Avian coronavirus mRNA vaccine, plasmid and preparation method thereof

Abstract

The invention discloses an avian coronavirus mRNA (messenger Ribonucleic Acid) vaccine, a plasmid and a preparation method thereof, and aims to provide the avian coronavirus mRNA vaccine which is high in immunogenicity and good in safety, the avian coronavirus mRNA vaccine is formed by wrapping mRNA with nucleotide lipid nanoparticles, and the mRNA is mRNA-N with a nucleotide sequence as shown in SEQ ID NO.1; or the nucleotide sequence of the mRNA-N-Mosaic is shown as SEQ ID NO.2. The invention belongs to the technical field of vaccines.

Description

Technical Field

[0001] The present invention discloses an antigen, and also discloses a vaccine prepared from the antigen, specifically an mRNA vaccine against avian coronavirus IBV. The present invention also discloses the avian coronavirus mRNA vaccine, belonging to the technical field of vaccines. Background Art

[0002] It was developed by continuously passaging the IBV field isolate in chicken embryos; however, the safety, attenuation effect and immune effect of the live attenuated vaccine prepared by this method cannot be guaranteed, especially in terms of immune age, vaccine dose, bird type and vaccination route; in addition, the gene of the live attenuated vaccine is also unstable and has a tendency to revert to virulence. Moreover, the circulation of live attenuated virus in the environment provides an environment in which the virus population may mutate and recombine, resulting in the emergence of mutant viruses.

[0003] An inactivated vaccine refers to first culturing a virus or bacterium and then inactivating it with heat or a chemical agent. It is usually inoculated into laying hens and breeders by injection. Currently, studies have shown that when the IBV inactivated vaccine is used to immunize chickens, it will not cause a strong immune response to the circulating virulent strain IBV in chickens. However, the production and management of inactivated vaccines are time-consuming and costly, thus limiting their application. Therefore, there is an urgent need to develop an alternative IBV vaccine. Summary of the Invention

[0004] Aiming at the above deficiencies, the object of the present invention is to provide a plasmid of avian coronavirus; the present invention also provides an avian source coronavirus mRNA vaccine prepared from the plasmid and a preparation method of the vaccine, which has high immunogenicity and good safety.

[0005] For this reason, the first technical solution provided by the present invention is as follows: An avian coronavirus mRNA vaccine, wherein the avian coronavirus mRNA vaccine is mRNA encapsulated by nucleotide lipid nanoparticles, and the mRNA is mRNA-N with a nucleotide sequence as shown in SEQ ID NO.1; or mRNA-N-Mosaic with a nucleotide sequence as shown in SEQ ID NO.2.

[0006] Furthermore, for the above avian coronavirus mRNA vaccine, the nucleotide sequence as shown in SEQ ID NO.1 is obtained by adding a Kozak sequence to the N protein nucleotide sequence as shown in SEQ ID NO.6 at the head, a T7 promoter with a nucleotide sequence as shown in SEQ ID NO.9, a 5'UTR sequence of β-globin with a nucleotide sequence as shown in SEQ ID NO.10, and tandemly connecting two 3'UTR sequences of β-globin with a nucleotide sequence as shown in SEQ ID NO.11 at the tail.

[0007] Furthermore, for the above-mentioned avian coronavirus mRNA vaccine, the nucleotide sequence such as SEQ ID NO.2 is obtained by adding a Kozak sequence to the head of the N-mosaic protein nucleotide sequence shown by the nucleotide sequence such as SEQ ID NO.3, adding a T7 promoter shown by the nucleotide sequence such as SEQ ID NO.9, adding a 5'UTR sequence of β-globin shown by the nucleotide sequence such as SEQ ID NO.10, and tandemly connecting two 3'UTR sequences of β-globin shown by the nucleotide sequence such as SEQ ID NO.11 at the tail.

[0008] Even further, for the above-mentioned avian coronavirus mRNA vaccine, the sequence shown by SEQ ID NO.6 is a sequence obtained after optimization by the sequence shown by the nucleotide sequence such as SEQ ID NO.4.

[0009] Even further, for the above-mentioned avian coronavirus mRNA vaccine, the sequence shown by SEQ ID NO.3 is a sequence obtained after optimization by the sequence shown by the nucleotide sequence such as SEQ ID NO.5.

[0010] The second technical solution of the present invention is to provide an mRNA plasmid, which is a plasmid with a nucleotide sequence shown by SEQ ID NO.12 synthesized by connecting the nucleotide sequence shown by SEQ ID NO.1 in the first technical solution to the PUC57 vector; or a plasmid with a nucleotide sequence shown by SEQ ID NO.13 synthesized by connecting the nucleotide sequence shown by SEQ ID NO.2 to the PUC57 vector; The preparation method of the avian coronavirus mRNA vaccine described in the second technical solution successively includes the following steps: 1) Construct a plasmid with a nucleotide sequence shown by SEQ ID NO.12 or a plasmid with a nucleotide sequence shown by SEQ ID NO.13; 2) Amplify the plasmid constructed in step 1) to obtain a plasmid template. 3) Perform PCR amplification on the plasmid template obtained in step 2). 4) Perform in vitro transcription, capping, and tailing on the PCR amplification product obtained in step 3) to synthesize mRNA. 5) Synthesize the avian coronavirus mRNA vaccine by combining the in vitro transcribed mRNA molecule with a cationic lipid nanoparticle material.

[0011] Another technical solution of the present invention is the application of the above-mentioned avian coronavirus mRNA vaccine as a preparation for preventing diseases caused by avian coronavirus.

[0012] Compared with the prior art, the technical solutions provided by the present invention have the following technical advantages 1. Compared with commercial inactivated vaccines, the mRNA vaccine provided by the present invention can not only effectively activate cellular immunity, but also enhance the level of humoral immunity. It can produce sufficient immune protection effects with only a dose of 15 μg, can produce a more persistent antibody level, and the Mosaic vaccine also activates the immune response and has cross-protection.

[0013] 2. The preparation method of the mRNA vaccine provided by the present invention is simple in operation, has low requirements for instruments, can produce mRNA-LNP complexes of suitable size, and has a high encapsulation rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 is the antigenic epitope coverage rate of the N-MOSAIC sequence; Figure 2 is the prediction of the protein tertiary structure of the N and N-MOSAIC sequences; Figure 3 is the PCR amplification plasmid template diagram; Figure 4 is the indirect immunofluorescence detection result diagram; Figure 5 is the protein immunoblotting detection result diagram; Figure 6 is the standard curve diagram for detecting the encapsulation rate; Figure 7 is the particle size distribution diagram of the synthesized mRNA-LNP Figure 8 is the diagram of clinical symptoms, pathological dissection and sections after virus challenge; Figure 9 is the paraffin section diagram of tracheal pathology; Among them: Figure 9 a is the paraffin section diagram of tracheal pathology of the blank control group; Figure 9 b is the paraffin section diagram of tracheal pathology of the positive control group at 5 dpi after virus challenge Figure 9 c is the paraffin section diagram of tracheal pathology of the positive control group at 14 dpi after virus challenge; Figure 9 d is the paraffin section diagram of tracheal pathology of the commercial vaccine group (CV) at 5 dpi after virus challenge; Figure 9 f is the paraffin section diagram of tracheal pathology of the mRNA-LNP-N-Mosaic vaccine group at 14 dpi after virus challenge; Figure 9 g is the paraffin section diagram of tracheal pathology of the commercial vaccine group at 14 dpi after virus challenge; Figure 9h is the paraffin section of tracheal pathology at 14 dpi after challenge in the mRNA-LNP-N vaccine group; Figure 9 i is the paraffin section of tracheal pathology at 14 dpi after challenge in the mRNA-LNP-N-Mosaic vaccine group; Figure 10 It is the gating method and result analysis diagram of flow cytometry; Figure 11 It is the ELISA test result diagram. Specific implementation mode

[0015] The present invention will be further described below in conjunction with specific implementation modes. However, those skilled in the art should understand that without departing from the technical solution of the present invention, modifications or substitutions to the details and forms of the technical solution of the present invention fall within the protection scope of the present invention.

[0016] Example 1 Preparation of mRNA-LNP-N vaccine Step 1) Preparation of PUC57-mRNA-N plasmid 1) Plasmid construction and synthesis Download the N protein nucleotide sequence of IBV GI-1 strain (DQ834384.1) from the NCBI database. Its nucleotide sequence is as shown in SEQ ID NO.4. The sequence with the nucleotide sequence as shown in SEQ ID NO.4 is codon-optimized using the Yunzhou Biology codon optimization tool (https: / / www.vectorbuilder.cn / ) with chicken as the host. The optimized N protein nucleotide sequence of the sequence shown in SEQ ID NO.4 is as shown in SEQ ID NO.6. The sequence is used for protein tertiary structure prediction using the swissmodel tool (https: / / swissmodel.expasy.org / interactive) ( Figure 2 )

[0017] Add a Kozak sequence (gccaccatg), a T7 promoter (nucleotide sequence as shown in SEQ ID NO.9), and a 5'UTR sequence of β-globin (nucleotide sequence as shown in SEQ ID NO.10) to the head of the optimized N protein nucleotide sequence (nucleotide sequence as shown in SEQ ID NO.6), and tandem two 3'UTR sequences of β-globin (nucleotide sequence as shown in SEQ ID NO.11) at the tail to obtain the nucleotide sequence as shown in SEQ ID NO.1.

[0018] Then, the nucleotide sequence shown in SEQ ID NO.1 was ligated to the PUC57 vector to synthesize the plasmid PUC57-mRNA-N, and the nucleotide sequence of PUC57-mRNA-N is shown in SEQ ID NO.12); Plasmid amplification and extraction (using the Plasmid Midi Kit D6950 from OMEGA).

[0019] The specific steps are as follows: ① After resuspending the synthesized PUC57-mRNA-N plasmid in step 1) with LB medium, add it to a 100 mL conical flask (containing 40 mL of LB broth with ampicillin), and culture it in a shaker at 37 °C with a rotation speed of 220 rpm / min for 12 - 16 h; ② Aliquot the bacterial solution into 50 mL centrifuge tubes, centrifuge at 12000 g for 2 min, and discard the supernatant; ③ Add 2.5 mL of Solution Ⅰ / RNase A to each centrifuge tube to resuspend the precipitate; ④ Add 3.5 mL of Solution Ⅲ to each centrifuge tube, cover the centrifuge tube cap, gently shake the centrifuge tube for 2 min, and centrifuge at 12000 g for 10 min; ⑤ Pipette the supernatant after the previous centrifugation into a new centrifuge tube and discard the precipitate; ⑥ Take a new HiBind ® DNA mini adsorption column, place it in the waste liquid tube (outer tube), and transfer the supernatant to the HiBind ® DNA binding column with a 2 ml collection tube, centrifuge at 12000 g for 1 min, and discard the liquid in the waste liquid tube (if all the liquid cannot be loaded into the centrifuge column for centrifugation at one time, it can be centrifuged in batches); ⑦ Add 3 mL of HBC Buffer (10 mL of isopropanol needs to be added in advance according to the instructions) to the adsorption column, centrifuge at 12000 g for 1 min, and discard the liquid in the waste liquid tube; ⑧ Add 3.5 mL of DNA Wash Buffer (OMEGA D6950) (60 mL of absolute ethanol needs to be added in advance according to the instructions on the bottle) to the adsorption column, centrifuge at 12000 g for 1 min, and discard the liquid in the waste liquid tube; ⑨ Repeat step ⑧; ⑩ Without adding DNA washing buffer, centrifuge at 12000 g for 10 min, and discard the liquid in the waste liquid tube; ⑪ Open the lid of the adsorption column, dry the adsorption membrane in the adsorption column (usually takes 10 min), and transfer the adsorption column to a new 15 mL centrifuge tube; ⑫ Add 1 mL of deionized water, let it stand for 5 min, and centrifuge at 12000 g for 5 min; ⑬ Measure the concentration of the recovered product, record it, and store it in a -20 °C refrigerator.

[0020] Step 2) Preparation of Cap-mRNA-N-polyA 1) Perform PCR amplification on PUC57-mRNA-N to obtain an in vitro transcription template Use the synthesized plasmid PUC57-mRNA-N as a template for the PCR reaction system as shown in Table 1.

[0021] Table 1 Reagent Volume (μL) Prime STAR Max Premix(2X) 25 M13-47 1 mRNA-R2 1 Plasmid template 1 Deionized water 22 Total 50 The PCR reaction program is 98 °C for 3 min; 98 °C for 10 s; 55 °C for 5 s; 72 °C for 5 s; 72 °C for 5 min, 35 cycles; After the reaction, add 6 μL of 10× loading buffer (Vazyme P022-01) to the PCR product, perform electrophoresis using 1% agarose gel, and cut the target band ( Figure 3 ) and put it into a 2 mL centrifuge tube for gel recovery to detect the concentration.

[0022] The specific detection method is as follows: ① Weigh the cut gel strip, and add binding buffer to the cut gel strip according to the ratio of weight: volume = 1:1; ② Place the system containing the gel strip and binding buffer in a water bath at 55 °C - 60 °C for heating until all the gel strips are dissolved; ③ Take a new HiBind ® DNA mini binding column, place it in the waste liquid tube (outer tube), transfer the liquid after dissolving the gel strip into the adsorption column, centrifuge at 12000 g for 1 min, and discard the liquid in the waste liquid tube (if all the liquid cannot be loaded into the centrifuge column for centrifugation at one time, it can be centrifuged in batches); ④ Add 500 μL of binding buffer to the adsorption column, centrifuge at 12000 g for 1 min, and discard the liquid in the waste liquid tube; ⑤ Add 600 μL of washing buffer (OMEGA D2500-01) (100 mL of absolute ethanol needs to be added in advance according to the prompt) to the adsorption column, centrifuge at 12000 g for 1 min, and discard the liquid in the waste liquid tube; ⑥ Repeat step ⑤; ⑦ Without adding washing buffer, centrifuge at 12000 g for 2 min, and discard the liquid in the waste liquid tube; ⑧ Open the lid of the adsorption column, dry the adsorption membrane in the adsorption column (usually takes 2 min), and transfer the adsorption column to a new 1.5 mL centrifuge tube; ⑨ Add 30 μL of deionized water, let it stand for 2 min, and centrifuge at 12000 g for 2 min; ⑩ Measure the concentration of the recovered product, record it, and store it in a -20 °C refrigerator.

[0023] 2) In vitro synthesis of mRNA The experiment uses the Novoprotein T7 High Yield RNA Transcription Kit (N¹-Me-PseudoUTP) (DD4202-01) kit. All experiments are carried out using RNase-free consumables in an RNase-free environment. (After the reagent is melted on ice, vortex and mix well, then briefly centrifuge); The in vitro transcription reaction system is shown in Table 2.

[0024] Table 2 In vitro transcription reaction system Reagent Volume / μL 10×Transcription Buffer 2 UTP / N1-Me-PseudoUTP(ψUTP)(100 mM) 2 ATP Solution(100 mM) 2 GTP Solution(100 mM) 2 CTP Solution(100 mM) 2 T7 RNA Polymerase Mix 2 Murine RNase Inhibitor(40 U / μL) 2 PCR Gel Extraction Template 0.5 μg <![CDATA[RNase-free ddH2O]]> Add to 20 μL After preparing the system, vortex and briefly centrifuge, incubate in a 37 °C water bath for 3 h. After completion, add 1 μl of DNase I, incubate at 37 °C for 15 min to digest the excess gel-extracted DNA template to obtain the mRNA-N product.

[0025] 3) Purification using LiCL (referring to a 20 μL system) ① Add 30 μL of RNase-free ddH2O to dilute the mRNA-N product prepared in step 2) to 50 μL; ② Then add 30 μL of lithium chloride precipitation (7.5 M lithium chloride, 50 mM EDTA) to the diluted product; ③ After mixing, incubate at -20 °C for 30 min, centrifuge at 12000 rpm for 15 min, and discard the supernatant; ④ Add 500 μL of pre-cooled 75% ethanol to wash the RNA precipitate, centrifuge at 15000 rpm for 15 min at 4 °C, and discard the supernatant; ⑤ Repeat three times; ⑥ Open the lid and dry for 2 min, add 40 μL of RNase-free ddH2O to dissolve the RNA precipitate.

[0026] 4) Capping Use the Novoprotein Vaccinia Capping System (product number DD4109-01) kit to perform the capping reaction on the purified mRNA-N in step 3) (the experiment needs to be carried out using RNase-free experimental consumables in an RNase-free environment), as follows.

[0027] ① After the reagent is melted on ice, mix well and briefly centrifuge to collect the components to the bottom of the tube; ② Dilute SAM (32 mM) to 4 mM; ③ Put the RNA into a 65 °C water bath for thermal denaturation for 5 min, and then on ice for 5 min. This step is to open the complex structure at the 5' end of the RNA and improve the capping efficiency; ④ Add reagents according to the system in Table 3: Table 3 ⑤ Mix the prepared system evenly, put it into a 37 °C water bath, and incubate for 1 h; ⑥ Purify it using the LiCL purification method described in 3), detect the concentration, and store it in a -80 °C refrigerator for later use.

[0028] 5) Polyadenylation Since the replication process of the template plasmid containing the Poly(A) sequence is unstable in Escherichia coli and the A tail is prone to loss, the Novoprotein E.coli Poly(A) Polymerase (5 U / μl) (polyadenylation enzyme) kit is used for polyadenylation; ② Add reagents according to Table 4.

[0029] Table 4 Reagent Volume / μL 10×Polymerase Buffer 2 E.coli Poly(A) Polymerase (5 U / μl) 1 ATP(10 mM) 1 Murine RNase Inhibitor (40 U / μL) 2 Cap 1 RNA 10 μg <![CDATA[RNase-free ddH2O]]> Add to 20 μL After mixing the system, put it into a 37 °C water bath and incubate for 3 h, adding approximately 150 - 200 bases; ③ Purify it using the LiCL purification method described in 3) to obtain Cap-mRNA-N-polyA, measure the concentration, and store it in a -80 °C refrigerator for later use.

[0030] Step 3) Preparation of mRNA-LNP-N 1) Preparation of liposomes (LNP) S1: ① Dilute the ionizable lipid D-Link-MC3-DMA and PEG-DMG to 100 mM respectively using pharmaceutical-grade anhydrous ethanol; ② Dissolve DSPC and cholesterol to 10 mM using pharmaceutical-grade anhydrous ethanol; ③ Mix D-Link-MC3:DSPC:cholesterol:PEG-DMG in a molar ratio of 50:10:38.5:1.5 in sequence, and finally add pharmaceutical-grade anhydrous ethanol to dilute the mixture into a 20 mg / mL LNP solution; 2) mRNA-LNP synthesis ① Take out the LNP solution (20 mg / mL) prepared in step 1) from the -20 °C refrigerator and let it return to room temperature; ② Take out 300 μg of the Cap-mRNA-N-polyA synthesized in step (ii), add 20×citrate-sodium citrate buffer solution and DEPC-treated water for dilution, and dilute the mRNA into a solution with a pH of 4 and a concentration of 1 mg / mL (add 15 μL of 20×citrate-sodium citrate buffer solution, and the total volume of the solution is 300 μL); ③ Wash the customized annular microfluidic glass chip (width 200, depth 90) once with absolute ethanol; ④ Set the total injection volume of the dual-channel micro-injection pump to 300 μL, set the flow rate to 1.5 mL / min, and set the left and right limits; ⑤ Draw 300 μL of Cap-mRNA-N-polyA and 300 μL of LNP solution (LNP filled in the syringe with a black mark) into two 1 mL syringes respectively; ⑥ Install the syringes into the micro-injection pump, connect them to the microfluidic chip (properly raise the outlet of the chip), and click RUN; ⑦ The synthesized mRNA-LNP-N vaccine flows into a 15 mL centrifuge tube filled with PBS, invert and mix it, load the sample into an ultrafiltration centrifuge tube, centrifuge at 4000 g for 10 min, and collect the concentrate.

[0031] Example 2 Preparation of mRNA-LNP-N-Mosaic vaccine Step (i) PUC57-mRNA-N-mosaic plasmid 1) Construction and synthesis of plasmid Select the full-length N protein sequences of common epidemic strains of infectious bronchitis virus (GI-1, GI-19, GI-28, etc.). All sequences were obtained from NCIB. Upload the above sequences (193 in total) in Fasta format to the Mosaic vaccine design website Mosaic Vaccine Design (https: / / www.hiv.lanl.gov / content / sequence / MOSAIC / makeVaccine.html) (parameters: cocktail size at 3, epitope length at 9 aa, rareThreshold at 3, run time at 10 h, population size at 200, cycle time at 10, stall time at 10, and internal crossover probability at 0.5). Use the coverage assessment tool to determine the average epitope coverage of the mosaic antigen sequence in the native viral protein sequence (parameters: nominal epitopelength at 9 aa, the maximum amino acid mismatches to score at 2, and the minimum number of occurrences of the potential epitope in viral protein set to 3 aa) ( Figure 1 Left), analyze the CTL distribution of the mosaic antigen sequence (nucleotide sequence as shown in SEQ ID NO.5) in the native viral protein sequence through the position antigen determinant coverage assessment tool (Posicover) (parameters: nominalepitope length at 9 aa, and antigen counts to compute upper bounds at 3, 4) ( Figure 1 Right).

[0032] The nucleotide sequence shown in SEQ ID NO.5 was codon-optimized using the Yunzhou Biology Codon Optimization Tool (https: / / www.vectorbuilder.cn / ) with chicken as the host. The optimized Mosaic nucleotide sequence of the sequence shown in SEQ ID NO.5 is shown in SEQ ID NO.3. The tertiary structure prediction of the protein was performed on the sequence shown in SEQ ID NO.3 using the swissmodel tool (https: / / swissmodel.expasy.org / interactive) ( Figure 2 ).

[0033] The optimized Mosaic nucleotide sequence (the nucleotide sequence is shown in SEQ ID NO3) was added with a Kozak sequence (gccaccatg), a T7 promoter (the nucleotide sequence is shown in SEQ ID NO.9), and a 5'UTR sequence of β-globin (the nucleotide sequence is shown in SEQ ID NO.10) at the head, and two 3'UTR sequences of β-globin (the nucleotide sequence is shown in SEQ IDNO.11) were concatenated at the tail to obtain the nucleotide sequence shown in SEQ ID NO.2.

[0034] SEQ ID NO.2 was ligated to the PUC57 vector to synthesize a plasmid to obtain PUC57-mRNA-N-mosaic, and the nucleotide sequence of the PUC57-mRNA-N-mosaic is shown in SEQ ID NO.13) Amplification and extraction of the PUC57-mRNA-N-mosaic plasmid (using the Plasmid Midi Kit D6950 from OMEGA).

[0035] The specific steps are as follows: ① After resuspending the PUC57-mRNA-N-mosaic plasmid synthesized in step 1) with LB medium, add it to a 100 mL conical flask (containing 40 mL of LB broth with ampicillin), and culture it in a shaker at 37°C and 220 rpm / min for 12 - 16 h; ② Aliquot the bacterial solution into 50 mL centrifuge tubes, centrifuge at 12000 g for 2 min, and discard the supernatant; ③ Add 2.5 mL of SolutionⅠ / RNase A to each centrifuge tube to resuspend the pellet; ④ Add 3.5 mL of SolutionⅢ to each centrifuge tube, cover the centrifuge tube cap, gently shake the centrifuge tube for 2 min, and centrifuge at 12000 g for 10 min; ⑤ Pipette the supernatant after the previous centrifugation into a new centrifuge tube and discard the pellet; ⑥ Take a new HiBind ® DNA mini adsorption column, place it into the waste liquid tube (outer sleeve tube), and transfer the supernatant to the HiBind ® DNA binding column with a 2 ml collection tube sleeved. Centrifuge at 12,000 g for 1 min, and discard the liquid in the waste liquid tube (if not all the liquid can be loaded into the centrifuge column for centrifugation at one time, it can be centrifuged in batches); ⑦ Add 3 mL of HBC Buffer (10 mL of isopropanol needs to be added in advance according to the instructions) to the adsorption column, centrifuge at 12,000 g for 1 min, and discard the liquid in the waste liquid tube; ⑧ Add 3.5 mL of DNA Wash Buffer (OMEGA D6950) (60 mL of absolute ethanol needs to be added in advance according to the instructions on the bottle) to the adsorption column, centrifuge at 12,000 g for 1 min, and discard the liquid in the waste liquid tube; ⑨ Repeat step ⑧; ⑩ Without adding DNA washing buffer, centrifuge at 12,000 g for 10 min, and discard the liquid in the waste liquid tube; ⑪ Open the lid of the adsorption column, dry the adsorption membrane in the adsorption column (usually takes 10 min), and transfer the adsorption column to a new 15 mL centrifuge tube; ⑫ Add 1 mL of deionized water, let it stand for 5 min, and centrifuge at 12,000 g for 5 min; ⑬ Measure the concentration of the recovered product, record it, and store it in a -20 °C refrigerator.

[0036] Step 2) Prepare Cap-mRNA-N-mosaic-polyA 1) Perform PCR on PUC57-mRNA-N-mosaic to obtain an in vitro transcription template Use the synthesized PUC57-mRNA-N-mosaic plasmid as a template for the PCR reaction system as shown in Table 5.

[0037] Table 5 Reagent Volume (μL) Prime STAR Max Premix(2X) 25 M13-47 1 mRNA-R2 1 Plasmid template 1 Deionized water 22 Total 50 The PCR reaction program is 98 °C for 3 min; 98 °C for 10 s; 55 °C for 5 s; 72 °C for 5 s; 72 °C for 5 min, 35 cycles; After the reaction, add 6 μL of 10× loading buffer (Vazyme P022-01) to the PCR product, perform electrophoresis using 1% agarose gel, and cut the target band ( Figure 3 ) and load it into a 2 mL centrifuge tube for gel recovery to detect the concentration.

[0038] The specific detection method is as follows: ①Weigh the cut gel strips, and add binding buffer to the cut gel strips at a weight:volume ratio of 1:1. ②Place the system containing the gel strips and binding buffer in a water bath at 55 °C - 60 °C for heating until all the gel strips are dissolved. ③Take a new HiBind ® DNA mini binding column, place it in the waste liquid tube (outer sleeve), transfer the liquid after dissolving the gel strips into the adsorption column, centrifuge at 12000 g for 1 min, and discard the liquid in the waste liquid tube (if all the liquid cannot be loaded into the centrifuge column for centrifugation at one time, it can be centrifuged in batches). ④Add 500 μL of binding buffer to the adsorption column, centrifuge at 12000 g for 1 min, and discard the liquid in the waste liquid tube. ⑤Add 600 μL of washing buffer (OMEGA D2500 - 01) (100 mL of absolute ethanol needs to be added in advance according to the instructions) to the adsorption column, centrifuge at 12000 g for 1 min, and discard the liquid in the waste liquid tube. ⑥Repeat step ⑤. ⑦Without adding washing buffer, centrifuge at 12000 g for 2 min, and discard the liquid in the waste liquid tube. ⑧Open the lid of the adsorption column, air-dry the adsorption membrane in the adsorption column (usually takes 2 min), and transfer the adsorption column to a new 1.5 mL centrifuge tube. ⑨Add 30 μL of deionized water, let it stand for 2 min, and centrifuge at 12000 g for 2 min. ⑩Measure the concentration of the recovered product, record it, and store it in a -20 °C refrigerator.

[0039] In vitro synthesis of mRNA-N-mosaic The Novoprotein T7 High Yield RNA Transcription Kit (N¹-Me-PseudoUTP) (DD4202 - 01) was used in the experiment. All experiments were carried out using RNase-free consumables in an RNase-free environment. (After the reagent was melted on ice, it was vortexed and briefly centrifuged); The in vitro transcription reaction system is shown in Table 6.

[0040] Table 6 In vitro transcription reaction system Reagent Volume / μL 10×Transcription Buffer 2 UTP / N1-Me-PseudoUTP(ψUTP)(100 mM) 2 ATP Solution(100 mM) 2 GTP Solution(100 mM) 2 CTP Solution(100 mM) 2 T7 RNA Polymerase Mix 2 Murine RNase Inhibitor(40 U / μL) 2 PCR Gel Extraction Template 0.5 μg <![CDATA[RNase-free ddH2O]]> Add to 20 μL After preparing the system, vortex and centrifuge briefly, incubate in a 37 °C water bath for 3 h. After that, add 1 μl of DNase I, incubate at 37 °C for 15 min to digest the excess DNA template after gel recovery, and collect the mRNA-N-mosaic product.

[0041] 3) LiCL purification (referring to a 20 μL system) ① Add 30 μL of RNase-free ddH₂O to dilute the mRNA-N-mosaic product prepared in step 2) to 50 μL; ② Then add 30 μL of lithium chloride precipitation (7.5 M lithium chloride, 50 mM EDTA) to the diluted product; ③ After mixing, incubate at -20 °C for 30 min, centrifuge at 12000 rpm for 15 min, and discard the supernatant; ④ Add 500 μL of pre-cooled 75% ethanol to wash the RNA precipitate, centrifuge at 4 °C and 15000 rpm for 15 min, and discard the supernatant; ⑤ Repeat three times; ⑥ Open the lid and dry for 2 min, add 40 μL of RNase-free ddH₂O to dissolve the RNA precipitate.

[0042] 4) Capping Use the Novoprotein Vaccinia Capping System (product number DD4109-01) kit for the capping reaction. The experiment needs to be carried out using RNase-free experimental consumables in an RNase-free environment.

[0043] ① After the reagents are melted on ice, mix them well and centrifuge briefly to collect the components at the bottom of the tube; ② Dilute SAM (32 mM) to 4 mM; ③ Place the RNA in a 65 °C water bath for heat denaturation for 5 min, then on ice for 5 min. This step is to open the complex structure at the 5' end of the RNA and improve the capping efficiency; ④ Add reagents according to the system in Table 7: Table 7 ⑤ Mix the prepared system evenly, place it in a 37 °C water bath, and incubate for 1 h; ⑥ Purify it using the LiCL purification method described in 3), detect the concentration, and store it in a -80 °C refrigerator for later use.

[0044] 5) Tailing Since the template plasmid containing the Poly(A) sequence is unstable during replication in Escherichia coli and prone to A-tail loss, the Novoprotein E.coli Poly(A) Polymerase (5 U / μl) (tailing enzyme) kit is used for tailing; ② Add reagents according to Table 8.

[0045] Table 8 Reagent Volume / μL 10×Polymerase Buffer 2 E.coli Poly(A) Polymerase (5 U / μl) 1 ATP(10 mM) 1 Murine RNase Inhibitor (40 U / μL) 2 Cap 1 RNA 10 μg <![CDATA[RNase-free ddH2O]]> Add to 20 μL After the system is mixed evenly, place it in a 37 °C water bath and incubate for 3 h, adding approximately 150 - 200 bases; ③ Purify according to the LiCL purification method described in 3) to obtain Cap-mRNA-N-mosaic-polyA. After measuring the concentration, store it in a -80°C refrigerator for later use.

[0046] Step 3) Preparation of mRNA-LNP-N-Mosaic vaccine 1) Preparation of liposomes S1: ① Dilute the ionizable lipid D-Link-MC3-DMA and PEG-DMG to 100 mM respectively using pharmaceutical-grade anhydrous ethanol; ② Dissolve DSPC and cholesterol in pharmaceutical-grade anhydrous ethanol to 10 mM; ③ Mix D-Link-MC3:DSPC:cholesterol:PEG-DMG in a molar ratio of 50:10:38.5:1.5 in sequence. Finally, add pharmaceutical-grade anhydrous ethanol and dilute the mixture into a 20 mg / mL LNP solution; 2) Synthesis of mRNA-LNP-N-Mosaic vaccine ① Take out the LNP solution (20 mg / mL) prepared in step 1) from the -20°C refrigerator and let it return to room temperature; ② Take out 300 μg of the synthesized Cap-mRNA-N-mosaic-polyA and dilute it by adding 20×citrate-sodium citrate buffer and DEPC-treated water to dilute the mRNA into a solution with a pH of 4 and a concentration of 1 mg / mL (add 15 μL of 20×citrate-sodium citrate buffer, and the total volume of the solution is 300 μL); ③ Wash the customized annular microfluidic glass chip (width 200, depth 90) once with anhydrous ethanol; ④ Set the total injection volume of the dual-channel micro-injection pump to 300 μL, set the flow rate to 1.5 mL / min, and set the left and right limits; ⑤ Draw 300 μL of Cap-mRNA-N-mosaic-polyA and 300 μL of LNP solution (LNP filled in the syringe with a black mark) into two 1 mL syringes respectively; ⑥ Install the syringes into the micro-injection pump, connect them to the microfluidic chip (raise the chip outlet appropriately), and click RUN; ⑦ The synthesized mRNA-LNP-N-Mosaic flows into a 15 mL centrifuge tube containing a certain volume of PBS. Invert and mix it, then load the sample into an ultrafiltration centrifuge tube and centrifuge at 4000 g for 10 min to collect the concentrate.

[0047] Verification experiment 1. Cap-mRNA-N-polyA and Cap-mRNA-N-polyA cell transfection experiments Cell transfection of Cap-mRNA-N-polyA and Cap-mRNA-N-polyA was performed separately using Lipofectamine™ 2000 Reagent. The specific method is as follows: ① Take out the cryopreserved 293T cells in the liquid nitrogen tank for resuscitation and passage. After their growth is stable, plate them; ② Treat the 6-well plate with polylysine, then digest the cells in the dish, and dilute them with DMEM medium containing 10% fetal bovine serum (FBS) to 2×10 5 cells / mL for plating; ③ Place the cell plate in a 37℃ Thermo Scientific™ CO2 incubator (51032875) with 5% CO2 and culture until 80% cell confluence; ④ Refer to the Lipofectamine™ 2000 Reagent instruction manual for the plate of 293T cells. Dilute the liposome (lipo2000) with Opti-MEM to obtain the treated liposome dilution; dilute Cap-mRNA-N-polyA with Opti-MEM to obtain the Cap-mRNA-N-polyA dilution; dilute Cap-mRNA-N-mosaic-polyA with Opti-MEM to obtain the Cap-mRNA-N-mosaic-polyA dilution; Then mix according to the ratio of 1.5 μL of the treated liposome dilution to 1 μg of the Cap-mRNA-N-polyA dilution to obtain the Cap-mRNA-N-polyA liposome complex; incubate at room temperature for 20 min.

[0048] Then mix according to the ratio of 1.5 μL of the treated liposome dilution to 1 μg of the Cap-mRNA-N-mosaic-polyA dilution to obtain the Cap-mRNA-N-mosaic-polyA liposome complex and incubate at room temperature for 20 min ⑤ Carefully wash the cell plate twice with PBS, add Opti-MEM medium. After the incubation in the previous step ends, add the incubated Cap-mRNA-N-polyA liposome complex and Cap-mRNA-N-mosaic-polyA liposome complex in step ④ to the cell plate respectively, and place the cell plate in a 37℃ cell incubator with 5% CO2 for culture.

[0049] 2. Indirect immunofluorescence assay (IFA) ① Take out the cell plate transfected with the Cap-mRNA-N-polyA liposome complex and Cap-mRNA-N-mosaic-polyA liposome complex for 36 h, discard the medium, and wash 3 times with PBS; ② Treat with 4% paraformaldehyde for 15 min. After discarding the liquid, permeabilize with 0.1% Triton X-100 for 15 min; ③ Wash three times with PBS; ④ Block with TBST containing 1% BSA for 2 h; ⑤ Wash three times with PBS; ⑥ Dilute the IBV positive serum 1:200, add 200 μL per well, and incubate overnight at 4°C; ⑦ Wash three times with PBS; ⑧ Dilute the goat anti-chicken FITC-conjugated secondary antibody 1:400, incubate at 37°C in the dark for 45 min; ⑨ After washing three times with PBS, observe under an inverted fluorescence microscope, refer to Figure 4 ( Figure 4 In the left figure is the observation of the target protein N of Cap-mRNA-N-polyA under the fluorescence microscope, in the middle is the observation of the target protein N-mosaic of Cap-mRNA-N-mosaic-polyA under the fluorescence microscope, and in the right figure is the observation of the negative control group under the fluorescence microscope), through Figure 4 It can be seen that both the target protein N and the target protein N-mosaic are significantly expressed in 293T cells, while no fluorescence signal is detected in the negative control group, indicating successful transfection and good specificity, thus proving the expression efficiency and specificity of the mRNA vaccine of the present invention and providing evidence for subsequent immunogenicity research.

[0050] 3. Immunoblotting experiment (Western Blot, WB) ① Take out the cell plates transfected with Cap-mRNA-N-polyA liposome complex and Cap-mRNA-N-mosaic-polyA liposome complex for 24 h, discard the culture medium, wash once with PBS, add 100 μL of RIPA cell lysate and 1 μL of PMSF protease inhibitor to each well, and lyse on ice for 20 - 30 min; ② After sufficient lysis, collect the cells into a 1.5 mL EP tube and centrifuge at 14000 rpm for 10 min; ③ Transfer the supernatant to a new 1.5 mL EP tube, add 1 / 4 volume of 5×SDS, mix well, incubate in a metal bath at 100°C for 10 min, and centrifuge at 1200 g for 1 min; ④ Take 80 μL of IBV M41 strain virus solution (purchased from the China Institute of Veterinary Drug Control, AV1511), add 20 μL of 5×SDS protein loading buffer, incubate in a metal bath for 10 min, and centrifuge at 1200 g for 1 min as a positive control; ⑤Pre-prepared 10% SDS-PAGE gel, 30 μL of sample per well; ⑥After electrophoresis at 80 V for 30 min, adjust the voltage to 120 V and continue electrophoresis for 1 h; ⑦Transfer membrane: Use the wet transfer method. Take out the gel, cut it into appropriate size, and transfer the protein to the nitrocellulose (NC) membrane using a current of 400 mA for 1 h; ⑧Blocking: Put the transferred NC membrane into 5% BSA and block it at room temperature on a shaker for 2 h; ⑨Washing: Wash three times with TBST, 5 min each time; ⑩Dilute the positive serum 1:200 and incubate overnight at 4 °C; Washing: Wash three times with TBST, 5 min each time; ⑪Dilute the rabbit anti-chicken HRP fluorescent secondary antibody 1:20000 and incubate for 30 min at room temperature in the dark; ⑫Washing: Wash three times with TBST, 5 min each time; ⑬Develop using the developing kit and scan and image on the machine.

[0051] The electrophoresis pattern of the protein expression in cells after transfection is shown in Figure 5 , Figure 5 In it, 1 is the N protein, 2 is the Mosaic N protein, 3 is the negative control, and 4 is the positive control. It can be seen that both the N protein and the Mosaic N protein have obvious bands at 55 kDa, proving that the synthesized Cap-mRNA-N-polyA and Cap-mRNA-N-mosaic-polyA can successfully express the N protein and the Mosaic N protein in 293T cells; while no corresponding protein bands appear in the negative control group, verifying the specificity of the experimental results, thus ensuring that the subsequent prepared vaccine has a high immune response ability; the protein band of the positive control group serves as a reference for the experimental validity, helping to evaluate the expression level of the target protein.

[0052] Detection experiment 1. Particle size detection Use the Malvern Zetasizer Nano ZS90 nano particle size and zeta potential analyzer to measure the particle sizes of the prepared mRNA-LNP-N-Mosaic vaccine and mRNA-LNP-N vaccine. Take 10 μL of the sample, dilute it to 1 mL, add it to a quartz cuvette, set the parameters in the instrument, and then start the measurement and record the data.

[0053] 2) Plot the RNA concentration standard curve To measure the encapsulation efficiency of mRNA encapsulated by LNP, a standard curve needs to be established first. The concentration of different free RNAs in the liquid and the corresponding fluorescence intensity are detected by the Quant-iT™ RiboGreen™ RNA Assay Kit, and the standard curve is drawn ( Figure 6 ), Y = A*X + B. The encapsulation efficiency of the sample is calculated according to the standard curve. Each point of the standard curve is replicated three times, and the R 2 of the curve reaches 0.999, indicating a linear relationship between the fluorescence intensity and the sample concentration and its feasibility.

[0054] 3) Encapsulation efficiency detection ① After diluting the mRNA-LNP-N-Mosaic vaccine with PBS to 1500 ng / mL, then take 10 μL of the diluted mRNA-LNP-N-Mosaic vaccine and add it to two 1.5 mL EP tubes respectively. One EP tube is diluted to 1 mL with 1×TE Buffer; the other EP tube is diluted to 1 mL with 2% TritonX-100; ② After diluting the mRNA-LNP-N vaccine with PBS to 1500 ng / mL, then take 10 μL of the diluted mRNA-LNP-N vaccine and add it to two 1.5 mL EP tubes respectively. One EP tube is diluted to 1 mL with 1×TE Buffer; the other EP tube is diluted to 1 mL with 2% TritonX-100; ② Take out the RiboGreen solution in the kit, add 0.5 μL of the fluorescent solution to every 100 μL of 1×TE Buffer (pay attention to avoiding light); ③ Add the diluted mRNA-LNP-N vaccine and mRNA-LNP-N-Mosaic vaccine in ① to different wells of a black 96-well plate at a volume of 100 μL per well. Add the diluted RiboGreen solution in ② to the wells containing the mRNA-LNP-N vaccine and mRNA-LNP-N-Mosaic vaccine at a volume of 100 μL per well and incubate in the dark. Each group has 3 replicates. Detect the absorbance with a microplate reader. The results are shown in Figure 7 , and the encapsulation efficiency is calculated to be above 85% according to the RNA concentration standard curve in step 2). See Figure 7 .

[0055] Test results At a flow rate of 1.5 mL / min and a volume ratio of mRNA:LNP of 1:1, the finally measured particle size is between 200 - 300 nm, and the PDI < 0.2, and the encapsulation efficiency is above 85%. See Figure 7 .

[0056] SPF chicken immunization and challenge experiment Sixty specific pathogen free (SPF) chickens were divided into four groups, namely the PBS group, the mRNA-LNP-N vaccine group, the mRNA-LNP-N-Mosaic vaccine group, and the commercial vaccine group. The immunization method was intramuscular injection in the leg.

[0057] The immunization doses were as follows: PBS group: 300 μL of PBS per chicken; mRNA-LNP-N vaccine group: Each chicken was injected with 15 μg / 300 μL of the mRNA-LNP-N vaccine (i.e., 300 μL of PBS containing 15 μg of the mRNA-LNP-N vaccine); mRNA-LNP-N-Mosaic vaccine group: 15 μg / 300 μL of the mRNA-LNP-N-Mosaic vaccine per chicken (i.e., 300 μL of PBS containing 15 μg of the mRNA-LNP-N-Mosaic vaccine); Commercial vaccine group: 0.3 mL per chicken (the content of infectious bronchitis virus strain M41 before inactivation was ≥ 10 6.5 EID 50 / 0.1 mL) The challenge dose was: 10 6 EID 50 / 0.1 mL 0.2 mL.

[0058] The challenge method was: 100 μL by nasal drip and 100 μL by eye drop.

[0059] Immunization schedule: The first immunization was on day 14, followed by a second immunization, and the chickens were challenged 14 days later. 5) Clinical symptoms and pathological examination after challenge For the clinical symptoms and pathological examination results after challenge, refer to Figure 8 , after infection with the M41 strain in the challenged group, the clinical symptoms were not obvious, mainly manifested as not huddling together ( Figure 8 a), listlessness, and roaring, coughing, and sneezing occurred at 3 - 5 days post-challenge (dpc). During pathological examination, the trachea was congested, with bleeding points and excessive mucus ( Figure 8 b, c).

[0060] Paraffin pathological sections of chicken trachea were prepared at 5 days post-infection (dpi) and 14 dpi after challenge, and the results were as follows Figure 9 ; among them: Figure 9 a is the paraffin pathological section diagram of the trachea of the blank control group; Figure 9 b is the paraffin pathological section diagram of the trachea of the positive control group at 5 dpi after challenge; Figure 9 c is the paraffin pathological section diagram of the trachea of the positive control group at 14 dpi after challenge; Figure 9 d is the paraffin pathological section diagram of the trachea of the commercial vaccine group (CV) at 5 dpi after challenge; Figure 9Figure f shows the paraffin section of the trachea pathology at 14 dpi after challenge in the mRNA-LNP-N-Mosaic vaccine group; Figure 9 Figure g shows the paraffin section of the trachea pathology at 14 dpi after challenge in the commercial vaccine group; Figure 9 Figure h shows the paraffin section of the trachea pathology at 14 dpi after challenge in the mRNA-LNP-N vaccine group; Figure 9 Figure i shows the paraffin section of the trachea pathology at 14 dpi after challenge in the mRNA-LNP-N-Mosaic vaccine group; Through Figure 9 a, it can be seen that in the blank control group, goblet cells are irregularly arranged, the microvilli on the cell surface are clearly visible, the lamina propria is thin, and it is rich in fibrous connective tissue; Through Figure 9 b, it can be seen that in the positive control after challenge at 5 dpi, the tracheal mucosal epithelial cells are severely swollen, hyperplastic, deformed, and necrotic and shed (red arrow), the lamina propria hemorrhages (blue arrow), and a large number of inflammatory cells infiltrate (black arrow); Through Figure 9 c, it can be seen that in the positive control after challenge at 14 dpi, the tracheal mucosa is still swollen and hyperplastic, with exfoliation and necrosis (red arrow), and inflammatory cell infiltration (black arrow); Through Figure 9 d, it can be seen that in the commercial vaccine group (CV) after challenge at 5 dpi, the tracheal mucosal epithelial cells are swollen, hyperplastic, and their shape is damaged; Through Figure 9 e, it can be seen that in the mRNA-LNP-N vaccine group (N) after challenge at 5 dpi, the tracheal mucosal epithelial cells are slightly swollen and hyperplastic; Through Figure 9 f, it can be seen that in the mRNA-LNP-N-Mosaic vaccine group after challenge at 14 dpi, the tracheal mucosal epithelial cells are moderately swollen and hyperplastic, with partial necrosis and shedding, and there are many inflammatory cells infiltrating; Through Figure 9 g, it can be seen that in the commercial vaccine group after challenge at 14 dpi, the tracheal mucosal epithelial cells gradually recover, surface cilia appear, and mucus glands appear in the mucosal layer; Through Figure 9 h, it can be seen that in the mRNA-LNP-N vaccine group after challenge at 14 dpi, the tracheal mucosal epithelial cells are columnar, the surface microvilli are clear, and it is rich in fibrous tissue; Through Figure 9 i, it can be seen that in the mRNA-LNP-N-Mosaic vaccine group after challenge at 14 dpi, some tracheal mucosal epithelial cells recover, but there are still inflammatory cell infiltrations.

[0061] It is shown that the avian coronavirus mRNA vaccine provided by this application can effectively reduce the clinical symptoms and pathological damages caused by M41 strain infection.

[0062] 6) Detection of changes in chicken peripheral blood T cell subsets by flow cytometry Collect 2 mL of blood from the jugular vein of chickens 7 days and 14 days after the first immunization and the second immunization respectively, separate chicken peripheral blood lymphocytes. The TBD chicken peripheral blood lymphocyte separation liquid kit is purchased from Guangdong Zhanchen Biotechnology Co., Ltd.

[0063] Experimental steps: ① Collect 2 mL of heparinized anticoagulant blood from the jugular vein of chickens, and add 0.5 times the volume of sample diluent (2010C1119); ② Slowly add it to a centrifuge tube containing 4 mL of separation liquid, and centrifuge at 20 °C, 600 g for 25 min; ③ After centrifugation, aspirate the middle lymphocyte layer, and add 5 mL of washing liquid and centrifuge at 400 g for 10 min; ④ Suspend the cells with 0.4 mL of washing liquid, add 2 mL of red blood cell lysate, and incubate for 2 min. The lysis time can be adjusted according to the number of red blood cells; ⑤ After lysis is completed, add 10 mL of PBS to terminate lysis, and centrifuge at 300 g for 10 min to discard the supernatant; ⑥ Add 8 mL of PBS for washing, and centrifuge at 400 g for 10 min to discard the supernatant; ⑦ Add 2 mL of RPMI medium containing 5% FBS to resuspend the cells; ⑧ Take 20 μL of cell suspension, add 20 μL of AO / PI cell dye, and mix for cell counting; ⑨ Aliquot into 1.5 mL EP tubes, with 1×10 6 cells in each tube, and resuspend with 1 mL of PBS containing 2% FBS; ⑩ Prepare a staining solution by mixing 85 μL of PBS containing 2% FBS, 5 μL of FITC Anti-Swine CD3ε Antibody, 5 μL of PE Anti-Swine CD4 Antibody, and 5 μL of APC Anti-Swine CD8a Antibody to make 100 μL; ⑪ Centrifuge the aliquoted cells at 300 g for 5 min to discard the supernatant, add 100 μL of staining solution, and incubate at 4 °C in the dark for 30 min; ⑫ After staining, centrifuge at 300 g for 5 min and discard the supernatant; ⑬ Add 1 mL of PBS containing 2% FBS to resuspend the cells, centrifuge at 300 g for 5 min and discard the supernatant, and repeat once; ⑭ Add 200 μL of PBS containing 2% FBS to resuspend the cells and perform on-machine detection.

[0064] Note: If detection is not carried out immediately, 0.5% formaldehyde solution can be added and stored at 4 °C, but the storage time generally does not exceed 24 h.

[0065] 7) ELISA antibody detection method after immunization and challenge Sample requirements: Take whole animal blood and prepare serum by conventional methods. The serum should be clear and free of hemolysis. ① Preparation of washing solution: The concentrated washing solution should be restored to room temperature before use. If there are salt crystals, shake to dissolve the crystallized salt, and then make a 10-fold dilution with distilled water or deionized water. The diluted washing solution can be stored at 4°C for about one week.

[0066] ② Sample dilution: Dilute the serum to be tested in the serum plate at a volume ratio of 1:101 (for example: add 2 μL of the serum to be tested to 200 μL of the sample diluent).

[0067] Note: Negative and positive controls do not need to be diluted. Replace the pipette tip after taking each sample and accurately record the position of each sample on the plate; each sample should be thoroughly mixed before adding it to the micro-wells of the coated plate.

[0068] ③ Bring the kit to room temperature in advance and shake well before use. ④ Take the pre-coated test plate (it can be disassembled and used in batches according to the number of samples). Add 100 μL of the diluted serum to be tested to the test plate wells. At the same time, set up 1 well each for negative, positive, and blank controls. Add 100 μL each of the negative and positive controls to the reaction wells, and only add 100 μL of the sample diluent to the blank control. Gently shake the samples in the wells (do not overflow).

[0069] ⑤ Cover with a sealing plate film and incubate at 37°C for 30 minutes.

[0070] ⑥ Carefully remove the sealing plate film, discard the solution in the plate wells, add 350 μL of the working concentration washing solution to each well, let it stand for 1 minute after filling the liquid, then discard the liquid in the wells. Repeat the above steps 3 times, and finally pat dry on clean absorbent paper. Add 100 μL of the enzyme conjugate to each well, cover with a sealing plate film, and incubate at 37°C for 30 minutes.

[0071] ⑦ Carefully remove the sealing plate film, discard the solution in the plate wells, and wash 3 times. The method is the same as step 3.

[0072] ⑧ Add 50 μL of substrate solution A to each well first, then add 50 μL of substrate solution B, mix well, cover with a sealing plate film, and develop color at 37°C in the dark for 10 minutes.

[0073] ⑨ Add 50 μL of the stop solution to each well to terminate the reaction and measure the results within 10 minutes.

[0074] ⑩ Result determination: Refer to Figure 7, zero the blank control and read the absorbance value A at 450 nm (using 630 nm as the reference wavelength) with an enzyme-linked immunosorbent assay reader. The condition for the test to be valid is that the A450 value of the positive control well is greater than or equal to 0.5, and the A450 of the negative control well must be less than 0.15. If the A450 value of the sample is greater than 0.15 + the average value of the negative control wells, it is judged as positive; if the average value of the negative control wells is less than 0.05, it is calculated as 0.05.

[0075] Compared with the commercial inactivated vaccine, the mRNA vaccine provided by the present invention can not only effectively activate cellular immunity, but also enhance the level of humoral immunity. It produced sufficient immune protection effects with only a dose of 15 μg, could generate a more persistent antibody level, and the Mosaic vaccine also activated the immune response and had cross-protection.

[0076] The method for preparing the mRNA vaccine provided by the present invention is simple to operate, requires relatively low instrumentation, can produce mRNA-LNP complexes of suitable size, and has a high encapsulation rate.

Claims

1. An avian coronavirus mRNA vaccine, characterized in that: The avian coronavirus mRNA vaccine is mRNA encapsulated in nucleotide lipid nanoparticles, and the mRNA is mRNA-N with a nucleotide sequence as shown in SEQ ID NO.1; or mRNA-N-Mosaic with a nucleotide sequence as shown in SEQ ID NO.

2.

2. The avian coronavirus mRNA vaccine according to claim 1, characterized in that The nucleotide sequence such as SEQ ID NO.1 is obtained by adding a Kozak sequence to the head of the N protein nucleotide sequence shown in SEQ ID NO.6, a T7 promoter shown in SEQ ID NO.9, and a 5'UTR sequence of β-globin shown in SEQ ID NO.10, and concatenating two nucleotide sequences such as the 3'UTR sequence of β-globin shown in SEQ ID NO.11 at the tail.

3. The avian coronavirus mRNA vaccine according to claim 1, characterized in that The nucleotide sequence such as SEQ ID NO.2 is obtained by adding a Kozak sequence to the head of the N-mosaic protein nucleotide sequence shown in SEQ ID NO.3, a T7 promoter shown in SEQ ID NO.9, and a 5'UTR sequence of β-globin shown in SEQ ID NO.10, and concatenating two nucleotide sequences such as the 3'UTR sequence of β-globin shown in SEQ ID NO.11 at the tail.

4. The avian coronavirus mRNA vaccine according to claim 2, characterized in that The sequence shown in SEQ ID NO.6 is a sequence obtained after the nucleotide sequence shown in SEQ ID NO.4 is optimized.

5. The avian coronavirus mRNA vaccine according to claim 3, characterized in that The sequence shown in SEQ ID NO.3 is a sequence obtained after the nucleotide sequence shown in SEQ ID NO.5 is optimized.

6. An mRNA plasmid, characterized in that A plasmid having a nucleotide sequence as shown in SEQ ID NO.12 synthesized by connecting the nucleotide sequence shown in SEQ ID NO.1 of claim 1 to the PUC57 vector; or a plasmid having a nucleotide sequence as shown in SEQ ID NO.13 synthesized by connecting the nucleotide sequence shown in SEQ ID NO.2 to the PUC57 vector.

7. The method for preparing the avian coronavirus mRNA vaccine according to claim 1, characterized in that: The steps are as follows: 1) constructing a plasmid having a nucleotide sequence as shown in SEQ ID NO.12 or a plasmid having a nucleotide sequence as shown in SEQ ID NO.13; 2) Amplify the plasmid constructed in step 1) to obtain a plasmid template, 3) performing PCR amplification on the plasmid template obtained in step 2); 4) performing in vitro transcription, capping, and tailing of the PCR amplification product obtained in step 3) to synthesize mRNA; 5) Synthesize avian coronavirus mRNA vaccine by combining in vitro transcribed mRNA molecules with cationic liposome nanomaterials.

8. Use of the avian coronavirus mRNA vaccine according to claim 1 as a preparation for preventing diseases caused by avian coronavirus.

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