Broad-spectrum subunit vaccine for H3 subtype avian influenza and preparation method of broad-spectrum subunit vaccine

Abstract

The invention discloses an H3 subtype avian influenza broad-spectrum subunit vaccine and a preparation method thereof, and belongs to the technical field of gene engineering. The rHA-b protein of the H3 subtype avian influenza virus is screened out, and the protein is used as an antigen to prepare the H3 subtype avian influenza broad-spectrum subunit vaccine. The H3 subtype avian influenza subunit vaccine disclosed by the invention has broad spectrum, and an immune potency test shows that the antigen is safe, has no adverse reaction, can induce an organism to generate an antibody level equivalent to that of a whole-virus inactivated vaccine, can effectively inhibit infection and replication of different strains, and has a relatively good broad-spectrum immune effect.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering technology, and in particular relates to an H3 subtype avian influenza broad-spectrum subunit vaccine and a preparation method thereof. Background Art

[0002] Based on the serological reactivity of the viral surface hemagglutinin (HA) and neuraminidase (NA), avian influenza viruses (AIVs) are classified into 16 HA subtypes and 9 NA subtypes. The widespread infection and prevalence of subtypes H5, H7, H9, and H3 in chickens in my country has caused significant economic losses to the poultry industry. The increasingly sophisticated commercial vaccines for H5, H7, and H9 AIVs have made significant contributions to the effective immunization and prevention of H5, H7, and H9 subtypes of avian influenza. However, H3 AIV has been continuously isolated in poultry and wild birds since 2000, indicating that H3 AIV is circulating dynamically across a wide range of species in my country. Among the combinations of H3 HA and different NA subtypes, H3N2 and H3N8 predominate. These subtypes are not only circulating dynamically in poultry and wild birds in neighboring countries but are also prevalent in domestic chickens in my country. Their continued evolution and mutation further increase the risk of viral spillover to humans.

[0003] In April 2022, my country reported its first human infection with H3N8 AIV. Since then, reports of human infection with H3N8 AIV have continued, raising concerns about whether H3N8 AIV poses a public health threat. Serological surveys have shown that H3N2-positive sera react poorly with the newly emerged H3N8, suggesting little antigenic cross-reactivity between H3N2 and H3N8, and little pre-existing immunity to the newly emerged H3N8 AIV. Sporadic infection of H3N8 AIV in humans can continuously drive dynamic host adaptation, allowing for horizontal transmission among humans without cross-immune protection. The 1968 pandemic, caused by a human-adapted H3N2 AIV, provides a precedent for the potential public health threat posed by H3AIV.

[0004] H3N8 AIV not only demonstrates cross-species transmission but can also serve as a donor virus for the formation of novel influenza viruses, such as equine influenza, canine influenza, swine influenza, and the 1968 pandemic H3N2 virus. The persistent infection of H3N8 viruses in chickens, coupled with frequent mutations and recombination of antigenic genes, increases genetic diversity associated with changes in viral virulence, transmissibility, and mammalian adaptability, posing a significant threat to animal and human health. However, there are currently no H3 subtype avian influenza control products specifically for poultry, both domestically and internationally, necessitating the development of H3 subtype avian influenza vaccines. Traditional whole-virus inactivated vaccine antigen preparation relies heavily on chicken embryos, the quality of which significantly impacts vaccine antigen quality. Furthermore, whole-virus inactivated vaccines also carry the potential safety risk of incomplete inactivation. Therefore, this study employed genetic engineering techniques to express and prepare H3 subtype avian influenza vaccine antigens. Summary of the Invention

[0005] The purpose of the present invention is to overcome the deficiencies in the prior art and to provide an H3 subtype avian influenza broad-spectrum subunit vaccine and a preparation method thereof.

[0006] The purpose of the present invention can be achieved through the following technical solutions:

[0007] In a first aspect, the present invention claims protection for an rHA-b protein of an H3 subtype avian influenza virus, which is as follows (a1) or (a2) or (a3):

[0008] (a1) a protein with an amino acid sequence as shown in SEQ ID NO. 1;

[0009] (a2) a fusion protein obtained by attaching a protein tag to the N-terminus or / and C-terminus of the amino acid sequence shown in SEQ ID NO.1;

[0010] (a3) A protein having 90% or more identity and the same function as the protein shown in (a1) obtained by substituting and / or deleting and / or adding one or more amino acid residues in the amino acid residue sequence shown in SEQ ID NO.1.

[0011] In a second aspect, the present invention seeks protection for the gene encoding the above protein.

[0012] Furthermore, the above-mentioned gene is a DNA molecule of any one of the following (b1)-(b4):

[0013] (b1) a DNA molecule whose coding region includes the nucleotide sequence shown in SEQ ID NO. 2;

[0014] (b2) a DNA molecule whose nucleotide sequence is shown in SEQ ID NO. 2;

[0015] (b3) a DNA molecule that hybridizes under stringent conditions with the DNA sequence defined in (b1) or (b2) and encodes a protein that can serve as an antigen for a broad-spectrum subunit vaccine for H3 subtype avian influenza;

[0016] (b4) A DNA molecule that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology to the DNA sequence defined in (b1) or (b2) and encodes a protein that can serve as an antigen for a broad-spectrum subunit vaccine for H3 subtype avian influenza.

[0017] In a third aspect, the present invention seeks to protect biological materials containing the above-mentioned genes, which are expression cassettes, recombinant vectors, recombinant microorganisms or transgenic cell lines.

[0018] In a fourth aspect, the present invention claims a method for preparing rHA-b protein of H3 subtype avian influenza virus, wherein the rHA-b protein is obtained by culturing a recombinant baculovirus carrying the aforementioned rHA-b protein encoding gene.

[0019] Furthermore, the method for constructing the aforementioned recombinant baculovirus comprises the following steps:

[0020] S61, add a Kozak sequence to the 5' end of SEQ ID NO: 2, and add a GS Linker and Leu zip sequence to the 3' end;

[0021] S62, inserting the product obtained in step S61 into the baculovirus transfer vector pFastBac1 to obtain a recombinant baculovirus transfer plasmid;

[0022] S63, transforming the recombinant baculovirus transfer plasmid into Escherichia coli DH10Bac competent cells to obtain a recombinant baculovirus shuttle plasmid;

[0023] S64. Transfect the recombinant baculovirus shuttle plasmid into insect cells Sf9 to obtain a recombinant baculovirus expressing the rHA-b protein.

[0024] Furthermore, the recombinant baculovirus was cultured with High Five cells to obtain the recombinant protein rH3HA-b.

[0025] In a fifth aspect, the present invention seeks to protect the use of the aforementioned rHA-b protein, the aforementioned gene, or the aforementioned biological material in the preparation of a broad-spectrum subunit vaccine for H3 subtype avian influenza.

[0026] In a sixth aspect, the present invention seeks to protect a broad-spectrum subunit vaccine for H3 subtype avian influenza, which uses the inactivated recombinant protein rH3HA-b of the aforementioned rHA-b protein as an antigen.

[0027] In a seventh aspect, the present invention claims protection for a method for preparing the aforementioned H3 subtype avian influenza broad-spectrum subunit vaccine, the method comprising the following steps:

[0028] S91, taking the inactivated recombinant protein rH3HA-b as an antigen, the antigen and sterilized Tween 80 were mixed in a volume ratio of 96:4, and emulsified to obtain an aqueous phase;

[0029] S92, mixing white oil for injection and Siben 80 in a volume ratio of 94:6, sterilizing by high pressure at 121° C. for 15 min, and cooling to room temperature to obtain an oil phase;

[0030] S93. Mix and emulsify the aqueous phase and the oil phase in a volume ratio of 1:3 to obtain a broad-spectrum subunit vaccine of the H3 subtype avian influenza recombinant HA protein.

[0031] Furthermore, the above antigen was obtained by inactivating the recombinant protein rH3HA-b using 0.1% (v / v) formaldehyde at 37°C for 24 hours.

[0032] Beneficial effects of the present invention:

[0033] The H3 subtype avian influenza broad-spectrum subunit vaccine of the present invention has broad spectrum properties. Immune efficacy tests have shown that the antigen is safe and has no adverse reactions. It can induce the body to produce antibody levels similar to those of the whole virus inactivated vaccine, can effectively inhibit the infection and replication of different strains, and has a good broad-spectrum immune effect.

[0034] The HA protein antigen gene of the broad-spectrum H3 subtype avian influenza subunit vaccine of the present invention is cloned into a baculovirus expression system to obtain the broad-spectrum antigen, which is simple and efficient to prepare. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is the PCR identification result. Lane 1 is DNA Marker and lane 2 is PCR product, which is about 4500 bp in size. Figure 2 The results of the indirect immunofluorescence assay of the present invention are shown in Figure 3; (A) is a fluorescence diagram of Sf9 cells infected with recombinant baculovirus rBV-H3HA-b; (B) is a fluorescence diagram of Sf9 cells infected with wild-type baculovirus.

[0036] Figure 3The following are the results of a microhemagglutination test of the recombinant protein rH3HA of the present invention. The dilution multiples of the recombinant protein in each well are indicated above each well, namely 1log2, 2log2, 3log2, 4log2, 5log2…16log2; wherein: (A) shows the expression product of High Five cells infected with rBv-H3HA-b; (B) shows the expression product of High Five cells infected with rBv-H3HA-a; (C) shows the expression product of High Five cells infected with wild-type baculovirus; (D) shows a normal High Five cell culture; and (E) shows an H3N8 AIV control. DETAILED DESCRIPTION

[0037] The present invention will be further described with reference to the accompanying drawings and embodiments.

[0038] The test materials used in the following examples are as follows:

[0039] SPF chicken embryos and SPF chickens were purchased from Boehringer Ingelheim Biotech Co., Ltd., and various restriction endonucleases, T4 ligase, rTaq enzyme, etc. were purchased from Takara. Primers and gene sequences were synthesized by Qingke Biotechnology Co., Ltd. Transfection kits and insect cell culture media were purchased from Invitrogen. FITC-labeled goat anti-chicken fluorescent antibody was purchased from Thermo. H3 subtype avian influenza detection antigen and H3 subtype avian influenza virus positive serum were prepared and stored in our laboratory. Recombinant HA protein antiserum is a high-immune serum obtained by immunizing SPF chickens after preparing a vaccine using recombinant HA protein. The M13 universal primer was synthesized according to the Bac-to-Bac system instructions (Invitrogen).

[0040] Example 1 Design and synthesis of H3 subtype avian influenza broad-spectrum vaccine antigens and construction of recombinant baculovirus vectors.

[0041] Based on the H3 subtype avian influenza virus hemagglutinin protein gene published by GISAID, multiple H3 subtype avian influenza vaccine antigens were designed. It was found that only the rHA-b protein exhibited good broad-spectrum immunogenicity. The amino acid sequence of the rHA-b protein is shown in SEQ ID NO: 1, and the nucleotide sequence of the encoded rHA-b protein is shown in SEQ ID NO: 2.

[0042] The rHA-b protein gene sequence was designed as follows: a BamHI restriction site and a Kozak sequence were added to the 5' end of the gene sequence encoding the rHA-b protein, and a GS linker, a Leu zip sequence, and an EcoRI restriction site were added to the 3' end. The gene was sent to Qingke Biotechnology Company for synthesis and cloned into the recombinant baculovirus transfer vector pFastBac1, which was named the baculovirus transfer vector pFastBac1-H3HA-b.

[0043] The recombinant baculovirus transfer vector pFastBac1-H3HA-b was transformed into Escherichia coli DH10Bac competent cells and cultured on LB plates containing kanamycin (50 μg / mL), gentamicin (7 μg / mL), tetracycline (10 μg / mL), X-gal (100 μg / mL) and IPTG (40 μg / mL) for 24-48 hours. Three generations were screened by blue-white staining and single white colonies were picked. The white colonies were inoculated into liquid LB culture medium containing kanamycin (50 μg / mL), gentamicin (7 μg / mL) and tetracycline (10 μg / mL). The recombinant shuttle plasmid was identified by PCR using M13 universal primers. The PCR fragment size was consistent with the expected size. The correctly identified recombinant baculovirus shuttle plasmid was named rBacmid-H3HA-b. Figure 1 shown.

[0044] Example 2: Obtaining recombinant baculovirus, plaque purification and expression of recombinant protein.

[0045] 1. Obtaining recombinant baculovirus

[0046] According to the instructions of the transfection reagent, rBacmid-H3HA-b was transfected into Sf9 cells in the logarithmic growth phase and cultured in a 27°C incubator. After the cells showed obvious pathological changes, the cells and supernatant were collected to obtain the first generation recombinant baculovirus. The recombinant virus was subcultured on Sf9 cells to the fourth generation, and the fourth generation cells and supernatant were harvested. After repeated freezing and thawing, the cells were centrifuged at 1000 rpm for 5 minutes, and the supernatant was collected and stored as the fourth generation recombinant baculovirus. The TCID of the recombinant virus was determined according to the Reed-Muench method. 50 , with a titer of 10 7.25 TCID 50 / mL.

[0047] 2. Plaque Purification

[0048] Plaque purification steps are as follows: Sf9 cells were seeded in 6-well plates, with approximately 1×10 cells per well. 6To each well, 1 mL of Grace's medium containing 10% fetal bovine serum was added. The cells were cultured for 24 hours until they reached the logarithmic growth phase. The medium was discarded, and the cells were washed three times with Grace's medium. The diluted 4th-generation recombinant baculovirus solution was slowly added to the cells, along with a normal cell control, and incubated at 27°C for 1 hour. The virus solution was aspirated from the wells, and an agarose solution (prepared by mixing 10 mL of 4% agarose solution, 20 mL of 20% Grace's medium, and 10 mL of sterile ultrapure water) was slowly added to the cell surface along the edge of the wells. After the agar solidified, the cell plate was placed in an incubator and cultured for 7-10 days. Cytopathic effects were observed, and individual plaques were picked and added to Grace's medium. After repeated shaking, the supernatant was inoculated into Sf9 cells and cultured for another 96-120 hours. The cells and supernatant were harvested to obtain the purified recombinant baculovirus, designated rBV-H3HA-b. The recombinant baculovirus rBV-H3HA-b was propagated to the sixth generation in Sf9 cells, and the virus TCID 50 , with a titer of 10 8 TCID 50 / mL.

[0049] 3. Expression of Recombinant Protein

[0050] The 6th generation recombinant baculovirus was inoculated at a concentration of 1×10 6 The cells were cultured in a constant temperature shaker at 27°C for 96 to 120 hours, and the cell suspension was collected, lysed by ultrasonication, and the supernatant was collected by centrifugation to obtain the recombinant rH3HA-b protein.

[0051] Example 3 Indirect immunofluorescence assay (IFA) was used to identify the expression of the recombinant protein, microhemagglutination assay (HA) was used to identify the hemagglutination activity of the recombinant protein, and microhemagglutination inhibition assay (HI) was used to identify the cross-hemagglutination inhibition activity of the recombinant protein.

[0052] 1. Identification of recombinant protein expression by indirect immunofluorescence assay (IFA)

[0053] The recombinant baculovirus rBV-H3HA-b was inoculated into Sf9 cells and infected for 72 to 96 hours. The cell supernatant was discarded, the cells were washed once with PBS, fixed with 4% paraformaldehyde solution for 15 minutes at room temperature, and dried naturally. A 1:300 dilution of H3 subtype avian influenza positive serum was used as the primary antibody, incubated at 37°C for 1 hour, and washed 3 times with PBS; a 1:500 dilution of FITC-labeled goat anti-chicken fluorescent antibody was used as the secondary antibody, incubated at 37°C for 1 hour, washed 3 times with PBS, and the results were observed under a fluorescence microscope; at the same time, a H9 subtype avian influenza positive serum was set as a negative control. The results showed that Sf9 cells infected with rBV-H3HA-b could react with H3 subtype avian influenza positive serum, producing specific bright green fluorescence, but did not react with H9 subtype avian influenza positive serum, indicating that rH3HA-b was correctly expressed in Sf9 cells. Figure 2 As shown in Figures A and B.

[0054] 2. Microhemagglutination assay (HA) to identify the hemagglutination activity of recombinant protein

[0055] The recombinant baculovirus rBV-H3HA-b was inoculated into High Five cells and cultured with shaking for 96 to 120 hours. The cell suspension was collected, ultrasonically lysed, and centrifuged at 1000 rpm for 5 minutes. The supernatant was collected to obtain the recombinant protein rH3HA-b. The hemagglutination activity of the recombinant protein was detected by microhemagglutination assay, while H3 subtype avian influenza virus and PBS were used as controls. The results showed that the HA titer was 14Log2, indicating that the recombinant protein rH3HA-b has high hemagglutination activity. Figure 3 As shown in Figures (A), (B), (C), (D) and (E).

[0056] 3. Microhemagglutination inhibition test (HI) to identify the cross-hemagglutination inhibition activity of recombinant proteins

[0057] Recombinant rH3HA-b was used as the HI detection antigen. Cross-reactivity between rH3HA-b and positive sera from different H3 subtype avian influenza virus strains was tested using HI. Antiserum against recombinant rH3HA-a served as a positive serum control, negative serum from SPF chickens and sterilized PBS served as negative controls, and inactivated antigens from different H3 subtype avian influenza virus strains served as antigen controls. The results showed that recombinant rH3HA-b exhibited strong hemagglutination inhibition with all sera. Inactivated antigens from different H3 subtype avian influenza virus strains also exhibited strong hemagglutination inhibition with strain-specific sera, but reactivity with positive sera from other strains was relatively weak. These results demonstrate that recombinant rH3HA-b exhibits a broader spectrum of activity than prevalent strains, as shown in Table 1.

[0058] Table 1. Results of cross-hemagglutination inhibition test of recombinant protein rH3HA-b

[0059]

[0060] Example 4 Preparation of recombinant protein rH3HA-b and proliferation, inactivation and vaccine preparation of H3 subtype avian influenza virus. 1. Preparation of recombinant protein rH3HA-b and proliferation of H3 subtype avian influenza virus

[0061] 1. Preparation of recombinant protein rH3HA-b

[0062] The recombinant baculovirus rBV-H3HA-b was inoculated into High Five cells at an MOI of 1 and cultured on a shaker at 27°C for 96-120 hours. The cell suspension was ultrasonically lysed and the supernatant was collected after centrifugation to obtain the recombinant protein rH3HA-b.

[0063] 2. Proliferation of the H3N8 avian influenza virus

[0064] The H3N8 virus ZJ07, AH12 and JS13 strains were diluted 10,000 times with sterile PBS and inoculated into 10-day-old SPF chicken embryos at 0.1 mL / embryo. The cells were incubated at 37°C for 96 h. The hemagglutination titer was determined and the chicken embryo allantoic fluid with high hemagglutination titer was collected.

[0065] 2. Inactivation of recombinant protein rH3HA-b and H3N8 virus

[0066] Antigens were inactivated using 0.1% (v / v) formaldehyde at 37°C for 24 hours. Inactivation assays were performed to measure the HA titer of the antigen, the infectivity of the recombinant protein solution to Sf9 cells, and the infectivity of H3AIV to SPF chicken embryos. The results showed that the hemagglutination titers of the inactivated recombinant protein, ZJ07, AH12, and JS13 were 14 Log2, 8 Log2, 9 Log2, and 10 Log2, respectively. The inactivated recombinant protein solution and the whole virus antigen solution did not contain infectious virions.

[0067] 3. The preparation method of H3 subtype avian influenza recombinant baculovirus broad-spectrum subunit vaccine and H3N8 avian influenza inactivated vaccine comprises the following steps:

[0068] 1. Take the inactivated recombinant protein rH3HA-b and H3N8 (ZJ07 / AH12 / JS13) antigens and mix them with sterilized Tween 80 at a volume ratio of 96:4, emulsify, and obtain an aqueous phase;

[0069] 2. Mix the white oil for injection and Siben 80 in a volume ratio of 94:6, sterilize by high pressure at 121°C for 15 minutes, and cool to room temperature to obtain an oil phase.

[0070] 3. Mix the water phase and the oil phase in a volume ratio of 1:3 and emulsify at high speed to obtain an oil emulsion vaccine.

[0071] Example 5 Immune efficacy test of H3 subtype avian influenza broad-spectrum subunit vaccine.

[0072] 150 21-day-old SPF chickens were divided into five groups, 30 each. The first group (rH3HA group) received a subcutaneous injection of a broad-spectrum subunit vaccine for the H3 subtype avian influenza (prepared in Example 4) at 0.3 mL per chicken; the second group (AH12 group) received a subcutaneous injection of an inactivated vaccine for the H3 subtype avian influenza (AH12 strain) at 0.3 mL per chicken; the third group (ZJ07 group) received a subcutaneous injection of an inactivated vaccine for the H3 subtype avian influenza (ZJ07 strain) at 0.3 mL per chicken; the fourth group (JS13 group) received a subcutaneous injection of an inactivated vaccine for the H3 subtype avian influenza (JS13 strain) at 0.3 mL per chicken; and the fifth group served as a blank control group. Peripheral blood was collected from the test chickens 14, 21, and 28 days after immunization, and serum was separated. Serum antibody titers were measured using rH3HA and H3N8 JS13 as detection antigens, respectively. The results showed that serum antibody HI titers could be detected in all groups of experimental chickens 14 days after immunization, and the serum antibody HI titers continued to rise on days 14, 21, and 28. The recombinant protein immunization group reacted well with all the tested antigens. The antibody levels of the recombinant protein immunization group and the JS13 strain whole virus inactivated vaccine group were higher than those of the AH12 strain immunization group and the ZJ07 immunization group, as shown in Table 2.

[0073] Table 2. Results of antibody HI titer test in immune efficacy test

[0074]

[0075]

[0076] Example 6 H3 subtype avian influenza broad-spectrum subunit vaccine challenge cross-protection efficacy test.

[0077] 1. Clinical observation

[0078] In Example 5, each group of experimental chickens were challenged with poison 28 days after immunization. Each immune group and blank control group were divided into 3 challenge groups, each with 10 chickens, each with 10 6 EID 50 The AH12 (GenBank: PQ625902.1), ZJ07 (GenBank: PQ625894.1), and JS13 (GenBank: PQ625915.1) strains were inoculated intravenously with 0.2 mL of the virus. The chickens were observed for 14 consecutive days, and their mental state, food intake, and water consumption were recorded. The results showed that SPF chickens in all immunized groups showed no signs of depression or ruffled fur, and their food and water consumption were normal. In the blank control group (referred to as the blank challenge group), their food intake decreased from 4 to 6 days after challenge.

[0079] 2. Detoxification test

[0080] Throat and cloacal swabs were collected from each group of test chickens on days 3, 5, and 7 after challenge. The swabs were stored in 1 mL of pre-chilled sterile PBS containing tetraclonal antibiotics (penicillin, streptomycin, gentamicin, and kanamycin), shaken for 30 seconds, and centrifuged at 12,000 rpm for 15 minutes at 4°C. The supernatant was aseptically aspirated and inoculated into the allantoic cavity of 10-day-old SPF chicken embryos. Three swabs were inoculated per swab sample, with 0.2 mL per swab, and incubated at 37°C for 120 hours. Embryos were illuminated every 24 hours, and dead embryos within 24 hours were discarded and promptly refrigerated at 4°C. All embryos were tested for HA titer using a microhemagglutination (HA) assay; negative results were blindly propagated for one generation.

[0081] The results of the detoxification test are shown in Table 3. After infection with the H3 subtype avian influenza virus AH12 strain, the recombinant protein rH3HA immunization group and the AH12 immunization group did not detoxify; the ZJ07 immunization group and the JS13 immunization group both detoxified to varying degrees; the blank group detoxified on the 3rd, 5th, and 7th days, and the detoxification rate on the 5th and 7th days exceeded 90%.

[0082] After infection with the H3 subtype avian influenza virus ZJ07 strain, the recombinant protein rH3HA immunization group and the ZJ07 immunization group did not excrete toxins; the AH12 immunization group and the JS13 immunization group excreted toxins, and the blank group excreted toxins on the 3rd, 5th and 7th days after infection, and the excretion rate on the 5th and 7th days exceeded 80%.

[0083] After infection with the H3 subtype avian influenza virus JS13 strain, the recombinant protein rH3HA immunization group and the JS13 immunization group did not excrete toxins; the AH12 immunization group and the ZJ07 immunization group all excreted toxins on the 3rd, 5th and 7th days after infection; the blank group all excreted toxins on the 3rd, 5th and 7th days after infection, and the excretion rate on the 5th and 7th days exceeded 90%.

[0084] Table 3. Results of the challenge and protection test for detoxification

[0085]

[0086]

[0087] Sequence Listing

[0088] SEQ ID NO: 1

[0089] ATMKTIIALNYILCLAFGQNLPGKDSSTATLCLGHHSVPNGTIVKTITDDQIEVTNATELVQNSSTGKICNNPHKVLDGRDCTLIDAMLGDPHCDVFQDEKWDLFVERSSAFSNCYPYDVPDYASLRSLIASSGTLDFITESFTWAGVSQNGGSSACKRGPANGFFSRLNWLTKSGSSYPLLNVTMPNNYNFDKLYIWGVHHPSTNQEQTNLYVQASGRVTVSTRRSQQTIVPNIGSRPWVRGQSGRISIYWTIVKPGDVLVINSNGNLIAPRGFFKIRTGRSSIMRSDAPIETCISECITPNGSIPNDKPFQNVNKITYGACPKYVKQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQINGKLNRVIEKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKVDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDIYRDEALNNRFQIRGVELKSGYKDWILWISFAISCLLLCVVLLGFIMWACQKGNIRCNICIGGGGGGSSSSSSSSSRMKQIEDKIEEILSKIYHIGNEIARIKKLVGERGNTNSGGSTTTITNNNSGTNSSSTTYTVKSGDTLWGISQRYGISVAQIQSANNLKSTIIYIGQKLVLTGSASSTNSGGSNNSASTTPTTSVTPAKPTSQTTHHHHHH

[0090] SEQ ID NO:2

[0091]

Claims

1. An rHA-b protein of an H3 subtype avian influenza virus, which is (a1) or (a2) or (a3): (a1) a protein with an amino acid sequence as shown in SEQ ID NO. 1; (a2) a fusion protein obtained by attaching a protein tag to the N-terminus or / and C-terminus of the amino acid sequence shown in SEQ ID NO.1; (a3) A protein having 90% or more identity and the same function as the protein shown in (a1) obtained by substituting and / or deleting and / or adding one or more amino acid residues in the amino acid residue sequence shown in SEQ ID NO.

1.

2. A gene encoding the protein according to claim 1.

3. The gene according to claim 2, wherein: The gene is a DNA molecule of any one of the following (b1)-(b4): (b1) a DNA molecule whose coding region includes the nucleotide sequence shown in SEQ ID NO. 2 in the sequence listing; (b2) the nucleotide sequence is a DNA molecule as shown in SEQ ID NO. 2 in the sequence listing; (b3) a DNA molecule that hybridizes under stringent conditions with the DNA sequence defined in (b1) or (b2) and encodes a protein that can serve as an antigen for a broad-spectrum subunit vaccine for H3 subtype avian influenza; (b4) A DNA molecule that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology to the DNA sequence defined in (b1) or (b2) and encodes a protein that can serve as an antigen for a broad-spectrum subunit vaccine for H3 subtype avian influenza.

4. The biological material containing the gene according to claim 2 or 3, characterized in that: The biological material is an expression cassette, a recombinant vector, a recombinant microorganism or a transgenic cell line.

5. A method for preparing rHA-b protein of H3 subtype avian influenza virus, characterized in that: The rHA-b protein is obtained by culturing a recombinant baculovirus carrying the rHA-b protein encoding gene according to claim 1.

6. The method according to claim 5, characterized in that: The method for constructing the recombinant baculovirus comprises the following steps: S61, add a Kozak sequence to the 5' end of SEQ ID NO: 2, and add a GS Linker and Leu zip sequence to the 3' end; S62, inserting the product obtained in step S61 into the baculovirus transfer vector pFastBac1 to obtain a recombinant baculovirus transfer plasmid; S63, transforming the recombinant baculovirus transfer plasmid into Escherichia coli DH10Bac competent cells to obtain a recombinant baculovirus shuttle plasmid; S64. Transfect the recombinant baculovirus shuttle plasmid into insect cells Sf9 to obtain a recombinant baculovirus expressing the rHA-b protein.

7. Use of the rHA-b protein of claim 1, the gene of claim 2 or 3, or the biological material of claim 4 in the preparation of a broad-spectrum subunit vaccine for H3 subtype avian influenza.

8. A broad-spectrum subunit vaccine for H3 subtype avian influenza, characterized by: The vaccine uses the inactivated recombinant protein rH3HA-b of the rHA-b protein described in claim 1 as an antigen.

9. The method for preparing the broad-spectrum subunit vaccine of H3 subtype avian influenza according to claim 8, characterized in that: The method comprises the following steps: S91, taking the inactivated recombinant protein rH3HA-b as an antigen, the antigen and sterilized Tween 80 were mixed at a volume ratio of 96:4, and emulsified to obtain an aqueous phase; S92, mixing white oil for injection and Siben 80 in a volume ratio of 94:6, sterilizing by high pressure, and cooling to room temperature to obtain an oil phase; S93. Mix and emulsify the aqueous phase and the oil phase in a volume ratio of 1:3 to obtain a broad-spectrum subunit vaccine of the H3 subtype avian influenza recombinant HA protein.

10. The method according to claim 9, characterized in that: The antigen is obtained by inactivating the recombinant protein rH3HA-b at 37° C. for 24 hours using 0.1% formaldehyde.

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