A recombinant adenovirus vector containing porcine epidemic diarrhea virus immune protein, strain, vaccine and application thereof

Abstract

The present invention provides a recombinant adenovirus vector and strain, vaccine and application comprising porcine epidemic diarrhea virus (PEDV) immune protein, belonging to the technical field of biological products. The present invention provides a recombinant adenovirus vector comprising porcine epidemic diarrhea virus immune protein, with a human replication-deficient adenovirus vector as a backbone vector, comprising a coding gene for S protein or S1 protein; the S protein or S1 protein is from PEDVGIIa strain CH / HBXT / 2018 or GIIb strain CH / HNPJ / 2017. The present invention constructs 4 vaccines for the prevention and control of PEDV, and in vivo evaluation in BALB / c mice shows that all 4 vaccines can induce strong cellular immunity and humoral immunity, providing an effective means for preventing infection with the currently prevalent highly virulent PEDV strains.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological products, and specifically relates to a recombinant adenovirus vector and strain, vaccine and application containing porcine epidemic diarrhea virus immune protein. Background Art

[0002] Porcine epidemic diarrhea virus (PEDV) is an acute, highly contagious porcine enteric coronavirus characterized by diarrhea, vomiting, dehydration and high mortality in suckling piglets. It belongs to the genus Alphacoronavirus of the family Coronaviridae. The symptoms are severe in suckling piglets, with high morbidity and mortality. [1,2] PEDV was first discovered in the UK in 1977 [3] In 1978, PEDV was identified as a coronavirus [4] In recent years, highly pathogenic variants of GII PEDV have gradually spread to many countries, posing an increasingly serious threat to the global pig industry. [5] .

[0003] PEDV is usually divided into classical strains (GI genogroup, including GIa and GIb subgroups) and variant strains (GII genogroup, including GIIa, GIIb and S-INDEL subgroups). [6] However, after 2010, the classic strains of the GI genogroup gradually disappeared in the field, and the virulent variants of the GII genogroup have become the dominant strains worldwide. In addition, studies have shown that there is a large variation in the main antigen S protein between the GI strain and the GII strain, and vaccines based on the GI genogroup strain only provide partial protection against the virulent variants of the GII genogroup. [7,8] Therefore, it is urgent to develop a new, effective and safe PEDV vaccine based on the virulence variants of the GII genogroup.

[0004] PEDV is an enveloped single-stranded positive-strand RNA virus with a genome length of about 28 kb, including: 5' untranslated region (UTR), 7 open reading frames (ORF) and 3' untranslated region (UTR). Among them, 4 open reading frames encode 4 structural proteins: spike protein (S), membrane protein (M), envelope protein (E) and nucleocapsid protein (N). [9-11]. The S protein is located on the outer layer of the virus particle and is a type I glycoprotein. It is a key factor in determining the host cell tropism and cell entry of PEDV. The S protein is divided into S1 and S2 domains. The S1 subunit mainly binds to the receptor to promote virus adhesion and contains rich neutralizing epitopes. The S2 subunit is relatively conservative and mainly promotes viral membrane fusion. Therefore, the S protein is a key target for host antibody response and a good candidate for coronavirus vaccine development. [12,13] .

[0005] Human adenovirus type 5 (Ad5) is highly safe and can stimulate strong systemic immune responses and mucosal immune responses. Currently, Ad5 vectors have been widely used in the development of vaccines for a variety of pathogens. [14-23] ). In addition, studies have shown that Ad5 vector vaccines can be expressed in porcine cell lines.

[24] . Summary of the invention

[0006] In view of this, the object of the present invention is to provide a recombinant adenovirus vector containing porcine epidemic diarrhea virus immune protein, wherein the recombinant adenovirus vector contains the S or S1 coding genes of two different strains of the GIIa PEDV strain, respectively. The constructed viral vaccine can induce strong humoral immunity and cellular immunity by intramuscular injection, and is expected to become an effective vaccine for preventing the currently prevalent highly virulent PEDV strains.

[0007] The present invention provides a recombinant adenovirus vector containing porcine epidemic diarrhea virus immune protein, using a human replication-deficient adenovirus vector as a backbone vector, and containing a gene encoding an S protein or an S1 protein;

[0008] The S protein or S1 protein is from PEDV GIIa strain CH / HBXT / 2018 or GIIb strain CH / HNPJ / 2017.

[0009] Preferably, the amino acid sequence of the S protein from the PEDV GIIa strain CH / HBXT / 2018 is shown in SEQ ID NO: 1, and the nucleotide sequence of the corresponding encoding gene is shown in SEQ ID NO: 3;

[0010] The amino acid sequence of the S1 protein from the PEDV GIIa strain CH / HBXT / 2018 is shown in SEQ ID NO: 2, and the nucleotide sequence of the corresponding encoding gene is shown in SEQ ID NO: 4.

[0011] Preferably, the amino acid sequence of the S protein from the PEDVGIIb strain CH / HNPJ / 2017 is shown in SEQ ID NO:5, and the corresponding nucleotide sequence of the encoding gene is shown in SEQ ID NO:7;

[0012] The amino acid sequence of the S1 protein from the PEDV GIIb strain CH / HNPJ / 2017 is shown in SEQ ID NO:6, and the nucleotide sequence of the corresponding encoding gene is shown in SEQ ID NO:8.

[0013] Preferably, the human replication-defective adenovirus vector is pDC316;

[0014] Preferably, the recombinant adenovirus vector comprises pDC-XT-tPA-Sopt;

[0015] The nucleotide sequence of pDC-XT-tPA-Sopt is as shown in SEQ ID NO:12.

[0016] The invention provides a recombinant adenovirus strain based on a human replication-deficient adenovirus vector, which is obtained by packaging the recombinant adenovirus vector.

[0017] Preferably, it comprises Ad-XT-tPA-Sopt;

[0018] The Ad-XT-tPA-Sopt is packaged by pDC-XT-tPA-Sopt.

[0019] The invention provides a recombinant porcine epidemic diarrhea virus vaccine, comprising the recombinant adenovirus strain and an adjuvant.

[0020] Preferably, the volume ratio of the recombinant adenovirus strain and the adjuvant is 1:1.

[0021] The present invention provides the use of the recombinant adenovirus strain in preparing a vaccine for preventing and controlling porcine epidemic diarrhea.

[0022] Preferably, the porcine epidemic diarrhea virus strain comprises a GIIa type strain and / or a GIIb type strain.

[0023] The recombinant adenovirus vector containing porcine epidemic diarrhea virus immune protein provided by the present invention uses a human replication-deficient adenovirus vector as a backbone vector and contains a coding gene for S protein or S1 protein; the S protein or S1 protein is from PEDVGIIa strain CH / HBXT / 2018 or GIIb strain CH / HNPJ / 2017. The present invention uses the recombinant adenovirus strain packaged by the recombinant adenovirus vector as an antigen to immunize animals. The results show that the four strains can induce strong humoral immunity and cellular immunity by intramuscular injection, especially the vaccine prepared by Ad-XT-tPA-Sopt is more likely to prevent the currently popular highly toxic PEDV strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1Construction and identification of PEDV recombinant adenovirus, (A) Schematic diagram of Ad-XT-tPA-Sopt and Ad-PJ-tPA-Sopt expressing S protein, and Ad-XT-oriSIP-S1opt and Ad-PJ-oriSIP-S1opt expressing S1 protein; (B) PCR identification of Ad-XT-tPA-Sopt, Ad-XT-oriSIP-S1opt, Ad-PJ-tPA-Sopt and Ad-PJ-oriSIP-S1opt. (1) Ad-Control, (2) Ad-PJ-tPA-Sopt, (3) Ad-XT-tPA-Sopt, (4) Ad-PJ-oriSIP-S1opt, (5) Ad-XT-oriSIP-S1opt. M, DL5000 marker; the number on the left is the size of DNA in bp; (C) Western blot detection of S and S1 protein expression in HEK293 and LLC-PK1 cells; Lane (1) and (4) Ad-Control, (2) Ad-PJ-tPA-Sopt, (3) Ad-XT-tPA-Sopt, (5) Ad-PJ-oriSIP-S1opt, (6) Ad-XT-oriSIP-S1opt; C1: HEK293 cells infected with Ad-PJ-tPA-Sopt or Ad-XT-tPA-Sopt, S protein expression was detected using mouse anti-6×His monoclonal antibody; C2: HEK293 cells infected with Ad-PJ-tPA-Sopt or Ad-XT-tPA-Sopt, mouse anti-PEDV monoclonal antibody was used to detect S protein expression; The expression of S protein was detected by S2 monoclonal antibody; C3: HEK293 cells infected with Ad-PJ-oriSIP-S1opt or Ad-XT-oriSIP-S1opt, and the expression of S1 protein was detected by mouse anti-6×His monoclonal antibody; C4: LLC-PK1 cells infected with Ad-PJ-tPA-Sopt or Ad-XT-tPA-Sopt, and the expression of S protein was detected by mouse anti-6×His monoclonal antibody; C5: LLC-PK1 cells infected with Ad-PJ-tPA-Sopt or Ad-XT-tPA-Sopt, and the expression of S protein was detected by mouse anti-PEDV S2 monoclonal antibody; C6: LLC-PK1 cells infected with Ad-PJ-oriSIP-S1opt or Ad-XT-oriSIP-S1opt, and the expression of S1 protein was detected by mouse anti-6×His monoclonal antibody.(D) Indirect immunofluorescence technology was used to detect the expression of S protein in LLC-PK1 cells infected with Ad-PJ-tPA-Sopt or Ad-XT-tPA-Sopt, and detected by mouse anti-PEDV S2 monoclonal antibody; (E) Transmission electron microscopy identified Ad-XT-tPA-Sopt, Ad-XT-oriSIP-S1opt, Ad-PJ-tPA-Sopt and Ad-PJ-oriSIP-S1opt;.

[0025] Figure 2 The titer of S1 protein-specific IgG antibody in the serum of mice immunized with Ad-XT-tPA-Sopt, Ad-XT-oriSIP-S1opt, Ad-PJ-tPA-Sopt and Ad-PJ-oriSIP-S1opt by intramuscular injection; (A) Experimental scheme: 6-8 week-old BALB / c female mice were immunized with Ad-Control, Ad-XT-tPA-Sopt, Ad-XT-oriSIP-S1opt, Ad-PJ-tPA-Sopt and Ad-PJ-oriSIP-S1opt by intramuscular injection. (B) The content of S1 protein-specific IgG antibody in the serum of mice immunized with Ad-XT-tPA-Sopt; (C) The content of S1 protein-specific IgG antibody in the serum of mice immunized with Ad-XT-oriSIP-S1opt; (D) The content of S1 protein-specific IgG antibody in the serum of mice immunized with Ad-PJ-tPA-Sopt; (E) The content of S1 protein-specific IgG antibody in the serum of mice immunized with Ad-PJ-oriSIP-S1opt; (F) 10 8 The levels of S1 protein-specific IgG antibodies in the serum of mice injected with Ad-XT-tPA-Sopt, Ad-XT-oriSIP-S1opt, Ad-PJ-tPA-Sopt and Ad-PJ-oriSIP-S1opt for the second time with an IFU dose; data are presented as means±sem

[0026] Figure 3 Serum neutralizing antibody levels in mice immunized with Ad-XT-tPA-Sopt, Ad-XT-oriSIP-S1opt, Ad-PJ-tPA-Sopt and Ad-PJ-oriSIP-S1opt by intramuscular injection; (A) Serum neutralizing antibody levels in mice immunized with Ad-XT-tPA-Sopt; (B) Serum neutralizing antibody levels in mice immunized with Ad-XT-oriSIP-S1opt; (C) 10 8Comparison of serum neutralizing antibody levels in mice immunized with Ad-XT-tPA-Sopt or Ad-XT-oriSIP-S1opt after a second intramuscular injection of IFU. Data are presented as means±sem; (D) Serum neutralizing antibody levels in mice immunized with Ad-PJ-tPA-Sopt. (E) Serum neutralizing antibody levels in mice immunized with Ad-PJ-oriSIP-S1opt; (F) 10 8 Comparison of serum neutralizing antibody levels in mice injected with Ad-PJ-tPA-Sopt or Ad-PJ-oriSIP-S1opt for the second time with IFU; data are expressed as means±sem;

[0027] Figure 4 For 10 8 Cellular immune response of mice injected with Ad-XT-tPA-Sopt, Ad-XT-oriSIP-S1opt, Ad-PJ-tPA-Sopt and Ad-PJ-oriSIP-S1opt twice intramuscularly at an IFU dose; (A) Splenic CD4 + and CD8 + T cell response; (B) serum cytokine levels in mice; data are expressed as means ± sem;

[0028] Figure 5 Figure 2. Humoral and cellular immune responses induced by different routes of immunization with Ad-XT-tPA-Sopt or inactivated vaccine in mice; (A) Experimental scheme: 6-8 week-old BALB / c female mice were immunized with Ad-XT-tPA-Sopt twice intramuscularly, intramuscularly first followed by oral gavage, or twice orally gavage, and then injected intramuscularly with Ad-Control or inactivated PEDV vaccine twice; (B) S1 protein-specific IgG and IgA antibody levels and neutralizing antibody levels; (C) Splenic CD4 + and CD8 + T cell and cytokine levels; data are expressed as means±sem. DETAILED DESCRIPTION

[0029] The present invention provides a recombinant adenovirus vector comprising porcine epidemic diarrhea virus immune protein, which uses a human replication-deficient adenovirus vector as a backbone vector and comprises a coding gene for an S protein or an S1 protein; the S protein or the S1 protein is from a PEDV GIIa strain CH / HBXT / 2018 or a GIIb strain CH / HNPJ / 2017.

[0030] In the present invention, the amino acid sequence of the S protein derived from the PEDV GIIa strain CH / HBXT / 2018 is preferably as shown in SEQ ID NO: 1 (MKSLTYFWLFLPVLSTLSLPQDVTRCSANTNFRR

[0031]

[0032]

[0033] TACTTCTGGCTGTTCCTGCCTGTGCTGAGCACCCTGAGTCTGCCTCAGGATGTGACCAGATGCAGCGCCAACACCAACTTCCGGCGGTTCTTCAGCAAGTTCAACGTGCAGGCTCCTGCCGTGGTGGTGCTCGGAGGATATCTGCCTATCGGCGAGAACCAGGGCGTGAACAGCACATGGTATTGCGCCGGACAGCACCCTACAGCCAGCGGAGTGCACGGCATCTTTCTGAGCCACATCAGAGGCGGCCACGGCTTCGAGATCGGCATCAGCCAAGAGCCTTTCGACCCTAGCGGCTACCAGCTGTATCTGCACAAGGCCACCAACGGCAACACCAATGCCACCGCCAGACTGCGGATCTGTCAGTTCCCCAGCATCAAGACAC

[0034]

[0035]

[0036]

[0037] In the present invention, in order to improve the expression level of the target protein, the original signal peptide before the protein is replaced with a human tissue plasminogen activator signal, and in order to improve the translation efficiency, a Kozak sequence is added before the start codon of the S and S1 sequences, and a His tag is added before the stop codon of the protein to facilitate the detection of the target protein.

[0038] In the present invention, the human replication-deficient adenovirus vector is preferably pDC316; the insertion site of the S protein or S1 protein coding gene in the backbone vector is the Cre / loxP gene site. The present invention has no particular limitation on the construction method of the human replication-deficient adenovirus vector, and the construction method of the virus vector known in the art can be used.

[0039] In the present invention, the recombinant adenovirus vector preferably includes pDC-XT-tPA-Sopt; the nucleotide sequence of the pDC-XT-tPA-Sopt is preferably SEQ ID NO:12.

[0040] The invention provides a recombinant adenovirus strain based on a human replication-deficient adenovirus vector, which is obtained by packaging the recombinant adenovirus vector.

[0041] In the present invention, the recombinant adenovirus strain preferably includes Ad-XT-tPA-Sopt. The Ad-XT-tPA-Sopt is packaged by pDC-XT-tPA-Sopt. The present invention has no particular limitation on the packaging method of the recombinant adenovirus strain, and the packaging method of adenovirus known in the art can be used.

[0042] In the present invention, experiments show that the expression level of Ad-XT-tPA-Sopt is significantly higher than that of Ad-PJ-tPA-Sopt. At the same time, the four strains Ad-XT-tPA-Sopt, Ad-XT-oriSIP-S1opt, Ad-PJ-tPA-Sopt and Ad-PJ-oriSIP-S1opt constructed by the present invention can effectively express the corresponding S or S1 protein in porcine cells.

[0043] The invention provides a recombinant porcine epidemic diarrhea virus vaccine, comprising the recombinant adenovirus strain and an adjuvant.

[0044] In the present invention, the volume ratio of the recombinant adenovirus strain to the adjuvant is preferably 1: 1. The present invention has no particular limitation on the type of the adjuvant, and any adjuvant known in the art can be used, such as Freund's incomplete adjuvant and Freund's complete adjuvant.

[0045] The present invention provides the use of the recombinant adenovirus strain in preparing a vaccine for preventing and controlling porcine epidemic diarrhea.

[0046] In the present invention, the porcine epidemic diarrhea virus strain preferably includes a GIIa type strain and / or a GIIb type strain.

[0047] In the present invention, the method of using the vaccine is preferably intramuscular injection. The method of inoculation of the vaccine is preferably a two-shot inoculation method, and the interval between the two immunizations is preferably 4 weeks. During the inoculation, the inoculation dose for each time is preferably 10 8 IFU. The experimental results show that the four vaccines are compared with 10 8 The results of IgG antibody test of mice immunized twice with IFU dose showed that the IgG antibody in the serum of mice in the Ad-XT-oriSIP-S1opt group was the highest, but there was no significant difference between them and Ad-XT-tPA-Sopt. The results of neutralizing antibody test showed that 10 8 Second intramuscular injection of Ad-XT-tPA-Sopt or Ad-PJ-tPA-Sopt at an IFU dose produced the highest level of neutralizing antibodies in mice.

[0048] In the present invention, the results of cellular immune response detection showed that the four recombinant adenoviruses could induce strong cellular immune responses, but 8 The best immune effect was achieved with an IFU dose of Ad-XT-tPA-Sopt, with a priming-boosting intramuscular injection at 4-week intervals.

[0049] The following is a detailed description of a recombinant new virus vector comprising porcine epidemic diarrhea virus immune protein, its strain, vaccine and application provided by the present invention in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0050] 1. Ethical Statement

[0051] All animal experiments were conducted in strict accordance with the Guidelines for the Management and Use of Laboratory Animals of the People's Republic of China and approved by the Animal Ethics Committee of the Chinese Society of Agronomy. 6-8-week-old SPF-grade (SPF) female BALB / c mice were purchased and raised at the Experimental Animal Center of Lanzhou Veterinary Research Institute.

[0052] 2. Description of cell lines and virus sources

[0053] HEK293 cells (human embryonic kidney cells, purchased from the Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences) and Vero cells (African green monkey cells, ATCC No: CCL-81) were cultured in DMEM medium (Gibco, USA). LLC-PK1 cells (porcine kidney cells, ATCC No.: CL-101) were cultured in MEM medium (Gibco, USA). The culture medium was supplemented with 10% fetal bovine serum (BBI, China) and the culture conditions were 37°C, 5% CO 2 PEDV GIIa strain CH / HBXT / 2018 (GenBank: MH816969.1) and GIIb strain CH / HNPJ / 2017 (GenBank: MF152604.1) were separated and passaged in Vero cells and stored at -80°C. Human Ad5 vector pDC316 (No: P1415) and adenovirus backbone plasmid (pBHGlox (delta) E1, 3Cre) were purchased from Wuhan Miaoling Biotechnology Co., Ltd. and Beijing Zhuangmeng International Biotechnology Co., Ltd., respectively.

[0054] 3. Statistical analysis of data

[0055] IBM SPSS Statistics 27.0 was used for analysis. Serum IgG and neutralizing antibody titer data were logarithmically transformed before analysis. One-way analysis of variance was used for comparisons among multiple groups, and independent sample t test was used for comparisons between two groups. P value < 0.05 indicated significance (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

[0056] Example 1

[0057] 1. Construction of replication-deficient Ad5 vector vaccines expressing PEDV S or S1 protein

[0058] The genes of the S protein (2-1322 amino acids, SEQ ID NO: 1) and the S1 protein (2-792 amino acids, SEQ ID NO: 2) of the GIIa PEDV strain CH / HBXT / 2018 were codon-optimized and synthesized to obtain codon-optimized sequences (SEQ ID NO: 3 and SEQ ID NO: 4). At the same time, the genes of the S protein (2-1326 amino acids, SEQ ID NO: 5) and the S1 protein (2-796 amino acids, SEQ ID NO: 6) of the GIIb PEDV strain CH / HNPJ / 2017 were codon-optimized and synthesized to obtain codon-optimized sequences (SEQ ID NO: 7 and SEQ ID NO: 8).

[0059] In order to improve protein expression, the original signal peptide of S protein (oriSIP, 2-18 amino acids) was replaced with human tissue plasminogen activator signal peptide (tPA, GenBank: E00896.1, position: 14-82 bp, amino acid sequence see SEQ ID NO: 9, nucleotide sequence see SEQ ID NO: 10). In addition, in order to improve translation efficiency, a Kozak sequence (gccgccacc) was added before the start codon of S and S1 sequences. A 6×His tag (CATCATCACCATCACCAT, SEQ ID NO: 11) was added before the stop codon to facilitate the detection of the target protein. The target sequence was connected to the pDC316 vector by seamless cloning and inserted into the Cre / loxP gene site.

[0060] The shuttle plasmids pDC-XT-tPA-Sopt, pDC-XT-oriSIP-S1opt, pDC-PJ-tPA-Sopt and pDC-PJ-oriSIP-S1opt were obtained and confirmed to be correct by sequencing. The nucleotide sequences are shown in SEQ ID NO: 12 to SEQ ID NO: 15. Then, under the mediation of Lipofectamine 2000 transfection reagent (Invitrogen, USA), the shuttle plasmid pDC-XT-tPA-Sopt, pDC-XT-oriSIP-S1opt, pDC-PJ-tPA-Sopt or pDC-PJ-oriSIP-S1opt and the backbone plasmid of adenovirus (pBHGloxΔE1,3Cre) were co-transfected into HEK293 cells. The shuttle plasmids pDC-XT-tPA-Sopt, pDC-XT-oriSIP-S1opt, pDC-PJ-tPA-Sopt and pDC-PJ-oriSIP-S1opt were co-transfected with the backbone plasmid pBHGlox (delta) E1,3Cre under the action of LipofectamineTM 2000 transfection reagent to package the recombinant adenovirus, and the pDC316 empty vector transfection group was added. The specific method is as follows:

[0061] (1) HEK 293 cells were cultured at 1×10 6 Cells were seeded into 6-well plates and incubated at 37°C and 5% CO 2 Culture in a cell culture incubator overnight;

[0062] (2) The next day, when the cells have grown to 80-90% confluence, 1 hour before transfection, replace the cell culture medium with 2% DMEM medium without antibiotics (preheated at 37°C), 2 mL / well;

[0063] (3) Prepare plasmid dilution solution: add 150 μL of Opti-MEM medium to a 1.5 mL EP tube, then add 1 μg of shuttle plasmid and 4 μg of pBHGlox(delta)E1,3Cre plasmid, and mix well;

[0064] (4) Dilute Lip2000 with 150 μL of Opti-MEM medium and mix well; after standing at room temperature for 5 minutes, slowly pipette the DNA solution and liposome solution to mix well, and stand at room temperature for 20 minutes;

[0065] (5) Add the plasmid and liposome mixture to a 6-well plate and shake the plate cross-well to mix well;

[0066] (6) The cells were cultured at 37°C and 5% CO 2 The cells were cultured in a cell culture incubator, and the culture medium was replaced with DMEM medium containing 2% FBS after 4 to 6 hours.

[0067] Recombinant adenoviruses Ad-XT-tPA-Sopt, Ad-XT-oriSIP-S1opt, Ad-PJ-tPA-Sopt and Ad-PJ-oriSIP-S1opt were packaged respectively. The transfected cells were collected and passaged until typical cytopathic effects (CPE) were observed. The recombinant adenoviruses were amplified and passaged in HEK293 cells, and the infectious titer (IFU) was determined using Adeno-XTM RapidTiter Kit (Clontech, USA) according to the instructions. The recombinant adenoviruses were sequenced and identified using pDC316-specific primers (pDC316F: 5'-acgtgggtataagaggcg-3', SEQ ID NO: 16; pDC316R: 5'-cgatgctagacgatccag-3', SEQ ID NO: 17).

[0068] 2. Detection of the expression of recombinant adenovirus S and S1 proteins

[0069] Western blotting was used to verify the expression of S protein by Ad-XT-tPA-Sopt and Ad-PJ-tPA-Sopt, and the expression of S1 protein by Ad-XT-oriSIP-S1opt and Ad-PJ-oriSIP-S1opt. HEK293 cells and LLC-PK1 cells were infected with Ad-XT-tPA-Sopt, Ad-PJ-tPA-Sopt, Ad-XT-oriSIP-S1opt or Ad-PJ-oriSIP-S1opt, respectively. 24 hours after infection, the cells were washed with pre-cooled PBS and lysed with RIPA lysis buffer. The collected supernatants were subjected to 8% SDS-PAGE gel electrophoresis under non-reducing conditions. After transferring the proteins on the SDS-PAGE gel to a nitrocellulose membrane, the nitrocellulose membrane was blocked with 5% skim milk powder for 2 hours at room temperature. The membrane was incubated with a mouse anti-6×His monoclonal antibody diluted 1:5000 or a mouse anti-PEDV S2 monoclonal antibody diluted 1:5000 at 4°C overnight. The next day, the membrane was incubated with a 1:10000 dilution of horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody at room temperature for 1 hour. Chemiluminescence reaction was performed using BeyoECLPlus (Biyuntian Biotechnology), and images were acquired using an Amersham Imager 600 multifunctional imaging system. β-actin was detected on the same membrane using β-actin monoclonal antibody diluted 1:5000, using β-actin as an internal control.

[0070] 3. Indirect immunofluorescence (IFA) was used to detect the expression of S protein of Ad-XT-tPA-Sopt and Ad-PJ-tPA-Sopt, and the expression of S1 protein of Ad-XT-oriSIP-S1opt and Ad-PJ-oriSIP-S1opt. LLC-PK1 cells were infected with Ad-XT-tPA-Sopt or Ad-PJ-tPA-Sopt. After 48 hours, the culture medium was aspirated and 1 mL of pre-cooled 100% methanol was added to each well for fixation at -20°C for 10 min. The cells were washed three times with PBS containing 1% BSA and incubated with mouse anti-PEDV S2 monoclonal antibody diluted 1:500 at room temperature for 2 hours. The cells were washed three times with PBS containing 1% BSA, and incubated with FITC-conjugated goat anti-mouse IgG monoclonal antibody and DAPI diluted 1:200 at room temperature for 2 hours in the dark. After washing the cells three times with PBS, they were observed and photographed under a fluorescence microscope.

[0071] 4. Electron Microscopic Observation of Recombinant Virus Particles

[0072] Transmission electron microscopy was used to image viral particles of Ad-XT-tPA-Sopt, Ad-XT-oriSIP-S1opt, Ad-PJ-tPA-Sopt, and Ad-PJ-oriSIP-S1opt. Samples were prepared according to existing methods.

[25] Ad-XT-tPA-Sopt, Ad-XT-oriSIP-S1opt, Ad-PJ-tPA-Sopt and Ad-PJ-oriSIP-S1opt were inoculated into HEK293 cells, respectively. When CPE exceeded 90%, the cells were collected and repeatedly frozen and thawed three times. The cells were centrifuged at 10,000 rpm for 30 minutes, and the supernatant was taken and filtered through a 0.22 μm filter. Then, the virus particles were negatively stained with 1% phosphotungstic acid for 3 minutes. Images were obtained under a transmission electron microscope.

[0073] Construction and identification of recombinant adenoviruses Ad-XT-tPA-Sopt, Ad-XT-oriSIP-S1opt, Ad-PJ-tPA-Sopt and Ad-PJ-oriSIP-S1opt

[0074] In order to prepare a new and effective PEDV vaccine, Ad5 vector vaccines Ad-XT-tPA-Sopt and Ad-XT-oriSIP-S1opt expressing GIIa PEDV strain CH / HBXT / 2018S or S1 protein, and Ad5 vector vaccines Ad-PJ-tPA-Sopt and Ad-PJ-oriSIP-S1opt expressing GIIb PEDV strain CH / HNPJ / 2017S or S1 protein were constructed. In order to increase the amount of antigen expression, the S and S1 genes were codon optimized, and the tPA signal peptide was introduced into the S gene to replace the original signal peptide ( Figure 1 (A).

[0075] All four recombinant adenoviruses were successfully rescued and propagated in HEK293 cells. The S gene fragment was detected from Ad-XT-tPA-Sopt and Ad-PJ-tPA-Sopt, and the S1 gene fragment was detected from Ad-XT-oriSIP-S1opt and Ad-PJ-oriSIP-S1opt. The S and S1 gene fragments were sequenced correctly ( Figure 1 (middle B).

[0076] The expression of S protein can be detected in HEK293 and LLC-PK1 cells infected with Ad-XT-tPA-Sopt or Ad-PJ-tPA-Sopt using mouse anti-6×His monoclonal antibody and mouse anti-PEDV S2 monoclonal antibody, and the expression level of Ad-XT-tPA-Sopt is significantly higher than that of Ad-PJ-tPA-Sopt. The expression of S1 protein can be detected in HEK293 and LLC-PK1 cells infected with Ad-XT-oriSIP-S1opt or Ad-PJ-oriSIP-S1opt using mouse anti-6×His monoclonal antibody, and the expression levels of the two are similar, but the molecular weight of S1 protein expressed by Ad-XT-oriSIP-S1opt is slightly lower than that of S1 protein expressed by Ad-PJ-oriSIP-S1opt, which may be related to the different degree of glycosylation modification of the protein ( Figure 1 Middle C).

[0077] IFA results also showed that Ad-XT-tPA-Sopt and Ad-PJ-tPA-Sopt could effectively express S protein in LLC-PK1 cells ( Figure 1 These results all indicate that Ad-XT-tPA-Sopt, Ad-XT-oriSIP-S1opt, Ad-PJ-tPA-Sopt, and Ad-PJ-oriSIP-S1opt can effectively express S and S1 proteins in porcine cells. In addition, complete viral particles of the four recombinant adenoviruses were observed under transmission electron microscopy ( Figure 1 Middle E).

[0078] Example 2

[0079] 1. Animal immunization

[0080] To determine the optimal immunization antigen, dose and time, 6-8 week old SPF BALB / c mice were randomly divided into 25 groups (5 mice in each group). Mice in groups 1-24 were injected intramuscularly with 10 8 , 10 7 or 10 6 Half of the mice were boosted once at week 4. A negative control group was also set up, with each mouse injected intramuscularly with 10 7 Ad-Control of IFU.

[0081] To determine the content of specific antibodies and cytokines, serum from all mice was collected every two weeks until the end of the experiment at week 8. 8The mice in the IFU-dosed and Ad-Control groups were euthanized at week 8, and the spleen cell immune response was detected.

[0082] In addition, to confirm the mucosal immune effect of Ad-XT-tPA-Sopt, female BALB / c mice aged 6 to 8 weeks were randomly divided into 5 groups (5 mice in each group). Groups 1 to 3 were immunized with different immunization routes for 10 8 IFU of Ad-XT-tPA-Sopt. Group 1 mice were immunized twice by intramuscular injection at week 0 and week 4, respectively; Group 2 mice were given an intramuscular injection of the first immunization at week 0 and oral gavage booster immunization at week 4; Group 3 mice were immunized twice by OG at week 0 and week 4, respectively; Groups 4 and 5 were injected intramuscularly with chemically inactivated PEDV (the virus titer before inactivation was 106.0TCID 50 / mL) and Ad5-Control (10 8 In order to determine the content of specific antibodies and cytokines, serum and intestinal fluid samples of mice in each group were collected every two weeks until the end of the experiment at week 8. At week 8, the mice were humanely euthanized to detect the immune response of spleen cells.

[0083] ELISA test

[0084] The indirect ELISA method based on PEDV S1 protein was used to detect the IgG and IgA antibody levels in mice immunized with recombinant adenovirus. 0.5μg / mL or 0.1μg / mL purified PEDV S1 protein was coated on the ELISA plate overnight at 4℃. After the plate was blocked with 5% skim milk at 37℃ for 2 hours, diluted serum or intestinal fluid was added and reacted at 37℃ for 40 minutes. 1:10000 dilution of biological HRP-conjugated goat anti-mouse IgG antibody or 1:20,000 dilution of HRP-conjugated goat anti-mouse IgA antibody was added and reacted at 37℃ for 45 minutes. Then TMB substrate was added and reacted at 37℃ in the dark for 15 minutes. 100μL sulfuric acid stop solution was added, and the absorbance was measured at 450nm. The serum IgG titer was defined as the highest serum dilution factor that was 2.1 times higher than the negative control. The intestinal fluid IgA titer was defined as the absorbance value at 450nm.

[0085] According to the instructions, a commercial double-antibody one-step sandwich ELISA kit (Xinbosheng) was used to detect the levels of serum cytokines (IL-2, IL-4, IL-13, IFN-γ and TNF-α).

[0086] Flow cytometry

[0087] Mouse spleen cell suspension was prepared. Mouse spleen cells were incubated with FITC-conjugated mouse anti-CD3 antibody (BioLegend, Cat.: 100204), PE-conjugated mouse anti-CD4 antibody (BioLegend, Cat.: 100408) and APC-conjugated mouse anti-CD8a antibody (BioLegend, Cat.: 100713) at 4°C for 30 minutes. After washing the cells twice with PBS, the spleen cells were analyzed using Beckman CytoFLEX LX 5L19C, and the results were processed using CytExpert flow analysis software.

[0088] PEDV neutralization test

[0089] Evaluation of neutralizing antibody titers in mouse sera using a microneutralization assay

[26] The serum was inactivated at 56°C for 30 minutes. The serially diluted inactivated serum was mixed with 200 TCID 50 PEDV strains CH / HBXT / 2018 or CH / HNPJ / 2017 were incubated at 37°C for 1 hour. Vero monolayer cells in 96-well plates were washed three times with PBS. The antibody-virus complex was added to the Vero monolayer cells in 96-well plates and incubated at 37°C, 5% CO 2 Incubate for 1 hour under the same conditions. After incubation, add 100 μL of MEM maintenance solution (containing 20 μg / mL trypsin) to each well, and record the results after 5 to 7 days. Use the Reed-Muench method to calculate the neutralization results, that is, the maximum serum dilution that can protect 50% of tissue culture cells from CPE.

[0090] Test results

[0091] In mice 10 8 IFU dose and second intramuscular injection of Ad-XT-tPA-Sopt induced the strongest humoral and cellular immune responses

[0092] First, to determine the optimal immunization antigen, dose, and time, female BALB / c mice aged 6 to 8 weeks were injected intramuscularly with one or two doses of 10 8 , 10 7 or 10 6 IFU of Ad-XT-tPA-Sopt, Ad-XT-oriSIP-S1opt, Ad-PJ-tPA-Sopt, or Ad-PJ-oriSIP-S1opt, or two doses of 10 7 IFU Ad-Control. Detection of anti-PEDV HNPJ strain S1 specific IgG antibody levels, anti-HBXT strain or HNPJ strain specific NAb antibody levels and cellular immune response ( Figure 2 (A).

[0093] 1. In mice 10 8 Second intramuscular injection of Ad-XT-oriSIP-S1opt or Ad-XT-tPA-Sopt at an IFU dose induced the strongest anti-PEDV CH / HNPJ / 2017 strain S1-specific IgG response

[0094] IgG antibodies were detected by indirect ELISA. Mice vaccinated with Ad-XT-tPA-Sopt produced IgG antibodies in a dose-dependent manner. After two immunizations, 10 8 The IgG antibody in the IFU group was the highest, while the 10 7 IFU and 10 6 There was no significant difference in IgG antibody levels between the IFU groups ( Figure 2 (B). Mice vaccinated with Ad-XT-oriSIP-S1opt produced IgG antibodies in a dose- and dosage-dependent manner ( Figure 2 C). Mice vaccinated with Ad-PJ-tPA-Sopt produced IgG antibodies in a dose-dependent manner, and after two immunizations, there was no significant difference in IgG antibodies among the three dose groups ( Figure 2 For Ad-PJ-oriSIP-S1opt, 10 8 The IgG antibody level of mice immunized with two intramuscular injections in the IFU group was higher than that of mice immunized with one injection. 7 IFU and 10 6 There was no significant difference in IgG titer between mice immunized twice and once with IFU. 8 The IgG antibody in the IFU group was higher than 10 7 IFU group and 10 6 IFU group IgG antibody ( Figure 2 (E) Comparison of four vaccines at 10 8 The results of mouse IgG antibody immunization twice with IFU dose showed that the IgG antibody in the serum of mice in the Ad-XT-oriSIP-S1opt group was the highest, with no significant difference between Ad-XT-tPA-Sopt and Ad-PJ-tPA-Sopt. The IgG antibody content in the serum of mice in the Ad-PJ-tPA-Sopt group was the lowest, lower than that in the serum of mice in the Ad-XT-tPA-Sopt group, but the difference between the two was not statistically significant, which was consistent with the results of S protein expression in WB ( Figure 2 Middle F).

[0095] 2.10 8 Second intramuscular injection of Ad-XT-tPA-Sopt or Ad-PJ-tPA-Sopt at an IFU dose produced the highest level of neutralizing antibodies in mice

[0096] The GIIa PEDV strain CH / HBXT / 2018-specific microneutralization assay was used to detect NAbs in mice immunized with Ad-XT-tPA-Sopt and Ad-XT-oriSIP-S1opt. NAbs were produced in mice immunized with Ad-XT-tPA-Sopt in a dose-dependent manner. After two immunizations, 10 8 The IFU group had the highest NAbs, while the 10 7 IFU and 10 6 There was no significant difference in NAbs levels between the IFU groups ( Figure 3 (A).

[0097] For Ad-XT-oriSIP-S1opt, 10 8 The NAbs level of mice immunized with two intramuscular injections in the IFU group was higher than that of mice immunized with one injection. 7 IFU and 10 6 There was no significant difference in the NAbs titer between mice immunized twice and once with IFU. 8 NAbs in the IFU group were higher than 10 7 IFU group and 10 6 NAbs in the IFU group ( Figure 3 (B) Comparison of two recombinant adenoviruses at 10 8 The serum NAbs content of mice immunized twice with IFU dose was found to be higher in the Ad-XT-tPA-Sopt group than in the Ad-XT-oriSIP-S1opt group ( Figure 3 Middle C).

[0098] The GIIb PEDV strain CH / HNPJ / 2017-specific microneutralization assay was also used to detect NAbs in mice immunized with Ad-PJ-tPA-Sopt and Ad-PJ-oriSIP-S1opt. NAbs were produced in mice immunized with Ad-PJ-tPA-Sopt in a dose-dependent manner. After two immunizations, 10 6 The IFU group had the lowest NAbs, while the 10 8 IFU and 10 7 There was no significant difference in NAbs levels between the IFU groups ( Figure 3 For Ad-PJ-oriSIP-S1opt, 10 8 IFU and 10 7 The NAbs level of mice immunized with two intramuscular injections in the IFU group was higher than that of mice immunized with one injection. 6NAbs in the IFU group were very low, and there was no significant difference in the titer of NAbs between mice immunized twice and once. NAbs were produced in mice immunized twice with Ad-PJ-oriSIP-S1opt in a dose-dependent manner ( Figure 3 (E) Comparison of two recombinant adenoviruses at 10 8 The serum NAbs content of mice immunized twice with IFU dose was found to be higher in the Ad-PJ-tPA-Sopt group than in the Ad-PJ-oriSIP-S1opt group ( Figure 3 These results suggest that two intramuscular injections of 10 8 The NAbs titers of Ad-XT-tPA-Sopt or Ad-PJ-tPA-Sopt in IFU were significantly higher than those in other vaccine groups.

[0099] 3. All four recombinant adenoviruses can induce strong cellular immune responses

[0100] To evaluate the efficacy of Ad-XT-tPA-Sopt, Ad-XT-oriSIP-S1opt, Ad-PJ-tPA-Sopt and Ad-PJ-oriSIP-S1opt in 10 8 The cellular immune response induced by two intramuscular injections of IFU was measured by measuring the CD4 + and CD8 + The percentage of T cells and serum cytokine content in the four vaccine groups were higher than those in the Ad-Control group. + T cells, serum IL-2 and TNF-α, so all four vaccine groups produced strong cellular immune responses. All groups of mice produced similar levels of IFN-γ. In addition, the CD4 + and CD8 + There was no significant difference in the contents of T cells, IL-2, TNF-α, and IFN-γ, which indicates that although the two recombinant adenoviruses have different IgG antibody contents due to differences in antigen expression, the cellular immune responses induced by the two are similar ( Figure 4 ). Based on all humoral and cellular immunity results, it is believed that 10 8 The best immune effect was achieved with an IFU dose of Ad-XT-tPA-Sopt, with a priming-boosting intramuscular injection 4 weeks apart, and the mucosal immune response was further evaluated.

[0101] Example 4

[0102] Oral gavage immunization with Ad-XT-tPA-Sopt failed to induce strong systemic and mucosal immune responses in mice

[0103] To determine the mucosal immune effect of Ad-XT-tPA-Sopt, 10 8 Female BALB / c mice aged 6 to 8 weeks were immunized twice with IFU / mouse. Group 1 was injected with IM, group 2 was injected with OG after IM injection, and group 3 was injected with OG. 8 IFU / Ad-Control (Group 5) was the control group. Serum and small intestinal fluid were collected at weeks 2, 4, 6, and 8 to evaluate anti-S1-specific IgG and IgA antibody responses and anti-CH / HBXT / 2018-specific NAb titers.

[0104] Cellular immune responses were also measured at week 8 ( Figure 5 (A).

[0105] The highest levels of anti-S1-specific IgG antibodies were produced in the sera of mice that were injected intramuscularly twice or with the inactivated vaccine, and there were significant differences in the IgG antibody content between the two groups. The IgG antibody content in the sera of mice in the OG group was the lowest, and the IgG antibody content in the sera of mice in the intramuscular injection and OG immunization groups was intermediate. None of the vaccines stimulated the production of mucosal antibodies, as there was no significant increase in the level of IgA antibodies in the intestinal fluid of mice in the IM injection of OG or OG immunization groups compared with IM injection and PEDV inactivated vaccine. The NAb titers of mice immunized with two intramuscular injections and inactivated vaccines were the highest, and there was no significant difference between the two groups, while the NAb levels induced by Ad-XT-tPA-Sopt after two OG immunizations were significantly lower than those of other vaccines ( Figure 5 Middle B). Due to the spleen CD8 + The percentage of T cells increased, so Ad-XT-tPA-Sopt IM injection group or IM injection of OG and inactivated vaccine group mice induced a strong cellular immune response, and the difference was not statistically significant. In addition, the mice in each vaccine group also produced different levels of serum IL-2, IL-4, IL-13, IFN-γ and TNF-α ( Figure 5 In summary, these data suggest that mice vaccinated with Ad-XT-tPA-Sopt in the OG do not develop robust systemic and mucosal immunity and that IM injection is the optimal route of Ad-XT-tPA-Sopt immunization.

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[0132] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A recombinant adenovirus vector comprising porcine epidemic diarrhea virus immune protein, It is characterized in that A human replication-deficient adenovirus vector is used as a backbone vector, and contains a gene encoding the S protein or the S1 protein; The S protein or S1 protein is from PEDV GIIa strain CH / HBXT / 2018 or GIIb strain CH / HNPJ / 2017; The amino acid sequence of the S protein from the PEDV GIIa strain CH / HBXT / 2018 is shown in SEQ ID NO: 1; The amino acid sequence of the S1 protein from the PEDV GIIa strain CH / HBXT / 2018 is shown in SEQ ID NO: 2; The amino acid sequence of the S protein from the PEDV GIIb strain CH / HNPJ / 2017 is shown in SEQ ID NO: 5; The amino acid sequence of the S1 protein from the PEDV GIIb strain CH / HNPJ / 2017 is shown in SEQ ID NO:6; The human replication-defective adenovirus vector is pDC316; The recombinant adenovirus vector includes pDC-XT-tPA-Sopt; The nucleotide sequence of pDC-XT-tPA-Sopt is as shown in SEQ ID NO:

12.

2. The recombinant adenovirus vector according to claim 1, It is characterized in that The nucleotide sequence of the gene encoding the S protein from the PEDV GIIa strain CH / HBXT / 2018 is shown in SEQ ID NO: 3; The nucleotide sequence of the gene encoding the S1 protein from the PEDV GIIa strain CH / HBXT / 2018 is shown in SEQ ID NO:

4.

3. The recombinant adenovirus vector according to claim 1, It is characterized in that The nucleotide sequence of the gene encoding the S protein from the PEDV GIIb strain CH / HNPJ / 2017 is shown in SEQ ID NO: 7; The nucleotide sequence of the S1 protein derived from the PEDV GIIb strain CH / HNPJ / 2017 is shown in SEQ ID NO:

8.

4. A recombinant adenovirus strain containing a human replication-defective adenovirus vector, It is characterized in that The recombinant adenovirus vector is packaged according to any one of claims 1 to 3.

5. A recombinant porcine epidemic diarrhea virus vaccine, It is characterized in that It comprises the recombinant adenovirus strain as claimed in claim 4 and an adjuvant.

6. The recombinant porcine epidemic diarrhea virus vaccine according to claim 5, It is characterized in that The volume ratio of the recombinant adenovirus strain and the adjuvant in claim 5 is 1:

1.

7. Use of the recombinant adenovirus strain according to claim 4 in the preparation of a vaccine for preventing and controlling porcine epidemic diarrhea.

8. The use according to claim 7, It is characterized in that The porcine epidemic diarrhea virus strains include GIIa type strains and / or GIIb type strains.

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