POLYPEPTIDE WITH FUNCTION OF 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 EQPDPGAVAAAAILRAILE 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.-15. (canceled)

16. A polypeptide for targeting recognition of immune cells, comprising: (a) an amino acid sequence containing a C-terminal fragment sequence EQPDPGAVAAAAILRAILE (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 substantially identical means that an amino acid sequence has more than 80% 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.

17. The polypeptide according to claim 16, wherein the amino acid sequences of (a) and (b) comprise 60 or less amino acid residues.

18. The polypeptide according to claim 17, wherein the amino acid sequences of (a) and (b) comprise 45 or less amino acid residues.

19. The polypeptide according to claim 16, wherein the amino acid sequence of (a) is EQPDPGAVAAAAILRAILE (SEQ ID NO.:1), LEQPDPGAVAAAAILRAILE (SEQ ID NO.:2), EQPDPGAVAAAAILRAILEVLQS (SEQ ID NO.:3), KNMEAGAGRASYISSARLEQPDPGAVAAAAILRAIL (SEQ ID NO.:4), or TKNMEAGAGRASYISSARLEQPDPGAVAAAAILRAILEVLQS (SEQ ID NO.:5.

20. The polypeptide according to claim 16, wherein the amino acid sequence of (b) is a sequence from a C-terminal fragment of a non-human Triokinase/FMN cyclase and has more than 80% sequence identity to the amino acid sequence as described in (a).

21. The polypeptide according to claim 20, wherein the amino acid sequence of (b) is LQPDPGAVAAAAVLRAVLEGLQG (SEQ ID NO.:6), DQPDPGAVAAAAIFRAILEVLQTKAA (SEQ ID NO.:7), DQPDPGAVAAAAILRTILEVLQSQGV (SEQ ID NO.:8), DQPDPGAVAAAAILRAILEVLQSQGA (SEQ ID NO.:9) or EQPDPGAVAAAAILRAILEVLQS (SEQ ID NO.:10).

22. The polypeptide according to claim 16, wherein the immune cells comprise lymphocytes, dendritic cells, monocyte precursors, monocytes, macrophages, basophilic granulocytes, eosinophilic granulocytes, and mastocytes.

23. The polypeptide according to claim 22, wherein the immune cells comprise monocyte precursors, monocytes and macrophages.

24. A nucleic acid sequence encoding the polypeptide for targeting recognition of immune cells according to claim 16.

25. A composition comprising the polypeptide for targeting recognition of immune cells according to claim 16.

26. The composition according to claim 25, further comprising a reporter.

27. The composition according to claim 26, wherein the reporter is linked to the N-terminus and/or C-terminus of the polypeptide.

28. The composition according to claim 25, wherein the polypeptide is linked to the reporter through a cysteine or lysine residue which is attached to the polypeptide.

29. 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.

30. The composition according to claim 26, wherein the composition is used for in vivo imaging of immune cells, or for immune-labeling and/or microscopy analysis of cells or tissues in vitro.

31. The composition according to claim 25, further comprising a drug molecule.

32. The composition according to claim 31, wherein the composition is used for targeted drug delivery to immune cells.

33. The composition according to claim 25, wherein the immune cells comprise lymphocytes, dendritic cells, monocyte precursors, monocytes, macrophages, basophilic granulocytes, eosinophilic granulocytes, and mastocytes.

34. A method for in vivo imaging immune cells, comprising incubating an effective amount of the composition according to claim 25 with cells or tissues to be labeled.

35. The method according to claim 34, wherein the immune cells comprise lymphocytes, dendritic cells, monocyte precursors, monocytes, macrophages, basophilic granulocytes, eosinophilic granulocytes, and mastocytes.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0058] 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.

[0059] FIG. 2 shows in vivo fluorescent labeled image obtained by staining mouse macrophage cell line RAW264.7 cultured in vitro. The mouse macrophage cell line RAW264.7 was stained with EK24 probe (shown in red in the original fluorescent color image) and Nucblue (shown in blue in the original fluorescent color image).

[0060] FIG. 3 shows in vivo fluorescent labeled image obtained by staining macrophages in a mouse abdominal cavity with EK24 fluorescent probe. The mouse peritoneal macrophages were first labeled with the EK24 fluorescent probe in vivo, and then removed from the mouse peritoneal cavity for Nucblue staining. FIG. 3A is an EK24-labeled image (shown in red in the original fluorescence color image), and 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. 6A and 6B.

[0061] FIG. 4 shows fluorescent labeled image obtained by staining macrophages, which were removed from the mouse abdominal cavity and cultured in vitro. FIGS. 4A and 4B show fluorescent labeled images obtained by using EK24 probe and F4/80 antibody, respectively. FIG. 4C is a merged image of FIGS. 7A and 7B.

[0062] FIG. 5 shows fluorescent labeled image obtained by staining macrophages from rat peritoneal cavity with EK24 fluorescent probe. FIG. 5A shows a direct fluorescently labeled image of macrophages from a rat abdominal cavity obtained by using the EK24 fluorescent probe. FIG. 5B shows a fluorescent labeled image of cultured macrophages isolated from rat peritoneal cavity with EK24 probe.

[0063] FIG. 6 shows a fluorescent labeled image obtained by staining cultured monocyte precursors isolated from mouse bone marrow using, with EK24 fluorescent probe (shown in red in the original fluorescence color image) and Nucblue (shown in blue in the original fluorescence color image).

[0064] FIG. 7 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).

[0065] FIG. 8 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).

[0066] FIG. 9 shows fluorescent labeled image obtained by staining cultured peritoneal macrophages and mesenteric digestive cells with EK24 fluorescent probe. FIG. 9A is a labeled image obtained by using EK24 fluorescent probe, and FIG. 9B is a nuclear-staining image obtained by using Nucblue, and FIG. 9C is a merged image of FIGS. 9A and 9B.

[0067] FIG. 10 shows in vivo fluorescent labeled image obtained by staining macrophages in a mouse abdominal cavity with KS24 fluorescent probe. 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. 10A is a fluorescent labeled image obtained by using the KS24 fluorescent probe (shown in red in the original fluorescence color image), and FIG. 10B is a nuclear-staining image obtained by using Nucblue (shown in blue in the original fluorescence color image), and FIG. 10C is a merged image of FIGS. 10A and 10B.

[0068] FIG. 11 shows in vivo fluorescent labeled image obtained by staining macrophages in a mouse abdominal cavity with d-KV27 fluorescent probe. The mouse peritoneal macrophages were first labeled with d-KV27 fluorescent probe in vivo, and then removed from mouse peritoneal cavity for Nucblue staining. FIG. 11A is a d-KV27-labeled image (shown in red in the original fluorescence color image), and FIG. 11B is a nuclear-staining image with Nucblue (shown in blue in the original fluorescence color image), and FIG. 11C is a merged image of FIGS. 11A and 11B.

[0069] FIG. 12 shows in vivo fluorescent labeled image obtained by staining macrophages in a mouse abdominal cavity with d-KV27 fluorescent probe. The mouse peritoneal macrophages were first labeled with d-KV27 fluorescent probe in vivo, and then removed from mouse peritoneal cavity for Nucblue staining. FIG. 12A is a d-KV27-labeled image (shown in red in the original fluorescence color image), and FIG. 12B is a nuclear-staining image with Nucblue (shown in blue in the original fluorescence color image), and FIG. 12C is a merged image of FIGS. 12A and 12B.

[0070] FIG. 13 shows in vivo fluorescent labeled image 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. 13A is a TS42-labeled image (shown in green in the original fluorescence color image), and FIG. 13B is a nuclear-staining image with Nucblue (shown in blue in the original fluorescence color image), and FIG. 13C is a merged image of FIGS. 13A and 13B.

[0071] FIG. 14 shows in vivo fluorescent labeled image obtained by staining macrophages in a mouse abdominal cavity with KnNL36 fluorescent probe. The mouse peritoneal macrophages were first labeled with the KnNL36 fluorescent probe in vivo, and then removed from mouse peritoneal cavity for Nucblue staining. FIG. 14A is a KnNL36-labeled (shown in red in the original fluorescence color image), and FIG. 14B is a nuclear-staining image with Nucblue (shown in blue in the original fluorescence color image), and FIG. 14C is a merged image of FIGS. 14A and 14B.

[0072] FIG. 15 shows in vivo fluorescent labeled image obtained by staining macrophages in a mouse abdominal cavity with LK24 fluorescent probe. 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. 15A is a LK24-labeled image (shown in red 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.

[0073] FIG. 16 shows in vivo fluorescent labeled image obtained by staining macrophages in a mouse abdominal cavity with LEK21 fluorescent probe. The mouse peritoneal macrophages were first labeled with LEK21 fluorescent probe in vivo, and then removed from mouse peritoneal cavity for Nucblue staining. FIG. 16A is a LEK21-labeled image (shown in red 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.

[0074] FIG. 17 shows in vivo fluorescent labeled image obtained by staining macrophages in a mouse abdominal cavity with r-KA27 fluorescent probe. The mouse peritoneal macrophages were first labeled with r-KA27 fluorescent probe in vivo, and then removed from mouse peritoneal cavity for Nucblue staining. FIG. 17A is a r-KA27-labeled image (shown in red in the original fluorescence color image), and FIG. 17B is a nuclear-staining image with Nucblue (shown in blue in the original fluorescence color image), and FIG. 17C is a merged image of FIGS. 17A and 17B.

[0075] FIG. 18 shows the fluorescent labeled image obtained by staining mouse primary monocyte precursors with KS24 fluorescent probe at different incubation times. FIGS. 18A, 18B, 18C, and 18D show KS24-labeled images to incubate for 10 min, 20 min, 40 min and 80 min, respectively. In FIGS. 18A, 18B, 18C, and 18D, i) is a KS24-labeled image (shown in red in the original fluorescence color image), and ii) is a nuclear-staining image with Nucblue (shown in blue in the original fluorescence color image), and iii) is a merged image of i) and ii).

[0076] FIG. 19 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. 19A is a EK24-labeled image (shown in red in the original fluorescent color image), FIG. 19B is a CD68 antibody labeled image (shown in green in the original fluorescent color image), FIG. 19C is a nuclear-staining image with Nucblue (shown in blue in the original fluorescent color image), and FIG. 19D is a merged image of FIGS. 19A, 19B, and 19C.

DETAILED DESCRIPTION

[0077] 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 EK24 Fluorescent Probe

[0078] The polypeptides used in 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.

[0079] In this example, the polypeptide having the following amino acid sequence was synthesized:

TABLE-US-00001 EQPDPGAVAAAAILRAILEVLQSK.

[0080] 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 the pH, reacted for 1-2 hours, purified by HPLC to obtain the EK24 fluorescence probe. The chemical structure of the obtained fluorescent probe is as follows:

TABLE-US-00002 EQPDPGAVAAAAILRAILEVLQS-Y (EK24),
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 EK24 Fluorescent Probe Solution

[0081] 1 mg of EK24 fluorescent probe was dissolved in 87 μL of DMSO (dimethyl sulfoxide), which was then added to 6873 μL serum-free DMEM (Hyclone) medium, mixing well to give 50 NM of EK24 fluorescent probe solution. As required, 50 μM of EK24 fluorescent probe solution can be diluted by adding the serum-free DMEM medium during use.

Example 3 In Vitro Fluorescence Labeled Staining of a Mouse Macrophage Cell Line

[0082] 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. 2.

[0083] FIG. 2 shows images of in vitro cultured mouse macrophage cell line that were fluorescent labeled using the EK24 fluorescent probe (shown in red in the original fluorescence color image) and Nucblue (shown in blue in the original fluorescence color image).

[0084] It can be seen from FIG. 2 that the macrophage cell line RAW264.7 can be labeled with the EK24 fluorescent probe (shown in red in the original fluorescence color image).

Example 4 In Vivo Fluorescent Labeling Macrophages in a Mouse Abdominal Cavity with EK24 Fluorescent Probe

[0085] Dilute 300 μL of 50 μM EK24 fluorescent probe solution with serum-free DMEM incomplete medium to obtain 1 mL of solution, then inject into the abdominal cavity of 5-8 weeks 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 a 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. Add appropriate amount of cells to a 96-well plate and double-stain with a nuclear dye Nucblue (Invitrogen). Observe under fluorescence microscope (light source 10%, exposure 200 ms, gain 5) after the suspended cells were settled down at the bottom of the plate. The results are shown in FIG. 3.

[0086] FIG. 3A is an EK24-labeled image (shown in red in the original fluorescence color image), FIG. 2B is a nuclear-staining image obtained by using Nucblue (shown in blue in the original fluorescence color image), FIG. 3C is a merged image of FIGS. 3A and 3B.

[0087] It can be seen from FIG. 3 that the macrophages extracted from the mouse abdominal cavity can be labeled with the EK24 fluorescent probe (shown in red in the original fluorescence color image).

Example 5 In Vitro Fluorescent Labeling of Cultured Macrophages Isolated from Mouse Peritoneal Cavity by EK24 Probe

[0088] 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 bottom of the well, and other cells would be washed off. The cells were cultured for more than 2 days until the cells attached 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 Example 4. 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. 4.

[0089] FIG. 4A shows a fluorescent labeled image obtained by using EK24 fluorescent probe (shown in red in the original fluorescence color image) and nuclear stained image obtained by using Nucblue (shown in blue in the original fluorescence color image).

[0090] FIG. 4B shows a fluorescent labeled image obtained by using the F4/80 antibody (shown in green in the original fluorescence color image) and nuclear stained image obtained by using Nucblue (shown in blue in the original fluorescence color image). F4/80 antigen is currently recognized as a surface characteristic marker of mouse peritoneal macrophages. It can be seen from FIG. 4B that almost all the cells obtained by intraperitoneal massage were labeled with F4/80 antibody, indicating that these labeled cells were macrophages.

[0091] FIG. 4C is a merged image of FIGS. 4A, and 4B. 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 6 In Vitro Fluorescent Labeling of Primary Macrophages from Rat Peritoneal Cavity by EK24 Fluorescent Probe

[0092] With the same process as in Example 5, 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 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. 5.

[0093] FIG. 5A shows images obtained from direct fluorescent labeling of macrophages from a rat abdominal cavity with EK24 fluorescent probe (shown in red in the original fluorescence color image) and Nucblue (shown in blue in the original fluorescence color image). FIG. 5B 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 EK24 fluorescent probe (shown in red in the original fluorescence color image) and Nucblue (shown in blue in the original fluorescence color image).

[0094] It can be seen from FIG. 5 that the EK24 fluorescent probe can effectively label the in vitro cultured primary macrophages from rat peritoneal cavity.

Example 7 In Vitro Fluorescence Labeling of Mouse Primary Monocyte Precursors by EK24 Probe

[0095] 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.

[0096] In a bio-safety hood, take the femur of a Kunming mice, 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 it to a 96-well plate for culture. After culturing for more than 2 days, until the cells attached the bottom firmly, the cells remaining in the wells were basically monocyte precursors. Treat with reference to Example 5. Then the cells were observed under a fluorescence microscope (200×, light source 50%, exposure 200 ms, gain 5). The results are shown in FIG. 6.

[0097] FIG. 6 shows images obtained with direct fluorescent labeling of monocyte precursors from the mesenchyme of mouse with EK24 fluorescent probe (shown in red in the original fluorescence color image) and Nucblue (shown in blue in the original fluorescence color image). It can be seen that the EK24 fluorescent probe (shown in red in the original fluorescence color image) is capable of labeling monocyte precursors from the mesenchyme of mouse bone marrow.

Example 8 In Vitro Fluorescent Labeling of Rat Primary Monocyte Precursors by EK24 Fluorescent Probe

[0098] With a similar process to Example 7, 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. 7.

[0099] FIG. 7 shows images obtained by fluorescent labeling of monocyte precursors from rat bone marrow mesenchyme with EK24 fluorescent probe (shown in red in the original fluorescence color image) and Nucblue (shown in blue in the original fluorescence color image). It can be seen that the EK24 fluorescent probe (shown in red in the original fluorescence color image) is also capable of labeling monocyte precursors from rat bone marrow mesenchyme.

Example 9 In Vitro Fluorescent Labeling of Primary Monocyte Precursors of New Zealand White Rabbit by EK24 Fluorescent Probe

[0100] With a similar process to Example 7, 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. 8.

[0101] FIG. 8 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 in the original fluorescence color image) and Nucblue (shown in blue in the original fluorescence color image). It can be seen that the EK24 fluorescent probe (shown in red in the original fluorescence color image) is also capable of labeling monocyte precursors from New Zealand White Rabbit bone marrow mesenchyme.

Example 10 EK24 Fluorescent Probe Selective Labeling and Staining of Cultured Peritoneal Macrophages Among Other Cells Released from Enzyme-Digested Mesenterium

[0102] 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 a 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. 9.

[0103] FIG. 9A is an image obtained by labeling with EK24 fluorescent probe (shown in red in the original fluorescence color image), and FIG. 9B is an image obtained by nuclear-staining with Nucblue (shown in blue in the original fluorescence color image), and FIG. 9C is a merged image of FIGS. 9A and 9B. 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 but no red fluorescence).

Example 11 Fluorescent Labeling of Macrophages by the Probes which are Derived from the C-Terminal Fragmentses 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-00003 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 imaging Species Sequence effect Figure Name Chickens LQPDPGAVA + FIG. 15 LK24 (Gallus AAAVLRAVL gallus) EGLQG-Y Rats X-DQPDPGA +++ FIG. 17 r-KA27 (Rattus VAAAAIFRA norvegicus) ILEVLQTKA A Dogs X-DQPDPGA ++ FIG. 11 d-KV27 (canis VAAAAILRT lupus) ILEVLQSQG V Pigs X-DQPDPGA +++ FIG. 12 b-KA27 (sus VAAAAILRA scrofa) ILEVLQSQG A Cattles X-DQPDPGA +++ FIG. 12 b-KA27 (bos VAAAAILRA taurus) ILEVLQSQG A Rhesus EDQPDPGAV +++ FIG. 2-9 EK24 monkeys AAAAILRAI (macaca LEVLQS-Y mulatta) Humans EDQPDPGAV +++ FIG. 2-9 EK24 (Homo AAAAILRAI sapiens) LEVLQS-Y Humans X-EDQPDPG +++ FIG. 10 KS24 (Homo AVAAAAILR sapiens) AILEVLQS Humans LEQPDPGAV + FIG. 16 LEK21 (Homo AAAAILRAI sapiens) LE-Y Humans X-TKNMEAG ++ FIG. 13 TS42 (Homo AGRASYISS sapiens) ARLEQPDPG AVAAAAILR AILEVLQS Humans X-KNMEAGA ++ FIG. 14 KnNL36 ( Homo GRASYISSA sapiens) RLEQPDPGA VAAAAILRA IL  X, Y = reporter or cysteine/lysine + reporter (rhodamine or FITC)

[0105] From the results in Table 1, it can be seen that all the polypeptide fragments from the C-terminal fragmentses of Triokinase/FMN cyclases of different animals can targeted recognize monocytes/macrophages. Specifically, 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, EK24 derived from rhesus monkey Triokinase/FMN cyclase and EK24, KS24, LEK21, TS42 and KnNL36 derived from human Triokinase/FMN cyclase, can effectively recognize macrophages in vivo. It indicates that highly conserved polypeptide fragments from the C-terminal fragmentses of Triokinases/FMN cyclases of different species can be used as probes to label macrophages. The following examples provide further relevant experimental results.

Example 12 Fluorescent Labeling of Macrophages in a Mouse Abdominal Cavity by KS24 Fluorescent Probe

[0106] Referring to Example 4, using the same treatment process, the mouse peritoneal macrophages were first labeled in vivo using KS24 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 10%, exposure 200 ms, gain 5). The results are shown in FIG. 10.

[0107] FIG. 10A is a labeled image obtained by using KS24 fluorescent probe (shown in red in the original fluorescence color image), and FIG. 10B is a nuclear-staining image obtained by using Nucblue (shown in blue in the original fluorescence color image), and FIG. 10C is a merged image of FIGS. 10A and 10B.

[0108] It can be seen from FIG. 10 that the macrophages extracted from the mouse abdominal cavity can be labeled with KS24 fluorescent probe.

Example 13 Fluorescent Labeling of Macrophages in a Mouse Abdominal Cavity by d-KV27 Fluorescent Probe

[0109] Referring to Example 4, using the same treatment process, the mouse peritoneal macrophages were first labeled in vivo using d-KV27 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. 11.

[0110] FIG. 11A is a labeled image obtained by using d-KV27 fluorescent probe (shown in red in the original fluorescence color image), and FIG. 11B is a nuclear-staining image obtained by using Nucblue (shown in blue in the original fluorescence color image), and FIG. 11C is a merged image of FIGS. 11A and 11B.

[0111] It can be seen from FIG. 11 that the macrophages extracted from the mouse abdominal cavity can be labeled with d-KV27 fluorescent probe.

Example 14 Fluorescent Labeling of Macrophages in a Mouse Abdominal Cavity by b-KA27 Fluorescent Probe

[0112] Referring to Example 4, using the same treatment process, the mouse peritoneal macrophages were first labeled in vivo using b-KA27 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. 12.

[0113] FIG. 12A is a labeled image obtained by using b-KA27 fluorescent probe (shown in red in the original fluorescence color image), and FIG. 12B is a nuclear-staining image obtained by using Nucblue (shown in blue in the original fluorescence color image), and FIG. 12C is a merged image of FIGS. 12A and 12B.

[0114] It can be seen from FIG. 12 that the macrophages extracted from the mouse abdominal cavity can be labeled with b-KA27 fluorescent probe.

Example 15 Fluorescent Labeling of Macrophages in a Mouse Abdominal Cavity by TS42 Fluorescent Probe

[0115] Referring to Example 4, using the same treatment process, the mouse peritoneal macrophages were first labeled in vivo using TS42 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 50%, exposure 200 ms, gain 5). The results are shown in FIG. 13.

[0116] FIG. 13A is a labeled image obtained by using TS42 fluorescent probe (shown in green in the original fluorescence color image), and 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.

[0117] It can be seen from FIG. 13 that the macrophages extracted from the mouse abdominal cavity can be labeled with TS42 fluorescent probe.

Example 16 Fluorescent Labeling of Macrophages in a Mouse Abdominal Cavity by KnNL36 Fluorescent Probe

[0118] Referring to Example 4, using the same treatment process, the mouse peritoneal macrophages were first labeled in vivo using KnNL36 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. 14.

[0119] FIG. 14A is a labeled image obtained by using KnNL36 fluorescent probe (shown in red in the original fluorescence color image), and FIG. 14B is a nuclear-staining image obtained by using Nucblue (shown in blue in the original fluorescence color image), and FIG. 14C is a merged image of FIGS. 14A and 14B.

[0120] It can be seen from FIG. 14 that the macrophages extracted from the mouse abdominal cavity can be labeled with KnNL36 fluorescent probe.

Example 17 Fluorescent Labeling of Macrophages in a Mouse Abdominal Cavity by LK24 Fluorescent Probe

[0121] Referring to Example 4, using the same treatment process, the mouse peritoneal macrophages were first labeled in vivo using LK24 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×). The results are shown in FIG. 15.

[0122] FIG. 15 shows the image observed under a fluorescence microscope (light source 50%, exposure 200 ms, gain 5). Wherein FIG. 15A is a labeled image obtained by using LK24 fluorescent probe (shown in red in the original fluorescence color image), and FIG. 15B is a nuclear-staining image obtained by using Nucblue (shown in blue in the original fluorescence color image), and FIG. 15C is a merged image of FIGS. 15A and 15B.

[0123] It can be seen from FIG. 15 that the macrophages extracted from the mouse abdominal cavity can be labeled with LK24 fluorescent probe.

Example 18 Fluorescent Labeling of Macrophages in a Mouse Abdominal Cavity Using LEK21 Fluorescent Probe

[0124] Referring to Example 4, using the same treatment process, the mouse peritoneal macrophages were first labeled in vivo using LEK21 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×). The results are shown in FIG. 16.

[0125] FIG. 16 shows the image observed under a fluorescence microscope (light source 50%, exposure 200 ms, gain 5). Wherein FIG. 16A is a labeled image obtained by using LEK21 fluorescent probe (shown in red in the original fluorescence color image), and FIG. 16B is a nuclear-staining image obtained by using Nucblue (shown in blue in the original fluorescence color image), and FIG. 16C is a merged image of FIGS. 16A and 16B.

[0126] It can be seen from FIG. 16 that the macrophages extracted from the mouse abdominal cavity can be labeled with LEK21 fluorescent probe.

Example 19 Fluorescent Labeling of Macrophages in a Mouse Abdominal Cavity by r-KA27 Fluorescent Probe

[0127] Referring to Example 4, using the same treatment process, the mouse peritoneal macrophages were first labeled in vivo using r-KA27 fluorescent probe, 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. 17.

[0128] FIG. 17A is a labeled image obtained by using r-KA27 fluorescent probe (shown in red in the original fluorescence color image), and FIG. 17B is a nuclear-staining image obtained by using Nucblue (shown in blue in the original fluorescence color image), and FIG. 17C is a merged image of FIGS. 17A and 17B.

[0129] It can be seen from FIG. 17 that the macrophages extracted from the mouse abdominal cavity can be labeled with r-KA27 fluorescent probe.

Example 20 Fluorescence Labeling of Primary Monocyte Precursors of Mouse by KS24 Fluorescent Probe at Different Incubation Times

[0130] Referring to Example 7, using the same treatment process, the monocyte precursors from mouse bone marrow were obtained. After culturing for 6 days, the cultured cells were labeled with KS24 fluorescent probe over 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. 18.

[0131] FIG. 18A shows the fluorescent labeled results obtained by incubating with the KS24 fluorescent probe for 10 min. FIG. 18B shows the fluorescent labeled results obtained by incubating with the KS24 fluorescent probe for 20 min. FIG. 18C shows the fluorescent labeled results obtained by incubating with the KS24 fluorescent probe for 40 min. FIG. 18D shows the fluorescent labeled results obtained by incubating with the KS24 fluorescent probe for 80 min. Nucblue was further used for nuclear staining. In each of FIGS. 18A, 18B, 18C, and 18D, i) is an image labeled with the KS24 fluorescent probe (shown in red in the original fluorescence color image), and ii) is an image obtained by nuclear staining with Nucblue (shown in blue in the original fluorescence color image), and iii) is a merged image of i) and ii).

[0132] It can be seen from FIG. 18 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 monocyte precursors of mouse.

Example 21 In Vivo Fluorescent Labeling of Macrophages on Mouse Abdominal Wall by EK24 Fluorescent Probe and CD68 Antibody

[0133] Referring to Example 4, using the same drug injection process, inject EK24, and FITC anti-mouse CD68 antibody (Biolegend) which was diluted with PBS from 2 μL to 500p. 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. 19.

[0134] FIG. 19A is a labeled image obtained by labeling with EK24 fluorescent probe (shown in red in the original fluorescent color image), FIG. 19B is an CD68 antibody labeled image (shown in green in the original fluorescent color image), FIG. 19C is an image obtained by nuclear staining with Nucblue (shown in blue in the original fluorescent color image), and FIG. 19D is a merged image of FIGS. 19A, 19B and 19C.

[0135] It can be seen from FIG. 19 that the macrophages on mouse abdominal wall can be labeled with the EK24 fluorescent probe, while other cells cannot be labeled.

[0136] It can be concluded from the above embodiments that the probe derived from the C-terminal fragmentses of Triokinases/FMN cyclases of human and non-human animals can effectively recognize monocyte precursors and monocytes/macrophages.