Porcine epidemic diarrhea virus mRNA vaccine and construction method thereof

Abstract

The invention provides a porcine epidemic diarrhea virus mRNA vaccine and a construction method thereof. The mRNA vaccine is synthesized from a signal peptide containing 5 'UTR and human PLAT, a porcine epidemic diarrhea virus S protein truncation (amino acids at 477th to 629th sites of PEDV S protein), a Flag tag, a termination codon, 3' UTR and poly (A) tail nucleic acid. According to the invention, lipid nanoparticles are used as a delivery system of mRNA. The constructed mRNA vaccine is intramuscularly injected into a 6-8-week-old BALB / c mouse, the mRNA vaccine can remarkably improve IgG titer in serum of the immunized mouse, induce humoral immunity and generate a specific antibody aiming at a target protein, and also can induce cellular immunity, so that an effective technical means is provided for prevention and control of a new variant strain.

Description

Technical Field

[0001] The present invention belongs to the technical field of RNA vaccines, and in particular, relates to a porcine epidemic diarrhea virus mRNA vaccine and a construction method thereof. Background Art

[0002] Porcine epidemic diarrhea virus can cause the infectious intestinal disease porcine epidemic diarrhea, which causes intestinal cell destruction and villous atrophy, leading to watery diarrhea and vomiting. The mortality rate of newborn piglets after infection is as high as 100%. In 2010, a serious diarrhea epidemic broke out in large-scale pig farms in my country. Sequencing analysis found that the pathogen was PEDV, and its S gene had insertions, deletions or mutations. These mutations have increased the virulence of the virus strain, and the original vaccine cannot provide sufficient immune protection. Nucleic acid vaccines are simple to design, have a short production cycle, are low-cost and can be mass-produced. They can be used for gene therapy, tumor treatment, and prevention of infectious diseases.

[0003] The outbreak of the COVID-19 pandemic has promoted the rapid development of mRNA vaccines. In response to global challenges, mRNA vaccines have demonstrated their advantages. In December 2020, the U.S. FDA authorized two mRNA vaccines, mRNA-1273 and BNT 162b2, which showed an efficacy of more than 95% in Phase III clinical trials. The sudden emergence of PEDV mutant strains has greatly reduced the prevention and disease control effects of existing traditional vaccines. Traditional vaccines such as inactivated vaccines and attenuated vaccines generally have defects such as long research and development cycles, narrow range of prevention strains, unstable immune effects, and short duration of immunity. Based on the isolation of clinical epidemic strains and the mastery of molecular epidemiological information data of PEDV field strains, the present invention focuses on the development of a PEDV mRNA vaccine with good safety, strong immunogenicity, and broad-spectrum immune protection efficacy, providing reference and necessary material reserves for the development of PEDV nucleic acid vaccines in my country.

[0004] The purpose of mRNA vaccine template design is mainly to improve protein expression efficiency, and the main methods are to optimize 5'UTR, 3'UTR, add Kozak sequence, codon optimization, add Cap-1, optimize poly (A), use modified nucleotides to reduce immunogenicity, etc. At present, UTR mainly uses the UTR of natural protein, such as 5'UTR mainly from globin, heat shock protein 70 and other genes, 3'UTR mainly from hemoglobin α subunit or β subunit genes, but there are no reports on the screening of UTR of porcine protein. Summary of the invention

[0005] The purpose of the present invention is to provide a porcine epidemic diarrhea virus mRNA vaccine and a construction method thereof.

[0006] The present invention uses two isolated strains as research objects to develop mRNA vaccines. The screening of UTR sequences will focus on UTRs with relatively high expression levels; a signal peptide sequence is added to the N-terminus of the target protein to induce the extracellular secretion expression of the target protein to stimulate the body's humoral immunity; a Kozak sequence is added after the 5'UTR and before the CDS sequence to increase protein expression; a Flag tag is added to the C-terminus of the target protein to facilitate the identification of the subsequent expressed protein. The specific structure of the template is as follows Figure 1 shown.

[0007] Lipid nanoparticles are a new type of nucleic acid delivery system. The organic phase containing lipids and the inorganic phase containing mRNA are quickly mixed, and the lipids and nucleic acids will spontaneously assemble to form lipid nanoparticles. The lipids that make up lipid nanoparticles are divided into main lipids, auxiliary lipids, cholesterol, and PEGylated lipids. The ratio of the four types of lipids has a great influence on the physical and chemical properties of lipid nanoparticles. The optimal ratio can be determined by the nitrogen-phosphorus ratio (N / P).

[0008] In order to achieve the purpose of the present invention, in the first aspect, the present invention provides a porcine epidemic diarrhea virus mRNA vaccine, which is synthesized using a sequence containing a 5'UTR, a human PLAT (plasminogen activator) signal peptide, a porcine epidemic diarrhea virus S protein truncate, a Flag tag, a stop codon, a 3'UTR and a poly (A) tail nucleic acid connected in sequence.

[0009] The porcine epidemic diarrhea virus S protein truncation corresponds to the 477th to 629th amino acids of the porcine epidemic diarrhea virus S protein.

[0010] Furthermore, the sequences of the 5'UTR and 3'UTR are derived from the porcine protein TBCC. The nucleic acid sequences of the 5'UTR and 3'UTR are shown in SEQ ID NOs: 3 and 4, respectively.

[0011] Furthermore, the stop codon is 5'-TGATAA-3'.

[0012] Furthermore, the poly(A) tail consists of 89 A bases.

[0013] Furthermore, the nucleic acid sequence of the signal peptide of human PLAT is shown in SEQ ID NO:2.

[0014] Furthermore, the nucleic acid sequence of the porcine epidemic diarrhea virus S protein truncation after codon optimization is shown in SEQ ID NO:5.

[0015] In a specific embodiment of the present invention, the nucleic acid sequence of the mRNA vaccine without a poly (A) tail is shown as SEQ ID NO: 1.

[0016] In a second aspect, the present invention provides a method for constructing the mRNA vaccine, which comprises: selecting 5'UTR, Kozak sequence, signal peptide of human PLAT, 3'UTR, truncated S protein of porcine epidemic diarrhea virus, Flag tag, 3'UTR, and termination codon nucleic acid sequence to synthesize the nucleic acid sequence of the mRNA vaccine, and constructing it on a vector, transfecting bacteria to obtain recombinant bacteria, extracting plasmids from the recombinant bacteria to obtain recombinant plasmids, and enzymatically cutting, transcribing, and purifying the recombinant plasmids to obtain mRNA vaccine nucleic acid.

[0017] In a third aspect, the present invention provides a porcine epidemic diarrhea virus nucleic acid vaccine, comprising an mRNA vaccine nucleic acid prepared according to the above method and a delivery system;

[0018] The delivery system is lipid nanoparticles.

[0019] In a fourth aspect, the present invention provides a method for preparing the nucleic acid vaccine, which comprises: dissolving ionizable lipids Dlin-MC3-DMA, DSPC, DMG-PEG2000 and cholesterol in anhydrous ethanol as an organic phase, dissolving the mRNA vaccine nucleic acid in a sodium acetate solution at pH 4.0 and 25 mM or a sodium citrate solution at pH 4.0 and 25 mM as an inorganic phase, and preparing lipid nanoparticles encapsulated with mRNA using a microfluidic chip at a flow rate ratio of 1:3 between the organic phase and the inorganic phase and a total flow rate of 4 mL / min.

[0020] Furthermore, when the nitrogen-phosphorus ratio (N / P) is selected to be 10, the encapsulation efficiency of lipid nanoparticles can reach 85.44%. The particle size of the prepared lipid nanoparticles is between 40nm and 60nm, and the uniformity is good.

[0021] Preferably, the molar ratio of the ionizable lipid Dlin-MC3-DMA, DSPC, cholesterol and DMG-PEG2000 is 50:10:38.5:1.5.

[0022] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0023] The present invention selects ionizable nanoparticles as the delivery system of candidate vaccine mRNA, and the lipids used include ionizable lipid Dlin-MC3-DMA, auxiliary lipid DSPC, cholesterol, DMG-PEG2000, and ionizable lipids are electrically neutral in a body fluid environment, so the surface charge of ionizable lipid nanoparticles is electrically neutral, which reduces cytotoxicity. The particle size of lipid nanoparticles will affect the diffusion of particles in the body. Generally speaking, particles with a diameter less than 10nm are mainly transported through capillaries, and particles with a diameter of 20nm-200nm are directly transported to lymphatic vessels, and particles with a diameter greater than 200nm will limit their diffusion and convection, making it difficult to enter lymphatic vessels. Therefore, lipid nanoparticles with a particle size less than 200nm can better play a biological role.

[0024] The constructed mRNA vaccine was injected intramuscularly into 6-8 week old BALB / c mice. The mRNA vaccine can significantly increase the IgG titer in the serum of the immune mice, induce humoral immunity and produce specific antibodies against the target protein, and can also induce cellular immunity, among which the cellular immunity level induced by 5 μg mRNA vaccination is the highest. The present invention provides an effective technical means for the prevention and control of new mutant strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a schematic diagram of the in vitro transcription template of the mRNA vaccine nucleic acid of the present invention.

[0026] Figure 2 This is a schematic diagram of in vivo immunization in BALB / c mice in a preferred embodiment of the present invention.

[0027] Figure 3 The results of UTR, RBD and template amplification in the preferred embodiment of the present invention are shown in Figure 1. M. DL2000 Plus DNA Marker; 1. LDHB 5'UTR; 2. LDHB 3'UTR; 3. RBD sequence; 4. In vitro transcription template LDHB-RBD.

[0028] Figure 4 The protein expression detected by IFA in the preferred embodiment of the present invention. A. LLC-PK1 cells transfected with mRNA; B. Negative control.

[0029] Figure 5 The expression of the target protein in the preferred embodiment of the present invention. A. WB identification result of protein expression; B. Grayscale quantification of protein expression; LR. mRNA-LDHB-RBD; LY. mRNA-LDHB-RBDYH; GR. mRNA-GAPDH-RBD; GY. mRNA-GAPDH-RBDYH; NC. Negative control; RBD represents the original sequence, and RBDYH represents the codon-optimized sequence.

[0030] Figure 6 The effect of different lengths of 5'UTR on protein expression in the preferred embodiment of the present invention. A. UTR gradient WB results; B. Quality identification results of 5 kinds of mRNA C. Grayscale analysis results; NC: negative control; 1, 2, 3, 4, 5: 5'UTR of genes LDHB, ACTB, LDHC, HBB, GAPDH respectively; M. ss RNALadder. Note: The poly (A) length of the enzymatically tailed mRNA may differ from the theoretical length. The lengths (theoretical) of the 5 mRNAs are 920nt, 986nt, 1035nt, 1095nt, and 1227nt respectively.

[0031] Figure 7 The results of protein expression in different phases are shown in the preferred embodiment of the present invention. A. IFA results of protein expression in different phases; B. WB results of protein expression in different phases.

[0032] Figure 8 The test results of target protein secretion expression in the preferred embodiment of the present invention are as follows. A. Analysis of mRNA protein expression of 4 different signal peptides; B. Protein secretion detection; C. 4 mRNA quality detections; D. Western Blot grayscale analysis quantification; M. ssRNALadder; 1, 2, 3, 4. mRNA constructed by signal peptides of genes IL-4, IL-13, TLR9, and PRL; PLAT. mRNA constructed by signal peptide of gene human plasminogen activator; NC. Negative control.

[0033] Fig. 9 The results of the quality identification of 19 mRNAs in the preferred embodiment of the present invention are shown in FIG. 1. M.ss RNA Ladder; 1-19. represent the UTRs of different genes.

[0034] Fig.10 The expression of 19 mRNA proteins in the preferred embodiment of the present invention is shown in Figure 1. A. Indirect immunofluorescence detection of 19 mRNAs; B. WB detection of 19 mRNAs; C. Fluorescence intensity quantitative analysis; D. WB grayscale quantitative analysis.

[0035] Fig.11 It is constructed from the recombinant plasmid pUC57 A60G60 in the preferred embodiment of the present invention.

[0036] Fig.12 The results of plasmid linearization and homologous recombination fragment PCR in the preferred embodiment of the present invention are as follows: M. DL2000 Plus DNA Marker; 1. Plasmid pUC57 60G60 linearization; 2. LDHB-EGFP homologous recombination fragment.

[0037] Fig.13The results of the quality test of mRNA-LDHB-EGFP in the preferred embodiment of the present invention are shown in FIG.

[0038] Fig.14 The fluorescence observation of lipid nanoparticles with different N / P ratios in the preferred embodiment of the present invention. AN / P=6 lipid nanoparticle transfection fluorescence; BN / P=6 lipid nanoparticle white light; CN / P=10 lipid nanoparticle transfection fluorescence; DN / P=10 lipid nanoparticle white light; EN / P=20 lipid nanoparticle transfection fluorescence; FN / P=20 lipid nanoparticle white light; G. negative control; H. negative control group white light.

[0039] Fig.15 This is an analysis of protein expression after lipid nanoparticle transfection at different N / P values ​​in a preferred embodiment of the present invention.

[0040] Fig.16 This is the RNA standard curve in the preferred embodiment of the present invention.

[0041] Fig.17 The particle size and potential results of lipid nanoparticles in the preferred embodiment of the present invention.

[0042] Fig.18 This is the cryo-transmission electron microscopy result of lipid nanoparticles in a preferred embodiment of the present invention.

[0043] Fig.19 The EGFP positive cell rate after transfection in the preferred embodiment of the present invention. A. Negative control; BN / P=6 lipid nanoparticle transfection efficiency; CN / P=10 lipid nanoparticle transfection efficiency; DN / P=20 lipid nanoparticle transfection efficiency; E. Summary of transfection positive rate in 4 groups.

[0044] Fig. 20 The results of homologous recombination and mRNA quality analysis in the preferred embodiment of the present invention are shown in Figure 1. DL2000 Plus DNA Marker; M2. ss RNA Ladder; 1. Linearization of plasmid pUC57 60G60; 2. TBCC-RBD homologous recombination fragment; 3. mRNA-TBCC-RBD quality identification.

[0045] Fig.21 The in vitro validation results of mRNA-TBCC-RBDYH in the preferred embodiment of the present invention are as follows: A. BHK-21 cells transfected with mRNA-TBCC-RBDYH; B. Negative control; C. WB detection of protein expression.

[0046] Fig. 22 This is the spleen status of mice after immunization in a preferred embodiment of the present invention.

[0047] Fig.23 The results of mouse serum specific antibody detection in the preferred embodiment of the present invention. M: protein marker; 1: BHK-21 cells transfected with mRNA-TBCC-RBDYH; 2: BHK-21 cells not transfected with mRNA-TBCC-RBDYH. Note: The mouse serum of each group was mixed and diluted.

[0048] Fig.24 The following are the results of mouse T lymphocyte subset analysis in the preferred embodiment of the present invention. AT lymphocyte subset analysis results; BT lymphocyte subset quantitative graph. Note: *, P < 0.05; **, P < 0.01.

[0049] Fig.25 The results of the analysis of the total IgG titer of mouse serum in the preferred embodiment of the present invention. Note: ns, P>0.1; ****, P<0.0001.

[0050] Fig.26 The results of specific T lymphocyte detection in the preferred embodiment of the present invention. A. Control group; B. 5μg immunization group; C. 15μg immunization group; D. Spot number statistics; Nonspecific stimulation. PMA (final concentration 50ng / mL) + Ionomycin (final concentration 0.5μg / mL); Specific stimulation. PEDV S protein (SDLY2020, No. OL762458, 304aa-808aa) Note: ns, P>0.1; **, P<0.01. DETAILED DESCRIPTION

[0051] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the examples are all based on conventional experimental conditions, such as Sambrook et al. Molecular Cloning Laboratory Manual (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or the conditions recommended by the manufacturer's instructions.

[0052] PMA and Ionomycin used in the following examples were purchased from Shenzhen Dakoway Biotechnology Co., Ltd.

[0053] Example 1 Preparation of porcine epidemic diarrhea virus mRNA vaccine and optimization of its process conditions

[0054] 1. Materials

[0055] 1.1 Cells, plasmids, strains and their sequences

[0056] LLC-PK1 cells and BHK-21 cells are maintained by the Key Laboratory of Animal Epidemiology, Ministry of Agriculture and Rural Affairs.

[0057] DH-5α competent cells and pUC57 plasmid were purchased from Beijing Qingke Biotechnology Co., Ltd.

[0058] The strain used in the experiment was isolated and obtained by the present invention and was not uploaded to GenBank; the sequence selected was the PEDV S gene RBD (receptor binding domain) (amino acids 477 to 629, SEQ ID NO: 6), and codon optimization was performed to improve protein expression efficiency. The optimized sequence was synthesized by Beijing Qingke Biotechnology Co., Ltd. The specific sequences are shown in Tables 1 and 2:

[0059] Table 1 PEDV S gene RBD sequence (5′-3′)

[0060]

[0061]

[0062] 1.2 Experimental animals

[0063] Female BALB / c mice aged 6-8 weeks were purchased from Beijing Weitonglihua Experimental Animal Co., Ltd.

[0064] 1.3 Main Reagents

[0065] Restriction enzymes Sap I, Eco I, Nco I, T4 DNA ligase (#M0202), HiScribe TM T7ARC AmRNA (with tailing) kit (#E2060S), E. coli Poly (A) Polymerase (#M0276S), RNA Cleanup Kit (50 μg, #T2040L) was purchased from NEB (Beijing).

[0066] DL2000 Plus DNAMarker was purchased from Beijing Juhemei Biotechnology Co., Ltd.

[0067] RPMI 1640 medium, DMEM medium, fetal bovine serum (FBS), TRIzol Regent, transfection reagent Lipofectamine TM 3000, Pierce TM BCA protein quantification kit (#23227) was purchased from Thermo Fisher Scientific.

[0068] 30% (w / v) acrylamide, 1.5 M Tris-HCl (pH 8.8), 1 M Tris-HCl (pH 6.8), and agarose gel affinity chromatography medium rProtein A / G were purchased from Beijing Solebao Biotechnology Co., Ltd.

[0069] 2×RNALoading and 10×MOPS RNA electrophoresis buffers were purchased from Shanghai Biotech Biotechnology Co., Ltd.

[0070] Electrochemiluminescence (ECL) colorimetric solution was purchased from Beijing Ingen Biotechnology Co., Ltd.

[0071] Dlin-MC3-DMA and DMG-PEG2000 were purchased from Avituo (Shanghai) Pharmaceutical Technology Co., Ltd.

[0072] DSPC and cholesterol were purchased from MCE.

[0073] RNase-Free sodium acetate (pH 4.0) and RNase-Free water were purchased from Beijing Coolbo Technology Co., Ltd.

[0074] Anhydrous ethanol was purchased from Sinopharm Chemical Reagent Co., Ltd.

[0075] Ribogreen nucleic acid dye was purchased from Shanghai Shanjing Molecular Biotechnology Co., Ltd.

[0076] T7 High Yield RNA Transcription Kit(N 1 -Me-PseudoUTP), Vaccinia Capping System, and mRNACap2'-O-Methyltransferase were purchased from Nanjing Novozyme Biotechnology Co., Ltd.

[0077] Diethyl pyrocarbonate (DEPC), Seamless Cloning Kit, and 8M LiCl (DNase / RNase free) were purchased from Biotime Biotechnology Co., Ltd.

[0078] Platinum TM SuperFi II PCR high-fidelity enzyme (#12368010) was purchased from Thermo Fisher Scientific.

[0079] Total cholesterol (TC) content detection kit, red blood cell lysis buffer, mouse spleen lymphocyte separation buffer kit, 200-mesh nylon cell sieve, and Mouse IgG (Total) ELISA KIT were purchased from Beijing Solebow Biotechnology Co., Ltd.

[0080] Mouse IFN-γprecoated ELISPOT Kit, Serum-free Medium ELISPOT, universal were purchased from Shenzhen Dakoway Bioengineering Co., Ltd.

[0081] The PEDV S protein was expressed and purified by the Key Laboratory of Animal Epidemiology, Ministry of Agriculture and Rural Affairs.

[0082] 1.4 Antibodies

[0083] Flag tag antibody and β-actin mouse monoclonal antibody were purchased from Abima Biopharmaceuticals (Shanghai) Co., Ltd.

[0084] Horseradish peroxidase-labeled goat anti-mouse IgG (H+L) was purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.

[0085] Anti-Mouse CD3ε, FITC, Anti-Mouse CD4, PE were purchased from Hangzhou Lianke Biotechnology Co., Ltd.

[0086] PerCP / Cyanine5.5 anti-mouse CD8a antibody was purchased from BIOLEGEND (Beijing) Biotechnology Co., Ltd.

[0087] 1.6 Solution preparation

[0088] Ampicillin: Take 10g of ampicillin powder and add 90mL of ultrapure water. After complete dissolution, dilute to 100mL, then filter and aliquot for use.

[0089] LB liquid medium (for bacteria): Take 10g NaCl, 10g tryptone, and 5g yeast extract and add them to 900mL double distilled water. After complete dissolution, make up to 1L and sterilize under high pressure for later use. When screening for resistance, add ampicillin at a ratio of 1:1000.

[0090] Tryptone soy agar solid culture medium: Take 40g soy agar and 5g yeast extract, add 900mL double distilled water, dilute to 1L after complete dissolution, sterilize by high pressure, wait for it to cool to 40℃, add ampicillin at a ratio of 1:1000, shake well and pour into a bacterial culture dish, and invert at 4℃ after solidification for later use.

[0091] Electrophoresis buffer: Dissolve 3.02 g Tris-Base, 1 g sodium dodecyl sulfate, and 18.8 g glycine in distilled water and make up to 1 L.

[0092] Transfer solution: Use distilled water to dissolve 1.93 g Tris-Base and 9 g glycine and make up to 800 mL. Then add 200 mL methanol and precool at -20°C for later use.

[0093] 1.5% RNase-free TritonX-100: Take 1.5mL TritonX-100 and add 90mL ultrapure water. Dissolve it at 42℃ until it is completely dissolved, then make up to 100mL. Then add DEPC at a ratio of 1:1000 and treat it at room temperature overnight. Remove DEPC under high temperature and high pressure to obtain 1.5% RNase-free TritonX-100.

[0094] 2. Methods

[0095] 2.1 Construction and expression of in vitro transcription template for mRNA vaccine

[0096] 2.1.1 Selection of different functional regions of mRNA

[0097] The 5'UTR and 3'UTR (candidate 5'UTR, 3'UTR) of a small number of proteins were screened by consulting the relevant databases Uniprot and Ensembl genome broswer, and then the sequences were spliced ​​with the CDS sequences and determined through mirbase.org whether there were micro RNA binding sites. Finally, the RBD was codon optimized in order to increase protein expression. The sequence after codon optimization is shown in Table 2, and the original sequence of RBD is shown in Table 1.

[0098] 2.1.2 Construction of PCR template

[0099] The selected 5'UTR and 3'UTR are spliced ​​with the CDS sequence through fusion PCR, and then the required pre-template is amplified through PCR, recovered and purified, and then connected, transformed, picked, and sent for testing. Then, PCR amplification is performed using the correctly sequenced bacterial solution as a template to obtain an in vitro transcription template (PCR product), and care is taken to avoid RNase contamination.

[0100] In addition, when searching for a shorter 5'UTR, if the 3'UTR of the gene is longer or no data is available, the 3'UTR of the gene LDHB is used.

[0101] 2.1.2.1 Primer design

[0102] Corresponding primers were designed according to the UTR (5'UTR and 3'UTR) sequences obtained, and the primers are detailed in Table 24; UTR (5'UTR and 3'UTR) was amplified using porcine cell DNA as a template, and then a complete in vitro transcription template (PCR product) was obtained by fusion PCR; if the UTR length was shorter, it was added by primers.

[0103] 2.1.2.2 Construction of CDS sequence and fragment purification

[0104] Take mRNA-LDHB-RBD as an example to design in vitro transcription template:

[0105] Acquisition of fragment LDHB5: Use cDNA or plasmid to amplify the RBD fragment by PCR, use primers RBD-LDHB5-F1, RBD-LDHB5-F2, RBD-LDHB5-F3 and RBD-LDHB5-F4 to add the signal peptide PLAT, Kozak sequence, LDHB 5'UTR and T7 promoter to the 5' end of the RBD fragment, use primer RBD-R to add the 3' end homology arm, Flag tag and stop codon to the RBD fragment, and the fragment is named LDHB5 after recovery.

[0106] Acquisition of fragment LDHB3: Primers LDHB 3-F and LDHB 3-R were used to amplify LDHB3'UTR using porcine cell DNA as a template, and primers RBD-LDHB3-F and LDHB 3-R were used to add homology arms to LDHB 3'UTR. The recovered fragment was named LDHB3.

[0107] Acquisition of in vitro transcription template: Primers LDHB 5-F and LDHB 3-R were used to amplify the required in vitro transcription template with fragments LDHB5 and LDHB3 as templates, and named as LDHB-RBD.

[0108] The primers required for PCR are shown in Table 3; after PCR amplification, 1% agarose gel electrophoresis was performed, and the corresponding size bands were cut out and purified using EZNAGel Extraction Kit (omega) and marked. The specific steps are as follows:

[0109] Cut the target band into a 1.5 mL centrifuge tube and add an appropriate amount of Binding Buffer to dissolve at 65°C. Take 700 μL of the liquid and add it to the HiBind column. Centrifuge at 12,000 rpm for 1 min. Wash twice with 700 μL of SPW buffer, centrifuge at 12,000 rpm for 1 min each time, wash twice, and leave in the air for 1 min. Then dry the HiBind column at room temperature to completely remove the ethanol, dissolve it with 20 μL of enzyme-free water, and collect it by centrifugation in a new centrifuge tube and mark it for later use.

[0110] Table 3 Primers required for in vitro transcription template construction

[0111]

[0112]

[0113] 2.1.2.3 Ligation, transformation, and sequencing

[0114] The fusion fragment LDHB-RBD obtained in 2.1.2.2 was connected to pCE2 TA / Blunt-Zero using 5min TA / Blunt-Zero Cloning Kit (Vazyme). The reaction system is shown in Table 4:

[0115] Table 4 Ligation reaction system

[0116]

[0117] After mixing the liquid, centrifuge briefly at low speed at 25°C for 5 min, and then place the ligation product on ice.

[0118] Take 50 μL of DH-5α competent cells and add 5 μL of the connected vector, pipette to mix, ice bath for 30 min, heat shock at 42 °C for 60 s, ice bath for 3 min, add 1 mL of antibiotic-free liquid LB medium to activate for 1 h, take an appropriate amount of bacterial solution and evenly spread it on the corresponding resistance LB solid medium, invert and culture at 37 °C until the colony is the size of a pinprick, pick the colony and expand it with liquid LB medium, identify it by bacterial solution PCR, and send the bacterial solution with the target band to Beijing Qingke Biotechnology Co., Ltd. for sequencing.

[0119] 2.1.2.4 Preparation of in vitro transcription template (PCR product)

[0120] Use the correctly sequenced bacterial solution as template, use primers LDHB 5-F and LDHB 3-R for PCR, and then purify the fragments. For the fragment purification method, refer to 2.1.2.2. Be careful not to have RNase contamination in the obtained in vitro transcription template.

[0121] 2.1.3 In vitro transcription

[0122] Using HiScribe TM T7 ARCA mRNA (with tailing) kit was used for in vitro transcription. The in vitro transcription reaction system is shown in Table 5-1 and Table 5-2. The reaction conditions are: in vitro transcription, 37°C, 30 min; in vitro transcription template removal, 37°C, 15 min; Capped-RNA tailing system is shown in Table 6. The reaction conditions are: 37°C, 30 min.

[0123] Table 5-1 In vitro transcription reaction system

[0124]

[0125]

[0126] Table 5-2 In vitro transcription reaction system

[0127]

[0128] Table 6 Capped-RNA tailing reaction system

[0129]

[0130] 2.1.4 mRNA purification

[0131] use RNA Cleanup Kit was used to purify mRNA. 100 μL RNACleanup Binding Buffer was added to the tailing system, 150 μL anhydrous ethanol was added, and the mixture was mixed by pipetting. The Monarch RNA Cleanup Column was inserted into the Monarch Collection TubesⅡ and the mixed liquid was transferred to the Monarch RNA Cleanup Column. The mixture was centrifuged at 13000 rpm for 1 min, and the filtrate in the collection tube was discarded. 500 μL RNA Cleanup Wash Buffer was added, and the mixture was centrifuged at 13000 rpm for 1 min. The filtrate in the collection tube was discarded, and the mixture was washed once. The mixture was centrifuged at 13000 rpm for 1 min, and the filtrate in the collection tube was discarded. The mixture was washed once again, and the mixture was centrifuged at 13000 rpm for 1 min. The mixture was dried at room temperature for 5 min to remove the ethanol, and 50 μL nuclease-free water was added to dissolve the mRNA. The Monarch RNA Cleanup Column was transferred to a new centrifuge tube to collect the purified mRNA. Finally, the mRNA concentration and purity were determined using Nanodrop Lite.

[0132] 2.1.5 RNA denaturation electrophoresis

[0133] RNA denaturing electrophoresis was used to verify the integrity, homogeneity and fragment size of the prepared mRNA. The specific steps are as follows:

[0134] Prepare 1.5% agarose denaturing gel: 36 mL of nuclease-free water, 0.75 g of nuclease-free agarose, 5 mL of 10×MOPS RNA electrophoresis buffer, and 9 mL of formaldehyde. Heat to completely dissolve the agarose, then add 5 μL of EB dye, mix well, pour into a mold, and cool for later use.

[0135] Preparation of RNA samples: 1 μg of purified mRNA was added to an equal volume of 2× RNA loading buffer, mixed and denatured at 65°C for 5 min, and then placed on ice for 3 min; ss RNA Ladder was prepared according to the manufacturer's instructions.

[0136] After the samples are prepared, RNA denaturation electrophoresis is performed, and then imaging is performed using a gel imager to analyze the bands.

[0137] 2.1.6 mRNA in vitro cell transfection

[0138] Use transfection reagent Lipofectamine TM 3000 for in vitro cell transfection experiments, LLC-PK1 cells were evenly spread on the cell plate, cultured at 37°C, 5% CO2, and cell transfection was performed when the cell confluence reached 70%. TM 3000 was mixed with mRNA at a volume-mass ratio of 3 μL:1 μg to prepare mRNA liposome complexes, and the mixed solution was Opti-MEM medium. After mixing for 15 minutes, the mRNA liposome complexes were added to the cell wells and shaken evenly, and then cultured at 37°C and 5% CO2; 1.25 μg was transfected in 12-well plates and 2.5 μg was transfected in 6-well plates.

[0139] 2.1.7 Indirect immunofluorescence

[0140] The prepared mRNA was transfected into LLC-PK1 cells. After 12 hours, the expression of the target protein was detected by indirect immunofluorescence. The Flag tag antibody was used as the primary antibody. After washing away the secondary antibody, the cell nucleus was stained. The PBS in the cell plate was completely aspirated, 150 μL of DAPI dye was added to the cell wells, incubated in the dark for 10 minutes, the DAPI dye was discarded, and the cells were washed 33 times with PBS, each time for 5 minutes. The cells were observed and photographed under a fluorescence microscope.

[0141] 2.1.8 Western Blot

[0142] The prepared mRNA was transfected into LLC-PK1 cells. After a certain period of time, all cells in the wells (including dead cells in the culture medium) were collected using a scraper. Then, an appropriate amount of NP-40 cell lysis buffer (with protease inhibitor added at 1:100) was used to lyse the cells. The cells were lysed in an ice bath for 30 minutes and centrifuged at 12,000 rpm and 4°C for 20 minutes. The supernatant was taken into a new centrifuge tube for protein concentration determination and sample boiling. The protein concentration determination was determined by referring to the instructions of the BCA protein concentration determination kit. Sample boiling: 5×SDS loading buffer was added to the cell lysate, and the sample was boiled in boiling water for 10 minutes after being blown and mixed. Then, the sample was placed on ice for 15% SDS-PAGE gel electrophoresis. The specific steps of gel electrophoresis are as follows:

[0143] Electrophoresis: Use 80V voltage to flatten the protein sample. After the protein marker is dispersed, adjust the voltage to 120V. After the protein sample is separated, transfer to the membrane.

[0144] Transfer: Use methanol to activate the PVDF membrane, sandwich it in the order of "black-gel-membrane" for wet transfer, 100V, 120min;

[0145] Blocking: PVDF membrane was blocked with 5% skim milk (w / v) at room temperature for 2 h;

[0146] Primary antibody incubation: After blocking, wash the membrane with PBS to remove the residual blocking solution, and then incubate with 1:5000 diluted Flag tag monoclonal antibody at 4°C overnight;

[0147] Washing: Wash 3 times with 0.1% PBST, 5 min each time;

[0148] Secondary antibody incubation: Use horseradish peroxidase-labeled goat anti-mouse IgG (H+L) monoclonal antibody diluted 1:10000 for incubation at room temperature for 1 hour;

[0149] Washing: Wash 3 times with 0.1% PBST, 5 min each time;

[0150] Exposure: Prepare ECL luminescent working solution according to the instructions, and image the film with an imager after allowing it to act on the film for 1 minute.

[0151] 2.1.9 Protein immunoprecipitation

[0152] mRNA-LDHB-RBD was transfected into BHK-21 cells, with 2.5 μg of mRNA transfected into each well. Before transfection, the complete medium in the wells was replaced with serum-free DMEM medium, with 1 mL per well. After 24 hours, all the liquid in the wells was collected and centrifuged at 12,000 rpm and 4°C for 5 minutes. The supernatant was taken for immunoprecipitation. After antibody binding according to the instructions, agarose beads were incubated with the supernatant at 4°C for 4 hours. The agarose beads were collected by brief centrifugation and washed twice with washing solution. The washing solution was discarded, and the beads were resuspended with an appropriate amount of PBS and boiled with 5× loading buffer. The samples were then tested by Western Blot. For specific steps, see 2.1.8.

[0153] 2.2 Preparation and evaluation of lipid nanoparticles

[0154] 2.2.1 Construction of recombinant plasmid pUC57A60G60

[0155] 2.2.1.1 Primer design

[0156] Use primers pUC57 A60-G-60-F and pUC57-A60-G-60-R1 to add restriction sites EcoRⅠ, NcoⅠ, HindⅢ, XbaⅠ, and SapⅠ to the plasmid, and then use primers pUC57-A60-G-60-F and pUC57-A60-G-60-R2 to add 30A at both ends of the plasmid; take 50mM of primers 60-A and 60-A respectively, blow and mix, denature at 98℃, and anneal at 55℃ to form double-stranded DNA fragments.

[0157] 2.2.1.2 Plasmid PCR and purification

[0158] Using plasmid pUC57 as a template, the above primers were used to add segmented poly(A) sequences and restriction sites to the plasmid; the primers required for PCR are shown in Table 7, the PCR reaction system is shown in Table 8, and the reaction procedure is shown in Table 9.

[0159] Table 7 Primers required for constructing recombinant plasmid pUC57 A60G60

[0160]

[0161] Table 8 Plasmid PCR reaction system

[0162]

[0163] Table 9 Plasmid PCR reaction program

[0164]

[0165] 2.2.1.3 Fragment ligation, transformation, extraction and sequencing of recombinant plasmids

[0166] Use T4 DNA ligase to connect the linearized plasmid and fragment 60G60 at 16°C overnight, then transform the ligation product into DH5α and sequence the bacteria. See 2.1.2.3 for the method.

[0167] 2.2.2 Large-scale preparation of mRNA-LDHB-EGFP

[0168] The mRNA-LDHB-EGFP in vitro transcription template was constructed according to method 2.1.2. The fragment EGFP was obtained by plasmid pEGFP-N2 PCR, using the 5'UTR and 3'UTR of the gene LDHB. The primers are shown in Table 10.

[0169] Table 10 Primers required for mRNA-LDHB-EGFP template construction

[0170]

[0171] Note: Bold italics are restriction endonuclease recognition sites, and underlined are homologous sequences.

[0172] 2.2.2.1 Construction of plasmid pUC57 60G60-EGFP

[0173] The plasmid pUC5760G60 was double-digested with restriction endonucleases EcoI and NcoI, separated by agarose gel, and the concentration was measured after purification for later use; the homologous recombination fragment LDHB-EGFP was obtained by PCR amplification using primers UC-EGFP-F and UC-EGFP-R, with the mRNA-LDHB-EGFP in vitro transcription template as a template, and the concentration was measured after agarose gel separation and purification for later use. Homologous recombination was performed to construct the plasmid pUC5760G60-EGFP according to the molar ratio of vector: fragment = 1:3. The plasmid double enzyme digestion system is shown in Table 11, and the homologous recombination system is shown in Table 12. The homologous recombination product was transformed and picked, and the method is shown in 2.1.2.3.

[0174] Table 11 Plasmid double restriction enzyme digestion system

[0175]

[0176] Table 12 Seamless cloning reaction system

[0177]

[0178] Reaction conditions: 50°C, 15 min.

[0179] 2.2.2.2 Large-scale plasmid preparation

[0180] Take an appropriate amount of the correctly sequenced bacterial liquid and inoculate it into 150 mL of ampicillin-resistant liquid LB medium. Cultivate it at 37°C with shaking for 15 hours, then perform plasmid extraction to obtain a large amount of recombinant plasmids. The steps are as follows:

[0181] Pour 150mL of bacterial solution into a 250mL medium-extraction bucket, balance it, centrifuge it at 4℃, 6000rpm for 15min, discard the upper culture medium and wipe the LB culture medium on the side wall of the medium-extraction bucket clean, then add 7mL of cell resuspension solution (CRA), vortex and oscillate to resuspend the bacteria, add 7mL of cell lysis solution (CLA), shake evenly, lyse for 3min, add 10mL of neutralization solution (NSB) and shake evenly, you can see flocculent white precipitate, then centrifuge it at 4℃, 8000rpm for 20min, pour the supernatant into the filter column, drain the liquid by suction, then add 5mL of endotoxin removal solution (Endotoxin Removal Wash) to the adsorption column to remove endotoxin, add 20mL of column washing solution (Column 200), and add 10mL of ... After aspiration, continue aspiration for 5 min to completely evaporate the ethanol in the adsorption column, add 500 μL of preheated nuclease-free water and let stand for 2 min, then aspirate the liquid in the adsorption column into a 1.5 mL centrifuge tube, and finally use Nanodrop to measure the plasmid concentration.

[0182] 2.2.2.3 Linearization of recombinant plasmid pUC-5760G60-EGFP

[0183] Use type IIS nuclease Sap I to linearize the plasmid. The reaction system is shown in Table 13. The reaction conditions are 37°C for 2 hours. Then separate and recover by agarose gel electrophoresis. The method is shown in 2.1.2.2. Measure the concentration for later use.

[0184] Table 13 Plasmid linearization restriction enzyme system

[0185]

[0186] 2.2.2.4 Large-scale preparation of mRNA-EGFP

[0187] Use the Novozymes T7 High Yield RNA Transcription Kit (N 1 -Me-PseudoUTP) to obtain a large amount of RNA, the reaction system is shown in Table 14, the reaction conditions are 37°C, 2h, DNase I is added, the plasmid template is removed at 37°C for 15min, the RNA is purified by lithium chloride precipitation, the concentration is measured after dissolving in nuclease-free water, and stored at -80°C for later use.

[0188] Table 14 In vitro transcription system (large-scale preparation)

[0189]

[0190] Capped-mRNA was prepared by RNA capping using the Vaccinia Capping System. The capping system is shown in Table 15. The reaction conditions were 37°C for 1 h. RNA was then purified by lithium chloride precipitation. The concentration was measured after dissolution in nuclease-free water and stored at -80°C for later use.

[0191] Table 15 Capping system

[0192]

[0193]

[0194] 2.2.2.5 Lithium chloride precipitation

[0195] Use RNase free water to dilute 8M lithium chloride to 7.5M for later use. The specific steps are as follows:

[0196] The volume of the above system (in vitro transcription system, capping system) was expanded to 50 μL, and then 25 μL of 7.5 M lithium chloride was added and mixed by pipetting, precipitated at -20°C for 30 min, and then centrifuged at 12,000 rpm and 4°C for 30 min, the lithium chloride was discarded, and pre-cooled 70% ethanol was added to wash the RNA, centrifuged at 12,000 rpm and 4°C for 5 min, washed twice, and 70% ethanol was discarded. The RNA was air-dried for 5 min to remove the ethanol, and an appropriate amount of nuclease-free water was used to dissolve the RNA. After measuring the concentration, capping was performed or stored at -80°C for later use.

[0197] 2.2.3 Preparation of lipid nanoparticles

[0198] Lipid nanoparticles were prepared using a fishbone microfluidic chip, mRNA was dissolved in a sodium acetate solution at pH 4.0 and diluted to the required concentration according to N / P; lipids were dissolved in anhydrous ethanol at a molar ratio of Dlin-MC3-DMA:DSPC:cholesterol:DMG-PEG2000=50:10:38.5:1.5; a syringe was used to mix the lipid nanoparticles at a flow rate ratio of aqueous phase (mRNA):organic phase (lipid)=3:1, and the prepared lipid nanoparticles were immediately diluted 30 times with 0.01MPBS to reduce the ethanol concentration, and then concentrated by an ultrafiltration tube, and finally the concentrated lipid nanoparticles were filtered and sterilized using a 0.22μm filter.

[0199] 2.2.4 Determination of encapsulation efficiency and effective nucleic acid concentration

[0200] The RNA sample was diluted to 10 ng / μL to prepare an RNA standard (RNA Standard), and the RNA standard curve was established according to Table 16. According to Table 17, the lipid nanoparticles were demulsified using RNase-free 1.5% TritonX-100 to measure the total RNA amount in the sample, and the free RNA amount in the sample was measured without demulsification, and then the encapsulation efficiency was calculated according to formula (1).

[0201] Table 16 RNA standard curve

[0202]

[0203] Reaction conditions: reaction at room temperature for 5 minutes; measurement conditions: excitation light 485nm, emission light 530nm.

[0204] Table 17 RNA sample determination

[0205]

[0206] The reaction conditions and assay conditions were the same as above.

[0207]

[0208] 2.2.5 Particle size and Zeta potential

[0209] The particle size and potential analysis were performed using dynamic light scattering (DLS) technology. After the sample was diluted, 1 mL of the sample was added to the cuvette, which was then inserted into the test hole for testing. The test molecules were selected as biomacromolecules, and the dispersant was selected as 0.01 M PBS. The test was performed 14 times. 1 mL of the sample was added to the potential test sample pool to determine the Zeta potential, and the test was performed 100 times.

[0210] 2.2.6 Cryo-TEM

[0211] After the copper mesh is hydrophilized, the diluted sample is dripped onto the copper mesh. After waiting for a while, most of the sample is absorbed using filter paper. The copper mesh is then placed in liquid ethane for quick freezing. The quick-frozen copper mesh is then placed in a TEM four-hole sample box that has been cooled by liquid nitrogen in advance for testing.

[0212] 2.2.7 Cell flow cytometry

[0213] BHK-21 cells were evenly plated in 6-well plates. When the confluence was 70%, lipid nanoparticles containing 2.5 μg of effective nucleic acid were added to each well. After 24 hours, trypsin was used for digestion for 1 min. After discarding the trypsin, the cells were resuspended in PBS and centrifuged at 2500 rpm and 4°C for 5 min. After discarding the supernatant, the cells were resuspended in 400 μL PBS. The cell suspension was filtered into the flow tube using a 200-mesh filter and vortexed before being put on the machine. The voltage was adjusted so that the cells fell in the appropriate position, and then the cell group was selected for de-adhesion. The voltage was adjusted so that the maximum fluorescence intensity of the negative control group was maintained at 10 3 Then carry out sample measurement.

[0214] 2.3 Construction of PEDV mRNA vaccine and in vivo immune evaluation

[0215] 2.3.1 Construction of recombinant plasmid pUC57 60G60-RBD

[0216] The method is as described in 2.2.2.1 for the construction of plasmid pUC57 60G60-EGFP, and the primers used are shown in Table 18.

[0217] Table 18 Primers required for constructing recombinant plasmid pUC57 60G60-RBD

[0218]

[0219] Note: Bold italics are restriction endonuclease recognition sites, and underlined are homologous sequences.

[0220] 2.3.2 Large-scale preparation of mRNA-TBCC-RBDYH and lipid nanoparticles

[0221] For large-scale preparation of mRNA-TBCC-RBDYH, see Method 2.2.2.3 and Method 2.2.2.4; for mRNA purification, see Method 2.2.2.5; for the preparation method of lipid nanoparticles LNP-mRNA-TBCC-RBD, see 2.2.3.

[0222] 2.3.3 Vaccine Immunization

[0223] The effective nucleic acid concentration of the prepared lipid nanoparticles was determined, and the method is shown in 2.2.4. The encapsulation efficiency and effective nucleic acid concentration were determined by intramuscular injection of mice. The specific grouping and immunization dose are shown in Table 19, and the immunization strategy is shown in Figure 2 .

[0224] Table 19 Immunization groups and immunization doses

[0225]

[0226] 2.3.4 Indirect immunofluorescence

[0227] After the mouse blood was coagulated at room temperature to separate the serum, it was centrifuged at 6000×g for 5 min, and the supernatant was taken for later use;

[0228] Vero CCL81 cells were evenly plated in a 96-well plate. When the cell confluence reached 100%, the PEDVSD2020-1 strain was inoculated at an MOI of 0.1. IFA was performed 24 hours later. The method is shown in 2.2.4.2.

[0229] 2.3.5 Isolation of mouse peripheral blood lymphocytes

[0230] A mouse peripheral blood lymphocyte separation solution kit was used to separate peripheral blood lymphocytes. The specific steps are as follows:

[0231] Mouse anticoagulated peripheral blood was diluted with an equal volume of PBS, 3 mL of separation solution was added to a centrifuge tube, and then the diluted blood was spread evenly on the surface of the separation solution, keeping the interface between the two liquid surfaces clear. Centrifuge horizontally at room temperature and 800 g for 30 min. After centrifugation, the liquid was clearly stratified, with the upper layer being diluted plasma, the middle being a transparent separation solution layer, the white film between the plasma and the separation solution being the lymphocyte layer, and the bottom being red blood cells and granulocytes. The lymphocyte layer was carefully aspirated into a 15 mL centrifuge tube, and then 10 mL of PBS was used to wash the lymphocytes, and 250 g was centrifuged for 10 min. Then 2 mL of red blood cell lysis solution was added, and the cells were lysed on ice for 15 min, and then centrifuged at 400 g for 10 min. The supernatant was discarded and 5 mL of PBS was used to wash the lymphocytes, and the cells were centrifuged at 400 g for 10 min. This was repeated once, and the cells were resuspended in 500 μL PBS for later use.

[0232] 2.3.6 Isolation of mouse spleen lymphocytes

[0233] Preparation of spleen single cell suspension: Take the mouse spleen in the clean bench, remove the spleen capsule and cut it into small pieces, then use a 200-mesh nylon sieve to grind it. Keep the spleen in a liquid environment during the grinding process. After grinding is complete, rinse the sieve with PBS, collect the cell suspension, and filter it once with a sieve to make a spleen single cell suspension.

[0234] Separation of spleen lymphocytes: Take 3mL of separation solution and add it to the centrifuge tube, carefully add the spleen single cell suspension on the separation solution, keep the interface between the two liquid surfaces clear, centrifuge at room temperature, 800g for 30min, the centrifuge tube is divided into four layers after centrifugation, the first layer from top to bottom is the diluent layer; the second layer is the milky white lymphocyte layer; the third layer is the membrane separation liquid layer; the fourth layer is the red blood cell layer, carefully pipette the second layer of milky white lymphocytes into a 15mL centrifuge tube, then use 10mL PBS to wash the lymphocytes, 250g, centrifuge for 10min, add 2mL red blood cell lysis solution, lyse on ice for 15min, centrifuge at 400g for 10min, discard the supernatant, use 5mL PBS to wash the lymphocytes, 250g, centrifuge for 10min, repeat once, use 1mL RPMI 1640 basic culture medium to resuspend the cells for use.

[0235] 2.3.7 Flow cytometry

[0236] The separated peripheral blood lymphocytes and spleen lymphocytes were marked and placed on ice. Anti-MouseCD3ε, Anti-MouseCD4 and anti-mouseCD8a antibodies were added to each sample. The antibody dosage was the dosage recommended in the instruction manual. + CD8 + The double-negative group and three single-staining groups were incubated in an ice bath in the dark for 30 min, centrifuged at 3500 rpm and 4°C for 5 min, and then resuspended and washed twice with 700 μL PBS. After washing, the cells were resuspended with 1 mL PBS and passed through a 200-mesh sieve for flow cytometry detection.

[0237] 2.3.8 Determination of total IgG in mouse peripheral blood

[0238] The mouse serum was diluted 10-fold using PBS and the standard was diluted.

[0239] Mouse IgG (Total) ELISA KIT kit was used to measure the total IgG in mouse peripheral blood. First, the ELISA plate was soaked in washing solution for 30 seconds, then the washing solution was discarded and patted dry, and repeated twice. 100 μL of sample or standard was added to each well, incubated at 25°C for 120 minutes, then the liquid in the well was discarded and washed 4 times with 300 μL of washing solution, and the liquid in the well was patted dry each time. After washing, 100 μL of enzyme-labeled antibody was added to each well, incubated at 25°C for 45 minutes, then the liquid in the well was discarded and washed 5 times with 300 μL of washing solution, and the liquid in the well was patted dry each time. Then 100 μL of chromogenic substrate TMB was added to each well, protected from light, and color developed at 25°C for 15 minutes, then 50 μL of stop solution was added to each well, mixed, and dual-wavelength detection was performed to measure OD 450 and OD 630 .

[0240] A standard curve was drawn according to the concentration and absorbance of the standard, and then the IgG concentration in the sample was calculated.

[0241] 2.3.10 ELISA

[0242] After separation, spleen lymphocytes were counted and then diluted to 4 × 10 6 / mL for use, and the protein stimulator is diluted to 400μg / mL for use.

[0243] Add 200 μL serum-free ELISPOT medium to each well to activate the coated plate and let it stand at room temperature for 10 min. Discard the medium in the wells and add 4×10 5 cells, add 10 μL (4 μg) protein stimulant, do 3 replicates, put into the cell culture incubator, continue to culture for 24 hours, and do negative and positive controls at the same time. Add 10 μL RPMI 1640 culture medium to the negative control and 10 μL positive stimulant (PMA, Ionomycin) to the positive control.

[0244] After the culture is completed, discard the culture medium in the well. Add 200μL pre-cooled deionized water to each well, lyse the cells at 4℃ for 10min; discard the liquid in the well, add 300μL Washing Buffer, let it stand at room temperature for 1min, discard the liquid, and repeat 5 times; add 100μL Biotinylated Antibody working solution to each well, incubate at 37℃ for 1 hour, discard the liquid in the well, add 300μL Washing Buffer, let it stand at room temperature for 1min, discard the liquid, and repeat 5 times; add 100μL Streptavidin-HRP working solution to each well, incubate at 37℃ for 1 hour, discard the liquid in the well, add 300μL Washing Buffer, let it stand at room temperature for 1min, discard the liquid, and repeat 5 times. Buffer, let stand at room temperature for 1 min, discard the liquid, repeat 5 times, then wash the bottom of the membrane with deionized water, and dry the water on the bottom of the membrane with absorbent paper; pat dry the liquid in the wells, add freshly prepared AEC color development solution to each well, 100 μL per well, incubate at 37°C in the dark for 15 min; discard the color development solution in the wells, wash the wells and the bottom of the membrane with 300 μL deionized water, wash 3 times to stop the color development; pat dry the liquid in the wells, dry the water on the bottom of the membrane with absorbent paper, air-dry the bottom membrane in the wells in a cool place at room temperature, and finally count the ELISPOT plate spots.

[0245] 3. Results

[0246] 3.1 Construction of in vitro transcription template

[0247] LDHB 5'UTR was introduced by primer design, and fusion PCR technology was further used to obtain LDHB 5'UTR+RBD+LDHB 3'UTR fragments. PCR identification results showed that the target fragment of about 720 bp could be amplified, which was consistent with the expected fragment size. Figure 3 The sequencing results showed that it was consistent with the target sequence, indicating that the in vitro transcription template was successfully constructed.

[0248] 3.2 In vitro transfection

[0249] 3.2.1 Immunofluorescence detection of target protein expression

[0250] LLC-PK1 cells were transfected with mRNA-LDHB-RBD, and IFA was performed 12 h later. Figure 4 ). The results showed that after transfection, obvious specific green fluorescence could be observed in the cytoplasm of LLC-PK1 cells, indicating that the transfected mRNA-LDHB-RBD could be normally translated and expressed in LLC-PK1 cells.

[0251] 3.2.2 Western Blot analysis of target protein expression

[0252] Using the UTR of the genes LDHB and GAPDH, mRNA-LDHB-RBD, mRNA-GAPDH-RBD, mRNA-LDHB-RBDYH, and mRNA-GAPDH-RBDYH were constructed and transcribed. They were transfected into LLC-PK1 cells, and Western Blot was performed 12 hours later to analyze the expression of the target protein. The results are shown in Figure 5 shown.

[0253] The 3'UTR lengths of the genes LDHB and GAPDH are similar. When the 5'UTR length is 24bp (gene LDHB), the expression of the target protein can be detected; when the 5'UTR length is 334bp (gene GAPDH), the expression of the target protein cannot be detected. After codon optimization, the expression of the target protein increased significantly.

[0254] 3.3 Determination of the optimal 5'UTR length

[0255] Without changing the CDS (coding sequence) and 3'UTR, different lengths of 5'UTR were set to analyze their effects on protein expression. Among them, the 5'UTR length of the LDHB gene is 24bp, the 5'UTR length of the ACTB gene is 90bp, the 5'UTR length of the LDHC gene is 140bp, the 5'UTR length of the HBB gene is 199bp, and the 5'UTR length of the GAPDH gene is 334bp.

[0256] The purified mRNA was subjected to RNA denaturation electrophoresis to identify the quality of the mRNA. Figure 6 As shown in B. It can be seen that the band is single, without smearing or tailing. This shows that the purified mRNA has good homogeneity and integrity, and the length is in line with expectations.

[0257] WB detection of target protein expression with different 5'UTR lengths ( Figure 6 A) The results showed that as the length of 5'UTR continued to decrease, the expression of the target protein increased significantly. When the length of 5'UTR exceeded 140bp, the expression of the target protein was not detected. Figure 6 C is the grayscale analysis of protein expression.

[0258] 3.4 Determination of the optimal time for protein expression

[0259] LLC-PK1 cells were transfected with mRNA-LDHB-RBDYH. After 3h, 6h, 9h, and 12h, the cells were fixed for IFA. Meanwhile, all cells in the wells were scraped for WB detection. The results were as follows: Figure 7 shown.

[0260] The results showed that specific green fluorescence signals could be observed in the cytoplasm 3 hours after transfection, indicating that the target protein had been expressed in the host cells. The number of positive cells was the largest and the fluorescence signal was the strongest at 9-12 hours. Figure 7 A). WB results showed that the expression level of the target protein reached its peak 12 hours after transfection ( Figure 7 B).

[0261] 3.5 Detection of target protein secretion expression

[0262] To determine the effects of different porcine signal peptides on the secretion and expression of target proteins, we tested the expression of target proteins under the conditions of signal peptides such as IL-4, IL-13, TLR9, and PRL. Figure 8 As shown in A.

[0263] After transfecting LLC-PK1 cells with mRNA carrying different signal peptides, the expression levels of the target proteins were significantly different. Figure 8 D is the grayscale analysis of protein expression. When the signal peptide is IL-13, the expression level of the target protein is the highest.

[0264] Figure 8 C is the quality verification of the purified mRNA. The results show that the four mRNA bands are single and of good quality.

[0265] Considering that LLC-PK1 cells die after mRNA transfection, leading to false positive results of expressed protein release, we used BHK-21 cells to verify protein secretory expression.

[0266] The signal peptide of human plasminogen activator (PLAT) verified in the literature was selected to construct LDHB-sp(PLAT)-RBDYH and transfect BHK-21 cells. After 24 hours, the supernatant was collected for protein immunoprecipitation (IP) to verify the presence of secretory expression target protein in the supernatant. At the same time, the cells were collected for WB. The results are shown in Figure 8 As shown in B. WB results show that the expressed target protein can be detected in the cell lysate, but not in the supernatant. However, after immunoprecipitation enrichment, the target protein expressed in secreted form in the cell supernatant can be detected. This indicates that the signal peptide can guide the target protein to be secreted outside the cell.

[0267] 3.6 UTR screening of in vitro transcription templates

[0268] In order to determine the most suitable UTR for the candidate vaccine, in vitro transcription templates of 19 different UTRs were constructed. The specific information is shown in Table 20.

[0269] Table 20 Chinese and English names of genes used in 19 templates

[0270]

[0271] The purified mRNA was subjected to RNA denaturation electrophoresis to determine the quality of the mRNA. Fig. 9 As shown in the figure, the mRNA produced is single and the nucleotide length is consistent with the expected one.

[0272] LLC-PK1 cells were transfected with 19 mRNAs. After 12 hours, all cells in the wells were collected for WB and grayscale analysis. The results are shown in Figure 2. Fig.10 B and Fig.10 D. Except for mRNA-GAPDH-RBDYH and mRNA-PDYN-RBDYH, the expression of target proteins can be detected in the other 17 mRNAs. The expression levels of target proteins under different UTR conditions are different. Among them, the protein expression level of mRNA-TBCC-RBDYH is relatively the highest.

[0273] IFA verification results are as follows Fig.10 A and Fig.10 As shown in C. Consistent with the WB results, no specific fluorescence signal was observed in the cells transfected with mRNA-GAPDH-RBDYH and mRNA-PDYN-RBDYH.

[0274] 3.7 Preparation of mRNA-LHDB-EGFP

[0275] The recombinant plasmid pUC57 60G60 was constructed. The sequencing results are shown in Fig.11The results showed that the recombinant plasmid contained a poly(A) structure of 89 bp in length, and no base G was found in the poly(A) structure.

[0276] The plasmid pUC57 60G60 was double-digested with restriction endonucleases EcoI and NcoI, and homologous recombination was performed after recovery. The results of plasmid electrophoresis and homologous recombination fragment PCR amplification after restriction digestion are as follows Fig.12 As shown, a target band with a size consistent with the expected fragment can be amplified from the obtained homologous recombination fragment.

[0277] Prepare a large amount of mRNA-LDHB-EGFP. Perform RNA denaturation electrophoresis on the prepared mRNA to detect the quality of mRNA. It can be seen that the mRNA band is single and has no tail. This shows that the prepared mRNA is relatively pure and can be used for subsequent experiments ( Fig.13 ).

[0278] 3.8 Lipid Nanoparticle Transfection Effect

[0279] Three groups of N / P=6, N / P=10, and N / P=20 were set up to package and produce LNP-mRNA-LDHB-EGFP, which were then transfected into BHK-21 cells after ultrafiltration concentration and filtration sterilization. The culture medium was discarded after 24 hours, and the intracellular fluorescence signal was observed under a fluorescence microscope ( Fig.14 ). The results showed that when N / P=6 or 10, the intensity of the intracellular fluorescence signal was not much different; when N / P=20, the intensity of the intracellular fluorescence signal was significantly weakened.

[0280] WB verification results are as follows Fig.15 When N / P=6 or 10, the expression level of the target protein after the lipid nanoparticles were transfected into cells was significantly higher than that of N / P=20. Therefore, the nitrogen-phosphorus ratio for the preparation of lipid nanoparticles was temporarily set at 6 or 10.

[0281] 3.9 Encapsulation efficiency determination

[0282] The 2005 edition of the Chinese Pharmacopoeia stipulates that after liposome separation, the encapsulation rate is calculated according to the following formula, and the encapsulation rate should be greater than 80%. The calculation formula is shown in formula (1). According to the RNA standard curve (Table 21 and Fig.16 ) and Table 22 to calculate the total RNA content and free RNA content in the sample, and the encapsulation efficiency was calculated by formula (1).

[0283] As shown in Table 23, the encapsulation efficiency of different N / Ps is different. When N / P=6, the encapsulation efficiency is 79.55%; when N / P=10, the encapsulation efficiency is 85.44%; when N / P=20, the encapsulation efficiency is 92.63%. When N / P≥10, the encapsulation efficiency is greater than 80%, which meets the requirements of the Chinese Pharmacopoeia. Combined with the transfection effect, the lipid nanoparticles were finally prepared when N / P=10.

[0284] Table 21 RNA standard curve fluorescence intensity determination

[0285]

[0286] Table 22 Results of encapsulation efficiency determination of different N / P lipid nanoparticles

[0287]

[0288]

[0289] Table 23 Encapsulation efficiency of different N / P lipid nanoparticles

[0290]

[0291] 3.10 Particle size and Zeta potential results

[0292] The particle size potential analysis was performed using dynamic light scattering (DLS) technology. Fig.17 As shown, the average particle size of the prepared lipid nanoparticles is 85.42nm, and the polydispersity index (PdI) is 0.158, indicating that the particles are well dispersed and the particle size is relatively uniform, and 97.9% of the nanoparticles have a particle size less than 200nm.

[0293] Take 1mL of sample and add it to the potential test sample cell to measure the Zeta potential. The results show that the particle surface is basically close to neutrality, and the Zeta potential is 0.161±5.01mV. The ionizable lipids are not charged in the dispersant PBS, so it can be considered that the surface of the nanoparticles is neutral. The nanoparticles are not negatively charged, which is conducive to cell uptake and do not have the cytotoxicity of cationic lipids.

[0294] 3.11 Morphological observation of lipid nanoparticles

[0295] The morphology of lipid nanoparticles was observed using cryo-transmission electron microscopy. Fig.18 As shown. Under the electron microscope, the vast majority of lipid nanoparticles are between 40nm and 60nm in size (indicated by the red arrow), and there are also particles of about 90nm in size (indicated by the green arrow). Particles with small particle sizes are homogeneous solid spheres, while particles with large particle sizes are cavitated with clear edges. The results of cryo-TEM show that lipid nanoparticles were successfully produced.

[0296] 3.12 Lipid nanoparticle transfection efficiency

[0297] The LNP-mRNA-LDHB-EGFP with different N / P ratios was transfected into BHK-21 cells, and the transfection efficiency was determined by flow cytometry 24 hours later. Fig.19 ).

[0298] The results showed that the transfection efficiency of lipid nanoparticles prepared when N / P=10 was the highest, which was 46.4%; when N / P=6, the transfection efficiency of lipid nanoparticles was 29.1%; when N / P=20, the transfection efficiency of lipid nanoparticles was 14.1%.

[0299] 3.13 Preparation and quality inspection of mRNA-TBCC-RBDYH

[0300] According to method 2.2.2, mRNA-TBCC-RBDYH was prepared in large quantities, plasmid templates were constructed, and mRNA was produced. The results of linearized plasmid and homologous recombination fragment identification are shown in Fig. 20 The linearized plasmid and the expected recombinant fragment were obtained.

[0301] The prepared mRNA-TBCC-RBDYH was verified for quality by RNA denaturation electrophoresis ( Fig. 20 ), transfected into BHK-21 cells, and protein expression was detected by IFA and WB. The results are as follows Fig.21 As shown. Specific green fluorescence signals can be observed in the cytoplasm of transfected cells, and the efficient expression of the target protein is detected. This indicates that the prepared mRNA can complete the protein translation process after entering the cell.

[0302] 3.14 Mouse spleen status

[0303] 14 days after the second immunization, the mice were killed and the spleen morphology was observed. Fig. 22 As shown in the figure, the spleen of mice in the control group was small and bright red; the spleen of mice in the 5μg and 15μg groups was enlarged and dark red; and as the immunization dose increased, the spleen also enlarged. This suggests that mRNA vaccines can stimulate mice to produce immune responses, and the higher the dose, the stronger the immune response.

[0304] 3.15 Specific antibody detection

[0305] mRNA-TBCC-RBDYH was transfected into BHK-21 cells, and the cells were harvested after 24 hours. The immune mouse serum was collected and diluted 1:1000, used as the primary antibody, and WB was performed. The results are as follows Fig.23 shown.

[0306] The sera of mice immunized in the 5μg and 15μg groups could react with the RBD region of the S protein expressed in BHK-21 cells, indicating that the sera of immunized mice contained specific antibodies that could recognize the RBD region of the PEDV S protein; and the higher the mRNA immunization dose, the higher the level of antibodies stimulated to be produced by the body.

[0307] 3.16 Mouse T lymphocyte subset detection

[0308] Flow cytometry was used to detect T lymphocyte subsets in mouse peripheral blood and spleen. The voltage was adjusted to make lymphocytes fall in the appropriate position, the lymphocyte gate was selected, cell adhesion was removed, and then CD3 positive cells were selected for T lymphocyte subset analysis. The results are shown in Figure 2. Fig.24 Compared with the control group, the CD3 + CD4 + The proportion of cells decreased, but the difference was not significant. + CD8 + The cell ratio increased significantly, with the 5μg group being 3.99 times that of the control group and the 15μg group being 3.17 times that of the control group. This indicates that mRNA vaccines can induce mice to produce CD8 + T lymphocyte response.

[0309] 3.17 Determination of total IgG titer in mouse serum

[0310] Mouse sera were collected 2, 3, and 5 weeks after vaccination to measure the total IgG titer. The results are as follows: Fig.25 As shown. Compared with the control group, 2 weeks after the first immunization, the total IgG titer in the serum of mice in the 15μg immunization group increased significantly, and there was a significant difference. After the booster immunization, the total IgG titer in the serum of mice in the 5μg immunization group also increased significantly, showing a significant difference compared with the control group.

[0311] 3.18 ELISpot assay

[0312] The spleen of mice was collected and the ELISA test was performed. Fig.26 As shown. When no stimulant was added, the spleen lymphocytes of the three groups of mice were not activated and no spots were produced. When stimulated with a non-specific lymphocyte stimulator, the spleen lymphocytes of the three groups of mice were activated and a large number of spots were produced. When stimulated with a specific protein stimulator, the spleen lymphocytes of the control group mice were not activated or slightly activated, and a small number of spots or no spots were produced. The spleen lymphocytes of the mice in the 5μg immunization group and the 15μg immunization group were activated and a large number of spots were produced ( Fig.26 ). The results of differential analysis showed that there was a significant difference in the activation status of spleen lymphocytes between the 5μg immunization group and the control group, but there was no significant difference in the activation status of spleen lymphocytes between the 15μg immunization group and the control group.

[0313] The 5'UTR and 3'UTR of the porcine protein gene were added to both ends of the RBD sequence of the PEDV S protein to construct an in vitro transcription template; the preparation strategy of mRNA-TBCC-RBDYH containing the 5'UTR and 3'UTR of the porcine protein TBCC and the signal peptide of the gene PLAT was determined; the ionizable lipids Dlin-MC3-DMA, DSPC, DMG-PEG2000, and cholesterol were dissolved in anhydrous ethanol as the organic phase, and the mRNA was dissolved in a pH 4.0, 25mM sodium acetate solution as the inorganic phase. Lipid nanoparticles were prepared using a microfluidic chip at a flow rate ratio of 1:3 and a total flow rate of 4mL / min; lipid nanoparticles were prepared when the nitrogen-phosphorus ratio (N / P) was 10, and the encapsulation efficiency was 85.44%. The particle size of the lipid nanoparticles produced was between 40nm and 60nm, with good uniformity; the constructed mRNA vaccine was injected intramuscularly into 6-8 week-old BALB / c mice. The mRNA vaccine was able to significantly increase the IgG titer in the serum of the immune mice, induce humoral immunity and produce specific antibodies against the target protein, and also induce cellular immunity, among which the cellular immunity level induced by 5μg mRNA vaccination was the highest.

[0314] Table 24 Primers required for in vitro transcription template construction

[0315]

[0316]

[0317]

[0318] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A porcine epidemic diarrhea virus mRNA vaccine, characterized in that: The mRNA vaccine is synthesized using a sequence containing a 5'UTR, a signal peptide of human PLAT, a truncated S protein of porcine epidemic diarrhea virus, a Flag tag, a stop codon, a 3'UTR, and a poly(A) tail nucleic acid connected in sequence; The porcine epidemic diarrhea virus S protein truncation corresponds to the 477th to 629th amino acids of the porcine epidemic diarrhea virus S protein.

2. The mRNA vaccine according to claim 1, characterized in that The sequences of the 5'UTR and 3'UTR are derived from the porcine protein TBCC.

3. The mRNA vaccine according to claim 1, characterized in that The nucleic acid sequence of the signal peptide of human PLAT is shown in SEQ ID NO:

2.

4. The mRNA vaccine according to claim 2, characterized in that The nucleic acid sequences of 5'UTR and 3'UTR are shown in SEQ ID NOs: 3 and 4, respectively; The stop codon is 5'-TGATAA-3'; The poly(A) tail consists of 89 A bases.

5. The mRNA vaccine according to claim 1, characterized in that The nucleic acid sequence of the codon-optimized porcine epidemic diarrhea virus S protein truncation is shown in SEQ ID NO:

5.

6. The mRNA vaccine according to claim 1, characterized in that The nucleic acid sequence of the mRNA vaccine without a poly(A) tail is shown in SEQ ID NO:

1.

7. The method for constructing the mRNA vaccine according to any one of claims 1 to 6, characterized in that: The method comprises: selecting 5'UTR, signal peptide of human PLAT, 3'UTR, truncated S protein of porcine epidemic diarrhea virus, Flag tag, stop codon, 3'UTR, and poly(A) tail nucleic acid sequence to synthesize the nucleic acid sequence of mRNA vaccine, constructing it on a vector, transfecting bacteria to obtain recombinant bacteria, picking monoclonal colonies for sequencing, extracting plasmids from recombinant bacteria with correct sequencing to obtain recombinant plasmids, and enzymatically cutting, transcribing, capping, and purifying the recombinant plasmids to obtain mRNA vaccine nucleic acid.

8. A porcine epidemic diarrhea virus nucleic acid vaccine, characterized in that: Comprising an mRNA vaccine nucleic acid prepared according to the method of claim 7 and a delivery system; The delivery system is lipid nanoparticles.

9. The method for preparing the nucleic acid vaccine according to claim 8, characterized in that: The method includes: dissolving ionizable lipids Dlin-MC3-DMA, DSPC, DMG-PEG2000 and cholesterol in anhydrous ethanol as an organic phase, dissolving the mRNA vaccine nucleic acid in a pH 4.0, 25mM sodium acetate solution or a pH 4.0, 25mM sodium citrate solution as an inorganic phase, and using a microfluidic chip to prepare lipid nanoparticles encapsulated with mRNA at a flow rate ratio of 1:3 between the organic phase and the inorganic phase at a total flow rate of 4mL / min.

10. The method according to claim 9, characterized in that The molar ratio of the ionizable lipid Dlin-MC3-DMA, DSPC, cholesterol and DMG-PEG2000 is 50:10:38.5:1.5.

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