QX type avian infectious bronchitis virus mRNA vaccine and application thereof

Abstract

The invention provides a QX type avian infectious bronchitis virus mRNA vaccine and application thereof. The gene sequence of the mRNA vaccine molecule is shown as SEQ ID NO.9 or SEQ ID NO.13. The invention also provides a preparation method of the QX type avian infectious bronchitis virus mRNA vaccine. The preparation method of the QX-type avian infectious bronchitis virus mRNA vaccine molecule provided by the invention is simple to operate, preparation can be performed only by knowing a gene sequence, and compared with an inactivated vaccine, the production price is low, and the dosage is small; compared with attenuated live vaccines, the vaccine is short in production period and safe.

Description

Technical Field

[0001] This invention belongs to the field of biological products and vaccinology, specifically relating to a QX type avian infectious bronchitis virus mRNA vaccine and its application. Background Technology

[0002] mRNA vaccines mainly consist of five structures: 5′UTR, 3′UTR, CDS, 5′cap, and Poly(A) tail. Among these, the UTR, 5′cap, and poly(A) tail play crucial roles in mRNA stability and translation. Currently, cap1 (m7GpppNm) is the most widely used cap analog, with significantly higher protein translation yield than cap0. For UTR selection, the UTR of a highly expressed gene from the same species is typically chosen as a candidate UTR. The CDS, encoding the antigen protein, has the highest length among all functional elements. Its design must balance translational capability and in vivo stability, and optimization usually favors sequences with high CAI and low MFE. The Poly(A) tail is a long, repetitive polyadenylated (A) nucleotide that protects the cap structure from degradation and initiates protein translation. Spacing the Poly(A) tail during selection can significantly reduce recombination events during plasmid DNA amplification. The length and distribution of the A tail are also important.

[0003] Since the outbreak of COVID-19, mRNA vaccines have entered a period of rapid development, making a significant contribution to the global fight against the pandemic. Compared to traditional vaccines, mRNA vaccines have significant advantages in terms of development cycle and manufacturing process. They only require obtaining the virus's gene sequence to design and produce the corresponding vaccine in a short time, greatly shortening the vaccine preparation and production cycle. Furthermore, mRNA vaccines can quickly adapt to viral mutations by adjusting the gene sequence encoding the antigen, ensuring the vaccine's sustained protective effect. Compared to traditional vaccines, mRNA vaccines are faster to develop, have higher safety profiles, and do not replicate or integrate into the host genome, significantly reducing the risk of infection.

[0004] mRNA vaccines are a rising star in vaccinology with broad application prospects. In recent years, mRNA vaccines have been used in numerous medical fields, including infectious diseases, cancer, genetic diseases, and regenerative medicine. Currently, mRNA vaccines for various pathogens have entered clinical trials, including those for COVID-19, influenza virus, Zika virus, rabies virus, and human immunodeficiency virus. Research on mRNA vaccines in livestock and poultry is also progressing rapidly. For example, in 2020, a research team developed a candidate mRNA vaccine based on PRV gD. Immunized mice showed extremely high titers of specific IgG and high levels of neutralizing antibodies within 8 weeks, especially two weeks after the second injection, with a significant increase in antibody levels. Experimental results showed that 95% of immunized mice did not show obvious clinical symptoms after exposure to PRV, demonstrating the good protective effect of this candidate vaccine. For PEDV, scientists used Venezuelan equine encephalitis virus (VEEV) as a vector, cleverly allowing mRNA to express the PEDV spike protein (S protein) in pigs, stimulating sows to produce neutralizing antibodies and passing this protection on to their piglets. Experimental results showed that this candidate vaccine not only effectively reduced viral shedding in sows but also significantly reduced mortality in suckling piglets. In studies targeting H7N9 and H9N2 avian influenza, scientists used chitosan nanoparticles to encapsulate the HA2-HA1 and M2e antigens. Experimental results showed that chickens vaccinated with two doses of mRNA exhibited lower viral loads and stronger immune responses after viral exposure, fully demonstrating the effectiveness of this candidate vaccine.

[0005] The emergence of veterinary mRNA vaccines has brought new opportunities and challenges to the animal health industry. With the continuous maturation of the technology and the gradual promotion of its application, mRNA vaccines will play an increasingly important role in preventing and controlling animal infectious diseases, ensuring the healthy development of animal husbandry, and maintaining public health security. Avian infectious bronchitis virus (IBV) is a Class III animal disease as defined by the Ministry of Agriculture and Rural Affairs of my country, with a persistently high detection rate. Among them, the QX type IBV is the most widespread genotype in my country, causing various harms such as kidney enlargement, tracheal hemorrhage, and decreased egg production, posing a serious threat to the development of my country's poultry industry. However, there is currently a lack of vaccines targeting the QX type IBV on the market. The IBV S protein mediates the binding of the virus to host-specific receptors, thus serving as an important protective antigen. The N protein is the main structural protein of the virus, possessing good immunogenicity and a large number of antigenic epitopes, and is therefore often used as a target protein in diagnostic reagents and vaccine development. Based on the above conditions… This study designed a mRNA vaccine for the prevention and / or treatment of QX-type avian infectious bronchitis by optimizing the sequence of the prevalent QX-type avian infectious bronchitis virus in China, aiming to provide technical support for the prevention and control of QX-type avian infectious bronchitis virus. Summary of the Invention

[0006] This invention provides a QX-type avian infectious bronchitis virus mRNA vaccine and its application, thereby overcoming the shortcomings of the prior art.

[0007] The present invention first provides an mRNA fragment that can be used to prepare a QX type avian infectious bronchitis virus mRNA vaccine, wherein the sequence of the mRNA fragment is SEQ ID NO:9 or SEQ ID NO:13; The present invention also provides one use of the mRNA fragment, namely, its application in the preparation of mRNA vaccines.

[0008] The present invention also provides a recombinant plasmid, wherein the recombinant plasmid is an expression vector in which the above-mentioned mRNA fragment is inserted; As a specific example, the expression vector has the sequence SEQ ID NO: 10; The recombinant plasmid described herein, as a specific example, has the sequence SEQ ID NO: 11 or SEQ ID NO: 14; The present invention also provides a QX type avian infectious bronchitis virus mRNA vaccine, which is prepared by in vitro co-transcription and capping of the above-mentioned recombinant plasmid; Furthermore, the mRNA vaccine is encapsulated using LNP.

[0009] Compared with the prior art, the technical solution provided by the present invention has the following technical advantages: 1. The present invention provides a gene sequence of an mRNA vaccine molecule for the prevention and / or treatment of QX type avian infectious bronchitis virus, which can induce the body to produce high levels of antibodies and provide protection.

[0010] 2. The method for preparing QX type avian infectious bronchitis virus mRNA vaccine provided by the present invention is simple to operate, requiring only knowledge of the gene sequence. Compared with inactivated vaccines, it is cheaper to produce and requires less dosage; compared with live attenuated vaccines, it has a shorter production cycle and is safer. Attached Figure Description

[0011] Figure 1 Plasmid map of mRNA vaccine IBV-mRNA-S; Figure 2 : Plasmid diagram of mRNA vaccine IBV-mRNA-SN; Figure 3 Figure 1: Results of IBV-mRNA-S plasmid amplification and linearization; Figure 4Figure 1: Results of IBV-mRNA-SN plasmid amplification and linearization; Figure 5 Mass spectrometry of IBV-mRNA-S capping rate detection; Figure 6 Mass spectrometry of IBV-mRNA-SN capping rate detection; Figure 7 Mass spectrometry distribution map of IBV-mRNA-S tailing rate detection; Figure 8 Mass spectrometry distribution map of IBV-mRNA-SN tailing rate detection; Figure 9 : Immunoblotting results of IBV-mRNA-S protein; Figure 10 : Immunoblotting results of IBV-mRNA-SN protein; Figure 11 Diameter detection image of LNP encapsulation example of IBV-mRNA-S; Figure 12 Diameter detection image of LNP encapsulation example of IBV-mRNA-SN; Figure 13 Survival curve; Figure 14 : Observation of lesions after necropsy following viral infection; Figure 15 Tracheal ciliary score chart; Figure 16 Antibody level graph; Figure 17 HE staining image of tissue sections. Detailed Implementation

[0012] This invention presents an mRNA vaccine for the prevention or treatment of QX-type infectious bronchitis by optimizing the sequence of the prevalent QX-type avian infectious bronchitis virus in China, aiming to provide technical support for the prevention and control of QX-type avian infectious bronchitis virus.

[0013] The present invention will now be described in detail with reference to the embodiments and accompanying drawings.

[0014] Example 1: Preparation of IBV-mRNA-S and IBV-mRNA-SN vaccines Step (I) Preparation of IBV-mRNA-S plasmid 1. Through epidemiological surveillance of IBV in 2024, the laboratory obtained a large number of QX-type avian infectious bronchitis virus sequences. After sequence alignment, most sequences could be aligned to avian infectious bronchitis virus with GenBank accession number MT460496.1. The S protein nucleotide sequence of MT460496.1 was downloaded from NCBI. The S protein nucleotide sequence of MT460496.1 was optimized using the codon optimization tool provided by GenScript (https: / / www.genscript.com.cn / gensmart-free-gene-codon-optimization.html) with chickens as the host. The nucleotide sequence shown in SEQ ID NO:4 is the optimized sequence of SEQ ID NO:1.

[0015] 2. The optimized S protein nucleotide sequence (nucleotide sequence as shown in SEQ ID NO: 4) is followed by the addition of a T7 promoter (nucleotide sequence as shown in SEQ ID NO: 2) and the UTR sequence of the chicken high-expression gene HBE1 (nucleotide sequence as shown in SEQ ID NO: 3) to the head. Four nucleotide sequences are then tandemly appended to the tail: a stop codon (nucleotide sequence as shown in SEQ ID NO: 5) to effectively terminate the translation of the S protein; a 3' UTR of the chicken high-expression gene HBE1 (nucleotide sequence as shown in SEQ ID NO: 6); a 110nt polyadenylate tail (nucleotide sequence as shown in SEQ ID NO: 7) to initiate the protein translation process; and a BsQI IIS type restriction endonuclease sequence (nucleotide sequence as shown in SEQ ID NO: 8) to ensure the integrity of the Poly(A) tail. This yields the nucleotide sequence shown in SEQ ID NO: 9.

[0016] 3. The nucleotide sequence shown in SEQ ID NO: 9 was ligated into the vector shown in SEQ ID NO: 10 to synthesize the complete plasmid as shown in SEQ ID NO: 11. Figure 1 ).

[0017] 4. The synthesized plasmid was transformed into *E. coli*, and plasmid was extracted after fermentation in Kanamycin-resistant LB medium at 37°C and 220 rpm for 16 hours. (Plasmid extraction was performed using the TIAN GEN endotoxin-free plasmid kit DP210831). The specific steps for plasmid extraction are as follows: A. Column equilibration procedure: Add 2.5 ml of equilibration solution BL to the adsorption column CP6 (place the adsorption column in a 50 ml collection tube), centrifuge at 8,000 rpm for 2 min, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0018] B. Take 100 ml of the overnight culture and aliquot it into three 50 ml centrifuge tubes. Centrifuge at 8,000 rpm for 3 min at room temperature and collect the combined bacteria into one centrifuge tube.

[0019] C. Try to remove as much supernatant as possible. To ensure that all the supernatant is removed, please use clean absorbent paper to wipe away any water droplets on the bottle.

[0020] D. Add 8 ml of solution P1 to the centrifuge tube containing the bacterial cell pellet, and thoroughly suspend the bacterial cell pellet using a pipette or vortex mixer.

[0021] E. Add Bml of solution P2 to the centrifuge tube, and immediately and gently invert it 6-8 times to fully lyse the bacteria. Let it stand at room temperature for 5 minutes.

[0022] F. Add 8 ml of solution P4 to the centrifuge tube, immediately and gently invert it 6-8 times to mix thoroughly until a white, dispersed flocculent precipitate appears. Then, let it stand at room temperature for about 10 minutes, centrifuge at 8,000 rpm (~8, 228Xg) for 5-10 minutes to allow the white precipitate to settle to the bottom of the tube. Carefully pour all the solution into filter CS1, slowly push the push handle to filter, and collect the filtrate in a clean 50 ml tube.

[0023] G. Add 0.3 times the volume of isopropanol to the filtrate, mix by inverting the container, and then transfer it to the adsorption column CPG.

[0024] Centrifuge at 8,000 rpm (~8,228 Xq) for 2 min at room temperature, discard the waste liquid in the collection tube, and put the adsorption column CP6 back into the collection tube.

[0025] H. Add 10 ml of wash buffer PW to the adsorption column CP6, centrifuge at 8,000 rpm for 2 min, discard the wash buffer in the collection tube, and put the adsorption column back into the collection tube.

[0026] I. Repeat step H.

[0027] J. Add 3 ml of anhydrous ethanol to the adsorption column CP6, centrifuge at 8,000 rpm for 2 min at room temperature, and discard the waste liquid.

[0028] K. Place the adsorption column CP6 back into the collection tube and centrifuge at 8,000 rpm for 5 min to remove any residual wash solution from the adsorption column.

[0029] L. Place the adsorption column CP6 in a clean 50 ml collection tube, add 1-2 ml of elution buffer TB dropwise to the middle of the adsorption membrane, incubate at room temperature for 5 min, and then centrifuge at 8,000 rpm for 2 min at room temperature. Transfer all the elution buffer from the 50 ml centrifuge tube to a clean 1.5 ml centrifuge tube and store at -20°C.

[0030] 5. The extracted plasmid was linearized by BspQⅠ restriction endonuclease digestion (50℃, 0.5 h), and electrophoresis was performed on a 0.8% TAE agarose gel. The results showed that the plasmid digestion resulted in a single band. Figure 3 ) Step (II) Preparation of IBV-mRNA-SN plasmid 1. The S and N protein sequences of avian infectious bronchitis virus (GenBank ID MT460496.1) were optimized using a codon optimization tool with chickens as the host. After antigen epitope screening and verification, epitopes with a score of 0.90 or higher were selected and linked with a linker peptide. The nucleotide sequence shown in SEQ ID NO: 12 is the linked sequence.

[0031] 2. The selected SN epitope nucleotide sequence (nucleotide sequence as shown in SEQ ID NO: 12) is followed by the addition of a T7 promoter (nucleotide sequence as shown in SEQ ID NO: 2), the UTR sequence of the chicken high-expression gene HBE1 (nucleotide sequence as shown in SEQ ID NO: 3), and a start codon (nucleotide sequence as shown in SEQ ID NO: 16). Four nucleotide sequences are tandemly appended to the tail, including a stop codon (nucleotide sequence as shown in SEQ ID NO: 5), effectively terminating the translation of the S protein. The 3'UTR of the chicken high-expression gene HBE1 (nucleotide sequence as shown in SEQ ID NO: 6), the 110nt polyadenylate tail (nucleotide sequence as shown in SEQ ID NO: 7), and the BsQI IIS type restriction endonuclease sequence (nucleotide sequence as shown in SEQ ID NO: 8) ensure the integrity of the Poly(A) tail. This yields the nucleotide sequence shown in SEQ ID NO: 13.

[0032] 3. The nucleotide sequence shown in SEQ ID NO: 13 was ligated into the vector shown in SEQ ID NO: 10 to synthesize a complete plasmid as shown in SEQ ID NO: 14. Figure 2 ).

[0033] 4. The synthesized plasmid was transformed into *E. coli*, and plasmid was extracted after fermentation in Kanamycin-resistant LB medium at 37°C and 220 rpm for 16 hours. (Plasmid extraction was performed using the TIAN GEN endotoxin-free plasmid kit DP210831). The specific steps for plasmid extraction are as follows: A. Column equilibration procedure: Add 2.5 ml of equilibration solution BL to the adsorption column CP6 (place the adsorption column in a 50 ml collection tube), centrifuge at 8,000 rpm for 2 min, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0034] B. Take 100 ml of the overnight culture and aliquot it into three 50 ml centrifuge tubes. Centrifuge at 8,000 rpm for 3 min at room temperature and collect the combined bacteria into one centrifuge tube.

[0035] C. Remove as much supernatant as possible. To ensure that all the supernatant is removed, use clean absorbent paper to wipe away any water droplets on the bottle.

[0036] D. Add 8 ml of solution P1 to the centrifuge tube containing the bacterial cell pellet, and thoroughly suspend the bacterial cell pellet using a pipette or vortex mixer.

[0037] E. Add Bml of solution P2 to the centrifuge tube, and immediately and gently invert it 6-8 times to fully lyse the bacteria. Let it stand at room temperature for 5 minutes.

[0038] F. Add 8 ml of solution P4 to the centrifuge tube, immediately and gently invert it 6-8 times to mix thoroughly until a white, dispersed flocculent precipitate appears. Then, let it stand at room temperature for about 10 minutes, centrifuge at 8,000 rpm (~8, 228Xg) for 5-10 minutes to allow the white precipitate to settle to the bottom of the tube. Carefully pour all the solution into filter CS1, slowly push the push handle to filter, and collect the filtrate in a clean 50 ml tube.

[0039] G. Add 0.3 times the volume of isopropanol to the filtrate, mix by inverting the container, and then transfer it to the adsorption column CPG.

[0040] Centrifuge at 8,000 rpm (~8,228 Xq) for 2 min at room temperature, discard the waste liquid in the collection tube, and put the adsorption column CP6 back into the collection tube.

[0041] H. Add 10 ml of wash buffer PW to the adsorption column CP6, centrifuge at 8,000 rpm for 2 min, discard the wash buffer in the collection tube, and put the adsorption column back into the collection tube.

[0042] I. Repeat step H.

[0043] J. Add 3 ml of anhydrous ethanol to the adsorption column CP6, centrifuge at 8,000 rpm for 2 min at room temperature, and discard the waste liquid.

[0044] K. Place the adsorption column CP6 back into the collection tube and centrifuge at 8,000 rpm for 5 min to remove any residual wash solution from the adsorption column.

[0045] L. Place the adsorption column CP6 in a clean 50 ml collection tube, add 1-2 ml of elution buffer TB dropwise to the middle of the adsorption membrane, incubate at room temperature for 5 min, and then centrifuge at 8,000 rpm for 2 min at room temperature. Transfer all the elution buffer from the 50 ml centrifuge tube to a clean 1.5 ml centrifuge tube and store at -20°C.

[0046] 5. The extracted plasmid was linearized by BspQⅠ restriction endonuclease digestion (50℃, 0.5 h), and electrophoresis was performed on a 0.8% TAE agarose gel. The results showed that the plasmid digestion resulted in a single band. Figure 4 ) Step (III) Co-transcription capping and capping / tailing detection 1. Co-transcription capping To improve the stability of the mRNA vaccine and prevent its degradation, a Cap1 (3'OMe AG) cap analog was added to the 5'UTR region of the enzyme-digested plasmid using a co-transcription capping method. (The co-transcription T7 in vitro transcription reagent HBP001509 from HanHai New Enzyme Company was used). The specific steps are as follows: A. After shaking and mixing each reagent thoroughly, place it on ice for later use. The reaction system and the order of sample addition are shown in Table 1 below.

[0047] Table 1: Reaction System and Order of Addition

[0048] B. Mix the reagents by pipetting or shaking, briefly centrifuge for 3-5 seconds, and incubate at 37°C for 2 hours. 2. Capping rate detection A. Based on the design principles of mRNA vaccine probes, a 14-30 bp probe was designed at the 5' end of the mRNA using the online website of nearshore proteins (https: / / www.novoprotein.com.cn / tool-class). The probe contained 4-6 DNA bases near the 5' end or in the middle of the probe, with the remaining positions consisting of 2'-O-methyl modified RNA bases. The 3' end carried a Biotin TEG tag, and the GC% was between 40% and 70%. The designed probe sequence was AACGATGAGUAGGAAGUCCU.

[0049] B. Take out 4xRNase H Mix and the probe and thaw them on ice. In a 200 μL nuclease-free PCR tube, prepare the system in the following order at room temperature, and react at 50°C for 15 min to complete the incubation of mRNA and probe and the enzyme digestion reaction.

[0050] Table 2: Probe incubation and enzyme digestion reaction system Components volume Nuclease-free water Add to 100μL 4xRNase H Mix 25μL mRNA sample to be tested X μL (100 pmol) 10μM probe 20μL C. The enzymatically digested samples were purified using magnetic beads. The molecular weight of the purified samples was determined by LC-MS. The IBV-mRNA-S capping rate was measured (see [reference needed]). Figure 5 IBV-mRNA-SN capping rate detection can be found in [link to relevant documentation]. Figure 6 The results showed that the capping rate of IBV-mRNA-S was 94.50% and the capping rate of IBV-mRNA-S was 95.60%.

[0051] 3. Tail length detection RNase T1 enzyme was added to the mRNA vaccine stock solution for enzymatic digestion. The digested sample was then purified using a packing material. The molecular weight of the purified sample was determined by LC-MS. The IBV-mRNA-S tailing rate was measured as follows: Figure 7 IBV-mRNA-SN tailing rate detection is shown in [link to relevant documentation]. Figure 8 .

[0052] Step (III) Cell Expression Validation 1. Cell transfection A. 293T cell resuscitation: The 293T cells preserved in the laboratory were resuscitated and passaged. The initial resuscitation was carried out in DMEM medium containing 20% ​​fetal bovine serum (FBS) to improve the cell growth capacity. After the cells were stabilized, they were passaged. Stable passage was carried out in DMEM medium containing 10% FBS.

[0053] B. Cell seeding: Once the cells are in good condition, take 2 × 10⁶ cells... 5 pcs·mL -1 Cells were seeded in 24-well plates at a density of 500 μL per well. The plates were then incubated in a 37°C incubator with 5% CO2. Cells were ready for transfection experiments when they reached 80% confluence.

[0054] C. Mix the mRNA and transfection reagent, gently pipette to mix thoroughly, and let stand at room temperature for 10 minutes. D. Add the complex to a 24-well plate, gently mix by pipetting, and incubate for 48 hours before assessing transfection efficiency. 2. Western blot validation of protein expression A. 48 h after transfection, discard the culture medium, wash once with PBS, add RIPA lysis buffer (Shanghai Sangon Biotech C500005) to each well, and place on ice for 5-10 min. To promote cell lysis, gently pipette the cells for 10 seconds each time, 2-3 times, with 5-second intervals between each time. Gently tilt the culture dish to allow the lysis products to flow to one side or corner of the dish, and then transfer it to a 1.5 mL centrifuge tube. Place on ice for 5 min, shaking vigorously during the process, and centrifuge at 12000 rpm, 4℃ for 5 min. Collect the supernatant for Western blotting.

[0055] B. A 10% SDS-PAGE protein gel was prepared for protein electrophoresis. The gel was transferred using a Bio-Rad wet transfer apparatus and blocked with 5% skim milk solution on a shaker at room temperature for 1 h. IBV-mRNA-S was incubated with IBV Spike antibody (a laboratory-prepared polyclonal antibody, preserved by the Poultry Disease Monitoring Laboratory of the China Animal Health and Epidemiology Center, diluted 1:5000), an internal control antibody (β-actin antibody), and HRP-labeled goat anti-chicken IgG. The protein band corresponding to IBV-mRNA-S translation was between 95-140 kDa, consistent with the theoretical protein molecular weight. However, due to the presence of multiple glycosylation sites in IBV Spike, the actual molecular weight is likely larger. A band above 190 kDa was observed, possibly indicating a glycosylated protein (…). Figure 9 IBV-mRNA-SN was incubated with rabbit antibodies against the S and N protein tandem epitopes synthesized by Qingke and HRP-labeled goat anti-rabbit IgG. The protein band corresponding to the translation of IBV-mRNA-SN was approximately 20 kDa, and the protein band corresponding to the translation of the S and N protein tandem epitopes was approximately 19 kDa, which is consistent with the theoretical protein molecular weight. Figure 10 ).

[0056] Step (IV) LNP Encapsulation and Encapsulation Ratio Detection 1. The SM102 LNP formulation comprises four lipid components: cationic lipids (SM102), neutral phospholipids (DSPC), cholesterol, and PEG2000-DMG. The amount of each lipid component was calculated based on the molar ratio to prepare the organic phase. The molar ratio of cationic lipids (SM102):neutral phospholipids (DSPC):cholesterol:PEG2000-DMG is 50:10:38.5:1.5.

[0057] 2. Based on the formula for calculating the nitrogen-phosphorus ratio, the commonly used N / P ratios are 4, 6, or 8, depending on the amount of mRNA and LNP used.

[0058] 3. Lipids and nucleic acids are dissolved in organic phases (ethanol, isopropanol) and aqueous phases (acetate, citrate), respectively. The two phase solutions are injected into the two inlet channels of the preparation system. The two phases are completely mixed within one millisecond, which causes a change in solvent polarity and triggers the self-assembly of nucleic acid-rich LNPs.

[0059] 4. Sample concentration and encapsulation efficiency were determined using a fluorescent dye method, and particle size and PDI were determined using a particle size analyzer. Formulation concentration, encapsulation efficiency, particle size, and PDI are shown in Table 3 below. Particle size and PDI chromatograms are shown in [reference needed]. Figure 11 and Figure 12 .

[0060] Table 3: Formulation Concentration, Encapsulation Efficiency, Particle Size, and PDI Sample Name mRNA concentration in LNP formulations Encapsulation rate Particle size PDI IBV-mRNA-S 329.80 μg / mL 97.28% 85.50nm 0.067 IBV-mRNA-SN 315.99 μg / mL 96.89% 95.37nm 0.166 Example 2: Vaccine Challenge Protection Experiment 1. Animal Immunization and Challenge Protocol: Sixty 2-day-old SPF chickens were randomly divided into four groups: IBV-mRNA-S vaccine group (n=15), IBV-mRNA-SN vaccine group (n=15), LaSota+QXL87 bivalent vaccine group (n=15), and PBS group (n=15). The IBV-mRNA-S candidate vaccine group was immunized via intramuscular injection (10 μg / chicken); the LaSota+QXL87 bivalent vaccine group was immunized via nasal or ocular drops according to the product instructions. The initial and booster immunizations were performed at 2 days and 15 days of age, respectively. Two weeks after the second immunization, a challenge experiment was conducted: the virulent IBV strain Y1077 was challenged via nasal or ocular drops at a dose of 0.1 mL. 5 ELD50 / 0.1mL.

[0061] 2. Mortality observation: In the Y1077 challenge group, one chicken died on day 5 and day 6 after challenge; in the IBV-mRNA-S vaccine group, one chicken died on day 4 after challenge; no deaths were observed in the IBV-mRNA-SN vaccine group and the LaSota+QXL87 bivalent vaccine group. Autopsy of the deceased chickens revealed enlarged kidneys and urate deposits in the renal tubules. Figure 13 , Figure 14 ).

[0062] 3. Symptom observation: The onset criteria are determined by meeting any two of the following: a. Lethargy, ruffled feathers, and decreased appetite; b. Obvious respiratory symptoms, such as cough, rales, abnormal tracheal secretions or bleeding; c. Obvious gastrointestinal symptoms, such as diarrhea, dehydration, and pathological changes such as kidney enlargement or "mottled kidney"; d. Specific death. After 15 days of post-challenge rearing, the mental state and mortality of the chicks were observed and recorded. The results showed that the Y1077 challenge group exhibited depression, tracheal rales, and decreased feed intake 3-11 days after challenge, with a morbidity rate as high as 90%. The IBV-mRNA-S vaccine group exhibited depression and tracheal rales 3-7 days after challenge, with a morbidity rate of 50%. The LaSota+QXL87 bivalent vaccine group showed depression 2-6 days after challenge, with some chicks exhibiting rales, and a morbidity rate of 30%. The IBV-mRNA-SN vaccine group did not show obvious signs of lethargy and had a protection rate as high as 90%.

[0063] Table 4: Statistics on the Incidence Rate of Vaccines Grouping Number of cases Incidence rate Protection rate Virus attack team 9 / 10 90% 10% IBV-mRNA-S 5 / 10 50% 50% LaSota+QXL87 3 / 10 30% 70% IBV-mRNA-SN 1 / 10 10% 90% 4. Tracheal Ciliary Inhibition Experiment: 15 days after challenge, all chickens in each group were culled, and necropsies were performed. The trachea, trachea, and kidney tissues were examined, and tracheal rings were collected for the tracheal ring experiment: The entire trachea segment was removed, washed, and placed in 37°C physiological saline. Using sterile scissors, the trachea was cut into small segments: three segments from the upper part, four segments from the middle part, and three segments from the lower part. Ciliary activity was observed under a microscope. The scoring criteria were: 0 points: no damage, good ciliary activity; 1 point: 67%-100% ciliary activity; 2 points: 33%-67% ciliary activity; 3 points: 0-33% ciliary activity; 4 points: all ciliary movements ceased.

[0064] The results showed that the tracheal ciliary integrity of the immunized chickens reached 67%-100%; the tracheal ciliary movement of the challenged chickens was severely inhibited, the movement stopped, the cilia fell off, and the integrity was less than 33%. Figure 15 ).

[0065] 5. Antibody level monitoring: Blood was collected from chickens in each group weekly after the first immunization. After standing at room temperature for 2 hours, the blood was centrifuged at 2000×g for 10 minutes, and the supernatant serum was collected. The antibody levels of IBV in different immunization groups were detected using the ID. Vet infectivity in bronchitis virus indirect ELISA antibody detection kit 2.0.

[0066] The specific steps are as follows: A. On the dilution plate, dilute the sample to be tested 500 times with diluent No. 14 and mix well; B. Add 100 μl of negative control to wells A1 and B1 of the ELISA plate, 100 μl of positive control to wells C1 and D1, and 100 μl of diluted test sample to the remaining wells. C. Incubate at 21°C (±5°C) for 30±3 minutes; D. Dilute the concentrated enzyme conjugate (10X) 10 times with diluent No. 3 to obtain enzyme conjugate (1X); E. Discard the liquid in the wells. Add 300 μl of washing buffer (1X) and wash the plate 3 times; F. Add 100 μL of enzyme conjugate (1X) to each well; G. Incubate at 21℃ (±5℃) for 30±3 minutes; H. Discard the liquid in the wells. Add 300 μl of washing buffer (1X) and wash the plate 3 times; I. Add 100 μL of substrate solution to each well; J. Incubate at 21℃ (±5℃) in the dark for 15±2 minutes; K. Add 100 μl of stop solution to each well to terminate the reaction; L. Absorbance values ​​were read and recorded at a wavelength of 450 nm.

[0067] The results showed that IBV antibodies could be detected 7 days after immunization, regardless of whether it was an mRNA vaccine or a commercial vaccine. The LaSota+QXL87 bivalent vaccine group had significantly higher antibody levels in the early stages of immunization than the IBV-mRNA-SN vaccine group at days 7, 14, 21, and 28, and significantly higher antibody levels at days 21 and 28 than the IBV-mRNA-S vaccine group. This indicates that IBV-mRNA-SN can produce high levels of antibodies in vivo and has a long duration of action. Figure 16 ).

[0068] 6. Monitoring of in vitro virus shedding time: Oropharyngeal and cloacal swabs were collected from chickens in each group on days 3, 6, 9, 12, and 15 post-challenge using sterile cotton swabs to detect virus shedding. Results showed that from day 3 to day 15 post-challenge, all 10 chickens in the control group continuously shed the virus from their oropharynx and cloaca. In the IBV-mRNA-S vaccine group and the LaSota+QXL87 bivalent vaccine group, not all 10 chickens shed the virus. On day 9 post-challenge, virus shedding from the oropharynx and cloaca ceased in the IBV-mRNA-S vaccine group, while two chickens in the LaSota+QXL87 bivalent vaccine group continued to shed the virus through their oropharynx until day 12 when shedding ceased. The positive rate of oropharyngeal and cloacal swabs in the IBV-mRNA-S vaccine group was also lower than that in the LaSota+QXL87 bivalent vaccine group.

[0069] Table 5: Virus Detection Table for Oropharyngeal and Cloaca Swabs

[0070] 7. Monitoring of virus distribution in vivo: On days 3, 6, 9, 12 and 15 after challenge, one chicken from each group was dissected and lung, trachea and kidney tissues were collected to detect the replication of the virus in vivo. The results showed that from the third day to the fifteenth day after challenge, viral replication continued in the organs of the control group. On the third day after challenge, the virus was detectable in the kidneys, lungs, and trachea of ​​the IBV-mRNA-S vaccine group, the LaSota+QXL87 bivalent vaccine group, and the IBV-mRNA-SN vaccine group. On the sixth day after challenge, the IBV-mRNA-SN vaccine group successfully stopped viral replication in the kidneys, while the IBV-mRNA-S vaccine group and the LaSota+QXL87 bivalent vaccine group were still shedding the virus. On the ninth day after challenge, the virus was only detectable in the trachea of ​​the IBV-mRNA-SN vaccine group, the kidneys of the IBV-mRNA-S vaccine group stopped shedding the virus, and the lungs of the LaSota+QXL87 bivalent vaccine group stopped shedding the virus. On the fifteenth day after challenge, the virus was not detectable in any of the three organs of the IBV-mRNA-SN vaccine group and the LaSota+QXL87 bivalent vaccine group, while viral shedding was still detectable in the trachea of ​​the IBV-mRNA-S vaccine group.

[0071] Table 6: Organ Detoxification Test Data Table Grouping Days kidney lung trachea Virus attack team 3 + + + IBV-mRNA-S 3 + + + LaSota+QXL87 3 + + + IBV-mRNA-SN 3 + + + Virus attack team 6 + + + IBV-mRNA-S 6 + + + LaSota+QXL87 6 + + + IBV-mRNA-SN 6 - + + Virus attack team 9 + + + IBV-mRNA-S 9 - + + LaSota+QXL87 9 + - + IBV-mRNA-SN 9 - - + Virus attack team 12 + + + IBV-mRNA-S 12 - - + LaSota+QXL87 12 + - + IBV-mRNA-SN 12 - - - Virus attack team 15 + + + IBV-mRNA-S 15 - - + LaSota+QXL87 15 - - - IBV-mRNA-SN 15 - - - 8. Histopathological observation: 15 days after challenge, the trachea and kidney tissues of chickens in each challenge group were dissected, fixed in 10% neutral formalin solution, and prepared as paraffin sections for HE staining to observe histopathological changes. Results showed that the control group exhibited mild overall structural abnormalities in the trachea, with abundant inflammatory cell infiltration in the mucosa; the kidney tissue showed mild structural abnormalities, with abundant inflammatory cell infiltration within the tissue. The IBV-mRNA-S vaccine group showed intact kidney tissue structure, mild overall structural abnormalities in the trachea, and the mucosa was covered with stratified ciliated columnar epithelium with goblet cells. In contrast, the IBV-mRNA-SN vaccine group showed intact tracheal and kidney tissue structures. Figure 17 ).

Claims

1. An mRNA fragment, characterized in that, The sequence of the mRNA fragment is SEQ ID NO:

9.

2. The mRNA fragment as described in claim 1, characterized in that, The sequence of the mRNA fragment is SEQ ID NO:

13.

3. The use of the mRNA fragment according to claim 1 or 2 in the preparation of mRNA vaccines.

4. A recombinant plasmid, characterized in that, The recombinant plasmid is an expression vector into which the mRNA fragment of claim 1 or 2 is inserted.

5. The recombinant plasmid as described in claim 4, characterized in that, The sequence of the expression vector is SEQ ID NO:

10.

6. The recombinant plasmid as described in claim 5, characterized in that, The recombinant plasmid has the sequence SEQ ID NO:

11.

7. The recombinant plasmid as described in claim 5, characterized in that, The recombinant plasmid has the sequence SEQ ID NO:

14.

8. A QX-type avian infectious bronchitis virus mRNA vaccine, characterized in that, The mRNA vaccine described herein uses an antigen prepared by in vitro co-transcription and capping of the recombinant plasmid as described in claim 5.

9. The mRNA vaccine of claim 5 as described in claim 8, characterized in that, The mRNA vaccine described herein is encapsulated using LNP.

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