Modified pig IFN (interferon)-lambda3 mutant as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a modified pig IFN (interferon)-lambda3 mutant as well as a preparation method and application thereof. The mutant comprises an amino acid sequence as shown in SEQ ID NO: 27. The preparation method of the mutant comprises the following steps: transforming a recombinant plasmid into an escherichia coli competent cell, carrying out IPTG (isopropyl-beta-d-thiogalactoside) induced expression, and purifying to obtain a mutant protein; the recombinant plasmid contains a nucleotide sequence for coding the mutant. Compared with a wild type pig III type interferon lambda3, the pig III type interferon lambda3 mutant constructed by the invention has higher antiviral activity, and can better inhibit the proliferation of PRV, PDCoV, PoRV and PRRSV in cells, so that the pig III type interferon lambda3 mutant has better clinical effects of treating and preventing PRV, PDCoV, PoRV and PRRSV infection pigs.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a modified porcine IFN-λ3 mutant, its preparation method, and its application. Background Technology

[0002] The type III interferon (IFN-λ) family includes four subtypes: IFN-λ1, IFN-λ2, IFN-λ3, and IFN-λ4. However, only IFN-λ1, IFN-λ3, and IFN-λ4 are expressed in pigs. As a key cytokine in the host's antiviral defense, IFN-λ has multiple functions, including antiviral, antitumor, and immunomodulatory effects. Its antiviral mechanism is complex, mainly including the following aspects: 1) By specifically binding to the heterodimer receptor composed of IFNLR1 and IL10Rβ, it activates the JAK-STAT signaling pathway, induces the expression of interferon-stimulated genes (ISGs), and exerts a direct antiviral effect; 2) It upregulates the expression of major histocompatibility complex (MHC) molecules, enhancing antigen presentation; 3) It promotes apoptosis of infected cells; 4) It activates innate immune effector cells such as natural killer (NK) cells; 5) It induces the production of chemokines and cytokines, recruits and activates immune cells to the site of infection, establishes an antiviral environment, and synergistically clears the virus.

[0003] Although almost all nucleated cells express IFN-λ, the distribution of its receptor IFNLR1 is relatively limited, mainly found in epithelial cells of the respiratory tract, gastrointestinal tract, reproductive tract, liver, and kidneys, as well as in specific immune cell subsets (such as B cells, T cells, NK cells, and monocytes). Therefore, the primary target cells for IFN-λ are epithelial cells. Due to the limited distribution of the IFN-λ receptor IFNLR1, the risk of IFN-λ inducing systemic inflammatory responses (such as fever and leukopenia, common side effects of type I interferon) is significantly reduced, making it safer for antiviral therapy. Furthermore, compared to type I interferon, the inherently low immunogenicity of IFN-λ effectively reduces the likelihood of antibody production in the body. These characteristics give IFN-λ great potential as a long-acting antiviral therapeutic agent.

[0004] Recent studies have shown that among the three IFN-λ types in pigs (pIFN-λ1, pIFN-λ3, and pIFN-λ4), pIFN-λ3 exhibits the strongest antiviral activity. It shows some inhibitory activity against several important viruses that seriously threaten the pig industry, such as porcine reproductive and respiratory syndrome virus (PRRSV), porcine epidemic diarrhea virus (PEDV), classical swine fever virus (CSFV), and pseudorabies virus (PRV), but its efficacy still needs improvement. The unsatisfactory antiviral effect of pIFN-λ3 may be due to the low affinity of wild-type pIFN-λ3 for its receptor IFNLR1. By resolving the structure of the pIFN-λ3-receptor complex (IFNLR1 / IL10Rβ), identifying key amino acid sites affecting binding affinity, and performing site-directed mutagenesis, it is hoped that the binding affinity with IFNLR1 can be enhanced, thereby improving the antiviral effect of pIFN-λ3. Summary of the Invention

[0005] To enhance the antiviral effect of pIFN-λ3, this invention provides a modified porcine IFN-λ3 mutant, its preparation method, and its application.

[0006] This invention is achieved through the following technical solution:

[0007] In a first aspect, the present invention provides a modified porcine IFN-λ3 mutant comprising the amino acid sequence shown in SEQ ID NO:27: ARGCHLAQFKSLSPRALQAFKRAKDAFEESLLEDWNCSSRIFPRSRDLKQLQVWERPVALEAEVALTLSVLESLANSSLHSSLDQPLHTLRHIHAQLQACVPAQPMAGPRPRGRLHRWLHRLQEAQKKEPQSCLEASVMFNLFRLLARDLKCVASGDLCE. Compared to the wild-type porcine IFN-λ3 protein, it exhibits the following mutations: Q15R, G72E, H117R, T147A, and V160E. The mutant possesses higher antiviral activity compared to porcine type III interferon λ3. Specifically, using vesicular stomatitis virus as a model virus, the antiviral activity units of the porcine IFN-λ3 mutant were measured on MDBK cells. The results showed that the antiviral activity units of the mutant were significantly higher than those of wild-type porcine IFN-λ3. In a further embodiment, the amino acid sequence of the mutant is shown in SEQ ID NO:6.

[0008] Secondly, the present invention provides a nucleic acid encoding the above-mentioned mutant. Further, the nucleic acid sequence encoding the mutant is shown in SEQ ID NO:14.

[0009] Thirdly, the present invention provides biological materials containing the above-mentioned nucleic acids, wherein the biological materials are expression cassettes, transposons, plasmid vectors, viral vectors or host cells.

[0010] Fourthly, the present invention provides a method for preparing a modified porcine type III interferon λ3 mutant, comprising the following steps:

[0011] The recombinant plasmid was transformed into competent *E. coli* cells, expressed by IPTG, and purified to obtain the mutant protein. The recombinant plasmid contains the nucleic acid sequence encoding the mutant. Fifthly, this invention provides the application of a porcine type III interferon λ3 mutant in the evaluation of viral infection models. This recombinant protein exhibits significantly enhanced antiviral activity in different viral models. The viruses include VSV, PRV, PDCoV, PoRV, and PRRSV.

[0012] Sixthly, this invention provides the application of porcine type III interferon λ3 mutants in the preparation of antiviral drugs. The viruses include VSV, PRV, PDCoV, PoRV, and PRRSV.

[0013] This invention investigated the effects of two different concentrations of wild-type porcine type III interferon λ3 and a mutant porcine type III interferon λ3 on the proliferation of PRV, PDCoV, PoRV, and PRRSV at the cellular level. The results showed that both wild-type porcine type III interferon λ3 and the mutant porcine type III interferon λ3 inhibited the proliferation of the four viruses in a dose-dependent manner, and the inhibitory effect of the mutant porcine type III interferon λ3 was significantly stronger than that of wild-type porcine type III interferon λ3.

[0014] This invention evaluated the effects of wild-type porcine type III interferon λ3 and porcine type III interferon λ3 mutant on PRV proliferation in mice. The results showed that both wild-type porcine type III interferon λ3 and porcine type III interferon λ3 mutant could effectively inhibit PRV proliferation in mice, and the effect of porcine type III interferon λ3 mutant was significantly better than that of wild-type porcine type III interferon λ3.

[0015] The beneficial effects of this invention are as follows:

[0016] The porcine type III interferon λ3 mutant constructed in this invention has higher antiviral activity than wild-type porcine type III interferon λ3, and can better inhibit the proliferation of PRV, PDCoV, PoRV and PRRSV in cells. Therefore, it has better clinical efficacy in treating and preventing PRV, PDCoV, PoRV and PRRSV infection in pigs. Attached Figure Description

[0017] Figure 1The diagram shows the amino acid sequence comparison of pIFN-λ3 and hIFN-λ3 (A) and the amino acid site mutation diagram of the pIFN-λ3 mutant (B). In Figure B, the first row represents the amino acid sites of wild-type pIFN-λ3, the second row represents the mutation sites of pIFN-λ3-4, and the third to seventh rows represent the mutation sites of pIFN-λ3-D, pIFN-λ3-E, pIFN-λ3-H, pIFN-λ3-R, and pIFN-λ3-K, respectively.

[0018] Figure 2 SDS-PAGE electrophoresis images of wild-type and mutant recombinant pIFN-λ3 proteins. M: Protein Marker; I: Expression bacteria after induction; SP: Precipitate after sonication and centrifugation; SS: Supernatant after sonication and centrifugation.

[0019] Figure 3 SDS-PAGE electrophoresis (A) and Western blotting (B) images of purified pIFN-λ3 wild-type and mutant recombinant proteins. M: Protein Marker; WT: pIFN-λ3-WT; 4: pIFN-λ3-4; D: pIFN-λ3-D; E: pIFN-λ3-E; H: pIFN-λ3-H; K: pIFN-λ3-K; R: pIFN-λ3-R.

[0020] Figure 4 : This is a graph showing the determination of antiviral activity units of wild-type and mutant recombinant pIFN-λ3 protein. WT: pIFN-λ3-WT; 4: pIFN-λ3-4; D: pIFN-λ3-D; H: pIFN-λ3-H; K: pIFN-λ3-K; R: pIFN-λ3-R; E: pIFN-λ3-E. Figure 5 : for qPCR or RT-qPCR and TCID 50 Graphs showing the inhibitory effects of wild-type pIFN-λ3 and the mutant pIFN-λ3-E on viral replication. A: PRV; B: PDCoV; C: PoRV; D: PRRSV. Untreated: Virus control; L: 100 ng / ml recombinant protein; H: 1000 ng / ml recombinant protein; WT: pIFN-λ3-WT; E: pIFN-λ3-E.

[0021] Figure 6 : Gross lesion images of mouse organs and tissues. MOCK: Blank control group mice; PRV: Dead control group mice; WT: Surviving mice in the pIFN-λ3-WT treatment group; E: Surviving mice in the pIFN-λ3-E treatment group; Brain, Lung, Liver, and Kidney represent the brain, lung, liver, and kidney, respectively.

[0022] Figure 7 Figure 1: Results of qPCR detection of viral load in mouse blood. PRV: Virus control group; WT: pIFN-λ3-WT treatment group; E: pIFN-λ3-E treatment group. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of the embodiments of this invention will be clearly described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0024] PRV CH / HNSMX / 2012 strain, PDCoV DHeB1 strain, PoRV RShanD1 strain, and PRRSV WuH3 strain were all isolated, identified, and preserved by the National Key Laboratory for Discovery and Utilization of Agricultural Microbial Resources, Huazhong Agricultural University. Vesicular stomatitis virus (VSV) was kindly donated by Researcher Bu Zhigao of the Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0025] MDBK, PK-15, LLC-PK1, IPEC-J2, and Marc-145 cells were all purchased from the China Center for Type Culture Collection.

[0026] SPF-grade female Babl / C mice were purchased from Hubei Provincial Center for Disease Control and Prevention, ethical code HZAUMO-2025-0165.

[0027] Example 1: Amino acid sequence alignment of pIFN-λ3 and hIFN-λ3 and design of pIFN-λ3 mutants

[0028] The amino acid sequences of porcine IFN-λ3 (pIFN-λ3, GeneID: NM_001166490.1; amino acid sequence as shown in SEQ ID NO:1) and human IFN-λ3 (hIFN-λ3, GeneID: NM_001346937; amino acid sequence as shown in SEQ ID NO:2) were compared using Mega X software. The results are as follows: Figure 1As shown, the amino acid sequence identity between the two is 74.8%, the similarity is 82.8%, and the key functional domains are highly conserved. Studies have confirmed that the antiviral activity of the hIFN-λ3 mutant H11 (mutation sites are Q15R, E73D, H120R, T150A and V163E, amino acid sequence as shown in SEQ ID NO:10) is significantly improved (Mendoza JL, Schneider WM, Hoffmann HH, Vercauteren K, Jude KM, Xiong A, Moraga I, Horton TM, Glenn JS, de Jong YP, Rice CM, Garcia KC. The IFN-λ-IFN-λR1-IL-10Rβ Complex Reveals Structural Features Underlying Type III IFN Functional Plasticity. Immunity. 2017, 46(3):379-392.). Comparative analysis of the amino acid sequences of pIFN-λ3 and hIFN-λ3 revealed five sites corresponding to amino acid positions 15, 73, 120, 150, and 163 of hIFN-λ3 (the corresponding amino acid sites are marked with bold numbers, e.g., ...). Figure 1 In (as shown in A), pIFN-λ3 differs only in the second amino acid site, which is glycine (G72).

[0029] This embodiment designed and constructed 6 mutants of pIFN-λ3 ( Figure 1 B): (1) A mutant containing four conserved mutation sites (Q15R, H117R, T147A, and V160E) is named pIFN-λ3-4; (2) Based on the mutations at the four sites, G72 is mutated into five polar charged amino acids (G72D, G72E, G72H, G72R, and G72K) to obtain five mutants with five 5-point mutations, named pIFN-λ3-D, pIFN-λ3-E, pIFN-λ3-H, pIFN-λ3-R, and pIFN-λ3-K, respectively. Among them, the 15th, 72nd, 117th, 147th, and 160th sites are... Figure 1The position of each site in the pIFN-λ3 sequence (SEQ ID NO: 26) is used to denote the pIFN-λ3. Both the wild-type and mutant pIFN-λ3 have the first 35 amino acids (including the signal peptide and a strongly hydrophobic sequence) deleted from the N-terminus, and a 6×His tag (HHHHHH) introduced at the N-terminus to facilitate its expression in E. coli and subsequent detection.

[0030] Genes encoding wild-type and mutant pIFN-λ3 were synthesized and inserted into the pET-15b(+) vector to construct prokaryotic expression plasmids. Sequencing confirmed that all seven recombinant plasmids were constructed correctly. The nucleotide sequences of wild-type and mutant pIFN-λ3 (pIFN-λ3-4, pIFN-λ3-D, pIFN-λ3-E, pIFN-λ3-H, pIFN-λ3-R, and pIFN-λ3-K) are shown in SEQ ID NO:11-17, and the corresponding amino acid sequences are shown in SEQ ID NO:3-9.

[0031] Example 2: Expression and expression pattern analysis of wild-type and mutant recombinant pIFN-λ3 protein in Escherichia coli

[0032] Seven recombinant plasmids and pET-15b(+) were transformed into E. coli BL21(DE3) competent cells. The cells were plated on LB agar plates containing 100 mg / L ampicillin (Amp) and incubated upside down at 37°C for 12–14 h until single colonies formed. A single colony was picked and inoculated into 1 ml of LB liquid medium containing 100 mg / L Amp, and cultured at 37°C with shaking at 200 rpm for 12 h to prepare a seed culture. 1% of the seed culture was transferred to 5 ml of LB liquid medium containing 100 mg / L Amp and cultured at 37°C with shaking at 200 rpm until the logarithmic growth phase (OD50) was reached. 600 =0.6), then add IPTG to a final concentration of 1 mM for induction (37℃, 200 rpm). After 12 h of induction, collect the bacterial culture and centrifuge at 4℃, 12000 rpm for 5 min. Resuspend the bacterial pellet in PBS, sonicate on ice, and centrifuge at 4℃, 12000 rpm for 5 min after disruption. Take the supernatant and pellet for SDS-PAGE (12.5% ​​separating gel) analysis.

[0033] After electrophoresis, the sample was stained with Coomassie Brilliant Blue, and the results were as follows: Figure 2 As shown, both wild-type and mutant recombinant plasmids of pIFN-λ3 showed a specific band at approximately 18 kDa in E. coli, consistent with the theoretical size of pIFN-λ3, and the band mainly existed in the form of inclusion bodies. However, no band of the corresponding size was observed in bacteria transformed with the empty vector. This indicates that both wild-type and mutant recombinant proteins of pIFN-λ3 were expressed in E. coli and mainly existed in the form of inclusion bodies.

[0034] Example 3: Large-scale expression, purification, and validation of pIFN-λ3 wild-type and mutant recombinant proteins

[0035] 1. High expression of pIFN-λ3 wild-type and mutant recombinant protein

[0036] Add 10 μl of seed culture expressing pIFN-λ3 wild-type or mutant to 10 ml of LB liquid medium containing 100 mg / L Amp, mix well, and incubate at 37°C with shaking at 200 rpm for 12 h. Then add it to 1000 ml of LB liquid medium containing 100 mg / L Amp, mix well, and incubate at 37°C with shaking at 200 rpm until the bacterial culture reaches OD. 600 =0.6, add IPTG to a final concentration of 1mM for induction, and incubate at 37℃ for 12h. Collect the bacterial culture, centrifuge at 12000rpm for 5min at 4℃, and collect the bacterial cells. Resuspend the bacterial cells in 100ml PBS, and perform high-pressure disruption 5 times at 800bar, centrifuge at 12000rpm for 10min at 4℃, discard the supernatant, and collect the precipitate. Wash the precipitate twice with washing buffer (1% v / v Triton X-100, 5mM EDTA, 20mM Tris-HCl, 100mM NaCl, pH=8.5), centrifuge at 12000rpm for 10min at 4℃, discard the supernatant, and collect the precipitate, which is the inclusion body. Add 10ml of dissolving buffer (8M urea, 50mM Tris-HCl, 250mM NaCl, pH=8.5) to the collected inclusion bodies, and dissolve the inclusion bodies by shaking at 160rpm for 2h at 37℃. Centrifuge at 4℃ and 15000 rpm for 30 min, collect the supernatant and filter it through a 0.45 μm filter membrane. Collect the filtrate, which is the protein expressed in large quantities.

[0037] 2. Purification of wild-type and mutant recombinant pIFN-λ3 protein

[0038] The gravity column was equilibrated with 2 column volumes of dissolving buffer (8M urea, 50mM Tris-HCl, 250mM NaCl, pH=8.5). A large amount of expressed protein solution was added to the column. After the protein flowed through the column, the gravity column was washed with 3-4 column volumes of washing buffer (50mM PBS, 25mM imidazole, pH=7.4) to remove impurities. The column was then washed with elution buffer (50mM PBS, 250mM imidazole, pH=7.4). The eluted protein was collected, and its concentration was determined. The protein was then diluted with elution buffer to 1 mg / ml. The protein was diluted to 0.33 mg / ml with diluent (6M urea, 50mM PBS, pH=7.4) and placed in a dialysis bag. Dialysis was then performed sequentially with refolding solution 1 (4M urea, 50mM PBS, 0.5M L-arginine, 0.5% v / v Triton X-100, 2mM reduced glutathione, 0.5mM oxidized glutathione, pH=7.4), refolding solution 2 (same composition and concentration as refolding solution 1 except for 2M urea, pH=7.4), and refolding solution 3 (same composition and concentration as refolding solution 1 except for 1M urea, pH=7.4). Each refolding solution was used for 8 hours of dialysis. Finally, dialysis was performed three times with PBS (pH=7.4), each time for 8 hours.

[0039] After dialysis, the protein solution was collected and concentrated to 1 mg / ml using an ultrafiltration tube. It was then aliquoted and stored below -70°C. Simultaneously, samples were taken for SDS-PAGE analysis. The results showed that the purity of both the wild-type and mutant recombinant pIFN-λ3 proteins was above 95%. Figure 3 A). Further Western blot analysis of the expressed recombinant protein using an Anti-His-tagged antibody (1:5000) as the primary antibody showed correctly sized specific reaction bands in all cases. Figure 3 B) indicates that highly pure and correctly expressed pIFN-λ3 wild-type and mutant recombinant proteins were obtained.

[0040] Example 4: Determination of antiviral activity units of pIFN-λ3 wild-type and mutant recombinant protein

[0041] MDBK cells that had grown into a monolayer were digested and prepared into a cell suspension. After counting, the suspension was adjusted to 2 × 10⁻⁶ cells. 5 cells / ml, seeded into 96-well plates, 100 μl per well (2 × 10⁻⁶ cells / ml). 4Cells / well). When cells reached 90% confluence, the purified pIFN-λ3 wild-type and mutant recombinant protein from Example 3 was diluted to 100 ng / ml using DMEM containing 2% FBS, and then serially diluted 2-fold to 0.2 ng / ml (50 ng / ml, 25 ng / ml, 12.5 ng / ml, 6.3 ng / ml, 3.2 ng / ml, 1.6 ng / ml, 0.8 ng / ml, 0.4 ng / ml, 0.2 ng / ml). The cell culture medium in the 96-well plate was aspirated, and the cells were washed twice with sterile PBS. Interferon at each dilution was added to the cell plate, 100 μl per well for each dilution, and incubated at 37°C for 24 h. The liquid in the wells was aspirated, and the cells were washed twice with sterile PBS. Interferon diluted to 100 TCID⁻¹ was then inoculated into the wells. 50 100 μl of vesicular stomatitis virus (VSV) was injected into each well, with a virus control (VSV inoculated without interferon incubation) and a normal cell control. Cytopathic effects were observed daily after virus inoculation. When all cells in the virus control wells showed cytopathic effects and all cells in the normal cell control wells remained normal, the number of CPE wells was recorded, and the antiviral activity units were calculated using the Reed-Muench method. Results showed that the mutant pIFN-λ3-E had an antiviral activity unit of 4.35 × 10⁻⁶ units. 7 The concentration of U / mg was significantly higher than that of pIFN-λ3-WT (6.75×10). 6 The antiviral activity units (U / mg) of the mutant were 6.4 times that of pIFN-λ3-WT, while the antiviral activity units of the other five mutants were not significantly different from those of pIFN-λ3-WT. Figure 4 ).

[0042] Example 5: Effects of pIFN-λ3-WT and pIFN-λ3-E on the proliferation of PRV, PDCoV, PoRV, and PRRSV in cells

[0043] PK-15, LLC-PK1, IPEC-J2, and Marc-145 cells that had grown into monolayers were digested and prepared into cell suspensions. After counting, the suspensions were adjusted to 2 × 10⁻⁶ cells / mL. 5 Cells / ml, seeded into 24-well plates, 500 μl (1×10⁻⁶) per well. 5(cells / well). When the cells reached 90% confluence, the purified pIFN-λ3 wild-type and mutant recombinant proteins from Example 3 were diluted to 1000 ng / ml and 100 ng / ml respectively in DMEM medium containing 1% FBS. The solutions were added to cell wells, with 500 μl of each of the four cell types seeded into three wells at each dilution. The cells were incubated at 37°C for 24 h. The liquid in the wells was aspirated, and the cells were washed twice with sterile PBS. PRVCH / HNSMX / 2012 strain was seeded into PK-15 cells at 0.1 MOI, PDCoV DHeB1 strain into LLC-PK1 cells at 0.01 MOI, PoRV RShanD1 strain into IPEC-J2 cells at 0.01 MOI, and PRRSV WuH3 strain into Marc-145 cells at 0.1 MOI. A virus control (without interferon incubation, only virus seeding) and a normal cell control were also set up. Cytopathic effects were observed daily after virus inoculation. Once complete cytopathic effects were observed in the control wells, samples were collected, subjected to two freeze-thaw cycles at -20°C, and centrifuged at 12,000 rpm for 5 minutes at 4°C. The supernatant was collected, and TCID was determined. 50 Simultaneously, 200 μl of sample was taken for DNA or RNA extraction, and the viral load was detected by qPCR or RT-qPCR using the primers shown in Table 1. TCID 50 Both qPCR and RT-qPCR results showed that pIFN-λ3-WT and pIFN-λ3-E effectively inhibited the proliferation of PRV, PDCoV, PoRV, and PRRSV in cells in a dose-dependent manner, but the inhibitory effect of pIFN-λ3-E was significantly stronger than that of pIFN-λ3-WT. Figure 5 ).

[0044] Table 1. Primer sequences

[0045] Primer name Serial Number PRV-F SEQ ID NO: 18 PRV-R SEQ ID NO: 19 PDCoV-F SEQ ID NO: 20 PDCoV-R SEQ ID NO: 21 PoRV-F SEQ ID NO: 22 PoRV-R SEQ ID NO: 23 PRRSV-F SEQ ID NO: 24 PRRSV-R SEQ ID NO: 25

[0046] Example 6: Effects of pIFN-λ3-WT and pIFN-λ3-E on PRV proliferation in mice

[0047] Thirty-two 6-8 week old SPF-grade female Babl / C mice were randomly divided into four groups (n=8 per group): pIFN-λ3-WT treatment group, pIFN-λ3-E treatment group, virus control group, and blank control group. Mice in the pIFN-λ3-WT and pIFN-λ3-E treatment groups were intraperitoneally injected with pIFN-λ3-WT or pIFN-λ3-E, respectively, at a dose of 10 μg (100 μl). Twenty-four hours after inoculation, each mouse was injected bilaterally with 200 TCID2 via the paw pads of its hind limbs. 50(100 μl) of PRV CH / HNSMX / 2012 strain was administered. The virus control group was inoculated with an equal volume of PRV, and the blank control group was inoculated with an equal volume of DMEM. Specific grouping details are shown in Table 2. Three mice from each group were sacrificed on day 6 post-infection; the remaining mice were observed until day 12 post-infection. Mouse survival rates were recorded, and necropsies were performed on the deceased and surviving mice. Before each sacrifice, blood was collected via orbital sampling into sodium citrate anticoagulant tubes. After DNA extraction, the viral load in the blood was detected by qPCR using the primers shown in Table 1.

[0048] Table 2. Grouping of Animal Experiments

[0049]

[0050] Survival rate statistics showed that the survival rate of the virus control group was 20% (4 / 5 mice died), the survival rate of the pIFN-λ3-WT treatment group was 80% (1 / 5 mice died), and the survival rate of mice in the pIFN-λ3-E treatment group and the blank control group was 100%.

[0051] Autopsy results showed that the dead mice in the virus control group exhibited typical viral pathological features: meningeal vasodilation with significant congestion, multifocal hemorrhage on the surface of the brain parenchyma; widespread dark red hemorrhagic consolidation in the lungs; diffuse pinpoint hemorrhages and congestion under the liver capsule; and softening and loss of elasticity of the renal parenchyma with a dull capsule. In contrast, no characteristic pathological damage was observed in the surviving mice in the blank control group, the pIFN-λ3-WT treatment group, and the pIFN-λ3-E treatment group. Figure 6 As shown.

[0052] Viral load testing results showed that on days 6 and 12 post-PRV infection, the viral load in the blood of mice in both the pIFN-λ3-WT and pIFN-λ3-E treatment groups was significantly lower than that in the virus control group, and the viral load in the pIFN-λ3-E treatment group was significantly lower than that in the pIFN-λ3-WT treatment group. Figure 7 ).

[0053] The results above indicate that both pIFN-λ3-WT and pIFN-λ3-E can effectively inhibit the proliferation of PRV in mice, and the antiviral effect of pIFN-λ3-E is better than that of pIFN-λ3-WT.

[0054] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A modified porcine IFN-λ3 mutant, characterized in that, The amino acid sequence of the mutant is shown in SEQ ID NO:

27.

2. The modified porcine IFN-λ3 mutant according to claim 1, characterized in that, The amino acid sequence of the mutant is shown in SEQ ID NO:

6.

3. The nucleic acid encoding the mutant of claim 1 or 2.

4. The nucleic acid according to claim 3, characterized in that, The sequence of the nucleic acid is shown in SEQ ID NO:

14.

5. A biomaterial containing the nucleic acid of claim 3 or 4, characterized in that, The biological material is an expression cassette, transposon, plasmid vector, viral vector, or host cell.

6. The method for preparing the mutant according to claim 1 or 2, characterized in that, include: The recombinant plasmid was transformed into competent E. coli cells, expressed by IPTG, and purified to obtain the mutant protein; the recombinant plasmid contained the nucleic acid sequence encoding the mutant.

7. The application of the mutant of claim 1 or 2, the nucleic acid of claim 3 or 4, and the biological material of claim 5 in the evaluation of viral infection models.

8. The application according to claim 7, characterized in that, The viruses mentioned include VSV, PRV, PDCoV, PoRV, and PRRSV.

9. The use of the mutant of claim 1 or 2, the nucleic acid of claim 3 or 4, and the biomaterial of claim 5 in the preparation of antiviral drugs.

10. The application according to claim 9, characterized in that, The viruses mentioned include VSV, PRV, PDCoV, PoRV, and PRRSV.

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