Near-infrared fluorescent protein and its application

Abstract

The present application relates to near-infrared two-region fluorescent proteins and applications thereof.The near-infrared two-region fluorescent protein of the present application is a kind of fluorescent protein covalently combined with fluorescent small molecule FD1080 at its cysteine position.The emission spectrum of the near-infrared two-region fluorescent protein of the present application far exceeds the maximum emission peak (720 nm) of the existing near-infrared fluorescent protein, exhibits centimeter-level tissue penetration ability, is the strongest near-infrared fluorescent protein in terms of tissue penetration at present, and therefore has significant advantages in imaging applications in terms of penetration depth and spatial resolution, signal-to-noise ratio, and the like.Therefore, the near-infrared two-region fluorescent protein of the present application has broad prospects in the development of living body deep tissue marker imaging reagents.

Description

Technical Field

[0001] This application belongs to the field of protein technology, specifically relating to a near-infrared II (NIR-II) fluorescent protein and its applications. Background Technology

[0002] Genetically encoded fluorescent proteins have revolutionized our ability to observe life at the molecular level. However, despite decades of technological advancements, their application in living organisms remains limited to cells, thin tissue sections, or invasive in vivo manipulations—methods that only allow for localized and interfering observations of superficial tissues. This limitation stems from the fact that their emission spectra are primarily concentrated in the visible light band, where light scattering, absorption, and tissue autofluorescence rapidly attenuate the signal intensity in deeper tissues. Therefore, non-invasive observation of dynamic biological processes in intact living animals remains difficult.

[0003] Near-infrared (NIR) spectroscopy, particularly the near-infrared II (NIR-II) window (1000–1700 nm), offers a powerful alternative. Light in this band can penetrate several centimeters into tissue with extremely low scattering and background interference, enabling high-resolution, non-invasive deep tissue imaging. However, no genetically encoded fluorescent proteins have yet been reported to function in the NIR-II band. Existing NIR-II probes—such as quantum dots, carbon nanotubes, rare-earth-doped nanoparticles, and organic dyes—while capable of deep imaging, lack gene targeting and biocompatibility. These materials also suffer from poor water solubility, unsatisfactory pharmacokinetics, potential toxicity, or low quantum yields in aqueous environments. Therefore, developing a bright, stable, and genetically encoded NIR-II fluorescent protein remains a pressing need to advance in vivo deep tissue imaging. Summary of the Invention

[0004] Technical Purpose One of the technical objectives of this invention is to provide a near-infrared II fluorescent protein, which has the advantages of small molecular weight, high brightness, red-shifted emission peak, and strong penetration into deep tissues.

[0005] Another object of the present invention is to provide the application of the near-infrared II fluorescent protein in the field of near-infrared II imaging.

[0006] Technical solution On one hand, the present invention provides a near-infrared II fluorescent protein, wherein the near-infrared II fluorescent protein comprises an amino acid sequence selected from SEQ ID No.: 5-8, and each of the near-infrared II fluorescent proteins covalently binds a fluorescent small molecule FD1080 at its cysteine ​​position.

[0007] In some embodiments, the near-infrared II fluorescent protein may further include a tag sequence, particularly a His tag sequence, such as an 8x His tag sequence (HHHHHHHH), to facilitate the purification of the near-infrared II fluorescent protein.

[0008] In a specific embodiment, the amino acid sequence of the near-infrared II fluorescent protein is selected from SEQ ID No: 1-4.

[0009] In a specific embodiment, the amino acid sequence of the near-infrared II fluorescent protein is selected from SEQ ID No: 1 and 5.

[0010] On the other hand, the present invention provides a fusion protein comprising the aforementioned near-infrared II fluorescent protein and other proteins.

[0011] In a specific embodiment, the other protein is a CD276-binding protein or an EGFR-binding protein. In this case, the fusion protein can be used for targeted labeling of cells that highly express CD276 or cells that highly express EGFR.

[0012] In a specific embodiment, the other protein is the CNA35 protein. In this case, the fusion protein can be used to specifically recognize excessively deposited collagen in fibrotic tissue (e.g., kidney or non-fibrotic tissue).

[0013] In a specific embodiment, the amino acid sequence of the fusion protein is selected from SEQ ID No.:9-11.

[0014] On the other hand, the present invention provides a nucleic acid molecule that encodes the above-mentioned near-infrared fluorescent protein or the above-mentioned fusion protein.

[0015] On the other hand, the present invention provides a recombinant vector comprising the above-mentioned nucleic acid molecules.

[0016] On the other hand, the present invention provides a host cell comprising the above-described recombinant vector.

[0017] On the other hand, the present invention provides the application of the above-mentioned near-infrared II fluorescent protein, 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 near-infrared II fluorescent imaging reagents.

[0018] In a specific embodiment, the near-infrared II fluorescence imaging reagent can achieve near-infrared II fluorescence imaging at the cellular, tissue (especially deep tissue), and in vivo levels.

[0019] On the other hand, the present invention provides the application of the above-mentioned near-infrared II fluorescent protein in the preparation of cachexia monitoring reagents or near-infrared II angiography probes.

[0020] On the other hand, the present invention provides the application of the fusion protein of the above-mentioned near-infrared II fluorescent protein and CD276 binding protein in the preparation of a targeted labeling reagent for cells that highly express CD276.

[0021] On the other hand, the present invention provides the application of the fusion protein of the above-mentioned near-infrared II fluorescent protein and EGFR-binding protein in the preparation of a targeted labeling reagent for cells that highly express EGFR.

[0022] On the other hand, the present invention provides the application of the fusion protein of the above-mentioned near-infrared II fluorescent protein and CNA35 protein in the preparation of imaging reagents for detecting kidney and lung fibrosis.

[0023] Beneficial effects This invention utilizes protein design methods to design and obtain a series of near-infrared II fluorescent proteins with red-shifted emission spectra, small molecular weight, bright color, stable chemical properties, and monomeric nature, targeting the FD1080 small molecule.

[0024] Specifically, the near-infrared II fluorescent protein of this application has the following advantages: 1. The emission spectrum is located in the range of 1085-1103 nm, which far exceeds the maximum emission peak of existing near-infrared fluorescent proteins (720 nm), showing centimeter-level tissue penetration ability. It is the near-infrared fluorescent protein with the strongest tissue penetration at present. Therefore, it has significant advantages in terms of penetration depth, spatial resolution, and signal-to-noise ratio in imaging applications.

[0025] 2. It can achieve high-contrast real-time imaging of the whole body and local vascular systems, with extremely low liver accumulation.

[0026] 3. The protein size (approximately 14 kDa) is far below the kidney filtration threshold (approximately 40 kDa), allowing for rapid systemic clearance through the kidneys.

[0027] 4. It can be used as a tool to directly and in real time report glomerular filtration, enabling non-invasive and dynamic assessment of kidney function.

[0028] In addition, the near-infrared II fluorescent protein fusion protein of this application specifically labels deep tissues and exhibits excellent in vivo deep tissue imaging, making it the near-infrared fluorescent protein with the strongest penetration into in vivo tissues to date.

[0029] In summary, this invention is the first to report a near-infrared II fluorescent protein, which solves the common technical problems of current near-infrared fluorescent proteins, namely, the emission wavelength is limited to the near-infrared I region, the brightness is low, and the penetration into deep tissues is poor. Therefore, the near-infrared II fluorescent protein of this application has broad application prospects in imaging and in vivo deep tissue labeling imaging. Attached Figure Description

[0030] Figure 1 Flowchart of the design scheme for near-infrared fluorescent proteins combined with FD1080 (A); Cys–FD1080 nucleophilic substitution reaction mechanism (B); Absorption and emission peak diagrams of dsFP11, dsFP32, dsFP36, and dsFP46 (C).

[0031] Figure 2 Chromatographic results of dsFP11 (A), fluorescence spectrum (B), and mass spectrum of dsFP11 incubated with FD1080 small molecule (C), as well as fluorescence spectra of dsFP11 protein under different depths of Intralipid coverage (D) and under different thicknesses of chicken breast coverage (E).

[0032] Figure 3 Fluorescence images of EGFRb-dsFP11 and HM9-dsFP11 proteins (A, C) and cellular fluorescence images after 2 hours of incubation of EGFRb-dsFP11 and HM9-dsFP11 proteins (B, D).

[0033] Figure 4 Fluorescence image of dsFP11 protein in vivo angiography (A) and renal excretion kinetic analysis (B).

[0034] Figure 5 Schematic diagram of renal function monitoring in tumor-bearing cachexia mouse model (A), time-series NIR-II imaging of bladder fluorescence signal after dsFP11 injection (B), and hematoxylin-eosin (H&E) staining of kidney tissue (C).

[0035] Figure 6 Figure A shows a schematic diagram of dsFP11 fusion protein targeting different organs. Figure B shows fluorescence imaging of dsFP11 fusion protein targeting the kidney (B), lung (C), and liver (D). In Figure A, purple represents dsFP11; brown represents the binding reagent; and green represents the target site. Detailed Implementation

[0036] The technical content of this application is described in detail below through specific embodiments to enable those skilled in the art to better understand the present invention; however, these embodiments are not intended to limit the scope of the present invention.

[0037] Preparation Example 1: Protein Design, Expression, Purification, and Fluorescence Characterization This application establishes a novel rational design strategy for small molecule binding proteins. First, conformational sampling and energy optimization of the fluorescent small molecule FD1080 are performed using quantum mechanical calculations to obtain its stable conformational state under physiological conditions. Based on these conformations, a diverse protein scaffold capable of specifically recognizing different FD1080 conformations is constructed using a self-developed de novo protein design platform. Subsequently, the Rosetta energy function and ProteinMPNN deep neural network are combined to synergistically optimize sequence foldability and structure-chemical complementarity with ligands. Finally, structure prediction is performed using AlphaFold2, and candidate proteins whose predicted structures are highly consistent with the design model are screened. Figure 1 A). This process successfully yielded a novel protein scaffold capable of efficiently covalently binding to FD1080, providing an important molecular tool for small molecule probe-based biosensing and in vivo imaging technologies.

[0038] To express the designed FD1080 binding protein dsFPs, its gene sequence was synthesized and cloned into the pET-28a(+) vector (C-terminal 8×His tag, NcoI–XhoI site), and transformed into E. coli BL21(DE3) (EC1002, WEIDIBio) stably expressing HO1. Single colonies were picked and cultured in LB medium at 37°C and 220 rpm until OD600 ≈ 0.8. Then, 0.5 mM IPTG (206-703-0, MACKLIN), 2 mM glucose (C16827205, MACKLIN), and 25% HCl were added. M5-ALA (P232516, Damas-bata) induced protein expression, and the cells were cultured overnight at 37°C. The bacterial cells were collected by centrifugation at 8,000 × g, resuspended in lysis buffer (25 mM Tris-HCl pH 8.0, 150 mM NaCl), sonicated (70% power, 15 min), and centrifuged at 12,000 × g. The supernatant was collected. The target proteins (referred to in this paper as dsFP11, dsFP32, dsFP36, and dsFP46, respectively, with amino acid sequences SEQ ID No. 1-4) were enriched by Ni-NTA (QIAGEN) affinity chromatography.

[0039] Next, the collected target protein was incubated with FD1080 (molar ratio 1:3) at 37 °C for 12 h, dialyzed with a 3000 Da dialysis tape for 5 h, and then further purified by size exclusion chromatography (SEC) on a Superdex 75 10 / 300 GL (29148721, Cytiva) to obtain the target protein that further covalently binds to FD1080. (Note: The aforementioned target protein forms a covalent coupling with FD1080 at its respective cysteine ​​sites. Specifically, the cysteine ​​thiol in the protein attacks the chloroalkyl group of FD1080, replacing the chloride ion to form a stable covalent bond. The resulting structure can be found in [link to structural diagram].) Figure 1 China B and Figure 2 (C)

[0040] The absorption spectra (900–1200 nm range) of these proteins were measured using a UV-Vis-NIR spectrophotometer (UV-3600, Shimadzu), and the maximum absorption peak was adjusted to 0.1 for normalization analysis. The results showed that the maximum absorption peaks of these proteins covalently bound to FD1080 were located in the range of 1050 nm–1063 nm. Figure 1 C). Simultaneously, its emission spectrum was measured using a fluorescence spectrometer (Edinburgh FLS1000 + FS5 + PicoQuant Pico100), and the corresponding maximum emission peak was detected at 1084 nm - 1103 nm. Figure 1 (C)

[0041]

[0042] In the above sequences, the bolded M represents the starting amino acid M, which does not participate in functional regulation. The underline GSGHHHHHHHH indicates that GSG connects the amino acid and the 8 his, and it also does not participate in functional regulation.

[0043] The following SEQ ID Nos. 5-8 correspond to the functional sequences of SEQ ID Nos. 1-4 above, that is, the sequences of dsFP11, dsFP32, dsFP36, and dsFP46 whose N-terminus does not contain M and whose C-terminus does not contain GSGHHHHHHHH.

[0044]

[0045] As shown above, the proteins designed in this application consist of only 120 amino acids, making them the smallest near-infrared fluorescent proteins developed to date.

[0046] Note: All proteins and / or fusion proteins used to measure imaging performance in the following text are covalently bound to FD1080.

[0047] Test Example 1: Evaluation and Analysis of dsFP11's Tissue Penetration Depth Sufficient tissue penetration capability has always been one of the key challenges in in vivo fluorescence imaging technology; however, this performance is crucial for resolving biological structures and dynamic life activities in deep tissues. In this embodiment, dsFP11 was selected for tissue penetration depth evaluation analysis. SEC results showed that dsFP11 eluted with a single peak, corresponding to the monomer state ( Figure 2 (A). This result indicates that dsFP11 can be solublely expressed and stably forms monomers in *E. coli*, exhibiting good structural homogeneity, making it suitable for subsequent functional and structural studies. Further fluorescence imaging of the purified protein was performed using an InGaAs CCD small animal in vivo imaging system (AniView1000, BLT) under matched excitation and emission conditions. The results showed that dsFP11 possessed a significant and stable fluorescence signal in the near-infrared II (NIR-II) region. Figure 2 (B), confirming its excellent fluorescence properties. Mass spectrometry results show that dsFP11 can covalently bind to the small molecule FD1080 (B) after in vitro incubation. Figure 2 (C). The above data indicate that the designed dsFP11 protein possesses ideal optical properties at wavelengths of 1063 / 1085 nm, making it suitable for high-sensitivity fluorescence imaging applications in deep tissues.

[0048] To test the tissue penetration potential of dsFP11, this embodiment used a 1% Intralipid (HY-131370B, MCE) solution with a depth of 0-13 mm as the tissue optical simulation medium. A capillary encapsulated with dsFP11 was placed at the bottom of the 1% Intralipid solution for fluorescence imaging analysis. Figure 2 As shown in Figure D, in the near-infrared II (NIR-II, >1300 nm) emission window, although the fluorescence signal intensity decreases to some extent with the increase of Intralipid solution thickness, the morphological boundary of the capillary can still be clearly distinguished; even under the condition of simulating tissue depth up to 1 cm, its outline still maintains good visibility and spatial resolution.

[0049] To further verify the imaging performance of dsFP11 in a real biological tissue environment, its penetration ability was further evaluated in a chicken breast muscle tissue model. Figure 2 As shown in Figure E, when the tissue thickness does not exceed 7 mm, the covering tissue has little impact on the fluorescence signal, and the apparent diameter of the imaged capillary is highly consistent with the measurement value under the condition of no tissue coverage. It is worth noting that when the tissue coverage thickness increases to 1 cm, the outline structure of the capillary can still be clearly identified.

[0050] The above results demonstrate that dsFP11 exhibits excellent tissue penetration performance in the NIR-IIb (>1300 nm) imaging window, possessing the potential to achieve centimeter-level depth in vivo fluorescence imaging, and is expected to provide a new molecular tool for high-resolution optical imaging of deep tissues.

[0051] Test Example 2: Imaging Analysis of dsFP11 Protein in Mammalian Cells To evaluate the performance of dsFP11 as a fluorescently labeled probe, it was fused to the C-terminus of CD276-binding protein (CD276b, i.e., HM9) and EGFR-binding protein (EGFRb), respectively, to construct expression plasmids pET-CD276b-dsFP11 and pET-EGFRb-dsFP11, which were then purified via prokaryotic expression. The amino acid sequences of the expressed fusion proteins HM9-dsFP11 and EGFRb-dsFP11 are SEQ ID No:9 and SEQ ID No:10, respectively.

[0052] The purified fusion protein was incubated with FD1080 (molar ratio 1:3) at 37 °C for 12 h to form a covalent coupling as shown in Example 1. Fluorescence signals were detected using an InGaAs CCD small animal in vivo imaging system (AniView1000, BLT) under matched excitation and emission conditions. Figure 3 China A Figure 3 As shown in Figure C, at equimolar concentrations, the fluorescence intensity of dsFP11 is consistent with that of the two fusion proteins, indicating that its luminescence performance is not affected by the fusion tag and that it possesses good structural stability and optical fidelity.

[0053] Further cell-level validation was performed using SK-N-BE(2) cells (NCACC, TcHu200) highly expressing CD276 and A431 cells (NCACC, TcHu188) highly expressing EGFR. 293F cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C in a 5% CO2 humidified incubator to a density of 80–90%. After fixation (4% PFA, 20 min), they were incubated with CD276b-dsFP11 or EGFRb-dsFP11 at room temperature for 2 h, washed with PBS, and subjected to NIR-II fluorescence imaging. The results showed that target cells exhibited strong and specific fluorescence signals, non-target cells had low background, and signal contrast was clear. Figure 3 B, Figure 3 (D).

[0054] In summary, dsFP11 maintains excellent fluorescence performance in fusion expression and cell-targeted labeling, demonstrating high stability and specificity, confirming its potential as a highly efficient near-infrared II mammalian cell marker protein.

[0055] Test Example 3: Analysis of dsFP11 protein in in vivo angiography The vascular system plays a crucial role in systemic nutrient delivery and organ function maintenance. High-quality angiography is essential for the accurate identification of intraoperative vascular lesions and the assessment of postoperative blood flow restoration. Therefore, this study presents a systematic preclinical NIR-II vascular imaging study of the dsFP11.

[0056] like Figure 4 As shown in Figure A, after intravenous injection of 30 μmol dsFP11 into the tail vein of 8-week-old nude mice (Shanghai Southern Model Organism), dsFP11 demonstrated superior imaging contrast and resolution compared to FD1080 small molecule dye and AIE1010 (FNP2A / B03, Boluteng) in systemic vascular imaging, without local enrichment in organs. Following intravenous injection, dsFP11 enabled clear differentiation between arteries and veins and supported dynamic tracking of major blood vessels throughout the body. Its effective angiography time window was approximately 1 hour, after which the signal was rapidly cleared from the bloodstream and gradually enriched in the bladder via renal metabolism. Figure 4 (B) These results indicate its efficient urinary excretion capacity—a key characteristic for clinical safety and repeat dosing.

[0057] In summary, dsFP11 combines excellent angiography performance with good pharmacokinetics and rapid renal excretion kinetics, demonstrating its potential as a highly efficient and safe NIR-II zone angiography probe for clinical application.

[0058] Test Example 4: Monitoring and Evaluation of Renal Function in Tumor-Bearing Cachexia Mice using dsFP11 Protein The pharmacokinetic properties demonstrated in the previous embodiment make dsFP11 not only a safe contrast agent but also a real-time biomarker of glomerular filtration function. Currently used clinical markers of kidney injury—such as serum creatinine, cystatin C, and the urinary albumin-to-creatinine ratio (UACR)—are all indirect, static indicators that reflect cumulative functional impairment rather than real-time changes in filtration kinetics. These indicators are often insensitive to early lesions and are easily affected by non-renal factors such as muscle mass, inflammation, and diet. In contrast, the molecular size of dsFP11 (approximately 14 kDa) is highly matched to the clinical marker cystatin C (approximately 13 kDa), allowing it to serve as a direct, real-time reporter molecule for glomerular filtration, enabling non-invasive, dynamic assessment of kidney function.

[0059] Cancer cachexia is a systemic metabolic syndrome characterized by weight loss (including loss of fat and muscle) and kidney damage, directly contributing to the death of at least 20% of cancer patients. Current imaging techniques have not yet enabled early detection and quantitative assessment of kidney damage in cancer cachexia. This embodiment constructs a xenograft cancer cachexia model and its early detection protocol for kidney damage. Figure 5 (A)

[0060] Establishment of a xenograft cancer cachexia model: 6-8 week old male nude mice (Shanghai model organism) were fasted for 12 hours preoperatively. After intraperitoneal anesthesia, the skin was fixed and disinfected. The abdominal cavity was incised along the midline to expose the liver. The liver lobes were pulled out of the incision with cotton swabs, and then a needle was inserted into the liver at an oblique angle for about 3 mm. Then, 1×10⁻⁶ mmol / L was slowly injected. 6 SK-N-BE(2) / PC-3 tumor cells. Finally, the liver was gently placed back into the abdominal cavity and sutured layer by layer. Mice carrying liver tumors were monitored, and mice exhibiting a tumor-free weight loss of ≥6% from baseline, accompanied by muscle wasting, were classified as cachexia.

[0061] All animal experiments were conducted in strict accordance with ethical guidelines and approved by the Animal Care and Ethics Committee of Westlake University (ethics approval number: AP#25-042-2-CLX-2). Laboratory mice were housed individually under controlled conditions, with the room temperature maintained at 22-24°C and a 12-hour light-dark cycle. Throughout the experiments, animals had access to ample water and standard feed to ensure their health and minimize stress.

[0062] In a xenograft model of cancer cachexia (30 days) (PC-3 and SK-N-BE(2) cells (Cell Bank)), a significant acceleration of renal filtration was observed after tail vein injection of 30 μmol dsFP11. Control mice (day 0) showed bladder signals approximately 70 minutes after injection, while tumor-bearing mice (day 20), despite stable body weight, showed strong fluorescence within 30-40 minutes. By day 30, with the progression of cachexia, excretion further accelerated, and the signal detection time shortened to 10-20 minutes. To our knowledge, this is the first real-time observation of increased glomerular permeability before the onset of overt wasting, suggesting that impaired renal filtration barrier function may be an early event in the pathogenesis of cachexia. Figure 5 (Middle B). Hematoxylin and eosin (H&E) staining of kidney sections from tumor-bearing mice before cachexia further confirmed early glomerular damage, observing damage characterized by renal tubular dilation, vacuolation, and brush border loss. Figure 5 (C). These findings reveal a previously unknown link between systemic metabolic decline and kidney function, providing a new window for the early detection and intervention of cachexia.

[0063] Example 6: Imaging analysis of dsFP11 protein in deep organ tissues of live mice Reliable molecular visualization is crucial for elucidating biological functions and monitoring disease occurrence, development, and treatment response. Although indocyanine green (ICG) has been used in clinical lymphangiography, its optical properties in the NIR-I and limited NIR-IIa windows (<1100 nm) restrict tissue penetration depth and imaging resolution, limiting the potential of near-infrared fluorescence imaging in deep tissue applications. To overcome this bottleneck, this embodiment evaluates the ability of the dsFP11 fluorescent protein in the NIR-IIb (1300–1700 nm) region for molecular imaging of deep organs. Figure 6 (A)

[0064] Fibrosis is a common pathological feature of many chronic diseases, with renal and pulmonary fibrosis having a significant impact on global morbidity and mortality. Current imaging techniques still suffer from insufficient sensitivity in the early detection and quantitative assessment of fibrosis, necessitating the development of non-invasive, high-resolution in vivo monitoring methods. To this end, this application constructs a collagen-binding protein CNA35-dsFP11 (SEQ ID No:11) and incubates it with FD1080 (molar ratio 1:3) at 37 °C for 12 h to achieve covalent binding with FD1080 (see Example 1).

[0065] Used to specifically identify excessive collagen deposits in fibrotic tissue.

[0066] In a mouse model of unilateral ureteral obstruction (UUO), 12 to 24 hours after tail vein injection of 30 μmol of CNA35-dsFP11 covalently bound to FD1080, NIR-IIb imaging showed that the fluorescence signal of the fibrotic kidney was significantly higher than that of the contralateral healthy kidney tissue, and the probe clearance rate was significantly delayed, suggesting enhanced retention in the lesion area. Figure 6 (Middle B). Similar results were also validated in pulmonary fibrosis models ( Figure 6 (C)

[0067] In a nude mouse model of SK-N-BE(2) liver xenograft tumors, after intravenous injection of 30 μmol of CD276b-dsFP11 covalently bound to FD1080 (prepared as described in Example 3 above), emission signals at 1300–1700 nm were acquired using a wide-field imaging system with a 1064 nm excitation and a 2D InGaAs camera. The results showed that, compared to the control, significant and well-defined fluorescence enrichment was observed at the tumor site 12 hours post-injection, while background signals in the normal liver and kidneys were extremely low. Even after 24 hours, the target signal remained highly visible, indicating that the dsFP11 fusion probe possesses excellent in vivo stability, targeting specificity, and a high signal-to-noise ratio, making it suitable for long-term dynamic molecular imaging. Figure 6(D).

[0068] In summary, the dsFP11-based NIR-II molecular probe not only enables high-contrast imaging of deep tumors, but also allows for sensitive and non-invasive dynamic monitoring of the progression of renal and pulmonary fibrosis, demonstrating its broad application prospects in the precise diagnosis and efficacy evaluation of major chronic diseases.

Claims

1. A near-infrared II fluorescent protein, wherein, The near-infrared II fluorescent protein comprises an amino acid sequence selected from SEQ ID No.:5-8, and each of the near-infrared II fluorescent proteins covalently binds the fluorescent small molecule FD1080 at its cysteine ​​position.

2. The near-infrared II fluorescent protein according to claim 1, wherein, The near-infrared II fluorescent protein further includes a tag sequence.

3. The near-infrared II fluorescent protein according to claim 1, wherein, The amino acid sequence of the near-infrared II fluorescent protein is selected from SEQ ID No: 1-4, preferably, the amino acid sequence of the near-infrared II fluorescent protein is selected from SEQ ID No: 1 and 5.

4. A fusion protein comprising a near-infrared II fluorescent protein as described in any one of claims 1-3 and other proteins.

5. The fusion protein according to claim 4, wherein, The other proteins are selected from CD276-binding protein, EGFR-binding protein, and CNA35 protein.

6. The fusion protein according to claim 4, wherein, The amino acid sequence of the fusion protein is selected from SEQ ID No.:9-11.

7. A nucleic acid molecule encoding a near-infrared fluorescent protein as described in any one of claims 1-3 or a fusion protein as described in claim 5 or 6.

8. A recombinant vector comprising the nucleic acid molecule as described in claim 7.

9. The use of the near-infrared II fluorescent protein as described in any one of claims 1-3, the fusion protein as described in any one of claims 4-6, the nucleic acid molecule as described in claim 7, or the recombinant vector as described in claim 8 in the preparation of near-infrared II fluorescence imaging reagents.

10. The use of the near-infrared II fluorescent protein as described in any one of claims 1-3 in the preparation of a cachexia monitoring reagent or a near-infrared II angiography probe.

11. The use of the fusion protein as described in claim 5 in the preparation of a targeting labeling reagent for cells highly expressing CD276, wherein, The fusion protein is a fusion protein of near-infrared II fluorescent protein and CD276 binding protein as described in any one of claims 1-3.

12. The use of the fusion protein as described in claim 5 in the preparation of a targeting labeling reagent for cells highly expressing EGFR, wherein, The fusion protein is a fusion protein of near-infrared II fluorescent protein and EGFR-binding protein as described in any one of claims 1-3.

13. The use of the fusion protein of claim 5 in the preparation of imaging reagents for detecting renal and pulmonary fibrosis, wherein, The fusion protein is a fusion protein of near-infrared II fluorescent protein and CNA35 protein as described in any one of claims 1-3.

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