POLYPEPTIDE FOR TARGETING RECOGNITION OF IMMUNE CELLS AND APPLICATION THEREOF

Abstract

The present disclosure relates to a polypeptide recognizing immune cells, the polypeptide includes the following amino acid sequences: (a) an amino acid sequence containing C-terminal fragment sequence AILEVLQS of human Triokinase/FMN cyclase and its homologous sequence; or (b) an amino acid sequence that is substantially identical to the amino acid sequence described in (a), the substantially identical means 70% or more sequence identity to the amino acid sequence described in (a). The present invention also relates to a nucleic acid sequence encoding the polypeptide; a polypeptide probe used for targeting recognition of immune cells and containing the polypeptide described above and a reporter; a kit containing the probe described above; and, related applications of the polypeptide or probe described above.

Claims

1.-16. (canceled)

17. A polypeptide for targeting recognition of immune cells, wherein the polypeptide comprises: (a) an amino acid sequence containing the C-terminal fragment sequence AILEVLQS (SEQ ID NO.:1) of human Triokinase/FMN cyclase; or (b) an amino acid sequence that is substantially identical to the amino acid sequence described in (a), wherein the term “substantially identical” means that an amino acid sequence has 70% or more sequence identity to the amino acid sequence described in (a), wherein the amino acid sequences of (a) and (b) comprise 80 or less amino acid residues.

18. The polypeptide according to claim 17, wherein the amino acid sequences of (a) and (b) comprise 60 or less amino acid residues

19. The polypeptide according to claim 18 wherein the amino acid sequences of (a) and (b) comprise 45 or less amino acid residues.

20. The polypeptide according to claim 17, wherein the amino acid sequence of (a) is AILEVLQS (SEQ ID NO.: 1), LRAILEVLQS (SEQ ID NO.: 2), ILRAILEVLQS (SEQ ID NO.: 3), AAILRAILEVLQS (SEQ ID NO.: 4), EQPDPGAVAAAAILRAILEVLQS (SEQ ID NO.: 5), PGAVAAAAILRAILEVLQ (SEQ ID NO.: 6) or TKNMEAGAGRASYISSARLEQPDPGAVAAAAILRAILEVLQS (SEQ ID NO.: 7).

21. The polypeptide according to claim 17, wherein the amino acid sequence of (b) is a sequence from a C-terminal fragment of a non-human Triokinase/FMN cyclase and has 70% or more sequence identity to the amino acid sequence of (a).

22. The polypeptide according to claim 21, wherein the amino acid sequence of (b) is AVLEVLQG (SEQ ID NO.: 8), VLRAVLEVLQG (SEQ ID NO.: 9), EQPDPSAVAAAAILRAILEVLQG (SEQ ID NO.: 10), LQPDPSAVAAAAVLRAVLEVLQG (SEQ ID NO.: 11), LQPDPGAVAAAAVLRAVLEGLQG (SEQ ID NO.:12), DQPDPGAVAAAAIFRAILEVLQTKAA (SEQ ID NO.: 13), DQPDPGAVAAAAILRTILEVLQSQGV (SEQ ID NO.: 14), DQPDPGAVAAAAILRAILEVLQSQGA (SEQ ID NO.: 15), or EQPDPGAVAAAAILRAILEVLQS (SEQ ID NO.: 16).

23. The polypeptide according to claim 17, wherein the immune cells comprise lymphocytes, dendritic cells, monocyte precursors, monocytes/macrophages, basophils, eosinophils, and mastocytes

24. The polypeptide according to claim 17, wherein the immune cells are monocyte precursors or monocytes/macrophages.

25. A nucleic acid sequence encoding the polypeptide for targeted recognition of immune cells according to claim 17.

26. A composition comprising the polypeptide for targeted recognition of immune cells according to claim 17.

27. The composition according to claim 26, further comprising a reporter.

28. The composition according to claim 27, wherein the reporter group is linked to the N terminus and/or C-terminus of the polypeptide.

29. The composition according to claim 26, wherein the polypeptide is linked to the reporter through a cysteine residue or a lysine residue which is attached to the polypeptide.

30. The composition according to claim 26, wherein the reporter is a chromogenic enzyme, a fluorescent labeling group, a chemiluminescent labeling group, an isotope, or a magnetic functional group, wherein, the chromogenic enzyme is preferably a peroxidase or an alkaline phosphatase; the fluorescent labeling group is preferably fluorescent proteins, rhodamines, fluoresceins, anthocyanin dyes, cyanine dyes, AlexaFluor dyes and/or quantum dots fluorophores; the magnetic functional group is preferably a group having magnetic resonance imaging and changing relaxation efficiency, preferably a paramagnetic group; and the isotope is preferably a radionuclide.

31. The composition according to claim 27, wherein the composition is used for targeting recognition of monocyte precursors and monocytes/macrophages, wherein the composition is used for in vivo labeling and imaging of monocyte precursors and monocytes/macrophages, and more preferably pulmonary macrophages, or for in vitro immunostaining for immunoassay and/or microscopy examination analysis of cells or tissues.

32. The composition according to claim 36, further comprising a drug molecule.

33. The composition according to claim 32, wherein the composition is used for targeted drug delivery to immune cells.

34. The composition according to claim 33, wherein the composition is used for targeted drug delivery to monocyte precursors and monocytes/macrophages, and pulmonary macrophages.

35. A method for in vivo targeting immune cells, comprising administering an effective amount of the composition according to claim 26 to a subject in need thereof.

36. The method according to claim 35, wherein the composition is administered via subcutaneous injection, intravenous injection, intramuscular injection or pulmonary inhalation.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0066] FIG. 1 shows the high homology of the C-terminal fragments of Triokinase/FMN cyclases among different animal species. FIG. 1A shows that the C-terminal fragments of the Triokinase/FMN cyclases are highly conserved among different species. FIG. 1B shows that the C-terminal fragments of the Triokinase/FMN cyclase proteins have 100% sequence identity among 20 mammalian species.

[0067] FIG. 2 shows in vivo fluorescent labeled images obtained by staining macrophages in a mouse abdominal cavity with AK9 fluorescent probe. Wherein the mouse peritoneal macrophages were first labeled with the AK9 fluorescent probe in vivo, and then removed from the mouse peritoneal cavity for F4/80 antibody and Nucblue labeling. FIG. 2A is an AK9-labeled image (shown in red in the original fluorescent color image); FIG. 2B is a F4/80-labeled image (shown in green in the original fluorescent color image); FIG. 2C is a nuclear stained image obtained by using Nucblue (shown in blue fluorescence in the original fluorescent color image); and FIG. 2D is a merged image of FIGS. 2A, 2B, and 2C.

[0068] FIG. 3 shows in vivo fluorescent labeled images obtained by staining macrophages in a mouse abdominal cavity with LK11 fluorescent probe. Wherein the mouse peritoneal macrophages were first labeled with the LK11 fluorescent probe in vivo, and then removed from the mouse peritoneal cavity for Nucblue staining. FIG. 3A is a LK11-labeled image (shown in red in the original fluorescence color image); FIG. 3B is a nuclear-staining image obtained by using Nucblue (shown in blue in the original fluorescence color image); and FIG. 3C is a merged image of FIGS. 3A and 3B.

[0069] FIG. 4 shows in vivo fluorescent labeled images obtained by staining macrophages in a mouse abdominal cavity with AK14 fluorescent probe. Wherein the mouse peritoneal macrophages were first labeled with the AK14 fluorescent probe in vivo, and then removed from mouse peritoneal cavity for Nucblue staining. FIG. 4A is a labeled image obtained by using the AK14 fluorescent probe (shown in red in the original fluorescence color image); FIG. 4B is a nuclear-staining image obtained by using Nucblue (shown in blue in the original fluorescence color image); and FIG. 4C is a merged image of FIGS. 4A and 4B.

[0070] FIG. 5 shows an in vitro fluorescent labeled image obtained by staining mouse macrophage cell line RAW264.7 with EK24 probe. The macrophage cell line RAW264.7 was stained with the EK24 probe (shown in red in the original fluorescence color image) and Nucblue (shown in blue in the original fluorescence color image).

[0071] FIG. 6 shows in vivo fluorescent labeled images obtained by staining macrophages in a mouse abdominal cavity with EK24 probe. The mouse peritoneal macrophages were first labeled with the EK24 probe in vivo, and then removed from mouse peritoneal cavity for Nucblue staining. FIG. 6A is a fluorescent labeled image obtained by using the EK24 fluorescent probe (shown in red in the original fluorescence color image), and FIG. 6B is a nuclear-staining image obtained by using Nucblue (shown in blue in the original fluorescence color image), and FIG. 6C is a merged image of FIGS. 6A and 6B.

[0072] FIG. 7 shows fluorescent labeled images obtained by staining macrophages, which were removed from the mouse abdominal cavity and cultured in vitro, with EK24 probe. FIGS. 7A and 7B show fluorescent labeled images obtained by using EK24 probe and F4/80 antibody, respectively; and FIG. 7C is a merged image of FIGS. 7A and 7B.

[0073] FIG. 8 shows fluorescent labeled images obtained by staining macrophages from rat peritoneal cavity with EK24 fluorescent probe. FIG. 8A shows a direct fluorescently labeled image of macrophages from a rat abdominal cavity obtained by using the EK24 fluorescent probe; FIG. 8B shows a fluorescent labeled image of cultured macrophages isolated from rat peritoneal cavity with EK24 probe.

[0074] FIG. 9 shows a fluorescent labeled image obtained by staining cultured monocyte precursors isolated from mouse bone marrow, with EK24 fluorescent probe (shown in red in the original fluorescence color image) and Nucblue (shown in blue in the original fluorescence color image).

[0075] FIG. 10 shows a fluorescent labeled image obtained by staining cultured monocyte precursors isolated from rat bone marrow, with EK24 fluorescent probe (shown in red in the original fluorescence color image) and Nucblue (shown in blue in the original fluorescence color image).

[0076] FIG. 11 shows a fluorescent labeled image obtained by staining cultured monocyte precursors isolated from New Zealand White Rabbit bone marrow, with EK24 fluorescent probe (shown in red in the original fluorescence color image) and Nucblue (shown in blue in the original fluorescence color image).

[0077] FIG. 12 shows fluorescent labeled images obtained by staining cultured peritoneal macrophages and other cells released from enzyme-digested mesenterium with EK24 fluorescent probe. FIG. 12A is a labeled image obtained by using the EK24 fluorescent probe, and FIG. 12B is a nuclear-staining image obtained by using Nucblue, and FIG. 12C is a merged image of FIGS. 12A and 12B.

[0078] FIG. 13 shows in vivo fluorescent labeled images obtained by staining macrophages in a mouse abdominal cavity with KS24 fluorescent probe. Wherein the mouse peritoneal macrophages were first labeled with the KS24 fluorescent probe in vivo, and then removed from mouse peritoneal cavity for Nucblue staining. FIG. 13A is a fluorescent labeled image obtained by using the KS24 fluorescent probe (shown in red in the original fluorescence color image), FIG. 13B is a nuclear-staining image obtained by using Nucblue (shown in blue in the original fluorescence color image), and FIG. 13C is a merged image of FIGS. 13A and 13B.

[0079] FIG. 14 shows the fluorescent labeled images obtained by staining mouse primary monocyte precursors with KS24 fluorescent probe at different incubation times. FIGS. 14A, 14B, 14C, and 14D show fluorescent labeled images obtained by using the KS24 fluorescent probe to incubate for 10 min, 20 min, 40 min and 80 min, respectively. In each of FIGS. 14A, 14B, 14C, and 14D, i) is a KS24-labeled image (shown in red fluorescence in the original fluorescence color image), and ii) is a nuclear-staining image with Nucblue (shown as blue fluorescence in the original fluorescence color image), and iii) is a merged image of i) and ii).

[0080] FIG. 15 shows in vivo fluorescent labeled images obtained by staining macrophages in a mouse abdominal cavity with PK20 fluorescent probe. The mouse peritoneal macrophages were first labeled with the PK20 fluorescent probe in vivo, and then removed from mouse peritoneal cavity for Nucblue staining. FIG. 15A is a PK20-labeled image (shown in red fluorescence in the original fluorescence color image), and FIG. 15B is a nuclear-staining image with Nucblue (shown in blue in the original fluorescence color image), and FIG. 15C is a merged image of FIGS. 15A and 15B.

[0081] FIG. 16 shows in vivo fluorescent labeled images obtained by staining macrophages in a mouse abdominal cavity with TS42 fluorescent probe. The mouse peritoneal macrophages were first labeled with the TS42 fluorescent probe in vivo, and then removed from mouse peritoneal cavity for Nucblue staining. FIG. 16A is a TS42-labeled image (shown in green in the original fluorescence color image), and FIG. 16B is a nuclear-staining image with Nucblue (shown in blue in the original fluorescence color image), and FIG. 16C is a merged image of FIGS. 16A and 16B.

[0082] FIG. 17 shows in vivo fluorescent labeled image obtained by staining macrophages in a mouse abdominal cavity with LK24 fluorescent probe. Wherein the mouse peritoneal macrophages were first labeled with the LK24 fluorescent probe in vivo, and then removed from mouse peritoneal cavity for Nucblue staining. FIG. 17A is a LK24-labeled image (shown in red fluorescence in the original fluorescence color image), and FIG. 17B is a nuclear-staining image with Nucblue (shown in blue fluorescence in the original fluorescence color image), and FIG. 17C is a merged image of FIGS. 17A and 17B.

[0083] FIG. 18 shows in vivo fluorescent labeled image obtained by staining macrophages in a mouse abdominal cavity with r-KA27 fluorescent probe. Wherein the mouse peritoneal macrophages were first labeled with r-KA27 fluorescent probes in vivo, and then removed from mouse peritoneal cavity for Nucblue staining. FIG. 18A is a r-KA27-labeled image (shown in red fluorescence in the original fluorescence color image), and FIG. 18B is a nuclear-staining image with Nucblue (shown in blue in the original fluorescence color image), and FIG. 18C is a merged image of FIGS. 18A and 18B.

[0084] FIG. 19 shows in vivo fluorescent labeled image obtained by staining macrophages in a mouse abdominal cavity with d-KV27 fluorescent probe. Wherein the mouse peritoneal macrophages were first labeled with d-KV27 fluorescent probes in vivo, and then removed from mouse peritoneal cavity for Nucblue staining. FIG. 19A is a d-KV27-labeled image (shown in red fluorescence in the original fluorescence color image), and FIG. 19B is a nuclear-staining image with Nucblue (shown in blue fluorescence in the original fluorescence color image), and FIG. 19C is a merged image of FIGS. 19A and 19B.

[0085] FIG. 20 shows in vivo fluorescent labeled image obtained by staining macrophages in a mouse abdominal cavity withe b-KA27 fluorescent probe. Wherein the mouse peritoneal macrophages were first labeled with b-KA27 fluorescent probes in vivo, and then removed from mouse peritoneal cavity for Nucblue staining. FIG. 20A is a b-KA27-labeled image (shown in red fluorescence in the original fluorescence color image), and FIG. 20B is a nuclear-staining image with Nucblue (shown in blue in the original fluorescence color image), and FIG. 20C is a merged image of FIGS. 20A and 20B.

[0086] FIG. 21 shows in vivo fluorescent labeled image of macrophages in a mouse abdominal cavity using the KESG24 fluorescent probe. Wherein the mouse peritoneal macrophages were first labeled with the KESG24 fluorescent probe in vivo, and then removed from mouse peritoneal cavity for Nucblue staining. FIG. 21A is a KESG244-labeled image (shown in red in the original fluorescence color image), and FIG. 21B is a nuclear-staining image with Nucblue (shown in blue in the original fluorescence color image), and FIG. 21C is a merged image of FIGS. 21A and 21B.

[0087] FIG. 22 shows in vivo fluorescent labeled image obtained by staining macrophages in a mouse abdominal cavity with Cx-LK24 fluorescent probe. Wherein the mouse peritoneal macrophages were first labeled with Cx-LK24 fluorescent probes in vivo, and then removed from mouse peritoneal cavity for Nucblue staining. FIG. 22A is a Cx-LK24-labeled image (shown in red fluorescence in the original fluorescence color image), and FIG. 22B is a nuclear-staining image with Nucblue (shown in blue in the original fluorescence color image), and FIG. 22C is a merged image of FIGS. 22A and 22B.

[0088] FIG. 23 shows fluorescent labeled images obtained by staining mouse abdominal wall muscle cells with the EK24 fluorescent probe and FITC anti-mouse CD68 antibody (Biolegend). FIG. 23A is a EK24-labeled image (shown in red fluorescence in the original fluorescent color image), FIG. 23B is a CD68 antibody labeled image (shown in green in the original fluorescent color image), FIG. 23C is a nuclear-staining image with Nucblue (shown in blue in the original fluorescent color image), and FIG. 23D is a merged image of FIGS. 23A, 23B, and 23C.

[0089] FIG. 24 shows in vivo imaging obtained by staining pulmonary macrophages with EK24 fluorescent probe and Alexa Fluor®488 anti-mouse CD11c antibody (Biolegend) after pulmonary administration to mice. FIG. 24A is an EK24-labeled image, and FIG. 24B is a CD11c-labeled image (shown in green in the original fluorescence color image), and FIG. 24C is a merged image of FIGS. 24A and 24B.

[0090] FIG. 25 shows in vivo fluorescent labeled image obtained by staining macrophages in a mouse abdominal cavity with the Hx-AVGK9 fluorescent probe. The mouse peritoneal macrophages were first labeled with the Hx-AVGK9 fluorescent probe in vivo, and then removed from mouse peritoneal cavity for Nucblue staining. FIG. 25A is a Hx-AVGK9-labeled image (shown in red fluorescence in the original fluorescence color image), and FIG. 25B is a nuclear-staining image with Nucblue (shown in blue in the original fluorescence color image), and FIG. 25C is a merged image of FIGS. 25A and 25B.

[0091] FIG. 26 shows in vivo fluorescent labeled image obtained by staining macrophages in a mouse abdominal cavity with the IK12 fluorescent probe. Wherein the mouse peritoneal macrophages were first labeled with the IK12 fluorescent probe in vivo, and then removed from mouse peritoneal cavity for Nucblue staining. FIG. 26A is an IK12-labeled image (shown in red fluorescence in the original fluorescence color image), and FIG. 26B is a nuclear-staining image with Nucblue (shown in blue in the original fluorescence color image), and FIG. 26C is a merged image of FIGS. 26A and 26B.

[0092] FIG. 27 shows in vivo fluorescent labeled image obtained by staining macrophages in a mouse abdominal cavity with Hx-VVGK12 fluorescent probe. Wherein the mouse peritoneal macrophages were first labeled with the Hx-VVGK12 fluorescent probe in vivo, and then removed from mouse peritoneal cavity for Nucblue staining. FIG. 27A is a Hx-VVGK12-labeled image (shown in red in the original fluorescence color image), and FIG. 27B is a nuclear-staining image with Nucblue (shown in blue in the original fluorescence color image), and FIG. 27C is a merged image of FIGS. 27A and 27B.

DETAILED DESCRIPTION

[0093] Hereafter, the present disclosure will be described in detail with reference to the specific embodiments. However, it should be understood that the present disclosure will not be limited to the following embodiments. The protection scope of the present disclosure is defined by the appended claims, and the following embodiments of the present disclosure can be arbitrarily changed and combined without departing from the scope of the present disclosure.

Example 1. Preparation of AK9 Fluorescent Probe

[0094] The polypeptides used herein were obtained by means of conventional solid phase peptide chemical synthesis using a CEM fully automated microwave peptide synthesizer according to operating instructions provided by the supplier. The polypeptides used herein were derived from the C-terminal fragment of human or non-human Triokinase/FMN cyclases.

[0095] In this example, the polypeptide having the following amino acid sequence was synthesized: AILEVLQS K.

[0096] According to the manufacturer's instructions, the synthesized polypeptide was mixed with a reaction reagent of HOOK™ Dye Rhodamine Labeling Kit (Cat. #786-142, Biosciences), adjusted pH, reacted for 1-2 hours, purified by HPLC to obtain the AK9 fluorescence probe. The chemical structure of the obtained fluorescent probe is as follows:

AILEVLQS-Y (AK9),

[0097] wherein, Y is a lysine+a fluorescent reporter which is a rhodamine fluorescent labeling group linked to an amino group on the side chain of lysine.

Example 2. Preparation of AK9 Fluorescent Probe Solution

[0098] 1 mg of AK9 fluorescent probe was dissolved in 177 μL of DMSO (dimethyl sulfoxide), which was then added to 13981 μL serum-free DMEM (Hyclone) medium, mixing well to give 50 μM of AK9 fluorescent probe solution. As required, 50 μM of AK9 fluorescent probe solution can be diluted by adding serum-free DMEM medium during use.

Example 3. In Vivo Fluorescent Labeling Macrophages in a Mouse Abdominal Cavity with the AK9 Fluorescent Probe

[0099] Dilute 300 μL of 50 μM AK9 fluorescent probe solution with serum-free DMEM incomplete medium to obtain 1 mL of solution, then inject into the abdominal cavities of 5-8 week old Kunming mice. After 4 hours, inject 5 mL of 1×PBS, and massage. After 10 minutes, sacrifice the mice by cervical vertebra dislocation. Cut the abdominal cavity skin of the mice, pierce the abdominal wall muscle by syringe, and extract the fluid in the abdominal cavity of the mice. Centrifuge the extracted peritoneal fluid at 1000 rpm for 5 min, wash twice with 1×PBS. Double-stain an appropriate number of cells with Alexa Fluor®488 anti-mouse F4/80 antibody (Biolegend). Add the stained cells to a 96-well plate and stain with a nuclear dye Nucblue (Invitrogen). Observe under EVOS® FL Auto fluorescence microscope (Life Technologies, 20×, light source 50%, exposure 200 ms, gain 10) after the suspended cells were settled down at the bottom of the plate. The results are shown in FIG. 2.

[0100] FIG. 2A is an AK9-labeled image (shown in red fluorescence in the original fluorescence color image).

[0101] FIG. 2B is a F4/80 antibody labeled image (shown in green fluorescence in the original fluorescent color image). F4/80 antigen is currently recognized as a membrane biomarker of mouse peritoneal macrophages.

[0102] FIG. 2C is a Nucblue-stained nuclear image (shown in blue fluorescence in the original fluorescence color image).

[0103] FIG. 2D is a merged image of FIGS. 2A, 2B, and 2C. From this figure, it can be seen that both the AK9 fluorescent probe and the F4/80 antibody are labeled on the same cells. This result indicates that cells labeled with F4/80 antibody are also labeled with the AK9 fluorescent probe, which confirms the AK9 fluorescent probe can targeting recognition of macrophages.

Example 4. Fluorescent Labeling of Macrophages by the Probes which are Derived from the C-Terminal Fragments of Triokinases/FMN Cyclases of Different Mammal Species

[0104] Table 1 lists probes, which contain the polypeptide fragments from the C-terminal fragments of Triokinases/FMN cyclases of different species and were prepared by a similar process as described in Examples 1 and 2, and in vivo imaging results of mouse peritoneal macrophages using these probes.

TABLE-US-00001 TABLE 1 Probes from C-terminal fragments of Triokinases/FMN cyclases of different animal species and in vivo imaging results of mouse peritoneal macrophages using the probes. In vivo targeted Species Sequences imaging effect Figures Names Human AILEVLQS-Y + FIG. 2 AK9 (Homo sapiens) Human LRAILEVLQS-Y +++ FIG. 3 LK11 (Homo sapiens) Human ILRAILEVLQS-Y +++ FIG. 26 IK12 (Homo sapiens) Human AAILRAILEVLQS-Y ++++ FIG. 4 AK14 (Homo sapiens) Rhesus monkey EQPDPGAVAAAAILRAILEVLQS-Y +++ FIG. 5-12 EK24 (Macaca mulatto) Human EQPDPGAVAAAAILRAILEVLQS-Y +++ FIG. 5-12 EK24 (Homo sapiens) Human X-EQPDPGAVAAAAILRAILEVLQS +++ FIG. 13-14 KS24 (Homo sapiens) Human PGAVAAAAILRAILEVLQS-Y ++++ FIG. 15 PK20 (Homo sapiens) Human X- ++ FIG. 16 TS42 (Homo sapiens) TKNMEAGAGRASYISSARLEQPDPGAVAA AAILRAILEVLQS Chickens LQPDPGAVAAAAVLRAVLEGLQG-Y + FIG. 17 LK24 (Gallus gallus) Rat X-DQPDPGAVAAAAIFRAILEVLQTKAA +++ FIG. 18 r-KA27 (Rattus norvegicus) Dog X-DQPDPGAVAAAAILRTILEVLQSQGV ++ FIG. 19 d-KV27 (Canis lupus) Pig X-DQPDPGAVAAAAILRAILEVLQSQGA +++ FIG. 20 b-KA27 (Sus scrofa) Cow X-DQPDPGAVAAAAILRAILEVLQSQGA +++ FIG. 20 b-KA27 (Bos Taurus) Modified sequence AVLEVLQG-Y + FIG. 25 Hx- AVGK9 Modified sequence VLRAVLEVLQG-Y ++++ FIG. 27 Hx- VVGK12 Modified sequence X-EQPDPSAVAAAAILRAILEVLQG +++ FIG. 21 KESG24 Modified sequence LQPDPSAVAAAAVLRAVLEVLQG-Y +++ FIG. 22 Cx-LK24 X, Y = reporter or cysteine/lysine + reporter (rhodamine or FITC)

[0105] From the results in Table 1, it can be seen that all the polypeptides from the C-terminal fragments of Triokinase/FMN cyclases of different animal species or modified sequences thereof can specifically target monocytes/macrophages. Specifically, AK9, LK11, IK12, AK14, PK20, EK24, KS24, TS42 derived from human Triokinase/FMN cyclase, LK24 derived from chicken Triokinase/FMN cyclase, r-KA27 derived from rat Triokinase/FMN cyclase, d-KV27 derived from dog Triokinase/FMN cyclase, b-KA27 derived from pig and cattle Triokinase/FMN cyclase and EK24 derived from rhesus monkey Triokinase/FMN cyclase as well as the modified sequences can effectively recognize macrophages in vivo. It indicates that highly conserved polypeptide fragments from the C-terminal fragments of Triokinases/FMN cyclases of different species can be used as probes to label monocytes/macrophages. The following examples provide further relevant experimental results.

Example 5. Fluorescent Labeling of Macrophages in a Mouse Abdominal Cavity by the LK11 Fluorescent Probe

[0106] Referring to Example 3, using the same treatment method, the mouse peritoneal macrophages were first labeled in vivo using the LK11 fluorescent probe, and then removed from the mouse abdominal cavity for nuclear fluorescence labeling with Nucblue. The resulted macrophages were observed under a fluorescence microscope (20×, light source 50%, exposure 200 ms, gain 5). The results are shown in FIG. 3.

[0107] FIG. 3A is a labeled image obtained by using the LK11 fluorescent probe (shown in red fluorescence in the original fluorescence color image), and FIG. 3B is a nuclear-staining image obtained by using Nucblue (shown in blue fluorescence in the original fluorescence color image), and FIG. 3C is a merged image of FIGS. 3A and 3B.

[0108] It can be seen from FIG. 3 that the macrophages extracted from the mouse abdominal cavity can be labeled with the LK11 fluorescent probe.

Example 6. Fluorescent Labeling of Macrophages in a Mouse Abdominal Cavity by the AK14 Fluorescent Probe

[0109] Referring to Example 3, using the same treatment method, the mouse peritoneal macrophages were first labeled in vivo using the AK14 fluorescent probe, and then removed from the mouse abdominal cavity for Nuclear fluorescence labeling with Nucblue. The macrophages were observed under a fluorescence microscope (20×, light source 10%, exposure 200 ms, gain 5). The results are shown in FIG. 4.

[0110] FIG. 4A is a labeled image obtained by using the AK14 fluorescent probe (shown in red fluorescence in the original fluorescence color image), and FIG. 4B is a nuclear-staining image obtained by using Nucblue (shown in blue fluorescence in the original fluorescence color image), and FIG. 4C is a merged image of FIGS. 4A and 4B.

[0111] It can be seen from FIG. 4 that the macrophages extracted from the mouse abdominal cavity can be labeled with the AK14 fluorescent probe.

Example 7. In Vitro Fluorescence Labeled Staining of a Mouse Macrophage Cell Line by the EK24 Probe

[0112] Culture the mouse macrophage cell line RAW264.7 in a 96-well plate, wash the wells once with 1×PBS buffer or a serum-free DMEM incomplete medium, remove the washing liquid, add EK24 solution of 10 μM which had been diluted with 100 μL of serum-free DMEM incomplete medium, and incubate in a cell culture incubator for 1 h. After that, wash the wells twice with 1×PBS buffer or the serum-free DMEM incomplete medium. Then, add DMEM complete medium containing 10% FBS and 1% streptomycin-penicillin, and double-stain the cells using nuclear dye Nucblue and observe under EVOS® FL Auto fluorescence microscope (Life Technologies, light source 10%, exposure 200 ms, gain 5). The results are shown in FIG. 5.

[0113] FIG. 5 shows images of in vitro cultured mouse macrophage cell line that were fluorescent labeled using the EK24 fluorescent probe (shown in red fluorescence in the original fluorescence color image) and Nucblue (shown in blue in the original fluorescence color image).

[0114] It can be seen from FIG. 5 that the macrophage cell line RAW264.7 can be labeled with the EK24 fluorescent probe (shown in red fluorescence in the original fluorescence color image).

Example 8. In Vivo Fluorescent Labeling of Macrophages in a Mouse Abdominal Cavity by the EK24 Fluorescent Probe

[0115] Referring to Example 3, using the same processing method, the mouse peritoneal macrophages were first labeled in vivo using the EK24 fluorescent probe, and then removed from the mouse abdominal cavity for nuclear fluorescence labeling with Nucblue. Observe the macrophages under a fluorescence microscope (20×, light source 10%, exposure 200 ms, gain 5). The results are shown in FIG. 6.

[0116] FIG. 6A is a fluorescent labeled image obtained by using the EK24 fluorescent probe (shown in red fluorescence in the original fluorescence color image), and FIG. 6B is a nuclear stained image obtained by using Nucblue (shown in blue fluorescence in the original fluorescence color image), and FIG. 6C is a merged image of FIGS. 6A and 6B.

[0117] It can be seen from FIG. 6 that the macrophages extracted from the mouse abdominal cavity can be labeled with the EK24 fluorescent probe.

Example 9. In Vitro Fluorescent Labeling of Cultured Macrophages Isolated from Mouse Peritoneal Cavity by EK24 Probe

[0118] Inject 5 mL of serum-free DMEM incomplete medium into the abdominal cavity of 5 to 8 weeks old mice, massage the abdominal cavity gently for 10 min, and sacrifice the mice by cervical vertebra dislocation. Extract the fluid from the abdominal cavity gently with a syringe, inject into a 5 mL centrifuge tube and centrifuge at 1000 rpm for 5 min, remove the supernatant, and resuspend the collected ex-vivo cells in DMEM/F12 medium containing 10% FBS, then add into a 96-well plate for culture. Generally, if cells are taken intraperitoneally for in vitro culture, macrophages would tend to adhere to the bottom of the well, and other cells would be washed off. The cells were cultured for more than 2 days until the cells attached to the bottom firmly. At this time, the cells remaining in the wells were basically macrophages. Select two wells, one as a control and the other as an experimental well. The cells of the control were nuclear stained with Nucblue. The experimental well was added with a premix of AlexaFluor®488 anti-mouse F4/80 antibody (Biolegend) and EK24 fluorescent probe, and then incubated for 1 h at 37° C. The premix contained 10 μM of EK24 solution and 2 μL of Alexa Fluor®488 anti-mouse F4/80 antibody diluted with 100 μL of serum-free DMEM incomplete medium. The remaining treatment steps can refer to the above Example 3. The cells were observed under a fluorescence microscope (20× objective lens, light source 10%, exposure 200 ms, gain 5). The results are shown in FIG. 7.

[0119] FIG. 7A shows a fluorescent labeled image obtained by using the EK24 fluorescent probe (shown in red fluorescence in the original fluorescence color image) and nuclear stained image obtained by using Nucblue (shown in blue fluorescence in the original fluorescence color image).

[0120] FIG. 7B shows a fluorescent labeled image obtained by using the F4/80 antibody (shown in green fluorescence in the original fluorescence color image) and nuclear stained image obtained by using Nucblue (shown in blue fluorescence in the original fluorescence color image). It can be seen from FIG. 7B that almost all the cells obtained by intraperitoneal massage were labeled with F4/80 antibody, indicating that these labeled cells were macrophages.

[0121] FIG. 7C is a merged image of FIGS. 7A, and 7B. From this figure, it can be seen that both the EK24 fluorescent probe label and the F4/80 antibody label are co-localized on the same cells. This result indicates that cells labeled with the F4/80 antibody are also labeled with the EK24 fluorescent probe, which further indicates that the EK24 fluorescent probe can target and recognize macrophages.

Example 10. In Vitro Fluorescent Labeling of Primary Macrophages from Rat Peritoneal Cavity by EK24 Fluorescent Probe

[0122] With the same process as in Example 9, extract cells from the abdominal cavity of 5 to 8 weeks old SD rats using 20 mL serum-free DMEM incomplete medium and culture the cells in a 96-well plate. After culturing for more than 2 days until the cells attached to the bottom firmly, a well was selected and added with EK24 fluorescent probe for incubation. Then the cells were observed under a fluorescence microscope (20×, light source 10%, exposure 200 ms, gain 5). The results are shown in FIG. 8.

[0123] FIG. 8A shows images obtained from direct fluorescent labeling of macrophages from a rat abdominal cavity with the EK24 fluorescent probe (shown in red fluorescence in the original fluorescence color image) and Nucblue (shown in blue fluorescence in the original fluorescence color image). FIG. 8B shows images obtained by fluorescent labeling of primary macrophages, which were taken from rat peritoneal cavity and cultured for more than 2 days in vitro, with the EK24 fluorescent probe (shown in red fluorescence in the original fluorescence color image) and Nucblue (shown in blue fluorescence in the original fluorescence color image).

[0124] It can be seen from FIG. 8 that the EK24 fluorescent probe can effectively label the in vitro cultured primary macrophages from rat peritoneal cavity.

Example 11. In Vitro Fluorescence Labeling of Mouse Primary Monocyte Precursors by EK24 Probe

[0125] As described above, macrophages were derived from monocytes in blood, and monocytes were transformed from monocyte precursors in bone marrow. In order to verify whether the EK24 fluorescent probe can recognize monocyte precursors, the EK24 fluorescent probe and Nucblue were used to label monocytes from the mesenchyme of bone marrow.

[0126] In a bio-safety hood, take the femur of a Kunming mouse, cut off both ends of the femur, wash medulla out with PBS, and collect cells by centrifugation. The collected ex-vivo cells were resuspended in F12/DMEM medium containing 10% FBS and 1% streptomycin-penicillin, and add to a 96-well plate for culture. After culturing for more than 2 days until the cells attached to the bottom firmly, the cells remaining in the wells were basically monocyte precursors. Treat with reference to Example 9. Then the cells were observed under a fluorescence microscope (200×, light source 50%, exposure 200 ms, gain 5). The results are shown in FIG. 9.

[0127] FIG. 9 shows images obtained with direct fluorescent labeling of monocyte precursors from the mesenchyme of mouse bone marrow with the EK24 fluorescent probe (shown in red fluorescence in the original fluorescence color image) and Nucblue (shown in blue fluorescence in the original fluorescence color image). It can be seen that the EK24 fluorescent probe (shown in red fluorescence in the original fluorescence color image) is capable of labeling monocyte precursors from the mesenchyme of mouse bone marrow.

Example 12. In Vitro Fluorescent Labeling of Rat Primary Monocyte Precursors by the EK24 Fluorescent Probe

[0128] With a similar process to Example 11, mononuclear cell precursors from rat femoral bone marrow were taken and cultured, and then labeled by using EK24 fluorescent probe and Nucblue. Then the cells were observed under a fluorescence microscope (20×, light source 10%, exposure 200 ms, gain 5). The results are shown in FIG. 10.

[0129] FIG. 10 shows images obtained by fluorescent labeling of monocyte precursors from rat bone marrow mesenchyme with the EK24 fluorescent probe (shown in red fluorescence in the original fluorescence color image) and Nucblue (shown in blue fluorescence in the original fluorescence color image). It can be seen that the EK24 fluorescent probe (shown in red fluorescence in the original fluorescence color image) is also capable of labeling monocyte precursors from mesenchyme of rat bone marrow.

Example 13. In Vitro Fluorescent Labeling of Primary Monocyte Precursors of New Zealand White Rabbit by the EK24 Fluorescent Probe

[0130] With a similar process to Example 11, bone marrow monocyte precursors of New Zealand White Rabbit were taken and cultured, then labeled by using EK24 fluorescent probe and Nucblue. Then the cells were observed under a fluorescence microscope (20×, light source 10%, exposure 200 ms, gain 5). The results are shown in FIG. 11.

[0131] FIG. 11 shows images obtained from directly fluorescent labeling of monocyte precursors from mesenchyme of New Zealand White Rabbit bone marrow with the EK24 fluorescent probe (shown in red fluorescence in the original fluorescence color image) and Nucblue (shown in blue fluorescence in the original fluorescence color image). It can be seen that the EK24 fluorescent probe (shown in red fluorescence in the original fluorescence color image) is also capable of labeling monocyte precursors from New Zealand White Rabbit bone marrow mesenchyme.

Example 14. EK24 Fluorescent Probe Selective Labeling and Staining of Cultured Peritoneal Macrophages Among Other Cells Released from Enzyme-Digested Mesenterium

[0132] Take 5 to 8-week-old Kunming mice, inject intraperitoneally with 5 mL of serum-free DMEM incomplete medium. After 1 hour, sacrifice the mice by cervical vertebra dislocation. Make an incision on the skin of abdominal cavity, pierce the abdominal wall muscle using the syringe, and extract the fluid in the abdominal cavity of the mice. Wash twice with 1×PBS, collect peritoneal cells by centrifugation. Also take the mesenteries of the Kunming mice and purge twice with 1×PBS in centrifuge tubes. Incubate the obtained peritoneal cells and mesenteries in 0.25% trypsin solution and digest for 30 minutes. Enrich the cells by centrifugation, and then culture for 3 days in a 96-well plate using F12/DMEM culture solution containing 10% FBS and 1% streptomycin-penicillin. Referring to Example 5, label with the EK24 fluorescent probe and Nucblue. Then the cells were observed under a fluorescence microscope (20×, light source 10%, exposure 200 ms, gain 5). The results are shown in FIG. 12.

[0133] FIG. 12A is an image obtained by labeling with the EK24 fluorescent probe (shown in red fluorescence in the original fluorescence color image), and FIG. 12B is an image obtained by nuclear staining with Nucblue (shown in blue fluorescence in the original fluorescence color image), and FIG. 12C is a merged image of FIGS. 12A and 12B. It can be seen from the figures that the EK24 fluorescent probe only stains macrophages (as shown by the solid arrow in FIG. 12C, cells labeled with both blue and red fluorescences), and not stain non-macrophages (as shown by the hollow arrow in FIG. 12C, cells labeled with only blue fluorescence without red fluorescence).

Example 15. In Vivo Fluorescent Labeling of Macrophages in a Mouse Abdominal Cavity by the KS24 Fluorescent Probe

[0134] Referring to Example 3, using the same treatment process, the mouse peritoneal macrophages were first labeled in vivo using the KS24 fluorescent probe, and then removed from the mouse abdominal cavity for Nuclear fluorescence labeling with Nucblue. Then the labeled macrophages were observed under a fluorescence microscope (20×, light source 10%, exposure 200 ms, gain 5). The results are shown in FIG. 13.

[0135] FIG. 13A is an image obtained by labeling with the KS24 fluorescent probe (shown in red fluorescence in the original fluorescence color image), and FIG. 13B is an image obtained by nuclear staining with Nucblue (shown in blue fluorescence in the original fluorescence color image), and FIG. 13C is a merged image of FIGS. 13A and 13B.

[0136] It can be seen from FIG. 13 that the macrophages extracted from the mouse abdominal cavity can be labeled with the KS24 fluorescent probe.

Example 16. In Vitro Fluorescent Labeling of Mouse Peritoneal Macrophages by the EK24 Fluorescent Probe at Different Incubation Times

[0137] Referring to Example 9, using the same treatment process, the macrophages from mouse abdominal cavity were obtained. After culturing for 6 days, the cultured peritoneal cells were labeled with KS24 fluorescent probes for different incubation times. Then the cells were observed under a fluorescence microscope (20×, light source 20%, exposure 200 ms, gain 5). The results are shown in FIG. 14.

[0138] FIG. 14A shows the fluorescent label results obtained by incubating with the KS24 fluorescent probe for 10 min. FIG. 14B shows the fluorescent label results obtained by incubating with the KS24 fluorescent probe for 20 min. FIG. 14C shows the fluorescent label results obtained by incubating with the KS24 fluorescent probe for 40 min. FIG. 14D shows the fluorescent label results obtained by incubating with the KS24 fluorescent probe for 80 min. Nucblue (blue fluorescence) was further used for nuclear staining. In each of FIGS. 14A, 14B, 14C, and 14D, i) is an image labeled with the KS24 fluorescent probe (shown in red fluorescence in the original fluorescence color image), and ii) is an image obtained by nuclear staining with Nucblue (shown in blue fluorescence in the original fluorescence color image), and iii) is a merged image of i) and ii).

[0139] It can be seen from FIG. 14 that the KS24-labeled fluorescence of the cells gradually becomes stronger with the incubation time. The fluorescence can be even detected with microscope when incubating only for 10 min. It is sufficient to show that KS24 fluorescent probe can quickly and efficiently label mouse peritoneal macrophages.

Example 17. In Vivo Fluorescent Labeling of Macrophages in a Mouse Abdominal Cavity by the PK20 Fluorescent Probe

[0140] Referring to Example 3, using the same treatment method, the mouse peritoneal macrophages were first labeled in vivo using the PK20 fluorescent probe, and then removed from the mouse abdominal cavity for Nuclear fluorescent labeling with Nucblue. Then the macrophages were observed under a fluorescence microscope (20×, light source 10%, exposure 200 ms, gain 5), and the results are shown in FIG. 15.

[0141] FIG. 15A is an image obtained from fluorescent labeling using PK20 fluorescent probe (shown in red fluorescence in the original fluorescence color image), and FIG. 15B is an image obtained by nuclear staining with Nucblue (shown in blue fluorescence in the original fluorescence color image), and FIG. 15C is a merged image of FIGS. 15A and 15B.

[0142] It can be seen from FIG. 15 that the macrophages extracted from the mouse abdominal cavity can be labeled with the PK20 fluorescent probe.

Example 18. In Vivo Fluorescent Labeling of Macrophages in a Mouse Abdominal Cavity by the TS42 Fluorescent Probe

[0143] Referring to Example 3, using the same treatment process, the mouse peritoneal macrophages were first labeled in vivo using the TS42 fluorescent probe (carrying the FITC group), and then removed from the mouse abdominal cavity for nuclear fluorescence labeling with Nucblue. Then the macrophages were observed under a fluorescence microscope (20×, light source 50%, exposure 200 ms, gain 5). The results are shown in FIG. 16.

[0144] FIG. 16A is an image by labeling with the TS42 fluorescent probe (shown in green fluorescence in the original fluorescence color image), and FIG. 16B is an image by nuclear staining with Nucblue (shown in blue fluorescence in the original fluorescence color image), and FIG. 16C is a merged image of FIGS. 16A and 16B.

[0145] It can be seen from FIG. 16 that the macrophages extracted from the mouse abdominal cavity can be labeled with the TS42 fluorescent probe.

Example 19. In Vivo Fluorescent Labeling of Macrophages in a Mouse Abdominal Cavity by the LK24 Fluorescent Probe

[0146] Referring to Example 3, using the same treatment process, the mouse peritoneal macrophages were first labeled in vivo using the LK24 fluorescent probe, and then removed from the mouse abdominal cavity for nuclear fluorescence labeling with Nucblue. Then the macrophages were observed under a fluorescence microscope (20×). The results are shown in FIG. 17.

[0147] FIG. 17 shows the images observed under a fluorescence microscope (light source 50%, exposure 200 ms, gain 5). FIG. 17A is an image obtained by labeling with the LK24 fluorescent probe (shown in red fluorescence in the original fluorescence color image), and FIG. 17B is an image obtained by nuclear staining with Nucblue (shown in blue fluorescence in the original fluorescence color image), and FIG. 17C is a merged image of FIGS. 17A and 17B.

[0148] It can be seen from FIG. 17 that the macrophages extracted from the mouse abdominal cavity can also be labeled with the LK24 fluorescent probe.

Example 20. In Vivo Fluorescent Labeling of Macrophages in a Mouse Abdominal Cavity by the r-KA27 Fluorescent Probe

[0149] Referring to Example 3, using the same treatment process, the mouse peritoneal macrophages were first labeled in vivo using the r-KA27 fluorescent probe, and then removed from the mouse abdominal cavity for Nuclear fluorescence labeling with Nucblue. Then the macrophages were observed under a fluorescence microscope (20×, light source 10%, exposure 200 ms, gain 5). The results are shown in FIG. 18.

[0150] FIG. 18A is an image obtained by labeling with the KA27 fluorescent probe (shown in red fluorescence in the original fluorescence color image), and FIG. 18B is an image obtained by nuclear staining with Nucblue (shown in blue fluorescence in the original fluorescence color image), and FIG. 18C is a merged image of FIGS. 18A and 18B.

[0151] It can be seen from FIG. 18 that the macrophages extracted from the mouse abdominal cavity can be labeled with the r-KA27 fluorescent probe.

Example 21. In Vivo Fluorescent Labeling of Macrophages in a Mouse Abdominal Cavity by the d-KV27 Fluorescent Probe

[0152] Referring to Example 3, using the same treatment process, the mouse peritoneal macrophages were first labeled in vivo using the d-KV27 fluorescent probe, and then removed from the mouse abdominal cavity for nuclear fluorescence labeling with Nucblue. Then the macrophages were observed under a fluorescence microscope (20×, light source 10%, exposure 200 ms, gain 5). The results are shown in FIG. 19.

[0153] FIG. 19A is an image obtained by labeling with the d-KV27 fluorescent probe (shown in red fluorescence in the original fluorescence color image), and FIG. 19B is an image obtained by nuclear staining with Nucblue (shown in blue fluorescence in the original fluorescence color image), and FIG. 19C is a merged image of FIGS. 19A and 19B.

[0154] It can be seen from FIG. 19 that the macrophages extracted from the mouse abdominal cavity can be labeled with the d-KV27 fluorescent probe.

Example 22. In Vivo Fluorescent Labeling of Macrophages in a Mouse Abdominal Cavity by the b-KA27 Fluorescent Probe

[0155] Referring to Example 3, using the same treatment process, the mouse peritoneal macrophages were first labeled in vivo using the b-KA27 fluorescent probe, and then removed from the mouse abdominal cavity for nuclear fluorescence labeling with Nucblue. Then the macrophages were observed under a fluorescence microscope (20×, light source 10%, exposure 200 ms, gain 5). The results are shown in FIG. 20.

[0156] FIG. 20A is an image by labeling with the b-KA27 fluorescent probe (shown in red fluorescence in the original fluorescence color image), and FIG. 20B is an image obtained by nuclear staining with Nucblue (shown in blue fluorescence in the original fluorescence color image), and FIG. 20C is a merged image of FIGS. 20A and 20B.

[0157] It can be seen from FIG. 20 that the macrophages extracted from the mouse abdominal cavity can be labeled with the b-KA27 fluorescent probe.

Example 23. In Vivo Fluorescent Labeling of Macrophages in a Mouse Abdominal Cavity by the KESG24 Fluorescent Probe

[0158] Referring to Example 3, using the same treatment process, the mouse peritoneal macrophages were first labeled in vivo using the KESG24 fluorescent probe, and then removed from the mouse abdominal cavity for nuclear fluorescence labeling with Nucblue. Then the macrophages were observed under a fluorescence microscope (20×, light source 50%, exposure 200 ms, gain 5). The results are shown in FIG. 21.

[0159] FIG. 21A is an image obtained by labeling with the KESG244 fluorescent probe (shown in red fluorescence in the original fluorescence color image), and FIG. 21B is an image obtained by nuclear staining with Nucblue (shown in blue fluorescence in the original fluorescence color image), and FIG. 21C is a merged image of FIGS. 21A and 21B.

[0160] It can be seen from FIG. 21 that the macrophages extracted from the mouse abdominal cavity can be labeled with the KESG24 fluorescent probe.

Example 24. In Vivo Fluorescent Labeling of Macrophages in a Mouse Abdominal Cavity by the Cx-LK24 Fluorescent Probe

[0161] Referring to Example 3, using the same treatment process, the mouse peritoneal macrophages were first labeled in vivo using the Cx-LK24 fluorescent probe, and then removed from the mouse abdominal cavity for nuclear fluorescence labeling with Nucblue. Then the macrophages were observed under a fluorescence microscope (20×, light source 50%, exposure 200 ms, gain 5). The results are shown in FIG. 22.

[0162] FIG. 22A is an image obtained by labeling with the Cx-LK24 fluorescent probe (shown in red fluorescence in the original fluorescence color image), and FIG. 22B is an image obtained by nuclear staining with Nucblue (shown in blue fluorescence in the original fluorescence color image), and FIG. 22C is a merged image of FIGS. 22A and 22B.

[0163] It can be seen from FIG. 22 that the macrophages extracted from the mouse abdominal cavity can be labeled with the Cx-LK24 fluorescent probe.

Example 25. In Vivo Fluorescent Labeling of Macrophages on Mouse Abdominal Wall by the EK24 Fluorescent Probe and CD68 Antibody

[0164] Referring to Example 3, using the same drug injection process, inject EK24, and FITC anti-mouse CD68 antibody (Biolegend) which was diluted with PBS from 2 μL to 500 μL. Remove mouse abdominal wall muscle for Nucblue double-staining and then observing under a fluorescence microscope (20×, light source 50%, exposure 200 ms, gain 5). The results are shown in FIG. 23.

[0165] FIG. 23A is an image obtained by labeling with the EK24 fluorescent probe (shown in red fluorescence in the original fluorescent color image), FIG. 23B is an CD68 antibody labeled image (shown in green fluorescence in the original fluorescent color image), FIG. 23C is an image obtained by nuclear staining with Nucblue (shown in blue fluorescence in the original fluorescent color image), and FIG. 23D is a merged image of FIGS. 23A, 23B and 23C.

[0166] It can be seen from FIG. 23 that the macrophages on mouse abdominal wall can be labeled with the EK24 fluorescent probe, while other cells cannot be labeled.

Example 26. In Vivo Fluorescent Labeling of Mouse Pulmonary Macrophages by the EK24 Fluorescent Probe and CD11c Antibody

[0167] Spray 100 μL of 50 μM EK24-drug into the lungs of 4- to 8-week-old mice through the trachea thereof, and after 18 hours, dilute 5 μL of Alexa Fluor®488 anti-mouse CD11c antibody (Biolegend) to 100 μL and then spray into the lung for 1.5 h. Dissect the mice, and take the whole lungs for observing under a fluorescence microscope (20×, light source 50%, exposure 200 ms, gain 5). The results are shown in FIG. 24.

[0168] FIG. 24A is an image obtained by labeling with the EK24 fluorescent probe (shown in red fluorescence in the original fluorescence color image), and FIG. 24B is an image obtained by labeling with CD11c antibody (shown in green fluorescence in the original fluorescence color image), and FIG. 24C is a merged image of FIGS. 24A and 24B.

[0169] It can be seen from FIG. 24 that both the EK24 fluorescent probe and CD11c antibody recognize the same cells in mouse alveoli, i.e. mouse alveolar macrophages.

Example 27. In Vivo Fluorescent Labeling of Macrophages in a Mouse Abdominal Cavity by the Hx-AVGK9 Fluorescent Probe

[0170] Referring to Example 3, using the same treatment process, the mouse peritoneal macrophages were first labeled in vivo using the Hx-AVGK9 fluorescent probe, and then removed from the mouse abdominal cavity for nuclear fluorescence labeling with Nucblue. Then the macrophages were observed under a fluorescence microscope (20×, light source 60%, exposure 300 ms, gain 10). The results are shown in FIG. 25.

[0171] FIG. 25A is an image obtained by labeling with the Hx-AVGK9 fluorescent probe (shown in red fluorescence in the original fluorescence color image), and FIG. 25B is an image obtained by nuclear staining with Nucblue (shown in blue fluorescence in the original fluorescence color image), and FIG. 25C is a merged image of FIGS. 25A and 25B.

[0172] It can be seen from FIG. 25 that the macrophages extracted from the mouse abdominal cavity can be labeled with the Hx-AVGK9 fluorescent probe.

Example 28. In Vivo Fluorescent Labeling of Macrophages in a Mouse Abdominal Cavity by the IK12 Fluorescent Probe

[0173] Referring to Example 3, using the same treatment process, the mouse peritoneal macrophages were first labeled in vivo using the IK12 fluorescent probe, and then removed from the mouse abdominal cavity for nuclear fluorescence labeling with Nucblue. Then the macrophages were observed under a fluorescence microscope (20×, light source 29%, exposure 200 ms, gain 5). The results are shown in FIG. 26.

[0174] FIG. 26A is an image obtained by labeling with the IK12 fluorescent probe (shown in red fluorescence in the original fluorescence color image), and FIG. 26B is an image obtained by nuclear staining with Nucblue (shown in blue fluorescence in the original fluorescence color image), and FIG. 26C is a merged image of FIGS. 26A and 26B.

[0175] It can be seen from FIG. 26 that the macrophages extracted from the mouse abdominal cavity can be labeled with the IK12 fluorescent probe.

Example 29. In Vivo Fluorescent Labeling of Macrophages in a Mouse Abdominal Cavity by the Hx-VVGK12 Fluorescent Probe

[0176] Referring to Example 3, using the same treatment method, the mouse peritoneal macrophages were first labeled in vivo using the Hx-VVGK12 fluorescent probe, and then removed from the mouse abdominal cavity for nuclear fluorescence labeling Nucblue. Then the macrophages were observed under a fluorescence microscope (20×, light source 50%, exposure 200 ms, gain 5). The results are shown in FIG. 27.

[0177] FIG. 27A is an image obtained by labeling with the Hx-VVGK12 fluorescent probe (shown in red fluorescence in the original fluorescence color image), and FIG. 27B is an image obtaining by nuclear staining with Nucblue (shown in blue fluorescence in the original fluorescence color image), and FIG. 27C is a merged image of FIGS. 27A and 27B.

[0178] It can be seen from FIG. 27 that the macrophages extracted from the mouse abdominal cavity can be labeled with the Hx-VVGK12 fluorescent probe.

[0179] It can be concluded from the above embodiments that the probes derived from the C-terminal fragments of Triokinases/FMN cyclases of human and non-human animals can effectively recognize monocyte precursors and monocytes/macrophages.