Application of chicken interferon ChIFN-υ in antiviral

Abstract

The application discloses application of chicken type IV interferon ChIFN-v in antiviral, and belongs to the technical field of biology.The amino acid sequence of the chicken type IV interferon ChIFN-v is shown as SEQ ID NO.4.The application provides evidence of existence of the chicken type IV interferon ChIFN-v, and proves that the chicken type IV interferon ChIFN-v has strong antiviral activity through in-vitro cell protection experiments and animal infection treatment experiments.The application also verifies the synergistic effect of the ChIFN-v protein and the chicken type I interferon ChIFN-alpha in antiviral, provides new drug resources and solid data support for prevention and control of poultry diseases, has a clear commercial transformation path, and has remarkable social and economic benefits and wide market prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to the application of chicken type IV interferon ChIFN-υ in antiviral. BACKGROUND

[0002] Compared with mammals, the IFN system of chicken has fundamental differences, and multiple key molecules such as RIG-I, IRF3 and IRF9 are missing in its signaling pathway. This unique immune background suggests that a unique immune mechanism may have evolved in chickens. Currently, the IFNs identified in chicken genome include type I, type II and type III, which constitute the material basis for existing chicken antiviral technology and application. However, the antiviral spectrum and activity of these IFN molecules disclosed in the prior art are limited in different infection scenarios, and it is still difficult to fully meet the industrial demand for efficient disease prevention and control. Therefore, exploring IFN with novel structure and more optimal biological activity is a clear and unsolved technical demand in the field.

[0003] The direction of this technical demand is more clear due to the recent discovery of IFN family in lower vertebrates. Researchers have identified a class of type IV IFN (IFN-υ) that is independent of the classical type in sequence and gene structure in species such as zebrafish and African clawed frog, and confirmed its antiviral function. Although these findings reveal the existence of type IV IFN in vertebrates, the prior art strictly limits it to specific species such as fish and amphibians. Therefore, in the key avian species of chicken, whether type IV IFN really exists, what is its specific gene and amino acid sequence, and whether it can be prepared into an active protein and applied in the field of antiviral, are still the core technical gaps in the field. The direct consequence of this gap is that the technical personnel in the field cannot know and utilize this potential immune molecule resource, neither can they evaluate whether it has stronger or broader spectrum of antiviral activity than the existing IFN, nor can they develop a new generation of antiviral drugs, immune adjuvants or cultivate avian strains with strong innate resistance through genetic engineering means, so that the green prevention and control means for avian diseases are facing a bottleneck. SUMMARY

[0004] The purpose of the present application is to provide the application of chicken type IV interferon ChIFN-υ in antiviral, in order to solve the problems existing in the prior art. The present application provides evidence for the existence of chicken type IV interferon ChIFN-υ, and proves its strong antiviral activity through in vitro cell protection experiment and animal infection treatment experiment. The present application also verifies the synergistic effect of ChIFN-υ protein and type I interferon ChIFN-α in antiviral, providing new drug resources and solid data support for the prevention and control of avian diseases, with clear commercial transformation path, significant social and economic benefits and broad market prospect.

[0005] To achieve the above object, the present application provides the following scheme:

[0006] The present application provides application of chicken type IV interferon ChIFN-υ in preparation of medicine for preventing and / or treating viral infection, wherein the amino acid sequence of the chicken type IV interferon ChIFN-υ is shown as SEQ ID NO. 4.

[0007] The present application also provides application of substance expressing chicken type IV interferon ChIFN-υ in preparation of medicine for preventing and / or treating viral infection, wherein the amino acid sequence of the chicken type IV interferon ChIFN-υ is shown as SEQ ID NO. 4.

[0008] The substance expressing chicken type IV interferon ChIFN-υ includes a recombinant vector or a recombinant microorganism containing a coding gene of the chicken type IV interferon ChIFN-υ.

[0009] Further, the nucleotide sequence of the coding gene of the chicken type IV interferon ChIFN-υ is shown as SEQ ID NO. 3.

[0010] The present application also provides application of chicken type IV interferon ChIFN-υ combined with chicken type I interferon ChIFN-α in preparation of medicine for preventing and / or treating viral infection, wherein the amino acid sequence of the chicken type IV interferon ChIFN-υ is shown as SEQ ID NO. 4.

[0011] Optionally, the virus includes vesicular stomatitis virus and H9N2 subtype avian influenza virus.

[0012] The present application also provides medicine for preventing and / or treating viral infection, wherein the medicine takes chicken type IV interferon ChIFN-υ as effective component, or takes combination of chicken type IV interferon ChIFN-υ and chicken type I interferon ChIFN-α as effective component.

[0013] The amino acid sequence of the chicken type IV interferon ChIFN-υ is shown as SEQ ID NO. 4.

[0014] Optionally, the virus includes vesicular stomatitis virus and H9N2 subtype avian influenza virus.

[0015] The present application also provides application of substance knocking out coding gene of chicken type IV interferon ChIFN-υ in any one of the following:

[0016] A1. preparation of product for improving sensitivity of cell or animal to virus;

[0017] A2. preparation of viral infection model;

[0018] A3. Screening antiviral drugs;

[0019] The nucleotide sequence of the coding gene of the chicken type IV interferon ChIFN-υ is shown as SEQ ID NO. 3.

[0020] Optionally, the virus includes vesicular stomatitis virus and H9N2 subtype avian influenza virus.

[0021] Optionally, the substance for knocking out the coding gene of the chicken type IV interferon ChIFN-υ includes sgRNA with the coding gene of the chicken type IV interferon ChIFN-υ as a target, or a substance for knocking down the expression of the coding gene of the chicken type IV interferon ChIFN-υ, or an inhibitor of the coding gene of the chicken type IV interferon ChIFN-υ.

[0022] The present application discloses the following technical effects:

[0023] The present application provides evidence for the existence of chicken type IV interferon ChIFN-υ, fills the dual blank of field cognition and material basis, fundamentally breaks through the traditional cognition that the chicken IFN system is only composed of types I, II and III, adds a new member to the IFN family of birds and even vertebrates, and provides the most basic material prerequisite for subsequent theoretical research and application development.

[0024] The present application successfully constructs a complete preparation process including "inclusion body denaturation-arginine assisted renaturation-nickel column affinity purification" for the chicken type IV interferon ChIFN-υ, and can stably and repeatedly prepare ChIFN-υ protein with correct natural conformation. The successful establishment of the process provides a key and reliable material preparation guarantee for ChIFN-υ from gene sequence to functional research and practical application.

[0025] Through in vitro cell protection experiments and animal infection treatment experiments, it is confirmed that the ChIFN-υ protein has strong antiviral activity, and the titer of the ChIFN-υ protein against vesicular stomatitis virus (VSV) is as high as 1.17*10 4 IU / mg, and the titer of the ChIFN-υ protein against H9N2 subtype avian influenza virus (H9N2 subtype AIV) is as high as 4.68*10 4 IU / mg. The present application elucidates the mechanism of action of ChIFN-υ protein from the level of signal activation, provides theoretical guidance and scientific basis for its in-depth application. In addition, the present application also verifies the synergistic effect of ChIFN-υ protein and type I interferon ChIFN-α in antiviral aspect, provides new drug resources and solid data support for the prevention and control of avian diseases, has a clear commercial transformation path, has significant social and economic benefits, and has a broad market prospect. BRIEF DESCRIPTION OF DRAWINGS

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 The image shows the preliminary prediction and identification results of the ChIFN-υ gene;

[0028] Figure 2 The image shows the identification results of the ChIFN-υ gene amplification products; lanes 1-6 represent the amplification results of samples from different sources, lanes 1-2 are all spleen tissue, lanes 3-4 are all tracheal tissue, and lanes 5-6 are all DF-1 cells.

[0029] Figure 3 Phylogenetic tree diagram of the ChIFN-υ gene;

[0030] Figure 4 This is a tissue expression map of the ChIFN-υ gene in healthy chickens; where A represents a one-day-old healthy chicken and B represents a four-week-old healthy chicken.

[0031] Figure 5 The images show the Western Blot results of the ChIFN-υ recombinant protein; lane 1 shows the results of the first batch of ChIFN-υ recombinant protein; lane 2 shows the results of the second batch of ChIFN-υ recombinant protein.

[0032] Figure 6 Figure showing the results of the study on the activation of the downstream ISRE promoter by the ChIFN-υ recombinant protein;

[0033] Figure 7 Cytopathic effects of ChIFN-υ recombinant protein at different dilutions against VSV are shown in the figure; where A represents ChIFN-υ recombinant protein 4. 5 Cytopathic effects at 100-fold dilution; B represents recombinant ChIFN-γ protein 4. 6 Cytopathic effects at 100-fold dilution; C represents ChIFN-υ recombinant protein 4. 7 Cytopathic effect at 4-fold dilution; D = 4 8 Cytopathic effects at 100-fold dilution; E is the negative control; F is the positive control; scale bar is 100 μm.

[0034] Figure 8 Figure 4 shows the cytopathic effect of ChIFN-υ recombinant protein at different dilutions against H9N2 subtype AIV; where A represents ChIFN-υ recombinant protein 4.5 Cytopathic effects at 100-fold dilution; B represents recombinant ChIFN-γ protein 4. 6 Cytopathic effects at 100-fold dilution; C represents ChIFN-υ recombinant protein 4. 7 Cytopathic effect at 4-fold dilution; D = 4 8 Cytopathic effects at 100-fold dilution; E is the negative control; F is the positive control; scale bar is 100 μm.

[0035] Figure 9 Sequencing results for ChIFN-υ gene knockout cell lines (ChIFN-υ-KO1 and ChIFN-υ-KO2) and wild-type cells;

[0036] Figure 10 The results of viral titer detection in ChIFN-υ gene knockout cell lines (ChIFN-υ-KO1 and ChIFN-υ-KO2) and wild-type cells (ChIFN-υ-wt) are shown in the figure.

[0037] Figure 11 Figure 1 shows the in vivo antiviral test results of the ChIFN-υ recombinant protein; where A represents the titer of H9N2 subtype AIV virus in chicken lung tissue; B represents the titer of H9N2 subtype AIV virus in chicken tracheal tissue; C represents the titer of H9N2 subtype AIV virus in chicken pharyngeal swabs; and D represents the titer of H9N2 subtype AIV virus in chicken cloacal swabs. Detailed Implementation

[0038] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0039] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0040] All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains unless otherwise specifically defined herein. Although preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe in full the methods and / or materials which are described herein. In case of conflict between the content of the specification and that of any document incorporated herein by reference, the content of the specification prevails.

[0041] Many modifications and variations of this application can be made in the light of the above teachings without departing from the spirit or scope of the application. Other implementations of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given are exemplary only.

[0042] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean including, but not limited to.

[0043] The experimental methods in the following examples are all routine methods unless otherwise specified. The instruments and equipment used in the following examples are all routine laboratory instruments and equipment unless otherwise specified. The test materials used in the following examples are all purchased from routine biochemical reagent stores unless otherwise specified.

[0044] Example 1 Identification, cloning and bioinformatics analysis of ChIFN-υ gene

[0045] 1. Gene identification and sequence acquisition

[0046] Bioinformatics analysis: Based on the reported gene characteristics of IFN-υ, homologous search was performed on the chicken genome database (assembly version GRCg6a) using the BLASTP and TBLASTN programs of NCBI. At the same time, the specific region of chicken chromosome 2 was analyzed for potential exon-intron structure using gene prediction software such as FGENESH for de novo gene prediction.

[0047] Candidate gene determination: In a previously unannotated region of chicken chromosome 2, a candidate gene with a typical signal peptide and a conserved domain of the IFN family was identified, which was named ChIFN-υ (see Figure 1 ).

[0048] 2. Gene cloning

[0049] Template preparation: Spleen and trachea tissues of SPF chickens infected with H9N2 subtype AIV and DF-1 cells were collected, and total RNA was extracted using the TRIzol method.

[0050] Reverse transcription and amplification: Genomic DNA removal and cDNA first strand synthesis were performed using PrimeScript RT reagent Kit. Specific primers were designed to contain the complete open reading frame. PCR amplification was performed using high fidelity DNA polymerase with the following reaction conditions: 98°C pre-denaturation for 2 min; 98°C for 10 s, 60°C for 30 s, 72°C for 1 min, for 35 cycles; 72°C final extension for 5 min.

[0051] The specific primer sequences are as follows:

[0052] Upstream primer: 5'-GAATTCATGTATTGCCCCAGAAGCTT-3' (EcoRI site was introduced); SEQ ID NO. 1;

[0053] Downstream primer: 5'-CTCGAGATGTGTTGATCTTCTCCTGA-3' (XhoI site was introduced); SEQ ID NO. 2.

[0054] The amplification results are shown in Figure 1. A single band with the predicted size was successfully amplified in the above tissues and cells. Figure 2

[0055] 3. Sequence evolution analysis

[0056] Sequence confirmation: The PCR product was cloned into pMD19-T vector for sequencing. It was finally confirmed that the CDS full length of ChIFN-υ gene was 528 bp, and its sequence is shown as SEQ ID NO. 3. The gene sequence has been submitted to GenBank, and the accession number is PP465981. The gene encodes a protein of 175 amino acids, and its sequence is shown as SEQ ID NO. 4, with a predicted molecular weight of about 19.5 kDa and an isoelectric point of about 8.5.

[0057] SEQ ID NO. 3:

[0058] ​ATGTATTGCCCCAGAAGCTTCTTCGTCTTGACTGTTTGCTTTATTACCACATCCCTCGGTAATCGACAAAGCAACATCCACAAATGCTTTGAAAGTGTGCAGTGGAGTGAACTTTTAAATGAGATTGAGAAACTTAACAATAGAAGCTATTCTTTGGAGTGTGATGAAGTGAGCAACGAAGAATTGTGCTTCCCTGAGAAAATGCTAAAAGCAGTTAAACATGATCACGCAGCCATTGTTCATATAGTACATGAAATTGCTGAGTTTTTCAAGAGAACAGATGCACCTTTTCAAAATAAGAACATTTTTCTAAGAGAAATATATGAAGCTCATGCCCAACTCAAGACTTGTCTGAAGCCCAAACTTAACGTCATTCATGAGTCTATAGTGAAAGAGTGCTTCCAAAAAATGGATACATTTGTGTCCAAGACAAATGATCAGTGCACTTGGCAAGAGATCCATGCACAATCAAGAGAGCTACTTCAAAGAGTTGAAAACTACTCTTTCAGGAGAAGATCAACACATTAA;

[0059] SEQ ID NO. 4:

[0060] MYCPRSFFVLTVCFITTSLGNRQSNIHKCFESVQWSELLNEIEKLNNRSYSLECDEVSNEELCFPEKMLKAVKHDHAAIVHIVHEIAEFFKRTDAPFQNKNIFLREIYEAHAQLKTCLKPKLNVIHESIVKECFQKMDTFVSKTNDQCTWQEIHAQSRELLQRVENYSFRRRSTH.

[0061] Molecular genetic evolution analysis of ChIFN-υ: The genetic evolution analysis of IFN genes of different species was performed using MEGA-X software, the phylogenetic tree was constructed by neighbor-joining method, and 1000 times bootstrap verification was performed. The results are shown in Figure 3 As shown in the table, unlike the currently known type I, type II and type III IFN, ChIFN-υ gene is in the same independent evolution branch as the type IV IFN identified in zebrafish, grass carp and African clawed toad, indicating that the gene is a homologous gene of type IV IFN.

[0062] Gene structure analysis: By alignment analysis with chicken genome sequence, it was confirmed that the CDS of ChIFN-υ gene was composed of 5 exons and 4 introns, the ORF sequence contained 528 bp, encoding a protein of 175 amino acids. The multi-exon structure was one of the characteristic features of type IV IFN, which was different from the single-exon structure of chicken type I IFN.

[0063] Example 2 Tissue expression analysis of ChIFN-υ gene

[0064] Experimental animals: 3 one-day-old and 3 four-week-old SPF chickens.

[0065] Sample collection and processing: After autopsy, the heart, liver, spleen, lung, kidney, muscle, trachea, small intestine, brain and other tissues were rapidly collected and immediately placed in liquid nitrogen for quick freezing, then transferred to -80℃ for storage. The total RNA was extracted using TRIzol method.

[0066] qPCR analysis: PrimeScript RT reagent Kit was used for genomic DNA removal and cDNA first strand synthesis. ChIFN-υ specific primers and SYBR Green qPCR Master Mix were used for real-time fluorescent quantitative PCR. Chicken GAPDH gene was used as an internal reference, and the relative expression in each tissue was calculated by comparative Ct method (2^ –ΔΔCt ) and normalized to the lowest expression tissue.

[0067] ChIFN-υ specific primer sequences are as follows:

[0068] Upstream primer: 5'-ATGATCACGCAGCCATTGTT-3'; SEQ ID NO. 5;

[0069] Downstream primer: 5'-TTCAGACAAGTCTTGAGTTG-3'; SEQ ID NO. 6.

[0070] The results are shown in Figure 4 , ChIFN-υ gene showed extensive but differential tissue expression in healthy chicken tissues, with the highest expression in mucosa-associated lymphoid tissues (such as trachea and small intestine) in one-day-old chicks; in four-week-old young chickens, the highest expression was in systemic immune organs (spleen) and respiratory organs (lungs).

[0071] Example 3 Prokaryotic expression, renaturation and purification of ChIFN-υ recombinant protein

[0072] 1. Construction of expression vector

[0073] The sequenced ChIFN-υ CDS sequence was directionally cloned into the multiple cloning site of the prokaryotic expression vector pET-28b(+) through EcoRI and XhoI restriction enzyme sites (the vector introduced a 6xHis tag sequence at the N and C termini), to construct the recombinant plasmid pET-28b-ChIFN-υ, and the successful construction was confirmed by sequencing.

[0074] 2. Recombinant protein induction expression and inclusion body acquisition

[0075] Transformation and culture: the recombinant plasmid was heat-shocked and transformed into E. coli BL21(DE3) competent cells, and positive clones were selected on LB agar plates containing 50 μg / mL kanamycin. A single colony was inoculated into 5 mL of LB liquid medium (containing kanamycin) and incubated at 37°C, 220 rpm overnight as a seed solution. The seed solution was transferred to fresh LB medium at a ratio of 1:100, and the culture was continued until the OD 600 reached 0.6-0.8.

[0076] Induction of expression: IPTG was added to the bacterial solution to a final concentration of 0.8 mM, and expression was induced at 37°C for 6h. SDS-PAGE analysis showed that an obvious induced expression band was visible at about 20 kDa, and most of it existed in the form of inclusion bodies.

[0077] Cell disruption: the cells were collected by centrifugation at 8000 g for 10 min at 4°C, resuspended with pre-cooled buffer (20 mM Tris-HCl, 100 mM NaCl, pH 8.0), and disrupted using an ultrasonic disrupter (power 300W, ultrasonic 5s, stop 5s, total time 30 min) under ice water bath conditions.

[0078] 3. Inclusion body denaturation, renaturation and purification

[0079] Inclusion body washing: the inclusion body precipitate was collected by centrifugation at 12000 g for 15 min at 4°C. To reduce impurities, the precipitate was washed twice with buffer containing 2M urea.

[0080] Denaturation and dissolution: the washed inclusion body precipitate was fully dissolved with denaturation buffer (8M urea, 20 mM Tris-HCl, 100 mM NaCl, 10 mM imidazole, pH 9.0), incubated at room temperature for 1h, then centrifuged at 12000 g for 30 min at 4°C to obtain the supernatant, which was the denatured protein solution.

[0081] Dilution refolding: The denatured protein solution was slowly added dropwise into pre-cooled refolding buffer (0.3 M L-arginine, 50 mM Tris-HCl, 150 mM NaCl, 5% (v / v) glycerol, 3 mM reduced glutathione, 0.3 mM oxidized glutathione, pH 8.0) so that the final protein concentration was no higher than 100 μg / mL, and the refolding was carried out at 4°C for 24 h with gentle stirring.

[0082] Dialysis and concentration: The refolded protein solution was loaded into a dialysis bag with a molecular weight cut-off of 10 kDa, and multiple dialysis was performed at 4°C using dialysis buffer (20 mM Tris-HCl, 150 mM NaCl, 10% glycerol, pH 8.0) to remove urea and L-arginine.

[0083] Affinity purification: The dialyzed sample was loaded onto a Ni-NTA affinity chromatography column that had been previously equilibrated with binding buffer (20 mM Tris-HCl, 150 mM NaCl, 20 mM imidazole, pH 8.0). Unbound impurities were washed out with 10 column volumes of binding buffer, and then stage elution was performed using elution buffer containing 250 mM imidazole, and the elution peak was collected.

[0084] The above process was repeated to obtain two batches of ChIFN-υ recombinant protein. Western Blot verification was performed using anti-His tag mouse monoclonal primary antibody and HRP-labeled goat anti-mouse secondary antibody, as shown in FIG. 2, and it can be seen that specific bands appeared at the expected position in both batches of recombinant protein, indicating that the ChIFN-υ preparation process established in this example was stable and highly reproducible. The protein concentration was determined using the BCA method, and the aliquots were stored at -80°C. Figure 5

[0085] Example 4 Research on activation of downstream ISRE promoter by ChIFN-υ recombinant protein

[0086] ​To investigate the ChIFN-υ recombinant protein on the downstream of the induction of anti-viral signaling pathway, the dual luciferase reporter gene system was used to detect the ChIFN-υ recombinant protein on the activation of ChISRE promoter. DF-1 cells were seeded in 24-well plates overnight to 80% confluence. According to the manufacturer's instructions (Promega, USA), 100 ng / well of pGL3-chISRE-Luc (chicken ISRE promoter sequence: TAGAAAATGAAACCAGGGAAACCGAAACTGAGTTTCACTTTCCTAGTTTCACTTTCCCTAGAAACTGAAACAGTGGAAAGTGAAACCTGGGAAAATGAAACTCGGGAAAATGAAACTGGGGAAAACGAAACTGAGGAAATAGAAACTTAGGAAAAGGAAACTGGAGAAACCGAAACTATGGTTTCAGTTTTCCTGGTTTCATTTTCTACAGTTTCTGTTTCCTCAGTTTCGGTTTCCCGAGTTTCATTTCTTCAGGTTTCGTTTCTGCCTGTTTCAGTTTCTAAGGTTTCACTTTCCAGAGTTTCATTTTCCCCAGTTTCATTTTCCCCGCTTTCGTTTCCTCCAGTTTCGTTTTCCCAAGTTTCTATTTCCTCAGTTTCCTTTTCCTTATGTTTCGGTTTCTCTGGTTTCGTTTCCTC (SEQ ID NO. 7) inserted into the pGL3-Basic vector to construct) and 10 ng / well of pRL-TK were co-transfected using FuGENE HD transfection reagent. Cells transfected with 100 ng / well of pGL3-basic and 10 ng / well of pRL-TK were used as a control group. After 24 h of transfection, cells were treated with 1000 IU / ml ChIFN-υ recombinant protein or PBS, respectively. After 12 h of treatment, the luciferase activity was measured using the dual luciferase reporter assay system (Promega, USA), and the luminescence value was recorded by the fluorescence / multifunctional microplate detector (BioTek, USA). The firefly luciferase activity was normalized by the renilla luciferase luminescence value. The experimental results were taken as the average of three independent detections.

[0087] The results are shown in Figure 6 ChIFN-υ recombinant protein can effectively activate ChISRE promoter. It is shown that ChIFN-υ can strongly activate the downstream anti-viral signaling pathway, and then promote the establishment of anti-viral state in cells.

[0088] Example 5 Anti-virus function verification of ChIFN-α recombinant protein

[0089] 1. Anti-virus activity determination in vitro (cytopathic effect inhibition method)

[0090] Cells and viruses: DF-1 cells were maintained in DMEM complete medium (containing 10% FBS). Both vesicular stomatitis virus (VSV) and H9N2 subtype avian influenza virus (H9N2 subtype AIV) were isolated and preserved by the Animal Germplasm Resources and Utilization Laboratory of the Agricultural Biogene Research Center, Guangdong Academy of Agricultural Sciences.

[0091] Titer determination: DF-1 cells were seeded in 96-well plates at a density of 1×10 4 cells / well and cultured to 90% confluence. The old medium was discarded, and ChIFN-α recombinant protein was added after 4-fold serial dilution with DMEM complete medium (containing 2% FBS) (the initial concentration was 1 mg / mL). Four replicate wells were set for each dilution, and cell controls (negative controls, without any treatment) and virus controls (positive controls, only infected) were also set. After 24 h of incubation, the medium containing IFN was discarded, and the cells were gently washed once with PBS, and then 100 TCID 50 of VSV or H9N2 subtype AIV virus solution was added. The plates were incubated in a 37°C, 5% CO2 incubator, and the cytopathic effect (CPE) was observed and recorded under an inverted microscope every 12 h.

[0092] Titer calculation: When about 90% CPE appeared in the virus control wells, the CPE of each well was recorded. The half-protective concentration was calculated by the Reed-Muench method, and the anti-virus titer of ChIFN-α recombinant protein was calculated accordingly.

[0093] The results are shown in Figure 7 and Figure 8 . It can be seen that ChIFN-α recombinant protein still has anti-VSV activity when diluted 4 6 times ( Figure 7 B), while the cells have obvious lesions when diluted 4 7 times ( Figure 7 C), the negative and positive controls are established, the IFN titer is calculated by the Reed-Muench method, and the anti-VSV virus titer of ChIFN-α recombinant protein is 1.17×10 4 IU / mg. ChIFN-α recombinant protein still has anti-H9N2 subtype AIV activity when diluted 4 7 times ( Figure 8 C), while the cells have obvious lesions when diluted 4 8 times ( Figure 8ChIFN-υ recombinant protein was 4.68 x 10 4 IU / mg.

[0094] 2. Effect of ChIFN-υ knockout on virus replication

[0095] The sgRNA targeting the first exon region of ChIFN-υ gene was synthesized by GenScript and inserted into a special vector. Through vector transfection, antibiotic screening and single clone screening, two DF-1 cell lines stably knocking out ChIFN-υ gene were successfully obtained.

[0096] The target sequence of sgRNA was TGCTTTATTACCACATCCCT; SEQ ID NO. 8.

[0097] The sequencing results of ChIFN-υ-KO1 and ChIFN-υ-KO2 cells are shown in Figure 9 The knockout cells (ChIFN-υ-KO1 and ChIFN-υ-KO2) and wild-type cells (ChIFN-υ-wt) were inoculated into 12-well plates, and the wild-type cells and knockout cells were infected with 0.1 MOI of H9N2 subtype AIV. The cells were harvested at 12 h, 24 h, 36 h and 48 h after infection, and the virus titers were detected by TCID 50 The results are shown in Figure 10 The virus titers in ChIFN-υ-KO1 cells and ChIFN-υ-KO2 cells were significantly higher than those in wild-type cells, indicating that the replication ability of virus in DF-1 cells was significantly enhanced after knocking out ChIFN-υ gene.

[0098] 3. In vivo anti-virus experiment

[0099] Experimental design and grouping: 30 four-week-old SPF chickens were randomly divided into two groups: ChIFN-υ recombinant protein treatment group and PBS positive control group, 15 chickens in each group.

[0100] Pretreatment and challenge: The ChIFN-υ recombinant protein treatment group was given 1 x 10 3 IU of ChIFN-υ recombinant protein (dissolved in 100 μL PBS), and the PBS positive control group was given the same amount of PBS. After 24 h, all chickens were challenged with 200 μL of 10 6 EID 50 H9N2 subtype AIV by nasal and eye drop route.

[0101] Tissue virus load detection: On the 3rd, 5th, 7th day after challenge, 3 chickens were killed from each group, and lung and trachea tissues were collected aseptically. The tissues were homogenized and centrifuged, and the supernatant was diluted 10 times in series, inoculated into 9-11 day-old SPF chicken embryo allantoic cavity, and the allantoic fluid was collected after 72 h of incubation. The virus was determined to be positive by hemagglutination test, and the virus titer in the tissue was calculated by Reed-Muench method (expressed as EID 50 / g).

[0102] Detoxification monitoring: On the 3rd, 5th, 7th day after challenge, 6 chickens were fixed from each group, and oropharyngeal and cloaca swabs were collected in 1 mL PBS. After vortexing, the virus titer in the swabs was calculated by chicken embryo inoculation and hemagglutination test (expressed as EID 50 / mL).

[0103] The results are shown in Figure 11 The lung and trachea tissue virus load of the ChIFN-υ recombinant protein treatment group chickens, and the oropharyngeal and cloaca detoxification amount were significantly lower than the PBS control group on the 3rd and 5th day after infection (p < 0.05), and by the 7th day, some indicators (such as tracheal virus load) still showed an inhibitory trend.

[0104] Example 6 Verification of the synergistic antiviral function of ChIFN-υ recombinant protein and type I interferon ChIFN-α

[0105] Experimental materials: ChIFN-υ recombinant protein, type I interferon ChIFN-α (reference literature "Dai M, Wu S, Feng M, et al. Recombinant chicken interferon-alpha inhibits the replication of exogenous avian leukosis virus (ALV) in DF-1 cells [J]. Molecular Immunology, 2016, 76:62-69. DOI:10.1016 / j.molimm.2016.06.012." prepared and preserved by the laboratory); DF-1 cells were maintained in DMEM complete medium (containing 10% FBS); viruses were vesicular stomatitis virus (VSV) and H9N2 subtype avian influenza virus (H9N2 subtype AIV).

[0106] Determination of synergistic effect titer: DF-1 cells were inoculated with ChIFN-υ recombinant protein at 1×10 4The 96-well plates were seeded at a density of wells, and cultured to 90% confluence. Old liquid was discarded, and ChIFN-υ single treatment group, ChIFN-α single treatment group, ChIFN-υ+ChIFN-α combined treatment group (the initial concentration was 1 mg / mL; the mass ratio of ChIFN-υ and ChIFN-α in the combined treatment group was 1:1) were treated with 4-fold serial dilution using maintenance medium (containing 2% FBS), 4 duplicate wells were set for each dilution, and cell control (negative control, without any treatment) and virus control (positive control, only infected) were set. After 24h incubation, the medium containing IFN was discarded, and the cells were gently washed once with PBS, and then 100 TCID 50 of VSV or H9N2 subtype AIV virus liquid was added. The plates were incubated in a 37℃, 5% CO2 incubator, and the cytopathic effect was observed and recorded under an inverted microscope every 12h, and the IFN titer was calculated using the Reed-Muench method.

[0107] The results are shown in Table 1. The ChIFN-υ+ChIFN-α combined treatment group had higher antiviral titer against VSV and H9N2 subtype AIV than the ChIFN-υ single treatment group and the ChIFN-α single treatment group, indicating that the two had a synergistic antiviral effect.

[0108] Table 1. Antiviral titer detection results of each group

[0109]

[0110] The above-described embodiments are only to describe the preferred modes of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those of ordinary skill in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. Use of chicken type I interferon ChIFN-υ for the preparation of a medicament for the treatment of viral infections, characterized in that, The amino acid sequence of the chicken type IV interferon ChIFN-υ is shown as SEQ ID NO.

4. The viruses are vesicular stomatitis virus and H9N2 subtype avian influenza virus.

2. Use of a substance expressing chicken type IV interferon ChIFN-υ for the preparation of a medicament for the treatment of viral infections, characterized in that, The amino acid sequence of the chicken type IV interferon ChIFN-υ is shown as SEQ ID NO.

4. The substance expressing the chicken type IV interferon ChIFN-υ comprises a recombinant vector or a recombinant microorganism comprising a coding gene of the chicken type IV interferon ChIFN-υ. The viruses are vesicular stomatitis virus and H9N2 subtype avian influenza virus.

3. Use according to claim 2, characterized in that, The nucleotide sequence of the coding gene of the chicken type IV interferon ChIFN-υ is shown as SEQ ID NO.

3.

4. The use of chicken interferon type IV ChIFN-υ in combination with chicken interferon type I ChIFN-α in the preparation of a medicament for the treatment of viral infections, characterized in that, The amino acid sequence of the chicken type IV interferon ChIFN-υ is shown as SEQ ID NO.

4. The viruses are vesicular stomatitis virus and H9N2 subtype avian influenza virus.

5. A medicament for treating viral infection, characterized by comprising the compound of claim 1. The medicine comprises a combination of chicken type IV interferon ChIFN-υ and chicken type I interferon ChIFN-α as effective components. The amino acid sequence of the chicken type IV interferon ChIFN-υ is shown as SEQ ID NO.

4. The viruses are vesicular stomatitis virus and H9N2 subtype avian influenza virus.

6. Use of a substance knocking out the coding gene of chicken type IV interferon ChIFN-υ in the preparation of a cell or animal model with improved virus sensitivity. The nucleotide sequence of the coding gene of the chicken type IV interferon ChIFN-υ is shown as SEQ ID NO.

3. The substance knocking out the coding gene of chicken type IV interferon ChIFN-υ is an sgRNA targeting the coding gene of the chicken type IV interferon ChIFN-υ; the nucleotide sequence of the sgRNA is shown as SEQ ID NO.

8. The viruses are H9N2 subtype avian influenza virus.

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