Virus-like particles of H5N6, H5N8 and H7N9 as well as application and vaccine thereof

Abstract

The invention belongs to the technical field of biology, and discloses virus-like particles of H5N6, H5N8 and H7N9 and application and vaccine.The virus-like particles of H5N6, H5N8 and H7N9 are jointly connected to plasmids through HA, NA and M1 genes of H5N6, H5N8 and H7N9 and transferred into competent cells to obtain recombinant baculovirus plasmids, then the recombinant baculovirus plasmids are transfected and expressed to obtain the corresponding virus-like particles, and in addition, the virus-like particles of H5N6, H5N8 and H7N9 can be used for preparing the vaccine. The trivalent vaccines prepared from the virus-like particles do not generate antagonism among different antigens.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to virus-like particles of H5N6, H5N8 and H7N9 and uses thereof, vaccines. BACKGROUND

[0002] Inactivated vaccines are the main strategy for the prevention and control of avian influenza. However, these vaccines have some limitations, including a long production cycle, insufficient immunogenicity, suboptimal induction of cellular immune responses, and limited broad-spectrum protection against rapidly evolving viral strains. In addition, the production of inactivated vaccines relies on egg-based culture systems, which not only increases production costs but also poses a risk of antigenic variation due to adaptive mutations of the virus. Therefore, there is an urgent need to develop new vaccine platforms to provide enhanced immunogenicity, broader cross-protection, scalable production capacity, and improved safety.

[0003] Virus-like particles (VLPs) have emerged as a cutting-edge vaccine platform due to their unique ability to mimic viral structures and elicit strong immune responses. VLPs are self-assembled from viral structural proteins such as hemagglutinin (HA), neuraminidase (NA), and matrix protein (M1), forming particles that closely resemble native viruses in morphology and structure. This high degree of similarity enables VLPs to effectively mimic the viral infection process, thereby inducing strong humoral and cellular immune responses, while the absence of viral genetic material in VLPs further enhances their safety. Additionally, VLPs can be engineered to develop multivalent vaccines, providing broader cross-protection. HA is the main target of neutralizing antibodies and is the primary antigen for designing avian influenza vaccines. NA is crucial for virus release and transmission and is less variable, containing conserved epitopes that help provide broader protection. The highly conserved M1 is essential for viral particle assembly and morphology, further enhancing the breadth and durability of immune responses. Furthermore, M1 can act as an immunological adjuvant during viral infection, making vaccine design more effective. Due to their superior immunogenicity, safety, and engineering flexibility, VLPs are ideal candidates for developing multivalent vaccines against various pandemic avian influenza subtypes. The baculovirus expression vector system (BEVS) as a high-efficiency platform for VLP production enables the rapid generation of high-purity and high-yield VLPs in a short time. This capability significantly facilitates the rapid development and large-scale production of avian influenza vaccines.

[0004] Ting Zhang, Yufei Xu, Liang Qiao, Youchun Wang, Xueling Wu, Dongsheng Fan, Qinglin Peng, Xuemei Xu et al. in Trivalent Human Papillomavirus (HPV) VLP vaccine covering HPV type 58 can elicit high level of humoral immunity but also induce immune interference among component types pointed out that monovalent vaccines often cannot provide sufficient cross-protection against antigenically diverse H5 and H7 avian influenza viruses, thus developing multivalent vaccines targeting these key subtypes is crucial for enhancing overall protective efficacy and improving pandemic preparedness. However, immune interference between different antigens in multivalent vaccines is an important issue. For example, a trivalent vaccine against human papillomavirus (HPV) containing HPV types 16, 18, and 58 induces high levels of humoral immunity, but immune interference between different antigens is also observed, which becomes more pronounced when increasing the number of antigen types; Therefore, it can be seen that the interference between different antigens is a problem that needs to be paid attention to in the preparation process of multivalent vaccines, and it is of great significance to study the immune interference between different antigens, because it directly affects the overall effectiveness and protection range of the vaccine. Understanding and overcoming this interference can help optimize multivalent vaccine design, and improve the immune response of the vaccine by adjusting the antigen dose and adding appropriate adjuvants. This not only helps to improve the effectiveness of existing vaccines, but also promotes the development of new broad-spectrum vaccines, so as to more effectively cope with the threat of multiple pathogens and enhance public health protection capabilities.

[0005] The technical problem of the present scheme is how to develop a protective and broad-spectrum avian influenza (H5+H7) trivalent vaccine virus-like particle. SUMMARY

[0006] The purpose of the present application is to provide a broad-spectrum avian influenza vaccine virus-like particle, and through experiments it is found that when the virus-like particles of H5N6, H5N8 and H7N9 are used to prepare a trivalent vaccine, there is no significant difference in the immune effect on the parent strain compared with the monovalent vaccine of the three virus-like particles, and it can be seen that there is no immune interference phenomenon between the antigens of the three virus-like particles in the vaccine.

[0007] To achieve the above object, the application discloses virus-like particles of H5N6, H5N8 and H7N9, the virus-like particle of H5N6 is obtained by jointly connecting HA, NA and M1 genes of H5N6 virus to a plasmid and transferring into competent cells to obtain a recombinant baculovirus plasmid; Then the recombinant baculovirus plasmid is transfected into insect cells and expressed to obtain the virus-like particle of H5N6; The virus-like particle of H5N8 is obtained by jointly connecting HA, NA and M1 genes of H5N8 virus to a plasmid and transferring into competent cells to obtain a recombinant baculovirus plasmid; Then the recombinant baculovirus plasmid is transfected into insect cells and expressed to obtain the virus-like particle of H5N8; The virus-like particle of H7N9 is obtained by jointly connecting HA, NA and M1 genes of H7N9 virus to a plasmid and transferring into competent cells to obtain a recombinant baculovirus plasmid; Then the recombinant baculovirus plasmid is transfected into insect cells and expressed to obtain the virus-like particle of H7N9; The amino acid sequence of the H5N6 virus HA protein is shown as SEQ ID NO: 1; The amino acid sequence of the H5N6 virus NA protein is shown as SEQ ID NO: 2; The amino acid sequence of the H5N6 virus M1 protein is shown as SEQ ID NO: 3; The amino acid sequence of the H5N8 virus HA protein is shown as SEQ ID NO: 4; The amino acid sequence of the H5N8 virus NA protein is shown as SEQ ID NO: 5; The amino acid sequence of the H5N8 virus M1 protein is shown as SEQ ID NO: 6; The amino acid sequence of the H7N9 virus HA protein is shown as SEQ ID NO: 7; The amino acid sequence of the H7N9 virus NA protein is shown as SEQ ID NO: 8; The amino acid sequence of the H7N9 virus M1 protein is shown as SEQ ID NO: 9.

[0008] In addition, the application also discloses the use of the virus-like particles of H5N6, H5N8 and H7N9 as described above for preparing vaccines.

[0009] In addition, the application also discloses a H5+H7 trivalent vaccine prepared by taking the virus-like particles of H5N6, the virus-like particles of H5N8 and the virus-like particles of H7N9 as antigens.

[0010] Preferably, the virus-like particle of H5N6 is obtained by jointly connecting the HA, NA and M1 genes of H5N6 virus to a plasmid and transforming into competent cells to obtain a recombinant baculovirus plasmid; The recombinant baculovirus plasmid is then transfected into insect cells and expressed to obtain the virus-like particle of H5N6; The virus-like particle of H5N8 is obtained by jointly connecting the HA, NA and M1 genes of H5N8 virus to a plasmid and transforming into competent cells to obtain a recombinant baculovirus plasmid; The recombinant baculovirus plasmid is then transfected into insect cells and expressed to obtain the virus-like particle of H5N8; The virus-like particle of H7N9 is obtained by jointly connecting the HA, NA and M1 genes of H7N9 virus to a plasmid and transforming into competent cells to obtain a recombinant baculovirus plasmid; The recombinant baculovirus plasmid is then transfected into insect cells and expressed to obtain the virus-like particle of H7N9.

[0011] Preferably, the nucleotide sequence encoding the HA protein of H5N6 virus is as shown in SEQ ID NO: 10; The nucleotide sequence encoding the NA protein of H5N6 virus is as shown in SEQ ID NO: 11; The nucleotide sequence encoding the M1 protein of H5N6 virus is as shown in SEQ ID NO: 12; The nucleotide sequence encoding the HA protein of H5N8 virus is as shown in SEQ ID NO: 13; The nucleotide sequence encoding the NA protein of H5N8 virus is as shown in SEQ ID NO: 14; The nucleotide sequence encoding the M1 protein of H5N8 virus is as shown in SEQ ID NO: 15; The nucleotide sequence encoding the HA protein of H7N9 virus is as shown in SEQ ID NO: 16; The nucleotide sequence encoding the NA protein of H7N9 virus is as shown in SEQ ID NO: 17;

[0012] The nucleotide sequence encoding the M1 protein of H7N9 virus is as shown in SEQ ID NO: 18.

[0013] The beneficial effects of the present application are: the broad-spectrum avian influenza vaccine virus-like particles provided by the present application, through experiments, it is found that when the virus-like particles of H5N6, H5N8 and H7N9 are used to prepare a trivalent vaccine, the immune effect on the parent strain of the trivalent vaccine is not significantly different from that of the monovalent vaccine of the three virus-like particles, which shows that there is no immune interference phenomenon between the antigens of the three virus-like particles in the vaccine; Further, the antibody titer produced by the trivalent vaccine prepared by the three viruses described above is even higher than that of the commercial vaccine in a short time, which shows that the trivalent vaccine has the advantage of being able to rapidly induce an immune response. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 A schematic diagram of the rH5N6-pFastBac1-NA-HA-M1 plasmid is shown in the following figure: Figure 2 A schematic diagram of the rH5N8-pFastBac1-NA-HA-M1 plasmid is shown in the following figure: Figure 3 A schematic diagram of the rH7N9-pFastBac1-NA-HA-M1 plasmid is shown in the following figure: Figure 4 A fluorescence comparison chart of Sf9 control cell wells and Sf9 cell wells infected with recombinant baculovirus rBV-H5N6-NA-HA-M172h is shown in the following figure: Figure 5 A fluorescence comparison chart of Sf9 control cell wells and Sf9 cell wells infected with recombinant baculovirus rBV-H5N8-NA-HA-M172h is shown in the following figure: Figure 6 A fluorescence comparison chart of Sf9 control cell wells and Sf9 cell wells infected with recombinant baculovirus rBV-H7N9-NA-HA-M172h is shown in the following figure: Figure 7 A Western Blot identification chart of H5N6-VLP is shown in the following figure, Figure 7 In the figure a, PAGE is used to identify the expression of H5N6-VLP; Figure 7 In the figure b, the primary antibody is a His-tag monoclonal antibody incubated with H5N6-VLP; Figure 7 In the figure c, the primary antibody is a H5 subtype HA monoclonal antibody incubated with H5N6-VLP; Figure 8 A chart of PAGE used to identify the expression of H5N8-VLP and H7N9-VLP is shown in the following figure, Figure 8 In the figure a, PAGE is used to identify the expression of H5N8-VLP; Figure 8 In the figure b, PAGE is used to identify the expression of H7N9-VLP; and lane 1 is the target sample; lane 2 is the High Five cell sample negative control; Figure 9 A chart of Western blot used to identify the expression of H5N8-VLP and H7N9-VLP is shown in the following figure, Figure 9 In the figure a, the primary antibody is a His-tag monoclonal antibody incubated with H5N8-VLP; Figure 9H5N8-VLP identified by incubation with H5 subtype HA monoclonal antibody; Figure 9 H7N9-VLP identified by incubation with His tag monoclonal antibody, and lane 1 is the sample of interest; lane 2 is the sample of High Five cells negative control; Figure 10 Purification and identification of H5N6-VLP, Figure 10 a is the schematic diagram of sucrose purification; Figure 10 b is PAGE (H5N6-VLP in sucrose layer 20%~30%); Figure 10 c is the incubated His monoclonal antibody; Figure 10 d is the incubated H5 subtype avian influenza HA monoclonal antibody; Figure 11 Purification and identification of H5N8-VLP and H7N9-VLP, Figure 11 a is the schematic diagram of sucrose purification of H5N8-VLP; Figure 11 b is PAGE (H5N8-VLP in sucrose layer 20%~30%); Figure 11 c is the incubated His monoclonal antibody to identify H5N8-VLP; Figure 11 d is the incubated H5 subtype avian influenza HA monoclonal antibody to identify H5N8-VLP; Figure 11 e is the schematic diagram of sucrose purification of H5N8-VLP; Figure 11 f is PAGE (H7N9-VLP in sucrose layer 20%~30%); Figure 11 g is the incubated His monoclonal antibody to identify H7N9-VLP, and lane 1 is the sample of interest; lane 2 is the sample of High Five cells negative control; Figure 12 Electron micrograph of morphological characteristics of H5N6-VLP; Figure 13 Electron micrograph of morphological characteristics of H5N8-VLP; Figure 14 Electron micrograph of morphological characteristics of H7N9-VLP; Figure 15 Detection chart of HI antibody titer against H5N6-GD154 strain after immunization of SPF chickens with H5N6-VLP vaccine; Figure 16 Detection chart of MN antibody titer against H5N6-GD154 strain after immunization of SPF chickens with H5N6-VLP vaccine; Figure 17 Detection chart of survival rate of chickens in H5N6-GD154 strain challenge experiment after immunization of SPF chickens with H5N6-VLP vaccine; Figure 18Figure for comparison of HI antibody titers against parental strains after immunization of SPF chickens with H5N6-H5N8-H7N9-VLP vaccine, Figure 18 Figure a for comparison of H5N6-H5N8-H7N9-VLP and H5N6-VLP, Figure 18 Figure b for comparison of H5N6-H5N8-H7N9-VLP and H5N8-VLP, Figure 18 Figure c for comparison of H5N6-H5N8-H7N9-VLP and H7N9-VLP; Figure 19 Figure for detection of HI antibody titers against H5 subtype epidemic strains after immunization of SPF chickens with H5N6-H5N8-H7N9-VLP vaccine, Figure 19 Figure a for detection of antibody titers against H5N2-GD285 challenge strain; Figure 19 Figure b for detection of antibody titers against H5N1-GD105 challenge strain; Figure 20 Figure for detection of HI antibody titers against H7N9 subtype epidemic strains after immunization of SPF chickens with H5N6-H5N8-H7N9-VLP vaccine, Figure 20 Figure a for detection of antibody titers against H7N9-C5F-145 challenge strain; Figure 20 Figure b for detection of antibody titers against H7N9-GD014 challenge strain; Figure 21 Figure for comparison of survival rates of chickens in parental strain challenge experiment after immunization of SPF chickens with H5N6-H5N8-H7N9-VLP vaccine, Figure 21 Figure a for comparison of H5N6-H5N8-H7N9-VLP and H5N6-VLP; Figure 21 Figure b for comparison of H5N6-H5N8-H7N9-VLP and H5N8-VLP; Figure 21 Figure c for comparison of H5N6-H5N8-H7N9-VLP and H7N9-VLP. DETAILED DESCRIPTION

[0015] The present application will be described in detail below with reference to embodiments thereof, and in the description of the application, it should be noted that where no specific conditions are mentioned in the embodiments, the conventional conditions or the conditions recommended by the manufacturers are used. Where no manufacturers of the reagents or instruments are mentioned, the conventional products that can be commercially purchased are used.

[0016] Example 1 Construction of recombinant baculovirus plasmid 1.1 Design of recombinant transfer plasmid The HA, NA and M1 genes derived from A / Chicken / Guangdong / 154 / 2022 (H5N6-GD154), A / Chicken / Guangdong / 110 / 2022 (H5N8-GD110) and A / Chicken / Guangdong / 144 / 2022 (H7N9-C5E-144) were codon-optimized, synthesized and then connected to the baculovirus transfer plasmid pFastBac1 to obtain recombinant transfer plasmids rH5N6-pFastBac1-NA-HA-M1, rH5N8-pFastBac1-NA-HA-M1 and rH7N9-pFastBac1-NA-HA-M1, respectively. The plasmid construction map is shown in Figures 1-3 Figure 1 The rH5N6-pFastBac1-NA-HA-M1 plasmid design schematic is shown in Figure 2 The rH5N8-pFastBac1-NA-HA-M1 plasmid design schematic is shown in Figure 3 The rH7N9-pFastBac1-NA-HA-M1 plasmid design schematic is shown in The nucleotide sequences of the codon-optimized HA, NA and M1 genes of the H5N6 subtype are shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, and the amino acid sequences are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3.

[0017] The nucleotide sequences of the codon-optimized HA, NA and M1 genes of the H5N8 subtype are shown in SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, and the amino acid sequences are shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.

[0018] The nucleotide sequences of the codon-optimized HA, NA and M1 genes of the H7N9 subtype are shown in SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, and the amino acid sequences are shown in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9.

[0019] 1.2 Construction of recombinant baculovirus plasmid ​The recombinant transfer plasmids rH5N6-pFastBacl-NA-HA-M1, rH5N8-pFastBacl-NA-HA-M1 and rH7N9-pFastBacl-NA-HA-M1 were transformed into DH10bac competent cells, respectively, and the specific steps were as follows: 50 ng of recombinant transfer plasmid was added to 50 μL of DH10Bac competent cells, mixed slightly, and then incubated in an ice bath for 30 min, heated at 42°C for 45 s, and then incubated in an ice bath for 5 min. 900 μL of LB liquid medium without antibiotics was added to an EP tube, and then the tube was incubated at 37°C on a shaker at 220 r / min for 4 h. The bacterial solution was diluted 10 times with LB liquid medium without antibiotics to obtain bacterial solutions with dilution degrees of 10-1, 10-2 and 10-3. 400 μL of each dilution of the bacterial solution was uniformly coated on a blue-white spot screening LB culture plate, and then the plate was incubated in a 37°C incubator for 48 h. After that, a white single colony was picked and expanded, and then the plasmid was extracted after PCR identification. Finally, the recombinant baculovirus plasmid was obtained, and was named as Bacmid-H5N6-NA-HA-M1, Bacmid-H5N8-NA-HA-M1 and Bacmid-H7N9-NA-HA-M1, respectively.

[0020] Example 2: Obtaining of recombinant baculovirus 2.1 The recombinant baculovirus plasmid prepared in Example 1 was transfected into sf9 insect cells (Spodoptera frugiperda cells) (Invitrogen Company) by using a liposome-mediated transfection method. The specific steps of transfection were as follows: (1) Sf9 cells (1.6-1.8 x 10 6 cell / mL) in the logarithmic growth phase were plated in a six-well plate, and then incubated at 27°C for 1 h. After the cells adhered to the wall, transfection was performed; (2) 6-8 μL of Cellfectin® II was diluted in 100 μL of Grace medium and vortexed to mix. 1 μL of baculovirus DNA was diluted in 100 μL of Grace medium and mixed gently. The diluted DNA and diluted Cellfectin® II were mixed, and then incubated at room temperature for 15-30 min to obtain a DNA-liposome mixture; (3) 800 μL of Grace medium was added to the DNA-liposome mixture to make up 1 mL. The DNA-liposome mixture was added to the six-well plate, and then the cells were incubated at 27°C for 3-5 h; (4) The transfection mixture was removed, 2 mL of HF-SFM was added, and then the plate was incubated at 27°C for 72 h. During the incubation, the cytopathic effect was observed. After 72 h of incubation, the cell culture supernatant was collected, and then the first generation of recombinant baculovirus was obtained, and was named as rBV-H5N6-NA-HA-M1; The method for preparing recombinant baculovirus of H5N8 and H7N9 is the same as above.

[0021] 2.2 Detecting the expression of target gene using indirect immunofluorescence method (IFA) Sf9 cells were infected with P3 generation recombinant baculovirus (rBV-H5N6-NA-HA-M1, rBV-H5N8-NA-HA-M1 and rBV-H7N9-NA-HA-M1) at MOI = 0.1, respectively, and indirect immunofluorescence test was performed 48 h after infection, with the brief steps as follows: (1) Discard the liquid in the six-well plate, and add 1 mL of cold methanol solution to each well, and fix in the 4°C refrigerator for 15 min; (2) Discard the fixing solution, and add PBS solution, and repeat the washing for 3 times; (3) Add 1: 200 diluted mouse-derived anti-His tag monoclonal antibody, and incubate overnight at 4°C; (4) Recover the primary antibody, and add PBST buffer for washing, 5 min each time, and repeat the washing for 3 times; (5) Add diluted FITC-conjugated goat anti-mouse IgG antibody, and incubate at 37°C for 1 h in the dark; (6) Discard the secondary antibody, and add PBST buffer for washing, 5 min each time, and repeat the washing for 3 times; (7) Use fluorescence microscope to observe, and take Sf9 cells as control, and the results show that specific fluorescence can be detected in the wells infected with recombinant baculovirus, while no specific fluorescence is generated in the Sf9 control cell wells, and the specific fluorescence contrast graphs of Sf9 control cell wells and Sf9 cell wells infected with recombinant baculovirus rBV-H5N6-NA-HA-M1 72h are shown in FIG. 2, wherein, Figures 4-6 Figure 4 is the fluorescence contrast graph of Sf9 control cell wells and Sf9 cell wells infected with recombinant baculovirus rBV-H5N6-NA-HA-M1 72h; Figure 5 is the fluorescence contrast graph of Sf9 control cell wells and Sf9 cell wells infected with recombinant baculovirus rBV-H5N8-NA-HA-M1 72h; Figure 6 is the fluorescence contrast graph of Sf9 control cell wells and Sf9 cell wells infected with recombinant baculovirus rBV-H7N9-NA-HA-M1 72h.

[0022] Example 3 Assembly and purification of H5N6-VLP, H5N8-VLP and H7N9-VLP 3.1 Assembly and harvesting of virus-like particles rBV-H5N6-NA-HA-M1 virus liquid at different MOI was inoculated on well-grown High Five ++ ​Cells were cultured in suspension at 27℃, 115r / min for 5 days. The supernatant was collected by centrifugation at 2000 g for 20 min, and the H5N6-VLP was collected by centrifugation at 12000 g for 60 min with PBS. The results showed that the virus-like particles in the extracellular culture supernatant and the hemagglutination titers in the intracellular broken supernatant were the highest when the MOI was 0.1, indicating that the MOI of 0.1 was the best infection condition for producing virus-like particles (Table 1). Similarly, rBV-H5N8-NA-HA-M1 and rBV-H7N9-NA-HA-M1 with an MOI of 0.1 were inoculated into High Five ++ Insect cells for preparing H5N8-VLP and H7N9-VLP.

[0023] Table 1: Screening of MOI MOI High Five cell density Culture volume Hemagglutination titer (log2) of VLP 0.05 2.5 x 10 6 ]]> 50 mL 12 0.1 2.5 x 10 6 ]] 50 mL 14 0.5 2.5 x 10 6 ]] 50 mL 13 1 2.5 x 10 6 ]]> 50 mL 13

[0024] 3.2 Identification of virus-like particles The virus-like particles harvested in step 1 were subjected to SDS-PAGE and Western blot analysis. In the Western blot analysis, the primary antibody for identifying the HA protein of H5N6-VLP and H5N8-VLP was H5 subtype influenza hemagglutinin HA monoclonal antibody (Sino Biological, Beijing), and the primary antibody for identifying the HA, NA and M1 proteins was His-tag (4C2) mouse monoclonal antibody (Bioword Company), and the supernatant of normal High five insect cells was used as a negative control. The results are shown in Figures 7-9 that there were obvious bands at about 72 kDa, 60 kDa and 28.5 kDa in H5N6-VLP, corresponding to the HA, NA and M1 proteins, respectively. There were obvious bands at about 72 kDa, 55 kDa and 28.5 kDa in H5N8-VLP and H7N9-VLP, corresponding to the HA, NA and M1 proteins, respectively; It should be noted that, Figure 7 Figure for Western Blot identification of H5N6-VLP, wherein a: PAGE; b: incubation of the primary antibody of His-tag monoclonal antibody for identifying H5N6-VLP; c: incubation of the primary antibody of H5 subtype HA monoclonal antibody for identifying H5N6-VLP.

[0025] Figure 8 Figure for PAGE identification of the expression of H5N8-VLP and H7N9-VLP, Figure 8 a is the PAGE identification of the expression of H5N8-VLP; Figure 8 b is the PAGE identification of the expression of H7N9-VLP; and lane 1 is the target sample; lane 2 is the High Five cell sample negative control; Figure 9 Figure 2 shows the expression profile of H5N8-VLP and H7N9-VLP identified by Western blot, Figure 9 Figure 2a shows the identification of H5N8-VLP incubated with His-tag monoclonal antibody; Figure 9 Figure 2b shows the identification of H5N8-VLP incubated with H5 subtype HA monoclonal antibody; Figure 9 Figure 2c shows the identification of H7N9-VLP incubated with His-tag monoclonal antibody; and lane 1 is the sample of interest; lane 2 is the sample of High Five cells negative control;

[0026] 3.3 Purification of virus-like particles Prepare sucrose solutions of different concentrations: prepare 20%, 30%, 45%, and 60% (w / v) sucrose solutions, filter through a 0.22 μm filter; add the sucrose solutions from top to bottom in centrifuge tubes, with the uppermost being the sample of virus-like particles, centrifuge at 100000 x g at 4°C for 1 h; after centrifugation, collect the white transparent band between the sucrose layers; remove the sucrose by centrifugation at 100000 x g at 4°C for 1 h; resuspend the sample of virus-like particles in PBS buffer, and perform PAGE and Western Blot identification, the results are shown in Figures 10-11 Figure 3, which confirms that the HA, NA, and M1 proteins are expressed in the purified H5N6-VLP, H5N8-VLP, and H7N9-VLP, and that the HA titers of the H5N6-VLP, H5N8-VLP, and H7N9-VLP can reach 13 log2; It should be noted that, Figure 10 Figure 4 shows the purification and identification of H5N6-VLP, Figure 10 Figure 4a shows a schematic diagram of sucrose purification; Figure 10 Figure 4b shows PAGE (H5N6-VLP in the sucrose layer of 20% to 30%); Figure 10 Figure 4c shows incubation with His monoclonal antibody; Figure 10 Figure 4d shows incubation with H5 subtype avian influenza HA monoclonal antibody; Figure 11 Figure 5 shows the purification and identification of H5N8-VLP and H7N9-VLP, Figure 11 Figure 5a shows a schematic diagram of sucrose purification of H5N8-VLP; Figure 11 Figure 5b shows PAGE (H5N8-VLP in the sucrose layer of 20% to 30%); Figure 11 Figure 5c shows incubation with His monoclonal antibody to identify H5N8-VLP; Figure 11 Figure 5d shows incubation with H5 subtype avian influenza HA monoclonal antibody to identify H5N8-VLP; Figure 11 Figure 5e shows a schematic diagram of sucrose purification of H5N8-VLP; Figure 11f in the middle represents PAGE (20%–30% of the sucrose layer is H7N9-VLP); Figure 11 In lane g, the His monoclonal antibody was incubated to identify H7N9-VLP, and lane 1 was the target sample; lane 2 was a High Five cell sample as a negative control.

[0027] 3.4 Observation of the morphology and structure of virus-like particles using transmission electron microscopy The virus-like particle sample harvested in step 3 was dropped onto a carbon-coated copper grid for adsorption and incubated at room temperature for 2 minutes. Excess liquid on the copper grid was gently blotted away with absorbent paper. After drying, 3% uranium acetate was dropped onto the copper grid, and after 3 minutes, excess staining liquid was blotted away with a small piece of filter paper. The grid was then allowed to air dry at room temperature. The results were observed using a transmission electron microscope as follows: Figures 12-14 As shown ( Figure 12 Electron micrographs of the morphological characteristics of H5N6-VLP. Figure 13 Electron micrographs of the morphological characteristics of H5N8-VLP. Figure 14 (Electron micrograph of the morphological characteristics of H7N9-VLP): Particle size is about 100-120 nm, and fibrous structures are visible on the surface.

[0028] Example 4: Evaluation of the Homologous Protective Efficacy of H5N6-VLP Vaccine 4.1 Vaccine preparation H5N6-VLP and inactivated vaccines with different gradient hemagglutination titers (7log2, 9log2, 11log2) were prepared in a 1:1 ratio with ISA 78 adjuvant.

[0029] 4.2 Immunization schedule for vaccines Forty-five 21-day-old SPF chickens were randomly divided into four groups of ten each, with five chickens receiving PBS buffer. All groups were immunized via subcutaneous injection in the neck. Group 1 received 0.3 mL of H5N6-VLP vaccine with a hemagglutination titer of 7 log2 per chicken. Group 2 received 0.3 mL of H5N6-VLP vaccine with a hemagglutination titer of 9 log2 per chicken. Group 3 received 0.3 mL of H5N6-VLP vaccine with a hemagglutination titer of 11 log2 per chicken. Group 4 received 0.3 mL of H5N6 inactivated vaccine with a hemagglutination titer of 9 log2 per chicken. Group 5 served as a blank control with PBS buffer. The animal groupings and immunization protocols are shown in Table 2.

[0030] Table 2: Animal grouping and immunization regimen for evaluating the homologous protective efficacy of H5N6-VLP vaccine

[0031] 4.3 Serum antibody test results The blood of all test chickens was collected to separate serum at the 2nd and 3rd weeks after immunization, and antibody detection was carried out by using conventional serum inhibition (HI) micro-neutralization (MN) antibody detection test. The homologous four-unit antigen (four-unit antigen preparation: the hemagglutination titer of avian influenza virus antigen was determined, and the highest dilution multiple of complete hemagglutination was taken as the end point, and the end point dilution multiple was divided by 4 to obtain the dilution multiple of 4 HAU, and the antigen was diluted to 4 HAU using PBS buffer. 4 HAU review: prepare a 96-well V-shaped plate, add 25 µL of PBS buffer to each well; add 25 µL of 4 HAU to the first well, and continuously dilute by 2 times to the third well; add 25 µL of 1% chicken red blood cell suspension, and observe the results after standing at room temperature for 30 min) was prepared from H5N6-GD154 strain. The HI antibody detection results are shown in Figure 15 ( Figure 15 The HI antibody titer detection graph of SPF chickens immunized with H5N6-VLP vaccine against H5N6-GD154 strain). The results show that the HI antibody titer can be detected in each vaccine group at 2 weeks after immunization, and the average HI titers of the 7log2 group, the 9log2 group, the 11log2 group and the inactivated vaccine group at 2 weeks after immunization are 4.3log2, 5.7log2, 6.3log2 and 3.8log2 respectively, and the average HI titers at 3 weeks after immunization are 6.4log2, 7log2, 8log2 and 7.2log2 respectively. The MN antibody detection results are shown in Figure 16 ( Figure 16 The MN antibody titer detection graph of SPF chickens immunized with H5N6-VLP vaccine against H5N6-GD154 strain). The results show that the average MN antibody titers of each group of test chickens at 3 weeks after immunization are higher than those at 2 weeks after immunization. At 3 weeks after immunization, the MN antibody titer of the 11log2 group is 1:1920, and there is no significant difference between the MN antibody titer (1:1120) of the 9log2 group and the MN antibody titer (1:960) of the inactivated vaccine group.

[0032] 4.4 Homologous challenge protection experiment results Nose drop challenge was carried out using H5N6-GD154 avian influenza virus strain at 3 weeks after immunization, and the challenge dose was 0.2 mL per chicken (10 6.0 EID 50 ). The test chickens were observed daily for illness or death after challenge and recorded in time, and the observation was continued for 14 days. Throat swabs and cloaca swabs were collected at 3 days, 5 days and 7 days after challenge. The results show that all chickens in the PBS control group died within 2 days, showing typical clinical symptoms, including lethargy, movement retardation and nervous system abnormalities. Reference Figure 17 ( Figure 17The survival rate of the chickens in the H5N6-GD154 AIV lethal dose challenge experiment after immunization with the H5N6-VLP vaccine was 100% after 14 days. The virus isolation results showed (Table 3) that 2 chickens were detected to be shedding on the 3rd day after challenge, and 1 chicken was detected to be shedding on the 5th day after challenge. No shedding was detected in the other immunization groups on the 3rd day, 5th day and 7th day after challenge.

[0033] The above results show that the H5N6-VLP vaccine with a dose of 9log2 can effectively protect the SPF chickens from the attack of the lethal dose of H5N6-GD154 virus.

[0034] Table 3: Virus isolation results of the H5N6-GD154 strain challenge experiment after immunization of SPF chickens with the H5N6-VLP vaccine

[0035] Note: NA: no sample was collected due to the death of the experimental chicken; dpc: days post challenge.

[0036] Example 5 Immune efficacy evaluation of avian influenza (H5+H7) trivalent virus-like particle vaccine 5.1 Preparation of vaccine The high-speed homogenizer was washed with sterile PBS, and 150 μL of each of the previously prepared H5N6-VLP, H5N8-VLP and H7N9-VLP antigens were mixed uniformly. This step ensured that the amount of each antigen was the same to ensure the uniformity of the final mixture. The mixed antigens were mixed with 450 μL of ISA 78 adjuvant, and the volume ratio of antigen to adjuvant was 1:1. After emulsification by the homogenizer, 10 μL of the vaccine was detected, and the first drop was added after the second drop presented an oil film. If the vaccine was successfully prepared within 30 seconds without dispersion, the vaccine was obtained, which was called H5N6-H5N8-H7N9-VLP vaccine. The final vaccine had a hemagglutination titer of 9log2.

[0037] 5.2 Immunization program of vaccine A total of 205 21-day-old SPF chickens were randomly divided into 6 groups, and the specific allocation scheme is shown in Table 4. All groups were immunized by subcutaneous injection in the neck. The H5N6-VLP, H5N8-VLP, H7N9-VLP and PBS groups were injected with 0.3 mL per chicken; the commercial inactivated vaccine was injected according to the instructions, 0.3 mL per chicken; the H5N6-H5N8-H7N9-VLP vaccine group was injected with 0.9 mL per chicken.

[0038] Table 4: Animal grouping and immunization scheme for evaluating the immune efficacy of H5N6-H5N8-H7N9-VLP vaccine Group Vaccine Adjuvant Volume ratio Number (only) 1 H5N6 VLP ISA 78 1:1 10 2 H5N8 VLP ISA 78 1:1 10 3 H7N9 VLP ISA 78 1:1 10 4 H5N6-H5N8-H7N9-VLP ISA 78 1:1 70 5 Commercial inactivated vaccine - - 70 6 PBS - - 35

[0039] 5.3 Serum antibody detection results

[0040] 5.3.1 Antibody detection of H5N6-H5N8-H7N9-VLP vaccine against parental strains To evaluate the difference of H5N6-H5N8-H7N9-VLP trivalent vaccine in inducing antibodies compared with corresponding monovalent VLP vaccine and commercial inactivated vaccine, H5N6-VLP group, H5N8-VLP group, H7N9-VLP group, H5N6-H5N8-H7N9-VLP trivalent vaccine group and commercial inactivated vaccine group were set up for immunization, and serum was isolated at 2 weeks and 3 weeks after immunization for HI antibody detection against parental strains. The results of HI antibody detection are shown in Table 5. Figure 18 The results show that, for H5N6-GD154 strain, the average HI antibody titers of H5N6-VLP group, H5N6-H5N8-H7N9-VLP group and commercial inactivated vaccine group at 2 weeks after immunization are 5.5 log2, 5.8 log2 and 4.1 log2, respectively; and the average HI antibody titers at 3 weeks after immunization are 7.5 log2, 7.6 log2 and 8 log2, respectively (Table 5a). For H5N8-GD110 strain, the average HI antibody titers of H5N8-VLP group, H5N6-H5N8-H7N9-VLP group and commercial inactivated vaccine group at 2 weeks after immunization are 6.5 log2, 6.7 log2 and 7.1 log2, respectively; and the average HI antibody titers at 3 weeks after immunization are 8.2 log2, 8.5 log2 and 8.6 log2, respectively (Table 5b). For H7N9-C5e-144 strain, the average HI antibody titers of H7N9-VLP group, H5N6-H5N8-H7N9-VLP group and commercial inactivated vaccine group at 2 weeks after immunization are 5.6 log2, 5.8 log2 and 4.6 log2, respectively; and the average HI antibody titers at 3 weeks after immunization are 7.5 log2, 7.5 log2 and 8 log2, respectively (Table 5c). Figure 18 Figure 18 Figure 18 It should be noted that, Figure 18 the comparison chart of HI antibody titers against parental strains after immunization of SPF chickens with H5N6-H5N8-H7N9-VLP vaccine compared with H5N6-VLP, H5N8-VLP and H7N9-VLP is shown in Table 5a, Table 5b and Table 5c, respectively. Figure 18 Table 5a is the comparison chart of H5N6-H5N8-H7N9-VLP and H5N6-VLP. Figure 18 Table 5b is the comparison chart of H5N6-H5N8-H7N9-VLP and H5N8-VLP. Figure 18 Table 5c is the comparison chart of H5N6-H5N8-H7N9-VLP and H7N9-VLP.​​​

[0041] The above results show that all vaccine groups produced high levels of antibodies at 3 weeks post-immunization, and there was no significant difference in HI antibody titers between the monovalent VLP vaccine and the H5N6-H5N8-H7N9-VLP trivalent vaccine at 2 and 3 weeks post-immunization. At 3 weeks post-immunization, the HI antibody titers of the H5N6-H5N8-H7N9-VLP vaccine were not significantly different from those of the commercial inactivated vaccine. This shows that the H5N6-H5N8-H7N9-VLP vaccine has good immunization effects, comparable to those of the corresponding monovalent VLP vaccine and the commercial inactivated vaccine, and there is no immune interference between multiple antigens.

[0042] 5.3.2 Antibody detection of H5N6-H5N8-H7N9-VLP vaccine against prevalent strains The cross-protection of the H5N6-H5N8-H7N9-VLP vaccine against 2 H5 subtypes and 2 H7N9 subtypes of AIV strains prevalent in 2024 was detected by the HI antibody detection method. The results are shown in FIGS. 5A and 5B. Figure 19 Figure 19 FIG. 5A shows the antibody titer detection graph for H5N6-H5N8-H7N9-VLP vaccine immunized SPF chickens against H5 subtype prevalent strains, where a is the antibody titer detection graph for H5N2-GD285 challenge strain, and b is the antibody titer detection graph for H5N1-GD105. Figure 19 Figure 19 The average HI antibody titers of the H5N6-H5N8-H7N9-VLP group and the commercial inactivated vaccine group were 3.4 log2 and 3.0 log2, respectively, at 2 weeks post-immunization, and 5.2 log2 and 5.7 log2, respectively, at 3 weeks post-immunization, against the H5N2-GD285 strain. The average HI antibody titers of the H5N6-H5N8-H7N9-VLP group and the commercial inactivated vaccine group were 2.9 log2 and 2.3 log2, respectively, at 2 weeks post-immunization, and 4.4 log2 and 5.2 log2, respectively, at 3 weeks post-immunization, against the H5N1-GD105 strain. All immunized groups showed 100% HI antibody positive conversion at 3 weeks post-immunization in terms of immune response against H5 subtype AIV prevalent strains. In terms of immunization effect, the antibody level of the H5N6-H5N8-H7N9-VLP vaccine group was higher than that of the commercial inactivated vaccine group at 2 weeks post-immunization, but the antibody titer of the commercial inactivated vaccine group was significantly higher than that of the H5N6-H5N8-H7N9-VLP vaccine group at 3 weeks post-immunization. This shows that the H5N6-H5N8-H7N9-VLP vaccine can induce immune response quickly and has good cross-protection.

[0043] ​​The cross-protection of H5N6-H5N8-H7N9-VLP vaccine against H7N9 subtype AIV epidemic strain was further evaluated by HI antibody detection technology, and the results are shown in Figure 20 Figure 20 The HI antibody titers against H7N9 subtype epidemic strain after immunizing SPF chickens with H5N6-H5N8-H7N9-VLP vaccine are shown in Figure 20 In a, the antibody titer detection graph for the challenge strain H7N9-C5F-145; Figure 20 In b, the antibody titer detection graph for the challenge strain H7N9-GD014.For the H7N9-C5F-145 strain, the average HI antibody titers of the H5N6-H5N8-H7N9-VLP group and the commercial inactivated vaccine group were 4.6 log2 and 5.8 log2, respectively, at 2 weeks after immunization; and the average HI antibody titers of the two groups were 5.3 log2 and 6.4 log2, respectively, at 3 weeks after immunization. For the H7N9-GD014 strain, the average HI antibody titers of the H5N6-H5N8-H7N9-VLP group and the commercial inactivated vaccine group were 3.2 log2 and 4.8 log2, respectively, at 2 weeks after immunization; and the average HI antibody titers of the two groups were 4.8 log2 and 5.5 log2, respectively, at 3 weeks after immunization. For different strains of H7N9 subtype, all immunized groups reached 100% antibody positive conversion rate at 3 weeks after immunization.

[0044] In summary, the H5N6-H5N8-H7N9-VLP vaccine can rapidly induce an immune response in the early stage and has good cross-protection. However, in the late stage after immunization (3 weeks), the antibody response of the vaccine to heterologous strains is weaker than that of the commercial vaccine.

[0045] 5.3.3 Challenge protection experiment results of H5N6-H5N8-H7N9-VLP vaccine (1) Challenge protection results of H5N6-H5N8-H7N9-VLP vaccine against parent strain The protection efficacy of H5N6-H5N8-H7N9-VLP trivalent vaccine against parent strain was evaluated by challenge protection test, together with corresponding monovalent vaccine and commercial inactivated vaccine. The challenge was performed at 3 weeks after vaccine immunization, and the death of test chickens was recorded every day, and the results were observed continuously for 14 days Figure 21 Figure 21 The survival curve of chickens in the parent strain challenge experiment after immunizing SPF chickens with H5N6-H5N8-H7N9-VLP vaccine is shown in Figure 21 In a, the survival curve of chickens in the parent strain challenge experiment after immunizing SPF chickens with H5N6-H5N8-H7N9-VLP and H5N6-VLP; Figure 21 ​Figure 6. Survival curves of chickens immunized with H5N6-H5N8-H7N9-VLP and challenged with H5N6, H5N8 and H7N9. Figure 21 Figure 6. Survival curves of chickens immunized with H5N6-H5N8-H7N9-VLP and challenged with H5N6, H5N8 and H7N9.

[0046] Table 5. Virus isolation results of H5N6-GD154 challenge test after SPF chickens were immunized with H5N6-H5N8-H7N9-VLP

[0047] Note: NA: experimental chickens died and no samples were collected; dpc: post-challenge.

[0048] Table 6. Virus isolation results of H5N8-GD110 challenge test after SPF chickens were immunized with H5N6-H5N8-H7N9-VLP

[0049] Note: NA: experimental chickens died and no samples were collected; dpc: post-challenge.

[0050] Table 7. Virus isolation results of H7N9-C5e-114 challenge test after SPF chickens were immunized with H5N6-H5N8-H7N9-VLP

[0051] Note: NA: experimental chickens died and no samples were collected; dpc: post-challenge.

[0052] (2) Challenge protection results of H5N6-H5N8-H7N9-VLP against epidemic strains The protection efficacy of the H5N6-H5N8-H7N9-VLP trivalent vaccine and the commercial inactivated vaccine against the currently prevalent H5 subtype (H5N2-GD285 and H5N1-GD105) and H7 subtype (H7N9-C5F-145 and H7N9-GD014) highly pathogenic avian influenza epidemic strains was evaluated by a challenge protection test. Throat swabs and cloacal swabs of the test chickens were collected at 5 days and 7 days after the challenge for virus detection, and the results are shown in Tables 8-9. The PBS group all died within 3 days after the strain challenge, and symptoms such as dyspnea, diarrhea, staggering, listlessness, fluffed feathers, and listlessness were observed. After the challenge of the H5 subtype and H7 subtype HPAIV epidemic strains, the H5N6-H5N8-H7N9-VLP trivalent vaccine and the commercial inactivated vaccine all survived without obvious symptoms and virus detection within the entire observation period. It is indicated that the H5N6-H5N8-H7N9-VLP vaccine and the commercial inactivated vaccine can provide broad protection against 2 strains of H5 subtype and 2 strains of H7N9 subtype HPAIV epidemic strains.

[0053] Table 8: Virus isolation results after H5 subtype HPAIV epidemic strain challenge of test chickens

[0054] Note: NA: experimental chickens died and no samples were collected; dpc: after challenge.

[0055] Table 9: Virus isolation results after H7N9 subtype HPAIV epidemic strain challenge of test chickens for 5 days and 7 days

[0056] Note: NA: experimental chickens died and no samples were collected; dpc: after challenge.

[0057] All the above animal test results show that the present application develops an avian influenza virus-like particle vaccine (H5N6-H5N8-H7N9-VLP) covering H5N6, H5N8, and H7N9 three subtypes. The trivalent vaccine exhibits good immunization effect, and the protection efficacy is equivalent to that of the monovalent VLP vaccine and the commercial inactivated vaccine, and no immunological interference occurs between different antigens. The vaccine can produce good cross-protection against the parent strain and the main epidemic strain in 2024. The avian influenza (H5+H7) trivalent virus-like particle vaccine prepared by the present application provides a new vaccine selection in the prevention and control of avian influenza.

[0058] Comparative Example 1 Referring to CN113862284B, the subject is a gene encoding recombinant avian influenza virus HA protein, virus-like particles, vaccines and preparation and application. The vaccine can provide complete clinical protection and significantly inhibit virus shedding against lethal challenge of homologous and wild-type H7N9 subtype highly pathogenic avian influenza virus.

[0059] Comparative Example 1 is the prior application of the present applicant. By comparison with Comparative Example 1, it can be seen that the virus-like particle vaccine prepared in Comparative Example 1 is prepared by co-infection expression, which requires 3 kinds of insect baculovirus to co-infect during production; the vaccine in Comparative Example 1 is a monovalent virus-like particle vaccine of H7N9 subtype, which has limited protection ability. However, the virus-like particle vaccine prepared in the present application is prepared by co-expression, which can achieve high-level protein expression in the production with only one kind of insect baculovirus. Moreover, the present application is a trivalent virus-like particle vaccine of avian influenza (H5+H7), which covers three subtypes of avian influenza, i.e., H5N6, H5N8 and H7N9; in terms of vaccine cross-protection, in addition to the parent strain, the present application also selects the epidemic strain in 2024, which can better illustrate that the trivalent virus-like particle vaccine has a relatively broad protective efficacy.

Claims

1. Virus-like particles of H5N6, H5N8 and H7N9, characterized in that, The virus-like particle of H5N6 is obtained by connecting HA, NA and M1 genes of H5N6 virus to a plasmid and transferring into competent cells to obtain a recombinant baculovirus plasmid; The virus-like particle of H5N6 is obtained by connecting HA, NA and M1 genes of H5N6 virus to a plasmid and transferring into competent cells to obtain a recombinant baculovirus plasmid; The virus-like particle of H5N8 is obtained by connecting HA, NA and M1 genes of H5N8 virus to a plasmid and transferring into competent cells to obtain a recombinant baculovirus plasmid; The virus-like particle of H5N8 is obtained by connecting HA, NA and M1 genes of H5N8 virus to a plasmid and transferring into competent cells to obtain a recombinant baculovirus plasmid; The virus-like particle of H7N9 is obtained by connecting HA, NA and M1 genes of H7N9 virus to a plasmid and transferring into competent cells to obtain a recombinant baculovirus plasmid; The virus-like particle of H7N9 is obtained by connecting HA, NA and M1 genes of H7N9 virus to a plasmid and transferring into competent cells to obtain a recombinant baculovirus plasmid; The amino acid sequence of the H5N6 virus HA protein is shown as SEQ ID NO: 1; The amino acid sequence of the H5N6 virus NA protein is shown as SEQ ID NO: 2; The amino acid sequence of the H5N6 virus M1 protein is shown as SEQ ID NO: 3; The amino acid sequence of the H5N8 virus HA protein is shown as SEQ ID NO: 4; The amino acid sequence of the H5N8 virus NA protein is shown as SEQ ID NO: 5; The amino acid sequence of the H5N8 virus M1 protein is shown as SEQ ID NO: 6; The amino acid sequence of the H7N9 virus HA protein is shown as SEQ ID NO: 7; The amino acid sequence of the H7N9 virus NA protein is shown as SEQ ID NO: 8; The amino acid sequence of the H7N9 virus M1 protein is shown as SEQ ID NO:

9.

2. The use of the virus-like particles of H5N6, H5N8 and H7N9 vaccines according to claim 1.

3. A H5 + H7 trivalent vaccine, characterized in that, The virus-like particles of H5N6, H5N8 and H7N9 are prepared as antigens.

4. The vaccine of claim 3, characterized in that, The virus-like particle of H5N6 is obtained by connecting HA, NA and M1 genes of H5N6 virus to a plasmid and transferring into competent cells to obtain a recombinant baculovirus plasmid; The virus-like particle of H5N6 is obtained by connecting HA, NA and M1 genes of H5N6 virus to a plasmid and transferring into competent cells to obtain a recombinant baculovirus plasmid; The virus-like particle of H5N8 is obtained by connecting HA, NA and M1 genes of H5N8 virus to a plasmid and transferring into competent cells to obtain a recombinant baculovirus plasmid; The virus-like particle of H5N8 is obtained by connecting HA, NA and M1 genes of H5N8 virus to a plasmid and transferring into competent cells to obtain a recombinant baculovirus plasmid; The virus-like particle of H7N9 is obtained by connecting HA, NA and M1 genes of H7N9 virus to a plasmid and transferring into competent cells to obtain a recombinant baculovirus plasmid; The virus-like particle of H7N9 is obtained by connecting HA, NA and M1 genes of H7N9 virus to a plasmid and transferring into competent cells to obtain a recombinant baculovirus plasmid.

5. The H5+H7 trivalent vaccine of claim 4, characterized in that, The nucleotide sequence encoding the H5N6 virus HA protein is shown as SEQ ID NO: 10; The nucleotide sequence encoding the H5N6 virus NA protein is shown as SEQ ID NO: 11; The nucleotide sequence encoding the M1 protein of the H5N6 virus is shown as SEQ ID NO: 12; The nucleotide sequence encoding the HA protein of the H5N8 virus is shown as SEQ ID NO: 13; The nucleotide sequence encoding the NA protein of the H5N8 virus is shown as SEQ ID NO: 14; The nucleotide sequence encoding the M1 protein of the H5N8 virus is shown as SEQ ID NO: 15; The nucleotide sequence encoding the HA protein of the H7N9 virus is shown as SEQ ID NO: 16; The nucleotide sequence encoding the NA protein of the H7N9 virus is shown as SEQ ID NO: 17; The nucleotide sequence encoding the M1 protein of the H7N9 virus is shown as SEQ ID NO: 18.

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