A human metapneumovirus virus-like particle, preparation method, application and vaccine

Abstract

This invention discloses a human metapneumovirus virus-like particle, its preparation method, its application, and a vaccine. The human metapneumovirus virus-like particle is assembled from the M1 protein of influenza virus H2N2 and the F protein of human metapneumovirus. The nucleic acid sequence encoding the M1 protein is shown in SEQ ID NO:1, and the nucleic acid sequence encoding the F protein is shown in SEQ ID NO:2. The preparation method includes (1) constructing a recombinant plasmid: cloning the M1 gene of influenza virus H2N2 and the F gene sequence of human metapneumovirus into the pFastBacDual vector to obtain the recombinant plasmid pFastBacDual-M1-F; (2) transfection: transfecting the recombinant plasmid pFastBacDual-M1-F into sf9 insect cells to obtain recombinant sf9 cells; (3) isolation and purification: culturing the recombinant sf9 cells and centrifuging to purify and obtain human metapneumovirus virus-like particles (VLPs). This invention uses a baculovirus insect cell expression system to prepare virus-like particles based on HMPV F protein and influenza virus M1 protein, which have immunogenicity and excellent immunoprotective effects, providing theoretical data and laying the foundation for the development of HMPV vaccines, especially VLP vaccines.

Description

Technical Field

[0001] This invention belongs to the field of virology, specifically relating to a human metapneumovirus virus-like particle, its preparation method, its application, and a vaccine. Background Technology

[0002] Human metapneumovirus (HMPV) is a new member of the Pneumovirinae subfamily of the Pneumoviridae family, first isolated in 2001. It can infect people of all ages, with higher infection rates in infants, the elderly, and immunocompromised individuals. Serological studies show that the HMPV antibody positivity rate is 100% in 5-year-old children. HMPV mainly circulates during the winter and spring seasons. Currently, no HMPV vaccine has been approved for marketing. HMPV vaccines under investigation include live attenuated vaccines, subunit protein vaccines, formalin-inactivated vaccines, and CD8+ vaccines. + T-cell (TCD8) epitope vaccines. However, live attenuated vaccines are contraindicated in immunocompromised individuals, subunit protein vaccines have lower immunogenicity than live attenuated and inactivated vaccines, TCD8 epitope vaccines do not completely protect against viral infection, and HMPV inactivated vaccines can lead to antibody-dependent enhancement.

[0003] Virus-like particles (VLPs) are particulate substances formed by the self-assembly of viral proteins. They do not contain viral nucleic acids and have a morphological structure similar to viruses. VLPs not only have good safety profiles but also retain the spatial conformation of natural viral particles and antigenic epitopes that induce neutralizing antibodies, effectively inducing humoral and cellular immune responses. Therefore, VLPs are promising novel vaccine candidates, and VLP vaccines such as those for human papillomavirus (HPV) and hepatitis B virus (HBV) are already in clinical use.

[0004] Therefore, if a human metapneumovirus virus-like particle with good immunoprotective effect could be provided, it would have significant theoretical and social implications.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The technical problem this invention aims to solve is to overcome the shortcomings of existing technologies and provide a human metapneumovirus (HMPV) virus-like particle, its preparation method, its application, and a vaccine. This invention uses a baculovirus insect cell expression system to prepare virus-like particles based on HMPV F protein and influenza virus M1 protein, which exhibit immunogenicity and excellent immunoprotective effects, providing theoretical data and laying the foundation for the development of HMPV vaccines, especially VLP vaccines.

[0007] To solve the above-mentioned technical problems, the basic concept of the technical solution adopted by the present invention is as follows:

[0008] The first objective of this invention is to provide a human metapneumovirus virus-like particle (HMPV VLP) assembled from the M1 protein of influenza virus H2N2 and the F protein of human metapneumovirus.

[0009] The fusion protein F of human metapneumovirus (HMPV F protein) is a membrane glycoprotein that mediates viral penetration and cell fusion. It is a major protective antigen of the virus, and the neutralizing antibodies it induces can protect the body from infection by different subtypes of the virus. The matrix protein M1 of influenza virus H2N2 plays a crucial role in influenza virus replication, acting from viral entry to assembly.

[0010] In this invention, human metapneumovirus virus-like particles are assembled from the M1 protein of influenza virus H2N2 and the F protein of human metapneumovirus. They are structurally stable during expression, have high preparation efficiency, and the prepared virus-like particles have good immunogenicity, thus achieving excellent immunoprotective effects.

[0011] A further embodiment is provided, with the nucleic acid sequence encoding the M1 protein shown in SEQ ID NO:1 and the nucleic acid sequence encoding the F protein shown in SEQ ID NO:2.

[0012] In this invention, the coding sequence of the protein was optimized according to the codon preference of sf9 insect cells, which is more conducive to the preparation of HMPV VLP using the baculovirus insect cell expression system, and can improve the expression level and obtain HMPV VLP better.

[0013] A second objective of this invention is to provide a method for preparing human metapneumovirus virus-like particles as described above, comprising the following steps:

[0014] (1) Construction of recombinant plasmid: The M1 gene of influenza virus H2N2 and the F gene sequence of human metapneumovirus were cloned into the pFastBacDual vector to obtain the recombinant plasmid pFastBacDual-M1-F;

[0015] (2) Transfection: The recombinant plasmid pFastBacDual-M1-F was transfected into sf9 insect cells to obtain recombinant sf9 cells;

[0016] (3) Separation and purification: Recombinant sf9 cells were cultured and purified by centrifugation to obtain human metapneumovirus virus-like particles (VLPs).

[0017] This invention utilizes a baculovirus insect cell expression system to prepare HMPV VLPs. The baculovirus insect cell expression system is one of the four major expression systems in modern genetic engineering, characterized by safety, high efficiency, and structural and functional similarity between the expressed product and the natural protein. It has been successfully used to construct various virus-like particles. Although baculovirus expression systems are becoming increasingly mature, factors such as multiplicity of infection, infection time, and cell density still significantly impact large-scale production. Therefore, this invention focuses on exploring and optimizing the factors affecting baculovirus system expression.

[0018] In a further step, in step (3), recombinant sf9 cells are cultured using adherent culture to prepare VLP.

[0019] This invention utilizes two methods, adherent culture and suspension culture of sf9 insect cells, to express HMPV F protein in order to explore the optimal expression mode of the target protein. Ultimately, it was found that HMPVVLP can be better prepared by adhering culture of sf9 cells.

[0020] In a further step, in step (3), the recombinant sf9 cells are subjected to multiple generations of expanded culture and purification.

[0021] Preferably, it has undergone at least four generations of expansion culture, namely P1, P2, P3, and P4.

[0022] Further options, including expanding the culture and purification methods, include:

[0023] (1) After transfecting sf9 insect cells with recombinant plasmid, the supernatant of diseased cells was harvested 72 hours later, which is the P1 generation recombinant baculovirus;

[0024] (2) P1 generation recombinant baculovirus was added to sf9 insect cells in the logarithmic growth phase, cultured at 27°C for 72 hours, and the recombinant baculovirus was passaged to P4 generation. The supernatant and cells of the diseased cells were harvested.

[0025] (3) Collect the supernatant and cells of P4 generation, concentrate them by ultracentrifugation, and then resuspend them in PBS to obtain purified human metapneumovirus virus-like particles (VLPs).

[0026] The HMPV virus-like particle (VLP) was successfully expressed in sf9 cells. The expanded culture and purification method of this invention can realize the large-scale production of HMPV VLP, which is conducive to further promoting large-scale production.

[0027] A third objective of this invention is to provide a recombinant vector expressing human metapneumovirus virus-like particles as described above, using pFastBacDual as the expression vector, and inserting the M1 gene of influenza virus H2N2 and the F gene sequence of human metapneumovirus.

[0028] A fourth objective of this invention is to provide a recombinant cell comprising the carrier described above;

[0029] Preferably, the recombinant cells are sf9 insect cells containing the vector described above.

[0030] The fifth objective of this invention is to provide the use of the human metapneumovirus virus-like particles as described above in the preparation of drugs for the prevention and / or treatment of human metapneumovirus;

[0031] Preferred application of human metapneumovirus virus-like particles in the preparation of human metapneumovirus vaccines.

[0032] The sixth objective of this invention is to provide a human metapneumovirus vaccine comprising human metapneumovirus virus-like particles as described above.

[0033] Further options include pharmaceutically acceptable excipients for human metapneumovirus vaccines.

[0034] Preferably, the excipients include adjuvants;

[0035] In a preferred embodiment, the human metapneumovirus vaccine comprises human metapneumovirus virus-like particles and aluminum hydroxide adjuvant.

[0036] This invention demonstrates that intramuscular injection of HMPV VLP into mice induces specific humoral and cellular immune responses and produces good immunoprotective effects after HMPV challenge. Aluminum hydroxide adjuvant can enhance the immunogenicity and immunoprotective effects of HMPV VLP.

[0037] By adopting the above technical solution, the present invention has the following beneficial effects compared with the prior art:

[0038] 1. The human metapneumovirus virus-like particles of the present invention are assembled from the optimized M1 protein of influenza virus H2N2 and the F protein of human metapneumovirus, and have immunogenicity. Intramuscular injection of HMPV VLP into mice can induce humoral and cellular immune responses, and has excellent immunoprotective effect.

[0039] 2. Currently, there are no reports domestically or internationally on the preparation of HMPV VLPs using baculovirus insect cell expression systems. This invention establishes a method for preparing VLPs using baculovirus insect cells, explores and optimizes expression conditions, determines the optimal experimental conditions, and obtains HMPV VLPs. Specifically, this invention utilizes adherent sf9 cell culture, and through P1, P2, P3, and P4 generation expansion culture, can obtain a large quantity of HMPV VLPs.

[0040] 3. The combination of human metapneumovirus virus-like particles (HMPV) and aluminum hydroxide adjuvant in this invention can enhance the level of immune response. After HMPV virus challenge, VLP can produce effective immune protection.

[0041] The specific embodiments of the present invention will now be described in further detail with reference to the accompanying drawings. Attached Figure Description

[0042] The accompanying drawings, as part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments and descriptions of the invention are used to explain the invention, but do not constitute an undue limitation of the invention. Obviously, the drawings described below are merely some embodiments, and those skilled in the art can obtain other drawings based on these drawings without creative effort. In the drawings:

[0043] Figure 1 This is the PCR identification result of the recombinant plasmid pFastBacDual-M1;

[0044] Figure 2 These are the results of double enzyme digestion identification of plasmids pMD18-TF and pFastBacDual-M1; from left to right: pMD18-TF plasmid; pMD18-TF double enzyme digestion; pFastBacDual-M1 plasmid; pFastBacDual-M1 double enzyme digestion; DNAMarker15000;

[0045] Figure 3 This is the PCR identification result of the F gene of pFastBacDual-M1-F;

[0046] Figure 4 These are the PCR identification results of pFastBacDual-M1-F using the M13 primer; the two wells on the left: pFastBacDual-M1-F gene (4194bp); the two wells in the middle: pFastBacDual (2560bp); the last well: DNAmarker 15000;

[0047] Figure 5 This is a lesion image of pFastBacDual-M1-F transfected sf9 cells;

[0048] Figure 6 These are the results of Western blotting identification of influenza virus M1 protein (a) and HMPV F protein (b);

[0049] Figure 6 From left to right in b, the results are: 1 μg transfection with pFastBacDual-M1-F plasmid; 2 μg transfection with pFastBacDual-M1-F plasmid;

[0050] Figure 7 HMPV expression in sf9 insect cells was observed under a VLP electron microscope.

[0051] Figure 8 Serum IgG antibody levels in mice immunized with HMPV VLP;

[0052] Figure 9 The number of IFN-γ and IL-4 lymphocytes stimulated in HMPV VLP-immunized mice;

[0053] Figure 10 This refers to the change in body weight of mice immunized by intramuscular injection after HMPV challenge;

[0054] Figure 11 This involves the determination of viral load in the lungs of mice after HMPV challenge.

[0055] Figure 12 This is an immunohistochemical analysis of lung tissue from HMPV VLP-immunized mice.

[0056] It should be noted that these accompanying drawings and textual descriptions are not intended to limit the scope of the invention in any way, but rather to illustrate the concept of the invention to those skilled in the art by referring to specific embodiments. Detailed Implementation

[0057] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments will be clearly and completely described below with reference to the accompanying drawings. The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0058] Example 1: Preparation of HMPV VLP using a baculovirus insect cell expression system

[0059] (1) Construction of recombinant plasmid pFastBacDual-M1-F: The M1 gene sequence of influenza A / Ann Arbor / 6 / 1960 (H2N2) and the HMPV F gene were optimized according to the codon preference of sf9 insect cells. The M1 gene and F gene were synthesized and ligated into pMD18-T, respectively, to obtain pMD18-T-M1 and pMD18-TF. pMD18-T-M1 and pFastBacDual were digested with SphI and NheI, and then the digestion products were ligated. The resulting recombinant plasmid was named pFastBacDual-M1, and pFastBacDual-M1 was identified by PCR. pMD18-TF and pFastBacDual-M1 were digested with BamHI and NotI, and the digestion products were ligated and named pFastBacDual-M1-F. Then, PCR and enzyme digestion identification were performed.

[0060] The gene sequence of influenza A / Ann Arbor / 6 / 1960 (H2N2) M1 is shown in SEQ ID NO:1, and the sequence of HMPV F gene is shown in SEQ ID NO:2. Primers for the M1 and F genes are shown in Table 1.

[0061] Results: pMD18-T-M1 and pFastBacDual were double-digested with SphI and NheI, and the digestion products were ligated. PCR confirmed successful ligation (pFastBacDual-M1, e.g., ...). Figure 1 (As shown). pMD18-TF and pFastBacDual-M1 were double-digested with BamHI and NotI as shown. Figure 2 As shown, the enzyme digestion products were ligated overnight, and the PCR identification results of the ligation products are as follows. Figure 3 As shown, specific bands are visible, indicating that the recombinant baculovirus plasmid pFastBacDual-M1-F was successfully constructed.

[0062] Table 1 Primers

[0063]

[0064] (2) Recombinant plasmid pFastBacDual-M1-F was transformed into DH10 Bac competent cells.

[0065] pFastBacDual-M1-F and pFastBacDual were transformed into DH10Bac competent cells. The cells were then plated onto polyclonal LB agar plates at three dilutions: 1:10, 1:100, and 1:1000. F gene-positive colonies were screened by PCR identification using the M13 primer and sequencing. The M13 primer PCR identification results for pFastBacDual-M1-F are shown below. Figure 4 As shown.

[0066] (3) Transfection with recombinant baculovirus plasmid pFastBacDual-M1-F

[0067] The recombinant plasmid pFastBacDual-M1-F was transfected into sf9 cells, and VLP expression was performed using adherent sf9 cells. The expression of M1 and F proteins was expanded by P1, P2, P3 and P4 culture, and the expression of M1 and F proteins was identified by Western blot.

[0068] Specific methods for expanding cultivation:

[0069] (1) After transfecting sf9 insect cells with recombinant plasmid, the supernatant of diseased cells was harvested 72 hours later, which is the P1 generation recombinant baculovirus;

[0070] (2) P1 generation recombinant baculovirus was added to sf9 insect cells in the logarithmic growth phase, cultured at 27°C for 72 hours, and the recombinant baculovirus was passaged to P4 generation. The supernatant and cells of the diseased cells were harvested.

[0071] (3) Collect the supernatant and cells of P4 generation, concentrate them by ultracentrifugation, and resuspend them in PBS to obtain purified human metapneumovirus virus-like particles (VLPs). The morphology and structure of VLPs were identified by transmission electron microscopy.

[0072] Results: Microscopic observation revealed significant cytopathic effects in the pFastBacDual-M1-F transfection wells (e.g., Figure 5 As shown). Western blot analysis confirmed successful expression of influenza M1 protein (28 kDa) and HMPV F protein (62 kDa) using a rabbit-derived HMPV M1 protein polyclonal antibody as the primary antibody and goat anti-rabbit HRP-labeled IgG as the secondary antibody; and a mouse-derived HMPV F protein monoclonal antibody as the primary antibody and goat anti-mouse HRP-labeled IgG as the secondary antibody). Figure 6 a and 6b).

[0073] M1 and F proteins were expressed through culture expansion in P1, P2, P3, and P4. HMPV VLPs were obtained through purification, and the VLPs were visible under a negative-stain electron microscope. Figure 7 As shown.

[0074] Example 2: Preparation and Immunogenicity Study of HMPV VLP Vaccine

[0075] 1. Method

[0076] (1) Preparation of HMPV VLP vaccine

[0077] A. HMPV VLP vaccine:

[0078] The adjuvant-containing group: each mouse received an immunization dose of 5 μg VLP and 1 mg / ml Al(OH)3.

[0079] The immunization dose per mouse in the adjuvant-free group was 5 μg VLP.

[0080] B. PBS-Adjuvant Control: Contains no antigen, only 1 mg / ml Al(OH)3 adjuvant.

[0081] (2) Mouse immunization

[0082] BALB / C mice were randomly divided into six groups of 10 mice each. The vaccine prepared in (1) was administered to BALB / C mice via intramuscular injection on days 0, 21 and 42.

[0083] The experimental group included an immunization group containing adjuvant and VLP, an immunization group containing only VLP without adjuvant (A), and a control group containing only adjuvant without antigen (B).

[0084] (3) Sample collection and testing

[0085] Five mice were used in each group. Serum was collected on days 21, 42, and 56 after immunization. The serum total IgG antibody level and neutralizing antibody titer were detected by ELISA, and the distribution of IgG1, IgG2a, IgG2b, and IgG3 antibody subtypes was analyzed. Bronchoalveolar lavage fluid and spleen lymphocytes were collected on day 56. The sIgA level was detected by ELISA, and the cellular immune response was detected by ELISPOT.

[0086] 2. Test Results

[0087] 2.1 Humoral immunity level

[0088] (1) Detection of serum-specific total IgG antibody levels: such as Figure 8 As shown, with the increase of immunization times, the serum IgG antibody level induced by HMPV VLP immunization in mice gradually increased. The serum total IgG antibody level in the adjuvanted HMPV VLP group was higher than that in the adjuvant-free VLP group. No serum IgG antibody was produced in the adjuvant control group. This indicates that intramuscular injection of HMPV VLP can produce humoral immunity, and aluminum hydroxide adjuvant can enhance the humoral immune response.

[0089] (2) Serum-specific total IgG subtype detection: The serum of HMPV VLP-immunized mice was diluted 100 times with PBS. 80 μL of the diluted serum was added to the detection well of the mouse antibody subtype rapid detection card. Multiple heavy chain lines and multiple light chain lines appeared. The serum antibody was mainly IgG2b subtype based on the intensity of the red lines.

[0090] (3) Detection of serum neutralizing antibodies: A mixture of serially diluted immunized mouse serum and HMPV virus (GenBank: MN745087.1) was added to LLC-MK2 cells and cultured for 7 days before detecting the viral copy number. The results showed that the serum of mice immunized by intramuscular injection of HMPV VLP containing adjuvant had a virus neutralization efficiency of 73.86% at a dilution of 1:80, while the serum of the VLP immunized group without adjuvant had a virus neutralization efficiency of 35.75%, and the serum of the adjuvant control group had a virus neutralization efficiency of 6.1%, indicating that mice immunized by intramuscular injection of HMPV VLP can produce serum neutralizing antibodies.

[0091] 2.2 Cellular immune level

[0092] For the detection of memory B lymphocytes, the number of IFN-γ spots in the adjuvanted HMPV VLP intramuscular injection immunization group was higher than that in the non-adjuvanted HMPV VLP immunization group and the adjuvant control group (P < 0.05 and P = 0.01, respectively). The number of IL-4 spots in the adjuvanted HMPV VLP immunization group was higher than that in the non-adjuvanted HMPV VLP immunization group and the adjuvant control group (P ≤ 0.01). The results are as follows: Figure 9 As shown in the figure. The results indicate that intramuscular injection of HMPV VLP into mice induces a cellular immune response, and aluminum hydroxide adjuvant enhances the level of cellular immunity.

[0093] Example 3: Detection of HMPV challenge and VLP immunoprotective effect

[0094] On day 28 after the third immunization of mice, they were challenged with HMPV (GenBank: MN745087.1) via the nasal mucosa, and then the following tests were performed:

[0095] (1) Clinical observation and survival analysis: Monitor the survival rate, mental state and weight changes of mice 0-14 days after infection.

[0096] (2) Detection of viral load in the lungs: Five days after HMPV challenge, three mice in each group were taken, their lungs were aseptically removed, and nucleic acid was extracted after homogenization with DMEM. The viral load of HMPV was detected by real-time PCR.

[0097] (3) HE staining for lung pathological examination: Lung tissue was collected 5 days after viral challenge, and lung pathological examination was performed by HE staining, including analysis of eosinophils in the lungs. The pathological condition of the lung tissue was assessed by analyzing the number of inflammatory cells around the small trachea, the number of inflammatory cells in the alveolar cavity, and the number and thickness of inflammatory cells in the lung interstitium.

[0098] (4) Lung Immunohistochemical Detection: Five days after HMPV challenge, lung tissue was extracted, fixed with paraformaldehyde, and subjected to immunohistochemical analysis based on enzyme-linked immunosorbent assay (ELISA) to determine the HMPV infection status in the lung tissue. The immunohistochemical positivity rate is generally based on two aspects: the percentage of positive cells and the intensity of positivity. The cumulative optical density value is the integral of the optical density of all positive signals, divided by the area of ​​the tissue to be tested to reflect the quantity and depth of positivity, which is directly proportional to the positivity rate. The average optical density of each group was analyzed.

[0099] result:

[0100] (1) Clinical observation and survival analysis: Mice in both the HMPV VLP experimental group and the adjuvant control group survived, with a survival rate of 100%. The body weight of mice began to decrease from day 3 after viral challenge, and gradually increased from day 4, returning to pre-challenge weight by day 14. Figure 10 As shown.

[0101] Pulmonary viral load detection

[0102] (2) Detection of viral load in the lungs: The viral load in the lung tissue was measured to determine whether viral replication in the lungs of the immunized mice was inhibited. The results showed that on day 5 of viral infection, compared with the adjuvant control group, the viral load in the HMPVVLP immunization groups with and without adjuvant was significantly reduced (e.g., ...). Figure 11 As shown in the figure, the viral load in the HMPV VLP immunization group containing aluminum hydroxide adjuvant was lower than that in the VLP immunization group without adjuvant, indicating that HMPV VLP immunization produced a protective effect.

[0103] (3) HE staining for lung pathological examination: Lung tissue was collected 5 days after viral challenge. The pathological condition of the lung tissue was assessed by analyzing the number of inflammatory cells around the small trachea, the number of inflammatory cells in the alveolar cavity, the number of inflammatory cells in the lung interstitium, and the alveolar wall thickness. The statistical scores of each inflammatory cell in the sample were determined as follows: 0-1 cells / 20×field of view was 0; 2≤ / 20×field of view <5 cells was 1; 5≤ / 20×field of view <10 cells was 2; 10≤ / 20×field of view <20 cells was 3; and >20×field of view ≥10 cells was 4.

[0104] In this invention, the mean lung tissue pathological values ​​of mice immunized by intramuscular injection of HMPV VLP without adjuvant and with adjuvant were 5.6 and 5, respectively, while the adjuvant control group had a mean value of 8. This indicates that the lung pathological damage induced by intramuscular injection of HMPV VLP in mice was weaker than that in the adjuvant control group. The results are shown in Table 2.

[0105] Table 2. Pathological analysis results of lung tissue in mice immunized with HMPV-challenged VLP via intramuscular injection.

[0106]

[0107] (4) Immunohistochemical detection of lung tissue: Lung tissue was fixed with paraformaldehyde and then subjected to immunohistochemical analysis. The immunoprotective effect of HMPV VLP was preliminarily determined by the optical density value. Figure 12 It can be seen that the mean optical density of lung tissue in mice immunized with adjuvanted HMPV VLP was lower than that in the adjuvant-free VLP group and the adjuvant control group, with P values ​​of P > 0.05 and P < 0.05, respectively. This indicates that after HMPV challenge, the viral load in the lung tissue of mice immunized with adjuvanted VLP via intramuscular injection was reduced, while the viral load in the adjuvant control group was higher than that in the VLP experimental group. Therefore, HMPV VLP provides immunoprotection after HMPV challenge.

[0108] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-described technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A human metapneumovirus virus-like particle, characterized in that, Human metapneumovirus virus-like particles are assembled from the M1 protein of influenza virus H2N2 and the F protein of human metapneumovirus; the nucleic acid sequence encoding the M1 protein is shown in SEQ ID NO:1, and the nucleic acid sequence encoding the F protein is shown in SEQ ID NO:

2. The M1 gene of influenza virus H2N2 and the F gene sequence of human metapneumovirus were cloned into the pFastBacDual vector. The M1 gene was inserted between the SphI and NheI restriction sites of the pFastBacDual vector, and the F gene was inserted between the BamHI and NotI restriction sites of the pFastBacDual vector, resulting in the recombinant plasmid pFastBacDual-M1-F. Then, the recombinant plasmid pFastBacDual-M1-F was transfected into sf9 insect cells to obtain recombinant sf9 cells; Recombinant sf9 cells were cultured and purified by centrifugation to obtain human metapneumovirus virus-like particles (VLPs).

2. A method for preparing human metapneumovirus virus-like particles as described in claim 1, characterized in that, Includes the following steps: (1) Construction of recombinant plasmid: The M1 gene of influenza virus H2N2 and the F gene sequence of human metapneumovirus were cloned into the pFastBacDual vector to obtain the recombinant plasmid pFastBacDual-M1-F; (2) Transfection: The recombinant plasmid pFastBacDual-M1-F was transfected into sf9 insect cells to obtain recombinant sf9 cells; (3) Separation and purification: Recombinant sf9 cells were cultured and purified by centrifugation to obtain human metapneumovirus virus-like particles (VLPs).

3. The preparation method according to claim 2, characterized in that, In step (3), recombinant sf9 cells are cultured using adherent culture to prepare VLP.

4. The preparation method according to claim 2, characterized in that, In step (3), the recombinant sf9 cells are subjected to multiple generations of expanded culture and purification.

5. The preparation method according to claim 4, characterized in that, In step (3), the recombinant sf9 cells are expanded through at least P1, P2, P3, and P4 generations.

6. The preparation method according to claim 4 or 5, characterized in that, Large-scale culture purification methods include: (1) After transfecting sf9 insect cells with recombinant plasmid, the supernatant of diseased cells was harvested 72 hours later, which is the P1 generation recombinant baculovirus; (2) P1 generation recombinant baculovirus was added to sf9 insect cells in the logarithmic growth phase, cultured at 27°C for 72 hours, and the recombinant baculovirus was passaged to P4 generation. The supernatant and cells of the diseased cells were harvested. (3) Collect the supernatant and cells of P4 generation, concentrate them by ultracentrifugation, and then resuspend them in PBS to obtain purified human metapneumovirus virus-like particles (VLPs).

7. A recombinant vector expressing human metapneumovirus virus-like particles as described in claim 1, characterized in that, Using pFastBacDual as the expression vector, the M1 gene of influenza virus H2N2 and the F gene sequence of human metapneumovirus were inserted.

8. A recombinant cell, characterized in that, The recombinant cells contain the recombinant vector of claim 7.

9. The recombinant cell according to claim 8, characterized in that, The recombinant cells are sf9 insect cells containing the recombinant vector of claim 7.

10. The use of human metapneumovirus virus-like particles as described in claim 1 in the preparation of human metapneumovirus vaccines.

11. A human metapneumovirus vaccine, characterized in that, It contains human metapneumovirus virus-like particles as described in claim 1.

12. The human metapneumovirus vaccine according to claim 11, characterized in that, It also includes pharmaceutically acceptable excipients.

13. The human metapneumovirus vaccine according to claim 12, characterized in that, The excipients are adjuvants.

14. The human metapneumovirus vaccine according to claim 11, characterized in that, The human metapneumovirus vaccine described herein is composed of human metapneumovirus virus-like particles and aluminum hydroxide adjuvant.

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