Influenza H5 subtype recombinant virus based on VSV vector, inactivated vaccine and preparation method of influenza H5 subtype recombinant virus
By constructing a recombinant virus with VSV as a vector, inserting the NA and HA genes of the H5 subtype avian influenza virus strain, and saving it through reverse genetic technology, the problems of immune safety and immunogenicity of VSV vector vaccine were solved, and an inactivated vaccine preparation with efficient expression and immune safety were achieved.
Patent Information
- Application Number
- CN202411888247.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-20
- Publication Date
- 2025-03-28
AI Technical Summary
The prior art is difficult to improve the immune safety of the recombinant virus vaccine with VSV vector, and the immunogenicity of the inactivated virus is closely related to the abundance and conformation of the antigen, but no research on the preparation of inactivated vaccines with VSV vector.
By inserting the NA and HA genes of the H5 subtype avian influenza virus strain into the vesicular stomatitis virus genome that lacks the surface capsule protein gene, a recombinant virus with VSV as a vector was constructed, and rescued by reverse genetic technology, a recombinant virus that can effectively express the NA and HA protein of the H5 subtype avian influenza virus, and an inactivated vaccine was prepared by physical or chemical inactivation methods.
The efficient expression and immunogenicity of the recombinant virus are achieved. The inactivated vaccine has good immune safety and immunogenicity, and can induce high levels of hemagglutination inhibitory titers of avian influenza virus with the assistance of specific adjuvants.
Smart Images
Figure CN119709648A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of biological vaccine research and development, and in particular to a VSV vector-based influenza H5 subtype recombinant virus, an inactivated vaccine and a preparation method thereof. Background Art
[0002] In March 2024, the U.S. Department of Agriculture reported the first detection of highly pathogenic avian influenza A (H5N1) virus in dairy cows and unpasteurized milk samples from four dairy herds in Texas and Kansas. Subsequently, 15 farms in other states also confirmed the presence of the virus. The epidemic not only spread among cattle, but also infected cats, poultry and even workers on the farm. The H5 subtype highly pathogenic avian influenza virus continues to recombine and mutate in nature, posing severe challenges to the comprehensive prevention and control of avian influenza worldwide, and even seriously threatening human health. The development of vaccines against influenza viruses remains a top priority.
[0003] Vesicular Stomatitis Virus (VSV) belongs to the Rhabdoviridae family and can infect a variety of animals and insects. Because it can spread across synapses in the interganglionic direction, it has potential neurotoxicity. Due to its potential neurotoxicity, the development of recombinant virus live vaccine products with VSV as a vector is limited. Currently, only the Ebola recombinant virus vaccine with VSV as a vector has been approved. In the usual research on recombinant vector vaccines, researchers have focused on how to achieve VSV vector attenuation and reduce neurotoxicity as the main research direction, but the immune safety of live vaccine products cannot be ignored.
[0004] Live viruses can be inactivated after inactivation and are safe, but whether the inactivated virus has good immunogenicity is closely related to the abundance of antigens on the surface of virus particles and the conformation of antigens. There are no reports on the preparation of inactivated vaccines using replicative recombinant virus inactivated viruses with VSV as a carrier. Summary of the invention
[0005] The technical problem to be solved by the present invention is how to improve the immune safety of recombinant virus vaccines with VSV as a carrier, and to provide an influenza H5 subtype recombinant virus based on a VSV vector, an inactivated vaccine and a preparation method thereof.
[0006] In order to solve the above problems, the present invention proposes the following technical solutions:
[0007] In the first aspect, the present invention provides a recombinant virus, the genome of which comprises a vesicular stomatitis virus genome lacking a surface envelope protein gene, and the NA (neuraminidase) gene and HA (hemagglutinin) gene of an H5 subtype avian influenza virus strain; the NA gene and HA gene are located between the M gene and the L gene of the vesicular stomatitis virus genome, and the NA gene and HA gene are connected in series in a 5′-3′ direction.
[0008] The recombinant virus constructed by the present invention uses VSV as a vector, and replaces the surface envelope protein (G protein) gene expression box element in the VSV vector genome expression box with the NA gene expression box element and the HA gene expression box element of the H5 subtype avian influenza virus strain. It is experimentally verified that the recombinant virus rescued by reverse genetic technology can effectively express the NA protein and HA protein of the H5 subtype avian influenza virus and has immunogenicity.
[0009] It should be noted that the VSV vector genome expression cassette described above includes a VSV genome transcription initiation element, a gene expression open reading frame and a VSV genome terminator, and the NA protein gene and HA protein gene of the H5 subtype avian influenza virus strain in the expression cassette are connected in series in the 5′-3′ direction.
[0010] Furthermore, the H5 subtype avian influenza virus strain is the A / Texas / 37 / 2024 strain of H5N1 highly pathogenic avian influenza virus.
[0011] Furthermore, the amino acid sequence encoded by the HA gene includes the amino acid sequence shown in SEQ ID NO.1; and the amino acid sequence encoded by the NA gene includes the amino acid sequence shown in SEQ ID NO.2.
[0012] Furthermore, the recombinant virus has the ability to replicate and proliferate after being rescued by reverse genetic technology.
[0013] Furthermore, the recombinant virus comprises vesicular stomatitis virus nucleoprotein, vesicular stomatitis virus phosphoprotein, vesicular stomatitis virus matrix protein, vesicular stomatitis virus RNA polymerase, and NA protein and HA protein of H5 subtype avian influenza virus strain, and does not comprise vesicular stomatitis virus surface envelope protein.
[0014] In a second aspect, the present invention provides a method for preparing a recombinant virus as described in the first aspect, comprising: using a vesicular stomatitis virus lacking a surface envelope protein gene as a vector, inserting the NA gene and the HA gene in the form of an expression cassette between the M gene and the L gene of the vector, and rescuing the recombinant virus by reverse genetics technology.
[0015] Furthermore, the method for preparing the recombinant virus also includes inoculating the obtained recombinant virus into susceptible cells for cultivation, and harvesting the cell culture fluid containing the recombinant virus.
[0016] Furthermore, the susceptible cells include baby hamster kidney cells, such as BHK-21.
[0017] In a third aspect, the present invention further provides any of the following uses of the recombinant virus described in the first aspect or the recombinant virus obtained by the preparation method of the second aspect:
[0018] 1) For the preparation of vaccines or drugs for the treatment or prevention of H5 subtype avian influenza virus infection and related diseases caused by such infection;
[0019] 2) Used for preparing H5 subtype avian influenza virus infection detection reagents or kits;
[0020] 3) Used to prepare vaccine vectors.
[0021] In a fourth aspect, the present invention provides an inactivated avian influenza vaccine for mammals, comprising an inactivated recombinant virus and a vaccine adjuvant, wherein the recombinant virus is the recombinant virus described in the first aspect or the recombinant virus obtained by the preparation method of the second aspect.
[0022] Furthermore, the vaccine adjuvants include AddaS03, ADJ206, and AddaVax.
[0023] In a fifth aspect, the present invention provides a method for preparing the inactivated avian influenza vaccine composition for mammals according to the fourth aspect, comprising the following steps:
[0024] (1) inoculating the recombinant virus described in the first aspect or the recombinant virus obtained by the preparation method of the second aspect into susceptible cells for cultivation to obtain a virus liquid;
[0025] (2) The virus liquid obtained in step (1) is harvested, inactivated, and then mixed with a vaccine adjuvant in a certain proportion.
[0026] Furthermore, the susceptible cells include baby hamster kidney cells, such as BHK-21.
[0027] Furthermore, the inactivation method includes physical inactivation or chemical inactivation, and the chemical inactivation method preferably includes adding an inactivating agent for chemical inactivation.
[0028] The inactivator is specifically β-propiolactone, which acts directly on the viral nucleic acid. This method does not destroy the protein structure on the surface of the virus and can effectively ensure the abundance and conformation of antigens on the surface of viral particles.
[0029] Compared with the prior art, the technical effects achieved by the present invention include:
[0030] The recombinant virus provided by the present invention uses molecular biological technology to delete the surface envelope protein encoding gene in the genome of vesicular stomatitis virus, and inserts the NA gene and HA gene expression box of the H5 subtype avian influenza virus strain. The recombinant virus rescued by reverse genetic technology can effectively express the NA antigen and HA antigen of the H5 subtype avian influenza virus, and can be used as a new vaccine candidate.
[0031] The method for preparing the recombinant virus provided by the present invention uses reverse genetic technology to rescue the recombinant virus, and the rescued recombinant virus is inoculated into susceptible cells for culture, and replication and proliferation can be completed without adding exogenous trypsin, and high-titer virus can be harvested, which will effectively reduce the cost of producing purified virus in the industrial production process.
[0032] The inactivated avian influenza vaccine for mammals provided by the present invention comprises an inactivated recombinant virus and a vaccine adjuvant. The recombinant virus has lost its replication ability after being inactivated. With the assistance of a specific adjuvant, its surface NA protein and HA protein can still exert an immune effect. After immunizing mice, it can induce the production of a high level of avian influenza virus hemagglutination inhibition titer, thereby ensuring the immune safety of the vaccine while having good immunogenicity.
[0033] The method for preparing an inactivated avian influenza vaccine for mammals provided by the present invention uses a recombinant virus rescued by a reverse genetic system, which has the advantages of high virus yield, high foreign protein expression level, and easy mass preparation, and can refer to mature virus inactivation and purification processes, thereby improving the production safety of the vaccine. BRIEF DESCRIPTION OF THE DRAWINGS
[0034] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying any creative work.
[0035] Figure 1 This is a diagram of the gene recombination strategy for constructing the recombinant virus in Example 1 of the present invention.
[0036] Figure 2 This is the result of the recombinant virus constructed in Example 1 of the present invention infecting BHK-21 cells.
[0037] Figure 3 This is the virus growth curve of the recombinant virus in Example 2 of the present invention in BHK-21 cells.
[0038] Figure 4 These are the HI results of mice 21 days after immunization with the inactivated vaccines in Example 3 of the present invention. DETAILED DESCRIPTION
[0039] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments. Obviously, the embodiments described below are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0040] It should be understood that when used in this specification and the appended claims, the terms "include" and "comprises" indicate the presence of described features, integers, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or combinations thereof.
[0041] It should also be understood that the terms used in this description of the embodiments of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the embodiments of the present invention. As used in the description of the embodiments of the present invention and the appended claims, unless the context clearly indicates otherwise, the singular forms of "a", "an" and "the" are intended to include plural forms.
[0042] Example 1
[0043] Influenza H5 subtype recombinant virus based on VSV vector and its preparation method (virus construction and rescue)
[0044] See also Figure 1 , which is a schematic diagram of the structure of influenza H5 subtype recombinant virus constructed by VSV vector in the present invention. In this embodiment, vesicular stomatitis virus (VSV) is used as a vector, its surface envelope protein (G protein) gene is deleted, and the NA gene and HA gene expression box of H5 subtype avian influenza virus strain are inserted, and the influenza H5 subtype recombinant virus is constructed by reverse genetic technology. In the embodiment of the present invention, the influenza H5 subtype recombinant virus is used to prepare an inactivated recombinant vaccine for preventing H5 subtype influenza.
[0045] This embodiment takes the highly pathogenic avian influenza virus A / Texas / 37 / 2024 strain H5N1 as an example to introduce the H5 subtype recombinant virus constructed by the present invention. In some embodiments, other H5 subtype avian influenza virus strains can also be used to construct the H5 subtype recombinant virus.
[0046] The HA and NA protein sequences of this example are derived from the H5N1 A / Texas / 37 / 2024 strain. The amino acid sequences of the HA protein (GeneBank Accession no. PP577943.1) and the NA protein (GeneBank Accession no. PP577945.1) are SEQ ID NO.1 and SEQ ID NO.2, respectively. The genes were synthesized after codon optimization, and the nucleotide sequences after codon optimization were SEQ ID NO.3 and SEQ ID NO.4, respectively.
[0047] The vesicular stomatitis virus strain in this example is the Indiana strain.
[0048] After gene synthesis, the above synthetic fragments and the VSV vector lacking the surface envelope protein were PCR amplified according to the instructions of PrimerStar enzyme. The primer sequence information for amplification is shown in Table 1.
[0049] The coding gene sequences of the above-mentioned codon-optimized HA and NA proteins SEQ ID NO.3 and SEQ ID NO.4 were cloned into the G protein gene position of the viral vector VSV by homologous recombination (Uniclone One Step SeamLess Cloning Kit), and the ORF region of the G protein gene was deleted. The two proteins were connected by the non-coding region between the VSV genes, and the NA protein gene and the HA protein gene were connected in series in the 5′-3′ direction. The nucleotide sequence used for the series connection was SEQ ID NO.5, forming a recombinant virus full-length plasmid of VSV carrying both H5N1-HA protein and H5N1-NA protein, named pVSV-H5N1-HA+NA.
[0050] Table 1 Amplification primer sequences
[0051]
[0052]
[0053] The specific plasmid construction process is as follows:
[0054] 1. Use DNA polymerase (Primer Star) to perform PCR amplification to obtain the corresponding fragment;
[0055] Second, each fragment was recombined using homologous recombinase (Uniclone One Step Seamless Cloning Kit) and transformed into competent cells;
[0056] 3. Pick a single colony and use universal vector primers and Taq enzyme to perform bacterial solution PCR, and send the PCR product with the correct band size for testing;
[0057] 4. Extract plasmids from colony clones that have been sequenced correctly.
[0058] The specific process of constructing the recombinant plasmid and the auxiliary plasmid is prior art, and the present invention does not limit this.
[0059] The virus rescue method is as follows: BHK-21 cells are infected with poxvirus expressing T7 polymerase, followed by transfection of full-length plasmids and auxiliary plasmids expressing VSV-N, VSV-P, VSV-L, and VSV-G. After 48 hours, cells and supernatants are collected, and supernatants are taken for standby use after filtration through a 0.22 μm filter membrane to obtain a virus stock solution. The virus stock solution is inoculated into new BHK-21 cells, and the cultured cells are continuously passaged to observe whether lesions occur. If lesions occur, the cells are collected again and repeatedly frozen and thawed three times, filtered and aliquoted at 0.45 μm, and then frozen at -80°C to obtain a virus stock solution. The collected recombinant virus stock solution is continuously passaged and amplified on BHK-21 cells. After stable amplification, the virus genome is extracted for RT-PCR sequencing to confirm that the virus rescue is successful.
[0060] It has been determined that the supernatant of the above-mentioned passaged cells can be successfully rescued and amplified in BHK-21 cells to obtain recombinant viruses. The recombinant virus obtained by passage and amplification in BHK-21 cells is named VSV-H5N1-HA+NA in the present invention.
[0061] After inoculation of the cell line, the cytopathic effect was observed Figure 2 .
[0062] like Figure 2 The results showed that the recombinant virus VSV-H5N1-HA+NA can be propagated and amplified in BHK-21 cells and has the ability to replicate and amplify.
[0063] Example 2 Determination of recombinant virus growth kinetic curve
[0064] In order to verify the replication kinetics of the recombinant virus VSV-H5N1-HA+NA in cells, we established the virus growth curve in BHK-21 cells.
[0065] The specific steps are as follows: The recombinant virus was inoculated into a 6-well cell plate with a monolayer of cells at a dose of MOI = 0.1, and the infected cells were cultured at 37°C. The corresponding cell plate was repeatedly frozen and thawed three times at 24h, 48h, 72h, and 96h after infection, and the virus solution was collected. It was frozen at -80°C. The virus solution at each time point was diluted 10 times and inoculated into a 96-well plate with a monolayer of BHK-21 cells. The virus TCID was calculated according to the Reed-Muench method.50 , a one-step virus growth curve was drawn with time as the horizontal axis and virus titer as the vertical axis.
[0066] The growth kinetics curve of the recombinant virus is as follows Figure 3 The virus titer of VSV-H5N1-HA+NA was the highest at 72h after infection, even exceeding 10 8.5 TCID 50 This indicates that the recombinant virus obtained in the embodiment of the present invention has the advantages of high virus yield and high exogenous protein expression level.
[0067] Example 3 Inactivated vaccine preparation
[0068] Collect the amplified virus stock solution and measure the titer on BHK21 cells. The virus titer should be greater than 10^6.5TCID50 / mL. Introduce the virus stock solution into the inactivation tank, add β-propiolactone with a final concentration of 0.05%, mix thoroughly, and inactivate at 2-8°C and 100rpm / min for 24h. The inactivated virus solution is hydrolyzed at 37°C for 2h and stored at 2-8°C. The storage should not exceed 2 months.
[0069] The criterion for judging virus inactivation is that the inactivated virus is continuously inoculated and passaged on BHK21 cells for three generations without causing cell damage.
[0070] Vaccine adjuvants are added to the inactivated virus stock solution in proportion. The selection of vaccine adjuvants in the embodiment of the present invention covers common classical adjuvants including human vaccines. The vaccine configuration methods of different vaccine adjuvants are as follows:
[0071] 1) Vaccine with aluminum gel adjuvant: The volume of aluminum adjuvant is 15% of the total volume. After the inactivated virus stock solution and aluminum gel adjuvant are mixed at a volume ratio of 3:1, the rest is made up with sterile PBS solution. During the preparation process, the inactivated virus is first added to the sterile PBS solution, placed on a magnetic stirrer for stirring, and the aluminum gel adjuvant is slowly added dropwise, and stirred at room temperature for 30 minutes.
[0072] 2) Vaccine with AddaS03 (AS03-like) adjuvant: Add 3.2 mL of inactivated virus stock solution and 1.8 mL of normal saline, mix well, add 5 mL of AddaS03 adjuvant (warm to room temperature before use), (antigen: adjuvant volume ratio = 1:1), and then incubate at room temperature with rotation for 30 minutes.
[0073] 3) Vaccine with AddaVax (MF59-like) adjuvant: Add 3.2 mL of inactivated virus stock solution and 1.8 mL of normal saline, mix well, add 5 mL of AddaS03 adjuvant (warm to room temperature before use), (antigen: adjuvant volume ratio = 1:1), and then incubate at room temperature with rotation for 30 minutes.
[0074] 4) Vaccine with ISA71VG adjuvant: Gently add 6.4 mL of inactivated virus stock solution into 14 mL of ISA71VG (generally speaking, the volume ratio of antigen: adjuvant = 3:7), homogenize and disperse at 3000 rpm / min, slowly add the virus stock solution, and high-speed shear at 12000 rpm / min for 5-10 minutes. If it overheats in the middle, stop and cool down (6.4 mL of virus stock solution is added to this inactivated vaccine to keep the antigen content in each group of vaccines consistent, which can be adjusted during actual use).
[0075] 5) Vaccine with ADJ701 adjuvant: Mix 6.4 mL of inactivated virus stock solution + 0.3 mL of normal saline, and gently add to 13.3 mL of ADJ701 (antigen: adjuvant volume ratio = 1:2). Generally, first homogenize and disperse at 3000 rpm / min while adding slowly. After adding the virus solution, high-speed shear at 12000 rpm / min for 5-10 minutes. If it overheats in the middle, stop and cool down.
[0076] 6) Vaccine with ADJ206 adjuvant: Preheat the antigen and adjuvant to 33°C; mix 3.2 mL of inactivated virus stock solution + 1.3 mL of normal saline, then add 5.5 mL of ADJ206 (antigen: adjuvant mass ratio 1:1, volume ratio approximately 45:55), stir with a magnetic stirrer at 400-600 rpm for 20 min.
[0077] 7) Vaccine with HS801 (high molecular polymer) adjuvant: Add 3.2 mL of inactivated virus stock solution and 1.8 mL of normal saline, mix well, add 5 mL of HS801 adjuvant (warm to room temperature before use), (antigen: adjuvant volume ratio = 1:1), and then incubate at room temperature with rotation for 30 minutes.
[0078] At the same time, since influenza virus is sensitive to temperature, based on the above inactivated virus preparation, this study used a physical method, namely 65°C for 30 minutes to inactivate the recombinant virus VSV-H5N1-HA+NA, forming a high-temperature inactivated recombinant virus liquid.
[0079] Example 4 Immunogenicity Evaluation of Inactivated Vaccine
[0080] Babl / c mice aged 4-5 weeks were selected, with 5 mice in each group. The control group was set up with an inactivated virus group without adjuvant and a PBS group. The inactivated virus included pure virus liquid inactivated by β-propiolactone and pure virus liquid inactivated by high temperature, with a total of 3 groups; the experimental group was the inactivated vaccine obtained by adding different adjuvants to the above virus liquid inactivated by β-propiolactone, with a total of 7 groups. Immunization was performed on day 0 and day 14, with 100 μL of each mouse's left and right hind limbs immunized, and serum was collected on day 21 for hemagglutination inhibition experiments.
[0081] The hemagglutination inhibition test method is as follows:
[0082] Prepare four units of HA antigen, the dilution multiple of the virus antigen is equal to the virus agglutination titer / 4, and inactivate the mouse serum to be tested at 56°C for 30 minutes in advance, add 25 μL of the serum to be tested to the first well of the 96-well plate, dilute the immune serum in a gradient dilution ratio of 1:2, add 25 μL of four units of antigen to the diluted serum, react at room temperature for 30 minutes, add 25 μL of 1% chicken red blood cell suspension to each well, and observe the test results after standing at room temperature for 30 minutes, and record the highest dilution at which hemagglutination occurs, which is the HI (hemagglutination inhibition) level.
[0083] The HI results of each experimental group 21 days after immunization are shown in Figure 4 .
[0084] From the HI results, we can see that, except for the A3-A8 groups, the inactivated vaccines or inactivated viruses in the other groups did not produce hemagglutination inhibition. From the results of the A1-A2 groups, we can see that, regardless of whether physical or chemical inactivation is used, the inactivated viruses themselves have almost no ability to induce specific immune responses. In the A3 to A8 test groups, the HI titer of mice in the A3 group reached 2 6 and above, the positive rate reached 100%, and the HI titer of mice in groups A4 and A7 reached 2 5 The positive rate of mice in the A9 group and above was 100%, and the positive rate of hemagglutination inhibition in the other groups was 20%. Combined with the results of group A9, the widely used general aluminum gel adjuvant cannot induce a specific immune response after mixing with inactivated viruses.
[0085] In summary, the influenza H5 subtype recombinant virus based on the VSV vector provided by the present invention, after being inactivated by physical or chemical means, lacks the assistance of adjuvants and cannot successfully induce the body to produce an effective immune response. At the same time, not all classic adjuvants can cause sufficiently strong specific immune effects after mixing with the inactivated virus of the present invention. This study found that the immune effect is best after the inactivated virus is mixed with AddaS03 adjuvant. Secondly, the immune effect is second best after the inactivated virus is mixed with ADJ206 adjuvant or AddaVax adjuvant.
[0086] In the above embodiments, the description of each embodiment has its own emphasis. For parts that are not described in detail in a certain embodiment, reference can be made to the relevant descriptions of other embodiments.
[0087] The above is a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can easily think of various equivalent modifications or replacements within the technical scope disclosed by the present invention, and these modifications or replacements should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope of the claims.
Claims
1. A recombinant virus, characterized in that: The genome of the recombinant virus comprises a vesicular stomatitis virus genome lacking a surface envelope protein gene, and an NA gene and an HA gene of an H5 subtype avian influenza virus strain; the NA gene and the HA gene are located between an M gene and an L gene of the vesicular stomatitis virus genome, and the NA gene and the HA gene are connected in series in a 5′-3′ direction; and the recombinant virus has the ability to replicate and proliferate after being rescued by reverse genetic technology.
2. The recombinant virus according to claim 1, characterized in that The H5 subtype avian influenza virus strain includes the A / Texas / 37 / 2024H5N1 strain.
3. The recombinant virus according to claim 2, characterized in that The amino acid sequence encoded by the HA gene includes the amino acid sequence shown in SEQ ID NO.1; the amino acid sequence encoded by the NA gene includes the amino acid sequence shown in SEQ ID NO.
2.
4. A method for preparing a recombinant virus according to any one of claims 1 to 3, characterized in that: include: The vesicular stomatitis virus lacking the surface envelope protein gene is used as a vector, the NA gene and the HA gene are inserted between the M gene and the L gene of the vector in the form of an expression box, and the recombinant virus is rescued by reverse genetic technology.
5. The method for preparing a recombinant virus according to claim 4, characterized in that: The method also includes inoculating the obtained recombinant virus into susceptible cells for cultivation, and harvesting the cell culture fluid containing the recombinant virus.
6. Any of the following uses of the recombinant virus according to any one of claims 1 to 3 or the recombinant virus prepared by the preparation method according to any one of claims 4 to 5: 1) For the preparation of vaccines or drugs for the treatment or prevention of H5 subtype avian influenza virus infection and related diseases caused by such infection; 2) Used for preparing H5 subtype avian influenza virus infection detection reagents or kits; 3) Used to prepare vaccine vectors.
7. An inactivated avian influenza vaccine for mammals, characterized in that: The invention comprises an inactivated recombinant virus and a vaccine adjuvant, wherein the recombinant virus is the recombinant virus according to any one of claims 1 to 3 or the recombinant virus prepared by the preparation method according to any one of claims 4 to 5.
8. The inactivated avian influenza vaccine for mammals according to claim 7, characterized in that The vaccine adjuvant includes at least one of AddaS03, ADJ206 or AddaVax.
9. A method for preparing an inactivated avian influenza vaccine composition for mammals as claimed in claim 7 or 8, characterized in that: The steps include: (1) inoculating the recombinant virus according to any one of claims 1 to 3 or the recombinant virus prepared by the preparation method according to any one of claims 4 to 5 into susceptible cells for culturing to obtain a virus liquid; (2) The virus liquid obtained in step (1) is harvested, inactivated, and then mixed with a vaccine adjuvant in a certain proportion.
10. The preparation method according to claim 9, characterized in that: The susceptible cells include baby hamster kidney cells.