present invention has the advantages of red-shifted emission spectrum, small molecular weight, brightness, and stable chemical properties. Specifically, the near-infrared fluorescent protein is only 120 amino acids in size, and the emission spectrum is within the range of 672 to 738 nm, far exceeding the maximum emission peak (720 nm) of existing near-infrared fluorescent proteins, and the fluorescence intensity remains almost unchanged in the extreme environment of 0 to 100 ° C, pH 2 to 10, and 0-8 M denaturant.

Description

Technical Field

[0001] The present application belongs to the field of protein technology, and specifically relates to a near-infrared fluorescent protein and its application. Background Art

[0002] In general, near-infrared fluorescent proteins (NIRFPs) facilitate non-invasive imaging in living animals due to their exceptional light penetration within the NIR optical window (650-900 nm). This is primarily due to the minimal effect of hemoglobin, water, and lipids on tissue absorption within this optical window, resulting in reduced light scattering. Consequently, NIRFPs have become the preferred choice for protein labels and genetically encoded indicators in in vivo imaging applications.

[0003] Currently, near-infrared fluorescent proteins (NIRFPs) are primarily engineered from naturally occurring biliverdin-binding proteins (bacterial phytochromes and cyanobacterial phycobiliproteins) by covalently attaching biliverdin (BV) as a fluorophore. Biliverdin, an intermediate in mammalian heme metabolism, is abundant in mammalian cells, making these biliverdin-binding fluorescent proteins ideal for use as fluorescent tags without the need for exogenous fluorophore supply. Biliverdin is inherently nonfluorescent in aqueous solution, but binding to these proteins results in fluorescence due to rigidification processes that inhibit energy dissipation through molecular rotation, heating, photochemistry, internal conversion, or solvent quenching. Despite numerous efforts to optimize the photophysical and biochemical properties of these naturally occurring protein-derived fluorescent proteins through random mutagenesis-based direct evolution, structure-based mutagenesis, and rational design, challenges remain, including low brightness, unintended oligomerization, and relatively low pH / chemical stability, limiting their widespread use in real-world imaging applications. Summary of the Invention

[0004] One of the technical objectives of the present invention is to provide a near-infrared fluorescent protein or a mutant thereof, which has the advantages of low molecular weight, high brightness and stable chemical properties.

[0005] Another object of the present invention is to provide applications of the near-infrared fluorescent protein or its mutants in the field of near-infrared imaging.

[0006] In one aspect, the present invention provides a near-infrared fluorescent protein or a mutant thereof, wherein the near-infrared fluorescent protein comprises an amino acid sequence selected from SEQ ID No.: 21-40, and the mutant thereof has a sequence identity of more than 81% relative to the near-infrared fluorescent protein.

[0007] In a specific embodiment, the mutant has a sequence identity of 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more to the near-infrared fluorescent protein.

[0008] In a specific embodiment, the near-infrared fluorescent protein can be obtained through directed evolution technology.

[0009] In a specific embodiment, the near-infrared fluorescent protein comprises the amino acid sequence of SEQ ID No.: 37 or 40, or the amino acid sequence of the near-infrared fluorescent protein is selected from SEQ ID No. 37 or 40.

[0010] In a specific embodiment, the amino acid sequence of the near-infrared fluorescent protein is selected from SEQ ID No.: 21-40.

[0011] In some embodiments, the near-infrared fluorescent protein may further comprise an 8x His tag sequence (HHHHHHHH) to facilitate the purification of the near-infrared fluorescent protein.

[0012] In a specific embodiment, the near-infrared fluorescent protein comprises an amino acid sequence selected from SEQ ID No.: 1-20.

[0013] In a specific embodiment, the amino acid sequence of the near-infrared fluorescent protein is selected from SEQ ID No.: 1-20.

[0014] In a specific embodiment, the near-infrared fluorescent protein or its mutant is a monomeric near-infrared fluorescent protein, whose emission spectrum is within the range of 672-738 nm, and whose fluorescence intensity remains almost unchanged in extreme environments such as 0-100°C, pH 2-10, and denaturant up to 8M. In other words, its fluorescence intensity in extreme environments such as 0-100°C, pH 2-10, and denaturant up to 8M is comparable to its fluorescence intensity at 25°C, neutral conditions, and without denaturant.

[0015] In another aspect, the present invention provides a fusion protein comprising the aforementioned near-infrared fluorescent protein and other proteins.

[0016] In a specific embodiment, the other proteins are nuclear localized proteins and cytoskeletal proteins.

[0017] In a specific embodiment, the other protein is histone H2B or tubulin.

[0018] In a specific embodiment, the amino acid sequence of the fusion protein comprises an amino acid sequence selected from any one of SEQ ID No.: 41-44.

[0019] In a specific embodiment, the amino acid sequence of the fusion protein is selected from any one of SEQ ID No.: 41-44.

[0020] In another aspect, the present invention provides a nucleic acid molecule encoding the aforementioned near-infrared fluorescent protein or its mutant or the aforementioned fusion protein.

[0021] In another aspect, the present invention provides a recombinant vector comprising the above nucleic acid molecule.

[0022] In another aspect, the present invention provides a host cell comprising the above-mentioned recombinant vector.

[0023] On the other hand, the present invention provides the use of the above-mentioned near-infrared fluorescent protein or its mutant, the above-mentioned fusion protein, the above-mentioned nucleic acid molecule, the above-mentioned recombinant vector or the above-mentioned host cell in the preparation of a near-infrared fluorescent imaging agent or a tumor marker imaging agent.

[0024] In a specific embodiment, the near-infrared fluorescence imaging agent can realize near-infrared fluorescence imaging at the cellular, tissue and in vivo levels.

[0025] Beneficial effects

[0026] The present invention utilizes protein design methods to design and obtain a series of near-infrared fluorescent proteins with red-shifted emission spectra, small molecular weight, brightness, stable chemical properties and monomericity for endogenous biliverdin (BV).

[0027] Specifically, the emission spectra of the near-infrared fluorescent proteins are located in the range of 672 to 738 nm, which far exceeds the maximum emission peak of existing near-infrared fluorescent proteins (720 nm). Therefore, they have significant advantages in penetration depth, spatial resolution, and signal-to-noise ratio in imaging applications.

[0028] Furthermore, the near-infrared fluorescent protein described in SEQ ID No.: 21-40 has only 120 amino acids and is currently the smallest near-infrared fluorescent protein in terms of molecular weight.

[0029] In addition, the fluorescence intensity of the near-infrared fluorescent protein remains almost unchanged in an extreme environment of 0-100° C., pH 2-10, and 0-8M denaturant, making it the near-infrared fluorescent protein with the most stable chemical properties currently.

[0030] Therefore, the present invention solves the common technical problems of current near-infrared fluorescent proteins, namely, large molecular weight, low brightness, and poor chemical stability. Therefore, the near-infrared fluorescent protein of the present application has broad application prospects in cell imaging and in vivo tumor labeling imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Flowchart showing the design of near-infrared fluorescent proteins conjugated to BV.

[0032] Figure 2Shown are the structural schematics (A) and chromatographic results (B) of near-infrared fluorescent proteins Pr2-37, Pfr21, Pr1-88, and Pfr86.

[0033] Figure 3 Shown are the absorption and emission spectra of near-infrared fluorescent proteins Pr2-37, Pfr21, Pr1-88, and Pfr86 (A) and the protein fluorescence images under the corresponding absorption and emission spectra (B).

[0034] Figure 4 The relevant properties of Pr1-88.4 after five rounds of directed evolution of Pr1-88 are shown. Fluorescence images of E. coli expressing the near-infrared fluorescent protein Pr1-88 under corresponding excitation and emission spectra (A), light microscopy images of E. coli Pr1-88.4 obtained after five rounds of directed evolution of Pr1-88 (B, left) and fluorescence images under corresponding excitation and emission spectra (B, right), chromatographic results and absorption and emission spectra of Pr1-88.4 (left and middle panels of C), protein fluorescence images of Pr1-88.4 under corresponding absorption and emission spectra (right panel of C), mass spectrum of Pr1-88.4 unbound to BV (left panel of D), and mass spectrum of Pr1-88.4 bound to BV (right panel of D).

[0035] Figure 5 The results of the stability test of the near-infrared fluorescent protein Pr1-88.4 are shown. The temperature dependence (A), pH dependence (B), denaturant dependence (C, D) and time analysis of in vitro and in vivo maturation of the near-infrared fluorescent protein Pr1-88.4 are shown.

[0036] Figure 6 The infrared fluorescent protein Pr1-88.4 and Pfr21 fusion protein were used for imaging in mammalian cells. The near-infrared fluorescent proteins Pr1-88.4 and Pfr21 were used to label nucleosomal proteins and tubulin in HT1080 and HEK293F cells.

[0037] Figure 7 Figure 2 shows imaging analysis of the near-infrared fluorescent proteins Pr1-88.4 and Pfr21 in deep tissues of living mice. Fluorescence imaging of a Pr1-88.4 stably transfected cell line (A), fluorescence imaging of the near-infrared fluorescent proteins Pr1-88.4 and Pfr21 in deep tissues of living mice (B), and fluorescence imaging of the near-infrared fluorescent proteins mi670 and Pr1-88.4 in deep tissues of living mice (C). DETAILED DESCRIPTION

[0038] The technical contents of the present application are described in detail below through specific embodiments so that those skilled in the art can better understand the present invention. However, these embodiments are not intended to limit the contents of the present invention.

[0039] Example 1: Protein design, expression and purification

[0040] This application develops a new design method for small molecule binding proteins. Biliverdin molecules in different conformations have different electron cloud conjugation systems, which will produce different spectral absorption and fluorescence emission characteristics. First, quantum mechanics optimization methods are used to generate biliverdin molecules with different conformations, and then the protein design tools independently developed by the laboratory are used to generate proteins with different conformations that can specifically bind to different conformations of biliverdin. The amino acid sequence is then designed using Rosetta based on energy functions and ProteinMPNN based on deep neural networks to optimize the foldability of the protein itself, as well as the structural and chemical complementarity with the biliverdin molecule (biliverdin, BV). Finally, the AlphaFold2 structure prediction tool is used to predict the structure of the designed amino acid sequence, and the biliverdin binding protein ( Figure 1 ).

[0041] The designed Pr and Pfr forms of BV binding protein (120 amino acids) gene sequences were synthesized by SynbioB (Jiutian Gene) into pET-28a (+) NcoI ~ XhoI with an 8×His tag at the C-terminus. The cloned plasmid was chemically transformed into Escherichia coli BL21 (DE3) (EC1002, WEIDIBio) stably expressing the HO1 gene. After incubation, heat shock, recovery, and plating, the strains were grown overnight. The well-grown single clones were picked and cultured in LB broth at 37°C.

[0042] The cells were cultured at 220 RPM until OD600 reached 0.8, and IPTG (206-703-0, MACKLIN) with a final concentration of 0.5 mM, glucose (C16827205, MACKLIN), and 25 M 5-ALA (P232516, Damas-bata) were added, and the cells were cultured at 37°C and 220 RPM overnight. The cells were collected by centrifugation at 8000 RPM for 5 minutes, resuspended in 25 ml of protein buffer (25 mM Tris-HCl (pH 8.0) + 150 mM NaCl), and sonicated for 15 minutes (power 70%) using an ultrasonic disruptor. The whole-cell lysate was separated by centrifugation at 12000 RPM for 30 minutes, and the supernatant was collected and the target protein was purified using Ni-NTA (QIAGEN) resin. The target protein was further purified using molecular sieve chromatography Superdex75 10 / 300GL column (29148721, Cytiva) in (25 mM Tris-HCl (pH 8.0) + 150 mM NaCl).

[0043] The purified target proteins were Pr1-2, Pr1-20, Pr1-28, Pr1-37, Pr1-74, Pr1-78, Pr1-86, Pr1-88, Pr2-9, Pr2-19, Pr2-22, Pr2-37, Pr2-44, Pr2-78, Pfr16-v5, Pfr16, Pfr21, Pfr34, and Pfr86, and their corresponding amino acid sequences were SEQ ID No. 1-19 (as shown in the table below).

[0044]

[0045]

[0046]

[0047] In the above sequence, the bold MG represents the starting amino acid M and the linking amino acid G, which are not involved in the regulation of function. GSGHHHHHHHH GSG indicates that it connects amino acids and 8 his residues and is not involved in the regulation of function.

[0048] The following SEQ ID No. 21-39 correspond to the functional sequences of SEQ ID No. 1-19, respectively, that is, Pr1-2, Pr1-20, Pr1-28, Pr1-37, Pr1-74, Pr1-78, Pr1-86, Pr1-88, Pr2-9, Pr2-19, Pr2-37, Pr2-22, Pr2-44, Pr2-78, Pfr16-v5, Pfr16, Pfr21, Pfr34, and Pfr86 do not contain MG at the N-terminus and do not contain MG at the C-terminus. GSGHHHHHHHH of each sequence.

[0049]

[0050]

[0051]

[0052] As shown above, the proteins designed by the present application only include 120 amino acids and are therefore the smallest near-infrared fluorescent proteins currently developed.

[0053] The structural diagrams of Pr2-37, Pfr21, Pr1-88, and Pfr86 are shown in Figure 2 A, chromatogram is shown in Figure 2 Middle B shows that the size of the purified protein is consistent with the designed protein size, and both are in a monomeric state.

[0054] Example 2: Fluorescence property analysis of near-infrared fluorescent protein

[0055] The collected purified protein was measured for absorption and emission spectra using an ultraviolet spectrophotometer (UV2700, Daojin) and a fluorescence spectrometer (FS5, Edinburgh). The protein absorption spectrum was measured between 300 and 800 nm using an ultraviolet spectrophotometer, and the maximum absorption peak of the spectrum was adjusted to 0.1. The results showed that the maximum absorption peak of the designed protein was distributed in the range of 665 to 728 nm ( Figure 3 A, Table 1), the corresponding maximum emission peak is distributed in the range of 699 to 738 nm ( Figure 3 The collected and purified proteins were further detected using the Biospace Optima (Photon Imager Optima, Biospace Lab) small animal in vivo imaging system and found to exhibit strong fluorescence characteristics under the corresponding absorption and emission conditions of the protein ( Figure 3 Middle B).

[0056] Table 1

[0057]

[0058] Figure 3 The absorption and emission peaks of the four near-infrared fluorescent proteins shown are Pr2-37: 655 / 686 nm, Pfr21: 720 / 729 nm, Pr1-88: 665 / 701 n and Pfr86: 705 / 738 nm, and their amino acid sequences are SEQ ID No.: 12, SEQ ID No.: 17, SEQ ID No.: 8, and SEQ ID No.: 19, respectively.

[0059] The more red-shifted the emission spectrum, the higher the resolution, deeper the penetration, and the lower the autofluorescence. The most red-shifted near-infrared fluorescent protein in the prior art, miRFP720, which binds to BV, has a maximum emission peak of 720 nm, and the 720 nm limit cannot be broken through by directed evolution. The proteins designed in this application, Pr1-37 (SEQ ID No.: 4), Pfr16-v5 (SEQ ID No.: 15), Pfr16 (SEQ ID No.: 16), Pfr34 (SEQ ID No.: 18), Pfr21 (SEQ ID No.: 17), and Pfr86 (SEQ ID No.: 19), have emission peaks of 726 nm, 725 nm, 727 nm, 725 nm, 729 nm, and 738 nm, all exceeding the current maximum emission peak of 720 nm (Table 1), further red-shifting and expanding the emission spectrum of near-infrared fluorescent proteins.

[0060] Example 3: Directed evolution of near-infrared fluorescent protein

[0061] In this example, the Pr1-88 (SEQ ID No.: 8) protein in Example 2 was selected for directed evolution.

[0062] Directed evolution technology is used to evolve a near-infrared fluorescent protein with stronger fluorescence brightness. Before conducting directed evolution screening, it is necessary to determine whether Pr1-88 has the characteristics of a near-infrared fluorescent protein in the Escherichia coli monoclonal clone. The results are as follows Figure 4 As shown in Figure A, a single E. coli clone expressing Pr1-88 exhibited excellent near-infrared fluorescence at the corresponding excitation and emission wavelengths. After five rounds of directed evolution of Pr1-88, Pr1-88.4 (SEQ ID No.: 20, and its corresponding untagged sequence is SEQ ID No.: 40) was screened for better near-infrared fluorescent protein properties. Its absorption and emission wavelengths are 670 / 692nm, and its extinction coefficient and quantum yield are significantly improved. Its brightness is enhanced by 4.25 times, reaching 51% of the relative brightness of eGFP, making it the brightest near-infrared fluorescent protein with an emission wavelength greater than 670nm. Figure 4 , Table 2). In addition, Pr1-88.4 has 13 amino acid mutations compared to the original sequence, with a sequence identity of 89% and an identity of 81% with the core region (amino acid 6 to amino acid 116). Mass spectrometry results showed that Pr1-88.4 could bind to the small molecule biliverdin ( Figure 4 Middle D).

[0063]

[0064] Table 2

[0065]

[0066] Example 4: Stability detection of near-infrared fluorescent protein

[0067] In this example, the Pr1-88.4 (SEQ ID No.: 20) protein in Example 3 was selected for protein stability testing.

[0068] The purified Pr1-88.4 protein was collected and its near-infrared fluorescence stability was detected.

[0069] Temperature stability:

[0070] First, Pr1-88.4 was heated to 25℃, 50℃, 75℃, and 100℃ for 5 minutes. Then, the near-infrared fluorescence stability of Pr1-88.4 at different temperatures was measured using a spectrofluorometer. The results showed that the near-infrared fluorescence of Pr1-88.4 was extremely stable within 100℃. Figure 5 Middle A).

[0071] Denaturant stability:

[0072] The denaturant tolerance of Pr1-88.4 near-infrared fluorescence was further determined: Pr1-88.4 was dissolved in 1-8M urea and guanidine hydrochloride, respectively. The results showed that the near-infrared fluorescence of Pr1-88.4 showed almost no change in 1-8M urea, and the near-infrared fluorescence remained stable in 7M guanidine hydrochloride ( Figure 5 (C, D).

[0073] pH stability:

[0074] The Pr1-88.4 protein was eluted with a pH 2-10 buffer (25 mM Tris-HCL (pH 8.0) + 150 mM NaCl). After elution, its fluorescence intensity was measured. The results showed that the near-infrared fluorescence of Pr1-88.4 was stable between pH 2.0 and 9.0, and it showed strong resistance to strong acids and strong bases ( Figure 5 Middle B).

[0075] Pr1-88.4 can be fully matured after being expressed in Escherichia coli for 2 hours and incubated with biliverdin for 10 minutes in vitro, showing good fluorescent protein properties ( Figure 5 (E, F).

[0076] These results indicate that Pr1-88.4 near-infrared fluorescent protein has extremely strong chemical stability.

[0077] Example 5: Imaging analysis of near-infrared fluorescent proteins in mammalian cells

[0078] In this example, the Pr1-88.4 (SEQ ID No.: 20) protein in Example 3 and the Pfr21 (SEQ ID No.: 17) protein in Example 2 were selected to analyze the imaging performance of mammalian cells.

[0079] To test the performance of Pr1-88.4 and Pfr21 as fluorescent probes for labeling intracellular structures, Pr1-88.4 and Pfr21 were cloned into the C-terminus of pCDNA3.1-H2B (constructed in our laboratory) and the N-terminus of pCDNA3.1-Tublin (constructed in our laboratory), respectively, to construct pCDNA3.1-H2B-Pr1-88.4, pCDNA3.1-H2B-Pfr21, pCDNA3.1-Pr1-88.4-Tublin, and pCDNA3.1-Pfr21-Tublin fusion protein plasmids.

[0080] HT1080 (ATCC, CCL-121) and HEK293F (ATCC, CRL-3249) cells were cultured in a humidified incubator containing 10% fetal bovine serum (WISENT) and 1% streptomycin / penicillin (Thermo Fisher) at 37°C and 5% CO2. Experiments were performed in 12-well plates. Before plating the cells, a slide (18125, LABSELECT) was placed in each well, with 50,000 cells plated per well. Transfection was performed after 12 hours of incubation. 2.5 μg of pCDNA3.1-H2B-Pr1-88.4, pCDNA3.1-H2B-Pfr21, pCDNA3.1-Pr1-88.4-Tublin, and pCDNA3.1-Pfr21-Tublin plasmids were transiently transfected into HT1080 and HEK293F cells grown on slides using PEI (PolyScience). The medium was changed every 24 hours, and the cells on the slides were collected after 48 hours.

[0081] The transfected cells were collected and sealed with a mounting medium containing DAPI (ZL0221, Vectorlabs), and the cells were analyzed using a Leica Stellaris 8 super-resolution confocal microscope (ST8, Lecia). The results show that under the corresponding excitation and emission conditions in Table 2, the cells showed specific fluorescence localization in both HT1080 and HEK293F cells. The red fluorescence of H2B-Pr1-88.4 (SEQ ID No.: 41) and H2B-Pfr21 (SEQ ID No.: 42) was only concentrated in the cell nucleus and highly overlapped with the cell nucleus labeled with DAPI; Tublin labeled with Pr1-88.4-Tublin (SEQ ID No.: 43) and Pfr21-Tublin (SEQ ID No.: 44) was evenly distributed in the cytoplasm, perfectly staggered with the cell nucleus labeled with DAPI, and the fluorescence intensity of the group with the addition of 25M small molecule BV (30891, Sigma) was higher ( Figure 6 The results showed that all protein fluorescence in both HT1080 and HEK293F cells showed correct localization and clear separation of Pr1-88.4 and Pfr21 fluorescence signals, indicating that Pr1-88.4 and Pfr21 are good mammalian fluorescent marker proteins.

[0082] Example 6: Imaging and analysis of deep tissues of living mice using near-infrared fluorescent proteins

[0083] In this example, the Pr1-88.4 (SEQ ID No.: 20) proteins described in Example 3 and the Pfr21 (SEQ ID No.: 17) proteins described in Example 2 were selected for analysis of their in vivo imaging performance in mammals. To test the applicability of Pr1-88.4 and Pfr21 for in vivo imaging, in vivo imaging was performed using a xenograft tumor mouse model.

[0084] Construction of a stable expression vector of mi670(PC) using a lentiviral packaging system

[0085] (WEKWiKGENE, 0000006), Pr1-88.4, and Pfr21 cell lines. mi670 (PC), Pr1-88.4, and Pfr21 were constructed into the EcoRI-BamHI region of the lentiviral vector pLVX-IRES-Neo (constructed in our laboratory) to generate the pLVX-mi670-IRES-Neo, pLVX-Pr1-88.4-IRES-Neo, and pLVX-Pfr21-IRES-Neo plasmids. Add 4 μg of lentiviral vector, 400 ng of PVSGV (stored in our laboratory), and 4 μg of PSPAX2 (stored in our laboratory) to 500 μL of serum-free culture medium and vortex to mix thoroughly. Add 3 times the weight of PEI solution and vortex to mix thoroughly. Let stand at room temperature for 15 minutes before adding to 1080 cells in a 10 cm culture dish. After 6-8 hours, replace the cell culture medium with serum-supplemented full medium. After 48-72 hours, the cell culture medium was collected, centrifuged, and filtered using a 0.45 μm filter. The cell culture medium containing the virus was added to 1080 cells in a 12-well plate and cultured for 6-8 hours before changing the medium. After 72 hours, neomycin (1 mg / mL) was added to screen the stable cell lines until a stable cell line with good growth was obtained ( Figure 7 Center A, left side).

[0086] HT1080 cells (1×10 6 HT1080 cells (1x10 6 100 (500 cells) were injected into the fifth pair of left mammary glands as a control. After cell implantation, tumors were allowed to grow for one week and imaged using a Biospace Optima small animal in vivo imaging system (Photon Imager Optima, Biospace Lab). Photoactivated Pr1-88.4 and Pfr21 fluorescence signals were detected one week after cell injection using the corresponding excitation and emission wavelengths shown in Table 2.

[0087] The results showed that the control tumors and tumors expressing Pr1-88.4 and Pfr21 showed strong fluorescence signal contrast. The control tumors did not show fluorescence, while the tumors expressing Pr1-88.4 and Pfr21 showed strong red fluorescence ( Figure 7 Since the emission peaks of mi670 (emi: 670nm) and Pr1-88.4 (emi: 692nm) are close, the fluorescence intensities of mi670 and Pfr21 were compared in vivo. The results showed that the fluorescence intensity of Pr1-88.4 was significantly higher than that of mi670 (( Figure 7 C), and is consistent with the results in stable cells ( Figure 7 (Center A, right). These in vivo results demonstrate that Pr1-88.4 and Pfr21 function well in deep tissue imaging.

Claims

1. A near-infrared fluorescent protein, wherein the amino acid sequence of the near-infrared fluorescent protein is selected from any one of SEQ ID Nos: 21-40.

2. A near-infrared fluorescent protein, wherein: The amino acid sequence of the near-infrared fluorescent protein is selected from any one of SEQ ID Nos: 1-20.

3. A fusion protein consisting of: (i) The near-infrared fluorescent protein according to claim 1 or 2; and (ii). other proteins.

4. The fusion protein according to claim 3, wherein The other protein is a nuclear localization protein or a cytoskeleton protein.

5. The fusion protein according to claim 4, wherein The amino acid sequence of the fusion protein is selected from any one of SEQ ID Nos: 41-44.

6. A nucleic acid molecule encoding the near-infrared fluorescent protein according to claim 1 or 2 or the fusion protein according to any one of claims 3 to 5. A recombinant vector comprising the nucleic acid molecule according to claim 6 . A host cell comprising the recombinant vector according to claim 7 .

9. Use of the near-infrared fluorescent protein according to claim 1 or 2, the fusion protein according to any one of claims 3 to 5, the nucleic acid molecule according to claim 6, the recombinant vector according to claim 7, or the host cell according to claim 8 in the preparation of a near-infrared fluorescent imaging agent or a tumor marker imaging agent, wherein: The near-infrared fluorescence imaging agent can realize near-infrared fluorescence imaging at the cell, tissue and living body levels.

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