Tat-recombinant avian beta defensin fusion protein and its coding gene and application

Abstract

The application discloses a TAT-recombinant avian beta defensin fusion protein and a coding gene and application thereof, and belongs to the technical field of variation or genetic engineering. The application firstly fuses TAT with a recombinant avian beta defensin (AvBD) to construct a TAT-recombinant avian beta defensin fusion protein, the efficient cell penetration ability of TAT is fused with the antiviral ability of the recombinant avian beta defensin, and the inhibiting effect on virus infection is significantly enhanced. The fusion protein shows excellent antiviral activity in an in-vitro cell model and an in-vivo live chicken experiment, and can be applied as an antiviral biological preparation. The application uses a cell-penetrating peptide TAT to prepare a transmembrane delivery system, realizes intracellular delivery of a protein in one step, significantly inhibits in-vitro and in-vivo proliferation of avian viruses such as ALV-J, REV, MDV, CIAV and the like, is safe and efficient, can be developed into a broad-spectrum antiviral biological preparation, and simultaneously creates a new idea for research on prevention and treatment of avian viruses.

Description

Technical Field

[0001] This invention relates to the field of mutation or genetic engineering technology, specifically to the TAT-recombinant avian β-defensin fusion protein and its encoding gene and applications. Background Technology

[0002] Viral infections have become a persistent threat to the global livestock and poultry industry. Among them, retroviruses such as Avian leukosis virus J subtype (ALV-J) and reticuloendotheliosis virus (REV) can cause immunosuppression and tumor development in chicken flocks through vertical transmission. Marek's disease virus (MDV), as a tumorigenic herpesvirus, can induce avian T-cell lymphoma, and chicken infectious anemia virus (CIAV) can cause aplastic anemia and lymphoid atrophy in chicks. These viruses cause direct economic losses exceeding tens of billions of US dollars annually to the global livestock industry. Current antiviral treatments still face multiple challenges: chemical drugs easily induce drug-resistant mutations in viruses (e.g., the sensitivity of nucleoside reverse transcriptase inhibitors to ALV-J is declining year by year); the development of broad-spectrum antiviral drugs is limited by the highly diverse viral replication mechanisms. Therefore, discovering natural antiviral molecules with strong targeting and low resistance rates has become a key direction for overcoming the current predicament.

[0003] In the host's innate immune defense system, β-defensins, as a class of cysteine-rich cationic antiviral peptides, have become a research hotspot due to their broad-spectrum antiviral activity and species specificity. Among them, members of the avian β-defensin (AvBDs) family have shown good performance in combating avian viral infections, exhibiting broad-spectrum antibacterial activity against fungi, bacteria, viruses, and various pathogenic microorganisms, while also possessing certain immunomodulatory effects. However, β-defensins have difficulty crossing cell membrane barriers due to insufficient membrane permeability; furthermore, the presence of non-essential domains in β-defensins increases steric hindrance, reducing their effective antiviral activity. This leads to the problem of excessively high dosages in practical applications, hindering their further promotion. Summary of the Invention

[0004] To address the aforementioned limitations of existing technologies, the present invention aims to provide a TAT-recombinant avian β-defensin fusion protein, its encoding gene, and its applications. This invention, for the first time, recombines and tandemly expresses the antiviral domains of avian β-defensin 2 (AvBD2), avian β-defensin 6 (AvBD6), and avian β-defensin 9 (AvBD9), and then fuses the cell-penetrating peptide TAT with recombinant avian β-defensin (AvBD) to construct the TAT-recombinant avian β-defensin fusion protein. Through the highly efficient cell penetration capability of TAT and the antiviral activity of recombinant avian β-defensin, the fusion protein's inhibition rate against viral infection is increased by 2-3 times. Furthermore, this invention establishes a TAT-based targeted delivery platform, providing a novel technological paradigm for the intervention of intracellular viral infections and overcoming the technical limitations of traditional antiviral drugs' inability to penetrate cell membranes. Moreover, this fusion protein possesses advantages such as strong targeting, low resistance to drug resistance, and broad-spectrum efficacy, providing a novel biological agent for antiviral treatment in livestock and poultry, and is expected to overcome the technical bottlenecks of existing chemical drugs and traditional vaccines.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides a TAT-recombinant avian β-defensin fusion protein, wherein the TAT-recombinant avian β-defensin fusion protein is a protein as shown in (A1) or (A2) below:

[0007] (A1) A protein consisting of the amino acid sequence shown in SEQ ID NO.3 of the sequence listing;

[0008] (A2) The protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein defined in (A1).

[0009] In the aforementioned proteins, a protein tag refers to a polypeptide or protein fused with the target protein using in vitro DNA recombination technology for expression, detection, tracking, and / or purification of the target protein. Specifically, to facilitate purification of the protein in (A1), a tag can be attached to the amino or carboxyl terminus of the protein in (A1). The tag can be Poly-His (typically six HHHHHH), HA (YPYDVPDYA), FLAG (DYKDDDDK), or c-myc (EQKLISEEDL), etc.

[0010] As a preferred embodiment, the TAT-recombinant avian β-defensin fusion protein is a protein defined in (A1) above with 6 His tags attached to its C-terminus, and its amino acid sequence is shown in SEQ ID NO.5.

[0011] The TAT-recombinant avian β-defensin fusion protein of this invention is based on "eGFP fluorescent tag + TAT + recombinant avian β-defensin". TAT (YGRKKRRQRRR) is a cell-penetrating peptide that can rapidly bind to the negatively charged cell membrane via its positively charged arginine-rich sequence, helping β-defensins breach the cell membrane barrier and reach the target sites of intracellular replicating viruses such as ALV-J and REV. This significantly improves the intracellular accumulation rate and concentration of β-defensins, solving the problem of "high dosage" caused by insufficient membrane permeability in traditional β-defensins, and laying the foundation for reducing the actual application dosage. The recombinant avian β-defensin is derived from functional fragments of three defensins. This reduces steric hindrance and improves the membrane penetration efficiency after fusion with TAT. Moreover, recombinant avian β-defensin, as a cysteine-rich cationic antiviral peptide, is a natural fit with the cationic properties of TAT. Both are positively charged, avoiding mutual repulsion and ensuring the structural stability of the fusion protein, while also enhancing its binding ability to the cell membrane. In addition to serving as a visual indicator protein for the transmembrane delivery system, the eGFP fluorescent tag can also guide the entire fusion protein to adopt the correct conformation, reducing the probability of the target protein forming inclusion bodies or misfolding during expression. Furthermore, the stable structure of eGFP is equivalent to providing an anchor point for the fusion protein, improving the structural stability of the entire fusion protein.

[0012] This invention fuses eGFP-TAT-recombinant avian β-defensin together, which can efficiently penetrate cell membranes and bind to the genomes of various viruses to inhibit viral replication.

[0013] In a second aspect, the present invention provides the use of the above-mentioned TAT-recombinant avian β-defensin fusion protein in the preparation of a medicament for inhibiting viral infection.

[0014] In the above applications, the virus is one or more of avian leukosis virus subtype J (ALV-J), reticuloendotheliosis virus (REV), Marek's disease virus (MDV), and chicken infectious anemia virus (CIAV).

[0015] In the above applications, the TAT-recombinant avian β-defensin fusion protein can inhibit viral proliferation both in vitro and in vivo. When used in vitro, the effective concentration of the TAT-recombinant avian β-defensin fusion protein is 0.01 mg / mL - 0.1 mg / mL; when used in vivo, the dosage of the TAT-recombinant avian β-defensin fusion protein is 1 mg / kg of the inoculated subject.

[0016] A third aspect of the invention provides a gene encoding a TAT-recombinant avian β-defensin fusion protein, said gene being any of the nucleic acid molecules shown in (i)-(iv) below:

[0017] (i) Nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO.4;

[0018] (ii) Nucleic acid molecules that encode the amino acid sequence shown in SEQ ID NO.3, except for (i);

[0019] (iii) Nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO. 6;

[0020] (iv) Nucleic acid molecules that encode the amino acid sequence shown in SEQ ID NO.5, except for (iii).

[0021] In a fourth aspect, the present invention provides the use of the gene encoding the above-mentioned TAT-recombinant avian β-defensin fusion protein in the preparation of a medicament for inhibiting viral infection.

[0022] In the above applications, the virus is one or more of ALV-J, REV, MDV, and CIAV.

[0023] A fifth aspect of the invention provides the use of a substance that promotes the expression of the gene encoding the TAT-recombinant avian β-defensin fusion protein in the preparation of a medicament for inhibiting viral infection; wherein the virus is one or more of ALV-J, REV, MDV, and CIAV.

[0024] In the above applications, the substance is any one of the following:

[0025] C1) An expression cassette containing the gene encoding the TAT-recombinant avian β-defensin fusion protein;

[0026] C2) A recombinant vector containing the gene encoding the TAT-recombinant avian β-defensin fusion protein, or a recombinant vector containing the expression cassette described in C1);

[0027] C3) Recombinant microorganisms containing the encoding gene of TAT-recombinant avian β-defensin fusion protein, or recombinant microorganisms containing the expression cassette described in C1), or recombinant microorganisms containing the recombinant vector described in C2).

[0028] The beneficial effects of this invention are:

[0029] (1) The core innovation of this invention lies in the first-time recombinant tandem expression of the antiviral functional domains of avian β-defensin 2 (AvBD2), avian β-defensin 6 (AvBD6), and avian β-defensin 9 (AvBD9), followed by the fusion of the cell-penetrating peptide TAT with recombinant avian β-defensin to construct a TAT-recombinant avian β-defensin fusion protein. Truncation of the antiviral functional domains reduces steric hindrance, removes non-essential domains that may be present in the full-length defensin, improves the stability and penetration efficiency after fusion with TAT, and simultaneously reduces immunogenicity. Through the efficient cell penetration capability of TAT and the antiviral activity of recombinant avian β-defensin, the inhibition rate of the fusion protein against viral infection is increased by 2-3 times. Furthermore, this invention establishes a TAT-based targeted delivery platform, providing a novel technical paradigm for the intervention of intracellular viral infection and overcoming the technical limitation of traditional antiviral drugs' inability to penetrate the cell membrane.

[0030] (2) The TAT-recombinant avian β-defensin fusion protein of the present invention has two technical advantages: 1) Highly efficient transmembrane and targeted binding ability, achieving rapid cell internalization through TAT, and specifically recognizing viral nucleic acids or envelope proteins through the conserved cysteine ​​domain of recombinant avian β-defensin; 2) Strong antiviral activity. In vitro experiments show that the viral load of ALV-J, REV, MDV, and CIAV is reduced to 1 / 20-1 / 10 of the viral load of the control group; in vivo experiments show that the viral load of ALV-J, REV, MDV, and CIAV in the liver, kidney, spleen, and bursa of Fabricius is reduced to 1 / 10-1 / 5 of the viral load of the control group. This fusion protein lays the foundation for highly specific broad-spectrum antiviral biological agents and has important translational value in the fields of avian disease prevention and control and early warning of zoonotic diseases. Attached Figure Description

[0031] Figure 1 The image shows the cell viability of DF-1 cells 24 h after treatment with TAT-recombinant avian β-defensin fusion protein.

[0032] Figure 2 The image shows the cell viability of DF-1 cells after 48 h of treatment with TAT-recombinant avian β-defensin fusion protein.

[0033] Figure 3 The image shows the cell viability of DF-1 cells after 72 h of treatment with TAT-recombinant avian β-defensin fusion protein.

[0034] Figure 4 Comparative graph of TAT, recombinant avian β-defensin and TAT-recombinant avian β-defensin transmembrane function on DF-1 as detected by laser confocal microscopy.

[0035] Figure 5The image shows the Western blot results of TAT-recombinant avian β-defensin fusion protein after transduction into DF-1 cells at 12 h, 24 h, 48 h, and 72 h.

[0036] Figure 6 The graph shows the mRNA expression level of TAT-recombinant avian β-defensin fusion protein after 48 hours of treatment with ALV-J, as detected by quantitative real-time PCR.

[0037] Figure 7 A graph showing the mRNA expression level of TAT-recombinant avian β-defensin fusion protein after 48 hours of REV treatment, as detected by quantitative real-time PCR.

[0038] Figure 8 A graph showing the mRNA expression level of TAT-recombinant avian β-defensin fusion protein after 48 hours of treatment with MDV, as detected by quantitative real-time PCR.

[0039] Figure 9 A graph showing the mRNA expression level of TAT-recombinant avian β-defensin fusion protein after 48 hours of treatment with CIAV by quantitative real-time PCR.

[0040] Figure 10 The graph shows the mRNA expression level of TAT-recombinant avian β-defensin fusion protein in vivo acting on ALV-J 3d, as detected by quantitative real-time PCR.

[0041] Figure 11 A graph showing the mRNA expression level of TAT-recombinant avian β-defensin fusion protein in vivo at REV 3d, as detected by quantitative real-time PCR.

[0042] Figure 12 A graph showing the mRNA expression level of TAT-recombinant avian β-defensin fusion protein in vivo as an in vivo agent of MDV 3d, as detected by quantitative real-time PCR.

[0043] Figure 13 A graph showing the mRNA expression level of TAT-recombinant avian β-defensin fusion protein in vivo acting on CIAV 3d, as detected by quantitative real-time PCR. Detailed Implementation

[0044] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0045] As mentioned earlier, β-defensins suffer from problems such as poor membrane permeability and high dosage during practical use. Therefore, this invention conducts an in-depth study of the fourteen defensins identified in chickens, namely avian β-defensins 1 to 14 (AvBD1-14). The antiviral mechanisms of different types of β-defensins vary, among which:

[0046] The antiviral mechanism of avian β-defensin 2 (AvBD2): AvBD2 specifically recognizes and binds to the genomic RNA of ALV-J and REV (especially the packaging signal sequence in the 5' untranslated region) through its positively charged α-helical domain, forming a stable RNA-peptide complex. This process not only interferes with the assembly of the viral nucleocapsid but also inhibits the binding and elongation of template RNA by reverse transcriptase (RT) through steric hindrance, resulting in a reduction of viral cDNA synthesis efficiency by more than 50%. At the same time, avian β-defensin 2 can reduce the transcription and translation of ALV-J and REV envelope protein ENV by downregulating the activation of the NF-κB signaling pathway in host cells, ultimately reducing the amount of viral particles released by 60%-80%.

[0047] The antiviral mechanism of avian β-defensin 6 (AvBD6): Against the DNA virus MDV, avian β-defensin 6 targets and binds to the origin of replication (Ori) region and the promoter sequences of immediate early genes (IE1 / IE2) of the viral genome DNA through its conserved β-sheet domain. It competitively inhibits the binding of viral DNA polymerase to the initiation protein, thereby blocking the initiation of MDV DNA replication. In addition, avian β-defensin 6 (AvBD6) can interfere with the folding and transport of the viral envelope glycoprotein gB by binding to the N-terminal glycosylation site of gB, while downregulating the expression levels of gE (a key protein for intercellular transmission) and pp38 (a viral replication regulatory protein), thereby reducing the plaque-forming units (PFU) of MDV in DF-1 cells by 1-2 orders of magnitude.

[0048] The antiviral mechanism of avian β-defensin 9 (AvBD9): Against chicken infectious anemia virus (CIAV), it mainly achieves its antiviral effect through the synergistic action of direct viral inactivation and immunomodulation. As a cationic antiviral peptide, avian β-defensin 9 (AvBD9) can target and bind to the non-enveloped capsid protein (VP1) of CIAV by electrostatic attraction, thereby disrupting the structural integrity of the viral particle and blocking the adsorption and invasion of the virus into host cells (such as bone marrow hematopoietic stem cells and T lymphocytes). At the immunomodulatory level, avian β-defensin 9 (AvBD9) can significantly upregulate the expression levels of key cytokines such as interferon-gamma (IFN-γ) and interleukin-12 (IL-12) in central lymphoid organs such as the thymus and bursa of Fabricius in chicks, and promote CD4+ expression. + / CD8 +The proliferation and differentiation of T lymphocytes can reverse the immunosuppressive microenvironment induced by CIAV. In addition, avian β-defensin 9 (AvBD9) can effectively reduce viral load by inhibiting the replication initiation stage of the viral single-stranded circular DNA genome in the host cell nucleus, thereby alleviating bone marrow hematopoietic dysfunction and systemic lymphoid tissue atrophy caused by CIAV infection, and ultimately significantly improving the survival rate of infected chickens and alleviating clinical symptoms.

[0049] The core basis for selecting AvBD2, AvBD6, and AvBD9 in this invention is their broad-spectrum antiviral activity, coverage of key tissue distribution sites, and synergistic functional effects. All three exhibit high efficiency and specificity in antiviral activity, and their combination can cover various avian tumor virus infection scenarios. In terms of tissue distribution, AvBD2 is mainly distributed in the respiratory tract and urogenital mucosa, AvBD6 is enriched in immune organs such as bone marrow, and AvBD9 is widely distributed in mucosal tissues, matching key sites of immune defense. Functionally, the three have complementary mechanisms, exerting their effects by disrupting the viral envelope, interfering with replication, and blocking adsorption; their combined use can also reduce the risk of viral resistance.

[0050] Furthermore, this invention optimized and screened the antiviral functional domains of AvBD2, AvBD6, and AvBD9, selecting their core antiviral functional domains for tandem to construct recombinant avian β-defensin (AvBD), the amino acid sequence of which is shown in SEQ ID NO. 1. By combining the core functional domains, the steric hindrance caused by non-essential structural domains in the defensin is reduced, thereby lowering immunogenicity.

[0051] Regarding the selection of cell-penetrating peptides, TAT peptide, as a typical representative of cationic CPPs, possesses highly efficient cell-penetrating properties that are not energy-dependent. It can rapidly bind to the negatively charged cell membrane through a positively charged sequence rich in arginine, helping β-defensins to break through the cell membrane barrier and reach the target sites of intracellular replicating viruses such as ALV-J and REV. Its penetration efficiency ranks among the top of CPPs, which can significantly improve the intracellular accumulation rate and concentration of β-defensins, solve the problem of "high dosage" caused by insufficient membrane permeability of traditional β-defensins, and lay the foundation for reducing the actual application dosage. β-defensin, a cysteine-rich cationic antiviral peptide, is a natural fit with the cationic properties of TAT. Both are positively charged, avoiding mutual repulsion and ensuring the structural stability of the fusion protein. At the same time, it enhances the binding ability to the cell membrane. Furthermore, the delivery function of TAT and the antiviral activity of β-defensin do not interfere with each other, forming a combined effect of "targeted delivery + direct antiviral". In particular, it significantly improves the clearance efficiency of intracellular viruses. In addition, the low toxicity and broad-spectrum properties of TAT can avoid interfering with the antibacterial / antiviral activity of β-defensin itself, ensuring the biosafety of the fusion protein.

[0052] To visualize the transmembrane delivery process and improve the overall stability of the fusion protein, this invention also adds an eGFP fluorescent tag to the fusion protein.

[0053] Therefore, the TAT-recombinant avian β-defensin fusion protein of the present invention is based on "eGFP fluorescent tag + TAT + recombinant avian β-defensin", and its amino acid sequence is shown in SEQ ID NO.3. To facilitate the expression and purification of the fusion protein, a purification protein tag can be added to one end. In this invention, six His tags were added to the C-terminus, and the amino acid sequence of the fusion protein after adding the His tag is shown in SEQ ID NO.5.

[0054] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0055] The test materials used in the embodiments of the present invention, unless otherwise specified, are all conventional test materials in the art and can be purchased through commercial channels.

[0056] Example 1: Preparation and purification of TAT-recombinant avian β-defensin fusion protein

[0057] 1. Constructing recombinant expression vectors

[0058] The gene fragment encoding the TAT-recombinant avian β-defensin fusion protein (eGFP fluorescent tag + TAT + recombinant avian β-defensin + His tag) shown in SEQ ID NO.6 was ligated into the pET-22b(+) vector, and the recombinant expression vector pET22b-TAT-recombinant avian β-defensin was constructed by sequencing verification.

[0059] The gene fragment encoding the TAT fusion protein (eGFP fluorescent tag + TAT + His tag) shown in SEQ ID NO.8 was ligated into the pET-22b(+) vector, and the recombinant expression vector pET22b-TAT was constructed by sequencing verification.

[0060] The gene fragment encoding the recombinant avian β-defensin fusion protein (eGFP fluorescent tag + recombinant avian β-defensin + His tag) shown in SEQ ID NO.10 was ligated into the pET-22b(+) vector, and the recombinant expression vector pET22b-recombinant avian β-defensin was constructed by sequencing verification.

[0061] 2. Protein expression

[0062] The constructed recombinant expression vectors pET22b-TAT-recombinant avian β-defensin, pET22b-TAT, and pET22b-recombinant avian β-defensin were transformed into E. coli Rosetta DE3 to construct recombinant bacteria expressing TAT-recombinant avian β-defensin fusion protein, recombinant bacteria expressing TAT fusion protein, and recombinant bacteria expressing recombinant avian β-defensin fusion protein, respectively.

[0063] The recombinant bacteria constructed above were added to 10 mL of solution containing 100 ng / L AMP. + Incubate overnight at 37°C and 200 rpm in LB broth. Transfer 2 mL of the bacterial culture to 200 mL of fresh AMP solution containing 100 ng / L. + The culture was carried out in LB liquid medium at 37°C and 220 rpm for 4 h. IPTG, the inducer, was added to a final concentration of 1.5 mg / L, and expression was induced for another 4 h. The bacterial culture was then removed and centrifuged at 12,000 rpm for 20 min at 4°C. The supernatant was discarded, and the culture was resuspended in PBS. This process was repeated three times at 12,000 rpm at 4°C. The supernatant was discarded, and the precipitate was retained. The precipitate was resuspended in 1 mL of PBS, and the culture was sonicated at 200 W for 3 seconds followed by a 3-second pause, then sonicated on ice until clear. The culture was then centrifuged at 12,000 rpm for 15 min at 4°C, and the supernatant and precipitate were collected.

[0064] The collected supernatant and precipitate were analyzed for concentration using the BCA method according to the manufacturer's instructions. The supernatant and precipitate were thoroughly mixed with SDS-PAGE protein loading buffer and then denatured in a 100°C metal bath for 10 min. Identification was performed using Coomassie Brilliant Blue staining. The results showed that the protein was expressed in inclusion body form.

[0065] 3. Protein purification

[0066] Add protein inclusion bodies to lysis buffer and sonicate for 30 min at 200 W for 3 s on, 3 s off, and sonicate again. Centrifuge at 10000 g for 20 min at 4 °C. Discard the precipitate, collect the supernatant of bacterial lysis buffer, and place on ice. Take an appropriate amount of the well-mixed denaturing agent, centrifuge at 4 °C (1000 g × 60 s), discard the stock solution, add one column volume of lysis buffer to the gel, mix well to equilibrate the gel, centrifuge at 4 °C (1000 g × 60 s), discard the supernatant, repeat equilibration 2-3 times, and discard the liquid. Mix the bacterial lysis buffer and gel, and shake gently on a shaker at 4 °C for 60 min. Load the mixture of lysis buffer and gel into an appropriate empty column tube, open the bottom cap of the column, and allow the liquid in the column to flow out under gravity. Collect 1 mL of the flow-through for subsequent analysis. Wash the column 5 times, 1 mL of lysis buffer each time, and collect approximately 1 mL each time for subsequent analysis. Wash the column 5 times, using 1 mL of washing buffer each time, and collect approximately 1 mL of each eluent for subsequent analysis. Elute the target protein 5 times, using 1 mL of eluent each time, and collect the eluent from each wash.

[0067] 4. Protein refolding

[0068] The purified fusion protein was measured using the BCA method to maintain a protein concentration of approximately 0.7 mg / mL. A serial dilution method was used for protein renaturation. A renaturation solution was prepared by adding 6M urea, 0.5M L-arginine, 10% glycerol, and 50M Tris-HCl to a final volume of 500 mL, adjusting the pH to 8.0. The dilution gradient was from 6M to 4M to 2M to 0M, with each step lasting 12 hours. After renaturation, the protein concentration was determined using the BCA method.

[0069] 5. Protein Concentrate

[0070] After protein refolding, the concentration was too low and needed to be concentrated. Prepare a ready-to-use dialysis bag with a 25 kDa cutoff. Select a dialysis bag of appropriate size and secure one end firmly with a dialysis bag clamp. Then, place the refolded protein into the dialysis bag at the other end and dialyze the protein using polyethylene glycol 20000. After dialysis, carefully remove the protein from the dialysis bag and measure the protein concentration again. The final concentration was 0.8 mg / mL.

[0071] Sequencing confirmed that the amino acid sequences of the prepared TAT-recombinant avian β-defensin fusion protein (eGFP fluorescent tag + TAT + recombinant avian β-defensin + His tag) are shown in SEQ ID NO.5; the amino acid sequences of the TAT fusion protein (eGFP fluorescent tag + TAT + His tag) are shown in SEQ ID NO.7; and the amino acid sequences of the recombinant avian β-defensin fusion protein (eGFP fluorescent tag + recombinant avian β-defensin + His tag) are shown in SEQ ID NO.9.

[0072] Example 2: Toxicity assay of TAT-recombinant avian β-defensin fusion protein

[0073] 1. Test Methods

[0074] First, DF-1 cells were cultured until the cell density reached 3 × 10⁻⁶. 5 When the cell density is 100 cells / mL, the cells can be seeded into a plate. Add 3.3 μL of cell suspension to each well of a 96-well plate, and then add 96.7 μL of serum-containing culture medium to each well. Do not seed cells in a ring around the perimeter of the 96-well plate. Add PBS for liquid sealing.

[0075] When the cell density reached 80%, experimental, control, and blank groups were set up, including:

[0076] The experimental group was given different concentrations (0.01 mg / ml, 0.05 mg / ml, 0.1 mg / ml, 0.15 mg / ml, 0.2 mg / ml, 0.3 mg / ml) of the TAT-recombinant avian β-defensin fusion protein prepared in Example 1, and cultured in 2% serum medium.

[0077] DF-1 cells cultured normally without the addition of TAT-recombinant avian β-defensin fusion protein were used as the control group; 2% serum culture medium was used as the blank group.

[0078] Each group was repeated three times, and cell viability was detected by CCK-8 assay at 24h, 48h and 72h after culture.

[0079] 2. Test Results

[0080] The results are as follows Figures 1-3 As shown, the results indicate that the TAT-recombinant avian β-defensin fusion protein at a concentration of 0.1 mg / mL has no toxic effect on cells.

[0081] Example 3: Determination of the membrane-penetrating ability of TAT-recombinant avian β-defensin fusion protein

[0082] 1. Test Methods

[0083] The transduction of TAT-recombinant avian β-defensin fusion protein in cells was detected using laser confocal microscopy, as detailed below:

[0084] First, DF-1 cells were digested and pipetted, and 200 μL was transferred to a glass-bottomed culture dish. The dish was incubated for 30 min, and then carefully supplemented with 10% DMEM. When the cells reached 80% confluence, TAT, recombinant avian β-defensin, and TAT-recombinant avian β-defensin fusion protein at concentrations of 0.01 mg / mL, 0.05 mg / mL, and 0.1 mg / mL (prepared in Example 1) were added, and the dish was incubated for 4 hours. The culture dish was then removed from the incubator and fixed. The culture medium was discarded, and 2 mL of room-temperature PBS was slowly added for washing, three times at 5 min intervals. Finally, the PBS was drained, and 2 mL of 4% paraformaldehyde was added, maintaining the solution for 25 min. After this, 2 mL of PBS was used for washing, three times at 5 min intervals. 0.3% Triton solution was prepared and 2 mL was added to the culture dish, incubated in the dark for 15 min, and then washed three times with 2 mL of PBS at 5 min intervals. Finally, remove the clean PBS, add 200 μL of anti-fluorescence quenching mounting solution (containing DAPI) to each dish, and incubate overnight at 4°C.

[0085] Western blot was used to detect the intracellular retention of TAT-recombinant avian β-defensin fusion protein in DF-1 cells 12 h, 24 h, 48 h, and 72 h after transduction with TAT-recombinant avian β-defensin fusion protein.

[0086] 2. Test Results

[0087] Laser confocal detection results are as follows Figure 4 As shown, the results indicate that TAT-recombinant avian β-defensin can successfully penetrate the DF-1 cell membrane. Western blot results are as follows. Figure 5 As shown, the results indicate that TAT-recombinant avian β-defensin remained in DF-1 cells 12 h, 24 h, 48 h, and 72 h after transduction.

[0088] Example 4: Effect of TAT-recombinant avian β-defensin fusion protein on virus inhibition in vitro

[0089] 1. Test Methods

[0090] (1) Effect on ALV-J proliferation

[0091] DF-1 cells were plated and, when the cells reached 80% confluency, they were inoculated with ALV-J virus solution. After incubation for 2 hours, the medium was replaced with DMEM containing 2% serum. The cells were then divided into the following treatment groups:

[0092] Mock group: No fusion protein was added, serving as a control;

[0093] TAT group: After changing the medium, the TAT fusion protein prepared in Example 1 was added to make its final concentration in the culture medium 0.1 mg / mL;

[0094] Recombinant avian β-defensin group: After changing the medium, the recombinant avian β-defensin fusion protein prepared in Example 1 was added to make its final concentration in the culture medium 0.1 mg / mL;

[0095] TAT-recombinant avian β-defensin group: After changing the medium, add the TAT-recombinant avian β-defensin fusion protein prepared in Example 1 to make its final concentration in the culture medium 0.1 mg / mL.

[0096] All other culture conditions remained the same for each treatment group, and the cells were cultured for another time. Cells were harvested at 24h and 48h, respectively, and DNA / RNA / protein were extracted. RT-qPCR was then performed to detect the replication status of ALV-J virus.

[0097] (2) Effects on the proliferation of REV, MDV and CIAV

[0098] DF-1 cells were plated and inoculated when the cells reached 80% confluency. Equal amounts of REV, MDV and CIAV virus solutions were added to each well. After incubation for 2 hours, the medium was replaced with DMEM containing 2% serum. After the medium was replaced, the cells were treated according to the grouping method in the previous step “(1) Effect on ALV-J proliferation”.

[0099] All other culture conditions remained the same for each treatment group. After culturing for another 48 hours, cells were harvested, and DNA / RNA was extracted. RT-qPCR was then performed to detect the replication status of REV, MDV, and CIAV viruses.

[0100] 2. Test Results

[0101] The results of 48-hour quantitative PCR detection of TAT-recombinant avian β-defensin fusion protein acting on ALV-J are as follows: Figure 6 As shown, the results indicate that the TAT-recombinant avian β-defensin fusion protein can significantly inhibit the proliferation of ALV-J. Moreover, compared with the treatment of TAT fusion protein and recombinant avian β-defensin fusion protein, the TAT-recombinant avian β-defensin fusion protein, which fuses TAT ​​and recombinant avian β-defensin together, has a synergistic inhibitory effect on ALV-J, achieving a 1+1>2 effect.

[0102] The 48-hour quantitative PCR results of TAT-recombinant avian β-defensin fusion protein acting on REV, MDV, and CIAV are as follows: Figure 7 , Figure 8 and Figure 9As shown, the results indicate that the TAT-recombinant avian β-defensin fusion protein has a good inhibitory effect on different types of viruses.

[0103] Example 5: Detection of TAT-recombinant avian β-defensin fusion protein against ALV-J, REV, MDV and CIAV infection in live chickens

[0104] 1. Test Methods

[0105] Three-day-old SPF Leyhang chickens were used as experimental subjects and divided into 9 groups of 10 birds each. The experimental groups were further divided into 8 subgroups as follows: ALV-J, REV, MDV, CIAV, TAT-AvBD+ALV-J, TAT-AvBD+REV, TAT-AvBD+MDV, and TAT-AvBD+CIAV. The first four groups were virus-inoculated only at 3 days of age; the latter four groups were virus-plus-protein inoculated, receiving the virus at 3 days of age and the TAT-recombinant avian β-defensin fusion protein prepared in Example 1 at 7 days of age. The virus inoculation concentration was as follows: ALV-J (avian leukosis virus J subgroup) concentration was 10... 3 TCID 50 / animal, REV (reticuloendotheliosis virus) concentration was 10 4 TCID 50 / animal, MDV (Marek's disease virus) concentration is 10 3 PFU / bird, CIAV (Chicken Infectious Anemia Virus) 10 5 TCID 50 / animal; the protein inoculation concentration was 1 mg / kg, calculated based on the experimental subject's body weight. The control group was raised under the same conditions as the experimental group, but was not inoculated with virus or protein.

[0106] At 10 days old, liver, kidney, spleen and bursa of Fabricius were collected from each group, and DNA / RNA was extracted. The replication status of ALV-J, REV, MDV and CIAV viruses was detected by RT-qPCR.

[0107] 2. Test Results

[0108] The results of quantitative real-time PCR detection of the TAT-recombinant avian β-defensin fusion protein's in vivo action on ALV-J, REV, MDV, and CIAV are as follows: Figure 10 , Figure 11 , Figure 12 and Figure 13 The results showed that the TAT-recombinant avian β-defensin fusion protein had a good inhibitory effect on different types of viruses in vivo.

[0109] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A TAT-recombinant avian β-defensin fusion protein, characterized in that, The TAT-recombinant avian beta defensin fusion protein is a protein as shown in (A1) or (A2) below: (A1) a protein with an amino acid sequence as shown in SEQ ID NO. 3; (A2) a protein obtained by connecting a protein tag to the C-terminus of the protein defined in (A1).

2. The TAT-recombinant avian beta defensin fusion protein according to claim 1, characterized in that, The amino acid sequence of the protein as shown in (A2) is as shown in SEQ ID NO.

5.

3. Use of the TAT-recombinant avian β defensin fusion protein according to claim 1 or 2 for the manufacture of a medicament for inhibiting viral infection, characterized in that, The virus is one or more of ALV-J, REV, MDV or CIAV.

4. A gene encoding a TAT-recombinant avian β defensin fusion protein, characterized by, The coding gene is a nucleic acid molecule as shown in any one of (i)-(iv) below: (i) a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO. 4; (ii) a nucleic acid molecule other than (i) that encodes the amino acid sequence as shown in SEQ ID NO. 3; (iii) a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO. 6; (iv) a nucleic acid molecule other than (iii) that encodes the amino acid sequence as shown in SEQ ID NO.

5.

5. Use of the gene encoding the TAT-recombinant avian β defensin fusion protein according to claim 4 for the preparation of a medicament for inhibiting viral infection, characterized in that, The virus is one or more of ALV-J, REV, MDV or CIAV.

6. Use of a substance that promotes expression of a gene encoding a TAT-recombinant avian β defensin fusion protein in the manufacture of a medicament for inhibiting viral infection, characterized in that, The coding gene of the TAT-recombinant avian beta defensin fusion protein is the coding gene of claim 4; and the virus is one or more of ALV-J, REV, MDV and CIAV. The substance for promoting the expression of the coding gene of the TAT-recombinant avian beta defensin fusion protein is any one of the following: C1) an expression cassette containing the coding gene of the TAT-recombinant avian beta defensin fusion protein; C2) a recombinant vector containing the coding gene of the TAT-recombinant avian beta defensin fusion protein, or a recombinant vector containing the expression cassette of C1); C3) a recombinant microorganism containing the coding gene of the TAT-recombinant avian beta defensin fusion protein, or a recombinant microorganism containing the expression cassette of C1), or a recombinant microorganism containing the recombinant vector of C2).

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