Bovine interferon-tau type i-ferritin fusion proteins, mutants and uses thereof

Abstract

The application discloses a bovine type I interferon- ferritin fusion protein and a mutant and application thereof. The bovine type I interferon is fused with a ferritin subunit, the self-assembly characteristics of the ferritin are utilized, the interferon molecules are highly repeated and orderly displayed on the surface of the ferritin nanocage, a conformation suitable for the function of the interferon is formed, and the expression level, structural stability and antiviral activity of the interferon are significantly improved. The fusion protein is subjected to rational design mutation to obtain a unit point or multi-point mutant with significantly improved antiviral activity and stability. The fusion protein or the mutant is expressed by using a silkworm or insect cell eukaryotic expression system, the expression system is safe in operation, simple in procedure, low in cost and extremely beneficial to large-scale industrial production, and the prepared fusion protein or mutant nanoparticles can be applied to the preparation of various drugs for preventing or treating bovine viral infection, tumors and immune system diseases and an immune adjuvant for vaccine compounding.

Description

Technical Field

[0001] This invention relates to a fusion protein of bovine type I interferon, and more particularly to a fusion protein obtained by fusing bovine type I tau interferon and ferritin subunits, as well as mutants of the fusion protein, their preparation methods and applications, belonging to the field of fusion proteins of bovine interferon and ferritin, their mutants and applications. Background Technology

[0002] Interferons (IFNs) are highly active, multifunctional glycoproteins induced in specific cells by certain inducers, possessing antiviral, antitumor, and immunomodulatory effects. Based on their origin and acid tolerance, they are classified into types I, II, and III, mainly including INFα, β, ω, κ, τ, δ, and γ. Interferons have multiple functions, including antiviral, antitumor, and immunomodulatory effects, and show broad application prospects in veterinary clinical practice.

[0003] Ferritin nanoparticles are naturally occurring nanoparticle proteins with self-assembly properties. They consist of 24 ferritin subunits that self-assemble into a hollow cage-like structure with highly stable cavities and modifiable outer surfaces. They can be used to internally load or externally display targets through in vitro fusion expression and other methods. Ferritin also has considerable biocompatibility and certain targeted delivery capabilities, making it an ideal nanoplatform. Therefore, it is widely used in fields such as vaccine development, targeted drug delivery, biosensing, and catalysis.

[0004] In the prevention and treatment of viral diseases, the antiviral activity and stability of interferon determine its effectiveness.

[0005] Interferon-τ (IFN-τ) is a type I interferon specifically secreted by trophoblast cells in ruminant embryos. The crystal structure of IFN-τ is similar to other type I interferons, possessing five α-helices. It is known as a pregnancy recognition signal and plays a crucial biological role in maintaining the corpus luteum and establishing pregnancy. Furthermore, IFN-τ exhibits antiviral activity without cytotoxicity and possesses various immunomodulatory functions. However, bovine type I τ interferon has drawbacks such as low antiviral activity and short in vivo retention time, affecting its efficacy in the prevention or treatment of bovine viral diseases and limiting its widespread clinical application. Summary of the Invention

[0006] One of the objectives of this invention is to provide a fusion protein (Ferritin-BoIFN-τ) that combines bovine type I τ interferon (BoIFN-τ) and ferritin subunits.

[0007] A second objective of this invention is to provide a mutant of the fusion protein of bovine type I τ interferon and ferritin subunit;

[0008] The third objective of this invention is to provide a method for preparing a fusion protein of bovine type I tau interferon and ferritin subunit or a mutant thereof;

[0009] The fourth objective of this invention is to apply the fusion protein of bovine type I τ interferon and ferritin subunit or its mutant to the preparation of drugs or reagents for the prevention or treatment of bovine viral diseases or for immunomodulation.

[0010] To achieve the above objectives, the technical solution adopted by the present invention includes:

[0011] To address the shortcomings of bovine type I tau interferon, such as low antiviral activity and short in vivo retention time, this invention fuses bovine type I tau interferon with ferritin subunit nanoparticles to obtain a fusion protein with significantly enhanced antiviral activity. The ferritin subunit nanoparticle carrier increases the in vivo retention time of bovine type I tau interferon and reduces the frequency of administration. Furthermore, this invention further mutates the fusion protein to obtain single-site or multi-site mutants with significantly enhanced antiviral activity. In addition, this invention provides a method for preparing the fusion protein or its mutants, thus completing this invention.

[0012] One aspect of the present invention is to provide a fusion protein of bovine type I tau interferon and ferritin subunits, wherein the fusion protein is obtained by linking the C-terminus of the monomeric ferritin subunit to the N-terminus of bovine type I tau interferon through a flexible linker peptide with high conformational flexibility, thereby exhibiting bovine type I tau interferon on the surface of ferritin nanostructures.

[0013] The amino acid sequence of bovine type I tau interferon or ferritin subunit described in this invention can be an amino acid sequence derived from NCBI.

[0014] In a preferred embodiment of the present invention, the monomeric ferritin subunit includes any one of bacterial ferritin subunit, plant ferritin subunit, algal ferritin subunit, insect ferritin subunit, fungal ferritin subunit, or vertebrate ferritin subunit; preferably, the monomeric ferritin subunit of the present invention is bovine ferritin monomer, wherein the bovine ferritin monomeric subunit is obtained by deleting amino acids 163 to 181 from the amino acid sequence of the bovine ferritin monomeric subunit.

[0015] In a preferred embodiment of the present invention, the amino acid sequence number of bovine type I tau interferon is AAX98268.1. After analyzing its amino acid sequence, it was found that its signal peptide region is positions 1-23. Therefore, the signal peptide of bovine type I tau interferon is removed to obtain the amino acid sequence of bovine type I tau interferon as shown in SEQ ID No.1. Then, the nucleotide sequence of its encoding gene is optimized and modified according to the codon preference of silkworm using OptimumGene™ technology to obtain the optimized nucleotide sequence of the gene shown in SEQ ID No.2.

[0016] In a preferred embodiment of the present invention, the amino acid sequence of the flexible linker peptide with high conformational flexibility is shown in SEQ ID No. 3.

[0017] In a preferred embodiment of the present invention, a monomeric ferritin subunit with 15 amino acids removed from its C-terminus is linked to the N-terminus of bovine type I tau interferon (as shown in SEQ ID No. 3) via a linker peptide, resulting in a fusion protein. This allows bovine type I tau interferon to be displayed on the surface of ferritin nanoparticles. The amino acid sequence of the resulting fusion protein, which combines bovine type I tau interferon and the ferritin subunit, is shown in SEQ ID No. 4. Furthermore, the encoding gene of the fusion protein is codon-optimized using OptimumGene™ technology to target the codon preference of silkworms, resulting in a codon-optimized gene with the nucleotide sequence shown in SEQ ID No. 5.

[0018] Another aspect of the present invention is to provide a single-site mutant or a multi-site mutant with significantly enhanced antiviral activity or potency compared to the fusion protein.

[0019] To enhance the antiviral activity or potency of bovine type I interferon-ferritin subunit fusion protein, this invention involves site-directed or multi-site mutations of the fusion protein and screening these mutants to obtain site-directed or multi-site mutants with significantly enhanced antiviral activity or potency. Specifically, this includes: site-directed rational mutation of the amino acids in the bovine type I interferon-ferritin subunit fusion protein with the amino acid sequence shown in SEQ ID No. 4, based on its IFNAR2 receptor binding site, to obtain site-directed mutants using any one of the following amino acid site-directed mutation methods: L205R, K209R, L223R, L293R, K296R, Q300N, or Y311E; or site-directed mutation of the amino acid sequence shown in SEQ ID No. 4. The amino acids of the bovine type I interferon-ferritin subunit fusion protein shown in SEQ ID No. 4 are obtained by double-site mutation of any one of L205R-K209R, L205R-L223R, K209R-L223R, L293R-K296R, L293R-Y311E, L293R-Q300N, or Q300N-Y311E; or the amino acids of the bovine type I interferon-ferritin subunit fusion protein with the amino acid sequence shown in SEQ ID No. 4 are obtained by triple-site mutation of L205R-K209R-L223R or L293R-Q300N-Y311E. The antiviral activity or titer of these single-site mutants, double-site mutants, or triple-site mutants is significantly improved or enhanced compared with the bovine type I interferon-ferritin subunit fusion protein.

[0020] In this invention, the meaning of "L209R" single point mutation refers to the mutation of leucine at position 209 of the fusion protein shown in SEQ ID No.4 to arginine, and the meanings of the other single point mutations are deduced by analogy.

[0021] The meaning of the "L205R-K209R" double-site mutation mentioned in this invention refers to the mutation of leucine at position 205 of the fusion protein shown in SEQ ID No.4 to arginine, and lysine at position 209 to arginine. The meanings of other double-site mutations are deduced by analogy.

[0022] The meaning of the "L205R-K209R-L223R" three-point mutant in this invention refers to the mutation of leucine at position 205 to arginine, lysine at position 209 to arginine, and leucine at position 223 to arginine in the fusion protein shown in SEQ ID No.4. The meanings of the other three-point mutations are deduced by analogy.

[0023] Another aspect of the present invention provides a coding gene for the fusion protein or a mutant thereof, and an expression vector containing the coding gene; wherein the expression vector is preferably a eukaryotic expression vector.

[0024] Another aspect of the present invention provides a method for preparing the bovine type I τ-ferritin subunit fusion protein or a mutant thereof.

[0025] In this invention, the single-site mutants, two-site mutants, or three-site mutants of the above-mentioned fusion proteins were expressed in the silkworm eukaryotic expression system. The expression results showed that the antiviral activity of the fusion proteins expressed by these mutant sequences was significantly improved compared with the original fusion proteins.

[0026] As a preferred embodiment, the present invention provides a method for preparing the bovine type I τ-interferon-ferritin subunit fusion protein or a mutant thereof, the method comprising:

[0027] (1) Construct a baculovirus eukaryotic expression transfer vector containing the coding gene of the fusion protein or its mutant; (2) Co-transfect the constructed baculovirus eukaryotic expression transfer vector with baculovirus genomic DNA into insect cells to obtain recombinant baculovirus; (3) Infect insect hosts or cells with the recombinant baculovirus, express the fusion protein or its mutant in insect cells or live insects, purify and renature to obtain the baculovirus.

[0028] Another aspect of the present invention is to apply the bovine type I τ interferon-ferritin fusion protein or its mutant to the preparation of drugs or reagents for the prevention or treatment of bovine viral diseases; wherein the bovine viral diseases include, but are not limited to, any one of bovine viral diarrhea (BVD), bovine foot-and-mouth disease (FMD), bovine respiratory disease (BRD), bovine mastitis, or bovine endometritis.

[0029] Those skilled in the art can prepare bovine type I tau interferon-ferritin fusion protein or its mutants into various drug preparations or reagents for the prevention or treatment of bovine viral diseases using conventional pharmaceutical preparation methods in the art. These methods are well known to those skilled in the art.

[0030] Compared with the prior art, the present invention has the following advantages or effects:

[0031] 1. This invention connects bovine interferon with ferritin subunits via a flexible linker to achieve surface display of the interferon protein, forming a conformation suitable for its function, effectively improving the expression level and activity of bovine interferon; at the same time, based on the inherent characteristics of the tandem ferritin nanoparticles, it effectively improves the stability of bovine interferon and prolongs its half-life.

[0032] 2. The bovine type I τ interferon-ferritin subunit fusion protein or its mutant provided by this invention contains interferon, which is a protein component naturally present in animals. It has a simple structure, low toxicity to animals, weak antigenicity, and high antiviral activity. It can be used to prepare a variety of drugs for the prevention or treatment of viral infections, tumors, immune system diseases, as well as immune adjuvants for vaccines. Its high stability and long half-life overcome the disadvantages of traditional interferon applications, such as multiple administrations and time-consuming and labor-intensive processes, which is very beneficial for its promotion and application in the cattle breeding industry.

[0033] 3. This invention utilizes the silkworm baculovirus expression system to express fusion proteins or their mutants, which has strict host specificity. Compared with traditional interferon production methods, it is safer, simpler to operate, and produces a large expression level, making it suitable for rapid large-scale production.

[0034] Definitions of terms involved in this invention

[0035] Unless otherwise defined, 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 invention pertains.

[0036] The term "interferon" (IFN) refers to an important class of cytokines whose activity is regulated and controlled by the cellular genome, involving the synthesis of RNA and proteins. The IFN protein family is classified into type I, type II, and type III interferons based on the sequence of their encoding genes, chromosomal location, and receptor specificity.

[0037] The term "recombinant protein" refers to proteins produced using recombinant DNA technology, which can be used to clone and express genes in a variety of hosts, including bacteria, mammalian cells, insect cells, and plants, to produce proteins.

[0038] The terms "host cell" or "recombinant host cell" refer to a cell containing the polynucleotides of the present invention, regardless of the method used for insertion to produce a recombinant host cell, such as direct uptake, transduction, or other methods known in the art. The exogenous polynucleotides may remain as, for example, non-integrating vectors of plasmids or may be integrated into the host genome.

[0039] The term "transfection" refers to the process by which a host cell acquires a new genetic marker due to the incorporation of exogenous DNA. Attached Figure Description

[0040] Figure 1 The results of double enzyme digestion identification of recombinant plasmids pBR-BoIFN-τ and pBR-Ferritin-BoIFN-τ show that the large fragment is the 7618bp pBR transfer vector, and the small fragments are the 540bp BoIFN-τ and 1062bp Ferritin-BoIFN-τ target genes, respectively.

[0041] Figure 2 Serum drug metabolism diagrams for BoIFN-τ and Ferritin-BoIFN-τ. Detailed Implementation

[0042] The present invention will be further described below with reference to specific embodiments or test examples, and the advantages and features of the present invention will become clearer with the description. However, it should be understood that the embodiments or test examples are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but such modifications or substitutions all fall within the protection scope of the present invention.

[0043] Example 1: Construction and expression of bovine interferon protein, the fusion protein of bovine interferon and ferritin, and its mutant expression vectors.

[0044] Bovine ferritin and bovine interferon sequences were obtained from the NCBI database and used to generate the sequences using OptimumGene. TM After technical optimization, the amino acid sequence of bovine interferon obtained is shown in SEQ ID No. 1, and the protein is named BoIFN-τ. MCYLSEDHMLGARENLRLLARMNRLSPHPCLQDRKDFGLPQEMVEGSQLQKDQAISVLHEMLQQCFNLFHIEHSSAAWNTTLLEQLCTGLQQQLEDLDACLGPVMGEKDSDMGRMGPILTVKRYFQDIHVYLKEKEYSDCAWEIIRVEMMRALSSSTTLQKRLRKMGGDLNSL* (SEQ ID No. 1).

[0045] The amino acid sequence shown in SEQ ID No. 1 was input into the Codon Optimizer software, along with the E. coli genome sequence information. Codon optimization was performed based on the codon preference of the silkworm, and the silkworm Kozak sequence GCCAAC was added upstream to obtain the nucleotide sequence corresponding to bovine interferon-taurine protein (BoIFN-τ) (SEQ ID No. 2).

[0046] Using the linker peptide GGGSGGGGSGGGS (SEQ ID No. 3), bovine interferon-tau (SEQ ID No. 1) was fused to amino acid position 162 of the bovine ferritin subunit (i.e., amino acids 163-181 were deleted). The amino acid sequence of the resulting bovine interferon-tau ferritin fusion protein is shown in SEQ ID No. 4. This fusion protein was named Ferritin-BoIFN-tau. GGGSGGGGSGGGS CYLSEDHMLGARENLRLLARMNRLSPHPCLQDRKDFGLPQEMVEGSQLQKDQAISVLHEMLQQCFNLFHIEHSSAAWNTTLLEQLCTGLQQQLEDLDACLGPVMGEKDSDMMGRMGPILTVKRYFQDIHVYLKEKEYSDCAWEIIRVEMMRALSSSTTLQKRLRKMGGDLNSL* (SEQ ID No. 4).

[0047]

[0048] Introduced upstream and downstream of SEQ ID No. 2 and SEQ ID No. 5 respectively Bam H Ⅰ / Eco After RⅠ restriction site digestion, the whole gene was synthesized to obtain pUC57-BoIFN-τ (pUC57 with SEQ ID No. 2 inserted) and pUC57-Ferritin-BoIFN-τ plasmid (pUC57 with SEQ ID No. 5 inserted), respectively. After double restriction enzyme digestion, the plasmid was ligated into the pBR vector stored in the laboratory to construct pBR-Ferritin-BoIFN-τ plasmid.

[0049] Using pBR-Ferritin-BoIFN-τ plasmid as a template, a large number of single point mutations were performed using fusion PCR. In this experiment, only the effective single point mutations and the corresponding primers for mutation are listed in Table 1.

[0050] Table 1 Primer sequences used for effective point mutations

[0051]

[0052] The effective mutation sites (L205, K209, L223, L293, K296, Q300, Y311) in the amino acid sequence shown in SEQ ID No. 4 were sequentially subjected to single-point mutations using fusion PCR. The resulting effective mutant was named Ferritin-BoIFN-τ. mut 1 (L205R, K209R, L223R, L293R, K296R, Q300N, Y311E); Based on obtaining effective single-site mutations, various combinations of two-site mutations were further performed, and the better two-site combination mutants were named Ferritin-BoIFN-τ. mut 2 (L205R-K209R, L205R-L223R, K209R-L223R, L293R-K296R, L293R-Y311E, L293R-Q300N, K296R-Y311E or Q300N-Y311E); After analyzing the excellent combined mutants, further three-site combined mutagenesis was performed, named Ferritin-BoIFN-τ mut 3 (L205R-K209R-L223R, L293R-Q300N-Y311E). Since the two sites in the L205R-K209R and L293R-K296R double-site mutation combinations are relatively close, new intermediate and downstream primers were redesigned based on Ferritin-BoIFN-τ-L205R and Ferritin-BoIFN-τ-L293R, respectively, and are listed in Table 2.

[0053] Table 2. Primer design for L205R-K209R and L293R-K296R double mutants.

[0054]

[0055] The PCR reaction system is shown in Table 3.

[0056] Table 3 PCR reaction system

[0057]

[0058] PCR parameters were set as follows: 95℃, 30 s; 95℃, 15 s, 64℃, 15 s, 72℃, 60 s, for a total of 29 cycles; 72℃, 5 min.

[0059] PCR product recovery: The PCR products were subjected to agarose gel electrophoresis. The target band was cut out under UV light and placed in an EP tube. Three volumes of 6 M sodium iodide were added and the mixture was melted in a 55°C water bath. 8 μL of glass milk was added, mixed, and incubated on ice for 10 min, shaking every three minutes. The mixture was centrifuged at 12000 r / min for 10 s and the supernatant was discarded. 800 μL of New Wash was added and the mixture was gently washed, repeated three times. The supernatant was discarded and the mixture was dried in a 37°C oven for 5 min. 20 μL of 0.1×TE was added, mixed, and centrifuged at 12000 r / min for 5 min. The supernatant was collected and stored at -20°C.

[0060] Enzyme digestion and recovery: using restriction endonucleases Bam H Ⅰ and Eco R Ⅰ The above PCR products and pUC57-BoIFN-τ and pUC57-Ferritin-BoIFN-τ were double digested and inactivated at 65℃ for 10 min. The DNA was then recovered using the Tiangen agarose gel DNA recovery kit and stored at -20℃ for later use.

[0061] Ligation: The target fragment was ligated with T4 DNA ligase to the double-digested and inactivated baculovirus transfer vector pBR. The ligation product was transformed into E. coli competent cells Trans5τ, colonies were selected for culture, plasmids were extracted, and... Bam HI and Eco R-I double enzyme digestion identified positive clones containing a 7618 bp pBR transfer vector as the large fragment, and 582 bp and 1107 bp fragments of the target gene, respectively. Electrophoresis results are shown below. Figure 1 The correctly identified recombinant plasmids were sequenced, and the correctly sequenced plasmids were named pBR-BoIFN-τ and pBR-Ferritin-BoIFN-τ.

[0062] Table 4 Connection System

[0063]

[0064] BmN cells were resuscitated, passaged, and recombinant viruses were screened according to methods reported in existing literature. One day before co-transfection, BmN cells were dispersed into single-cell suspensions from cell culture flasks and seeded into six-well plates at 2 mL per well. Transfection was performed when cell confluence reached approximately 90%. For each sample, 2 μL of liposomes and 50 μL of sterile ultrapure water were mixed thoroughly and incubated at room temperature for 5 min. 1 µg of BmBac DNA from the laboratory-preserved silkworm baculovirus parent strain was added to a centrifuge tube, and 40 μL of sterile ultrapure water was added to dissolve the viral genome. The mixture was incubated at room temperature for 5 min. Then, the mixture was aliquoted into 1.5 mL centrifuge tubes, labeled, and 5 μg of pBR-BoIFN-τ and pBR-Ferritin-BoIFN-τ plasmids were added to the centrifuge tubes. The mixed liposome dilution was added dropwise to the plasmid dilution, and the mixture was incubated at room temperature for 20 min. After washing the cells in the six-well plate twice with serum-free insect cell culture medium, add 2 mL of serum-free medium. Add the mixed liposome-plasmid complex solution dropwise to the six-well plate, labeling each well with the sample name and transfection date. Seal the plate with sealing film and place it in a 27°C cell culture incubator. Replace the medium with complete medium 4 hours after transfection. Seal the plate again with sealing film and incubate at 27°C for 4–5 days until the cells detach and float. Collect the cell culture medium to obtain the recombinant viruses BmBac (BoIFN-τ) and BmBac (Ferritin-BoIFN-τ) containing the target gene.

[0065] The purification and amplification method for recombinant silkworm baculovirus is as follows: An appropriate amount of cells (approximately 80-90%) are inoculated into 35mm petri dishes. After cell adhesion, the culture medium is aspirated. The collected cell culture medium is diluted to different concentrations, and 1mL is gently added to the adherent cells, ensuring even distribution. After infection at 27℃ for 1 hour, the infection medium is aspirated. 2% low-melting-point agarose gel is melted in a 60℃ water bath, cooled to 40℃, and mixed thoroughly with 4mL of preheated 2×TC-100 medium (containing 20% ​​FBS). 4mL of gel is added to each petri dish, and after solidification, the dish is sealed with sealing film and returned to the incubator. The dish is incubated upside down at 27℃ for 3-5 days. After plaque formation, plaques are picked, and the above steps are repeated. After 2-3 rounds of purification, pure recombinant silkworm baculovirus BmBac (BoIFN-τ) and BmBac (Ferritin-BoIFN-τ) are obtained.

[0066] Normally growing BmN cells were infected with recombinant silkworm baculoviruses BmBac (BoIFN-τ) and BmBac (Ferritin-BoIFN-τ). After culturing for 3-5 days, the supernatant was collected, which contained a large number of recombinant viruses BmBac (BoIFN-τ) and BmBac (Ferritin-BoIFN-τ).

[0067] The recombinant virus culture medium was prepared at a ratio of 10... 5 Inject 5th instar silkworms or silkworm pupae with PFU / head and culture them at 27℃ and 70%~80% humidity. In the late stage of silkworm larval development, bovine interferon and bovine interferon-ferritin fusion proteins are highly expressed under the action of the polyhedrome gene promoter. About 3.5 to 4.5 days after inoculation, symptoms such as swelling of the silkworm larvae's body segments, abnormal behavior, and decreased appetite can be observed. When the larvae are observed to be significantly reduced in size and stop feeding, hemolymph is collected and stored at -20℃ for later use.

[0068] Experimental Example 1: Detection of antiviral activity of bovine interferon and bovine interferon-ferritin fusion protein

[0069] Using the same method as the national standard for mammalian interferon (GB / T 45138-2024), the antiviral activity of bovine type I τ interferon (BoIFN-τ) and the fusion protein of ferritin subunit (Ferritin-BoIFN-τ) expressed in the silkworm samples of Example 1 was detected on the VERO / VSV*GFP system by the micro-cytopathic inhibition method.

[0070] healthy VERO cells were injected with 5.0 × 10⁻⁶ cells. 5 Silkworm samples were seeded at a density of cells / mL in 96-well plates. The silkworm samples, which had been sonicated and filtered for sterilization, were prepared into solutions of different dilutions using DMEM / F12 medium containing 2% fetal bovine serum. 100 μL of the diluted samples were seeded per well into wells already contaminated with VERO cells. Each dilution and control sample had at least eight replicates. A cell control group without silkworm hemolymph and VSV*GFP and a virus control group with VSV*GFP were also included. The plates were incubated at 37°C and 5% CO2 for 18–24 h. VSV*GFP virus diluted to 100 TCID50 was added at 100 μL per well to wells where the supernatant had been removed. The plates were then incubated at 37°C and 5% CO2. Under an inverted fluorescence microscope, when a suitable number of cells in the virus control group showed fluorescence, while the cells in the cell control group remained fully grown and showed no fluorescence, the control system was considered fully qualified and ready for observation.

[0071] The results of the antiviral potency assay of bovine interferon and bovine interferon-ferritin fusion protein are listed in Table 5.

[0072] Table 5. Results of antiviral activity assay of bovine interferon and bovine interferon-ferritin fusion protein

[0073]

[0074] According to the antiviral activity assay results, the antiviral potency of the bovine interferon-ferritin fusion protein prepared in Example 1 is significantly improved compared with that of bovine interferon.

[0075] Experimental Example 2: Detection of the antiviral activity of bovine interferon-ferritin fusion protein and its mutants.

[0076] The antiviral activity of bovine interferon-ferritin fusion protein was detected using the same method as in Experiment Example 1. The antiviral activity of effective single-site or multi-site mutants of bovine interferon-ferritin fusion protein was tested. The antiviral efficacy results of some single-site or multi-site mutants are listed in Table 6 (Note: Most mutants whose antiviral activity or efficacy was not increased or decreased compared to before the mutation are not listed in the table).

[0077] Table 6. Results of antiviral activity assay of bovine interferon-ferritin fusion protein and its mutants.

[0078]

[0079] According to the antiviral activity assay results in Table 6, the unit point mutants obtained by mutating any one of the amino acid sequences of the bovine type I τ-interferon-ferritin subunit fusion protein (SEQ ID No. 4) using L205R, K209R, L223R, L293R, K296R, Q300N, or Y311E showed significantly enhanced or improved antiviral activity or titer compared to the bovine type I τ-interferon-ferritin subunit fusion protein. The antiviral activity or potency of the two-site mutants obtained by mutating the amino acids of the bovine type I interferon-ferritin subunit fusion protein shown in SEQ ID No. 4 at any one of the following two-site mutations (L205R-K209R, L205R-L223R, K209R-L223R, L293R-K296R, L293R-Y311E, L293R-Q300N, or Q300N-Y311E) is significantly enhanced or improved compared to the bovine type I interferon-ferritin subunit fusion protein. The antiviral activity or potency of the three-site mutants L205R-K209R-L223R or L293R-Q300N-Y311E obtained by combining the amino acids of the bovine type I interferon-ferritin subunit fusion protein with the amino acid sequence shown in SEQ ID No. 4 at three different sites is significantly enhanced or improved compared to the bovine type I interferon-ferritin subunit fusion protein.

[0080] Experimental Example 3: Serum Stability Assay of Bovine Interferon and Bovine Interferon-Ferritin Fusion Protein

[0081] Eighteen healthy male rats weighing 180-220 g were randomly divided into three groups. The rats were intravenously injected with a single dose (10 million IU / kg) of BoIFN-τ, Ferritin-BoIFN-τ fusion protein, and a corresponding control group, respectively. Serum samples were collected at 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 6.0, 8.0, and 12.0 hours post-injection. The anti-VSV-GFP activity was determined using the method described in Example 1.

[0082] The results showed that the half-life (T1 / 2) of BoIFN-τ in vivo was approximately 2.0 hours, while that of Ferritin-BoIFN-τ and Ferritin-BoIFN-τ... mut3 (Ferritin-BoIFN-τ (L293R-Q300N-Y311E)) is slowly eliminated in vivo and has a long distribution phase. Its half-life (T1 / 2) values ​​are 5.53 and 5.38 hours, respectively, which are more than 2.5 times longer than those of BoIFN-τ. Figure 2 ).

Claims

1. A mutant of a fusion protein obtained by fusing type I τ interferon and ferritin subunits, characterized in that, The mutants are selected from: a single point mutant obtained by mutating the amino acids of the fusion protein with the amino acid sequence of SEQ ID No. 4 using the L293R single point mutation method; or a two-site mutant obtained by mutating the amino acids of the fusion protein with the amino acid sequence of SEQ ID No. 4 using any one of the two-site mutations L293R-K296R, L293R-Y311E or L293R-Q300N; or a three-site mutant obtained by mutating the amino acids of the fusion protein with the amino acid sequence of SEQ ID No. 4 using the L293R-Q300N-Y311E three-site mutation.

2. A method for preparing the mutant according to claim 1, characterized in that, include: (1) Construct a baculovirus eukaryotic expression transfer vector containing the coding gene of the mutant; (2) Co-transfect the constructed baculovirus eukaryotic expression transfer vector with baculovirus genomic DNA into insect cells to obtain recombinant baculovirus; (3) Infect insect hosts or cells with recombinant baculovirus, express the mutant in insect cells or live insects, purify and renature to obtain the final product.

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