H7N9 subtype avian influenza recombinant strain and H7N9 subtype avian influenza virus vaccine

Abstract

The invention belongs to the technical field of avian influenza vaccines, and particularly relates to an H7N9 subtype avian influenza recombinant strain and an H7N9 subtype avian influenza virus vaccine. The H7N9 subtype avian influenza recombinant virus is formed by an HA gene and an NA gene which are transformed from an H7N9 subtype avian influenza epidemic strain and six internal genes PB2, PB1, PA, NP, NS and M of an H5N2 subtype avian influenza virus D7 strain, and the H7N9 subtype avian influenza recombinant virus is obtained by further using a chicken liver cancer suspension cell system as a carrier for proliferation. The HA titer of the obtained proliferated virus is higher than that of a traditional chick embryo proliferated virus, the virus yield is remarkably increased, after the prepared vaccine is used for immunizing SPF chickens, the HI antibody titer is remarkably higher than that of a chick embryo source vaccine, the challenge protection rate reaches 100%, and no virus is discharged. The technology provides an efficient and reliable solution for prevention and control of avian influenza.

Description

Technical Field

[0001] The present invention belongs to the technical field of avian influenza vaccines, and more specifically, relates to a recombinant H7N9 subtype avian influenza virus strain and an H7N9 subtype avian influenza virus vaccine. Background Art

[0002] Since its initial discovery in 2013, the H7N9 subtype of avian influenza virus has posed a serious threat to global public health security due to its high mortality rate (approximately 40%) and potential for human-to-human transmission. While traditional inactivated vaccines have been used clinically, they suffer from issues such as insufficient immunogenicity and weak cellular immune responses. Furthermore, the frequent variability of viral strains limits the effectiveness of vaccines over time.

[0003] In recent years, recombinant virus vaccines based on reverse genetics technology have become a research hotspot due to their advantages such as precise regulation of viral antigen epitopes and retention of natural conformations. However, existing technologies still face two major bottlenecks: First, although influenza vaccines produced based on mammalian cell line suspension cells have shown good immune protection effects in animal models, the production of avian influenza viruses using mammalian cell lines will lead to adaptive mutations, resulting in differences in vaccine antigenicity, which in turn will affect the antigenicity and protective efficacy of the vaccine; second, the preparation process of avian influenza virus vaccines relies on chicken embryos as proliferation carriers, which has defects such as a fragile supply system, high production costs, and residual impurity proteins.

[0004] Therefore, developing a new vaccine preparation platform that combines immune protection efficacy, process stability and cost-effectiveness has become a key scientific issue to break through the bottleneck of avian influenza vaccine research and development. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of the existing vaccine, such as the frequent mutation of virus strains that leads to the ineffectiveness of the protective effect of existing vaccines, the easy induction of adaptive mutations during the production process of mammalian cell lines, which in turn affects the antigenicity and protective efficacy of the vaccine, and the fragile supply system, high production cost and residual impurity proteins of traditional chicken embryos, and to provide a recombinant H7N9 subtype avian influenza virus.

[0006] The purpose of the present invention is to provide a recombinant poultry-derived H7N9 subtype avian influenza virus vaccine candidate strain.

[0007] Another object of the present invention is to provide the use of the H7N9 subtype avian influenza recombinant virus or the recombinant all-poultry-source H7N9 subtype avian influenza virus vaccine candidate strain in the preparation of a vaccine for preventing the H7N9 subtype avian influenza virus.

[0008] Another object of the present invention is to provide an inactivated antigen of H7N9 subtype avian influenza virus.

[0009] Another object of the present invention is to provide an H7N9 subtype avian influenza virus vaccine.

[0010] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0011] The present invention protects a recombinant H7N9 subtype avian influenza virus, which is composed of a modified HA gene and NA gene of an H7N9 subtype avian influenza epidemic strain, and six internal genes PB2, PB1, PA, NP, NS, and M of the H5N2 subtype avian influenza virus D7 strain; the modified HA gene sequence is shown in SEQ ID NO: 1, and the NA gene sequence is shown in SEQ ID NO: 2; the nucleotide sequences of the six genes PB2, PB1, PA, NP, NS, and M of the D7 strain are shown in SEQ ID NOs: 3 to 8, respectively; the H7N9 subtype avian influenza epidemic strain is A / chicken / sichuan / 23-17 / 2023.

[0012] To address the problem of frequent variability of avian influenza virus strains leading to the loss of vaccine protection, the present invention rescues H7N9 subtype recombinant viruses based on reverse genetics. The obtained H7N9 subtype recombinant viruses are conducive to the development of avian influenza virus vaccines with higher immune protection efficacy.

[0013] Furthermore, the H5N2 subtype avian influenza virus D7 strain is the world's first all-poultry-sourced waterfowl-specific H5N2 subtype avian influenza inactivated vaccine developed by South China Agricultural University in 2013.

[0014] Furthermore, the H7N9 subtype avian influenza recombinant virus is prepared by the following steps: the HA gene and NA gene after modification of the H7N9 subtype avian influenza epidemic strain are rescued together with the six internal genes PB2, PB1, PA, NP, NS and M of the H5N2 subtype avian influenza virus D7 strain to obtain the recombinant virus.

[0015] Specifically, the method includes constructing six plasmids, namely PB2, PB1, PA, NP, NS and M, which respectively express the D7 system, and constructing two plasmids that respectively express the modified HA gene of the sequence described in SEQ ID NO: 1 and the NA gene of the sequence described in SEQ ID NO: 2, mixing the eight plasmids and adding them to 293T cells with a transfection reagent, and obtaining the H7N9 subtype avian influenza recombinant virus strain after culture.

[0016] Furthermore, the transfection reagent is PEI transfection reagent.

[0017] Preferably, the mass ratio of the transfection reagent to the plasmid is 1:(2-5), preferably 1:2.5.

[0018] As a preferred embodiment, the method for preparing the H7N9 subtype avian influenza recombinant virus specifically comprises the following steps:

[0019] (1) Extraction of avian influenza virus RNA

[0020] RNA was extracted from the H7N9 subtype avian influenza epidemic strain with reference to conventional RNA extraction methods.

[0021] Specifically, refer to the operating instructions of the Fijet total RNA extraction kit, take 200 μL of the allantoic fluid of the H7N9 subtype avian influenza epidemic strain, add the lysis buffer, mix thoroughly, and then transfer to the adsorption column, centrifuge to remove impurities, and wash twice with decontamination buffer and ethanol respectively; finally, elute the total RNA with 30 μL of elution buffer, determine the concentration, and store at -80°C after aliquoting.

[0022] (2) Reverse transcription

[0023] The RNA extracted in step (1) above is reverse transcribed using influenza virus-specific reverse primers to obtain double-stranded cDNA.

[0024] (3) Amplification of HA and NA gene segments of recombinant viruses

[0025] The alkaline cleavage site on the HA gene fragment of the 23-17 avian influenza virus was deleted from the double-stranded cDNA obtained in step (2). Overlap PCR was used to perform targeted modification of the sequence of consecutive basic amino acids on the HA fragment. Simultaneously, universal primers were used to amplify the full-length sequence of the NA gene. The modified fragments were designated 23-7-HA and 23-7-H-NA. The 23-7-HA gene sequence is shown in SEQ ID NO: 1, and the 23-7-H-NA gene sequence is shown in SEQ ID NO: 2.

[0026] (4) Construction and extraction of plasmids

[0027] The 23-7-HA and 23-7-H-NA obtained in step (3) were ligated to the pHW2000 vector, i.e., the 23-7-HA and 23-7-H-NA were ligated to the pHW2000 vector using the restriction endonuclease BsmBI to prepare a recombinant plasmid. After sequencing and identification, the recombinant plasmids were successfully prepared. The resulting recombinant plasmids were named pHW2000-23-17D-HA and pHW2000-23-17-NA.

[0028] (5) Rescue of H7N9 subtype avian influenza recombinant virus

[0029] The pHW2000-23-17D-HA and pHW2000-23-17-NA obtained in step (4) were co-transfected into 293T cells with pHW2000-D7-PB2, pHW2000-D7-PB1, pHW2000-D7-PA, pHW2000-D7-NP, pHW2000-D7-M, and pHW2000-D7-NS from the D7 system (the acquisition of the plasmids of the six genes of the D7 strain refers to the authorized patent 202110564096.4, in which the connection vector is replaced with pHW2000). After 60 hours, the transfected cell supernatant was collected and inoculated into chicken embryos for passage. After 60 hours, the allantoic fluid with hemagglutination activity was harvested, and the obtained virus was confirmed by PCR and sequencing to be the target recombinant strain D7-23-17, that is, the H7N9 subtype avian influenza recombinant strain.

[0030] The present invention also protects a recombinant all-poultry-source H7N9 subtype avian influenza virus vaccine candidate strain, comprising an immune amount of the H7N9 subtype avian influenza recombinant virus.

[0031] The present invention protects the use of the H7N9 subtype avian influenza recombinant virus or the recombinant all-poultry-source H7N9 subtype avian influenza virus vaccine candidate strain in the preparation of a vaccine for preventing the H7N9 subtype avian influenza virus.

[0032] The present invention protects the use of the H7N9 subtype avian influenza recombinant virus or the recombinant all-poultry-source H7N9 subtype avian influenza virus vaccine candidate strain in the preparation of a vaccine for preventing the H7N9 subtype avian influenza virus.

[0033] The present invention protects an H7N9 subtype avian influenza virus inactivated antigen, which contains the inactivated H7N9 subtype avian influenza recombinant virus or the recombinant all-poultry-source H7N9 subtype avian influenza virus vaccine candidate strain.

[0034] The present invention protects an inactivated antigen of the H7N9 subtype avian influenza virus. The antigen is obtained by inactivating the recombinant H7N9 subtype avian influenza virus through culture with chicken hepatoma (LMH) suspension cells. LMH suspension cells are derived from an avian cell line and can be cultured in low-serum or serum-free or suspension cultures. Using the LMH suspension cell system to produce an inactivated avian influenza vaccine results in high antigen yield, low production cost, and high safety.

[0035] Furthermore, the method of culturing chicken liver cancer suspension cells includes the following steps: taking the chicken liver cancer suspension cells after subculture and performing proliferation culture of the H7N9 subtype avian influenza recombinant virus in an inoculation system including TPCK trypsin and penicillin-streptomycin solution, and harvesting the virus after sufficient culture.

[0036] Furthermore, the chicken liver cancer suspension cells are LMH cells obtained through virus sensitivity screening and susceptible to H7N9 subtype avian influenza virus.

[0037] Furthermore, the infection ratio of the H7N9 subtype avian influenza recombinant virus is ≥5%.

[0038] Preferably, the inoculation ratio of the H7N9 subtype avian influenza recombinant virus is 5% to 20%, more preferably 5%.

[0039] Furthermore, the concentration of the TPCK pancreatic enzyme is ≥10 μg / mL.

[0040] Preferably, the concentration of the TPCK pancreatic enzyme is 10-90 μg / mL, more preferably 10 μg / mL.

[0041] Furthermore, the cell density of the chicken liver cancer suspension cells is ≥2.0×10 6 .

[0042] Preferably, the cell density of the chicken liver cancer suspension cells is (2-30)×10 6 , more preferably (20-30)×10 6 , and more preferably 25×10 6 .

[0043] Furthermore, the time for harvesting the virus is ≥24h.

[0044] Preferably, the time for harvesting the virus is 24 to 72 hours, more preferably 48 to 72 hours, and most preferably 48 hours.

[0045] Preferably, the subcultured cell generation is the fifth generation (F5), that is, the chicken liver cancer suspension cells after subculture are F5 cell lines.

[0046] Furthermore, the concentration of penicillin and streptomycin is 1 vol%.

[0047] Furthermore, the inactivation method is to use chemical means (such as formaldehyde) to destroy the genetic material of the virus, so that it loses its replication ability and pathogenicity.

[0048] Furthermore, the inactivation is to inactivate the H7N9 subtype avian influenza virus using formaldehyde. Preferably, the concentration of the formaldehyde is 0.1 vol% formaldehyde solution.

[0049] The invention protects an H7N9 subtype avian influenza virus vaccine, and mixes an aqueous phase containing the H7N9 subtype avian influenza virus inactivated antigen and an oil phase, and fully emulsifies them to obtain the H7N9 subtype avian influenza virus inactivated vaccine.

[0050] Furthermore, the method for preparing the aqueous phase comprises the following steps: mixing the inactivated antigen and Tween 80 to obtain the aqueous phase.

[0051] Preferably, the concentration of the inactivated antigen in the aqueous phase is 3.5 vol%.

[0052] Furthermore, the oil phase includes white mineral oil and S-Ben-80.

[0053] Preferably, the volume ratio of the white mineral oil to SIBEN-80 is 94:6 (v / v).

[0054] Furthermore, the preparation method of the oil phase comprises the following steps: mixing the components of the oil phase in proportion, sterilizing the mixture, and fully emulsifying the mixture to obtain the oil phase.

[0055] Furthermore, as an optional embodiment, in the method for preparing the oil phase, the condition for sufficient emulsification is: centrifugal emulsification at 10,000 rpm for 5 minutes.

[0056] Furthermore, as an optional embodiment, in the method for preparing the inactivated vaccine of H7N9 subtype avian influenza virus, the condition for sufficient emulsification is: centrifugal emulsification at 20,000 rpm for 7 minutes.

[0057] Preferably, the volume ratio of the water phase to the oil phase is 1:2.

[0058] Compared with existing technologies, the present invention has the following advantages: The present invention combines the modified HA and NA genes of the H7N9 subtype avian influenza epidemic strain with the six internal genes PB2, PB1, PA, NP, NS, and M of the H5N2 subtype avian influenza virus D7 strain to form an H7N9 subtype avian influenza recombinant virus. The resulting H7N9 subtype avian influenza recombinant virus is further propagated using a chicken hepatoma suspension cell system as a vector. The resulting propagated virus has a higher HA titer than viruses propagated using traditional chicken embryos, significantly increasing virus yield. After immunization of SPF chickens with the prepared vaccine, the HI antibody titer is significantly higher than that of chicken embryo-derived vaccines, and the challenge protection rate reaches 100% with no virus shedding. This technology provides a highly efficient and reliable solution for avian influenza prevention and control. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 This is the electrophoresis result of the D7-23-17 recombinant avian influenza virus obtained in Example 1.

[0060] Figure 2 This is a statistical diagram of the survival rate of SPF chickens after infection in Example 5. DETAILED DESCRIPTION

[0061] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0062] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0063] The LMH suspension cells and MDCK adherent cell lines used in the present invention are both preserved by the National Avian Influenza Professional Laboratory (Guangzhou). The present invention uses the isolated epidemic strain A / chicken / sichuan / 23-17 / 2023 (H7N9, abbreviated as 23-17, isolated, identified and preserved by the National Avian Influenza Professional Laboratory (Guangzhou)) to transform the HA gene (gene sequence as shown in SEQ ID NO: 1) and the NA gene (gene sequence as shown in SEQ ID NO: 2) and the six internal genes D7-PB2 (gene sequence shown in SEQ ID NO: 3), D7-PB1 (gene sequence shown in SEQ ID NO: 4), D7-PA (gene sequence shown in SEQ ID NO: 5), D7-NP (gene sequence shown in SEQ ID NO: 6), D7-NS (gene sequence shown in SEQ ID NO: 7), and D7-H5N2 (H5N2 subtype, D7 strain, see https: / / baike.baidu.com / item / %E7%A6%BD%E6%B5%81%E6%84%9F%E7%81%AD%E6%B4%BB%E7%96%AB%E8%8B%97%EF%BC%88H5N2%E4%BA%9A%E5%9E%8B%EF%BC%8CD7%E6%A0%AA%EF%BC%89 / 53972364) of the vector. NO: 7) and D7-M (gene sequence shown in SEQ ID NO: 8) as the backbone to rescue the D7-23-17 avian influenza recombinant virus. All experiments involving highly pathogenic pathogens were conducted in a P3-level laboratory.

[0064] Table 1 Main primer sequences involved in the present invention

[0065]

[0066] Example 1 Rescue of H7N9 subtype recombinant virus based on reverse genetics

[0067] 1. Experimental Methods

[0068] 1. Extraction of avian influenza virus RNA

[0069] Extraction of avian influenza virus 23-17 RNA: Follow the instructions of the Filige total RNA extraction kit. Take 200 μL of viral allantoic fluid, add lysis buffer, mix thoroughly, and transfer to an adsorption column. Centrifuge to remove impurities and wash twice with decontamination buffer and ethanol respectively. Finally, elute the total RNA with 30 μL of elution buffer, determine the concentration, and store at -80°C after aliquoting.

[0070] 2. Reverse transcription

[0071] Reverse transcription: The RNA extracted in step 1 above was reverse transcribed according to the instructions for use of reverse transcriptase (M-MLV) provided by Takara. Reverse transcription was performed using an influenza virus-specific 12-unit reverse transcription primer with the primer sequence: AGCAAAAGCAGG (5'-3'). The reverse transcription system is shown in Table 2.

[0072] Table 2 Reverse transcription system

[0073]

[0074] To a 200 μL nuclease-free EP tube, add the components listed in Table 2 in the order specified. Vortex to mix thoroughly, then centrifuge briefly. Place the reaction tube in a constant-temperature water bath and incubate at 42°C for 120 min. Aliquot the cDNA product into pre-chilled centrifuge tubes and transfer to a -20°C freezer for short-term storage. For long-term storage, store at -80°C and avoid repeated freeze-thaw cycles.

[0075] 3. Target gene amplification

[0076] Amplification of the entire gene: The alkaline cleavage site on the 23-17 avian influenza virus HA gene fragment was deleted from the resulting double-stranded cDNA, and the sequence of consecutive alkaline amino acids on the HA fragment was deleted using Overlap PCR. The designed specific primers S23-17-R and S23-17-F were used, with HF-HA-F and S23-17-R used to amplify the upstream sequence of the alkaline cleavage site, and S23-17-F and HF-HA-R used to amplify the downstream sequence. The full-length HA gene with the alkaline cleavage site deleted was amplified by fusion PCR to obtain the HA target gene of the virus (named 23-7-HA). Specific primers HF-NA-F and HF-NA-R were used for PCR amplification to obtain the NA target gene of the virus (named 23-7-H-NA).

[0077] 4. Construction and extraction of plasmids

[0078] Enzyme digestion: Use BsmBI endonuclease to digest the gel-recovered product of the target fragment (23-7-HA or 23-7-H-NA) and the pHW2000 vector. The enzyme digestion system is shown in Table 3.

[0079] Table 3 Enzyme digestion system

[0080]

[0081] The reaction system was prepared according to the sequence in Table 3 in a 500 μL EP tube. After vortexing and centrifugation, the mixture was incubated in a 55°C water bath for 2 hours, followed by an inactivation treatment at 80°C for 20 minutes. The reaction products were then separated by 1% agarose gel electrophoresis. The gel region corresponding to the target band was excised and the fragment was purified using a gel extraction kit. The fragment concentration was determined, aliquoted, and stored at -20°C until further use.

[0082] Ligation: Use T4 ligase to ligate the target fragments after digestion to the pHW2000 vector. The ligation system is shown in Table 4.

[0083] Table 4 Connection system

[0084]

[0085] Pipette the digested target fragments and the linearized pHW2000 vector into a 200 μL EP tube according to the ratio in Table 4. Add T4 DNA ligase and the corresponding buffer. Mix the reaction system by gently pipetting. Then, place it at room temperature for 2 hours to obtain the ligation product.

[0086] Transformation: Thaw DH5α chemically competent cells on ice for approximately 5 minutes. Mix the ligation product with the cell suspension at a 1:5 volume ratio. After 30 minutes of ice-cooling, heat shock the cells in a 42°C water bath for 90 seconds, then quickly cool the cells on ice for 5 minutes to terminate the reaction. Add 500 μL of antibiotic-free LB medium and transfer the cells to a 37°C shaker at 200 rpm / min for 1 hour. Once the resuspension solution becomes slightly turbid, centrifuge at 3000 rpm / min for 5 minutes. Discard 300 μL of the supernatant and resuspend the cells. Apply the resuspended solution evenly to a preheated LB agar plate containing ampicillin. Gently spread the solution repeatedly with a spreader to ensure uniformity. Incubate the plate upside down at 37°C overnight in a 37°C incubator for 12 hours.

[0087] Bacterial liquid identification: After 12 hours, allow the colonies in the LB culture dish to grow to an appropriate size. Pick a single colony and transfer it to a 1.5 mL EP tube containing 500 μL of ampicillin-resistant LB broth. Incubate at 37°C, shaking at 200 rpm / min, for 3–6 hours. Once the culture becomes turbid, use the culture as a template for PCR identification using primers pHW2000-F and pHW2000-R. The PCR system is shown in Table 5.

[0088] Table 5 Bacterial liquid PCR system

[0089]

[0090]

[0091] The reaction system was prepared according to the sequence in Table 5 in a 200 μL EP tube and amplified using a PCR instrument. The PCR amplification program was as follows: 95°C for 3 minutes (initial denaturation); 30 cycles of 95°C for 15 seconds (denaturation); 55°C for 15 seconds (annealing); 72°C for 2 minutes (extension); and 72°C for 5 minutes (final extension). Amplified products were analyzed by 1% agarose gel electrophoresis, and strains with clear target bands were selected for sequencing verification.

[0092] Plasmid Extraction: Single colonies verified by sequencing were inoculated into 20 mL of LB liquid medium containing ampicillin in a clean bench and cultured at 37°C with a shaker at 200 rpm / min for 12 hours. The culture was then collected in a 50 mL centrifuge tube and centrifuged at 3000 rpm / min for 5 minutes. Lysis, binding, washing, and elution procedures were performed according to the instructions for the Plasmid Miniprep Kit (Tiangen). Plasmid DNA concentration was determined and stored at -20°C. The resulting plasmids were pHW2000-23-17D-HA and pHW2000-23-17-NA.

[0093] 5. Transfection

[0094] 293T cells were cultured at 5 × 10 4 The cells were seeded into 12-well plates at a density of 1 / well and cultured at 37°C under 5% CO2 until the cell density reached 90%. According to the "6+2" model, pHW2000-23-17D-HA, pHW2000-23-17-NA and reverse genetic plasmids carrying the six genes of the D7 strain (pHW2000-D7-PB2, pHW2000-D7-PB1, pHW2000-D7-PA, pHW2000-D7-NP, pHW2000-D7-M and pHW2000-D7-NS, the reverse genetic plasmids of the six genes of the D7 strain were obtained by referring to the authorized patent 202110564096.4, in which the connection vector was replaced with pHW2000) were added to 250 μL Opti-MEM medium at a concentration of 500 ng per plasmid, a total of 4 μg plasmid and 10 μg PEI transfection reagent, and gently mixed. After culturing for 1 hour, the medium was replaced with maintenance medium containing 1% BSA, 0.1 μL g / mL The cells were cultured for 60 h and the cell supernatant was collected for subsequent virus hemagglutination titer (HA) determination and chicken embryo inoculation.

[0095] 6. Recombinant virus passage and detection

[0096] After 60 hours of culture, the cell plate was removed and placed in a freeze-thaw zone at -80°C. Any cells that had not yet detached were pipetted to dislodge, and the entire cell fluid was collected. The cell fluid was diluted 10-fold with sterile PBS. The original solution, the 10-fold dilution, and the 100-fold dilution were selected for inoculation. 0.2 mL was inoculated into each SPF chicken embryo. The wells were sealed with paraffin wax and incubated in a 37°C incubator, with the embryos illuminated every 12 hours for 72 hours. After 72 hours of inoculation, the allantoic fluid was collected in a clean bench and the HA titer of the allantoic fluid was determined by hemagglutination assay. The virus fluid with hemagglutination titer was collected and aliquoted. The viral RNA in the allantoic fluid was extracted and reverse engineered. PCR amplification of the target fragment was performed and sequencing was performed to confirm the successful rescue of the recombinant influenza virus.

[0097] 2. Experimental Results

[0098] Identification of recombinant virus: RNA of the successfully rescued virus was extracted and whole genome sequencing was performed using RT-PCR. After correct identification, the rescued recombinant virus was named D7-23-17.

[0099] The electrophoresis results are as follows Figure 1 As shown, PCR successfully amplified the HA, NA, and M segments of the D7-23-17 avian influenza recombinant virus, further demonstrating that the D7-23-17 avian influenza recombinant virus was successfully rescued. Therefore, the D7-23-17 recombinant avian influenza virus can be successfully rescued according to the above method.

[0100] Example 2 Screening and subculture of LMH suspension cell monoclones

[0101] 1. Experimental Methods

[0102] 1. Obtaining monoclonal cells by limiting dilution method

[0103] 100 μL of cell suspension was collected from the LMH adherent cell system and mixed with trypan blue stain. Viability and density were measured, and cell viability was maintained above 95%. Cells were diluted to 50 cells / 10 mL by limiting dilution. After pipetting and mixing, 100 μL of cell suspension was plated per well of a 96-well plate. The 96-well plate was incubated at 37°C with 5% CO2.

[0104] Place the 96-well plate in a 37°C, 5% CO2 incubator and incubate statically to avoid vibration. Change half the medium every three days and observe the formation of single-cell colonies under a microscope. Transfer the single-cell colonies to a 48-well plate. When the colony has expanded to approximately 1000 cells, gradually expand to 24-well and then 6-well plates to expand the culture of the sorted LMH cells.

[0105] 2. Virus sensitivity screening

[0106] The D7-23-17 avian influenza recombinant virus was subjected to a tissue culture infection dose assay to determine the virus titer (TCID 50 ).

[0107] The expanded LMH cell monoclonal clones were seeded into 24-well plates (1.0×10 5 cells / mL cells), and each well was inoculated with 0.001MOI 23-17H7N9 avian influenza virus for virus propagation experiment. 24 hours after infection, the supernatant was collected for half tissue culture infection dose determination and the virus titer (TCID 50 ). According to the virus TCID 50 , screen out the LMH cell clones with the highest pre-susceptibility.

[0108] 3. Suspension culture acclimation of LMH cells highly sensitive to avian influenza virus

[0109] Use low-serum medium (5% FBS) to gently pipette adherent cells until partially suspended. Transfer to a shake flask and shake at low speed (80 rpm), gradually extending the suspension time to 24 hours. Completely remove the adherent surface, increase the shaking speed to 120 rpm, and gradually reduce the FBS to 2%. Passage at a 1:3 ratio every 3-4 days to maintain a cell density of 2.0×10 5 ~1.0×10 6 Use serum-free suspension culture medium, adjust dissolved oxygen and pH (DO>30%, pH 7.0-7.4), and gradually increase the culture volume.

[0110] The acclimated LMH suspension cells were expanded and frozen for future use. All subsequent LMH suspension cells used were acclimated LMH suspension cells.

[0111] 4. LMH suspension cell recovery treatment

[0112] Open the thermostatic water bath in advance, set the temperature to 37°C, and place all items in the biosafety cabinet for disinfection. Quickly remove the frozen LMH suspension cells from the liquid nitrogen and quickly place them in a water bath for thawing. After about 5 minutes, they are completely thawed. Pipette the completely thawed cell suspension and add it to a 15mL centrifuge tube containing 5mL of serum-free medium. Gently pipette to mix it and centrifuge it at 1000rpm for 5 minutes. Discard the supernatant and resuspend the cells with an appropriate volume of cell culture medium. Transfer it to a 125mL shake flask and fill the volume of the medium in the shake flask to 15mL. Take 100μL of the cell suspension and mix it with trypan blue dye for viability and density detection. Place the shake flask in a constant temperature shaking incubator at 37°C, 5% CO2, and 120rpm for incubation. After 48 hours of incubation, fill the volume of each shake flask to 50mL.

[0113] 5. Cell subculture

[0114] The routine subculture operation of LMH suspension cells follows the principle of density-dependent growth regulation. When the cell density reaches 1.0×10 7 cells / mL to 3.0×10 7 When the cell suspension is 100 cells / mL, transfer a certain volume of cell suspension to a 50 mL sterile conical centrifuge tube and centrifuge at 1000 rpm for 5 min. Discard the supernatant and add serum-free medium preheated to 37°C to the cell pellet. Gently pipette and resuspend until no clumps are visible. Adjust the subculture ratio according to the cell proliferation rate. High-viability cells (survival rate ≥ 95%) are subcultured at a ratio of 1:4 to 1:6. The initial density after subculture is approximately 2.0×10 6 cells / mL, while slow-growing or suboptimal cells were passaged at a ratio of 1:2 to 1:3 to maintain metabolic activity.

[0115] 2. Experimental Results

[0116] Table 6 LMH suspension cell passage results

[0117]

[0118] The results are shown in Table 6. Frozen LMH suspension cells were thawed and passaged, with cell density and viability recorded 24 hours after each passage. The cell viability of the F5 generation of the cell line exceeded 99%. Subsequent viral propagation was performed using F5 generation LMH suspension cells.

[0119] Example 3 Specific method of inoculating LMH suspension cells with viruses and culturing avian influenza viruses

[0120] 1. Experimental Methods

[0121] 1. LMH suspension cell culture of avian influenza virus

[0122] Use the selected LMH suspension cell system to propagate the avian influenza virus. Cultivate the suspension cells to a certain density, and keep the cell viability above 95%, then you can carry out the virus propagation experiment. The infection system includes: a certain infection ratio of virus, TPCK trypsin (the weak virus with the alkaline cleavage site deleted after recombination needs to be added) and penicillin-streptomycin solution. After mixing the above infection systems, inoculate them into a cell shake flask, which contains a suspension cell system with adjusted cell density. Continuously shake and culture at 120 rpm under 37°C and 5% CO2.

[0123] 2. Exploration of the optimal inoculation ratio for avian influenza virus propagation in LMH suspension cells

[0124] To investigate the effect of the virus inoculation ratio on the hemagglutination titer (HA titer) of the suspension cells and the cell suspension culture system when the avian influenza virus is propagated, the virus inoculation ratios in the inoculation system were grouped and then the virus was inoculated under the same inoculation conditions. 6 Cells were passaged at a starting density of 10 cells / mL. After the subculture system was expanded, the cell suspension was centrifuged and discarded, and resuspended in serum-free cell culture medium. 100 μL of cell suspension was taken from the LMH suspension cell system and mixed with trypan blue dye to test the viability and density. The cell density of the LMH suspension cell system was adjusted to 2.0×10 6 cells / mL. Subsequently, the D7-23-17 avian influenza recombinant virus (HA titer 1:512) was added, including virus solution and 1% penicillin-streptomycin solution added in different proportions, using a 250mL cell culture shake flask, and the total volume of the cell suspension after infection was 50mL. The infection ratio of the infection system was adjusted to 1%, 5%, 10%, 15% and 20% of the total culture volume for a total of 5 experimental groups, and a blank control group was set up. The cell suspension was collected 24h and 48h after infection to monitor the dynamic changes in the HA titer, cell density and cell viability of the avian influenza virus proliferated in the LMH suspension cell system.

[0125] 3. Optimal TPCK trypsin concentration for propagation of avian influenza virus in LMH suspension cells

[0126] To investigate the effect of TPCK trypsin after virus inoculation on the proliferation of suspension cells and the hemagglutination titer (HA) of the virus in cell suspensions when D7-23-17 recombinant avian influenza virus was propagated in a suspension cell culture system, the TPCK trypsin in the inoculation system was set to a gradient density to group the cells, and then the inoculation was performed under the same inoculation conditions. 6 Cells were passaged at a starting density of 10 cells / mL. After the subculture system was expanded, the cell suspension was cultured for 48 hours, the cell suspension was centrifuged and discarded, and resuspended in serum-free cell culture medium. 100 μL of cell suspension was taken from the LMH suspension cell system and mixed with trypan blue dye to test the viability and density. The cell density of the LMH suspension cell system was adjusted to 2.0×10 6cells / mL. Subsequently, the recombinant D7-23-17 avian influenza virus (HA titer 1:512) was diluted according to the optimal inoculation ratio of different suspension culture systems. The inoculation system included virus solution, TPCK trypsin and 1% penicillin-streptomycin solution. A 250mL cell culture shake flask was used, and the total volume of the cell suspension after inoculation was 50mL. The TPCK trypsin concentration in the inoculation system was adjusted to 7 gradients of 1μg / mL, 5μg / mL, 10μg / mL, 15.0μg / mL, 30μg / mL, 60μg / mL and 90.0μg / mL, and a blank control group was set up. The cell suspension was collected 24h and 48h after inoculation to monitor the dynamic changes of cell density, cell viability and HA titer of avian influenza virus in the LMH suspension cell system.

[0127] 4. Optimal cell density for propagation of avian influenza virus in LMH suspension cells

[0128] To investigate the effect of cell density at the time of virus inoculation on the proliferation of suspension cells and the hemagglutination titer of the virus in cell suspensions when D7-23-17 recombinant avian influenza virus was propagated in a suspension cell culture system, the cell system was divided into groups by setting a gradient density at the time of inoculation, and then the virus was inoculated under the same inoculation conditions. 6 Cells were passaged at a starting density of 10 cells / mL. After the culture system was expanded through passage, the cell suspension was centrifuged and discarded, and resuspended in serum-free cell culture medium. 100 μL of cell suspension was taken from the LMH suspension cell system and mixed with trypan blue dye to test the viability and density. According to the measured cell density, the cell density of the LMH suspension cell system at the time of infection was set to 2.0×10 6 , 5.0×10 6 , 1.0×10 7 , 1.5×10 7 , 2.0×10 7 , 2.5×10 7 and 3.0×10 7 Seven gradients of 1:1 cells / mL were used, along with a blank control group. D7-23-17 avian influenza recombinant virus (HA titer 1:512) was inoculated using an optimal inoculation ratio, TPCK trypsin concentration, and 1% penicillin-streptomycin solution. A 250mL cell culture shake flask was used, and the total volume of the cell suspension after inoculation was 50mL. Cell suspensions were harvested 24h and 48h after inoculation, and the dynamic changes in cell density, cell viability, and HA titer of the avian influenza virus were monitored in the LMH suspension cell system.

[0129] 5. The best time to collect samples for LMH suspension cell propagation of avian influenza virus

[0130] To investigate the effect of the time of virus sample harvest on the proliferation of suspension cells and the hemagglutination titer (HA) of the virus in cell suspension culture when D7-23-17 recombinant avian influenza virus was propagated in suspension cell culture system, we first used 2.0×10 6 Cells were passaged at a starting density of 100 cells / mL. After the subculture system was expanded and cultured for 48 hours, the cell suspension was centrifuged and resuspended in serum-free cell culture medium. 100 μL of cell suspension was collected from the LMH suspension cell system and mixed with trypan blue stain for viability and density. The LMH suspension cell system was adjusted to the optimal cell density based on the measured cell density. Subsequently, the D7-23-17 avian influenza recombinant virus (HA titer 1:512) was inoculated at the optimal ratio, TPCK trypsin concentration, and 1% penicillin-streptomycin solution in a 250 mL shake flask. Samples were collected at five time points: 12, 24, 48, 72, and 96 hours after inoculation. Dynamic changes in cell density, cell viability, and HA titer of the avian influenza virus were monitored in the LMH suspension cell system.

[0131] 2. Experimental Results

[0132] 1. Results of the study on the optimal inoculation ratio of avian influenza virus in LMH suspension cells

[0133] Table 7 Results of hemagglutination test based on LMH suspension cell to explore infection ratio

[0134]

[0135] The results of the hemagglutination test are shown in Table 7. When the infection ratio of the LMH suspension cell system was 5%, the HA titer in the cell fluid reached a maximum of 1:64, which was the optimal infection ratio.

[0136] 2. Results of the exploration of the optimal TPCK trypsin concentration for propagation of avian influenza virus in LMH suspension cells

[0137] Table 8 TPCK trypsin concentration results based on LMH suspension cells

[0138]

[0139] By setting a gradient density of TPCK trypsin in the inoculation system at the time of inoculation, the groups were divided and the inoculation was carried out under the same inoculation conditions. The changes in cell density and cell viability of the avian influenza virus propagated in the LMH suspension cell system at 24 hours and the HA titer at 48 hours were monitored. The results are shown in Table 8. The suspension cells in the blank control group grew normally at 48 hours, while all the cells in the other experimental groups died after inoculation. According to the comparison of the HA titer results of the cell suspension collected at 48 hours, it was found that when the D7-23-17 recombinant avian influenza virus was propagated in LMH suspension cells, the TPCK trypsin concentration was 10μg / mL and the virus HA titer in the cell fluid reached a maximum of 1:2 6 .

[0140] 3. Results of the exploration of the optimal cell density for propagation of avian influenza virus in LMH suspension cells

[0141] Table 9 Results of hemagglutination test based on different cell densities when LMH suspension cells were infected

[0142]

[0143] By setting different groups according to the cell density when the culture system was infected with the virus, the results are shown in Table 9. 72 hours after the infection, the suspension cells in the blank control group grew normally, while all the cells in the other experimental groups died after the infection. According to the comparison of the HA titer results of the cell suspension collected at 72 hours, it was found that the cell density was 2.5×10 7 cells / mL, the viral HA titer in the cell fluid collected at 48 h reached a maximum of 1:1024, and as the cell density increased during infection, the viral HA titer in the cell fluid finally harvested showed a downward trend.

[0144] 4. Results of the study on the optimal sampling time for LMH suspension cell propagation of avian influenza virus

[0145] Table 10 Results of hemagglutination test at different times based on LMH suspension cells

[0146]

[0147] By setting different time points for sampling of avian influenza virus propagated based on suspension cell culture system, and performing inoculation under the same other inoculation conditions, the HA titer results of the cell suspensions collected at different time points were compared, as shown in Table 10, it was found that the HA titer of the D7-23-17 recombinant avian influenza virus propagated in the LMH suspension cell system reached a maximum value of 1:1024 at 48 hours. With the increase of the sampling time point, the HA titer of the virus in the cell fluid of the LMH suspension cell system decreased to 1:64 at 96 hours. The results showed that the optimal sampling time point based on the LMH suspension cell system was 48 hours after inoculation.

[0148] Example 4 Preparation of Inactivated Avian Influenza Vaccine

[0149] 1. Experimental Methods

[0150] 1. Inactivated antigen

[0151] Supernatants containing the D7-23-17 avian influenza recombinant virus were prepared according to the method of Example 3. The HA titer of the D7-23-17 avian influenza recombinant virus produced in the LMH suspension cell system and the SPF chicken embryo system was determined. 1 / 1000 of formaldehyde was added to the supernatant containing the D7-23-17 avian influenza recombinant virus and the virus was transferred to a 37°C shaking incubator at 200 rpm / min for 48 hours to inactivate the supernatant to prepare the antigen. The HA titer of the D7-23-17 avian influenza recombinant virus propagated in SPF chicken embryos was determined. 1 / 1000 (0.1 vol%) of formaldehyde was added to the supernatant containing the D7-23-17 avian influenza recombinant virus and the virus was transferred to a 37°C shaking incubator at 200 rpm / min for 24 hours to inactivate the virus. The HA titer of each inactivated antigen was determined. After inactivation, 0.2 mL of each antigen was inoculated into three chicken embryos and placed in a 37°C incubator for 48 hours to test the inactivation efficacy. Take 0.1 mL of the culture medium and inoculate it into the antibiotic-free LB medium, place it in a 37°C incubator for 24 h, and perform a sterility test.

[0152] 2. Vaccine Preparation

[0153] HA titers were measured using antigens prepared from inactivated D7-23-17 avian influenza recombinant viruses propagated in different culture systems. Each inactivated antigen was adjusted to the same HA titer using serum-free medium and uninoculated SPF embryonic chick allantoic fluid, resulting in a titer of 1:256. The inactivated antigen was then mixed with Tween 80 at a final concentration of 3.5 vol% in a total volume of 5 mL and thoroughly mixed using a vortex mixer. The oil phase was prepared by mixing Marcol-52 white mineral oil with Span-80 in a 94:6 (v / v) ratio. After autoclaving, 10 mL of the oil phase was centrifuged and emulsified at 10,000 rpm for 5 minutes. The inactivated antigen-Tween mixture was then mixed with the oil phase in a 1:2 volume ratio and emulsified at 20,000 rpm for 7 minutes until a uniform and stable milky white emulsion was formed. The prepared vaccine is dripped onto the surface of cold water. If the droplets remain intact and undispersed, exhibiting typical characteristics of a water-in-oil (W / O) formulation, this indicates complete emulsification. Sterility testing is performed using the LB broth culture method: an appropriate amount of inactivated antigen sample is inoculated into non-resistant LB broth and incubated at 37°C, 200 rpm, and shaker for 12 hours. Changes in the culture turbidity are observed to confirm the sterility of the vaccine.

[0154] 2. Experimental Results

[0155] Table 11 Results of inactivated antigen HA titer produced based on different systems

[0156]

[0157] The experimental results, shown in Table 11, measured the HA titers of D7-23-17 avian influenza recombinant viruses propagated in different culture systems before and after inactivation. The HA titer of LMH-D7-23-17 was superior to that of SPF embryo-D7-23-17, indicating that the D7-23-17 avian influenza recombinant virus produced in the LMH suspension cell system had a higher yield. Using serum-free medium and uninoculated SPF embryo allantoic fluid, the inactivated antigens were adjusted to the same level, resulting in an HA titer of 1:256. HA testing of each inactivated antigen confirmed satisfactory inactivation.

[0158] The prepared vaccine was dripped onto the surface of cold water. The droplets remained intact and did not disperse, exhibiting a typical water-in-oil (dose form characteristic). The vaccine was then inoculated in non-resistant LB broth to confirm its sterility. All vaccine formulations passed the sterility test.

[0159] Example 5 Comparison of immune efficacy of inactivated avian influenza vaccines

[0160] 1. Experimental Methods

[0161] 1. Immunization of SPF chickens

[0162] Twenty-seven SPF chickens were randomly divided into three groups, each containing nine birds: two experimental groups and one control group. The SPF chickens were immunized at 21 days of age. Each immunized group received a subcutaneous injection of 0.3 mL of inactivated vaccine into the neck, while the control group received a subcutaneous injection of 0.3 mL of sterile PBS into the neck. Prior to vaccination, 10 SPF chickens were randomly selected for wing vein blood sampling, serum isolation, and testing for antibodies against the H7N9 virus. The specific groupings and immunization doses are shown in Table 12.

[0163] Table 12 Immunization schedule

[0164]

[0165] 2. Serum antibody titer detection

[0166] On days 7, 14, and 21 after immunization, blood was collected from the subwing vein of SPF chickens in each vaccine group and the PBS control group. Serum was separated and assayed for HI antibodies according to the standard operating procedures of the National Influenza Center. Four hemagglutination units (4HAU) of the D7-23-17 avian influenza recombinant virus were prepared from two different sources: LMH cells and SPF chicken embryos. After determining the antigen titer using a hemagglutination (HA) assay, the antigen was diluted to 4HAU using sterile PBS buffer, and the titer was verified to ensure accuracy. During the experiment, 25μL of PBS buffer was pre-loaded into each well of a V-bottom 96-well microtiter plate. Subsequently, 25μL of the serum sample to be tested was added to the first well, and serial dilutions were performed using a multichannel micropipette. The 25μL dilution from the last well was discarded. 25μL of 4HAU antigen was added to all wells, mixed thoroughly, and incubated at room temperature for 30 minutes. Then, 25 μL of 1% chicken erythrocyte suspension was added to each well, and a negative control (PBS buffer) was set up at the same time. The reaction plate was placed at room temperature for 20 minutes, and the results were determined by vertical observation.

[0167] 3. Virus attack and detoxification detection

[0168] After 21 days of immunization, each immune group and the control group were challenged with A / Chicken / Sichuan / 23-17 / 2023 (H7N9) at a dose of 10 6 EID 50 The virus was inoculated via nasal and ocular instillation at a concentration of 200 μL / 200 μL. Throat and cloacal swabs were collected from SPF chickens in each group on days 3, 5, and 7 post-challenge. The swabs were stored in 1 mL of sterile PBS containing penicillin and streptomycin. Each swab sample was then inoculated into three 10-day-old embryonated chicken embryos. Embryos were illuminated every 12 hours, and allantoic fluid was collected 72 hours after challenge. HA titers were determined to monitor virus excretion after challenge.

[0169] 4. Tissue and organ viral load determination

[0170] On the third day after the challenge, three SPF chickens were randomly killed in each experimental group to observe the macroscopic lesions of each organ. Then, the same part of the lung tissue was collected under sterile conditions for tissue viral load determination. The collected organ tissues were weighed and mixed with 0.5 g of tissue and 1 mL of DMEM medium containing 5000 U / mL penicillin-streptomycin. The tissue was fully ground using ultrasonic oscillation. The tissue slurry was centrifuged at 8000 r / min, 4°C, for 5 min, and the supernatant was collected and diluted 10 times (10 -1 to 10 -9200 μL of each dilution was inoculated into 9- to 11-day-old normal chicken embryos, with three replicates. After inoculation, the cells were incubated in a 37°C incubator for 72 hours. After incubation, allantoic fluid was collected for hemagglutination (HA) assay to measure viral hemagglutination activity. Tissue and organ viral loads were calculated using the Reed-Muench method.

[0171] 2. Experimental Results

[0172] 14 and 21 days after immunization, blood was collected from the wing vein of the SPF chickens in each experimental group to separate the serum. The HI titer results are shown in Tables 13 to 15 below.

[0173] Table 13 HI titer results of SPF chickens on day 14 after immunization

[0174]

[0175] Table 14 HI titer results of SPF chickens on day 21 after immunization

[0176]

[0177] Table 15 Statistical analysis of HI titer results after immunization

[0178]

[0179] The experimental results, shown in Table 13, show that 14 days after immunization of SPF chickens, antibody titers varied among the groups using different HI antigen sources. When using LMH cell-derived antigens, the average titers for the LMH-D7-23-17 and SPF embryo-D7-23-17 groups were 6.7 log2 and 6.2 log2, respectively; when using SPF embryo-derived antigens, the titers were 6.1 log2 and 5.8 log2, respectively. Regardless of the antigen source, the LMH-D7-23-17 group had higher titers than the SPF embryo-D7-23-17 group, indicating that the LMH group had an advantage in antibody response.

[0180] The experimental results, shown in Table 14, show that 21 days after immunization with SPF chickens, antibody titers varied among the groups using different HI antigen sources. When LMH cell-derived antigens were used, the average titers for the LMH-D7-23-17 and SPF embryo-D7-23-17 groups were 8.4 log2 and 7.7 log2, respectively; when the antigen was derived from SPF embryos, the average titers were 8.4 log2 and 8 log2, respectively. Antibody levels in all groups were significantly elevated on day 21 compared to day 14, with the inactivated LMH-D7-23-17 vaccine group showing the highest antibody levels, indicating optimal immunogenicity.

[0181] Statistical analysis of the experimental results is shown in Table 15. 14 days and 21 days after immunization of SPF chickens, when the source of HI antigen is consistent with the source of vaccine preparation, the HI antibody titer of SPF chickens immunized with LMH vaccine reached 6.7±0.5log2 (5.8±0.8log2 for chicken embryo vaccine) and 8.4±0.5log2 (8±1log2 for chicken embryo vaccine) in the second and third weeks after immunization. There are differences in the antibody titers of each group, indicating that the vaccine prepared using LMH suspension cells of the present invention has a better immune response effect.

[0182] The experimental results are as follows Figure 2 As shown, the SPF chickens in the experimental group vaccinated with the inactivated vaccine remained alive throughout the observation period, with a survival rate of 100%. No abnormal clinical symptoms were observed during the period, and the chickens were in an active mental state and had normal food intake. The SPF chickens in the PBS control group showed typical pathological signs after infection, manifesting as systemic symptoms such as mental depression and loss of appetite, accompanied by local characteristic reactions such as hemorrhagic lesions on the foot scales, cyanotic swelling of the comb, and edema of the head and face. Deaths began to occur in the SPF chickens in the PBS control group within 3 days after infection, and all the SPF chickens in the PBS control group had died by the 5th day after infection. The above results show that the vaccine prepared using LMH suspension cells of the present invention has good immune protection efficacy.

[0183] Table 16 Detoxification results of SPF chicken animal experiments

[0184]

[0185] Note: “-” indicates that all the experimental chickens have died at this time point.

[0186] All SPF chickens in each experimental group were challenged with A / Chicken / Sichuan / 23-17 / 2023(H7N9). All SPF chickens in the control group died within 3-5 days. Throat and cloacal swabs collected from immunized SPF chickens on days 3, 5, and 7 were negative for virus isolation, while throat and cloacal swabs collected from control SPF chickens on day 3 were positive for virus isolation. The experimental results (Table 16) show that no virus shedding was detected in the vaccine groups on day 3 after challenge. All inactivated vaccine groups had good immune protection, and the antibodies produced provided effective protection to the chickens.

[0187] Table 17 Determination of virus load in organs of SPF chickens in the D7-23-17 immunization group

[0188]

[0189] The experimental results showed (Table 17) that on the third day after the challenge, the average lung virus titer of the inactivated vaccine challenge group was 0, and the average lung virus titer of the PBS control group was 2.75×10 5EID 50 / mL, indicating that each inactivated vaccine group has good immune protection efficacy, and the antibodies produced by the inactivated vaccine can provide effective protection for chickens.

[0190] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A recombinant H7N9 subtype avian influenza virus, characterized in that: The invention is composed of the modified HA gene and NA gene of the H7N9 subtype avian influenza epidemic strain and the six internal genes PB2, PB1, PA, NP, NS and M of the H5N2 subtype avian influenza virus D7 strain; the modified HA gene sequence is shown in SEQ ID NO: 1, and the NA gene sequence is shown in SEQ ID NO: 2; the nucleotide sequences of the six genes PB2, PB1, PA, NP, NS and M of the D7 strain are shown in SEQ ID NOs: 3 to 8, respectively; the H7N9 subtype avian influenza epidemic strain is A / chicken / sichuan / 23-17 / 2023.

2. A recombinant poultry-derived H7N9 subtype avian influenza virus vaccine candidate strain, characterized in that: The invention comprises an immune amount of the H7N9 subtype avian influenza recombinant virus according to claim 1.

3. Use of the H7N9 subtype avian influenza recombinant virus according to claim 1 or the recombinant poultry-derived H7N9 subtype avian influenza virus vaccine candidate strain according to claim 2 in the preparation of a vaccine for preventing H7N9 subtype avian influenza virus.

4. An inactivated antigen of H7N9 subtype avian influenza virus, characterized in that: Contains the inactivated H7N9 subtype avian influenza recombinant virus of claim 1 or the recombinant whole-poultry-source H7N9 subtype avian influenza vaccine candidate strain of claim 2.

5. An inactivated antigen of H7N9 subtype avian influenza virus, characterized in that: The H7N9 subtype avian influenza recombinant virus according to claim 1 is obtained by inactivating the virus after culturing chicken liver cancer suspension cells.

6. The inactivated antigen of H7N9 subtype avian influenza virus according to claim 5, characterized in that The method of culturing chicken liver cancer suspension cells comprises the following steps: taking the chicken liver cancer suspension cells after subculture and performing proliferation culture of the H7N9 subtype avian influenza recombinant virus according to claim 1 in an inoculation system including TPCK trypsin and penicillin-streptomycin solution, and harvesting the virus after sufficient culture.

7. The inactivated antigen of H7N9 subtype avian influenza virus according to claim 6, characterized in that The inoculation ratio of the H7N9 subtype avian influenza recombinant virus is ≥5%.

8. The inactivated antigen of H7N9 subtype avian influenza virus according to claim 6, characterized in that The concentration of the TPCK trypsin is ≥10 μg / mL.

9. The inactivated antigen of H7N9 subtype avian influenza virus according to claim 6, characterized in that The cell density of the chicken hepatoma suspension cells is ≥2.0×10 6 .

10. An H7N9 subtype avian influenza virus vaccine, characterized in that: The aqueous phase and the oil phase containing the inactivated H7N9 subtype avian influenza virus antigen according to any one of claims 4 to 9 are mixed and fully emulsified to obtain an inactivated H7N9 subtype avian influenza virus vaccine.

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