Virus-like particle carrier based on porcine paternal expression gene 10 and its application in mRNA delivery

Abstract

The application discloses a virus-like particle carrier based on a porcine paternal expression gene 10 and application thereof in mRNA delivery, and belongs to the technical field of genetic engineering and biological medicine. The virus-like particle is formed through self-assembly after the nucleocapsid domain of the porcine paternal expression gene 10 (SsPEG10) is deleted and replaced with a lambda phage lambda N peptide segment or an MS2 phage coat protein MCP in situ. The target mRNA is specifically packaged into the VLP interior by using the high-specificity RNA-protein interaction between the lambda N peptide segment and the 12xBoxB RNA stem loop structure or the MCP and the 12xMS2 RNA stem loop structure, and VSV-G is co-expressed during the VLP assembly process, so that the VLP is endowed with the membrane fusion ability mediated by endocytosis. Finally, the recombined VLP carrier capable of efficiently packaging the target mRNA is successfully constructed, the target mRNA can be delivered into target cells, and efficient expression and translation of the target protein are realized.

Description

Technical Field

[0001] This invention relates to a recombinant virus-like particle (VLP) vector based on porcine paternal expression gene 10 (SsPEG10), its preparation method, and its application in RNA delivery. This invention belongs to the fields of genetic engineering and biomedical technology. Background Technology

[0002] mRNA vaccines work by introducing mRNA encoding antigens into host cells, where the cells synthesize antigen proteins using their own translation system, inducing specific humoral and cellular immune responses. Compared to traditional live attenuated vaccines, inactivated vaccines, and subunit vaccines, mRNA vaccines offer advantages such as shorter development cycles, standardized production processes, and non-integration into the host genome, making them valuable for infectious disease control. The effectiveness of mRNA depends on a highly efficient and safe delivery system. Currently, the most widely used clinical delivery system, lipid nanoparticles (LNPs), while offering high mRNA encapsulation rates, has inherent limitations: excessive liver targeting leading to significant toxicity to non-target organs, poor formulation stability requiring strict low-temperature cold chain transportation, and high cost, making it difficult to meet the large-scale application needs of livestock and poultry mRNA vaccines.

[0003] Virus-like particles (VLPs) retain the morphology and cellular invasion capabilities of natural viruses, do not contain infectious viral genomes, and offer high safety, making them a hot topic in next-generation drug and gene delivery vector development. Among them, the mammalian endogenously expressed paternal 10 (PEG10) protein can spontaneously assemble into virus-like particles and package mRNA, exhibiting low immunogenicity potential and supporting repeated dosing. However, natural PEG10 VLPs suffer from poor mRNA packaging specificity and low efficiency.

[0004] To address the aforementioned issues, this invention employs two RNA-protein specific interaction systems, λN-BoxB and MCP-MS2, to enrich target RNA: λN peptide is a 22-amino acid short peptide derived from λ phage, which binds with high affinity to the stem-loop structure of BoxB RNA and has a small molecular weight, resulting in low interference with host protein self-assembly; MCP protein is the capsid protein of MS2 phage, which can specifically recognize the stem-loop structure of MS2 RNA.

[0005] Currently, research on PEG10 VLPs mainly focuses on human PEG10, and there are no reports of combining porcine SsPEG10 VLPs with λN-BoxB or MCP-MS2 targeted packaging systems for mRNA delivery. Therefore, it is still impossible to meet the urgent need for efficient transduction of porcine cells and the development of livestock and poultry mRNA vaccines. Summary of the Invention

[0006] In view of the above-mentioned defects in the existing technology, the purpose of this invention is to provide a recombinant endogenous virus-like particle (VLP) mRNA delivery system based on porcine SsPEG10, which can efficiently load target mRNA and realize its delivery and functional expression in target cells.

[0007] To achieve the above objectives, the present invention employs the following technical means:

[0008] The porcine paternal gene 10 (SsPEG10) is an endogenous retroviral protein encoded by the porcine genome. Naturally, through a frameshift mechanism at the -1 ribosome, the same transcript is translated into two protein isoforms: PEG10-RF1 and PEG10-RF1 / RF2. RF1 encodes a Gag-like protein with a CCHC-type zinc finger structure. RF1 / RF2 is translated through a frameshift mechanism to produce a Gag-Pol-like fusion protein. Both can spontaneously assemble into virus-like particles and have mRNA packaging capabilities. This invention involves targeted molecular modification of porcine SsPEG10 protein: deleting its nucleocapsid domain (NC) and replacing it with a λ phage λN peptide or an MS2 phage capsid protein (MCP), resulting in recombinant fusion proteins SsPEG10ΔNC-λN and SsPEG10ΔNC-MCP. Utilizing the highly specific RNA-protein interaction between the λN peptide and the 3' untranslated region (3'UTR) of the cargo mRNA (a 12×BoxBRNA stem-loop structure), or between MCP and a 12×MS2 RNA stem-loop structure, the target cargo mRNA is specifically packaged into a VLP formed by the self-assembly of SsPEG10. Simultaneously, vesicular stomatitis virus G glycoprotein (VSV-G) is co-expressed as an envelope protein during VLP assembly, and pseudotype modification endows the VLP with endocytosis-mediated membrane fusion capability. Ultimately, a recombinant SsPEG10 VLP capable of efficiently packaging cargo mRNA and possessing cellular delivery activity was successfully constructed. The vector can deliver cargo mRNA into target cells, enabling targeted enrichment and specific packaging and delivery of target mRNA, as well as efficient expression and translation of target proteins.

[0009] Based on the above research, this invention first proposes a virus-like particle based on porcine paternal expression gene 10. The virus-like particle is formed by deleting the nucleocapsid (NC) domain of porcine paternal expression gene 10 and replacing it in situ with λ phage λN peptide or MS2 phage capsid protein (MCP) to obtain a recombinant fusion protein, which is then self-assembled.

[0010] Preferably, the amino acid sequence of the recombinant fusion protein obtained by deleting the nucleocapsid (NC) domain of the porcine paternal expression gene 10 and replacing it in situ with the λ phage λN peptide is shown in SEQ ID NO.6; the amino acid sequence of the recombinant fusion protein obtained by deleting the nucleocapsid (NC) domain of the porcine paternal expression gene 10 and replacing it in situ with the MS2 phage capsid protein (MCP) is shown in SEQ ID NO.8.

[0011] Preferably, the method further includes co-expressing vesicular stomatitis virus G glycoprotein (VSV-G) as an envelope protein during the assembly of the virus-like particles, thereby endowing the virus-like particles with endocytosis-mediated membrane fusion capability through pseudotype modification.

[0012] Furthermore, the present invention also proposes the application of the aforementioned virus-like particles in the preparation of mRNA delivery vectors.

[0013] Preferably, a 12×BoxB RNA stem-loop structure or a 12×MS2 RNA stem-loop structure is introduced into the 3' untranslated region of the mRNA to specifically package the mRNA into the interior of the virus-like particle through highly specific RNA-protein interactions.

[0014] Preferably, the nucleotide sequences of the 12×BoxB RNA stem-loop structure and the 12×MS2 RNA stem-loop structure are shown in SEQ ID NO.10 and 12, respectively.

[0015] Furthermore, this invention also proposes a virus-like particle mRNA delivery system based on porcine SsPEG10, the system comprising:

[0016] (1) Capsid protein granules: The capsid protein granules are plasmids expressing the modified porcine paternal expression gene 10. The modified porcine paternal expression gene 10 is obtained by deleting the nucleocapsid (NC) domain gene and replacing it in situ with λ phage λN peptide or MS2 phage capsid protein (MCP) gene based on the original gene.

[0017] (2) Cargo protein granules: The cargo protein granules are plasmids carrying target mRNA, wherein the 3' untranslated region of the mRNA contains a 12×BoxB RNA stem-loop structure or a 12×MS2 RNA stem-loop structure;

[0018] (3) Lentiviral packaging plasmid: The lentiviral packaging plasmid is the pMD2.G plasmid expressing vesicular stomatitis virus G glycoprotein (VSV-G).

[0019] Preferably, the capsid protein particles are prepared by the following method:

[0020] (1) The full-length SsPEG10 sequence was amplified using SsPEG10-F / R primers. The GenBank accession number for the SsPEG10 sequence is NM_001302462.1. The recombinant vector was constructed into the eukaryotic expression vector pcDNA3.1 using T4-DNA ligase and named pcDNA3.1-SsPEG10. The primer sequences are as follows:

[0021] SsPEG10-F: 5'-AAGCTTGGCTAGCGTTTGCCACCATGGGGCCCGACTGCCCACC-3'

[0022] SsPEG10-R:

[0023] 5'-CGAGGCTGATCAGCGGGTTTTTACATGGCGCTGTAAGACGGT-3'

[0024] (2) Using pcDNA3.1-SsPEG10 plasmid as template and SsPEG10ΔNC-F / R as primers, the SsPEG10 sequence with the nucleocapsid domain deleted from the Gag protein was amplified and ligated to pcDNA3.1 using homologous recombinase. The resulting recombinant vector was named pcDNA3.1-SsPEG10ΔNC. The primer sequences are as follows:

[0025] SsPEG10ΔNC-F: 5'-TGCATCCACATCGAGCCTGCCAAGGCCTCGAAG-3'

[0026] SsPEG10ΔNC-R: 5'-AGGCTCGATGTGGATGCACTGGCCGATCAGGGC-3'

[0027] (3) Using pcDNA3.1-SsPEG10ΔNC as a template, the SsPEG10ΔNC sequence missing the SsPEG10 NC domain was amplified using SsPEG10ΔNC-ZT-F / R primers for homologous recombination with λN and MCP. The MCP sequence was amplified using pMCP-RecT plasmid as a template and MCP-F / R primers. In addition, the λN sequence was generated by denaturation and annealing with primer λN-F / R. Subsequently, the amplified SsPEG10ΔNC sequence missing the SsPEG10 NC domain was ligated with the MCP sequence and λN sequence respectively by homologous recombination to obtain SsPEG10ΔNC-MCP and SsPEG10ΔNC-λN, which were then ligated with pcDNA3.1 using homologous recombination enzymes. The primer sequences are as follows:

[0028] SsPEG10ΔNC-ZT-F: 5'-CCTGCCAAGGCCTCGAAG-3'

[0029] SsPEG10ΔNC-ZT-R: 5'-CTCGATGTGGATGCACTGGC-3'

[0030] λN-F: 5'-gccagtgcatccacatcgagGGATCCGGCGGCGGCGGCAGCAAG-3'

[0031] λN-R: 5'-gccttcgaggccttggcaggCTCGAGATTGGCGGCTTCCACTG-3'

[0032] MCP-F: 5'-gccagtgcatccacatcgagACTCCCTCTAGAGGTGGTGGGG-3'

[0033] MCP-R: 5'-gccttcgaggccttggcaggCTCGAGGTAGATACCTGAGTTAGCGGC-3'.

[0034] Preferably, the GagΔNC-λN nucleotide and its encoded protein sequence in SsPEG10ΔNC-MCP and SsPEG10ΔNC-λN are shown in SEQ ID NO.5 and 6, respectively; the GagΔNC-MCP nucleotide and its encoded protein sequence are shown in SEQ ID NO.7 and 8, respectively; and the Pol nucleotide and its encoded protein sequence are shown in SEQ ID NO.3 and 4, respectively.

[0035] Compared with the prior art, the beneficial effects of the present invention are:

[0036] (1) This invention provides a novel recombinant VLP mRNA delivery system with well-defined base and amino acid sequences. With the help of the λN / BoxB and MCP / MS2 high-specificity RNA-protein interaction system, the target mRNA can be efficiently and specifically packaged and protected to prevent it from being degraded by extracellular nucleases.

[0037] (2) The core framework of the delivery system of the present invention is derived from the endogenous protein of pigs, contains no virus replication-related genes, and has no risk of infection; compared with exogenous viral vectors such as adenovirus and lentivirus, it has significantly lower immunogenicity, can effectively reduce inflammatory response and clearance of anti-vector antibodies, and supports repeated administration.

[0038] (3) The system of the present invention is a modular universal delivery platform. It can deliver different targets by simply replacing the antigen coding sequence of the target mRNA. There is no need to remodel the VLP core skeleton, which facilitates the rapid expansion of application scenarios and the development of multivalent vaccines. Attached Figure Description

[0039] Figure 1 This diagram illustrates the construction of the three plasmid components of the PEG10 delivery system of the present invention and the recombinant SsPEG10ΔNC-λN and SsPEG10ΔNC-MCP expression plasmids; wherein, A represents the three plasmid components; and B represents the plasmid construction diagram.

[0040] Figure 2 The figure shows the results of Western blotting detection of recombinant SsPEG10 protein expression in the whole lysate of HEK-293T cells and the cell supernatant; where A is the whole lysate and B is the cell supernatant.

[0041] Figure 3 The morphological structure of purified VSV-G pseudoformed SsPEG10ΔNC-λN and SsPEG10ΔNC-MCP VLPs observed by transmission electron microscopy (TEM) is shown in the figure.

[0042] Figure 4 Figure showing the expression of green fluorescent protein in HEK-293T cells after delivery of EGFP reporter gene mRNA by SsPEG10 VLP, as detected by fluorescence microscopy.

[0043] Figure 5 For PRRSV GP3 delivery performance testing. Detailed Implementation

[0044] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0045] The schematic diagram of the construction of the three plasmid components of the PEG10 delivery system of the present invention and the recombinant SsPEG10ΔNC-λN and SsPEG10ΔNC-MCP expression plasmids is shown below. Figure 1 As shown, a vector based on the SsPEG10 VLP RNA delivery system was prepared by deleting the SsPEG10 ΔNC domain to remove its ability to bind its own RNA, followed by inserting λN and MCP RNA-binding proteins into the original NC domain to achieve modular assembly with the target mRNA. In specific delivery applications, the SsPEG10-λN and SsPEG10-MCP delivery systems can achieve efficient mRNA delivery in various cell types.

[0046] Example 1: Preparation of a virus-like particle mRNA delivery system based on porcine SsPEG10

[0047] 1. Plasmid construction

[0048] (1) Construction and modification of SsPEG10 expression vector

[0049] The full-length SsPEG10 sequence (GenBank: NM_001302462.1) was amplified using SsPEG10-F / R primers and constructed into the eukaryotic expression vector pcDNA3.1 using T4-DNA ligase. The resulting recombinant vector was named pcDNA3.1-SsPEG10. Using the pcDNA3.1-SsPEG10 plasmid as a template, SsPEG10ΔNC-F / R primers were used for amplification to obtain the SsPEG10 sequence with the NC (nucleocapsid) domain deleted from the Gag protein (where the GagΔNC nucleotide and its encoded protein sequence are shown in SEQ ID NO. 1 and 2, respectively, and the Pol nucleotide and its encoded protein sequence are shown in SEQ ID NO. 3 and 4, respectively). This sequence was then ligated to pcDNA3.1 using homologous recombinase, and the resulting recombinant vector was named pcDNA3.1-SsPEG10ΔNC. Using pcDNA3.1-SsPEG10ΔNC as a template, the SsPEG10ΔNC sequence, lacking the SsPEG10 NC domain, was amplified using SsPEG10ΔNC-ZT-F / R primers for homologous recombination ligation with λN and MCP. The MCP sequence was amplified using pMCP-RecT plasmid (purchased from the Miaoling plasmid platform) as a template and MCP-F / R primers. Additionally, the λN sequence was generated by denaturation and annealing using primers λN-F / R. Subsequently, the amplified SsPEG10ΔNC sequence lacking the SsPEG10 NC domain was ligated to the MCP and λN sequences via homologous recombination, yielding SsPEG10ΔNC-MCP and SsPEG10ΔNC-λN, which were then ligated to pcDNA3.1 using homologous recombination enzymes. The nucleotide and encoded protein sequences of GagΔNC-λN are shown in SEQ ID NO. 5 and 6, respectively, and the nucleotide and encoded protein sequences of GagΔNC-MCP are shown in SEQ ID NO. 7 and 8, respectively. The primers for cloning the SsPEG10 gene and constructing mutants are shown in Table 1, and the plasmid construction diagram is shown in Figure 1. Figure 1 As shown.

[0050] Table 1. Primer sequences

[0051]

[0052] (2) Construction of 12×BoxB recombinant vector

[0053] The recombinant vector was synthesized by BGI Genomics. 12×BoxB was constructed into the eukaryotic expression vector pEGFP-C3, and the recombinant vector was named pEGFP-12×BoxB. A schematic diagram of the sequence construction is shown below. Figure 1As shown in B. The nucleotide sequence of the pEGFP-12×BoxB vector is shown in SEQ ID NO.9. The nucleotide sequence of 12×BoxB is shown in SEQ ID NO.10.

[0054] (3) Construction of 12×MS2 modified recombination vector

[0055] The recombinant vector was synthesized by BGI Genomics. The 12×MS2 sequence was synthesized into the eukaryotic expression vector pEGFP-C3, and the recombinant vector was named pEGFP-12×MS2. The sequence construction diagram is shown below. Figure 1 As shown in B. The nucleotide sequence of the pEGFP-12×MS2 vector is shown in SEQ ID NO.11. The nucleotide sequence of 12×MS2 is shown in SEQ ID NO.12.

[0056] (4) Construction of PRRSV GP3-12×BoxB cargo carrier

[0057] The cargo vector was optimized and synthesized by BGI Genomics and named PRRSV GP3-12×BoxB. The nucleotide sequence of the recombinant vector is shown in SEQ ID NO.13.

[0058] Example 2: SsPEG10 VLP Packaging and Testing

[0059] 1. SsPEG10 VLP plasmid transfection and secretion detection

[0060] (1) Cell transfection: Cells were seeded in 6-well plates and transfected when the cell density reached 40%. The ratio of plasmid to transfection reagent was 1:3. The transfection ratio and dosage are shown in Table 2 below.

[0061] Table 2 Transfection ratio and dosage

[0062]

[0063] Six hours after transfection, the cells were replaced with fresh DMEM containing 10% FBS. Forty-eight hours after transfection, the cell supernatant was collected. The cells were centrifuged at 8000 rpm for 10 min at 4°C and filtered through a 0.45 μm filter to obtain SsPEG10ΔNC-MCP or SsPEG10ΔNC-λ VLP containing EGFP-encoded mRNA. Virus-like particles were obtained by expressing GagΔNC-λN or GagΔNC-MCP. The nucleotide and encoded protein sequences of GagΔNC-λN are shown in SEQ ID NO. 5 and 6, respectively, and the nucleotide and encoded protein sequences of GagΔNC-MCP are shown in SEQ ID NO. 7 and 8, respectively. By utilizing the highly specific RNA-protein interaction between the λN peptide and the 12×BoxB RNA stem-loop structure introduced into the 3' untranslated region (3'UTR) of EGFP mRNA, or the MCP and the 12×MS2 RNA stem-loop structure, pEGFP-12×BoxB or pEGFP-12×MS2 is specifically packaged into the self-assembled SsPEG10ΔNC-λN VLP and SsPEG10ΔNC-MCP VLP. At the same time, vesicular stomatitis virus G glycoprotein (VSV-G) is used as the envelope protein, and pseudotype modification is used to endow the VLP with endocytosis-mediated membrane fusion ability for later use.

[0064] (2) Western blot detection

[0065] Cell supernatant was collected 48 hours after transfection for analysis. Rabbit anti-HA tag was used as the primary antibody, and HRP goat anti-rabbit was used as the secondary antibody. Results are as follows: Figure 2 As shown.

[0066] 2. SsPEG10 VLP electron microscopy detection

[0067] Sample preparation and observation for transmission electron microscopy (TEM): SsPEG10ΔNC-λN VLP and SsPEG10ΔNC-MCP VLP samples, purified by sucrose density gradient centrifugation, were filtered through a 0.22 μm filter to remove impurities. 10 μL of sample was dropped onto a hydrophilicized carbon-supported copper mesh and incubated at room temperature for 10 min. Excess liquid was then blotted away with filter paper. Subsequently, 2% phosphotungstic acid solution (pH 6.8) was added for negative staining for 1 min. After absorbing the stain with filter paper, the sample was allowed to air dry. The samples were observed using a Hitachi TEM (80 kV) at 60,000x magnification. The results are as follows: Figure 3 As shown, SsPEG10ΔNC-λN and SsPEG10ΔNC-MCP can be efficiently assembled into spherical VLPs with uniform morphology and a diameter of about 20 nm.

[0068] 3. SsPEG10 VLP delivery performance testing

[0069] 3.1 Detection of transduction effect (fluorescence microscopy)

[0070] HEK-293T cells in the logarithmic growth phase were seeded into 24-well cell culture plates, with 1 × 10⁶ cells per well. 5 Cells were cultured in DMEM complete medium containing 10% fetal bovine serum at 37°C with 5% CO2. After 12 hours of plating, transduction experiments were performed, with the following groups: the negative control group received an equal volume of serum-free DMEM medium; the MCP experimental group received a 2-fold diluted SsPEG10ΔNC-MCP VLP sample containing EGFP-encoded mRNA; and the λN experimental group received a 2-fold diluted SsPEG10ΔNC-λN VLP sample containing EGFP-encoded mRNA. Cells were incubated at 37°C for 24 hours, after which the VLP-containing medium was discarded and replaced with fresh DMEM complete medium, and cultured for another 24 hours. After culture, the expression of EGFP green fluorescence was observed and photographed under a fluorescence microscope.

[0071] The results are as follows Figure 4 As shown, SsPEG10ΔNC-λ delivers better results than SsPEG10ΔNC-MCP.

[0072] Example 3: Detection of PRRSV antigen delivery efficiency (Western Blot method)

[0073] SsPEG10ΔNC-λ, which has better delivery performance, was selected for PRRSV antigen cell transduction validation.

[0074] (1) Cell transfection: Cells were seeded in 6-well plates and transfected when the cell density reached 40%. The ratio of plasmid to transfection reagent was 1:3. The transfection ratio and dosage are shown in Table 3 below.

[0075] Table 3 Transfection ratio and dosage

[0076]

[0077] Six hours after transfection, the cells were replaced with fresh DMEM containing 10% FBS. Forty-eight hours after transfection, the cell supernatant was collected. The cells were centrifuged at 8000 rpm for 10 min at 4°C and filtered through a 0.45 μm filter to obtain SsPEG10ΔNC-λVLPs containing GP3-encoding mRNA. The GagΔNC-λN nucleotide and its encoded protein sequences are shown in SEQ ID NO. 5 and 6, respectively. Utilizing the highly specific RNA-protein interaction between the λN peptide and the 12×BoxB RNA stem-loop structure introduced into the 3' untranslated region (3'UTR) of GP3 mRNA, PRRSV GP3-12×BoxB was specifically packaged into VLPs self-assembled by SsPEG10. Simultaneously, vesicular stomatitis virus G glycoprotein (VSV-G) was used as the envelope protein, and pseudotype modification was employed to endow the VLPs with endocytosis-mediated membrane fusion capability for later use.

[0078] Take PK-15 cells in the logarithmic growth phase and distribute them at a rate of 1 × 10⁶ cells per well. 5 Two groups were established: a negative control group was inoculated with an equal volume of serum-free DMEM medium, and the SsPEG10 VLP group was inoculated with packaged SsPEG10ΔNC-λ VLP carrying GP3 encoding mRNA for transduction. Western blotting was performed using Flag-tagged mouse anti-mouse antibody as primary antibody and HRP-tagged goat anti-mouse antibody as secondary antibody. The results are shown in Figure 5. The SsPEG10 VLP transduction group showed a specific expression band of GP3 protein.

Claims

1. A virus-like particle based on porcine paternally expressed gene 10 (SsPEG10), characterized in that, The virus-like particles are formed by deleting the nucleocapsid (NC) domain of porcine paternal expression gene 10 and replacing it in situ with λ phage λN peptide or MS2 phage capsid protein (MCP) to obtain a recombinant fusion protein, which is then self-assembled.

2. The virus-like particle as described in claim 1, characterized in that, The amino acid sequence of the recombinant fusion protein obtained by deleting the nucleocapsid (NC) domain of porcine paternal expression gene 10 and replacing it in situ with the λ phage λN peptide is shown in SEQ ID NO. 6; the amino acid sequence of the recombinant fusion protein obtained by deleting the nucleocapsid (NC) domain of porcine paternal expression gene 10 and replacing it in situ with the MS2 phage capsid protein (MCP) is shown in SEQ ID NO.

8.

3. The virus-like particle as described in claim 1, characterized in that, It also includes co-expressing vesicular stomatitis virus G glycoprotein (VSV-G) as an envelope protein during the assembly of the virus-like particles, and endowing the virus-like particles with endocytosis-mediated membrane fusion capability through pseudotype modification.

4. The use of the virus-like particles according to any one of claims 1-3 in the preparation of mRNA delivery vectors.

5. The application as described in claim 4, characterized in that, Introducing a 12×BoxBRNA stem-loop structure or a 12×MS2 RNA stem-loop structure into the 3' untranslated region of the mRNA allows for highly specific RNA-protein interactions, enabling the mRNA to be specifically packaged into the interior of the virus-like particle.

6. The application as described in claim 5, characterized in that, The nucleotide sequences of the 12×BoxB RNA stem-loop structure and the 12×MS2 RNA stem-loop structure are shown in SEQ ID NO.10 and 12, respectively.

7. A virus-like particle mRNA delivery system based on porcine SsPEG10, characterized in that, The system includes: (1) Capsid protein granules: The capsid protein granules are plasmids expressing the modified porcine paternal expression gene 10. The modified porcine paternal expression gene 10 is obtained by deleting the nucleocapsid (NC) domain gene and replacing it in situ with λ phage λN peptide or MS2 phage capsid protein (MCP) gene based on the original gene. (2) Cargo protein particle: The cargo protein particle is a plasmid carrying the target mRNA, wherein the 3' untranslated region of the target mRNA contains a 12×BoxB RNA stem-loop structure or a 12×MS2 RNA stem-loop structure; (3) Lentiviral packaging plasmid: The lentiviral packaging plasmid is the pMD2.G plasmid expressing vesicular stomatitis virus G glycoprotein (VSV-G).

8. The mRNA delivery system as described in claim 7, characterized in that, The capsid protein particles are prepared by the following method: (1) The full length of the SsPEG10 sequence was amplified using SsPEG10-F / R as primers. The GenBank accession number of the SsPEG10 sequence is NM_001302462.

1. It was then constructed into the eukaryotic expression vector pcDNA3.1 using T4-DNA ligase. The resulting recombinant vector was named pcDNA3.1-SsPEG10. The primer sequences are as follows: SsPEG10-F: 5'-AAGCTTGGCTAGCGTTTGCCACCATGGGGCCCGACTGCCCACC-3' SsPEG10-R: 5'-CGAGGCTGATCAGCGGGTTTTTACATGGCGCTGTAAGACGGT-3' (2) Using pcDNA3.1-SsPEG10 plasmid as template and SsPEG10ΔNC-F / R as primers, the SsPEG10 sequence with the nucleocapsid domain deleted from the Gag protein was amplified and ligated to pcDNA3.1 using homologous recombinase. The resulting recombinant vector was named pcDNA3.1-SsPEG10ΔNC. The primer sequences are as follows: SsPEG10ΔNC-F: 5'-TGCATCCACATCGAGCCTGCCAAGGCCTCGAAG-3' SsPEG10ΔNC-R: 5'-AGGCTCGATGTGGATGCACTGGCCGATCAGGGC-3' (3) Using pcDNA3.1-SsPEG10ΔNC as a template, the SsPEG10ΔNC sequence missing the SsPEG10 NC domain was amplified using SsPEG10ΔNC-ZT-F / R primers for homologous recombination with λN and MCP. The MCP sequence was amplified using pMCP-RecT plasmid as a template and MCP-F / R primers. In addition, the λN sequence was generated by denaturation and annealing with primer λN-F / R. Subsequently, the amplified SsPEG10ΔNC sequence missing the SsPEG10 NC domain was ligated with the MCP sequence and λN sequence respectively by homologous recombination to obtain SsPEG10ΔNC-MCP and SsPEG10ΔNC-λN, which were then ligated with pcDNA3.1 using homologous recombination enzymes. The primer sequences are as follows: SsPEG10ΔNC-ZT-F: 5'-CCTGCCAAGGCCTCGAAG-3' SsPEG10ΔNC-ZT-R: 5'-CTCGATGTGGATGCACTGGC-3' λN-F: 5'-gccagtgcatccacatcgagGGATCCGGCGGCGGCGGCAGCAAG-3' λN-R: 5'-gccttcgaggccttggcaggCTCGAGATTGGGCGGCCTTCCACTG-3' MCP-F: 5'-gccagtgcatccacatcgagACTCCCTCTAGAGGTGGTGGGG-3' MCP-R: 5'-gccttcgaggccttggcaggCTCGAGGTAGATACCTGAGTTAGCGGC-3'.

9. The mRNA delivery system as described in claim 8, characterized in that, The sequences of GagΔNC-λN nucleotides and their encoded proteins in SsPEG10ΔNC-MCP and SsPEG10ΔNC-λN are shown in SEQ ID NO.5 and 6, respectively. The sequences of GagΔNC-MCP nucleotides and their encoded proteins are shown in SEQ ID NO.7 and 8, respectively. The sequences of Pol nucleotides and their encoded proteins are shown in SEQ ID NO.3 and 4, respectively.

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