Near-infrared fluorescent protein and application thereof

Abstract

The invention relates to a near-infrared fluorescent protein and application thereof. The near-infrared fluorescent protein comprises an amino acid sequence selected from SEQ ID No. 21 to SEQ ID No. 40. The invention also relates to a fusion protein containing the near-infrared fluorescent protein. The near-infrared fluorescent protein disclosed by the invention has the advantages of red shift of emission spectrum, small molecular weight, brightness and stable chemical property, specifically, the size of the near-infrared fluorescent protein is only 120 amino acids, and the emission spectrum is in a range of 672-738nm and far exceeds the maximum emission peak value (720nm) of the existing near-infrared fluorescent protein; and the fluorescence intensity is almost unchanged in the extreme environment of 0-100 DEG C, pH of 2-10 and 0-8M 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 are advantageous for non-invasive imaging in living animals due to their exceptional light penetration within the near-infrared optical window (650-900nm). This is primarily due to the minimal combined effects of hemoglobin, water, and lipids on tissue absorption and reduced light scattering within this optical window. Therefore, near-infrared fluorescent proteins have become the first choice for protein labels and genetically encoded indicators in in vivo imaging applications.

[0003] Currently, near-infrared fluorescent proteins are mainly modified from natural biliverdin-binding proteins (bacterial phytochromes and cyanobacterial phycobiliproteins) to covalently link biliverdin (BV) as a fluorophore. Biliverdin is an intermediate in mammalian heme metabolism and is abundant in mammalian cells, so these biliverdin-binding fluorescent proteins are very suitable for use as fluorescent tags without the need for exogenous fluorophore supply. Biliverdin is inherently non-fluorescent in aqueous solution, but the binding of biliverdin to these proteins produces fluorescence due to the rigidification process that inhibits energy dissipation through molecular rotation, heating, photochemistry, internal conversion, or solvent quenching. Despite the optimization of the photophysical and biochemical properties of these natural protein-derived fluorescent proteins through countless random mutagenesis-based direct evolution, structure-based mutagenesis, and rational design, problems still exist, 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 application of the near-infrared fluorescent protein or its mutant 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 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more sequence identity 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 contain 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 in the range of 672-738 nm, and whose fluorescence intensity is almost unchanged in extreme environments such as 0-100°C, pH 2-10, and denaturant up to 8 M. That is, its fluorescence intensity in extreme environments such as 0-100°C, pH 2-10, and denaturant up to 8 M is equivalent to the fluorescence intensity at 25°C, neutral conditions, and without denaturant.

[0015] On the other hand, the present invention provides a fusion protein, wherein the fusion protein comprises the above-mentioned near-infrared fluorescent protein and other proteins.

[0016] In a specific embodiment, the other proteins are nuclear localization 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] On the other hand, the present invention provides a nucleic acid molecule encoding the above-mentioned near-infrared fluorescent protein or its mutant or the above-mentioned 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 cell, tissue and living body levels.

[0025] Beneficial Effects

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

[0027] Specifically, the emission spectra of the near-infrared fluorescent proteins are respectively within the range of 672 to 738 nm, which far exceeds the maximum emission peak value (720 nm) of existing near-infrared fluorescent proteins. Therefore, they have significant advantages in terms of 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 near-infrared fluorescent protein with the smallest 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, and is currently the near-infrared fluorescent protein with the most stable chemical properties.

[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 marking imaging. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0032] Figure 2Shown are the structural schematic diagrams (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 5 rounds of directed evolution of Pr1-88 are shown. Fluorescence image of Escherichia coli expressing near-infrared fluorescent protein Pr1-88 under corresponding excitation and emission spectra (A), light microscopy image of Escherichia coli Pr1-88.4 obtained after 5 rounds of directed evolution of Pr1-88 (B, left) and fluorescence image under corresponding excitation and emission spectra (B, right), chromatographic result image and absorption and emission spectrum image of Pr1-88.4 (left and middle images of C), protein fluorescence image of Pr1-88.4 under corresponding absorption and emission spectra (right image of C), mass spectrum of Pr1-88.4 not bound to BV (left image of D), mass spectrum of Pr1-88.4 bound to BV (right image 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 vivo and in vitro maturation of the near-infrared fluorescent protein Pr1-88.4 (E, F).

[0036] Figure 6 The infrared fluorescent protein Pr1-88.4 and Pfr21 fusion protein imaging analysis in mammalian cells. The near-infrared fluorescent protein Pr1-88.4 and Pfr21 labeled nucleosomal proteins and tubulin imaging of HT1080 and HEK293F.

[0037] Figure 7 The imaging analysis of near-infrared fluorescent proteins Pr1-88.4 and Pfr21 in deep tissues of living mice is shown. Fluorescence imaging of Pr1-88.4 stable cell lines (A), fluorescence imaging of near-infrared fluorescent proteins Pr1-88.4 and Pfr21 in deep tissues of living mice (B), and fluorescence imaging of 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 completely 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 structure 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 a biosynthetic company (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) that stably expressed the HO1 gene, incubated, heat-shocked, revived, plated and grown overnight, and the well-growing monoclonal strains were picked and incubated 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 RMP for 5 minutes, resuspended in 25 ml protein buffer (25 mM Tris-HCL (pH 8.0) + 150 mM NaCl), and ultrasonically lysed 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 are 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 are SEQ ID No.1-19 (as shown in the table below).

[0044]

[0045]

[0046]

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

[0048] The following SEQ ID No.21-39 correspond to the functional sequences of the above SEQ ID No.1-19, 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 in 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 In Figure B, the figure 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 absorption spectrum of the protein between 300 and 800 nm was measured 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 by Biospace Optima (Photon ImagerOptima, 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 (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 / 686nm, Pfr21: 720 / 729nm, Pr1-88: 665 / 701n and Pfr86: 705 / 738nm, 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 the emission spectrum is red-shifted, the imaging has the significant advantages of higher resolution, deeper penetration and lower autofluorescence. The most red-shifted near-infrared fluorescent protein miRFP720 combined with BV in the prior art has a maximum emission peak of 720nm, and the 720nm limit cannot be broken through by directed evolution. The proteins 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) designed by the present application have emission peaks of 726nm, 725nm, 727nm, 725nm, 729nm and 738nm, all exceeding the current maximum emission peak of 720nm (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 near-infrared fluorescent proteins with stronger fluorescence brightness. Before conducting directed evolution screening, it is necessary to determine whether Pr1-88 has near-infrared fluorescent protein characteristics in Escherichia coli monoclonal clones. The results are as follows Figure 4 As shown in Figure A, the E. coli monoclone expressing Pr1-88 exhibited good near-infrared fluorescence at the corresponding excitation and emission wavelengths. After 5 rounds of directed evolution of Pr1-88, Pr1-88.4 (sequence is as follows: SEQ ID No.: 20, and its corresponding sequence without a tag is SEQ ID No.: 40) with better near-infrared fluorescent protein characteristics was screened out. The absorption and emission wavelengths are 670 / 692nm, the extinction coefficient and quantum yield are significantly improved, and its brightness is enhanced by 4.25 times, reaching 51% of the relative brightness of eGFP. It is currently 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 with 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 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 min, and 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 (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 had almost no change in 1-8M urea, and the near-infrared fluorescence was still 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), and its fluorescence intensity was measured after elution. The results showed that the near-infrared fluorescence of Pr1-88.4 was stable between pH 2.0 and 9.0, and showed strong resistance to strong acids and strong bases ( Figure 5 (B).

[0075] Pr1-88.4 can be fully matured after being expressed in Escherichia coli for 2 h and incubated with biliverdin in vitro for 10 min, 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) 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] In order 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] The cells used were HT1080 (ATCC, CCL-121) and HEK293F (ATCC, CRL-3249) cells, which were cultured in a humidified incubator containing 10% fetal bovine serum (WISENT) and 1% streptomycin / penicillin (Thermo Fisher) at 37°C and 5% CO2. The experiment was conducted using a 12-well plate, and a slide (18125, LABSELECT) was placed in each culture well before the cells were plated, with 50,000 cells per well, and transfection was performed after the cells were cultured for 12 hours. 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, sealed with a sealing agent containing DAPI (ZL0221, Vectorlabs), and 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 nucleus and highly overlapped with the 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 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 the 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 analysis of near-infrared fluorescent proteins in deep tissues of living mice

[0083] 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 for analysis of mammalian in vivo imaging performance. In order to test the applicability of Pr1-88.4 and Pfr21 in in vivo imaging, a xenograft tumor mouse model was used for in vivo imaging.

[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 interval of the lentiviral vector pLVX-IRES-Neo (constructed in this laboratory) to obtain pLVX-mi670-IRES-Neo, pLVX-Pr1-88.4-IRES-Neo and pLVX-Pfr21-IRES-Neo plasmids. 4μg lentiviral vector, 400ng PVSGV (stored in this laboratory), 4μg PSPAX2 (stored in the laboratory) were added to 500L serum-free culture medium and vortexed. Add 3 times the mass of PEI solution and vortex to mix, let stand at room temperature for 15 minutes, and then add it to 1080 cells in a 10cm culture dish. After 6-8h, the cell culture medium was replaced with complete culture medium containing serum. 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 (1x10 6 HT1080 cells (1x10 6 ) were injected into the fifth pair of left mammary glands as controls. After cell implantation, tumors were allowed to grow for 1 week and imaged using the Biospace Optima small animal in vivo imaging system (Photon Imager Optima, Biospace Lab). The photoactivated Pr1-88.4 and Pfr21 fluorescence signals were detected 1 week after cell injection using the corresponding excitation and emission lights in Table 2, respectively.

[0087] The results showed that the control tumors and tumors expressing Pr1-88.4 and Pfr21 showed a strong contrast in fluorescence signals. 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 intensity of mi670 and Pfr21 in vivo imaging was compared. 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 indicate that Pr1-88.4 and Pfr21 perform well in deep tissue imaging.

Claims

1. 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.

2. The near-infrared fluorescent protein or a mutant thereof according to claim 1, wherein 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.

3. The near-infrared fluorescent protein or a mutant thereof according to claim 1, wherein 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.

4. The near-infrared fluorescent protein or a mutant thereof according to claim 1, wherein The amino acid sequence of the near-infrared fluorescent protein is selected from SEQ ID No.: 21-40.

5. The near-infrared fluorescent protein or a mutant thereof according to claim 1, wherein The near-infrared fluorescent protein comprises an amino acid sequence selected from SEQ ID No.: 1-20, or the amino acid sequence of the near-infrared fluorescent protein is selected from SEQ ID No.: 1-20.

6. A fusion protein, comprising: (i) the near-infrared fluorescent protein or a mutant thereof according to any one of claims 1 to 5; and (ii) other proteins.

7. The fusion protein according to claim 6, wherein The other proteins are nuclear localization proteins and cytoskeletal proteins.

8. The fusion protein according to claim 7, wherein The amino acid sequence of the fusion protein comprises an amino acid sequence selected from any one of SEQ ID Nos.: 41-44, or the amino acid sequence of the fusion protein is selected from any one of SEQ ID Nos.: 41-44.

9. A nucleic acid molecule encoding the near-infrared fluorescent protein or a mutant thereof as described in any one of claims 1 to 5 or the fusion protein as described in any one of claims 6 to 8.

10. A recombinant vector comprising the nucleic acid molecule according to claim 9. A host cell comprising the recombinant vector according to claim 10 .

12. Use of the near-infrared fluorescent protein or mutant thereof according to any one of claims 1 to 5, the fusion protein according to any one of claims 6 to 8, the nucleic acid molecule according to claim 9, the recombinant vector according to claim 10 or the host cell according to claim 11 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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