Adenoviral vector recombinant porcine epidemic diarrhea syndrome coronavirus vaccine

Abstract

The application belongs to the technical field of veterinary medicine, and particularly relates to an adenovirus vector recombinant pig acute diarrhea syndrome coronavirus vaccine. The application constructs a replication-defective adenovirus vector vaccine (rAd-SADS-S) expressing a full-length SADS-CoV S protein, realizes immune induction of high-efficiency humoral and cellular immunity, and provides passive protection for milk mice through maternal antibody transmission, and overcomes the defects of no available vaccine for SADS-CoV, low protection rate in existing research, and biological safety risk.

Description

Technical Field

[0001] This invention belongs to the field of veterinary technology, specifically relating to an adenovirus vector recombinant porcine acute diarrhea syndrome coronavirus vaccine. Background Technology

[0002] Swine Acute Diarrhea Syndrome Coronavirus (SADS-CoV) belongs to the genus Alphacoronavirus and is a newly emerging, highly pathogenic enteric pathogen that mainly causes acute vomiting and diarrhea in newborn piglets and a mortality rate as high as 80%-100%. There is currently no licensed SADS-CoV vaccine.

[0003] The limitations of the SADS-CoV vaccine technology currently under research are reflected in the following aspects:

[0004] Live vaccine: The SADS-CoV CN / GDWT / 2017 strain was passaged 83 times. Although its pathogenicity decreased, it was still pathogenic to piglets (Sun Y, Cheng J, Luo Y, Yan XL, Wu ZX, He LL, Tan YR, Zhou ZH, Li QN, Zhou L, Wu RT, Lan T, Ma JY. Attenuation of a virulent swine acute diarrhea syndrome coronavirus strain via cell culture passage. Virology. 2019 Dec; 538:61-70. doi:10.1016 / j.virol.2019.09.009.Epub 2019Sep 21.PMID:31580972; PMCID:PMC7112038.). Immunizing female mice with live SADS-CoV virus can also induce an immune response against SADS-CoV in mice. The suckling mice produced by immunized mice can develop resistance to SADS-CoV, but the protective effect is not 100%, and there are biosafety risks associated with using live virus for immunization (Chen Y, Jiang RD, Wang Q, Luo Y, Liu MQ, Zhu Y, Liu X, He YT, Zhou P, Yang XL, Shi ZL. Lethal Swine Acute Diarrhea Syndrome Coronavirus Infection in Suckling Mice. J Virol. 2022 Sep 14;96(17):e0006522. doi:10.1128 / jvi.00065-22. Epub 2022 Aug 22. PMID:35993737;PMCID:PMC9472626.).

[0005] Inactivated vaccines: It is generally believed that inactivated vaccines cannot effectively induce cellular immune responses, and also have problems such as unstable viral titers and high production costs.

[0006] Recombinant viral vaccines: such as the recombinant SADS-CoV described in Chinese invention patent application publication number CN118360258A, enhance mucosal immunity by replacing the ORF3a gene with the COE+S1D epitope of PEDV. Although such vaccines can induce local immunity, they are based on live virus modification, which poses biosafety risks (such as virulence reversion), and the production process is complex. A recombinant virus constructed by expressing the SADS-CoV S protein using vesicular stomatitis virus (VSV) as a backbone was used to immunize mice and produce neutralizing antibodies against SADS-CoV. However, no challenge evaluation test was performed (Zhu Z, Han Y, Gong M, Sun B, Zhang R, Ding Q. Establishment of replication-competent vesicular stomatitis virus recapitulating SADS-CoV entry. J Virol. 2024 May 14;98(5):e0195723.doi:10.1128 / jvi.01957-23.Epub 2024Apr 1.PMID:38557247;PMCID:PMC11092325.), and the VSV vector used was a reproducible vector.

[0007] Adenovirus vectors, due to their high safety, strong immunogenicity, and ability to induce dual immunity (humoral and cellular immunity), have been widely used in coronavirus vaccine development, specifically as follows:

[0008] For example, Chinese invention patent application CN116676340A utilizes human adenovirus type 5 (Ad5) to express the S or S1 protein of PEDV, successfully inducing an immune response in mice and pigs, with a single immunization producing highly effective protection. CanSino Biologics' Ad5-nCoV COVID-19 vaccine further validates the advantages of this vector in single-dose immunization, simultaneously stimulating high-titer neutralizing antibodies and T-cell immunity. The Ad5 vector is already widely used in human medicine, and there are also application cases in veterinary medicine, such as Chinese invention patent application CN101966341A.

[0009] The spike protein (S protein) of SADS-CoV is a key structural protein mediating viral invasion of host cells and a major target of neutralizing antibodies. Generally, coronavirus S proteins are difficult to express in vitro. Furthermore, although adenovirus vectors have proven effective in treating coronaviruses such as PEDV, no adenovirus vaccine research has been reported for SADS-CoV. Additionally, existing coronavirus vaccines mostly focus on a single antigen (such as the S1 subunit), making them susceptible to immune evasion due to viral mutations. Summary of the Invention

[0010] This invention constructs a replication-deficient adenovirus vector vaccine (rAd-SADS-S) expressing the full-length SADS-CoV S protein, achieving highly efficient humoral and cellular immunity through immune induction, and providing passive protection to suckling mice through maternal antibody delivery. This overcomes the shortcomings of the lack of available SADS-CoV vaccines and the low protection rate and biosafety risks in existing studies.

[0011] To achieve the above objectives, the present invention adopts the following technical solution:

[0012] In a first aspect, the present invention provides a recombinant viral plasmid containing a nucleic acid molecule expressing the S protein of porcine acute diarrhea syndrome coronavirus (PASCoV), the amino acid sequence of which is shown in SEQ ID NO:1. Furthermore, the sequence of the nucleic acid molecule expressing the S protein of PASCoV is shown in SEQ ID NO:2. The recombinant viral plasmid is a recombinant adenovirus plasmid, and the adenovirus is a replication-defective adenovirus, more specifically, a human adenovirus type 5 lacking the E1 and E3 regions.

[0013] Furthermore, the method for preparing the recombinant viral plasmid includes:

[0014] Step 1: The SADS-CoV S gene expression cassette is cloned into the shuttle plasmid to obtain the recombinant shuttle plasmid; furthermore, the SADS-CoV S gene expression cassette also contains a flag tag, and the base sequence of the S gene expression cassette of porcine acute diarrhea syndrome coronavirus is shown in SEQ ID NO:3.

[0015] Step 2: The recombinant shuttle plasmid and adenovirus plasmid were co-transformed into competent E. coli cells for homologous recombination, and the recombinant adenovirus plasmid was obtained by screening.

[0016] In a second aspect, the present invention provides a recombinant virus, which is obtained by packaging the recombinant virus plasmid. Specifically, the packaging involves linearizing the recombinant virus plasmid and then transfecting it into HEK-293A cells to rescue the recombinant virus.

[0017] In a third aspect, the present invention provides an adenovirus vector recombinant porcine acute diarrhea syndrome coronavirus vaccine, wherein the vaccine contains a recombinant adenovirus, the recombinant adenovirus being obtained by packaging a recombinant adenovirus plasmid containing a nucleic acid molecule expressing the S protein of porcine acute diarrhea syndrome coronavirus.

[0018] The method for preparing the recombinant adenovirus plasmid includes:

[0019] Step 1: The S gene expression cassette of porcine acute diarrhea syndrome coronavirus was cloned into the shuttle plasmid to obtain the recombinant shuttle plasmid;

[0020] Step 2: The recombinant shuttle plasmid and adenovirus plasmid were co-transformed into competent E. coli cells for homologous recombination, and the recombinant adenovirus plasmid was obtained by screening.

[0021] In a fourth aspect, this invention provides the use of the aforementioned recombinant viral plasmid, recombinant virus, or vaccine in the preparation of a medicament for the prevention and / or treatment of porcine acute diarrhea syndrome coronavirus (SADS-CoV). This invention has discovered that passive immunization of female mice with a recombinant adenovirus vaccine can confer complete protection against lethal SADS-CoV infection in newborn suckling mice.

[0022] The beneficial effects of this invention are as follows:

[0023] (1) Antigen selection and expression optimization: This invention uses the full-length sequence of the S protein of SADS-CoV (which includes the S1 and S2 subunits) as the target antigen, thus providing more comprehensive coverage of antigenic epitopes. In addition, this invention also optimizes the codons of the SADS-CoV S protein to achieve high expression.

[0024] (2) Innovation in immune strategy: The embodiments of this invention demonstrate that maternal immunity can induce maternal antibodies to be transferred through breast milk to protect suckling mice.

[0025] In summary, the rAd5-SADS-S constructed in this invention is characterized by safety, high efficiency, and replication defects, and the immunized mother can provide complete protection against lethal SADS-CoV challenge to newborn individuals. Attached Figure Description

[0026] Figure 1 Construction of a recombinant porcine acute diarrhea syndrome coronavirus vaccine rAd5-SADS-S using an adenovirus vector.

[0027] A. Schematic diagram of recombinant virus genome; B. Pathogenic effects produced by recombinant virus in HEK 293A cells; C. PCR identification of recombinant virus; D. WB identification of recombinant virus.

[0028] Figure 2 Immunogenicity evaluation of the adenovirus vector recombinant porcine acute diarrhea syndrome coronavirus vaccine rAd5-SADS-S in mice.

[0029] A. Animal experimental procedure for evaluating the immunogenicity of rAd5-SADS-S; B. SADS-CoV IgG antibodies in serum; C. SADS-CoV neutralizing antibodies in serum; D. Splenic lymphocyte proliferation assay; E. ELISPOT assay for splenic lymphocytes.

[0030] Figure 3 Evaluation of the protective efficacy of the adenovirus vector recombinant porcine acute diarrhea syndrome coronavirus vaccine rAd5-SADS-S in suckling mice.

[0031] A. Procedural data of suckling mice; B. SADS-CoV IgG antibodies in the stomach of suckling mice; C. SADS-CoV IgG antibodies in the intestines of suckling mice; D. Survival curve of suckling mice; E. Weight change curve of suckling mice; F. SADS-CoV viral load in suckling mouse tissues; G. Pathological changes in suckling mouse tissues; H. Immunofluorescence of suckling mouse tissues, green indicating SADS-CoV N protein antigen; I. Appearance of suckling mice. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of the embodiments of this invention will be clearly described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0033] Adenovirus plasmid pAdEasy-1 and shuttle plasmid pShuttle were purchased from Zhuangmeng Biotechnology. pcDNA3.1-SADS-S-flag was obtained by cloning the SADS-S-flag gene (as shown in SEQ ID NO:3) with a flag tag fused to its C-terminus into pcDNA3.1. The SADS-S sequence was codon-optimized based on the S gene sequence of the SADS-CoV GDS04 strain. The empty plasmid pcDNA3.1 was a commercially available product and was stored in the inventor's laboratory. BJ5183 E. coli competent cells were purchased from Shanghai Weidi Biotechnology Co., Ltd. HEK-293A cells were a commercially available product and were stored in the inventor's laboratory.

[0034] The SADS-CoV GDS04 strain was donated by Professor Yongchang Cao of Sun Yat-sen University (Gong L, Li J, Zhou Q, Xu Z, Chen L, Zhang Y, Xue C, Wen Z, Cao YA New Bat-HKU2-like Coronavirus in Swine, China, 2017. Emerg Infect Dis. 2017 Sep; 23(9):1607-9. doi:10.3201 / eid2309.170915. Epub2017 Sep 17. PMID:28654418; PMCID:PMC5572857.).

[0035] Example 1: Construction and identification of recombinant adenovirus rAd5-SADS-S

[0036] 1.1 Construction of recombinant adenovirus plasmid

[0037] Using pShuttle as a template and Shuttle F and Shuttle R (see Table 1) as primers, a fragment of approximately 6600 bp was amplified and used as a vector. Using pcDNA3.1 and pcDNA3.1-SADS-S-flag as templates and CMV F and bGH R as primers, respectively, the empty CMV promoter expression cassette and the SADS-S-flag expression cassette were amplified, with amplification product lengths of 1018 bp and 4367 bp, respectively. The amplified pShuttle was ligated to the empty CMV promoter expression cassette and the SADS-S-flag expression cassette using the ClonExpress II One Step Cloning Kit (Novizan product) to obtain pShuttle-CMV-EV and pShuttle-SADS-S plasmids, respectively. pShuttle-CMV-EV and pShuttle-SADS-S were linearized with the restriction endonuclease PmeI (NEB product) and then co-transformed with pAdEasy-1 into BJ5183 E. coli competent cells for homologous recombination. Recombinant adenovirus plasmids pAd-CMV-EV and pAd-SADS-S were screened for kanamycin resistance.

[0038] 1.2 Recombinant adenovirus rescue

[0039] pAd-CMV-EV and pAd-SADS-S were linearized using the restriction endonuclease PacI (NEB product), and the DNA was recovered using a DNA extraction reagent (phenol:chloroform:isoamyl alcohol (25:24:1)). The linearized DNA was transfected into monolayer HEK-293A cells, and obvious cytopathic effects (CPE) appeared after 7 days. The cell supernatant was collected and seeded into newly passaged HEK-293A cells for expansion culture to obtain recombinant adenovirus rAd5-SADS-S and control virus rAd5-CMV-EV.

[0040] 1.3 Identification of Recombinant Adenovirus

[0041] The constructed adenovirus was identified by CPE observation, PCR experiment, and WB experiment.

[0042] Figure 1 A is a schematic diagram of the viral genomes of recombinant adenovirus rAd5-SADS-S and empty vector control virus rAd5-CMV-EV. The viral backbone used is human adenovirus type 5 (commercial backbone that can replicate in cells expressing E1 and E3 but not in normal cells) that lacks the E1 and E3 regions.

[0043] When F5 generation recombinant viruses rAd5-CMV-EV and rAd5-SADS-S were inoculated into HEK-293A cells, significant CPE was observed in both rAd5-CMV-EV and rAd5-SADS-S cells inoculated for 48 hpi. Figure 1 B).

[0044] DNA was extracted from F5 recombinant viruses rAd5-CMV-EV and rAd5-SADS-S as templates, and PCR amplification was performed using CMV F and bGH R as primers. The expected fragment size was obtained in both the rAd5-CMV-EV and rAd5-SADS-S groups. Figure 1 C).

[0045] F5 generation recombinant viruses rAd5-CMV-EV and rAd5-SADS-S were inoculated into HEK-293A cells. Cells were collected at 36 hpi and Western blot (WB) experiments were performed using anti-Flag antibody (Proteintech product) and anti-β-actin antibody (Servicebio product) as primary antibodies. β-actin was detected in cells that were not inoculated with rAd5-CMV-EV or rAd5-SADS-S virus, while only the rAd5-SADS-S group showed a SADS-CoV S-Flag band. Figure 1 D).

[0046] Table 1. Primers used in the examples

[0047]

[0048] Example 2: Immunogenicity of recombinant adenovirus rAd5-SADS-S

[0049] 2.1 Animal Experiment Design

[0050] Five-week-old SPF BALB / c mice were provided by the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (Lanzhou Branch of the China Animal Health and Epidemiology Center). Animal experiments were conducted in the animal facility of the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (Lanzhou Branch of the China Animal Health and Epidemiology Center). Female BALB / c mice were divided into three groups of eight mice each: a blank control group (DMEM), a vector control group (rAd5-CMV-EV), and a vaccine group (rAd5-SADS-S). Figure 2 Procedure A involves immunizing the vector control group and the vaccine group with 100 μL of 10 [units of something] via intramuscular injection. 6 TCID 50The mice were immunized three times with rAd5-CMV-EV and rAd5-SADS-S, with each immunization occurring two weeks apart. The blank control group received 100 μL of DMEM culture medium each time. One week after the second immunization, two mice each of the rAd5-CMV-EV and rAd5-SADS-S immunizations were housed together with male mice (for the third immunization according to the prescribed procedure); the newborn mice were used for SADS-CoV challenge evaluation. Two weeks after the third immunization, blood was collected from each group of mice to separate serum for the detection of SADS-CoV-specific antibodies and neutralizing antibodies; three mice from each group were dissected, and lymphocytes were isolated from the spleen for lymphocyte proliferation assays and Elispot assays.

[0051] 2.2 SADS-CoV-specific antibody detection

[0052] The purified SADS-CoV GDS04 strain was diluted to 2 μg / mL, and 50 μL was added to each well of an ELISA plate and incubated overnight at 4°C. The plates were then washed four times with PBST, and enzyme stabilizer (Baiditai product) was added, followed by incubation at 37°C for 1 h. After washing four times with PBST, 50 μL of serum sample (diluted 1:50 with PBS) was added to each well, and the plates were incubated at 37°C for 30 min. After washing four times with PBST, 50 μL of HRP-conjugated goat anti-mouse IgG (Abcam product) (diluted 1:20000 with PBS) was added to each well, and the plates were incubated at 37°C for 30 min. After washing four times with PBST, 50 μL of TMB (Surmodics product) was added to each well for color development, and the plate was incubated for 10 min. Finally, ELISA stop solution (Solarbio product) was added, and the OD value at 450 nm was read using a microplate reader.

[0053] The results showed that immunization of mice with rAd5-SADS-S induced the production of ELISA antibodies against SADS-CoV. Figure 2 B).

[0054] 2.3 SADS-CoV Neutralizing Antibody Detection

[0055] The isolated serum was inactivated at 56°C for 30 min, and then serially diluted 2-fold with DMEM, with an initial dilution of 1:4. SADS-CoV GDS04 was diluted with DMEM to 2000 TCID. 50 / mL, take the same volume of virus and serum samples, mix them well, and incubate at 37℃ for 1h. SADS-CoV and serum mixture was inoculated into a monolayer of Huh-7 cells and cultured in a cell incubator for 48 h. The SADS-CoV infection status was determined by indirect immunofluorescence and the neutralizing titer was calculated using a monoclonal antibody that recognizes SADS-CoV N as the primary antibody (Cao L, Kong X, Li X, Suo X, Duan Y, Yuan C, Zhang Y, Zheng H, Wang Q. A Customized Novel Blocking ELISA for Detection of Bat-Origin Swine Acute Diarrhea Syndrome Coronavirus Infection. Microbiol Spectr. 2023 Aug 17;11(4):e0393022.doi:10.1128 / spectrum.03930-22.Epub 2023 Jun 5.PMID:37272819;PMCID:PMC10434073.).

[0056] The results showed that immunization of mice with rAd5-SADS-S induced the production of neutralizing antibodies against SADS-CoV. Figure 2 C).

[0057] 2.4 Lymphocyte proliferation assay

[0058] Six weeks after the initial immunization, three mice in each group were euthanized, and spleen lymphocytes were isolated using mouse spleen lymphocyte separation medium (Dakoi product). The cells were then diluted to 2 × 10⁻⁶ cells / mL with RPMI-1640 medium containing 10% FBS. 6 Cells / mL. Mouse lymphocytes were seeded into 96-well cell culture plates at 100 μL per well, with each cell group consisting of a non-stimulated group and a SADS-CoV stimulated group. The stimulated group used 10 μL of SADS-CoV virus per well at a dose of 10 μL / well. 3 TCID 50 / well, the non-stimulated group was supplemented with 10 μL / well of RPMI-1640 medium. A control group was also included, consisting of wells containing only RPMI-1640 medium without cells. The cell culture plates were sealed and incubated for 60 h, then the cell OD value was measured using a CCK8 assay kit (Beyotime product). The formula for calculating lymphocyte proliferation stimulation was: SI = (OD stimulation group - OD control group) / (OD non-stimulated group - OD control group).

[0059] The results showed that upon SADS-CoV restimulation, the proliferation of splenic lymphocytes in rAd5-SADS-S immunized mice was significantly higher than that in control mice treated with rAd5-CMV-EV or DMEM medium. Figure 2 D).

[0060] 2.5 Enzyme-linked immunospot assay (ELISPOT)

[0061] Using the mouse IFN-γ ELISpot kit (Darkow), cells were treated according to the grouping and stimulation methods of the lymphocyte proliferation assay, cultured for 20 h, and the number of antigen-specific IFN-γ spots was detected according to the product instructions.

[0062] The results showed that the IFN-γ secreted by splenic lymphocytes in rAd5-SADS-S immunized mice was significantly higher than that in splenic lymphocytes from control mice treated with rAd5-CMV-EV or DMEM medium. Figure 2 E).

[0063] In summary, rAd5-SADS-S immunization of mice can induce both humoral and cellular immunity.

[0064] Example 3: Immunoprotection of neonatal mice produced from BALB / c mice immunized with recombinant adenovirus rAd5-SADS-S against SADS-CoV challenge.

[0065] Passive immunization and SADS-CoV challenge evaluation were performed on suckling mice produced from mice immunized with rAd5-CMV-EV and rAd5-SADS-S at 2 days of age. Figure 3 A). Specifically, three suckling mice were dissected in each group, and their stomachs and intestines were rinsed with PBS. SADS-CoV-specific IgG antibodies in the wash solution were detected using the method described in Example 2. Each suckling mouse was orally administered 20 μL of SADS-CoV GDS04 with a viral load of 2000 TCID. 50The number of suckling mice was 13 in the rAd5-CMV-EV group and 11 in the rAd5-SADS-S group. Mice were observed for 14 days after challenge, during which time their weight and survival were recorded. Seven days after challenge, three mice from each group were dissected to detect the SADS-CoV load in the brain and intestines, and to observe pathological damage and tissue immunofluorescence. SADS-CoV viral load was detected using RT-qPCR to determine the copy number of the N gene. Primers were found in the published article (Duan Y, Yuan C, Suo X, Li Y, Shi L, Cao L, Kong X, Zhang Y, Zheng H, Wang Q. Bat-Origin Swine Acute Diarrhea Syndrome Coronavirus Is Lethal to Neonatal Mice. J Virol. 2023 Mar 30;97(3):e0019023. doi:10.1128 / jvi.00190-23. Epub 2023 Mar 6. PMID:36877051;PMCID:PMC10062167.). Pathological section preparation and observation were performed using standard procedures. Immunofluorescence of tissues used a monoclonal antibody against SADS-CoV N (Cao L, Kong X, Li X, Suo X, Duan Y, Yuan C, Zhang Y, Zheng H, Wang QA Customized). NovelBlocking ELISA for Detection of Bat-Origin Swine Acute Diarrhea SyndromeCoronavirus Infection.Microbiol Spectr.2023Aug 17;11(4):e0393022.doi:10.1128 / spectrum.03930-22.Epub 2023Jun 5. PMID: 37272819; PMCID: PMC10434073.).

[0066] Experimental results: SADS-CoV-specific IgG antibodies were detected in the gastric and intestinal washings of suckling mice born to mothers immunized with rAd5-SADS-S. Figure 3 B and Figure 3 C), while no cases were detected in the rAd5-CMV-EV immunized group. All suckling mice in the rAd5-CMV-EV group died 12 days after challenge, while suckling mice in the rAd5-SADS-S group did not die or show clinical symptoms until 14 days after challenge. Figure 3 D). The body weight of suckling mice in the rAd5-CMV-EV group was significantly lower than that in the rAd5-SADS-S group (D). Figure 3 E). Seven days after challenge, high copies of the SADS-CoV viral genome were detected in the brains of suckling mice in the rAd5-CMV-EV group, followed by the intestines. In the rAd5-SADS-S group, the viral copy numbers detected in both the brains and intestines were significantly lower than those in the rAd5-CMV-EV group. Figure 3 F) Correspondingly, pathological examination of tissue sections revealed significant pathological changes in the brain and intestines of the rAd5-CMV-EV group of suckling mice, while the rAd5-SADS-S group showed normal results. Figure 3 G); Immunofluorescence observation showed that the brains of suckling mice in the rAd5-CMV-EV group contained a large amount of SADS-CoV antigen, and the intestines contained a small amount of SADS-CoV antigen, while no SADS-CoV antigen was observed in the rAd5-SADS-S group. Figure 3 H). Eleven days after challenge, the pups in the rAd5-CMV-EV group were noticeably thinner and more stunted than those in the rAd5-SADS-S group. Figure 3 I). These data indicate that rAd5-SADS-S can confer complete protection against lethal SADS-CoV infection in newborn mice through passive immunization.

[0067] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A recombinant viral plasmid, characterized in that, The recombinant virus plasmid contains a nucleic acid molecule expressing S protein of porcine acute diarrhea syndrome coronavirus, and the amino acid sequence of the S protein of the porcine acute diarrhea syndrome coronavirus is shown as SEQ ID NO:

1. The recombinant virus plasmid is a recombinant adenovirus plasmid, the adenovirus is a replication-deficient adenovirus, and the replication-deficient adenovirus is a human type 5 adenovirus with deleted E1 and E3 regions.

2. The recombinant viral plasmid according to claim 1, wherein, The sequence of the nucleic acid molecule expressing the S protein of the porcine acute diarrhea syndrome coronavirus is shown as SEQ ID NO:

2.

3. The recombinant viral plasmid according to claim 1, wherein, The preparation method of the recombinant virus plasmid comprises: Step 1: cloning the S gene expression cassette of the porcine acute diarrhea syndrome coronavirus into a shuttle plasmid to obtain a recombinant shuttle plasmid; Step 2: co-transforming the recombinant shuttle plasmid and the adenovirus plasmid into an E. coli competent cell for homologous recombination to obtain the recombinant virus plasmid.

4. The recombinant viral plasmid according to claim 3, wherein, In step 1, the base sequence of the S gene expression cassette of the porcine acute diarrhea syndrome coronavirus is shown as SEQ ID NO:

3.

5. A recombinant virus, wherein, The recombinant virus is obtained by packaging the recombinant virus plasmid of any one of claims 1-4.

6. An adenoviral vector recombinant porcine epidemic diarrhea syndrome coronavirus vaccine, characterized by, The vaccine contains a recombinant adenovirus, and the recombinant adenovirus is obtained by packaging the recombinant adenovirus plasmid containing the nucleic acid molecule expressing the S protein of the porcine acute diarrhea syndrome coronavirus, and the amino acid sequence of the S protein of the porcine acute diarrhea syndrome coronavirus is shown as SEQ ID NO:

1. The adenovirus is a replication-deficient adenovirus, and the replication-deficient adenovirus is a human type 5 adenovirus with deleted E1 and E3 regions.

7. The adenoviral vector recombinant porcine epidemic diarrhea syndrome coronavirus vaccine of claim 6, characterized in that, The sequence of the nucleic acid molecule expressing the S protein of the porcine acute diarrhea syndrome coronavirus is shown as SEQ ID NO:

2.

8. The adenoviral vector recombinant porcine epidemic diarrhea syndrome coronavirus vaccine of claim 6, characterized in that, The preparation method of the recombinant adenovirus plasmid comprises: Step 1: cloning the S gene expression cassette of the porcine acute diarrhea syndrome coronavirus into a shuttle plasmid to obtain a recombinant shuttle plasmid; Step 2: co-transforming the recombinant shuttle plasmid and the adenovirus plasmid into an E. coli competent cell for homologous recombination to obtain the recombinant adenovirus plasmid.

9. Use of the recombinant virus plasmid of any one of claims 1-4, the recombinant virus of claim 5, or the vaccine of any one of claims 6-8 in the preparation of a porcine acute diarrhea syndrome coronavirus preventive drug.

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