Porcine coronavirus type delta recombinant DNA vaccine and construction method and application thereof

Abstract

The application belongs to the technical field of biotechnology, and provides a porcine deltacoronavirus recombinant DNA vaccine, a construction method and application thereof, the porcine deltacoronavirus recombinant DNA vaccine comprising a porcine deltacoronavirus NTD antigen gene or a porcine deltacoronavirus RBD antigen gene; wherein the nucleotide sequence of the porcine deltacoronavirus NTD antigen gene is shown as SEQ ID NO. 1, and the nucleotide sequence of the porcine deltacoronavirus RBD antigen gene is shown as SEQ ID NO. 2. The application firstly fuses the immunogenic gene fragments NTD and RBD of PDCoV with IgG1 Fc fragments respectively, constructs a recombinant DNA vaccine, and evaluates the safety, immunogenicity and protective efficacy of the recombinant DNA vaccine through experiments, and verifies that the PDCoV recombinant DNA vaccine has good effects in preventing porcine deltacoronavirus infection.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to a recombinant DNA vaccine against porcine dinococcal coronavirus, its construction method, and its application. Background Technology

[0002] Porcine PDCoV, a member of the δ-coronavirus genus, can infect piglets through the respiratory and digestive tracts. Infected piglets typically exhibit symptoms such as vomiting, watery diarrhea, and severe dehydration, causing significant economic losses to the livestock industry. The PDCoV genome encodes four structural proteins: spike protein (S), envelope protein (E), membrane protein (M), and nucleocapsid protein (N). The S protein plays a crucial role in receptor binding, cell membrane fusion, and viral invasion. The S protein contains an N-terminal domain (NTD) and a receptor-binding domain (RBD). The NTD contains abundant antigenic epitopes that can interact with certain carbohydrate receptors on the host cell membrane surface, assisting in initial viral attachment. The RBD is a key region mediating the specific binding of the virus to host cell receptors; therefore, both are of great significance for coronavirus vaccine development.

[0003] DNA vaccines have significant advantages in preventing emerging pathogens, with rapid development, stable production, and the ability to induce humoral and cellular immunity. Furthermore, when combined with adjuvants, they have the potential to enhance the immunogenicity of DNA vaccines by improving antigen presentation and prolonging immune stimulation. Summary of the Invention

[0004] The purpose of this invention is to provide a recombinant DNA vaccine against porcine dinococcal coronavirus, aiming to solve the problems mentioned in the background art.

[0005] The present invention is implemented as follows: a porcine d-coronavirus recombinant DNA vaccine, wherein the porcine d-coronavirus recombinant DNA vaccine includes the porcine d-coronavirus NTD antigen gene or the porcine d-coronavirus RBD antigen gene;

[0006] The nucleotide sequence of the porcine d-coronavirus NTD antigen gene is shown in SEQ ID NO.1, and the nucleotide sequence of the porcine d-coronavirus RBD antigen gene is shown in SEQ ID NO.2.

[0007] Another objective of this invention is to provide a method for constructing a recombinant DNA vaccine against porcine dinococcal coronavirus, comprising the following steps:

[0008] Total viral RNA of PDCoV CC23 was extracted and reverse transcribed into cDNA.

[0009] Using cDNA as a template, PCR amplification was performed using PDCoV-NTD primers or PDCoV-RBD primers to obtain the amplification product, which was then purified by gel extraction to obtain PDCoV-NTD or PDCoV-RBD.

[0010] The PDCoV-NTD was inserted into the pFUSE-hIgG1e1-Fc1 plasmid to obtain the recombinant plasmid PDCoV-NTD-Fc, the base sequence of which is shown in SEQ ID NO.3;

[0011] Alternatively, PDCoV-RBD can be inserted into the pFUSE-hIgG1e1-Fc1 plasmid to obtain the recombinant plasmid PDCoV-RBD-Fc, the base sequence of which is shown in SEQ ID NO.4;

[0012] The recombinant plasmid was transformed into competent cells and cultured.

[0013] Another objective of this invention is to provide the application of a recombinant DNA vaccine against porcine d-coronavirus in the preparation of a drug for the prevention of porcine d-coronavirus.

[0014] This invention, for the first time, fuses the PDCoV immunogenic gene fragments NTD and RBD with the tag gene IgG1 Fc fragment in the pFUSE-hIgG1e1-Fc1 plasmid to construct a recombinant DNA vaccine. The safety, immunogenicity, and protective efficacy of the recombinant DNA vaccine were evaluated through experiments, verifying that the PDCoV recombinant DNA vaccine has a good effect in preventing porcine coronavirus infection. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of the structure of the porcine dinocoronavirus S protein and the construction of the vaccine provided in Embodiment 1 of the present invention;

[0016] Figure 2 This is a schematic diagram of the PDCoV S protein monomer structure provided in Example 1 of the present invention;

[0017] Figure 3 The vaccine construction results provided in Example 1 of this invention (B is the amplification and PCR verification diagram of the PDCoV-NTD target gene, C is the amplification and PCR verification diagram of the PDCoV-RBD target gene, and D is the expression of PDCoV-NTD-Fc and PDCoV-RBD-Fc after transfection into 293T cells).

[0018] Figure 4The purification results of PDCoV-NTD protein and PDCoV-RBD protein provided in Example 2 of the present invention are shown in Figure 2. (A is the purification result of PDCoV-NTD protein, B is the SDS-PAGE detection result of PDCoV-NTD protein after refolding, C is the purification result of PDCoV-RBD protein, and D is the SDS-PAGE detection result of PDCoV-NTD protein after refolding.)

[0019] Figure 5 The following graphs show the body temperature and weight of mice provided in Example 3 of this invention (A is a graph showing the body temperature changes of mice vaccinated with PDCoV-NTD-Fc within 35 days, B is a graph showing the weight changes of mice vaccinated with PDCoV-NTD-Fc within 35 days, C is a graph showing the body temperature changes of mice vaccinated with PDCoV-RBD-Fc within 35 days, and D is a graph showing the weight changes of mice vaccinated with PDCoV-RBD-Fc within 35 days).

[0020] Figure 6 , Figure 7 The results of changes in mouse cytokine secretion levels provided in Example 3 of the present invention ( Figure 6 In Figure A, the result of serum TNF-α (tumor necrosis factor-α) detection is shown. Figure 6 C represents the serum IL-2 (interleukin-2) detection result. Figure 7 B represents the serum IFN-γ (interferon-γ) detection result. Figure 7 (D represents the result of serum IL-4 (interleukin-4) detection).

[0021] Figure 8 The mouse-specific IgG antibody level provided in Example 3 of the present invention (A is the result of serum-specific IgG antibody detection, and B is the result of serum neutralizing antibody detection);

[0022] Figure 9 The mouse spleen lymphocyte status provided in Example 3 of the present invention (A is the result of spleen lymphocyte proliferation detection, and B is the result of spleen lymphocyte subtype detection).

[0023] Figure 10 This is a flowchart of mouse immunization and challenge provided in Example 4 of the present invention;

[0024] Figure 11 The results of the mouse immunization and challenge experiments provided in Example 4 of this invention are as follows: (A is the monitoring results of weight change after challenge; B is the detection results of PDCoV N gene mRNA expression level in the intestine after challenge; C is the detection of PDCoV positive signal in the duodenum, jejunum and ileum of mice by immunofluorescence; D is the results of pathological changes in the duodenum, jejunum and ileum of mice observed by H&E pathological tissue sections).

[0025] in, Figures 6 to 9In this text, lowercase letters a, b, c, d, and e represent differences. If there is no difference between two groups, the same letter is used. For example, if both columns are 'a', and there is a difference (P < 0.05) with the next group, then 'b' is used to indicate the next group. 'ab' indicates that there is no difference with either 'a' or 'b' alone. "Indicates dosage from low to high." Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0027] Unless otherwise specified, all reagents or instruments used in the embodiments of this invention are conventional products that can be purchased commercially.

[0028] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0029] Example 1: PDCoV-NTD-Fc and PDCoV-RBD-Fc recombinant DNA vaccines, the vaccines comprising the NTD antigen gene and RBD antigen gene from the spike (S) protein of porcine diclofenac coronavirus, as shown in the example. Figure 1 As shown, the monomer structure is as follows Figure 2 As shown, its construction method includes the following steps:

[0030] Total viral RNA of PDCoV CC23 (PDCoV CC23 strain, GenBank: PQ539659.1, isolated in the laboratory) was extracted using an RNA extraction kit and reverse transcribed into cDNA. Using cDNA as a template, the total PCR reaction volume was 25 μL (Prime STAR Max 12.5 μL, cDNA template 1 μL, upstream primer 1 μL, downstream primer 1 μL, ddH2O 9.5 μL). The reaction program was 98 ℃ for 2 min; 98 ℃ for 10 s, 58 ℃ for 30 s, 72 ℃ for 10 s, 34 cycles; extension at 72 ℃ for 5 min. Primer sequences are shown in Table 1. Amplification was performed to obtain the amplified products, which were then purified by gel electrophoresis.

[0031] Table 1. Primer sequences for PDCoV-NTD / RBD

[0032]

[0033] PDCoV NTD and RBD fragments were ligated into the pFUSE-hIgG1e1-Fc1 vector via homologous recombination. After transformation, the vector was transferred into Trans1-T1 competent cells, and successful ligation was confirmed by colony PCR. Figure 3 The sequence (shown in B and C) was then sent to Changchun Sangon Biotech Co., Ltd. for sequencing. After confirming that the sequence matched the original target fragment sequence, the plasmid was extracted and expressed on 293T cells for verification. The results are as follows: Figure 3 As shown in Figure D, the PDCoV-NTD-Fc protein has a size of 50 kDa, and the PDCoV-RBD-Fc protein has a size of 40 kDa.

[0034] Example 2: Purification of PDCoV-NTD protein

[0035] Following the method in Example 1, the amplified PDCoV NTD and RBD fragments were ligated and transformed into the pET-32a prokaryotic expression plasmid. 500 μL of bacterial culture containing either the PDCoV-NTD-Fc or PDCoV-RBD-Fc recombinant plasmid was added to 200 mL of LB broth containing ampicillin. After incubation at 37°C for 6 h, IPTG (isopropyl-β-D-thiogalactopyranoside) was added to a final concentration of 1 mM and the mixture was incubated overnight at 16°C. The NTD and RBD proteins were purified using a Beyotime protein purification kit. After refolding, the proteins were concentrated via ultrafiltration. The results were observed using SDS-PAGE and Coomassie Brilliant Blue staining. Figure 4 As shown in Figures A and C, the PDCoV-NTD protein is 44 kDa in size, and the PDCoV-RBD protein is 32 kDa in size; both proteins were stably expressed after renaturation, as detected by SDS-PAGE (e.g., ...). Figure 4 (As shown in B and D).

[0036] Example 3: Safety and immunogenicity analysis of PDCoV-NTD and PDCoV-RBD recombinant DNA vaccines in mice:

[0037] Fifty-five 3-week-old SPF-grade female BALB / c mice (purchased from Liaoning Changsheng Biotechnology Co., Ltd., China) were randomly divided into 11 groups (n=5 per group) and vaccinated twice via intramuscular injection on days 0 and 21. Blood samples were collected from the orbital venous plexus on days 0, 14, and 35 after the initial vaccination. The specific grouping method is shown in Table 2.

[0038] Table 2. Grouping and Dosage of Mice Vaccinated with DNA

[0039]

[0040] Mice were monitored for 35 days after vaccination, including body temperature, weight, mental state, and food and water intake. Results showed that the mice tolerated the vaccine well, with normal body temperature fluctuations and steady daily weight gain (e.g., ...). Figure 5 As shown in Figure AD), this demonstrates the safety of the PDCoV-NTD-Fc and PDCoV-RBD-Fc recombinant DNA vaccines;

[0041] The changes in the secretion levels of TNF-α, IFN-γ, IL-2, and IL-4 in mice after vaccination were assessed using an enzyme-linked immunosorbent assay (ELISA). After collecting mouse blood samples, the supernatant serum was separated after standing at room temperature for 1 hour. The serum sample and standards were diluted using a universal diluent, and 100 μL was added to a 96-well plate of a mouse ELISA kit (TNF-α, IFN-γ, IL-2, IL-4). The procedure was performed according to the manufacturer's instructions. After adding the stop solution, the OD was measured using a microplate reader. 450nm Value, result as Figure 6 , Figure 7 As shown in the AD diagram, compared with the unvaccinated group, all vaccine groups were able to induce varying degrees of increase in Th1 and Th2 cytokines, and the increase was dose-dependent, with the high-dose PDCoV-RBD-Fc combined with adjuvant showing the most significant effect;

[0042] The level of specific IgG antibodies in mouse serum was detected by ELISA. Purified PDCoV RBD or NTD protein (5 μg / mL) was mixed with antigen coating solution at a 1:1 ratio. 200 μL of the mixture was added to a 96-well plate and incubated overnight. After blocking, 100 μL of mouse serum was added to each well, and the plate was incubated at 37 °C for 1 h. The plate was washed three times with PBST and incubated with secondary antibody at 37 °C for 1 h. After washing with PBST, the plate was developed using 3,3',5,5'-tetramethylbenzidine (TMB) reagent, and the OD was detected using a microplate reader. 450nm Value, result as Figure 8 As shown in Figure A, the DNA vaccine effectively induced the production of specific antibodies against PDCoV, and the antibody titer increased with increasing vaccination dose. The endpoint titer of specific antibodies reached a high level after booster immunization. The level of neutralizing antibodies in mouse serum was detected using a neutralization titration method. Serum collected on day 35 was heat-inactivated at 56 °C for 30 min, serially diluted twofold using DMEM culture medium, and the diluted serum samples were then mixed with 100 TCID50. 50 The PDCoV mixture was incubated at 37°C for 1 h. The incubated virus-serum mixture was then transferred to the corresponding cell plate wells, gently shaken to ensure thorough mixing and full contact with the cells, and the cytopathic effect (CPE) was observed under a microscope. Results are as follows: Figure 8As shown in Figure B, the neutralizing antibody titers induced by NTD ranged from 1:9 to 1:84, while those induced by RBD ranged from 1:11 to 1:169, indicating that the antibody production level induced by RBD was significantly better than that of the NTD group.

[0043] Thirty-five days after the initial vaccination, mice were anesthetized and euthanized by cervical dislocation. Spleens were collected in a clean bench, ground, and then mixed with lymphocyte separation medium to collect splenic lymphocytes. These cells were stimulated with NTD and RBD proteins (2.5 μg / mL). The positive control group received concanavalin A (ConA, 2.5 μg / mL), while the negative control group received 50 μL of serum-free DMEM. Cells were incubated for 72 h, followed by incubation for another 4 h with the addition of thiazolyl blue (MTT). OD values ​​were then measured using a microplate reader. 570nm Value, result as Figure 9 As shown in Figure A, splenic lymphocytes in all vaccine-treated mice showed varying degrees of proliferation upon stimulation by exogenous proteins. The lymphocyte proliferation level in the RBD vaccine group was significantly higher than that in the NTD vaccine group. The collected splenic lymphocytes were stained with PE-labeled anti-CD8 and FITC-labeled anti-CD4 antibodies, and the results are shown in Figure A. Figure 9 As shown in Figure B, CD4 levels in the spleen of mice after vaccination... + and CD8 + The number of T cells increased significantly, especially in the high-dose RBD+GEL01 group. In summary, the results indicate that PDCoV recombinant DNA vaccine did not have an adverse effect on the health status of mice and could effectively activate humoral and cellular immune responses by inducing antibody production and promoting lymphocyte proliferation. In addition, the immune effect increased in a dose-dependent manner with the increase of DNA vaccine dose and the combined use of adjuvants.

[0044] Example 4: Experimental analysis of the protective effect of PDCoV recombinant DNA vaccine on mice:

[0045] Twenty-five mice were randomly divided into five groups (n=5 per group): Naïve (untreated), PBS (PBS+PDCoV), Fc (Fc+PDCoV), NTD+GEL01 (high-dose NTD+GEL01+PDCoV), and RBD+GEL01 (high-dose RBD+GEL01+PDCoV). On day 35 post-initial vaccination, the mice were treated with 10... 4.2 TCID 50PDCoV was administered to mice via gavage. Body weight changes were monitored for 7 days post-challenge. Mice were euthanized on day 7 post-challenge, and their duodenum, jejunum, and ileum were collected. The protective efficacy of the vaccine in mice was assessed using qRT-PCR, immunofluorescence staining (IFA), and H&E staining. The specific procedure is as follows: Figure 10 As shown, the results indicated that the unvaccinated groups (PBS group and Fc group) experienced weight loss or slow weight gain on the second day after challenge (e.g. Figure 11 (As shown in Figure A); Further detection of viral load in the small intestine of mice by qRT-PCR showed that the expression level of PDCoV mRNA in the intestine of the vaccine-immunized group was significantly lower than that in the non-immunized group (PBS group and Fc group) (as shown in Figure A). Figure 11 (As shown in Figure B); PDCoV N protein antigen positive signals were detected in different segments of the small intestine using IFA. The results showed that after immunization with the DNA vaccine, the PDCoV positive signals in the duodenum, jejunum, and ileum of mice were significantly reduced (e.g., ...). Figure 11 (As shown in C); H&E staining results showed that although PDCoV was detected in the mouse intestine, no obvious pathological changes were observed in the intestinal tissue (e.g., ...). Figure 11 As shown in Figure D, the reason is that the binding ability of PDCoV virus to mouse aminopeptidase N (mAPN) is weaker than that to piglet aminopeptidase N (pAPN), resulting in low infection efficiency in mouse intestinal epithelial cells. That is, the virus is present but replication is not active, and therefore no obvious histopathological changes are induced. The results of mouse challenge experiments show that the recombinant DNA vaccine can effectively inhibit PDCoV infection in mouse intestines.

[0046] In summary, the PDCoV-NTD-Fc vaccine and PDCoV-RBD-Fc vaccine constructed in this embodiment of the invention have good safety profiles. No adverse reactions such as fever, depression, or weight loss were observed in the tested mice after vaccination. In the immunogenicity experiment, each vaccine group effectively induced humoral and cellular immune responses in mice. Especially after administration of high-dose vaccines and adjuvants, high titers of specific IgG and neutralizing antibodies were induced in mice, and a strong cellular immune response was activated. In the challenge protection experiment, the vaccine significantly inhibited viral replication in the intestines after immunization, providing effective protection for mice.

[0047] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A recombinant DNA vaccine against porcine d-coronavirus, characterized in that, The porcine d-coronavirus recombinant DNA vaccine includes the porcine d-coronavirus NTD antigen gene or the porcine d-coronavirus RBD antigen gene; The nucleotide sequence of the porcine d-coronavirus NTD antigen gene is shown in SEQ ID NO.1, and the nucleotide sequence of the porcine d-coronavirus RBD antigen gene is shown in SEQ ID NO.

2. The porcine d-coronavirus recombinant DNA vaccine also includes a plasmid vector, wherein the plasmid vector is pFUSE-hIgG1e1-Fc1; The method for constructing the porcine d-coronavirus recombinant DNA vaccine includes the following steps: Total viral RNA of PDCoV CC23 was extracted and reverse transcribed into cDNA. Using cDNA as a template, PCR amplification was performed using PDCoV-NTD primers or PDCoV-RBD primers to obtain amplification products. These products were then purified via gel extraction to obtain PDCoV-NTD or PDCoV-RBD. The PDCoV-NTD primers include PDCoV-NTD-F and PDCoV-NTD-R, with the sequence of PDCoV-NTD-F shown in SEQ ID NO. 5 and the sequence of PDCoV-NTD-R shown in SEQ ID NO.

6. Similarly, the PDCoV-RBD primers include PDCoV-RBD-F and PDCoV-RBD-R, with the sequence of PDCoV-RBD-F shown in SEQ ID NO. 7 and the sequence of PDCoV-RBD-R shown in SEQ ID NO.

8. The PDCoV-NTD was inserted into the pFUSE-hIgG1e1-Fc1 plasmid to obtain the recombinant plasmid PDCoV-NTD-Fc, the base sequence of which is shown in SEQ ID NO.3; Alternatively, PDCoV-RBD can be inserted into the pFUSE-hIgG1e1-Fc1 plasmid to obtain the recombinant plasmid PDCoV-RBD-Fc, the base sequence of which is shown in SEQ ID NO.4; The recombinant plasmid was transformed into competent cells and cultured.

2. The porcine d-coronavirus recombinant DNA vaccine according to claim 1, characterized in that, The porcine d-coronavirus recombinant DNA vaccine also includes pharmaceutically acceptable vectors or excipients.

3. The porcine d-coronavirus recombinant DNA vaccine according to claim 1, characterized in that, The competent cells are Trans1-T1 competent cells.

4. The use of a recombinant DNA vaccine against porcine d-coronavirus as described in any one of claims 1-2 in the preparation of a medicament for the prevention of porcine d-coronavirus.

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