Construction method and application of IBRV vector vaccine with gD gene extracellular region modification

Abstract

This invention discloses a method for constructing and applying an IBRV vector vaccine modified with the extracellular region of the gD gene, belonging to the field of veterinary biological products technology. Using bovine infectious rhinotracheitis virus strain LN01 / 08 as a backbone, the full-length coding sequence of its own gD gene is replaced through homologous recombination technology to construct a recombinant virus. This recombinant virus exhibits good proliferation characteristics in MDBK suspension cells, with a viral titer reaching 10. 8.5 TCID 50 The recombinant virus, when immunized in cattle, induces higher levels of neutralizing antibodies, achieving 100% protection against virulent IBRV challenge. It also possesses gD antibody diagnostic marker characteristics, distinguishing naturally infected animals from vaccine-immunized animals. The vector vaccine provided by this invention exhibits good safety and high immunogenicity, and can be used to establish differential diagnostic methods, demonstrating promising application prospects.

Description

Technical Field

[0001] This invention belongs to the field of veterinary biological products technology, specifically relating to a bovine infectious rhinotracheitis virus vector vaccine modified with the extracellular region of the gD gene, its construction method, and its application. Background Technology

[0002] Infectious bovine rhinotracheitis (IBR) is caused by bovine herpesvirus type 1 (BoHV-1), also known as bovine infectious rhinotracheitis virus. Infectious bovine rhinotracheitis virus IBRV (Infectious Disease of Bovine Species) is an acute, febrile, and contagious disease of cattle. It presents with diverse clinical manifestations, including respiratory, reproductive tract, meningitis, and ophthalmitis forms, and can lead to slowed weight gain in fattening cattle and decreased milk production in dairy cows, causing severe economic losses to the cattle industry.

[0003] Currently, prevention of infectious bovine rhinotracheitis (BBT) mainly relies on vaccination. Commercially available vaccines primarily fall into two categories: live attenuated vaccines and inactivated vaccines. Inactivated vaccines have relatively good safety profiles, but the induced immune response is shorter-lasting, requiring multiple doses, and they primarily induce humoral immunity, making it difficult to effectively induce cellular immune responses. Live attenuated vaccines offer better immunization efficacy, but there is a potential risk of virulence reversion, which is detrimental to serological monitoring and eradication of the disease. Summary of the Invention

[0004] This invention provides a method for constructing a recombinant bovine infectious rhinotracheitis virus modified with the gD gene, comprising the following steps: Provides the recombinant gD gene, the upstream homologous arm of the gD gene, and the downstream homologous arm of the gD gene; The upstream homologous arm of the gD gene, the recombinant gD gene, and the downstream homologous arm of the gD gene are sequentially cloned into a shuttle vector to construct a recombinant transfer vector containing the upstream homologous arm of the gD gene, the recombinant gD gene, and the downstream homologous arm of the gD gene. The recombinant transfer vector was co-transfected with the genomic DNA of bovine infectious rhinotracheitis virus strain LN01 / 08 into MDBK cells. Through homologous recombination, the recombinant gD gene replaced the original gD gene in the genome of bovine infectious rhinotracheitis virus strain LN01 / 08. Recombinant viruses were screened and purified to obtain recombinant bovine infectious rhinotracheitis virus with gD gene modification.

[0005] Preferably, the recombinant gD gene is as shown in SEQ ID NO:3.

[0006] Preferably, the upstream homologous arm of the gD gene is shown in SEQ ID NO:5; and the downstream homologous arm of the gD gene is shown in SEQ ID NO:6.

[0007] Preferably, the shuttle carrier is a pUC19-derived carrier.

[0008] Preferably, the co-transfection is performed using liposome-mediated or electroporation methods.

[0009] Preferably, the screening and purification are performed using plaque purification.

[0010] The present invention also provides a recombinant bovine infectious rhinotracheitis virus vector vaccine modified with the gD gene, wherein the vector vaccine comprises recombinant bovine infectious rhinotracheitis virus obtained by the construction method.

[0011] Preferably, the viral titer of the recombinant bovine infectious rhinotracheitis virus on MDBK suspension cells is not less than 10. 8.0 TCID 50 / mL.

[0012] The present invention also provides the application of the recombinant bovine infectious rhinotracheitis virus obtained by the above construction method in the preparation of vaccines or drugs for the prevention of bovine infectious rhinotracheitis.

[0013] This invention also provides the application of the recombinant bovine infectious rhinotracheitis virus obtained by the aforementioned construction method in virus identification and detection. Using a gD protein monoclonal antibody, the recombinant bovine infectious rhinotracheitis virus of this invention can be distinguished from the parental wild-type IBRV at the antigenic level of infected cells; cells infected with the recombinant virus show no fluorescence or very weak fluorescence after staining with this gD monoclonal antibody, while cells infected with the parental wild-type virus show clear and specific fluorescence.

[0014] Compared with the prior art, the present invention has the following advantages: 1. This invention, for the first time, uses bovine infectious rhinotracheitis virus strain LN01 / 08 as a backbone and conducts targeted modification of its gD gene using homologous recombination technology to successfully construct a recombinant vector vaccine rIBRV-LN01 / 08. This recombinant virus possesses monoclonal antibody distinguishing marker characteristics at the level of infected cell antigens; staining with a specific gD protein monoclonal antibody can differentiate the recombinant virus from its parent wild-type IBRV.

[0015] 2. The present invention features dual optimization of gD expression elements: the natural secretion signal peptide is replaced with a high-secretion-efficiency tPA signal peptide, a Kozak sequence is added to the 5' end, and bovine cell synonymous codons are optimized throughout the genome, which significantly increases the extracellular secretion of gD protein; immunological experiments have confirmed that the level of neutralizing antibodies induced by the recombinant virus is slightly higher than that of the parental LN01 / 08 strain, and the immune response is faster.

[0016] 3. The recombinant vector vaccine constructed in this invention retains the good safety profile and MDBK suspension cell proliferation characteristics of the parental attenuated strain, with a viral titer reaching 10. 8.5 TCID 50 / mL or higher, suitable for large-scale industrial production.

[0017] 4. The recombinant virus retains the integrity and safety of the parent attenuated strain, with a 100% animal challenge protection rate, and can effectively prevent and control bovine infectious rhinotracheitis. Attached Figure Description

[0018] Figure 1 The image shows the electrophoretic identification pattern, including lanes for the recombinant transfer vector pUC19-gDUP-OptgD-gDDOWN and its enzyme digestion fragments, lanes for identifying the recombinant virus, and lanes for identifying the parent virus.

[0019] Figure 2 This is a typical CPE diagram for the recombinant virus IBRV.

[0020] Figure 3 Indirect immunofluorescence images of the parent virus (top) and recombinant virus (bottom).

[0021] Figure 4 This is a comparison of the growth curves of the recombinant virus rIBRV-LN01 / 08 of this invention and the parental LN01 / 08 strain on MDBK suspension cells.

[0022] In this application, the parent strain IBRV LN01 / 08 (taxonomically named bovine infectious rhinotracheitis virus) was deposited on April 20, 2012, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 6033, and the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. Detailed Implementation

[0023] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0024] The technical solution of the present invention will be described in detail below with reference to the embodiments. The following embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. Experimental methods in the embodiments that do not specify specific conditions are generally carried out under conventional conditions or under the conditions recommended by the manufacturer.

[0025] Example 1: Construction of a recombinant transfer vector 1. Recombinant gD gene sequence In this embodiment, the full-length coding sequence of the gD gene of strain LN01 / 08 (provided by the isolation and identification of bovine infectious rhinotracheitis virus strain LN01 / 08 and its main functional gene sequence analysis, published in the master's thesis on June 1, 2010, shown in SEQ ID NO:1) is used as the basis, and SEQ ID NO:2 is the amino acid sequence of the gD protein.

[0026] Using the natural gD gene SEQ ID NO:1 (protein SEQ ID NO:2) of strain LN01 / 08 as a template, the following modifications were made: (1) Signal peptide replacement: The natural N-terminal 1-18aa signal peptide MQGPTLAVLGALLAVAVS was replaced with the tPA high-efficiency secretion signal peptide MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRR; (2) Codon synonym optimization: The entire coding region was modified according to the codon preference of MDBK bovine cells, changing only the codons and not the encoded amino acids; (3) Translational regulatory elements: A GCCACC Kozak enhancement sequence was added to the 5' end, and a TAA stop codon was added to the 3' end. The modified recombinant gD gene SEQ ID NO:3 corresponds to the protein SEQ ID NO:4; Except for the first 30 tPA signal peptide amino acids, the remaining mature gD protein sequence of SEQ ID NO:4 is completely the same as that of the natural SEQ ID NO:2.

[0027] 2. Synthesis The recombinant gD gene shown in SEQ ID NO:3 was synthesized using a whole-genome synthesis method. The upstream homologous arm of the gene is SEQ ID NO:5 (GenBank accession number AJ004801:118096..118895) and the downstream homologous arm of the gene is SEQ ID NO:6 (GenBank accession number AJ004801:120150..120949).

[0028] 3. PCR amplification Amplification of the upstream homologous arm of the gD gene: Using the DNA shown in SEQ ID NO:5 as a template, the upstream homologous arm of the gD gene was amplified by PCR using primers gD-UP-F (containing the EcoRI site, SEQ ID NO:7) and gD-UP-R (containing the BamHI site, SEQ ID NO:8). The PCR product was recovered after double digestion with EcoRI and BamHI for later use.

[0029] Amplification of the recombinant gD gene: Using the DNA shown in SEQ ID NO:3 as a template, the recombinant gD gene was amplified by PCR using primers gD-F (containing the BamHI site, SEQ ID NO:11) and gD-R (containing the HindIII site, SEQ ID NO:12). The PCR product was recovered after double digestion with BamHI and HindIII for later use.

[0030] Amplification of the downstream homologous arm of the gD gene: Using the DNA shown in SEQ ID NO:6 as a template, the downstream homologous arm of the gD gene was amplified by PCR using primers gD-DOWN-F (containing the HindIII site, SEQ ID NO:9) and gD-DOWN-R (containing the Xbal site, SEQ ID NO:10). The PCR product was recovered after double digestion with HindIII and Xbal for later use.

[0031] 4. Enzyme digestion and ligation The pUC19 vector was digested with EcoRI and XbaI, and the linearized vector fragment (approximately 2686 bp) was recovered.

[0032] The linearized pUC19 vector (EcoRI-XbaI), the upstream homologous arm of gD (EcoRI-BamHI), the recombinant gD gene (BamHI-HindIII), and the downstream homologous arm of gD (HindIII-XbaI) were mixed in a molar ratio of 1:3:3:3, and T4 DNA ligase was added. Ligation was performed overnight at 16°C. The ligation product was transformed into *E. coli* DH5α competent cells, plated on LB agar plates containing ampicillin, and cultured overnight at 37°C.

[0033] 5. Screening and identification of recombinant plasmids Single clones were selected for preliminary screening using bacterial culture PCR. Positive clones were subjected to plasmid extraction for EcoRI / XbaI double digestion and sequencing verification. The correctly constructed recombinant transfer vector was named pUC19-gDUP-OptgD-gDDOWN.

[0034] 6. Test Results EcoRI / XbaI double digestion confirmed that the recombinant transfer vector pUC19-gDUP-OptgD-gDDOWN digestion yielded a vector fragment of approximately 2686 bp and a target insert fragment of approximately 2902 bp, consistent with the expected size. Figure 1 Sequencing results confirmed that the fragments were correctly linked and the sequences were error-free.

[0035] Example 2: Rescue and purification of recombinant bovine infectious rhinotracheitis virus rIBRV-LN01 / 08 1. Preparation of MDBK cells After digestion, well-grown MDBK cells were seeded into 6-well cell culture plates, approximately 5 × 10⁶ cells per well.5 Cells were cultured overnight at 37°C in a 5% CO2 incubator until the cell density reached 80%-90%, at which point they were used for transfection.

[0036] 2. Extraction of genomic DNA from strain LN01 / 08 The LN01 / 08 strain F3 generation virus solution was inoculated into well-grown MDBK monolayer cells (T75 cell culture flask, cell density approximately 80%). After adsorption at 37°C for 1 hour, maintenance medium (DMEM containing 2% horse serum) was added, and the cells were incubated at 37°C in a 5% CO2 incubator. Cytopathic effect (CPE) was observed daily. When CPE reached 80% or higher, the virus solution was harvested, and after three freeze-thaw cycles at -80°C, the supernatant was collected by centrifugation at 12,000 rpm for 10 min. Genomic DNA of the LN01 / 08 strain was extracted according to the instructions of the viral genomic DNA extraction kit and stored at -20°C for later use.

[0037] 3. Co-transfection Mix 2 μg of recombinant transfer vector pUC19-gDUP-OptgD-gDDOWN and 1 μg of LN01 / 08 strain genomic DNA, and add to 125 μL of Opti-MEM medium. Following the Lipofectamine™ 3000 kit instructions, add 6 μL of P3000 reagent (1 μg DNA corresponds to 2 μL P3000 reagent) to the DNA mixture and mix thoroughly. Separately, add 5 μL of Lipofectamine™ 3000 to 125 μL of Opti-MEM medium. Mix the two solutions and incubate at room temperature for 15 min. Add the transfection complex dropwise to 6-well MDBK cells and incubate at 37°C in a 5% CO2 incubator. Six hours after transfection, replace the medium with DMEM maintenance medium containing 2% horse serum and continue culturing.

[0038] 4. Screening of recombinant viruses Observe the cytopathic effect of the virus daily after transfection. When typical herpesvirus CPE (cell shrinkage, aggregation, and shedding) appears, harvest the culture supernatant and cells, freeze and thaw three times at -80℃, centrifuge at 12,000 rpm for 10 min and collect the supernatant as the crude extract of recombinant virus.

[0039] 5. Plaque purification The crude recombinant virus extract was serially diluted 10-fold and inoculated into well-grown MDBK monolayer cells in 6-well plates. After adsorption at 37°C for 1 hour, the virus solution was discarded, and DMEM covering medium containing 1% low-melting-point agarose and 2% horse serum was added to each well. After solidification, the plates were incubated at 37°C with 5% CO2. Plaques appeared after 3-5 days. Individual plaques were picked up using a sterile pipette tip under an inverted microscope and inoculated into 24-well MDBK cells for amplification. Plaque purification was repeated 3-5 times until purified recombinant virus was obtained.

[0040] 6. Large-scale culture of recombinant viruses The purified recombinant virus was inoculated into MDBK cells in T75 cell culture flasks for amplification, and the viral fluid was harvested, aliquoted, and stored at -80°C.

[0041] 7. Test Results like Figure 2 As shown, typical IBRV cytopathic effects (CPE) appeared on day 3 after co-transfection, characterized by shrunken cells clustered in grape-like patterns, followed by cell detachment. After five rounds of plaque purification, purified recombinant virus was obtained and named rIBRV-LN01 / 08.

[0042] Example 3 Identification of recombinant virus rIBRV-LN01 / 08 1. PCR identification Genomic DNA of recombinant virus rIBRV-LN01 / 08 was extracted and amplified by PCR using primer pairs as shown in SEQ ID NO:13~14.

[0043] Genomic DNA was extracted from the parental strain LN01 / 08 and amplified by PCR using primer pairs as shown in SEQ ID NO:15~16.

[0044] 2. PCR identification results The genomic DNA of the recombinant virus rIBRV-LN01 / 08 can amplify a full-length band of the gD gene of approximately 1300 bp. Figure 1 The genomic DNA of the parent virus can be amplified to produce a full-length band of the gD gene of approximately 1250 bp. Figure 1 All tests were performed and the results met expectations.

[0045] 3. Indirect immunofluorescence identification Recombinant virus rIBRV-LN01 / 08 and parental strain LN01 / 08 were inoculated into 96-well MDBK cells and cultured at 37°C for 24-48 hours. After CPE (cytopathic effect) appeared, the culture medium was aspirated, and the cells were washed twice with PBS. Pre-chilled methanol-acetone (1:1) fixative was added, and the cells were fixed at -20°C for 20 min. After washing three times with PBS, IBRV-positive serum (1:200 dilution) and gD protein monoclonal antibody (1:500 dilution) were added, and the cells were incubated at 37°C for 1 hour. After washing three times with PBS, FITC-labeled goat anti-mouse IgG secondary antibody (1:1000 dilution) was added, and the cells were incubated at 37°C in the dark for 45 min. After washing three times with PBS, the cells were observed under a fluorescence microscope.

[0046] 4. Results of indirect immunofluorescence identification When stained with IBRV-positive serum, cells infected with both the recombinant virus and the parent virus showed specific green fluorescence, indicating that the recombinant virus successfully expressed the IBRV antigen. Figure 3 As shown, when stained with a gD protein monoclonal antibody, the parental virus exhibits clear fluorescence, while the recombinant virus shows no fluorescence or very weak fluorescence. This phenomenon indicates that the spatial conformation of the gD protein expressed by the recombinant virus has changed, resulting in the loss of the conformational epitope (non-linear epitope) recognized by the monoclonal antibody. It is speculated that this is due to changes in protein folding and conformational epitope caused by signal peptide substitution. Based on this, recombinant virus and parental wild-type IBRV can be distinguished at the antigen level of infected cells.

[0047] Example 4: Proliferation characteristics of recombinant virus rIBRV-LN01 / 08 on MDBK suspension cells 1. Preparation of MDBK suspension cells Cells were resuscitated from the MDBK suspension cell bank and expanded in shake flasks using serum-free MDBK suspension cell culture medium. Culture conditions: 37℃, 5% CO2, shaker speed 120 rpm. Cells were cultured until the density reached 2.0-3.0 × 10⁶ cells / year. 6 When the number of cells / mL is reached, it can be used for passage or for inoculation.

[0048] 2. Virus inoculation Take well-grown MDBK suspension cells and use 1.5 × 10⁻⁶ cells. 6 Cells were seeded at a density of 2.5 × 10⁶ cells / mL in shake flasks and cultured for 24 hours until the cell density reached 2.5 × 10⁶ cells / mL. 6 At a concentration of [number] cells / mL, recombinant virus rIBRV-LN01 / 08 and parental strain LN01 / 08 were inoculated with an MOI of 0.01, respectively. After inoculation, the cells were cultured at 37°C, 5% CO2, and 120 rpm.

[0049] 3. Virus harvesting and titer determination Samples were collected every 12 hours after virus inoculation, and the sampling was continued until 96 hours. The samples were repeatedly frozen and thawed 3 times at -80°C, then centrifuged at 12000 rpm for 10 min to collect the supernatant, and the Reed-Muench method was used to determine the virus TCID 50 . The specific method: The virus solution was serially diluted 10-fold with DMEM maintenance medium, and 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 5 dilution levels were inoculated into MDBK cells that had been cultured for 16-20 hours in 96-well plates, 8 wells were inoculated for each dilution, 100 μL per well, and normal cell controls were set at the same time. The cells were cultured in a 37°C, 5% CO2 incubator for 4-5 days, the number of wells with CPE was recorded, and the TCID was calculated according to the Reed-Muench method 50 .

[0050] 4. Test results The growth curves of the recombinant virus rIBRV-LN01 / 08 and the parental LN01 / 08 strain on MDBK suspension cells are shown in Figure 4 . Both viruses began to show significant proliferation 24 hours after inoculation, and reached the peak virus titer at 48-60 hours. The peak virus titer of the recombinant virus rIBRV-LN01 / 08 was 10 8.5 TCID 50 / mL, and the peak virus titer of the parental LN01 / 08 strain was 10 8.3 TCID 50 / mL, there was no significant difference between the two (P>0.05). The results showed that the modification of the gD gene did not affect the proliferation ability of the virus on MDBK suspension cells, and the recombinant virus maintained the good cell adaptability of the parental strain.

[0051] Example 5: Safety test of recombinant virus rIBRV-LN01 / 08 in mice. 1. Test materials SPF grade BALB / c male mice, weighing 18-22g, with the license number SYXK (Su) 2018-0024. Recombinant virus rIBRV-LN01 / 08 (F2 generation virus solution, titer 10 8.5 TCID 50 / mL), parental LN01 / 08 strain (F3 generation virus solution, titer 10 8.3 TCID 50 / mL), DMEM medium.

[0052] 2. Test methods 40 BALB / c mice were randomly divided into 4 groups, 10 mice in each group: Group 1 (High-dose recombinant virus group): Intraperitoneal injection of 0.5 mL / animal of rIBRV-LN01 / 08 virus solution (approximately 5 × 10⁻⁶ cells / animal). 7 TCID 50 / Only); Group 2 (low-dose recombinant virus group): Intraperitoneal injection of 0.1 mL / animal of rIBRV-LN01 / 08 virus solution (approximately 1 × 10⁻⁶). 7 TCID 50 / Only); Group 3 (parental virus control group): Intraperitoneal injection of 0.5 mL / animal of LN01 / 08 strain virus fluid (approximately 5 × 10⁸) 7 TCID 50 / Only); Group 4 (blank control group): 0.5 mL of DMEM culture medium per animal was injected intraperitoneally.

[0053] Mice were observed for 14 consecutive days after inoculation, and their clinical symptoms (mental state, appetite, activity level, and mortality) were recorded daily, along with their body weight (before inoculation and on days 3, 7, 10, and 14 post-inoculation). After the observation period, all mice were euthanized, and the pathological changes of the major organs (heart, liver, spleen, lungs, and kidneys) were observed through dissection.

[0054] 3. Test Results During the observation period, no obvious clinical symptoms (lethargy, decreased appetite, reduced activity, etc.) were observed in any of the experimental groups of mice, and no mice died. Regarding weight changes, all groups of mice showed a normal weight gain trend, and there was no significant difference in weight gain among the groups on day 14 post-inoculation (P>0.05). Autopsy observation revealed no obvious pathological changes visible to the naked eye in the major organs of any group of mice.

[0055] The results showed that the recombinant virus rIBRV-LN01 / 08 had good safety in mice, comparable to the safety of the parental LN01 / 08 strain.

[0056] Example 6 Immunogenicity test of recombinant virus rIBRV-LN01 / 08 1. Test materials Thirty healthy IBRV antigen-antibody negative calves aged 3-7 months were provided by a cattle farm in a non-epidemic area of ​​Jiangsu Province. The following were prepared: recombinant virus rIBRV-LN01 / 08 (F3 generation virus solution), parental LN01 / 08 strain (F3 generation virus solution), virulent IBRV LN01 / 08 strain challenge virus (preserved by Huawit (Jiangsu) Biopharmaceutical Co., Ltd.), and PBS solution.

[0057] 2. Test methods (1) Animal grouping and immunization: 30 calves were randomly divided into 3 groups of 10 each: Recombinant virus immunization group: intramuscular injection of rIBRV-LN01 / 08 strain, with a dose of 10 mg / L per animal. 6.0 TCID 50 Parental virus immunization group: LN01 / 08 strain was injected intramuscularly, with a dose of 10 mg / L per animal. 6.0 TCID 50 Blank control group: 1 mL PBS injected intramuscularly per head.

[0058] (2) Serum collection and neutralizing antibody detection: Serum was collected before immunization and on days 7, 14, 21, and 28 after immunization. The serum neutralizing antibody titer was detected using a micro-volume serum neutralization test (VN). Specific method: The serum to be tested was inactivated at 56℃ for 30 min, serially diluted 2-fold with DMEM, and mixed with an equal volume containing 100-200 TCID50. 50 Mix the IBRV (LN01 / 08 strain) virus solution and neutralize at 37℃ for 40-60 min. Inoculate the mixture into 96-well MDBK cells, 100 μL per well for each dilution, incubate at 37℃, 5% CO2, and observe for 4-5 days. Record CPE and calculate the neutralizing antibody titer (expressed as the highest serum dilution that inhibits 50% CPE) according to the Reed-Muench method.

[0059] (3) Challenge protection test: On day 28 after immunization, 5 calves were randomly selected from each group, and 4 mL of virulent strain IBRVLN01 / 08 (F3 generation) was sprayed into the nostrils of each calf (containing 10... 6.0 TCID 50 Clinical symptoms of calves were observed and recorded daily for 14 consecutive days after challenge, including body temperature, mental state, appetite, respiratory symptoms, and changes in the nose and eyes. Nasal swab samples were collected on days 3, 5, 7, 10, and 14 after challenge for virus isolation. The criteria for determining protection after challenge were as follows: Calves with a persistent body temperature exceeding 40.0℃, exhibiting typical IBR clinical symptoms (nasal discharge, cough, lethargy, etc.), and a positive nasal swab virus isolation were considered unprotected; calves with normal or transiently slight body temperature (not exceeding 40.0℃), no typical clinical symptoms or mild symptoms, and a negative nasal swab virus isolation were considered protected.

[0060] (4) Assessment of immune response capacity Peripheral blood was collected from calves in each group on day 14 after immunization. Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood using density gradient centrifugation with lymphocyte separation medium. The isolated PBMCs were seeded into 24-well plates as follows: experimental group: PBMCs + IBRV whole virus inactivated antigen (specific stimulation); positive control group: PBMCs + ConA (concanavalin A, non-specific stimulation); negative control group: PBMCs + equal volume of PBS. The plates were incubated at 37°C in a 5% CO2 incubator for 48 hours.

[0061] Cell culture supernatants from each group were collected, and the concentration of IFN-γ in the supernatant was detected using a bovine IFN-γ ELISA kit, strictly following the instructions. IFN-γ is a key cytokine in Th1-type cellular immune responses, and its secretion level directly reflects the strength of cellular immunity.

[0062] 3. Test Results (1) Neutralizing antibody detection results Serum neutralizing antibody tests at various time points post-immunization showed that all calves were negative for neutralizing antibodies (<1:2) before immunization. On day 7 post-immunization, neutralizing antibodies began to appear in both the recombinant virus immunization group and the parental virus immunization group, with average titers of 1:8 and 1:4, respectively. On day 14 post-immunization, antibody levels in both groups significantly increased, with average titers of 1:32 in both the recombinant virus and parental virus immunization groups. On day 28 post-immunization, the average neutralizing antibody titer reached 1:64 in both the recombinant virus and parental virus immunization groups. The blank control group remained negative for neutralizing antibodies throughout the entire experiment.

[0063] The results showed that the recombinant virus rIBRV-LN01 / 08 could stimulate the body to produce a good neutralizing antibody response.

[0064] (2) Results of the challenge protection test The results of the challenge protection test are shown in Table 1.

[0065] Table 1. Results of the protection test of calves challenged with recombinant virus rIBRV-LN01 / 08.

[0066] Following viral challenge, calves in the blank control group began exhibiting typical IBR clinical symptoms on days 2-3, including fever (up to 41.2℃), lethargy, loss of appetite, serous or purulent nasal discharge, and coughing. Virus isolation from nasal swabs was positive in all cases. Calves in the recombinant virus immunization group and the parental virus immunization group showed normal or only transiently mild fever (<39.8℃) after viral challenge, with no obvious IBR clinical symptoms observed, and virus isolation from nasal swabs was negative in both groups. The protection rate in both immunization groups was 100%.

[0067] (3) IFN-γ detection results Negative and positive controls: The concentration of IFN-γ in the negative control group of all groups was below the detection limit; high levels of IFN-γ were detected in the positive control group, indicating good cell viability.

[0068] Recombinant virus immunization group: After stimulation with IBRV antigen, the average concentration of IFN-γ in the culture supernatant of PBMCs was 850 pg / mL.

[0069] Parental virus immunization group: After stimulation with IBRV antigen, the average concentration of IFN-γ in the culture supernatant of PBMCs was 420 pg / mL.

[0070] Blank control group: No significant level of IFN-γ was detected.

[0071] The results of this embodiment demonstrate that, compared with the parental viral vaccine, the recombinant viral vaccine rIBRV-LN01 / 08 of this invention can induce a significantly stronger specific cellular immune response (IFN-γ secretion is increased by approximately 2 times). Stronger cellular immunity is crucial for clearing virus-infected cells and establishing long-term immune memory, proving that the vaccine of this invention has a more comprehensive and efficient immune protection mechanism, exhibiting outstanding technical advantages.

[0072] Example 7: Persistence of Immunity Assay for Recombinant Virus rIBRV-LN01 / 08 1. Test materials Thirty healthy IBRV calves aged 3-7 months that were negative for antigen and antibody. Recombinant virus rIBRV-LN01 / 08 (F3 generation virus liquid), parental virus strain LN01 / 08 (F3 generation virus liquid), and virulent IBRV strain LN01 / 08 strain for challenge.

[0073] 2. Test methods The 20 calves were randomly divided into 3 groups of 10 each: Recombinant virus immunization group: Recombinant virus rIBRV-LN01 / 08 was injected intramuscularly, with a dose of 10 mg / L per animal. 6.0 TCID 50 ; Parental virus immunization group: Parental virus LN01 / 08 was injected intramuscularly, with a dose of 10 mg / L per animal. 6.0 TCID 50 ; Blank control group: 1 mL PBS injected intramuscularly per head.

[0074] Serum samples were collected on days 30, 90, 180, 270, and 360 post-immunization to detect neutralizing antibody titers.

[0075] On days 180 and 360 post-immunization, five calves from each of the immunized and control groups were selected for challenge experiments. On day 360 post-immunization, five calves from each of the remaining surviving calves were randomly selected for challenge experiments. The challenge procedure was the same as in Example 6. The protective effect of the challenge was observed and recorded after the challenge.

[0076] 3. Test Results The average geometric mean titer of neutralizing antibodies in each group of calves at different time points is shown in Table 2 below.

[0077] Table 2. Geometric mean titer of neutralizing antibodies in calves of each group at different time points.

[0078] The results showed that the recombinant virus immunization group had higher levels of neutralizing antibodies at all time points after immunization than the parental virus immunization group, and the antibody decline rate was slower. At 360 days post-immunization, the recombinant virus group maintained a protective antibody level of 1:32, while the antibodies in the parental virus group had essentially disappeared.

[0079] The results of the challenge protection test are shown in Table 3 below.

[0080] Table 3 Results of the challenge protection test

[0081] At challenge 180 days post-immunization, both vaccine groups provided 100% protection. However, at challenge 360 ​​days post-immunization, the recombinant virus immunization group maintained 100% protection, with no clinical symptoms observed in any calves and negative virus isolation from nasal swabs; while the protection rate of the parent virus immunization group dropped to 40%, with most calves exhibiting typical IBR clinical symptoms.

[0082] The results of this embodiment demonstrate that the recombinant viral vaccine rIBRV-LN01 / 08 provided by this invention not only induces the body to produce higher levels of neutralizing antibodies, but also provides a significantly longer duration of immune protection than the parent viral vaccine. The recombinant viral vaccine still provides complete protection one year after immunization, while the protective efficacy of the parent viral vaccine declines significantly after one year. This proves that by modifying the gD gene, the vaccine of this invention can induce a more durable and effective immune memory, thereby achieving a longer period of immune protection—a significant technological advancement and advantage.

[0083] The above description is merely a preferred embodiment of this application and is not intended to limit this application. 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 method for constructing a recombinant bovine infectious rhinotracheitis virus modified with the gD gene, characterized in that, Includes the following steps: Provides the recombinant gD gene, the upstream homologous arm of the gD gene, and the downstream homologous arm of the gD gene; The upstream homologous arm of the gD gene, the recombinant gD gene, and the downstream homologous arm of the gD gene are sequentially cloned into a shuttle vector to construct a recombinant transfer vector containing the upstream homologous arm of the gD gene, the recombinant gD gene, and the downstream homologous arm of the gD gene. The recombinant transfer vector was co-transfected with the genomic DNA of bovine infectious rhinotracheitis virus strain LN01 / 08 into MDBK cells. Through homologous recombination, the recombinant gD gene replaced the original gD gene in the genome of bovine infectious rhinotracheitis virus strain LN01 / 08. Recombinant viruses were screened and purified to obtain recombinant bovine infectious rhinotracheitis virus with gD gene modification.

2. The construction method according to claim 1, characterized in that, The recombinant gD gene is shown in SEQ ID NO:

3.

3. The construction method according to claim 1, characterized in that, The upstream homologous arm of the gD gene is shown in SEQ ID NO:5; the downstream homologous arm of the gD gene is shown in SEQ ID NO:

6.

4. The construction method according to claim 1, characterized in that, The shuttle carrier is a pUC19-derived carrier.

5. The construction method according to claim 1, characterized in that, The co-transfection was performed using liposome-mediated or electroporation methods.

6. The construction method according to claim 1, characterized in that, The screening and purification were performed using plaque purification.

7. A recombinant bovine infectious rhinotracheitis virus vector vaccine modified with the gD gene, characterized in that, The vector vaccine comprises recombinant bovine infectious rhinotracheitis virus obtained by the construction method according to any one of claims 1-6.

8. The vector vaccine according to claim 7, characterized in that, The viral titer of the recombinant bovine infectious rhinotracheitis virus on MDBK suspension cells is not less than 10. 8.0 TCID 50 / mL.

9. The use of the recombinant bovine infectious rhinotracheitis virus obtained by the construction method according to any one of claims 1-6 in the preparation of a vaccine or drug for the prevention of bovine infectious rhinotracheitis.

10. The application of the recombinant bovine infectious rhinotracheitis virus obtained by the construction method according to any one of claims 1-6 in virus identification and detection.

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