An antiviral fusion peptide with the effect of inhibiting BPIV3 and BoHV-1 infection and its application

Abstract

The present invention discloses an antiviral fusion peptide and its application with the effect of inhibiting BPIV3 and BoHV-1 infection. The antiviral fusion peptide is obtained by fusing TAT membrane-penetrating peptide with bovine Viperin (BoViperin) protein. The present invention confirms that overexpression of BoViperin protein in bovine cells can inhibit the replication of BPIV3 and BoHV-1; TAT membrane-penetrating peptide mediates BoViperin protein and can be transduced into bovine cells and mouse lungs, which can inhibit the replication of BPIV3 and BoHV-1, indicating that BoViperin protein can quickly establish an antiviral infection state at the cellular level and animal level. The present invention confirms that the antiviral fusion peptide obtained by fusing TAT membrane-penetrating peptide with BoViperin has the advantages of simple preparation and convenient delivery, and can lay the foundation for the development of efficient and broad-spectrum cattle antiviral drugs.

Description

Technical Field

[0001] The present invention relates to an antiviral fusion peptide and its application, and in particular to an antiviral fusion peptide composed of a TAT transmembrane peptide fused to a BoViperin protein and its application in inhibiting BPIV3 and BoHV-1 infection. The present invention belongs to the field of biotechnology. Background Art

[0002] Bovine respiratory disease (BRD) is a common disease in large-scale cattle farming. With its rapid onset and high mortality rate, it poses a serious threat to cattle health and causes significant economic losses to the cattle industry. The disease is caused by a complex interaction between numerous pathogens, environmental factors, and host factors. Viral factors have the highest infection rate, spread rapidly, and pose a serious threat. The viruses primarily implicated in BRD include bovine parainfluenza virus type 3 (BPIV3), bovine herpesvirus type 1 (BoHV-1), and bovine respiratory syncytial virus (BRSV). Bovine herpesvirus type 1 is also known as bovine infectious rhinotracheitis virus.

[0003] BPIV3 infection in cattle can cause respiratory tract infections, with clinical symptoms primarily including fever, depression, anorexia, serous nasal and ocular discharge, and rales in the lungs and trachea. Pneumonia caused solely by BPIV3 is usually subclinical, and clinical mortality from BPIV3 infection alone is rare. However, complicated pneumonia caused by BPIV3 is often accompanied by bacterial infections such as Mannheimer's bacterium, Pasteurella multocida, and Haemophilus somnus. Despite the availability of numerous vaccines and antimicrobial agents, pneumonia still results in significant morbidity and mortality.

[0004] BoHV-1 infection in cattle can cause upper respiratory tract disorders, conjunctivitis, genital inflammation, and immune suppression. BoHV-1 causes infectious rhinotracheitis (IBR) in cattle, with symptoms mainly characterized by fever, rhinitis, dyspnea, and upper respiratory tract inflammation, leading to abortion and pneumonia in dairy cows. Mixed infection of the virus with bacteria can delay the growth and weight gain of fattening cattle, affecting the milk production and fertility of adult cattle. If not treated in time, it often leads to death. The younger the age, the more severe the clinical symptoms and pathological damage.

[0005] Diagnosis of bovine viral respiratory disease primarily involves virus isolation and identification, viral nucleic acid detection, and serological diagnosis. Viral isolation and identification primarily involves collecting nasal swabs from infected or deceased cattle, or by collecting specimens from autopsy lesions or isolating the pathogen. Viral nucleic acid detection technology offers high sensitivity and specificity. Serological diagnosis is primarily accomplished through enzyme-linked immunosorbent assay (ELISA), a simple, rapid, and accurate method favoring clinical application.

[0006] Vaccination plays a crucial role in preventing and controlling the spread of BRD. BPIV3 is administered intranasally to calves after birth to initiate a mucosal immune response. A booster shot is given intramuscularly two to three months after birth, when maternal antibodies begin to wane. Infectious rhinotracheitis can be treated with either an inactivated or attenuated bovine infectious rhinotracheitis vaccine. During vaccination, the vaccine dose is determined based on the cattle's weight to boost immunity and reduce the incidence of respiratory disease. However, there are currently no vaccines available in China to prevent BPIV3 and BRSV infections, forcing the calf to rely on imports.

[0007] Currently, there is no specific drug to treat BPIV3 and BoHV-1 infection, and symptomatic treatment and auxiliary treatment are the only options. Therefore, the development of safe and effective antiviral drugs is of great significance for controlling the epidemic. Summary of the Invention

[0008] The present invention aims to provide an antiviral fusion peptide having the ability to inhibit BPIV3 and BoHV-1 and its application.

[0009] In order to achieve the above object, the present invention adopts the following technical means:

[0010] The present invention first transfects the eukaryotic expression vector pcDNA3.1-3HA-BoViperin expressing BoViperin into bovine testicular cells (Bovine testicular cells BT), bovine lung fibroblasts (Bovine lung cells, BL). Western blot results show that, during transfection 36h, the expression of BoViperin protein is the highest in BT cells and BL cells. On this basis, BT cells and BL cells are infected respectively with BPIV3 and BoHV-1, and the viral load and viral titer of the virus in different infection time cell samples are detected using qPCR method and virus titer determination method. The test result shows that, compared with the control group, the overexpressed BoViperin can significantly suppress the replication of BPIV3 and BoHV-1. Secondly, TAT membrane-penetrating peptide is introduced, prokaryotic expression plasmids pET-30a-TAT-BoViperin, pET-32a-TAT-BoViperin and pGEX-6p-TAT-BoViperin are constructed, and are transformed into Rosetta competent cells by changing induction time, induction temperature and IPTG concentration. The results showed that only a small amount of TAT-BoViperin protein was expressed in pET-30a-TAT-BoViperin and pET-32a-TAT-BoViperin, while TAT-BoViperin protein was successfully expressed in pGEX-6p-TAT-BoViperin. 2+ The expressed product was purified by -NTA agarose gel column to remove endotoxin from the purified TAT-BoViperin protein.

[0011] The antiviral activity of BoViperin was evaluated in both cellular and animal models. The TAT-penetrating peptide was used to mediate transmembrane transduction of BoViperin into bovine cells and mice. CCK-8 assays demonstrated that TAT-BoViperin at a concentration of 45 μL / mL had a weak inhibitory effect on MDBK cells and mild cytotoxicity. TAT-BoViperin at concentrations of 4.5 μL / mL and above had some inhibitory effect on BL cells and showed some cytotoxicity. MDBK and BL cells were transduced with various concentrations of TAT-BoViperin and then infected with BPIV3 and BoHV-1, respectively. The results demonstrated that TAT-BoViperin significantly inhibited BPIV3 / BoHV-1 replication in both MDBK and BL cells. C57BL / 6 mice were immunized with TAT-BoViperin protein and then artificially infected with BPIV3 12 hours after immunization. The effects of TAT-BoViperin on BPIV3 replication in mice were analyzed by observing clinical anatomical and pathological changes in the lungs and by qPCR. The results showed that TAT-BoViperin protein could inhibit BPIV3 replication in mice.

[0012] Based on the above research, the present invention proposes an antiviral fusion peptide that has the effect of inhibiting bovine parainfluenza virus type 3 and bovine herpes virus type 1. The antiviral fusion peptide is obtained by fusing the human immunodeficiency virus transactivator factor penetrating peptide with the bovine interferon-induced endoplasmic reticulum-associated virus inhibitory protein (Viperin).

[0013] Among them, preferably, the amino acid sequence of the antiviral fusion peptide is shown as SEQ ID NO.1.

[0014] The polynucleotide encoding the antiviral fusion peptide, the expression vector containing the polynucleotide, and the host cell containing the expression vector also fall within the protection scope of the present invention.

[0015] Preferably, the nucleotide sequence of the polynucleotide is as shown in SEQ ID NO.2.

[0016] Among them, preferably, the expression vector is a vector containing the polynucleotide and carrying a GST or other lytic tag.

[0017] Among them, preferably, the host cell is a Rosetta (DE3) competent cell.

[0018] Furthermore, the present invention also proposes the use of the antiviral fusion peptide in the preparation of drugs against parainfluenza virus type 3 and / or bovine herpes virus type 1.

[0019] Furthermore, the present invention also provides a vaccine composition, wherein the vaccine contains the antiviral fusion peptide.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] This study demonstrates that overexpressing BoViperin in bovine cells can inhibit the replication of BPIV3 and BoHV-1. The TAT-BoViperin protein was successfully expressed using the pGEX-6p prokaryotic expression system. The TAT-penetrating peptide mediated the transduction of BoViperin into bovine cells and mouse lungs, demonstrating that the TAT-BoViperin protein can inhibit the replication of BPIV3 and BoHV-1 at the cellular level and BPIV3 in infected mice, indicating that BoViperin can rapidly establish an antiviral infection state at both the cellular and animal levels. The antiviral fusion peptide, obtained by fusing the TAT-penetrating peptide to BoViperin, has advantages such as simple preparation and convenient delivery, and can lay the foundation for the development of highly effective, broad-spectrum bovine antiviral drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the sequence analysis diagram of bovine Viperin;

[0023] Note: The signal cleavage site is marked with an arrow, the conserved leucine is indicated by a gray shadow plus a circle; the α-helical domain is indicated by a gray shadow, the SAM middle domain is indicated by a light gray shadow, and the conserved C-terminal domain is indicated by a gray shadow plus a dotted line;

[0024] Figure 2 This is a multiple comparison of Viperin amino acid sequences between different species;

[0025] Figure 3 This is a graph showing the expression level of BoViperin in MDBK cells infected with BPIV3 / BoHV-1;

[0026] Among them, A: BoViperin expression level in MDBK cells infected with BPIV3; B: BoViperin expression level in MDBK cells infected with BoHV-1;

[0027] Figure 4 Comparison of normal MDBK cells and pathological MDBK cells (100×);

[0028] Among them, A: normal MDBK cells; B: cytopathic morphology of MDBK cells 36 hours after BPIV3 infection; C: cytopathic morphology of MDBK cells 24 hours after BoHV-1 infection;

[0029] Figure 5 One-step growth curves for BPIV3 and BoHV-1;

[0030] Among them, A: one-step growth curve of BPIV3; B: one-step growth curve of BoHV-1;

[0031] Figure 6 This is an identification diagram of the expression level of overexpressed BoViperin protein;

[0032] A: Identification diagram of the expression level of overexpressed BoViperin protein in HEK293T cells; B: Identification diagram of the expression level of overexpressed BoViperin protein in BT cells; C: Identification diagram of the expression level of overexpressed BoViperin protein in BL cells;

[0033] Figure 7 This is a graph showing the effect of BoViperin on BPIV3 replication;

[0034] A, B: BoViperin's effect on BPIV3 viral load; C, D: BoViperin's effect on BPIV3 titer;

[0035] Figure 8 This is a graph showing the effect of BoViperin on BoHV-1 replication;

[0036] Among them, A, B: the results of the effect of BoViperin on BoHV-1 viral load; C, D: the results of the effect of BoViperin on BoHV-1 titer;

[0037] Figure 9 This is the result of BoViperin gene amplification;

[0038] Among them, M.Trans 2000bp DNA Marker; 1. Amplified product of BoViperin gene; 2. Negative control;

[0039] Figure 10 Double enzyme digestion and PCR identification of recombinant plasmids

[0040] Among them, M. Trans 2K Plus II DNA Marker; 1. Double enzyme digestion identification of recombinant plasmid (Eco RI / Xho Ⅰ;); 2. PCR identification of recombinant plasmid; 3. PCR negative control;

[0041] Figure 11 Figure 2 is the SDS-PAGE analysis result of pET-30a-TAT-BoViperin induced expression;

[0042] Wherein, M.Page RulerMarker; 1. pET-30a(+) transformed E.coli pre-induction lysis product; 2. pET-30a(+) transformed E.coli post-induction lysis product 3. pET-30a(+)-TAT-BoViperin transformed E.coli pre-induction lysis product; 4. pET-30a(+)-TAT-BoViperin transformed E.coli post-induction lysis product; 5,6 pET-30a(+)-TAT-BoViperin transformed E.coli post-induction lysis product supernatant and precipitate;

[0043] Figure 12 Figure 2 shows the SDS-PAGE analysis results of pET-30a-TAT-BoViperin after optimizing the induction conditions;

[0044] Among them, A: SDS-PAGE analysis results of induction time exploration; C: SDS-PAGE analysis results of different low-temperature induction times;

[0045] M. Unstained protein molecular weight marker; 1. Lysate of E. coli transformed with pET-30a(+) after induction; 2. Lysate of E. coli transformed with pET-30a(+)-BoViperin after induction;

[0046] 3. pET-30a (+) -TAT-BoViperin transformed E. coli pre-induction lysis product; 4-8.

[0047] Lysate of E. coli transformed with pET-30a(+)-TAT-BoViperin after induction;

[0048] B: SDS-PAGE analysis results of IPTG concentration exploration M. Unstained protein molecular weight marker; 1. Lysate of E. coli transformed with pET-30a(+) before induction; 2. Lysate of E. coli transformed with pET-30a(+) after induction; 3. Lysate of E. coli transformed with pET-30a(+)-TAT-BoViperin before induction;

[0049] 4-7. Lysis product of E. coli transformed with pET-30a(+)-TAT-BoViperin after induction;

[0050] Figure 13 Figure 2 is the SDS-PAGE analysis result of pET-32a-TAT-BoViperin induced expression;

[0051] M. Page Ruler Marker; 1. Lysis product of E. coli transformed with pET-32a(+) after induction; 2. Lysis product of E. coli transformed with pET-32a(+)-TAT-BoViperin before induction; 3.

[0052] Lysate of E. coli transformed with pET-32a(+)-TAT-BoViperin after induction; 4, 5.

[0053] The supernatant and precipitate of the lysate after induction of pET-30a(+)-TAT-BoViperin transformation E. coli;

[0054] Figure 14 Figure 2 is the SDS-PAGE analysis result of pGEX-6p-TAT-BoViperin induced expression;

[0055] Wherein, A: SDS-PAGE analysis results of TAT-BoViperin protein expression; M: Page Ruler Marker; 1. Lysate of E. coli transformed with pGEX-6p(+) after induction; 2. Lysate of E. coli transformed with pGEX-6p-TAT-BoViperin before induction; 3. Lysate of E. coli transformed with pGEX-6p-TAT-BoViperin after induction; 4, 5. Supernatant and precipitate of lysate of E. coli transformed with pGEX-6p-TAT-BoViperin after induction;

[0056] B: SDS-PAGE analysis results of IPTG concentration exploration M. Page Ruler Marker; 1. Lysate of E. coli transformed with pGEX-6p(+) after induction; 2. Lysate of E. coli transformed with pGEX-6p-TAT-BoViperin before induction; 3-6. Lysate of E. coli transformed with pGEX-6p-TAT-BoViperin after induction;

[0057] Figure 15 This is the Western blot identification result of TAT-BoViperin protein;

[0058] Among them, M. Page RulerMarker; 1. Lysate of E. coli transformed with pGEX-6p(+) after induction; 2. Lysate of E. coli transformed with pGEX-6p-TAT-BoViperin before induction; 3. Lysate of E. coli transformed with pGEX-6p-TAT-BoViperin after induction; 4, 5. Supernatant and precipitate of lysate of E. coli transformed with pGEX-6p-TAT-BoViperin after induction;

[0059] Figure 16 This is the SDS-PAGE analysis result of TAT-BoViperin protein purification;

[0060] Wherein, A: M. Page Ruler Marker; 1. Loading solution; 2. Effluent; 3-5. PBS wash solution; 6-9. GST eluate; B: M: Premixed Protein Ladder; 1. Loading solution; 2. Effluent; 3-7. 20mM imidazole wash solution; 8-10. 500mM imidazole eluate;

[0061] Figure 17 This is the SDS-PAGE analysis result of TAT-BoViperin protein concentration;

[0062] Figure 18 This is a diagram showing the identification results of endotoxin removal from TAT-BoViperin protein;

[0063] Figure 19 This is a diagram of the cytotoxicity analysis of TAT-BoViperin;

[0064] Figure 20 This figure shows the effect of TAT-BoViperin on the replication of BPIV3 / BoHV-1 in MDBK cells;

[0065] Among them, A: Exploration of the optimal protein concentration of TAT-BoViperin protein against BPIV3; B: Determination of BPIV3 titer at different infection times; C: Exploration of the optimal protein concentration of TAT-BoViperin protein against BoHV-1; D: Determination of BoHV-1 titer at different infection times;

[0066] Figure 21 This is a graph showing the effect of BoViperin on BPIV3 / BoHV-1 replication in BL cells;

[0067] Among them, A: Exploration of the optimal protein concentration of TAT-BoViperin protein against BPIV3; B: Determination of BPIV3 titer at different infection times; C: Exploration of the optimal protein concentration of TAT-BoViperin protein against BoHV-1; D: Determination of BOHV-1 titer at different infection times;

[0068] Figure 22 This figure shows the effect of TAT membrane-penetrating peptide on BPIV3 replication;

[0069] Figure 23 This is a diagram showing clinical pathological changes in lung tissue after BPIV3 infection in mice;

[0070] Figure 24 The pathological changes of the lungs of infected mice were observed by HE staining (200×);

[0071] Figure 25 This is the result of detecting BPIV3 mRNA transcription level in mouse lung tissue;

[0072] Figure 26 This is a graph showing changes in viral load in the lungs of mice infected with BPIV3;

[0073] Figure 27 This is a graph showing the detection results of cytokine mRNA transcription levels in mouse lungs;

[0074] Among them, A: IL-4; B: IL-6; C: IL-10; D: IL-1β; E: IL-12; F: TNF-α; G: IFN-γ. DETAILED DESCRIPTION

[0075] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the present invention, not all of the embodiments. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are also within the scope of protection of the present invention.

[0076] Example 1 Preparation of TAT-BoViperin recombinant protein and identification of its antiviral activity at the cellular level

[0077] 1 Materials and Methods

[0078] 1.1 Test materials

[0079] 1.1.1 Viruses, cells, and animals

[0080] Bovine parainfluenza virus type 3 (BPIV3) and bovine herpesvirus type 1 (BoHV-1) were provided by the Laboratory of Microbiology and Immunology, Northeast Agricultural University (hereinafter referred to as this laboratory). Madin Darby bovine kidney cells (MDBK), bovine lung fibroblasts (BL), bovine testicular cells (BT), and human embryonic kidney 293T cells (HEK293T) were also generated and maintained in this laboratory.

[0081] 1.1.2 Vectors and plasmids

[0082] The prokaryotic expression vectors pET-30a(+), pET-32a(+), pGEX-6p-1(+) vectors, competent cells E. coli DH5α, E. coli Rosetta and recombinant plasmid pcDNA3.1-3HA-BoViperin were all prepared and preserved in our laboratory.

[0083] 1.1.3 Preparation of main solutions

[0084] (1)PBS(T):

[0085] Prepare 20x PBS stock solution: 2.74M NaCl, 54mM KCl, 0.4M Na2HPO4·12H2O and 35mM KH2PO4. Dilute to 1x with water before use and add 0.05% (v / v) Tween-20.

[0086] (2) LB medium:

[0087] Dissolve 0.5% yeast extract, 1% NaCl and 1% tryptone in deionized water, make up to 1 L, sterilize by autoclaving and store at room temperature until use.

[0088] (3) DMEM cell culture basal medium

[0089] Dissolve 10.4 g DMEM (GIBCO) and 3.7 g NaCO3 in deionized water, adjust the pH to 7.2-7.4, make up to 1 L, filter sterilize, and store at 4°C until use.

[0090] (4) 0.25% pancreatic enzyme solution

[0091] Dissolve 0.25 g of trypsin and 0.025 g of EDTA in 1× PBS solution, adjust the pH to 7.4, and make up to 100 mL. Filter sterilize and store at -20°C until use.

[0092] (5)SDS-PAGE:

[0093] Stacking gel: deionized water, 30% Acr-Bis (29:1), 1 M Tris (pH 6.8), 10% (w / v) SDS, 10% (w / v) ammonium persulfate, and TEMED. Resolving gel: deionized water, 30% Acr-Bis (29:1), 1.5 M Tris (pH 8.8), 10% (w / v) SDS, 10% (w / v) ammonium persulfate, and TEMED.

[0094] (6) Transfer buffer:

[0095] 0.04 M glycine, 0.04 M Tris-Base, 1 mM SDS, and 5% (v / v) methanol. The reducing electrophoresis transfer buffer does not contain SDS.

[0096] (7) Electrophoresis buffer:

[0097] 94g C2H5NO2, 15.1g Tris-Base, 5g SDS, dilute to 1L, and store at 4℃.

[0098] 1.1.4 Main primer sequences

[0099] The main sequences are shown in Table 1.

[0100] Table 1 PCR primers

[0101]

[0102]

[0103] 1.2 Test methods

[0104] 1.2.1 Analysis of the Genetic Biological Characteristics of BoViperin

[0105] The molecular characteristics of BoViperin were analyzed based on the BoViperin reference sequence published by NCBI (accession number MN585200), and the nucleotide and amino acid sequences of BoViperin were compared with those of Viperin from other species.

[0106] 1.2.2 Effects of BPIV3 and BoHV-1 on BoViperin expression

[0107] MDBK cells were infected with 0.1 MOI of BPIV3 and BoHV-1, respectively. Cell cultures were collected at 0 h, 1 h, 3 h, 6 h, 12 h, 24 h, and 36 h after virus infection. RNA of the cell samples was extracted with TRIzol, and the expression level of BoViperin was determined by qPCR.

[0108] 1.2.3 Study on the antiviral activity of BoViperin

[0109] 1.2.3.1 Propagation and culture of BPIV3 and BoHV-1

[0110] BPIV3 and BoHV-1 were propagated in MDBK cells. When the cell density reached 70-80%, the cells were washed three times with sterile PBS. BPIV3 and BoHV-1 were diluted in serum-free DMEM at an MOI of 0.01 and inoculated into cell flasks. The cells were cultured at 37°C in 5% CO2. Pathological changes were observed daily, and the virus was harvested when 80% of the cells showed pathological changes. The culture flasks were placed in a -40°C freezer and repeatedly frozen and thawed three times to disrupt the cells and release the virus. The culture medium was centrifuged at 5000 rpm for 10 minutes at 4°C. Cell debris was discarded, and the supernatant containing the virus was stored at -70°C.

[0111] 1.2.3.2 Plotting of one-step growth curves for BPIV3 and BoHV-1

[0112] The virus was inoculated into a monolayer of MDBK cells at an MOI of 0.01, and 2% fetal bovine serum DMEM medium was used as a normal cell control. The cells were harvested at 1, 3, 6, 12, 24, 36, 48, 60, and 72 h after virus infection. The collected cell-virus mixture was repeatedly frozen and thawed 3 times, centrifuged at 12,000 r / min for 15 min, and the supernatant was filtered through a 0.22 μm filter. The viral titers of BPIV3 and BoHV-1 at the above 9 time points were measured according to the Reed-Muench method, and a one-step growth curve was drawn.

[0113] 1.2.3.3 Determination of BPIV3 and BoHV-1 Virus Titers

[0114] Dilute the virus solution to 10 -1 -10 -10The cells were seeded into 96-well cell culture plates at a density of 70% MDBK cells. Each dilution was replicated in eight wells, with 100 μL per well. Two negative controls were also set up. The 96-well plates were incubated in a 37°C, 5% CO2 incubator for 1.5 hours. The virus solution was discarded and replaced with DMEM medium supplemented with 2% serum. The cells were incubated at 37°C, 5% CO2 for 48-72 hours. The cytopathic effect (CPE) was observed daily, and the TCID50 of each virus was calculated using the Reed-Muench method.

[0115] 1.2.3.4 Identification of BoViperin's antiviral activity

[0116] The plasmid pcDNA3.1-3HA-BoViperin stored in the laboratory was first transformed, picked, cultured in large doses, and then the plasmid was extracted using a plasmid midi extraction kit and the plasmid concentration was measured to meet the transfection plasmid concentration requirements.

[0117] HEK293T cells were collected at 1×10 6 The cells were plated into 12-well cell culture plates and transfected into HEK293T cells within 24 h. The cells were identified by Western blot. Then, BT cells and BL cells were cultured at 1×10 6 Each well was plated into a 12-well cell culture plate and transfected within 24 hours. The transfection reagent was lipo2000, and the operation was performed according to the instructions of the transfection reagent. 1.2 μg of plasmid was transfected into BL cells and BT cells, and Western blot was used for identification to determine the optimal time for protein expression. After transfection again, the cells were infected with 0.5 MOI of BPIV3 and BoHV-1 for 12 hours, 24 hours, and 36 hours, respectively, and cell samples were collected. Cell culture RNA was extracted from one group of cells, and the viral load at different infection times was determined by qPCR; the other group of cells was repeatedly frozen and thawed three times, and the virus liquid in the cell sample was collected to detect the titer of the virus in the cell sample. The steps for determining the virus titer are the same as 1.2.3.3.

[0118] 1.2.4 Construction of TAT-BoViperin recombinant expression vector

[0119] 1.2.4.1 Primer design

[0120] According to the BoViperin (MN585200) sequence published in GenBank, the corresponding specific primers were designed, and a TAT short peptide sequence, a histidine sequence (His), a protective base, and a restriction endonuclease recognition site were introduced into the primer sequence. The primers were synthesized by Kumei Biogene Co., Ltd.

[0121] 1.2.4.2 Amplification and recovery of target genes

[0122] Using the pcDNA3.1-3HA-BoViperin plasmid as a template, PCR was performed using primers TAT-BoViperin-F, BoViperin.REV+His, and BoViperin.REV. (For fragments ligated to pET-30a or pET-32a, primers TAT-BoViperin-F and BoViperin.REV (without a His tag) were used; for fragments ligated to the pGEX-6p vector, primers TAT-BoViperin-F and BoViperin.REV+His were used.) The BoViperin-encoding nucleic acid was amplified. The PCR product was recovered from the gel using a DNA purification kit.

[0123] 1.2.4.3 Double enzyme digestion and ligation of target gene and vector

[0124] The recovered amplified product and vector were double-digested with the restriction endonucleases corresponding to the plasmids. The digestion conditions were both incubated at 37°C for 2 hours. The products were then recovered using a DNA purification kit. The digested products were mixed with the vector plasmid and ligated in a metal bath at 16°C overnight.

[0125] Use 10 μL of ligation product to transform competent cells E. coli DH5α, use 10 μL of ligation product to transform competent cells E. coli DH5α, ice bath for 30 min, heat shock at 42 ° C for 90 s, ice bath for 5 min, add 200 μL of sterile LB liquid culture to the competent cells, shake and culture at 37 ° C, 220 r / min for 1 h, then spread the culture product on LB agar plate containing 30 μg / mL ampicillin (Amp) and culture at 37 ° C overnight.

[0126] 1.2.4.4 Enzyme digestion and identification of TAT-BoViperin recombinant plasmid

[0127] From the transformed plate, single colonies were picked from pET-30a-TAT-BoViperin and pET-32a-TAT-BoViperin, respectively, and incubated in LB medium containing 10 μg / mL Amp. After 12 hours of incubation, recombinant plasmids pET-30a-TAT-BoViperin, pET-32a-TAT-BoViperin, and pGEX-6p-TAT-BoViperin were extracted using a small-dose plasmid preparation kit and identified by double enzyme digestion with EcoRI and XhoI, as described in 1.2.4.3. After successful enzyme digestion, the recombinant plasmids were sequenced.

[0128] 1.2.4.5 PCR identification of TAT-BoViperin recombinant plasmid

[0129] The recombinant plasmids pET-30a-TAT-BoViperin, pET-32a-TAT-BoViperin, and pGEX-6p-TAT-BoViperin were identified by PCR. The PCR system and procedure were the same as in 1.2.4.1. After the PCR, the plasmids were identified by nucleic acid gel.

[0130] 1.2.5 Preparation of TAT-BoViperin Protein

[0131] 1.2.5.1 Expression of TAT-BoViperin Protein

[0132] Transform E. coli Rosetta (DE3) with pET-30a-TAT-BoViperin, pET-32a-TAT-BoViperin, or pGEX-6p-TAT-BoViperin recombinant plasmids TM Pick a single colony and inoculate it into 5mL liquid LB medium (Kan + / Amp + ), 37°C, 220r / min shaking culture overnight. The overnight culture was added to liquid LB medium (containing 10μg / mL Amp or 30μg / mL Kan) at a ratio of 2%, and the induction conditions such as induction time, induction temperature, and IPTG concentration of the TAT-BoViperin protein were optimized. The culture was centrifuged at 5000r / min for 10min and the supernatant was discarded. The bacterial pellet was resuspended in PBS and sonicated in an ice bath for 1h. Each sonication time was 5s, with an interval of 3s. The sonicated product was centrifuged at 5000r / min for 10min. The supernatant was collected and the pellet was dissolved with PBS containing 8M urea.

[0133] 1.2.5.2 Identification of TAT-BoViperin Protein

[0134] The sample was mixed with 2×SDS loading buffer at a ratio of 1:1, incubated in a boiling water bath for 10 min, and analyzed for target protein expression by SDS-PAGE. Western blot analysis was then performed using mouse anti-His tag monoclonal antibody and mouse anti-GST tag monoclonal antibody as primary antibodies (1:5000) and HRP-labeled goat anti-mouse IgG as secondary antibody (1:5000) to identify the expression pattern of BoViperin protein.

[0135] 1.2.5.3 Purification of TAT-BoViperin Protein

[0136] There are two ways to purify the successfully expressed TAT-BoViperin protein: one is to use GST purification medium, and the other is to use His tag Ni 2+ -NTA affinity chromatography was used for purification.

[0137] 1.2.5.4 Dialysis and Concentration of TAT-BoViperin Protein

[0138] Purified TAT-BoViperin protein was dialyzed against a gradient of 6 M urea and 10% glycerol in TGE buffer to remove urea and imidazole. The buffer was changed every 4-6 hours using 6 M urea TGE buffer, 4 M urea TGE buffer, 2 M urea TGE buffer, and finally urea-free TGE dialysate. The protein was concentrated using sucrose. The concentrated protein was analyzed by 10% SDS-PAGE and the protein concentration was determined using a BCA protein assay kit.

[0139] 1.2.5.5 Removal of endotoxin from TAT-BoViperin protein

[0140] (1) Before loading the protein sample, filter it with a 0.22 μm or 0.45 μm filter membrane to reduce impurities, improve protein purification efficiency, and prevent column clogging. The sample pH is best controlled at 7-8, which is the optimal pH condition for endotoxin binding to the column. It is best to control the sample at an appropriate ionic strength to reduce nonspecific adsorption, such as 0.15-0.5 M NaCl.

[0141] (2) Activate the resin: Place the pre-packed column on an iron stand and fix it vertically. Remove the cover on the top of the pre-packed column and open the flow controller to allow the protective liquid to flow out under the action of gravity. Add 5 ml of regeneration buffer (cooled) and adjust the flow controller to maintain the flow rate at 0.25 ml / min (or 1 drop / 6 s). After the regeneration buffer has flowed out, add 5 ml of regeneration buffer (pre-cooled) and repeat the operation twice.

[0142] (3) Equilibration of resin: After activation, add 6 ml of equilibration buffer at a flow rate of 0.5 ml / min (or 1 drop / 3 s), and repeat the operation twice.

[0143] (4) Endotoxin removal: Add 1.5 ml of protein sample to the equilibrated column, adjust the flow rate to 0.25 ml / min (or 1 drop for 6 seconds), and do not collect the effluent. When the protein sample has flowed out, continue to add protein sample. Add 1.5 ml of equilibration solution to the protein sample and combine it with the previous effluent.

[0144] 1.2.6 Cytotoxicity Analysis of TAT-BoViperin Protein

[0145] The toxicity of TAT-BoViperin protein on MDBK and BL cells was detected using the CCK-8 kit. MDBK cells and BL cells were seeded in 96-well plates, respectively. When the cells grew to about 70%, the cells were treated with 10-fold diluted TAT-BoViperin protein and incubated in a 37°C cell culture incubator for 72 hours. CCK-8 (10 μL / well) was added and incubated for another 1-4 hours. The absorbance at 450 nm was then measured using a microplate reader. Three wells were repeated for each treatment group.

[0146] 1.2.7 Identification of TAT-BoViperin's antiviral activity at the cellular level

[0147] MDBK cells and BL cells were cultured at a rate of 1×10 6 Four 6-well cell culture plates were plated per well to explore the optimal concentration of TAT-BoViperin protein for antiviral activity. MDBK cells and BL cells were incubated with TAT-BoViperin protein at concentrations of 1 μg / ml, 100 ng / ml, 50 ng / ml, 10 ng / ml, and 1 ng / ml for 12 hours. MDBK cells and BL cells were then infected with BPIV3 and BoHV-1 at a 0.5 MOI, respectively. Negative and positive controls were also established. Viral titers were measured in cell samples 12 hours, 24 hours, and 36 hours after infection. The steps for determining viral titers were the same as in 1.2.2.3.

[0148] To investigate the effect of TAT membrane-penetrating peptide on viral replication, MDBK cells were incubated with 1 μg / ml of TAT-BoViperin and TAT-mScarlet proteins for 12 h, respectively. The cells were then infected with 0.5 MOI of BPIV3 for 12, 24, and 36 h. The virus fluid in the cell samples was collected, and the virus titer in the cell samples was detected 12, 24, and 36 h after infection. The steps for determining the virus titer were the same as those in 1.2.2.3.

[0149] 1.2.8 Statistical methods

[0150] The experiments in this study were repeated three times and the results were analyzed using Graphpad Prime 8.0 software. The data are expressed as mean ± standard deviation (Mean ± SD). p>0.05 indicates no significant difference (ns), *p<0.05, **p<0.01, ***p<0.001 indicate significant difference.

[0151] 2 Results

[0152] 2.1 Analysis of the Genetic Biological Characteristics of Bovine Viperin (BoViperin)

[0153] Bovine Viperin consists of 1092 bases, encoding 363 amino acids, with a protein molecular weight of 41.5kDa and an isoelectric point of 6.88. Analysis of the bovine Viperin sequence revealed that the nucleotide similarity between bovine Viperin and ovine Viperin is 96.5%, and the nucleotide similarity between bovine Viperin and other animal Viperin is 24.7-96.5%. Bovine Viperin has three structural domains, such as Figure 1 As shown. The N-terminal domain contains an α-helix, the signal peptide is located in the first 18 amino acids, and five conserved leucine sites have been identified; the middle SAM domain contains four Motif characteristic motifs, and both the SAM domain and the C-terminal domain are relatively conserved. Homology analysis results show that ( Figure 2 The amino acid similarity of bovine Viperin with those of sheep and rabbit is 99.2% and 92.5%, respectively, and the similarity with the amino acid of other animals is 67.1-99.2%. The sequence differences between species mainly exist in the N-terminal domain.

[0154] Effects of BPIV3 and BoHV-1 on BoViperin expression

[0155] After MDBK cells were infected with BPIV3 and BoHV-1 at 0.1 MOI, the expression of BoViperin was detected within 24 h of BPIV3 infection and 36 h of BoHV-1 infection. Figure 3 As shown. Both BPIV3 and BoHV-1 can significantly induce the production of BoViperin, and the expression level of BoViprin is the highest after 6h infection with BPIV3 and 24h infection with BoHV-1.

[0156] 2.3 Study on the antiviral activity of BoViperin

[0157] 2.3.1 Proliferation of BPIV3 and BoHV-1

[0158] 3.3.1.1 Cytopathic Effects of BPIV3 and BoHV-1

[0159] Both BPIV3 and BoHV-1 can proliferate well in MDBK cells, and MDBK cells can develop lesions 24h-48h after infection with BPIV3 and BoHV-1. The main characteristics of the lesions are that the cells become rounded, wrinkled, detached, or even broken, which are different from the morphology of normal cells, such as Figure 4 shown.

[0160] 2.3.1.2 Plotting of one-step growth curves for BPIV3 and BoHV-1

[0161] One-step growth curves of BPIV3 and BoHV-1, such as Figure 5 As shown. BPIV3 grows fastest 24h-60h after infection, reaches a peak at 60h after infection, and the BPIV3 virus titer begins to decline after 60h of infection. BoHV-1 grows fastest 12h-48h after infection in MDBK cells, and the growth rate gradually slows down from 48h to 60h after infection, reaching a peak at 60h after infection, and the BoHV-1 virus titer begins to decline after 60h of infection.

[0162] 2.3.1.3 Determination of BPIV3 and BoHV-1 Virus Titers

[0163] The BPIV3 and BoHV-1 virus stock solutions were diluted 10-fold, and the TCID50 of BPIV3 and BoHV-1 were determined. The following data were calculated using the Reed-Muench method: TCID50 of BPIV3 = 10 -7.445 / 0.1mL; TCID50 of BoHV-1 = 10 -8.4 / 0.1ml; see Table 2 and Table 3.

[0164] Table 2 TCID50 assay results for BPIV3

[0165]

[0166] Calculate TCID50 using the Reed-Muench method:

[0167] Distance ratio d = (66.7% - 50%) / (66.7% - 29.2%) = 0.445

[0168] TCID50=-7+0.445×(-1)=-7.445

[0169] TCID50 of BPIV3 = 10 -7.445 / 0.1mL.

[0170] Table 3 TCID50 determination results of BoHV-1

[0171]

[0172]

[0173] Calculate TCID50 using the Reed-Muench method:

[0174] Distance ratio d = (66.7% - 50%) / (66.7% - 25%) = 0.4

[0175] TCID50 = -8 + 0.4 × (-1) = -8.4

[0176] TCID50 of BoHV-1 = 10 -8.4 / 0.1ml

[0177] 2.3.2 Western blot identification of overexpressed BoViperin

[0178] The recombinant plasmid pcDNA3.1-3HA-BoViperin was transiently transfected into 293T cells at a dose of 1.2 μg / well, and protein samples were collected at 12 h and 24 h for Western blot identification. Figure 6 The BoViperin protein was significantly expressed. The recombinant plasmid was then transfected into BT cells and BL cells at a dose of 1.2 μg / well. After 36 hours of expression, the expression level of BoViperin reached its highest level.

[0179] 2.3.3 Effect of BoViperin Overexpression on BPIV3 Replication

[0180] BT and BL cells were transfected with the eukaryotic expression plasmid pcDNA3.1-3HA-BoViperin at a dose of 1.2 μg / well for 36 hours. The cells were then infected with BPIV3 at a 0.5 MOI for 12 hours, 24 hours, and 36 hours. The viral load and titer in the cell samples were then measured. Figure 7 When BPIV3 was infected in BT and BL cells for 12 and 24 hours, the viral load of BPIV3 in the experimental group decreased compared with the control group, and the viral load decreased by nearly 10 times. The viral titer of the experimental group was significantly decreased compared with the control group. The results show that BoViperin overexpression can inhibit the replication of BPIV3.

[0181] 2.3.4 Effect of overexpression of BoViperin on BoHV-1 replication

[0182] The eukaryotic expression plasmid pcDNA3.1-3HA-BoViperin was transfected into BT cells and BL cells at a dose of 1.2 μg / well for 36 hours. The cells were infected with BoHV-1 at 0.5 MOI for 12 hours, 24 hours, and 36 hours, and the cell samples were collected. The viral load and virus titer in the cell samples were detected. The results showed Figure 8As shown. When BT cells were infected with BoHV-1 for 12h and 24h, the viral load and viral titer of BoHV-1 in the experimental group decreased compared with the control group; when BL cells were infected with BoHV-1 for 12h, 24h, and 36h, the viral load and viral titer of BoHV-1 in the experimental group decreased compared with the control group. The difference was most significant when infected with BoHV-1 for 36h, and the viral load and viral titer of BoHV-1 in the experimental group decreased by nearly 100 times. The above results indicate that Viperin overexpression can inhibit the replication of BoHV-1.

[0183] 2.4 Construction of TAT-BoViperin recombinant expression vector

[0184] 2.4.1 BoViperin gene amplification

[0185] Using pcDNA3.1-3HA-BoViperin plasmid as template, PCR was performed with TAT-BoViperin-F primer, BoViperin.REV+His primer, and BoViperin.REV. The PCR product was 1165 bp, as shown in Figure 2. Figure 9 shown.

[0186] 2.4.2 Identification of TAT-BoViperin recombinant plasmid

[0187] Recombinant expression plasmids pET-30a-TAT-BoViperin, pET-32a-TAT-BoViperin, and pGEX-6p-TAT-BoViperin were constructed, and the prokaryotic recombinant plasmids were identified by enzyme digestion and PCR. The results were consistent with expectations, such as Figure 10 shown.

[0188] 2.5 Preparation of TAT-BoViperin Protein

[0189] 2.5.1 Expression of TAT-BoViperin Protein

[0190] The successfully constructed prokaryotic recombinant plasmid pET-30a-TAT-BoViperin was used to TM Competent cells were transformed and induced to express TAT-BoViperin protein. When the bacterial solution was cultured at 37℃ to an OD600nm of 0.4-0.6, 1mM IPTG was added and the shaker speed was 220rpm. The induction was carried out for 4 hours. The results of SDS-PAGE analysis were as follows: Figure 11 The expected size of TAT-BoViperin protein is 50 KDa, but no specific band was found at the 50 KDa position, indicating that TAT-BoViperin protein was not expressed.

[0191] The induction time, IPTG concentration and induction temperature of TAT-BoViperin protein were explored. When the induction temperature was 37°C and the IPTG concentration was 1mM, the induction time was explored for 3h, 4h, 5h, 6h and 8h. Figure 12 . The results showed that a small amount of TAT-BoViperin protein was expressed when the induction time was 6h (lane 7). When the induction temperature was 37°C and the induction time was 6h, IPTG concentrations of 0.5mM, 0.8Mm, 1.0mM, and 1.2mM were explored. The results showed that under induction at concentrations of 0.5mM, 0.8Mm, 1.0mM, and 1.2mM IPTG, only a small amount of TAT-BoViperin protein was expressed at the 50KDa position. The induction temperature was changed to induce expression of pET-30a-TAT-BoViperin. When the induction temperature was 27°C and the IPTG concentration was 1mM, the expression was induced for 8h, 10h, 12h, 14h, and 16h. The results showed that only a small amount of TAT-BoViperin protein was expressed. In summary, changing the induction time, induction temperature, and IPTG concentration could not significantly increase the expression level of TAT-BoViperin protein, and only a small amount of TAT-BoViperin protein was expressed.

[0192] The successfully constructed prokaryotic recombinant plasmid pET-32a-TAT-BoViperin was used to TM Induce the expression of TAT-BoViperin protein, the expected protein size is about 60KDa, such as Figure 13 After optimization of the induction conditions, the expression level of TAT-BoViperin protein was still very low.

[0193] The successfully constructed prokaryotic recombinant plasmid pGEX-6p-TAT-BoViperin was isolated and purified by E. coli Rosetta (DE3) TM Induce the expression of TAT-BoViperin protein. When the bacterial solution is cultured at 37℃ to an OD600nm of 0.4-0.6, add 1mM IPTG and shake at 220rpm for 6h. SDS-PAGE analysis results show that Figure 14 A, a non-specific band was found at the 70KDa position, which was consistent with the expected protein size. In order to increase the expression of TAT-BoViperin protein, the IPTG concentration was optimized. The results of SDS-PAGE analysis showed that Figure 14B, TAT-BoViperin protein expression is high under 0.6 mM IPTG induction conditions. Therefore, TAT-BoViperin expression was performed under the induction conditions of 37°C, 0.6 mM IPTG concentration, and 6 h induction time.

[0194] 2.5.2 Identification of TAT-BoViperin Protein

[0195] The successfully expressed TAT-BoViperin protein was identified. Western blot results showed that a band consistent with the expectation was detected at the 70 kDa position, and its expression form was determined to be inclusion bodies. Figure 15 The amino acid sequence of TAT-BoViperin protein is shown in SEQ ID NO.1, and its encoding nucleotide sequence is shown in SEQ ID NO.2.

[0196] 2.5.3 Purification of TAT-BoViperin Protein

[0197] Since TAT-BoViperin protein carries GST tag and His tag, there are two ways to purify TAT-BoViperin protein: one is to purify it using GST purification medium, and the other is to purify it using His tag Ni 2+ -NTA affinity chromatography method for purification, such as Figure 16 As shown. TAT-BoViperin protein can be purified using both GST tag and His tag, but using His tag Ni 2+ -NTA affinity chromatography is more efficient, so the His tag Ni 2+ -NTA affinity chromatography was used for purification.

[0198] 2.5.4 Dialysis and Concentration of TAT-BoViperin Protein

[0199] When dialyzing the TAT-BoViperin protein, a gradient dialysis was performed using PBS buffer containing 6M urea and 5% glycerol. However, the TAT-BoViperin protein precipitated during the dialysis process. Therefore, the TAT-BoViperin protein was diluted and then dialyzed using TGE buffer containing 6M urea and 10% glycerol to remove urea and imidazole. The TAT-BoViperin protein was concentrated using sucrose. The concentrated protein was analyzed using 10% SDS-PAGE for protein concentration. Figure 17 The purity of TAT-BoViperin protein can reach more than 90%, and the concentration is 1 mg / mL.

[0200] 2.5.5 Detection of Endotoxin in TAT-BoViperin Protein

[0201] Endotoxin removal kit was used to efficiently remove endotoxin from TAT-BoViperin protein, and the endotoxin levels in TAT-BoViperin protein before and after endotoxin removal were detected and compared. Figure 18 As shown in Figure 3, the endotoxin content in TAT-BoViperin protein decreased significantly after endotoxin removal.

[0202] 2.6 Cytotoxicity Analysis of TAT-BoViperin Protein

[0203] According to the results, TAT-BoViperin protein acted on MDBK cells and BL cells for 72 hours. Among them, high-dose (45μL / mL) TAT-BoViperin protein had a weak proliferation inhibition and slight cytotoxicity on MDBK cells; 4.5μL / mL and above BoViperin protein had a certain proliferation inhibition and cytotoxicity on BL cells. Figure 19 .

[0204] 2.7 Analysis of the antiviral activity of TAT-BoViperin protein in cells

[0205] According to the observation and analysis results, we can get Figure 20 As shown in the results, in MDBK cells, both 1 μg / mL and 100 ng / mL TAT-BoViperin proteins could reduce the viral titers of BPIV3 and BoHV-1, and the difference between 1 μg / mL TAT-BoViperin protein and 1 μg / mL TAT-BoViperin protein was more significant. Therefore, 1 μg / mL TAT-BoViperin protein was selected to further study the effect of TAT-BoViperin on BPIV3 / BoHV-1 replication.

[0206] After incubating MDBK cells with 1 μg / mL TAT-BoViperin protein for 12 hours, the MDBK cells were infected with 0.5 MOI of BPIV3 / BoHV-1 for 12 hours, 24 hours, and 36 hours, respectively. Cell samples were collected and the virus titers in the cell samples were detected. The results showed that when MDBK cells were infected with BPIV3 for 12 hours and 24 hours, the virus titer of BPIV3 in the protein experimental group was significantly decreased compared with the control group. When MDBK cells were infected with BoHV-1 for 12 hours, the virus titer of BoHV-1 in the protein experimental group was significantly decreased compared with the control group, indicating that 1 μg / mL TAT-BoViperin protein can inhibit the replication of BPIV3 and BoHV-1 in MDBK cells, and only inhibits the replication of BoHV-1 in the early stage of BoHV-1 infection.

[0207] According to the observation and analysis results, we can get Figure 21 As shown, in BL cells, 100ng / mL TAT-BoViperin protein can reduce the viral titer of BPIV3 and BoHV-1. After incubating BL cells with 100ng / mL TAT-BoViperin protein for 12h, BL cells were infected with 0.5MOI of BPIV3 / BoHV-1 for 12h, 24h and 36h, respectively, and cell samples were collected to detect the viral titer in the cell samples. The results showed that when infected with BPIV3 for 12h, 24h, and 36h, the viral titer of BPIV3 in the protein experimental group was significantly lower than that in the control group. When infected with BoHV-1 for 12h, the viral titer in the protein experimental group was significantly lower than that in the control group. However, there was no significant difference between the protein experimental group and the control group at 24h and 36h of infection. The above results indicate that 100ng / mL TAT-BoViperin protein can inhibit the replication of BPIV3 and BoHV-1 in BL cells, and only inhibits the replication of BoHV-1 in the early stage.

[0208] To investigate whether TAT membrane-penetrating peptide has an effect on viral replication, TAT-mScarlet protein was used as a control. Figure 16 As shown, the virus titer of the TAT-BoViperin protein group was lower than that of the control group, while the virus titer of the TAT-mScarlet protein group and the control group was basically the same ( Figure 22 The results showed that BoViperin protein has the ability to inhibit the replication of BPIV3 virus, while TAT membrane-penetrating peptide has no inhibitory effect on BPIV3 virus.

[0209] Example 2 Analysis of Anti-BPIV3 Activity of TAT-BoViperin Protein in Mice

[0210] 1. Experimental Animals

[0211] 6-8 week old SPF-grade C57BL / 6 mice were purchased from Liaoning Changsheng Biotechnology Co., Ltd. All animal experiments were conducted in accordance with the Animal Welfare and Ethics Standards of Northeast Agricultural University. TAT-BoViperin protein was prepared as in Example 1.

[0212] 2. Methods

[0213] (1) Ultracentrifugation of BPIV3

[0214] 400 mL of BPIV3 virus was cultured according to the BPIV3 propagation culture method described in 1.2.3.1 of Example 1. The virus solution was subjected to differential centrifugation. The virus solution was first centrifuged at 3000 rpm for 30 min. The supernatant of the virus solution was then transferred to a new sterile centrifuge tube and centrifuged again at 5000 rpm for 30 min. The supernatant of the virus solution was collected and concentrated by ultracentrifugation. The virus solution was placed in an ultracentrifuge tube, capped and balanced, and placed in an angle rotor. Centrifuged at 35,000 rpm for 2.5 h at 4°C, the supernatant discarded, and the virus pellet resuspended in pre-chilled PBS. The BPIV3 pellet was dissolved in PBS and stored at -80°C. The titer of the BPIV3 after ultracentrifugation was determined according to the method described in 1.2.3.3 of Example 1.

[0215] (2) Animal grouping and immunization

[0216] 6-8 week old C57 BL / 6 mice were selected and divided into three groups: blank control group, BPIV3 group, and TAT-BoViperin group. TAT-BoViperin group mice were immunized with TAT-BoViperin protein (100 μg) 12 hours later and injected into the trachea for 10 9 / 0.1mL of BPIV330μL, and the blank control group was not injected.

[0217] (3) Collection of disease samples

[0218] Clinical manifestations of the challenged mice were observed after infection. Lung tissue was collected from three mice in each group at 6, 12, 24, 36, 48, and 72 hours after infection. A small amount of lung tissue was soaked in 4% paraformaldehyde and stored at room temperature for pathological sectioning. A portion of the lung was ground and pulverized in a small amount of liquid nitrogen. Genomic RNA was extracted using the Trizol method, and RNA levels of BPIV3 and several cytokines in the lung tissue were measured.

[0219] (4) Observation of pathological changes

[0220] Observe lung pathological changes. Lung tissue was preserved by immersion in 4% paraformaldehyde fixative, and pathological sections were prepared and stained with hematoxylin-eosin (HE). Histopathological changes were observed under both visual and microscopic examinations.

[0221] (5) Quantitative RT-PCR was used to detect the transcription level of BPIV3 in lung tissue, and the relative content of BPIV3 mRNA in lung tissue of mice in each group was analyzed and compared.

[0222] (6) Quantitative RT-PCR was used to detect the transcription levels of cytokines in lung tissues and analyze the relative mRNA contents of cytokines in lung tissues of mice in each group.

[0223] 3. Results

[0224] 3.1 Clinical anatomical changes of the lung

[0225] Compared with the lung tissues of mice in the blank control group, no obvious pathological changes were observed in each group at 36h, 12h, 48h and 72h after BPIV infection. At 24h after BPIV infection, a small amount of hemorrhage spots appeared in the lungs of the BPIV3 group, and the lungs were slightly swollen, while the TAT-BoViperin group did not show obvious pathological features. At 36h after BPIV infection, the lungs of the BPIV3 group were swollen, with obvious congestion and dark red color, while the TAT-BoViperin group had milder symptoms as observed with the naked eye, such as Figure 23 .

[0226] 3.2 Pathological changes in lung tissue

[0227] When infected with BPIV3 for 6-12 hours, enlarged alveolar septa and capillary congestion were observed under the lung microscope in the BPIV3 group, while mild capillary congestion was observed in the TAT-BoViperin group. When infected with BPIV3 for 24-36 hours, varying degrees of alveolar epithelial cell proliferation, capillary congestion and hemorrhage in the alveolar septa, significant thickening of the alveolar walls, infiltration of macrophages and lymphocytes in the alveoli and their septa, and damage to the alveolar structure were observed in the BPIV3 group, leading to interstitial pneumonia. In contrast, capillary congestion, widened alveolar septa, and a small amount of inflammatory cell infiltration were found in the TAT-BoViperin group. When infected with BPIV3 for 48 hours, capillary congestion, hemorrhage, and inflammatory cell infiltration were observed in the BPIV3 group, while vascular hemorrhage, thickening of the alveolar walls, and widening of the alveolar septa were observed in the TAT-BoViperin group. At 72h after BPIV3 infection, the BPIV3 group showed capillary bleeding, inflammatory cell infiltration, and epithelial cell shedding, while the TAT-BoViperin group showed widening of the alveolar wall septa, thickening of the tube wall, and inflammatory cell infiltration. In summary, at 6-48h after infection, the pathological changes in the lungs of mice in the TAT-BoViperin group were lower than those in the BPIV3 group. Figure 24 .

[0228] 3.3 Comparative analysis of BPIV3 mRNA levels in lung tissue

[0229] To investigate the protective effect of TAT-BoViperin protein on mice, the BPIV3 mRNA level in lung tissues of each group was detected by Quantitative RT-PCR. Figure 25As shown. In lung tissues at 6h, 12h, 24h, 36h, and 48h after infection, the BPIV3 mRNA levels in the TAT-BoViperin group were significantly lower than those in the BPIV3 group, with the greatest significant difference in lung tissue at 36h after infection. In lung tissues at 72h after infection, there was no significant difference in BPIV3 mRNA levels between the TAT-BoViperin group and the BPIV3 group. In lung tissues at all infection time periods, there was no significant difference in BPIV3 mRNA levels between the BPIV3 group and the PBS group. These results indicate that when BPIV3 infects mice, TAT-BoViperin protein can inhibit BPIV3 replication in mice.

[0230] 3.4 Detection and analysis of cytokines in lung tissue

[0231] Th1 cells secrete IFN-γ and TNF-α, mediating macrophage activation and killing intracellular pathogens, thus playing a role in cellular immunity. Th2 cells secrete IL-4, IL-6, and IL-10, mediating eosinophil activation and combating extracellular pathogens. A balanced Th1-Th2 ratio maintains homeostasis; an imbalance can lead to the development and progression of various diseases. Th1 cells and the cytokines they secrete are generally considered to have pro-inflammatory functions, while Th2 cells and the cytokines they secrete have anti-inflammatory functions.

[0232] To detect the cytokine response in the mouse lungs, Quantitative RT-PCR was used to detect the mRNA transcription levels of IL-4, IL-6, IL-10, IL-1β, IL-12, IFN-γ, and TNF-α in the lungs of mice in the TAT-BoViperin group, BPIV3 group, and blank control group. The results are shown in Figure 2. Figure 27 As shown, during BPIV3 infection, the transcription levels of IL-6, IL-10, IL-1β, IL12, IFN-γ, and TNF-α were significantly higher in the BPIV3 group than in the control group. The expression levels of IL-6 and IL-1β in the BPIV3 group decreased with increasing infection time, while the expression levels of IL-10, IL12, and IFN-γ in the BPIV3 group first decreased and then increased with increasing infection time. The mRNA transcription levels of IL6, IL-10, IL-1β, IL-12, and IFN-γ in the BPIV3 group were significantly higher than those in the TAT-BoViperin group.

[0233] After infection with BPIV3, the increased mRNA transcription levels of IL6, IL-10, IL-1β, IL-12, IFN-γ, and TNF-α in the BPIV3 group are a normal response of the body to resist the virus, while the mRNA transcription levels of IL6, IL-10, IL-1β, IL-12, and IFN-γ in the TAT-BoViperin group were significantly lower than those in the BPIV3 group, indicating that the degree of BPIV3 infection in the TAT-BoViperin group was lighter than that in the BPIV3 group, and TAT-BoViperin protein can inhibit the replication of BPIV3 in mice.

Claims

1. Use of an antiviral fusion peptide in the preparation of a drug that is simultaneously effective against bovine parainfluenza virus type 3 and bovine herpes virus type 1, wherein the antiviral fusion peptide is obtained by fusing a human immunodeficiency virus transactivator transmembrane peptide with a bovine interferon-induced endoplasmic reticulum-associated virus inhibitory protein, Viperin. The amino acid sequence of the bovine interferon-induced endoplasmic reticulum-associated virus inhibitory protein, Viperin, is shown in SEQ ID NO.

1.

2. Use of an antiviral fusion peptide in the preparation of an anti-bovine herpesvirus type 1 drug, the antiviral fusion peptide being obtained by fusing a human immunodeficiency virus transactivator transmembrane peptide with a bovine interferon-induced endoplasmic reticulum-associated virus inhibitory protein, Viperin, the amino acid sequence of which is shown in SEQ ID NO.1.

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