Viral vector delivery system for porcine respiratory tract and digestive tract and use thereof

Abstract

Provided is a viral vector delivery system for the porcine respiratory tract and digestive tract. The viral vector delivery system comprises a backbone plasmid obtained by inserting full-length cDNA of porcine enterovirus type B into a pUC57 plasmid, and a helper plasmid obtained by inserting a green fluorescent protein-encoding gene into plasmid pCAG-T7-polymerase. The virus titer of the viral vector delivery system can reach 107.75TCID50 per mL, and porcine respiratory tract and digestive tract pathogenic epitopes are inserted into the backbone plasmid, which has a good immune effect and can be used for constructing candidate strains of porcine respiratory tract and digestive tract vaccines.

Description

A viral vector delivery system for pig respiratory and digestive tracts and its application Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a virus vector delivery system for pig respiratory tract and digestive tract and application thereof. Background Art

[0002] Viruses are infectious agents that are associated with a variety of diseases and are parasitic only on living organisms. Therefore, they are generally considered to be extremely harmful to humans and mammals. However, research has shown that there is great potential in using viruses as therapeutic agents. As a commonly used biological vector, viral vectors can serve as an attractive vaccine platform due to their ability to induce strong antigen-specific humoral and cellular immune responses and their high safety. In addition to being a tool to deliver exogenous genes or genetic material into organisms or cells, viral vectors are currently also widely used in human and veterinary vaccination and gene therapy. Although non-viral gene delivery vectors such as liposomes and cationic polymers are also used in gene therapy and the construction of candidate vaccine strains, viruses are usually the preferred delivery vector because their gene delivery efficiency in the body is much higher than that of non-viral gene delivery vectors.

[0003] African swine fever virus (ASFV) is a highly contagious pathogen that causes severe hemorrhage and necrosis in the respiratory and digestive tracts of domestic and wild pigs. The African swine fever it causes has a serious impact on the economy and people's livelihoods. Digestive tract diseases, or gastrointestinal infections, refer to viral, bacterial, or parasitic infections that can cause gastroenteritis, leading to inflammation of the gastrointestinal tract, including the stomach and small intestine. Symptoms include diarrhea, vomiting, and abdominal pain. The main viral pathogens that cause digestive tract symptoms in pigs include porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), and porcine deltacoronavirus (PDCoV). These viruses can cause contact enteric infections such as vomiting, diarrhea, and dehydration in pigs. Clinically, PDCoV can infect pigs of all ages, primarily causing acute diarrhea and vomiting in suckling piglets, leading to rapid dehydration and death. Clinically, PDCoV is often co-infected with other viruses such as PEDV, TGEV, and PRRSV, which brings greater challenges to the prevention and control of enteroviruses, because the occurrence of these diseases will greatly reduce the production performance of pig herds.

[0004] The existing technology lacks an efficient viral vector delivery system for porcine respiratory and digestive tract vaccines.

[0005] Summary of the Invention

[0006] The purpose of the present invention is to provide a viral vector delivery system for pig respiratory and digestive tracts, which has high construction efficiency, can quickly produce cytopathic effects after infecting cells, and the virus titer can reach 10 7.75 TCID 50 / mL and maintain high stability.

[0007] Another object of the present invention is to provide the application of the viral vector delivery system in the construction of pig respiratory and digestive tract vaccine antigens. The vaccine antigen obtained after inserting pig respiratory and digestive tract pathogen epitopes into the backbone plasmid is non-pathogenic, has good stability, and has a high virus titer. After being inoculated into pigs, it has a good immune effect.

[0008] The purpose of the present invention is achieved by adopting the following technical solutions:

[0009] A viral vector delivery system for both the respiratory and digestive tracts of pigs comprises a backbone plasmid and a helper plasmid: the backbone plasmid is obtained by inserting the full-length cDNA of porcine enterovirus type B into the pUC57 plasmid; the helper plasmid is obtained by inserting the green fluorescent protein encoding gene into the plasmid pCAG-T7-polymerase.

[0010] In the present invention, a T7 promoter is inserted into one end of the full-length cDNA of porcine enterovirus type B PEVB in the backbone plasmid, and a His tag and a polyA tail are inserted into the other end.

[0011] The present invention also provides a method for constructing the viral vector delivery system, comprising the following steps: separately constructing a backbone plasmid and constructing an auxiliary plasmid, and then co-transfecting the backbone plasmid and the auxiliary plasmid into monkey kidney cells or porcine bronchial epithelial cells.

[0012] In the present invention, the method for constructing the backbone plasmid is as follows: using the full-length cDNA of porcine enterovirus type B PEV-B-KOR strain as a template, introducing a T7 promoter at the 5' end of the full-length cDNA by PCR, introducing a his-tag label and a ployA tail at the 3' end, and then inserting it into pUC57 to obtain a backbone plasmid.

[0013] In the present invention, the method for constructing the auxiliary plasmid is as follows: inserting the green fluorescent protein encoding gene into the plasmid pCAG-T7-polymerase to obtain the auxiliary plasmid.

[0014] In the present invention, the backbone plasmid and the helper plasmid are co-transfected into monkey kidney veroE6 cells using liposome Lipofectamin 3000, and the virus plaques producing green fluorescence are picked to obtain the viral vector delivery system.

[0015] The present invention also provides application of the viral vector delivery system in constructing porcine respiratory tract and digestive tract vaccine antigens.

[0016] In the present invention, the respiratory and / or digestive tract antigen epitope encoding gene is inserted between the VP4 gene and 5'UTR gene of the backbone plasmid, and then co-transfected with the helper plasmid into monkey kidney veroE6 cells or porcine bronchial epithelial cells hTERT-PBECs using liposome Lipofectamin 3000 to obtain the vaccine antigen.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] (1) This application is the first to introduce full-length PEVB cDNA into the T7 promoter through PCR, ultimately obtaining an infectious clone carrying the T7 promoter, which was then linearized and extracted to serve as a backbone vector. A helper plasmid containing T7 RNA polymerase and a green fluorescent marker was also constructed. The DNA co-transfection strategy eliminates the risk of RNA degradation during the transfection process in traditional RNA in vitro transcription methods, significantly improves transfection efficiency, and saves time. This system is highly efficient, easy to operate, and economical.

[0019] (2) In the presence of a helper plasmid, the recombinant porcine enterovirus r-PEVB of the present invention can infect immortalized porcine bronchial epithelial cells and begin to produce cytopathic effects 36-60 hours after infection. The titer of the obtained r-PEVB virus can reach 10 7.75 TCID 50 / mL, and the infection efficiency and titer were significantly higher than those on African green monkey kidney cells, veroE6. The proliferation titer of the recombinant porcine enterovirus r-PEVB on immortalized porcine bronchial epithelial cells was 0.75 titers higher than that of the wild-type parent strain on immortalized porcine bronchial epithelial cells, and 0.75 titers higher than that of the wild-type parent strain cultured on veroE6, and remained stable.

[0020] (3) The recombinant porcine enterovirus r-PEVB of the present invention, which incorporates porcine respiratory and digestive tract pathogen epitopes, exhibits no pathogenicity, good stability, and high viral titers, resulting in a good immune response after inoculation in pigs. Therefore, the viral vector delivery system of the present invention can be used in the future research of various porcine respiratory and digestive tract vaccine candidate strains and multi-combination vaccines, possessing significant theoretical significance and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is a schematic diagram of the structure of the recombinant plasmid pUC57-PEVB, wherein T7 promoter is the T7 promoter and His tag is the His tag.

[0022] Figure 2 is an electrophoretic diagram of target fragments 1-3 obtained by RT-PCR using RNA from positive clone 1 carrying the recombinant plasmid pUC57-PEVB as a template and primers 1, 2, or 3, respectively. M represents DNA Marker DL5000.

[0023] Figure 3 Schematic diagram of the construction of the eukaryotic helper plasmid pCAG-T7 RNA polymerase-EGFP containing T7 RNA polymerase and green fluorescent protein, wherein pCAG is the enhancer in the plasmid pCAG-T7-polymerase and pCMV is the eukaryotic promoter in the plasmid pCAG-T7-polymerase.

[0024] Figure 4 PCR identification of the auxiliary plasmid pCAG-T7 RNA polymerase-EGFP recombinant plasmid. Lane 1 is DNA marker DL1500, lane 2 is DNA marker DL5000, lanes 3-7 are the amplification products of the picked single colonies 1, 2, 3, 4, and 5 for the fragment T7 RNA polymerase, lanes 8-12 are the amplification products of the picked single colonies 1, 2, 3, 4, and 5 for the enhancer fragment pCA, and lanes 13-17 are the amplification products of the picked single colonies 1, 2, 3, 4, and 5 for the EGFP fragment.

[0025] Figure 5 Fluorescence identification of veroE6 co-transfected with linearized backbone plasmid and auxiliary plasmid at a mass ratio of 3:2, wherein Figure A is an image of the co-transfected cells observed under white light, and Figure B is an image of the co-transfected cells observed under fluorescence light.

[0026] Figure 6 shows electron micrographs of the cytopathic effect of the recombinant porcine enterovirus r-PEVB, passage P30, on veroE6 and hTERT-PBECs. The first row shows the cytopathic effect of the r-PEVB, passage P30, on veroE6 cells at 24 and 48 hours after infection, as well as the normal veroE6 cell control (NC). The second row shows the cytopathic effect of the r-PEVB, passage P30, on hTERT-PBECs at 24 and 48 hours after infection, as well as the normal hTERT-PBEC cell control (NC).

[0027] Figure 7 shows the growth curve of the recombinant porcine enterovirus r-PEVB, where p-PEVB is the parent strain PEV-B-KOR.

[0028] Figure 8 is an SDS-PAGE electrophoresis of recombinant VP4 protein after prokaryotic mass expression. Lane 1 is the protein marker, lane 2 is the uninduced VP4-32a lysate, and lanes 3 and 4 are the precipitates of the induced VP4-32a lysate.

[0029] Figure 9 shows large-scale purification of recombinant VP4 protein after inclusion body extraction. Lanes from left to right are: protein marker, precipitate after ultrasonic lysis of induced bacteria, supernatant after ultrasonic lysis of induced bacteria, eluate collected from Ni column after elution with 20 mM imidazole, eluate collected from Ni column after elution with 50 mM imidazole, eluate collected from Ni column after elution with 100 mM imidazole, eluate collected from Ni column after elution with 250 mM imidazole, eluate collected from Ni column after elution with 300 mM imidazole, and VP4 protein purified and refolded from inclusion bodies after elution with 500 mM imidazole.

[0030] Figure 10 IFA inter-immune verification of recombinant porcine enterovirus r-PEVB using rabbit anti-VP4 protein polyclonal antibody as primary antibody, Figure A: recombinant porcine enterovirus r-PEVB; Figure B: negative control; Figure C: blank control.

[0031] Figure 11 is the Western Blotting verification of the recombinant porcine enterovirus r-PEVB using rabbit anti-VP4 protein polyclonal antibodies and His antibodies, wherein lane M in Figure A is a protein marker, and lanes 2-3 are the Western Blotting verification results of hTERT-PBECs proteins infected with recombinant virus r-PEVB and hTERT-PBECs proteins not infected with recombinant virus r-PEVB using rabbit anti-VP4 protein polyclonal antibodies, respectively; lane M in Figure B is a protein marker, and lane 1 is the Western Blotting verification result of hTERT-PBECs proteins infected with recombinant virus r-PEVB using rabbit His antibodies.

[0032] Figure 12 shows the results of enzyme digestion identification of recombinant expression plasmids ATr-PEVB, Mr-PEVB, and AMr-PEVB. Figure A shows the results of enzyme digestion of recombinant expression plasmid ATr-PEVB, with lane M being the DL 10000 marker, and lanes 1 and 2 being the electrophoretic images of recombinant expression plasmid ATr-PEVB (undigested) and recombinant expression plasmid ATr-PEVB digested with EcoRI, respectively. Figure B shows the results of enzyme digestion of AMr-PEVB, with lane M being the DL 10000 marker, and lanes 1 and 2 being the electrophoretic images of AMr-PEVB (undigested) and AMr-PEVB digested with EcoRI, respectively. Figure C shows the electrophoretic image of Mr-PEVB digested with EcoRI, with lane M being the DL 10000 marker, and lanes 1 and 2 being the electrophoretic images of Mr-PEVB (undigested) and Mr-PEVB digested with EcoRI, respectively.

[0033] Figure 13 Fluorescence identification images of the recombinant expression plasmids ATr-PEVB, Mr-PEVB and AMr-PEVB co-transfected with auxiliary plasmids into veroE6 at a mass ratio of 2:1. The first row shows the transfected cells observed under a white light source, and the second row shows the transfected cells observed under a fluorescent light source. From left to right, they are the fluorescence identification images of the recombinant expression plasmids ATr-PEVB, Mr-PEVB, AMr-PEVB co-transfected with auxiliary plasmids into veroE6, and the fluorescence identification images of the positive control PC and the negative control NC.

[0034] Figure 14 shows the cytopathic effects of P14 generation recombinant viruses ATr-PEVB, Mr-PEVB and AMr-PEVB on hTERT-PBECs. From left to right, they are the cytopathic effects of recombinant viruses ATr-PEVB, Mr-PEVB, and AMr-PEVB 24 hours after infection of hTERT-PBECs. NC is the hTERT-PBECs cell control that is not infected with the virus.

[0035] Figure 15 shows the indirect immunofluorescence IFA identification results of recombinant virus Mr-PEVB against Mhp P97R1 monoclonal antibody and PEVB VP4 polyclonal antibody, respectively. P97-R1 on the left indicates that the corresponding sample was detected using Mhp P97R1 monoclonal antibody, and Vp4 on the left indicates that the corresponding sample was detected using PEVB VP4 polyclonal antibody. NC indicates that the corresponding sample is a negative control (pig bronchial epithelial cells hTERT-PBECs without recombinant porcine enterovirus r-PEVB infection). DAPI above indicates DAPI channel, RED indicates RED channel, and MERGE indicates merge.

[0036] Figure 16 Indirect immunofluorescence IFA identification results of recombinant virus ATr-PEVB against African swine fever virus positive serum antibodies and rabbit anti-VP4 protein polyclonal antibodies respectively; Vp4 on the left indicates that the corresponding sample was detected using rabbit anti-VP4 protein polyclonal antibodies; Virus on the left indicates that the corresponding sample was detected using African swine fever virus positive serum antibodies, NC indicates porcine bronchial epithelial cells hTERT-PBECs without recombinant porcine enterovirus r-PEVB infection, DAPI above indicates DAPI channel, RED indicates RED channel, and MERGE indicates merge.

[0037] Figure 17 Indirect immunofluorescence IFA identification results of recombinant virus AMr-PEVB against Mhp P97R1 monoclonal antibody, rabbit anti-VP4 protein polyclonal antibody and African swine fever virus positive serum antibody, respectively. P97-R1 on the left indicates that the corresponding sample was detected using Mhp P97R1 monoclonal antibody, Vp4 on the left indicates that the corresponding sample was detected using rabbit anti-VP4 protein polyclonal antibody, Virus on the left indicates that the corresponding sample was detected using African swine fever virus positive serum antibody, NC indicates that the corresponding sample is a negative control (pig bronchial epithelial cells hTERT-PBECs without recombinant porcine enterovirus r-PEVB infection), DAPI above indicates DAPI channel, RED indicates RED channel, and MERGE indicates merge.

[0038] Figure 18 shows the Western blotting identification results of recombinant viruses AMr-PEVB, ATr-PEVB and Mr-PEVB against rabbit anti-VP4 protein polyclonal antibody, where lane M is the protein marker, and lanes 1-4 are AMr-PEVB, ATr-PEVB, Mr-PEVB and negative control (hTERT-PBECs not infected with the virus), respectively.

[0039] Figure 19 shows the Western blotting results of recombinant viruses AMr-PEVB and Mr-PEVB against Mhp P97R1 monoclonal antibody, wherein lane M is the protein marker, and lanes 1-3 are AMr-PEVB, Mr-PEVB and negative control (hTERT-PBECs not infected with the virus), respectively.

[0040] Figure 20 shows the Western blotting identification results of recombinant viruses AMr-PEVB and ATr-PEVB against African swine fever virus-positive serum antibodies, where lane M is the protein marker, and lanes 1-3 are AMr-PEVB, ATr-PEVB and negative control (hTERT-PBECs not infected with the virus), respectively.

[0041] Figure 21 shows the HE results of the ileum of pigs infected with the recombinant virus r-PEVB and the parental strain PEV-B-KOR, respectively. Figure A shows the HE results of the ileum of pigs infected with the recombinant virus r-PEVB, and Figure B shows the HE results of the ileum of pigs infected with the PEV-B-KOR strain.

[0042] Figure 22 shows the serum IgG antibody levels after immunization with the recombinant virus ATr-PEVB, where Figures AF are the serum IgG antibody levels against the six antigens of African swine fever virus, CD2V-N, P10, P30, P22, P54 and B602L, respectively. The ordinate is the OD450 value for detecting the serum IgG antibody level, and the abscissa is the blood collection time after the first immunization, where 0dpi is the blood collection day of the first immunization, and the negative control is the negative control group (pigs in immune culture medium).

[0043] Figure 23 shows the expression level of sIgA in oral fluid after immunization with recombinant virus ATr-PEVB. The horizontal axis represents the sampling time starting from the first immunization. The negative control is the negative control group (oral fluid of pigs in immune culture medium).

[0044] Figure 24 shows the secretion levels of various inflammatory cytokines 14 and 28 days after immunization with recombinant virus ATr-PEVB. The vertical axis represents the relative cytokine expression level induced by immunization with recombinant virus ATr-PEVB compared with pigs immunized with culture medium (set to 1), and the horizontal axis represents the sampling time starting from the first immunization.

[0045] Figure 25 shows the T cell typing results of peripheral blood lymphocytes of pigs after immunization with the recombinant virus ATr-PEVB. In the horizontal axis, "ATr-PEVB" and "NC" represent the indicators of pigs immunized with the recombinant virus ATr-PEVB and the negative control group, respectively. D14 and D28 represent the 14th and 28th days after the first immunization, respectively.

[0046] Figure 26 shows the apparent lesions in the lungs of pigs immunized with the recombinant virus Mr-PEVB.

[0047] Figure 27 Electron microscopic observation of bronchial ciliary structure in pigs immunized with recombinant virus Mr-PEVB.

[0048] Figure 28 shows the serum IgG antibody levels of pigs immunized with the recombinant virus Mr-PEVB. D0 indicates sampling before the first vaccination, and D70 indicates sampling on the 70th day after the first vaccination (i.e., after the infection). "*" indicates p<0.05, and the difference is significant. "***" indicates p<0.01, and the difference is extremely significant.

[0049] Figure 29 shows the levels of mucosal sIgA antibodies in nasal swabs of pigs after immunization with the recombinant virus Mr-PEVB. D0 indicates sampling before the first vaccination, and D70 indicates sampling on the 70th day after the first vaccination (i.e., after the virus attack). "*" indicates p<0.05, and the difference is significant. "***" indicates p<0.01, and the difference is extremely significant.

[0050] Figure 30 shows the DNA copy number of Mhp in the alveolar lavage fluid of pigs immunized with the recombinant virus Mr-PEVB. The vertical axis is the DNA copy number of Mycoplasma hyopneumoniae in the lung tissue based on log10. "***" indicates p<0.01, which means the difference is extremely significant. DETAILED DESCRIPTION

[0051] The porcine bronchial epithelial cell line hTERT-PBEC used in the present invention has a deposit number of CCTCC NO: C201749 and is disclosed in CN107338225A. It can be stably passaged for more than 60 generations and retains the characteristics of primary cells. It can be used to study the pathogenic mechanism of porcine respiratory pathogen infection.

[0052] The present invention will be further described below with reference to specific examples. It should be understood that these examples are only for illustrative purposes and in no way limit the scope of protection of the present invention.

[0053] Example 1: Construction of a viral vector delivery system for both the respiratory and digestive tracts of pigs

[0054] 1. Construction of backbone plasmid and acquisition of linearized fragments

[0055] Total RNA was extracted from 200 μL of frozen porcine enterovirus type B (PEV-B-KOR) cell culture suspension (published in GenBank, NCBI GenBank sequence number JQ818253) according to the instructions of the Viral RNA / DNA Extraction Kit (Axygen, Cat. No. AP-MN-BF-VNA-250, NY, USA). cDNA was obtained from the extracted total RNA using a Viral RNA Reverse Transcription Kit (Vazyme, Cat. No. R223-01).

[0056] Based on the restriction enzyme cleavage sites of the virus and the pUC57 E. coli vector plasmid, a forward primer, PEntero-F, containing a T7 promoter, and a reverse primer, PEntero-R, containing a ployA tail, were designed. Using cDNA as a template and PEntero-F and PEntero-R as primers, a full-length cDNA of porcine enterovirus type B (PEV-B-KOR) strain was amplified, incorporating a T7 promoter at the 5' end and a his-tag and ployA tail at the 3' end. The amplified full-length cDNA and the pUC57 E. coli vector plasmid were digested with EcoRV and then ligated with T4 DNA ligase. This resulted in the insertion of the full-length PEVB cDNA (7393 bp), his-tag, ployA tail, and T7 promoter into the pUC57 plasmid, creating a recombinant plasmid named pUC57-PEVB. The recombinant plasmid pUC57-PEVB was introduced into the competent cell state of Escherichia coli XL-10, and the positive clones carrying the recombinant plasmid pUC57-PEVB were screened using LB plates containing ampicillin resistance.

[0057] The sequence of the forward primer PEntero-F is as follows:

[0058] 5'-AATAATACGACTCACTATAGGGTTAAAACAGCCTGTGGGTTGTTCCCA-3';

[0059] The sequence of the reverse primer PEntero-R is as follows:

[0060] 5'-AATTTTTTTTTTTTTTCACCCCATCCGGTGGGTGTATTGAAT-3'.

[0061] The complete genome sequence of the PEV-B-KOR strain was divided into target segments 1, 2, and 3 (see Table 1 for details). Three pairs of specific primers (see Table 1) were designed using Primer Premier 5.0 software to amplify target segments 1-3, respectively. The primers were synthesized at Nanjing GenScript Biotechnology Co., Ltd. RT-PCR amplification was performed using RNA from a positive clone carrying the recombinant plasmid pUC57-PEVB as a template, using each specific primer pair with a one-step RT-PCR kit (Beijing Quanshijin Biotechnology Co., Ltd.). The total RT-PCR volume was 20 μL, including 10 μL of 2× R-Mix Buffer, 0.5 μL each of upstream and downstream primers, 0.4 μL of E-Mix, and 2 μL of RNA template. RNase-free water was used to bring the volume to 20 μL. Amplification protocol: reverse transcription at 45°C for 30 minutes; 94°C for 5 minutes; 94°C for 30 seconds, 55-60°C for 30 seconds (annealing temperatures for each primer during amplification are shown in Table 1); 72°C for 2 minutes, 35 cycles; and 72°C for 10 minutes. Target fragments 1-3 were purified and recovered separately, then transformed into E. coli XL-10 cells. They were identified and sequenced using colony PCR. Sequencing results revealed that the complete viral genome sequence of target fragments 1-3, obtained by RT-PCR using RNA from positive clone 1 carrying the recombinant plasmid pUC57-PEVB as a template, exhibited 100% sequence homology to the PEV-B-KOR sequence, confirming the successful construction of the recombinant plasmid pUC57-PEVB (backbone plasmid). A schematic diagram of the structure is shown in Figure 1, and the PCR identification results of target fragments 1-3 are shown in Figure 2. The recombinant plasmid pUC57-PEVB in positive clone 1 was digested with PacI and recovered using an OMEGA fragment recovery kit to obtain a linearized infectious clone plasmid containing PEVB (i.e., a linearized backbone plasmid), which was named L-pUC57-PEVB.

[0062] Table 1 Primers used for whole genome amplification of PEV-B-KOR strain

[0063] 2. Construction of a helper plasmid containing T7 RNA polymerase and a green fluorescent marker

[0064] Using the eukaryotic expression vector plasmid pEGFG-C1 (purchased from Clontech) as a template and EF and ER (Table 2) as primers, the green fluorescent protein encoding gene EGFP fragment was amplified. Using the CE II One Step Cloning Kit (purchased from Vazyme, catalog number Vazyme.C112-01), the amplified EGFP fragment was inserted into pCAG-T7-polymerase, which contains the gene encoding T7 RNA polymerase, using homologous recombination. The specific method is as follows: the amplified EGFP fragment encoding the green fluorescent protein gene was digested with HindIII and then ligated with pCAG-T7-polymerase, also digested with HindIII, using T4 DNA ligase to generate a eukaryotic helper plasmid containing T7 RNA polymerase and green fluorescent protein, designated pCAG-T7 RNApolymerase-EGFP. A schematic diagram of the construction is shown in Figure 3. The ligation system includes 100 μL of 5× CE II buffer, μL of enhanced recombinase Exnase II, 5 μL of the digested product of the amplified EGFP fragment encoding the green fluorescent protein gene, and 5 μL of linearized pCAG-T7-polymerase.

[0065] Table 2 Primers for auxiliary plasmid construction

[0066] Note: In Table 2, the lowercase sequence is the recombinant sequence added at the 5' end to achieve homologous recombination, and the underline is the restriction enzyme cutting site: AAGCTT.

[0067] The eukaryotic helper plasmid pCAG-T7 RNA polymerase-EGFP was transformed into a competent DH5a cell and plated onto LB plates containing kanamycin and ampicillin. Single colonies 1-5 were selected and amplified using the three primer pairs listed in Table 2. The amplified fragment was then confirmed by electrophoresis and sequencing. The results, shown in Figure 4, indicate that the size of the amplified fragment from single colony 1 was consistent with the expected size and sequenced correctly. Therefore, single colony 1 was a positive clone carrying the eukaryotic helper plasmid pCAG-T7 RNA polymerase-EGFP.

[0068] 3. Cell Co-transfection, Acquisition and Passaging Identification of Recombinant Porcine Enterovirus

[0069] Using conventional methods, the linearized backbone plasmid obtained in Title 1 of this Example and the helper plasmid pCAG-T7 RNA polymerase-EGFP obtained in Title 2 of this Example were co-transfected into African green monkey kidney (veroE6) cells using Lipofectamin 3000. The mass ratio of the linearized backbone plasmid to the helper plasmid was 3:2. The transfected cells were subcultured on veroE6 cells and observed under ultraviolet light at a wavelength of 488 nm. Cytopathic plaques emitting green fluorescence were selected and transferred to freshly prepared veroE6 monkey kidney cells for culture. The green plaques were then repeatedly selected (Figure 5) to obtain purified recombinant porcine enterovirus (r-PEVB), which is the P0 generation.

[0070] Improper liposome concentration or transfection ratio during transfection can lead to massive cell death, significantly impacting transfection efficacy and reducing the success rate, as liposomes themselves are inherently toxic. Extensive experiments have shown that a 3:2 ratio of linearized backbone plasmid to helper plasmid provides the highest transfection efficiency, with post-transfection fluorescence accounting for over 85-90%.

[0071] The recombinant porcine enterovirus (r-PEVB) was passaged at P0 on immortalized porcine bronchial epithelial cells (hTERT-PBECs) (disclosed in Chinese Invention Patent ZL201710556520.4, deposited as CCTCC NO: C201749). Cell culture medium was prepared by supplementing DMEM / F12 medium (Gibco) with a final concentration of 5% (volume percentage) FBS (Australian fetal bovine serum), 200 μg / mL of the antibiotic G418, and the complete set of epithelial cell regulatory factors (LONZA, cat. no. CC-4175) from the BEGM kit. The concentrations of the various substances in the complete set of epithelial cell regulatory factors in the cell culture medium in the BEGM kit are as follows: 26 μg / mL BPE (bovine pituitary extract), 15.5 ng / mL hEGF (human epithelial growth factor), 5 μg / mL human insulin, 1.4 μM hydrocortisone, 2.7 μM epinephrine, 9.7 nM iodothyronine, 0.3 nM retinoic acid, and 10 μg / mL transferrin. Immortalized porcine bronchial epithelial cells hTERT-PBECs were cultured in cell culture medium at 37°C in a 5% CO2 incubator. When the cells reached 85%-90% confluence, 10 cells were plated. 4 TCID 50The P0 generation recombinant porcine enterovirus r-PEVB was incubated for 2 hours, washed twice with serum-free DMEM medium, and replaced with DMEM / F12 medium containing 2% (volume percentage concentration) FBS. After culturing for 72-96 hours, the cell supernatant was collected and centrifuged at 1500 rpm at 4°C. The supernatant was used as the virus liquid; the cells were then continuously passaged according to the same method for more than 30 generations. The cytopathic effect (CPE) during the passage process was observed, and the CPE of the P30 generation on immortalized cells is shown in Figure 6. RNA was extracted from recombinant porcine enterovirus r-pEVB at different passages, and the 5'UTR gene was assayed by one-step real-time PCR using the HiScript III U+One Step qRT-PCR kit (purchased from Vazyme, catalog number Q611) from Novozymes Biotech. The primers used were PEVB-F and PEVB-R, and the probe was PEVB-probe. The PEVB-probe was labeled with FAM at the 5' end and BHQ at the 3' end. The specific nucleotide sequences of the primers and probe are shown in Table 3. The CT values ​​of the recombinant r-PEVB fluorescence quantitative PCR for passages P0-P30 are shown in Table 4. As shown in Table 4, the recombinant r-PEVB virus was still positive until passage P30, indicating that the recombinant porcine enterovirus r-PEVB can be continuously passaged for more than 30 generations.

[0072] Table 3 Nucleotide sequences of real-time PCR primers and probes

[0073] Table 4 CT values ​​of recombinant virus r-PEVB of P0-P30 generations (every 2 generations) by real-time PCR

[0074] Note: NC in Table 4 is a negative control, in which DMEM / F12 medium was used instead of viral RNA for real-time PCR. Undetermined refers to no detection value.

[0075] 3. Identification and biological characteristics of recombinant porcine enterovirus r-PEVB

[0076] 3.1 TCID of recombinant TERT in porcine bronchial epithelial cells hTERT-PBECs 50 Determination

[0077] The growth curves of the parent strain of porcine enterovirus (type B porcine enterovirus PEV-B-KOR strain) and the recombinant porcine enterovirus r-PEVB were determined on porcine bronchial epithelial cells hTERT-PBECs. 3 TCID 50hTERT-PBECs were infected with the recombinant porcine enterovirus r-PEVB (P25 generation) and the parental strain porcine enterovirus type B PEV-B-KOR strain (P5 generation). After incubation for two hours, the medium was changed to DMEM / F12 medium containing 2% (volume percentage concentration) FBS and cultured in a 37°C incubator with 5% CO2. Virus samples were collected at 24h, 48h, 72h, 96h and 120h after inoculation, and the TCID of the virus samples at each time point was determined after repeated freezing and thawing three times. 50 The one-step growth curve of the recombinant porcine enterovirus r-PEVB was drawn based on the virus titer, and the parent strain was used as a control to compare the differences in growth kinetics between the two. The results (Figure 7) show that the growth titer of the recombinant porcine enterovirus r-PEVB is generally higher than that of the parent strain. At 96 hours after infection, the TCID 50 The titer is 10 7.75 TCID 50 / mL, 1 TCID higher than that of PEV-B-KOR strain 50 The titer of the recombinant porcine enterovirus (r-PEVB) was 0.75 higher than that of the wild-type parent strain when cultured on veroE6 cells and remained stable. Furthermore, the infection efficiency and titer of the recombinant porcine enterovirus (r-PEVB) on immortalized porcine bronchial epithelial cells were significantly higher than those on veroE6 cells of African green monkey kidney. These results indicate that the constructed recombinant porcine enterovirus has a stronger replication ability.

[0078] 4.2 Identification of recombinant porcine enterovirus r-PEVB

[0079] The VP4 protein gene of PEVB (sequence shown in SEQ ID NO: 1) was inserted into the protein expression vector pET-32a and then introduced into the E. coli host to generate recombinant VP4-32a. After IPTG induction, the recombinant VP4 protein (with a his-tag) was expressed. A large amount of the target protein was found in the precipitate after lysis of the recombinant VP4-32a (Figure 8), indicating that the target protein was expressed primarily in the form of inclusion bodies. A high-affinity Ni column (purchased from GenScript, Cat. No. L00666) was used according to the instructions. The precipitate after lysis of the induced recombinant VP4-32a was loaded onto the Ni column and eluted with 20mM imidazole, 50mM imidazole, 100mM imidazole, 250mM imidazole, 300mM imidazole, and 500mM imidazole solutions, sequentially. The eluates from each imidazole solution were collected, and recombinant VP4 protein was found in the 500mM imidazole eluate. The recombinant VP4 protein was renatured using a refolding solution according to conventional methods. Refolding solution preparation: Add L-Arg HCl (400 mM), 2 mM EDTA, 5 mM glutathione (GSH), 1 mM oxidized glutathione (GSSG), and 0.5 mM PMSF to a final concentration of 100 mM Tris buffer, pH 8.0. As shown in Figure 9, a specific band at 25 kD was observed for the refolded recombinant VP4 protein, indicating significant removal of contaminants.

[0080] New Zealand white rabbits were inoculated with renatured recombinant VP4 protein as an immunogen to generate rabbit anti-VP4 protein polyclonal antibodies. ELISA antibody testing (3 μg of renatured recombinant VP4 protein was coated per well) revealed an ELISA antibody titer exceeding 100,000. This rabbit anti-VP4 protein polyclonal antibody was subsequently used for indirect immunofluorescence (IFA) and immunoblotting (WB) analysis of r-PEVB (P25).

[0081] 4.2.1 IFA Identification

[0082] Recombinant porcine enterovirus r-PEVB was cultured with porcine bronchial epithelial cells (hTERT-PBECs) for 96 hours, fixed with paraformaldehyde, and washed three times with phosphate-buffered saline (PBS). The cells were permeabilized with 0.2% Triton X-100 aqueous solution for 5 minutes at room temperature and washed three times with PBS. The cells were blocked with 3% BSA aqueous solution for 1 hour at room temperature and washed three times with PBS. Rabbit anti-VP4 protein polyclonal antibody was used as the primary antibody, incubated overnight at 4°C, and washed three times with PBS. The cells were then incubated with Alexa Fluor 555-labeled donkey anti-rabbit IgG (H+L) (purchased from Beyotime, Cat. No. A0453) at 37°C in the dark for 1 hour, washed three times with PBS, and observed under a fluorescence microscope. Porcine bronchial epithelial cells (hTERT-PBECs) without recombinant porcine enterovirus r-PEVB infection served as a negative control, and porcine bronchial epithelial cells without recombinant porcine enterovirus r-PEVB infection but with PBS buffer instead of rabbit anti-VP4 protein polyclonal antibody served as a blank control. As shown in FIG10 , the recombinant porcine enterovirus r-PEVB culture showed red specific fluorescence, confirming that the recombinant virus r-PEVB had been successfully constructed.

[0083] 4.2.2 Western Blotting (WB) Identification

[0084] Porcine bronchial epithelial cells hTERT-PBECs (normal control), porcine bronchial epithelial cells hTERT-PBECs infected with recombinant porcine enterovirus r-PEVB, and porcine bronchial epithelial cells hTERT-PBECs infected with the parental strain (porcine enterovirus type B PEV-B-KOR strain) were collected, and the total protein of each cell was extracted using RIPA protein lysis buffer (catalog number: P0013B) from Beyotime Company. After SDS-PAGE electrophoresis, the membrane was transferred to PVDF membrane. After transfer, the membrane was removed and washed 3 times with PBST; blocked with 5% skim milk solution at 37°C for 2h and washed 3 times with PBST; incubated with rabbit anti-VP4 protein polyclonal antibody (prepared in title 4.2 of this example) at 4°C overnight and washed 3 times with PBST; incubated with horseradish peroxidase-labeled goat anti-rabbit IgG (H+L) (purchased from Beyotime Company, catalog number: A0208) at room temperature for 1h, washed 3 times with PBST, and then developed with ECL. In addition, hTERT-PBECs infected with recombinant porcine enterovirus r-PEVB were validated by Western blotting with his antibodies. The method was the same as that used for Western blotting with rabbit anti-VP4 protein polyclonal antibody, except that his antibodies (purchased from Boster, Cat. No.: M30975-) were used instead of rabbit anti-VP4 protein polyclonal antibody. The specific results are shown in Figure 11. The results showed that the proteins of hTERT-PBECs infected with recombinant porcine enterovirus r-PEVB could specifically react with rabbit anti-VP4 protein polyclonal antibody and his antibodies, and specific bands appeared in the lanes, confirming that the PEVB recombinant virus r-PEVB had been successfully constructed.

[0085] Example 2 Application of viral vectors for both the respiratory and digestive tracts of pigs

[0086] 1. Study on the characteristics of recombinant porcine enterovirus r-PEVB as a viral vector

[0087] The gene sequence of ASFV (African swine fever virus) T cell epitope (SEQ ID NO: 2) was inserted between the VP4 gene and the 5'UTR gene of the recombinant porcine enterovirus r-pEVB to obtain the recombinant expression plasmid ATr-PEVB. The amino acid sequence of the ASFV (African swine fever virus) T cell epitope is shown in SEQ ID NO: 3. The gene fragment of the ASFV (African swine fever virus) T cell epitope was synthesized by KingScript. The specific method for constructing the recombinant expression plasmid ATr-PEVB is as follows: the recombinant plasmid pUC57-PEVB constructed in Example 1 is double-digested with EcoR1 and HindIII, and then connected with the gene fragment of the synthesized ASFV (African swine fever virus) T cell epitope by conventional methods using T4 DNA ligase to obtain the recombinant expression plasmid ATr-PEVB.

[0088] The B cell epitope gene (SEQ ID NO:4) of the major protective adhesion factor antigen P97R1 of Mhp (Mycoplasma hyopneumoniae) was inserted between the VP4 gene and the 5'UTR gene of the recombinant porcine enterovirus r-pEVB to obtain the recombinant expression plasmid Mr-PEVB. The amino acid sequence of the B cell epitope of the major protective adhesion factor antigen P97R1 of Mhp (Mycoplasma hyopneumoniae) is shown in SEQ ID NO:5. The specific method for constructing the recombinant expression plasmid Mr-PEVB is as follows: the recombinant plasmid pUC57-PEVB constructed in Example 1 was double-digested with EcoR1 and HindIII, and then ligated with the synthesized P97R1 B cell epitope gene fragment using T4 DNA ligase using conventional methods to obtain the recombinant expression plasmid Mr-PEVB.

[0089] A gene fragment containing the T cell epitope of ASFV and the B cell epitope of P97R1 of Mhp was inserted between the VP4 gene and the 5'UTR gene of the recombinant porcine enterovirus r-pEVB, the sequence of which is shown in SEQ ID NO:6, to obtain the recombinant expression plasmid AMr-PEVB. The amino acid sequence containing the T cell epitope of ASFV and the B cell epitope of P97R1 of Mhp is shown in SEQ ID NO:7. The specific method for constructing the recombinant expression plasmid AMr-PEVB is as follows: the recombinant plasmid pUC57-PEVB constructed in Example 1 was double-digested with EcoR1 and HindIII, and then ligated with the synthesized gene fragment with the sequence shown in SEQ ID NO:6 using T4 ligase using conventional methods to obtain the recombinant expression plasmid AMr-PEVB.

[0090] The above-mentioned recombinant expression plasmids were digested with EcoRI and then electrophoresed for identification. The results are shown in Figure 12, which are consistent with expectations. The recombinant expression plasmids ATr-PEVB, Mr-PEVB, and AMr-PEVB were digested with Pac I restriction endonuclease to obtain the corresponding linearized fragments LATr-PEVB, LMr-PEVB, and LAMr-PEVB. Each linearized fragment was co-transfected with the auxiliary plasmid pCAG-T7-polymerase-EGFP using liposome lipo3000 at a mass ratio of 2:1 into monkey kidney veroE6 cells. At the same time, a positive control group PC (only the auxiliary plasmid was co-transfected into monkey kidney veroE6 cells using liposome lipo3000) and a negative control group NC (monkey kidney veroE6 cells were co-transfected with ultrapure water and liposome lipo3000) were set up. Each transfected cell was subcultured and observed under ultraviolet light at a wavelength of 488 nm. The cytopathic plaques emitting green fluorescence were picked and transferred to freshly prepared monkey kidney cells veroE6 for culture. Then, the green spots were picked repeatedly (Figure 13) to obtain the purified recombinant toxins ATr-PEVB, Mr-PEVB and AMr-PEVB, which were recorded as the P0 generation.

[0091] The recombinant viruses ATr-PEVB, Mr-PEVB and AMr-PEVB can be continuously passaged on hTERT-PBECs and can be stably passaged for more than 25 generations. During the passage process, cytopathic CPE was observed, and the cytopathic CPE of the P14 generation on the immortalized cells hTERT-PBECs is shown in Figure 14. Cytotoxicity of different generations of each recombinant virus was collected, RNA was extracted, and HiScript III U+One Step qRT-PCR (purchased from Vazyme, product number Q611) was used to perform one-step real-time PCR detection according to the method in Title 3 of Example 1. Each recombinant virus P0-P30 generation was detected by fluorescent quantitative PCR every 5 generations, and the corresponding CT values ​​are shown in Table 5. Among them, it was stable before the P25 generation, and the virus titer could reach up to 10 7.25 TCID50 / mL.

[0092] Table 5 CT values ​​of recombinant viruses at different passages P0-P30 (tested every 5 passages) by real-time PCR

[0093] Note: NC in Table 5 is the negative culture medium control.

[0094] The applicant has also selected multiple sites to insert the cellular epitope genes of the above three antigens, and found that the transfection efficiency is very low, less than 10%, for example, inserting between the 2A gene and the VP1 gene.

[0095] 2. Identification of Recombinant Viruses

[0096] hTERT-PBECs were cultured according to the method described in Title 3 of Example 1, and recombinant ATr-PEVB, Mr-PEVB, and AMr-PEVB were inoculated for proliferation. The cells were cultured for 72-96 hours, frozen and thawed three times, and centrifuged at 10,000 rpm for 10 minutes at 4°C. Indirect immunofluorescence (IFA) and Western blotting were performed for identification. Negative controls were uninfected hTERT-PBECs. First, a rabbit anti-VP4 protein polyclonal antibody was used as the primary antibody for PEVB VP4 protein, and AlexaFluor 555-labeled donkey anti-rabbit IgG (H+L) (purchased from Beyotime, Catalog No. A0453) was used as the secondary antibody to identify recombinant ATr-PEVB, Mr-PEVB, and AMr-PEVB by indirect immunofluorescence (IFA) and Western blotting. Then, the recombinant virus Mr-PEVB was detected by indirect immunofluorescence IFA and Western blotting using Mhp P97R1 antibody (published in Xie Xing et al, 2022, Construction of a telomerase-immortalized porcine tracheal epithelial cell model for swine-origin mycoplasma infection, Journal of Integrative Agriculture 2022, 21(2): 504–520) as the primary antibody and Alexa Fluor 555-labeled donkey anti-mouse IgG (H+L) (purchased from Beyotime, catalog number A0460) as the secondary antibody. The recombinant virus ATr-PEVB was detected by indirect immunofluorescence IFA and Western blotting using African swine fever virus positive serum antibody as the primary antibody and FITC-labeled goat anti-swine fluorescent secondary antibody (purchased from Beijing Biolong, catalog number BD9448) as the secondary antibody. Indirect immunofluorescence (IFA) and Western blotting were performed for AMr-PEVB using antibodies against serum positive for African swine fever virus as the primary antibody and a FITC-labeled goat anti-swine fluorescent secondary antibody (purchased from Beijing Biolong, Cat. No. BD9448) as the secondary antibody. Indirect immunofluorescence (IFA) and Western blotting were performed using antibodies against Mhp P97R1. Negative controls for both indirect immunofluorescence (IFA) and Western blotting were porcine bronchial epithelial cells (hTERT-PBECs) not infected with recombinant porcine enterovirus r-PEVB.

[0097] Indirect immunofluorescence (IFA) results (Figures 15-17) showed that recombinant ATr-PEVB carrying the T cell epitope of African swine fever virus, recombinant Mr-PEVB carrying the B cell epitope of Mycoplasma hyopneumoniae P97R1, and recombinant AMr-PEVB carrying both the T cell epitope of African swine fever virus and the B cell epitope of Mycoplasma hyopneumoniae P97R1 were successfully constructed. Compared with the negative control (hTERT-PBECs without recombinant porcine enterovirus r-PEVB infection), they exhibited specific green, red, and red fluorescence, respectively. When using indirect immunofluorescence (IFA) to identify recombinant porcine enterovirus PEVB, a rabbit VP4 protein polyclonal antibody was used as the primary antibody and Alexa Fluor 555-labeled donkey anti-rabbit IgG (H+L) as the secondary antibody. The successfully constructed recombinant porcine enterovirus PEVB emitted red fluorescence under fluorescence. When using indirect immunofluorescence (IFA) to identify African swine fever epitopes, positive serum antibodies against African swine fever are used as the primary antibody, and FITC-labeled goat anti-swine fluorescent antibodies are used as the secondary antibody. If the result is positive, specific green fluorescence will appear after microscopic observation of the IFA test. When using indirect immunofluorescence (IFA) to identify the P97R1 epitope, a monoclonal antibody against the P97R1 protein is used as the primary antibody, and Alexa Fluor 555-labeled donkey anti-mouse IgG (H+L) is used as the secondary antibody. If the result is positive, red fluorescence will be emitted.

[0098] As can be seen from Figures 18-20, compared with the negative control (hTERT-PBECs not infected with the virus), each recombinant virus showed obvious specific bands. The Western blotting identification results of the recombinant viruses AMr-PEVB, ATr-PEVB and Mr-PEVB against rabbit anti-VP4 protein polyclonal antibodies in Figure 18 showed that a specific band appeared at 25KD, indicating that the above viruses are recombinant enteroviruses; the Western blotting identification results of the recombinant viruses AMr-PEVB and Mr-PEVB against Mhp P97R1 monoclonal antibodies in Figure 19 showed that a specific band appeared at a size of 29KD, indicating that the Mhp P97R1 antigen epitope was successfully inserted into the recombinant virus; the Western blotting identification results of the recombinant viruses AMr-PEVB and ATr-PEVB against African swine fever virus positive serum antibodies in Figure 20 showed that two specific bands appeared at 70KD and 55KD, indicating that the African swine fever antigen epitope was successfully inserted. The negative control showed no bands or only very few weak and shallow bands, indicating that the recombinant viruses were successfully constructed, confirming that the r-PEVB recombinant virus vector expression strain with ASFV and / or Mhp antigens inserted was successfully obtained.

[0099] 3. Immunogenicity of each recombinant virus in pigs (Mhp and ASFV) and evaluation of protection against challenge (Mhp)

[0100] A comprehensive evaluation was conducted on the feasibility of using recombinant r-PEVB as a viral vector for both respiratory and digestive tract vaccines in pigs.

[0101] The preparation methods for each recombinant virus were as follows: First, the recombinant virus r-PEVB, which had been passaged on hTERT-PBECs to passage 14 (P14) and stored at -70°C, was amplified on hTERT-PBECs. After 96 hours of culture and when obvious CPE was observed, the virus was harvested and frozen and thawed three times. The supernatant was then centrifuged and used as the recombinant r-PEVB. The recombinant viruses ATr-PEVB and Mr-PEVB were prepared using the same method as the recombinant r-PEVB; therefore, all recombinant viruses used for immunization were at passage 15.

[0102] (1) Safety evaluation of the recombinant virus r-PEVB and the parent strain PEV-B-KOR in pigs

[0103] Put 10 6.5 TCID50 / mL of recombinant r-PEVB and 10 6.5 The PEV-B-KOR strain with a TCID50 / mL PCR dose of 2 mL / pig was injected intramuscularly into 10-day-old SPF piglets. Clinical symptoms were observed within 14 days, and an autopsy was performed 14 days later. The results showed that the recombinant virus r-PEVB, like the parental strain, did not cause any clinical symptoms or diarrhea in pigs after intramuscular injection. Intestinal pathological sections (Figure 21) showed that everything was normal, indicating that the recombinant virus r-PEVB is non-pathogenic and safe as a live vaccine vector.

[0104] (2) Immunogenicity of recombinant virus ATr-PEVB against ASFV in pigs

[0105] 28-day-old SPF piglets were selected for the experiment, with 5 pigs in each group. They were randomly divided into two groups and inoculated with recombinant virus ATr-PEVB and DMEM / F12 culture medium (negative control group) respectively. The time of the first immunization was day 0, recorded as 0dpi. The recombinant virus ATr-PEVB was immunized twice and three times. The second immunization was 14 days after the first immunization, and the third immunization was 21 days after the first immunization. In each immunization, immunization was performed by nasal drops and intramuscular injection at the same time. The vaccination dose for each piglet was 10 7.0 The virus solution with TCID50 / mL is 1mL, so the total inoculation dose for each pig is 2mL, and the virus content is 10 7.0 For the negative control group, each pig was inoculated with 2 mL of DMEM / F12 medium.

[0106] Immunogenicity was assessed in piglets inoculated with the recombinant virus ATr-PEVB. First, serum IgG antibody levels against the full ASFV viral protein, ASFV p30, p22, p10, p54, cd2v-N, and B602L proteins were compared before and after immunization. The method for detecting serum antibodies to ASFV p30 is described in Xie Qinyun et al., 2023, Dynamics of Serological and Mucosal Antibody Responses against African Swine Fever Viruses in Experimentally Infected Pigs. Transboundary and emerging diseases, Article ID 9959847. For the detection of serum antibodies to other proteins, only the envelope protein was replaced with the corresponding protein, and the rest of the method was the same as for detecting serum antibodies to ASFV p30. Secondly, the levels of sIgA antibodies in oral fluid before and after immunization were measured (the ELISA plate was coated with African swine fever virus p22 and p30 proteins at a protein concentration ratio of 2:1. Other steps were the same as Xie Qinyun et al., 2023, Dynamics of Serological and Mucosal Antibody Responses against African Swine Fever Viruses in Experimentally Infected Pigs. Transboundary and emerging diseases, Article ID 9959847). The expression levels of cellular immunity-related and pro-inflammatory cytokines were measured by qPCR. The qPCR measurement method for the expression levels of cellular immunity-related and pro-inflammatory cytokines is as follows: Fluorescent quantitative primers for pig pro-inflammatory cytokines (see the table below) are used to extract RNA from the serum and then use a reverse transcription kit III RT SuperMix for qPCR (+gDNA wiper) (purchased from Vazyme, product number R323-01) was reverse transcribed into cDNA. The β-actin gene was used as an internal control. -ΔΔCTMethods (Livak and Schmittgen, 2001, Analysis of relative gene expression data using real-time quantitative PCR and the 2(T)(-Delta Delta C) method. Methods 25, 402-408.) The fold changes in porcine cytokine gene mRNA expression between the immunized group and the negative control group were determined.

[0107] Table 1 Fluorescence quantitative primers for porcine proinflammatory cytokines

[0108] Secondly, the T cell populations of pig peripheral blood lymphocytes (PBMCs) were detected (for specific methods, see: Chung CJ, Cha SH, Grimm AL, Ajithdoss D, Rzepka J, Chung G, et al. (2018) Pigs that recover from porcine reproduction and respiratory syndrome virus infection develop cytotoxic CD4+CD8+ and CD4+CD8-T-cells that kill virus infected cells. PLoS ONE 13(9): e0203482) to comprehensively evaluate the immunogenicity of the recombinant virus ATr-PEVB carrying T cell epitopes of ASFV in pigs.

[0109] Results: The serum IgG antibody levels against five antigens (CD2V-N, P10, P30, P54, and B602L) were significantly increased 28 days after immunization compared with the control group. The serum IgG antibody level against P22 was significantly increased 14 days after immunization compared with the control group, as shown in Figure 22. The oral fluid sIgA antibody level was significantly increased 14 and 28 days after immunization compared with the pre-immunization level, as shown in Figure 23. In terms of inflammatory cytokine expression levels, IFN-α, IFN-γ, and IL-1β were significantly downregulated 14 days after immunization compared with the negative control group, and IL-10 was significantly upregulated 28 days after immunization, as shown in Figure 24. Compared with the negative control group, the percentage of CD8+CD3+ cells in peripheral blood lymphocytes (PBMC) was significantly decreased on the 28th day after the first immunization; the CD4+ / CD8+T cell ratio in PBMC was significantly increased on the 14th day after the first immunization compared with the negative control group; and the CD4+ / CD8+T cell ratio was significantly increased on the 28th day after the first immunization compared with the negative control group; indicating that the recombinant virus ATr-PEVB can weaken CD8+T cell immunity on the 28th day after the first immunization, causing the body to produce an immune response biased towards CD4+T cells. The above results all indicate that the recombinant virus ATr-PEVB has good immunogenicity in pigs after immunization.

[0110] (3) Evaluation of the immunogenicity and protection against Mhp in pigs immunized with the recombinant virus Mr-PEVB

[0111] 28-day-old SPF pigs were randomly divided into three groups (immune challenge group, challenge control group and negative control group), with 5 pigs in each group. The immune challenge group was immunized three times, with the first immunization time recorded as day 0, recorded as 0 dpi, the second immunization time as 14 days after the first immunization, and the third immunization time as 28 days after the first immunization. In each immunization, 1 mL of virus with a content of 10 7.0 The TCID50 / mL Mr-PEVB virus solution was immunized by nasal drops and intramuscular injection. Each pig was immunized with 2mL of virus containing 10 7.0 The negative control group was immunized three times according to the same method as above, except that DMEM / F12 medium was used instead of Mr-PEVB virus solution. On the 42nd day after the first immunization, the immune challenge group was challenged with 5mL of Mycoplasma hyopneumoniae 168 virus solution in the trachea, with a challenge dose of 10×10 9.5CCU / head / time. The negative control group (denoted as the KM2 control group) was injected with KM2 liquid culture medium through the trachea for three immunizations, and the volume of each immunization was the same as that of Mr-PEVB virus liquid, without virus attack. The virus attack control group was injected with the same volume of KM2 liquid culture medium through the trachea for three immunizations, and injected with 168 strains of Mycoplasma hyopneumoniae through the trachea on the 42nd day after the first immunization, and the virus attack dose was the same as that of the immune virus attack group. During the entire experiment, each group of pigs was observed every day for the occurrence of respiratory clinical symptoms such as coughing, and the pigs were killed on the 28th day after the virus attack, that is, the 70th day after the first immunization. Lung lesions were scored after autopsy in the immunization, challenge, and negative control groups (for scoring methods, see Madec F, Kobisch M, Bilaan Lesionnel des poumons des porcs chaarcutiers alabattoir, Journees de la Recherche Porcine en France, 1982, 14:405-412). Serum antibodies against Mhp P97R1 and mucosal sIgA antibodies in bronchoalveolar lavage fluid were measured before and after immunization and challenge by ELISA (for detection methods, see Gan & Xie et al., 2020, Establishment of a model of Mycoplasma hyopneumoniae infection using Bama miniature pigs, Food Production, Processing and Nutrition (2020) 2:19). Pulmonary cilia were observed before and after immunization and challenge by electron microscopy (for specific methods, see Gan & Xie et al., 2020, Establishment of a model of Mycoplasma hyopneumoniae infection). hyopneumoniae infection using Bama miniature pigsFood Production,Processing and Nutrition(2020)2:19), and comprehensively evaluated the immunogenicity and immune protection against Mhp after immunization of pigs with recombinant virus Mr-PEVB.

[0112] During the entire challenge immunization experiment, no pathological changes were found at the injection site, indicating that the recombinant virus Mr-PEVB is relatively safe as a candidate viral vector vaccine strain for Mhp.

[0113] As shown in Figure 26, the results of apparent lung lesions after autopsy showed that the challenged control group showed very typical and strong Mhp lesions, with typical red to purple flesh-like lesions in the tip and heart lobes, and the 28-point score could reach between 18 and 20 points. The difference between the immune challenged group and the negative control group was not much, with scores of 3 and 0 respectively, indicating that Mr-PEVB can have a more obvious immune protection effect on the immune challenged group.

[0114] After necropsy, bronchial rings were excised from each pig, fixed in glutaraldehyde, and examined for cilia under an electron microscope. The results, shown in Figure 27, show that the challenge control group experienced 90% cilia loss, with the cilia losing their morphology and becoming bald. While the Mr-PEVB-immunized group also experienced approximately 5-10% cilia loss, the cilia retained a relatively intact morphology. The negative control group retained a very intact cilia morphology, with no cilia loss observed.

[0115] Before and after the first vaccination, 3 mL of whole blood was collected from each pig to prepare serum. Nasal swabs were also collected to measure serum IgG antibodies against Mhp and mucosal sIgA antibodies against Mhp in the nasal swabs. As shown in Figures 28 and 29, the levels of serum IgG and mucosal sIgA antibodies in the challenge group were significantly higher than those in the negative control group, with the difference in mucosal antibody levels being even more significant. These results indicate that the Mr-PEVB challenge group did have a certain protective effect at the antibody level.

[0116] After autopsy, bronchoalveolar lavage fluid (BALF) was collected from each pig. The Mhp copy number in the BALF of each pig was determined by fluorescence quantitative PCR using the constructed standard positive plasmid PMD-T-P97 for the Mycoplasma hyopneumoniae P97 antigen (the P97 sequence of the Mycoplasma hyopneumoniae strain 168 (GeneBank accession number NC_017509.1) was inserted into the PMD-18T vector to finally obtain a standard positive plasmid containing the Mycoplasma hyopneumoniae P97 antigen). For specific detection methods, see Wu, Y., Ishag, H.Z.A., Hua, L., Zhang, L., Liu, B., Zhang, Z., et al. (2019). Establishment and application of a real-time, duplex PCR method for simultaneous detection of Mycoplasma hyopneumoniae and Mycoplasma hyorhinis. Kafkas Universitesi Veterinary Fakultesi Dergisi, 25, 405–414. As shown in Figure 30, compared with the challenge control group, the DNA copy number in the immunized group decreased significantly, while no Mhp was detected in the negative control group. These results indicate that immunization with the recombinant Mr-PEVB virus provides significant protection against Mhp infection in pigs.

[0117] In summary, Mr-PEVB is safe to use as a recombinant viral vector expression strain of Mycoplasma hyopneumoniae and has a good immune protection effect against Mycoplasma hyopneumoniae infection.

[0118] In summary, r-PEVB is used as a respiratory and gastrointestinal vaccine carrier with good safety and good immune protection against Mycoplasma hyopneumoniae and African swine fever.

Claims

1. A viral vector delivery system for both the respiratory tract and digestive tract of pigs, characterized in that: It includes a backbone plasmid and an auxiliary plasmid: the backbone plasmid is obtained by inserting the full-length cDNA of type B porcine enterovirus into the pUC57 plasmid; the auxiliary plasmid is obtained by inserting the green fluorescent protein coding gene into the plasmid pCAG-T7-polymerase.

2. The viral vector delivery system according to claim 1, characterized in that In the backbone plasmid, a T7 promoter is inserted at one end of the full-length cDNA of type B porcine enterovirus PEVB, and a His tag and a polyA tail are inserted at the other end.

3. The method for constructing the viral vector delivery system according to claim 1, characterized in that The method comprises the following steps: constructing a backbone plasmid and an auxiliary plasmid respectively, and then co-transfecting the backbone plasmid and the auxiliary plasmid into monkey kidney cells or pig bronchial epithelial cells.

4. The construction method according to claim 3, characterized in that The method for constructing the backbone plasmid is as follows: using the full-length cDNA of porcine enterovirus type B PEV-B-KOR strain as a template, introducing a T7 promoter at the 5' end of the full-length cDNA by PCR, introducing a his-tag label and a ployA tail at the 3' end, and then inserting it into pUC57 to obtain a backbone plasmid.

5. The construction method according to claim 4, characterized in that The method for constructing the auxiliary plasmid is as follows: inserting the green fluorescent protein encoding gene into the plasmid pCAG-T7-polymerase to obtain the auxiliary plasmid.

6. The construction method according to claim 5, characterized in that The backbone plasmid and the auxiliary plasmid were co-transfected into monkey kidney veroE6 cells using liposome Lipofectamin 3000, and the virus plaques producing green fluorescence were picked to obtain the virus vector delivery system.

7. Use of the viral vector delivery system according to claim 1 in constructing porcine respiratory and digestive tract vaccine antigens.

8. The use according to claim 7, characterized in that The respiratory and / or digestive tract antigen epitope encoding gene is inserted between the VP4 gene and the 5'UTR gene of the backbone plasmid, and then co-transfected with the auxiliary plasmid into monkey kidney veroE6 cells or porcine bronchial epithelial cells hTERT-PBECs using liposome Lipofectamin 3000 to obtain the vaccine antigen.

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