High-efficiency expression method of recombinant canine interleukin-2 and application thereof

Abstract

The application discloses a high-efficiency expression method of recombinant canine interleukin-2 and application thereof. In view of the technical defects that wild-type canine interleukin-2 is prone to non-specific hydrophobic aggregation in the expression of an engineering bacterium and the renaturation process of an inclusion body and has a low soluble yield, the application targets to eliminate hydrophobic patches on the surface of the protein by rationally designing molecules and mutating extremely hydrophobic leucines at the 42th and 101th positions in the amino acid sequence of the protein into hydrophilic serines (L42S / L101S). The method comprises codon optimization, recombinant vector construction, Escherichia coli fermentation expression and high-efficiency denaturation and renaturation purification of an inclusion body. Results show that the mutant significantly reduces the intermolecular aggregation tendency on the premise of perfectly retaining the functions of wild-type receptor binding and immune activation, and the soluble purification yield of the mutant is more than 4 times higher than that of the wild type.

Description

Technical Field

[0001] This invention relates to the fields of bioengineering and veterinary biological products, and more specifically, to a highly efficient expression method for recombinant canine interleukin-2 and its application. Background Technology

[0002] Interleukin-2 (IL-2) is a key pleiotropic cytokine that plays a central role in regulating the body's immune system. Canine interleukin-2 can specifically stimulate the proliferation and activation of canine peripheral blood mononuclear cells (PBMCs), T lymphocytes, and natural killer cells, showing great clinical application potential in canine anti-tumor immunotherapy, antiviral infection treatment, and as an adjuvant for novel veterinary vaccines. With the booming development of the pet medical market, the industry's demand for low-cost, large-scale production of high-purity, highly biologically active recombinant canine interleukin-2 is increasingly urgent.

[0003] Currently, the use of E. coli prokaryotic expression systems is the mainstream approach for large-scale production of recombinant cytokines. However, wild-type canine interleukin-2 (CaIL2-WT) readily forms biologically inactive, insoluble inclusion bodies when expressed at high density in engineered bacteria. The core drawback lies in the presence of significant hydrophobic patches on the three-dimensional surface of wild-type CaIL2, with some highly hydrophobic residues (such as leucine) highly exposed. During inclusion body denaturation and refolding, as well as high-concentration liquid fermentation, these exposed hydrophobic residues readily induce non-specific hydrophobic aggregation and precipitation between molecules. This inherent structural defect results in extremely low soluble yields of wild-type protein during refolding and poor thermal stability in liquid formulations, becoming a core technological bottleneck restricting its industrial scale-up.

[0004] To address the aforementioned technical challenges of low soluble yield and easy aggregation and precipitation, existing technologies mostly focus on optimizing external processes, such as simply adjusting the refolding buffer formulation or optimizing fermentation parameters. However, these conventional methods often only treat the symptoms, not the root cause, and cannot overcome the inherent strong hydrophobic aggregation tendency of wild-type sequences. Therefore, there is an urgent need in this field for a fundamental modification strategy based on the molecular structure level. That is, without destroying the protein's core α-helical backbone and receptor binding activity, through rational molecular structure design and site-directed mutagenesis, to target and eliminate hydrophobic plaques on the surface, thereby weakening the protein's non-specific aggregation tendency at its source, and thus developing a highly efficient method for preparing recombinant canine interleukin-2 with both high stability and high soluble yield. Summary of the Invention

[0005] To address the technical problems of non-specific hydrophobic aggregation and precipitation, low soluble yield, and poor thermal stability of recombinant wild-type canine interleukin-2 during high-density expression and inclusion body refolding in engineered bacteria, this invention provides a highly efficient expression method for recombinant canine interleukin-2 and its application.

[0006] To address the aforementioned problems, this invention first provides a recombinant canine interleukin-2 mutant. In some embodiments, the recombinant canine interleukin-2 mutant is based on wild-type canine interleukin-2 (amino acid sequence shown in SEQ ID NO:1), with both highly hydrophobic leucine (Leu) residues at positions 42 and 101 mutated to hydrophilic serine (Ser) residues. This dual-site mutation (L42S / L101S) introduces hydrophilic groups with less steric hindrance in the side chain, reshaping the local hydrophilic hydrogen bond network without disrupting the core α-helix backbone, and targeting and eliminating hydrophobic patches on the surface of the wild-type protein. The amino acid sequence of the mutant is shown in SEQ ID NO:2.

[0007] The present invention also provides a preparation method, the method comprising the following steps: (1) constructing a recombinant expression vector containing the recombinant canine interleukin-2 mutant gene sequence and transforming it into Escherichia coli host cells to obtain recombinant engineered bacteria; (2) fermenting and inducing expression of the recombinant engineered bacteria, collecting and breaking the bacterial cells, and separating inclusion body precipitates; (3) washing, denaturing and dissolving, static refolding and affinity chromatography purification of the inclusion body precipitates in sequence to obtain the recombinant canine interleukin-2 mutant protein.

[0008] Finally, this invention provides an application in which the recombinant canine interleukin-2 mutant is used in the preparation of veterinary vaccine adjuvants or canine immunotherapeutic drugs. In some embodiments, the recombinant canine interleukin-2 mutant can be used as a core active ingredient, co-prepared with veterinarily acceptable excipients, diluents, or carriers to form a pharmaceutical composition.

[0009] Compared with the prior art, the present invention has at least the following beneficial effects: This invention, for the first time, completely solves the problem of hydrophobic aggregation of wild-type canine interleukin-2 in prokaryotic expression at the molecular structure level. Molecular dynamics simulations confirm that the mutant exhibits a folding free energy change (ΔΔG) of -2.15 kcal / mol, demonstrating significantly enhanced spontaneous folding ability and thermodynamic stability. Experiments confirm that the mutant successfully avoids nonspecific flocculent precipitation during inclusion body refolding, resulting in a final soluble purification yield that is 4.15 times higher than that of the wild type, breaking through the technical bottleneck of low-cost mass production. Attached Figure Description

[0011] Figure 1 : This is an SDS-PAGE gel electrophoresis image of the recombinant canine interleukin-2 wild-type (CaIL2-WT) and mutant (CaIL2-mut) proteins obtained by affinity chromatography purification and desalting process in Example 2 of this invention.

[0012] Figure 2 This is a comparison of the total protein expression levels of recombinant canine interleukin-2 wild-type and mutant strains in whole bacterial lysate after IPTG induction in Example 3 of the present invention.

[0013] Figure 3 This is a comparison chart of the yield of soluble protein from wild-type and mutant recombinant canine interleukin-2 in Example 3 of the present invention.

[0014] Figure 4 This study aims to detect the affinity activity of recombinant canine interleukin-2 mutant and wild-type canine peripheral blood mononuclear cells (PBMCs) in vitro during PBMC proliferation in Example 4 of this invention. Detailed Implementation

[0015] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0016] Example 1: Rational design and molecular docking simulation of a canine interleukin-2 mutant (CaIL2-mut) with high stability and high expression levels This embodiment provides a rational design method for recombinant canine interleukin-2 mutants, aiming to solve the technical defects of wild-type protein in engineered bacteria expression, such as easy non-specific aggregation and poor thermal stability, without changing its immune activation and receptor binding biological activities.

[0017] Template sequence acquisition and 3D structural modeling: The amino acid sequence of wild-type canine interleukin-2 (CaIL2-WT) ([Canis lupus familiaris], GenBank: CAJ87635.1) was obtained as follows: MYKMQLLSCIPLTLVLVANSAPITSSSTKETEQQMEQLLLDLQLLLNGVNNYQNPQLSRMLTFKFYTPKKATEFTHLQCLAKELKNLEEVLGLPQSKNVHLSYFNESLFHKKGGRRAPTEKKKNSTWEQEDKKATITEFMREWITVRQRIYKN (SEQ ID NO:1). The above sequence was used to perform 3D structural homology modeling with AlphaFold2 and SWISS-MODEL software to obtain a high-precision spatial conformation model.

[0018] Structural Defect Analysis and Mutation Site Design: The above model was imported into Discovery Studio software to calculate its solvent-accessible surface area (SASA), surface charge, and distribution of hydrophobic residues. The results showed that obvious hydrophobic patches existed on the three-dimensional surface of wild-type CaIL-2. Specifically, the local regions containing leucine residues at positions 42 and 101 were highly exposed on the protein surface, and both residues had extremely strong hydrophobic side chains. During high-density fermentation expression or inclusion body refolding, these exposed strongly hydrophobic residues easily induce non-specific hydrophobic aggregation and precipitation between molecules, which is the core reason for its low soluble yield and poor thermal stability in liquid formulations. Therefore, this invention, through computer-aided design, introduced two site-directed mutations into the parent sequence: mutating leucine residue at position 42 to serine (L42S) and mutating leucine residue at position 101 to serine (L101S). The design aims to target and eliminate hydrophobic plaques on the surface and reshape the local hydrophilic hydrogen bond network by introducing serine, which has less steric hindrance and is highly hydrophilic, without disrupting the core α-helical backbone of canine interleukin-2.

[0019] Mutant canine interleukin-2 (CaIL2-mut): MYKMQLLSCIPLTLVLVANSAPITSSSTKETEQQMEQLLLDSQLLLNGVNNYQNPQLSRMLTFKFYTPKKATEFTHLQCLAKELKNLEEVLGLPQSKNVHSSYFNESLFHKKGGRRAPTEKKKNSTWEQEDKKATITEFMREWITVRQRIYKN (SEQ ID NO: 2).

[0020] Quantitative analysis of mutant structure: To verify the actual impact of the above-mentioned double-site mutation (L42S / L101S) on the protein spatial conformation and stability, molecular dynamics (MD) simulations were performed on the wild-type (CaIL2-WT) and mutant (CaIL2-mut) models at 300K for 50 ns, respectively, using GROMACS software, as shown in Table 1.

[0021] Table 1: Comparison of molecular dynamics and thermodynamic calculation results between wild-type and mutant recombinant canine interleukin-2 Detection indicators and parameters Wild type (CaIL2-WT) Mutant (CaIL2-mut) Structural-functional analysis and conclusions Change in folding free energy (ΔΔG) 0 (Benchmark Reference) -2.15 kcal / mol ΔΔG<0 indicates that the thermodynamic stability of the mutant is significantly improved and its spontaneous folding ability is enhanced. Global conformational stability (50 ns average RMSD) 0.38 ± 0.03nm 0.24 ± 0.02nm The mutant's RMSD value decreased significantly, indicating that its global three-dimensional structure is more compact and less prone to loosening in an aqueous environment. Hydrophobic surface area (SASA) 55.8 nm² 43.2 nm² The hydrophobic area decreased significantly, which weakened the tendency of nonspecific hydrophobic aggregation during high-concentration expression and refolding from the source. Local flexibility of the receptor-binding region (RMSF, key interface region) 0.18 ± 0.02nm 0.18 ± 0.01nm There was no significant difference before and after the mutation (it remained at around 0.18 nm), confirming that the mutation perfectly preserved the conformational basis of receptor binding.

[0022] FoldX calculations showed that the folding free energy change (ΔΔG) of the mutant was -2.15 kcal / mol, indicating a significantly enhanced spontaneous folding ability and a substantial improvement in thermodynamic stability. MD trajectory analysis further revealed that the root mean square deviation (RMSD) of the mutant over 50 ns decreased from 0.38 nm in the wild type to 0.24 nm, confirming a more compact global structure in aqueous solution. Simultaneously, the surface hydrophobic accessible area (SASA) of the mutant significantly decreased, effectively reducing the tendency for non-specific aggregation. Most importantly, the root mean square fluctuation (RMSF) of the key interface region responsible for IL-2 receptor binding showed no significant difference before and after the mutation (both remained around 0.18 nm), confirming that the modification perfectly preserved the conformational basis for receptor binding, providing solid theoretical support for the subsequent preparation of highly active, high-yield recombinant proteins.

[0023] Example 2: High-efficiency expression and parallel preparation process of recombinant canine interleukin-2 mutant and wild type This embodiment details the process flow for E. coli codon optimization, expression vector construction, fermentation-induced expression, inclusion body denaturation and renaturation, and Ni column / ultrafiltration purification to obtain purified protein. Wild-type and mutant proteins were prepared in parallel under identical process parameters.

[0024] The amino acid sequence of wild-type canine interleukin-2 (CaIL2-WT) ([Canis lupus familiaris], GenBank: CAJ87635.1) predicted based on the Signal Peptide online website is: APITSSSTKETEQQMEQLLLDLQLLLNGVNNYQNPQLSRMLTFKFYTPKKATEFTHLQCLAKELKNLEEVLGLPQSKNVHLSYFNESLFHKKGGRRAPTEKKKNSTWEQEDKKATITEFMREWITVRQRIYKN (SEQ ID NO:3).

[0025] The mutant canine interleukin-2 (CaIL2-mut) predicted based on the Signal Peptide online website is: APITSSSTKETEQQMEQLLLDSQLLLNGVNNYQNPQLSRMLTFKFYTPKKATEFTHLQCLAKELKNLEEVLGLPQSKNVHSSYFNESLFHKKGGRRAPTEKKKNSTWEQEDKKATITEFMREWITVRQRIYKN (SEQ ID NO:4) 1. Codon Optimization and Expression Vector Construction: Based on the codon preference of *Escherichia coli*, the nucleotide sequences of wild-type canine interleukin-2 (CaIL2-WT) and its mutant (CaIL2-mut) without the signal peptide were optimized. A 6×His tag sequence was designed and fused to the 3' end of the target gene, and the whole gene was synthesized by GenScript. The synthesized gene fragment was digested with Nco I and Xho I (New England Biolabs, NEB) and then directionally cloned into the prokaryotic expression plasmid pET-28a(+) (Novagen). After the successfully constructed recombinant expression vector was verified by Sanger sequencing, it was heat-shock transformed into *E. coli* BL21(DE3) competent cells (TransGen Biotech, Beijing), plated on LB agar plates containing 50 μg / mL kanamycin, and incubated overnight at 37°C inverted. Positive recombinant engineered bacteria were then screened.

[0026] 2. Fermentation Culture and Induction of Expression: Single-clone recombinant engineered bacteria were selected and inoculated into 5 mL of LB liquid medium containing 50 μg / mL kanamycin. The culture was incubated at 37℃ and 220 rpm for 12 hours with shaking to obtain the seed culture. The seed culture was then inoculated into 1 L of LB fermentation medium at a 1:100 volume ratio and cultured at 37℃ and 250 rpm for scale-up. When the optical density (OD600) of the bacterial culture reached 0.6-0.8, isopropyl-β-D-thiogalactoside (IPTG, Sigma-Aldrich) was added to the medium at a final concentration of 0.5 mM to induce expression. After induction at 37℃ for another 4 hours, the bacterial cells were collected by centrifugation at 6,000 × g and 4℃ for 15 minutes using a refrigerated centrifuge (Thermo Fisher Scientific, Sorvall LYNX 6000) and cell wall disruption was performed.

[0027] 3. Cell Disruption and Inclusion Body Separation and Washing: Collected bacterial cells were resuspended at a ratio of 1 g wet cells in 10 mL of lysis buffer (50 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, pH 8.0) and placed in an ice bath. The cells were homogenized three times using an ATS Engineering (AH-1500) at 800 bar to separate the inclusion body precipitate. The disrupted solution was centrifuged at 10,000 × g, 4 °C for 30 minutes, and the bottom precipitate was collected. Subsequently, the precipitate was resuspended and washed twice with a detergent-containing washing buffer (50 mM Tris-HCl, 100 mM NaCl, 1% Triton X-100 (Sigma-Aldrich), 2 M urea, pH 8.0), and finally washed once with a buffer without Triton X-100 to thoroughly remove host proteins and endotoxins.

[0028] 4. Inclusion body denaturation, dissolution, and renaturation: The inclusion bodies were dissolved using a denaturing buffer containing a denaturing agent. Specifically, the washed inclusion bodies were suspended at a 1:10 (w / v) ratio in denaturing buffer (50 mM Tris-HCl, 8 M urea, 50 mM DTT, pH 8.0), and magnetically stirred at room temperature for 2 hours until fully dissolved. The supernatant was then collected by centrifugation at 12,000 × g for 30 minutes. Using a rapid dilution method, the denatured protein solution was slowly and continuously added dropwise at a 1:20 ratio to renaturation buffer (50 mM Tris-HCl, 0.5 M L-arginine, 1 mM GSH, 0.1 mM GSSG, 5% glycerol, pH 8.5) for renaturation. The entire renaturation process was carried out at 4°C for 48 hours.

[0029] 5. Ni-column affinity purification and ultrafiltration desalting: After clarification by filtration through a 0.45 μm microporous membrane (Millipore), the refolded solution was loaded into a pre-equilibrated 5 mL HisTrap HP affinity chromatography column using an ÄKTA pure chromatography system (Cytiva). Non-specifically bound proteins were washed sequentially with buffers containing 20 mM and 50 mM imidazole. Finally, the target protein peak was collected using elution buffer containing 250 mM imidazole. The collected solution was transferred to Amicon Ultra-15 ultrafiltration centrifuge tubes (Merck Millipore) with a molecular weight cutoff of 10 kDa and centrifuged at 4,000 × g at 4°C. Three ultrafiltration cycles were performed using PBS buffer (pH 7.4) to ensure complete desalting, ultimately yielding high-purity recombinant canine interleukin-2 mutant and wild-type proteins. (See [link to documentation]). Figure 1 .

[0030] Figure 1The images show SDS-PAGE gel electrophoresis results of the recombinant canine interleukin-2 mutant (CaIL2-mut) and wild-type (CaIL2-WT) proteins obtained by purification process in Example 2 of this invention. The results show that the purity of the target proteins is greater than 95%, and the molecular weight is consistent with the expectations.

[0031] Example 3: Comparison of expression levels and soluble yield of recombinant canine interleukin-2 mutant and wild type To quantitatively evaluate the actual improvement effect of the rationally designed dual-site mutation (L42S / L101S) in Example 1 on the expression performance and final soluble yield of recombinant canine interleukin-2 in engineered bacteria, this example systematically compared the wild-type (CaIL2-WT) and mutant (CaIL2-mut) prepared according to the same fermentation and purification process as in Example 2.

[0032] Total expression level and target protein percentage analysis: After fermentation culture (IPTG induction for 4 hours), 1 mL of fermentation broth from CaIL2-WT and CaIL2-mut was collected by centrifugation and completely lysed using an ultrasonic homogenizer. Equal volumes of whole-cell lysate were subjected to SDS-PAGE electrophoresis, stained with Coomassie Brilliant Blue R-250, and then scanned using a gel imaging system (Bio-Rad, model: ChemiDoc MP, catalog number: 12003154). The target protein bands were analyzed using Image Lab software for grayscale scanning and quantification. (See attached image). Figure 2 .

[0033] Figure 2 The results showed that CaIL2-WT and CaIL2-mut accounted for 26.5% and 28.2% of the total protein in the whole bacterial cell, respectively. This indicates that the introduction of the double mutation did not increase the expression load or cytotoxicity of E. coli, and both achieved high-density basal expression.

[0034] Refolding efficiency and final soluble yield determination: The final purified protein solution after Ni column affinity purification and ultrafiltration desalting in Example 2 was collected and accurately quantified using a BCA protein concentration assay kit (Thermo Fisher Scientific, Pierce™ BCA Protein Assay Kit, catalog number: 23225). A standard curve was plotted using bovine serum albumin (BSA standard, Thermo Fisher Scientific, catalog number: 23209), and the absorbance was measured at 562 nm using a multi-functional microplate reader (Molecular Devices, model: SpectraMax M5). The average purification yield of three independent fermentations (1 L shake flask fermentation level) was calculated, see [see figure]. Figure 3 .

[0035] Figure 3 The results showed that the wild-type protein (CaIL2-WT) exhibited severe nonspecific white flocculent precipitation upon standing in inclusion body refolding buffer, with a large amount of protein irreversibly lost due to hydrophobic aggregation, resulting in extremely low refolding efficiency. After affinity purification, the final soluble yield of the wild-type protein was only 115.4 ± 12.3 mg / L fermentation broth. In contrast, the mutant (CaIL2-mut) showed excellent solubility in refolding buffer, with almost no visible aggregates during the 48-hour refolding process. After purification and quantification, the final soluble yield of the mutant was as high as 478.6 ± 21.5 mg / L fermentation broth, approximately 4.15 times that of the wild-type.

[0036] The experimental results above demonstrate that by mutating the highly hydrophobic leucine residues at positions 42 and 101 to serine residues, which have less steric hindrance and are highly hydrophilic, the hydrophobic plaques on the surface of wild-type canine interleukin-2 were successfully targeted and eliminated. This molecular-level modification significantly reduces the non-specific intermolecular hydrophobic aggregation tendency of recombinant canine interleukin-2 during inclusion body folding and refolding. Without affecting the basic fermentation expression level, the mutant significantly overcomes the technical deficiency of low soluble yield of wild-type protein, laying a solid foundation for the low-cost industrial-scale production of this recombinant protein.

[0037] Example 4: Determination of the proliferation activity and specific activity of recombinant canine interleukin-2 mutant in vitro stimulated canine peripheral blood mononuclear cells (PBMCs) This embodiment aims to quantitatively evaluate the biological activity of high-purity, high-yield recombinant canine interleukin-2 mutant (CaIL2-mut) obtained according to Examples 2 and 3 in vitro to stimulate the proliferation of PBMCs in healthy dogs. This is to verify that the mutation of leucine (Leu) at positions 42 and 101 in the rational design to serine (Ser) eliminates surface hydrophobic plaques and improves soluble yield without negatively affecting the core functions—receptor binding and immune activation activity.

[0038] Experimental materials and instruments: (1) Experimental cells: peripheral blood mononuclear cells (Canine PBMCs) of healthy beagle dogs, purchased from a commercial cell bank (purchased from IQ Biosciences (USA), Beagle Canine PBMCs, SKU: IQB-CPB102).

[0039] (2) Main reagents: RPMI-1640 medium (Gibco, catalog number: 11875093); high-quality fetal bovine serum (FBS, Gibco, catalog number: 10099141); CCK-8 cell proliferation assay kit (Dojindo, catalog number: CK04).

[0040] (3) Experimental consumables and instruments: 96-well cell culture plate (Corning, catalog number: 3599); multifunctional microplate reader (Molecular Devices).

[0041] Experimental steps: (1) Cell plating: Resuspend well-recovered canine PBMCs in RPMI-1640 complete medium containing 10% FBS and adjust the cell density to 2×10⁶ cells / cm². 5 Cells / mL. Add 100 μL of cell suspension to each well of a 96-well cell culture plate and incubate at 37°C in a 5% CO2 incubator for 2 hours.

[0042] (2) Sample serial dilution: The CaIL2-WT protein and CaIL2-mut protein to be tested were serially diluted using sterile PBS buffer (containing 0.1% BSA). The initial maximum concentration was set at 1000 ng / mL, followed by 3-fold serial dilutions, for a total of 8 concentration gradients. A blank control group with only complete culture medium was also set up.

[0043] (3) Cell stimulation culture: Add protein dilutions of different concentration gradients to the 96-well plates containing PBMCs, according to the grouping, 100 μL per well (final volume 200 μL / well), and set 3 replicates for each concentration. Incubate the culture plates at 37℃ and 5% CO2 for 72 hours.

[0044] (4) Proliferation activity detection: After incubation, add 20 μL of CCK-8 detection reagent to each well in the dark, and gently shake on a shaker for 1 minute to mix. After incubating the culture plate at 37°C for another 4 hours, measure the absorbance (OD) of each well at 450 nm using a microplate reader. 450 ).

[0045] 3. Data Processing and Experimental Results: Using GraphPad Prism 9.0 software, the measured OD values ​​were processed... 450 The mean and the corresponding logarithmic protein concentration were fitted using a four-parameter logistic regression curve, see [link to figure]. Figure 4 .

[0046] Figure 4The results showed that the mutant prepared by this invention had no significant difference in biological activity and affinity activity compared with the wild type, indicating that the introduced mutation site perfectly preserved the natural biological function of the protein while increasing the soluble yield.

[0047] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A highly efficient expression method for recombinant canine interleukin-2, characterized in that, The recombinant canine interleukin-2 is a canine interleukin-2 mutant, and its amino acid sequence is shown in SEQ ID NO:

2. The efficient expression method includes the following steps: (1) constructing a recombinant expression vector containing the gene sequence of the canine interleukin-2 mutant; (2) transforming the recombinant expression vector into Escherichia coli host cells to obtain recombinant engineered bacteria; (3) fermenting and inducing expression in the recombinant engineered bacteria, collecting and breaking the bacterial cells, and separating inclusion body precipitates; (4) washing, denaturing, renaturing and purifying the inclusion body precipitates by affinity chromatography to obtain the recombinant canine interleukin-2 mutant protein.

2. The method for efficient expression of recombinant canine interleukin-2 according to claim 1, characterized in that, The canine interleukin-2 mutant is based on the amino acid sequence of wild-type canine interleukin-2, with leucine at positions 42 and 101 both mutated to serine; the amino acid sequence of wild-type canine interleukin-2 is shown in SEQ ID NO:

1.

3. A recombinant canine interleukin-2 mutant, characterized in that, The mutant is prepared by the efficient expression method according to claim 1 or 2, or the amino acid sequence of the mutant is as shown in SEQ ID NO:

2.

4. A pharmaceutical composition or veterinary vaccine adjuvant, characterized in that, The pharmaceutical composition or veterinary vaccine adjuvant comprises the recombinant canine interleukin-2 mutant of claim 3, and pharmaceutically or veterinarily acceptable excipients, diluents or carriers.

5. The use of the recombinant canine interleukin-2 mutant of claim 3, or the pharmaceutical composition or veterinary vaccine adjuvant of claim 4, in the preparation of canine immunotherapy drugs or veterinary vaccines.

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