A recombinant chimeric vaccine of H9 subtype avian influenza and its preparation method

Abstract

The present invention belongs to the field of immunology, and specifically discloses a recombinant chimeric vaccine of H9 subtype avian influenza and a preparation method thereof. While retaining the head structure in the HA skeleton of the main protective antigen of H9 subtype avian influenza, the neck region of the vaccine H9 is replaced with the influenza HA neck region with broad-spectrum immune protection, thereby constructing a recombinant chimeric HA. The recombinant chimeric HA of the present invention is consistent with the natural H9 trimeric protein in structure and molecular weight, and by breaking the advantages of the head structure, the immune response to the conservative stem region is enhanced, and the immune response is concentrated to the stem region with sub-immunity, and the combined action stimulates the production of more protective antibodies. The present invention provides a method for obtaining a high-purity, high-expression, and active H9 chimeric H1 protein, which successfully achieves protection against the invasion of the H9N2 subtype strain of avian influenza virus. It has high expression yield, simple cost, safe process, and can effectively cope with the spread of the H9N2 influenza subtype strain.

Description

Technical Field

[0001] The invention belongs to the field of immunology, and specifically relates to a H9 subtype avian influenza recombinant chimeric vaccine and a preparation method thereof. Background Art

[0002] H9N2 subtype avian influenza virus (Avian influenza virus, AIV) is classified as low pathogenic AIV according to the pathogenicity. However, due to its wide host range, fast transmission speed, long epidemic time, it is very easy to co-infect poultry with other bacteria or viruses, resulting in reduced production performance, which seriously affects the economic value of poultry. H9N2 subtype avian influenza virus (AIV) is widely distributed worldwide and is generally divided into two major lineages, the North American lineage and the Eurasian lineage. Specifically, the Eurasian lineage further multiplies into various virus clusters, represented by BJ / 94-like or Y280-like, G1-like, Y439-like, F / 98-like, etc.

[0003] In China, G1-like, which is mainly spread in quail, has a geographical advantage in the southern region, while BJ / 94-like and F / 98-like, which are prevalent in chickens, are dominant in the northern and eastern regions, respectively. The latest epidemiological studies in recent years also suggest that the S genotype accounts for the highest proportion. Compared with other genotypes, the G57 genotype (equivalent to the S genotype) is more contagious and has been dominant in China since 2010, causing serious damage to poultry farming. AIVs contained in wild birds are often highly pathogenic when transmitted across species. Studies have found that the six internal genes of the H7N9, H10N8 and H5N6 subtype AIVs that have emerged in my country and caused human infections are all from the H9N2 subtype AIV, which also provides a strong environment for the outbreak of HPAIV. Therefore, we need to pay enough attention to the epidemic potential of the H9N2 subtype AIV, develop an effective H9N2 subtype AIV vaccine, effectively prevent and control the epidemic of the virus, and provide protection for our public health security.

[0004] Many countries have monitored H9N2 AIV, especially in China and other Asian countries. Since 2016, most research teams in my country have collected tens of thousands of samples in 37 cities in 23 provinces, municipalities and autonomous regions in China, and identified the strains of HxNy subtypes through next-generation sequencing (NGS), finding that H9N2 is the dominant subtype. Serological survey reports also showed that the positive rate of H9N2 virus antibodies in the general population was 1.3% to 1.4%, and the positive rate in retail poultry workers exceeded 15%, confirming human infection with H9N2 virus, and finding that live poultry markets (LPM) are an important exposure risk factor for human infection with avian influenza virus.

[0005] Combining phylogenetic and antigenic analysis of H9N2 isolates from the latest samples collected from 2013 to 2018, it was found that the recent isolates were mainly clustered in subgroup II and subgroup III, far away from the vaccine strains (mainly in subgroup I). The homology of HA proteins in different antigenic clusters was analyzed to evaluate the amino acid residues that may cause antigenic drift. It was found that 11 amino acid residues were conserved in the vaccine cluster viruses, but mutated in the subgroup II and subgroup III lineage viruses; and there were 10 mutated amino acid residues in the HA protein between subgroup II and subgroup III, which may potentially affect the protective efficacy of the H9N2 AIV inactivated vaccine.

[0006] At present, the whole virus inactivated avian influenza vaccine is the most widely used vaccine in the world, and vaccination is also the main measure to prevent avian influenza. Shao Huabin et al. (Shao Huabin, Wen Guoyuan, Luo Ling, et al. Recombinant Newcastle disease heat-resistant vaccine strain expressing truncated HA protein of H9 subtype avian influenza virus and preparation method, 2016.) selected the HA1 region (1-1041nt) with strong immunogenicity in the HA protein and inserted it into the Newcastle disease virus heat-resistant vector, which can specifically enhance the immune protection effect of the vaccine and greatly reduce the dependence on the cold chain system. It can be used to prepare H9 avian influenza and Newcastle disease two-in-one heat-resistant live vaccine. The article also determined the pathogenicity of the recombinant virus, maintaining the low toxicity of the parent strain, and the insertion of exogenous genes did not change the pathogenicity of the recombinant virus. Krammer et al. (Krammer F, et al. Chimeric hemagglutinin influenza virus vaccine constructs elicit broadly protective stalk-specific antibodies. J Virol. 2013 Jun; 87(12): 6542-50.; Krammer F, et al. H3stalk-based chimeric hemagglutinin influenza virus constructs protect mice from H7N9 challenge. J Virol. 2014 Feb; 88(4): 2340-3.) used the H1 stem of influenza A (H1N1) viruses from different epidemic seasons for chimerization, and then used the heads of H5, H6 and H9 influenza viruses and the chimeric stem to construct cHAs vaccines respectively. Studies have shown that mice that were continuously immunized with the vaccine had a 100% survival rate after being attacked by multiple viruses such as H5N1, H6N1 and H7N9, which was significantly higher than that of the control groups, and produced high titers of stem-reactive antibodies; his team successively immunized ferrets with cHA with heads of H9, H5 and H6. After being vaccinated with cHA with heads of H5 and H6, the serum reactivity of ferrets to the pandemic H1N1 virus increased by 4 and 8 times respectively compared with the previous time.

[0007] Common avian influenza vaccines include whole virus inactivated vaccines, attenuated live vaccines, genetically engineered subunit vaccines, genetically recombinant live vector vaccines, nucleic acid vaccines, RNA replicon vaccines, universal influenza vaccines, transgenic plant vaccines, etc. However, farms currently still use inactivated avian influenza vaccines to prevent H9 subtype avian influenza virus infection in poultry. However, due to antigenic drift caused by its immune pressure, H9 subtype avian influenza virus is still mutating and spreading. H9N2 subtype avian influenza virus also actively participates in gene rearrangement in other subtypes of influenza virus, producing new influenza viruses such as H5N2, H6N1, H7N7, H7N9, and H10N8, threatening human health. The H7N9 influenza that broke out in 2013 was the result of mixing in chickens. Its external genes came from H7 subtype influenza virus, while its internal genes came from H9N2 subtype influenza virus.

[0008] In addition, the inactivated vaccine has problems such as large amount of chicken embryos, high premature death rate of chicken embryos, small average single embryo harvest, unstable potency and high cost in the production process. The application of inactivated vaccine cannot distinguish between naturally infected chickens and vaccinated chickens, thus interfering with the monitoring of avian influenza epidemics and epidemiological investigations; attenuated live vaccines may undergo gene rearrangement with other influenza viruses to obtain reassortant viruses with restored virulence, and it has also been found that cold-adapted attenuated live vaccines have the risk of causing disease in immunodeficient people; genetic engineering vaccines have been found to have shortcomings such as short antibody duration and high cost; recombinant live vector vaccines can only last for a short time for chickens that have been immunized, and there are certain restrictions on their use for chickens that have been vaccinated or infected with virus vectors; researchers have found that the vectors of nucleic acid vaccines often carry antibiotic genes, which may bring difficulties to the prevention and treatment of bacterial diseases, and the internal expression efficiency of nucleic acid vaccines is not high. The ideal avian influenza virus vaccine should be highly safe for poultry, be able to distinguish between naturally infected poultry and vaccine-immunized poultry, have good production safety and long duration, etc. At present, no vaccine has all the above characteristics. The design concept of the universal influenza vaccine is to be effective against various influenza subtypes during the avian influenza pandemic stage or the virus transition and mutation stage.

[0009] In addition, continuous vaccination will also lead to antigenic mutation and immune escape to varying degrees. Clinically, the replacement speed of inactivated vaccine strains cannot keep up with the pace of viral antigen mutation, resulting in the continued spread and mutation of antigen escape strains. In this way, the avian influenza virus is not only not effectively controlled, but there is also a trend of continued spread and expansion of the virus.

[0010] Therefore, the development and evaluation of vaccines with cross-immune protection against antigenic variant strains or different subtypes of avian influenza viruses is very necessary for the prevention and control of H9 subtype viruses. Summary of the invention

[0011] The present invention constructs a novel recombinant protein vaccine based on the trimeric mammalian expression protein of the key influenza surface antigen HA. That is, by retaining the head structure in the HA skeleton of the main protective antigen HA of the H9 subtype avian influenza, the neck region of the vaccine H9 is replaced with the influenza HA neck region with broad-spectrum immune protection after structural biology design and transformation, thereby constructing a recombinant chimeric HA. The recombinant chimeric HA is consistent with the natural H9 trimer protein in structure and molecular weight. The chimeric vaccine of the present invention breaks the advantage of the head structure, enhances the immune response to the conservative stem region, concentrates the immune response to the immune sub-dominant stem region, and works together to stimulate the production of more protective antibodies.

[0012] Therefore, the present invention provides a H9 subtype avian influenza recombinant chimeric vaccine, which includes the head region in the H9 subtype avian influenza antigen HA skeleton and the influenza HA neck region with broad-spectrum immune protection. Preferably, positions 1-921 calculated from ATG are the HA head region; positions 922-1548 are the HA neck region, wherein positions 922-957 are the active peptide structure of the neck region.

[0013] The present invention firstly provides a H9 subtype avian influenza recombinant chimeric vaccine antigen protein, which is obtained by replacing the neck region with an influenza HA neck region having broad-spectrum immune protection on the basis of retaining the head structure region in the H9 subtype avian influenza antigen HA skeleton.

[0014] Specifically, the head structure region in the H9 subtype avian influenza antigen HA skeleton is specifically from position 1 to 307 of the protein amino acid sequence; the influenza HA neck region is specifically from position 308 to 516 starting from ATG of the HA protein amino acid sequence.

[0015] Preferably, the amino acid sequence of the head structure region in the H9 subtype avian influenza antigen HA skeleton is as shown in SEQ ID NO:1.

[0016] In addition, preferably, the amino acid sequence of the influenza HA neck region with broad-spectrum immune protection is shown in SEQ ID NO: 2.

[0017] The present invention thus provides nucleic acids encoding the chimeric vaccine antigen proteins.

[0018] Preferably, the nucleotide sequence of the head structure region in the H9 subtype avian influenza antigen HA skeleton is from positions 1 to 921 of the HA nucleotide sequence; and the influenza HA neck region is from positions 922 to 1548 of the HA nucleotide sequence.

[0019] More preferably, the nucleotide sequence of the head structure region in the H9 subtype avian influenza antigen HA skeleton is as shown in SEQ ID NO:3.

[0020] Preferably, the nucleotide sequence of the influenza HA neck region is as shown in SEQ ID NO:4.

[0021] More preferably, the full-length encoding nucleotide sequence of the H9 chimeric H1 protein is as shown in SEQ ID NO:5.

[0022] The present invention also provides an expression vector containing the nucleic acid, preferably a PCAGGS vector.

[0023] The present invention further provides a method for preparing a recombinant chimeric vaccine of H9 subtype avian influenza, which comprises the following steps:

[0024] The first step: the nucleic acid synthesized in full sequence with sticky ends (specifically, the sticky ends are EcoRⅠ and XhoⅠ) and the NA gene nucleic acid fragment (preferably, the two are fused with different purification tag peptides) are respectively connected to the vector (specifically, the starting vector is PCAGGS vector) after double enzyme digestion (corresponding to EcoRⅠ and XhoⅠ) to construct a recombinant expression vector.

[0025] Step 2: Co-transfect the plasmid HA and NA into 293T mammalian cells at a ratio of 1:1 for secretory expression, collect the supernatant, and purify the target HA protein by affinity chromatography based on the purification tag.

[0026] Step 3: Emulsify the HA protein and adjuvant in a ratio (preferably 1:1 by volume) to prepare a recombinant chimeric vaccine.

[0027] Finally, the present invention particularly provides a H9 subtype avian influenza recombinant chimeric vaccine, which comprises the chimeric vaccine antigen protein and optionally further comprises a pharmaceutically acceptable adjuvant.

[0028] The present invention designs a method for obtaining a high-purity, high-expression, and active H9 chimeric H1 protein, and successfully realizes the protection of the H9 chimeric H1 vaccine against the invasion of the H9N2 subtype strain of avian influenza virus. The H9 chimeric H1 protein of the present invention has high expression yield, simple cost, and safe process, and can effectively cope with the spread of the H9N2 subtype strain of avian influenza, providing support for the preparation of an efficient and universal avian influenza subunit vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Typical molecular sieve map and SDS-PAGE gel image of H9 chimeric H1 protein, the arrow points to the protein marker 72KD.

[0030] Figure 2 Results of hemagglutination inhibition tests of H1N1 strain (A) and H3N2 strain (B) on different groups.

[0031] Figure 3A and Figure 3B It is the result of HI antibody test. Figure 3A Experimental groups: 1, serum from the non-immunized group; 2, serum from the positive control group (inactivated avian influenza virus H9 subtype A / Chicken / Hebei / G / 2012 (H9N2 strain)); 3, serum from the experimental group H9 chimeric H1 ISA 71 vaccine group; 4, serum from the single adjuvant control group. Figure 3B The experimental groups were set up as follows: 1. serum from the non-immunized group; 2. serum from the positive control group (commercially available Pleco H9 inactivated vaccine 032101001A); 3. serum from the experimental group H9 chimeric H1 white oil vaccine group;

[0032] Figure 4A and Figure 4B ELISA detects specific antibodies in chicken serum. Figure 4A Experiment C: The immune response of the positive control group (H9+Newcastle disease bivalent vaccine) serum and the experimental group H9 chimeric H1 Freund's vaccine group serum to different influenza HAs; the coating proteins are H1 full-length protein, H5ORI protein, and H9ORI protein. Figure 4B Experiment D: The immune response of the single adjuvant control group serum (Group 1), the positive control (H9+Newcastle disease bivalent vaccine) group serum (Group 2), and the experimental group H9 chimeric H1 Freund's vaccine group serum (Group 3) to the envelope protein H9 chimeric H1 protein, H1 stem protein, and H9ORI protein.

[0033] Figure 5 Blood suppression test results.

[0034] Figure 6 Statistical results of antibody titer. Among them: 1, serum from the single adjuvant control group; 2, serum from the positive control (H9+Newcastle disease bivalent vaccine) group; 3, serum from the experimental group H9 chimeric H1 Freund's vaccine group. DETAILED DESCRIPTION

[0035] The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and substance of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention all fall within the scope of the present invention.

[0036] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0037] Example 1 Construction of HA gene of H9 subtype and NA gene plasmids for assisting HA expression

[0038] The designed HA or NA nucleic acid sequence is sent to the company for full-sequence synthesis of DNA fragments with sticky ends (the sticky ends are the restriction sites of EcoRⅠ and XhoⅠ). The vector is selected as the PCAGGS vector with ampicillin resistance, the vector is double-digested, the fully synthesized DNA fragment is connected to the vector, and the following HA gene and NA gene nucleic acid fragments (both fused with different purification tag peptides) with full-sequence synthesis and sticky ends (specifically, the sticky ends are EcoRⅠ and XhoⅠ) are connected to the PCAGGS vector after double digestion (corresponding to EcoRⅠ and XhoⅠ) to construct a recombinant expression vector.

[0039] The HA gene sequence structure is: EcoRI restriction site + Kozak sequence + signal peptide sequence + HA head region sequence + HA neck region sequence + thrombin restriction site sequence + trimer tag sequence + histidine tag sequence + XhoI restriction site.

[0040] EcoRI restriction site: GAATTC;

[0041] Kozak sequence: GCCACC;

[0042] Signal peptide amino acid sequence: METVSLITILLVVTVSNA

[0043] Signal peptide nucleotide sequence: ATGGAGACAGTATCACTAATAACTATACTACTAGTAGTAACAGTAAGCAATGCA;

[0044] Thrombin cleavage site amino acid sequence: LVPRGS

[0045] Thrombin cleavage site nucleotide sequence: CTGGTGCCAAGAGGCTCT;

[0046] Trimer tag sequence: CCTGGCAGCGGCTATATTCCTGAGGCTCCCAGAGATGGCCAGGCCTACGTTAGAAAGGATGGCGAGTGGGTGCTGCTGAGCACCTTTCTGGGA;

[0047] His-tag amino acid sequence: HHHHHH;

[0048] His-tag nucleotide sequence: CACCACCACCATCACCAC;

[0049] XhoI restriction site: CTCGAG.

[0050] Amino acid sequence of the HA head region (SEQ ID NO: 1): METVSLITILLVVTVSNADKICIGYQSTNSTETVDTLTE NNVPVTHAKELLHTEHNGMLCATSLGHPLILDTCTIEGLIYGNPSCDLLLGGREWSYIVERPSAVNGLCYPGNVENLEELRSLFSSARSYQRIQIFPDTIWNVSYSGTSKACSDSFYRSMRWLTQKNNAYPIQDAQYTNNQEKNILFMWGINHPPTDTAQTNLYTRTDTTTSVATEEINRTFKPLIGPRPLVNGLQGRIDYYWSVLKPGQTLRIRSNGNLIAPWYGHILSGESHGRILKTDLKRGSCTVQCQTEKGGLNTTLPFQNVS

[0051] HA head region nucleotide sequence (SEQ ID NO: 3): ATGGAGACAGTATCACTAATAACTATACTACTAGTAGTAACAGTAAGCAATGCAGATAAAATCTGCATCGGCTATCAATCAACAAACTCCACAGAAACTGTAGACACACTAACAGAAAACAACGTCCCTGTGACACATGCCAAAGAATTGCTCCACACAGAGCATAATGGGATGCTGTGTGCAACAAGCTTGGGACACCCTCTTATTCTAGACACCTGTACCATTGAAGGACTAATCTATGGCAATCCTTCTTGTGATCTATTGTTGGGAGGAAGAGAATGGTCCTATATCGTCGAGAGACCATCAGCTGTTAACGGATTGTGTTATCCCGGGAATGTAGAAAATCTAGAAGAGCTAAGGTCACTTTTTAGTTCTGCTAGGTCTTATCAAAGGATCCAGATTTTCCCAGACACAATCTGGAATGTGTCTTACAGTGGGACAAGCAAAGCATGTTCAGATTCATTCTACAGAAGCATGAGATGGTTGACTCAAAAGAACAATGCTTACCCTATTCAAGACGCCCAATACACAAATAATCAAGAAAAGAACATTCTTTTCATGTGGGGCATAAATCACCCACCCACCGATACTGCGCAGACAAATCTGTACACAAGAACCGACACAACAACGAGTGTGGCAACAGAAGAAATAAATAGGACCTTCAAACCATTGATAGGACCAAGGCCTCTTGTCAACGGTTTGCAGGGAAGAATTGATTATTATTGGTCGGTATTGAAACCGGGTCAAACACTGCGAATAAGATCTAATGGGAATCTAATAGCTCCATGGTATGGACACATTCTTTCAGGAGAGAGCCACGGAAGAATCCTGAAGACTGATTTAAAAAGGGGTAGCTGCACAGTGCAATGTCAGACAGAAAAAGGTGGATTAAACACAACATTGCCATTCCAAAACGTAAGT

[0052] HA neck region amino acid sequence (SEQ ID NO: 2):

[0053] KYAIGDCPKYVKQNTLKLATGLRNIPSIQSRGLFGAIAGFTEGGWTGMVDGLYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQYTAVGKEFNKSERRMENLNKKVDDGKIDLWSYNAELLVALENQHTIDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKR

[0054] Nucleotide sequence of the HA neck region (SEQ ID NO: 4):

[0055] AAGTATGCCATCGGCGACTGCCCCAAATACGTGAAGCAGAATACCCTGAAGCTGGCCACCGGCCTGAGAAACATCCCCAGCATCCAGAGCAGAGGCCTGTTCGGAGCCATTGCCGGCTTTACTGAAGGCGGCTGGACAGGCATGGTGGATGGCCTGTATGGCTATCACCACCAGAATGAGCAAGGCAGCGGATACGCCGCTGACCAGAAGTCTACCCAGAACGCTATCAATGGCATCACCAACAAAGTGAACTCCGTGATCGAGAAGATGAACACCCAGTACACCGCCGTGGGCAAAGAGTTCAACAAGAGCGAGCGGCGGATGGAAAACCTGAACAAGAAGGTGGACGACGGCAAGATCGACCTGTGGTCCTACAATGCCGAACTGCTGGTGGCCCTGGAAAACCAGCACACCATCGACTTCCACGACAGCAACGTGAAGAACCTGTACGAGAAAGTGAAGTCCCAGCTGAAGAACAACGCCAAAGAGATCGGCAACGGCTGCTTCGAGTTCTACCACAAGTGCAACGACGAGTGCATGGAAAGCGTGAAGAATGGCACCTACGACTACCCCAAGTACAGCGAGGAATCCAAGCTGAACCGCGAGAAAATCGACGGCGTGAAGAGA。

[0056] The full-length coding full-length nucleotide sequence of the H9 chimeric H1 protein (SEQ ID NO: 5) is:

[0057] GAATTCGCCACCATGGAGACAGTATCACTAATAACTATACTACTAGTAGTAACAGTAAGCAATGCA

[0058] GATAAAATCTGCATCGGCTATCAATCAACAAACTCCACAGAAACTGTAGACACACTAACAGAAAAC

[0059] AACGTCCCTGTGACACATGCCAAAGAATTGCTCCACACAGAGCATAATGGGATGCTGTGTGCAACAA

[0060] GCTTGGGACACCCTCTTATTCTAGACACCTGTACCATTGAAGGACTAATCTATGGCAATCCTTCTTGTG

[0061] ATCTATTGTTGGGAGGAAGAGAATGGTCCTATATCGTCGAGAGACCATCAGCTGTTAACGGATTGTGTT

[0062] ATCCCGGGAATGTAGAAAATCTAGAAGAGCTAAGGTCACTTTTTAGTTCTGCTAGGTCTTATCAAAGG

[0063] ATCCAGATTTTCCCAGACACAATCTGGAATGTGTCTTACAGTGGGACAAGCAAAGCATGTTCAGATTC

[0064] ATTCTACAGAAGCATGAGATGGTTGACTCAAAAGAACAATGCTTACCCTATTCAAGACGCCCAATACA

[0065] CAAATAATCAAGAAAAGAACATTCTTTTCATGTGGGGCATAAATCACCCACCCACCGATACTGCGCAG

[0066] ACAAATCTGTACACAAGAACCGACACAACAACGAGTGTGGCAACAGAAGAAATAAATAGGACCTTC

[0067] AAACCATTGATAGGACCAAGGCCTCTTGTCAACGGTTTGCAGGGAAGAATTGATTATTATTGGTCGGT

[0068] ATTGAAACCGGGTCAAACACTGCGAATAAGATCTAATGGGAATCTAATAGCTCCATGGTATGGACACA

[0069] TTCTTTCAGGAGAGAGCCACGGAAGAATCCTGAAGACTGATTTAAAAAGGGGTAGCTGCACAGTGCA

[0070] ATGTCAGACAGAAAAAGGTGGATTAAACACAACATTGCCATTCCAAAACGTAAGTAAGTATGCCATCG

[0071] GCGACTGCCCCAAATACGTGAAGCAGAATACCCTGAAGCTGGCCACCGGCCTGAGAAACATCCCCAG

[0072] CATCCAGAGCAGAGGCCTGTTCGGAGCCATTGCCGGCTTTACTGAAGGCGGCTGGACAGGCATGGTG

[0073] GATGGCCTGTATGGCTATCACCACCAGAATGAGCAAGGCAGCGGATACGCCGCTGACCAGAAGTCTA

[0074] CCCAGAACGCTATCAATGGCATCACCAACAAAGTGAACTCCGTGATCGAGAAGATGAACACCCAGTA

[0075] CACCGCCGTGGGCAAAGAGTTCAACAAGAGCGAGCGGCGGATGGAAAACCTGAACAAGAAGGTGG

[0076] ACGACGGCAAGATCGACCTGTGGTCCTACAATGCCGAACTGCTGGTGGCCCTGGAAAACCAGCACAC

[0077] CATCGACTTCCACGACAGCAACGTGAAGAACCTGTACGAGAAAGTGAAGTCCCAGCTGAAGAACAA

[0078] CGCCAAAGAGATCGGCAACGGCTGCTTCGAGTTCTACCACAAGTGCAACGACGAGTGCATGGAAAG

[0079] CGTGAAGAATGGCACCTACGACTACCCCAAGTACAGCGAGGAATCCAAGCTGAACCGCGAGAAAATC

[0080] GACGGCGTGAAGAGACTGGTGCCCAGAGGCTCTCCTGGCAGCGGCTATATTCCTGAGGCTCCCAGAG

[0081] ATGGCCAGCCTACGTTAGAAAGGATGGCGAGTGGGTGCTGCTGAGCACCTTTCTGGGACACCACCACCATCACCACTGACTCGAG.

[0082] The sequence structure of the NA gene is: EcoRI restriction site + signal peptide sequence + Flag tag sequence + tetramer tag sequence + thrombin restriction site sequence + 09NA sequence + XhoI restriction site.

[0083] EcoRI restriction site: GAATTC;

[0084] Signal peptide amino acid sequence: MGAGATGRAMDGPRLLLLLLLGVSLGGA;

[0085] Signal peptide nucleotide sequence: ATGGGGGCAGGTGCCACCGGCCGCGCCATGGACGGGCCGCGCCTGCTGCTGTTGCTGCTTCTGGGGGTGTCCCTTGGAGGTGCC;

[0086] Flag tag amino acid sequence: DYKDDDDK;

[0087] Flag tag nucleotide sequence: GATTATAAGGATGATGATGATAAG;

[0088] Tetramer tag amino acid sequence: SSSDYSDLQRVKQELLEEVKKELQKVKEEIIEAFVQELRKRGS;

[0089] Tetramer tag nucleotide sequence: AGCTCCAGTGATTACTCGGACCTACAGAGGGTGAAACAGGAGCTTCTGGAAGAGGTGAAGAAGGAATTGCAGAAAGTGAAAGAGGAAATCATTGAAGCCTTCGTCCAGGAGCTGAGGAAGCGGGGTTCT;

[0090] Thrombin cleavage site amino acid sequence: LVPRGS;

[0091] Thrombin cleavage site nucleotide sequence: CTGGTACCACGAGGTAGT;

[0092] 09NA amino acid sequence (SEQ ID NO: 6): PSRSVKLAGNSSLCPVSGWAIYSKDNSVRIGSKGDVFVIREPFISCSPLECRTFFLTQGALLNDKHSNGTIKDRSPYRTLMSCPIGEVPSPYNSRFESVAWSASACHDGINWLTIGISGPDNGAVAVLKYNGIITDTIKSWRNNILRTQESECACVNGSCFTVMTDGPSNGQASYKIFRIEKGKIVKSVEMNA PNYHYEECSCYPDSSEITCVCRDNWHGSNRPWVSFNQNLEYQIGYICSGIFGDNPRPNDKTGSCGPVSSNGANGVKGFSFKYGNGVWIGRTKSISSRNGFEMIWDPNGWTGTDNNFSIKQDIVGINEWSGYSGSFVQHPELTGLDCIRPCFWVELIRGRPKENTIWTSGSSISFCGVNSDTVGWSWPDGAELPFTIDK;

[0093]

[0094] XhoI restriction site: CTCGAG.

[0095] Example 2 Expression and purification of H9 subtype HA protein

[0096] Transform the recombinant expression vector plasmid into Top10 competent cells, draw an appropriate amount of bacterial solution and apply it on a solid LB plate (LB plate formula: 1% NaCl, 1% tryptone, 0.5% yeast extract, 1.5% agar powder; ampicillin is used at 1:1000) with ampicillin resistance (ampicillin stock concentration: 100 mg / ml) by streaking, and culture overnight at 37°C. Pick a single clone, shake 5ml of bacterial solution (ampicillin resistance), and then shake 300ml overnight, and use the endotoxin-free plasmid extraction kit (TIANGEN, DP117) to obtain influenza virus antigen plasmid.

[0097] 293T cells were cultured in a 37°C, 5% CO2 incubator with DMEM containing 10% FBS. When the confluence of 293T cells reached about 70%, the influenza virus antigen plasmid (HA: NA = 20ug: 20ug / plate) was transfected into the 293T cells using PEI transfection reagent. After 4-6 hours of transfection, the cell culture supernatant was collected after the culture was replaced with serum-free DMEM for 3 days. Serum-free DMEM was added to continue the culture for 4 days, and the cell culture supernatant was collected. When HA is expressed in mammalian cells, sialic acid will adhere to it, resulting in the inability to bind to the receptor, so the expression of neuraminidase NA was also performed. NA only assists the expression of HA protein. The 09NA gene was selected and a FLAG tag was added for easy detection. Since the purification tags of HA and NA are different, we chose an affinity column with a His tag to purify HA protein, so that the protein we finally purified was only HA protein. We used western blot to verify that the amount of HA protein obtained after HA+NA co-transfection was more than that obtained after HA single transfection. In order to purify a large amount of HA protein, the HA designed later was co-transfected with NA for secretory expression.

[0098] The collected cell culture supernatant was filtered through a 0.22 μm filter membrane and then TMExcel (GE) was used for overnight binding at 4°C, and protein elution buffer A (20 mM Tris, 150 mM NaCl, pH 8.0) and buffer B (20 mM Tris, 150 mM NaCl, pH 8.0, 1 M imidazole) were prepared. The His column was then washed with buffer A to remove non-specifically bound proteins, and then 2% buffer B (20mM Tris, 150mM NaCl, pH 8.0, 20mM imidazole) was used to remove impurities. Finally, the target protein was eluted from the His column with 30% B solution (20mM Tris, 150mM NaCl, pH 8.0, 300mM imidazole), and the solution was replaced with buffer A (20mM Tris, 150mM NaCl, pH 8.0) in a 30KD cutoff protein concentrator to remove the imidazole concentration in the protein solution. Finally, the protein solution was concentrated to 0.5ml, and thrombin (1mg protein plus 5ul thrombin) was added for enzyme digestion at 4°C overnight. The protein solution after enzyme digestion was further purified by molecular sieve, using AKTA-purifier (GE) and Column Hiload 16 / 60superdex 200PG molecular sieve (GE). The molecular sieve was balanced with buffer A (20mM Tris, 150mM NaCl, pH 8.0), and 1ml loop was loaded. The ultraviolet absorption value at 280nm was monitored at the same time, and the target protein was collected and the protein purity was identified by SDS-PAGE. The typical molecular sieve map and SDS-PAGE analysis of the target protein are shown in Figure 1. Figure 1 shown.

[0099] The protein peak position in Column Hiload 16 / 60superdex 200PG molecular sieve is 62.5ml. Since a trimer tag was added after the thrombin cleavage site (LVPR↓GS, the arrow indicates the amino acid that can recognize and cleave this site) during the construction, the main peak of the protein is in the form of a trimer. The trimer tag falls off from HA after thrombin cleavage, and the monomer size is about 70KD. The lanes of the SDS-PAGE gel map correspond to the trimer and monomer of the H9 chimeric H1 protein in the molecular sieve map.

[0100] Example 3: Vaccine preparation

[0101] The purified H9 chimeric H1 protein is mixed with different adjuvants to prepare a vaccine, and the emulsification is carried out in a volume ratio of 1:1 until the bath water does not dissolve for animal immunization. The H1 stem protein is the neck region protein of the influenza H1 subtype, which has a broad-spectrum immune protection effect. On this basis, we designed the H9 chimeric H1 protein, which has an immune response effect on different influenza HAs, as shown in Example 6. The specific vaccine preparation method is shown in Example 3; the functional verification of the influenza HA neck region (H1stem) with broad-spectrum immune protection is reflected by the immunization mouse experiment, as shown in Example 4; the prepared H9 chimeric H1 vaccine immunization SPF chicken test is shown in Example 5; the prepared H9 chimeric H1 vaccine immunization Hy-Line White laying hens test is shown in Example 6.

[0102] 1. Preparation of H1stem vaccine

[0103] The concentration of H1 stem protein was 10 mg / ml, PBS was used as diluent, and the immune adjuvant was MF59. The H1 stem protein of the test group was mixed with an equal volume of MF59 adjuvant, shaken until completely emulsified and not dissolved in the bath water for animal immunization.

[0104] The amino acid sequence of the H1stem vaccine (SEQ ID NO: 8), in which positions 194-262 are conserved peptides: MYRMQLLSCIALSLALVTNSTYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSKQSQGLFGAIAGFTEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQYTAIGCEYNKSERCMKQIEDKIEEIESKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQIEGRHHHHHH

[0105] The nucleotide sequence of the H1stem vaccine (SEQ ID NO: 9), wherein positions 580 to 786 are conserved peptide segments:

[0106] ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACGAATTCAACCTACGCCGACACCATTTGCATCGGCTACCACGCCAACAACAGCACCGACACCGTGGACACCGTGCTGGAGAAGAACGTGACCGTGACCCACAGCGTGAATCTGCTGGAGAACGGAGGAGGCGGCAAATACGTCTGCAGCGCCAAACTGAGGATGGTGACCGGACTGAGGAACAAGCCCAGCAAGCAGAGCCAGGGACTGTTCGGAGCCATTGCCGGATTCACCGAGGGAGGTTGGACAGGAATGGTGGACGGTTGGTACGGCTACCACCACCAGAACGAGCAGGGAAGCGGATACGCCGCCGATCAGAAAAGCACCCAGAACGCCATCAACGGCATCACCAACAAGGTCAACAGCGTGATCGAGAAGATGAACACCCAGTACACCGCCATCGGTTGCGAGTACAACAAGAGCGAGCGCTGCATGAAGCAGATCGAGGACAAGATCGAGGAGATCGAGAGCAAGATCTGGTGCTACAACGCCGAACTGCTGGTGCTGCTGGAGAACGAGAGGACCCTGGACTTCCACGACAGCAACGTGAAGAACCTGTACGAGAAGGTCAAGAGCCAGCTGAAGAACAACGCCAAGGAGATCGGCAACGGCTGCTTCGAGTTCTACCACAAGTGCAACGACGAGTGCATGGAGAGCGTGAAGAACGGCACCTACGACTACCCCAAGTACAGCGAGGAGAGCAAGCTGAACCGGGAGAAGATCGACGGCGTGAAGCTGGAGAGCATGGGCGTGTACCAGATCGAGGGCAGACATCACCACCACCACCATCATTAG。

[0107] 2. Preparation of H9 chimeric H1 ISA71 vaccine (adjuvant is Montanide TM ISA 71VG)

[0108] The concentration of H9 chimeric H1 protein is 10 mg / ml, PBS is used as the diluent, and the immune adjuvant is MontanideTM ISA 71VG.

[0109] The vaccine is prepared by adding the adjuvant to the aqueous antigen medium in a mass ratio of 7:3. At room temperature or below room temperature, the aqueous antigen medium is added to the Montanide TM ISA 71VG was mixed under vigorous stirring to obtain a stable preparation for animal inoculation, labeled, and stored at 2-8°C.

[0110] 3. Preparation of H9 chimeric H1 white oil vaccine (adjuvant is mineral white oil)

[0111] The concentration of H9 chimeric H1 protein is 10mg / ml, PBS is the diluent, and the immune adjuvant is mineral white oil. Preparation of oil phase: Take 94 parts of white oil for injection, 2 parts of aluminum stearate, heat to 80℃, then add 6 parts of Siben-80, maintain for 30min when the temperature reaches 116℃, and set aside after cooling. Preparation of water phase: Take 4 parts of Tween-80, sterilize, cool, and add to 96 parts of inactivated antigen solution, stirring while adding until Tween-80 is completely dissolved. Emulsification: Take 2 parts of oil phase, start the motor to stir slowly, then slowly add 1 part of water phase, and emulsify at 2800-3200r / min for 30min after adding. Quantitative packaging, cover and seal, label, and store at 2-8℃.

[0112] 4. Preparation of H9 chimeric H1 Freund's vaccine (the adjuvant is Freund's adjuvant)

[0113] The concentration of H9 chimeric H1 protein was 10 mg / ml, PBS was used as diluent, and the immune adjuvant was Freund's adjuvant. The H9 chimeric H1 protein was completely emulsified with an equal volume of Freund's complete adjuvant until the bath water did not dissolve for animal vaccination. Freund's complete adjuvant was used for the first immunization, and Freund's incomplete adjuvant was used for the second immunization.

[0114] Example 4 Animal Experiment 1: Functional Verification of Influenza HA Neck Region (H1 Stem) with Broad Spectrum Immune Protection

[0115] 1. Experimental animals: 4-6 week old BALB / c female mice, 6 mice in each group, immunized by intramuscular injection.

[0116] 2. Group setting: ① negative control (adjuvant MF59); ② positive control (influenza virus split vaccine TIV); ③ experimental group H1stem vaccine.

[0117] 3. Test steps:

[0118] 3.1 Animal Immunization:

[0119] The vaccine preparation process refers to Example 3, 1. Preparation of H1 stem vaccine. The positive group immunization dose was 100 μL per animal; the test group immunization dose was 20 μg / 100 μL per animal, the protein was diluted with PBS, each group had 6 animals, immunized 3 times, and the immunization interval was 14 days.

[0120] 3.2 Serum separation:

[0121] Bleeding was taken from the eyeballs of mice in the above immunization groups to prepare serum, which was treated with RED. For every 1 volume of serum, 4 volumes of RDE were added, incubated in a 37°C water bath for 18 h, and inactivated at 56°C for 30 min for subsequent experiments.

[0122] 3.3 TCID analysis of H1N1 and H3N2 strains 50 Determination

[0123] a, Dilute the virus stock solution using 10-fold serial dilution.

[0124] b. Prepare a 96-well plate 18-24 hours in advance and add 100 μL (2×10 5 The cells were cultured in a 37°C, 5% CO2 incubator.

[0125] c. Virus inoculation: Wash the cells once with PBS, 150 μL per well, then add the diluted virus solution in the first step to a 96-well plate, inoculate one vertical row for each dilution, 100 μL per well, set dilution controls in columns 11 and 12, and culture in a 37°C incubator.

[0126] d. Observe and record the results daily. After 72 hours, calculate the results according to the Reed-Muench method.

[0127] 3.4 Neutralization experiment:

[0128] a. Virus dilution: dilute to 200 TCID using virus culture medium 50 / 50μL.

[0129] b, serum negative control (only cell maintenance fluid was added)

[0130] Serum dilution: The initial dilution of mouse serum was 1:40, and the neutralization test was performed at a 2-fold dilution ratio.

[0131] c. Virus incubation: dilute the virus (200 TCID 50 )50 μl was added to 50 μl of serum, mixed evenly, and incubated at 37°C for 1 h.

[0132] d. MDCK cell adsorption: MDCK cells were washed once with 150 μL of PBS and then discarded. 100 μL of virus-serum mixture was added to each well. Three wells were placed in parallel in a cell culture incubator and cultured for 72 h.

[0133] e. The results of the blood suppression experiment were observed and calculated. The results are as follows Figure 2 shown.

[0134] The results showed that the positive vaccine TIV had a complete neutralizing effect on influenza virus H1N1 at a dilution of 1:40, and had a complete neutralizing effect on influenza virus H3N2 at a dilution of 1:160, while the sera of the control group MF59 group could not prevent the destruction of red blood cells by influenza viruses H1N1 and H3N2. The sera of the H1stem group had a complete neutralizing effect on H1N1 and H3N2 at a dilution of 1:40, and when the serum was diluted to 1:1280, some blood cells remained intact, indicating that antibodies at high dilutions of serum can still prevent the destruction of red blood cell membranes by influenza strains H1N1 and H3N2, indicating that H1stem can produce specific antibodies against influenza viruses H1N1 and H3N2, providing reference data for the research and development of universal vaccines. Example 5 Animal Test 2: Immune protection test of H9 chimeric H1 vaccine on SPF chickens

[0135] 1. Experimental animals: 3-week-old SPF chickens, immunized by subcutaneous injection in the neck.

[0136] 2.A experimental group settings: 1, non-immunized group; 2, positive control group (inactivated avian influenza virus H9 subtype A / Chicken / Hebei / G / 2012 (H9N2 strain)); 3, experimental group H9 chimeric H1ISA71 vaccine group; 4, single adjuvant control group; 10 animals in each group; B experimental group settings: 1, non-immunized group; 2, positive control group (commercialized Pleco H9 inactivated vaccine 032101001A); 3, experimental group H9 chimeric H1 white oil vaccine group.

[0137] 3. Test steps:

[0138] 3.1 Animal Immunization:

[0139] A test: The vaccine preparation process is detailed in Example 3, 2. Preparation of H9 chimeric H1ISA71 vaccine (adjuvant is Montanide TM ISA 71VG). The immunization dose of the control group was used according to the specifications; the injection dose of each animal in the experimental group was 20ug / bird, the number of immunizations was 10 birds per group, and the immunizations were repeated twice, with an interval of 14 days between each immunization. Blood was collected from the heart 3 weeks after the second immunization, and serum was separated for subsequent experiments.

[0140] B test: The vaccine preparation process is detailed in Example 3. Preparation of H9 chimeric H1 white oil vaccine (adjuvant is mineral white oil). The immunization dose of the control group is used according to the specifications; the injection dose of each animal in the test group is 30ug / bird, the number of immunizations is 10 birds per group, the immunization method is subcutaneous injection in the neck, immunization is 2 times, each immunization interval is 3 weeks, and heart blood is collected 3 weeks after the second immunization, and serum is separated for subsequent experiments.

[0141] 3.2 Serum separation:

[0142] Blood was collected from the heart 3 weeks after the second immunization, centrifuged at low temperature, and serum was collected and stored in aliquots at -80°C.

[0143] 3.3HI antibody titer detection:

[0144] According to the standard operation specified in the national standard GB / T18936-2020, 25 μL of PBS was added to wells 1-12 of the 96-well microhemagglutination plate, and 25 μL of serum 3 weeks after immunization was added to the first well, and diluted to the 10th well with a 25 μL pipette, and then 25 μL of 4HAU (four-unit virus solution) was added, and PBS and hemagglutinin controls were set; incubate at 37°C for 10 minutes, add 25 μL of 1% SPF chicken red blood cells to each space, mix well and incubate at room temperature for 30 minutes, and then calculate the SPF chicken HI antibody titer of the immunized group and the non-immunized group.

[0145] The results are as follows Figure 3A The test results shown in A: the HI antibody detection titer of the non-immunized group (Group 1) and the single adjuvant control group (Group 4) was 0, the HI antibody titer of the positive vaccine group (Group 2) was about 11log2, and the HI antibody titer of the experimental group H9 chimeric H1 ISA 71 vaccine group (Group 3) was also about 11log2. Compared with the positive vaccine group (Group 2), the antibody titer mean of the chimeric vaccine of the present invention may be slightly higher, indicating that the chimeric vaccine designed by the present invention can effectively induce more antibodies to neutralize the H9N2 strain like the commercial vaccine. Figure 3B The results of test B are as follows: the HI antibody titer after the second immunization is higher than that after the first immunization. The titer of the commercial H9 vaccine (Group 2) is very high after both immunizations. The HI antibody titer of the H9 chimeric H1 white oil vaccine (Group 3) increased significantly after the second immunization, and there was no significant difference compared with the commercial H9 vaccine. Both can neutralize the infection of the H9N2 subtype avian influenza virus very well.

[0146] 3.4 Experimental animals challenged with viruses after immunization

[0147] The strain is: H9 subtype WD strain. SPF chickens were challenged with the virus after one vaccination. There were 5 chickens in each of the commercial H9 vaccine control group, the non-immunized group and the experimental group (H9 chimeric H1 white oil vaccine). Each SPF chicken was intravenously injected with a 1:10 diluted avian influenza H9 subtype strain (0.5 ml / chicken). On the 5th day after the challenge, the cloaca and throat swabs of each chicken were collected for virus isolation, and the number of virus isolation positives in the immunized group and the non-immunized group was compared.

[0148] The cloaca and tracheal swabs of the same chicken were mixed as one sample. Each sample was inoculated into 5 SPF chicken embryos aged 9 to 11 days through the allantoic cavity, with 0.2 ml per embryo. After incubation and observation for 5 days, the HA titer of the chicken embryo fluid was determined embryo by embryo. As long as the HA titer of the embryo fluid of one of the 5 chicken embryos inoculated with each sample is ≥1:16 (micro-dosage method), it can be judged as positive for virus isolation. For samples with negative virus isolation, they should be blindly transmitted once before judgment. There should be at least 4 chickens with negative virus isolation in the immunization group, and at least 4 chickens with positive virus isolation in the control group.

[0149] The results showed that after the H9 subtype WD strain was challenged according to the regulations, both the Pleco vaccine and the H9 chimeric H1 white oil vaccine groups could provide 100% protection to the immunized chickens, and the challenged but unimmunized group met the requirements.

[0150] Table 1: Results of the immunized and non-immunized groups after challenge

[0151]

[0152]

[0153] Example 6 Animal Experiment 3: Immune protection test of H9 chimeric H1 vaccine on Hy-Line white laying hens

[0154] 1. Experimental animals: 7-day-old Hy-Line white laying hens, immunized by subcutaneous injection in the neck.

[0155] 2. Group settings: 1, single adjuvant control group; 2, positive control (H9 + Newcastle disease bivalent vaccine) group; 3, experimental group H9 chimeric H1 Freund's vaccine group; 7 mice in each group.

[0156] 3. Experimental steps:

[0157] 3.1 Animal immunization:

[0158] The process of preparing the vaccine is detailed in Example 3, 4. Preparation of H9 chimeric H1 Freund's vaccine (the adjuvant is Freund's adjuvant). The single adjuvant control group immunized 200ul per animal; the experimental group immunized 60ug / 200ul / feather per animal, the protein was diluted with PBS, 7 doses per group, and the immunization method was subcutaneous injection in the neck, 2 immunizations, each immunization interval was 2 weeks, and heart blood was collected 3 weeks after the second immunization, and serum was separated for subsequent experiments.

[0159] 3.2 Serum separation:

[0160] Blood was collected from the heart 3 weeks after the second immunization, centrifuged at low temperature, and serum was collected and stored in aliquots at -80°C.

[0161] 3.3ELISA detection of specific antibodies in laying hen serum:

[0162] a, 200 ng of protein per well was coated at 4°C overnight, and the coated proteins were H1 full-length protein, H1 stem protein, H5ORI protein, H9ORI protein, and H9 chimeric H1 protein;

[0163] b, Washing: The next day, wash the coated plate with PBST containing 0.05% Tween-20 for 4 times, 5 min each time;

[0164] c, blocking: add 200ul of blocking solution (5% skim milk powder, prepared with PBST) and block at room temperature for 3h;

[0165] d, washing: wash the plate to be tested with PBST for 4 times, 5 min each time;

[0166] e, primary antibody incubation: dilute the serum to be tested with PBST (1:50 and 1:100), add 100ul of the serum dilution to be tested to each well, and incubate at 37°C for 1h; PBST was used as a negative control;

[0167] f, washing: rinse the coated plate with PBST, wash 5 times, 5 min each time;

[0168] g, secondary antibody incubation: HRP-labeled goat anti-chicken secondary antibody was diluted with PBST (1:30000), 100ul was added to each well, and incubated at 37°C for 1h;

[0169] h, washing: rinse the coated plate with PBST, wash 5 times, 5 min each time;

[0170] i. Color development: Add TMB substrate buffer for color development, 50ul / well, develop at 37℃ in the dark for 3min, then add ELISA stop solution to terminate the reaction, 50ul / well, at OD 450nm Next reading.

[0171] The results are as follows Figure 4A and Figure 4B As shown: the difference between H9 chimeric H1 protein and H9ORI protein is that the head region is the same but the neck region is different; H1stem is the neck region protein of influenza H1 subtype, and its conserved sequence is the same as that of the neck region of H9 chimeric H1 protein. Figure 4AThe results of the C experiment showed that compared with the control group, the serum of the H9 chimeric H1 Freund's vaccine group produced an immune response to influenza H1, H5, and H9 subtypes, indicating that the H9 chimeric H1 Freund's vaccine stimulated the body to produce antibodies against different influenza HAs in the serum after immunization, providing theoretical support for the design of a universal influenza vaccine. Figure 4B The results of the D experiment show that when the coating protein is H1stem, the H9 chimeric H1 Freund's vaccine group serum (Group 3) can stimulate more neck area antibodies than the positive control group serum (H9+Newcastle disease bivalent vaccine) (Group 2), breaking the traditional advantage of head area binding antibodies; the comparison of the H9 chimeric H1 Freund's vaccine group serum (Group 3) on the coating protein H9 chimeric H1 protein and H1stem protein found that the H9 chimeric H1 Freund's vaccine group serum (Group 3) can stimulate more head and neck area antibodies to improve the body's immunity.

[0172] 3.4 Hemagglutination test

[0173] a. Add 25ul PBS to each well of the hemagglutination plate. Add 25ul of inactivated H9N2 subtype avian influenza virus to the first well, and make 2-fold serial dilutions in sequence. At the same time, set up a negative control well.

[0174] b. Add 25ul of 1% chicken red blood cell suspension to each well, shake on a horizontal shaker for 1-2 minutes to mix, and let stand at 37℃ for 30 minutes before judging the result. Result: The maximum dilution of the virus with 100% agglutination (++++) is the hemagglutination titer of the virus, which is one agglutination unit. The hemagglutination titer of H9N2 subtype avian influenza virus is 1:2 7 .

[0175] 3.5 Hemagglutination inhibition test

[0176] Prepare four units of virus solution according to the results in 3.4;

[0177] a. Add 25ul PBS to each well of the blood coagulation plate, add 25ul of the serum to be tested (original serum) to the first well, and make 2-fold gradient dilutions in sequence. At the same time, set up a negative control well (PBS);

[0178] b. Except for the negative control well, add 25ul of four-unit virus solution to each well: shake on a horizontal shaker for 12min, and then stand at 37℃ for 15min;

[0179] c. Add 25ul of 1% chicken red blood cell suspension to each well, shake on a horizontal shaker for 1 to 2 minutes to mix, and let it stand at 37℃ for 30 minutes before judging the result.

[0180] result Figure 5As shown: The blood inhibition titer of H9 chimeric H1 Freund's vaccine (Group 3) after immunization was about 5log2, which could stimulate more antibodies to neutralize H9N2 subtype avian influenza virus compared with the single adjuvant control group (Group 1), and there was no significant difference compared with the commercial H9+Newcastle disease bivalent vaccine (Group 2), providing theoretical support for the development of new recombinant protein vaccines.

[0181] 3.5 Experimental animals challenged with viruses after immunization

[0182] Three weeks after the second vaccination, A / chicken / Shanxi / 1.23TGRL003-O / 2019H9N2 was administered intranasally, with a dose of 300ul (4HAU) per laying hen. Samples were collected from the mouth and anus of each chicken on the 7th day after the challenge with a cotton swab, stored in 1ml of virus preservation solution, and inoculated into 10-day-old SPF chicken embryos after shaking. After 72 hours, allantoic fluid was collected to detect the excretion of chicken embryos.

[0183] The pharyngeal and anal swabs collected on the 7th day after the virus attack were used for virus isolation. Six laying hens were selected from each group to inoculate the virus solution isolated from 10-day-old SPF chicken embryos. Three replicate groups were set up for each group, and the incubation and observation were carried out for 72 hours. The allantoic fluid was collected for HA determination. Regardless of dead or live embryos, the hemagglutination titer of the allantoic fluid of the chicken embryos should be determined, and the number of virus-positive chickens isolated from the immunized group and the control group was compared. As long as the HA titer of one chicken embryo fluid among the three chicken embryos inoculated with each sample is ≥1:16 (micro-method), it can be judged as positive for virus isolation. For samples with negative virus isolation, they should be blindly transmitted once before judgment. The results are shown in Table 2.

[0184] Table 2: Results of different groups after challenge

[0185]

[0186] The results showed that the single adjuvant control group showed that the H9N2 virus could be detected in all samples, while the experimental vaccine group could inhibit the reproduction of the H9N2 virus in the laying hen samples compared with the control group, which was consistent with the effect of the positive vaccine group. The vaccine designed by the present invention can effectively resist the invasion of the H9N2 virus.

Claims

1. A recombinant chimeric vaccine antigen protein of H9 subtype avian influenza, characterized in that: The head structure region in the HA skeleton of the H9 subtype avian influenza antigen is retained, and its neck region is replaced with the influenza HA neck region with broad-spectrum immune protection; The amino acid sequence of the head structure region in the H9 subtype avian influenza antigen HA skeleton is shown in SEQ ID NO: 1, and the amino acid sequence of the influenza HA neck region is shown in SEQ ID NO: 2; The amino acid sequences of the H9 subtype avian influenza recombinant chimeric vaccine antigen protein are SEQ ID NO: 1 and SEQ ID NO: 2 from N-terminus to C-terminus.

2. A nucleic acid encoding the chimeric vaccine antigen protein as claimed in claim 1.

3. The nucleic acid according to claim 2, characterized in that The nucleotide sequence of the head structure region in the H9 subtype avian influenza antigen HA skeleton is shown in SEQ ID NO: 3, and the nucleotide sequence of the influenza HA neck region is shown in SEQ ID NO:

4.

4. The nucleic acid according to claim 3, characterized in that The full-length nucleotide sequence is shown in SEQ ID NO: 5 。 5. An expression vector containing the nucleic acid according to any one of claims 2 to 4.

6. The expression vector according to claim 5, characterized in that The starting vector is the PCAGGS vector.

7. A method for preparing a recombinant chimeric vaccine of H9 subtype avian influenza, characterized in that: The steps include: The first step: connecting the fully synthesized nucleic acid with sticky ends as described in any one of claims 2 to 4 and the NA gene nucleic acid fragment into the vector after double enzyme digestion to construct a recombinant expression vector; Step 2: Co-transfect the plasmid HA and NA into 293T mammalian cells for secretory expression, collect the supernatant, and purify the target HA protein by affinity chromatography according to the purification tag; Step 3: Emulsify the HA protein and adjuvant to prepare a recombinant chimeric vaccine.

8. The method according to claim 7, characterized in that In the second step, the mass ratio of plasmid HA and NA was 1:1; in the third step, the volume ratio of HA protein to adjuvant was 1:

1.

9. The method according to claim 7, characterized in that The vector is a PCAGGS vector, and the sticky ends are EcoR Ⅰ and Xho Ⅰ.

10. A recombinant chimeric vaccine of H9 subtype avian influenza, characterized in that: It comprises the chimeric vaccine antigen protein as claimed in claim 1, and a pharmaceutically acceptable adjuvant.

11. The H9 subtype avian influenza recombinant chimeric vaccine according to claim 10, characterized in that: Its adjuvant is Montanide TM ISA 71 VG, mineral white oil, or Freund's adjuvant.

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