Bovine interferon-alpha type i-ferritin fusion protein, mutants thereof, and methods of making and using same

Abstract

The application discloses a bovine type I alpha interferon-ferritin fusion protein, a mutant thereof, and a preparation method and application thereof. The bovine type I alpha 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, and the expression level, structural stability and antiviral activity of the interferon are significantly improved. The fusion protein is further subjected to rational design mutation at a unit point or multiple points, and a mutant with significantly improved antiviral activity and stability is obtained. The fusion protein or the mutant thereof 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 nanometer particle has application prospects in the preparation of medicines or reagents for preventing or treating bovine viral diseases.

Description

Technical Field

[0001] This invention relates to fusion proteins of interferon and ferritin and their mutants, particularly to fusion proteins obtained by fusing bovine type I α 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 interferon and ferritin and their mutants and applications. Background Technology

[0002] Interferons (IFNs) are highly active, multifunctional glycoproteins induced in specific cells by certain inducing agents, 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. Due to their high species specificity and multiple functions including antiviral, antitumor, cell proliferation inhibition, and immunomodulation, interferons can inhibit both RNA and DNA viruses. Therefore, they are widely used in veterinary clinical practice for the treatment and prevention of various viral infectious diseases, effectively reducing the use of antibiotics.

[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 efficacy. Bovine type I alpha interferon has defects such as low antiviral activity, short retention time in the body, and lack of targeting, which affect its efficacy in the prevention or treatment of bovine viral diseases and needs to be improved. Summary of the Invention

[0005] One of the objectives of this invention is to provide a fusion protein (Ferritin-BoIFN-α) obtained by fusing bovine type I α interferon (BoIFN-α) and ferritin subunits.

[0006] A second objective of this invention is to provide single-site or multi-site mutants of the fusion protein of bovine type I α interferon and ferritin subunit;

[0007] The third objective of this invention is to provide a method for preparing a fusion protein of bovine type I α interferon and ferritin subunit or a mutant thereof;

[0008] The fourth objective of this invention is to apply the fusion protein of bovine type I α interferon and ferritin subunit or its mutant to the preparation of drugs or reagents for the prevention or treatment of bovine viral diseases.

[0009] To achieve the above objectives, the technical solution adopted by the present invention includes:

[0010] To address the shortcomings of bovine type I alpha interferon, such as low antiviral activity, short in vivo retention time, and lack of targeting, this invention fuses bovine type I alpha 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 alpha interferon, reducing the frequency of administration and imparting a certain degree of targeting. Furthermore, the fusion protein is further mutated to obtain single-site or multi-site mutants with significantly enhanced antiviral activity. In addition, a method for preparing the fusion protein or its mutants is provided, thus completing this invention.

[0011] One aspect of the present invention is to provide a fusion protein of bovine type I α-interferon and ferritin subunits, wherein the fusion protein is obtained by connecting the C-terminus of the monomeric ferritin subunit to the N-terminus of bovine type I α-interferon through a linker with a highly conformationally flexible linker, thereby exhibiting bovine type I α-interferon on the surface of the ferritin nanostructure.

[0012] The amino acid sequence of bovine type I α-interferon or ferritin subunit described in this invention can be an amino acid sequence derived from NCBI.

[0013] 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 162 to 176 from the amino acid sequence of the bovine ferritin monomeric subunit.

[0014] In a preferred embodiment of the present invention, the amino acid sequence of bovine type I α interferon is shown in SEQ ID No. 1, and the nucleotide sequence of its encoding gene is shown in SEQ ID No. 2.

[0015] In a preferred embodiment of the present invention, the amino acid sequence of the linker peptide is shown in SEQ ID No. 3.

[0016] In one 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 α-interferon (SEQ ID No. 1) via the linker peptide shown in SEQ ID No. 3 to obtain a fusion protein, thereby displaying bovine type I α-interferon on the surface of ferritin nanoparticles. The amino acid sequence of the resulting fusion protein of bovine type I α-interferon and ferritin subunit is shown in SEQ ID No. 4. Furthermore, the encoding gene of the fusion protein is codon-optimized according to the codon preference of silkworms to obtain a codon-optimized gene with the nucleotide sequence shown in SEQ ID No. 5.

[0017] In order to improve the antiviral activity or titer of bovine type I α interferon-ferritin subunit fusion protein, this invention performs single point mutations or multi-site mutations on the fusion protein and screens these mutants to obtain single point mutants or multi-site mutants with significantly improved antiviral activity or titer.

[0018] Therefore, another aspect of the present invention provides a single-site mutant or multi-site mutant of bovine type I α-interferon-ferritin subunit fusion protein, including: a single-site mutant obtained by single-site mutation of the amino acids of the bovine type I α-interferon-ferritin subunit fusion protein with the amino acid sequence shown in SEQ ID No. 4 according to any one of K248R, A251G, K282Q, E312K or S335R; or a double-site mutant obtained by double-site mutation of the amino acids of the bovine type I α-interferon-ferritin subunit fusion protein with the amino acid sequence shown in SEQ ID No. 4 according to any one of K248R-A251G, K282Q-E312K, K282Q-S335R or E312K-S335R, wherein the antiviral activity or titer of these single-site mutants or double-site mutants is significantly enhanced or improved compared with the bovine type I α-interferon-ferritin subunit fusion protein.

[0019] In this invention, the meaning of the "K248R" single point mutation refers to the mutation of lysine at position 248 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.

[0020] The meaning of the "K248R-A251G" double-site mutation mentioned in this invention refers to the mutation of lysine at position 248 of the fusion protein shown in SEQ ID NO.4 to arginine and simultaneously the mutation of alanine at position 251 to glycine. The meanings of other double-site mutations are deduced by analogy.

[0021] In this invention, the single-site mutants or two-site mutants of the above-mentioned fusion proteins were expressed in the silkworm eukaryotic expression system. The expression results showed that the expression levels of these single-site mutants or two-site mutant sequences were significantly increased compared with the original fusion proteins.

[0022] Another aspect of the present invention provides a method for preparing the bovine type I α interferon-ferritin subunit fusion protein or a single-site or multi-site mutant thereof, comprising:

[0023] (1) Construct a baculovirus eukaryotic expression transfer vector containing the coding gene of the fusion protein or its single-site or multi-site 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.

[0024] Another aspect of the present invention is to use the bovine type I α-interferon-ferritin fusion protein or its mutants in 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.

[0025] Those skilled in the art can prepare bovine type I α-interferon-ferritin fusion protein or its mutants into various pharmaceutical preparations or reagents for the prevention or treatment of bovine viral diseases using conventional pharmaceutical preparation methods in the art, all of which are known to those skilled in the art.

[0026] Compared with the prior art, the present invention has the following advantages or effects:

[0027] 1. The fusion protein provided by this invention connects bovine alpha interferon and ferritin subunits through a flexible linker to achieve surface display of the interferon protein, forming a conformation suitable for its function. This effectively increases the expression level and activity of bovine alpha interferon, while also improving the stability of bovine alpha interferon and prolonging its half-life, thus effectively extending the degradation time of bovine alpha interferon in vivo.

[0028] 2. The bovine type I α-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, small molecular weight, strong permeability, 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, and immune adjuvants. Its small molecular weight and high stability make it suitable for the prevention or treatment of viral diseases in cattle.

[0029] 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.

[0030] Definitions of terms involved in this invention

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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, f-pairing, 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.

[0035] 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

[0036] Figure 1 shows the double enzyme digestion identification results of recombinant plasmids pBR-BoIFN-α and pBR-Ferritin-BoIFN-α; the large fragment is the 7618bp pBR transfer vector, and the small fragments are the 582bp BoIFN-α and 1107bp Ferritin-BoIFN-α target genes, respectively.

[0037] Figure 2 is a schematic diagram showing the statistical control of cytopathic effects in fluorescent wells in the Vero / VSV*GFP system.

[0038] Figure 3 Serum drug metabolism diagrams for BoIFN-α and Ferritin-BoIFN-α. Detailed Implementation

[0039] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, it should be understood that the embodiments described 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.

[0040] Example 1: Construction and expression of bovine alpha interferon protein, bovine alpha interferon-ferritin fusion protein, and their mutant expression vectors.

[0041] Bovine ferritin and bovine alpha interferon sequences were obtained from the NCBI database and used to generate the sequences using OptimumGene. TM After technical optimization, the resulting bovine α-interferon amino acid sequence is shown in SEQ ID No. 1, and the protein is named BoIFN-α. MAPAWSFLLALLLLSCNAICSLGCHLPHTHSLANRRVLTLLRQLRRVSPSSCLQDRNDFAFPQEALGGSQLQKAQAISVLHEVTQHTFQLFSTEGSAAVWDESLLDKLRAALDQQLTDLQACLRQEEGLRGAPLLKEDSSLAVRKYFHRLTLYLQEKRHSPCAWEVVRAEVMRAFSSSTNLQERFRRKD* (SEQ ID No. 1).

[0042] 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 α-interferon protein (BoIFN-α) (SEQ ID No. 2).

[0043] Using the linker peptide GGGSGGGGSGGGS (SEQ ID NO.3), bovine alpha interferon (SEQ ID NO.1) was fused to amino acid position 161 of the bovine ferritin subunit (i.e., amino acids 162-176 were deleted). The amino acid sequence of the resulting bovine alpha interferon-ferritin fusion protein is shown in SEQ ID No.4. This fusion protein was named Ferritin-BoIFN-α. MTTASPSQVRQNYHQDSEAAINRQINLELYASYVYLSMSYYFDDDVALKNFAKYFLHQSHEEREHAERLMKLQNQRGGRIFLQDIKKPDRDDWENGLTAMECALCLERSVNQSLLELHKLATEKNDPHLCDFIETHYLNEQVEAIKELGDHITNLRKMGAPGGGSGGGGSGGGSMAPAWSFLL ALLLLSCNAICSLGCHLPHTHSLANRRVLTLLRQLRRVSPSSCLQDRNDFAFPQEALGGSQLQKAQAISVLHEVTQHTFQLFSTEGSAAVWDESLLDKLRAALDQQLTDLQACLRQEEGLRGAPLLKEDSSLAVRKYFHRLTLYLQEKRHSPCAWEVVRAEVMRAFSSSTNLQERFRRKD* (SEQ ID No.4).

[0044]

[0045] 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-α (pUC57 with SEQ ID No. 2 inserted) and pUC57-Ferritin-BoIFN-α 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-α plasmid.

[0046] Using pBR-Ferritin-BoIFN-α plasmid as a template, a large number of single point mutations were performed using fusion PCR. The effective single point mutations and the corresponding primers used for mutation are listed in Table 1.

[0047] Table 1 Primer sequences used for effective point mutations

[0048]

[0049] The effective mutations (K248, A251, K282, E312, or S335) in the amino acid sequence shown in SEQ ID NO.4 were progressively mutated using fusion PCR. The resulting effective mutant was named Ferritin-BoIFN-α. mut 1 (K248R, A251G, K282Q, E312K, S335R); Based on obtaining effective single-point mutations, various combinations of two-site mutations were further performed, and the better two-site combination mutants were named Ferritin-BoIFN-α. mut 2 (K248R-A251G, K282Q-E312K, K282Q-S335R, or E312K-S335R). Because the two sites of K248R-A251G are relatively close, new intermediate upstream and downstream primers were redesigned based on Ferritin-BoIFN-α-K248R and are listed in Table 2.

[0050] Table 2. Primer Design for K248R-A251G Double Mutant

[0051]

[0052] The PCR reaction system is shown in Table 3.

[0053] Table 3 PCR reaction system

[0054]

[0055] 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.

[0056] Product recovery: The PCR product was 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.

[0057] Enzyme digestion and recovery: using restriction endonucleases Bam H Ⅰ and Eco R Ⅰ The above PCR products and pUC57-BoIFN-α and pUC57-Ferritin-BoIFN-α were double digested and inactivated at 85℃ for 10 min. The DNA was then recovered using the Tiangen agarose gel DNA recovery kit and stored at -20℃ for later use.

[0058] 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 Trans5α, colonies were selected for culture, plasmids were extracted, and... Bam HI and Eco RI double enzyme digestion identified positive clones containing a 7618 bp pBR transfer vector as the large fragment, and target gene fragments of 582 bp and 1107 bp, respectively. Electrophoresis results are as follows: Figure 1 The correctly identified recombinant plasmids were sequenced, and the correctly sequenced plasmids were named pBR-BoIFN-α and pBR-Ferritin-BoIFN-α.

[0059] Table 4 Connection System

[0060]

[0061] 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 from cell culture flasks into single-cell suspensions 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-α and pBR-Ferritin-BoIFN-α 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-α) and BmBac (Ferritin-BoIFN-α) containing the target gene.

[0062] 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-α) and BmBac (Ferritin-BoIFN-α) are obtained.

[0063] Normally growing BmN cells were infected with recombinant silkworm baculoviruses BmBac (BoIFN-α) and BmBac (Ferritin-BoIFN-α). After culturing for 3-5 days, the supernatant was collected, which contained a large number of recombinant viruses BmBac (BoIFN-α) and BmBac (Ferritin-BoIFN-α).

[0064] 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 alpha interferon and bovine alpha 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 have significantly shrunk in size and have stopped feeding, collect the hemolymph and store it at -20℃ for later use.

[0065] Experimental Example 1: Detection of antiviral activity of bovine alpha interferon and bovine alpha interferon-ferritin fusion protein

[0066] Using the same method as the national standard for mammalian interferon determination, the antiviral activity of bovine type I α interferon (BoIFN-α) and the fusion protein of ferritin subunit (Ferritin-BoIFN-α) expressed in the silkworm samples of Example 1 was detected on the VERO / VSV*GFP system using the micro-cytopathic effect inhibition method.

[0067] healthy VERO cells were injected with 5.0 × 10⁻⁶ cells. 5 Silkworm samples were inoculated at a density of 100 cells / mL into 96-well plates. The sonicated and filtered silkworm samples were prepared into different dilutions using DMEM / F12 medium containing 2% fetal bovine serum. 100 μL of the diluted samples were inoculated 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 28°C and 5% CO2 for 18–24 h. VSV*GFP virus diluted to 100 TCID50 was added at 100 μL / well to wells after the supernatant had been removed, and the plates were incubated at 28°C and 5% CO2. Under an inverted fluorescence microscope, when a suitable number of cells in each well of the virus control group showed fluorescence, while the cells in the cell control group remained fully grown and showed no fluorescence, it indicated that the control system was fully qualified and could be fully observed. Figure 2 is a schematic diagram of the control of the degree of cell lesions in the fluorescence wells of the Vero / VSV*GFP system. The results of the antiviral activity assay of bovine α-interferon and bovine α-interferon-ferritin fusion protein are listed in Table 5.

[0068] Table 5. Results of antiviral activity assay of bovine interferon alpha and bovine interferon alpha-ferritin fusion protein

[0069]

[0070] The results of the antiviral activity assay showed that the antiviral activity of bovine alpha interferon-ferritin fusion protein was significantly higher than that of bovine alpha interferon.

[0071] Experimental Example 2: Detection of the antiviral activity of bovine alpha interferon-ferritin fusion protein and its mutants

[0072] The antiviral activity of bovine α-interferon-ferritin fusion protein and its effective single-site or multi-site mutants was determined in the same way as in Experiment 1. The antiviral titer test results of some single-site or multi-site mutants are listed in Table 6.

[0073] Table 6. Results of antiviral activity assay of bovine alpha interferon-ferritin fusion protein and its mutants.

[0074]

[0075] As shown in Table 7, the antiviral activity results indicate that the antiviral titers of the five single-site mutants (K248R, A251G, K282Q, E312K, and S335R) and the four double-site mutants (K248R-A251G, K282Q-E312K, K282Q-S335R, and E312K-S335R) are significantly higher than those of the bovine interferon-alpha ferritin fusion protein. The antiviral titers of the remaining single-site mutants or double-site mutants are lower than those of the bovine interferon-alpha ferritin fusion protein.

[0076] Experimental Example 3: Plasma stability assay of bovine alpha interferon and bovine alpha interferon-ferritin fusion protein

[0077] Eighteen healthy male rats weighing 180-220 g were randomly divided into three groups and intravenously injected with a single dose (10 million IU / kg). -1 Bovine interferon alpha (BoIFN-α) and bovine interferon alpha-ferritin (Ferritin-BoIFN-α) fusion proteins, as well as a control group without interferon treatment, were included. Serum samples were collected immediately after injection (0 h), and at 0.5, 2.0, 4.0, 8.0, 12, 24, 48, 72, 96, 120, and 144 h. Their anti-VSV-GFP activity was determined using the method described in Example 1.

[0078] The results showed that the half-life (T1 / 2) of BoIFN-α in vivo was 4.56 h, while that of Ferritin-BoIFN-α was 26.32 h, which was 5.77 times longer than that of BoIFN-α. Figure 3 ).

Claims

1. A mutant of a fusion protein obtained by fusing bovine type I α-interferon and ferritin subunits, characterized in that, The mutant is a single-point mutant obtained by mutating the amino acids of the bovine type I α-interferon-ferritin subunit fusion protein with the amino acid sequence shown in SEQ ID No. 4 using the E312K amino acid single-point mutation method; or the mutant is a two-site mutant obtained by mutating the amino acids of the bovine type I α-interferon-ferritin subunit fusion protein with the amino acid sequence shown in SEQ ID No. 4 using any one of the two-site mutations K282Q-E312K or E312K-S335R.

2. The coding gene of the mutant according to claim 1.

3. An expression vector containing the encoding gene as described in claim 2.

4. A host cell containing the expression vector of claim 3.

5. The 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 described above; (2) The constructed baculovirus eukaryotic expression transfer vector and baculovirus genomic DNA were co-transfected into insect cells to obtain recombinant baculovirus; (3) The recombinant baculovirus was used to infect insect hosts or cells, and the fusion protein or its mutant was expressed in insect cells or live insects. The protein was then purified and renatured to obtain the baculovirus.

6. Use of the mutant of claim 1, the encoding gene of claim 2, or the expression vector of claim 3 in the preparation of a drug or reagent for the prevention or treatment of bovine viral diseases.

7. The use according to claim 6, characterized in that, The bovine viral diseases mentioned include any one of bovine viral diarrhea, foot-and-mouth disease, bovine respiratory disease, bovine mastitis, or bovine endometritis.

Images