Recombinant glycoprotein targeting siglec-9 and preparation method and application thereof

Abstract

The application discloses a recombinant glycoprotein targeting Siglec-9 and a preparation method and application thereof. The recombinant protein contains an amino acid sequence as shown in SEQ ID NO:1 or a sequence with more than 90% homology thereto. The recombinant protein targets and stimulates Siglec-9, serves as a Siglec-9 receptor stimulant, has strong binding force with Siglec-9, high activity, and has potential for treating excessive inflammatory diseases.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a recombinant glycoprotein targeting Siglec-9, its preparation method, and its application. Background Technology

[0002] Sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9) is a receptor expressed on the surface of myeloid cells that specifically recognizes sialic acid-glycan ligands. It plays an important role in some physiological functions in the human body, including the recognition of self and non-self and the local regulation of osteoclast activity. [1] , Maintenance of neutrophil homeostasis in the blood [2] And so on. They also participated in infectious disease control. [3] Autoimmune diseases [4] Cardiovascular diseases [5] and the occurrence and development of tumors. [6,7] In recent years, Siglec-9 has been increasingly studied as a target for various diseases, especially in the fields of anti-inflammatory immunity and oncology, and the number of related research reports has shown a year-on-year increasing trend.

[0003] Siglec-9 is widely expressed in myeloid cells such as monocytes, neutrophils, dendritic cells, natural killer cells, and platelets, as well as a small number of lymphocyte subsets. In peripheral blood, Siglec-9 is primarily expressed on neutrophils, followed by NK cells, B cells, and monocytes. [8] Siglec-9 can mediate cell interactions and signaling functions through cis and trans interactions with sialyl glycan ligands (i.e., intracellular or intercellular). [9] It inhibits the activation of inflammation-related cells and reduces the level of inflammatory response. Although Siglec-9 is expressed at low levels on cells in a "resting" state, it is rapidly upregulated on the surface of cells exhibiting an inflammatory response, thereby inhibiting related inflammatory signaling pathways. [10,11] This suggests that the Siglec-9 signaling pathway has a low impact on physiological state, but it has good specificity for excessive inflammatory responses, making it a controllable and effective new target for preventing and treating related diseases caused by excessive inflammatory responses.

[0004] The biological activity of Siglec-9 / E requires activation by its ligands; therefore, glycan ligands are agonists of the Siglec-9 / E pathway. Since activation of the Siglec-9 signaling pathway requires the simultaneous binding of the same agonist molecule to multiple Sigglc-9 molecules on the cell membrane, physiological Siglec-9 / E agonists are primarily high-molecular-weight glycoproteins (often tens of kDa) expressed on the cell membrane. Currently, antibodies and small-molecule ligands designed to target Siglec-9 only exert inhibitory effects, and research on related agonists is limited. Reported agonists include pS9L.

[12] It can inhibit the activation of macrophages and neutrophils, and has the potential to suppress excessive inflammatory responses.

[13] However, pS9L is a synthetically produced large lipopeptide glycoside, belonging to exogenous macromolecular compounds. Its preparation process is complex, and it may also be immunogenic. Therefore, exploring and developing novel Siglec-9 agonists has significant scientific value and potential clinical significance in the prevention and treatment of diseases related to excessive inflammation. Simply put, activating Siglec-9 is a novel and effective anti-inflammatory strategy, but currently there are no suitable agonists. The main reason is that activating Siglec-9 requires a glycoprotein with a sufficiently large molecular weight.

[0005] Physiological ligands of Siglec-9 are widely distributed in the human body, but they are difficult to develop into Siglec-9 agonists. For example, MUC5B is a highly viscous, high-molecular-weight (>150 kDa), highly O-glycosylated mucoprotein with a "centipede"-like structure and extremely dense glycosylation modifications. This glycoprotein presents significant obstacles in drug delivery and glycosylation modification, making its modification as a Siglec-9 agonist ligand virtually impossible. Sialidized glycoprotein A on erythrocytes, in addition to interacting with Siglec-9, participates in several physiological functions, including maintaining erythrocyte morphology, stability, immune escape, and the ABO and Rh blood group systems.

[0006] If attempts are made to modify them into Siglec-9 agonist ligands, different individuals require different types of glycosylation modifications; otherwise, severe immune responses may be triggered. Gangliosides, as relatively small lipids, can interact with multiple members of the Siglec family in addition to binding to Siglec-9, which may lead to non-specific immunosuppression.

[0007] [1] Andes F T, Adam S, Hahn M, et al. The human sialic acid-bindingimmunoglobulin-like lectin Siglec-9 and its murine homolog Siglec-E controlosteoclast activity and bone resorption[J]. Bone, 2021, 143: 115665. [2] Dou C, Zhen G, Dan Y, et al. Sialylation of TLR2 initiatesosteoclast fusion[J]. Bone Res, 2022, 10(1): 24. [3] Karmakar J, Mandal C. Interplay between sialic acids, Siglec-E,and Neu1 regulates MyD88- and TRIF-dependent pathways for TLR4-activationduring leishmania donovani infection[J]. Front Immunol, 2021, 12: 626110. [4] Wu Y, Yang D, Liu R, et al. Selective response to bacterialinfection by regulating Siglec-E expression[J]. iScience, 2020, 23(9):101473. [5] Raich-Regue D, Resa-Infante P, Gallemi M, et al. Role of siglecsin viral infections: A double-edged sword interaction[J]. Mol Aspects Med,2023, 90: 101113. [6] Tsai T Y, Huang M T, Sung P S, et al. Siglec-3 (CD33) serves asan immune checkpoint receptor for HBV infection[J]. J Clin Invest, 2021, 131(11): e141965. [7] Zhao D, Jiang X, Xu Y, et al. Decreased Siglec-9 expression onnatural killer cell subset associated with persistent HBV replication[J].Front Immunol, 2018, 9: 1124. [8] Adeniji O S, Kuri-Cervantes L, Yu C, et al. Siglec-9 defines andrestrains a natural killer subpopulation highly cytotoxic to HIV-infectedcells[J]. PLoS Pathog, 2021, 17(11): e1010034. [9] Costa A F, Campos D, Reis C A, et al. Targeting glycosylation: Anew road for cancer drug discovery[J]. Trends Cancer, 2020, 6(9): 757-766.

[10] Stowell S R, Ju T, Cummings R D. Protein glycosylation in cancer[J]. Annu Rev Pathol, 2015, 10: 473-510.

[11] Rodriguez E, Boelaars K, Brown K, et al. Sialic acids inpancreatic cancer cells drive tumor-associated macrophage differentiation via the Siglec receptors Siglec-7 and Siglec-9[J]. Nat Commun, 2021, 12(1):1270.

[12] Schmassmann P, Roux J, Buck A, et al. Targeting the Siglec–sialic acid axis promotes antitumor immune responses in preclinical models ofglioblastoma[J]. Sci Transl Med, 2023, 15(705): eadf5302.

[13] Zeng Z, Li M, Wang M, et al. Increased expression of Siglec-9 inchronic obstructive pulmonary disease[J]. Sci Rep, 2017, 7(1): 10116. Summary of the Invention

[0008] The purpose of this invention is to provide a recombinant glycoprotein targeting Siglec-9, its preparation method, and its application.

[0009] To achieve the objectives of this invention, the following implementation scheme is provided.

[0010] In one embodiment, the present invention provides a recombinant glycoprotein that targets Siglec-9, said recombinant protein targeting and activating Siglec-9. In some embodiments, the recombinant glycoprotein described above contains an amino acid sequence as shown in SEQ ID NO: 1 or a sequence that is 90% or more homologous to it.

[0011] In some embodiments, the recombinant glycoprotein described above comprises a sequence that is 98% or more homologous to SEQ ID NO: 1, preferably a sequence with 99% homology.

[0012] The amino acid sequence of the recombinant glycoprotein (SEQ ID NO: 1) is as follows: ATMYRMQLLSCIALSLALVTNSGTTEGDKVRATRTVVKFPTKAHTTPWGLFYSWSTASLDSQMPSSLHPTQESTKEQTTFPPRWTPNFTLHMESITFSKTPKSTTEPTPSPTTS EPVPEPAPNMTTLEPTPSPTTPEPTSEPAPSPTTPEPTSEPAPSPTTPEPTSEPAPSPTTPEPTPIPTIATSPTILVSATSLITPKSTFLTTKPVSLLESTKKTIPELDQLEGG GSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0013] In some embodiments, a polynucleotide is provided that encodes the recombinant glycoprotein of the present invention described above. Preferably, the recombinant glycoprotein contains an amino acid sequence as shown in SEQ ID NO: 1 or a sequence that is more than 90% homologous to it.

[0014] In some embodiments, the polynucleotide described above has a nucleotide sequence as shown in SEQ ID NO: 2.

[0015] The nucleotide sequence (SEQ ID NO: 2) is as follows:

[0016] In some embodiments, a plasmid vector is provided containing a polynucleotide with a nucleotide sequence as shown in SEQ ID NO: 2.

[0017] In some implementations, the plasmid vector is pCAGGS, with an Fc tag (IgG1) linked to the C-terminus of a 300-500 nucleotide sequence selected from GPIbα via the linker peptide GGGS. This tag can be specifically captured and recognized by Protein A filler. A Kozak sequence is linked to the N-terminus of the nucleotide sequence to enhance eukaryotic gene translation efficiency, and an IL-2 sequence is directly linked after the Kozak sequence to promote the secretion of the target protein. EcoRI and BglII restriction endonuclease sites are also designed.

[0018] In some implementations, a host cell is provided containing the plasmid vector described above.

[0019] In some embodiments, a method for preparing the recombinant glycoprotein of the present invention is provided, comprising inserting a polynucleotide into a plasmid vector, then transforming and expressing it in a host cell, and then isolating and purifying it.

[0020] In one specific embodiment, the method for preparing the recombinant glycoprotein of the present invention includes the following steps: 1) The plasmid was transformed and extracted using E. coli; 2) Protein expression: Culture expression cells, mix plasmid with PEI and add it dropwise to the cultured expression cells, continue culturing, and collect the supernatant; 3) Purification: The collected supernatant is centrifuged and passed through a column.

[0021] Preferably, the above preparation method further includes adding non-energy sugars to the expression system 1-3 hours before the plasmid in step 2 is added to the cultured expression cells.

[0022] In some preferred embodiments, the above preparation method uses fucose, sialic acid, and mannose as the supplementary non-energy sugar.

[0023] In some embodiments, the invention provides the use of the recombinant glycoprotein targeting Siglec-9 in the manufacture of a medicament for treating inflammatory diseases, preferably, the inflammatory disease being a hyperinflammatory disease.

[0024] In some preferred embodiments, the recombinant glycoprotein of the present invention is M. HG protein.

[0025] The recombinant glycoprotein (M) of the present invention HGProtein) is M NG The recombinant protein expressed under optimized conditions exhibited significantly altered glycosylation levels, enhanced affinity for Siglec-9, and increased recombinant glycoprotein activity. This recombinant glycoprotein shows potential for treating inflammatory diseases, particularly hyperinflammatory diseases. Attached Figure Description

[0026] Figure 1 pCAGGS-M NG Plasmid design diagram.

[0027] Figure 2 pCAGGS-M NG -IL2ΔSP plasmid design diagram.

[0028] Figure 3 pCAGGS-M NG His 1 plasmid design diagram.

[0029] Figure 4 pCAGGS-M NG His 2 plasmid design diagram.

[0030] Figure 5 The electrophoresis diagram is used to verify the recombinant plasmid gel electrophoresis and enzyme digestion.

[0031] Figure 6 The figure shows the protein expression and purification results, where (A)M NG Elution chromatogram of Protein A column, 0.1 M pH 3.36 glycine-hydrochloric acid buffer; (B) M NG Protein expression results SDS-PAGE gel electrophoresis with Coomassie brilliant blue staining. Lane 1: Supernatant before Protein A column loading; Lanes 2 & 3: Supernatant after first and second Protein A column loading; Lanes 4-10; Glycine-hydrochloric acid elution tubes 1-7; (C)M NG Eluent passed through a desalting column, PBS displacement diagram; (D) Final purified M NG SDS-PAGE gel electrophoresis image with Coomassie brilliant blue staining.

[0032] Figure 7 For M NG The results of the in vitro binding assay with Siglec-9 / E, including (A) the determination of M by ELISA. NG (a) Siglec-E binding curve (n=3, Mean±SD); (b) Determination of M by non-denaturing, non-reducing gel electrophoresis Western Blot. NG The glycoprotein binds to Siglec-9His and is incubated on a PVDF membrane by Siglec-9His.

[0033] Figure 8 M marked for FITC NG Immunofluorescence image of protein binding to Thp-M macrophages, green represents FITC-labeled M. NG Protein, M NG + Fc group is pre-added with untagged Fc protein to prevent the Fc tag of the recombinant protein from binding to the cell membrane surface in a manner not associated with Siglec-9.

[0034] Figure 9 shows the FITC-marked M. NG Immunofluorescence image of protein binding to mouse neutrophils, green represents FITC-labeled M NG Proteins were categorized into Control group (no stimulation with nigrain) and Model group (stimulation with nigrain). The group marked "+Fc" was pre-added with untagged Fc protein to prevent the recombinant protein's Fc tag from binding to the cell membrane surface in a manner unrelated to Siglec-9.

[0035] Figure 10 For M NG Immunofluorescence images of mouse neutrophil activation inhibition, including (A) neutrophil immunofluorescence image; (B) Cit-H3 fluorescence intensity statistics, ns = no statistical difference, * p < 0.05, (n = 3, Mean ± SD).

[0036] Figure 11 The survival curves for the eukaryotic expression system HEK 293F cells are shown, where (A)M NG With M HG Cell viability in the sugar-added group; (B)M NG With M HG Changes in the number of groups with added sugar. (Compared to M) NG Compared with the previous group, **** p < 0.0001 (n=3, Mean ± SD).

[0037] Figure 12 The binding of each optimized group to Siglec-9 / E was determined by non-denaturing, non-reducing gel Western blotting. Glycoproteins were incubated with Siglec-9 His on PVDF membranes; ***p < 0.001, ****p < 0.0001 (n=3, Mean ± SD).

[0038] Figure 13 M marked for FITC HG Immunofluorescence image of protein binding to Thrp-M macrophages. Green represents FITC-labeled M. HG Protein, MHG The + Fc group is pre-added with untagged Fc protein to prevent the Fc tag of the recombinant protein from binding to the cell membrane surface in a manner unrelated to Siglec-9.

[0039] Figure 14 The graph shows the results of sialic acid content determination, where (A) is the standard curve of sialic acid content; and (B) is the determination of sialic acid content in different proteins, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (n = 4, Mean ± SD).

[0040] Figure 15 The image shows the sialic acid type determination of recombinant ligand proteins. In (A), the lectin blot results for Maackia Amurensis Lectin II are shown, with the statistical value being the relative M on the same membrane. NG The values, *p < 0.05, **p < 0.01, ***p < 0.001, (n = 4, Mean ± SD), (B) Sambucus NigraLectin lectin imprint results, statistical values ​​are relative to M on the same membrane. NG The value of p < 0.01 (n = 3, Mean ± SD); (C) Coomassie brilliant blue staining.

[0041] Figure 16 For M HG Excitation of Siglec-9 leads to upregulation of p-SHP-1 levels, including (A) WB bands and their statistical results; (B) band statistical results. Detailed Implementation

[0042] The following embodiments are provided to describe the present invention in more detail. However, these embodiments are provided only to help further understand the present invention and are not intended to limit the present invention. Those skilled in the art should understand that equivalent substitutions or corresponding improvements made to the content of the present invention still fall within the protection scope of the present invention.

[0043] Example 1: Design, expression, and purification of recombinant glycoprotein particles To obtain high purity M NG The recombinant protein was subjected to plasmid transformation, amplification, identification, and eukaryotic expression and purification.

[0044] 1. Plasmid construction and identification A mucin-like protein sequence from the 300-500 region of GPIbα was selected, designed, and named M. NG M NG Four types of plasmids were designed.

[0045] (1) MNG Plasmid: To successfully obtain this protein segment, it was cloned into the pCAGGS vector for eukaryotic expression. An Fc tag (IgG1) was designed at the C-terminus of the target nucleic acid sequence (300-500 nucleic acid sequences selected from GpIba), and the two were linked by the artificial linker peptide GGGS. This tag can be specifically captured and recognized by Protein A filler. In addition, a Kozak sequence was designed and linked at the N-terminus of the target nucleic acid sequence (300-500 nucleic acid sequences selected from GpIba) to enhance the eukaryotic gene translation efficiency, and an IL-2 sequence was directly linked after the Kozak sequence to promote the secretion of the target protein. EcoRI and BglII restriction endonuclease sites were designed for the insertion of the target sequence. The plasmid map is shown in Figure 1.

[0046] (2) M NG -IL2ΔSP: In M NG The IL-2 sequence was removed from the plasmid, while the rest of the sequence remained unchanged. See the plasmid map below. Figure 2 .

[0047] (3) M NG His 1 plasmid: The 300-500 sequence was cloned into the pcDNA3.1 (+) vector for eukaryotic expression. An 8 × His tag was designed at the C-terminus of the pcDNA3.1 (+) vector, and a Kozak sequence was designed at the N-terminus of the protein. BamHI and XhoI restriction endonuclease sites were designed for the insertion of the target sequence. The map is shown in Figure 3.

[0048] (4) M NG His 2 plasmid: Partial mutation and deletion of sequence 300-500, the rest of the design is the same as M. NG His 1 plasmid, the map is shown in Figure 4.

[0049] ①M NG Nucleic acid sequence (SEQ ID NO: 2):

[0050] ②M NG -IL2ΔSP nucleic acid sequence (SEQ ID NO: 3):

[0051] ③M NG His 1 nucleic acid sequence (SEQ ID NO: 4): ggatccgccaccatggatgcaatgaagagagggctctgctgtgtgctgctgctgtgtggagcagtcttcgtttcgcccgatgtgaaagccatgaccagcaacgtggccagcgtgcagtgcgacaacagcgacaagtttccagtgtataagtaccccggcaagggctgtcctaccctgggcgacgagggagataccgacctgtacgactactaccctgaggaagatacagagggcgacaaagtgcgggccacaagaaccgtggtgaagttccctacaaaggcccacaccacaccttggggcctgttctacagctggtccaccgcctctctggacagccaaatgccttctagcctgcaccccacacaggagtctaccaaggaacagaccacattcccccccagatggacccctaacttcaccctgcacatggaaagcatcaccttcagcaagaccccaaagtccaccactgaacctacccctagccctacaacaagcgagcccgtccccgagcctgctcctaatatgacccaccaccaccaccaccaccaccactagctcgag。

[0052] ④M NG His 2 nucleic acid sequence (SEQ ID NO: 5): .

[0053] Detection was performed by recombinant plasmid gel electrophoresis and enzyme digestion, such as Figure 5 As shown in the results, all four plasmids were successfully constructed. Figure 5 In the middle, (A) is M NG Recombinant plasmid, lane 1: DNA marker, lane 2: M NG Plasmid DNA, lane 3: plasmid digested with KpnI-XhoI; (B) is M NG -IL2ΔSP recombinant plasmid, lane 1: DNA marker, lane 2: EcoRI BglII digested plasmid, lane 3: M NG -IL2ΔSP plasmid DNA; (C) is M NG His 1 recombinant plasmid, lane 1: DNA marker, lane 2: M NG His 1 plasmid DNA, lane 3: plasmid digested with BamHI and XhoI; (D) is M NGHis 2 recombinant plasmid, lane 1: DNA marker, lane 2: plasmid digested with BamHI and XhoI, lane 3: M NG His 2 plasmid DNA.

[0054] 2. Plasmid transformation and extraction Take 4 μg M NG The plasmid powder was dissolved in ddH2O and the concentration was adjusted to 100 ng / μL. The plasmid was added to DH5α competent *E. coli* cells, and after a 30-min ice bath, a 90-s heat shock at 42 °C, and a 3-min ice bath, antibiotic-free LB medium was added, and the cells were incubated at 37 °C and 180 rpm for 40 min. After centrifugation and discarding some of the supernatant, the bacterial culture was resuspended and plated on LB agar plates containing ampicillin, and incubated overnight at 37 °C. Single colonies were picked and inoculated into LB liquid medium containing ampicillin for amplification and culture for 12–16 h, followed by scaling up to 100 mL. The plasmid was extracted according to the plasmid large-scale extraction kit instructions and sequenced to confirm correct plasmid construction.

[0055] 3.M NG protein expression HEK293F cells were cultured at a concentration of 0.3 × 10⁻⁶. 6 Cells / mL were seeded in serum-free medium and cultured at 37 ℃, 5% CO2, and 115 rpm. Transfection was performed when cells were in logarithmic growth phase and viability >98%. 150 mL of OPM medium and HEK293F cells were added to a 500 mL shake flask to achieve a final density of 1.2 × 10⁻⁶ cells / mL. 6 cells / mL. Transient transfection was performed using the PEI (polyethyleneimine) method. The plasmid and PEI were mixed at a mass ratio of 150:350, incubated at room temperature for 15 min, and then added dropwise to the cells. The cells were cultured at 37 ℃ and 115 rpm for 7 days. The supernatant was collected when the cell viability was below 70%.

[0056] 4.M NG Protein purification Cell culture supernatant was collected, centrifuged at 4 °C to remove cells and contaminating proteins, and filtered through a 0.22 μm filter membrane. An AKTA Pure protein purification system was used, with a 5 mL Protein A column attached. After equilibration with PBS, the sample was loaded for affinity purification. The column was washed with 1 × PBS, followed by elution with 0.1 M glycine-hydrochloric acid solution (pH 3.0–3.5), and immediately neutralized with 1 M Tris-HCl (pH 9.0). The elution peak was collected and analyzed by SDS-PAGE electrophoresis. After 10% polyacrylamide gel electrophoresis and Coomassie brilliant blue staining, the target band was clearly visible (e.g., ...). Figure 6 ).

[0057] 5. Protein desalting, concentration, and preservation The eluent containing the target protein was desalted and transferred to a PBS buffer system, followed by concentration by ultrafiltration at 4 °C. After filtration through a 0.22 μm filter membrane, the eluent was aliquoted and packaged. Store at 80°C.

[0058] Using the same method described above, plasmid M NG Replace with M NG -IL2ΔSP、M NG His 1 and M NG His 2, respectively, were used for protein expression and purification.

[0059] The results, confirmed by column chromatography and gel electrophoresis (see Table 1), indicate that M... NG and M NG -IL2ΔSP protein was successfully expressed and the target protein was obtained, of which M NG The expression yield was 11.7 mg / L, significantly higher than that of M. NG -IL2ΔSP (0.8 mg / L). M NG His 1 and M NG His 2 plasmid did not yield the target protein (see Table 1). This suggests M... NG The recombinant plasmid was successfully constructed and can be efficiently expressed in HEK293F cells, yielding high-purity recombinant M. NG protein.

[0060] Table 1. Protein Expression Yield Table

[0061] Example 2: In vitro determination of recombinant protein M NG Binding capability with Siglec-9 / E 1. ELISA method for determining M NG Binding with Siglec-E (a mouse homologue of Siglec-9) Prepare 0.05 mol / L, pH 9.6 carbonate buffer (CBB) as the coating buffer. Dilute Siglec-E stock solution (100 μg / mL) with CBB to 2 μg / mL, add 100 μL to each well, and incubate overnight at 4 °C for coating. The next day, discard the coating buffer and wash three times with PBST. Block with 1% BSA (prepared in PBS) at 37 °C for 2 h, wash three times, and then add different concentrations of M... NG Proteins (10, 50, 100, 1000, 5000, 10000, 20000 ng / mL) were added at 100 μL / well and incubated at 37 ℃ for 1 h. After washing, goat anti-human IgG1 HRP-labeled secondary antibody (1:6000) was added and incubated at 37 ℃ for 30 min. After washing again, 100 μL of TMB chromogenic solution was added and the reaction was carried out in the dark for 12 min. The reaction was terminated by adding 2 mol / L sulfuric acid. The absorbance at 450 nm was measured using a microplate reader, and the data were analyzed using GraphPad Prism 10 software. Positive controls, negative controls, and blank controls were set up, and each group had 3 replicates. The results showed that M NG It binds to Siglec-E in a concentration-dependent manner (see...) Figure 7 A).

[0062] 2. Determination of M by non-denaturing gel electrophoresis (Western Blot) NG Combination with Siglec-9 His M NG Protein concentration was adjusted to 0.2 mg / mL with PBS. Samples were treated with non-denaturing, non-reducing loading buffer at a loading volume of 10 μL. A 10% polyacrylamide gel was prepared for electrophoresis under non-denaturing PAGE conditions. After electrophoresis, the membrane was transferred and blocked. Siglec-9 His protein (1:500 diluted in DPBST) was premixed with Anti-His HRP-labeled secondary antibody (1:2000) for 30 min and incubated overnight at 4 °C. The membrane was washed, developed, and exposed the next day. ImageJ software was used to analyze the band grayscale values, and the M... NG Normalization is performed based on the baseline.

[0063] The results showed that, under non-denaturing and non-reducing conditions, M NG It forms a stable complex with Siglec-9 His, resulting in distinct specific bands (see...). Figure 7 B). Explanation of M NG The protein can specifically bind to Siglec-9 and Siglec-E.

[0064] Example 3: Cell-level determination of recombinant protein M NG Binding capability with Siglec-9 / E 1.M NG Protein FITC fluorescent group conjugation and verification Prepare 1 M sodium bicarbonate buffer: Dissolve 0.84 g sodium bicarbonate in 9 mL of deionized water, adjust the pH to 9.0 with 1 M sodium hydroxide solution, and add deionized water to a final volume of 10 mL. Calculation: The amount of active dye used in each reaction depends on the concentration of the protein to be labeled.

[0065] reactive dye stock solution (μL) =

[0066] Where MW is the molecular weight of the protein, 0.2 mL is the volume of the protein solution, 389 is the molecular weight of the reactive dye, 100 is the unit conversion factor, and MR is the molar ratio of dye to protein in the reaction mixture. Transfer 200 μL of protein solution to a 2 mL reaction tube containing a stir bar. Add 20 μL of freshly prepared 1M sodium bicarbonate solution to the reaction tube containing the protein.

[0067] Preparation of 10 mg / mL reactive dye stock solution: Heat DMSO and FITC reactive dye to room temperature. Before the reaction begins, add 37 μL of DMSO to the reactive dye. Use a pipette to stir up and down to ensure complete dissolution. While stirring the protein solution in the reaction tube, add 5.26 μL of reactive dye and discard the remaining reactive dye. Incubate the reaction mixture at room temperature in the dark and stir for 1 h.

[0068] Then follow the instructions for the Thermo Fisher FITC protein fluorescent labeling kit.

[0069] Protein preservation and validation: Aliquot the protein in the dark and store at -80 ℃. Take a small amount of protein for concentration determination and gel electrophoresis analysis.

[0070] 2.M NG Experimental verification of protein binding to Thp-M0 macrophages Take healthy Thp-1 cells, centrifuge at 1000 rpm for 5 min, and seed them in 24-well plates (each well containing a cell spreader), adjusting the cell density to 1×10⁶ cells / well. 4cells / mL. 1 mg / mL phorbol 12-myristate 13-acetate (PMA) was diluted to 100 ng / mL with 1640 complete medium and used to stimulate Thrp-1 cells for approximately 36 h. The medium was then removed, and the cells were washed once with PBS. M NG + For the Fc group, unlabeled Fc protein was diluted to 12 μg / mL with 1640 complete medium and incubated for 30 min. Labeled M was then added. NG The final protein concentration was 10 μg / mL, and the mixture was incubated in the dark for 30 min.

[0071] Cells were removed, culture medium discarded, and cells were gently washed once with 500 μL PBS. 200 μL of immunofluorescence fixative was added to each well for 15 min. Cells were washed twice with PBS, and 200 μL of immunofluorescence blocking buffer was added for 30 min. WGA was diluted 1:1000 with PBS, 200 μL per well, and incubated at room temperature for 1 h. Cells were washed three times with PBST, 5 min each time. The control group was incubated with FITC-labeled goat anti-mouse secondary antibody (1:500) for 1 h, and a separate secondary antibody group was set up. Then, DAPI and allophycocyanin (APC) were mixed according to the instructions, 200 μL per well, and stained for 20 min. Cells were washed three times with PBST, slides were prepared, two smears were placed on each slide, and 2 μL of antifluorescence quencher was added to each slide. After slightly air-drying, the slides were fixed with nail polish around the perimeter. Images were captured using a laser confocal microscope, data were saved, results were processed, and images were exported.

[0072] Results: Fluorescent label M NG The protein can clearly bind to the surface of THP-1 derived macrophages ( Figure 8 ).

[0073] 3.M NG Experiments verifying the binding of protein to mouse neutrophils (1) Extraction and isolation of mouse neutrophils Neutrophils derived from mouse bone marrow were isolated using the Percoll density gradient centrifugation method. First, Percoll separation solution was prepared: 45 mL of Percoll was added to 5 mL of 10×PBS to prepare Percoll working solution, which was then diluted with 1×PBS to prepare 80%, 65%, and 55% Percoll working solutions, respectively. Eight-week-old male C57 mice were anesthetized with isoflurane and euthanized by cervical dislocation. The mice were disinfected with 75% alcohol and soaked for 5 min. A midline incision was made ventrally, the muscles of both hind limbs were removed, and the femur and tibia were separated and placed in pre-cooled 1×PBS. The ends of the bones were cut open, and the bone marrow was washed with basal culture medium into centrifuge tubes. After filtering through a 70 μm cell sieve, the cells were centrifuged at 300 g for 5 min, the supernatant was discarded, and the cells were resuspended in culture medium containing 1% FBS. In a 15 mL centrifuge tube, 2 mL of 80%, 65%, and 55% Percoll separation buffer were added sequentially to create a density gradient. Bone marrow cells were slowly added to the top layer, and the tube was centrifuged at 1000 g for 18 min at 4 °C. After centrifugation, the cell layer between the 80% and 65% Percoll interfaces was aspirated, washed twice with PBS (400 g for 10 min, 300 g for 5 min), and finally resuspended in RPMI 1640 medium containing 1% FBS and counted to obtain mouse neutrophils.

[0074] (2) Immunofluorescence verification of M NG Protein binding to mouse neutrophils The isolated neutrophils were diluted to 3 × 10⁻⁶. 5 Cells / mL were seeded into 24-well plates pre-prepared with cell spreaders, 250 μL per well. The Model group was stimulated with 25 μM nigrain for 1 h, while the Control group received an equal volume of anhydrous ethanol. Subsequently, Fc receptors on the cell surface were blocked for 30 min with or without the addition of 0.6 mg / mL Fc protein. Then, fluorescently labeled M... NG The protein was incubated for 30 min. After incubation, it was fixed and prepared using standard methods, and observed and images were acquired using a laser confocal microscope.

[0075] The results showed that the fluorescent label M NG The protein can bind significantly to the surface of mouse neutrophils. Furthermore, activated mouse neutrophils can bind even more M protein to their surface. NG Protein (see) Figure 9 ).

[0076] Example 4 Recombinant protein M NG Suppressing neutrophil inflammatory response Mouse neutrophils were isolated and extracted using Percoll density gradient centrifugation. The obtained neutrophils were seeded into 24-well culture plates for later use. Recombinant protein M was then added. NG A protein solution of 480 μg / mL was prepared using RPMI-1640 medium containing 1% FBS. 0.5 mL was added to each well to achieve a final concentration of 240 μg / mL, and pre-incubated for 0.5 h. Subsequently, 12.5 μM Nigericin was added to each well for 4 h to induce a neutrophil inflammatory activation model. The control group was treated with an equal volume of medium. Immunofluorescence staining was performed after stimulation. First, primary antibody incubation was performed: anti-Cit-H3 (goat-derived) and anti-MPO (rabbit-derived) were mixed according to the manufacturer's instructions and prepared using immunofluorescence primary antibody dilution buffer, then incubated overnight at 4 ℃. After washing, secondary antibody incubation was performed: AF488-labeled donkey anti-goat secondary antibody and AF594-labeled donkey anti-rabbit secondary antibody were mixed at a 1:1000 ratio in immunofluorescence secondary antibody dilution buffer and incubated at room temperature in the dark for 1.5 h. The remaining steps were performed according to standard immunofluorescence slide preparation methods. After staining, images were taken using a laser confocal microscope. Fluorescence intensity was quantitatively analyzed using ImageJ software, and statistical analysis and graph generation were performed using GraphPad Prism 10 software. Experimental results are expressed as mean ± standard deviation (Mean ± SD). The t-test was used for comparisons between two groups, and P < 0.05 was considered statistically significant.

[0077] The results showed, as shown in Figure 10, that in the model group, neutrophils stimulated with Nigerian styracin showed significantly increased Cit-H3 expression and marked changes in cell morphology; while those stimulated with M... NG After treatment, the cell morphology remained relatively intact, and the expression level of Cit-H3 was significantly reduced, showing a statistically significant difference compared to the model group (P<0.05). This indicates that the recombinant protein M... NG It can significantly inhibit nigra-induced neutrophil activation and inflammatory response in mice.

[0078] Example 5: Optimization of expression conditions and functional evaluation of recombinant proteins To improve M NG The expression yield and biological activity of the protein were measured, the expression conditions were optimized, and the binding activity and function of the optimized protein were verified.

[0079] 1. Optimization of expression conditions and detection of cell state Referring to the expression method in Example 1, non-energy sugars were added to the original expression system: fucose and sialic acid were added to 5 mM, and mannose was added to 15 mM, named M. HG Group; only fucose and sialic acid (both 5mM) were added, named M. MG Group. No group has been added (M). NG Group. Non-energy sugars are added 1-3 hours before transfection.

[0080] For M NG Group and M HG Daily sampling was performed on each group. 20 μL of cells were diluted 5-fold with OPM medium, and cell density and viability were measured using an automated cell counter (Countstar IE1000). Cell survival curves were plotted by recording the cell status of both groups. Figure 11 When cell viability drops below 70%, stop culturing and collect the supernatant. Subsequent protein expression and purification steps are the same as described above.

[0081] Table 2. Protein Optimization Expression Yield Table

[0082] 2. Detection of the binding ability of the optimized group with Siglec-9 The binding affinity of each protein group to Siglec-9 His was detected by non-denaturing, non-reducing gel electrophoresis (Western Blot), using the same method as before. The results showed that the optimized M... HG Histone binding bands to Siglec-9 His were more pronounced (see...) Figure 12 ).

[0083] 3.M HG Detection of binding with THP-M0 cells M was detected by immunofluorescence method HG The binding of the protein to THP-M0 cells was investigated using the same method as before. Results showed that M... HG It exhibits clear binding to cells, see Figure 13 .

[0084] 4. Determination of protein sialic acid content using the resorcinol colorimetric method Following the methods described in the Chinese Pharmacopoeia (2020 edition), the resorcinol-hydrochloric acid colorimetric method was used to determine the sialic acid content in proteins. A sialic acid standard curve (20-200 μg / mL) was prepared. The protein concentration in each group was uniformly adjusted to 0.3 mg / mL. After color development and extraction, the absorbance was measured at 580 nm. The standard curve was plotted using GraphPad Prism 10, and the relative sialic acid content in the proteins was calculated. Figure 14 The results showed that M HGHistone sialic acid content was significantly higher than that of M NG Group.

[0085] 5. Sialic acid type detection Western blotting was performed using Maackia Amurensis Lectin II (MAL II) and Sambucus Nigra Lectin (SNA) lectins to analyze the α-2,3 and α-2,6 linked sialic acid types. Fluorescence imaging was performed, and band grayscale analysis was conducted using Image J. The results showed that M... HG - The level of sialic acid modification in the group was significantly enhanced ( Figure 15 ).

[0086] 6.M HG Effects on the THP-M1 macrophage signaling pathway After THP-1 cells were induced to differentiate by PMA, an M1 macrophage model was established using PMA+LPS stimulation. Subsequently, 20 μg / mL M1 macrophages were added to the cell line. HG Stimulation was performed for 1 h. Cell lysates were collected, and the expression levels of p-SHP-1 and β-actin were detected by Western blotting. Results showed... Figure 16 M HG The group significantly upregulated p-SHP-1 expression levels. Statistical analysis was performed using t-tests or one-way ANOVA (P<0.05 was considered statistically significant).

[0087] The results show that M obtained through expression condition optimization HG The protein was significantly superior to M in terms of yield, sialic acid modification level, and Siglec-9 binding activity. NG protein.

Claims

1. A recombinant glycoprotein targeting Siglec-9, characterized in that, The recombinant glycoprotein targets and activates Siglec-9.

2. The recombinant glycoprotein of claim 1, comprising an amino acid sequence as shown in SEQ ID NO: 1 or a sequence having 90% or 98% or more homology with it.

3. A polynucleotide encoding the recombinant glycoprotein of claim 1 or 2.

4. The polynucleotide of claim 3, having a nucleotide sequence as shown in SEQ ID NO:

2.

5. A plasmid vector containing the polynucleotide of claim 3 or 4.

6. The plasmid vector of claim 5, wherein the vector is pCAGGS, an Fc tag is linked to the C-terminus of a 300-500 nucleic acid sequence selected from GPIbα via a linker peptide GGGS, a Kozak sequence is linked to the N-terminus, and an IL-2 sequence is directly linked after the Kozak sequence.

7. A host cell containing the plasmid vector of claim 5 or 6.

8. A method for preparing the recombinant glycoprotein of claim 1 or 2, comprising inserting the nucleotide of claim 3 or 4 into a plasmid vector, then transforming it into a host cell, transforming it, expressing the protein, and separating and purifying it.

9. A method for preparing the recombinant glycoprotein of claim 1 or 2, comprising the following steps: 1) Plasmid transformation and extraction: The plasmid of claim 7 was transformed and extracted using Escherichia coli; 2) Protein expression: Culture expression cells, supplement the culture medium with non-energy sugars, mix the transformed plasmid with PEI and add it dropwise to the cultured expression cells, continue culturing, and collect the supernatant; 3) Purification: The collected supernatant is centrifuged and filtered through a column. The non-energy sugar is two or three of the following: fucose, sialic acid, and mannose.

10. The use of the recombinant glycoprotein of claim 1 or 2 in the manufacture of a medicament for treating inflammatory diseases, preferably, wherein the inflammatory disease is a hyperinflammatory disease, or the recombinant glycoprotein is M. HG protein.

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