H9N2 subtype avian influenza virus virus-like particle vaccine and preparation method thereof

Abstract

The invention discloses an H9N2 subtype avian influenza virus virus-like particle. The virus-like particle is formed by assembling M1 protein of an H5N1 subtype avian influenza virus and HA protein and NA protein of the H9N2 subtype avian influenza virus; the M1 protein, the HA protein and the NA protein are respectively coded by genes M1, HA and NA, and the genes M1, HA and NA respectively have base sequences shown as SEQ ID NO.1 to SEQ ID NO.3 in a sequence table. Accordingly, the inventor also establishes a corresponding preparation method, i.e., HA, NA and M1 proteins are expressed by using a Bac-to-Bac baculovirus expression system, and the three proteins are self-assembled into the virus-like particles. Researches show that the expressed HA-M1 and NA recombinant protein can be self-assembled in Sf9 cells to form virus-like particles, the concentrated virus-like particles can agglutinate 1% of chicken erythrocytes, and the hemagglutination titer is 1: 64. Therefore, the invention lays a foundation for further research on the virus-like particle vaccine of the H9N2 subtype avian influenza virus.

Description

Technical Field

[0001] The invention belongs to the technical field of virus vaccines, and in particular relates to a virus-like particle vaccine of H9N2 subtype avian influenza virus and a preparation method thereof. Background Art

[0002] Avian influenza (AI) is an infectious disease of poultry (domestic and wild birds) caused by avian influenza virus (AIV) of the genus influenza virus A of the family Orthomyxoviridae. Since AIV can infect a variety of hosts, it is easy to undergo genetic recombination to produce new subtypes or new combinations, which poses a serious threat to public health and safety. It is reported that genetic recombination can occur between different subtypes of AIV, such as the H7N9 and H10N8 subtypes of AIV that can infect humans contain gene segments of the H9N2 subtype. As the most widely distributed low pathogenic AIV (LPAIV) in the world, the H9N2 subtype AIV can not only cause poultry disease and cause serious economic losses to the poultry farming industry, but also serve as a gene pool for genetic recombination of other subtypes of AIV. Due to its special status in the evolution of AIV, H9N2 has gradually attracted attention in recent years. In order to reduce the harm caused by the H9N2 subtype AIV, many regions use traditional inactivated vaccines as the main means of preventing H9N2. However, inactivated vaccines also have many disadvantages, such as poor ability to induce cellular immunity and mucosal immunity, low immune efficiency, high production cost, complex process, and possible antigen drift. Therefore, new AIV vaccines have become a new direction of current research and development, mainly subunit vaccines, recombinant live virus vector vaccines, universal influenza vaccines, virus-like particle vaccines, DNA vaccines, etc.

[0003] Virus-like particles (VLPs) are structured hollow particles self-assembled by viral structural proteins. In terms of morphology, VLPs are similar to natural virus particles and have immunogenicity, but they do not contain viral genetic material, are non-infectious, and do not have the ability to self-replicate. At the same time, the high-density antigenic epitopes on the surface of VLPs can induce the body's immune response, and foreign genes or gene fragments can be inserted. VLPs have become the focus of vaccine research and development due to their advantages such as short production cycle, high safety, and good immune effect. At present, influenza viruses mainly use glycoproteins HA, NA and matrix proteins M1 and M2 to construct VLPs. The M1 protein plays an important role in the packaging and morphogenesis of influenza viruses, and the immunity of this protein can induce good cellular immunity, which can provide cross-protection for different subtypes of influenza viruses; HA and NA proteins, as the main glycoproteins, play a key role in their virulence. HU et al. constructed a VLP vaccine using the HA, NA and M1 proteins of the H7N9 subtype AIV, and found that it could significantly reduce lung lesions and induce cellular immunity after immunization of chickens and mice. ZHU et al. successfully constructed a VLP vaccine using the HA and M1 proteins of H6 AIV, and tested the immunogenicity and protective efficacy when used in combination with adjuvants in a chicken model. The results showed that it could effectively reduce viral replication and produce specific serum antibodies IgG. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a virus-like particle vaccine of H9N2 subtype avian influenza virus and a preparation method thereof.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0006] The invention relates to virus-like particles of H9N2 subtype avian influenza virus, which are assembled from the M1 protein of H5N1 subtype avian influenza virus and the HA protein and NA protein of H9N2 subtype avian influenza virus; the M1 protein, HA protein and NA protein are encoded by genes M1, HA and NA respectively, and the genes M1, HA and NA respectively have the base sequences of SEQ.ID.NO.1 to 3 in the sequence table.

[0007] Gene M1 comes from the recombinant avian influenza virus H5 subtype bivalent inactivated vaccine, and genes HA and NA come from strain A / chicken / Guangxi / 116c4 / 2012 (H9N2).

[0008] The hemagglutination titer of the virus-like particles was 1:64.

[0009] The method for preparing the virus-like particles of the H9N2 subtype avian influenza virus utilizes the Bac-to-Bac baculovirus expression system to express HA, NA and M1 proteins, and the three proteins self-assemble into virus-like particles.

[0010] The above preparation method first constructs two recombinant baculoviruses rBV-HA-M1 and rBV-NA through the Bac-to-Bac baculovirus expression system, then co-infects insect Sf9 cells with rBV-HA-M1 and rBV-NA, collects the cell suspension, and obtains virus-like particles after treatment and concentration.

[0011] The recombinant baculoviruses rBV-HA-M1 and rBV-NA were of the P3 generation.

[0012] rBV-HA-M1 and rBV-NA were co-infected in insect Sf9 cells at an MOI of rBV-HA-M1 :MOI rBV-NA The quantity ratio is 1.5:1.5.

[0013] The time for co-infection of insect Sf9 cells with rBV-HA-M1 and rBV-NA was 72h.

[0014] The use of the virus-like particles or the virus-like particles obtained by the above preparation method in the preparation of H9N2 subtype avian influenza virus virus-like particle vaccines.

[0015] The virus-like particle vaccine of H9N2 subtype avian influenza virus contains the above-mentioned virus-like particles or the virus-like particles obtained by the above-mentioned preparation method.

[0016] In view of the existing problems in the prevention of H9N2 subtype AIV, the inventors designed and prepared a virus-like particle of H9N2 subtype avian influenza virus, which is assembled from the M1 protein of H5N1 subtype avian influenza virus and the HA protein and NA protein of H9N2 subtype avian influenza virus; the M1 protein, HA protein and NA protein are encoded by genes M1, HA and NA, respectively, and the genes M1, HA and NA have the base sequences of SEQ.ID.NO.1 to 3 in the sequence table, respectively. Based on this, the inventors also established a corresponding preparation method, that is, using the Bac-to-Bac baculovirus expression system to express HA, NA and M1 proteins, and the three proteins self-assemble into virus-like particles. By taking hemagglutinin (HA), neuraminidase (NA) and matrix protein (M1) as the objects, two recombinant baculoviruses, namely rBV-HA-M1 and rBV-NA, were constructed by Bac-to-Bac baculovirus expression system, and the immune activity of the recombinant proteins was identified by IFA and Western-blot, and then rBV-HA-M1 and rBV-NA were co-infected with insect Sf9 cells at the optimal infection multiplicity ratio, and virus-like particles were obtained after concentration, and hemagglutination titer determination and transmission electron microscopy observation were performed at the same time. The transmission electron microscopy observation results showed that the expressed HA-M1 and NA recombinant proteins can self-assemble to form virus-like particles in Sf9 cells, and the concentrated virus-like particles can agglutinate 1% of chicken red blood cells, and the hemagglutination titer is 1:64. Therefore, the present invention lays a foundation for further research on H9N2 subtype avian influenza virus virus-like particle vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a diagram of the amplification results of HA, NA and M1 genes, in which: M: Trans2K Plus II DNA Marker; 1-2: amplification products of HA gene; 3-4: amplification products of NA gene; 5-6: amplification products of M1 gene.

[0018] Figure 2 This is a diagram of the enzyme digestion identification results of the recombinant plasmids pFastBacDual-HA-M1 and pFastBacDual-NA, in which: M: Trans2K Plus II DNA Marker; A1: digestion product of the HA gene in the pFastBacDual-HA-M1 plasmid, A2: digestion product of the M1 gene in pFastBacDual-HA-M1; B1 and B2: digestion products of pFastBacDual-NA.

[0019] Figure 3This is a diagram showing the PCR identification results of the recombinant bacmids rBacmid-HA-M1, rBacmid-NA and rBacmid-Dual, wherein: M: Trans2K Plus II DNA Marker; 1: amplification product of rBacmid-HA-M1; 2: amplification product of rBacmid-NA; 3: amplification product of rBacmid-Dual.

[0020] Figure 4 This is a diagram showing the cytopathic effect of Sf9 insect cells transfected with rBacmid-HA-M1, rBacmid-NA and rBacmid-Dual for 72 hours, wherein: A: rBacmid-HA-M1; B: rBacmid-NA; C: rBacmid-Dual; D: normal cells.

[0021] Figure 5 This is a graph showing the PCR identification results of rBV-HA-M1, rBV-NA and rBV-Dual, wherein: M: Trans2K PlusII DNA Marker; 1: amplification product of rBV-HA-M1 using HA-specific primers; 2: amplification product of rBV-NA using NA-specific primers; 3: amplification product of rBV-HA-M1 using M1-specific primers; 4: rBV-Dual blank control.

[0022] Figure 6 This is an indirect immunofluorescence assay (IFA) result diagram, in which: A: HA protein mouse monoclonal antibody; B: HisTag mouse monoclonal antibody; C: M1 protein mouse monoclonal antibody; D: rBV-Dual control.

[0023] Figure 7 This is a Western-blot identification result of HA, NA and M1 proteins at 24, 48, 72 and 96 hours when rBV-HA-M1 and rBV-NA co-infected Sf9 cells, in the figure: M: 6.5-270kDa Marker; A2~A5: expression of HA protein at 24h, 48h, 72h and 96h respectively; B2~B5: expression of NA protein at 24h, 48h, 72h and 96h respectively; C2~C5: expression of M1 protein at 24h, 48h, 72h and 96h respectively; A1, B1 and C1: cell supernatant 72 hours after rBV-HA-M1 and rBV-NA co-infection; 6: rBV-Dual cell lysate.

[0024] Figure 8This is a Western-blot identification result diagram of HA and NA proteins under different treatment conditions, in which: M: 6.5-270kDa Marker; A: NA protein; B: HA protein; 1: protein treated with PNGase F; 2: untreated protein.

[0025] Fig. 9 The results of the expression of HA, NA and M1 in Sf9 cells co-infected with rBV-HA-M1 and rBV-NA at different MOI ratios are shown in the figure. In the figure: M: 6.5-270kDa Marker; A: expression of NA protein at different MOI ratios of rBV-HA-M1 and rBV-NA; B: expression of HA and M1 proteins at different MOI ratios of rBV-HA-M1 and rBV-NA; 1-9: MOI rBV-HA-M1 :MOI rBV-NA =1:1, 1:1.5, 1:2, 1.5:1, 1.5:1.5, 1.5:2, 2:1, 2:1.5, 2:2.

[0026] Fig.10 This is a graph showing the results of VLPs hemagglutination titer determination, in which: 1 to 3 are the hemagglutination titers of the same VLPs.

[0027] Fig.11 This is the morphological observation result of VLPs under transmission electron microscope.

[0028] Fig.12 This is a graph showing the HI titer of mouse serum.

[0029] Fig.13 This is the OD value result graph of IgG and IgG1 in mouse serum. DETAILED DESCRIPTION

[0030] 1 Materials and methods

[0031] 1.1 Materials

[0032] 1.1.1 Plasmids, antibodies, cells and strains The insect cell expression vector pFastBacDual and anti-H9N2 subtype AIVHA protein monoclonal antibody are deposited by the applicant; the HA and NA genes are derived from the strain A / chicken / Guangxi / 116c4 / 2012 (H9N2); the Sf9 insect cells are deposited by the applicant; the M1 gene in the M1-18T plasmid is obtained by extracting the recombinant avian influenza virus (H5 subtype) bivalent inactivated vaccine (H5N6 H5-Re13 strain + H5N8 H5-Re 14 strain) and then using gene cloning technology.

[0033] 1.1.2 Main reagents Escherichia coli DH5α and Trans2K Plus II DNA Marker were purchased from Beijing Quanshijin Biotechnology Co., Ltd., Escherichia coli DH10Bac, sf-900II SFM medium and Cellfectin transfection kit were purchased from Invitrogen; Nhe I, BamHⅠ, HindⅢ, Kpn I, T4 ligase and Premix Taq TM (LATaq TM Version 2.0 plus dye) were purchased from Bio-Rad Biotechnology (Beijing) Co., Ltd., SDS-PAGE gel rapid preparation kit was purchased from Nanjing GenScript Biotechnology Co., Ltd.; endotoxin-free plasmid extraction kit and nucleic acid gel recovery reagent were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; protein molecular weight standard BeyoColor TM Color prestained protein molecular weight standard (6.5-270kD), nitrocellulose membrane (PVDF membrane), BCIP / NBT alkaline phosphatase colorimetric kit, FITC-goat anti-mouse IgG antibody and His Tag mouse monoclonal antibody were purchased from Shanghai Biotech Biotechnology Co., Ltd.; anti-influenza A virus M1 protein monoclonal antibody was purchased from abcam; mouse antibody subtype identification kit was purchased from Sigma.

[0034] 1.1.3 Primers The primers used to amplify the HA and NA genes of H9N2 subtype AIV and the primers used to amplify the M1 gene are shown in Table 1 below. The size of the HA gene is 1719 bp, the size of the NA gene is 1418 bp, and the size of the M1 gene is 780 bp. The reverse complementary bases encoding 6 histidine codons were added to the 5' end of the BDNA-R primer. The primers were synthesized by BGI Biotechnology Co., Ltd.

[0035] Table 1 Primer information

[0036]

[0037]

[0038] The underline "__" indicates the restriction site, and the bold part is the reverse complementary base encoding the 6 histidine codons

[0039] 1.2 Methods

[0040] 1.2.1 Amplification of HA, NA and M1 genes A / chicken / Guangxi / 116c4 / 2012(H9N2) cDNA and M1-18T plasmid were used as templates, and HA, NA and M1 genes were amplified by PCR using BDH9-F / R, BDNA-F / R and BDM1F / R, respectively. PCR amplification reaction conditions: pre-denaturation at 95℃ for 3min; denaturation at 95℃ for 30s, annealing at 55℃ for 30s, extension at 72℃ for 2min, for a total of 35 cycles; and extension at 72℃ for 5min. PCR products were subjected to agarose gel electrophoresis and gel recovery.

[0041] 1.2.2 Construction and identification of recombinant transfer vectors pFastBacDual-M1-HA and pFastBacDual-NA The HA gene gel recovery product and the pFastBacDual vector were double-digested with restriction endonucleases BamHⅠ and HindⅢ, and the HA gene was ligated to the pFastBacDual vector according to the T4 ligase operating instructions to obtain the recombinant transfer vector pFastBacDual-HA; the gel recovery products of pFastBacDual-HA and M1 gene were double-digested with restriction endonucleases Nhe I and Kpn I, respectively, the double-digested products were purified and recovered, and pFastBacDual-HA and M1 gene were ligated with T4 ligase to construct the recombinant transfer vector pFastBacDual-HA-M1. The pFastBacDual vector and the gel recovery product of the NA gene were double-digested with restriction endonucleases Xho I and Kpn I, respectively, and the double-digested products of the pFastBacDual vector and the NA gene were recovered. The pFastBacDual vector and the NA gene after digestion were connected with T4 ligase to obtain the recombinant transfer vector pFastBacDual-NA. The two recombinant transfer vectors were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The plasmids with correct sequencing were double-digested with the corresponding restriction endonucleases, and the digested products were subjected to electrophoresis.

[0042] 1.2.3 Transposition to construct recombinant bacmid DNA The constructed recombinant transfer vectors pFastBacDua-NA, pFastBacDua-HA-M1 and the blank control pFastBacDual were transformed into Escherichia coli DH10Bac competent cells respectively. The method of reference (Zhang Minxiu, Xie Zhixun, Huang Li, et al. Expression of HA gene of H9N2 subtype avian influenza virus in insect cells [J]. Genomics and Applied Biology, 2017, 36(03): 859-864.) was used to screen rBacmid-HA-M1, rBacmid-NA and rBacmid-Dual recombinant bacmids. The universal primers PUC-M13-F and PUC-M13-R were used to perform PCR identification on the bacterial solution containing the third-generation recombinant bacmid. The recombinant bacmids in the third-generation positive bacterial solution were extracted to obtain the recombinant bacmids rBacmid-HA-M1, rBacmid-NA and rBacmid-Dual.

[0043] 1.2.4 Preparation of recombinant baculovirus According to the operating steps of the Cellfectin transfection kit manual, 3 μg of recombinant bacmids rBacmid-HA-M1, rBacmid-NA and rBacmid-Dual were transfected into Sf9 cells respectively, and cultured at 27°C. The cytopathic effect was observed every day, and the first generation of recombinant virus (P1) was collected on the 6th day of culture. P1 was further inoculated into Sf9 insect cells, and recombinant viruses rBV-HA-M1, rBV-NA and rBV-Dual were obtained after purification to the third generation. The third generation inoculated Sf9 cells and cell supernatants were collected to obtain recombinant virus rBV-HA-M1, rBV-NA and rBV-Dual cell suspensions. The recombinant virus suspension was extracted with viral DNA according to the operating instructions of the EasyPure Viral DNA Extraction Kit, and the extracted DNA was identified by PCR using specific primers for amplifying HA, NA and M1 genes.

[0044] 1.2.5 Indirect immunofluorescence (IFA) identification of HA, M1 and NA proteins The expression of HA, M1 and NA proteins was verified using IFA experiments. P3 generation rBV-HA-M1, rBV-NA, and rBV-Dual were inoculated into Sf9 cells, respectively, and the supernatant was discarded after culture for 72 hours. The cells were fixed with 4% paraformaldehyde at room temperature for 30 minutes, washed with PBS, and blocked with 5% skim milk powder at 37°C for 1 hour; washed with PBS three times, and Sf9 cells infected with rBV-NA were incubated with anti-HisTag mouse monoclonal antibody as the primary antibody (1:500), and Sf9 cells infected with rBV-HA-M1 were incubated with anti-M1 protein and anti-HA protein mouse monoclonal antibodies as the primary antibody (1:500), both of which were incubated at 37°C for 1 hour, washed with PBS three times, and FITC-goat anti-mouse IgG antibody (1:1000) was added and incubated at 37°C for 1 hour; washed with PBST, and placed under an inverted microscope to observe fluorescence and take pictures.

[0045] 1.2.6 Optimization of conditions for co-infection of Sf9 cells with rBV-HA-M1 and rBV-NA and detection of glycosylation of HA and NA proteins Sf9 cells were co-infected with rBV-HA-M1 and rBV-NA of P3 generation at MOI=1. When infected for 24, 48, 72 and 96 hours, the cell suspensions at different time points after co-infection were collected. The cells infected with rBV-Dual for 72 hours were collected as negative controls. Western-blot was used to identify the expression of the three proteins at different time points. The collected cells were processed as follows: the cell suspension was centrifuged at 1000r / min for 10 minutes, the cell supernatant and cell pellet were collected respectively, the cell pellet was lysed with cell lysis buffer to obtain cell lysate; the cell lysate and cell supernatant were electrophoresed with 12% protein gel SDS-PAGE, the gel was transferred to a PVDF membrane, and blocked with 3% bovine serum albumin (3% BSA) at 4°C overnight to obtain anti-His Tag mouse monoclonal antibody, anti-M1 protein and anti-HA protein mouse monoclonal antibody were used as primary antibody (1:1000), incubated at 37℃ for 1h, washed and then alkaline phosphatase-labeled goat anti-mouse IgG (H+L) was used as secondary antibody (1:2000). The cell lysate infected with rBV-Dual was used as negative control for Western-blot analysis to identify the expression of HA, NA and M1 proteins at different time points after co-infection of rBV-HA-M1 and rBV-NA. The sizes of M1, NA and HA proteins were approximately 28ku, 52ku and 62ku, respectively. Image J software was used to analyze the grayscale of Western-blot protein bands. The cell lysate collected after 72 hours of rBV-HA and rBV-NA infection of Sf9 cells was treated with the instructions of the peptide N-glycosidase F (PNGase F) product to remove the sugar chains on the HA and NA proteins. The treated products and untreated HA and NA proteins were subjected to SDS-PAGE electrophoresis at the same time and analyzed according to the above Western-blot steps.

[0046] 5×10 5 Sf9 cells / mL were plated into 6-well cell culture plates, and P3 rBV-HA-M1 and rBV-NA were co-infected into Sf9 cells at an MOI of rBV-HA-M1 :MOI rBV-NA The molecular weight ratios were 1:1, 1:1.5, 1:2, 1.5:1, 1.5:1.5, 1.5:2, 2:1, 2:1.5, and 2:2, respectively. After 72 hours of co-infection with rBV-HA-M1 and rBV-NA, the cell suspension was collected and centrifuged at 1000 r / min for 10 min. The cell pellet was collected and lysed with cell lysis buffer to obtain cell lysate. Western-blot was used to identify the expression of the three proteins at different MOIs.

[0047] 1.2.7 Concentration of VLPs and MOI rBV-HA-M1 :MOI rBV-NA After 72 hours of co-infection of Sf9 insect cells with rBV-HA-M1 and rBV-NA at a ratio of 1.5:1.5, the supernatant of the suspended cells was collected, centrifuged twice at 4°C to remove cell debris and retain the supernatant, 30% PEG6000 was added until a precipitate appeared, and placed at 4°C overnight. The obtained solution was centrifuged at 19000r / min for 20min, the supernatant was discarded, and the precipitate was dissolved with an appropriate amount of PBS; after dissolution, it was centrifuged at 35000r / min for 3h, the supernatant was discarded, and the precipitate was dissolved with an appropriate amount of PBS, all of which needed to be completed at 4°C.

[0048] 1.2.8 Determination of hemagglutination titer of VLPs The hemagglutination titer was determined in accordance with the national standard GB / T 18936-2020 for diagnostic technology of highly pathogenic avian influenza, and the HA titer of concentrated VLPs was detected using 1% chicken red blood cell suspension in a 96-well V-type hemagglutination plate.

[0049] 1.2.9 Transmission electron microscopy observation of VLPs The concentrated VLPs were collected and sent to Beijing Zhongke Baice Technology Co., Ltd. for transmission electron microscopy observation.

[0050] 1.2.10 Preliminary analysis of the immunogenicity of VLPs In this study, 8-week-old BALB / c mice were selected as immunized animals to preliminarily evaluate the immunogenicity of the obtained VLPs. Ten mice were divided into two groups, with 5 mice in each group. One group was injected after emulsification with PBS and an equal amount of adjuvant; the other group was the VLPs group. Immunization was performed twice, with an interval of 2 weeks between the two immunizations. Before immunization, VLPs were mixed and emulsified with the same volume of Freund's complete adjuvant. After emulsification, the emulsified VLPs vaccine was subcutaneously immunized according to the immunization dose of 50 μg VLPs per mouse. At the same time, a control group was set up, which was emulsified and injected with PBS in the same way; on the 15th day after the first immunization, the second immunization was performed in the same way as the first immunization. Blood was collected from the orbital venous plexus of mice 7 days and 14 days after the first and second immunizations, and the serum was separated. The serum antibody titers after the first and second immunizations were determined by hemagglutination inhibition test. On the 21st day after the second immunization, the IgG and IgG1 in the serum were determined using a mouse antibody subtype identification kit to evaluate the immune effect of VLPs vaccine on mice.

[0051] 2 Results

[0052] 2.1 Genetic evolution analysis of HA and NA genes The nucleotide sequences of HA and NA genes uploaded to NCBI from 2020 to 2023 were selected for sequence comparison with the strains used in this article. The evolutionary tree results showed that the strain A / chicken / Guangxi / 116c4 / 2012 (H9N2) used in this experiment was genetically close to other strains and was an epidemic strain.

[0053] 2.2 Amplification results of HA, NA and M1 genes Agarose gel electrophoresis of the amplified products of HA, NA and M1 genes showed that the sizes of the amplified fragments were consistent with expectations, with the HA gene being approximately 1719 bp, the NA gene being approximately 1430 bp, and the M1 gene being approximately 780 bp ( Figure 1 ).

[0054] 2.3 Construction and identification of recombinant transfer vectors pFastBacDual-HA-M1 and pFastBacDual-NA The positive recombinant plasmids pFastBacDual-HA-M1 and pFastBacDual-NA were double-digested with BamHI and HindⅢ, NheI and Kpn I, and XhoI and Kpn I, respectively. The double-digestion products of pFastBacDual-HA-M1 with BamHI and HindⅢ were about 1719 bp and 6120 bp ( Figure 2 A), the double digestion products of pFastBacDual-HA-M1 with Nhe I and Kpn I are about 780 bp and 7059 bp ( Figure 2 A), pFastBacDual-NA digestion products are about 1430 bp and 5340 bp ( Figure 2 B), which is consistent with expectations, indicating that the HA and M1 genes were co-ligated to the pFastBacDual vector, and the NA gene was successfully ligated to the pFastBacDual vector.

[0055] 2.4 Identification of recombinant bacmid DNA rBacmid-HA-M1, rBacmid-NA and rBacmid-Dual The recombinant bacmid DNA rBacmid-HA-M1, rBacmid-NA and rBacmid-Dual were identified by recombinant bacmid universal primers and then agarose gel electrophoresis showed ( Figure 3 ), rBacmid-HA-M1 obtained a target fragment of about 5059 bp, rBacmid-NA PCR product size was about 3990 bp, and rBacmid-Dual obtained a target fragment of about 2560 bp. Recombinant bacmids rBacmid-HA-M1, rBacmid-NA and rBacmid-Dual were successfully constructed.

[0056] 2.5 Preparation of recombinant baculovirus After rBacmid-HA-M1, rBacmid-NA and rBacmid-Dual were transfected into Sf9 cells, the cells began to show cytopathic effects at 48 h, and the cells became round and enlarged. Figure 4The cell lesions were 72 hours after transfection. The DNA of the third-generation recombinant viruses rBV-HA-M1, rBV-NA and rBV-Dual were verified by PCR using specific primers for amplifying HA, NA and M1 genes. The electrophoresis results showed that rBV-Dual had no specific target bands, and the DNA amplification of rBV-HA-M1 and rBV-NA obtained amplified bands of HA, NA and M1 genes, which was consistent with expectations ( Figure 5 ).

[0057] 2.6 IFA identification of HA, M1 and NA proteins Figure 6 It can be seen that after the third-generation recombinant viruses rBV-HA-M1 and rBV-NA reacted with the corresponding monoclonal antibodies, bright green fluorescence could be observed under an inverted fluorescence microscope, while no green fluorescence was observed in Sf9 cells infected with the third-generation rBV-Dual.

[0058] 2.7 Optimization of conditions for co-infection of Sf9 cells with rBV-HA-M1 and rBV-NA and detection of glycosylation of HA and NA proteins Western-blot results showed that HA, M1 and NA proteins were expressed after rBV-HA-M1 and rBV-NA co-infected Sf9 cells, but the HA, M1 and NA proteins were not secreted into the cell supernatant; mouse monoclonal antibodies against HA and M1 proteins could specifically react with HA and M1 proteins expressed in Sf9 cells. The sizes of HA and M1 proteins were approximately 62 ku and 28 ku, respectively ( Figure 7 A and 7C). Western-blot identification of NA protein was performed using anti-His Tag mouse monoclonal antibody ( Figure 7 B), the results showed that the NA protein was larger than 52 ku, which may be due to the presence of glycosylation sites causing the size of the NA protein to be larger than the actual protein size ( Figure 7 B). Image J software was used to analyze the expression of HA, M1 and NA proteins at different time points. The results showed that the expression of HA, M1 and NA proteins was optimal at 72h, 96h and 48h of infection, respectively. Subsequent experiments collected cell samples 72h after infection. Western-blot analysis of the products of HA and NA proteins treated with PNGase F and untreated HA and NA proteins revealed that the untreated NA protein band was located between 58ku and 80ku of the protein marker ( Figure 8 A), the NA protein band after PNGase F treatment is located at 50ku~58ku ( Figure 8 A), which is consistent with the actual size of NA protein; while the size of HA protein after PNGase F treatment did not change significantly compared with that before treatment, indicating that HA protein was not glycosylated ( Figure 8 B) Western-blot analysis of different MOIsrBV-HA-M1 :MOI rBV-NA The results of co-infection of Sf9 cells showed that MOI rBV-HA-M1 :MOI rBV-NA The optimal infection multiplier ratio is 1.5:1.5 ( Fig. 9 ).

[0059] 2.8 VLPs hemagglutination titer determination results The hemagglutination titer of concentrated VLPs was determined, and the results showed that the hemagglutination titer of concentrated VLPs was 1:64 ( Fig.10 ).

[0060] 2.9 Transmission electron microscopy observation results are as follows Fig.11 As shown, under a transmission electron microscope, virus-like particles with a diameter of about 100 nm, a capsule but no nucleic acid, and spikes on the surface can be seen.

[0061] 2.10 Preliminary analysis of immunogenicity of VLPs

[0062] Determine the HI titer of the sera of immunized mice. Fig.12 As shown in the figure, the HI titer of the immunized group increased slowly 7-14 days after the first immunization, and increased rapidly on the 21st day after the second immunization. The average HI titer was as high as 256 on the 21st day after the second immunization (the 35th day after the first immunization), and the HI titer decreased slightly on the 42nd day. The indirect ELISA method was used to detect the IgG and IgG1 antibodies in the mouse serum on the 14th day after the second immunization, and the OD values ​​were obtained. The average OD values ​​of IgG and IgG1 antibodies in the immunized group were 1.036 and 0.830 ( Fig.13 ), which was significantly higher than that of the control group (P<0.05), indicating that VLP immunization induced mice to produce good humoral immunity.

[0063] 3 Discussions

[0064] At present, vaccination is the main means of preventing and controlling the spread of H9N2 subtype AIV in my country, but the isolates used to prepare the vaccine are still the H9N2 strains that were prevalent in the early 1990s. As AIV continues to mutate, its protective effect gradually weakens. In addition, studies have found that the widespread use of commercial vaccines with strain / subtype specificity is an important reason for the continuous mutation of AIV and the emergence of immune escape mutants. H9N2 subtype AIV can not only cause avian respiratory infectious diseases, but also provide internal segment templates for other influenza viruses through cross-recombination, posing a threat to public health and human safety. Therefore, it is crucial to develop a new H9N2 subtype AIV vaccine that is efficient, broad-spectrum, safe, and non-mutagenic.

[0065] As a self-assembling viral protein complex, VLPs are very similar to natural virus particles in conformation, can effectively stimulate the body to produce an immune response, and are not contagious or pathogenic. Currently, VLPs can be produced in a variety of different expression host systems such as bacteria, insects, yeast or mammalian cells. At the same time, compared with traditional inactivated vaccines, VLPs can induce more specific immune responses based on the antigens used in production. Based on the above advantages, VLPs have become a hot spot for the development of new vaccines. VLPs have been successfully constructed for various subtypes of AIV and have produced protective immune responses. For example, Prel et al. made VLPs using HA, NA and M1 proteins, and vaccinated ducks with the H5N3 VLP vaccine. The results showed that the ducks were protected; El-Husseiny et al. used the Bac-to-Bac baculovirus expression system to construct VLPs of the highly pathogenic H5N1 subtype AIV, and demonstrated the immune protection of VLPs by vaccinating SPF chickens; Lee et al. constructed H9N2 VLPs using HA and M1 proteins, and identified the efficacy of the vaccine through animal experiments and ELISA.

[0066] In this study, the construction of H9N2 subtype AIV VLPs was completed by expressing three main proteins, HA, NA and M1. The experimental results showed that VLPs can still be self-assembled by concentrating cell culture HA, NA and M1 proteins, and have hemagglutination activity. The hemagglutination titer of VLPs is only 1:64, which may be related to the fact that VLPs have not been purified and the total amount of concentration is small. At the same time, the Bac-to-Bac baculovirus expression system used in this study has been proven to have high immunogenicity in the production of VLP vaccines. The experiment further optimized the optimal infection multiplicity ratio of the Bac-to-Bac baculovirus expression system, so that HA, NA and M1 proteins are fully expressed, which is more conducive to the assembly of VLPs and the improvement of immune effect. By immunizing mice with VLPs, the results showed that a higher antibody titer was obtained, and the IgG and IgG1 antibody titers in the serum of the immune group were significantly higher than those of the control group, indicating that the VLPs can induce good humoral immunity in mice. This study is a phased result, and the immune protection test of SPF chickens will be further conducted to evaluate its immune effect in chickens.

[0067] In summary, this study co-expressed the M1 protein in the H5N1 AIV vaccine and the HA and NA proteins of the H9N2 subtype AIV in Sf9 cells, allowing the three proteins to form VLPs in insect cells with hemagglutination activity, laying a foundation for the development of new vaccines for the H9N2 subtype AIV and the effective prevention and control of the disease. It also provides data references for future explorations of the preparation of VLPs based on different proteins of different subtypes of AIV, in order to obtain an effective broad-spectrum virus-like particle AI vaccine.

Claims

1. A virus-like particle of H9N2 subtype avian influenza virus, Features The virus-like particle is assembled from the M1 protein of the H5N1 subtype avian influenza virus and the HA protein and NA protein of the H9N2 subtype avian influenza virus; the M1 protein, HA protein and NA protein are encoded by genes M1, HA and NA respectively, and the genes M1, HA and NA respectively have the base sequences of SEQ.ID.NO.1 to 3 in the sequence table.

2. The H9N2 subtype avian influenza virus virus-like particle according to claim 1, Features: The gene M1 comes from a recombinant avian influenza virus H5 subtype bivalent inactivated vaccine, and the genes HA and NA come from the strain A / chicken / Guangxi / 116c4 / 2012 (H9N2).

3. According to the H9N2 subtype avian influenza virus virus-like particle according to claim 2, Features The hemagglutination titer of the virus-like particles was 1:

64.

4. The method for preparing virus-like particles of H9N2 subtype avian influenza virus according to claim 1, Features The Bac-to-Bac baculovirus expression system was used to express HA, NA and M1 proteins, which self-assembled into virus-like particles.

5. The preparation method according to claim 4, Features: First, two recombinant baculoviruses, rBV-HA-M1 and rBV-NA, were constructed using the Bac-to-Bac baculovirus expression system. Then, rBV-HA-M1 and rBV-NA were co-infected into insect Sf9 cells. The cell suspension was collected and concentrated to obtain virus-like particles.

6. The preparation method according to claim 4, Features: The recombinant baculoviruses rBV-HA-M1 and rBV-NA are of the P3 generation.

7. The preparation method according to claim 4, Features: The rBV-HA-M1 and rBV-NA co-infected insect Sf9 cells at an MOI of rBV-HA-M1 :MOI rBV-NA The quantity ratio is 1.5:1.

5.

8. The preparation method according to claim 3, Features: The time for the rBV-HA-M1 and rBV-NA to co-infect insect Sf9 cells was 72 hours.

9. Use of the virus-like particles according to claims 1 to 3 or the virus-like particles obtained by the preparation methods according to claims 4 to 8 in preparing virus-like particle vaccines of H9N2 subtype avian influenza virus.

10. A virus-like particle vaccine of H9N2 subtype avian influenza virus, Features Contains the virus-like particles described in claims 1 to 3 or the virus-like particles obtained by the preparation method described in claims 4 to 8.

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