A bovine parainfluenza virus type 3 and bovine viral diarrhea virus combined adenovirus vector vaccine and its application

Abstract

The present invention provides a bovine parainfluenza virus type 3 and bovine viral diarrhea virus bivalent adenovirus vector vaccine, prepared by cloning the BPIV3C F gene and the BVDV-1E2 gene into a replication-deficient human adenovirus type 5 vector. The BPIV3C F gene sequence is shown in SEQ ID No. 1, and the BVDV-1E2 gene sequence is shown in SEQ ID No. 2. The bivalent vaccine can stimulate the body to produce strong cellular and humoral immunity, with good immune efficacy and high safety.

Description

Technical Field

[0001] The present invention belongs to the field of veterinary biological products, and specifically relates to a bovine parainfluenza virus type 3 and bovine viral diarrhea virus binary adenovirus vector vaccine and its application. Background Art

[0002] Bovine parainfluenza virus type 3 (BPIV3) and bovine viral diarrhea virus (BVDV) are important pathogens that cause bovine respiratory disease complex (BRDC). They are widespread in cattle and are a global epidemic, contributing to the economic losses of the global cattle industry. Currently, there is no specific treatment, and vaccination is one of the preventive measures. However, there are currently no commercially available bivalent vaccines targeting these two pathogens in China. Therefore, accelerating the development of safe, effective, and novel genetically engineered vaccines is crucial for better prevention and control of BRDC.

[0003] BPIV3 belongs to the genus Respirovirus in the family Paramyxoviridae and is a single-stranded negative-strand RNA virus. BPIV3 can be divided into three genotypes: A, B, and C. Genotype C was first identified in China. Epidemiological surveys have shown that BPIV3C has become one of the main prevalent genotypes in cattle herds in China. BVDV belongs to the genus Pestivirus in the family Flaviviridae and is a single-stranded positive-strand RNA virus. Epidemiological surveys have shown that BVDV is divided into three genotypes, namely BVDV-(1, 2, and 3). Domestic BVDV etiological monitoring results show that BVDV-1 has become one of the main prevalent genotypes in cattle herds.

[0004] The F and E2 proteins are the primary structural proteins of BPIV3 and BVDV, respectively, and are also the primary antigenic proteins that induce neutralizing antibodies. Numerous studies both domestically and internationally have confirmed that the F and E2 proteins are important antigenic targets for the development of subunit and genetically engineered vaccines. Currently, only subunit and genetically engineered vaccines targeting these two proteins have been developed; there are no dual vaccines that protect against both pathogens simultaneously. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention provides a bovine parainfluenza type 3 virus (BPIV3C) and bovine viral diarrhea virus (BVDV-1) dual recombinant adenovirus vector vaccine and its preparation method and application.

[0006] The present invention provides a bovine parainfluenza virus type 3 and bovine viral diarrhea virus dual adenovirus vector vaccine, which is prepared by cloning the BPIV3C F gene and the BVDV-1E2 gene into a replication-deficient human adenovirus type 5 vector, wherein the BPIV3C F gene sequence is shown in SEQ ID No. 1, and the BVDV-1E2 gene sequence is shown in SEQ ID No. 2.

[0007] The BPIV3C F gene and the BVDV-1E2 gene were cloned into a replication-defective human adenovirus type 5 vector in the form of a fusion protein, wherein the nucleotide sequence of the fusion protein is shown in SEQ ID No.3.

[0008] The preparation method of the bivalent vaccine is as follows:

[0009] a. Synthesize the sequence of claim 1 or 2 and clone it into the adenovirus shuttle plasmid pDC316 to obtain the recombinant shuttle plasmid pDC316-F-P2A-E2;

[0010] b. Co-transfect the recombinant shuttle vector pDC316-F-P2A-E2 and the adenovirus backbone plasmid pBHGlox E1,3Cre into HEK293 cells, recombinant and packaged.

[0011] The present invention also provides the use of the above-mentioned bivalent vaccine in the preparation of biological products for preventing bovine parainfluenza type 3 and bovine viral diarrhea.

[0012] Wherein, the bivalent vaccine is prepared as an injection, nasal drops or inhaler.

[0013] Furthermore, the injection is an intramuscular injection.

[0014] The present invention constructs a dual recombinant adenovirus vector vaccine that can simultaneously express the full-length BPIV3C type F protein and the full-length BVDV-1 type E2 protein. The dual vaccine is immunized into mice through intramuscular injection and nasal drops, which can induce mice to produce strong humoral immunity and cellular immune responses, and can induce the body to produce high-titer antibodies. The effect is better than commercial vaccines, and there is no immunosuppression caused by simultaneous immunization with vaccines. It can simultaneously prevent and control bovine parainfluenza virus type 3 and bovine viral diarrhea virus, has good immune effect, high safety, and has good application prospects.

[0015] The present invention is further described below with reference to the accompanying drawings and specific embodiments of the present invention. However, the embodiments do not limit the present invention in any way. For those skilled in the art, any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered equivalent replacement methods and are included within the scope of protection of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Schematic diagram of recombinant adenovirus construction.

[0017] Figure 2 pDC316-F-P2A-E2 was identified by double enzyme digestion.

[0018] Figure 3 Recombinant adenovirus was packaged in HEK293 cells.

[0019] Figure 4 The recombinant adenovirus was identified by agarose gel electrophoresis.

[0020] Figure 5 IFA results of target antigens, where A1 / A2: HAd5-F-P2A-E2-infected cells; A3: blank cells; B1 / B2: obvious green fluorescence; B3: no green fluorescence.

[0021] Figure 6 Western blot results of target antigens, where M: pre-stained marker; 1: HAd5-F-P2A-E2 infected cells; 2: blank cells.

[0022] Figure 7 Western blot results of target antigen, where M: pre-stained marker; 1: HAd5-F infected cells; 2: blank cells.

[0023] Figure 8 Western blot results of target antigen, where M: pre-stained marker; 1: HAd5-E2 infected cells; 2: blank cells.

[0024] Figure 9 The IgG antibody titer against BPIV3C F protein in serum was detected by ELISA.

[0025] Figure 10 The IgG antibody titer against BVDV-1E2 protein in serum was detected by ELISA.

[0026] Figure 11 Cellular immune response detection diagram. DETAILED DESCRIPTION

[0027] The present invention is further described in detail below with reference to the examples, but the present invention is not limited thereto. The experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials described are commercially available unless otherwise specified.

[0028] Example 1 Preparation of vaccine

[0029] 1. Optimization and Synthesis of F and E2 Protein Genes

[0030] The target antigens of the recombinant adenovirus vaccine are the F protein of the BPIV3C strain CH / SC20 / 2020 (GenBank No. OM621819.1) and the E2 protein of the BVDV-1 strain CH / SWU-Z6 / 2016 (GenBank No. MF693403.1). By optimizing the F and E2 protein genes, their expression levels were increased, thereby enhancing the vaccine's immunogenicity.

[0031] Before gene optimization, the GC content of the F protein gene was 35.7%. After optimization, the GC content increased to 55.5%. Codon usage bias was adjusted to accommodate the highest expression profile of the target host, and the CAI (Codon Adaptation Index) was increased from 0.64 to 0.97. Before gene optimization, the GC content of the E2 protein gene was 44.2%. After optimization, the GC content increased to 61.9%, and the CAI (Codon Adaptation Index) was increased from 0.71 to 0.99 (a CAI of 0.8-1.0 is considered favorable for high expression).

[0032] After optimizing the F and E2 protein genes, a Kozak sequence was added before the translation start codon, and an EcoRI restriction site was inserted upstream of the entire sequence. The P2A polypeptide was introduced between the F and E2 genes, and a SalI restriction site was inserted downstream. The gene sequences were then synthesized. The sequence of the optimized fusion protein (with EcoRI and SalI restriction sites) is shown in SEQ ID NO. 3.

[0033] After optimization of F protein gene (SEQ ID NO.1):

[0034]

[0035] After optimization of E2 protein gene (SEQ ID NO.2):

[0036]

[0037] Fusion protein sequence (SEQ ID NO.3):

[0038]

[0039] The original gene sequence of F and E2 proteins (SEQ ID NO. 4, wherein the sequence from positions 13 to 1632 is the F gene, and the sequence from positions 1717 to 2844 is the E2 gene):

[0040]

[0041] 2. Vector Construction

[0042] The synthesized gene sequence was digested with EcoRI and SalI, respectively, to recover the target gene fragment, which was then ligated into the shuttle plasmid pDC316 of the AdMax adenovirus system (Microbix Biosystems Inc., Canada). The fragment was transformed into a DH5α competent cell and plated on an Amp LB plate. Single colonies were identified by colony PCR, and clones that tested positive by PCR were sequenced. The plasmid with optimized F and E2 protein genes was designated pDC316-F-P2A-E2. A schematic diagram of recombinant adenovirus construction is shown in the figure. Figure 1 As shown, pDC316-F-P2A-E2 double enzyme digestion was identified as Figure 2 shown.

[0043] 3. Recombinant Adenovirus Packaging, Preparation, and Identification

[0044] 3.1 Recombinant Adenovirus Packaging

[0045] The constructed vector pDC316-F-P2A-E2 and the backbone plasmid pBHGlox_E1,3Cre of the AdMax adenovirus system were co-transfected into HEK293 cells for recombinant adenovirus packaging. The process is as follows:

[0046] a. One day before transfection, HEK293 cells were seeded in six-well plates at 7×10 cells per well. 5 The cells were cultured in DMEM+10% FBS at 37℃ in a cell culture incubator containing 5% CO2 overnight.

[0047] b. When the cells have grown to 80-90% of their basal area, take the backbone plasmid (pBHGlox_E1,3Cre) and shuttle plasmid and transfect using jetPRIME transfection reagent according to the included instructions. Specific steps are:

[0048] (1) Take 1.6 μg of backbone plasmid and 0.4 μg of shuttle plasmid into each transfection well and mix them evenly; dilute the plasmid with 200 μL buffer medium.

[0049] (2) Take 2 μL of jetPRIME transfection reagent and add it to the diluted buffer and mix well.

[0050] (3) The transfection reagent and plasmid mixture was placed at room temperature for 10 minutes and then added to the cells.

[0051] c. The day after transfection, the cells were passaged on a 25cm 2Continue culturing in the cell culture flask with DMEM medium containing 5% FBS, observe every day, and when the cells have grown to the bottom of the flask, transfer them to a 75cm 2 In the cell culture flask, observe the signs of cell toxicity every day. The signs of toxicity are that the cells become larger and rounder, like grapes, and obvious plaques begin to appear ( Figure 3 The virus was collected when most of the cells became diseased and fell off from the bottom.

[0052] d. Place the toxin-detoxified cells in a 70°C refrigerator and a 37°C water bath and freeze-thaw three times.

[0053] The supernatant containing the virus was collected by centrifugation at 12000 g for 10 minutes, and the precipitate was discarded. The virus strain expressing the optimized sequence of the F-P2A-E2 full-length protein gene was labeled HAd5-F-P2A-E2.

[0054] 3.2 Identification of recombinant adenovirus

[0055] 3.2.1 PCR amplification of the full sequence of the F-P2A-E2 gene and sequencing identification

[0056] The full sequence of the F-P2A-E2 protein was amplified using universal primers for the pDC316 vector. The primer sequences are as follows:

[0057] pDC316-F:ACGTGGGTATAAGAGGCG

[0058] pDC316-R:CGATGCTAGACGATCCAG

[0059] Take 20 μL of vaccine candidate strain virus liquid, add 10 μL of proteinase K, digest at 58°C for 30 minutes to release the viral genome, and then heat at 100°C for 5 minutes, using this as a template to amplify the F-P2A-E2 protein gene sequence.

[0060] Agarose gel electrophoresis results showed that a single target band could be amplified and the fragment size was correct ( Figure 4 ), where 1, 2, and 3 represent P1, P2, and P3 of HAd5-F-P2A-E2, i.e., the first to third generation of HAd5-F-P2A-E2, respectively, and - represents a negative control. The target band was recovered from the gel and sequenced, and the alignment results showed that the sequencing sequence was completely correct.

[0061] 3.2.2 Target antigen expression identification

[0062] The recombinant adenovirus was used to infect HEK293 cells, and the target antigen was detected by IFA and Western blot 48 hours later. IFA could detect obvious green fluorescence, and WB could detect obvious expression of the target protein.

[0063] HEK293 cells were infected with the seed virus HAd5-F-P2A-E2. HEK293 cells not inoculated with the virus served as a negative control and were cultured for 48 hours. The culture medium was discarded, the cells were fixed with 80% acetone, and blocked with 5% skim milk powder at 37°C for 1 hour. Rabbit anti-BVDV-1E2 protein serum (1:100) and rabbit anti-BPIV3C polyclonal serum (1:100) were added as primary antibodies, respectively. Goat anti-rabbit IgG antibody (IgG-FITC) was then used as a secondary antibody (1:5000) in the dark. The expression of the target protein was observed under an inverted fluorescence microscope.

[0064] Sample Preparation: 48 hours after HAd5-F-P2A-E2 infection, carefully aspirate the culture medium, resuspend the cells in PBS, centrifuge at 500g for 5 minutes, and discard the supernatant. Resuspend the cells in 200 μL of RIPA buffer (Thermo Scientific, Prod. 89900) supplemented with appropriate amounts of protease inhibitors and nuclease inhibitors. Incubate on ice for 15 minutes, centrifuge at 12,000 rpm at 4°C for 5 minutes, remove the supernatant, add 1 / 4 volume of 5× SDS-PAGE loading buffer containing 200 mmol / L DTT, heat at 95°C for 5 minutes, and freeze for Western blotting.

[0065] Western blot analysis: SDS-PAGE was performed using a 10-well 12% SDS-PAGE gel, with 20 μL of sample loaded per well. Electrophoresis conditions: 80 V for 30 min; 120 V, until bromophenol blue just emerged from the gel. Proteins from the SDS-PAGE gel were transferred to a polyvinylidene difluoride (PVDF) membrane using an electrophoresis instrument at 200 mA for 1.5 h. After electrotransfer, the PVDF membrane was blocked with 5% skim milk powder for 1 h. The membrane was then incubated with rabbit anti-BVDV-1E2 protein polyclonal serum (1:1000) and rabbit anti-BPIV3C whole virus polyclonal serum (1:1000) as primary antibodies, respectively, at 4°C overnight. The membrane was washed four times with Western blotting buffer, each time for 10 min on a shaker. Then, HRP-labeled goat anti-rabbit IgG antibody (Bioss, bs-0296G-HRP) diluted 1:5000 in 5% skim milk powder was added and incubated at room temperature for 2 hours. The membrane was washed 4 times with WB washing solution, and chemiluminescence reaction was performed using BeyoECL Star (Beyotime, Cat.No.P0018AFT). Images with different exposure times were collected using a chemiluminescence imager. The IFA results and WB results are shown in Figure 2. Figure 5-8 .

[0066] 3.2.3 Recombinant adenovirus culture

[0067] HEK293 cells were cultured adherently at 37°C in 5% CO2. Forty-eight hours after virus inoculation, when cell viability dropped below 40%, the cell flask was frozen and thawed three times, first in a -70°C refrigerator and then in a 37°C water bath. The cells were centrifuged at 12,000 g for 10 minutes, and the virus-containing supernatant was collected and the pellet discarded.

[0068] 3.2.4 Recombinant adenovirus purification

[0069] Adenovirus particles were isolated and purified using a Diamond Layer 700BA (Borgron). The column was equilibrated with Solution A (PBS, pH 7.4). The sample was slowly introduced into a loop tube. After loading, the elution peak was collected in separate tubes at 0.6 mL / min and equilibrated to the UV baseline. The impurities were then washed with 100% Solution B (1 M NaOH + 1 M NaCl, pH 7.5).

[0070] 3.3 Identification and Titer Determination of HAd5-F-P2A-E2

[0071] 3.3.1 PCR amplification of the full sequence of the target protein gene and sequencing identification

[0072] The experimental method and process were the same as in 3.2.1. Agarose gel electrophoresis results showed that a single target band was amplified and the fragment size was correct. The target band was recovered from the gel and sequenced. Alignment results showed that the sequence was completely correct.

[0073] 3.3.2 Infectious titer determination

[0074] When the HEK293 cells reached a density of about 80-90%, the cells were digested and counted. A cell suspension was prepared using DMEM containing 5% FBS. Each plate required 10 mL of a 1×10 5 / mL cell suspension; 100 μL per well (i.e. 1×10 4 cells) were added to three 96-well plates; infection samples were prepared.

[0075] Inoculate samples: Add 100 μL of 5% FBS-containing DMEM to each of columns 11 and 12 of a 96-well plate as a negative control. Add 100 μL of each of the eight serial dilutions of sample solution labeled "A" to each of rows A, H, of the 96-well plate. Cover the first plate and incubate in a 37°C CO2 incubator. Repeat the same steps for the second and third plates.

[0076] The TCID of HAd5-F-P2A-E2 can be calculated 50 1×10 5.8 / mL.

[0077] 3.3.3 Virus Particle Count Determination

[0078] Virus sample treated with SDS was then subjected to A260nm measurement (UVSDS method). To determine the number of viral particles, 200 μL of HAd5-F-P2A-E2 injection solution was added to an equal volume of 0.2% SDS solution for lysis and vortexed to mix. The sample was placed in a 56°C water bath for 10 minutes. Once the temperature dropped to room temperature, the sample was centrifuged at 12,000 rpm for 5 minutes and the supernatant was collected. A blank control was prepared by mixing equal volumes of virus stabilization solution with 0.2% SDS solution. The absorbance at wavelengths of 260 nm and 280 nm was measured. This experiment was repeated twice.

[0079] Calculation formula: Number of virus particles = A260nm × dilution factor × 1.1 × 10 12 .

[0080] The number of virus particles of purified HAd5-F-P2A-E2 was 9.2×10 10 VP / mL.

[0081] The beneficial effects of the present invention are specifically described below through test examples:

[0082] Experimental Example 1: Immunization of mice with recombinant adenovirus HAd5-F-P2A-E2

[0083] 1. Vaccine humoral immune response testing

[0084] Thirty-five SPF female BALB / c mice (6-8 weeks old) were randomly divided into seven groups, with five mice in each group. The mice were immunized with HAd5-F-P2A-E2 according to the grouping shown in Table 1.

[0085] Among them, the HAd5-F intramuscular injection group and the HAd5-E2 intramuscular injection group both used the optimized sequences, and the construction process was the same as in Example 1.

[0086] For intramuscular injection, 200 μL was injected into the inner thigh. For intranasal immunization, mice were anesthetized with isoflurane and 200 μL was instilled into the nasal cavity. A second immunization was performed 14 days after the first immunization at the same dose.

[0087] Table 1 Mouse immunization grouping

[0088]

[0089] Blood was collected from mice at specific time points after immunization, and serum was separated. The IgG antibody titers against BPIV3C F protein and BVDV-1 E2 protein in the serum were detected by ELISA. Figure 9-10 shown.

[0090] The results of ELISA antibody level detection showed that intramuscular injection of HAd5-F-P2A-E2 in mice could produce higher serum IgG antibodies.

[0091] The antibody titer against BPIV3C-F protein reached its highest level 14 days after the second vaccination. There was no significant difference between the HAd5-F-P2A-E2 intramuscular injection group and the HAd5-F intramuscular injection group, but both were significantly higher than the HAd5-F-P2A-E2 intranasal drops group and the BPIV3C inactivated vaccine group (ns, p < 0.01, p < 0.001). There was no significant difference between the HAd5-F-P2A-E2 intranasal drops group and the BPIV3C inactivated vaccine group (ns) ( Figure 9 ).

[0092] The antibody titer against BVDV-E2 protein reached its highest level 14 days after the second vaccination. There was no significant difference between the HAd5-F-P2A-E2 intramuscular injection group and the HAd5-E2 intramuscular injection group, but both were significantly higher than those in the HAd5-F-P2A-E2 intranasal drop group and the BVDV-1 commercial inactivated vaccine group (ns, p < 0.01, p < 0.01). Among them, the antibody level in the HAd5-F-P2A-E2 intranasal drop vaccination group was significantly higher than that in the BVDV-1 commercial inactivated vaccine group (p < 0.05) ( Figure 10 ).

[0093] Therefore, the bivalent vaccine prepared by the present invention can induce the production of higher serum IgG antibodies after immunization without immunosuppression, and the antibody level is significantly higher than that of commercial inactivated vaccines.

[0094] 2. Cellular Immune Response Detection

[0095] Immunization was performed in the same manner as above. Mice were sacrificed 14 days after the second immunization and spleen lymphocytes were isolated. Splenic tissue was mechanically processed to form a single cell suspension and the cell concentration was adjusted to 4×10 6 In vitro stimulation medium, RPMI1640 medium (containing 10% fetal bovine serum, abbreviated as FBS), containing 100 ng / mL PMA, 1 μg / mL ionomysin, and 3 μM monomysin, was used for stimulation and culture in a 37°C incubator for 6 hours. Protein secretion blockers were also added to block cytokine secretion.

[0096] For IL-4 and IFN-γ flow cytometry, transfer 100 μl of cell suspension to a flow cytometer tube. Add 1 mL of Foxp3 fixation / permeabilization working solution to each tube and pulse-vortex and incubate at 4°C or room temperature for 40 minutes. Add 2 mL of 1X permeabilization buffer to each tube, centrifuge the sample at 500 g for 5 minutes at room temperature, discard the supernatant, and repeat this step once. Add 1 μg of fluorescent antibody against IL-4 or IFN-γ, incubate at 4°C for 30 minutes, resuspend the cells in 2 mL of PBS, centrifuge at 500 g, discard the supernatant, and resuspend the cells in 400 μl of PBS. Immediately analyze the samples using Everest software.

[0097] The results are as follows Figure 11 As shown, intramuscular injection of HAd5-F-P2A-E2 can produce higher IL-4 and IFN-γ expressions, and there is a significant difference between the intramuscular injection group of HAd5-F-P2A-E2 and the control group (PBS group) (p < 0.01).

[0098] CD4 + T lymphocytes, CD8 + For T lymphocyte detection, 100 μl of cell suspension was placed in a flow cytometry tube and CD4 + T, CD8 + Add 1 μg of each fluorescent antibody to the T-cells and incubate at 4°C for 30 min. Resuspend the cells in 2 ml of PBS and centrifuge at 300 g to discard the supernatant. Repeat this step once. Resuspend the cells in 400 μl of PBS and immediately analyze the cells. Analyze the results using Everest software.

[0099] The results are as follows Figure 11 As shown, intramuscular injection of HAd5-F-P2A-E2 can produce higher CD4 + T lymphocytes and CD8 + T lymphocyte expression, in which HAd5-F-P2A-E2 intramuscular injection group induced CD4 + T lymphocytes and CD8 + T lymphocytes were significantly higher than those in the control group (p<0.01) and (p<0.05).

[0100] Therefore, the bivalent vaccine prepared by the present invention can induce a higher cellular immune response after immunization, which is significantly higher than that of the control group.

[0101] In summary, the bovine parainfluenza virus type 3 and bovine viral diarrhea virus bivalent vaccine prepared using recombinant adenovirus vectors in the present invention can induce good humoral immunity and cellular immunity effects after immunization, and there is no immunosuppression caused by simultaneous immunization with vaccines. It can solve the problem that different vaccines cannot be used for simultaneous immunization in actual production, and is a relatively ideal bivalent adenovirus vector vaccine.

Claims

1. A bovine parainfluenza virus type 3 and bovine viral diarrhea virus combined adenovirus vector vaccine, characterized in that: The vaccine is prepared by cloning the BPIV3C F gene and the BVDV-1 E2 gene into a replication-deficient human adenovirus type 5 vector, wherein the BPIV3C F gene sequence is shown in SEQ ID No. 1, and the BVDV-1 E2 gene sequence is shown in SEQ ID No. 2; the BPIV3C F gene and the BVDV-1 E2 gene are cloned into the replication-deficient human adenovirus type 5 vector in the form of a fusion protein, wherein the nucleic acid sequence of the fusion protein is shown in SEQ ID No.

3.

2. The bivalent vaccine according to claim 1, characterized in that: The preparation method is as follows: a. Synthesizing the nucleic acid of the fusion protein of claim 1 and cloning it into the adenovirus shuttle plasmid pDC316 to obtain the recombinant shuttle plasmid pDC316-F-P2A-E2; b. Co-transfect the recombinant shuttle vector pDC316-F-P2A-E2 and the adenovirus backbone plasmid pBHGlox E1, 3Cre into HEK293 cells, recombinant and package.

3. Use of the bivalent vaccine according to any one of claims 1 to 2 in the preparation of biological products for preventing bovine parainfluenza type 3 and bovine viral diarrhea.

4. The use according to claim 3, characterized in that: The dual vaccine is prepared as an injection, nasal drops or inhalation.

5. The use according to claim 4, characterized in that: The injection is an intramuscular injection.

Images