Recombinant plasmid and application thereof

Abstract

The invention discloses a recombinant plasmid and application thereof, and belongs to the technical field of gene engineering. The invention constructs a recombinant plasmid which comprises a signal peptide, a receptor binding domain RBD, heptapeptide repetitive sequences HR1 and HR2 and a flexible connecting peptide. Wherein the signal peptide has the functions of guiding protein transportation and positioning, and can accurately guide related peptide fragments to proper cell positions. And the peptide fragments HR1 and HR2 can be automatically assembled into a 6-spiral bundle structure. The S1 protein plays a core role in virus infection and immune response, and correct folding of the S1 protein is a basis for ensuring functional integrity of the S1 protein. The S1 protein is prepared by the recombinant plasmid constructed by the invention, and the S1 protein can be correctly folded in cells and effectively anchored on cell membranes. Therefore, the antigen epitope of the S1 protein can be better displayed, and favorable conditions are provided for subsequent immunoreactions.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to a recombinant plasmid and its applications. Background Technology

[0002] Porcine epidemic diarrhea (PED) is a significant and challenging disease in the swine industry. Characterized by its acute onset and high contagiousness, it causes substantial economic losses to the swine sector. Infection with porcine epidemic diarrhea virus (PEDV) disrupts the digestive system of pigs, leading to symptoms such as loss of appetite, vomiting, and severe diarrhea. Even with drug treatment, piglets may suffer from poor growth and other serious consequences, resulting in significant economic losses for the swine industry. Therefore, the prevention and control of porcine epidemic diarrhea is a crucial task in the swine industry.

[0003] PEDV S protein is a type I transmembrane glycoprotein, approximately 1383 amino acids in length, comprising: S1 subunit: responsible for receptor binding (e.g., sialic acid), containing an N-terminal domain (NTD), a C-terminal domain (CTD), and multiple neutralizing epitopes. S2 subunit: mediates membrane fusion, containing a fusion peptide, a heptapeptide repeat region (HR1 / HR2), and a transmembrane domain. Trimerization domain: located in the HR1 / HR2 region of the S2 subunit, driving trimer assembly by forming a coiled-coil structure.

[0004] Different expression systems, due to differences in post-translational modification capabilities and folding mechanisms, significantly affect the correct assembly and stability of the PEDV S1 protein. Some research teams currently use prokaryotic expression systems to express and purify the PEDV-S1 protein; however, the recombinant protein obtained by this method lacks the native conformation and cannot mimic the PEDV-S1 protein molecule on the viral surface. Therefore, it is essential to develop a recombinant plasmid for the efficient preparation of porcine epidemic diarrhea virus S1 protein. Summary of the Invention

[0005] To address the aforementioned shortcomings of existing technologies, the present invention aims to provide a recombinant plasmid and its application for the efficient preparation of porcine epidemic diarrhea virus S1 protein.

[0006] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: A ZY-2 PEDV S1-RBD-HR / Trimer recombinant plasmid is provided, comprising a signal peptide, a receptor-binding domain RBD of the ZY-2-PEDV S1 subunit, heptapeptide repeat sequences HR1 and HR2 of ZY-2 PEDV S2, and a flexible linker peptide; the ZY-2 PEDV S1-RBD-HR / Trimer recombinant plasmid is formed by linking the signal peptide, RBD, and HR1 and HR2 in tandem via the flexible linker peptide; HR1 has any of the following nucleotide sequences: (A1) The nucleotide sequence shown in SEQ ID NO.4; (A2) A nucleotide sequence encoding a protein with the same function obtained by substituting, inserting or deleting one or more bases of the nucleotide sequence shown in SEQ ID NO.4; (A3) A nucleotide sequence that has at least 80% homology with the nucleotide sequence shown in SEQ ID NO.4; HR2 has any of the following nucleotide sequences: (B1) The nucleotide sequence shown in SEQ ID NO.5; (B2) A nucleotide sequence encoding a protein with the same function obtained by substituting, inserting or deleting one or more bases of the nucleotide sequence shown in SEQ ID NO.5; (B3) A nucleotide sequence having at least 80% homology with the nucleotide sequence shown in SEQ ID NO.5; The receptor-binding domain (RBD) has any of the following nucleotide sequences: (C1) The nucleotide sequence shown in SEQ ID NO.12; (C2) A nucleotide sequence encoding a protein with the same function obtained by substituting, inserting or deleting one or more bases as shown in SEQ ID NO.12; (C3) A nucleotide sequence having at least 80% homology with the nucleotide sequence shown in SEQ ID NO.12; Signal peptides are signal peptides that can guide the secretion and expression of proteins.

[0007] Furthermore, the signal peptide is the GP67 signal peptide, which has any of the following nucleotide sequences: (D1) The nucleotide sequence as shown in SEQ ID NO.3; (D2) A nucleotide sequence encoding a protein with the same function obtained by substituting, inserting or deleting one or more bases as shown in SEQ ID NO.3; (D3) A nucleotide sequence that has at least 80% homology with the nucleotide sequence shown in SEQ ID NO.3.

[0008] Furthermore, the flexible linker peptide has any of the following nucleotide sequences: (E1) The nucleotide sequence shown in SEQ ID NO.2; (E2) A nucleotide sequence encoding a protein with the same function obtained by substituting, inserting or deleting one or more bases as shown in SEQ ID NO.2; (E3) A nucleotide sequence that has at least 80% homology with the nucleotide sequence shown in SEQ ID NO.2.

[0009] Furthermore, the C-terminus of the ZY-2 PEDV S1-RBD-HR / Trimer recombinant plasmid is also fused with a histone tag.

[0010] Furthermore, ZY-2 PEDV S1-RBD-HR / Trimer has any of the following nucleotide sequences: (F1) The nucleotide sequence shown in SEQ ID NO.1; (F2) A nucleotide sequence encoding a protein with the same function obtained by substituting, inserting or deleting one or more bases as shown in SEQ ID NO.1; (F3) A nucleotide sequence that has at least 80% homology with the nucleotide sequence shown in SEQ ID NO.1.

[0011] This invention provides the application of the above-mentioned ZY-2 PEDV S1-RBD-HR / Trimer recombinant plasmid in the preparation of porcine epidemic diarrhea virus S1 protein.

[0012] This invention provides a method for preparing porcine epidemic diarrhea virus S1 protein, comprising the following steps: (1) Insert the above ZY-2 PEDV S1-RBD-HR / Trimer sequence into the XhoI / KpnI site of the Bac-to-Bac baculovirus vector pFastBac-Dual to construct a recombinant vector; (2) Transform the recombinant vector from step (1) into competent cells to obtain recombinant baculovirus; screen the recombinant baculovirus, and then transfect it into Sf9 cells to obtain recombinant baculovirus; (3) High Five cells were infected with the recombinant baculovirus from step (2), and the viral supernatant was collected, purified, and then prepared.

[0013] Furthermore, the construction of the recombinant vector in step (1) specifically includes the following steps: ① Using ZY-2 PEDV S1-RBD-HR / Trimer as a template, PEDV-S1-RBD-FL-F and PEDV-S1-RBD-FL-R were amplified using primer pairs; the nucleotide sequences of PEDV-S1-RBD-FL-F and PEDV-S1-RBD-FL-R are shown in SEQ ID NO.6 and SEQ ID NO.7, respectively. ② The pFastBac-Dual vector was digested with XhoI and KpnI enzymes. After purification and recovery, the digestion product was ligated into ZY-2 PEDV S1-RBD-HR / Trimer using a seamless clone and transformed into competent cells. The cells were then selectively cultured on amp+ medium. Positive clones were identified using primer pairs P10-F and P10-R to obtain the recombinant vector pFastBac-Dual-ZY-2PEDV-S1 / RBD-HR / Trimer. The nucleotide sequences of P10-F and P10-R are shown in SEQ ID NO.8 and SEQ ID NO.9, respectively.

[0014] Furthermore, the promoter of the recombinant vector pFastBac-Dual-ZY-2 PEDV-S1 / RBD-HR / Trimer is the baculovirus late promoter p10.

[0015] Furthermore, in step (3), High Five cells are infected when MOI = 0.1-10.

[0016] The present invention has the following beneficial effects: 1. Expression Advantages: This invention constructs a recombinant plasmid in which the signal peptide and the heptapeptide repeat sequences 1 and 2 (HR1 and HR2) based on the ZY-2 PEDV S2 subunit play a crucial role. The signal peptide guides protein transport and localization, accurately directing relevant peptides to appropriate cellular locations. The HR1 and HR2 peptides can automatically assemble into a 6-helix bundle structure. This unique structure is essential for the correct folding of the S1 protein. The S1 protein plays a central role in viral infection and immune responses, and its correct folding is fundamental to ensuring its functional integrity. Using the recombinant plasmid of this invention to prepare the S1 protein, it achieves correct folding within the cell and effectively anchors to the cell membrane. This not only allows the S1 protein to better display its antigenic epitopes but also provides favorable conditions for subsequent immune responses. The S1 protein on the cell membrane can interact more directly with immune system cells, thereby stimulating a more effective immune response.

[0017] 2. Significance of Dynamic Expression Regulation: This invention employs the baculovirus late promoter p10 to regulate the expression of the S1 gene, a design with significant biological implications. During gene expression, early expression of certain genes may have toxic effects on host cells. This is because early host cells may still be in critical stages of growth and metabolism, unable to withstand the large-scale synthesis and accumulation of certain gene products. However, the baculovirus late promoter p10 is specific, initiating S1 gene expression in the late stages of cell growth. At this point, the host cell has completed most of its growth and metabolic processes, exhibiting greater tolerance and adaptability. This dynamic expression regulation avoids host toxicity caused by early expression, ensuring normal growth and metabolism of the host cell, thereby providing a stable cellular environment for the efficient expression of the S1 protein.

[0018] 3. Safety Advantages: The technology involved in this invention has a high level of safety, eliminating the risk of PEDV attenuated virus reversion. In traditional vaccine development, the use of attenuated vaccines carries certain safety risks, as the attenuated virus may revert to its virulence under certain conditions, leading to the spread and transmission of the disease. This invention, however, utilizes genetic engineering technology and does not rely on the preparation method of attenuated vaccines. It expresses the immunogenic S1 protein by constructing a specific expression system, without involving the use of live virus. Therefore, it fundamentally avoids the possibility of PEDV attenuated virus reversion, providing a higher level of safety assurance for vaccine use.

[0019] 4. Native Conformation and Immunogenic Effect. The S1 protein prepared in this invention has a native conformation, which allows it to better mimic the immunogenic effect of PEDV virus. The native conformation of the S1 protein retains the antigenic epitope characteristics of the virus in its natural state, enabling it to be recognized more accurately by the immune system. When the body comes into contact with the native conformation of the S1 protein, the immune system recognizes it as a real viral invasion, thereby initiating a series of immune responses. This immunogenic effect, mimicking that of a real virus, can stimulate the body to produce a more comprehensive and effective immune response, including humoral immunity and cellular immunity. Humoral immunity can produce specific antibodies to neutralize the virus and prevent viral infection and transmission; cellular immunity can recognize and kill virus-infected cells, clearing the virus.

[0020] 5. Convenience of High-Tier Immunogen. The preparation of S1 protein using the recombinant plasmid of this invention yields a high-titer immunogen, which greatly facilitates vaccine use. A high-titer immunogen means that more antigenic material can be provided at the same dose, thereby more effectively stimulating an immune response. For injection administration, a high-titer immunogen can reduce the volume and number of injections, lowering the difficulty of the procedure and reducing stress on animals. Attached Figure Description

[0021] Figure 1 Schematic diagram of ZY-2 PEDV S1-RBD-HR / Trimer recombinant plasmid; Figure 2 Vector map of recombinant plasmid pFastBac-Dual-ZY-2 PEDV-S1 / RBD-HR / Trimer; Figure 3 The results of PCR identification of the recombinant plasmid pFastBac Dual-p10-PCV2dZY-2 PEDV S1-RBD-HR / Trimer in bacterial culture; Figure 4 Western blot results of ZY-2 PEDV S1-RBD-HR / Trimer protein (non-reduced sample) in SF9 and H5 cells; Figure 5 Western blot results of ZY-2 PEDV S1-RBD-HR / Trimer protein (reduced sample) in SF9 and H5 cells; Figure 6 The results of the immunogenicity evaluation of the ZY-2 PEDV S1-RBD vaccine. Detailed Implementation

[0022] The examples given below are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, conditions in the examples are performed under standard conditions or as recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0023] Example 1: Gene optimization and construction of ZY-2 PEDV S1-RBD-HR / Trimer recombinant plasmid (1) Codon optimization: Based on the codon preference of fall armyworm (Sf9) insect cells, the receptor-binding domain RBD gene of the S1 subunit of a specific strain of porcine epidemic diarrhea virus (ZY-2-PEDV S1) was optimized, and the high-frequency codon AGG was replaced with AGA (the nucleotide sequence of the optimized ZY-2 PEDV S1-RBD-HR / Trimer is shown in SEQ ID NO.1) to improve its expression efficiency.

[0024] (2) Design of signal peptides and transmembrane domains: ①N-terminal signal peptide: The baculovirus GP67 signal peptide (GP67, nucleotide sequence as shown in SEQ ID NO.3, amino acid sequence as shown in SEQ ID NO.10) is used, which can guide the protein secretion to the endoplasmic reticulum; ② C-terminal heptapeptide repeat region: The heptapeptide repeat sequences HR1 (nucleotide sequence shown in SEQ ID NO.4) and HR2 (nucleotide sequence shown in SEQ ID NO.5) of the ZY-2 PEDV S2 subunit can automatically assemble into a 6-helix bundle structure. Based on the rigid structure characteristics of the ZY-2 PEDV RBD and HR 6-helix bundle, it was deduced that a sufficiently long linker region needs to be set between the RBD subunit and the HR trimer tag to ensure the correct formation of the protein trimer. Therefore, a 15-amino acid flexible linker peptide (GGGGSGGGGSGGGGS, SEQ ID NO.2) was designed. The RBD, HR1, and HR2 of the ZY-2-PEDV S1 subunit are directly linked in tandem through the flexible linker peptide to enhance the protein membrane localization ability. At the same time, the introduction of a histone tag at the C-terminus is beneficial for subsequent purification processes and the detection of the target protein.

[0025] A schematic diagram of the constructed ZY-2 PEDV S1-RBD-HR / Trimer recombinant plasmid is shown below. Figure 1 .

[0026] Example 2: Construction of pFastBac-Dual-ZY-2 PEDV-S1 / RBD-HR / Trimer recombinant vector The ZY-2 PEDV S1-RBD-HR / Trimer sequence from Example 1 (as shown in SEQ ID NO.1) was inserted into the XhoI / KpnI site of the Bac-to-Bac baculovirus vector pFastBac-Dual (derived from Nanjing Genscript Biotech Co., Ltd.) to construct the recombinant vector pFastBac-Dual-ZY-2 PEDV S1-RBD-HR / Trimer. The specific steps are as follows: 1. Gene synthesis: The ZY-2 PEDV S1-RBD-HR / Trimer sequence was synthesized by Nanjing Genscript Biotech.

[0027] 2. Construction of recombinant baculovirus expression vector: Using the ZY-2 PEDV S1-RBD-HR / Trimer sequence as a template, PEDV-S1-RBD-FL-F and PEDV-S1-RBD-FL-R primers were used for amplification. The pFastBac-Dual vector was digested with XhoI and KpnI enzymes at 37°C. After purification and recovery, the digested products were ligated into seamless clones and transformed into E. coli Top10 competent cells. Cells were cultured on AMP+ medium, and positive clones were identified using primers P10-F and P10-R to obtain the recombinant vector pFastBac-Dual-ZY-2 PEDV-S1 / RBD-HR / Trimer (vector map shown). Figure 2 ).

[0028] The primer sequences are shown below: PEDV-S1-RBD-FL-F: 5'-CCGCTCGAGTTTAATGATCATTC-3' (SEQ ID NO. 6); PEDV-S1-RBD-FL-R: 5'-CGGGGTACCTTAGTCAGTGACACCTTC-3' (SEQ ID NO. 7); P10-F: 5'-CGGACCTTTAATTCAACCC-3' (SEQ ID NO. 8); P10-R: 5'-GGTATCGACAGAGTGCCAG-3' (SEQ ID NO. 9).

[0029] The results of PCR identification of the recombinant plasmid pFastBac Dual-p10-ZY-2 PEDV S1-RBD-HR / Trimer are shown in the figure. Figure 3 .Depend on Figure 3 It was found that the five selected white DH10Bac-pFastBac-Dual-ZY-2-S1 and -RBD colonies were completely identical to the target bands amplified by the positive control group, with a positive rate of 100%. Furthermore, the positive and negative controls were valid (where M: Novizan DL5000 marker; Paths 1-5: represent DH10Bac-pFastBac-Dual-ZY-2-S1 bacterial suspensions 1#-5#; Paths 8-12: represent DH10Bac-pFastBac-Dual-ZY-2-RBD bacterial suspensions 1#-5#; Paths 6 and 13: represent positive controls for pFastBac-Dual-ZY-2-S1 and pFastBac-Dual-ZY-2-RBD plasmids, respectively; Paths 7 and 14: represent the corresponding negative controls). Sanger sequencing confirmed that the positive plasmid sequence was 100% identical to the designed sequence.

[0030] Example 3: Preparation of Recombinant Baculovirus The recombinant vector prepared in Example 2 was transformed into DH10Bac competent cells, and recombinant baculids were screened. After transfection into Sf9 cells, recombinant baculovirus rBac-ZY-2 PEDV S1-RBD-HR was obtained. When MOI=0.2, High Five cells were infected and expanded to passage P3, with a viral titer reaching 1×10⁻⁶. 8 TCID50 / mL. The specific procedure is as follows: 1. Extraction of recombinant Bacmid: Take 10 mL of the transformed DH10Bac fermentation broth, centrifuge at 5000 rpm for 10 min, discard the supernatant, and resuspend the precipitate thoroughly with 300 μL of Solution I containing RNase; add 300 μL of Solution II and mix gently, and let stand at room temperature for 5 min; add 300 μL of 3M potassium acetate (pH 5.5, 4℃), mix gently, place on ice for 10 min, and centrifuge at 13,300 g for 15 min at 4℃; carefully transfer the supernatant to a new sterile EP tube (about 800 μL) in a laminar flow hood, add an equal volume of chloroform (frozen at -20℃), gently invert to mix, and maintain a low temperature environment of 4℃; centrifuge at 13,300 g for 5 min at 4℃, and carefully add the supernatant dropwise to an equal volume of isopropanol pre-chilled at -20℃ (about 700 μL), and place on ice for 10 min. Then, after centrifuging at 13,300g for 15 min at 4°C, discard the supernatant and slowly add 500 μL of 70% ethanol solution refrigerated at 4°C (without disturbing the DNA precipitate) along the tube wall; centrifuge at 13,300g for 5 min at 4°C and wash once more; carefully remove the ethanol, and allow the remaining portion to air dry naturally for 10 min in a laminar flow hood. Add 50 μL of sterile water to the precipitate and carefully dissolve the DNA. Store the extracted recombinant Bacmid at 4°C.

[0031] 2. Transfection: The recombinant Bacmid extracted above was transfected into Sf9 cells (MOI=2) using Cellfectin® II Reagent. Specifically: First, SF9 cells were counted at a rate of 1.0 × 10⁶ cells / cells. 6Inoculate 2 mL per well in a 6-well plate and incubate at 27°C for 1 hour to allow cell adhesion. Add 200 μL of Grace's Insect Medium Unsupplemented, 8 μL of Cellfectin® II Reagent, and 1 μg of rod granules, mix gently, and incubate for 30 minutes. Add 800 μL of Grace's Insect Medium Unsupplemented, mix gently, and incubate for several minutes. Gently wash the plate twice with Grace's Insect Medium Unsupplemented, 2 mL per well. Discard the wash solution and immediately add the transfection mixture dropwise, gently shaking the plate to evenly cover the cells. Incubate at 27°C for 5 hours. Discard the incubation solution and add 2 mL of SF-SFM complete medium (Suzhou Womei) to each well. Incubate at 27°C for 5 days. Under a microscope, the cells should exhibit polyhedral lesions, swelling and then fragmenting. The cell layer was detached by pipetting the cell tip, and the suspension was harvested. The suspension was frozen and thawed once, and centrifuged at 500g for 1 min to remove cells and large debris. The supernatant was collected, 2% (V / V) FBS was added, mixed well, and dispensed into 1 mL vials. The suspension was temporarily stored at 4℃ or stored long-term in liquid nitrogen.

[0032] 3. Titer determination: SF-SFM culture medium was used as the growth medium for SF9 cells, with a passage ratio of 1:3, or 2×10⁻⁶ cells / mL. 6 Passaged for 72 h at a density of 1 mL. Select SF9 cells in good growth condition, count them, dilute the cell suspension with growth medium, and then... 5 Seed 0.1 mL / well in a 96-well plate and incubate at 27°C. Prepare sterile 1.5 mL EP tubes according to the number of samples, arrange them in a row from left to right on a tube rack, and add 900 μL of SFM / H502C culture medium (containing 2% newborn calf serum) to each tube. Take 100 μL of the sample to be tested and perform a 10-fold serial dilution, with each dilution being 10... -1 10 -2 10 -3 10 -4 10 -5 10 -6 and 10 -7Select appropriate dilutions for cell seeding, seeding 6 wells per dilution, with 100 μL seeded per well. A negative control (SFM medium containing 2% newborn calf serum) and a positive control are also included, with a final volume of 200 μL per well. Incubate the 96-well plate at 27°C for 5 days. Discard the culture supernatant from the 96-well plate, add 50 μL of acetone-methanol (1:1 volume ratio) fixative per well, and incubate at -20°C for 30 min. After fixation, discard the acetone-methanol fixative and ventilate in a fume hood for 10 min. Add 50 μL of mouse anti-His primary antibody (1:1000) per well and incubate at 37°C for 1 h. Discard the primary antibody and wash three times with PBS. Add 50 μL of FITC-labeled goat anti-mouse IgG secondary antibody (1:200) per well and incubate at 37°C in the dark for 1 h. Discard the secondary antibody and wash three times with PBS. Under a fluorescence microscope, the number of fluorescent wells (n=6) was observed. The viral load of the sample, calculated using the Reed-Muench method, was 10⁻⁶. 8.00 TCID50 / mL.

[0033] 4. Virus amplification: The virus was passaged at an MOI of 2, and at the 3rd passage, it was used as seed virus for amplification culture. High Five cells were infected (MOI=0.2), and the viral supernatant was collected after 72 hours.

[0034] 5. Protein Expression and Identification: The expression levels of ZY-2 PEDV-S1 / RBD-HR / Trimer proteins were detected in SF9 and H5 cells using Western blot on reduced protein gels (containing SDS) and non-reduced gels (without SDS), respectively. The harvested virus solution was subjected to SDS-PAGE electrophoresis and then transferred to PVDF membranes. The membranes were blocked with 5% milk for 2 hours, incubated with PEDV rabbit positive serum primary antibody at room temperature for 2 hours, washed three times (10 min each time), and incubated with HRP-goat anti-rabbit IgG secondary antibody at room temperature for 1 hour, washed three times (10 min each time). Then, enhanced chemiluminescent substrate was added, and the membranes were developed and photographed in the dark.

[0035] Western blot results of ZY-2 PEDV S1-RBD-HR / Trimer protein (non-reduced sample) in SF9 and H5 cells are shown below. Figure 4 .Depend on Figure 4It was observed that ZY-2 PEDV S1-RBD-HR / Trimer produced bands of approximately 110 kDa, the theoretical size of the trimer, in both H5 cells (lanes 4 and 5) and SF9 cells (lanes 9 and 10), indicating successful protein expression. (Where M represents the pre-stained protein marker Blue Plus® II Protein Marker (14-120 kDa); Lane 1 represents the total protein in the supernatant after H5 cell lysis; Lane 2 represents the supernatant of P2-Bac-ZY-2-S1-5-1-H5; Lane 3 represents the supernatant of P2-Bac-ZY-2-S1-5-2-H5; Lane 4 represents the supernatant of P2-Bac-ZY-2-RBD-1-H5; Lane 5 represents the supernatant of P2-Bac-ZY-2-RBD-2-H5). Supernatant; Yongdao 6: represents total protein in the supernatant after SF9 cell lysis; Yongdao 7: P2-Bac-ZY-2-S1-5-1-SF9 supernatant; Yongdao 8: P2-Bac-ZY-2-S1-5-2-SF9 supernatant; Yongdao 9: P2-Bac-ZY-2-RBD-1-SF9 supernatant; Yongdao 10: P2-Bac-ZY-2-RBD-2-SF9 supernatant).

[0036] Western blot analysis results of ZY-2 PEDV S1-RBD-HR / Trimer protein (reduced sample) in SF9 and H5 cells are shown below. Figure 5 .Depend on Figure 5 It was observed that the ZY-2 PEDV S1-RBD-HR / Trimer protein produced bands approximately 34 kDa in both H5 cells (lanes 3 and 4) and SF9 cells (lanes 5 and 6), indicating successful protein expression. (Where, lane 1 represents total protein from the supernatant after SF9 cell lysis; lane 2 represents total protein from the supernatant after H5 cell lysis; lane 3 represents supernatant of total protein from the freeze-thaw lysis of ZY-2-S1-RBD-1-H5; lane 4 represents supernatant of total protein from the freeze-thaw lysis of ZY-2-S1-RBD-2-H5; lane 5 represents supernatant of total protein from the freeze-thaw lysis of ZY-2-S1-RBD-1-SF9; lane 6 represents supernatant of total protein from the freeze-thaw lysis of ZY-2-S1-RBD-2-SF9; M: Blue Plus® II Protein Marker (14-120)). (kDa) prestained protein marker).

[0037] Example 4: Immunogenicity Evaluation 1. Animal grouping: 20g BALB / c mice were divided into experimental group 1 (ZY-2 PEDV-S1-RBD / Trimer purified group), experimental group 2 (ZY-2 PEDV-S1-RBD / Trimer unpurified group), commercial PEDV inactivated vaccine group 1, commercial PEDV inactivated vaccine group 2 and control group (empty vector Bacmid).

[0038] 2. Immunization procedure: The purified and unpurified samples were centrifuged at 10,000 r / min for 10 minutes at 4℃. The supernatant was collected and mixed with aluminum hydroxide gel at a volume ratio of 1:1 to prepare the vaccine. At the same time, the control group and the commercial vaccine were injected intramuscularly into the right leg on days 0 and 21, and the clinical observation was carried out for 21 consecutive days.

[0039] 3. PEDV IgG Antibody Detection: 21 days after the second immunization (i.e., 42 days after initial immunization), PEDV IgG antibodies were detected using the PEDV-Ab ELISA kit. The detection procedure followed the IDEXX Procine Epidemic Diarrhea Virus (PEDV) IgA Antibody Test Kit, except that the anti-pig antibody used as the secondary antibody was replaced with an anti-mouse IgG antibody; all other steps remained the same. ELISA results are shown (see...). Figure 6 The immune response was initiated 21 days after administration of the ZY-2 PEDV S1-RBD vaccine. After the second immunization booster, IgG levels increased sharply and showed a very significant difference (p<0.001).

[0040] 4. Serum neutralizing antibody titer determination: Vero cells that had grown into a well-formed monolayer (72 hours after passage) were used to determine the titer using 5.0 × 10⁻⁶ serum neutralizing antibody titers. 4 Cells per well were seeded into 96-well cell culture plates and incubated at 37°C with 5% CO2. Serum was serially diluted 2-fold in 1.5 mL sterile centrifuge tubes with cell maintenance medium at dilution ratios of 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, 1:512, and 1:1024, and vortexed to mix. The PEDV-G2c virus solution with pre-determined viral load was diluted with cell maintenance medium to a concentration of 2.0 × 10⁻⁶ cells / well. 3TCID50 / mL. Mix equal volumes of diluted virus solution and diluted serum for testing, with both virus and normal cell controls included. Incubate at 37°C for 1 hour. Take a well-grown monolayer of 96-well NC-Vero cell culture plates (approximately 24 hours after passage), discard the cell growth medium, and rinse once with serum-free DMEM medium. Add the above virus-neutralized sample and control to 6 wells, 0.1 mL per well. Add the normal cell control wells first, then the virus neutralization wells, and finally the virus control wells. Incubate at 37°C, 5% CO2 for 1 hour. Discard the virus neutralization solution, rinse once with serum-free DMEM medium, and add 0.2 mL of cell maintenance medium to each well. Incubate at 37°C, 5% CO2 for 48 hours to determine the results. Observe the normal cell control wells and virus control wells. No cytopathic effect was observed in the normal cell control wells, while cytopathic effect was observed in the virus control wells, confirming the experimental results. Observe the wells of the sample to be tested and record the number of cell-protected wells, and determine the results. Calculate the neutralizing antibody titer of the sample according to the Reed-Muench method.

[0041] The results of neutralizing antibody titers against PEDV CV777 strain in mouse immune serum are shown in Table 1. As shown in Table 1, the protective function of neutralizing antibodies was observed in all experimental groups. A serum titer of 1:64 provided complete protection against neutralization. Neutralizing antibody titer test results showed that the neutralizing antibody titer in the control serum was 1:8, in group 1 of the commercially available PEDV inactivated vaccine was 1:64, in group 2 of the commercially available PEDV inactivated vaccine was 1:128, in the unpurified ZY-2 PEDV S1-RBD group was 1:64, and in the purified ZY-2 PEDV S1-RBD group was 1:512. This indicates that the serum from the third week after the initial immunization had a protective effect; details are shown in Table 1.

[0042] Table 1. Results of ZY-2 PEDV S1-RBD neutralizing antibody titer assay

[0043] The nucleotide and amino acid sequences involved in this invention are shown below: (1)Nucleotide sequence of the optimized ZY-2 PEDV S1-RBD-HR / Trimer: ATGCTACTTGTTAATCAGAGTCACCAGGGTTTTAATAAAGAGCATACGTCAAAGATGGTTTCGGCTATCGTGCTTTACGTTCTTCTCGCGGCAGCGGCACATAGCGCTTTCGCCTTTAATGACCACTCTTTTGTAAACATAACAGTTAGTGCTTCTTTTGGAGGCCATAGCGGAGCGAACTTAATCGCAAGTGACACAACCATTAACGGGCTATCTTCATTCTGCGTGGACACTCGGCAGTTCACTATCTCACTATTCTACAATGTAACGAACAGTTATGGCTATGTGTCGAAGTCGCAAGATAGCAACTGTCCTTTTACCCTGCAGAGTGTCAACGATTACCTGAGTTTTTCTAAGTTCTGTGTGAGCACCAGCTTATTAGCGTCGGCGTGCACTATAGACCTCTTCGGTTATCCAGAATTCGGCTCGGGGGTAAAATTCACATCTTTGTACTTTCAGTTTACGAAAGGCGAGTTGATAACAGGGACACCCAAGCCGCTTGAAGGGGTAACGGACGGGGGTGGCGGTTCCGGAGGTGGTGGCTCGGGGGGAGGTGGATCTAGGAATCAACAAATGCTCGCCGAGAGTTTCAATTCTGCCATTGGAAACATCACGTCAGCTTTTGAATCCGTGAAAGAGGCTATTAGCCAAACGTCAAAAGGGCTAAATACGGTTGCACACGCCCTGACCAAGGTACAGGAAGTCGTCAACTCACAAGGAGCTGCGCTGACTCAACTCACTGTCCAATTGCAGCACAACTTCCAGGCCATTTCGTCATCCATCGACGATATATACTCCCGTCGAACTGGGCCGTCCCTGTCCTTAGATGTCTTTAATGCAACCTATCTAAACTTAACTGGCGAAATAGCCGATCTTGAACAGCGCTCAGAGTCCTTGAGAAATACCACAGAGGAGTTGCAAAGCCTCCATCATCATCACCACCAC (SEQ ID NO.1); (2) Nucleotide sequence of GP67 signal peptide: atgctacttgttaatcagagtcaccagggttttaataaagagcatacgtcaaagatggtttcggctatcgtgctttacgttcttctcgcggcagcggcacatagcgctttcgcc (SEQ ID NO.3); (3) Nucleotide sequence of HR1: cggaaccagcaaatgcttgctgagtcttttaactctgctattggtaatataacttcagcctttgagagtgttaaagaggctattagtcaaacttccaagggtttgaacactgtgg ctcatgcgcttactaaggttcaagaggttgttaactcgcagggtgcagctttgactcaacttaccgtacagctgcaacacaacttccaagccatttctagttctattgatgacatttactctcga (seq ID NO.4); (4) Nucleotide sequence of HR2: agaactggtccaagtctttctctagatgtttttaatgccacttatctcaatctcactggtgaaattgcagatttagagcagcgttcagagtctctccgtaatactacagaagagctccaaagtctt (SEQ ID NO.5); (5) The amino acid sequence of the GP67 signal peptide: MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFA (SEQ ID NO.10); (6) The amino acid sequences of HR1 and HR2 in tandem are as follows: RNQQMLAESFNSAIGNITSAFESVKEAISQTSKGLNTVAHALTKVQEVVNSQGAALTQLTVQLQHNFQAISSSIDDIYSRRTGPSLSLDVFNATYLNLTGEIADLEQRSESLRNTTEELQSL (SEQ ID NO.11) (7) RBD optimized nucleotide sequence: tttaatgaccactcttttgtaaacataacagttagtgcttcttttggaggccatagcggagcgaacttaatcgcaagtgacacaaccat taacgggctatcttcattctgcgtggacactcggcagttcactatctcactattctacaatgtaacgaacagttatggctatgtgtcgaagtcgcaagatagcaac tgtccttttaccctgcagagtgtcaacgattacctgagtttttctaagttctgtgtgagcaccagctttattagcgtcggcgtgcactatagacctcttcggttat ccagaattcggctcgggggtaaaattcacatctttgtactttcagtttacgaaaggcgagttgataacagggacacccaagccgcttgaaggggtaacggac (seq ID NO.12).

[0044] 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, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A ZY-2 PEDV S1-RBD-HR / Trimer recombinant plasmid, characterized in that, The plasmid includes a signal peptide, the receptor-binding domain RBD of the ZY-2-PEDVS1 subunit, the heptapeptide repeat sequences HR1 and HR2 of ZY-2 PEDV S2, and a flexible linker peptide; the ZY-2 PEDV S1-RBD-HR / Trimer recombinant plasmid is formed by linking the signal peptide, RBD, and HR1 and HR2 in tandem via the flexible linker peptide. The HR1 has any of the following nucleotide sequences: (A1) The nucleotide sequence shown in SEQ ID NO.4; (A2) A nucleotide sequence encoding a protein with the same function obtained by substituting, inserting or deleting one or more bases of the nucleotide sequence shown in SEQ ID NO.4; (A3) A nucleotide sequence that has at least 80% homology with the nucleotide sequence shown in SEQ ID NO.4; The HR2 has any of the following nucleotide sequences: (B1) The nucleotide sequence shown in SEQ ID NO.5; (B2) A nucleotide sequence encoding a protein with the same function obtained by substituting, inserting or deleting one or more bases of the nucleotide sequence shown in SEQ ID NO.5; (B3) A nucleotide sequence having at least 80% homology with the nucleotide sequence shown in SEQ ID NO.5; The receptor-binding domain (RBD) has any of the following nucleotide sequences: (C1) The nucleotide sequence shown in SEQ ID NO.12; (C2) A nucleotide sequence encoding a protein with the same function obtained by substituting, inserting or deleting one or more bases as shown in SEQ ID NO.12; (C3) A nucleotide sequence having at least 80% homology with the nucleotide sequence shown in SEQ ID NO.12; The signal peptide is a signal peptide that can guide the secretion and expression of proteins.

2. The ZY-2 PEDV S1-RBD-HR / Trimer recombinant plasmid according to claim 1, characterized in that, The signal peptide is the GP67 signal peptide, which has any of the following nucleotide sequences: (D1) The nucleotide sequence as shown in SEQ ID NO.3; (D2) A nucleotide sequence encoding a protein with the same function obtained by substituting, inserting or deleting one or more bases as shown in SEQ ID NO.3; (D3) A nucleotide sequence that has at least 80% homology with the nucleotide sequence shown in SEQ ID NO.

3.

3. The ZY-2 PEDV S1-RBD-HR / Trimer recombinant plasmid according to claim 1, characterized in that, The flexible linker peptide has any of the following nucleotide sequences: (E1) The nucleotide sequence shown in SEQ ID NO.2; (E2) A nucleotide sequence encoding a protein with the same function obtained by substituting, inserting or deleting one or more bases as shown in SEQ ID NO.2; (E3) A nucleotide sequence that has at least 80% homology with the nucleotide sequence shown in SEQ ID NO.

2.

4. The ZY-2 PEDV S1-RBD-HR / Trimer recombinant plasmid according to claim 1, characterized in that, The C-terminus of the ZY-2 PEDV S1-RBD-HR / Trimer recombinant plasmid is also fused with a histone tag.

5. The ZY-2 PEDV S1-RBD-HR / Trimer recombinant plasmid according to any one of claims 1-4, characterized in that, The ZY-2 PEDV S1-RBD-HR / Trimer has any of the following nucleotide sequences: (F1) The nucleotide sequence shown in SEQ ID NO.1; (F2) A nucleotide sequence encoding a protein with the same function obtained by substituting, inserting or deleting one or more bases as shown in SEQ ID NO.1; (F3) A nucleotide sequence that has at least 80% homology with the nucleotide sequence shown in SEQ ID NO.

1.

6. The use of the ZY-2 PEDV S1-RBD-HR / Trimer recombinant plasmid according to any one of claims 1-5 in the preparation of porcine epidemic diarrhea virus S1 protein.

7. A method for preparing porcine epidemic diarrhea virus S1 protein, characterized in that, Includes the following steps: (1) Insert the ZY-2 PEDV S1-RBD-HR / Trimer sequence of any one of claims 1-5 into the XhoI / KpnI site of the Bac-to-Bac baculovirus vector pFastBac-Dual to construct a recombinant vector; (2) Transform the recombinant vector from step (1) into competent cells to obtain recombinant baculovirus; screen the recombinant baculovirus, and then transfect it into Sf9 cells to obtain recombinant baculovirus; (3) High Five cells were infected with the recombinant baculovirus from step (2), and the viral supernatant was collected, purified, and then prepared.

8. The preparation method according to claim 7, characterized in that, The construction of the recombinant vector in step (1) specifically includes the following steps: ① Using ZY-2 PEDV S1-RBD-HR / Trimer as a template, PEDV-S1-RBD-FL-F and PEDV-S1-RBD-FL-R were amplified using primer pairs; wherein, the nucleotide sequences of PEDV-S1-RBD-FL-F and PEDV-S1-RBD-FL-R are shown in SEQ ID NO.6 and SEQ ID NO.7, respectively; ② The pFastBac-Dual vector was digested with XhoI and KpnI enzymes. After purification and recovery, the digestion product was ligated into ZY-2 PEDV S1-RBD-HR / Trimer using a seamless clone and transformed into competent cells. The cells were then selectively cultured on amp+ medium. Positive clones were identified using primer pairs P10-F and P10-R to obtain the recombinant vector pFastBac-Dual-ZY-2 PEDV-S1 / RBD-HR / Trimer. The nucleotide sequences of P10-F and P10-R are shown in SEQ ID NO.8 and SEQ ID NO.9, respectively.

9. The preparation method according to claim 8, characterized in that, The promoter of the recombinant vector pFastBac-Dual-ZY-2PEDV-S1 / RBD-HR / Trimer is the baculovirus late promoter p10.

10. The preparation method according to claim 7, characterized in that, In step (3), HighFive cells are infected when MOI = 0.1-10.

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