A broad-spectrum H9 subtype avian influenza subunit vaccine and a preparation method thereof

Abstract

The application provides a broad-spectrum H9 subtype avian influenza subunit vaccine and a preparation method thereof, and belongs to the field of genetic engineering. The antigen in the broad-spectrum H9 subtype avian influenza subunit vaccine is a protein with an amino acid sequence as shown in SEQ ID NO: 3. The antigen of the broad-spectrum H9 subtype avian influenza subunit vaccine can specifically react with H9 subtype avian influenza virus positive serum of different branches or sub-branches, and the immunization efficacy test shows that the antigen is safe, has no adverse reactions, can induce the body to produce a higher antibody level, can resist the attack of multiple branches or sub-branches of H9 subtype avian influenza viruses, significantly inhibits toxin discharge, and has a good immunization effect.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, specifically relating to a broad-spectrum H9 subtype avian influenza subunit vaccine and its preparation method. Background Technology

[0002] H9N2 avian influenza virus (AIV) is a significant infectious disease threatening the development of my country's poultry industry. Since its initial discovery in Wisconsin, USA in 1966, H9N2 AIV has spread widely worldwide. It was first isolated in Guangdong Province, my country in 1994. Epidemiological studies show that the infection rate of H9N2 AIV has gradually increased since 2016, and it has now become the most prevalent subtype of avian influenza in my country. It causes severe infection in chickens, turkeys, and waterfowl, with a high virus isolation rate in young birds. Especially when secondary infections occur, the mortality rate can reach 90%, causing serious losses to the poultry industry. Recent antigenic evolution analysis results show that H9N2 AIV consists of multiple branches and subbranches, including h9.4.2.5, exhibiting high genotypic diversity and poor cross-protection between different genotypes.

[0003] The primary means of controlling H9 subtype avian influenza remains immunization. Currently, the only available technologies and products, both domestically and internationally, are inactivated vaccines prepared based on a single circulating strain. While these vaccines have reduced the economic losses caused by H9N2 to the poultry industry to some extent, they cannot prevent infected chickens from shedding the virus, nor can they provide effective protection against variant strains. H9AIV is frequently isolated from immunized flocks, mainly due to poor vaccine antigen matching leading to viral antigen mutations and immune escape. However, there are currently no broad-spectrum immunization technologies or products for H9AIV prevention and control, either domestically or internationally. Summary of the Invention

[0004] The purpose of this invention is to provide a highly effective and broad-spectrum H9 subtype avian influenza subunit vaccine, which provides better prevention and control for the prevention and control of H9 subtype avian influenza.

[0005] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0006] A broad-spectrum H9 subtype avian influenza subunit vaccine, wherein the antigen in the vaccine is a protein with an amino acid sequence as shown in SEQ ID NO:3.

[0007] In this invention, the vaccine antigen is prepared by culturing a recombinant baculovirus carrying the protein-coding gene.

[0008] In a preferred embodiment, the sequence of the protein-coding gene is shown in SEQ ID NO:4.

[0009] In this invention, the recombinant baculovirus is constructed using the following method: a kozak sequence is added to the 5' end of SEQ ID NO:4, a Leu zip sequence is added to the 3' end, and then inserted into the baculovirus transfer vector pFastBac1 to obtain a recombinant baculovirus transfer plasmid; the recombinant baculovirus transfer plasmid is transformed into Escherichia coli DH10Bac competent cells to obtain a recombinant baculovirus shuttle plasmid; the recombinant baculovirus shuttle plasmid is transfected into insect Sf9 cells to obtain a recombinant baculovirus expressing the antigen.

[0010] In a preferred embodiment, the recombinant baculovirus is cultured using High Five cells.

[0011] In a preferred embodiment, the antigen is inactivated, and 4% by volume of Tween-80 is added and emulsified to obtain an aqueous phase; 96 parts by volume of white oil and 4 parts by volume of Span-80 are mixed evenly to obtain an oil phase; the aqueous phase and the oil phase are mixed and emulsified at a volume ratio of 1:3 to obtain the vaccine.

[0012] In the preferred technical solution, formaldehyde is used to inactivate the antigen.

[0013] The H9 subtype avian influenza subunit vaccine of this invention has broad-spectrum activity and can specifically cross-react with positive sera of different branches of H9 subtype avian influenza (h9.1 branch A / Chicken / Heilongjiang / 35 / 00 (H9N2), h9.4.2.3 branch A / Chicken / Shandong / S / 96 (H9N2), h9.4.2.5 subbranch A / Chicken / Henan / 01 / 2019 (H9N2), h9.4.2.5 subbranch A / Chicken / Jilin / 01 / 2019 (H9N2), h9.4.2.5 subbranch A / Chicken / Liaoning / 01 / 2015 (H9N2)). Immunopotency tests show that the antigen is safe, has no adverse reactions, can induce the body to produce high antibody levels, and has good immunization effects. Challenge protection tests show that the antibodies induced by the antigen after immunization can effectively inhibit viral shedding.

[0014] The antigen gene in the broad-spectrum H9 subtype avian influenza subunit vaccine of this invention can be cloned into the baculovirus expression system (Invitrogen) to obtain the antigen. Therefore, the preparation method of the vaccine antigen of this invention is simple, efficient and low cost. Attached Figure Description

[0015] Figure 1The results of indirect immunofluorescence assays are shown. (A) Schematic diagram of Sf9 cells infected with recombinant baculovirus rBV-HA-a; (B) Schematic diagram of Sf9 cells infected with recombinant baculovirus rBV-HA-b; (C) Schematic diagram of Sf9 cells infected with wild-type baculovirus.

[0016] Figure 2 The results of the microhemagglutination assays for recombinant proteins HA-a and HA-b are shown. The dilution factor of the recombinant protein in each well is marked above the well: 1log2, 2log2, 3log2, 4log2, 5log2…13log2. (A) rBv-HA-a infection product of HighFive cells; (B) rBv-HA-b infection product of HighFive cells; (C) wild-type baculovirus infection product of HighFive cells; (D) normal HighFive cell culture.

[0017] Figure 3 The results show the cross-reactivity of recombinant proteins HA-a and HA-b with positive sera from different branches or sub-branches of H9 subtype avian influenza virus. The horizontal axis shows the detection antigens of four units of HA-a and HA-b, and the vertical axis shows the HI titer of serum antibodies.

[0018] Figure 4 The levels of HI antibodies against different subclades of H9AIV in SPF chicken serum at different time points after immunization are shown. (A) The HI antibody titer in serum of each group after immunization was detected using the H9.4.2.5 subclade HN01 strain virus as a four-unit antigen; (B) The HI antibody titer in serum of each group after immunization was detected using the H9.4.2.5 subclade LN01 strain virus as a four-unit antigen.

[0019] Figure 5 The graphs show the weight gain curves of the experimental chickens after challenge. The horizontal axis represents the time after challenge in days, and the vertical axis represents the weight gain rate on day 0 of challenge in %. (A) Weight gain of the experimental chickens in each group 14 days after challenge with strain H9AIVHN01 of subbranch h9.4.2.5; (B) Weight gain of the experimental chickens in each group 14 days after challenge with strain H9AIV LN01 of subbranch h9.4.2.5; (C) Weight gain of the experimental chickens in each group 14 days after challenge with strain H9AIV S of branch h9.4.2.3. Detailed Implementation

[0020] The present invention can be further understood through the specific implementation of the following experiments.

[0021] The experimental materials used in the following embodiments are as follows:

[0022] Various restriction endonucleases, T4 ligases, and rTaq enzymes were purchased from Takara Bio Inc. Primers were synthesized by Beijing Qingke Biotechnology Co., Ltd. Transfection kits were purchased from Invitrogen. Insect cell culture medium was purchased from Yishengke Biotechnology Co., Ltd. FITC-labeled goat anti-chicken fluorescent antibody was purchased from Thermo Fisher Scientific. Antigens and positive sera from different branches or sub-branches of H9 subtype avian influenza were prepared and preserved in our laboratory. SPF chicken embryos were purchased from Boehringer Ingelheim Viton Biotechnology Co., Ltd.

[0023] Example 1

[0024] 1. Design, synthesis, and construction of recombinant baculovirus vector for H9 subtype avian influenza vaccine antigens.

[0025] Based on the hemagglutinin protein genes of H9 subtype avian influenza viruses published by NCBI, Influenza Research Database, GISAID, etc. from 2010 to 2019, multiple H9 subtype avian influenza vaccine antigen genes, including HA-a and HA-b, were designed. Immunopotency tests revealed that only the HA-b recombinant protein exhibited broad-spectrum and high-efficiency efficacy.

[0026] The following sections of this embodiment describe the construction of a recombinant baculovirus vector expressing HA-b protein and the construction of a recombinant baculovirus vector expressing HA-a protein.

[0027] The amino acid sequence of the HA-a protein is shown in SEQ ID NO:1, and the sequence of the encoding gene is shown in SEQ ID NO:2. The amino acid sequence of the HA-b protein is shown in SEQ ID NO:3, and the sequence of the encoding gene is shown in SEQ ID NO:4.

[0028] A kozak sequence was added to the 5' end of the HA-a protein coding gene, a Leu zip sequence was added to the 3' end, a BamHI restriction site was added to the 5' end, and an EcoRI restriction site was added to the 3' end. The resulting fragment was then sent to Qingke Biotechnology Co., Ltd. for synthesis to obtain the HA-a protein gene synthetic fragment.

[0029] A kozak sequence was added to the 5' end of the HA-b protein encoding gene, a Leu zip sequence was added to the 3' end, a BamHI restriction site was added to the 5' end, and an EcoRI restriction site was added to the 3' end. The resulting fragment was then sent to Qingke Biotechnology Co., Ltd. for synthesis to obtain the HA-b protein gene synthesis fragment.

[0030] The pFastBac1 and HA-b protein gene fragments were double-digested with BamHI and EcoRI, respectively, and then ligated overnight at 4°C using T4 ligase. The ligation products were transformed into *E. coli* DH5α competent cells, and positive clones were selected for Amp resistance. The recombinant vector was extracted from the positive clones, identified by enzyme digestion, and sequenced. The recombinant baculovirus transfer plasmid that was correctly identified by enzyme digestion and sequencing was named pFastBac1-HA-b. Using the same method, HA-a was inserted into pFastBac1 to obtain the recombinant baculovirus transfer plasmid pFastBac1-HA-a.

[0031] 2. Construction and identification of recombinant baculovirus shuttle vectors

[0032] Recombinant baculovirus transfer plasmids pFastBac1-HA-a and pFastBac1-HA-b were transformed into *E. coli* DH10Bac competent cells and cultured for 24–48 h on LB agar plates containing kanamycin (50 μg / mL), gentamicin (7 μg / mL), tetracycline (10 μg / mL), X-gal (100 μg / mL), and IPTG (40 μg / mL). After three passages of blue-white screening, white colonies were picked. These white colonies were inoculated into liquid LB medium containing kanamycin (50 μg / mL), gentamicin (7 μg / mL), and tetracycline (10 μg / mL). Colony PCR was performed to detect positive clones, and the recombinant shuttle plasmid was extracted and identified by PCR. The recombinant baculovirus shuttle plasmid carrying the HA-a protein-coding gene was correctly identified and named rBacmid-HA-a, while the recombinant baculovirus shuttle plasmid carrying the HA-b protein-coding gene was named rBacmid-HA-b.

[0033] Example 2

[0034] This embodiment describes the acquisition of recombinant baculovirus, recombinant protein expression, and IFA identification.

[0035] 1. Obtaining recombinant baculovirus

[0036] Following the transfection kit instructions, rBacmid-HA-b was transfected into Sf9 cells in logarithmic growth phase and cultured at 27°C. After the cells developed cytopathic effects, the cells and supernatant were collected; this was the first-generation recombinant baculovirus, named rBV-HA-b. The recombinant baculovirus rBV-HA-b was passaged into Sf9 cells to the fourth generation. The fourth-generation cells and supernatant were harvested, subjected to repeated freeze-thaw cycles, and centrifuged at 1000 rpm for 5 min. The supernatant was collected as the fourth-generation recombinant baculovirus, and TCID was determined using the Reed-Muench method. 50 The rBV-HA-b viral titer is 10.7.8 TCID 50 / 0.1mL.

[0037] Following the transfection kit instructions, rBacmid-HA-a was transfected into Sf9 cells in logarithmic growth phase and cultured at 27°C. After the cells developed cytopathic effects, the cells and supernatant were collected; this was the first-generation recombinant baculovirus, named rBV-HA-a. The recombinant baculovirus rBV-HA-a was passaged into Sf9 cells to the fourth generation. The fourth-generation cells and supernatant were harvested, subjected to repeated freeze-thaw cycles, and centrifuged at 1000 rpm for 5 min. The supernatant was collected as the fourth-generation recombinant baculovirus, and TCID was determined using the Reed-Muench method. 50 The rBV-HA-a virus titer is 10. 7.5 TCID 50 / 0.1mL.

[0038] 2. Expression of recombinant proteins

[0039] The third-generation recombinant baculovirus rBV-HA-b was inoculated at an MOI of 1 to a concentration of 1×10⁻⁶. 7 The cells were cultured in High Five medium at 27°C for 110 h in a constant temperature shaker, lysed by sonication, and the supernatant was collected by centrifugation to obtain recombinant HA-b protein.

[0040] HA-a protein was prepared using the same method.

[0041] 3. Indirect immunofluorescence assay (IFA) to identify recombinant protein expression.

[0042] Recombinant baculovirus rBV-HA-b was inoculated into Sf9 cells. After 72 hours of infection, the supernatant was discarded, cells were washed once with PBS, fixed with 4% paraformaldehyde at room temperature for 20 minutes, and then air-dried. Primary antibody was prepared with 1:200 diluted H9 subtype avian influenza positive serum, incubated at 37°C for 1 hour, and washed three times with PBS. Secondary antibody was prepared with 1:400 diluted FITC-labeled goat anti-chicken fluorescent antibody, incubated at 37°C for 1 hour, and washed three times with PBS. Results were observed under a fluorescence microscope. The recombinant protein HA-a was identified using the same indirect immunofluorescence assay (IFA), except that recombinant baculovirus rBV-HA-a was used instead of rBV-HA-b. Wild-type baculovirus infection of Sf9 cells was used as a negative control. Results ( Figure 1 The results showed that Sf9 cells infected with recombinant baculovirus rBV-HA-a and rBV-HA-b exhibited specific bright green fluorescence, while Sf9 cells infected with wild-type baculovirus did not exhibit specific fluorescence, indicating that recombinant proteins HA-b and HA-a were correctly expressed in Sf9 cells.

[0043] Example 3

[0044] 1. Identification of hemagglutination activity of recombinant protein

[0045] High Five cells were inoculated with recombinant baculovirus rBV-HA-b. After 100 h of infection, cells and culture medium were collected, lysed by sonication, and centrifuged at 3000 rpm for 15 min. The supernatant was collected to obtain recombinant protein HA-b. Recombinant protein HA-a was prepared using the same method, except that recombinant baculovirus rBV-HA-a was used instead of recombinant baculovirus rBV-HA-b. The hemagglutination activity of recombinant proteins HA-b and HA-a was detected by a microhemagglutination assay. Wild-type baculovirus-infected High Five cell expression products and normal High Five cell cultures were used as controls. Results ( Figure 2 The results showed that the hemagglutination titer of recombinant protein HA-a was 9 Log2, and that of recombinant protein HA-b was 12 Log2, indicating that recombinant proteins HA-a and HA-b have good hemagglutination activity.

[0046] 2. Recombinant protein cross-reactivity test

[0047] Using recombinant proteins HA-a and HA-b as four-unit antigens, the microhemagglutination inhibition (HI) assay was used to detect the recombinant proteins in relation to strains h9.1 branch A / Chicken / Heilongjiang / 35 / 00(H9N2) (hereinafter referred to as HLJ35 strain), h9.4.2.3 branch A / Chicken / Shandong / S / 96(H9N2) (hereinafter referred to as S strain), and h9.4.2.5 subbranch A / Chicken / Henan / 01 / Cross-reactivity of positive sera from 2019(H9N2) (hereinafter referred to as HN01 strain), h9.4.2.5 subclade A / Chicken / Jilin / 01 / 2019(H9N2) (hereinafter referred to as JL01 strain), and h9.4.2.5 subclade A / Chicken / Liaoning / 01 / 2015(H9N2) (hereinafter referred to as LN01 strain) was studied. Recombinant protein HA-a and HA-b antisera were used as positive serum controls, and SPF chicken serum was used as a negative control. Results ( Figure 3 As shown in Table 1, recombinant proteins HA-a and HA-b exhibit good cross-reactivity with H9AIV positive sera from different branches or subbranches, indicating that recombinant proteins HA-a and HA-b have broad-spectrum cross-reactivity with H9 subtype avian influenza.

[0048] Table 1 Results of cross-reactivity assays for recombinant proteins

[0049] h9.1 branch HLJ35 strain positive serum 8 8 h9.4.2.3 Branch S strain positive serum 9 9 h9.4.2.5 branch HN01 strain positive serum 14 14 h9.4.2.5 branch JL01 strain positive serum 14 13 h9.4.2.5 branch LN01 strain positive serum 14 13 SPF chicken serum 0 0

[0050] Example 4

[0051] 1. Preparation and inactivation of recombinant proteins

[0052] Recombinant baculovirus rBV-HA-b was cultured using standard methods, centrifuged, and then resuspended in an SF-900 atmosphere. TM In II SFM (Invitrogen), a concentration of 1×10⁻⁶ was obtained. 7 High Five cell culture medium containing [number] cells / mL. Recombinant baculovirus rBV-HA-b was inoculated at an MOI of 1 to a concentration of 1 × 10⁻⁶ cells / mL. 7 Recombinant protein HA-b was obtained by culturing High Five cells at 27°C for 110 h in a shaker at a concentration of 1 / mL. Cells and culture supernatant were then lysed by sonication, centrifuged, and the supernatant was collected to yield recombinant protein HA-b with a hemagglutination titer of 12 Log2. Recombinant protein HA-a was prepared using the same method as HA-b, except that rBV-HA-a was used instead of rBV-HA-b. The resulting recombinant protein HA-a had a hemagglutination titer of 9 Log2.

[0053] Formaldehyde aqueous solution with a volume percentage of 37-40% was added to each recombinant protein, with four concentration gradients of 0.1%, 0.15%, 0.2%, and 0.3% formaldehyde aqueous solution, and the proteins were inactivated at 37°C for 24 hours. Hemagglutination (HA) titers and viral infectivity were then tested on the inactivated recombinant proteins. Results showed that at final volume percentages of 0.1%, 0.15%, 0.2%, and 0.3% formaldehyde aqueous solution, the hemagglutination titers of the inactivated HA-a antigen were 9 Log2, 9 Log2, 8.5 Log2, and 8 Log2, respectively, and the hemagglutination titers of the HA-b antigen were 12 Log2, 12 Log2, 11.5 Log2, and 10.5 Log2, respectively. Viral infectivity tests showed that the inactivated recombinant HA-a and HA-b protein solutions did not contain infectious viruses. Therefore, it was determined that formaldehyde aqueous solution with a final concentration (volume percentage) of 0.1% was added to recombinant proteins HA-b and HA-a for inactivation.

[0054] 2. Preparation of H9 subtype avian influenza subunit vaccine

[0055] The preparation of the H9 subtype avian influenza subunit vaccine HA-b using the recombinant protein HA-b prepared in Title 1 of this embodiment as the antigen includes the following steps:

[0056] (1) Mix 96 parts by volume of white oil for injection (see Appendix 343 of the Quality Standard for Veterinary Biological Products of the People's Republic of China) and 4 parts by volume of Span-80 evenly, sterilize at 121°C for 15 min, and cool to room temperature to obtain the oil phase.

[0057] (2) A 37-40% (v / v) formaldehyde aqueous solution was added to the recombinant protein HA-b (prepared in Title 1 of Example 4) to achieve a final v / v concentration of 0.1%. The protein was then inactivated at 37°C for 24 hours to obtain the inactivated recombinant protein. The inactivated recombinant protein HA-b was then emulsified with 4% (v / v) of Tween-80 to obtain an aqueous phase.

[0058] (3) Mix and emulsify the aqueous phase and oil phase at a volume ratio of 1:3 to obtain the H9 subtype avian influenza subunit vaccine HA-b.

[0059] Using the same method described above, the recombinant protein HA-a prepared in Title 1 of this embodiment was used as the antigen to prepare the H9 subtype avian influenza subunit vaccine HA-a.

[0060] Example 5

[0061] 1. Immunopotency Trial of H9 Subtype Avian Influenza Subunit Vaccine

[0062] One hundred and twenty 14-day-old SPF (Specific Pathogen Free) chickens were divided into four groups of 30 each. Group 1 (HA-a) received a subcutaneous injection of 0.5 mL / chicken of H9 subtype avian influenza subunit vaccine HA-a (prepared in Example 4) in the neck; Group 2 (HA-b) received a subcutaneous injection of 0.5 mL / chicken of H9 subtype avian influenza subunit vaccine HA-b in the neck; Group 3 (commercial vaccine group) received a subcutaneous injection of 0.3 mL / chicken of H9N2 commercial vaccine (containing H9N2 SZ strain); Group 4 served as a blank control group, inoculated with PBS buffer, 0.5 mL / chicken. Peripheral blood was collected from each group of chickens at 14, 21, and 28 days post-immunization. Serum was separated, and serum HI antibody titers were measured using the h9.4.2.5 branch HN01 strain and h9.4.2.5 branch LN01 strain as detection antigens. Results ( Figure 4 The results showed that serum HI titers were detectable in chickens of all immunization groups 14 days post-immunization, and these titers continued to rise at 14, 21, and 28 days. The rate of antibody production and its development trend in the HA-a group were not significantly different from those in the HA-b group and the commercial vaccine group. However, the HI antibody level induced in the HA-b group was significantly higher than that in the HA-a group and the commercial vaccine group. The serum antibody HI titer in the HA-a group was comparable to that in the commercial vaccine group. These results indicate that HA-b has better immunogenicity compared to HA-a and the commercial vaccine.

[0063] Example 6: Cross-protective efficacy of H9 subtype avian influenza subunit vaccine against viral challenge

[0064] 1. Clinical observation

[0065] In Example 5, the chickens in each group were challenged 28 days after immunization. Each immunization group and the blank control group were divided into three challenge groups, with 10 chickens in each challenge group. Each challenge group was treated with 10... 6 Chickens were intranasally challenged with strains S (h9.4.2.3 branch), HN01 (h9.4.2.5 subbranch), and LN01 (h9.4.2.5 subbranch) at ELD50 / 0.1 mL. Following challenge, the chickens were observed for 14 consecutive days, and their mental state, feed intake, and water consumption were recorded. Clinical observation results showed that no chickens in the immunized groups exhibited lethargy, ruffled feathers, or other abnormalities, and their feed and water intake remained normal. The blank control group (referred to as the blank challenge group) showed reduced feed and water intake on days 3-4 after challenge.

[0066] 2. Weight gain monitoring

[0067] Body weight of chickens in each group was monitored on days 0, 2, 4, 6, 8, 10, 12, and 14 post-infection. Body weight monitoring results ( Figure 5 The results showed that the experimental chickens in the HA-b group had the highest weight gain rate, while there was no significant difference in weight gain rate between the HA-a group and the commercial seedling group. The experimental chickens in the blank challenge group all showed a slowdown or decrease in weight gain on days 2-4 after challenge.

[0068] 2. Detoxification testing

[0069] Throat swabs and cloacal swabs were collected from chickens in each group on days 3, 5, and 7 after viral challenge. The swabs were stored in 1 mL of sterile PBS buffer containing four antibodies (penicillin, streptomycin, gentamicin, and kanamycin), homogenized under sterile conditions, centrifuged at 12,000 rpm for 15 min, and the supernatant was collected and inoculated into 10-day-old SPF chicken embryos. Three swab samples were inoculated per embryo, 0.2 mL per embryo. The embryos were incubated at 37°C, and candled every 12 hours. After 120 hours of observation, the embryos were cooled to 4°C, and the allantoic fluid titer (HA) was determined using a microhemagglutination test. A HA ≥ 4 log2 in the allantoic fluid was considered positive, indicating viral shedding in the chicken. If the HA < 4 log2, blind passage was required. A HA < 4 log2 after blind passage was considered negative, indicating that the chicken was not viral shedding.

[0070] The results of the virus shedding test (Table 2) showed that after attacking strain S, in the HA-a group, 4 / 10 were positive for throat shedding and 2 / 10 were positive for cloacal shedding on day 3; on days 5 and 7, both throat and cloacal shedding were negative. In the HA-b group, both throat and cloacal shedding were negative on days 3, 5, and 7. In the commercial seedling group, 10 / 10 were positive for throat shedding and 3 / 10 were positive for cloacal shedding on day 3; on day 5, 2 / 10 were positive for throat shedding and all were negative for cloacal shedding; on day 7, both throat and cloacal shedding were negative. In the blank challenge group, 10 / 10 were positive for throat and cloacal shedding on days 3 and 5, and both throat and cloacal shedding were negative on day 7.

[0071] Table 2. Results of virus shedding detection after challenge

[0072]

[0073] After attacking the h9.4.2.5 subbranch HN01 strain, in the HA-a group, 2 / 10 showed positive pharyngeal shedding and 1 / 10 showed positive cloacal shedding on day 3; both pharyngeal and cloacal shedding were negative on days 5 and 7. In the HA-b group, both pharyngeal and cloacal shedding were negative on days 3, 5, and 7. In the commercial seedling group, 10 / 10 showed positive pharyngeal shedding and 2 / 10 showed positive cloacal shedding on day 3; 1 / 10 showed positive pharyngeal shedding and all cloacal shedding were negative on day 5; both pharyngeal and cloacal shedding were negative on day 7. In the blank challenge group, 10 / 10 showed positive pharyngeal and cloacal shedding on days 3 and 5, and both pharyngeal and cloacal shedding were negative on day 7. After attacking the h9.4.2.5 subbranch LN01 strain, in the HA-a group, 3 / 10 showed positive pharyngeal shedding and 2 / 10 showed positive cloacal shedding on day 3; both pharyngeal and cloacal shedding were negative on days 5 and 7. In the HA-b group, throat and cloacal excretion were negative on days 3, 5, and 7. In the commercial vaccine group, 10 / 10 had positive throat excretion and 3 / 10 had positive cloacal excretion on day 3; 2 / 10 had positive throat excretion and negative cloacal excretion on day 5; and both throat and cloacal excretion were negative on day 7. In the blank challenge group, 10 / 10 had positive throat and cloacal excretion on days 3 and 5, and both throat and cloacal excretion were negative on day 7.

[0074] HA-a protein amino acid sequence (SEQ ID NO:1, 556aa):

[0075] METVSLITILLVATVSNADKICIGYQSTNSTETVDTLTENNVPVTHAKELLHTEHNGMLCATSLGHPLILDTCTIEGLIYGNPSCDLLLGGREWSYIVERPSAVNGLCYPGNVENLEELRSLFSSARSYQRIQIFPDTIWNVSYSGTSKACSDSFYRSMRWLTQKNNAYPIQDAQYTNNQGKNILFMWGINHPPTDTAQTNLYTRTDTTTSVATEEINRTFKPLIGPRPLVNGLMGRIDYYWSVLKPGQTLRIRSNGNLIAPWYGHILSGESHGRILKTDLKRGSCTVQCQTEKGGLNTTLPFQNVSKYAFGNCSKYIGIKSLKLAVGLRNVPRGLFGAIAGFIEGGWSGLVAGWYGFQHSNDQGVGMAADRDSTQKAIDKITSKVNNIVDKMNKQYEIIDHEFSEVETRLNMINNKIDDQIQDIWAYNAELLVLLENQKTLDEHDANVNNLYNKVKRALGSNAVEDGKGCFELYHKCDDQCMETIRNGTYNRRKYQEESKLERQKIEGVKLESEGTYKILTIYSTVASSLVIAMGFAAFLFWAMSNGSCRCNICI.

[0076] Nucleotide sequence of HA-a protein encoding gene (SEQ ID NO:2, 1671 nt):

[0077]

[0078] Amino acid sequence of HA-b protein (SEQ ID NO:3, 568aa):

[0079] METVSLITILLAATASNADKICIGYQSTNSTETVDTLTENNVPVTHAKELLHTEHNGMLCATSLGQPLILDTCTIEGLIYGNPSCDPLLEEREWSYIVERPSAVNGLCYPGNVENLEELRSLFSSARSYQRVQIFPDTIWNVSYDGTSNACSGSFYRNMRWLTQKNGNYPIQDTQYTNNQGKNILFMWGINHPPTDDTQRNLYTRTDTTTSVATEEINRIFKPLIGPRPLVNGLMGRINYYWSVLKPGQTLRIKSDGNLIAPWYGYILSGESHGRILRTDLKRGSCTVQCQTEKGGLNTTLPFQNVSKYAFGNCSKYIGIKSLKLAVGLRNVPSRSSRGLFGAIAGFIEGGWSGLVAGWYGFQHSNDQGVGIAADRESTQKAIDKITSKVNNIVDKMNKQYEIIDHEFSEVEARLNMINNKIDDQIQDIWAYNAELLVLLENQKTLDEHDANVNNLYNKVKRALGSNAVEDGKGCFELYHKCDDQCMETIRNGTYNRRKYQEESKLERQKIEGVKLESEGTYKILTIYSTVASSLVIAMGFAAFLFWAMSNGSCRCNVVYKVWRQYH.

[0080]

Claims

1. A broad-spectrum H9 subtype avian influenza subunit vaccine, characterized in that... The antigen in the vaccine is a protein with an amino acid sequence as shown in SEQ ID NO:

3.

2. The broad-spectrum H9 subtype avian influenza subunit vaccine according to claim 1, characterized in that... The vaccine antigen is prepared by culturing a recombinant baculovirus carrying the protein-coding gene.

3. The broad-spectrum H9 subtype avian influenza subunit vaccine according to claim 1 or 2, characterized in that... The sequence of the protein-coding gene is shown in SEQ ID NO:

4.

4. The broad-spectrum H9 subtype avian influenza subunit vaccine according to claim 3, characterized in that... The recombinant baculovirus was constructed using the following method: a kozak sequence was added to the 5' end of SEQ ID NO:4, and a Leu zip sequence was added to the 3' end. This was then inserted into the baculovirus transfer vector pFastBac1 to obtain a recombinant baculovirus transfer plasmid. The recombinant baculovirus transfer plasmid was transformed into *E. coli* DH10Bac competent cells to obtain a recombinant baculovirus shuttle plasmid. The recombinant baculovirus shuttle plasmid was then transfected into insect Sf9 cells to obtain a recombinant baculovirus expressing the antigen.

5. The broad-spectrum H9 subtype avian influenza subunit vaccine according to claim 4, characterized in that... The recombinant baculovirus was cultured using High Five cells.

6. The broad-spectrum H9 subtype avian influenza subunit vaccine according to claim 5, characterized in that... The antigen is inactivated, and 3-5% of the volume of Tween-80 after inactivation is added and emulsified to obtain an aqueous phase. White oil and Span-80 are mixed evenly at a volume ratio of 96:3-5 to obtain an oil phase. The aqueous phase and oil phase are mixed and emulsified at a volume ratio of 1:2.5-3.5 to obtain the vaccine.

7. The broad-spectrum H9 subtype avian influenza subunit vaccine according to claim 6, characterized in that... The antigen was inactivated using formaldehyde.

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