Bovine parainfluenza virus type 3 V protein deletion strain and application thereof

Abstract

The invention belongs to the technical field of virus construction, and particularly relates to a bovine parainfluenza virus type 3 V protein deletion strain and application thereof. The construction process of the strain comprises the following steps: by taking pBPIV3-SX full-length infectious clone plasmid as a template, respectively amplifying two gene segments before and after a base editing site of a V gene, and fusing the obtained amplified segments to obtain a segment containing V gene deletion; and replacing a fragment containing V gene deletion to BssHII and PacI restriction enzyme cutting sites of the pBPIV3-SX plasmid, and transfecting to carry out virus rescue, thereby obtaining the recombinant plasmid pBPIV3-SX. According to the invention, on the basis of a reverse genetics operation technology, a V protein deleted infectious clone plasmid is constructed, and a recombinant deleted virus is successfully rescued; meanwhile, the V protein is obtained through an escherichia coli expression system, a mouse is immunized to obtain a polyclonal antibody, a theoretical basis is provided for functional research of the BPIV3 non-structural V protein and development of genetic engineering vaccines, and the BPIV3 non-structural V protein has important scientific value and application value.

Description

Technical Field

[0001] This invention belongs to the field of virus construction technology, specifically relating to a bovine parainfluenza virus type 3 V protein deletion strain and its application. Background Technology

[0002] Bovine respiratory disease complex (BRDC) is a leading cause of bovine respiratory diseases worldwide, causing significant economic losses to the cattle industry annually. With the development of intensive farming, bovine respiratory diseases are now widespread in stall-fed cattle. Bovine parainfluenzavirus type 3 (BPIV3) is one of the important viral pathogens causing bovine respiratory diseases, inducing respiratory symptoms of varying degrees. Currently, BPIV3 is widespread globally, causing substantial economic losses to the livestock industry. In 1959, American researchers detected and isolated the first BPIV3 virus from a case of "transport fever." Subsequently, the virus has been continuously isolated and reported, and BPIV3 infection now has a global distribution. Epidemiological surveys show that the disease has occurred in many provinces in my country, causing significant economic losses and severely restricting the sustainable development of my country's cattle industry. Although domestic and international scholars have conducted extensive research on this pathogen, no targeted drug has yet been developed to treat the disease. According to virological classification, BPIV3 belongs to the paramyxovirus family (…). Paramyxoviridae Paramyxovirinae ( Paramyxovirus subfamily ), Respiratory Viruses ( Respiratory virus genus The virus is a member of the family of viruses. Viral particles are spherical, enveloped, and approximately 150–200 nm in diameter. The full-length genome is approximately 15 kb in length, and its genome structure is: 3' Leader-NPMF-HN-L-5' Trailer. At the 3' end of the genome is a non-coding leader sequence of approximately 110 nt, which plays a crucial role in initiating mRNA transcription and gene replication. At the 5' end is a crucial non-coding trailing sequence of 115 nt. Furthermore, the translation and assembly of the progeny viral genome begins with the binding of the NP protein to the last six nucleotides of the 5' Trailer, proceeding sequentially from 5' to 3' to complete the transcription process.

[0003] V protein, a multifunctional non-structural protein in paramyxoviruses, plays an important role in antagonizing host interferon and regulating viral replication. BPIV3, as a paramyxoviridae family... Paramyxoviridae) one of the members, the encoded V protein only in the function of antagonizing interferon preliminary study. V protein consists of about 410 amino acid residues, is a very important protein for bovine parainfluenza virus 3 replication, on the one hand, can be combined with NP protein to jointly control the virus replication, on the other hand, can affect the state of the host cell, such as regulating various signal pathways in the host cell, so that the virus can have sufficient replication time in the cell. However, whether the V protein can also play other roles, and the molecular mechanism of how to regulate the virus replication has not been elucidated. SUMMARY

[0004] Based on the above technical problems, the present application provides a bovine parainfluenza virus 3 type V protein deletion strain, which maintains effective replication ability in cells, and the virus titer is significantly reduced, which meets the characteristics of attenuated vaccine.

[0005] The specific technical scheme provided by the present application is as follows: In the first aspect of the present application, a bovine parainfluenza virus 3 type V protein deletion strain is provided, which is constructed according to the following steps: The two gene fragments before and after the base editing site of the V gene are amplified respectively by taking pBPIV3-SX full-length infectious clone plasmid as a template, and the obtained amplified fragments are fused to obtain a fragment containing V gene deletion; The fragment containing V gene deletion is replaced into pBPIV3-SX plasmid, and virus rescue is carried out by transfection, so that the bovine parainfluenza virus 3 type V protein deletion strain is obtained.

[0006] As a preferred embodiment of the present application, the primers used for amplification are SA-F, VKO-R and VKO-F, SB-R, and the sequences are as follows: SA-F: 5'-AGGCGCGCGTAATACGACTCACTATAGGGACCAAACAAGAGGAGAGAATTGTTTGG-3'; VKO-R: 5'-GCTTGCTTCCAAATATCCTCCTCACCCCCTTTTTTAATTTCTTTG-3'; VKO-F: 5'-CAAAGAAATTAAAAAAGGGGGTGAGGAGGATATTTGGAAGCAAGC-3'; SB-R: 5'-GGTTAATTAATCCAAGATGGACCATAAAGTTT-3'.

[0007] Further preferably, the fusion is carried out by using primers SA-F and SB-R for fragment fusion.

[0008] As a preferred embodiment of the present application, the process of replacing the V gene deletion-containing fragment into the pBPIV3-SX plasmid is as follows: Bss HII and P acI Both the V gene deletion-containing fragment and the pBPIV3-SX plasmid are double-digested with Bss

[0009] As a preferred embodiment of the present application, the transfection is the co-transfection of the product obtained after replacing the V gene deletion-containing fragment into the pBPIV3-SX plasmid and three helper plasmids containing viral NP, P and L genes into BHK-21 cells for virus rescue.

[0010] Further preferably, the mass ratio of the product and the three helper plasmids containing viral NP, P and L genes is 10:2:1:7.

[0011] More preferably, a plasmid containing T7 RNA polymerase is added during transfection, and the total mass of the product and the helper plasmids containing viral NP, P and L genes is 1.5:1.

[0012] In the second aspect of the present application, the V protein deletion strain of the bovine parainfluenza virus 3 is provided for use in the preparation of a product for treating bovine respiratory syndrome.

[0013] As a preferred embodiment of the present application, the product is a vaccine.

[0014] Compared with the prior art, the present application has the following beneficial effects: Based on the reverse genetic manipulation technology, the present application constructs an infectious clone plasmid with V protein deletion, and further successfully rescues the recombinant deletion virus; at the same time, the V protein is obtained through the E. coli expression system, and the polyclonal antibody is obtained by immunizing mice, which provides a theoretical basis for the functional research of BPIV3 non-structural V protein and the development of genetic engineering vaccine, and has important scientific value and application value.

[0015] The present application proves by virus titer determination that the virus titer of the constructed V protein deletion virus strain is significantly lower than that of the parent virus, and the virus still maintains good proliferation ability in cells, indicating that it can be used as a very potential attenuated vaccine candidate strain.

[0016] Biological material preservation information: The V protein deletion recombinant virus BPIV3-VS of bovine parainfluenza virus 3 was preserved in the China General Microbiological Culture Collection Center on September 23, 2025, with a preservation number of CGMCC NO. 46645. The preservation address is No. 3, Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0017] Figure 1 The results of PCR amplification of the BPIV3-V gene; Figure 2 Western blot results for pCDNA3-HA-V eukaryotic expression plasmid; 24 h after pCDNA3-HA-V transfection into HeLa cells; 24 h after pCDNA3-KHA empty plasmid transfection into HeLa cells; Figure 3 The results of indirect immunofluorescence detection of pCDNA3-HA-V eukaryotic expression plasmid; the subcellular localization results of pCDNA3-HA-V eukaryotic expression plasmid 24 h after transfection into HeLa cells, using HA tag antibody as the primary antibody. Figure 4 To investigate the effect of V protein overexpression on BPIV3 replication; A and C: Western blot analysis of BPIV3 NP protein expression levels; B and D: Detection of changes in viral load in cell supernatant after V gene overexpression; Figure 5 SDS-PAGE identification of recombinant V protein; lane 1 is the supernatant, lane 2 is the precipitate; Figure 6 Purification of recombinant V protein; Figure 7 Western blot identification of recombinant V protein; Figure 8 For the identification of polyclonal antibodies against protein V; Figure 9 A schematic diagram illustrating the construction of a strain with V protein deletion; Figure 10 Western blot identification of V protein-deleted strains; Figure 11 Sequencing identification results for the V protein deletion strain; Figure 12 To detect the expression of V gene deletion NP protein using Western blot; Figure 13 The effect of V gene deletion on BPIV3 viral titer. Detailed Implementation

[0018] To facilitate understanding of the present invention, a more comprehensive description is provided below, along with preferred embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0020] 1. Materials and Methods 1.1. Viruses, Cells, Plasmids and Antibodies BHK-21, HeLa and MDBK passage cell lines were purchased from ATCC cell bank.

[0021] BPIV3-SX-2021 (Genebank: ON804787) strain was isolated and preserved by the laboratory, see the literature "Han Y, Adam FEA, Zhang R, Gao Y, Lei J, Lu K, Wang X, Xiao S, Liu H, Yang Z. Isolation and Identification of a Genotype C Bovine Parainfluenza Virus Type 3 and Its Pathogenicity in Albino Guinea Pigs. Transbound Emerg Dis. 2023 Nov 23;2023:8854528. doi: 10.1155 / 2023 / 8854528. PMID: 40303758; PMCID: PMC12016689.".

[0022] pET30a prokaryotic expression plasmid and pCDNA3-KHA plasmid were purchased from Moli Plasmid Platform.

[0023] pcDNA3-KHA eukaryotic expression plasmid, BPIV3-SX-2021 strain full-length genome infectious clone plasmid pBPIV3-SX, auxiliary plasmid pCI-NP / P / L containing viral NP, P, L genes and pCAGGS-T7 plasmid containing T7 RNA polymerase sequence were preserved by the laboratory.

[0024] The construction process of the pBPIV3-SX full-length genome infectious clone plasmid pBPIV3-SX is described in the literature "Han Y, Lu K, Zhang R, Wei X, Guo H, Tong L, Wang X, Xiao S, Liu H, Yang Z. Construction and characterization of a reverse genetics system of bovine parainfluenza virus type 3c as a tool for rapid screening of antivirals in vitro. Front Vet Sci. 2024 Mar 12;11:1336663. doi: 10.3389 / fvets.2024.1336663. PMID: 38545559; PMCID: PMC10967227." The construction process of the pCAGGS-T7 plasmid containing the T7 RNA polymerase sequence is described in the literature "Han Y, Lu K, Zhang R, Wei X, Guo H, Tong L, Wang X, Xiao S, Liu H, Yang Z. Construction and characterization of a reverse genetics system of bovine parainfluenza virus type 3c as a tool for rapid screening of antivirals in vitro. Front Vet Sci. 2024 Mar 12;11:1336663. doi: 10.3389 / fvets.2024.1336663. PMID: 38545559; PMCID: PMC10967227."

[0025] The construction process of the helper plasmid pCI-NP / P / L containing the viral NP, P, and L genes is described in the literature "Han Y, Lu K, Zhang R, Wei X, Guo H, Tong L, Wang X, Xiao S, Liu H, Yang Z. Construction and characterization of a reverse genetics system of bovine parainfluenza virus type 3c as a tool for rapid screening of antivirals in vitro. Front Vet Sci. 2024 Mar 12;11:1336663. doi: 10.3389 / fvets.2024.1336663. PMID: 38545559; PMCID: PMC10967227."

[0026] HA and His-tag monoclonal antibodies were purchased from Wuhan Sanying Biological Co., Ltd.; BPIV3 NP protein polyclonal antibodies were prepared and preserved by the laboratory.

[0027] 1.2. Main reagents Various restriction enzymes and T4 ligase for constructing recombinant plasmids were purchased from NEB, USA; cell culture medium DMEM and fetal bovine serum were purchased from Gibco Biological Company; transfection reagent TurboFect™ Transfection Reagent was purchased from Thermo Fisher Scientific Company; Opti-MEM, fetal bovine serum, and DMEM were purchased from Gibco Company; PrimerSTARMAX high-fidelity polymerase and Trizol RNAiso Plus were purchased from TaKaRa Bio-Medical Technology Co., Ltd.; StarPrep rapid DNA gel recovery kit was purchased from Beijing Genestar; plasmid small extraction kit was purchased from Tiangen; viral DNA / RNA small purification kit was purchased from Invitrogen; Freund's complete and incomplete adjuvants were purchased from Sigma, USA; His-tag protein purification kit was purchased from Biyun Tian Biological Company; and the rest of the chemical reagents were all analytical pure.

[0028] 1.3. Main instruments Nucleic acid electrophoresis apparatus (Beijing Liyi Biological Technology Co., Ltd. DYY-6C type), protein electrophoresis apparatus (CAVOY PP-1150), micropipette (Eppendorf), gel imaging system (Shanghai Kinxiang Scientific Instrument Co., Ltd. GenoSens2000 series), laboratory ultrapure water machine (Chengdu Aikuo Water Treatment Equipment Co., Ltd.), fluorescence inverted microscope (OLYMPUS DP80), inverted optical microscope (UOP / AUPO DSZ2000X), digital display tilting plate shaker (SCILOGEX SLK-R3000-S), digital display circular shaker (SCILOGEX SLK-O3000-S), vortex mixer (Jiangsu Haimen Kelin Medical Instrument Factory), chemiluminescence imager (Suzhou MiniChemi610), desktop micro high-speed centrifuge (SCILOGEX CF1524R), heating type dry metal bath thermostat (Hangzhou Ruicheng Instrument Co., Ltd. DH300), gene amplifier (Hangzhou Bo Rizhong Technology Co., Ltd. TC-XP-D), electronic balance (Ohaus Instrument Co., Ltd. CP124C), water isolation type electric heating constant temperature incubator (Shanghai Yuejin Medical Instrument Factory PYX-DHS-50X65-BS-II), biological safety cabinet (Suzhou An Tai BHC-1300IIA2), super clean bench (Fuxia SW-CJ-2FD), super clean bench (Beijing Yataikelong Experimental Science and Technology Development Center YT-CJ-1N), high-pressure steam sterilization pot.

[0029] 1.4, Construction and verification of BPIV3-V protein eukaryotic expression plasmid According to the BPIV3-SX-2021 strain full genome sequence information, the genome fragment corresponding to the V gene was found to design primers. Since the V gene is formed by randomly introducing a G base at the base editing site of the P gene, thereby changing the coding order of the P gene, it is necessary to amplify the two gene fragments before and after the base editing site respectively, and to synthesize the complete fragment by fusion PCR. The primers were synthesized by Xi'an Qikai Biological Technology Co., Ltd. (Table 1). Specifically, the following steps are included:

[0030] According to the Trizol RNAiso Plus reagent instruction, the RNA of BPIV3-SX-2021 was extracted, and then according to the StarScript II First-strand cDNA Synthesis Mix reverse reagent kit instruction, the cDNA was obtained.

[0031] Using cDNA as template, the N-terminal and C-terminal domains of V gene were amplified by primers V-HA-F / VN-HA-R and VC-HA-F / V-HA-R, respectively, and then the nucleic acid fragments were purified. The fusion PCR was performed with V-HA-F / R primers, and the fusion fragment was purified and recovered as V gene.

[0032] The V gene was cloned into the pCDNA3-KHA eukaryotic expression vector, positive colonies were screened by bacterial liquid PCR for expansion culture, and the recombinant plasmid was extracted for sequencing. According to the TurboFect™ Transfection Reagent kit instructions, the correctly sequenced plasmid was transfected into HeLa cells, and the HA-tagged monoclonal antibody was used as a primary antibody for verification by IFA and Western blot. The correctly sequenced plasmid was named pCDNA3-HA-V.

[0033] Table 1 Primers for amplifying V protein gene sequence 1.5, Effect of overexpression of V protein on BPIV3 replication Before transfection, HeLa cells were digested and inoculated into a 12-well cell culture plate, and when the cell confluence reached 70%, 1 μg of pCDNA3-HA-V and empty plasmid pCDNA3-KHA were transfected into HeLa cells, respectively. After 24 h of transfection, 1 MOI of BPIV3-SX-2021 was infected, and cell and supernatant samples were harvested at 24 h and 36 h after viral infection, respectively. The expression of viral protein NP was detected by Western blot, and the virus titer of the cell supernatant was determined. In addition, to further explore whether the V protein has a dose-dependent effect on viral replication, 1 μg and 2 μg of plasmid were transfected, respectively, and 1 MOI of BPIV3 was inoculated, and cell and supernatant samples were harvested at 24 h after viral infection for NP protein detection and virus titer determination.

[0034] 1.6, Prokaryotic expression of BPIV3-V protein C-terminal domain Since the N-terminal domain of the V gene is the same as the P protein, the C-terminal domain was selected for prokaryotic expression. A pair of specific primers was designed according to the genomic sequence of the C-terminal domain of the V gene, and the primer information is shown in Table 2. The genomic fragment was cloned into the pET-30a prokaryotic expression vector, and the recombinant plasmid was verified by sequencing. The extracted correctly sequenced plasmid was transformed into BL21 (DE3) competent cells, and after resistance screening, the monoclonal colonies identified as positive by sequencing were expanded and induced for expression. The bacterial suspension was centrifuged, the culture supernatant was discarded, and the bacterial pellet was resuspended in PBS buffer. The suspension was placed in an ultrasonic disrupter for bacterial disruption, and the supernatant and precipitate were collected for SDS-PAGE and Western-Blot detection. The correctly identified target protein was purified according to the His-tag purification kit and stored at -70 °C for future use.

[0035] Table 2 Primer sequences for amplifying C-terminal domain of V gene 1.7. Preparation and identification of polyclonal antibody against C-terminal domain of V protein of BPIV3 The purified recombinant V protein was diluted to 2 μg / μL with physiological saline, and mixed with incomplete adjuvant in equal volume to emulsify. Each mouse was injected subcutaneously with 100 μL of the emulsion at multiple sites on the back. Fourteen days after the first immunization, the recombinant protein was mixed with complete Freund's adjuvant to emulsify for booster immunization. On day 21, blood was collected from the tail vein, and the titer of polyclonal antibody was determined by indirect ELISA. When the desired titer was reached, blood was collected from the eye, and the serum was separated and stored at -20 °C for later use. To verify whether the polyclonal antibody against the C-terminal domain of V protein could specifically bind to V protein, HeLa cells were seeded in a 12-well cell culture plate, and when the confluence reached 70%, 1 μg of pCDNA3-HA-V was transfected into the HeLa cells, and 1 MOI of BPIV3-SX-2021 strain was inoculated as a positive control. Cell samples were harvested 24 h after transfection and virus infection, respectively, and Western blot was performed using the polyclonal antibody against V protein as the primary antibody (1:5000 dilution).

[0036] 1.8. Construction of BPIV3-V protein deletion virus To construct the V protein deletion virus, a stop codon was introduced at the base editing site of the P gene to cause premature termination of the V gene during transcription, thereby causing deletion of the C-terminal domain without affecting the transcription of the P gene. Therefore, a pair of specific primers (VKO-F / R) was designed for site-directed mutagenesis, and the primer information is shown in Table 3. First, the pBPIV3-SX full-length infectious clone plasmid was used as the template, and SA-F / VKO-R and VKO-F / SB-R were used to amplify the fragments, respectively. The amplified PCR fragments were purified, and fragment fusion was performed using SA-F / SB-R. The product was purified and used for BssHII digestion. The digested product and the pBPIV3-SX plasmid were digested with BssHII and PciI, respectively. The fragment containing the V gene deletion was replaced into pBPIV3-SX, thereby obtaining pBPIV3-SX-VS. HII and P acI The fragment and the pBPIV3-SX plasmid were digested with BssHII and PciI, respectively. The fragment containing the V gene deletion was replaced into pBPIV3-SX, thereby obtaining pBPIV3-SX-VS.

[0037] Table 3 Primer sequences for amplifying site-directed mutagenesis of V gene 1.9. Rescue and identification of recombinant virus BHK-21 cells were seeded into 12-well cell culture plates before transfection, and the cells were used for transfection when they grew to 70% confluence. The mass ratio of pBPIV3-SX-VS plasmid and helper plasmids pCI-NP, pCI-P, pCI-L containing viral NP, P and L genes was 10:2:1:7, a total of 1.5 μg; in addition, the system is based on T7 promoter for transcription and translation, and T7 RNA polymerase is lacking in mammalian cells, so 1 μg of plasmid containing T7 RNA polymerase is additionally transfected for the work of the system. The transfected cells were collected after 72 h and placed at -70 ℃ for repeated freezing and thawing 3 times, denoted as F0 generation. 200 μL of F0 generation cell supernatant was inoculated into MDBK cells for blind passage, and the cell supernatant was harvested after 72 h and denoted as F1 generation. The blind passage was carried out according to this method for each generation until obvious cytopathic effect (CPE) was produced.

[0038] 2.0, Identification of V protein deletion virus To further identify whether the rescued virus encodes V protein, the parent virus and the rescued virus were inoculated into MDBK cells, and the cell samples were harvested 24 h after virus infection for Western blot identification to detect the expression of viral NP protein and V protein. In addition, the RNA of the rescued virus and the parent virus was extracted, and RT-PCR amplification was carried out using VD-F / R (Table 3) primers, and the PCR product was purified and sequenced for analysis.

[0039] 2.1, Determination of biological characteristics of V protein deletion virus To further explore the influence of V protein deletion on the biological characteristics of the virus, the V protein deletion virus and the parent virus were inoculated into MDBK cells at (MOI=1), and the cell samples and supernatants were collected at 12 h, 24 h and 36 h after virus infection, and the difference in virus titers and the expression level of NP protein of the two viruses at different time points were determined.

[0040] 3, Results 3.1, Identification of recombinant plasmid pCDNA3-HA-V The V gene fragment was obtained by PCR amplification of the N-terminal and C-terminal domains of the V gene and fusion of the fragments, and the results are shown in Figure 1 The V gene was cloned into the eukaryotic expression vector pCDNA3-KHA to construct the recombinant plasmid, which was identified by sequencing results. The recombinant plasmid with correct sequencing results was extracted in small amounts.

[0041] To verify whether the plasmid is expressed in cells, the plasmid is transfected into HeLa cells for Western blot and IFA detection. Since the N-terminal of the vector carries an HA tag, an HA tag antibody is used as a primary antibody for detection. The results show that the HA antibody can specifically recognize the target protein, proving that the recombinant plasmid can be correctly expressed in cells Figure 2 ).

[0042] In addition, the subcellular localization of V protein is observed by laser confocal observation, and the results show that V protein can be expressed in cytoplasm and nucleus Figure 3 ).

[0043] 3.2, V protein promotes the proliferation of BPIV3 The recombinant plasmid carrying the V gene is transfected into HeLa cells, and the cells are infected with virus 24 h after transfection, and the cell samples at different time points are collected for detection. The results show that the expression of NP protein in the experimental group overexpressing V gene is significantly increased compared with the control group 24 h and 48 h after BPIV3 infection. By determining the virus titer of cell supernatant, the results show that the virus titer of the experimental group is significantly higher than that of the control group Figure 4 ).

[0044] Further transfection with different doses of plasmid, and determination of the expression of NP protein and virus titer after infection. The results show that with the increase of transfection dose, the expression of NP protein and virus titer increase significantly Figure 4 ). The above results show that overexpression of V protein significantly promotes the proliferation of virus.

[0045] 3.3, identification of V gene C-terminal domain recombinant protein SDS-PAGE identified an obvious band at about 35 kDa consistent with the size of the target protein, which was expressed in the form of inclusion body Figure 5 ). Purified by Ni-NTA affinity chromatography, and finally the purified protein was concentrated and the protein concentration was determined and stored at -70 ℃ for standby use Figure 6 ).

[0046] The band can detect His-tag signal by Western blot Figure 7 ). The target protein is denatured and renatured, and is prepared for V protein C-terminal domain polyclonal antibody, and the recombinant protein is mixed with Freund's adjuvant for emulsification, and the blood is collected after 3 times of immunization of mice and the serum is separated and stored for standby use.

[0047] 3.4, identification of V gene C-terminal domain recombinant protein polyclonal antibody Western blot detection was performed 24 h after transfection of pCDNA3-HA-V and BPIV3 infection of HeLa cells using V protein polyclonal antibody as the first antibody. The results showed that the V protein transfection and virus infection cell samples could specifically bind to the V protein polyclonal antibody Figure 8 ).

[0048] 3.5, Rescue and identification of V protein deletion virus The pBPIV3-SX-VS plasmid and the helper plasmid were co-transfected into BHK-21 cells for virus rescue, and the rescued virus was obtained by blind passage in MDBK cells Figure 9 ). The rescued virus was passaged continuously, and the deletion virus could be stably passaged in MDBK cells.

[0049] The Western blot identification results showed that the expression of NP protein could be detected in both the parent virus and the rescued virus, proving that the rescued virus was BPIV3. The detection results of the virus V protein showed that the parent virus could specifically bind to the V protein polyclonal antibody, while the expression of V protein was not detected in the rescued virus Figure 10 ).

[0050] To further prove the deletion of V protein, the mutation region of the rescued virus and the parent virus was sequenced and identified, and the results showed that the mutation of the base editing site of the P gene of the rescued virus existed. In summary, the rescued virus is a virus with deletion of the C-terminal domain of V protein, named BPIV3-VS Figure 11 ), which was preserved in the China General Microbiological Culture Collection Center on September 23, 2025, and the preservation number was CGMCC NO. 46645.

[0051] 3.6, Biological characteristics of V protein deletion virus To explore the replication difference of BPIV3-VS and the parent virus in cells, the parent virus and the rescued virus were infected into HeLa cells, and the virus titers of the supernatants of the cells at different time points were determined. The results showed that the virus titer of the rescued virus was significantly lower than that of the parent virus Figure 13 ). In addition, the expression level of NP protein in the cells was detected, and the results showed that the expression level of the rescued virus was also lower than that of the parent virus Figure 12 ).

[0052] The technical features of the above-described embodiments can be combined in any manner. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described, but as long as the combinations of the technical features do not exist, they should be considered as the scope of the present disclosure.

[0053] The above embodiments only express several implementation manners of the present application, and the description is more specific and detailed, but it should not be understood as a limitation on the scope of the patent. It should be noted that for ordinary skilled persons in the art, without departing from the concept of the present application, several modifications and improvements can be made, which are within the scope of protection of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. A bovine parainfluenza virus type 3 V protein-deficient strain, characterized in that, It is constructed according to the following steps: Using the full-length infectious clone plasmid pBPIV3-SX as a template, the two gene fragments before and after the base editing site of the V gene were amplified respectively. The resulting amplified fragments were fused to obtain a fragment containing the deletion of the V gene. Replace the fragment containing the V gene deletion into the Bss of the pBPIV3-SX plasmid. HII and P acI By removing the enzyme cleavage site and transfecting the virus for rescue, the bovine parainfluenza virus type 3 V protein-deficient strain is obtained.

2. The bovine parainfluenza virus type 3 V protein-deficient strain according to claim 1, characterized in that, The primers used for amplification were SA-F, VKO-R and VKO-F, SB-R, with the following sequences: SA-F: 5'-AGGCGCCGTAATACGACTCACTATAGGGACCAAACAAGAGGAGAGAATTGTTTGG-3'; VKO-R: 5'-GCTTGCTTCCAAATATCCTCCTCACCCCCTTTTTTTAATTTCTTTG-3'; VKO-F: 5'-CAAAGAAATTAAAAAAGGGGGTGAGGAGGATATTTGGAAGCAAGC-3'; SB-R: 5'-GGTTAATTAATCCAAGATGGACCATAAAGTTT-3'.

3. The bovine parainfluenza virus type 3 V protein-deficient strain according to claim 2, characterized in that, The fusion is performed using primers SA-F and SB-R to fuse fragments.

4. The bovine parainfluenza virus type 3 V protein-deficient strain according to claim 1, characterized in that, The process of replacing the fragment containing the V gene deletion into the pBPIV3-SX plasmid is as follows: Using BSS HII and P acI The fragment containing the V gene deletion and the pBPIV3-SX plasmid were both double-digested with enzymes, and the digestion products were then ligated.

5. The bovine parainfluenza virus type 3 V protein-deficient strain according to claim 1, characterized in that, The transfection involved replacing the fragment containing the V gene deletion into the pBPIV3-SX plasmid, and then co-transfecting the resulting product with helper plasmids containing the viral NP, P, and L genes into BHK-21 cells for virus rescue.

6. The bovine parainfluenza virus type 3 V protein-deleted strain according to claim 5, characterized in that, The mass ratio of the product to the three helper plasmids containing the viral NP, P, and L genes is 10:2:1:

7.

7. The bovine parainfluenza virus type 3 V protein-deficient strain according to claim 6, characterized in that, During transfection, a plasmid containing T7 RNA polymerase is added for transfection, and the total mass ratio of the product and the three helper plasmids containing viral NP, P, and L genes to the mass ratio of the plasmid containing T7 RNA polymerase is 1.5:

1.

8. The use of the bovine parainfluenza virus type 3 V protein-deficient strain according to any one of claims 1 to 7 in the preparation of a product for treating bovine respiratory syndrome.

9. The application according to claim 8, characterized in that, The product in question is a vaccine.

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