A recombinant adenovirus vector expressing PEDV-TGEV receptor binding domain and a preparation method and application thereof

Abstract

The present application belongs to the technical field of bioengineering, and particularly relates to a recombinant adenovirus vector expressing PEDV-TGEV receptor binding domain and a preparation method and application thereof. The present application uses a human type 5 defective adenovirus as an expression vector, optimizes RBD gene fragments of PEDV and TGEV at the same time, constructs a vector capable of efficiently expressing TGEV-PEDV receptor binding domain antigen protein, has good immunogenicity, can induce mucosal immunity, cellular immunity and humoral immunity response, thereby significantly improving immunity, and has a wide application prospect in the field of immunotherapy of porcine transmissible gastroenteritis and porcine epidemic diarrhea.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a recombinant adenovirus vector expressing the PEDV-TGEV receptor binding domain, its preparation method, and its application. Background Technology

[0002] Transmissible gastroenteritis of swine (TGE) is one of the most serious diseases affecting the pig industry worldwide. It is a highly contagious intestinal disease caused by the transmissible gastroenteritis virus (TGEV). Affected piglets exhibit vomiting, diarrhea, dehydration, and high mortality. The severity of clinical signs caused by TGEV is inversely proportional to the age of the pig herd. Piglets under 2 weeks old are most susceptible to infection, exhibiting vomiting, watery diarrhea, and yellow feces, often accompanied by undigested curd, with a mortality rate as high as 100%.

[0003] Porcine epidemic diarrhea (PED) is a contagious intestinal disease in pigs caused by porcine epidemic diarrhea virus (PEDV), characterized by severe diarrhea, vomiting, and loss of appetite in suckling piglets. PEDV is primarily transmitted via the fecal-oral route, replicating in the small intestinal epithelial cells, leading to the destruction and necrosis of infected cells. Diarrhea and vomiting will be observed after a period of time, and it is highly lethal to piglets under 2 weeks of age.

[0004] Since both TGE and PED are highly lethal to piglets under 2 weeks old, it is necessary to vaccinate piglets under 2 weeks old against both TGE and PED in order to improve their survival rate. Currently, TGE and PED vaccines need to be administered separately, which is cumbersome and costly. Therefore, there is a need to provide a vaccine that can provide immunity against both TGE and PED simultaneously.

[0005] Both TGEV and PEDV belong to the genus Coronavirus within the family Coronaviridae, and both are single-stranded positive-sense RNA viruses. Since their infection process is primarily mediated by the spike protein (S protein) of the coronavirus, currently known recombinant vaccines typically embed the S protein. However, the inventors have discovered that simultaneously embedding the S proteins of both TGEV and PEDV can affect the strength of the immune response elicited by the vaccine. An excessively strong immune response can lead to certain side effects. Furthermore, the S protein is a large type I transmembrane glycoprotein, and embedding two excessively large S proteins simultaneously can reduce the stability of the recombinant vaccine and its expression efficiency in host cells, thus decreasing vaccine efficacy. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a recombinant adenovirus vector expressing the PEDV-TGEV receptor-binding domain, its preparation method, and its applications. This invention screens the optimal eukaryotic plasmid capable of expressing the PEDV-TGEV receptor-binding domain antigen protein by evaluating cellular and humoral immune responses in mice. A replication-deficient viral vector capable of efficiently expressing the PEDV-TGEV receptor-binding domain is further packaged, exhibiting good immunogenicity and the ability to induce mucosal, cellular, and humoral immune responses, thereby significantly enhancing immunity. This vector shows broad application prospects in the immunotherapy of porcine transmissible gastroenteritis and porcine epidemic diarrhea.

[0007] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a recombinant adenovirus vector expressing a PEDV-TGEV receptor binding domain, which contains a fusion gene; The fusion gene encodes the receptor-binding domains of TGEV and PEDV; The amino acid sequence of the receptor-binding domain of the TGEV is shown in SEQ ID NO:1; The amino acid sequence of the receptor-binding domain of the PEDV is shown in any one of SEQ ID NO:2 to SEQ ID NO:5.

[0008] The receptor-binding domain (RBD) is typically located in the extracellular region of the viral spike protein (S protein) or receptor-binding protein (RBP / G protein), and is the smallest functional unit mediating viral attachment to receptors on the surface of host cells. The RBD possesses immunogenicity, is a key region for viral binding to host cells, and has broad-spectrum protective potential, enhancing the immune response against potential variants. Therefore, this invention selects the RBDs of TGEV and PEDV as antigen genes for insertion into human type 5 defective adenovirus.

[0009] In existing technologies, there are reports on constructing expression vectors for the receptor domains of TGEV and PEDV separately. However, there are no studies on simultaneously expressing both in the same expression vector. This is because there are multiple viral strains of TGEV and PEDV with significantly different immunogenicity. Furthermore, the selection of the receptor-binding domain sequence segment also affects the expression of different fusion genes, thus impacting the therapeutic activity of the protein. Therefore, to obtain a vector for efficiently expressing the TGEV-PEDV receptor-binding domain, this invention selects amino acid sequences at different positions in the S1 protein of the Qingdao strain of PEDV as the RBD region and combines them with the wild-type TGEV-RBD to construct a polynucleotide expressing the TGEV-PEDV receptor-binding domain, in order to screen for polynucleotide sequences capable of efficiently expressing the RBD proteins of PEDV and TGEV.

[0010] Furthermore, the nucleotide sequence of the gene encoding the RBD protein derived from TGEV is shown in SEQ ID NO:6; the nucleotide sequence of the gene encoding the RBD protein derived from PEDV is shown in any one of SEQ ID NO:7 to SEQ ID NO:10.

[0011] Furthermore, the nucleotide sequence of the fusion gene is shown in any one of SEQ ID NO:11 to SEQ ID NO:14.

[0012] Furthermore, the nucleotide sequence of the fusion gene is shown in SEQ ID NO:14.

[0013] Secondly, the present invention provides a method for preparing the above-mentioned recombinant adenovirus vector, including the step of inserting a fusion gene into the vector.

[0014] Furthermore, the fusion gene is inserted into the restriction enzyme sites of the vector, wherein the restriction enzyme sites are EcoRI and BglII.

[0015] Furthermore, the vector is a human type 5 defective adenovirus vector.

[0016] Human type 5 defective adenovirus is an adenovirus lacking the E1 and E3 genes. The E1 gene is an essential gene for controlling viral replication, while the E3 gene is a non-essential gene for replication. Its deletion can increase the insertion capacity. Meanwhile, PEDV and TGEV mainly induce mucosal immunity in piglets, while human type 5 adenovirus has the characteristic of mucosal infection. Therefore, this invention selects human type 5 defective adenovirus as a vector, so that the vaccine can effectively simulate the interaction between the virus and host cells during natural infection, which is conducive to stimulating the body to produce a mucosal response and stimulating the immune system to produce a protective response, thereby achieving the purpose of preventing viral infection.

[0017] Furthermore, the human type 5 defective adenovirus vector is pDC316-mCMV.

[0018] Thirdly, the present invention provides a vaccine comprising the aforementioned recombinant adenovirus vector and adjuvant.

[0019] The present invention also provides a method for preparing the above-mentioned vaccine, comprising the following steps: Recombinant adenovirus strains were obtained by packaging the recombinant adenovirus vector described in this invention; The recombinant adenovirus strain is obtained by mixing it with an adjuvant.

[0020] Furthermore, the vaccine formulation is a live vaccine.

[0021] Fourthly, the present invention provides a composition comprising the above-described recombinant adenovirus vector.

[0022] Furthermore, the composition may include a buffer and may also include other substances suitable for the intended use.

[0023] Those skilled in the art can readily select a suitable buffer for the intended use, many of which are known in the art. In some cases, the composition may contain pharmaceutically acceptable excipients, many of which are known in the art and need not be discussed in detail herein.

[0024] Fifthly, the present invention provides the use of the above-mentioned recombinant adenovirus vector or vaccine or composition in the preparation of drugs for the prevention or treatment of porcine transmissible gastroenteritis and / or porcine epidemic diarrhea.

[0025] Furthermore, the present invention also provides a method for treating porcine transmissible gastroenteritis and / or porcine epidemic diarrhea, comprising administering to a subject an effective dose of a recombinant adenovirus vector or vaccine or composition as defined herein.

[0026] Furthermore, the subjects were mice or piglets.

[0027] As used herein, the term "effective dose" includes, within its meaning, a non-toxic but sufficient amount of recombinant adenovirus vector or composition to provide the desired therapeutic effect. The exact amount required will vary from subject to subject, depending on factors such as the species being treated, the subject's age and general condition, the severity of the condition being treated, the specific drug administered, and the method of administration. The effective dose is sufficient to induce an immune response in the subject, thereby preventing and / or treating porcine transmissible gastroenteritis and / or porcine epidemic diarrhea. Therefore, it is impossible to specify an exact "effective dose." However, for any given situation, a suitable "effective dose" can be determined by a person skilled in the art using only routine experimental methods.

[0028] The terms “treatment” and “curing” are used interchangeably in this article to mean the relief, reduction, alleviation, improvement or other suppression of a condition, including one or more symptoms of the condition.

[0029] The terms “treatment,” “cure,” and similar terms also include the reduction, decrease, relief, improvement, or other suppression of the effects of a condition for at least a period of time. It should also be understood that the terms “treatment,” “cure,” and similar terms do not imply a permanent reduction, decrease, relief, improvement, or other suppression of the condition or its symptoms, and therefore also include temporary reduction, decrease, relief, improvement, or other suppression of the condition or its symptoms.

[0030] The beneficial effects of this invention are as follows: This invention discloses a recombinant adenovirus vector, using a human type 5 defective adenovirus vector as the backbone vector, containing a fusion gene encoding a receptor-binding domain protein. The fusion gene includes an RBD gene fragment of PEDV and an RBD gene fragment of TGEV. Experiments have shown that the recombinant adenovirus vector of this invention can efficiently express the TGE-PED receptor-binding domain protein and has good immunogenicity. In animal model experiments, the antigen protein can induce mucosal immunity, cellular immunity, and humoral immunity responses, thereby significantly improving immunity. It has broad application prospects in the field of immunotherapy for porcine transmissible gastroenteritis and porcine epidemic diarrhea. Attached Figure Description

[0031] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0032] Figure 1 This is a schematic diagram showing the results of plasmid transfection under a fluorescence microscope in an embodiment of the present invention; wherein, A is the control plasmid pDC316-mCMV-GFP, B is the recombinant plasmid pDC316-mCMV-S11-TGEV-RBD, C is the recombinant plasmid pDC316-mCMV-S12-TGEV-RBD, D is the recombinant plasmid pDC316-mCMV-S13-TGEV-RBD, and D is the recombinant plasmid pDC316-mCMV-S14-TGEV-RBD.

[0033] Figure 2 The figure shows the activation results of mouse germinal center B cells using recombinant plasmids in this embodiment of the invention; This indicates that P < 0.05. P < 0.01, and ns indicates no significant difference.

[0034] Figure 3The figures show the mouse neutralizing antibody titers of the recombinant plasmid in this embodiment of the invention; This indicates that P < 0.01. P < 0.001, and ns indicates no significant difference.

[0035] Figure 4 The above are the results of quantitative fluorescence identification of recombinant adenovirus and control adenovirus in cell pellet and lysed cell supernatant in this embodiment of the invention. Ad-GFP and Ad-PT-RBD represent control adenovirus and recombinant adenovirus extracted from cell pellet, respectively, and Ad-GFP-m and Ad-PT-RBD-m represent control adenovirus and recombinant adenovirus extracted from lysed cell supernatant, respectively.

[0036] Figure 5 The figures show the expression results of β-actin (internal reference) and the expression results of the target protein of recombinant adenovirus obtained in the embodiments of the present invention. In the figure, 1 represents recombinant adenovirus that does not express the target gene, and 2 represents recombinant adenovirus that expresses the target gene.

[0037] Figure 6 The results of indirect immunofluorescence assay in this embodiment of the invention show the expression of recombinant adenovirus RBD-PEDV-TGEV and empty vector in infected HEK 293 A cells.

[0038] Figure 7 The figures show the results of flow cytometry analysis of B cells in the germinal centers of the mediastinal lymph nodes of mice after booster immunization with PBS, porcine transmissible gastroenteritis and porcine epidemic diarrhea bivalent inactivated vaccine, Ad-RBD-PEDV-TGEV recombinant adenovirus vector vaccine, and Ad-GFP recombinant adenovirus vector vaccine, respectively. This indicates that P < 0.01. P < 0.001, and ns indicates no significant difference.

[0039] Figure 8 In this embodiment of the invention, mice in the PBS group, porcine transmissible gastroenteritis and porcine epidemic diarrhea bivalent inactivated vaccine group, Ad-RBD-PEDV-TGEV recombinant adenovirus vector vaccine group, and Ad-GFP recombinant adenovirus vector vaccine group were boosted with IgA of mediastinal lymph node B cells by flow cytometry. + The test results are shown in the image. The expression ns indicates that there is no significant difference.

[0040] Figure 9The figures show the results of a neutralizing antibody assay used to detect the levels of mouse-specific neutralizing antibodies after booster immunization with mice in the PBS group, the porcine transmissible gastroenteritis and porcine epidemic diarrhea bivalent inactivated vaccine group, the Ad-RBD-PEDV-TGEV recombinant adenovirus vector vaccine group, and the Ad-GFP recombinant adenovirus vector vaccine group, respectively. This indicates that P < 0.01. P < 0.001, and ns indicates no significant difference.

[0041] Figure 10 The figure shows the clinical scoring results of diarrhea in piglets after PEDV challenge in this embodiment of the invention. P < 0.01, and ns indicates no significant difference. Detailed Implementation

[0042] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included within the scope of this invention. The upper and lower limits of these smaller ranges may be included independently within the range.

[0043] Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof. Experimental methods described in the following detailed embodiments, unless specific conditions are specified, are generally performed according to conventional methods and conditions in molecular biology within the art, which are fully explained in the literature. See, for example, the techniques and conditions described in Sambrook et al., *Molecular Cloning: A Laboratory Manual*, or according to the manufacturer's recommendations.

[0044] The present invention will be further illustrated with specific examples. These examples are for illustrative purposes only and do not limit the scope of the invention. Unless otherwise specified, experimental conditions not explicitly stated in the examples are generally performed under conventional conditions or as recommended by the selling company. Materials and reagents used in the examples, unless otherwise specified, are commercially available.

[0045] The experimental materials and reagents involved in the examples are as follows: Vectors, strains and cells *E. coli* Trans-T1 competent cells were purchased from Beijing TransGen Biotech Co., Ltd.; pDC316-mCMV-GFP and pBHGlox(delta)E13Cre were purchased from Wuhan Miaoling Biotechnology Co., Ltd.; and HEK-293A cells were purchased from Shanghai Saibaikang Biotechnology Co., Ltd. Wild-type TGEV and Qingdao PEDV strains were preserved in our laboratory. HEK293A cells were preserved in our laboratory.

[0046] laboratory animals Six-week-old SPF-grade female BALB / c mice were purchased from Spiefer Beijing Biotechnology Co., Ltd.; they were housed in the SPF-grade breeding room of the Innovation Building of Jilin Agricultural University and were tested after acclimatization for one week.

[0047] Enzymes and main experimental reagents Blastaq™ 2× qPCR MasterMix kit was purchased from abm; fluorescent nucleic acid staining reagent was purchased from TransGen Biotech Ltd.; total RNA extraction kit was purchased from Beijing Huaxin Kangxin Biotechnology Co., Ltd.; DNA kit and agarose gel DNA recovery kit were purchased from Tiangen Biotech; DEPC (RNA-free) H2O was purchased from BDBiosciencesbdbiosciences.com; penicillin-streptomycin mixture was purchased from Hyclone; 180kDa Prestained Protein Marker was purchased from Vazyme; 4% paraformaldehyde universal tissue fixative was purchased from Biosharp Biotechnology Co., Ltd.; DMEM / HIGHGLUCOSE was purchased from Gibco; anti-fluorescence attenuation mounting medium was purchased from Beijing Solarbio Science & Technology Co., Ltd.; X-treme GENE HPDNA Transfection Reagen transfection reagent was purchased from Jiangsu Kangcheng Bio-Tech Co., Ltd.; Premix Taq, DL2000, DL 10000 DNA Marker, and SDS-PAGE Loading Buffer were also purchased. (5×) endonucleases EcoRI and BglII were purchased from Takara; Albumin Bovine V was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; T4 DNA ligase was purchased from Takara; ProteinExt Mammalian Membrane Protein Extraction Kit and Trans1-T1 Phage Resistant Chemical Competent Cell were purchased from TransGen Biotech Co., Ltd.; Omni-Easy™ One-Step PAGE Gel Preparation Kit and Coomassie Brilliant Blue Rapid Staining Solution were purchased from Yamei Biopharmaceutical Technology Co., Ltd.; PBS phosphate buffer (powder) was purchased from Thermo Fisher Scientific (China) Co., Ltd. Mouse ear tags, scissors, forceps, 1.5 mL EP tubes, small gavage needles, blood collection tubes, PBS, 24-well cell culture plates, and Cell 1640 culture medium were all purchased from Beijing TransGen Biotech Co., Ltd.; flow cytometry antibodies CD3, CD4, CD8, CD11, CD80, CD86, B220, IgA, IFN-γ, and IL-4 were all purchased from BD Biosciences.

[0048] Main instruments and equipment The following equipment was used: Leica DMi8 inverted fluorescence microscope (Germany); Eppendorf mini high-speed centrifuge (Germany); Eppendorf 4°C refrigerated centrifuge (Centrifuge 5810R) (Germany); Eppendorf gradient PCR instrument (Germany); metal bath from Beijing Tiangen Biotech Co., Ltd. (Germany); Eppendorf pipettes (Germany); Sigma GR85DA fully automated autoclave (Germany); Eppendorf constant temperature shaker incubator (Germany); BIO-RAD gel imaging analysis system (Universal Hood II, USA); Mettler Toledo precision electronic balance (ME204, Switzerland); BIOTeK microplate spectrophotometer (Epoch 2, USA); Leica RM2245 fully automated paraffin embedding machine (Germany); Thermo Scientific CO2 incubator (HERACELL 240i, USA); BIO-RAD electrophoresis apparatus (USA); and BD LSRFortessa™ flow cytometer (USA). Low-temperature ultracentrifuge (Eppendorf 5810R); SW-CJ-2FD single-sided double-person clean bench (Shanghai Boxun); HRLM-80 fully automatic autoclave; BCD-649WDCE refrigerator (Haier).

[0049] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0050] Example 1: Preparation and validation of recombinant adenovirus vector expressing PED and TGE RBD proteins 1. Obtain the RBD encoded sequences of PEDV and TGEV: The gene sequence encoding the S1 protein of PEDV was found in GenBank. Primers were designed to obtain the partial S1 gene sequences of PEDV, as shown in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10. The gene sequence encoding the RBD protein of TGEV (SEQ ID NO:6) was found in GenBank.

[0051] Based on the above gene sequences, four fusion genes composed of the S1 gene sequence of Kozak and PEDV and the RBD gene sequence of TGEV were designed. These fusion genes were sent to a company for synthesis to obtain DNA fragments of the S11-TGEV-RBD (SEQ ID NO:11), S12-TGEV-RBD (SEQ ID NO:12), S13-TGEV-RBD (SEQ ID NO:13), and S14-TGEV-RBD (SEQ ID NO:14) fusion genes. Using the above fusion genes as templates, the primer sequences shown in Table 1 were designed for PCR amplification. The amplified fragments were then ligated into pEASY-Blunt Zero Cloning Vector (PLEZ) and sequenced to verify their correctness.

[0052] Table 1 Amplification Primer Sequences

[0053] Using the pEASY-Blunt Zero Cloning plasmid containing S11-TGEV-RBD, S12-TGEV-RBD, S13-TGEV-RBD, and S14-TGEV-RBD as templates, the target genes S11-TGEV-RBD, S12-TGEV-RBD, S13-TGEV-RBD, and S14-TGEV-RBD were obtained after double digestion with EcoRI and BglII and gel recovery. Using pDC-316-mCMV-HA as a template, the pDC316-mCMV gene fragment was obtained after double digestion with EcoRI and BglII and gel recovery. The S11-TGEV-RBD, S12-TGEV-RBD, S13-TGEV-RBD, and S14-TGEV-RBD gene fragments were ligated to the pDC316-mCMV gene fragment to obtain recombinant plasmids pDC316-mCMV-S11-TGEV-RBD, pDC316-mCMV-S12-TGEV-RBD, pDC316-mCMV-S13-TGEV-RBD, and pDC316-mCMV-S14-TGEV-RBD, respectively. Plasmid sequencing confirmed the correct ligation of these recombinant plasmids.

[0054] 2. Recombinant plasmid transfection The recombinant eukaryotic plasmids pDC316-mCMV-S11-TGEV-RBD, pDC316-mCMV-S12-TGEV-RBD, pDC316-mCMV-S13-TGEV-RBD, pDC316-mCMV-S14-TGEV-RBD, and pDC316-mCMV-GFP (control plasmid) were combined with Lipofectamine. TM3000X-treme GENE HP DNA Transfection Reagen was diluted with serum-free and antibiotic-free medium and transfected into HEK 293A cells using liposome-mediated transfection. Detailed transfection procedures were described in the Lipofectamine reference. TM Follow the instructions in 3000. The transfection results for each group were observed under a fluorescence microscope, and the results are as follows: Figure 1 As shown in Figures A, B, C, D, and E, fluorescent proteins were expressed in each group, proving that the plasmids in each group were successfully constructed.

[0055] 3. Immunizing mice Fifty-six BALB / c mice were randomly divided into seven groups (n=8): PBS immunization control group, commercial vaccine (Vaccine) positive control group, plasmid control group, pDC316-mCMV-S11-TGEV-RBD group (S11), pDC316-mCMV-S12-TGEV-RBD group (S12), pDC316-mCMV-S13-TGEV-RBD group (S13), and pDC316-mCMV-S14-TGEV-RBD group (S14). The immunization regimen is shown in Table 2. The immunization dose was 100 μL / mouse, and the concentration of each plasmid was 1 μg / μL, administered via intramuscular injection in the leg. The initial immunization and booster immunization were performed two weeks apart. Blood was collected from the tail of each mouse one day before the initial immunization and again two weeks after the booster immunization. Serum was separated and stored at -20℃ for later use.

[0056] Table 2 Immunization Schedule

[0057] The activation status of B cells in the germinal centers of mouse inguinal lymph nodes was detected using the following method: (1) Two weeks after booster immunization, four mice from each group were euthanized to obtain inguinal lymph nodes and prepare single-cell suspensions. 1×10n cells were taken from each sample. 6 / 100 μL. (2) Take out the intracellular single-label tube, ISO empty cell tube and 3 surface single-label tubes, put them in a 4℃ refrigerator, resuspend the remaining samples, stain with antibody, add 30 μL of mixed antibody to each of the 30 sample tubes, take out the 3 surface single-label tubes and add 10 μL of the corresponding antibody to each tube, and keep them in the dark at 4℃ for 20 min. (3) Add 1 mL of PBS with 2% serum to the 30 sample tubes and 3 surface single-label tubes and wash once, centrifuge at 2000 r / min for 5 min at 4℃, discard the supernatant and keep 100 μL. (4) Fix the 30 sample tubes washed in (3) and the 1 intracellular single-label tube placed in the 4℃ refrigerator, add 200 μL of formaldehyde to them, and keep them in the dark at 4℃ for 20 min. Add 1 mL of membrane penetration solution (1×) to the 31 fixed sample tubes, let stand at room temperature for 5 min, centrifuge at 2500 r / min for 5 min at 20℃, discard the supernatant and keep 100 μL. (5) Resuspend the 31 samples after membrane rupture in (4), add 10 μL of IgA antibody to each tube, and incubate at 4°C in the dark for 25 min. (6) Add 1 mL of PBS containing 2% serum and sodium azide, centrifuge at 2500 r / min for 5 min at 4°C, discard the supernatant, and keep 100 μL. (7) Take out the samples from (6) and the 3 single-label tubes placed in the 4°C refrigerator, add 200 μL of PBS containing 2% serum and 1% sodium azide to each tube, and pass them through a membrane for testing.

[0058] The results are as follows Figure 2 As shown, the recombinant plasmid pDC316-mCMV-S14-TGEV-RBD exhibits superior activation effects compared to other recombinant plasmids.

[0059] 4. Virus neutralization test (1) Seed Vero cells in 96-well cell culture plates and incubate at 37°C for 12 hours when the cell density reaches 80%. (2) Prepare a virus maintenance solution containing 20 mg / mL trypsin and 1% penicillin antibody. Serially dilute the collected serum samples with the prepared virus maintenance solution. (3) Mix the diluted solution with an appropriate amount of PEDV in a proportional manner. Incubate the mixture containing virus and serum in a 37°C incubator at 5% CO2 for 1 hour. (4) Discard the virus maintenance solution from (1), wash the cells three times with PBS, and then add the solution incubated for 1 hour to the 96-well cell culture plates from which the virus maintenance solution was discarded. Perform 5 replicates per group and incubate in a 37°C incubator at 5% CO2 for 1 hour. (5) Discard the liquid in the 96-well cell culture plate from (4), add the virus maintenance solution prepared in (2), and incubate in an incubator containing 5% CO2 at 37°C for 3-4 days. Record the lesion condition, analyze the neutralizing antibody titer using the Reed-Muench method, and calculate the highest serum dilution that can protect 50% of the cultured cells from CPEs.

[0060] The results are as follows Figure 3 As shown, the recombinant plasmid pDC316-mCMV-S14-TGEV-RBD exhibits superior neutralization performance compared to other recombinant plasmids.

[0061] Therefore, the recombinant plasmid pDC316-mCMV-S14-TGEV-RBD was selected for further experiments.

[0062] Example 2: Preparation and validation of recombinant adenovirus vector expressing PED and TGE RBD proteins 1. Packaging and TCID of recombinant adenovirus 50 Measurement (1) Cell passage: Discard the culture medium in the HEK-293A cell culture flask purchased from Shanghai Saibaikang Biotechnology Co., Ltd., add 3 mL PBS, wash away the serum, add 1 mL trypsin and place in a 37℃ incubator for about 1 min to digest. Immediately add 1 mL 10% FBS cell culture medium to stop digestion. Transfer the cells and supernatant to a 15 mL centrifuge tube, centrifuge at 1000 rpm for 5 min at 4℃, discard the supernatant, add 1 mL 10% FBS cell culture medium and slowly pipette the cells multiple times. Finally, transfer the cells to a T25 cell culture flask, add 4 mL 10% FBS culture medium to the T25 cell culture flask, and place in a 37℃ cell culture incubator for culture.

[0063] (2) Packaging of recombinant adenovirus: Transfection began when the density of 9th generation 293A cells reached approximately 30%. X-tremeGENE HP DNA Transfection Reagen, DNA (the eukaryotic plasmid pDC316-mCMV-S14-TGEV-RBD prepared in Example 1 and showing the best screening effect in animal experiments) were mixed with DMEM. 3 μg of recombinant plasmid (pDC316-mCMV-S14-TGEV-RBD) and 3 μg of backbone (pBHGlox(delta)E13Cre) were added to 600 μL of serum-free DMEM and gently mixed. X-tremeGENE HP DNA Transfection Reagen was directly added to the culture medium containing diluted DNA, gently mixed, and incubated for 15 min. The cell culture dish was then removed from the incubator, and the transfection complex was added dropwise directly to the cell culture dish.

[0064] Twenty-four hours after transfection, the cells were observed under a fluorescence microscope to detect green fluorescent protein expression. They were then incubated at 37°C for 14 days. During this period, the transfection efficiency and viral load were monitored by observing the expression of fluorescent protein and CPE (cell detachment). When approximately 70% of the cells detached, the adherent cells were repeatedly pipetted to collect the cells and supernatant into 15 mL centrifuge tubes. The tubes were centrifuged at 5000 rpm for 5 min at 5°C, and 4 mL of supernatant was discarded, leaving only 1 mL. The tubes were then repeatedly and slowly pipetted, and the cells were subjected to three freeze-thaw cycles at -80°C and 4°C. The supernatant collected after centrifugation at 12000 rpm for 2 min at 4°C was the first-generation virus. (Simultaneously, an empty adenovirus vector was packaged as a negative control.) (3) Amplification of recombinant adenovirus: 293A cells were seeded in T25 cell flasks. When the cell density reached approximately 70%, the culture medium in the cell flasks was replaced with maintenance medium. 100 μL of first-generation virus was premixed in 1 mL of maintenance medium. The premixed 1 mL of maintenance medium containing the virus was then added to the T25 cell flasks. Fluorescence was observed after 24 hours, and cytopathic effects were very pronounced 2 / 3 days after infection. After harvesting the virus, 293A cells in T75 cell flasks were reinfected. The collected virus solution was the third-generation virus (the recombinant adenovirus was named Ad-PT-RBD). It was frozen and stored at -80°C for later use.

[0065] (4) Recombinant adenovirus TCID 50 Assay: 293A cells were digested, passaged, and seeded into 96-well plates. Once the cell density reached 30%, two recombinant adenovirus strains were inoculated into two 96-well plates. The procedure was as follows: 990 μL of maintenance medium was added to a 1.5 mL centrifuge tube, and 10 μL of the second-generation virus solution was added. The mixture was then serially diluted 10-fold to a final volume. -10100 μL of diluted virus solution was added to each 96-well cell culture plate, with a blank control group included. Eight replicates were performed for each dilution gradient. The plates were incubated at 37°C. On day 7, the 96-well plates were removed and observed under a fluorescence microscope to monitor fluorescence expression at each dilution gradient. The number of fluorescent wells for each gradient was recorded, and the viral titer was calculated using the Reed-Muench method.

[0066] 2. Quantitative fluorescence identification of recombinant adenovirus DNA from two recombinant adenoviruses, Ad-GFP and Ad-PT-RBD, was extracted from cell pellets and lysed cell supernatants using a viral genome extraction kit. Triple replicates were performed for each group, followed by quantitative real-time PCR. Results are shown below. Figure 4 As shown, after 293A cells were infected with Ad-PT-RBD recombinant adenovirus, the PT-RBD gene could be detected in the cell pellet, but the PT-RBD gene level was even higher in the lysed cell supernatant.

[0067] 3. Identification results of the target protein of recombinant adenovirus Two flasks of monolayer 293A cells were prepared. When the cell count reached 80%, two different recombinant adenoviruses were inoculated separately. Cells were collected after 24 hours, and total cellular protein was extracted. The target protein of the recombinant adenovirus was detected using serum from immunized PEDV mice as the primary antibody and alpaca anti-mouse IgG antibody. The expression of the target protein of the recombinant adenovirus was then assessed. Results are as follows: Figure 5 As shown, PT-RBD fusion protein expression was detected at approximately 35.4 kDa in the recombinant adenovirus lane, consistent with the expected results.

[0068] 4. Indirect immunofluorescence assay to determine the expression levels of recombinant adenovirus vector and empty vector in infected HEK 293A cells. (1) Digest and passage 293A cells and seed them into 24-well cell plates, with 1.5 × 10⁶ cells per well. 6Cells, 1 mL of 10% medium, after 12 hours the cells were completely adhered to the plate. 5 μL of recombinant PEDV-TGEV-RBD and 5 μL of empty vector virus without the target gene were inoculated into well plates (premixed in 10 μL of 2% medium) and incubated at 37℃ for 24 hours. Two parallel test wells were set up for each group. (2) Discard the medium and wash the cell wells with about 500 μL of PBS. (3) Add 4% paraformaldehyde placed at 4 degrees to the cell wells for fixation at room temperature for 15 min. (4) Discard the fixative and then add 400 μL of pre-cooled formic acid for 10 min (methanol was placed at -20℃ 20 min in advance). (5) Wash 3 times with PBST, add 5% BSA, and block at room temperature for 1 h. (6) Discard the blocking solution and incubate 200 μL of primary antibody (using TGEV-infected mouse serum as the primary antibody) (1:100 diluted with 5% BSA) at 4°C overnight. (7) Discard the primary antibody, wash 3 times with PBST for 3 min each time, and add 100 μL of diluted secondary antibody to each well. Incubate in the dark for 1 h. (8) Discard the fluorescent secondary antibody, wash 3 times with PBST for 3 min each time, and add diluted DAPI to each well. Incubate at room temperature in the dark for 5 min. (9) Discard the DAPI, wash 3 times with PBST for 3 min each time, add a small amount of fluorescence quencher, and observe under a fluorescence microscope.

[0069] The results are as follows Figure 6 As shown, compared with the Ad-GFP group, the Ad-PT-RBD recombinant adenovirus exhibits significantly higher red fluorescence intensity, indicating that the Ad-PT-RBD recombinant adenovirus successfully expresses the fusion protein.

[0070] Example 3 1. Animal experiments Example 2 successfully constructed two recombinant adenovirus vectors and expressed them in vitro. In this example, BALB / c mice were selected as experimental animals for preliminary animal experiments. Mice were immunized by intramuscular injection. Changes in humoral immunity, cellular immunity, and mucosal immunity of immunized mice were detected by flow cytometry and neutralizing antibody assays, thereby studying the effect of the RBD recombinant adenovirus expressing PEDV and TGEV on the immunity of immunized animals.

[0071] Thirty-two mice were randomly divided into four groups of eight each. Each group received an intramuscular injection of PBS, porcine transmissible gastroenteritis and porcine epidemic diarrhea (TIG / PD) bivalent inactivated vaccine (purchased from Anhui Dongfang Diwei Biological Products Co., Ltd.), Ad-PT-RBD recombinant adenovirus vector vaccine, or Ad-GFP recombinant adenovirus vector vaccine via the inner thigh on day 0 and day 7, respectively. Each mouse was labeled with a number representing eight natural numbers in each group. The specific experimental grouping scheme is shown in Table 3.

[0072] Table 3 Animal Immunization and Grouping Scheme

[0073] Blood was collected from the parotid vein. Using a venipuncture needle, the mouse's parotid region was located, and the needle was gently rotated to insert. After bleeding, the blood was drained into a 1.5 mL EP tube, and 0.2 mL of blood was collected. Venous blood was collected every 14 days. Fecal collection was performed every 7 days. Flow cytometry analysis was conducted 7 days after the second booster immunization.

[0074] 2. Study on the immunogenicity of recombinant adenovirus in mice Preparation of spleen cell suspension and lung mediastinal lymph node suspension: (1) After blood was collected from the eyeballs of mice, they were sacrificed. The mice were sprayed with 75% alcohol for disinfection and placed in a large petri dish. The spleen and lung mediastinal lymph nodes were removed and placed on a small petri dish with a copper mesh. 1 mL of RPMI Medium 1640 culture medium containing 2% serum was added to them. (2) Gently grind the cells with the end of a 1 mL syringe until no tissue is visible (place on ice after grinding), then transfer the filtered cell solution from the petri dish into a 1.5 mL EP tube; wash once with 1 mL of RPMI Medium 1640 medium containing 2% serum (centrifuge at 2000 r / min for 5 min at 4℃), and keep 100 μL; (3) Add 1 mL of erythrocyte lysis buffer, lyse at room temperature in the dark for 10 min, then add 400 μL of RPMI Medium 1640 medium containing 2% serum to stop the lysis; centrifuge at 2000 r / min for 5 min at 4℃, discard the supernatant and keep 100 μL; (4) Wash once again with 1 mL of RPMI Medium 1640 medium containing 2% serum; (5) Resuspend the cells in 1 mL of RPMI Medium 1640 medium containing 2% serum, and the resulting solution is the original spleen cell solution.

[0075] Splenic cell and mediastinal lymph node counts: Splenic cells were diluted 50-fold (500 μL = 10 μL stock solution + 490 μL PBS), thoroughly resuspended, and counted. Mediastinal lymph node cells were diluted 10-fold (100 μL = 10 μL stock solution + 90 μL PBS), thoroughly resuspended, and counted.

[0076] 4. Detection of B cells in the germinal centers of mouse spleen (1) Take 34 labeled 1.5 mL EP tubes (30 samples, 3 single labels, 1 ISO empty cell, 34 tubes in total), and add 1×10 to each tube. 6Cell stock solution, then add 1 mL of PBS containing 2% serum to each tube, centrifuge at 2000 r / min for 5 min at 4℃, discard the supernatant and keep 100 μL; take out the ISO empty cell tube and put it in a 4℃ refrigerator. Premixed antibody. (2) Take out 3 surface single-label tubes, add 10 μL of the corresponding antibody to each tube, and then add 30 μL of the mixed antibody to each of the remaining 30 sample tubes, and keep them in the dark for 20 min at 4℃. (3) Add 1 mL of PBS containing 2% serum to the 30 sample tubes and 3 surface single-label tubes to wash once, centrifuge at 2000 r / min for 5 min at 4℃, discard the supernatant and keep 100 μL. (4) Take out the 33 sample tubes washed in (3) and 1 ISO empty cell tube placed in a 4℃ refrigerator, add 1 mL of PBS containing 2% serum and 1% sodium azide, centrifuge at 2500 r / min for 5 min at 4℃, discard the supernatant and keep 100 μL. (5) Add 200 μL of PBS containing 2% serum and 1% sodium azide to each tube, and pass it through a membrane for instrumentation.

[0077] The results are as follows Figure 7 As shown, the results indicated that, compared to the PBS group, the B220 level in the Ad-PT-RBD group, administered via intramuscular injection into the inner thigh, was significantly lower. + CD95 + GL7 + The proportion of GC B cells increased significantly (P<0.05).

[0078] 5. Detection of IgA in B cells of mediastinal lymph nodes in mice. + Expression of situation (1) Take 35 labeled 1.5 mL EP tubes (30 samples, 4 single labels, 1 ISO empty cell, 35 tubes in total), and add 1×10 to each tube. 6(1) Add 1 mL of PBS containing 2% serum to each tube, centrifuge at 2000 r / min for 5 min at 4℃, discard the supernatant, and keep 100 μL. (2) Take out the intracellular single-label tube, ISO empty cell tube and 3 surface single-label tubes, put them in a 4℃ refrigerator, resuspend the remaining samples, stain with antibody, add 30 μL of mixed antibody to each of the 30 sample tubes, take out the 3 surface single-label tubes and add 10 μL of the corresponding antibody to each tube, and protect from light at 4℃ for 20 min. (3) Add 1 mL of PBS containing 2% serum to the 30 sample tubes and 3 surface single-label tubes and wash once, centrifuge at 2000 r / min for 5 min at 4℃, discard the supernatant, and keep 100 μL. (4) Fix the 30 sample tubes washed in (3) and the 1 intracellular single-label tube placed in a 4℃ refrigerator, add 200 μL of formaldehyde, and protect from light at 4℃ for 20 min. Add 1 mL of membrane perforation solution (1×) to the 31 fixed sample tubes, let stand at room temperature for 5 min, centrifuge at 2500 r / min for 5 min at 20℃, discard the supernatant, and keep 100 μL. (5) Resuspend the samples after membrane perforation in (4), add 10 μL of LigA antibody to each tube, and incubate at 4℃ in the dark for 25 min. (6) Add 1 mL of PBS containing 2% serum and sodium azide, centrifuge at 2500 r / min for 5 min at 4℃, discard the supernatant, and keep 100 μL. (7) Take out the samples from (6) and the 3 single-label tubes placed in the 4℃ refrigerator, add 200 μL of PBS containing 2% serum and 1% sodium azide to each tube, and pass them through the membrane for testing.

[0079] The results are as follows Figure 8 As shown, compared with the PBS group, the Ad-PT-RBD group and the Vaccine group had B220 + IgA + The proportion of B cells was significantly increased (P<0.05). This result indicates that recombinant Ad-PT-RBD adenovirus promotes the growth of B220 cells in the mediastinal lymph nodes of mice. + IgA + B cell expression.

[0080] 6. Virus neutralization test (1) Seed Vero cells in 96-well cell culture plates and incubate at 37°C for 12 hours after the cell density reaches 80%. (2) Prepare a virus maintenance solution containing 20 mg / mL trypsin and 1% penicillin antibiotics. Serially dilute the collected serum samples with the prepared virus maintenance solution. (3) Mix the diluted solution with 200 TCID50 solutions. 50(3) Mix the PEDV in equal proportions and incubate the mixture containing virus and serum in a 5% CO2, 37°C incubator for 1 hour. (4) Discard the virus maintenance medium from (1), wash three times with PBS, and then add the liquid from the incubator after 1 hour to a 96-well cell culture plate from which the virus maintenance medium has been discarded. Perform 5 replicates per group and incubate for 1 hour in a 5% CO2, 37°C incubator. (5) Discard the liquid in the 96-well cell culture plate from (4), add the virus maintenance medium prepared in (2), and incubate in a 5% CO2, 37°C incubator for 3-4 days. Record the lesion condition, analyze the neutralizing antibody titer using the Reed-Muench method, and calculate the highest serum dilution that can protect 50% of the cultured cells from CPEs.

[0081] The results are as follows Figure 9 As shown, compared with the PBS group, the level of neutralizing antibodies produced by mice immunized in the Ad-PT-RBD group was significantly increased (P<0.01). The results indicate that recombinant Ad-PT-RBD adenovirus can induce the production of neutralizing antibodies against PEDV in animals.

[0082] 7. Evaluation of the protective effect on piglets Sixteen 3-day-old piglets were randomly divided into four groups of four. Each group received an intramuscular injection in the neck, with the following doses: 1 mL of PBS and 1 mL of a combined inactivated vaccine for porcine transmissible gastroenteritis and porcine epidemic diarrhea (containing 3×10⁻⁶ mg / L). 8 TCID 50 ), 1 mL of Ad-PT-RBD recombinant adenovirus (267 μL virus solution + 733 μL PBS, containing 3 × 10⁻⁶ ppm). 8 TCID 50 ), 1 mL of Ad-GFP recombinant adenovirus (containing 3 × 10⁻⁶) 8 TCID 50 On day 8 post-immunization, a challenge experiment was conducted, in which four groups of immunized piglets were orally infected with laboratory-preserved PEDV. Each piglet was infected with 10 [units of something - likely a specific type of virus]. 8 TCID 50 PEDV.

[0083] To monitor diarrhea in piglets after PEDV challenge, we observed and photographed the piglets daily to record their diarrhea status. Based on these observations, we clinically scored the diarrhea status of piglets on day 4 post-challenge, categorizing it into five levels: no diarrhea (0 points), mild diarrhea (1 point), moderate diarrhea (2 points), severe diarrhea (4 points), and watery diarrhea (5 points). Figure 10As shown, compared with the PBS group, the Ad-PT-RBD group and the Vaccine group showed significant differences (P<0.05) in the absence of diarrhea, with the piglets in the Ad-PT-RBD group showing the best condition.

[0084] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A recombinant adenovirus vector expressing the PEDV-TGEV receptor-binding domain, characterized in that, Contains fusion genes; The fusion gene encodes the receptor-binding domains of TGEV and PEDV; The amino acid sequence of the receptor-binding domain of the TGEV is shown in SEQ ID NO:1; The amino acid sequence of the receptor-binding domain of the PEDV is shown in SEQ ID NO:5; The nucleotide sequence of the fusion gene is shown in SEQ ID NO:

14.

2. The recombinant adenovirus vector as described in claim 1, characterized in that, The gene sequence encoding the receptor-binding domain of the TGEV is shown in SEQ ID NO:6; the gene sequence encoding the receptor-binding domain of the PEDV is shown in SEQ ID NO:

10.

3. The method for preparing the recombinant adenovirus vector according to any one of claims 1 to 2, characterized in that, This includes the step of inserting the fusion gene into the vector.

4. The preparation method according to claim 3, characterized in that, The fusion gene is inserted into the restriction enzyme site of the vector.

5. The preparation method according to claim 4, characterized in that, The vector is a human type 5 defective adenovirus vector.

6. A vaccine, characterized in that, It comprises the recombinant adenovirus vector and adjuvant as described in any one of claims 1 to 2.

7. The composition, characterized in that, It includes the recombinant adenovirus vector according to any one of claims 1 to 2.

8. The use of the recombinant adenovirus vector according to any one of claims 1 to 2, the vaccine according to claim 6, or the composition according to claim 7 in the preparation of a drug for the prevention or treatment of porcine transmissible gastroenteritis and / or porcine epidemic diarrhea.

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