A virus-like particle and its construction method and application

Abstract

The present invention relates to the field of biomedicine, and specifically to a virus-like particle and a construction method and application thereof. Among them, a virus-like particle can be composed of three proteins, membrane protein, envelope protein and nucleocapsid protein of a new coronavirus, or two proteins, the membrane protein and envelope protein, wherein the membrane protein is a complete membrane protein or a truncated membrane protein. The specific composition method includes plasmid construction, screening and amplification, plasmid extraction, cell transfection, and collection of virus-like particles, and the prepared virus-like particles can be used as a delivery tool for nucleic acid drugs such as RNA and DNA. The virus-like particles of the present invention are obtained by self-assembly. Compared with the active wild new coronavirus, they do not contain the genetic material of the virus itself. The virus-like particles assembled with truncated membrane proteins greatly reduce the possibility of the body's immune response to them while reducing the cell expression load, and the use of them as delivery systems for RNA, DNA, etc. is safer and more stable.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a virus-like particle and a construction method and application thereof. Background Art

[0002] Gene therapy drugs need to pass through the vascular endothelial barrier, extracellular matrix, and targeted tissue cell membranes to finally reach the target. Compared with small molecules, large molecule drugs such as nucleic acids and antibodies are more difficult to enter the target, so scientists have developed various delivery systems to help these drugs better reach the target location.

[0003] Currently, common mRNA delivery systems include viral systems and non-viral systems.

[0004] The viral system mainly includes adenovirus vectors (ADV), adeno-associated virus vectors (AAV) and lentivirus (LV) vectors. Among them, adenovirus vectors (ADV) are the most widely used viral vectors, but they are highly immunogenic and can easily cause immune responses in the body. Adeno-associated virus vectors (AAV) have a small loading capacity. Lentivirus (LV) vectors have the risk of insertion mutations and are prone to off-target effects, so their use in clinical practice is limited.

[0005] Non-viral delivery systems mainly include polymer materials, peptides, liposome nanoparticles (LNPs), etc. Among them, polymer materials are generally highly toxic and difficult to degrade in the body. Most of them are currently in the preclinical research stage. Peptide delivery carriers are mainly concentrated in cell-penetrating peptides, but this technology is not yet mature and has no targeting selectivity. mRNA preparations prepared with liposomes (LNPs) as carriers will accumulate in the liver and spleen, making it difficult to target other parts and prone to leakage.

[0006] Virus-Like Particles (VLPs) are a type of nanoparticles that can self-assemble, have the external characteristics of viruses but do not contain viral genetic material and have no infectious ability. VLPs can be obtained through a variety of expression systems, such as mammalian cells, plants, insects, and bacteria. VLPs have good biocompatibility, biodegradability, high safety of targeted delivery, large internal capacity, easy availability, and convenience of use.

[0007] VLP can deliver drugs (small molecules or nucleic acids) and can package the mRNA to be delivered. Unlike viral vectors, VLPs deliver gene editing agents instantaneously in the form of mRNA or protein rather than DNA, which greatly reduces the risk of off-target gene editing and viral genome integration. Existing commonly used delivery VLPs include bacteriophage virus-like particles (MS2VLP), hepatitis B virus-like particles (HBVVLP), papillomavirus (HPVVLP), etc. By modifying the VLP protein and mRNA, VLP can efficiently bind to mRNA and improve delivery efficiency.

[0008] The novel coronavirus (SARS-CoV-2) virus particle is about 100nm in diameter and has four structural proteins, including spike (S) protein, membrane (M) protein, envelope (E) protein and nucleocapsid (N) protein. The spike protein (1273AA in SARS-CoV-2) precursor can be cleaved into two glycosylated subunits S1 and S2. S1 binds to the host receptor ACE2, while S2 mediates the fusion of the virus and host membranes. The S protein easily causes a strong immune response in the body. The nucleocapsid protein (about 419AA in SARS-CoV-2) binds to the viral genomic RNA and forms a helical ribonucleocapsid, which is involved in genome protection, viral RNA replication, virion assembly and immune evasion. The membrane protein (about 222AA in SARS-CoV-2) is the most abundant structural component in the virion and its sequence is very conserved. The membrane protein mediates the assembly and budding of viral particles by recruiting other structural proteins to the "ER-Golgi intermediate region (ERGIC)". It interacts with the nucleocapsid protein to package the RNA into the virion. The envelope protein (about 75AA in SARS-CoV-2) is involved in virus assembly, budding, and pathogenesis. The envelope protein can form the viroporin of the homomeric pentameric ion channel and also interacts with M, N, 3a, and 7a. Existing studies have shown that coronaviruses require two or more structural proteins from M, E, S, and N to form VLPs. The new coronavirus-like particles composed of M, E, S, and N proteins are currently commonly used as mRNA vaccine vectors.

[0009] Coronavirus contains a large RNA genome (26-32kb), which can overcome the shortcomings of other vectors that carry mRNA and other nucleic acid drugs with short length. The simplest structure of the new coronavirus-like particle can be formed by two or three structural proteins of M, E and N, among which a large number of studies have reported that M and E proteins are necessary structural proteins for the formation of new coronavirus-like particles. The difficulty in whether the VLP loaded with nucleic acid drugs of SARS-CoV-2 can be used clinically is how to remove its antigenic epitopes outside the membrane and reduce immunogenicity. Therefore, in the self-assembly process of SARS-CoV-2 VLP containing only M and E or M, E, and N, truncating most of the transmembrane structure of M and the immunogenic structure outside the membrane is the main idea to solve this problem. To this end, the patent of this invention designed and verified for the first time that the truncated M protein can form the VLP of SARS-CoV-2, and can be used for drug delivery such as mRNA. Summary of the invention

[0010] The purpose of the present invention is to overcome the problems of small loading capacity, immunogenicity, and easy induction of immune response of the body in the virus delivery system of mRNA delivery system in the prior art, and to provide a virus-like particle and its construction method and application.

[0011] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0012] A virus-like particle, which is composed of three proteins, namely, membrane protein, envelope protein (E) and nucleocapsid protein (N) of the new coronavirus, or two proteins, namely, the membrane protein and envelope protein (E).

[0013] The membrane protein, envelope protein (E) and nucleocapsid protein (N) of the new coronavirus can be synthesized into virus-like particles (VLPs) through transfection. Among them, smaller virus-like particles (VLPs) can also be synthesized by only the membrane protein and envelope protein (E).

[0014] The sequence of SEQ ID No: 1 is:

[0015]

[0016] The sequence of SEQ ID No: 2 is:

[0017]

[0018]

[0019] The sequence of SEQ ID No: 3 is:

[0020]

[0021] The sequence of SEQ ID No: 4 is:

[0022]

[0023] Preferably, the membrane protein is an intact membrane protein (M) or a truncated membrane protein (Mt).

[0024] The complete membrane protein (M) includes the N-terminal extramembrane region, three transmembrane regions, and the C-terminal intramembrane region. The N-terminal extramembrane region is immunogenic and is easily recognized by antibodies outside the membrane, thereby being cleared by antibodies. The truncated membrane protein (Mt) cuts off the N-terminal extramembrane region and two transmembrane regions, solving the problem of synthetic VLPs being recognized by antibodies and thus cleared.

[0025] Preferably, the plasmid construction method of the truncated membrane protein (Mt) comprises the following steps:

[0026] T1, using KOD enzyme to perform PCR amplification on the existing pcDNA 3.4-M plasmid;

[0027] T2. Digest the PCR product with enzymes and perform electrophoresis with 1% agarose at 120V constant voltage for 30-40 minutes. Cut out a single target fragment under ultraviolet irradiation and place it in a clean EP tube for gel recovery and purification.

[0028] T3. Transform the ligation product together with competent cells, culture and amplify the transformed cells, spread the culture, screen the plasmid single colony, and obtain the plasmid of the truncated membrane protein (Mt).

[0029] The existing pcDNA 3.4-M plasmid was cut with KOD enzyme, and then the cut fragments were amplified by PCR. The DNA fragments were collected and transformed into competent cells for culture. After screening and purification, cells containing truncated membrane protein (Mt) plasmids were obtained.

[0030] Preferably, the plasmid ratio required for preparing the virus-like particles is one or more of the following: plasmid of complete membrane protein (M) gene: plasmid of envelope protein (E) gene: plasmid of nucleocapsid protein (N) gene = 4:0.2-4:0.2-4, plasmid of truncated membrane protein (Mt) gene: plasmid of envelope protein (E) gene: plasmid of nucleocapsid protein (N) gene = 4:0.2-4:0.2-4, plasmid of complete membrane protein (M) gene: plasmid of envelope protein (E) gene = 4:0.2-4, plasmid of truncated membrane protein (Mt) gene: plasmid of envelope protein (E) gene = 4:0.2-4.

[0031] Virus-like particles (VLPs) can be synthesized through the above-mentioned plasmid combinations of the new coronavirus.

[0032] A method for constructing virus-like particles comprises the following steps:

[0033] S1. Select the complete membrane protein (M), truncated membrane protein (Mt), envelope protein (E), nucleocapsid protein (N) and target gene of the new coronavirus, and construct a plasmid containing the complete membrane protein (M) gene, a plasmid containing the truncated membrane protein (Mt) gene, a plasmid containing the envelope protein (E) gene, a plasmid containing the nucleocapsid protein (N) gene, and a plasmid containing the target gene respectively;

[0034] S2, respectively introducing the above plasmids into competent cells, screening the competent cells in which each plasmid is successfully expressed, and amplifying the competent cells in which the plasmid is successfully expressed under constant temperature conditions;

[0035] S3, harvesting competent cells containing different plasmids respectively, and performing cell lysis to extract plasmids;

[0036] S4. Transfect mammalian cells with plasmids in the ratio of complete membrane protein (M) gene plasmid: envelope protein (E) gene plasmid: nucleocapsid protein (N) gene plasmid: target gene plasmid = 4: 0.2-4: 0.2-4: 0.2-4, truncated membrane protein (Mt) gene plasmid: envelope protein (E) gene plasmid: nucleocapsid protein (N) gene plasmid: target gene plasmid = 4: 0.2-4: 0.2-4, complete membrane protein (M) gene plasmid: envelope protein (E) gene plasmid: target gene plasmid = 4: 0.2-4: 0.2-4, truncated membrane protein (Mt) gene plasmid: envelope protein (E) gene plasmid: target gene plasmid = 4: 0.2-4: 0.2-4, and transfection time is 48-72 hours;

[0037] S5. Collect the supernatant of the transfected cells for ultrafiltration, and collect the purified virus-like particles.

[0038] S6. Use virus-like particles to infect normal cells and detect whether the target gene is expressed in the infected cells.

[0039] Plasmids capable of expressing four proteins are constructed respectively, and the plasmids are introduced into competent cells, competent cells expressing the plasmids normally are screened and amplified, the cells are lysed to extract the plasmids, and the protein plasmids and the target gene plasmids are transfected into mammalian cells according to a certain ratio, and cultured for 48-72 hours. The mammalian cells synthesize virus-like particles (VLPs) and secrete them into the culture environment, and the supernatant is collected for ultrafiltration and purification to obtain virus-like particles (VLPs), which contain mRNA of the target gene, and the virus-like particles (VLPs) are used to infect normal cells, and the expression of the target gene in the normal cells is detected by a detection method.

[0040] Preferably, the target gene is most eukaryotic DNA that can form mRNA, one or more of pcDNA3.1-mCherry, pEGFP-BBC3 and pEGFP-C1.

[0041] The target gene includes DNA fragments of all eukaryotic cells that can form mRNA, among which mCherry is a red fluorescent protein widely used as a tracer, and pEGFP can produce brighter enhanced green fluorescent protein in mammalian cells, both of which can be used as transfection markers to play a role in tracing and positioning; BBC3 is a kind of apoptosis gene, which will accelerate the apoptosis of the target cells after entering the target cells. The selection of target genes includes but is not limited to the above three. After the pcDNA3.1-mCherry plasmid, pEGFP-BBC3 plasmid or pEGFP-C1 plasmid is co-transfected with the plasmid that forms VLP, they are self-assembled into VLPs carrying mCherry-mRNA, pEGFP-mRNA or pEGFP-BBC3-mRNA.

[0042] Preferably, the mammalian cells in step S4 are one or more of HEK-293t cells, A549 cells and PC9 cells.

[0043] HEK-293t cells, which produce large amounts of recombinant proteins, can greatly increase the production of recombinant proteins; the A549 cell line is a widely used human lung adenocarcinoma cell line derived from primary lung tumors, is highly proliferative, and has been used in various research applications; the PC9 cell line is a cell line isolated and cultured from adenocarcinoma tissue of patients with non-small cell lung cancer.

[0044] Preferably, in step S4, the transfection process can achieve normal expression of the target gene in mammalian cells.

[0045] The supernatant of the transfected cell culture fluid contains virus-like particles (VLPs) secreted by the cells.

[0046] Preferably, in step S6, the detection method is one or more of: dynamic light scattering (DLS) analysis, protein immunoblotting (WB) detection and negative staining electron microscopy observation.

[0047] The particle size of the prepared VLP was determined by dynamic light scattering (DLS) analysis; the normal expression of the protein was detected by protein immunoblotting (WB); and the morphology of the VLP was observed by negative staining electron microscopy.

[0048] An application of a virus-like particle, wherein the virus-like particle can be used as a delivery vehicle for mRNA and drugs.

[0049] By infecting host cells with the VLP constructed as described above and culturing and expressing them, a virus-like particle delivery system encapsulating mRNA is obtained, thereby achieving efficient and safe delivery of mRNA and better exerting the therapeutic value of mRNA.

[0050] Compared with the prior art, the present invention has the following beneficial effects:

[0051] 1. The virus-like particles of the present invention are prepared by self-assembly. Compared with the active new coronavirus, they do not contain genetic material and are not easy to cause immune response in the body. They can be used as a delivery tool for nucleic acid drugs such as RNA and DNA. The delivery system has higher safety, better stability and higher effectiveness, and is not easy to cause immune response in the body.

[0052] 2. The method for constructing virus-like particles of the present invention utilizes the novel coronavirus membrane protein, envelope protein (E) and nucleocapsid protein (N) to self-assemble into virus-like particles (VLPs), truncates the complete membrane protein (M) sequence to form a truncated membrane protein (Mt), and then co-transfects into HEK-293t cells through sibling membrane protein plasmids, nucleocapsid protein plasmids and mCherry, and then infects HEK-293t cells with the supernatant to preliminarily determine whether virus-like particles are produced by fluorescence observation. The production and preparation is convenient and efficient, and can achieve good mRNA delivery effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 is a schematic diagram of an integral membrane protein (M).

[0054] Figure 2 Schematic diagram of truncated membrane protein (Mt).

[0055] Figure 3 This is a photo of 1% agarose gel electrophoresis results after Dpn I digestion. From left to right, they are: M: DNA Marker; 1, PCR product Dpn I digestion electrophoresis; 2, PCR product Dpn I digestion electrophoresis.

[0056] Figure 4 This is a photo of the 0.9% agarose gel electrophoresis result after Xba I digestion. From left to right, they are: M: DNAMarker; 1, pcDNA3.4-M plasmid; 2, pcDNA3.4-M plasmid Xba I digestion; 3, pcDNA3.4-M truncated plasmid Xba I digestion.

[0057] Figure 5This is the result of plasmid extraction. From left to right, they are M: DNA Marker; 1: pcDNA3.4-M plasmid; 2: pcDNA3.4-Mt plasmid; 3: pcDNA3.4-E plasmid; 4: pcDNA3.4-N plasmid; 5: pcDNA3.1-mCherry plasmid.

[0058] Figure 6 Figure 1 is the fluorescence staining result after plasmid transfection (×50 times). Among them, A and B are the bright field and fluorescence images of pcDNA3.4-M:pcDNA3.4-E:pcDNA3.4-N:pcDNA3.1-mCherry=4:1:1:1 plasmid co-transfection; C and D are the bright field and fluorescence images of pcDNA3.4-Mt:pcDNA3.4-E:pcDNA3.4-N:pcDNA3.1-mCherry=4:1:1:1 plasmid co-transfection; E and F are the bright field and fluorescence images of pcDNA3.4-M:pcDNA3.4-E:pcDNA3.1-mCherry=4:1:1 plasmid co-transfection; G and H are the bright field and fluorescence images of pcDNA3.4-Mt:pcDNA3.4-E:pcDNA3.1-mCherry=4:1:1 plasmid co-transfection.

[0059] Figure 7 The following are photos of the dynamic light scattering analysis test results. A is the MEN-mCherry particle size distribution obtained by dynamic light scattering analysis; B is the MtEN-mCherry particle size distribution obtained by dynamic light scattering analysis; C is the ME-mCherry particle size distribution obtained by dynamic light scattering analysis; and D is the MtE-mCherry particle size distribution obtained by dynamic light scattering analysis.

[0060] Figure 8 This is a photo of Western blotting (WB) test results. Among them, 1: purified MEN-mCherry; 2: purified ME-mCherry; 3: purified MtEN-mCherry; 4: purified MtE-mCherry; 5: empty cell pellet.

[0061] Fig. 9 These are negatively stained electron micrographs. A is MEN-mCherry; B is MtEN-mCherry; C is ME-mCherry; and D is MtE-mCherry.

[0062] Fig.10The nuclear staining images, fluorescence images and Merge images (×100 times) of HEK-293t cells infected with MEN-mCherry VLP, MtEN-mCherry VLP, ME-mCherry VLP and MtE-mCherryVLP and incubated for 48 hours.

[0063] Fig.11 The nuclear staining images, fluorescence images and Merge images (×100 times) of A549 cells infected with MEN-mCherry VLP, MtEN-mCherry VLP, ME-mCherry VLP and MtE-mCherryVLP and incubated for 48 hours.

[0064] Fig.12 The nuclear staining images, fluorescence images and Merge images (×100 times) were obtained after PC9 cells were infected with MEN-mCherry VLP, MtEN-mCherry VLP, ME-mCherry VLP and MtE-mCherry VLP and incubated for 48 hours.

[0065] Fig.13 The line graphs are the changes of cell activity over time after A549 cells or PC9 cells were infected with VLPs formed by pEGFP-C1 or pEGFP-BBC3 plasmids and MEN, ME, MtEN or NtE, respectively. Among them: A is the activity graph of A549 cells, and B is the activity graph of PC9 cells. DETAILED DESCRIPTION

[0066] In order to more clearly describe the invention purpose, technical scheme and technical effect advantages in the specific implementation case of the present invention, the scheme in the specific embodiment will be described in detail in conjunction with the drawings of the specification of the present invention. The specific technical scheme involved in the following specific embodiments is only for a clear and complete description of the innovative technical scheme of the present invention. It is only a part of the specific implementation scheme that can be adopted by the present invention, not all embodiments, and should not be understood as a limitation on the innovative scheme of the present invention. Any scheme adopting the same inventive concept of the present invention should be included in the protection scope of the present invention.

[0067] The relevant descriptions involving the drawings in the specific embodiments of the present invention are only for the convenience of technical personnel to understand the scheme of the present invention. Some details in the drawings are shown for the convenience of clearly presenting the technical scheme. It should not be considered that all technical features in the drawings must be included in the specific implementation cases, and the detailed features in the drawings cannot be regarded as additional limitations on the innovative technical scheme of the present invention.

[0068] Some of the reagents and materials used in the examples of the present invention are as follows, and commercially available raw materials are purchased and set aside.

[0069] Plasmids: pcDNA3.4-M, pcDNA3.4-E, pcDNA3.4-N, pcDNA3.1-mCherry, pEGFP-BBC3, and pEGFP-C1.

[0070] Cells: HEK-293t, A549, PC9.

[0071] Plasmid construction reagents: KOD-Plus-Neo (Toyobo, KOD-401), Dpn I (New England Biolabs, R0176V), MonClone TM Hi-Fusion Cloning Mix V2 (Monad, MC40101), Xba I (NEB, R0145V).

[0072] Transfection reagent: Lipo293 TM (Beyotime, C0521).

[0073] Culture medium: DMEM (Gibco, 11995065), FBS (ExCell, FND500).

[0074] Antibodies: Anti-SARS-CoV-2 Membrane Protein antibody (ProSci, 9165), Goat Anti-Rabbit IgG / HRP labeled (Zhongshan Jinqiao, ZB 2301).

[0075] Color developing solution: BeyoECLMoon (Beyotime, P0018FS).

[0076] CCK-8 reagent: Cell Counting Kit-8 (APExBIO, K1018).

[0077] The amino acid sequences of the complete membrane protein (M), truncated membrane protein (Mt), envelope protein (E), and nucleocapsid protein (N) of the new coronavirus are shown in Table 1.

[0078] Table 1 Membrane protein amino acid sequence table

[0079]

[0080]

[0081] Example 1

[0082] The laboratory currently has pcDNA3.4-M, pcDNA3.4-E, pcDNA3.4-N, pcDNA3.1-mCherry, pEGFP-BBC3 and pEGFP-C1 plasmids.

[0083] like Figure 1-2 As shown, the complete membrane protein (M) includes the N-terminal extramembrane region, three transmembrane regions and the C-terminal intramembrane region, wherein the amino acid chain outside the membrane is immunogenic and is easily recognized by antibodies outside the membrane, thereby being cleared. The truncated membrane protein (Mt) cuts off the N-terminal extramembrane region and two transmembrane regions, solving the problem of synthetic VLPs being recognized by antibodies and thus cleared.

[0084] The specific steps for constructing a truncated membrane protein (Mt) expression vector are as follows.

[0085] First, the existing pcDNA 3.4-M plasmid was amplified by PCR using KOD enzyme. The PCR system and reaction conditions are shown in Tables 2 and 3.

[0086] Table 2 PCR reaction system

[0087]

[0088] Table 3 PCR reaction conditions

[0089]

[0090] Then, agarose gel electrophoresis and gel recovery of target products: PCR products were digested with enzymes, electrophoresed with 1% agarose at 120V constant voltage for 30-40 minutes, and single target fragments were cut out under ultraviolet irradiation and placed in clean EP tubes for gel recovery and purification. The digestion conditions were 1 hour at room temperature. The specific reagents and their volumes are shown in Table 4.

[0091] Table 4 Enzyme digestion reaction system

[0092]

[0093] The results of Dpn I restriction enzyme electrophoresis are as follows Figure 3 As shown, from left to right are: M: DNA Marker; 1: PCR product Dpn I restriction enzyme electrophoresis; 2: PCR product Dpn I restriction enzyme electrophoresis.

[0094] Glue recovery product connection: The reaction system was placed at 50°C for 1 hour. The specific reagents and their volumes are shown in Table 5.

[0095] Table 5 Seamless cloning system

[0096]

[0097] * Make up to 10: Add ddH2O to a total volume of 10 μL.

[0098] Transformation of ligation product: The method for transformation of ligation product is as follows: take 100 μL competent cells and put them on ice to melt, take 5 μL ligation product and competent cells and mix them; ice bath for 30 minutes, heat shock in 42°C water bath for 45 seconds, and continue ice bath for 5 minutes; add 500 μL LB culture medium to the mixture, shake and culture in a shaker at 37°C, and culture at 200 rpm / min for 1 hour; after shaking, centrifuge at 3000 rpm for 2 minutes; remove part of the supernatant and keep about 200 μL of the mixed precipitate, apply it on the LB culture plate, turn the plate upside down, and culture in a 37°C constant temperature incubator for 12 to 16 hours to obtain a single colony of the recombinant plasmid.

[0099] Verification of recombinant plasmid: After the recombinant plasmid single colony was expanded by shaking, the recombinant plasmid was extracted and the recombinant plasmid was digested by enzymes. The digestion conditions were incubation at room temperature for 1 hour and incubation at 65°C for 20 minutes to inactivate the XbaⅠ enzyme. The specific reagents and their volumes are shown in Table 6.

[0100] Table 6 Recombinant plasmid verification system

[0101]

[0102]

[0103] * Make up to 10: Add ddH2O to a total volume of 10 μL.

[0104] The results of XbaⅠ restriction electrophoresis are as follows Figure 4 As shown, from left to right are: M: DNA Marker; 1: pcDNA3.4-M nucleic acid electrophoresis; 2: pcDNA3.4-M plasmid Xba I enzyme electrophoresis; 3: pcDNA3.4-Mt plasmid Xba I enzyme electrophoresis. From the 0.9% nucleic acid gel electrophoresis photo after Xba I enzyme digestion, it can be seen that the truncated membrane protein (Mt) plasmid pcDNA3.4-Mt was successfully prepared.

[0105] Example 2

[0106] Based on Example 1, virus-like particles (VLPs) were prepared by a self-assembly method, and the specific steps are as follows.

[0107] 1. Plasmid transformation:

[0108] Add 1 μL each of plasmids pcDNA3.4-M, pcDNA3.4-E, pcDNA3.4-N, pcDNA3.4-Mt and pcDNA3.1-mCherry plasmids to 100 μL of Escherichia coli competent TOP10, incubate on ice for 30 minutes, heat shock at 42°C for 90 seconds, incubate on ice for 120 seconds, add 700 μL of antibiotic-free LB, incubate at 37°C on a shaker at 220 rpm, and culture for 1 hour. Then, spread the bacterial solution on ampicillin or kanamycin-resistant LB plates and culture at 37°C overnight.

[0109] 2. Plasmid extraction:

[0110] Pick a single colony and inoculate it into 200 mL of ampicillin or kanamycin resistant LB medium, and culture it overnight at 37°C in a shaker at 220 rpm. 600 =0.5, and the bacterial culture was harvested and the plasmid was extracted using the alkaline lysis method.

[0111] Plasmid electrophoresis results are as follows Figure 5 As shown in the figure. From left to right, they are M: DNA Marker; 1: pcDNA3.4-M plasmid; 2: pcDNA3.4-Mt plasmid; 3: pcDNA3.4-E plasmid; 4: pcDNA3.4-N plasmid; 5: pcDNA3.1-mCherry plasmid. The electrophoresis results showed that the plasmid bands were in line with expectations, and the purity and concentration of the plasmids met the transfection requirements.

[0112] 3. Plasmid transfection:

[0113] Before transfection, observe the state of HEK-293t cells under a microscope to ensure that the cells are in good condition, the cell density of 10 cm culture dish is about 80%, and there is no contamination; replace 8 mL of fresh DMEM medium containing 10% FBS per culture dish; transfect the plasmids according to pcDNA3.4-M:pcDNA3.4-E:pcDNA3.4-N:pcDNA3.1-mCherry=4:1:1:1, pcDNA3.4-Mt:pcDNA3.4-E:pcDNA3.4-N:pcDNA3.1-mCherry=4:1:1:1, pcDNA3.4-M:pcDNA3.4-E:pcDNA3.1-mCherry=4:1:1, pcDNA3.4-Mt:pcDNA3.4-E:pcDNA3.1-mCherry=4:1:1. Observe the expression of the transfected plasmids by fluorescence microscopy 48-72 hours after transfection. The results are as follows: Figure 6 As shown in the figure, it can be clearly seen that after transfection, HEK-293t cells can normally express the target gene mCherry. At this time, the corresponding VLP can be obtained by collecting the cell supernatant.

[0114] 4.VLP purification:

[0115] 48 hours after transfection, the supernatant of HEK-293t cell culture fluid was collected once, and then 4 mL of culture medium was added to continue culturing for 72 hours, and the supernatant was collected again, about 15 mL / dish was placed in a sterile 50 mL centrifuge tube, centrifuged at 2000g for 10 minutes to remove cell debris, and then the supernatant was filtered with a 0.45μm filter membrane; 20% sucrose was prepared: 10 g of sucrose was dissolved in 40 mL of 1×PBS, and after dissolution, it was made up to 50 mL and filtered through a 0.22μm filter membrane for use.

[0116] The pretreated cell culture fluid was dispensed into sterile dry ultrafiltration tubes, 4 mL per tube, centrifuged at 4°C, 3000 rpm for 10 minutes, and the remaining liquid above the filter membrane was collected; 6 mL of 20% sucrose solution was added to the ultracentrifuge tube, and 3 mL of the supernatant after ultrafiltration and concentration was gently added using a Pasteur pipette; the supernatant was carefully aspirated, and 1×PBS was added to dissolve the precipitate by blowing to obtain VLP products, which were VLPs (MEN-mCherry) self-assembled by M protein, E protein, N protein and mCherry, VLPs (MtEN-mCherry) self-assembled by M protein, E protein, N protein and mCherry, VLPs (ME-mCherry) self-assembled by M protein, E protein and mCherry, and VLPs (MtE-mCherry) self-assembled by Mt protein, E protein and mCherry, and the VLPs were stored at 4°C.

[0117] 5. Dynamic Light Scattering (DLS) Analysis:

[0118] Take 20 μL of the sample after 20% sucrose ultracentrifugation and dilute it in 4 mL of deionized water. TM 500) to determine the particle size distribution, and the dynamic light scattering analysis results are as follows Figure 7 shown.

[0119] A, B, C, and D are the particle size distributions of MEN-mCherry, MtEN-mCherry, ME-mCherry, and MtE-mCherry VLPs, respectively, which are generally consistent with the particle size characteristics of typical VLPs.

[0120] 6.WB detection (Western blotting, protein blotting):

[0121] Take 10 μL of BCA quantitatively purified VLPs, add 2 μL SDS PAGE Loading Buffer (Sangon Biotech, C516031-0005), treat in a metal bath at 95°C for 10 minutes, centrifuge at 12000g for 10 minutes, take the supernatant and load it on SDS-PAGE protein gel (Beyotime, P0012A), protein electrophoresis 80-120v, 90 minutes. Transfer to PVDF membrane at 100v for 15-30 minutes, and block with 5% blocking solution for 1 hour. The primary antibody (Anti-SARS-CoV-2 Membrane Protein antibody) was diluted 1:1000 with 5% blocking solution and incubated overnight at 4°C. Wash 3 times with PBST, 10 minutes each time. The secondary antibody (Goat Anti-Rabbit IgG (H+L)) was diluted 1:3000 with 1% blocking solution and incubated at room temperature for 1 hour. Wash 3 times with TBST, 10 minutes each time. Prepare the color developing solution BeyoECLMoon (Beyotime, P0018FS). Use E-BLOT (eBLOT, Touch imager XLi) contact chemiluminescence imager for imaging.

[0122] WB results are as follows Figure 8 As shown. In the figure, 1: purified MEN-mCherry VLP; 2: purified ME-mCherry VLP; 3: purified MtEN-mCherry VLP; 4: purified MtE-mCherry VLP; 5: empty cell precipitate. The results of Western-blot identification of the purified samples using the Anti-SARS-CoV-2 Membrane Protein antibody showed that the presence of Membrane protein could be detected in lanes 1, 2, 3, and 4, indicating that the VLP was successfully assembled.

[0123] 7. Negative staining electron microscopy observation:

[0124] The sample purified by 20% sucrose ultracentrifugation was dropped onto the transmission electron microscope copper grid, incubated for 5 minutes, negatively stained with 1% phosphotungstic acid for 3 minutes, and observed under an electron microscope.

[0125] Negative staining electron microscopy results Fig. 9 As shown. A is MEN-mCherry VLP; B is MtEN-mCherry VLP; C is ME-mCherry VLP; D is MtE-mCherry VLP.

[0126] Example 3

[0127] Based on Example 1 and Example 2, virus-like particles (VLPs) were used for mRNA gene delivery through a self-assembly method.

[0128] VLPs were incubated with cells (infected) and observed:

[0129] 24 hours before the experiment, 5×10 3 HEK-293t, 5×10 3 A549 or 1×10 4 PC9 cells were seeded in each well with 5 × 10 3 5 μL / well of the VLPs harvested in Example 2 were inoculated into 24-well cell culture plates. The plates were placed in a 37°C, 5% CO2 incubator for culture. After 24 hours, the expression of mCherry was observed using a fluorescence microscope.

[0130] Infection results such as Fig.10 , Fig.11 and Fig.12 As shown, VLP infection fluorescence images ×100.

[0131] in, Fig.10 Fluorescence images after MEN-mCherry VLP, MtEN-mCherry VLP, ME-mCherry VLP and MtE-mCherry VLP were co-incubated with HEK-293t cells for 48 hours;

[0132] Fig.11 Fluorescence images after MEN-mCherry VLP, MtEN-mCherry VLP, ME-mCherry VLP and MtE-mCherryVLP were co-incubated with A549VLP cells for 48 hours;

[0133] Fig.12 Fluorescence images after MEN-mCherry VLP, MtEN-mCherry VLP, ME-mCherry VLP and MtE-mCherry VLP were co-incubated with PC9 cells for 48 hours;

[0134] The infection fluorescence results showed that HEK-293t, A549 and PC9 cells all expressed red fluorescent protein, indicating that the VLP delivery system of the present invention can efficiently deliver the loaded mRNA to a variety of cells including tumor cells.

[0135] Based on the experimental results of the above-mentioned embodiments 1 to 3, we summarize as follows:

[0136] 1. Virus-like particles can be produced by co-transfection of SARS-COV-2 M, E and N proteins, Mt, E and N proteins, M and E proteins, and Mt and E proteins. The simplest system and plasmid ratio for transfection to produce VLPs is Mt:E=4:1.

[0137] 2. Virus-like particles with a diameter of about 160 nm were successfully obtained by co-transfection of SARS-COV-2 Mt and E proteins.

[0138] 3. By co-transfecting SARS-COV-2 M, E, N proteins and mCherry proteins, Mt, E, N proteins and mCherry proteins, Mt, E proteins and mCherry proteins, and Mt, E proteins and mCherry proteins, the harvested VLPs can transfer the mCherry gene to HEK-293t, A549 and PC9 cells, and express mCherry in HEK-293t, A549 and PC9 cells.

[0139] The method of the present invention can obtain high-quality virus-like particles, which can be used in mRNA delivery systems, exert good tumor cell affinity, and achieve excellent targeted drug delivery effects.

[0140] Example 4

[0141] On the basis of Example 1, Example 2 and Example 3, the use of virus-like particles (VLP) for anti-tumor mRNA gene delivery was verified by a self-assembly method, and the specific steps are as follows.

[0142] 1. Plasmid transfection:

[0143] The plasmids pEGFP-C1 and pEGFP-BBC3 were extracted and transfected according to the method described in Example 2. The plasmids were transfected into HEK-293t cells according to the following ratios:

[0144] pcDNA3.4-M: pcDNA3.4-E: pcDNA3.4-N: pEGFP-C1=4:1:1:1;

[0145] pcDNA3.4-M: pcDNA3.4-E: pcDNA3.4-N: pEGFP-BBC3=4:1:1:1;

[0146] pcDNA3.4-Mt: pcDNA3.4-E: pcDNA3.4-N: pEGFP-C1=4:1:1:1;

[0147] pcDNA3.4-Mt: pcDNA3.4-E: pcDNA3.4-N: pEGFP-BBC3=4:1:1:1;

[0148] pcDNA3.4-M: pcDNA3.4-E: pEGFP-C1=4:1:1;

[0149] pcDNA3.4-M: pcDNA3.4-E: pEGFP-BBC3=4:1:1;

[0150] pcDNA3.4-Mt:pcDNA3.4-E:pEGFP-C1=4:1:1;

[0151] pcDNA3.4-Mt:pcDNA3.4-E:pEGFP-BBC3=4:1:1.

[0152] 48-72 hours after transfection, the cell supernatant was collected, and the supernatant contained the corresponding virus-like particles.

[0153] 2. Purification of VLPs:

[0154] 48 hours after transfection, the supernatant of HEK-293t cell culture fluid was collected once, and then 4 mL of culture medium was added to continue culturing for 72 hours, and the supernatant was collected again, about 15 mL / dish was placed in a sterile 50 mL centrifuge tube, centrifuged at 2000g for 10 minutes to remove cell debris, and then the supernatant was filtered with a 0.45μm filter membrane; 20% sucrose was prepared: 10 g of sucrose was dissolved in 40 mL of 1×PBS, and after dissolution, it was made up to 50 mL and filtered through a 0.22μm filter membrane for use.

[0155] The pretreated cell culture fluid was dispensed into sterile dry ultrafiltration tubes, 4 mL per tube, centrifuged at 4°C, 3000 rpm for 10 minutes, and the remaining liquid on the filter membrane was collected; 6 mL of 20% sucrose solution was added to the ultracentrifuge tube, and 3 mL of the ultrafiltration concentrated supernatant was gently added using a Pasteur pipette; centrifuged at 4°C, 30000 rpm for 3 hours in an ultracentrifuge; the supernatant was carefully aspirated, and 1×PBS was added to dissolve the precipitate by pipetting to obtain VLPs, which were stored at 4°C.

[0156] 3. Anti-tumor cell experiment (CCK-8):

[0157] 24 hours before the experiment, 2.5 × 10 3 A549 or PC9 cells. The medium was replaced with DMEM + 10% FBS for the last time. 0.5 μL / well of the VLPs harvested above were inoculated into a 96-well plate. The plate was cultured in a 37°C, 5% CO2 incubator. 10 μL of CCK8 reagent was added to each well after 24, 48, 72, and 96 hours of incubation. The absorbance was measured at 450 nm after incubation at 37°C for 1 hour. The experimental results are shown in the figure. Fig.13As shown, the results showed that VLPs carrying BBC3 had a significant killing effect on tumor cells in A549 and PC9 cells.

[0158] For those skilled in the art, when understanding the solutions described in the specific embodiments of the present invention, they can refer to the conventional technical manuals in the field. At the same time, for the places where the above-mentioned terms appear, they can make appropriate understandings or adjustments for reference, and deduce the implementation of the same or similar technical solutions without paying any creative work.

Claims

1. A virus-like particle, characterized in that The virus-like particles are composed of three proteins, namely, membrane protein, envelope protein and nucleocapsid protein of the new coronavirus, or are composed of two proteins, namely, membrane protein and envelope protein. The sequence of the envelope protein is SEQ ID No: 1, the sequence of the nucleocapsid protein is SEQ ID No: 2, the membrane protein is a truncated membrane protein, and the sequence of the truncated membrane protein is SEQ ID No:

4.

2. A virus-like particle according to claim 1, characterized in that: The plasmid construction method of the truncated membrane protein comprises the following steps: T1, using KOD enzyme to perform PCR amplification on the existing pcDNA 3.4-M plasmid; T2. Digest the PCR product with enzymes and perform electrophoresis on 1% agarose at 120V constant voltage for 30-40 minutes. Cut out a single target fragment under ultraviolet irradiation and place it in a clean EP tube for gel recovery and purification. T3. Transform the ligation product together with competent cells, culture and amplify the transformed cells, spread the culture, screen the plasmid single colony, and obtain the plasmid of the truncated membrane protein.

3. A virus-like particle according to claim 2, characterized in that: The plasmid ratio required for preparing the virus-like particles is one or more of the following: plasmid of complete membrane protein gene: plasmid of envelope protein gene: plasmid of nucleocapsid protein gene = 4: 0.2-4: 0.2-4, plasmid of truncated membrane protein gene: plasmid of envelope protein gene: plasmid of nucleocapsid protein gene = 4: 0.2-4: 0.2-4, plasmid of complete membrane protein gene: plasmid of envelope protein gene = 4: 0.2-4, plasmid of truncated membrane protein gene: plasmid of envelope protein gene = 4: 0.2-4.

4. A method for constructing a virus-like particle, characterized in that: The method for constructing a virus-like particle according to any one of claims 1 to 3 comprises the following steps: S1. Select the complete membrane protein, truncated membrane protein, envelope protein, nucleocapsid protein and target gene of the new coronavirus, and construct a plasmid containing the complete membrane protein gene, a plasmid containing the truncated membrane protein gene, a plasmid containing the envelope protein gene, a plasmid containing the nucleocapsid protein gene and a plasmid containing the target gene respectively; S2, respectively introducing the above plasmids into competent cells, screening the competent cells in which each plasmid is successfully expressed, and amplifying the competent cells in which the plasmid is successfully expressed under constant temperature conditions; S3, harvesting competent cells containing different plasmids respectively, and performing cell lysis to extract plasmids; S4. Transfect mammalian cells with plasmids in the ratio of complete membrane protein gene plasmid: envelope protein gene plasmid: nucleocapsid protein gene plasmid: target gene plasmid = 4: 0.2-4: 0.2-4: 0.2-4, truncated membrane protein gene plasmid: envelope protein gene plasmid: nucleocapsid protein gene plasmid: target gene plasmid = 4: 0.2-4: 0.2-4: 0.2-4, complete membrane protein gene plasmid: envelope protein gene plasmid: target gene plasmid = 4: 0.2-4: 0.2-4, truncated membrane protein gene plasmid: envelope protein gene plasmid: target gene plasmid = 4: 0.2-4: 0.2-4, and the transfection time is 48-72 hours; S5, collecting the supernatant of the transfected cells for ultrafiltration, and collecting the purified virus-like particles; S6. Use virus-like particles to infect normal cells and detect whether the target gene is expressed in the infected cells.

5. The method for constructing a virus-like particle according to claim 4, characterized in that: The target gene is a eukaryotic DNA capable of forming mRNA, and the target gene includes one or more of pcDNA3.1-mCherry, pEGFP-BBC3 and pEGFP-C1.

6. The method for constructing a virus-like particle according to claim 4, characterized in that: The mammalian cells in step S4 are one or more of HEK-293t cells, A549 cells and PC9 cells.

7. The method for constructing a virus-like particle according to claim 4, characterized in that: In step S4, the transfection process can achieve normal expression of the target gene in mammalian cells.

8. The method for constructing a virus-like particle according to claim 4, characterized in that: In step S6, the detection method is one or more of dynamic light scattering analysis, protein immunoblotting detection and negative staining electron microscopy observation.

9. An application of a virus-like particle, characterized in that: The virus-like particle as described in any one of claims 1 to 8 can be used as a delivery vehicle for nucleic acid drugs.

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