Composite IFNs-Fc fusion protein composition and pharmaceutical application thereof

Abstract

The invention relates to the technical field of genetic engineering, in particular to a composite IFNs-Fc fusion protein and application and a medicine thereof. The composite IFNs-Fc fusion protein comprises an IFN gamma-Fc fusion protein, an IFN alpha2-Fc fusion protein and an IFN lambda3-Fc fusion protein, and the coding sequences are sequentially shown as SEQ ID No.1-3. The composite IFNs-Fc can effectively inhibit the replication of ASFV on PAMs cells, and the inhibition rates of the ASFV on the PAMs cells in 24 hours and 48 hours are 87.44% and 93.63% respectively. And the compound also has a broad-spectrum antiviral effect on PRRSV, PEDV and PRV.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a composite IFNs-Fc fusion protein and its applications and pharmaceuticals. Background Technology

[0002] African swine fever (ASF) has caused enormous losses to the pig farming industry, and its prevention and control are extremely challenging. Currently, there are no commercially available effective vaccines or antiviral drugs. There is an urgent need to find emergency preventative drugs for ASFV infection. Types I, II, and III interferons (IFN) have broad-spectrum antiviral and immunomodulatory functions; however, whether the combination of these three substances can inhibit the occurrence and development of ASF remains unknown.

[0003] IFNs are a class of glycoproteins secreted by cells after viral infection, exerting their biological functions by activating various antiviral pathways and immunomodulatory functions. Studies have shown that type I IFNs inhibit ASFV replication and spread by affecting viral invasion, gene expression, and protein synthesis; type II IFNs enhance host resistance to ASFV by regulating host immunity; and IFN-γ has a significant inhibitory effect on both virulent and attenuated ASFV strains (GAO et al., 2022). Continuous treatment of ASFV-infected Vero cells with IFN-α can inhibit ASFV replication (FAN et al., 2023). The combined use of IFN-α and IFN-γ has a stronger anti-ASFV effect than using either IFN alone (AVAGYAN et al., 2022). It is currently unclear whether the combined use of type I, II, and III IFNs can inhibit the occurrence and development of ASFV infection, and traditional IFNs have drawbacks such as poor activity, low yield, and short half-life. Summary of the Invention

[0004] To address the aforementioned issues, this invention provides a composite IFNs-Fc fusion protein and its applications and pharmaceutical uses. This study employed the HEK 293T cell expression system to fuse IFN with the Fc fragment of human IgG1, thereby improving the soluble expression and protein stability of IFN. Three IFNs-Fc fusion proteins (types I, II, and III IFNs) were expressed: IFNγ-Fc, IFNα2-Fc, and IFNλ3-Fc. The IFNs-Fc fusion proteins were purified using the specific binding of the Fc fragment to Protein A. SDS-PAGE and Western blotting confirmed that the protein size matched the expected values, and mass spectrometry confirmed correct expression of IFNs-Fc. The Bradford method was used to determine the concentration of the IFNs-Fc fusion protein, calculating yields of 65.55 mg / L, 22.65 mg / L, and 122.7 mg / L, respectively. The titers of the three IFNs-Fc proteins were determined using a microcytopathic effect inhibition assay, and their in vitro antiviral activity was investigated. IFNγ-Fc(1.04×10 9 U / mg), IFNα2-Fc (1.63×10) 7 U / mg), IFNλ3-Fc (9.23×10) 9 U / mg), each of the three was 1.0×10 5 A composite IFNs-Fc was prepared by mixing U / mL, and cells were treated with 3000U for 24h, followed by 100 TCID. 50 Viral infection was investigated using real-time quantitative polymerase chain reaction (RT-qPCR). The results showed that the compound IFNs-Fc could effectively inhibit the replication and proliferation of ASFV, PRRSV, PEDV, and PRV on cells, indicating that it has good broad-spectrum antiviral activity and can be used for subsequent in vivo anti-ASFV infection studies.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] This invention provides a composite IFNs-Fc fusion protein, comprising IFNγ-Fc fusion protein, IFNα2-Fc fusion protein, and IFNλ3-Fc fusion protein;

[0007] The coding sequence of the IFNγ-Fc fusion protein is shown in SEQ ID No. 1;

[0008] The coding sequence of the IFNα2-Fc fusion protein is shown in SEQ ID No. 2;

[0009] The coding sequence of the IFNλ3-Fc fusion protein is shown in SEQ ID No. 3.

[0010] Preferably, the enzyme activity ratio of the IFNγ-Fc fusion protein, IFNα2-Fc fusion protein, and IFNλ3-Fc fusion protein is 1:1:1.

[0011] Preferably, the coding sequence of the IFNγ-Fc fusion protein is obtained by fusing the IFNγ sequence and the Fc sequence, wherein the Genebank sequence number of the IFNγ sequence is NM_213948.1 and the Genebank sequence number of the Fc sequence is AF150959.1;

[0012] The coding sequence of the IFNα2-Fc fusion protein is obtained by fusing the IFNα2 sequence and the Fc sequence. The Genebank sequence number of the IFNα2 sequence is MH538099.1, and the Genebank sequence number of the Fc sequence is AF150959.1.

[0013] The coding sequence of the IFNλ3-Fc fusion protein is obtained by fusing the IFNλ3 sequence and the Fc sequence. The Genebank sequence number of the IFNλ3 sequence is NM_001166490.1, and the Genebank sequence number of the Fc sequence is AF150959.1.

[0014] This invention also provides the application of the composite IFNs-Fc fusion protein described above in the preparation of drugs for treating African swine fever.

[0015] The present invention also provides the application of the composite IFNs-Fc fusion protein described in the above technical solution in the preparation of drugs for treating porcine reproductive and respiratory syndrome.

[0016] This invention also provides the application of the composite IFNs-Fc fusion protein described above in the preparation of drugs for treating porcine epidemic diarrhea.

[0017] This invention also provides the application of the composite IFNs-Fc fusion protein described above in the preparation of drugs for treating porcine pseudorabies.

[0018] The present invention also provides a drug for treating swine diseases, comprising the composite IFNs-Fc fusion protein described in the above technical solution.

[0019] Preferably, the swine diseases include one or more of African swine fever, porcine reproductive and respiratory syndrome, porcine epidemic diarrhea, and pseudorabies.

[0020] Preferably, the enzyme activity ratio of IFNγ-Fc fusion protein, IFNα2-Fc fusion protein, and IFNλ3-Fc fusion protein in the composite IFNs-Fc fusion protein is 1:1:1.

[0021] The beneficial effects of this invention are:

[0022] This invention utilizes the HEK 293T cell expression system to fuse IFN with the Fc fragment of human IgG1 to improve the soluble expression and protein stability of IFN. Three IFNs-Fc fusion proteins (types I, II, and III IFNs) were expressed: IFNγ-Fc, IFNα2-Fc, and IFNλ3-Fc, respectively. The IFNs-Fc fusion proteins were purified using the specific binding of the Fc fragment to Protein A. SDS-PAGE and Western blotting confirmed that the proteins matched the expected sizes, and mass spectrometry confirmed correct expression of IFNs-Fc. The Bradford assay determined the concentrations of the IFNs-Fc fusion proteins, calculating yields of 65.55 mg / L, 22.65 mg / L, and 122.7 mg / L, respectively. The titers of the three IFNs-Fc proteins were determined using a microcytopathic effect inhibition assay, and their in vitro antiviral activity was investigated. IFNγ-Fc (1.04 × 10⁻⁶) was used. 9 U / mg), IFNα2-Fc (1.63×10) 7 U / mg), IFNλ3-Fc (9.23×10) 9 U / mg), each of the three was 1.0×10 5 A composite IFNs-Fc was prepared by mixing U / mL, and cells were treated with 3000U for 24h, followed by 100 TCID. 50 Viral infection was investigated using real-time quantitative polymerase chain reaction (RT-qPCR). The results showed that the compound IFNs-Fc could effectively inhibit the replication and proliferation of ASFV, PRRSV, PEDV, and PRV on cells, indicating that it has good broad-spectrum antiviral activity and can be used for subsequent in vivo anti-ASFV infection studies.

[0023] Preparation and in vitro antiviral activity study of IFNs-Fc fusion protein

[0024] The porcine IFNs type I, II, and III genes were fused with Fc and expressed using the HEK 293T cell expression system, resulting in IFNγ-Fc, IFNα2-Fc, and IFNλ3-Fc, respectively. SDS-PAGE and WB results showed that the fusion sizes were consistent with the expected values, and mass spectrometry confirmed correct expression, with yields of 65.55 mg / L, 22.65 mg / L, and 122.7 mg / L, respectively. IFNγ-Fc, IFNα2-Fc, and IFNλ3-Fc exhibited high antiviral activity, with yields of 1.04 × 10⁻⁶. 9 U / mg, 1.63×10 7 U / mg and 9.23×109 U / mg. The three were mixed to prepare a compound IFNs-Fc for PAMs cell treatment, and then administered at 100 TCID50. 50 Infection with ASFV, compound IFNs-Fc can effectively inhibit ASFV replication on PAMs cells, with inhibition rates of 87.44% and 93.63% at 24 and 48 hours, respectively. It also has broad-spectrum antiviral activity against PRRSV, PEDV, and PRV.

[0025] IFN-Fc fusion proteins have broad application potential in drug development and treatment. Their main advantages include: ① Ease of expression and purification: The Fc fragment can bind to IFN, increasing the soluble expression and stability of antibodies; Fc exhibits high selectivity with affinity agents such as protein A or protein G, enabling efficient antibody purification and simplifying the purification process of IFN-Fc fusion proteins, thus improving purity and yield. ② Targeted action: IFN-Fc fusion proteins can selectively bind to antigens on the surface of target cells, increasing drug accumulation in vivo and reducing impact on non-pathological tissues. ③ Enhanced stability: IFN-Fc fusion proteins better maintain their structural integrity, enhancing protein stability and half-life, thereby further prolonging the duration of drug action. ④ Enhanced immune activity: Fc interacts with Fc receptors on immune cells, activating effector cells and enhancing the immune activity of the fusion protein. ⑤ Combining multiple functions, IFN-Fc fusion proteins can simultaneously exert multiple biological activities of antibodies and IFN. In addition to exerting the antiviral, antitumor, and immunomodulatory functions of IFN, they can also combine with other biological functions of antibodies, such as promoting cell apoptosis and inducing immunity. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0027] Figure 1 The diagrams are of the pMAL-IFNs-Fc vectors. Note: These are diagrams of pMAL-IFNγ-Fc (6102bp), pMAL-IFNα2-Fc (6147bp), and pMAL-IFNλ3-Fc (6189bp).

[0028] Figure 2 The results are for IFNs-Fc PCR. Note: M is the DNA molecular weight standard, IFNγ-Fc (470bp), IFNα1-Fc (554bp), IFNα2-Fc (546bp), and IFNλ3-Fc (588bp).

[0029] Figure 3SDS-PAGE results for expressing IFNs-Fc fusion proteins. Note: M is the protein molecular standard, IFNγ-Fc (54.7kDa), IFNα2-Fc (55.3kDa), IFNλ3-Fc (56.5kDa);

[0030] Figure 4 The results of Western Blot analysis of the IFNs-Fc fusion protein are shown. Note: M is the protein molecular standard, IFNγ-Fc (54.7 kDa), IFNα2-Fc (55.3 kDa), IFNλ3-Fc (56.5 kDa).

[0031] Figure 5 The absorption peak curve of the IFNs-Fc fusion protein;

[0032] Figure 6 The results of SDS-PAGE of the purified IFNs-Fc fusion protein are shown. Note: M represents the protein marker, IFNγ-Fc (54.7 kDa), IFNα2-Fc (55.3 kDa), and IFNλ3-Fc (56.5 kDa).

[0033] Figure 7 The results of Western blot analysis (WB) of the purified IFNs-Fc fusion protein are shown. Note: M is the protein molecular standard, IFNγ-Fc (54.7 kDa), IFNα2-Fc (55.3 kDa), and IFNλ3-Fc (56.5 kDa).

[0034] Figure 8 This is the protein concentration standard curve (Bradford method), where y (mg / mL) is the protein concentration and x is the absorbance of the protein at OD570 nm;

[0035] Figure 9 For the cytotoxicity of compound IFNs-Fc, note: Figure 9 The study investigated the cell proliferation toxicity of the compound IFNs-Fc (1000 U / μL of each component) on PAMs cells at different time points. The cell viability of cells treated with the compound IFNs-Fc for 24 hpi was 99.99%, and the cell viability of cells treated with the compound IFNs-Fc for 48 hpi was 99.59%. The compound IFNs-Fc showed no cytotoxicity to PAMs cells.

[0036] Figure 10To illustrate the effect of IFNs-Fc on ASFV replication and proliferation, note: a) IFNs-Fc inhibits ASFV replication at 24 h after infection; the IFNs-Fc treatment group significantly inhibited ASFV replication on PAMs, and the difference between the two groups was extremely significant (p<0.0001); b) IFNs-Fc inhibits ASFV replication at 48 h after infection; the IFNs-Fc treatment group significantly inhibited ASFV replication on PAMs, and the difference between the two groups was extremely significant (p<0.0001).

[0037] Figure 11 IFNs-Fc inhibits ASFV replication and proliferation (IFA). Note: Figure 11 To demonstrate the inhibitory effect of IFNs-Fc on ASFV replication after 48 hours of infection, a large amount of green fluorescence was observed in the ASFV group under green excitation light (eGFP), while a small amount of green fluorescence was observed in the IFNs-Fc treatment group; in the bright field, nuclear condensation and increased intercellular spaces were observed in the ASFV group. Scale bar: 100 μm.

[0038] Figure 12 For the inhibition of PRRSV replication by combined IFNs-Fc, note: Figure 12 The results showed that the combined IFNs-Fc inhibited the replication of PRRSV in MARC-145 cells. The viral copy number in the combined IFNs-Fc treatment group was lower than that in the PRRSV control group, and the difference between the two groups was significant (p<0.0001).

[0039] Figure 13 Combined IFNs-Fc inhibits PEDV replication. Note: Figure 13 The results showed that the combined IFNs-Fc inhibited the replication of PEDV in Vero cells. The viral copy number in the combined IFNs-Fc treatment group was lower than that in the PEDV control group, and the difference between the two groups was extremely significant (p<0.0001).

[0040] Figure 14 For the inhibition of PRV replication by combined IFNs-Fc, note: Figure 14 The results showed that the combined IFNs-Fc inhibited PRV replication in PK15 cells. The viral copy number in the combined IFNs-Fc treatment group was lower than that in the PRV control group, and the difference between the two groups was significant (**p<0.01). Detailed Implementation

[0041] This invention provides a composite IFNs-Fc fusion protein, comprising an IFNγ-Fc fusion protein, an IFNα2-Fc fusion protein, and an IFNλ3-Fc fusion protein; the coding sequence of the IFNγ-Fc fusion protein is shown in SEQ ID No. 1; the coding sequence of the IFNα2-Fc fusion protein is shown in SEQ ID No. 2; and the coding sequence of the IFNλ3-Fc fusion protein is shown in SEQ ID No. 3.

[0042] SEQ ID No. 1:

[0043] Gaattcgccaccatgagctacaccacctactttctggccttccagctgtgtgtgaccctgtgcttcag cggctcttattgccaggcccctttcttcaaggaaatcacaatcctgaaggactactttaacgccagcacaagcgac gtccccaacggcggaccactgttcctggaaatcctgaaaaactggaaggaagagtctgataagaagatcatccaga gccagatcgtgtccttctacttcaagttctttgagattttcaaagataaccaggctatccaaagaagcatggacgt gatcaaacaggacatgttccaaagattcctgaacggcagcagcggcaagctgaatgatttcgagaagctcatcaag atccctgtggacaacctgcagatccagcggaaggccatcagcgagctgatcaaggtgatgaacgacctgagcccta gatctaatctgagaaagcggaaaagatcccagaccatgtttcagggccagcgggcctctaagctggtgcccagagg cagcgctagccatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggccccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa; The underlined part is the codon-optimized IFNγ sequence, and the bold part is the Fc sequence.

[0044] SEQ ID No.2:

[0045] Gaattcgccaccatggcccctaccagcgccttcctgaccgccctggtcctgctgagctgcaatgccat ctgctgcctgggatgtgacctgcctcagacacacagcctggcccataccagagccctgagactgctggctcagatg cggagaatcagcccattttcttgtctggaccacagaagagacttcggcttcccccaggaggctctgggcggcaacc aggtgcagaaggcccaggccatggccctggtgcacgagatgctgcagcaaacatttcagctgttctctacagaggg cagcgccgctgcttgggacgagagcctgctgcaccagttctgcaccggcctggatcagcagctgcgggacctggaa gcctgcgtgatgcaggaggccggcctggaagcaacccctctgcttgaggaagatagcatcctggccgccagaaagt acttccaccggctcaccctgtacctgcaagaaaaaagctacagcccctgcgcctgggaaatcatccgggccgaggt gatgaggtccttctccagctctagaaacctgctggtgcccagaggcagcgctagccatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggccccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa; The underlined part is the codon-optimized IFNα2 sequence, and the bold part is the Fc sequence.

[0046] SEQ ID No.3:

[0047] gaattcgccaccatggccctgggaggcagcctggtgctggtgctggttctgatgaccgtggcccctcc acgcaccggcgccgtgcccgtccccgaggccctcagggccctgcctggcgccagaggctgtcacctggcccagttc aagagcctgagccctcaggccctgcaggcttttaaaagagctaaggacgccttcgaggaaagcctgctggaggact ggaactgcagctcccggatcttccccagaagcagagatctgaagcagctgcaggtgtgggagagacccgtggccct ggaagccgaggtggcccttacactgtccgtgctgggcagcctggctaatagcagcctccattcttctctggaccag cctctgcacacactgcggcacatccacgcccagctgcaagcatgcgtgccagcccaacctatggccggccctcggc ctcggggaagactgcaccactggctgcacagactgcaggaggcccagaaaaaggaacctcagagctgcctggaagc ttctgtgatgttcaacctgttcagactgctgaccagagatctgaagtgcgtggccagcggcgacctgtgtgtgctg gtgcccagaggcagcgctagccatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggccccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa; The underlined part is the codon-optimized IFNλ3 sequence, and the bold part is the Fc sequence.

[0048] In this invention, the enzyme activity ratio of the IFNγ-Fc fusion protein, IFNα2-Fc fusion protein, and IFNλ3-Fc fusion protein is preferably 1:1:1. In this invention, the coding sequence of the IFNγ-Fc fusion protein is preferably obtained by fusing an IFNγ sequence and an Fc sequence, wherein the Genebank sequence number of the IFNγ sequence is NM_213948.1, and the Genebank sequence number of the Fc sequence is AF150959.1. In this invention, the coding sequence of the IFNα2-Fc fusion protein is preferably obtained by fusing an IFNα2 sequence and an Fc sequence, wherein the Genebank sequence number of the IFNα2 sequence is MH538099.1, and the Genebank sequence number of the Fc sequence is AF150959.1. In this invention, the coding sequence of the IFNλ3-Fc fusion protein is preferably obtained by fusing the IFNλ3 sequence and the Fc sequence, wherein the Genebank sequence number of the IFNλ3 sequence is NM_001166490.1 and the Genebank sequence number of the Fc sequence is AF150959.1.

[0049] This invention also provides the application of the composite IFNs-Fc fusion protein described above in the preparation of drugs for treating African swine fever.

[0050] The present invention also provides the application of the composite IFNs-Fc fusion protein described in the above technical solution in the preparation of drugs for treating porcine reproductive and respiratory syndrome.

[0051] This invention also provides the application of the composite IFNs-Fc fusion protein described above in the preparation of drugs for treating porcine epidemic diarrhea.

[0052] This invention also provides the application of the composite IFNs-Fc fusion protein described above in the preparation of drugs for treating porcine pseudorabies.

[0053] This invention also provides a drug for treating swine diseases, comprising the composite IFNs-Fc fusion protein described in the above-described technical solution. In this invention, the swine diseases preferably include one or more of African swine fever, porcine reproductive and respiratory syndrome, porcine epidemic diarrhea, and pseudorabies. In this invention, the enzyme activity ratio of IFNγ-Fc fusion protein, IFNα2-Fc fusion protein, and IFNλ3-Fc fusion protein in the composite IFNs-Fc fusion protein is preferably 1:1:1.

[0054] To further illustrate the present invention, the following detailed description is provided in conjunction with embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0055] Example 1

[0056] 1.2.1 Construction of pMal-IFNs-Fc expression plasmid

[0057] Based on the fusion of the full-length porcine IFN sequences (IFN-γ, IFN-α2, IFN-λ3) with the Fc gene from GenBank (Table 1), the Hinge region of the Fc gene was used as the linker. Since there was no stop codon at the end of the IFNs, an EcoRI restriction site and a Kozak sequence were introduced upstream of the IFNs. A thrombin cleavage site and an NheI restriction site were introduced between the IFNs and the Fc gene to synthesize IFNs-Fc (IFNγ-Fc, IFNα2-Fc, IFNλ3-Fc). Codon optimization was performed based on mammalian codon preferences. All sequences were synthesized by Nanjing GenScript Biotech Co., Ltd.

[0058] Table 1 Gene Sequences

[0059] IFN Name Genebank Accession Number Classification Size (bp) Species IFN-γ NM_213948.1 Ⅱ 717 Porcine IFN-α2 MH538099.1 Ⅰ 546 Porcine IFN-λ3 NM_001166490.1 Ⅲ 588 Porcine Fc AF150959.1 IgG1 669 Human

[0060] The modified pMAL-cDNA3.0 was selected as the vector. A tandem IFN-Fc gene was inserted between the EcoRI and NheI restriction sites downstream of the CMV promoter. To enable the Fc and IFNs to be secreted as a fusion protein from mammalian cells, a Kozak sequence was introduced before the IFN gene sequence. The expression plasmids were named pMAL-IFNγ-Fc, pMAL-IFNα2-Fc, and pMAL-IFNλ3-Fc. Figure 1 After all plasmids are correctly sequenced, they are used for cell transfection. The specific steps are as follows:

[0061] 1) Double digestion and ligation: The IFN-Fc gene and pMAL vector were double-digested separately. The digestion system is shown in Table 2.

[0062] Table 2 Double enzyme digestion system

[0063] Component Volume (total 50 μL) IFN-Fc / pMAL Vector 25 EcoRI 2.5 NheI 2.5 10× Buffer 5 ddH2O 15

[0064] 2) Incubate at 37℃ for 2 hours, then purify and recover the IFN-Fc gene according to the instructions of the gel extraction kit. Ligate the double-digested IFN-Fc gene with the pMAL vector at 16℃ for 8 hours.

[0065] The connection system is shown in Table 3:

[0066] Table 3 Connection System

[0067] Component Volume (total 20 μL) IFN-Fc Double Digestion and Purification Product 8 Purified product of double digestion of pMAL 9 10×T4 Ligase Buffer 2 T4 Ligase 1

[0068] 3) Preparation of competent cells: Cryopreserved competent bacteria (E. coli DH5a) were prepared in a sterile laminar flow hood. One loopful of bacterial culture was picked up using a disposable inoculation loop and streaked onto an LB agar plate. The plate was incubated at 37°C for 16 hours. Single colonies were then picked from the plate and transferred to a test tube containing 3 mL of LB medium. The tube was then incubated overnight at 37°C and 220 rpm on a horizontal shaker. The following day, 1 mL of the bacterial culture was inoculated into 100 mL of LB medium and incubated for 4 hours at 37°C and 250 rpm on a shaker. Immediately after removing the test tube, incubate on ice for 15 minutes. Then, transfer the culture medium to a pre-cooled 50 mL centrifuge tube, centrifuge at 4000 rpm for 5 minutes, and discard the supernatant. Add 0.1 M CaCl2 solution to resuspend the bacterial cells, incubate on ice for 30 minutes, centrifuge at 4000 rpm for 5 minutes at 4°C, and discard the supernatant. All the above operations should be performed at 4°C or on ice. Finally, resuspend the bacterial cells in 4 mL of 0.1 M CaCl2 solution, add 80% sterile glycerol and mix well. Aliquot into 100 μL tubes and store at -80°C for later use.

[0069] 4) Transformation: Add 5 μL of ligation product to 100 μL of competent cells, gently swirl to mix, incubate on ice for 30 min, then immediately heat shock at 42°C for 90 s, followed by an immediate ice incubation for 3 min. Add 800 μL of antibiotic-free LB medium to each tube and incubate on a shaker at 37°C and 160 rpm for 45 min. Subsequently, spread the bacterial culture on LB agar plates containing antibiotics (50 μg / mL Amp+) and incubate overnight at 37°C.

[0070] 5) Plasmid Extraction: Extract plasmids according to the kit instructions. Centrifuge the collected bacterial culture at 12000 rpm for 1 min and remove the supernatant. Add 250 μL of P1 solution (containing RNase A) to the centrifuge tube and vortex to suspend the bacterial pellet. Add 250 μL of P2 solution to the centrifuge tube and gently mix up and down 6-8 times to fully lyse the bacteria. Add 350 μL of P3 solution to the centrifuge tube and quickly invert to mix thoroughly. Centrifuge at 12000 rpm for 10 min. Transfer the supernatant from centrifugation to the pre-treated CP3 adsorption column (place the adsorption column in the collection tube) and centrifuge at 12000 rpm for 1 min. Discard the waste liquid in the collection tube, return the CP3 adsorption column to the collection tube, add 600 μL of PW wash buffer (containing anhydrous ethanol) to the adsorption column, centrifuge at 12000 rpm for 1 min, discard the waste liquid, return the CP3 column to the collection tube, and repeat the wash once. After centrifugation, return the CP3 adsorption column to the collection tube and centrifuge at 12,000 rpm for 2 minutes to remove as much wash buffer as possible from the adsorption column. Transfer the centrifuged CP3 adsorption column to a new clean centrifuge tube, add 100 μL of EB elution buffer to the center of the column, and incubate at room temperature for 2 minutes. Centrifuge at 12,000 rpm for 2 minutes, then collect the plasmid solution into a centrifuge tube and store at -20°C.

[0071] 6) Identification of recombinant plasmids: The extracted plasmid DNA was identified by PCR, and the PCR products were analyzed and identified by agarose gel electrophoresis.

[0072] 1.2.2 Expression of IFNs-Fc fusion protein

[0073] HEK 293T cells were transfected using a liposome (LR)-mediated method. The expressed IFNs-Fc fusion protein was identified by SDS-PAGE and Western Blot, with the specific steps as follows:

[0074] 1) Cell pretreatment: HEK 293T cells were passaged in cell culture dishes at a cell density of 1×105mL and cultured in DMEM + 10% FBS medium for 12h. When the cells reached 70% confluence, they were transfected.

[0075] 2) Preparation of transfection reagent: Dilute Lipofecta mine 3000 reagent with Opti-MEM culture medium. For 24-well plates, use 250 μL of Opti-MEM culture medium in two tubes, add 7.5 μL and 1.5 μL of Lipofecta mine 3000 reagent respectively, and mix thoroughly.

[0076] 3) Prepare DNA premix: Dilute DNA with Opti-MEM culture medium, add 10 μg DNA to 500 μL of Opti-MEM culture medium, then add P3000 (2 μL / μg DNA) reagent and mix thoroughly.

[0077] 4) Binding of transfection reagent to DNA: Add diluted DNA to 250 μL of prepared Lipofecta mine 3000 reagent at a 1:1 ratio and incubate at room temperature for 15 min.

[0078] 5) Cell transfection: Use a 100 μL pipette to pick up the DNA-lipid complex (500 μL of the complex contains 5000 ng of DNA) and add it evenly to the 293T cells in the culture dish.

[0079] 5) Cell culture: The transfected cells from step 5 were cultured in a 37°C incubator for 7 days.

[0080] 6) Collect cells and supernatant, centrifuge at 10,000 rpm for 30 min for subsequent purification and identification.

[0081] 1.2.3 Purification of IFNs-Fc fusion protein

[0082] After high-level expression of the IFNs-Fc fusion protein, centrifugation was performed, and the supernatant was filtered through a 0.45 μm microporous ultrafiltration membrane. The expression product was then purified by affinity chromatography. The specific steps are as follows:

[0083] 1) Ultrafiltration: Collect a certain amount of supernatant from HEK 293T cells, centrifuge at 10,000 rpm for 30 min at 4℃, and then use a 0.45 μm Millipore ultrafiltration membrane to ultrafilter the supernatant. Concentrate the supernatant 10 times. Use an ultrafiltration cup to concentrate 300 mL of supernatant to 30 mL, add TBS buffer (pH 8.0) to 300 mL, and repeat the concentration to 30 mL.

[0084] 2) Protein A Affinity Chromatography: Protein A, acting as an affinity ligand, specifically binds to the Fc fragment of the IFNs-Fc fusion protein. By altering the pH of the elution buffer, the target protein is separated from the affinity medium, thus achieving purification. The affinity chromatography column is equilibrated with binding buffer (0.02M sodium phosphate, 3M NaCl, pH 7.0). A concentrated eluent (0.1M sodium citrate) is passed through the column at a flow rate of 1 mL / min. Elution buffer (0.1M sodium citrate, pH 3.0) is then added to elute the fusion protein bound to the Protein A affinity chromatography column at a flow rate of 1 mL / min. The target protein peak is collected. After elution, binding buffer (0.02M sodium phosphate, 3M NaCl, pH 7.0) is added to reequilibrate the affinity column. The entire process is monitored using an AKTA protein purification instrument, and instrument parameters are adjusted as needed.

[0085] 1.2.4 Identification and analysis of IFNs-Fc fusion protein

[0086] After expression and purification, the IFNs-Fc fusion protein was identified and analyzed by SDS-PAGE, Western Blot and mass spectrometry.

[0087] 1) SDS-PAGE identification: Prepare a 10% separating gel, pour it to an appropriate height, add a small amount of water for sealing, and allow the polymer to solidify for about 1 hour. Pour off the water and blot dry with filter paper. Prepare a 5% stacking gel, pour it to the top of the glass plate, insert a comb, and let it stand for about 40 minutes. After the gel solidifies, remove the comb and fill the electrophoresis tank with 1× electrophoresis buffer. Prepare a 1× mixture of expression product and loading buffer, boil it in boiling water for 5 minutes, cool it on ice, and add the prepared sample and protein marker in sequence. Set the electrophoresis apparatus voltage to 80V and electrophoresis for 30 minutes. When the bromophenol blue dye front enters the separating gel, adjust the voltage to 120V and electrophoresis for 60 minutes. When the dye reaches the bottom of the separating gel, turn off the power. The gel was immersed in Coomassie Brilliant Blue staining solution and stained overnight on a low-speed shaker. After pouring out the staining solution, destaining solution was added and destaining on a low-speed shaker for 2 hours. The destaining solution was changed every 30 minutes. After a clear blue protein band was observed, the gel was scanned and photographed using a gel imaging system.

[0088] 2) Western Blot: After SDS-PAGE electrophoresis, remove the gel, cut it to an appropriate size, and perform electrotransfer without staining. Nitrocellulose membranes (NC) are pre-cut to the same size as the gel. During electrotransfer, stack the membranes in the following order: negative electrode sponge (1 layer) + filter paper (2-3 layers) + gel + NC membrane + filter paper (2-3 layers) + sponge (1 layer) to positive electrode, ensuring no air bubbles between each layer. The top layer of filter paper should not contact the NC membrane, and each layer should be fully wetted with electrotransfer buffer. Set the power supply to 250mA and electrotransfer for 50-60 minutes. The membrane should be placed in an ice bath during transfer. After transfer, turn off the power, remove the NC membrane (pre-stained markers will be visible), and place the NC membrane in freshly prepared 5% BSA solution. Block the membrane at room temperature on a low-speed shaker (50 rpm) for 2 hours. Dilute the Anti-Fc antibody with PBST (1:10000 dilution), place the blocked NC membrane in the antibody dilution solution, and incubate overnight at 4°C. The membrane was washed four times with TBST on the second day, each time for 10 minutes. The developing solution was prepared and stored away from light. The developing solution was prepared fresh for each use. The developing solution was then dropped onto the NC membrane, and the imaging system automatically exposed and saved the image.

[0089] 3) Mass spectrometry identification: The target band after SDS-PAGE electrophoresis is cut out and sent to the mass spectrometry center for peptide fingerprint mass spectrometry identification. The reference sequence is shown in Table (1).

[0090] 1.2.5 Determination of the concentration and yield of IFNs-Fc fusion protein

[0091] The concentration of IFNs-Fc fusion protein was determined using the Bradford method. Bradford working solution was prepared, and standard proteins diluted to different concentrations were added. After standing for 5 minutes, the OD was measured using a UV spectrophotometer. 570A standard curve was plotted with protein content on the ordinate and absorbance on the abscissa. The IFNs-Fc sample to be tested was serially diluted 2-fold, Bradford working solution was added, and the mixture was thoroughly mixed. The mixture was then incubated at 37°C for 30 min. The OD value of the standard curve was recorded using tube 0 as a reference. 570 Value, where m is the protein content on the standard curve, according to Equation 1:

[0092]

[0093] Calculate the concentration of purified IFNs-Fc fusion protein and calculate the yield based on the number of cells collected per L of culture medium.

[0094] 1.2.6 Method for determining the titer of IFNs-Fc fusion protein by inhibiting cytopathic effects

[0095] The anti-VSV-GFP activity of IFNs-Fc (IFNγ-Fc, IFNα2-Fc, IFNλ3-Fc) was assessed using a cytopathic effect inhibition method based on the VSV-3D4 / 2 and VSV-PK15 systems, and the IFNs-Fc titer was calculated according to the Reed-Muench method. The specific steps are as follows:

[0096] 1) Passage well-grown 3D4 / 2 or PK15 cells and prepare a homogeneous cell suspension with a cell concentration of approximately 1×10⁻⁶. 6 / mL, transferred to 96-well cell culture plates, 100μl per well, and incubated at 37℃ in a 5% CO2 incubator for 24h.

[0097] 2) The initial concentration of all test samples was set at 10 μg / μL. The test samples were diluted with RPMI 1640 culture medium (containing 7% FBS) at a concentration of 4 μg / μL. 1 ~4 9 Eight dilution gradients were used, with six replicates for each dilution; a blank control (CK), a negative control (cells), and a positive control (VSV-GFP 100 TCID) were also included. 50 ).

[0098] 3) Add the diluted sample to the corresponding well, 100 μL per well, and incubate at 37°C in a 5% CO2 incubator for 24 h.

[0099] 4) Remove the liquid from the cell plate and wash twice with PBS.

[0100] Add 100 μL of 100 TCID solution prepared with RPMI 1640 (containing 2% FBS) to each well. 50 VSV-GFP virus solution was incubated at 37°C in a 5% CO2 incubator for 1 hour.

[0101] 5) Remove the cell culture plate and add 100 μL of RPMI 1640 culture medium (containing 2% FBS) to each well. Incubate at 37°C in a 5% CO2 incubator for 48 hours. When cytopathic effect (CPE) appears in all positive wells and stops changing, discard the liquid in the cell culture plate, wash twice with PBS, add 10 μL of CCK-8 working solution to each well, incubate at 37°C in a 5% CO2 incubator for 2 hours, and then record the OD using a microplate reader. 450 value.

[0102] 6) Data processing: An activity unit is defined as the amount of IFN that inhibits 50% of cytopathic effects. The Reed-Muench method is used to calculate the IFNs-Fc titer, which is the dilution factor that inhibits 50% of cytopathic effects. Substitute the values ​​from Table 2-4, where X is the cell negative control and Y is the virus positive control value, and calculate the IFNs-Fc titer according to Equation 2.

[0103] Formula 2-3: IFN titer to be tested (U / 0.1mL) = pre-dilution factor × 4[4+(Gn-0.5) / (Gn-Gn+1)]

[0104] Table 4. IFN Potency Calculation Table (Li Bing, 2018. Preparation and Functional Study of Canine Serum Albumin Fusion Interferon-γ. Doctoral Dissertation)

[0105]

[0106] Note: X in the table represents the OD of the cell negative control wells. 450 Value, Y is the OD of the virus positive control well. 450 value.

[0107] 1.2.7 Cell proliferation and toxicity analysis of combined IFNs-Fc

[0108] The CCK-8 assay was used to detect the proliferative toxicity of the combined IFNs-Fc compound on porcine primary alveolar macrophages (PAMs). PAMs were inoculated at 2.0 × 10⁻⁶ cells / cells. 5 Cells / mL were seeded in 24-well cell culture plates and incubated at 37°C in a 5% CO2 incubator for 12 h. IFNα2-Fc, IFNγ-Fc, and IFNλ3-Fc were each seeded at 1×10⁻⁶ cells / mL. 5The IFNs-Fc complex was prepared by mixing U / mL. 300 μL of the IFNs-Fc complex was used to treat PAMs cells. A negative control group (PAMs) and a blank control group (Blank) were also included. Each treatment group was replicated in triplicate. Cells were incubated at 37°C with 5% CO2 for 24 and 48 hours. At 24 and 48 hours, the culture liquid was removed from the wells, and the cells were washed twice with PBS. Then, 10 μL of CCK-8 working solution was added to each well, and the cells were incubated at 37°C with 5% CO2 for 2–4 hours. The absorbance (OD) values ​​were recorded using a microplate reader. 450 And according to formula (Equation 3):

[0109]

[0110] Calculate the cell proliferation toxicity of PAMs cells treated with combined IFNs-Fc at different time points.

[0111] 1.2.8 Inhibition of ASFV Replication by Composite IFNs-Fc

[0112] 1.2.8.1 Experimental treatment for ASFV inhibition by combined IFNs-Fc

[0113] To investigate the effect of combined IFNs-Fc on ASFV replication, IFNα2-Fc, IFNγ-Fc, and IFNλ3-Fc were combined at a ratio of 1×10⁻⁶. 5 The mixture was prepared by mixing U / mL to form a composite IFNs-Fc. 300 μL of the mixture was used to treat PAMs cells for 24 h with 100 TCID50. 50 Cells were infected with ASFV for 24 and 48 hours, and the cell supernatant was collected for RT-qPCR to detect viral load. The specific steps are as follows:

[0114] 1) Plating: PAMs cells were prepared into 2.0 × 10⁶ cells using complete culture medium (DMEM + 10% FBS). 5 Cell suspension of 300 μL / mL was seeded into 24-well cell culture plates. The plates were incubated at 37°C in a 5% CO2 incubator for 12 hours.

[0115] 2) Prepare composite IFNs-Fc, IFNα2-Fc, IFNγ-Fc and IFNλ3-Fc by first diluting them with diluent (7% FBS + DMEM) to a concentration of 1×10⁻⁶. 5 U / mL, and then take 100μL of each and mix them in equal volumes (1000U / well each of IFNα2-Fc, IFNγ-Fc and IFNλ3-Fc).

[0116] 3) Set up test wells (treated with compound IFNs-Fc), negative control (PAMs), and positive control (ASFVp72) in the cell culture plate. Add 300 μL of compound IFNs-Fc to the test wells, and add 7% FBS+DMEM to the negative and positive controls. Incubate at 37℃ in a 5% CO2 incubator for 24 h.

[0117] 4) Virus preparation: Dilute ASFV with DMEM culture medium containing 2% FBS, and add 100 TCID50. 50 / The dose of the virus was used to infect the test wells and positive control wells.

[0118] 5) After treating cells with compound IFNs-Fc for 24 hours, remove the compound IFNs-Fc from the test wells, and simultaneously add the pre-prepared virus solution to both the test wells and the positive control wells at 100 TCID50. 50 / well dose of virus challenge, placed in a 37℃, 5% CO2 incubator for 24 and 48 hours.

[0119] 6) Sample collection: Collect cell supernatant at 24 and 48 h after ASFV infection for subsequent RT-qPCR detection; at 48 h, fix the cells and perform indirect immunofluorescence experiments, with 3 replicates at each time point.

[0120] 1.2.1.2 RT-qPCR detection of viral load

[0121] The ASFV p72 gene in samples was detected by RT-qPCR using the OIE-recommended TaqMan method. Primers and probes were synthesized by Nanjing Genscript Biotech Co., Ltd. Viral copy number was calculated according to the detection method developed in our laboratory. The TaqMan RT-qPCR reaction system is shown in Table 5. The reaction program was: 95℃, 5 min, 95℃, 10 s, 60℃, 30 s, repeated for 40 cycles. After the program, the viral copy number corresponding to the Ct value of each sample was calculated using a standard curve.

[0122] Table 5 TaqMan RT-qPCR reaction system

[0123] Ingredient Volume (total 20 μL) 2×AceQ Qpcr Probe Master Mix 10 ASFV-F (10 μM) 0.4 ASFV-R (10 μM) 0.4 ASFV-Probe (10 μM) 0.2 50×ROX Reference Dye1 0.4 DNA 2 ddH2O 6.6

[0124] 1.2.8.3 Indirect Immunofluorescence Assay

[0125] To further investigate the effect of combined IFNs-Fc on ASFV replication, IFNα2-Fc, IFNγ-Fc, and IFNλ3-Fc were each added at a concentration of 1×10⁻⁶. 5 The mixture was prepared by mixing U / mL to form a composite IFNs-Fc. 300 μL of the mixture was used to treat PAMs cells for 24 h with 100 TCID50. 50Cells were infected with ASFV in each well for 48 hours, then fixed and subjected to indirect immunofluorescence (IFA) assays. The specific steps are as follows:

[0126] 1) Discard the supernatant in the cell culture wells, wash twice with pre-cooled PBS, add 4% paraformaldehyde to fix the cells, incubate at room temperature for 30 min, then discard the fixative, wash three times with PBS for 5 min each time.

[0127] 2) Add 0.1% Triton X-100, let stand at room temperature for 20 min, discard the solution, and wash with PBS 3 times, 5 min each time.

[0128] 3) Add 5% BSA blocking solution, block at 37°C for 1 hour, remove the blocking solution, and wash with PBS 3 times for 5 minutes each time.

[0129] 4) Add the primary antibody diluted with 5% BSA solution, incubate at 37°C for 1 hour, and wash with PBS 3 times for 5 minutes each time.

[0130] 5) Then add diluted fluorescently labeled antibody in the dark, block at 37°C in the dark for 1 hour, and wash with PBS 3 times, 5 minutes each time.

[0131] 6) Add anti-fluorescence quenching mounting solution for 10 seconds, discard the mounting solution, and then observe the protein expression and collect images using an inverted fluorescence microscope.

[0132] 1.2.9 Determination of the anti-PRRSV, PEDV, and PRV activity of the composite IFNs-Fc

[0133] This study investigated the broad-spectrum antiviral activity of compound IFNs-Fc against porcine reproductive and respiratory syndrome virus (PRRSV), porcine epidemic diarrhea virus (PEDV), and porcine pseudorabies virus (PRV). The inhibitory ability of compound IFNs-Fc on the replication of PRRSV, PEDV, and PRV was detected by RT-qPCR and IFA.

[0134] 1.2.9.1.1 RT-qPCR detection of PRRSV viral load

[0135] 1) Plating: Wash well-cultured MARC-145 cells twice with PBS, trypsin digest, collect cells, and plate them into 2.0 × 10⁶ cells using complete culture medium (DMEM + 10% FBS). 5 Cell suspension at a concentration of [number] cells / mL was seeded into 300 μL per well of a 24-well cell culture plate. The plates were incubated at 37°C in a 5% CO2 incubator for 12–24 h.

[0136] 2) Preparation of composite IFNs-Fc: IFNα2-Fc, IFNγ-Fc and IFNλ3-Fc were first diluted with diluent (7% FBS + DMEM) to a concentration of 1×10⁻⁶. 5 Take 100 μL of each of the U / mL liquids and mix them in equal volumes (1000 U / well each of IFNα2-Fc, IFNγ-Fc and IFNλ3-Fc).

[0137] 3) Set up test wells, negative control, and positive control in the culture plate. When the cells in the culture wells reach 80%, add 300 μL of compound IFNs-Fc to the test wells. Add 7% FBS+DMEM to the negative control and positive control. Incubate at 37℃ in a 5% CO2 incubator for 24 h.

[0138] 4) Virus preparation: Dilute PRRSV virus solution with DMEM culture medium containing 2% FBS, and add 100 TCID50. 50 / well dose challenge (for wells less than 300μL, supplement with 2% FBS DMEM culture medium to 300μL).

[0139] 5) After treating cells with compound IFN-Fc for 24 hours, remove the compound IFN-Fc liquid from the test wells. Simultaneously add the pre-prepared virus solution to both the test wells and the positive control wells at 100 TCID50. 50 / well dose of virus challenge, placed in a 37℃, 5% CO2 incubator for 48h.

[0140] 6) Sample collection: Cell supernatant and cell samples were collected 24 hours after challenge for RT-qPCR detection and IFA, with 3 replicates at each time point.

[0141] 1.2.9.1.2 Anti-PRRSV Indirect Immunofluorescence Assay

[0142] To further investigate the effect of the composite IFNs-Fc on PRRSV replication, IFNα2-Fc, IFNγ-Fc, and IFNλ3-Fc were mixed at 1000 U / μL to prepare the composite IFNs-Fc. MARC-145 cells were treated with this mixture for 24 h at 100 TCID50. 50 / Infect cells with PRRSV for 24 hours, fix the cells, and perform IFA experiments. The specific steps are the same as in 1.2.8.3.

[0143] 1.2.9.2 Composite IFNs-Fc resists PEDV replication

[0144] 1.2.9.2.1 RT-qPCR detection of PEDV viral load

[0145] Vero cells were treated with compound IFNs-Fc for 24 h at 100 TCID.50 Cells were infected with PEDV, and the ability of compound IFNs-Fc to inhibit PEDV replication was detected by RT-qPCR (SU et al., 2020). Other steps were the same as in 1.2.9.1.1.

[0146] 1.2.9.2.2 Indirect Immunofluorescence Assay for Anti-PEDV

[0147] To further investigate the effect of the composite IFNs-Fc on PEDV replication, IFNα2-Fc, IFNγ-Fc, and IFNλ3-Fc were mixed at 1000 U / μL to prepare the composite IFNs-Fc. Vero cells were treated with this mixture for 24 h and then injected with 100 TCID50. 50 / Infect cells with PRRSV for 24 hours, fix the cells, and perform IFA experiments. The specific steps are the same as in 1.2.9.1.2.

[0148] 1.2.9.3 Composite IFNs-Fc Anti-PRV Replication

[0149] 1.2.9.3.1 RT-qPCR detection of PRV viral load

[0150] PK15 cells were treated with compound IFNs-Fc for 24 h at 100 TCID. 50 Cells infected with PRV were tested for their ability to inhibit PRV replication by RT-qPCR (TU et al., 2021). Other steps were the same as in 1.2.9.1.1.

[0151] 1.2.9.3.2 Anti-PRV Indirect Immunofluorescence Assay

[0152] To further investigate the effect of the composite IFNs-Fc on PRV replication, IFNα2-Fc, IFNγ-Fc, and IFNλ3-Fc were mixed at 1000 U / μL to prepare the composite IFNs-Fc. PK15 cells were treated with this mixture for 24 h at 100 TCID50. 50 / Well PRV infected cells for 24 hours, cells fixed, and IFA experiment performed, the specific steps are the same as 2.2.10.1.2.

[0153] 1.3 Results

[0154] 1.3.1 Construction of IFNs-Fc expression vector

[0155] Based on the full-length porcine IFN sequences (IFN-γ, IFN-α2, IFN-λ3) in GenBank, their codons were optimized. The modified pMAL was selected as the eukaryotic expression vector. The optimized IFNs and Fc genes were introduced downstream of the CMV promoter to construct three expression plasmids, named pMAL-IFNγ-Fc, pMAL-IFNα2-Fc, and pMAL-IFNλ3-Fc. PCR identification results are shown below. Figure 2 The results showed that the PCR product sizes of IFNγ-Fc (470bp), IFNα2-Fc (546bp), and IFNλ3-Fc (588bp) were consistent with the theoretical values.

[0156] 1.3.2 Expression of IFNs-Fc fusion protein

[0157] HEK 293T cells were transfected with expression plasmids pMAL-IFNγ-Fc, pMAL-IFNα2-Fc, and pMAL-IFNλ3-Fc. After 7 days of expression, the supernatant was collected for SDS-PAGE and Western blotting. SDS-PAGE results showed (…). Figure 3 The sizes of the three fusion proteins, IFNγ-Fc (54.7 kDa), IFNα2-Fc (55.3 kDa), and IFNλ3-Fc (56.5 kDa), were consistent with theoretical values. Western blot results showed that ( Figure 4 All three fusion proteins expressed were reactive, and their sizes were consistent with theoretical values.

[0158] 1.3.3 Purification of IFNs-Fc fusion protein

[0159] Fc can specifically bind to Protein A. Affinity chromatography using a Protein A purification column was used to purify the expressed IFNγ-Fc, IFNα2-Fc, and IFNλ3-Fc fusion proteins. The purification process was observed using a curve showing protein absorption peaks. Figure 5 ).

[0160] The purification efficiency of the three fusion proteins was assessed by SDS-PAGE and Western blotting. SDS-PAGE results (…) Figure 6 The results showed that IFNγ-Fc (54.7 kDa), IFNα2-Fc (55.3 kDa), and IFNλ3-Fc (56.5 kDa). Western blotting results showed... Figure 7 The protein size matches the theoretical value, and the purity can reach over 90%.

[0161] 1.3.4 Identification of IFNs-Fc fusion protein profiles

[0162] The target band after SDS-PAGE electrophoresis was cut out and sent to a mass spectrometry center for peptide fingerprinting identification. The mass spectrometry results showed that all three IFNs-Fc fusion proteins were correctly expressed.

[0163] Fusion protein sequence: The underlined sequence is the codon-optimized IFN sequence, and the bolded sequence is human IgG1 Fc (AF150959.1).

[0164] IFNγ-Fc (SEQ ID No. 1):

[0165] >Sus scrofa interferon gamma(IFNG)NM_213948.1

[0166] Gaattcgccaccatgagctacaccacctactttctggccttccagctgtgtgtgaccctgtgcttcag cggctcttattgccaggcccctttcttcaaggaaatcacaatcctgaaggactactttaacgccagcacaagcgac gtccccaacggcggaccactgttcctggaaatcctgaaaaactggaaggaagagtctgataagaagatcatccaga gccagatcgtgtccttctacttcaagttctttgagattttcaaagataaccaggctatccaaagaagcatggacgt gatcaaacaggacatgttccaaagattcctgaacggcagcagcggcaagctgaatgatttcgagaagctcatcaag atccctgtggacaacctgcagatccagcggaaggccatcagcgagctgatcaaggtgatgaacgacctgagcccta gatctaatctgagaaagcggaaaagatcccagaccatgtttcagggccagcgggcctctaagctggtgcccagagg cagcgctagccatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggccccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa;

[0167] The corresponding amino acid sequence is shown in SEQ ID No. 4:

[0168] MSTPHSYTTYFLALQLCVTLCLSGSYCPAPFLQGNHNPEGLLLTPAQATSPTGGLPLFKKSKNWKEEPDKKIIQSQIVSFYFKFFEIFKDNQAIQRSMDV IKQDMFQRFLNGSSGKLNDFEKLIKIPVDNLQIQRKAISELIKVMNDLSPRSNLRKRKRSQTMFQGQRASKLVPRGSASHAHRAQHLNSWGDRQSSSSPKP KDTLMISRTPEVTCVVVDVSHEDPEWKFNWYVDGVEVHNACTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNCALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGPFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。

[0169] IFNα2-Fc(SEQ ID No.2):>Sus scrofa breed Congjiang Xiang interferonalpha 2(IFN-a2)

[0170] Gaattcgccaccatggcccctaccagcgccttcctgaccgccctggtcctgctgagctgcaatgccat ctgctgcctgggatgtgacctgcctcagacacacagcctggcccataccagagccctgagactgctggctcagatg cggagaatcagcccattttcttgtctggaccacagaagagacttcggcttcccccaggaggctctgggcggcaacc aggtgcagaaggcccaggccatggccctggtgcacgagatgctgcagcaaacatttcagctgttctctacagaggg cagcgccgctgcttgggacgagagcctgctgcaccagttctgcaccggcctggatcagcagctgcgggacctggaa gcctgcgtgatgcaggaggccggcctggaagcaacccctctgcttgaggaagatagcatcctggccgccagaaagt acttccaccggctcaccctgtacctgcaagaaaaaagctacagcccctgcgcctgggaaatcatccgggccgaggt gatgaggtccttctccagctctagaaacctgctggtgcccagaggcagcgctagccatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggccccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa;

[0171] The corresponding amino acid sequence is shown in SEQ ID No. 5:

[0172] MAPTSAFLTALVLLSCNAICCLGCDLPQTHSLAHTRALRLLAQMRRISPFSCLDHRRDFGFPQEALGGNQVQKAQAMALVHEMLQQTFQLFSTEGSAAAWDESLLHQFCTGLDQQLRDLEACVMQEAGLEATPLLEEDSILAARKYFHRLTLYLQEKSYSPCAWEIIRAEVMRSFSSSRNLLVPRGSASHAHRAQHLNSWGDRQSSSSPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGPFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。

[0173] IFNλ3-Fc(SEQ ID No.3):

[0174] >Sus scrofa IL-28bgene

[0175] Gaattcgccaccatggccctgggaggcagcctggtgctggtgctggttctgatgaccgtggcccctcc acgcaccggcgccgtgcccgtccccgaggccctcagggccctgcctggcgccagaggctgtcacctggcccagttc aagagcctgagccctcaggccctgcaggcttttaaaagagctaaggacgccttcgaggaaagcctgctggaggact ggaactgcagctcccggatcttccccagaagcagagatctgaagcagctgcaggtgtgggagagacccgtggccct ggaagccgaggtggcccttacactgtccgtgctgggcagcctggctaatagcagcctccattcttctctggaccag cctctgcacacactgcggcacatccacgcccagctgcaagcatgcgtgccagcccaacctatggccggccctcggc ctcggggaagactgcaccactggctgcacagactgcaggaggcccagaaaaaggaacctcagagctgcctggaagc ttctgtgatgttcaacctgttcagactgctgaccagagatctgaagtgcgtggccagcggcgacctgtgtgtgctg gtgcccagaggcagcgctagccatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggccccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa。

[0176] The corresponding amino acid sequence is shown in SEQ ID No. 6:

[0177] MALGGSLVLVLVLMTVAPPRTGAVPVPEALRALPGARGCHLAQFKSLSPQALQAFKRAKDAFEESLLEDWNCSSSRIFPRSRDLKQLQVWERPVALEAEVALTLSVL GSLANSSLHSSLDQPLHTLRHIHAQLQACVPAQPMAGPRPRGRLHHWLHRLQEAQKKEPQSCLEASVMFNLLRLLTRDLKCVASGDLCVLVPRGSHAHRAQHLNSW GDRQSSSSPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGPFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0178] 1.3.5 IFNs-Fc fusion protein concentration

[0179] The concentration of IFNs-Fc fusion protein was detected using the Bradford method, and the results were analyzed based on the plotted Bradford standard curve. Figure 8 The protein concentrations of each fusion protein were calculated using the formula: y = 0.6951x + 0.017. The calculation results (Table 6) show the concentrations of the fusion proteins as follows: IFNγ-Fc (4.37 mg / mL), IFNα2-Fc (1.51 mg / mL), and IFNλ3-Fc (8.18 mg / mL). The yields were IFNγ-Fc (65.55 mg / L), IFNα2-Fc (22.65 mg / L), and IFNλ3-Fc (122.7 mg / L), respectively.

[0180] Table 6 Protein Concentration and Yield

[0181] Name Concentration (mg / mL) Yield (mg / L) IFNγ-Fc 4.37 65.55 IFNα2-Fc 1.51 22.65 IFNλ3-Fc 8.18 122.7

[0182] 1.3.7 Assay of IFNs-Fc fusion protein titer

[0183] The anti-VSV-GFP activities of IFNγ-Fc, IFNα1-Fc, IFNα2-Fc, IFNω-Fc, IFNλ1-Fc, and IFNλ3-Fc were determined using the 3D4 / 2-VSV-GFP and PK15-VSV-GFP systems. The specific steps are as follows:

[0184] The initial concentrations of IFNγ-Fc, IFNα2-Fc, and IFNλ3-Fc were set to 10 μg / mL, and then 3D4 / 2 cells were treated for 24 h with 100 TCID45. 50 VSV-GFP infected cells; when all positive control wells showed green fluorescence, the OD of each well was detected using a CCK-8 assay. 450 Absorbance values ​​were used to calculate the antiviral titer of IFNs-Fc using the Reed-Muench method. Fluorescence observation showed no green fluorescence in the cell control group, while most cells in the virus control group (VSV-GFP) exhibited CPE and green fluorescence. Results from the IFNs-Fc treatment groups showed that IFNγ-Fc completely inhibited the virus at a 1:256 dilution; IFNα2-Fc completely inhibited the virus at a 1:4096 dilution; and IFNλ3-Fc completely inhibited the virus at a 1:1024 dilution. This indicates that high doses of IFNs-Fc completely inhibit VSV-GFP, while medium doses partially inhibit it, and the antiviral ability of IFNs-Fc is positively correlated with its concentration.

[0185] When green fluorescence is observed in all positive wells, the OD of each well is detected using a CCK-8 analyzer. 450 The absorbance was calculated according to the Reed-Muench method based on the method in Table 4. The results (Table 7) showed that all six IFN fusion proteins exhibited high antiviral activity in both the VSV-3D4 / 2 and VSV-PK15 systems, with titers of: IFNγ-Fc (1.04 × 10⁻⁶). 9 U / mg; 1.47×10 8 U / mg), IFNα2-Fc (1.63×10) 7 U / mg; 1.75×10 7 U / mg), IFNλ3-Fc (9.23×10) 9 U / mg; 9.97×10 7 (U / mg). IFNα2-Fc showed similar antiviral activity on 3D4 / 2 and PK15 cells; the other 5 fusion proteins all had higher titers on 3D4 / 2 cells than on PK15 cells.

[0186] Table 7. Titer of IFNs-Fc fusion protein detected in different cells.

[0187]

[0188]

[0189] 1.3.8 Cell proliferation toxicity of combined IFNs-Fc

[0190] To study composite IFNs-Fc (each component 1×10⁻⁶) 5 The proliferation toxicity of a mixture of IFNs-Fc (U / mL) to PAMs cells at different time points was investigated. The experiment included a compound IFNs-Fc treatment group, a negative control group (MOCK), and a blank control group (Blank), with three replicates for each treatment group. 300 μL of compound IFNs-Fc was added to well-growing PAMs cells and incubated at 37°C with 5% CO2 for 24 and 48 h. Then, 10 μL / well of CCK-8 working solution was added, and the cells were incubated at 37°C with 5% CO2 for 2 h. OD values ​​were recorded using a microplate reader. 450 Value. The results show ( Figure 9 The cell proliferation activity of the negative control group was set at 100%. The cell viability of the compound IFNs-Fc treatment group was 99.99% at 24 h and 99.59% at 48 h. This indicates that the compound IFNs-Fc has no cytotoxicity to PAMs cells, and the treatment time is not correlated with cell proliferation toxicity.

[0191] 1.3.9 Inhibition of ASFV Replication by Combined IFNs-Fc

[0192] 1.3.9.1 RT-qPCR detection of viral load

[0193] To investigate the effect of compound IFNs-Fc on ASFV replication in PAMs cells, IFNα2-Fc, IFNγ-Fc, and IFNλ3-Fc were mixed at a concentration of 1×10⁻⁶. 5 The IFNs-Fc complex was prepared by mixing U / mL. 300 μL of the complex IFNs-Fc was used to treat PAMs cells for 24 h, followed by treatment with 100 TCID50. 50 Cells were infected with ASFV for 24 and 48 hours, and cell supernatants were collected for RT-qPCR to detect viral load. Results are as follows: Figure 10 As shown, the ASFV infection results after 24 hours indicated that the average viral copy number in the combined IFNs-Fc treatment group was 2.269 × 10⁻⁶. 4 The viral copy number was lower than the average of 1.807 × 10⁻⁶ in the ASFV control group. 5 The difference between the two groups was extremely significant (p<0.0001). Figure 10 (a) Results of ASFV infection 48 hours later showed that the average viral copy number in the combined IFNs-Fc treatment group was 1.334 × 10⁻⁶. 5 The viral copy number was lower than the average of 2.094 × 10⁻⁶ in the ASFV control group. 6 The difference between the two groups was extremely significant (p<0.0001). Figure 10(b) This indicates that the compound IFNs-Fc can effectively inhibit the replication and proliferation of ASFV in PAMs cells, with inhibition rates of 87.44% and 93.63% at 24 and 48 hours, respectively.

[0194] 1.3.9.2 IFA detection of ASFV

[0195] To verify the inhibitory effect of the compound IFNs-Fc on ASFV replication in PAMs cells, IFNα2-Fc, IFNγ-Fc and IFNλ3-Fc were mixed at a concentration of 1×10⁻⁶. 5 The IFNs-Fc complex was prepared by mixing U / mL. 300 μL of the IFNs-Fc complex was used to treat PAMs cells for 24 h with 100 TCID50. 50 / well ASFV-infected cells for 48 h, cell samples were collected for IFA. Results showed ( Figure 11 ), with 100 TCID 50 After 48 hours of ASFV infection into PAMs cells, specific fluorescence targeting the ASFV structural protein p72 was detected. The IFNs-Fc combined treatment group showed only a few specific fluorescence samples targeting p72, with significantly fewer fluorescence samples compared to the ASFV group. Under white light, the ASFV group cells exhibited nuclear condensation and increased intercellular spaces. This indicates that the IFNs-Fc combined treatment effectively inhibits ASFV replication and proliferation in PAMs cells.

[0196] 1.3.10 Determination of the anti-PRRSV, PEDV, and PRV activity of the composite IFNs-Fc

[0197] The broad-spectrum antiviral activity of compound IFNs-Fc against PRRSV, PEDV, and PRV was studied. RT-qPCR and IFA were used to detect the ability of compound IFNs-Fc to inhibit the replication of PRRSV, PEDV, and PRV.

[0198] 1.3.10.1.1 RT-qPCR detection of PRRSV viral load

[0199] MARC-145 cells were treated with compound IFNs-Fc for 24 h, followed by 100 TCID50. 50 Cells were infected with PRRSV for 24 hours, and the inhibitory effect of compound IFNs-Fc on PRRSV replication was detected by RT-qPCR. The results showed that ( Figure 12 The average viral copy number in the combined IFNs-Fc treatment group was 3.59 × 10⁻⁶. 8 The viral copy number was lower than the average of 8.98 × 10⁻⁶ in the PRRSV control group. 8The difference between the two groups was extremely significant (p<0.0001). The inhibition rate of PRRSV by the compound IFNs-Fc was 60%, indicating that the compound IFNs-Fc can inhibit the replication and proliferation of PRRSV in MARC-145.

[0200] 1.3.10.2 Composite IFNs-Fc resists PEDV replication

[0201] 1.3.10.2.1 RT-qPCR detection of PEDV viral load

[0202] Vero cells were treated with compound IFNs-Fc for 24 h at 100 TCID. 50 Cells were infected with PEDV for 24 hours, and the inhibitory effect of compound IFNs-Fc on PEDV replication was detected by RT-qPCR. The results showed that ( Figure 13 The average viral copy number in the combined IFNs-Fc treatment group was 1.25 × 10⁻⁶. 6 The viral copy number was lower than the average of 2.0 × 10⁻⁶ in the PEDV control group. 7 The difference between the two groups was extremely significant (p<0.0001). The inhibition rate of PEDV by the compound IFNs-Fc was 93.73%, indicating that the compound IFNs-Fc can effectively inhibit the replication and proliferation of PEDV on Vero cells.

[0203] 1.2.10.3 Composite IFNs-Fc Anti-PRV Replication

[0204] 1.2.10.3.1 RT-qPCR detection of PRV viral load

[0205] PK15 cells were treated with compound IFNs-Fc for 24 h at 100 TCID. 50 The ability of compound IFNs-Fc to inhibit PRV replication was detected by RT-qPCR 24 h after PRV infection. Results indicate ( Figure 14 The average viral copy number in the combined IFNs-Fc treatment group was 1.63 × 10⁻⁶. 5 The viral copy number was lower than the average of 9.12 × 10⁻⁶ in the PRV control group. 5 The difference between the two groups was statistically significant (**p<0.01). The inhibition rate of PRV by the compound IFNs-Fc was 82.14%, indicating that the compound IFNs-Fc can inhibit the replication and proliferation of PRV in PK15 cells.

[0206] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A composite IFNs-Fc fusion protein, characterized in that, Including IFNγ-Fc fusion protein, IFNα2-Fc fusion protein, and IFNλ3-Fc fusion protein; The coding sequence of the IFNγ-Fc fusion protein is shown in SEQ ID No. 1; The coding sequence of the IFNα2-Fc fusion protein is shown in SEQ ID No. 2; The coding sequence of the IFNλ3-Fc fusion protein is shown in SEQ ID No.

3.

2. The composite IFNs-Fc fusion protein according to claim 1, characterized in that, The enzyme activity ratio of the IFNγ-Fc fusion protein, IFNα2-Fc fusion protein, and IFNλ3-Fc fusion protein is 1:1:

1.

3. The composite IFNs-Fc fusion protein according to claim 1, characterized in that, The coding sequence of the IFNγ-Fc fusion protein is obtained by fusing the IFNγ sequence and the Fc sequence. The Genebank sequence number of the IFNγ sequence is NM_213948.1, and the Genebank sequence number of the Fc sequence is AF150959.

1. The coding sequence of the IFNα2-Fc fusion protein is obtained by fusing the IFNα2 sequence and the Fc sequence. The Genebank sequence number of the IFNα2 sequence is MH538099.1, and the Genebank sequence number of the Fc sequence is AF150959.

1. The coding sequence of the IFNλ3-Fc fusion protein is obtained by fusing the IFNλ3 sequence and the Fc sequence. The Genebank sequence number of the IFNλ3 sequence is NM_001166490.1, and the Genebank sequence number of the Fc sequence is AF150959.

1.

4. The use of the composite IFNs-Fc fusion protein according to any one of claims 1 to 3 in the preparation of drugs for treating African swine fever.

5. The use of the composite IFNs-Fc fusion protein according to any one of claims 1 to 3 in the preparation of a drug for treating porcine reproductive and respiratory syndrome.

6. The use of the composite IFNs-Fc fusion protein according to any one of claims 1 to 3 in the preparation of a drug for treating porcine epidemic diarrhea.

7. The use of the composite IFNs-Fc fusion protein according to any one of claims 1 to 3 in the preparation of a drug for treating porcine pseudorabies.

8. A drug for treating swine diseases, characterized in that, Includes the composite IFNs-Fc fusion protein as described in any one of claims 1 to 3.

9. The medicament according to claim 8, characterized in that, The swine diseases mentioned include one or more of African swine fever, porcine reproductive and respiratory syndrome, porcine epidemic diarrhea, and pseudorabies.

10. The medicament according to claim 8, characterized in that, The enzyme activity ratio of IFNγ-Fc fusion protein, IFNα2-Fc fusion protein, and IFNλ3-Fc fusion protein in the composite IFNs-Fc fusion protein is 1:1:1.

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