Adenovirus vector recombinant porcine acute diarrhea syndrome coronavirus vaccine

Abstract

The invention belongs to the technical field of veterinary medicine, and particularly relates to an adenovirus vector recombinant porcine acute diarrhea syndrome coronavirus vaccine. According to the invention, by constructing a replication-deficient adenovirus vector vaccine (rAd-SADS-S) expressing the full length of SADS-CoV S protein, efficient body fluid and cell immunity is induced by immunity, passive protection is provided for suckling mice through maternal antibody transfer, and the defects that no SADS-CoV vaccine is available and the existing research protection rate is low and the biological safety risk is caused are overcome.

Description

Technical Field

[0001] The present invention belongs to the technical field of veterinary medicine, and in particular relates to an adenovirus vector recombinant porcine acute diarrhea syndrome coronavirus vaccine. Background Art

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

[0003] The limitations of SADS-CoV vaccine technology at the current research stage are reflected in the following aspects:

[0004] Live vaccine: The SADS-CoV CN / GDWT / 2017 strain was passaged 83 times. Although its pathogenicity was reduced, it was still pathogenic for 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 2019 Sep 21. PMID: 31580972; PMCID: PMC7112038.). Immunization of female mice with live SADS-CoV virus can also induce an immune response against SADS-CoV in mice, and the suckling mice produced by immunized mice can develop resistance to SADS-CoV, but the protection effect does not reach 100%, and there are biosafety risks in using live virus 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 there are problems such as unstable virus titers and high production costs.

[0006] Recombinant virus vaccines: For example, the recombinant SADS-CoV described in Chinese invention patent application publication number CN118360258A replaces the ORF3a gene with the COE+S1D epitopes of PEDV to enhance mucosal immunity. Although such vaccines can stimulate local immunity, they are based on live virus modifications, pose biosafety risks (such as reversion to virulence), and are complex to produce. A recombinant virus constructed using vesicular stomatitis virus (VSV) as a backbone to express the SADS-CoV S protein can produce neutralizing antibodies against SADS-CoV in mice immunized with this recombinant virus, but no virus 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 2024 Apr 1. PMID: 38557247; PMCID: PMC11092325.), and the VSV vector used is a replication-competent vector.

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

[0008] For example, the Chinese invention patent application with application publication number 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 providing 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 has been widely used in human medicine and has also been used in veterinary medicine, as exemplified by the Chinese invention patent application with application publication number CN101966341A.

[0009] The spike protein (S protein) of SADS-CoV is a key structural protein that mediates viral invasion of host cells and is also the main target of neutralizing antibodies. Generally speaking, the coronavirus S protein is difficult to express in vitro. In addition, although adenovirus vectors are mature in the application of coronaviruses such as PEDV, there are no reports on adenovirus vaccine research for SADS-CoV. In addition, existing coronavirus vaccines mostly focus on a single antigen (such as the S1 subunit), which is prone to immune escape due to viral mutations. Summary of the Invention

[0010] The present invention constructs a replication-deficient adenovirus vector vaccine (rAd-SADS-S) expressing the full-length SADS-CoV S protein to achieve immune induction of highly efficient humoral and cellular immunity, and provides passive protection for suckling mice through maternal antibody transmission, thereby overcoming the shortcomings of no available SADS-CoV vaccine, low protection rate and biosafety risks in existing research.

[0011] To achieve the above object, the present invention adopts the following technical solutions:

[0012] In a first aspect, the present invention provides a recombinant viral plasmid comprising a nucleic acid molecule expressing the S protein of porcine acute diarrhea syndrome coronavirus, 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 porcine acute diarrhea syndrome coronavirus 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 comprises:

[0014] Step 1: Clone the SADS-CoV S gene expression cassette into a shuttle plasmid to obtain a recombinant shuttle plasmid; further, 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 are co-transformed into E. coli competent cells for homologous recombination, and the recombinant adenovirus plasmid is screened.

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

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

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

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

[0020] Step 2: The recombinant shuttle plasmid and adenovirus plasmid are co-transformed into E. coli competent cells for homologous recombination, and the recombinant adenovirus plasmid is screened.

[0021] In a fourth aspect, the present 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. The present invention has found that passive immunization of female mice with a recombinant adenovirus vaccine can confer complete protection against lethal infection with SADS-CoV in newborn mice.

[0022] The beneficial effects of the present invention are:

[0023] (1) Antigen Selection and Expression Optimization: The present invention uses the full-length sequence of the SADS-CoV S protein (S protein includes S1 subunit and S2 subunit) as the target antigen, covering more comprehensive antigenic epitopes. In addition, the present invention also performs codon optimization on the SADS-CoV S protein to achieve high expression.

[0024] (2) Innovation in immunization strategy: The examples of the present invention demonstrate that maternal immunization can induce maternal antibodies to protect suckling mice through breast milk.

[0025] In general, the rAd5-SADS-S constructed in the present invention is safe, highly effective, and replication-deficient, and the immune mother can provide complete protection to newborn individuals against lethal SADS-CoV challenge. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0028] Figure 2 : Evaluation of immunogenicity of adenovirus vector recombinant porcine acute diarrhea syndrome coronavirus vaccine rAd5-SADS-S in mice.

[0029] A. Animal test process 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. Splenic lymphocyte ELISPOT assay.

[0030] Figure 3 Evaluation of the protective effect of adenovirus vectored recombinant porcine acute diarrhea syndrome coronavirus vaccine rAd5-SADS-S on suckling mice

[0031] A. Experimental process for suckling mice; B. SADS-CoV IgG antibodies in the stomach of suckling mice; C. SADS-CoV IgG antibodies in the intestine 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 indicates SADS-CoV N protein antigen; I. Appearance of suckling mice. DETAILED DESCRIPTION

[0032] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly described below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0033] Adenovirus plasmid pAdEasy-1 and shuttle plasmid pShuttle were purchased from Zhuangmeng Bio. pcDNA3.1-SADS-S-flag was obtained by cloning the SADS-S-flag gene (as shown in SEQ ID NO: 3) with a C-terminal fusion flag tag 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 is a commercially available product and was maintained in the inventor's laboratory. BJ5183 Escherichia coli competent cells were purchased from Shanghai Weidi Biotechnology Co., Ltd. HEK-293A cells are commercially available and were maintained in the inventor's laboratory.

[0034] The SADS-CoV GDS04 strain was a gift from Professor Cao Yongchang 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. Epub 2017 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 to obtain a vector. Using pcDNA3.1 and pcDNA3.1-SADS-S-flag as templates, and CMV F and bGHR as primers, the CMV promoter empty vector expression cassette and the SADS-S-flag expression cassette were amplified, respectively. The amplified products were 1018 bp and 4367 bp in length, respectively. The amplified pShuttle was ligated with the CMV promoter empty vector expression cassette and the SADS-S-flag expression cassette using the ClonExpress II One Step Cloning Kit (Novagen) to obtain pShuttle-CMV-EV and pShuttle-SADS-S plasmids. 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 Escherichia coli competent cells for homologous recombination. The recombinant adenovirus plasmids pAd-CMV-EV and pAd-SADS-S were selected by kanamycin resistance.

[0038] 1.2 Recombinant adenovirus rescue

[0039] pAd-CMV-EV and pAd-SADS-S were linearized with the restriction endonuclease PacI (NEB), and DNA was recovered using a DNA extraction reagent (phenol:chloroform:isoamyl alcohol (25:24:1)). The linearized DNA was transfected into HEK-293A cell monolayers. After 7 days of significant cytopathic effect (CPE), the cell supernatant was collected and inoculated into newly passaged HEK-293A cells for expansion to generate 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 genome of the recombinant adenovirus rAd5-SADS-S and the empty vector control virus rAd5-CMV-EV. The viral backbone used is a human adenovirus type 5 lacking the E1 and E3 regions (a commercial backbone that can replicate in cells expressing E1 and E3 but not in normal cells).

[0043] The F5 recombinant viruses rAd5-CMV-EV and rAd5-SADS-S were inoculated into HEK-293A cells. At 48 hpi, obvious CPE was observed in the cells inoculated with rAd5-CMV-EV and rAd5-SADS-S. Figure 1 B).

[0044] DNA of F5 recombinant viruses rAd5-CMV-EV and rAd5-SADS-S was extracted and used as templates. PCR amplification was performed using CMV F and bGHR as primers. The expected fragment size ( Figure 1 C).

[0045] HEK-293A cells were inoculated with F5 recombinant viruses rAd5-CMV-EV and rAd5-SADS-S. Cells were collected at 36 hpi and Western blotting was performed using anti-Flag antibody (Proteintech product) and anti-β-actin antibody (Servicebio product) as primary antibodies. The intracellular reference β-actin was detected in cells that were not infected and inoculated with rAd5-CMV-EV and rAd5-SADS-S viruses, while the SADS-CoV S-Flag band was only detected in the rAd5-SADS-S group ( 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 Chinese Center for Animal Health and Epidemiology). Animal experiments were conducted in the animal room of the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (Lanzhou Branch of the Chinese Center for Animal Health and Epidemiology). BALB / c female mice were divided into three groups, each with 8 mice: a blank control group (DMEM), a vector control group (rAd5-CMV-EV), and a vaccine group (rAd5-SADS-S). Figure 2 The vector control group and vaccine group were immunized by intramuscular injection of 100 μL containing 10 6 TCID 50Rats were immunized three times with rAd5-CMV-EV and rAd5-SADS-S, separated by two weeks. A blank control group received 100 μL of DMEM medium each time. One week after the second immunization, two mice each from rAd5-CMV-EV and rAd5-SADS-S were co-housed with male mice (for the third immunization according to the protocol). The resulting suckling mice were used for SADS-CoV challenge evaluation. Two weeks after the third immunization, blood was collected from mice in each group, and serum was isolated to detect SADS-CoV-specific and neutralizing antibodies. Spleens from three mice in each group were dissected, and lymphocytes were isolated for lymphocyte proliferation assays and Elispot assays.

[0051] 2.2 SADS-CoV-specific antibody detection

[0052] Purified SADS-CoV GDS04 strain was diluted to 2 μg / mL and added to ELISA plates at a volume of 50 μL per well for overnight incubation at 4°C. Plates were then washed four times with PBST and incubated with enzyme-linked stabilizer (Bio-Tel) at 37°C for 1 hour. After four washes with PBST, 50 μL of serum sample (diluted 1:50 in PBS) was added to each well and incubated at 37°C for 30 minutes. After four washes with PBST, 50 μL of HRP-conjugated goat anti-mouse IgG (Abcam) (diluted 1:20,000 in PBS) was added to each well and incubated at 37°C for 30 minutes. After four washes with PBST, 50 μL of TMB (Surmodics) was added to each well for color development for 10 minutes. ELISA stop buffer (Solarbio) was then added, and the OD 450 nm readings were read using a microplate reader.

[0053] The results showed that rAd5-SADS-S immunized mice could induce the production of ELISA antibodies against SADS-CoV ( Figure 2 B).

[0054] 2.3 SADS-CoV neutralizing antibody detection

[0055] The separated serum was inactivated at 56°C for 30 min and then diluted 2-fold with DMEM, with the initial dilution being 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, and incubate them at 37℃ for 1h. The mixture of SADS-CoV and serum was inoculated into a monolayer of Huh-7 cells and cultured in a cell culture incubator for 48 h. The SADS-CoV infection was determined by indirect immunofluorescence using a monoclonal antibody that recognizes SADS-CoV N as the primary antibody, and the neutralization titer was calculated (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 rAd5-SADS-S immunization of mice could induce the production of neutralizing antibodies against SADS-CoV ( Figure 2 C).

[0057] 2.4 Lymphocyte proliferation assay

[0058] Six weeks after the first immunization, three mice in each group were killed and spleen lymphocytes were separated using mouse spleen lymphocyte separation medium (Dakowei product). The spleen lymphocytes were diluted to 2×10 6 Mouse lymphocytes were inoculated into 96-well cell culture plates, with 100 μL per well. Each group of cells was divided into a non-stimulated group and a SADS-CoV stimulated group. The virus volume used in the stimulated group was 10 μL / well, and the dose was SADS-CoV 10 3 TCID 50 10 μL / well of RPMI-1640 medium was added to the unstimulated group. A control well containing only RPMI-1640 medium without cells was also established as a control group. The cell plates were sealed and placed in a cell culture incubator for 60 hours. The cell OD values ​​were then measured using a CCK8 kit (Biyuntian). Stimulated lymphocyte proliferation was calculated as: SI = (OD of stimulated group - OD of control group) / (OD of unstimulated group - OD of control group).

[0059] The results showed that when SADS-CoV was restimulated, the proliferation of spleen lymphocytes in mice immunized with rAd5-SADS-S 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] The mouse IFN-γ ELISpot kit (Dakoway) was used to treat the cells according to the grouping and stimulation method of the lymphocyte proliferation test, cultured for 20 hours, 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 spleen lymphocytes of mice immunized with rAd5-SADS-S was also significantly higher than that of spleen lymphocytes of 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 immunity and cellular immunity.

[0064] Example 3: Immunity protection of suckling mice produced by immunizing BALB / c mice with recombinant adenovirus rAd5-SADS-S against SADS-CoV challenge

[0065] The passive immunity test and SADS-CoV challenge evaluation were performed on the suckling mice produced by rAd5-CMV-EV and rAd5-SADS-S immunized mice at 2 days of age ( Figure 3 A). Specifically, three suckling mice were dissected from each group, and the stomach and intestine were washed with PBS. SADS-CoV-specific IgG antibodies in the washing solution were detected using the method described in Example 2. Each suckling mouse was orally fed 20 μL of SADS-CoV GDS04, with a virus content of 2000 TCID 50The number of suckling mice was 13 in the rAd5-CMV-EV group and 11 in the rAd5-SADS-S group. The mice were observed for 14 days after challenge, during which their weight and survival were recorded. Seven days after challenge, three mice in each group were dissected to measure SADS-CoV load in the brain and intestine, observe pathological lesions, and observe tissue immunofluorescence. The SADS-CoV load was detected by RT-qPCR to detect the copy number of the N gene. The primers were shown 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 sections were prepared and observed in the conventional manner. Tissue immunofluorescence was performed using 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 the suckling mice born to the mother mice immunized with rAd5-SADS-S ( Figure 3 B and Figure 3 C), while no clinical symptoms were detected in the rAd5-CMV-EV group. All the suckling mice in the rAd5-CMV-EV group died 12 days after the challenge, while the suckling mice in the rAd5-SADS-S group did not die or show clinical symptoms until 14 days after the challenge ( Figure 3 D). The body weight of the rAd5-CMV-EV group was significantly lower than that of the rAd5-SADS-S group ( 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. The viral copy numbers detected in the brains and intestines of the rAd5-SADS-S group were significantly lower than those in the rAd5-CMV-EV group ( Figure 3 F) Corresponding to this. Pathological section observation revealed that the rAd5-CMV-EV group had obvious pathological changes in the brain and intestines of the suckling mice, while the rAd5-SADS-S group was normal ( Figure 3 G); Immunofluorescence observation showed that there was a large amount of SADS-CoV antigen in the brain and a small amount of SADS-CoV antigen in the intestine of the rAd5-CMV-EV group, while no SADS-CoV antigen was observed in the rAd5-SADS-S group ( Figure 3 H). Eleven days after infection, the rAd5-CMV-EV group showed thinner and more delayed development than the rAd5-SADS-S group ( Figure 3 I). These data indicate that rAd5-SADS-S can fully protect neonatal mice against lethal infection with SADS-CoV 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, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

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

1.

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

2.

3. A recombinant viral plasmid according to claim 1, characterized in that The recombinant virus plasmid is a recombinant adenovirus plasmid, and the adenovirus is a replication-defective adenovirus.

4. A recombinant viral plasmid according to claim 3, characterized in that The replication-deficient adenovirus is a human adenovirus type 5 lacking the E1 and E3 regions.

5. A recombinant viral plasmid according to claim 3 or 4, characterized in that: The method for preparing the recombinant viral plasmid comprises: Step 1: cloning the S gene expression cassette of porcine acute diarrhea syndrome coronavirus into a shuttle plasmid to obtain a recombinant shuttle plasmid; preferably, the base sequence of the S gene expression cassette of porcine acute diarrhea syndrome coronavirus is as shown in SEQ ID NO: 3; Step 2: The recombinant shuttle plasmid and adenovirus plasmid are co-transformed into Escherichia coli competent cells for homologous recombination, and the recombinant virus plasmid is screened.

6. A recombinant virus, characterized in that The recombinant virus is obtained by packaging the recombinant virus plasmid according to any one of claims 1 to 5.

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

1.

8. The adenovirus vector recombinant porcine acute diarrhea syndrome coronavirus vaccine according to claim 7, characterized in that: The sequence of the nucleic acid molecule expressing the S protein of porcine acute diarrhea syndrome coronavirus is shown in SEQ ID NO:

2.

9. The adenovirus vector recombinant porcine acute diarrhea syndrome coronavirus vaccine according to claim 7, characterized in that: The method for preparing the recombinant adenovirus plasmid comprises: Step 1: Clone the S gene expression cassette of porcine acute diarrhea syndrome coronavirus into the shuttle plasmid to obtain a recombinant shuttle plasmid; Step 2: The recombinant shuttle plasmid and adenovirus plasmid are co-transformed into Escherichia coli competent cells for homologous recombination, and the recombinant adenovirus plasmid is screened.

10. Use of the recombinant viral plasmid according to any one of claims 1 to 5, the recombinant virus according to claim 6, or the vaccine according to any one of claims 7 to 9 in the preparation of a drug for the prevention and / or treatment of porcine acute diarrhea syndrome coronavirus.

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