Preparation method and application of bacteriophage capsid protein MS2 armored PRRSV antigen mRNA particles

Abstract

This invention relates to genetic engineering technology and aims to provide a method for preparing and applying bacteriophage capsid protein MS2-armored PRRSV antigen mRNA particles. The method includes: inserting the MS2 bacteriophage capsid protein gene and an mRNA expression cassette into a double multiple cloning site expression vector; transforming the recombinant expression plasmid into host bacteria; inducing expression to allow the MS2 capsid protein to express and self-assemble into virus-like particles; utilizing the specific recognition of the MS2 capsid protein on the packaging stem-loop structure, packaging the transcribed mRNA inside the virus-like particles to form MS2-armored PRRSV antigen mRNA particles; lysing the host bacteria, separating the supernatant, and enriching and purifying the particles. This invention significantly enhances the resistance of mRNA to nuclease degradation and improves formulation stability; it can increase the efficiency of mRNA entering antigen-presenting cells and enhance immunogenicity; it can be mass-produced with controllable processes and relatively low cost.

Description

Technical Field

[0001] This invention relates to the fields of veterinary biological products and genetic engineering technology, specifically to a bacteriophage capsid protein MS2 armored porcine reproductive and respiratory syndrome virus (PRRSV) mRNA vaccine, as well as the preparation method of the vaccine and its application in preventing or reducing PRRSV infection. Background Technology

[0002] Porcine reproductive and respiratory syndrome virus (PRRSV) is a highly dangerous pathogen that poses a significant threat to the pig industry. It can cause reproductive disorders, abortions, stillbirths, and severe respiratory symptoms in piglets, resulting in substantial economic losses. PRRSV is an enveloped, single-stranded, positive-sense RNA virus. Due to its rapid genetic mutation and frequent recombination, the circulating strains change rapidly, posing a continuous challenge to immunization efforts.

[0003] Currently, PRRSV control mainly relies on vaccination, but commercially available vaccines still have significant shortcomings in terms of safety and efficacy. While inactivated vaccines have relatively high biosafety, they often fail to completely block viral infection and shedding due to their difficulty in inducing effective cellular and mucosal immune responses, and their cross-protection against heterologous strains is extremely limited. Modified live vaccines (MLVs), although capable of inducing strong immune responses, have safety risks that remain a major concern in the industry. Numerous studies have shown that MLV strains exhibit genetic instability, with the potential for virulence reversion and vertical transmission in sows during field applications. More seriously, frequent gene recombination between vaccine strains and field wild-type strains often leads to the emergence of new highly pathogenic strains. Furthermore, given PRRSV's extremely high mutation rate, existing vaccine strains struggle to cover constantly emerging new prevalent lineages (such as NADC30-like), resulting in widespread "immunization failure." Therefore, there is an urgent need to develop novel vaccine strategies that combine high biosafety with broad-spectrum protection potential.

[0004] mRNA vaccines have attracted attention due to their advantages such as short development cycles, platform-based production, and no need for pathogen culture. However, their application in the veterinary field still faces key bottlenecks. For example, mRNA is easily degraded by environmental nucleases and has poor in vivo stability; naked mRNA has low efficiency in entering cells and requires delivery systems (such as lipid nanoparticles) for effective expression; at the same time, issues such as material safety, batch-to-batch consistency, cold chain transportation, and long-term storage of delivery systems limit the promotion of mRNA vaccines in large-scale farming scenarios.

[0005] Armored RNA (ARNA) technology encapsulates target RNA within virus-like particles (VLPs), protecting them from nuclease degradation and significantly enhancing particle stability. MS2 phage capsid proteins can self-assemble into nanoscale particles and load RNA by specifically recognizing packaging stem-loop / pac sites. Compared to chemical delivery systems such as liposomes, MS2 armored RNA particles offer advantages such as structural stability, scalable preparation, high biosafety, and ease of transport and storage, providing a new approach for constructing stable and efficient mRNA vaccine delivery platforms.

[0006] However, current research and applications of armored RNA technology mainly focus on nucleic acid detection quality control materials, standard substances, or in vitro stabilization and preservation. No publicly available literature documents research directly applying armored RNA technology to vaccine preparation. This is because mRNA vaccines require not only that the encapsulated RNA possess resistance to degradation, but also that it maintains translatable activity within host cells and completes effective delivery, intracellular release, antigen expression, and immune activation. The MS2 armored system is highly sensitive to RNA length, secondary structure, packaging signals, and particle assembly efficiency. For antigen mRNA containing 5′UTR, antigen coding sequence, 3′UTR, Poly(A), and packaging elements, there is a lack of research and reports on how to improve stability while simultaneously ensuring packaging efficiency, particle integrity, and in vivo expression activity. Especially in the PRRSV vaccine scenario, in addition to nucleic acid stability, it is also necessary to consider whether antigen expression can induce an effective immune response and the feasibility of production, preservation, and application of the formulation under veterinary conditions.

[0007] Therefore, given the publicly available armored RNA technology, those skilled in the art have been unable to find an MS2 armored mRNA vaccine strategy that can induce a PRRSV-specific immune response, and there are no publicly available reports on the successful development of MS2 armored PRRSV antigen-encoded mRNA vaccines in the existing technology. Summary of the Invention

[0008] The technical problem this invention aims to solve is to address the insufficient protection and safety risks of existing PRRSV vaccines, as well as the bottlenecks of mRNA vaccines such as easy degradation, low delivery efficiency, and high requirements for preservation and transportation. This invention provides a method for preparing and applying PRRSV antigen mRNA particles armored by the phage capsid protein MS2. By stably encapsulating the PRRSV antigen-encoding mRNA, this invention facilitates its preparation and preservation, and can induce an immune response against PRRSV in the body, thus proposing a novel concept for vaccine preparation and application.

[0009] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0010] A method for preparing phage capsid protein MS2 armored PRRSV antigen mRNA particles is provided, comprising the following steps:

[0011] (1) Constructing recombinant expression plasmids:

[0012] The MS2 phage capsid protein gene and mRNA expression cassette were inserted into the first multiple cloning site MCS1 and the second multiple cloning site MCS2 of the double multiple cloning site expression vector, respectively, to obtain the recombinant expression plasmid; the mRNA expression cassette contains a packaging stem-loop structure and a PRRSV antigen coding sequence.

[0013] (2) Induced expression and self-assembly:

[0014] The recombinant expression plasmid was transformed into the host bacteria, and the MS2 capsid protein was induced to be expressed in the host bacteria and self-assembled to form virus-like particles. During this process, the MS2 capsid protein was used to specifically recognize the packaging stem-loop structure to package the transcribed mRNA inside the virus-like particles, forming MS2 armored PRRSV antigen mRNA particles.

[0015] (3) Particle purification:

[0016] The host bacteria were broken, the supernatant was separated, and the MS2 armored PRRSV antigen mRNA particles were enriched and purified.

[0017] As a preferred embodiment of the present invention, the double multiple cloning site expression vector is pACYCDuet-1.

[0018] As a preferred embodiment of the present invention, the sequence of the MS2 phage capsid protein gene is shown in SEQ NO.1; when inserting this gene into the first multiple cloning site MCS1 of the double multiple cloning site expression vector, the specific primer sequences used are shown in SEQ ID NO.2 and SEQ ID NO.3.

[0019] As a preferred embodiment of the present invention, the mRNA expression cassette sequentially includes the 5′-UTR, 12×stemloop, PRRSV antigen coding sequence, 3′-UTR, and Poly(A) sequence as shown in SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.8, and SEQ ID NO.9; the PRRSV antigen coding sequence is the GP4 protein coding sequence of the PRRSV JXA1 strain.

[0020] As a preferred embodiment of the present invention, when inserting the mRNA expression cassette into the second multiple cloning site MCS2 of the dual multiple cloning site expression vector, the specific primer sequences used are shown in SEQ ID NO.10 and SEQ ID NO.11.

[0021] As a preferred embodiment of the present invention, the host bacterium is Escherichia coli BL21(DE3); the conditions for inducing expression are as follows: when the OD450 of the bacterial culture reaches 0.6-0.8, IPTG with a final concentration of 1mM is added, and the culture is induced at 16°C for 16-20 hours.

[0022] As a preferred embodiment of the present invention, the enrichment and purification steps include: preliminary enrichment of particles in the broken supernatant using PEG / NaCl precipitation; purification of the reconstituted precipitate using His-tagged nickel column affinity chromatography, followed by elution using an imidazole-containing buffer; and dialysis and nuclease treatment of the eluent to remove exogenous free nucleic acids.

[0023] The present invention further provides MS2-armored PRRSV antigen mRNA particles prepared by the aforementioned method. The microstructure of the particles has an icosahedral shell formed by the self-assembly of MS2 capsid protein, and the PRRSV antigen-encoded mRNA is encapsulated inside the shell, enabling the particles to resist nuclease degradation.

[0024] The present invention also provides the use of the aforementioned MS2 armored PRRSV antigen mRNA particles in the preparation of immunogenic compositions or medicaments for the prevention or treatment of porcine reproductive and respiratory syndrome (PRRS).

[0025] As a preferred embodiment of the present invention, the immunogenic composition comprises the MS2 armored PRRSV antigen mRNA particles, and a veterinary-acceptable carrier, excipient or immunoadjuvant.

[0026] Compared with the prior art, the present invention has at least the following beneficial effects:

[0027] (1) By physically encapsulating the target mRNA with the MS2 capsid, the resistance of mRNA to nuclease degradation is significantly enhanced, the stability of the formulation is improved, and the dependence on a strict cold chain is reduced;

[0028] (2) MS2 virus-like particles have a nanometer-sized particle size and a uniform structure, which can be used as a delivery carrier to improve the efficiency of mRNA entering antigen-presenting cells, thereby enhancing immunogenicity;

[0029] (3) The preparation process is based on prokaryotic expression and self-assembly, which can be mass-produced, with controllable process and relatively low cost, making it suitable for veterinary use.

[0030] (4) The particles do not contain replicable pathogens, have high biosafety, and can avoid the potential risks of reactivation and recombination of traditional live vaccines. Attached Figure Description

[0031] Figure 1This is a schematic diagram of the construction of the pACYCDuet-MS2-mGP4 recombinant plasmid.

[0032] Figure 2 This is a schematic diagram of the construction of the pACYCDuet-MS2-meGFP recombinant plasmid.

[0033] Figure 3 PCR identification diagram of pACYCDuet-MS2--mGP4 and pACYCDuet-MS2-meGFP recombinant plasmids.

[0034] Figure 4 This is a schematic diagram of the SDS-PAGE / Western blot detection results of the purified particles.

[0035] Figure 5 DLS particle size distribution of pACYCDuet-MS2-mGP4 purified particles.

[0036] Figure 6 The DLS particle size distribution of the pACYCDuet-MS2-meGFP purified particles is shown in the diagram.

[0037] Figure 7 TEM image of the purified pACYCDuet-MS2-mGP4 particles.

[0038] Figure 8 TEM image of the purified pACYCDuet-MS2-meGFP particles.

[0039] Figure 9 The image shows the results of qRT-PCR amplification of the target fragment mRNA for purified particles.

[0040] Figure 10 The image shows the ELISA results of antibody levels in the immune serum of mice in the MS2-mGP4 vaccine group (days 0-28).

[0041] Figure 11 The image shows the ELISA results of antibody levels in the immune serum of mice in the MS2-meGFP vaccine group (days 0-28).

[0042] Figure 12 The figure shows the results of IFA detection of PRRSV activity in mouse immune serum. Detailed Implementation

[0043] The present invention will be further described below with reference to embodiments. It should be understood that the embodiments are only used to explain the present invention and are not intended to limit the scope of protection of the present invention. Those skilled in the art can make various modifications or improvements to the present invention without departing from the spirit of the present invention, and all such modifications or improvements shall fall within the scope of protection of the present invention.

[0044] I. Overview of the Technical Solution of this Application

[0045] 1. Construction of recombinant plasmids: The MS2 phage capsid protein gene was inserted into the first multiple cloning site MCS1 of pACYCDuet-1 to obtain the pACYCDuet-MS2 recombinant plasmid, which was identified by PCR sequencing. An mRNA expression cassette was constructed in the second multiple cloning site MCS2 of the recombinant plasmid. The mRNA expression cassette sequentially included a 5′-UTR, a 12×stem loop (MS2 packaging stem-loop structure), a PRRSV JXA1 strain GP4 coding fragment (mGP4), a 3′-UTR, and a Poly(A) sequence to obtain the pACYCDuet-MS2-mGP4 recombinant plasmid. At the same time, the control plasmid pACYCDuet-MS2-meGFP was constructed by replacing mGP4 with eGFP.

[0046] 2. Preparation of recombinant virus-like particles: After transforming the recombinant plasmid into a host bacterium (preferably Escherichia coli BL21(DE3)), the MS2 capsid protein is expressed under induction conditions and self-assembled into virus-like particles within the bacteria. At the same time, the target mRNA containing 12×stem loop is specifically packaged into the particle to obtain MS2 armored PRRSV mRNA particles.

[0047] 3. Expression and purification of recombinant virus-like particles: The bacterial cells after induced expression were disrupted, and the virus-like particles were enriched and purified by PEG / salting precipitation and His-tagged nickel column affinity chromatography. The exogenous contaminating nucleic acids were removed by treatment with a totipotent nuclease to obtain a high-purity armored mRNA particle vaccine formulation.

[0048] 4. Identification of MS2 armored RNA particle morphology: Capsid protein expression was verified by SDS-PAGE / Western blot, the presence of mRNA and RNase resistance within the particles were verified by RT-PCR / quantitative PCR, and particle size distribution and morphological integrity were observed by dynamic light scattering (DLS) and transmission electron microscopy (TEM).

[0049] 5. Immunization of mice and evaluation of efficacy: The MS2 armored mGP4 mRNA particle vaccine was used for animal immunization (such as pre-evaluation in mice or immunization verification in pigs). Serum antibody levels, neutralizing capacity, cellular immune indicators and safety were detected to evaluate its immunization efficacy and protective potential.

[0050] The present invention also provides the use of the MS2 armored PRRSV mRNA particles in the preparation of veterinary vaccines, immunizing agents or related products for the prevention or reduction of PRRSV infection.

[0051] II. Implementation Examples of the Technical Solution of this Application

[0052] Example 1: Construction of recombinant plasmids

[0053] (1) Reconstruction of MS2 expression frames:

[0054] The MS2 phage capsid protein gene (CP), sequenced as shown in SEQ ID NO.1, was selected. PCR was performed using specific primers (F: sequence SEQ ID NO.2; R: sequence SEQ ID NO.3) and the pACYCDuet vector (purchased from Genewiz) as a template. The purified PCR product was inserted into the multiple cloning site MCS1 of pACYCDuet-1 using homologous recombination to obtain the recombinant plasmid pACYCDuet-MS2 containing the MS2 sequence.

[0055] The recombinant product was transformed into DH5α competent cells. Specifically, competent cells frozen at -80℃ were thawed on ice, and 8 μL of the recombinant product was added to every 100 μL of competent cell suspension. The mixture was then plated on 2×YT solid medium containing chloramphenicol, and positive clones were obtained through antibiotic selection. Single colonies were picked and cultured in 10 mL of 2×YT liquid medium containing chloramphenicol. Colony PCR with specific primers and sequencing were used to verify the correctness of the inserted fragment. Correctly inserted bacteria were then stored at -20℃.

[0056] (2) Construction of mRNA expression cassette:

[0057] Referring to the method for constructing the MS2 expression cassette described in step (1), specific primers (F: sequence SEQ ID NO.10; R: sequence SEQ ID NO.11) were used to insert the mRNA expression cassette sequence into the multiple cloning site MCS2 of pACYCDuet-MS2 to obtain the pACYCDuet-MS2-mGP4 recombinant plasmid.

[0058] The expression cassette sequentially contains: a 5′-UTR (SEQ ID NO. 4), a 12×stem loop (SEQ ID NO. 5), a GP4 coding sequence for PRRSV JXA1 strain (SEQ ID NO. 6), a 3′-UTR (SEQ ID NO. 8), and a Poly(A) (SEQ ID NO. 9) sequence; the 12×stem loop refers to an MS2 capsid protein binding sequence repeated 12 times in tandem, used to mediate the assembly of mRNA and MS2 protein to form armor particles. The mRNA expression cassette sequence in this embodiment was synthesized by Genewiz Biotechnology Co., Ltd.

[0059] In addition, to verify the immunogenicity of the delivery system and demonstrate that the MS2 armor mRNA can enter the body, express proteins, and induce antibodies, the GP4 coding sequence in the mRNA expression cassette sequence was replaced with the eGFP coding sequence (sequence SEQ ID NO. 7) to construct the control plasmid pACYCDuet-MS2-meGFP. The recombinant plasmid was amplified by PCR and sequenced to confirm the correctness of the capsid protein expression cassette and mRNA expression cassette sequences.

[0060] (3) Experimental verification and result analysis:

[0061] Figure 1 , 2 The map of the successfully constructed recombinant expression plasmid is shown, including the T7 dual promoter, MS2 capsid protein sequence, packaging stem-loop structure (12×stem loop), and target gene (…). Figure 1 The middle one is GP4; Figure 2 The expression cassette in the middle is eGFP.

[0062] PCR identification results as follows Figure 3 As shown: Amplification was performed using specific primers with the constructed recombinant plasmid as a template. Figure 3 (a) shows that a specific band was amplified at approximately 390 bp, consistent with the expected size of the MS2 coat protein, and a specific band was amplified at approximately 4178 bp, consistent with the expected size of the pACYCDuet vector gene fragment. Figure 3 (b) shows that a single bright band was amplified at approximately 1475 bp, consistent with the expected size of the eGFP mRNA expression cassette containing 12×stem loop, and a single bright band was amplified at approximately 4568 bp, consistent with the expected size of pACYCDuet-MS2. Figure 3 Image (c) shows that a specific band was amplified around 531 bp, consistent with the expected size of the PRRSV GP4 mRNA expression cassette containing a 12×stem loop. Sequencing results further confirmed that the inserted fragment sequence was a perfect match with the designed sequence, indicating the successful construction of the pACYCDuet-MS2-mGP4 and pACYCDuet-MS2-meGFP recombinant expression plasmids.

[0063] Example 2: Preparation, Expression and Purification of Recombinant Virus-like Particles

[0064] (1) Host bacteria construction:

[0065] pACYCDuet-MS2-mGP4 and pACYCDuet-MS2-meGFP were transformed into competent Escherichia coli BL21(DE3), and positive clones were obtained by resistance screening.

[0066] (2) Induced self-assembly and packaging:

[0067] Positive clones were inoculated into 200-400 mL of 2×YT liquid medium containing chloramphenicol and cultured to the logarithmic phase (OD450 = 0.6-0.8). The protein expression inducer IPTG was added to a final concentration of 1 mM, and the culture was induced at 16℃ and 120 rpm for 16-20 h. In the presence of IPTG, the positive bacteria were induced to express the MS2 capsid protein and self-assemble to form virus-like particles. At the same time, the target mRNA containing 12×stem loop was specifically packaged into the particles, resulting in bacterial products containing armored mRNA.

[0068] (3) Collection and disruption of bacterial cells:

[0069] The induced bacterial cells were collected and resuspended in 50 mM PBS buffer, lysed by sonication, centrifuged at 12,000 rpm for 10 minutes, and the supernatant was collected.

[0070] (4) Particle enrichment:

[0071] Virus-like particles in the supernatant were enriched using polyethylene glycol / sodium chloride (PEG8000 / NaCl). A 20% PEG8000 / NaCl solution was prepared and added 1:10 to the sonicated supernatant. The mixture was placed on ice for 2 hours until most of the virus-like particle proteins precipitated. After centrifugation at 12000 rpm, the precipitate was redissolved with 50 mM PBS. Higher purity virus-like particles were obtained using His-tagged nickel column affinity chromatography. The His-tagged affinity resin was washed with 50 mM PBS and transferred to the virus-like particle solution. The mixture was incubated at 4°C on a shaker for 4-6 hours. The suspension was then transferred to a chromatography column, impurities were aspirated with PBS containing 50 mM imidazole, and the target protein was eluted with PBS containing 500 mM imidazole. Imidazole was removed from the eluted protein solution by dialysis. The protein solution was placed in a dialysis bag, sealed, and then placed in PBS solution with a low imidazole concentration. The solution was gently vortexed and dialyzed at 4°C for 1 hour. The imidazole concentration was gradually reduced until finally dialyzed with 50 mM PBS for 2 hours.

[0072] (5) Removal of exogenous nucleic acids:

[0073] The purified virus-like particle solution was treated with omnipotent nucleases such as Benzonase (37°C for 20 min) to remove unencapsulated contaminating nucleic acids. The buffer was replaced with 50 mM PBS solution using an ultrafiltration tube to obtain the MS2 armored PRRSV mRNA particle vaccine stock solution.

[0074] Example 3: Identification of MS2 Armored RNA Particle Morphology and Packaging

[0075] (1) Protein expression identification:

[0076] Purified particles were subjected to SDS-PAGE or Western blot to detect the MS2 capsid protein band. Virus-like particles were separated and purified using 12% SDS-PAGE at a constant voltage of 140V for 1 hour. After separation, the SDS-PAGE gel was stained with rapid staining solution for 10 minutes and then destained with pure water. Protein purity was visually assessed, or Western blot was used to transfer the gel to a PVDF membrane. The membrane was blocked with 5% skim milk in PBST solution at room temperature for 2 hours, followed by incubation with anti-His-tagged monoclonal antibody at 37°C for 1 hour, and washed three times with PBST. The membrane was then incubated with HRP-labeled goat anti-mouse IgG at 37°C for 1 hour. After three washes, the immunoreaction signal was detected using a chemiluminescent substrate.

[0077] (2) Particle morphology and particle size:

[0078] Dynamic light scattering (DLS) was used to detect particle size distribution and dispersion, while transmission electron microscopy (TEM) was used to observe particle morphology, integrity, and uniformity.

[0079] (3) mRNA packaging identification:

[0080] Nucleic acid was extracted from the purified particles for qRT-PCR amplification of the target fragment. Total RNA was extracted from the purified particles using an RNA extraction kit according to the manufacturer's instructions, and reverse transcription was performed using a reverse transcription kit according to the manufacturer's instructions. The expression cassette mRNA was quantitatively detected by qRT-PCR using specific primers (GP4-F: SEQ ID NO.12; GP4-R SEQ ID NO.13; eGFP-F: SEQ ID NO.14; eGFP-R SEQ ID NO.15), and the amount of expression cassette mRNA was determined by CT value.

[0081] (4) Experimental verification and result analysis:

[0082] The electrophoretic results of the purified recombinant particle protein are as follows: Figure 4 As shown:

[0083] SDS-PAGE results ( Figure 4 (Upper middle side) As shown, the pACYCDuet-MS2-mGP4 and pACYCDuet-MS2-meGFP products purified by nickel column affinity chromatography both exhibited single, clear protein bands at approximately 15 kDa, indicating a significant removal of contaminating proteins.

[0084] Western blot results ( Figure 4As shown in the lower middle section, after incubation with an anti-His-tagged antibody, a strong positive blot signal was detected at the same location (~15 kDa), confirming that the purified protein is a His-tagged MS2 capsid protein. These results demonstrate that the purification process established in this invention can obtain high-purity recombinant virus-like particle protein.

[0085] Particle size distribution (DLS): Dynamic light scattering results as follows Figure 5 , 6 As shown, Figure 5 pACYCDuet-MS2-mGP4, Figure 6 The sample was pACYCDuet-MS2-meGFP. The purified particles exhibited a monodisperse distribution, with an average hydrodynamic diameter concentrated around 20-25 nm. The low polydispersity index (PDI) indicates that the particles were uniform in size in the solution and there was no obvious aggregation.

[0086] Morphological observation (TEM): Results of transmission electron microscopy observation are as follows Figure 7 , 8 As shown, Figure 7 pACYCDuet-MS2-mGP4, Figure 8 The sample is pACYCDuet-MS2-meGFP. At 100k magnification, numerous structurally intact, well-defined spherical particles with a diameter of approximately 25 nm are visible in the field of view. These particles exhibit typical icosahedral symmetry without obvious damage or deformation, confirming that the MS2 capsid protein successfully self-assembled to form virus-like particles (VLPs).

[0087] mRNA encapsulation verification (qRT-PCR): After removing free nucleic acids by treatment with a totipotent nuclease (RNase), the nucleic acids inside the particles were extracted and detected by qRT-PCR. The results are as follows: Figure 9 As shown, compared to the MS2 empty vector control, both the MS2-mGP4 and MS2-meGFP particle groups amplified specific GP4 and eGFP gene fragments, and the CT values ​​were significantly lower than those of the control group, while also meeting the requirement of a CT value less than 25. This confirms that the target mRNA with packaging signals (12×stem loop) is specifically encapsulated inside the MS2VLP, and that the MS2 capsid can effectively protect the internal mRNA from degradation by external nucleases, thus endowing the vaccine with excellent stability.

[0088] Example 5: Immunization of mice and evaluation of the effect

[0089] (1) Immunization grouping:

[0090] Six- to eight-week-old BALB / c mice were randomly divided into MS2-mGP4 group, MS2-meGFP group, and MS2 virus-like particle control group without mRNA packaging box. Each mouse was subcutaneously immunized with 10 μg of virus-like particles. Serum was collected at different time points after immunization. The mice were immunized again with the same dose at 14 dpi. The serum PRRSV GP4, eGFP protein-specific antibodies, and PRRSV neutralizing antibodies were detected.

[0091] (2) Evaluation of humoral immunity:

[0092] ELISA was used to detect specific IgG levels against PRRSV GP4 and eGFP antigens. 100 μL of 5 μg / mL prokaryotically expressed PRRSV GP4 antigen and eGFP protein were added to each well, and the plates were coated overnight at 4°C. After washing three times with PBST, 150 μL of PBST solution containing 5% skim milk was added to each well, and the plates were blocked at 37°C for 2 h. After washing, mouse serum diluted 1:100 was added as the primary antibody, and the plates were incubated at 37°C for 1 h. After washing three times, HRP-conjugated goat anti-mouse antibody diluted 1:5000 was added as the secondary antibody, and the plates were incubated at 37°C for 1 h. After washing, 100 μL of TMB chromogenic solution was added to each well, and the reaction was stopped after 10 min of development by adding 50 μL of 5% sulfuric acid solution to each well. The absorbance of the chromogenic solution at 450 nm was measured using a microplate reader to evaluate mouse serum PRRSV levels. GP4-specific antibody levels; the inhibitory effect of serum on PRRSV infection was assessed using a cellular-level neutralization assay. Marc-145 cells were cultured in 96-well cell culture plates until the cell confluence in each well reached approximately 80%. Serum from immunized mice was then used to neutralize the antibodies. 2 2 3 2 4 2 5 2 6 2 7 Dilute 100 μL of serum diluent with 100 TCID50 PRRSV JXA1 virus solution, incubate at 37°C for 2 h, then add 200 μL to each well onto cells. After 2 h, wash away serum and unbound virus with D-Hanks solution. After maintaining culture for 60 h, fix cells with 4% paraformaldehyde solution at room temperature. Detect PRRSV-infected cells using immunofluorescence. The primary antibody was laboratory-preserved PRRSV-N protein monoclonal antibody VH13 (dilution 1:1000), and the secondary antibody was Alexa Fluor 488 green fluorescent conjugate goat anti-mouse antibody (dilution 1:5000). Calculate serum neutralizing antibody levels based on cell infection results at each dilution.

[0093] (3) Experimental verification and result analysis:

[0094] Serum antibody levels (ELISA):

[0095] Serum antibody test results after mouse immunization are as follows Figure 10 , 11 As shown. Compared with the MS2 empty vector control group, mice in the MS2-mGP4 vaccine group showed detectable specific IgG antibodies against PRRSV GP4 protein 14 days after immunization (14 dpi); after the second immunization (28 dpi), antibody levels showed a significant upward trend. Figure 10 Similarly, compared with the MS2 empty vector control group, mice in the MS2-meGFP vaccine group showed detectable specific IgG antibodies against the eGFP protein 21 days after two immunizations (21 dpi), and the antibody levels showed a significant upward trend. Figure 11 This indicates that MS2 armored mRNA particles have good immunogenicity, can effectively deliver mRNA into the body and translate it into antigen proteins, and induce a high level of humoral immune response in the body.

[0096] Viral neutralizing activity (IFA):

[0097] Serum neutralization test results as follows Figure 12 As shown in the figure, immune sera at different dilutions (1:16, 1:32) were incubated with PRRSVJXA1 virus solution and then used to infect Marc-145 cells. Immunofluorescence observation revealed that at the 1:16 dilution, almost no green fluorescence signal was observed in the field of view of the MS2-mGP4 group, indicating that viral infection was completely blocked. At the 1:32 dilution, although a small number of green fluorescent spots appeared, the fluorescence intensity and the number of infected cells were significantly reduced compared to the virus control group (full field of view fluorescence). These results confirm that the serum produced by mice immunized with the MS2-mGP4 mRNA vaccine contains high titers of neutralizing antibodies, which can specifically bind to and neutralize PRRSV virus, effectively preventing viral invasion of target cells and exhibiting excellent antiviral biological activity.

[0098] The sequence contents of SEQ ID NO.1 to SEQ ID NO.15 mentioned above are summarized in Table 1 below.

[0099] Table 1. List of amino acid and / or nucleotide sequences in the instruction manual.

[0100] .

Claims

1. A method for preparing phage capsid protein MS2 armored PRRSV antigen mRNA particles, characterized in that, Includes the following steps: (1) Constructing recombinant expression plasmids: The MS2 phage capsid protein gene and mRNA expression cassette were inserted into the first multiple cloning site MCS1 and the second multiple cloning site MCS2 of the double multiple cloning site expression vector, respectively, to obtain the recombinant expression plasmid; the mRNA expression cassette contains a packaging stem-loop structure and a PRRSV antigen coding sequence. (2) Induced expression and self-assembly: The recombinant expression plasmid was transformed into the host bacteria, and the MS2 capsid protein was induced to be expressed in the host bacteria and self-assembled to form virus-like particles. During this process, the MS2 capsid protein was used to specifically recognize the packaging stem-loop structure to package the transcribed mRNA inside the virus-like particles, forming MS2 armored PRRSV antigen mRNA particles. (3) Particle purification: The host bacteria were broken, the supernatant was separated, and the MS2 armored PRRSV antigen mRNA particles were enriched and purified.

2. The method according to claim 1, characterized in that, The dual multiple cloning site expression vector is pACYCDuet-1.

3. The method according to claim 1, characterized in that, The sequence of the MS2 phage capsid protein gene is shown in SEQ NO.1; the specific primer sequences used when inserting this gene into the first multiple cloning site MCS1 of the double multiple cloning site expression vector are shown in SEQ ID NO.2 and SEQ ID NO.

3.

4. The method according to claim 1, characterized in that, The mRNA expression cassette sequentially includes the 5′-UTR, 12×stemloop, PRRSV antigen coding sequence, 3′-UTR, and Poly(A) sequence as shown in SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.8, and SEQ ID NO.9; the PRRSV antigen coding sequence is the GP4 protein coding sequence of the PRRSVJXA1 strain.

5. The method according to claim 1, characterized in that, When inserting the mRNA expression cassette into the second multiple cloning site MCS2 of the dual multiple cloning site expression vector, the specific primer sequences used are shown in SEQ ID NO.10 and SEQ ID NO.

11.

6. The method according to claim 1, characterized in that, The host bacterium is Escherichia coli BL21(DE3); the conditions for inducing expression are as follows: when the OD450 of the bacterial culture reaches 0.6-0.8, IPTG with a final concentration of 1 mM is added, and the culture is induced at 16°C for 16-20 h.

7. The method according to claim 1, characterized in that, The enrichment and purification steps include: preliminary enrichment of particles in the broken supernatant using PEG / NaCl precipitation; purification of the reconstituted precipitate using His-tagged nickel column affinity chromatography, followed by elution with an imidazole-containing buffer; and dialysis and nuclease treatment of the eluent to remove exogenous free nucleic acids.

8. MS2 armored PRRSV antigen mRNA particles prepared by the method according to any one of claims 1 to 7, characterized in that, The particle's microstructure features an icosahedral shell formed by the self-assembly of the MS2 capsid protein, with the PRRSV antigen-encoded mRNA encapsulated inside the shell, enabling the particle to resist nuclease degradation.

9. The use of the MS2 armored PRRSV antigen mRNA particles of claim 8 in the preparation of immunogenic compositions or medicaments for the prevention or treatment of porcine reproductive and respiratory syndrome.

10. The application according to claim 9, characterized in that, The immunogenic composition comprises the MS2 armored PRRSV antigen mRNA particles, and a veterinary-acceptable carrier, excipient, or immunoadjuvant.

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