A h1n1 broad-spectrum mRNA vaccine based on key amino acid mutations of ha and na protein as targets and a preparation method thereof

Abstract

The application provides a H1N1 broad-spectrum mRNA vaccine based on key amino acid mutation of HA and NA protein series as a target and a preparation method thereof, and relates to the technical field of vaccine preparation.The amino acid sequence of the mRNA vaccine is shown as SEQ ID NO.1, and the nucleic acid sequence is shown as SEQ ID NO.2, that is, on the basis of the HA conservative sequence, variation sites of amino acid properties across the vicinity of the main antigen site of the HA head and the receptor binding site are retained, the HA' amino acid sequence containing the key mutation site is obtained, and the HA' and the NA conservative amino acid sequence are connected by GGGSGGGSGGGSGGGS to obtain the mRNA vaccine.The application overcomes the defects of the prior art, improves the broad-spectrum protection effect on the H1N1 type influenza virus by retaining the key amino acid mutation site in the HA conservative sequence, reduces the vaccine production cost and the immunization cycle, and provides an effective response scheme for the influenza outbreak season.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of vaccine preparation, in particular to a H1N1 broad-spectrum mRNA vaccine with HA and NA protein as targets based on key amino acid mutations and a preparation method thereof. BACKGROUND

[0002] Influenza virus belongs to Orthomyxoviridae and is a segmented negative-strand RNA virus. The eight segmented negative-strand RNAs of the genome encode viral RNA polymerase complex (PB1, PB2, PA), hemagglutinin (HA), nucleoprotein (NP), neuraminidase (NA), matrix protein (M1), ion channel protein (M2), and a series of non-structural proteins (NS1, NS2, NS3). Among them, HA and NA are two important glycoproteins on the surface of the virus and are the more mainstream vaccine immunogens. HA is responsible for the binding of the virus to the host cell surface receptor and mediates the entry of the virus into the host cell; NA is responsible for hydrolyzing the sialic acid on the host surface to promote the release of the virus from the host cell.

[0003] Influenza virus has been widely concerned since the Spanish flu outbreak in 1918. At least 18 different subtypes of influenza A virus (IAV) are transmitted in animal hosts, and these viruses occasionally enter the human population and cause pandemics. At present, H1N1 and H3N2 IAV and one or two antigenically different influenza B virus (IBV) lineages are seasonally transmitted in the human population. Although the research of various epidemic surveillance projects and models has increased the understanding of influenza pandemics, it is still not possible to accurately predict which influenza subtype will cause the next pandemic. Therefore, the research of universal influenza vaccines has always been the focus of the vaccine industry. However, due to the large differences between different types of influenza viruses, combined with the high frequency of variation of influenza viruses through antigenic drift and antigenic shift, it is difficult to achieve broad-spectrum coverage with a monovalent vaccine and a single immunogen. Based on this, the design of a broad-spectrum mRNA vaccine is crucial. SUMMARY

[0004] In view of the deficiencies in the prior art, the present application provides a H1N1 broad-spectrum mRNA vaccine with HA and NA protein as targets based on key amino acid mutations and a preparation method thereof. By retaining key amino acid mutation sites in the conserved sequence of HA, the broad-spectrum protection effect on H1N1 type influenza virus is improved, the production cost of the vaccine is reduced, and the immune cycle is reduced, thereby providing an effective response scheme for the influenza outbreak season.

[0005] To achieve the above purpose, the present application is implemented by the following technical solutions:

[0006] A H1N1 broad-spectrum mRNA vaccine based on key amino acid mutations of HA and NA protein as targets, the amino acid sequence of the mRNA vaccine is shown as SEQ ID NO. 1, and the nucleic acid sequence is shown as SEQ ID NO. 2.

[0007] The preparation method of the mRNA vaccine comprises the following steps:

[0008] S1, based on the HA conservative sequence, the variation sites of amino acid property across near the main antigenic site of the HA head and the receptor binding site are retained to obtain the HA' amino acid sequence containing key mutation sites, wherein the HA' amino acid sequence is shown as SEQ ID NO. 3;

[0009] S2, the HA' amino acid sequence and the conservative NA amino acid sequence are connected by a flexible peptide to obtain the amino acid sequence shown as SEQ ID NO. 1;

[0010] S3, the obtained amino acid sequence is codon optimized according to the codon preference of human to obtain a DNA sequence, shown as SEQ ID NO. 2, and the sequence is constructed on a plasmid PUC57 containing a T7 promoter, 5'UTR, 3'UTR and 100 PolyA by enzyme digestion and connection, that is, PUC57-T7-5UTR-HA-NA-3UTR-PolyA, to complete the plasmid construction;

[0011] S4, the successfully constructed plasmid is amplified and transcribed to obtain the mRNA vaccine;

[0012] S5, the obtained mRNA is prepared into a lipid nanoparticle LNP by a microfluidic method.

[0013] Preferably, the flexible peptide in step S2 is four repeated GGGS, that is, the amino acid sequence of the flexible peptide is: GGGSGGGSGGGSGGGS.

[0014] Preferably, the nucleotide sequence of the 5'UTR in step S3 is shown as SEQ ID NO. 4, and the nucleotide sequence of the 3'UTR is shown as SEQ ID NO. 5.

[0015] Preferably, in step S4, the TSINGKE TSC-C01 Trelief® 5α Chemically Competent Cell is used for transformation and amplification after transformation, and BsaI is used for enzyme digestion and linearization of the DNA template after extraction of the plasmid, and 70% isopropanol is used for precipitation of the linearized template under the condition of-80℃, and 70% ethanol is used for washing.

[0016] Preferably, the linear template in step S4 is subjected to in vitro transcription by using a commercial kit Novizan, and before transcription, the transcription raw material UTP is completely replaced by N1-methyl pseudouridine triphosphate N1-Me-pUTP.

[0017] Preferably, the specific method for preparing the lipid nanoparticles LNP in step S5 comprises the following steps:

[0018] S5-1, dissolve the mRNA into a citric acid buffer solution with a pH of 4 and a concentration of 50mM, and obtain an mRNA-citric acid solution with a final concentration of 108 ng / μL according to the ratio of P element content to N element content in the mRNA, which is 1:8;

[0019] S5-2, prepare an anhydrous ethanol solution with a SM102 mass concentration of 50%, a DMG-PEG2000 mass concentration of 1.5%, a DSPC mass concentration of 10%, and a cholesterol mass concentration of 38.5%, to obtain a lipid mixture solution with a concentration of 16mM;

[0020] S5-3, filter the lipid mixture solution and the mRNA-citric acid solution through a 0.22μm microporous filter membrane respectively, and mix them using a microfluidic instrument to obtain the lipid nanoparticles LNP.

[0021] Preferably, the flow rate ratio of the mRNA-citric acid solution to the lipid mixture solution is 15mL / min:5mL / min when the microfluidic instrument is used for mixing in step S5-3.

[0022] The present application provides a H1N1 broad-spectrum mRNA vaccine based on the key amino acid mutation of HA and NA protein as a target, which has the following advantages compared with the prior art:

[0023] The present application designs a vaccine with HA and NA double targets for immune protection, and obtains HA' based on key amino acid mutations, and the influenza mRNA vaccine designed with HA' and NA double targets has certain immunogenicity and can induce good T cell immune response and has good protection efficacy, and by retaining the key amino acid mutation site in the original conserved sequence of HA', the broad-spectrum protection effect on H1N1 type influenza virus is improved, the production cost of the vaccine is reduced, and the immune cycle is shortened, which provides an effective response scheme for the influenza outbreak season. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 The present application provides a H1N1 broad-spectrum mRNA vaccine based on the key amino acid mutation of HA and NA protein as a target, which has the following advantages compared with the prior art:

[0025] Figure 2Figure for the experimental results of detecting the qualified vaccine in the embodiment of the present application, wherein (a) electrophoresis of in vitro transcribed RNA (lane 2); (b) encapsulation verification: lane 1: mRNA-LNP, lane 2: mRNA-LNP after fully releasing mRNA by treating with triton x-100; (c) particle size distribution of prepared mRNA-LNP;

[0026] Figure 3 Figure for the expression of K-1 vaccine transfected cells in 293T cells compared with RNA transfection by Mirus kit in the embodiment of the present application;

[0027] Figure 4 Figure for the immunization cycle of K-1 vaccine in the embodiment of the present application;

[0028] Figure 5 Figure for the antibody level after immunization of K-1 vaccine in the embodiment of the present application;

[0029] Figure 6 Representative pictures and counting results of ELISPOT experiment for secretion of IFN-γ in the embodiment of the present application;

[0030] Figure 7 Representative pictures and counting results of ELISPOT experiment for secretion of IL-2 in the embodiment of the present application;

[0031] Figure 8 Representative pictures and counting results of ELISPOT experiment for secretion of IL-4 in the embodiment of the present application;

[0032] Figure 9 Figure for the level of neutralizing antibodies produced by the vaccine design reference strain (A / Michigan / 45 / 2015 (H1N1)) after immunization of K-1 vaccine in the embodiment of the present application;

[0033] Figure 10 Figure for the level of neutralizing antibodies produced by the strain not included in the reference of the vaccine design (Influenza AH1N1 (A / Victoria / 25 / 2570 / 2019)) after immunization of K-1 in the embodiment of the present application;

[0034] Figure 11 Figure for the changes of body temperature and body weight within 7 days after challenge of A / Michigan / 45 / 2015 (H1N1) strain in the embodiment of the present application;

[0035] Figure 12 Figure for the changes of virus load in nasopharyngeal swab of mice within 7 days after challenge of A / Michigan / 45 / 2015 (H1N1) strain in the embodiment of the present application;

[0036] Figure 13Virus load level in lung, concha and trachea main tissues of mice after A / Michigan / 45 / 2015 (H1N1) strain attack for seven days in the embodiment of the present application;

[0037] Figure 14 HE staining (left) and pathological score (right) of lung tissue of mice after A / Michigan / 45 / 2015 (H1N1) strain attack in the embodiment of the present application;

[0038] Figure 15 Changes of body temperature and body weight of mice within seven days after Influenza A H1N1 (A / Victoria / 25 / 2570 / 2019) strain attack in the embodiment of the present application;

[0039] Figure 16 Virus load level of nasopharyngeal swab of mice within seven days after Influenza A H1N1 (A / Victoria / 25 / 2570 / 2019) strain attack in the embodiment of the present application;

[0040] Figure 17 Virus load level in lung, concha and trachea main tissues of mice after Influenza A H1N1 (A / Victoria / 25 / 2570 / 2019) strain attack for seven days in the embodiment of the present application;

[0041] Figure 18 HE staining (left) and pathological score (right) of lung tissue of mice after Influenza A H1N1 (A / Victoria / 25 / 2570 / 2019) strain attack in the embodiment of the present application. DETAILED DESCRIPTION

[0042] In order to make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme of the embodiments of the present application will be described clearly and completely below in combination with the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0043] In the following examples, 12 μg is used as the high-dose group, 5 μg is used as the low-dose group, and the blank solvent (Tirs HCl (pH=6.8)) is used as the control group. Example 1:

[0044] Preparation of H1N1 influenza virus broad-spectrum mRNA vaccine (K-1) with influenza hemagglutinin HA' and neuraminidase protein as targets while retaining key mutant genes:

[0045] Conserved HA amino acid sequence:

[0046] Conserved HA amino acid sequence:

[0047] MKAILVVLLYTFTTANADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTARSWSYIVETSNSDNGTCYPGDFINYEELREQLSSVSSFERFEIFPKTSSWPNHDSDKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKINQTYINDKGKEVLVLWGIHHPPTTADQQSLYQNADAYVFVGTSRYSKKFKPEIATRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFTMERNAGSGIIISDTPVHDCNTTCQTPEGAINTSLPFQNVHPITIGKCPKYVKSTKLRLATGLRNVPSIQSRGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDKITNKVNSVIEKMNTQFTAVGKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRNQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREKIDGVKLESTRIYQILAIYSTVASSLVLVVSLGAISFWMCSNGSLQCRICI;

[0048] Conserved NA amino acid sequence:

[0049] MNPNQKIITIGSICMTIGMANLILQIGNIISIWVSHSIQIGNQSQIETCNKSVITYENNTWVNQTYVNISNTNFAARQSVASVKLAGNSSLCPVSGWAIYSKDNSVRIGSKGDVFVIREPFISCSPLECRTFFLTQGALLNDKHSNGTIKDRSPYRTLMSCPIGEVPSPYNSRFESVAWSASACHDGTNWLTIGISGPDSGAVAVLKYNGIITDTIKSWRNNILRTQESECACVNGSCFTIMTDGPSDGQASYKIFRIEKGKIIKSVEMKAPNYHYEECSCYPDSSEITCVCRDNWHGSNRPWVSFNQNLEYQMGYICSGVFGDNPRPNDKTGSCGPVSSNGANGVKGFSFKYGNGVWIGRTKSISSRKGFEMIWDPNGWTGTDNKFSIKQDIVGINEWSGYSGSFVQHPELTGLDCIRPCFWVELIRGRPEENTIWTSGSSISFCGVDSDIVGWSWPDGAELPFTIDK;

[0050] On the basis of the HA conserved sequence, the variation sites of amino acid properties across the vicinity of the main antigenic sites of HA head and the receptor binding sites were reserved to obtain the HA' sequence containing the key mutation sites:

[0051] HA' sequence:

[0052] MKAILVVLLYTFTTANADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKLCKLGGVAPLHLGQCNIAGWILGNPECESLSTASSWSYIVETPSSDNGTCYPGDFINYEELREQLSSVSSFERFEIFPKTSSWPNHDSDNGVTAACSHAGAKSFYKNLIWLVKKGKSYPKLSKTYINDKGKEVLVLWGIHHPSTITDQESLYQNADAYVFVGTSRYSKKFKPEIAIRPKVRDQAGRMNYYWTLVEPGDKITFEATGNLVVPRYAFTMERNAGSGIIISDTPVHDCNTTCQTPEGAINTSLPFQNVHPITIGKCPKYVKSTKLRLATGLRNVPSIQSRGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDKITNKVNSVIEKMNTQFTAVGKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRNQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREKIDGVKLESTRIYQILAIYSTVASSLVLVVSLGAISFWMCSNGSLQCRICI;

[0053] The HA' and NA are connected by flexible peptide (GGGSGGGSGGGSGGGS) to obtain the amino acid sequence as shown in SEQ ID NO. 1, and the DNA sequence is codon optimized according to the human codon preference (as shown in SEQ ID NO. 2). The sequence is double enzyme cut by Spe I and Xho I, and the vector and the target fragment are connected under the T4 DNA enzyme to construct the plasmid PUC57 plasmid (PUC57-T7-5UTR-HA-NA-3UTR-PolyA) containing T7 promoter, 5'UTR (nucleotide sequence as shown in SEQ ID NO. 3), 3'UTR (nucleotide sequence as shown in SEQ ID NO. 4) and 100 PolyA. After the plasmid construction is completed, sequencing verification is carried out.

[0054] The constructed transformation was introduced into competent E. coli (TSINGKE TSC-C01 Trelief®5α Chemically Competent Cell) using heat shock method, and single colony was selected for culture. After correct sequencing of the bacterial solution, the plasmid was amplified by large-scale culture, and the plasmid was extracted and linearized DNA template was subjected to BsaI enzyme digestion. The linearized template was precipitated at -80°C using 70% isopropanol, and washed with 70% ethanol;

[0055] The linearized template was subjected to in vitro transcription using a commercial kit (Novagen). Before transcription, the transcription raw material UTP was completely replaced with N1-methyl pseudouridine triphosphate (N1-Me-pUTP). After transcription, the mRNA vaccine was obtained as shown in Figure 1 ;

[0056] The mRNA was dissolved in a citric acid buffer solution with a concentration of 50 mM at pH 4 to obtain an mRNA-citric acid solution with a final concentration of 108 ng / μL. Then, a lipid mixture solution was prepared: SM102, DMG-PEG2000, DSPC, and cholesterol were mixed and dissolved in anhydrous ethanol solution at a ratio of 50%, 1.5%, 10%, and 38.5%, respectively, to obtain a lipid mixture solution. The mRNA-citric acid solution and the lipid mixture solution were filtered through a 0.22 μm microporous filter, respectively. The mRNA and the N element content of SM102 were mixed at a ratio of 1:8 using a microfluidic instrument. The flow rate ratio of the mRNA-citric acid solution to the lipid mixture solution was 15 mL / min:5 mL / min to obtain mRNA-LNP.

[0057] Detection:

[0058] 1. The obtained mRNA-LNP was immediately diluted with 15 ml of the above citric acid buffer solution, and ultrafiltration was performed using a 100 KD ultrafiltration tube. The centrifugal force was 3000g. After ultrafiltration to 1 / 4 volume, 50 mM Tris-HCl buffer solution (pH 7.5) was added to 15 mL. After repeated twice, the vaccine was detected for qualification:

[0059] The specific results are shown in Figure 2 , wherein Figure 2 (a) Lane 2 is the electrophoresis map of in vitro transcribed RNA, Figure 2 (b) is the encapsulation verification electrophoresis map. Lane 1 is mRNA-LNP, and lane 2 is mRNA-LNP after being fully released by 1% tritonx-100 treatment; Figure 2 (c) is the particle size distribution of the prepared mRNA-LNP;

[0060] from Figure 2It can be seen that the transcribed RNA has no obvious degradation and impurity band, the LNP is successfully encapsulated and the particle size is about 100 nm; the mRNA vaccine quality is qualified.

[0061] 2. Verify the in vitro expression effect of mRNA-LNP (i.e. K-1 vaccine) in 293T cells: 293T cells are cultured in DMEM complete medium (add 10% FBS and 1% penicillin-streptomycin) at 37°C, 5% CO2; 293T cells in logarithmic growth phase are transferred to a 12-well plate at a ratio of 1:2, and cultured overnight; when the cell confluence reaches 80%, discard the complete culture medium, add 1ml Opti-MEM low serum medium to each well, use the Mirus MIR2225 kit to transfect mRNA (according to the instructions), and add appropriate mRNA-LNP to the vaccine group; 6h after transfection, replace the low serum medium with complete medium, continue to culture for 36h, and then extract cell protein using RIPA lysis buffer on ice for Western blot experiment to detect the in vitro expression level of the target protein.

[0062] The specific results are shown in Figure 3 After transfection of K-1 vaccine in 293T cells, the expression is good compared with the transfection of RNA by Mirus kit, and the negative control without transfection (-) has no target band.

[0063] BALB / c mice were immunized with K-1 vaccine, and the level of binding antibody in mouse serum was detected by enzyme-linked immunosorbent assay (ELISA): use ELISA antigen coating solution to coat antigen protein (HA and NA protein of A / Wisconsin / 67 / 2022 (H1N1) strain) at 0.8ug / well, 4°C overnight; after washing with PBST (0.05% Tween 20 in PBS solution), block with 2% BSA solution (prepared with PBS) for 1h; wash with PBST for three times, incubate the gradient diluted serum (1:400) for 1h; wash for three times, dilute the secondary antibody at 1:30000, incubate for 1h; add TMB substrate (3,3',5,5'-tetramethylbenzidine) and incubate in the dark for 15min, then add stop solution, detect the absorbance values at 450nm and 630nm, and the absorbance value at 450nm minus the absorbance value at 630nm>0.1 is judged as positive, and the reciprocal of the maximum dilution multiple of the positive well represents the binding antibody level.

[0064] The specific results are that after BALB / c mice are immunized with K-1 vaccine, the serum of the mice has a significantly increased HA and NA protein antigen binding antibody titer; the K-1 immunization cycle is as follows Figure 4As shown, the booster immunization was given at 28 days after the primary immunization, and the whole blood was collected at 7-day intervals starting from 14 days for the detection of the binding antibody level. The binding antibody level was determined by Figure 5 From the binding antibody level after the K-1 vaccine immunization, it can be seen that the binding antibodies against the HA and NA proteins were obviously produced starting from 14 days after the K-1 vaccine immunization, and the antibody level gradually increased thereafter. After the booster immunization at 28 days, the antibody level could be maintained at 10 5 The above.

[0065] 4. After the BALB / c mice were immunized with the K-1 vaccine, the ELISPOT experiment was performed:

[0066] According to the mouse immunization process, the spleen tissue was taken out at 49 days after the primary immunization to isolate the spleen lymphocytes for the ELISPOT experiment:

[0067] 1) 1 ml of the whole blood and tissue diluent was added to a plate, the spleen of the mouse was taken out under sterile conditions, the capsule of the spleen was torn off, and the spleen was cut into small pieces in the plate using an ophthalmic scissors. It was ensured that the spleen and the obtained cells were always in a liquid environment.

[0068] 2) The cell screen was placed on a 50 ml centrifuge tube, the spleen was placed on the screen, and the spleen was ground using a syringe piston until no red particles were visible.

[0069] 3) The cell screen was washed with 1 ml of the whole blood and tissue diluent.

[0070] 4) A centrifuge tube was taken, and an equal amount of the separation solution as the single-cell suspension of the spleen was added. The separation solution was not less than 3 ml at least, and the total volume was not more than two-thirds of the centrifuge tube.

[0071] 5) The single-cell suspension of the spleen was slowly added to the surface of the separation solution, and a clear interface was formed due to the difference in density.

[0072] 6) At room temperature, the horizontal rotor was used for centrifugation at a speed of 500 g for 20 min.

[0073] 7) After centrifugation, the liquid in the centrifuge tube was divided into four layers from top to bottom, which were the plasma layer, the milky white lymphocyte layer, the separation solution layer, and the red blood cell layer at the bottom.

[0074] 8) The second ring-shaped milky white lymphocyte layer was carefully sucked into another clean 15 ml centrifuge tube, 10 ml of cell washing solution was added, and the mixture was inverted to mix. The horizontal rotor was centrifuged at 250 g for 5 min.

[0075] 9) The supernatant was discarded, 5 ml of PBS was added, and the cell pellet was resuspended by blowing. The horizontal rotor was centrifuged at 250 g for 5 min.

[0076] 10) Calculate the number of wells per cell to be plated, using 50 μl per well plus 10 μl of ELISPOT special medium to resuspend the cells.

[0077] 11) Take 10 μl of the resuspended cells and add 90 μl of PBS and mix. Then take 10 μl of the cell suspension to count the cells (note that the value obtained here is the value after 10-fold dilution).

[0078] 12) Wash the plate 4 times with 200 μl of sterile PBS, and let it stand for 2 min each time. Add 200 μl of ELISPOT special medium per well to the washed plate, and incubate the plate at room temperature for 30 min to equilibrate the plate.

[0079] 13) Remove the medium from the plate, dilute the cells using ELISPOT special medium according to the count, and take the cell number that most of them can satisfy, generally 3.0 x 10 5 -6.0 x 10 5 cells per well. Add 50 μl of the cell suspension per well.

[0080] 14) Add the stimulator, 1 μg or 2 μg per well, to the reaction well, and add 50 μl per well. At the same time, set up the PHA positive stimulator control and the blank control without adding stimulator.

[0081] 15) Place the plate in a 37 °C, 5% CO2 incubator and incubate for 24-48 h, and do not move it in the middle.

[0082] 16) Discard the medium in the plate, add 200 μl of deionized water pre-cooled at 4 °C, soak for 2 min, and then discard.

[0083] 17) Wash the plate with 200 μl of PBS per well, soak for 2 min, and wash 5 times.

[0084] 18) Dilute the detection antibody (IFN-γ-biotin) to 1 μg / ml using 0.5% FBS PBS, add 100 μl per well, and incubate at room temperature for 2 h or at 37 °C for 1 h.

[0085] 19) Wash the plate.

[0086] 20) Dilute Streptavidin-ALP to 1:1000 using 0.5% FBS PBS, add 100 μl per well, and incubate at room temperature for 1 h.

[0087] 21) Wash the plate, step (9).

[0088] 22) Filter the chromogenic solution with 0.45 μm filter, add 100 μl per well until obvious spots appear, usually 5-10 min.

[0089] 23) Wash the plate with tap water until the color no longer becomes darker. Let the plate dry naturally in the air. Store the plate in the dark.

[0090] The specific results are shown in Figures 6-8 , and Figure 6 The left side is a representative result photo of the number of cells secreting cytokine IFN-γ detected by ELISPOT experiment using HA and NA proteins of A / Wisconsin / 67 / 2022 (H1N1) strain as single or combined stimulators; Figure 6 The right side is the spot count result of cells secreting IFN-γ; Figure 7 The left side is a representative result photo of the number of cells secreting cytokine IL-2 detected by ELISPOT experiment using HA and NA proteins of A / Wisconsin / 67 / 2022 (H1N1) strain as single or combined stimulators; Figure 7 The right side is the spot count result of cells secreting IFN-γ; Figure 8 The left side is a representative result photo of the number of cells secreting cytokine IL-4 detected by ELISPOT experiment using HA and NA proteins of A / Wisconsin / 67 / 2022 (H1N1) strain as single or combined stimulators; Figure 8 The right side is the spot count result of cells secreting IL-4;

[0091] It can be seen that K-1 vaccine immunization of mice can cause strong T cell immune response.

[0092] 5) Antibody production of BALB / c mice immunized with K-1 vaccine against vaccine design reference strains and strains not included in the reference range at the time of design:

[0093] 1) Add 4 times the volume of RDE (receptor destroying enzyme) to the serum and treat at 37°C for 16 h.

[0094] 2) Inactivate at 56°C for 30 min.

[0095] 3) Add 25ul PBS to the first column of the U-shaped disposable reaction plate, add 50ul serum and RDE mixture to the first column, and dilute step by step with 25ul.

[0096] 4) Add 4 hemagglutination units of virus solution, incubate at room temperature for 20 min.

[0097] 5) Add 50ul 1% guinea pig red blood cells, and observe the agglutination after standing at room temperature for 1h.

[0098] The whole blood of the mice was collected 42 days after the initial immunization, and the serum was separated and detected for neutralizing antibody levels by hemagglutination inhibition test. According to the relevant guidelines of the National Influenza Center Standard Operating Procedures, the protective effect is considered when the inhibition titer of the tested serum to the reference antigen is ≥40. Figure 9 The neutralizing antibody levels produced by the K-1 vaccine against one of the vaccine design reference strains (A / Michigan / 45 / 2015 (H1N1)) and Figure 10 The neutralizing antibody levels produced by the K-1 vaccine against one of the vaccine design reference strains (A / Michigan / 45 / 2015 (H1N1)) and

[0099] 6. Challenge protection experiment of BALB / c mice after immunization

[0100] After the mice completed the immunization process, the challenge protection experiment was performed. To verify the broad-spectrum of the vaccine against H1N1 type, two strain challenge protection experiments were performed, including the vaccine design reference strain A / Michigan / 45 / 2015 (H1N1) and the vaccine design strain not included in the reference Influenza A H1N1 (A / Victoria / 25 / 2570 / 2019); the challenge dose was 256 HAU, and the virus liquid with a volume of 50 ul was used for challenge experiment by nasal instillation.

[0101] The challenge protection experiment detected the body temperature and body weight changes of the mice during the challenge, the changes of the viral load in the nasopharyngeal swab, the viral load levels in the lung, turbinates and trachea on the seventh day, and the lung tissue pathological damage. The specific results are shown in Figures 11-18 The results show that the K-1 vaccine has good challenge protection effect against the two strains.

[0102] The above examples are only used to illustrate the technical solutions of the present application, but not to limit it; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that they can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A broad-spectrum H1N1 mRNA vaccine targeting the tandem mutations of key amino acid HA and NA proteins, characterized in that, The amino acid sequence of the mRNA vaccine is shown in SEQ ID NO.1, and the nucleic acid sequence is shown in SEQ ID NO.

2.

2. A method for preparing an mRNA vaccine as described in claim 1, characterized in that, The preparation method includes the following steps: S1. Based on the conserved HA sequence, the variant sites near the main antigenic sites in the HA head and the receptor binding site that cross amino acid properties are retained to obtain the HA' amino acid sequence containing the key mutation site, wherein the HA' amino acid sequence is shown in SEQ ID NO.3; S2. The HA' amino acid sequence and the conserved NA amino acid sequence are linked by a flexible peptide to obtain the amino acid sequence shown in SEQ ID NO.1; S3. The obtained amino acid sequence is codon optimized according to human codon preference to obtain a DNA sequence, as shown in SEQ ID NO.

2. This sequence is constructed into plasmid PUC57 containing T7 promoter, 5'UTR, 3'UTR and 100 PolyA by enzyme digestion and ligation, which is PUC57-T7-5UTR-HA-NA-3UTR-PolyA, and the plasmid construction is completed. S4. The plasmid successfully constructed above is amplified and transcribed to obtain an mRNA vaccine; S5. The mRNA obtained above is prepared into lipid nanoparticles (LNPs) using a microfluidic method.

3. The preparation method according to claim 2, characterized in that: In step S2, the flexible peptide is four repeating GGGS, that is, the amino acid sequence of the flexible peptide is: GGGSGGGSGGGSGGGS.

4. The preparation method according to claim 2, characterized in that: The nucleotide sequence of the 5'UTR in step S3 is shown in SEQ ID NO.4, and the nucleotide sequence of the 3'UTR is shown in SEQ ID NO.

5.

5. The preparation method according to claim 2, characterized in that: In step S4, after transformation with TSINGKE TSC-C01Trelief® 5α Chemically Competent Cell, the plasmid was amplified, and after plasmid extraction, the DNA template was linearized by BsaI restriction enzyme digestion. The linearized template was precipitated with 70% volume isopropanol at -80℃ and washed with 70% ethanol.

6. The preparation method according to claim 2, characterized in that: In step S4, the linearized template is transcribed in vitro using the commercially available kit Novizan. Before transcription, the transcription raw material UTP is completely replaced with N1-methylpseuuridine triphosphate N1-Me-pUTP.

7. The preparation method according to claim 2, characterized in that, The specific method for preparing lipid nanoparticles (LNPs) in step S5 includes the following steps: S5-1. Dissolve the mRNA in a 50mM citrate buffer solution with pH=4. The phosphorus content and nitrogen content in the mRNA are in a 1:8 ratio to obtain a final concentration of 108 ng / μL mRNA-citric acid solution. S5-2. Prepare an anhydrous ethanol solution with 50% SM102, 1.5% DMG-PEG2000, 10% DSPC, and 38.5% cholesterol to obtain a lipid mixed solution. S5-3. The lipid mixture and mRNA-citric acid solution were filtered separately through a 0.22 μm microporous membrane and mixed using a microfluidic instrument to obtain lipid nanoparticles (LNPs).

8. The preparation method according to claim 7, characterized in that: In step S5-3, the flow rate ratio for mixing using a microfluidic instrument is 15 mL / min for mRNA-citric acid solution and 5 mL / min for lipid mixture.

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