Application of antiviral protein immune enhancer in inactivated H9N2 avian influenza vaccine

Abstract

The present invention discloses the application of an antiviral protein immune enhancer in an inactivated H9N2 avian influenza vaccine. Among them, the immune enhancer is the chicken-derived antiviral protein IFITM1. The chicken-derived antiviral protein IFITM1 in this solution, as an immune enhancer, in combination with the inactivated H9N2 subtype avian influenza vaccine for immunization, can induce immunized chickens to produce higher levels of H9N2 subtype avian influenza serum HI antibodies and enhance the effect of suppressing virus excretion. Moreover, 21 days after the antiviral protein IFITM1 is subcutaneously inoculated alone in the neck as an immune enhancer, it can also effectively inhibit the virus excretion of chicken H9N2 subtype avian influenza virus, significantly reducing the tissue damage of chickens. At present, there is no inactivated vaccine of avian antiviral protein immune enhancer on the market in China. The avian immune enhancer in this solution equipped with the inactivated H9N2 subtype avian influenza vaccine can more effectively prevent and control avian influenza, and at the same time can also make up for the vacancy of immune enhancers in the current poultry market.

Description

Technical Field

[0001] The present invention relates to the technical fields of vaccine technology and veterinary biological products, and particularly to the application of an antiviral protein immune enhancer in an inactivated H9N2 avian influenza vaccine. Background Art

[0002] Although the H9N2 subtype avian influenza virus (AIV) belongs to a low pathogenic avian influenza virus, the pathogen has a wide prevalence range, can cause a decrease in egg production or mixed infection, causing huge economic losses to the poultry industry. In addition, the H9N2 AIV can also serve as an internal gene donor for other novel influenza viruses, accelerating mutation and promoting the emergence and prevalence of novel influenza viruses, posing a great potential threat to public health. Epidemiological investigation results in China in recent years show that H9N2 has replaced H5N6 and H7N9 as the main subtype of influenza virus in the poultry industry. Therefore, strengthening the prevention and control of this disease is of great significance for poultry farming and public health safety.

[0003] Currently, in response to the prevalence and harm of H9N2 AIV, different methods and measures have been adopted in various countries to control the spread of the disease. Among them, traditional inactivated vaccines still occupy the main market for immune prevention of this disease. However, commercial vaccines have poor protective effects against newly emerging strains and mutant strains. In addition, the prevalence of AIV often has certain regional characteristics, resulting in the current inability of many commercial vaccines to meet the needs of H9 immunoprophylaxis in China. On the other hand, novel influenza vaccines against the H9N2 subtype, such as subunit vaccines and recombinant vaccines, have not been successfully marketed due to various problems such as poor immune effects. Therefore, in order to improve the disadvantages commonly possessed by current inactivated H9N2 vaccines and novel vaccines, such as poor immunogenicity, weak immune response, and short immune memory, the research on safe and highly effective immune enhancers is particularly urgent.

[0004] With the continuous development of modern molecular biology, immunology, genetic engineering and other technologies, researchers have gradually carried out the research and preparation of a variety of new vaccine adjuvants, including nanomaterials, CpG oligodeoxynucleotides, immune ligands and other various biomolecular immune enhancers. Among them, cytokines are a class of soluble protein active molecules secreted by a variety of cells induced by immunogens, mitogens or other stimulants. They play an important role in cell signal transduction, can regulate innate immunity and adaptive immunity by binding to corresponding receptors, and have various functions such as regulating hematopoiesis, cell growth, and repair of damaged tissues. Because they are derived from animal organisms, they have the characteristics of safety, high efficiency, specificity and strong species specificity, can overcome the residues and side effects of many traditional adjuvants, and have obvious advantages as veterinary immune enhancers, which can fully guarantee the food safety of edible livestock and poultry. In addition, cytokines, as key participants in the immune response mediated by Toll-like receptors (TLRs), also play an important role in immune pathway signal transduction, stimulating cellular immunity and humoral immunity. Therefore, the research and development of cytokine immune enhancers is of great significance to social economy and the development of animal husbandry. At present, many studies have proved that cytokines have the potential to be used as adjuvants, and can enhance the immunogenicity of viral antigens in a variety of animals, but their immune enhancement effects are greatly related to the use conditions, antigen types and host characteristics. The interferon-induced transmembrane protein family (IFITMs) is an important member of interferon-stimulated genes (ISGs) and has been proven to be a key cytokine involved in a variety of viral immune responses and plays a certain role in resisting pathogenic microorganism infections.

[0005] There is currently no product on the market in China that is an antiviral protein immune enhancer derived from chickens. Therefore, the research and development of an efficient immune enhancer for use in combination with inactivated vaccines meets the current market demand in the poultry industry and is of great significance for the vaccine research and development of H9N2 subtype avian influenza and the prevention and control of chicken-source diseases in China. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to propose an application of an antiviral protein immune enhancer that is reliable and effective in implementation, convenient to prepare and can be used to enhance the immune efficacy of inactivated H9N2 subtype avian influenza vaccine in the inactivated H9N2 avian influenza vaccine.

[0007] In order to achieve the above technical objectives, the technical solutions adopted by the present invention are as follows:

[0008] Application of antiviral protein immune enhancer in inactivated H9N2 avian influenza vaccine. Among them, the antiviral protein immune enhancer is a chicken-derived antiviral protein, and this application includes its application in whole virus inactivated vaccine or recombinant inactivated vaccine of H9N2 subtype avian influenza virus.

[0009] As a possible implementation method, further, the antiviral protein immune enhancer is chicken-derived antiviral protein IFITM1.

[0010] As a possible implementation method, further, the antiviral protein immune enhancer is chicken-derived antiviral protein IFITM1 expressed prokaryotically by Escherichia coli, which is purified by affinity nickel column chromatography.

[0011] Based on the above scheme, the present invention also provides an inactivated H9N2 subtype avian influenza vaccine, which includes an antigen, a vaccine adjuvant and an immune enhancer. Among them, the immune enhancer is chicken-derived antiviral protein IFITM1.

[0012] As a preferred implementation option, preferably, the antigen is an inactivated antigen, which is inactivated influenza A virus strain FZ or a recombinant virus HA+NA / PR8 strain that contains the HA and NA genes of influenza A virus strain FZ and 6 internal genes of H1N1-PR8 strain rescued successfully through reverse genetics system. Among them, influenza A virus strain FZ is preserved in China Center for Type Culture Collection, the preservation address is Wuhan, China, the preservation number is CCTCC NO:V 202171, and the preservation date is September 26, 2021.

[0013] As a preferred implementation option, preferably, the antigen is inactivated with formaldehyde.

[0014] Based on the above scheme, the present invention also provides an application method of antiviral protein immune enhancer in inactivated H9N2 avian influenza vaccine, which includes the following steps:

[0015] 1) Dilute the avian influenza H9N2 subtype vaccine strain 1000 times with sterile PBS and inoculate it into the allantoic cavity of 9-11-day-old SPF chicken embryos, then incubate at 37°C. After 48 hours, harvest the allantoic fluid of chicken embryo virus, perform hemagglutination test with 1% chicken red blood cells, aseptically collect the allantoic fluid of virus with HA titer ≥ 256, and then store it in an environment of -80°C (such as a low-temperature refrigerator);

[0016] 2) Antigen inactivation: Add formaldehyde solution with a final concentration of 0.1% to the allantoic fluid of chicken embryo virus prepared in step 1), shake it while adding to make it fully mixed; then place it in a constant temperature shaker at 37°C and shake it at 200-220 r / min for 12 hours and then take it out to obtain the inactivated virus solution of avian influenza H9N2 subtype vaccine strain, and store it at 4°C (such as a refrigerator) for standby;

[0017] 3) Add the inactivated avian influenza H9N2 subtype vaccine virus liquid and vaccine adjuvant from step 2) in a ratio of 1:2.81, and then add antiviral protein IFITM1 as an immune enhancer to make its concentration in the vaccine 167 μg / mL. Use an HR-500 dispersion emulsifier to gradually start emulsification from gear A to gear D, and let it emulsify at 15000 r / min for 30 minutes under low-temperature conditions.

[0018] As a preferred implementation option, preferably, in step 3), the preparation method of the antiviral protein IFITM1 is as follows: Infect SPF chicks with H9N2 avian influenza virus. Five days after infection, take lung tissue; Design primers with reference to the coding region nucleotide sequence of the avian-derived IFITM1 gene published in GenBank, and perform PCR amplification of the target band. Sequence to identify the mRNA level expression and gene sequence of chicken-derived IFITM1; Then construct a recombinant expression plasmid of IFITM1, transform Escherichia coli BL21(DE3) competent cells. After induction with IPTG and ultrasonic disruption, analyze the expression of antiviral protein by SDS-PAGE electrophoresis, and purify it by nickel column affinity chromatography. The purified antiviral protein is stored at 4°C for standby.

[0019] Based on the above scheme, the present invention also provides the application of antiviral protein IFITM1, an avian immune enhancer, in the preparation of avian vaccines.

[0020] Adopting the above technical scheme, compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. The immune enhancer of the present invention's scheme is applied in the inactivated H9N2 avian influenza vaccine, which can enhance the inactivated H9N2 avian influenza vaccine to induce chickens to produce higher levels of serum antibodies. After co-immunization with the recombinant inactivated vaccine, it can improve the detoxification inhibition effect and can effectively prevent the prevalence of current prevalent strains.

[0022] 2. The immune enhancer of the present invention's scheme has the effect of inhibiting the detoxification of H9N2 avian influenza and reducing tissue damage 21 days after being inoculated alone.

[0023] 3. There is currently no chicken-derived antiviral protein immune enhancer product on the market in China. Therefore, the research and development of highly efficient immune enhancers for use in combination with inactivated vaccines meet the current market demand of the poultry industry and are of great significance for the vaccine research and development of H9N2 subtype avian influenza and the prevention and control of chicken-derived diseases in China. Description of the Drawings

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0025] Figure 1 For the detection of the mRNA level of the full gene of chicken-derived IFITM1 after H9N2 subtype avian influenza infection in the present invention's solution;

[0026] Figure 2 For the SDS-PAGE detection after purification of the recombinant chicken-derived IFITM1 antiviral protein in the present invention's solution;

[0027] Figure 3 It is a characterization diagram of the effect of protein purification using nickel column affinity chromatography in Example 1 of the present invention's solution.

[0028] Figure 4 It is the specific autopsy result after the experimental samples in Example 2 of the present invention's solution were challenged with the virus. Detailed implementation manners

[0029] The following will further describe the present invention in detail in conjunction with the drawings and embodiments. It should be particularly noted that the following embodiments are only used to illustrate the present invention, but do not limit the scope of the present invention. Similarly, the following embodiments are only some embodiments of the present invention rather than all embodiments. All other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0030] Example 1: Screening and preparation of antiviral protein IFITM1 as an immune enhancer

[0031] (1) Detection of the mRNA level expression of antiviral genes IFITM1, IFITM3, and Viperin after H9N2 subtype avian influenza virus infection

[0032] Use H9N2 avian influenza virus to infect SPF chicks by intravenous injection, with an infection dose of 10 6.0 EID 50 . Subsequently, 5 days after infection, take lung tissue. Design primers according to the coding region nucleotide sequences of avian-derived IFITM1, IFITM3, and Viperin genes published in GenBank, and perform PCR amplification of the target bands. According to the size of the target bands and the sequencing results, it is determined that H9N2 AIV can induce the mRNA level expression of IFITM1, IFITM3, and Viperin, and the results are as Figure 1 shown.

[0033] (II) Expression and Purification of Chicken-derived Antiviral Proteins IFITM1, IFITM3 and Viperin

[0034] Specific primers were designed and PCR was performed to amplify the target bands. The gene fragments of GIFITM1, GIFITM3 and GViperin obtained by PCR were double-digested with restriction enzymes XHOⅠ and BamHⅠ. At the same time, the pCOLD-TF vector was double-digested with the same enzymes. The recovered gene fragments were ligated with the corresponding vector fragments. After transformation, plasmids were extracted. It was confirmed by double-digestion identification and plasmid sequencing that the three prokaryotic expression plasmids pCOLD-GIFITM1, pCOLD-GIFITM3 and pCOLD-GViperin were successfully constructed.

[0035] The constructed prokaryotic expression plasmids pCOLD-GIFITM1, pCOLD-GIFITM2 and pCOLD-GViperin were transformed into BL21(DE3) competent cells. Colonies were picked and positive strains were obtained after colony PCR. The three positive strains were inoculated into liquid LB and cultured with shaking in a 37℃ shaker until the OD600 was about 0.6 - 1.0. Then, IPTG with a final concentration of 0.5 mmol / L was used to induce for 20 - 22 h at 15℃ and a rotation speed of 200 rpm (the optimal concentration of IPTG and the optimal temperature obtained from preliminary pre-experiments). SDS-PAGE was used to analyze the expression of total E. coli proteins: The recombinant plasmids pCOLD-GIFITM1, pCOLD-GIFITM2 and pCOLD-GViperin expressed 1 specific protein band at positions close to 72 kDa, 74 kDa and 101 kDa respectively, which was consistent with the expected size of the fusion proteins, indicating that the fusion proteins were successfully induced for expression. The three successfully induced fusion proteins were sonicated, and SDS-PAGE was used to analyze the solubility of the fusion proteins. The results were as Figure 2 shown. Three antiviral proteins were successfully expressed, and the three fusion proteins mainly existed in the supernatant of the bacterial cell lysate. Subsequently, nickel column affinity chromatography was used for protein purification, and the purification effect was good as Figure 3 shown.

[0036] Example 2. Detection of the Immune Efficacy of Antiviral Proteins IFITM1, IFITM3 and Viperin as Immune Enhancers in Combination with Inactivated Whole Virus Vaccine against H9N2 Subtype Avian Influenza

[0037] (I) Preparation of Seed Solution

[0038] The allantoic fluid of the purified H9N2 strain FZ (i.e., influenza A virus FZ strain, with the deposit address in Wuhan, China, deposit number CCTCC NO: V 202171, and deposit date September 26, 2021) was diluted and inoculated into the allantoic cavity of 10-day-old SPF chicken embryos at 0.1 mL / embryo, and then incubated in a constant temperature incubator at 37°C after inoculation. Observation was carried out every 12 h, and the chicken embryos that died within 24 h were discarded. The allantoic fluid of chicken embryos with a hemagglutination titer ≥ 256 was collected at 48 h, and generations 5 to 15 were collected as the seed lot and stored at -80°C.

[0039] (II) Amplification and culture of virus seeds

[0040] The allantoic fluid of the preserved H9N2 strain was thawed at room temperature and inoculated into the allantoic cavity of 9 - 11-day-old SPF chicken embryos at 0.1 mL / embryo, and then incubated in a constant temperature incubator at 37°C after inoculation. Observation was carried out every 12 h, and the chicken embryos that died within 24 h were discarded. The allantoic fluid of chicken embryos was collected at 48 - 72 h. The results showed that the hemagglutination titer of the collected allantoic fluid was greater than or equal to 256, and the virus antigen content was greater than or equal to 10 8.5 EID 50 / 0.1 mL.

[0041] (III) Inactivation of virus strain

[0042] Formaldehyde solution with a final concentration of 0.1% was added to the sterile allantoic fluid of H9N2 subtype AIV FZ strain virus prepared in step (II), and it was shaken immediately after addition to make it fully mixed. Then it was taken out after shaking and inactivating at 200 - 220 r / min in a constant temperature shaker at 37°C for 12 hours, and stored in a 4°C refrigerator for standby.

[0043] (IV) Inactivation test

[0044] Inactivated virus solution was inoculated into the allantoic cavity of 10-day-old SPF chicken embryos at a standard of 3 pieces / sample and 0.1 mL / piece, sealed and marked, and cultured in an incubator at 37°C. Observation was carried out continuously for 5 days, 2 times / day. After 5 days, the allantoic fluid of chicken embryos was harvested for HA determination. The results showed that the virus solution could be fully inactivated by using 0.1% formaldehyde and shaking and inactivating at 200 - 220 r / min in a constant temperature shaker at 37°C for 12 hours.

[0045] (V) Vaccine preparation

[0046] In a sterile container, the inactivated H9N2 subtype AIV FZ strain virus solution (the virus antigen content of the vaccine is 10 8.0 EID 50 / mL) and vaccine adjuvant were added in a ratio of 1:2.81, and antiviral protein IFITM1 was added to make its concentration in the vaccine 167 μg / mL, gradually start emulsification from gear A to gear E using an HR-500 dispersion emulsifier, and complete the preparation of vaccine B in 30 minutes; complete the preparation of vaccines C and D containing antiviral proteins IFITM3 and Viperin as immune enhancers in the same method; complete the preparation of vaccines F, G, and H without adding antigens (sterile PBS replaces antigens) in the same method; complete the preparation of vaccines A and E of other control groups in the same method. I and J are the blank group and the challenge control group respectively, as shown in Table 1.

[0047] (VI) Finished product inspection

[0048] 1. Appearance: Take 5 mL of each vaccine and place it in a clean glass tube, observe that the color of the vaccine has no change and there are no impurities, etc.

[0049] 2. Dosage form: Take a clean pipette, suck a small amount of the vaccine and drop it on the surface of cold water, observe that the vaccine does not spread.

[0050] 3. Stability: Suck 10 mL of each batch of vaccine and add it to a centrifuge tube, centrifuge at 3000 r / min for 15 minutes, observe that the vaccine does not show stratification.

[0051] 4. Sterility test: Conduct according to the appendix of the current "Chinese Veterinary Pharmacopoeia", and there is no bacterial growth.

[0052] 5. Purity test: Conduct the test according to the appendix of the current "Chinese Veterinary Pharmacopoeia", and all meet the regulations.

[0053] 6. Determination of formaldehyde residue: Conduct the determination according to the appendix of the current "Chinese Veterinary Pharmacopoeia", and the results show that it meets the regulations of the General Rules for Veterinary Biological Products.

[0054] 7. Safety test: Take 10 14-day-old SPF chickens, subcutaneously inject the inactivated vaccine containing immune enhancer in the present invention into the neck, 0.5 mL for each chicken, continuously observe for 14 days, and there are no obvious abnormalities in the spirit, food intake and drinking water of all test chickens, and the injected vaccines do not cause local or systemic adverse reactions.

[0055] 8. Serum antibody titer test: Ten 14-day-old SPF chickens were randomly divided into 10 groups of 10 each. Chickens in each group were subcutaneously injected with vaccines A, B, C, D, E, F, G, and H in the neck. Blood samples were collected from the chickens in each group on the 14th and 21st days after immunization, and sera were separated to measure the HI antibodies against H9N2 subtype avian influenza. The results of the HI antibodies against H9N2 subtype avian influenza are shown in Table 1. The average HI titer of the vaccine A group was 9 on the 14th day after immunization, and that of vaccine B was 10.6. On the 21st day after immunization, the average HI titer of the vaccine A group was 10.2, and that of vaccine B was 11.6. The addition of immune enhancers IFITM1, IFITM3, and Viperin increased the antibody level to varying degrees, among which IFITM1 had the best effect. In the control group of chickens without added antigen, the average HI titer was <2.

[0056] 9. Potency test: On the 21st day after immunization, 0.2 mL (10 6.0 EID 50 / mL) of the original H9N2 subtype avian influenza virus was inoculated intravenously. After challenge, throat swabs were collected from the chickens in each group for embryo inoculation detection of H9N2 subtype avian influenza. The results of the embryo inoculation detection of H9N2 subtype avian influenza virus excretion are shown in Table 1. Groups A, B, C, and D all had 100% inhibition of virus excretion, and the immune groups F, G, and H with only antiviral proteins added also showed good inhibitory effects on virus excretion, among which IFITM1 had the best inhibitory effect. The clinical observation results showed that on the 2nd day after infection, the challenge control groups E and J began to show mild respiratory symptoms, and obvious clinical manifestations appeared from the 3rd to 7th day, including head shaking, respiratory rales, and mild depression. Only individual chickens in groups F, G, and H showed mild respiratory symptoms. The specific autopsy results after challenge are shown in Figure 4 , in the challenge control group, there was bleeding or mucus secretion in the larynx and trachea, and in addition, there were no obvious lesions in other organs.

[0057] Table 1: Detection of antibodies and inhibitory virus excretion after immunization with immune enhancer combined with inactivated whole virus vaccine

[0058]

[0059] Example 3. Immunopotency detection of antiviral protein IFITM1 as an immune enhancer combined with inactivated recombinant H9N2 subtype avian influenza virus vaccine

[0060] Select the best-performing chicken-derived antiviral protein IFITM1 in Example 2 as an immune enhancer, and prepare it into an inactivated vaccine together with the allantoic fluid of the purified H9N2 recombinant virus HA+NA / PR8 strain (rescued by the reverse genetics system, containing the HA and NA genes of the H9N2 subtype avian influenza FZ strain and the 6 internal genes NP, M, NS, PA, PB1, and PB2 of the PR8 strain). The methods for preparing the seed liquid, amplifying and culturing the virus seed, inactivating the virus, inactivating inspection, and vaccine preparation are all the same as those in Example 2. As shown in Table 2, vaccines A, B, C, and D containing different components are prepared, and then the inactivated vaccine is inspected:

[0061] 1. Appearance: Take 5 mL of each vaccine and place it in a clean glass tube. Observe that the color of the vaccine does not change and there are no impurities, etc.

[0062] 2. Dosage form: Take a clean pipette, suck a small amount of the vaccine and drop it on the surface of cold water. Observe that the vaccine does not spread.

[0063] 3. Stability: Take 10 mL of each batch of vaccine and add it to a centrifuge tube. Centrifuge at 3000 r / min for 15 minutes. Observe that the vaccine does not show stratification.

[0064] 4. Sterility test: Conduct according to the appendix of the current "Chinese Veterinary Pharmacopoeia", and no bacteria grow.

[0065] 5. Purity test: Conduct the test according to the appendix of the current "Chinese Veterinary Pharmacopoeia", and all meet the regulations.

[0066] 6. Determination of formaldehyde residue: Conduct the determination according to the appendix of the current "Chinese Veterinary Pharmacopoeia", and the results show that it meets the regulations of the General Rules for Veterinary Biological Products.

[0067] 7. Safety test: Take 10 14-day-old SPF chickens, inject the inactivated vaccine containing the immune enhancer in the present invention subcutaneously in the neck, 0.5 mL per chicken. Continuously observe for 14 days, and there are no obvious abnormalities in the spirit, food intake, and drinking water of all test chickens. The injection of the vaccine does not cause local or systemic adverse reactions.

[0068] 8. Serum antibody titer test: Take 70 14-day-old SPF chickens, randomly divide them into 7 groups, with 10 chickens in each group. Inject vaccines A, B, C, D, and groups E and F as blank controls, and group G as a challenge control subcutaneously in the neck respectively. Blood samples are taken from the chickens in each group on the 14th and 21st days after immunization, the sera are separated, and the HI antibody against H9N2 subtype avian influenza is measured. The results of the HI antibody against H9N2 subtype avian influenza are shown in Table 2. The average HI titer of vaccine group A was 10 on the 14th day after immunization, and that of vaccine group B was 9. The average HI titer of vaccine group A was 11.6 on the 21st day after immunization, and that of vaccine group B was 10.2. The addition of the immune enhancer IFITM1 group increased the antibody level of the H9N2 recombinant inactivated vaccine. In the control group of chickens without added antigen, the average HI titer < 2.

[0069] 9. Efficacy test: Intravenous inoculation of 0.2 mL (10 6.0 EID 50 / mL) of the original H9N2 avian influenza virus. After virus challenge, throat swabs of each group of chickens were collected for embryo inoculation detection of H9N2 avian influenza. The results of embryo inoculation detection of H9N2 avian influenza are shown in Table 2. Adding chicken-derived antiviral protein IFITM1 as an immune enhancer can improve the virus excretion inhibition rate of the recombinant inactivated H9N2 avian influenza vaccine. The clinical observation results showed that on the 2nd day after infection, mild respiratory symptoms began to appear in the virus challenge control group. Only 1 chicken in Vaccine Group D showed mild respiratory symptoms. Autopsy showed that there was bleeding or mucus secretion in the larynx of the virus challenge control group, while only individual chickens in Vaccine Groups B and D showed mild laryngeal mucus. In addition, there were no obvious lesions in other organs.

[0070] Table 2: Detection of antibodies and virus excretion inhibition after immunization with immune enhancer combined with recombinant inactivated vaccine

[0071]

[0072] The above are only some embodiments of the present invention, and thus do not limit the protection scope of the present invention. Any equivalent device or equivalent process transformation made by using the content of the specification and drawings of the present invention, or directly or indirectly applied in other related technical fields, shall be equally included in the patent protection scope of the present invention.

Claims

1. An inactivated H9N2 subtype avian influenza vaccine, characterized in that: It includes an antigen, a vaccine adjuvant and an immune enhancer; The antigen is an inactivated antigen, which is an inactivated influenza A virus strain FZ or a recombinant virus HA+NA / PR8 strain containing the HA and NA genes of influenza A virus strain FZ and the 6 internal genes NP, M, NS, PA, PB1 and PB2 of H1N1-PR8 strain rescued by reverse genetics system. Among them, the influenza A virus strain FZ is deposited in the China Center for Type Culture Collection, with the deposit address being Wuhan, China, the deposit number being CCTCC NO: V 202171, and the deposit date being September 26, 2021; The immune enhancer is a chicken-derived antiviral protein IFITM1 expressed prokaryotically by Escherichia coli; The antigen is inactivated with formaldehyde.

2. Use of an antiviral protein immune enhancer in the preparation of an inactivated H9N2 avian influenza vaccine, characterized in that, It includes the following steps: 1) Dilute the avian influenza H9N2 subtype vaccine strain 1000-fold with sterile PBS and inoculate it into the allantoic cavity of 9-11-day-old SPF chicken embryos, then incubate at 37°C. After 48 hours, harvest the allantoic fluid of chicken embryo virus, perform a hemagglutination test using 1% chicken red blood cells, aseptically collect the allantoic fluid of virus with an HA titer ≥ 256, and then store it in an environment of -80°C; 2) Antigen inactivation: Add a formaldehyde solution with a final concentration of 0.1% to the allantoic fluid of chicken embryo virus prepared in step 1), shake it while adding to make it fully mixed; then place it in a constant temperature shaker at 37°C and shake it at 200-220 r / min for 12 hours and then take it out to obtain an inactivated avian influenza H9N2 subtype vaccine strain virus solution, and store it at 4°C for standby; 3) Add the inactivated avian influenza H9N2 subtype vaccine strain virus solution and the vaccine adjuvant prepared in step 2) at a ratio of 1:2.81, and then add the chicken-derived antiviral protein IFITM1 as an immune enhancer to make its concentration in the vaccine 167 µg / mL, and use an HR-500 dispersion emulsifier to gradually start emulsifying from gear A to gear D, and let it emulsify at 15000 r / min for 30 minutes under low temperature conditions.

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