A fusion protein of interferon alpha 2 and thymosin alpha 1 and its preparation method and application

Abstract

The application belongs to the technical field of biology, and particularly relates to a fusion protein of interferon alpha 2 and thymosin alpha 1 and a preparation method and application thereof. The fusion protein provided by the application has good antiviral activity, and shows an effect obviously superior to that of interferon alpha 2 or thymosin alpha 1 alone in terms of antiviral activity, and has the ability of enhancing T-lymphocyte immune response, thereby providing a safer and more effective treatment scheme for enhancing cell immunity for the livestock industry. In addition, the fusion protein can be efficiently expressed in Escherichia coli through specific conditions, and has significant biological activity after denaturation and renaturation to realize correct folding of the protein, and can be widely used in the development of antiviral veterinary drugs or drugs for enhancing immunity.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a fusion protein of interferon α2 and thymosin α1, its preparation method, and its application. Background Technology

[0002] In animal husbandry, viruses are often characterized by high concealment, strong pathogenicity, and rapid spread, making them a significant factor in economic losses. For example, vesicular stomatitis virus (VSV) is a virus that primarily hosts ruminants and cloven-hoofed animals such as horses, cattle, pigs, sheep, and camels. Infected animals develop grayish-white blisters and ulcers in the mouth, tongue, and hooves. Severe cases result in drooling, lameness, difficulty feeding, reduced egg production, decreased milk production, and weight loss. Although the mortality rate of VSV is not high, its recovery period is long, and there is currently no specific treatment. Furthermore, VSV infection symptoms are easily misdiagnosed as foot-and-mouth disease, leading to international trade embargoes and causing devastating economic losses. Chicken adenovirus (FAdV) is a virus that primarily hosts chickens. It can cause inclusion body hepatitis (IBH) or pericardial effusion syndrome (HHS), leading to weight loss and a sharp decline in egg production. The mortality rate of highly pathogenic adenoviruses can reach 30%-80%, making it one of the leading causes of death in the poultry industry. Although vaccines exist for FadV, the prevalent strains vary greatly across different regions, making vaccine selection difficult. Furthermore, viral mutations can lead to vaccine incompatibility, and high levels of maternal antibodies can interfere with the effectiveness of chick vaccination. Current strategies for combating FadV primarily rely on immune enhancement and adjuvant therapy, lacking antiviral drugs.

[0003] Interferon-α2 (IFNα2) is a biopharmaceutical effective in treating viral infections and various cancers. IFNα2 inhibits viral replication through mechanisms such as inducing interferon-stimulated genes (ISGs), activating JAK-STAT (Janus kinase / signal transduction and transcription activator) MAP kinase, and the PI3K-AKT-MTOR (phosphatidylinositol 3-kinase / protein kinase B / mammalian target of rapamycin) signaling pathway. However, for febrile alpha (FAdV), IFNα2 requires higher doses to produce significant antiviral activity.

[0004] Thymosin α1 (Tα1) is a 28-amino acid peptide that can regulate immune responses through Toll-like receptors (TLRs), enhancing the activity of NK cells, T cells, dendritic cells (DCs), and macrophages, thereby exerting an antiviral effect. Tα1 can also increase the expression of marker proteins on the surface of virus-infected cells, preventing these cells from evading recognition by immune cells. However, Tα1 cannot directly exert an effective inhibitory effect on viruses.

[0005] Therefore, it is necessary to develop more effective antiviral products to address the widespread problem of viral infection and transmission in animal husbandry. Summary of the Invention

[0006] To address the above problems, this invention provides a fusion protein of interferon α2 and thymosin α1, its preparation method, and its applications. The fusion protein provided by this invention enhances T-lymphocyte immune responses and exhibits excellent antiviral activity, significantly inhibiting the activity of viruses such as vesicular stomatitis virus (VSV) and chicken adenovirus (FAdV), and improving cell survival rate. Furthermore, this fusion protein can be expressed in a host cell and can be produced in a green, industrialized manner through biosynthesis.

[0007] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0008] The first aspect of the present invention provides a fusion protein of interferon α2 and thymosin α1, the fusion protein comprising interferon α2, a linker peptide and thymosin α1 in series.

[0009] This fusion protein, composed of interferon α2 and thymosin α1, has the ability to significantly enhance T-lymphocyte immune responses and can serve as an enhancer of animal immunogenicity. At the same time, it is significantly superior to the effect of interferon α2 or thymosin α1 alone at the same dose in terms of antiviral activity. It can effectively and directly inhibit the activity of viruses such as vesicular stomatitis virus and chicken adenovirus, improve cell survival rate, and thus effectively solve the problem of viral infection and transmission in animal husbandry.

[0010] Furthermore, this fusion protein can be highly expressed in E. coli and can be produced on a large scale and at low cost through biosynthesis, making it easier for its widespread application.

[0011] Preferably, the linker peptide is a flexible linker peptide.

[0012] Preferably, the amino acid sequence of the linker peptide is shown in SEQ ID No. 5.

[0013] Preferably, the amino acid sequence of the fusion protein is shown in SEQ ID No. 1. This amino acid sequence contains an interferon amino acid sequence derived from chicken, and a small molecule polypeptide amino acid sequence with specific immune effects isolated from calf thymus. It can promote T cell activity and immune balance, and also enhance the antigenic capacity of T cells, thereby improving the body's immunity.

[0014] More preferably, the gene sequence encoding interferon α2 is shown in SEQ ID No. 3.

[0015] More preferably, the gene sequence encoding thymosin α1 is shown in SEQ ID No. 4.

[0016] More preferably, the gene sequence encoding the linker peptide is shown in SEQ ID No. 6.

[0017] A second aspect of the present invention provides a recombinant expression vector pET21a-IFNα2-Tα1, wherein the recombinant expression vector pET21a-IFNα2-Tα1 expresses the above-mentioned fusion protein, or contains the coding gene of the above-mentioned fusion protein.

[0018] Preferably, the backbone vector of the recombinant expression vector pET21a-IFNα2-Tα1 is the pET-21a(+) plasmid.

[0019] A third aspect of the present invention provides a recombinant expression strain that expresses the above-mentioned recombinant expression vector pET21a-IFNα2-Tα1.

[0020] Preferably, the starting strain of the recombinant expression strain is *Escherichia coli*. E. coli BL21(DE3).

[0021] A fourth aspect of the present invention provides a method for expressing the above-mentioned fusion protein using the above-mentioned recombinant expression strain, specifically comprising the following steps: culturing the recombinant expression strain in a fermentation medium, and when OD... 600 When the concentration reaches 20-30, add IPTG to make the final concentration 1 mM, and induce at 37±3℃ for 10-14 h. Separate the solid and liquid, and collect the cells. Break the obtained cells, centrifuge, and collect the precipitated inclusion body proteins.

[0022] Preferably, the fermentation medium comprises: glucose, peptone, yeast extract, KH2PO4, K2HPO4, Na2HPO4, NH4Cl, (NH4)2SO4, MnSO4, CoCl2, Na2MoO4, ZnCl2, CuSO4, H3BO4, FeSO4, CaCl2, MgSO4, and an antifoaming agent, with a pH of 7.

[0023] More preferably, the fermentation medium comprises: 2 g / L glucose, 2 g / L peptone, 2 g / L yeast extract, 1 g / L KH2PO4, 2 g / L K2HPO4, 2 g / L Na2HPO4, 1 g / L NH4Cl, 0.3 g / L (NH4)2SO4, 0.001 g / L MnSO4, 0.001 g / L CoCl2, 0.002 g / L Na2MoO4, 0.05 g / L ZnCl2, 0.01 g / L CuSO4, 0.01 g / L H3BO4, 0.001 g / L FeSO4, 0.001 g / L CaCl2, 0.01 g / L MgSO4, and an antifoaming agent, with a pH of 7 and sterile water as the solvent.

[0024] Preferably, the conditions for culturing the recombinant expression strain in the fermentation medium are: controlling the glucose concentration to not exceed 1.5% and the dissolved oxygen (DO) to be controlled at 40% to 50%.

[0025] Preferably, the final concentration of IPTG is 1 mM.

[0026] Preferably, the induction time is 12 h.

[0027] Preferably, the centrifugation parameters are: centrifugation at 4℃ and 9000 rpm for 1 h.

[0028] Preferably, the method further includes washing the inclusion body proteins.

[0029] More preferably, the method for washing the inclusion body proteins is as follows: washing with washing buffer I and washing buffer II, then washing with 2 M urea and 1 M NaCl, centrifuging, and collecting the supernatant; the washing buffer I consists of 50 mM Tris, 5 mM EDTA, 0.8% NaCl, and pH 8.5; the washing buffer II consists of 0.8% NaCl, 0.02% KCl, 10 mM Na2HPO4, 1% Triton X-100, and 1.7 mM KH2PO4.

[0030] More preferably, the centrifugation parameters are: centrifugation at 9000 rpm for 30 min at 4°C.

[0031] Preferably, the method further includes a modification process and a remodeling process.

[0032] More preferably, the denaturation process is as follows: the inclusion bodies are fully dissolved in denaturation buffer at 4°C, centrifuged, and the supernatant is collected; the refolding process is as follows: refolding buffer is added to the supernatant obtained from the denaturation process, and the mixture is refolded at 4°C for 48 h. The centrifugation parameters are preferably 4°C, 9000 rpm, and 30 min; the denaturation buffer consists of 6 M guanidine hydrochloride, 2 mM EDTA, 50 mM Tris, and 10 mM DTT; the refolding buffer consists of 0.5 M arginine, 2 mM EDTA, 0.9 mM GSSG, 100 mM Tris, and 20% glycerol.

[0033] The denaturation process described above can fully reduce protein disulfide bonds and achieve correct protein folding in the refolding system, thereby significantly improving refolding efficiency and biological activity.

[0034] The fifth aspect of this invention provides the application of the above-mentioned fusion protein in the preparation of veterinary disinfectants, antiviral veterinary drugs, or medicines for improving the immunity of livestock and poultry.

[0035] Preferably, the veterinary disinfectant is an antiviral disinfectant for vesicular stomatitis.

[0036] Preferably, the veterinary disinfectant is an anti-chicken adenovirus disinfectant.

[0037] Preferably, the antiviral veterinary drug is a drug for treating vesicular stomatitis virus.

[0038] Preferably, the antiviral veterinary drug is an antiviral drug for chicken adenovirus.

[0039] The beneficial effects of this invention are as follows: The fusion protein provided by this invention is derived from the tandem link of the interferon α2 gene (IFNα2) and the thymosin α1 gene (Tα1), exhibiting advantages such as good antigenicity and high antiviral activity. This fusion protein not only enhances T-lymphocyte immune responses and can serve as an immunogenic enhancer in animals, but also possesses excellent antiviral replication capabilities, effectively inhibiting the activity of viruses such as vesicular stomatitis virus and chicken adenovirus, and improving cell survival rates, thereby reducing the economic losses to livestock caused by viral infection and transmission. Furthermore, compared with conventional chemically synthesized thymosin, the fusion protein provided by this invention can be expressed in the host, has high yield, and is environmentally friendly, reducing the production cost of synthesis and facilitating its large-scale production and widespread application. Attached Figure Description

[0040] Figure 1 The enzyme digestion detection results of the recombinant expression vector pET21a-IFNα2-Tα1 in Example 2 of the present invention are shown; lane 1 is the original plasmid pET-21a(+); lane 2 is the NdeI and XhoI double digestion product of the recombinant expression vector pET21a-IFNa2-Tα1; lane 3 is the DNA Marker. Figure 2 The recombinant IFNα2-Tα1 fusion protein in Example 1 of this invention was tested in Escherichia coli. E. coli The expression effect in BL21(DE3) is shown in the following diagram: lane 1 is the marker; lane 2 is the inclusion body protein obtained in step 1 of Example 3; lane 3 is the refolding solution of the refolded sample containing the fusion protein inclusion body obtained in step 3 of Example 3; and lane 4 is the purified IFNα2-Tα1 protein obtained in step 4 of Example 3. Figure 3 The results of Western blot analysis of the recombinant IFNα2-Tα1 fusion protein in Example 1 of this invention, performed using an anti-interferon α2 antibody, are shown. Lane 1 is the marker, and lane 2 is the purified protein containing the IFNα2-Tα1 fusion protein obtained in Example 3. Figure 4The results of the rosette experiment in Example 2 of this invention were used to detect the immunological activity of the recombinant IFNa2-Tα1 fusion protein. Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. All other embodiments obtained by those skilled in the art based on the implementation methods of this invention without inventive effort fall within the protection scope of this invention.

[0042] Viruses are a significant factor contributing to economic losses in livestock farming. For example, there is currently no specific treatment for vesicular stomatitis virus (VSV), and its symptoms are easily misdiagnosed as foot-and-mouth disease, leading to severe economic losses. Chicken adenovirus (FAdV) can cause a sharp decline in meat and egg production in chickens, and similarly, there is a lack of effective antiviral drugs. Interferon-α2 (IFNα2) has antiviral activity, but for FADV, higher doses are required to produce significant antiviral activity. Thymosin α1 (Tα1) can indirectly exert its antitumor effect by regulating immune cell activity, but it cannot directly and effectively inhibit viruses.

[0043] This invention provides a fusion protein of interferon α2 and thymosin α1, comprising interferon α2, a linker peptide, and thymosin α1 in tandem. This fusion protein significantly enhances T-lymphocyte immune responses and antiviral activity, effectively inhibiting the activity of viruses such as vesicular stomatitis virus and chicken adenovirus, and improving cell survival. Furthermore, this fusion protein can be highly expressed in *E. coli* and can be produced on a large scale and at low cost through biosynthesis.

[0044] This invention also provides a recombinant expression vector pET21a-IFNα2-Tα1 containing the coding gene of the above-mentioned fusion protein to express the fusion protein.

[0045] The present invention also provides a recombinant expression strain expressing the above-mentioned recombinant expression vector pET21a-IFNα2-Tα1, and a method for expressing the above-mentioned fusion protein using the recombinant expression strain.

[0046] This invention also provides the application of the above-mentioned fusion protein in the preparation of veterinary disinfectants, antiviral veterinary drugs, or medicines for improving the immunity of livestock and poultry.

[0047] The present invention will be described below through specific embodiments.

[0048] Unless otherwise specified, other reagents or instruments used in the following examples are all commercially available products. The methods used in the following examples are conventional methods in the art.

[0049] Example 1 This embodiment provides a fusion protein of interferon α2 and thymosin α1 (IFNα2-Tα1 fusion protein), the amino acid sequence of which is shown in SEQ ID No.1 and the encoding gene sequence is shown in SEQ ID No.2.

[0050] This fusion protein was synthesized by Shanghai Sangon Biotech Co., Ltd. by linking the 3' end of the IFNα2 gene of interferon α2 (as shown in SEQ ID No. 3) and the Tα1 gene of thymosin α1 (as shown in SEQ ID No. 4) with a flexible linker peptide (amino acid sequence as shown in SEQ ID No. 5, encoding gene sequence as shown in SEQ ID No. 6).

[0051] Example 2 This embodiment provides a recombinant expression vector pET21a-IFNα2-Tα1 for expressing the IFNα2-Tα1 fusion protein in Example 1, and its construction method is as follows: EcoRI and XhoRI restriction endonuclease sites were selected as the restriction sites for homologous recombination. Using the IFNα2-Tα1 fusion protein encoding gene shown in SEQ ID No. 2 as a template, specific amplification primers IFNα2-Tα1-F and IFNα2-Tα1-R were designed based on the sequences of the IFNα2 gene (as shown in SEQ ID No. 3), Tα1 gene (as shown in SEQ ID No. 4), linker peptide, and pET-21a(+) used in this patent. The restriction sites of EcoRI (restriction site: 5'-GAATTC-3') and XhoI (restriction site: 5'-CTCGAG-3') were inserted into the upstream primer IFNα2-Tα1-F (the sequence after inserting the EcoRI restriction site is shown in SEQ ID No. 7) and the downstream primer IFNα2-Tα1-R (the sequence after inserting the XhoI restriction site is shown in SEQ ID No. 8), respectively, to amplify the IFNα2-Tα1 fragment, thus achieving the fusion of Tα1 at the C-terminus of IFNα2.

[0052] Using homologous recombinase, the correctly synthesized IFNα2-Tα1 fragment was transferred into... E. coli BL21 was used to screen positive clones, from which plasmids were extracted and subjected to enzyme digestion detection (results are shown in the image). Figure 1The recombinant plasmid (as shown in SEQ ID No. 9) was subjected to DNA sequencing, and the sequencing results were completely consistent with the designed and synthesized sequence (including the promoter and terminator sequences, as shown in SEQ ID No. 9). Sequencing results showed that this embodiment successfully constructed the recombinant expression vector pET21a-IFNα2-Tα1 and the recombinant expression strain expressing this recombinant expression vector pET21a-IFNα2-Tα1.

[0053] Example 3 This embodiment provides a method for expressing the fusion protein of interferon α2 and thymosin α1 using the recombinant expression strain constructed in Example 2.

[0054] 1. Fermentation expression of recombinant expression strains Fermentation medium: 2 g / L glucose, 2 g / L peptone, 2 g / L yeast extract, 1 g / L KH2PO4, 2 g / L K2HPO4, 2 g / L Na2HPO4, 1 g / L NH4Cl, 0.3 g / L (NH4)2SO4, 0.001 g / L MnSO4, 0.001 g / L CoCl2, 0.002 g / L Na2MoO4, 0.05 g / L ZnCl2, 0.01 g / L CuSO4, 0.01 g / L H3BO4, 0.001 g / L FeSO4, 0.001 g / L CaCl2, 0.01 g / L MgSO4, and 0.5% v / v defoamer (KM-1040 high-efficiency polyether defoamer), pH 7, solvent: sterile water.

[0055] Fermentation culture conditions: The recombinant expression strain obtained in Example 2 was cultured in seed culture medium at 37℃, pH 6.8–7.2, dissolved oxygen (DO) 20%–50%, air flow rate 1 VVM–4.5 VVM, and stirring speed 200 rpm until the logarithmic growth phase. The inoculum size was 10%, and the seed culture medium was conventional LB medium containing 50 μg / mL of antibiotics. The cultured seed culture was added to the fermentation medium. During the culture process, the glucose concentration was controlled to not exceed 1.5%, and the dissolved oxygen (DO) was controlled to be 40%–50%. The culture was continued until the OD reached the growth phase. 600 When the concentration was 30, IPTG was added to a final concentration of 1 mM, and induction was performed at 37°C and 600 rpm for 12 h (OD). 600(Reaching a concentration of 60 or higher). Centrifuge at 4℃ and 9000 rpm for 20 min, discard the supernatant and collect the bacterial cells. Resuspend each gram of bacterial cells in 100 mL of PBS-bound buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.5), sonicate to disrupt the protein, and then centrifuge at 4℃ and 9000 rpm for 1 h, discard the supernatant and collect the inclusion body protein.

[0056] 2. Inclusion body washing process The solubility of inclusion bodies after washing was analyzed in different wash buffer solutions, and the degree of solubility in each solution was determined. Inclusion body proteins were washed sequentially with wash buffer I (50 mM Tris, 5 mM EDTA, 0.8% NaCl, pH 8.5) and wash buffer II (0.8% NaCl, 0.02% KCl, 10 mM Na₂HPO₄, 1% Triton X-100, 1.7 mM KH₂PO₄), followed by further washing with 2 M urea and 1 M NaCl to remove insoluble particles. Insoluble particles in the inclusion bodies were removed by centrifugation at 9000 rpm for 30 min at 4 °C. The clarified solutions were then used for denaturation and renaturation processes.

[0057] 3. Modification and remodeling processes Inclusion bodies were thoroughly dissolved in denaturing buffer (6 M guanidine hydrochloride, 2 mM EDTA, 50 mM Tris, 10 mM DTT) at 4°C at a volume ratio of 1:20 for 1 h. Insoluble particles were removed from the dissolved inclusion bodies by centrifugation (9000 rpm, 30 min, 4°C). The clarified solution (denatured sample containing fusion protein inclusion bodies) was then refolded. The clarified solution was then slowly added dropwise at 1 mL / min in refolding buffer (0.5 M arginine, 2 mM EDTA, 0.9 mM GSSG, 100 mM Tris, 20% glycerol) at 4°C at a volume ratio of 1:20 for 48 h to obtain the refolded solution (refolded sample containing fusion protein inclusion bodies).

[0058] 4. Purification The resulting refolded solution was concentrated by ultrafiltration and the pH was adjusted to 4.5 to precipitate most of the bacterial proteins. After centrifugation, the supernatant was collected. CM Sepharose FF was equilibrated with 20 mM NaAC-HAC equilibration buffer at pH 4.5, loaded onto the sample, and equilibrated again. Then, a gradient elution was performed using 20 mM NaAC-HAC elution buffer containing 0, 0.5, and 1 M NaCl for preliminary purification. Subsequently, the pH of the purified product was adjusted to 8.0, and QSepharose FF was equilibrated with 20 mM Tris-HCl pH 8.0 equilibration buffer, loaded onto the sample, and equilibrated again. A gradient elution was then performed using 30 mM Tris-HCl (pH 8.0) elution buffer containing 0, 0.5, and 1 M NaCl for further purification and concentration, yielding purified IFNα2-Tα1 protein containing the IFNα2-Tα1 fusion protein. Electrophoresis analysis showed that the purity of the obtained purified IFNα2-Tα1 protein reached over 95%.

[0059] Test Example 1 This test example performed Western blot analysis on the inclusion body protein obtained in step 1 of Example 3, the refolded fusion protein inclusion body sample obtained in step 3, and the purified IFNα2-Tα1 protein obtained in step 4.

[0060] The inclusion body proteins obtained in step 1 of Example 3, the refolding solution of the inclusion body containing fusion protein obtained in step 3, and the purified IFNα2-Tα1 protein obtained in step 4 were detected by conventional SDS-PAGE electrophoresis, and analyzed by a gel imaging system (Image Lab). The results are as follows: Figure 2 As shown.

[0061] The purified IFNα2-Tα1 protein was analyzed by SDS-PAGE electrophoresis and then transferred to a PVDF membrane. The expression product was then qualitatively and quantitatively analyzed using standard Western blotting. The Western blotting results of the recombinant IFNα2-Tα1 fusion protein in engineered bacteria are shown below. Figure 3 . Figure 3 The 25kDa region represents the purified IFNa2-Tα1 fusion protein designed for this experiment.

[0062] Test Example 2 This test example describes the immunoassay of the IFNα2-Tα1 fusion protein obtained in Example 3 by measuring its immunogenicity.

[0063] The immunomodulatory activity of purified IFNα2-Tα1 protein was determined using the standard rosette assay. In this experiment, piglet T lymphocytes adsorbed to three or more sheep erythrocytes were used as positive rosette cells, and 100 lymphocytes were randomly counted. Negative control: an equal volume of PBS was added; positive control: thymosin working concentrations of 1, 5, and 10 µg / mL; IFNa2-Tα1 purified protein group: IFNα2-Tα1 purified protein working concentrations of 1, 5, and 10 µg / mL (based on the amount of IFNα2-Tα1 fusion protein). Each experiment was performed in triplicate. The results are shown in Table 1. Microscopic observations of the IFNa2-Tα1 purified protein group are shown below. Figure 4 As shown.

[0064] Table 1 Results of the Rose Garland Experiment

[0065] Test Example 3 This test example measured the anti-vesicular stomatitis virus (VSV) activity of IFNα2, Tα1, and the purified IFNα2-Tα1 protein obtained in Example 3.

[0066] 2.5 × 10⁶ bovine serum albumin monolayers were cultured in 96-well plates. 5 ~3.5×10 5 / mL (100 μl per well); incubate for 4-6 h.

[0067] Sample group: After bovine serum-treated cells adhered, samples were added: interferon α2, thymosin α1, and purified IFNα2-Tα1 protein. Each sample was pre-diluted at a certain ratio and then serially diluted 10-fold, for a total of 3 dilutions, with 2-3 replicates per dilution (100 μl per well). The diluent was culture medium containing 7% serum. The pre-dilution factor needed to be adjusted based on the results of the interferon challenge protection experiment and this experiment. After 24 hours of incubation, the supernatant in the cell culture plate was discarded, and the culture medium was diluted to 100 CCID with 3% FBS. 50 VSV virus (100 μl per well, with 3% FBS culture medium added to cell control wells) was cultured at 37°C and 5% carbon dioxide for 24 hours (microscopic examination showed that 50% of the lesions in the standard solution were at 1 IU / mL).

[0068] Virus control: After bovine serum-containing cells adhered to the culture plate, medium containing 7% serum was added. After incubation for 24 hours, the supernatant in the cell culture plate was discarded, and 100 μl of culture medium diluted to 100 CCID with 3% FBS was added. 50 The VSV virus.

[0069] Cell control group: Bovine serum-treated cells were cultured in medium supplemented with 3% FBS after adhesion.

[0070] After the challenge was completed, the supernatant in the cell culture plate was discarded. 50 μl of staining solution was added to each well and incubated at room temperature for 30 min. The staining solution was then carefully rinsed off with running water and the residual water was blotted dry. 100 μl of destaining solution was added to each well and incubated at room temperature for 5 min. After mixing, the results were recorded at 570 nm using a microplate reader with 630 nm as the reference wavelength.

[0071] The results showed that under treatment with 100 IU / mL IFNα2-Tα1 fusion protein, cell viability increased to 63.8% ± 5.6%; when the concentration of IFNα2-Tα1 fusion protein increased to 1000 IU / mL, cell viability further increased to 85.3% ± 4.9%. In the 10000 IU / mL IFNα2-Tα1 fusion protein treatment group, cell viability reached 91.2% ± 3.8%, close to the level of the cell control group. These results indicate that the IFNα2-Tα1 fusion protein samples exhibited clear antiviral activity within the measured dilution range, significantly inhibiting VSV-induced cytopathic effects, and the inhibitory effect gradually weakened with increasing sample dilution factor. Under the same molar concentration (1000 IU / mL), the cell viability of the IFNα2-Tα1 fusion protein treatment group was 85.3% ± 4.9%, which was significantly higher than that of the interferon α2 group alone (78.6% ± 6.3%) (P<0.05). The cell viability of the thymosin α1 treatment group alone (1000 IU / mL) was 36.3% ± 4.3%, showing only a slight protective effect, but the difference was not statistically significant (P>0.05). This reveals that the IFNα2-Tα1 fusion protein has certain advantages in anti-VSV activity.

[0072] Test Example 4 This test example determined the anti-chicken adenovirus activity of the purified IFNα2-Tα1 protein obtained in Example 3.

[0073] The antiviral titer of the IFNα2-Tα1 fusion protein was determined using a chicken adenovirus (FAdV) infection inhibition assay. Chicken adenovirus-sensitive monolayers of chicken cells (CEF cells) were seeded in 96-well cell culture plates at a cell density of approximately 2.5 × 10⁻⁶ cells / well. 5 ~3.5×10 5 Cells / mL, 100 μL per well, incubated at 37℃ and 5% CO2 for 4-6 h until cells adhere well.

[0074] After cell adhesion, 100 μL of purified IFNα2-Tα1 protein, thymosin α1, and interferon α2 samples were added to each well according to the concentrations specified in Table 2. Cell control and virus control wells were also included. The samples were pre-diluted appropriately and then serially diluted 10-fold, with 2-3 parallel wells for each dilution. After 24 h of cell culture following sample pretreatment, chicken adenovirus challenge solution was added to all wells except the cell control wells to achieve a final concentration of 100 CCID. 50 / well; add the same volume of 3% FBS culture medium to the cell control wells.

[0075] Continue culturing for 48–72 h and observe cytopathic effects under a microscope. Use the degree of inhibition of cytopathic effects as the indicator. If necessary, use crystal violet staining to quantitatively determine cell viability. Record the results at 570 nm on a microplate reader.

[0076] The experimental results are shown in Table 2.

[0077] Table 2 Results of anti-chicken adenovirus virus activity assay

[0078] The cell viability in the virus control group was 34.7% ± 4.5%, exhibiting a significant cytopathic effect. Treatment with the IFNα2–Tα1 fusion protein significantly improved the survival rate of FAdV-infected cells in a dose-dependent manner. At treatments of 100, 1000, and 10000 IU / mL fusion protein, the cell viability was 64.9% ± 5.8%, 82.7% ± 4.6%, and 92.1% ± 3.3%, respectively, all significantly higher than that in the virus control group (P<0.05).

[0079] Under the same conditions, the cell viability of the interferon α2 treatment groups alone was 56.3% ± 6.2%, 73.8% ± 5.4%, and 85.6% ± 4.1%, which were significantly lower than those of the corresponding concentrations of fusion protein treatment groups (P<0.05).

[0080] The cell viability rates in the thymosin α1-treated groups were 36.3 ± 4.5%, 37.9% ± 5.1%, and 35.7 ± 5.8%, respectively, which were not significantly different from the virus control group (P>0.05).

[0081] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A fusion protein of interferon α2 and thymosin α1, characterized in that, The fusion protein comprises interferon α2, a linker peptide, and thymosin α1 in tandem.

2. The fusion protein according to claim 1, characterized in that, The linker peptide is a flexible linker peptide.

3. The fusion protein according to claim 1, characterized in that, The amino acid sequence of the linker peptide is shown in SEQ ID No. 5; and / or The amino acid sequence of the fusion protein is shown in SEQ ID No.

1.

4. The fusion protein according to claim 3, characterized in that, The gene sequence encoding interferon α2 is shown in SEQ ID No. 3; and / or The gene sequence encoding thymosin α1 is shown in SEQ ID No. 4; and / or The gene sequence encoding the linker peptide is shown in SEQ ID No.

6.

5. A recombinant expression vector pET21a-IFNα2-Tα1, characterized in that, The recombinant expression vector pET21a-IFNα2-Tα1 expresses the fusion protein of claim 1, or contains the encoding gene of the fusion protein of claim 1.

6. The recombinant expression vector pET21a-IFNα2-Tα1 according to claim 5, characterized in that, The backbone vector of the recombinant expression vector pET21a-IFNα2-Tα1 is the pET-21a(+) plasmid.

7. A recombinant expression strain, characterized in that, The recombinant expression strain expresses the recombinant expression vector pET21a-IFNα2-Tα1 as described in claim 5 or 6.

8. A method for expressing the fusion protein of any one of claims 1 to 4 using the recombinant expression strain of claim 7, characterized in that, Specifically, the following operations are included: culturing the recombinant expression strain in a fermentation medium, and when the OD... 600 When the concentration reaches 20-30, add IPTG to make the final concentration 1 mM, and induce at 37±3℃ for 10-14 h. Separate the solid and liquid, and collect the cells. Break the obtained cells, centrifuge, and collect the precipitated inclusion body proteins.

9. The method according to claim 8, characterized in that, The fermentation medium comprises: glucose, peptone, yeast extract, KH₂PO₄, K₂HPO₄, Na₂HPO₄, NH₄Cl, (NH₄)₂SO₄, MnSO₄, CoCl₂, Na₂MoO₄, ZnCl₂, CuSO₄, H₃BO₄, FeSO₄, CaCl₂, MgSO₄, and an antifoaming agent, with a pH of 7; and / or The conditions for culturing the recombinant expression strain in the fermentation medium were as follows: glucose concentration not exceeding 1.5%, dissolved oxygen (DO) controlled at 40%–50%; and / or The final concentration of the IPTG is 1 mM; and / or The induction time is 12 h; and / or The centrifugation parameters are: centrifugation at 4℃ and 9000 rpm for 1 h; and / or The method further includes washing the inclusion body proteins; and / or The method also includes a modification process and a remodeling process.

10. The use of the fusion protein according to any one of claims 1 to 4 in the preparation of veterinary disinfectants, antiviral veterinary drugs, or medicines for improving the immunity of livestock and poultry.

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