Pig IL-2-IFN-alpha-T alpha 1 interferon fusion protein as well as preparation method and application thereof

Abstract

The invention belongs to the field of biotechnology and veterinary immune preparations, and relates to a porcine IL-2-IFN-alpha-T alpha 1 interferon fusion protein and a preparation method and application thereof.The amino acid sequence of the interferon fusion protein is shown as SEQ ID NO.1, and the interferon fusion protein is composed of recombinant porcine interleukin 2, flexible connecting peptide, recombinant porcine alpha interferon, flexible connecting peptide and thymosin; the amino acid sequence of the recombinant porcine interleukin 2 is as shown in SEQ ID NO. 2; the amino acid sequence of the recombinant porcine alpha interferon is as shown in SEQ ID NO.3; the amino acid sequence of the thymosin is as shown in SEQ ID NO. 4; the amino acid sequence of the flexible connecting peptide is as shown in SEQ ID NO.5. The porcine IL-2-IFN-alpha-T alpha 1 interferon fusion protein provided by the invention has good biological activity and no cytotoxicity, has high antiviral property and immune regulation function, and can effectively improve the antiviral ability of pigs.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and veterinary immunomodulators, specifically relating to a porcine IL-2–IFN-α–Tα1 interferon fusion protein, its preparation method, and its application. Background Technology

[0002] With the increasing intensification of pig farming, viral infectious diseases have become one of the main bottlenecks restricting the healthy development of the industry. These diseases not only lead to a significant increase in morbidity and mortality rates in pigs, but also, due to the widespread presence of immunosuppressive viruses, severely damage the pig's immune system, causing a decline or even failure of conventional vaccine immunization, resulting in huge economic losses. Against this backdrop, developing safe and efficient new immunization and prevention strategies, based on technological advancements and the need for high-quality development of animal husbandry, is particularly urgent.

[0003] Immunostimulants, due to their potential to regulate the body's immune function, enhance vaccine potency, and increase antiviral capabilities, have gradually become a research hotspot in the field of animal disease prevention and control. An ideal immunostimulant should possess clearly defined properties, high efficiency and stability, and be non-toxic and environmentally friendly, representing a crucial direction for replacing antibiotics and achieving green farming. How to obtain such an ideal immunostimulant is a worthy research topic.

[0004] Interleukin-2 (IL-2) is a pleiotropic cytokine primarily produced by activated CD4+ T cells, playing a central role in adaptive immune responses. It exerts its biological functions by binding to a high-affinity trimeric receptor complex composed of IL-2Rα (CD25), IL-2Rβ (CD122), and a common γ chain (γc, CD132). As a Th1-type cytokine, IL-2 is a key growth factor for T lymphocytes, crucial for the survival and function of effector T cells, thus playing a central role in antiviral and antitumor immunity. Furthermore, IL-2 has important regulatory functions on innate immune cells. It significantly enhances the cytotoxic activity of natural killer cells and induces their proliferation, thereby maintaining and amplifying the killing effect of NK cells on virus-infected cells and tumor cells. In humoral immunity, IL-2 acts as a co-stimulatory signal, synergistically promoting the proliferation and differentiation of activated B cells and regulating antibody class switching, which is essential for generating effective antibody responses. Therefore, incorporating IL-2 into the design of fusion proteins aims to provide a powerful T-cell and NK-cell activation signal, thereby enhancing the overall cellular immune response.

[0005] Alpha-interferon (IFN-α) can regulate the activity of immune cells, exhibiting broad-spectrum antiviral, antitumor, and cellular immunity-enhancing functions. When IFN-α binds to its homodimer receptor, it activates receptor-associated JAK1 and TYK2 tyrosine kinases. These kinases subsequently phosphorylate transcription factors STAT1 and STAT2. Phosphorylated STAT1 and STAT2 form a heterotrimeric complex, namely interferon-stimulated gene factor 3 (ISGF3), with constitutively expressed interferon regulator 9. This ISGF3 complex rapidly translocates to the nucleus, recognizes and binds to interferon-stimulated response elements in the promoter regions of hundreds of interferon-stimulated genes, thereby initiating their transcription. The protein products encoded by these ISGs establish a broad antiviral state through multiple synergistic mechanisms. Furthermore, IFN-α can enhance antigen presentation by upregulating the expression of major histocompatibility complex (MMCC) molecules and directly promote the maturation of dendritic cells and the clonal expansion of CD8+ T cells, thus bridging innate and adaptive immunity and jointly strengthening the body's antiviral and antitumor defense capabilities.

[0006] Thymosin α1 (Tα1) is a 28-amino acid polypeptide initially isolated from thymic tissue and has been identified as a potent immunomodulator. One of its core functions is promoting the development, maturation, and functional differentiation of T lymphocytes. Studies have shown that Tα1 can enhance the differentiation and maturation of bone marrow-derived pre-T cells in the thymus, increasing the proportion of CD4+ and CD8+ T cells. More importantly, Tα1 can significantly enhance the expression of high-affinity IL-2 receptors on the surface of activated T cells by activating key signaling pathways downstream of T cell receptors. This effect synergizes with the function of IL-2: upregulated IL-2 receptors make T cells more sensitive to environmental and even low levels of IL-2, thereby greatly amplifying IL-2-driven T cell proliferation. In addition, Tα1 can promote the secretion of various Th1 cytokines, such as IFN-γ and IL-2 itself, while inhibiting excessive Th2 responses to some extent, thus shifting immune homeostasis towards cellular immunity that is conducive to clearing intracellular pathogens and tumors. This multi-target immunomodulatory property makes it an ideal component for enhancing the efficacy of vaccines and immunotherapies.

[0007] Natural polysaccharides are widely used as ideal materials for next-generation vaccine adjuvants due to their excellent biocompatibility, biodegradability, and low toxicity. Among them, cationic polysaccharide chitosan (CS) has a wide range of applications. Studies have shown that chitosan can be endocytosed by dendritic cells and then escape into the cytoplasm via the endosome / lysosome pathway. In the cytoplasm, chitosan can be recognized by the DNA sensor cGAS through interactions with endogenous nucleic acids. cGAS catalyzes the production of the second messenger cGAMP, which in turn activates the STING protein on the endoplasmic reticulum. Activation of the STING pathway ultimately leads to the production of type I interferon and pro-inflammatory cytokines, driving dendritic cell maturation and polarization into a Th1 immune response, which is crucial for resisting viruses and tumors. 2-Hydroxypropyltrimethylammonium chloride chitosan (HTCC) is synthesized by introducing a quaternary ammonium group onto the amino group of chitosan. HTCC, through structural modification by introducing a quaternary ammonium group, possesses several comprehensive properties superior to CS. This derivative possesses water solubility across the entire pH range, completely overcoming the poor solubility of chitosan under physiologically neutral conditions, and greatly expanding its applicability in formulations. Simultaneously, its permanent positive charge is unaffected by environmental pH, ensuring potent and stable interactions with antigens and cell membranes. Based on this, HTCC exhibits a more efficient and stable mucosal penetration enhancement effect, significantly promoting the transmucosal transport and absorption of antigens. Furthermore, the introduction of the quaternary ammonium structure endows the material with significantly enhanced antibacterial activity. Most importantly, while achieving the aforementioned performance improvements, HTCC fully inherits the inherent excellent biocompatibility and safety of chitosan. On the other hand, anionic polysaccharides such as dextran sulfate also exhibit strong adjuvant activity, effectively presenting antigens to dendritic cells via scavenger receptors and promoting the activation of inflammasomes, thereby inducing strong antibody and T-cell responses. Summary of the Invention

[0008] To address the problems existing in the background art, the present invention provides a porcine IL-2–IFN-α–Tα1 interferon fusion protein, its preparation method, and its application. This fusion protein has good biological activity, as well as high antiviral and immunomodulatory functions, and can effectively improve the antiviral ability of pigs.

[0009] To achieve the above objectives, the technical solution of the present invention is as follows:

[0010] A porcine IL-2–IFN-α–Tα1 interferon fusion protein, the amino acid sequence of which is shown in SEQ ID NO.1, is composed of recombinant porcine interleukin 2, a first flexible linker peptide, recombinant porcine α-interferon, a second flexible linker peptide, and thymosin.

[0011] The amino acid sequence of the recombinant porcine interleukin-2 is shown in SEQ ID NO.2;

[0012] The amino acid sequence of the recombinant porcine α-interferon is shown in SEQ ID NO.3;

[0013] The amino acid sequence of the thymosin is shown in SEQ ID NO.4;

[0014] The amino acid sequences of the first flexible and the second linker peptide are both shown in SEQ ID NO.5.

[0015] The method for preparing the porcine IL-2–IFN-α–Tα1 interferon fusion protein includes the following steps:

[0016] S1. Synthesize the gene fragment encoding the porcine IL-2–IFN-α–Tα1 interferon fusion, and digest it with restriction endonucleases EcoR I and Hind III to obtain the digested product;

[0017] S2. The pCold-TF plasmid was double-digested with restriction endonucleases EcoRI and HindIII to obtain a linearized vector;

[0018] S3. The enzyme digestion product of S1 is ligated with the linearized vector of S2 to construct a recombinant expression vector, which is then transformed into Escherichia coli DH5α. Positive clones are screened, and the recombinant plasmid pCold-IL-2–IFN-α–Tα1 is extracted.

[0019] S4. The recombinant plasmid was transformed into Escherichia coli BL21(DE3), and expression was induced by IPTG. The bacterial cells were collected, lysed, and crude fusion protein was obtained.

[0020] S5. The crude fusion protein is purified to obtain purified porcine IL-2–IFN-α–Tα1 interferon fusion protein.

[0021] The gene fragment described in S1 was synthesized and then amplified by PCR. The primer sequences used are as follows:

[0022] IL-2–IFN-α–Tα1-F: CGAATTCATGGGCGCTCCGA;

[0023] IL-2–IFN-α–Tα1-R:AAGCTTTCAGTTCTCCGCTTCTTC.

[0024] The induction conditions for S4 were: IPTG concentration of 1 mmol / L, temperature of 16℃, shaking speed of 180-220 rpm, and induction time of 18 h.

[0025] The purification described in S5 includes sequential nickel-agarose gel affinity chromatography and molecular sieve chromatography.

[0026] The application of the porcine IL-2–IFN-α–Tα1 interferon fusion protein in the preparation of antiviral drugs.

[0027] The antiviral drug comprises a fusion protein and a carrier, with a mass ratio of carrier to fusion protein of 7:(1-6). The carrier is nanoparticles of quaternized chitosan and dextran sulfate, wherein the mass ratio of dextran sulfate to quaternized chitosan is 2:5.

[0028] IL-2–IFN-α–Tα1 is an artificially designed fusion protein. Its design combines the immunomodulatory function of IL-2 with the antiviral activity of interferon-α (IFN-α) and the immunomodulatory properties of thymosin α1 (Tα1) to produce a synergistic antiviral effect.

[0029] This invention utilizes electrostatic adsorption to combine quaternized chitosan, dextran sulfate, and IL-2–IFN-α–Tα1, preparing an HTCC / DS / IL-2–IFN-α–Tα1 nanoparticle complex. Experiments demonstrate that HTCC / DS nanoparticles can effectively load IL-2–IFN-α–Tα1, protecting it from protease degradation. As a novel sustained-release drug, HTCC / DS / IL-2–IFN-α–Tα1 exhibits effective antiviral activity in vivo. Compared to IL-2–IFN-α–Tα1 alone, HTCC / DS / IL-2–IFN-α–Tα1 nanoparticles, as a novel sustained-release drug, possess superior mucosal immune activity.

[0030] This application has the following beneficial effects:

[0031] 1. This invention is the first to design a novel multifunctional fusion protein by tandemly linking porcine IL-2, IFN-α, and Tα1 with a flexible linker peptide. In vitro experiments have confirmed its significant anti-porcine pseudorabies virus (PRV) activity.

[0032] 2. This invention innovatively employs the pCold-TF expression system, utilizing its molecular chaperone (Trigger Factor, TF) tag and cold shock protein A (cspA) promoter to induce expression at low temperatures, significantly increasing the soluble expression ratio and yield of the fusion protein in E. coli. This solves the problem of easy inclusion body formation in such proteins, and the process has good reproducibility, making it suitable for large-scale preparation.

[0033] 3. This invention constructs nanoparticles from two complementary polysaccharides, chitosan and dextran sulfate, and loads the fusion protein onto HTCC / DS polyelectrolyte nanoparticles. Utilizing the mucosal adhesion and sustained-release properties of the nanoparticles, the drug's residence time at the site of action is prolonged. This nanodelivery system not only protects the protein from degradation, but its components, HTCC and DS, also possess immunoadjuvant activity, synergistically enhancing the mucosal and systemic immune responses induced by the fusion protein.

[0034] 4. Animal experiments have shown that HTCC / DS nanoparticles loaded with fusion protein, when administered intranasally, can effectively promote dendritic cell maturation, activate CD4+ and CD8+ T lymphocytes, and induce ISG expression, thereby providing significant protection against PRV attack, improving the survival rate of infected animals, and having no toxicity to major organs, demonstrating good safety. Attached Figure Description

[0035] Figure 1 This is the elution peak diagram of molecular sieve chromatography during the purification process in Example 1;

[0036] Figure 2 This is an SDS-PAGE image of the purified fusion protein from Example 1, where M is the marker.

[0037] Figure 3 This is a Western blot image of the purified fusion protein from Example 1, where M is the marker.

[0038] Figure 4 Morphological image of PK-15 cells treated with porcine IL-2–IFN-α–Tα1 fusion protein in Example 3 (no lesions were observed).

[0039] Figure 5 This is a diagram of PK-15 cell lesions in the positive control group of Example 3;

[0040] Figure 6 The following is a characterization diagram of the particle size (A) and polydispersity index (B) of HTCC / DS nanoparticles at different mass ratios in Example 4;

[0041] Figure 7 This is a transmission electron microscope (TEM) image of the HTCC / DS nanoparticles in Example 4;

[0042] Figure 8 The graph shows the changes in particle size (A), PDI (B), and Zeta potential (C) of HTCC / DS nanoparticles before and after loading the fusion protein in Example 4.

[0043] Figure 9 Example 5 illustrates the effect of in vivo imaging on prolonging the retention time of the model antigen OVA-Cy5 in the nasal cavity of mice by HTCC / DS nanoparticles.

[0044] Figure 10 Comparison of H&E stained pathological sections of major organs from mice in the treatment group and PBS control group in Example 6 (scale bar: 20 μm).

[0045] Figure 11 This is a scatter plot representing the CD4 and CD8 subsets of spleen T lymphocytes in the treatment group mice analyzed by flow cytometry in Example 6.

[0046] Figure 12 The graph shows the percentage of CD4+ (A) and CD8+ (B) T lymphocytes in the spleen of the treatment group and the PBS control group at different time points in Example 6.

[0047] Figure 13 This is a representative scatter plot showing the expression of co-stimulatory molecules CD80 and CD86 on the surface of nasal DCs cells in the treatment group of mice in Example 6, analyzed by flow cytometry.

[0048] Figure 14 This is a statistical graph showing the percentage of CD80+ and CD86+ cells in nasal DCs at different time points in the treatment group and PBS control group in Example 6.

[0049] Figure 15 The relative mRNA expression levels of interferon-stimulated genes ISG15 (A), IFIT1 (B), and IFIT2 (C) in the lungs of mice in the treatment group and the PBS control group in Example 6;

[0050] Figure 16 Survival curves for mice in the PBS control group, PRV infection group, and treatment group in Example 6 mouse experiment;

[0051] Figure 17 The relative transcription levels of PRV gE gene mRNA in the brain (A), spleen (B), and lung (C) tissues of mice in the PRV infection group and treatment group in Example 6;

[0052] Figure 18 Comparison of H&E stained pathological sections of brain, lung, and liver tissues from mice in the PBS control group, treatment group, and PRV infection group in Example 6 (scale bars are 100μm and 20μm). Detailed Implementation

[0053] The present invention will be further described in detail below with reference to specific embodiments.

[0054] Experimental methods not specified in the examples are generally performed in accordance with conventional manuals such as Molecular Cloning: A Laboratory Manual or the conditions recommended by the manufacturer.

[0055] Dextran sulfate (100 kDa) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., chitosan quaternary ammonium salt (98% substitution degree) was purchased from Shanghai Maclean Biochemical Technology Co., Ltd., and DH5α and BL21(DE3) receptor cells were purchased from Beijing Quanshi Gold Biotechnology Co., Ltd. (Beijing, China).

[0056] DNA extension and PCR amplification were performed using high-fidelity hot-start PrimeSTAR® HS (Premix) enzymes purchased from TaKaRa. Restriction endonucleases EcoR I and Hind III were purchased from NEB (New England Biolabs, Inc.).

[0057] The preparation method of ampicillin LB liquid culture medium includes: mixing ampicillin aqueous solution (ampicillin concentration of 50 mg / mL) with LB liquid culture medium at a volume ratio of 1:1000 to obtain ampicillin LB liquid culture medium.

[0058] Porcine kidney cells (PK-15) and porcine pseudorabies virus (PRV) were preserved in the laboratory of Henan Agricultural University.

[0059] Monoclonal antibodies against mouse CD3-APC, CD4-FITC, and CD8-PE were purchased from Wuhan Sanying Biotechnology Co., Ltd. BCA kits were purchased from Beyotime Biotechnology Co., Ltd., ovalbumin (OVA-Cy5) from Solarbio, and goat horseradish peroxidase-labeled IgG antibody from Abcam, UK. Balb / c mice were purchased from Liaoning Changsheng Biotechnology Co., Ltd.

[0060] Unless otherwise specified in this invention, conventional conditions shall apply; reagents or instruments whose manufacturers are not specified shall be obtained through commercial purchase.

[0061] Example 1: A porcine IL-2–IFN-α–Tα1 interferon fusion protein and its preparation method

[0062] The porcine IL-2–IFN-α–Tα1 interferon fusion protein, whose amino acid sequence is shown in SEQ ID NO.1, is composed of recombinant porcine interleukin-2, flexible linker peptide, recombinant α-interferon, flexible linker peptide, and thymosin.

[0063] SEQ ID NO.1:

[0064] MGAPTSSSTKNTKKQLEPLLLDLQLLLKEVKNYENADLSRMLTFKFYMPKQATELKHLQCLVEELKALEGVLNLGQSKNSDSANIKESMNNINVTVLELKGSETSFKCEYDDETVTAVEFLNKWITFCQSIYSTLTGGGGSGGGGSGGGGSCDLPQTHSLAHTRALRLLAQMRRISPFSC LDHRRDFGSPHEAFGGNQVQKAQAMALVHEMLQQTFQLFSTEGSAAAWNESLLHQFCTGLDQQLRDLEACVMQEAGLEGTPLLEEDSILAVRKYFHRLTLYLQEKSYSPCAWEIVRAEVMRSFSSSRNLQDRLRKKEGGGGSGGGGSGGGGSSDAAVDTSSEITTKDLKEKKEVVEEAEN.

[0065] The amino acid sequence of recombinant porcine interleukin-2 is shown in SEQ ID NO.2;

[0066] SEQ ID NO.2:

[0067] MGAPTSSSTKNTKKQLEPLLLDLQLLLKEVKNYENADLSRMLTFKFYMPKQATELKHLQCLVEELKALEGVLNLGQSKNSDSANIKESMNNINVTVLELKGSETSFKCEYDDETVTAVEFLNKWITFCQSIYSTLT.

[0068] The amino acid sequence of recombinant porcine alpha interferon is shown in SEQ ID NO.3;

[0069] SEQ ID NO.3:

[0070] CDLPQTHSLAHTRALRLLAQMRRISPFSCLDHRDFGSPHEAFGGNQVQKAQAMALVHEMLQQTFQLFSTEGSAAAWNESLLHQFCTGLDQQLRDLEACVMQEAGLEGTPLLEEDSILAVRKYFHRLTLYLQEKSYSPCAWEIVRAEVMRSFSSSTNLQDRLRKKE.

[0071] The amino acid sequence of thymosin is shown in SEQ ID NO.4;

[0072] SEQ ID NO.4: SDAAVDTSSEITTKDLKEKKEVVEEAEN.

[0073] The amino acid sequence of the flexible linker is shown in SEQ ID NO.5.

[0074] SEQ ID NO. 5: GGGGSGGGGSGGGGS.

[0075] The preparation method of porcine IL-2–IFN-α–Tα1 interferon fusion protein includes the following steps:

[0076] S1. Gene synthesis and expression vector construction

[0077] Based on the codon preference of *E. coli*, synonymous codon substitutions were performed on the encoding DNA sequence of the fusion protein (SEQ ID NO.1) without altering its amino acid sequence to improve gene expression efficiency in *E. coli*. The codon-optimized DNA sequence was then subjected to whole-genome chemical synthesis to obtain the complete coding gene fragment. Finally, using the chemically synthesized optimized gene fragment as a template, polymerase chain reaction (PCR) was used for amplification to obtain the target gene fragment.

[0078] The primers used for PCR amplification are: IL-2–IFN-α–Tα1-F: CGAATTCATGGGCGCTCCGA (SEQ ID NO. 6); IL-2–IFN-α–Tα1-R: AAGCTTTTCAGTTCTCCGCTTCTTC (SEQ ID NO. 7).

[0079] The recombinant porcine interleukin-2 gene with nucleotide sequence as shown in SEQ ID NO.2, the recombinant porcine α-interferon gene with nucleotide sequence as shown in SEQ ID NO.3, and the thymosin gene with nucleotide sequence as shown in SEQ ID NO.4 were linked together with a flexible linker peptide with amino acid sequence as shown in SEQ ID NO.5 to obtain the target gene, denoted as IL-2–IFN-α–Tα1.

[0080] The pCold-TF vector was double-digested with EcoRI and HindIII, respectively. The recombinant porcine IL-2–IFN-α–Tα1 interferon was then ligated into the pCold-TF vector using T4 ligase to obtain the recombinant expression vector, denoted as pCold&IL-2–IFN-α–Tα1. Sequencing confirmed the sequence was correct.

[0081] S2. Protein Induction Expression: 1 μL of the above recombinant expression vector was transformed into 50 mL of DH5α competent cells, and 500 μL of antibiotic-free LB medium was added for activation. 50 μL of the bacterial culture was spread onto an LB solid culture dish containing ampicillin and grown at 37℃ and 5% CO2 for 13 h. Single clones were then selected for identification. The above recombinant expression vector was identified using pCold-TF universal primers and sequencing, and was determined to be pCold&IL-2–IFN-α–Tα1.

[0082] 1 μL of the above recombinant expression vector plasmid was transferred to 50 μL of BL21(DE3) expression competent cells, and 500 μL of LB liquid medium was added for activation. 50 μL of the resulting system was spread onto an ampicillin LB solid culture dish and grown at 37℃ and 5% CO2 for 13 h to obtain the genetically engineered bacteria.

[0083] Single positive colonies of the above-mentioned genetically engineered bacteria were added to 5 ml of ampicillin LB liquid medium and cultured at 37℃ and 220 rpm on a shaker, then expanded to 500 mL. The culture was continued at 37℃ and 220 rpm until the colonies reached the logarithmic growth phase. When the OD600 of the colonies reached 0.6, IPTG (final concentration 1 mmol / L) was added, and expression was induced at 16℃ and 180 rpm for 18 h. The bacterial culture was then collected and centrifuged to obtain bacterial sludge. The bacterial sludge was resuspended in 20 mL of PB buffer and sonicated (30% power, sonication for 5 s, pause for 5 s). After centrifugation, bacterial pellet and supernatant were obtained. SDS-PAGE analysis was performed on the supernatant and pellet to determine whether the colonies could express the protein. The results showed a clear band in the supernatant. It was confirmed that the porcine IL-2–IFN-α–Tα1 interferon fusion protein was expressed in the form of the secretory supernatant.

[0084] S3. Protein Purification: The bacterial supernatant was centrifuged again and filtered through a 0.22 μm filter to obtain the protein solution. The protein solution was then passed through a nickel-NTA agarose gel and eluted with PB elution buffer containing different concentrations of imidazole. The eluted protein solution was collected. Then, molecular sieve chromatography was performed for purification. Results are shown below. Figure 1 .

[0085] The eluted protein solution was subjected to SDS-PAGE and Western blot analysis. The results are as follows: Figure 2 and Figure 3 As shown, the molecular weight of the expressed proteins is approximately 90 kDa. The concentration of the eluted protein solution was determined using a BCA protein assay kit. If the protein concentration is <0.5 mg / mL, a concentration tube (10 kDa) can be used to concentrate the protein and replace the buffer, resulting in an eluted protein solution concentration >2 mg / mL.

[0086] Example 2: A sustained-release antiviral drug and its preparation method

[0087] A sustained-release drug (antiviral drug) comprises an active ingredient and a carrier. The active ingredient is the porcine IL-2–IFN-α–Tα1 interferon fusion protein of Example 1, and the carrier is HTCC / DS nanoparticles. The mass ratio of the carrier to the active ingredient is 7:(1–6).

[0088] HTCC / DS nanoparticles are composed of quaternized chitosan (HTCC) and dextran sulfate (DS).

[0089] The preparation method of the above-mentioned sustained-release drug includes the following steps:

[0090] Step 1: Prepare HTCC / DS nanoparticles.

[0091] Step 1-a: Dissolve 200 μg / mL of dextran sulfate (DS) in deionized water, filter using a 0.22 μm diameter filter membrane, and dilute to obtain solution A;

[0092] Step 1-b: Dissolve 500 μg / mL of quaternized chitosan (HTCC) in deionized water, filter using a 0.22 μm diameter filter membrane, and dilute to obtain solution B;

[0093] Step 1-c: Slowly add solution B to solution A while stirring, and continue stirring to obtain a quaternized chitosan / dextran sulfate nanoparticle solution. The mass ratio of dextran sulfate to quaternized chitosan (HTCC) is 2:5. The molecular weight of dextran sulfate is 100 kDa, and the degree of quaternization of the quaternized chitosan is not less than 95%.

[0094] Step 2: Under the condition of stirring speed of 1300 r / min, the porcine IL-2–IFN-α–Tα1 interferon fusion protein is slowly added dropwise to the quaternized chitosan / dextran sulfate nanoparticle solution. The mass ratio of HTCC / DS nanoparticles to porcine IL-2–IFN-α–Tα1 interferon fusion protein is 7:(1-6). Mix and stir for 15-30 min to obtain the sustained-release drug.

[0095] Example 3: In vitro antiviral activity assay of interferon fusion protein

[0096] The antiviral activity of porcine IL-2–IFN-α–Tα1 interferon fusion protein was evaluated by its ability to inhibit cytopathic effects induced by PRV in PK-15 cells (i.e., the CPE inhibition method, cytopathic effect inhibition method).

[0097] When PK-15 cells in 96-well plates reached 90% coverage, the supernatant was removed, and the medium was replaced with 100 μL / well of fresh medium containing a 2-fold serially diluted recombinant interferon protein. The plates were then cultured for another 12 h. Subsequently, the cells were washed with PBS buffer and infected with PRV virus (100 TCID50). 50 / well). In a 96-well plate, 8 wells were virus-free as a negative control, and 8 wells contained virus but lacked porcine IL-2–IFN-α–Tα1 interferon fusion protein as a positive control. Observations were performed every 12 hours until 80% of the cells in the positive control wells showed cytopathic effects, at which point the culture was terminated, and antiviral activity was calculated using the Reed-Muench method (half-maximal effective dose cumulative method). Results showed that the antiviral activity of porcine IL-2–IFN-α–Tα1 interferon fusion protein against PRV was 1 × 10⁻⁶. 6 U / mg.

[0098] from Figure 4 , Figure 5 It can be seen that obvious cytopathic effects were observed in the positive control group, while no cytopathic effects were observed in PK-15 cells treated with the recombinant porcine IL-2–IFN-α–Tα1 interferon fusion protein. This indicates that the porcine IL-2–IFN-α–Tα1 interferon fusion protein exhibits good antiviral activity in PK-15 cells and can inhibit PRV replication.

[0099] Example 4 Characterization experiments of HTCC / DS nanoparticles and drug-loaded nanoparticles

[0100] HTCC and DS were dissolved in double-distilled water at a concentration of 500 μg / mL, and mixed at mass ratios of 1:1, 2:1, 3:1, 4:1, 5:1, and 5:2, respectively. The mixtures were stirred in an evaporating dish with a magnetic stirrer for 15-30 min to obtain HTCC / DS nanoparticles at different mass ratios. The particle size and PDI of the HTCC / DS nanoparticles at different volume ratios were determined using a Zetasizer NanosS laser particle size analyzer. The results are shown below. Figure 6 As shown in A and 6B.

[0101] After preparation, the sample was placed in a desktop transmission electron microscope for transmission scanning, and the results are as follows: Figure 7 As shown. From Figure 7 As can be seen, HTCC / DS nanoparticles are spherical. These positively charged particles can serve as excellent drug carriers, forming stable complexes with negatively charged antigen proteins / drugs through electrostatic interactions, thus providing strong support for subsequent drug loading and release.

[0102] Following the steps of Examples 2 and 3, the drug-loaded nanoparticles were characterized. The mass ratio of HTCC / DS nanoparticles to IL-2–IFN-α–Tα1 was 7:1. The particle size, PDI, and potential of the HTCC / DS nanoparticles and the drug-loaded nanoparticles were measured using a Zetasizer NanosS laser particle size analyzer, as shown below. Figure 8 A, 8B, and 8C.

[0103] In vitro characterization of HTCC / DS particles showed a particle size of 173±3 nm, a PDI of <0.3, and a Zate potential of 39±3 mV. Transmission electron microscopy revealed that the particles were spherical and uniformly dispersed. When the target protein was bound, the particle size increased to 290±5 nm, the PDI decreased to <0.3, and the Zate potential decreased to 27±3 mV, indicating that the target protein and nanoparticles were bound by electrostatics and that the particles were uniformly dispersed.

[0104] Example 5 Biodistribution of drugs loaded onto HTCC / DS nanoparticles

[0105] To evaluate the effect of HTCC / DS nanoparticle-loaded drug retention on the nasal mucosa, commercially available OVA-Cy5 was used as a standard. Mice were intranasally inoculated with both OVA-Cy5 and HTCC / DS-OVA-Cy5. In vivo fluorescence imaging was performed at 3 h, 6 h, 9 h, 12 h, 24 h, and 36 h post-administration using an IVIS® Lumina IIl, PerkinElmer, USA. The effect of HTCC / DS nanoparticles on the retention of OVA-Cy5 on the nasal mucosa was observed using the in vivo imaging system.

[0106] The results are as follows Figure 9 As shown, mice immunized with OVA-Cy5.5 via the nose exhibited a weak fluorescent signal in their nasal cavity, which is likely due to the clearance of the antigen by cilia within the nasal cavity. Conversely, immunization of mice with HTCC / DS-OVA-Cy5.5 resulted in a significantly enhanced fluorescent signal in the nasal cavity, which persisted for 24 hours. This indicates that HTCC / DS nanoparticles can effectively prolong the retention time of OVA-Cy5.5 in the mouse nasal cavity.

[0107] Example 6 Animal Experiment

[0108] Twenty-four BALB / c mice were randomly divided into a PBS control group and a treatment group. The treatment group received intranasal instillation of the sustained-release drug from Example 2 (50 μg / mouse / day, based on fusion protein) for 3 consecutive days. The control group received an equal volume of PBS. Organ and nasal cavity cells (n=3) were collected from mice on days 3, 5, and 7. Flow cytometry was used to detect changes in spleen lymphocytes (including CD4+ and CD8+ T lymphocytes). Simultaneously, nasal cavities were collected, ground, and nasal cavity cells were collected for flow cytometry analysis of DC cell uptake (including CD11C, CD80, and CD86). Heart, liver, spleen, lung, and kidney were also collected to evaluate organ safety and changes in ISG transcription levels in the lungs. Internal organs from each mouse were fixed in 10% neutral buffered formalin for 24–48 h and embedded in paraffin. 4 mm thick sections of internal organs were stained with H&E.

[0109] 1. Safety and immunological evaluation in mice

[0110] To evaluate the safety of sustained-release drugs in mouse viscera, visceral organs were harvested on day 7 after the first administration for H&E staining, photography, and pathological analysis.

[0111] The results are as follows Figure 10 As shown, neither the lungs of the treatment group nor the PBS control group exhibited enlargement or thickening. The liver, myocardium, spleen, and kidneys showed clear structures with intact cell morphology, abundant numbers, and uniform color, without obvious pathological changes. This indicates that the sustained-release drug is safe and can be used in intranasal drug delivery systems.

[0112] 2. Assessment of CD4+ and CD8+ T lymphocytes

[0113] Lymphocytes were isolated from mouse spleens using a mouse lymphocyte isolation kit. 5-10 mL of erythrocyte lysis buffer was added to completely lyse the erythrocytes. The cells were washed twice with PBS, centrifuged at 200g for 5 min each time. The cell pellet was resuspended in 500 μL of PBS buffer. 1 × 10⁻⁶ lymphocytes were collected from each mouse after obtaining the lymphocytes. 6 Cells were stained with fluorescent labels. APC-labeled Anti-Mouse CD3, FITC-labeled Anti-Mouse CD4, and PE-labeled Anti-Mouse CD8 were incubated separately. After incubation in the dark for 20–30 min, the cells were washed twice with PBS, centrifuged at 200g for 5 min each time. The cell pellet was resuspended in 500 μL of PBS buffer and immediately detected and analyzed by flow cytometry.

[0114] Test results as follows Figure 11 , Figure 12As shown, compared with the PBS control group, the IL-2–IFN-α–Tα1 treatment group significantly activated CD4+ and CD8+ T lymphocytes after treatment, and these numbers remained significantly higher than those in the control group until day 7. This indicates that IL-2–IFN-α–Tα1 can enhance specific cellular immune activation.

[0115] 3. Evaluation of local mucosal immune activation

[0116] Single-cell suspensions were collected from the mouse nasal cavity. 5-10 mL of lysis buffer was added to completely lyse the erythrocytes. The cells were washed twice with PBS, centrifuged at 200g for 5 min each time, to obtain nasal cavity cells. 1×10⁶ cells were collected from each mouse. 6 Cells were subjected to fluorescent staining. Nasal cavity cells were incubated with anti-CD80-BV421, anti-CD86-FITC, and anti-CD11c-APC antibodies, respectively, for fluorescent staining. After incubation in the dark for 20–30 min, the cells were washed twice with PBS, centrifuged at 200 g for 5 min each time. The resulting cell pellet was resuspended in 500 μL of PBS buffer and immediately detected and analyzed by flow cytometry.

[0117] Test results as follows Figure 13 , Figure 14 As shown in the figure, compared with the PBS control group, the number of CD80+ and CD86+ cells in the treatment group increased significantly on day 3; they decreased on days 5 and 7, but the proportion of CD80+ and CD86+ cells in the treatment group was still significantly higher than that in the control group.

[0118] 4. Evaluation of interferon-stimulated gene (ISG) induction

[0119] To evaluate the ability of sustained-release drugs to induce ISGs in mice, the expression of three ISGs—ISG-15, IFIT-1, and IFIT-2—in mouse lungs was analyzed. RNA was prepared from lung tissue using Trizol RNA extraction reagent (Thermo Fisher Scientific, USA). Each sample was tested in triplicate, and the mRNA levels of ISG-15, IFIT-1, IFIT-2, and gE genes in various mouse organs were detected by SYBR Green real-time PCR. To standardize each sample, β-actin was used as an internal control, and 2... −ΔΔCt The relative expression levels were analyzed using a method. Primers used for detecting ISG gene and viral transcription levels in vivo are shown in Table 1.

[0120] Table 1 Primers used for detecting ISG gene and viral transcription levels in vivo.

[0121]

[0122] The results are as follows Figure 15 As shown, the mRNA level of ISG was significantly increased in the treatment group compared to the PBS control group. The mRNA levels of ISG-15, IFIT-1, and IFIT-2 in the treatment group were all higher than those in the PBS control group at 3, 5, and 7 days post-treatment. This indicates that the sustained-release drug can induce ISG expression in mice.

[0123] 5. Anti-PRV virus infection protection experiment

[0124] BALB / c mice were randomly divided into a PBS control group, a PRV infection group, and a treatment group. Mice in the PRV infection and treatment groups received 100 μL of PRV intranasally at a titer of 10. 3 TCID 50 On day 1 after PRV infection, the treatment group received 50 μg / animal / day of the sustained-release drug from Example 2 via nasal drops for 3 consecutive days. The PBS control group was not infected with PRV and did not receive treatment with the sustained-release drug.

[0125] The results are as follows Figure 16 As shown, mice in the PRV-infected group exhibited severe clinical symptoms, including itching, skin biting, tissue damage, and bleeding. Mice in the treatment group also showed clinical symptoms, but to a lesser degree than those in the PRV-infected group. Furthermore, mice in both the PRV-infected and treatment groups began to die on days 4 and 5, with all mice in the PRV-infected group dying by day 5, while the survival rate in the treatment group was 60%. All mice in the PBS control group survived, maintained good mental condition, and had normal appetites.

[0126] To further investigate the effect of sustained-release drugs on PRV replication in different mouse tissues, three mice were randomly selected from each group, and spleen, lung, and brain tissues were harvested. Total RNA was extracted from the tissues, and the transcriptional level of the virus in the tissues was determined using qPCR. The results are as follows: Figure 17 As shown, the viral mRNA transcription levels in the brain, lungs, and spleen of mice in the treatment group were significantly lower than those in the PRV-infected group.

[0127] To further investigate the protective effect of sustained-release drugs against tissue and organ damage caused by PRV infection, pathological observations were performed on the brain, lung, and liver tissues of mice in each group, including H&E staining and organ morphology observation. All surviving mice were sacrificed on day 12, and their brains, lungs, and livers were collected. Tissues from mice that died during the experiment were collected immediately after death, and histopathological sections of the brain, lung, and liver tissues from each group were analyzed.

[0128] The results are as follows Figure 18As shown in the figures (scale bars at 100 μm and 20 μm), compared to the PBS control group, no significant pathological changes were observed in the spleen, lungs, and brain of the treated mice. Mice in the PRV-infected group exhibited encephalitis and neurotropism resulting from microglial proliferation; liver cells showed blurred boundaries, inflammatory infiltration, and vacuolar degeneration; lung tissue showed alveolar wall thickening, with obvious histological lesions and inflammatory infiltration. In contrast, no severe lesions were observed in the tissue sections of the treated mice, indicating significant protection. This suggests that the sustained-release drug can exert a good antiviral effect, protecting mice from PRV infection.

Claims

1. A porcine IL-2–IFN-α–Tα1 interferon fusion protein, characterized in that, Its amino acid sequence is shown in SEQ ID NO.1, and it consists of recombinant porcine interleukin-2, a first flexible linker peptide, recombinant porcine α-interferon, a second flexible linker peptide, and thymosin. The amino acid sequence of the recombinant porcine interleukin-2 is shown in SEQ ID NO.2; The amino acid sequence of the recombinant porcine α-interferon is shown in SEQ ID NO.3; The amino acid sequence of the thymosin is shown in SEQ ID NO.4; The amino acid sequences of the first flexible and the second linker peptide are both shown in SEQ ID NO.

5.

2. A method for preparing the porcine IL-2–IFN-α–Tα1 interferon fusion protein as described in claim 1, characterized in that, Includes the following steps: S1. Synthesize the gene fragment encoding the porcine IL-2–IFN-α–Tα1 interferon fusion, and digest it with restriction endonucleases EcoR I and Hind III to obtain the digested product; S2. The pCold-TF plasmid was double-digested with restriction endonucleases EcoRI and HindIII to obtain a linearized vector; S3. The enzyme digestion product of S1 is ligated with the linearized vector of S2 to construct a recombinant expression vector, which is then transformed into Escherichia coli DH5α. Positive clones are screened, and the recombinant plasmid pCold-IL-2–IFN-α–Tα1 is extracted. S4. The recombinant plasmid was transformed into Escherichia coli BL21(DE3), and expression was induced by IPTG. The bacterial cells were collected, lysed, and crude fusion protein was obtained. S5. The crude fusion protein is purified to obtain purified porcine IL-2–IFN-α–Tα1 interferon fusion protein.

3. The method for preparing the porcine IL-2–IFN-α–Tα1 interferon fusion protein according to claim 2, characterized in that, The gene fragment described in S1 was synthesized and then amplified by PCR. The primer sequences used are as follows: IL-2–IFN-α–Tα1-F: CGAATTCATGGGCGCTCCGA; IL-2–IFN-α–Tα1-R:AAGCTTTCAGTTCTCCGCTTCTTC.

4. The method for preparing the porcine IL-2–IFN-α–Tα1 interferon fusion protein according to claim 2, characterized in that, The induction conditions for S4 were: IPTG concentration of 1 mmol / L, temperature of 16℃, shaking speed of 180-220 rpm, and induction time of 18 h.

5. The method for preparing the porcine IL-2–IFN-α–Tα1 interferon fusion protein according to claim 2, characterized in that, The purification described in S5 includes sequential nickel-agarose gel affinity chromatography and molecular sieve chromatography.

6. The use of the porcine IL-2–IFN-α–Tα1 interferon fusion protein as described in claim 1 in the preparation of antiviral drugs.

7. The application according to claim 6, characterized in that, The antiviral drug comprises a fusion protein and a carrier, with a mass ratio of carrier to fusion protein of 7:(1-6). The carrier is nanoparticles of quaternized chitosan and dextran sulfate, wherein the mass ratio of dextran sulfate to quaternized chitosan is 2:5.

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