A cat granulocyte colony-stimulating factor mutant, fusion protein and application

Abstract

The present application belongs to the technical field of biological genetic engineering, and particularly relates to a cat granulocyte colony-stimulating factor mutant, a fusion protein and application. The mutant is obtained by performing amino acid site mutation on cat granulocyte colony-stimulating factor, and the amino acid sequences of the obtained mutant are shown in SEQ ID NO. 1-3. The fusion protein is obtained by fusing cat granulocyte colony-stimulating factor and its mutant with a protein tag after codon optimization, the nucleic acid sequences of the cat granulocyte colony-stimulating factor and its mutant after codon optimization are shown in SEQ ID NO. 5-8; the obtained mutant and fusion protein have high expression and high activity, the fusion tag protein is small, and injection use is not easy to produce antibodies, solving some heterologous reactions, low activity and the like in the prior art in which dog or other source G-CSF and its fusion protein are used for cat treatment.

Description

Technical Field

[0001] This invention belongs to the technical field of bioengineering, specifically relating to a cat granulocyte colony-stimulating factor mutant, fusion protein, and its applications. Background Technology

[0002] Granulocyte colony-stimulating factor (G-CSF) is a cytokine primarily produced by fibroblasts, bone marrow mesenchymal cells, monocytes / macrophages, and vascular endothelial cells. It selectively stimulates the proliferation and differentiation of hematopoietic stem cells into a specific lineage. It regulates the viability, proliferation, and differentiation of granulocyte precursor cells and the function of neutrophils, and is mainly used clinically to treat granulocytopenia caused by various factors (such as chemotherapy in cancer patients). Different species of G-CSF exhibit complete cross-activity. Multiple domestic and international guidelines and expert consensus have recommended G-CSF as the first-line drug for the prevention and treatment of neutropenia (FN). Furthermore, with ongoing research, new mechanisms of action of G-CSF are being discovered. For example, a foreign study suggests that G-CSF may be a promising treatment for recurrent miscarriage; G-CSF also appears to show promise in promoting myocardial angiogenesis in patients after myocardial infarction.

[0003] Recombinant human granulocyte-colony stimulating factor (rhG-CSF) is mainly used for neutropenia following antitumor therapy and is an important supportive treatment for cancer patients.

[0004] Currently, there are 16 domestically produced rhG-CSFs used clinically, mainly focusing on the expression and activity of colony-stimulating factors in dogs. However, there are still some differences between canine and feline colony-stimulating factors, and there are currently no dedicated feline colony-stimulating factor products on the market. According to the "2021 China Pet Industry White Paper," the number of pet cats kept by urban families in China has reached 58.06 million, therefore, the development of feline colony-stimulating factors has a promising market prospect.

[0005] Expressing and producing feline granulocyte colony-stimulating factor (GCSF) using genetic engineering and related modern molecular biology techniques is an economical and efficient method. Patent CN202110941898.2 discloses a recombinant fusion protein of a feline GCSF mutant, its preparation method, and its applications. This method connects a feline G-CSF mutant with a truncated peptide of ricin B to obtain a long-acting GCSF, which enhances the biological activity of GCSF and improves its protein stability in the blood, thereby increasing the drug's half-life in vivo. However, ricin B is a feline exogenous protein with a large molecular weight, making it prone to antibody production upon injection, and its activity is lower than that of unfused or fused small fragment proteins. Currently, most related products use E. coli expression systems, mainly because the activity of CSF-related proteins requires less post-translational modification. The advantages of E. coli expression systems are that they can obtain gene expression products quickly and at a relatively low cost. The method is also relatively simple, and a wide range of proteins can be expressed. Therefore, the expression and eventual industrial production of feline granulocyte colony-stimulating factor can be achieved in a prokaryotic expression system. Summary of the Invention

[0006] To address the above-mentioned problems, the present invention aims to provide a cat granulocyte colony-stimulating factor mutant, a fusion protein, and its applications.

[0007] The technical content of this invention is as follows:

[0008] This invention provides a mutant of feline granulocyte colony-stimulating factor (FCGF), which is obtained by mutating the amino acid sites of feline FCGF (amino acid sequence as shown in SEQ ID NO. 1). The mutation sites include mutating valine at position 21 to methionine (fG-CSF-mut1), valine at position 21 to methionine, valine at position 48 to methionine (fG-CSF-mut2), valine at position 24 to isoleucine, glutamate at position 45 to glutamine, and isoleucine at position 110 to valine (fG-CSF-mut3). The amino acid sequences of the resulting mutant are shown in SEQ ID NO. 1 to 3, respectively.

[0009] The present invention also provides a feline granulocyte colony-stimulating factor mutant for the preparation of recombinant feline granulocyte colony-stimulating factor protein.

[0010] The present invention also provides a feline granulocyte colony-stimulating factor fusion protein, wherein the fusion protein is obtained by fusing feline granulocyte colony-stimulating factor and its mutants with a protein tag after codon optimization.

[0011] The obtained fusion protein was synthesized into an expression vector, transformed into an expression strain, and then induced to express. The expression product was crudely extracted, refolded, and purified to obtain the purified fusion protein product.

[0012] The codon-optimized nucleic acid sequences of the feline granulocyte colony-stimulating factor and its mutants are shown in SEQ ID NO.5–8.

[0013] The refolding process involves refolding the crudely extracted expression product by dilution. The refolding solution used includes Tris, NaCl, Arg, GSH, and GSSH, with a pH of 7.0–9.0, a refolding temperature of 4–8°C, a dilution factor of 10–30 times, and a refolding time of 12–48 hours.

[0014] The expression vectors include, but are not limited to, pET-3a, pET-11a, pET-12a, pET-14b, pET-15b, pET-16b, pET-17b, pET-19b, pET-20b, pET-21a, pET-22b, pET-23a, pET-24a, pET-25b, pET-26b, pET-27b, pET-28a, pET-29a, pET-30a, pET-31b, pET-32a, pET-33b, p... ET-34b, pET-35b, pET-37b, pET-39b, pET-40b, pET-41a, pET-42a, pET-43.1a, pET-44a, pET-45b, pET-47b, pET-4 8b, one of pET-49b, pET-50b, pET-51b, pET-52b, pETDuet-1, pACYCDuet-1, pCDFDuet-1, pRSFDuet-1, and pColaDuet-1;

[0015] The expression strains include, but are not limited to, one of BL21, cBL21, BL21(DE3), BL21codonplus(DE3), BL21Star(DE3), B834(DE3), Tuner(DE3), Rosetta(DE3), Rosetta2(DE3), Antarctic Express(DE3), Origami(DE3), Origami 2(DE3), OrigamiB(DE3), SHuffle T7, ER2566, OverExpressC43(DE3), BLR(DE3), HMS174(DE3), GT115, MG1655, and NovaBlue(DE3).

[0016] This invention also provides a feline granulocyte colony-stimulating factor fusion protein for the preparation of drugs to treat leukopenia-related diseases.

[0017] The beneficial effects of this invention are as follows:

[0018] The feline granulocyte colony-stimulating factor mutant of the present invention is obtained by mutating the amino acid sites of feline granulocyte colony-stimulating factor and then expressing it in the expression system after codon modification to obtain a fusion protein. The mutant fusion protein has a better expression level, and the fusion protein obtained after purification and refolding has high activity. Although feline granulocyte colony-stimulating factor is expressed as inclusion bodies in E. coli, the present invention provides a high recovery rate and an easy-to-scale refolding and purification method. The mutant and fusion protein obtained by the present invention have high expression levels and high activity. The fusion tag protein is small and does not easily generate antibodies when injected, which solves the problems of heterologous reactions and low activity in the treatment of cats with canine or other source G-CSF and its fusion proteins in the prior art. Attached Figure Description

[0019] Figure 1 A schematic diagram illustrating the construction of the feline granulocyte colony-stimulating factor fusion protein;

[0020] Figure 2 PCR identification results of Escherichia coli bacterial culture containing the fusion protein of feline granulocyte colony-stimulating factor (FSGF);

[0021] Figure 3 Figure showing the SDS-PAGE analysis results of the feline granulocyte colony-stimulating factor fusion protein;

[0022] Figure 4 The image shows the results of Western blotting analysis of the feline granulocyte colony-stimulating factor fusion protein.

[0023] Figure 5 Figure 1 shows the SDS-PAGE analysis results of the purified recombinant feline granulocyte colony-stimulating factor fusion protein.

[0024] Figure 6 The figure shows the activity assay results of the purified recombinant feline granulocyte colony-stimulating factor fusion protein. Detailed Implementation

[0025] The present invention will be further described in detail below through specific implementation examples and accompanying drawings. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of protection of the present invention. After reading the present invention, any modifications of the present invention in various equivalent forms by those skilled in the art fall within the scope of the appended claims.

[0026] Unless otherwise specified, all raw materials and reagents used in this invention are from the conventional market.

[0027] Example

[0028] Construction and expression of a cat granulocyte colony-stimulating factor mutant and its fusion protein:

[0029] 1. Synthesis of whole-genome fragments and construction of recombinant plasmids

[0030] The cat granulocyte colony-stimulating factor (fG-CSF) protein sequence (NP_001009227.1) was retrieved from NCBI. It consists of 174 amino acids, and the amino acid sequence is shown in SEQ ID NO.4. The full sequence of fG-CSF was obtained by whole-genome synthesis.

[0031] The amino acid sites of fG-CSF were mutated. The mutated sites were fG-CSF-mut1, fG-CSF-mut2, fG-CSF-mut3, fG-CSF-mut4, fG-CSF-mut5, fG-CSF-mut6, fG-CSF-mut7, fG-CSF-mut8, fG-CSF-mut9, fG-CSF-mut1, fG-CSF-mut2, fG-CSF-mut3 ...

[0032] To facilitate purification, fG-CSF, fG-CSF-mut1, fG-CSF-mut2, and fG-CSF-mut3 were fused with histidine tags (6×His) to obtain fusion proteins. The amino acid sequences of fG-CSF and its mutants, as well as histidine, were modified with E. coli codons beforehand. The optimized nucleic acid sequences are shown in SEQ ID NO.5–9, and the amino acid sequences of the resulting fusion proteins are shown in SEQ ID NO.10–13.

[0033] The complete gene was synthesized in the *E. coli* expression vector pET30a(+) by Nanjing Genscript Biotech Co., Ltd., with cloning sites of NdeI and BamHI. A schematic diagram of the construction is shown below. Figure 1 As shown.

[0034] The expression vector may also be pET-11a, pET-12a, pET-14b, pET-15b, pET-16b, pET-17b, pET-19b, pET-20b, pET-21a, pET-22b, pET-23a, pET-24a, pET-25b, pET-26b, pET-27b, pET-28a, pET-29a, pET-30a, pET-31b, pET-32a, pET-33b, pET-3 4b, pET-35b, pET-37b, pET-39b, pET-40b, pET-41a, pET-42a, pET-43.1a, pET-44a, pET-45b, pET-47b, pET-48b , one of pET-49b, pET-50b, pET-51b, pET-52b, pETDuet-1, pACYCDuet-1, pCDFDuet-1, pRSFDuet-1, and pColaDuet-1.

[0035] 2. Construction of recombinant Escherichia coli expression strains

[0036] The feline granulocyte colony-stimulating factor fusion proteins pET30a(+)-fG-CSF-H, pET30a(+)-fG-CSF-mut1-H, pET30a(+)-fG-CSF-mut2-H, and pET30a(+)-fG-CSF-mut3-H were transformed into competent BL21(DE3) cells. The procedure is as follows:

[0037] 1) Remove competent expression cells (BL21(DE3)) from the -80℃ freezer, thaw them in an ice bath, add 1 μL of the constructed plasmid and 10 μL of competent cells to a new sterile 1.5 mL EP tube at a volume ratio of 1:10, gently pipette to mix, and incubate on ice for 30 min.

[0038] 2) Then subject to a 42℃ heat stress for 90 seconds, followed by an ice bath for 5 minutes;

[0039] 3) Add 200 μL of antibiotic-free LB liquid medium to the centrifuge tube and incubate at 37°C with gentle shaking for 60 min;

[0040] 4) Spread the resuspended bacterial solution evenly on LB solid medium containing 50 μg / mL Kana, place it in a 37℃ incubator and incubate upright for about 15 min until all liquid is absorbed, then invert the plate and incubate for 12-16 h.

[0041] The expression strain may also be one of BL21, cBL21, BL21codonplus(DE3), BL21 Star(DE3), B834(DE3), Tuner(DE3), Rosetta(DE3), Rosetta2(DE3), Antarctic Express(DE3), Origami(DE3), Origami 2(DE3), OrigamiB(DE3), SHuffle T7, ER2566, OverExpress C43(DE3), BLR(DE3), HMS174(DE3), GT115, MG1655, or NovaBlue(DE3).

[0042] 3. PCR identification of recombinant prokaryotic strains

[0043] Using a sterilized pipette tip, carefully pick up single colonies with Kana resistance growing on the above LB plates. Pick 3 colonies from each plate and transfer them into 2 mL of LB liquid medium containing 50 μg / mL Kana. Activate and culture at 37°C and 200 r / min for 4 h. Perform bacterial PCR identification of the cultured bacterial solution according to the system and procedure in the table below.

[0044] Table 1 PCR identification system

[0045]

[0046] Table 2. Procedure for PCR identification of recombinant bacterial cultures

[0047]

[0048] like Figure 2 As shown in the figure, M is a 5000bp DNA Marker, P is a positive control, N is a negative control, 1-3 are bacterial cultures of recombinant Escherichia coli BL21(DE3) / pET30a-fG-CSF-H, 4-6 are bacterial cultures of recombinant Escherichia coli BL21(DE3) / pET30a-fG-CSF-mut1-H, 7-9 are bacterial cultures of recombinant Escherichia coli BL21(DE3) / pET30a-fG-CSF-mut2-H, and 10-12 are bacterial cultures of recombinant Escherichia coli BL21(DE3) / pET30a-fG-CSF-mut3-H. As can be seen from the figure, all groups of plasmids are positive, indicating that the plasmids were successfully constructed.

[0049] 4. Induced expression of recombinant prokaryotic strains

[0050] 1) Inoculate the strains that were PCR positive and showed bright nucleic acid bands in the above steps into 100 mL of LB liquid medium containing 50 μg / mL Kana at a volume ratio of 1%, and incubate at 37°C with shaking at 200 r / min for 4 h until OD600 = 0.6-0.8;

[0051] 2) Add the appropriate concentration of IPTG to the above bacterial culture and incubate overnight at 37℃ and 200 r / min with shaking to induce incubation;

[0052] 3) Take the overnight expressed bacterial culture and sonicate it (ultrasonic conditions: 5s sonication, 5s interval, 10min, 30% power), centrifuge the crushed sample (12000rpm, 4℃, centrifugation for 10min) and collect the supernatant and precipitate.

[0053] Take an appropriate amount of the precipitate for SDS-PAGE to examine protein expression. Perform SDS-PAGE analysis, and the results are as follows: Figure 3 As shown in the figure, M is the low molecular weight protein marker, N is the negative control, 1-2 are recombinant E. coli BL21(DE3) / pET30a-fG-CSF-H induced proteins, 3-4 are recombinant E. coli BL21(DE3) / pET30a-fG-CSF-mut1-H induced proteins, 5-6 are recombinant E. coli BL21(DE3) / pET30a-fG-CSF-mut2-H induced proteins, and 7-8 are recombinant E. coli BL21(DE3) / pET30a-fG-CSF-mut3-H induced proteins. It can be seen that, compared with groups 1-2, the fG-CSF mutant fusion proteins in groups 3-8 were all highly expressed in E. coli.

[0054] 5. Western blot analysis

[0055] After recombinant bacterial expression was induced, the bacterial cells were collected by sonication and diluted 1 to 10 times before Western blot detection. The primary antibody was Anti-His Tag Mouse Monoclonal Antibody, and the secondary antibody was HRP Goat Anti-Mouse IgG.

[0056] Figure 4 In the figure, M represents the pre-stained low molecular weight protein marker, N represents the negative control, 1 represents the fG-CSF-H induced protein (undiluted stock solution), 2 represents the fG-CSF-mut1-H induced protein (10-fold dilution), 3 represents the fG-CSF-mut2-H induced protein (10-fold dilution), and 4 represents the fG-CSF-mut3-H induced protein (10-fold dilution). As shown in the figure, the results indicate that the protein size in the induced precipitate is normal, and the recombinant cat granulocyte colony-stimulating factor fusion protein was efficiently expressed in Escherichia coli.

[0057] 6. Expression of the crude extraction, refolding, chromatography, concentration, and dialysis of the product.

[0058] 1) Take 1g of wet bacterial cells and add 10g of lysis buffer (40mM Tris, 0.1-0.2M NaCl, pH 7.0-9.0), and stir magnetically for 30-60 minutes;

[0059] 2) After resuspending, the bacterial suspension was placed in an ice bath and then subjected to ultrasonic disruption. The disruption conditions were 65% power, ultrasonic for 5 seconds, pause for 5 seconds, and a total disruption time of 20 minutes.

[0060] 3) Centrifuge at 10000g, 4℃ for 20min, discard the supernatant, add the same volume of inclusion body washing solution (40mM Tris, 0.1M NaCl, 2~4M Urea, 0.5~1% Triton X-100, pH7.0~9.0) to the precipitate, and stir magnetically for 30~60min;

[0061] 4) Centrifuge at 10000g, 4℃ for 20min, discard the supernatant, add the same volume of inclusion body dissolution solution (40mM Tris, 0.1M NaCl, 6~8M Urea, pH7.0~9.0) to the precipitate, and stir magnetically at room temperature for 4~16h.

[0062] 5) Centrifuge at 10000g, 4℃ for 20min. Filter the supernatant through a 0.45μm filter and then refold. The refolding method is dilution refolding. Slowly add the refolding solution to the protein while stirring. The refolding solution is 40mM Tris, 0.1-0.3M NaCl, 0.3-0.5M Mg, 1-5mM GSH, 0.1-0.5mM GSSH (pH 7.0-9.0). The refolding temperature is 4-8℃, the dilution factor is 10-30 times, and the refolding time is 12-48h.

[0063] 6) After dilution and refolding, the sample was filtered through a 0.45 μm filter and then subjected to reducing agent-resistant Ni column affinity chromatography to obtain high-purity refolded active target product;

[0064] 7) After chromatography, the sample is concentrated and dialyzed through a 5KD ultrafiltration membrane to a final concentration of 1-2 mg / mL. The final solution is 0.1-0.3 M sorbitol, 0.05-0.1 M sodium acetate buffer, pH 4.0-5.0.

[0065] The samples processed using the above techniques were analyzed by SDS-PAGE, such as... Figure 5(M represents the pre-stained low molecular weight protein marker, N represents the negative control, 1 represents the purified fG-CSF-H protein, 2 represents the purified fG-CSF-mut1-H protein, 3 represents the purified fG-CSF-mut2-H protein, and 4 represents the purified fG-CSF-mut3-H protein.) As shown, there is a relatively single band around 20 kDa, which is consistent with the expected size of 19.56 kDa. The content of the purified protein is shown in Table 3.

[0066] Table 3. Protein content of purified expression products

[0067]

[0068] As shown in Table 3, relatively pure protein stock solutions can be obtained from fG-CSF-mut1-H, fG-CSF-mut2-H, and fG-CSF-mut3-H after crude extraction, refolding, chromatography, concentration, and dialysis purification processes.

[0069] 7. Bioactivity Analysis

[0070] The biological activity of G-CSF was detected based on the fact that the growth status of mouse bone marrow leukemia cells (NFS-60 cells) varies with the biological activity of human granulocyte-stimulating factor (G-CSF).

[0071] 1) Reagent preparation

[0072] (1) RPMI 1640 culture medium: Take one bag of RPMI 1640 culture medium powder (1L specification), add water to dissolve and dilute to 1000mL, add 105IU of penicillin and 105IU of streptomycin, then add 2.1g of sodium bicarbonate, dissolve, mix well, filter sterile, and store at 4℃.

[0073] (2) Basic culture medium: Measure 100 mL of newborn calf serum and add it to 900 mL of RPMI 1640 culture medium. Store at 4°C.

[0074] (3) Complete culture medium: Add human granulocyte colony-stimulating factor to the basic culture medium until the final concentration is 10-20 ng per 1 mL;

[0075] (4) PBS: Weigh 8g of sodium chloride, 0.2g of potassium chloride, 1.44g of disodium hydrogen phosphate and 0.24g of potassium dihydrogen phosphate, dissolve in water and dilute to 1000ml, then sterilize at 121℃ for 15 minutes;

[0076] (5) Thiazol blue (MTT) solution: Weigh 0.10 g of MTT powder, dissolve it in 20 mL of PBS to prepare a solution containing 5.0 mg per 1 mL, filter it through a 0.22 μm filter membrane for sterilization, and store it at 4 °C in the dark.

[0077] (6) Lysis buffer: Measure 14 mL of hydrochloric acid and 50 mL of Triton X-100 solution, add isopropanol, prepare a 500 mL solution, and store at room temperature away from light.

[0078] 2) Preparation of standard solutions

[0079] Take the human granulocyte colony-stimulating factor biological activity assay standard, reconstitute it according to the instructions, and dilute it with basal culture medium to a concentration of 50-300 IU per mL. Perform a 2-fold serial dilution in a 96-well cell culture plate, with a total of 8 dilutions. Perform 2 wells for each dilution, and retain 50 μL of standard solution in each well. Discard any excess solution in the wells.

[0080] The above procedures are performed under aseptic conditions.

[0081] 3) Preparation of the test solution

[0082] After reconstituted the four fusion protein test samples according to the labeled amount, they were diluted with basal culture medium to a concentration of 50-300 IU per mL. In a 96-well cell culture plate, a 2-fold serial dilution was performed, resulting in a total of 8 dilutions. Each dilution was performed in 2 wells, and 50 μL of the test sample solution was retained in each well. Excess solution in the wells was discarded.

[0083] The above procedures are performed under aseptic conditions.

[0084] Assay method: NFS-60 cell lines were cultured in complete culture medium at 37℃ and 5% carbon dioxide, and the cell concentration was controlled at 1.0 × 10⁻⁶ cells per mL. 5 ~4.0×10 5 10 cells were passaged and used for biological activity assays 24–36 hours after passage. The solutions used in the experiment were preheated to 37°C.

[0085] Take a sufficient amount of NFS-60 cell culture, centrifuge to collect NFS-60 cells, wash three times with RPMI 1640 culture medium, and then resuspend in basal culture medium to prepare a solution containing 2.0 × 10⁶ cells per mL. 5 Cell suspension of 1 cell was prepared and stored at 37°C. 50 μL of cell suspension was added to each well of a 96-well cell culture plate containing standard solution and test solution. The plate was cultured at 37°C and 5% CO2 for 40–48 hours. 20 μL of MTT solution was added to each well and the plate was cultured at 37°C and 5% CO2 for 5 hours.

[0086] The above procedures are performed under aseptic conditions;

[0087] Add 100 μL of lysis buffer to each well, mix well, place in a microplate reader, use 630 nm as the reference wavelength, and measure the absorbance at a wavelength of 570 nm. Record the measurement results.

[0088] The experimental data were processed using a computer program or a four-parameter regression method, and the results were calculated using the following formula:

[0089]

[0090] In the formula, Pr represents the biological activity of the standard, in IU / mL;

[0091] Ds is the pre-dilution factor of the test sample;

[0092] Dr represents the pre-dilution factor of the standard;

[0093] Es is the dilution factor of the test sample relative to half the effective amount of the standard;

[0094] Er is the dilution factor of the half-effective dose of the standard.

[0095] The results of the above activity tests are as follows: Figure 6 As shown, the activity of the purified sample is greater than 6 × 10⁻⁶. 7 IU / mg, the activity was greater than that of the standard.

Claims

1. A cat granulocyte colony-stimulating factor mutant, characterized in that, The mutant was obtained by mutating the amino acid sites of cat granulocyte colony-stimulating factor, and the amino acid sequences of the resulting mutants are shown in SEQ ID NO.1~3.

2. The feline granulocyte colony-stimulating factor mutant according to claim 1, characterized in that, The amino acid sequence of the feline granulocyte colony-stimulating factor is shown in SEQ ID NO.4; The mutant with the amino acid sequence shown in SEQ ID NO.1 is obtained by mutating the 21st valine of feline granulocyte colony-stimulating factor to methionine; The mutant with the amino acid sequence shown in SEQ ID NO.2 is obtained by mutating valine at position 21 to methionine and valine at position 48 to methionine in feline granulocyte colony-stimulating factor. The mutant with the amino acid sequence shown in SEQ ID NO.3 was obtained by mutating valine at position 24 of feline granulocyte colony-stimulating factor to isoleucine, glutamate at position 45 to glutamine, and isoleucine at position 110 to valine.

3. A feline granulocyte colony-stimulating factor fusion protein, characterized in that, The fusion protein is obtained by fusing the feline granulocyte colony-stimulating factor mutant of claim 1 or 2 with a protein tag after codon optimization. The nucleic acid sequences of the mutant feline granulocyte colony-stimulating factor after codon optimization are shown in SEQ ID NO.5~7.

4. The feline granulocyte colony-stimulating factor fusion protein according to claim 3, characterized in that, The obtained fusion protein was synthesized in an expression vector, transformed into an expression strain, and then induced to express. The expression product was then crudely extracted, refolded, and purified to obtain the purified fusion protein product.

5. The feline granulocyte colony-stimulating factor fusion protein according to claim 4, characterized in that, The refolding process involves diluting and refolding the crudely extracted expression product. The refolding solution used consists of Tris, NaCl, Arg, GSH, and GSSH, with a pH of 7.0–9.0, a refolding temperature of 4–8°C, a dilution factor of 10–30 times, and a refolding time of 12–48 hours.

6. The feline granulocyte colony-stimulating factor fusion protein according to claim 4, characterized in that, The expression vectors include pET-3a, pET-11a, pET-12a, pET-14b, pET-15b, pET-16b, pET-17b, pET-19b, pET-20b, pET-21a, pET-22b, pET-23a, pET-24a, pET-25b, pET-26b, pET-27b, pET-28a, pET-29a, pET-30a, pET-31b, pET-32a, pET-33b, pET... -34b, pET-35b, pET-37b, pET-39b, pET-40b, pET-41a, pET-42a, pET-43.1a, pET-44a, pET-45b, pET-47b, pET-48 b. One of pET-49b, pET-50b, pET-51b, pET-52b, pETDuet-1, pACYCDuet-1, pCDFDuet-1, pRSFDuet-1, and pColaDuet-1.

7. The feline granulocyte colony-stimulating factor fusion protein according to claim 4, characterized in that, The expressed strains include one of BL21 (DE3), BL21codonplus (DE3), BL21 Star (DE3), B834 (DE3), Tuner (DE3), Rosetta (DE3), Rosetta2 (DE3), Antarctic Express (DE3), Origami (DE3), Origami2 (DE3), OrigamiB (DE3), SHuffle T7, ER2566, OverExpress C43 (DE3), BLR (DE3), HMS174 (DE3), and NovaBlue (DE3).

8. The feline granulocyte colony-stimulating factor fusion protein according to claim 4, characterized in that, The expression strain includes one of BL21, GT115, and MG1655.

9. The use of the feline granulocyte colony-stimulating factor fusion protein according to any one of claims 3 to 8 in the preparation of a medicament for treating feline neutropenia.

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