CN116874607A - H9 subtype avian influenza recombinant chimeric vaccine and preparation method thereof - Google Patents

Abstract

The invention belongs to the field of immunology, and particularly discloses an H9 subtype avian influenza recombinant chimeric vaccine and a preparation method thereof. While retaining the head structure in the HA backbone of the major protective antigen of H9 subtype avian influenza, the neck region of vaccine H9 was replaced with influenza HA neck region with broad spectrum immunoprotection, thereby constructing recombinant chimeric HA. The recombinant chimeric HA of the invention is consistent with the natural H9 trimeric protein in structure and molecular weight, and by breaking the advantage of the head structure, the immune response to the conserved stem region is enhanced, the immune response is concentrated to the immunosub-dominant stem region, and the combined action excites the generation of more protective antibodies. The invention provides a method for obtaining high-purity, high-expression and active H9 chimeric H1 protein, which successfully realizes the protection of invasion of avian influenza virus H9N2 subtype strains. The method has the advantages of high expression yield, simple cost and safe process, and can effectively spread H9N2 influenza subtype strains.

Description

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

Technical field

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

Background technique

H9N2 subtype avian influenza virus (AIV) is classified as a low-pathogenic AIV according to its pathogenicity. However, because of its wide host range, fast transmission speed, and long epidemic time, it is easy to cooperate with other bacteria or viruses. Infected poultry leads to reduced production performance, seriously affecting the economic value of poultry. H9N2 subtype avian influenza virus (AIV) is widely distributed around the world and is generally divided into two lineages: North American lineage and Eurasian lineage. Specifically, the Eurasian lineage further multiplied into various virus clusters, represented by BJ/94-like or Y280-like, G1-like, Y439-like, F/98-like, etc.

In China, G1-like, which is mainly spread among quail, has a geographical advantage in the southern region, while BJ/94-like and F/98-like, which are prevalent in chicken flocks, 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. AIV contained in wild birds is often highly pathogenic when transmitted across species. Research has found that six internal genes of the H7N9, H10N8 and H5N6 subtypes of AIV that have emerged in my country and caused human infection are all derived from the H9N2 subtype of AIV, which also provides a powerful environment for the outbreak of HPAIV. Therefore, we need to pay enough attention to the epidemic potential of H9N2 subtype AIV, develop effective H9N2 subtype AIV vaccine, effectively prevent and control the epidemic of this virus, and provide guarantee for our public health security.

H9N2 AIV is monitored in many countries, especially in China and other Asian countries. Since 2016, most research teams in my country have sampled tens of thousands of samples from 37 cities in 23 provinces, municipalities, and ethnic autonomous regions in China. They have identified strains of the HxNy subtype through next-generation sequencing (NGS) and found that H9N2 is the dominant subtype. Serological survey reports also show that the positive rate of H9N2 virus antibodies in the general population is 1.3% to 1.4%, and the positive rate among retail poultry workers exceeds 15%, confirming human infection with the H9N2 virus, and found that live poultry markets (LPM) are Important exposure risk factors for human infection with avian influenza viruses.

The most recent samples collected from 2013 to 2018, combining phylogeny and antigenic analysis of H9N2 isolates, found that the most recent isolates clustered mainly in subgroup II and subgroup III, far away from the vaccine strains (mainly in subgroup I) Far. Analyze the homology of HA proteins from different antigenic clusters to evaluate amino acid residues that may contribute to antigenic drift. It was found that 11 amino acid residues are conserved among vaccine cluster viruses but mutated in subgroup II and subgroup III lineage viruses; and 10 mutated amino acid residues are found in the HA protein between subgroup II and subgroup III. , these mutated amino acids may potentially affect the protective efficacy of the H9N2 AIV inactivated vaccine.

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 its preparation method, 2016.) selected HA1, which has strong immunogenicity among the HA proteins region (1-1041nt), inserted into the heat-resistant vector of Newcastle disease virus, which can enhance the immune protection effect of the vaccine in a targeted manner and greatly reduce the dependence on the cold chain system. It can be used to prepare H9 avian influenza and Newcastle disease double combinations. For the heat-resistant live vaccine, the pathogenicity of the recombinant virus was also measured in the article and it maintained the low-virulence characteristics of the parent strain. The insertion of foreign genes did not change the pathogenicity of the recombinant virus. Krammer F, et al. Chimeric hemagglutinin influenza virus vaccine constructs elicit broadly protective stalk-specific antibodies. JVirol. 2013Jun; 87(12):6542-50.; Krammer F, et al. H3 stalk-based chimeric hemagglutinin influenza virus constructs protect mice from H7N9 challenge.JVirol.2014Feb; 88(4):2340-3.) The H1 stems of influenza A H1N1 viruses of different epidemic seasons were used for chimerization, and then the heads of H5, H6 and H9 influenza viruses were used to chimerize the Chimeric stems construct cHAs vaccines. Studies have shown that mice that were continuously immunized with the vaccine had a survival rate of 100% after being challenged by various viruses such as H5N1, H6N1 and H7N9, which was significantly higher than that of the control groups, and they produced high titers of stem-reactive antibodies. ; His team successively immunized ferrets with cHAs with H9, H5, and H6 heads. After inoculation with cHAs with H5 and H6 heads, the ferrets' serum reactivity to the pandemic H1N1 virus was higher than before. times increased by 4 and 8 times.

Common avian influenza vaccines include whole-virus inactivated vaccines, live attenuated vaccines, genetically engineered subunit vaccines, recombinant live vector vaccines, nucleic acid vaccines, RNA replicon vaccines, universal influenza vaccines, genetically modified plant vaccines, etc., but at present, breeding farms Inactivated avian influenza vaccines are still used to prevent infection of poultry by H9 subtype avian influenza viruses. However, due to antigenic drift caused by immune pressure, H9 subtype avian influenza viruses continue to mutate and become prevalent. The H9N2 subtype avian influenza virus also actively participates in gene rearrangements within other subtypes of influenza viruses, producing new influenza viruses such as H5N2, H6N1, H7N7, H7N9, and H10N8, which threaten human health. The H7N9 influenza that broke out in 2013 was the result of mixing in chickens. Its external genes came from the H7 subtype influenza virus, and its internal genes came from the H9N2 subtype influenza virus.

In addition, the production process of inactivated vaccines has problems such as a large amount of chicken embryos, a high premature mortality rate of chicken embryos, a small average harvest of single embryos, unstable potency, and high cost. The application of inactivated vaccines cannot distinguish between naturally infected chickens and vaccinated chickens, thus interfering with avian influenza epidemic surveillance and epidemiological investigations; live attenuated vaccines may undergo genetic rearrangement with other influenza viruses to achieve reassortment that restores virulence. strain of the virus, it was also found that cold-adapted live attenuated vaccines have disease risks when used in immunodeficient people; genetically engineered vaccines were found to have shortcomings such as short antibody duration and high cost; recombinant live vector vaccines are only effective for immunized chickens It lasts for a short time, and there are certain restrictions on the use of chickens that have been vaccinated or infected with viral vectors; researchers have found that the vectors of nucleic acid vaccines often contain antibiotic genes, which may cause difficulties in the prevention and treatment of bacterial diseases, and The internal expression efficiency of nucleic acid vaccines is not high. An ideal avian influenza vaccine should be highly safe for poultry, be able to differentiate between naturally infected poultry and vaccine-immune poultry, have good production safety, and have a long duration. Currently, there is no vaccine that has the above characteristics at the same time. Features. The design concept of the universal influenza vaccine is to be effective against various influenza subtypes during the avian influenza pandemic stage or during the transitional mutation stage of the virus.

In addition, continuous vaccine immunization will lead to immune escape due to antigenic variation to varying degrees. Clinically, it has also been found that the replacement rate of inactivated vaccine strains cannot keep up with the pace of viral antigenic variation, resulting in the continued prevalence and mutation of antigenic escape strains. In this way, poultry Not only has the influenza virus not been effectively controlled, there has also been a trend of the virus continuing to spread and its scope of transmission expanding.

Therefore, the development and evaluation of vaccines with cross-immune protective effects against antigenic variant strains or different subtypes of avian influenza viruses are very necessary for the prevention and control of H9 subtype viruses.

Contents of the invention

The present invention constructs a new 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 of H9 subtype avian influenza, and at the same time replacing the neck area of vaccine H9 with the influenza HA neck area with broad-spectrum immune protection that has been designed and modified through structural biology, Thus, recombinant chimeric HA (chimeric HA) was constructed. The recombinant chimeric HA is consistent in structure and molecular weight with the natural H9 trimer protein. The chimeric vaccine of the present invention breaks the advantages of the head structure, enhances the immune response to the conserved stem region, concentrates the immune response to the immunologically sub-dominant stem region, and works together to stimulate the production of more protective antibodies.

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, of which positions 922-957 are the active peptide structure of the neck region.

The present invention first provides an H9 subtype avian influenza recombinant chimeric vaccine antigen protein, which retains the head structural region in the H9 subtype avian influenza antigen HA skeleton and replaces its neck region with a broad-spectrum immune Protection is obtained from the influenza HA neck area.

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

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

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.

The invention thus provides nucleic acids encoding said chimeric vaccine antigen proteins.

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

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

At the same time, preferably, the nucleotide sequence of the influenza HA neck region is shown in SEQ ID NO: 4.

Further preferably, the full-length coding nucleotide sequence of the H9 chimeric H1 protein is shown in SEQ ID NO: 5.

The present invention thus also provides an expression vector containing the nucleic acid. Preferably, the starting vector is a PCAGGS vector.

The present invention further provides a method for preparing a recombinant chimeric vaccine for H9 subtype avian influenza, which includes the following steps:

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

Step 2: Co-transfect plasmids 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 through affinity chromatography according to the purification tag.

Step 3: emulsify HA protein and adjuvant in proportion (preferably 1:1 in volume) to prepare a recombinant chimeric vaccine.

Finally, the present invention particularly provides a recombinant chimeric vaccine for H9 subtype avian influenza, which includes the chimeric vaccine antigen protein and optionally pharmaceutically acceptable auxiliaries.

The present invention designs a set of methods to obtain 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 of avian influenza virus. The H9 chimeric H1 protein of the present invention has high expression yield, simple cost and safe process, can effectively respond to the spread of avian influenza H9N2 subtype strains, and provides support for the preparation of efficient universal avian influenza subunit vaccines.

Description of the drawings

Figure 1 Typical molecular sieve pattern and SDS-PAGE gel pattern of H9 chimeric H1 protein. The arrow points to the protein marker72KD.

Figure 2 Hemagglutination inhibition test results of H1N1 strain (A) and H3N2 strain (B) in different groups.

Figure 3A and Figure 3B show the HI antibody detection results. Among them, Figure 3A test groups: 1, serum from the unimmunized group; 2, serum from the positive control group (inactivated avian influenza virus H9 subtype A/Chicken/Hebei/G/2012 (H9N2 strain)); 3, test group H9 chimeric H1 ISA 71 vaccine group serum; 4, single adjuvant control group serum. Figure 3B Test group settings: 1. Serum from the unimmunized group; 2. Serum from the positive control group (commercial Pleco H9 inactivated vaccine 032101001A); 3. Serum from the test group H9 chimeric H1 white oil vaccine group;

Figure 4A and Figure 4B ELISA detects specific antibodies in chicken serum. Among them, Figure 4A shows the C test: the immune response of the serum of the positive control group (H9+ Newcastle disease two-combination vaccine) and the serum of the experimental group H9 chimeric H1 Freund's vaccine group to different influenza HAs; the packaging protein is H1 full-length protein, H5ORI protein, H9ORI protein. Figure 4B shows D test: serum from the single adjuvant control group (Group 1), serum from the positive control (H9+ Newcastle disease dual vaccine) group (Group 2), serum from the test group H9 chimeric H1 Freund's vaccine group (Group 3) Group) Immune response to package protein H9 chimeric H1 protein, H1 stem protein, and H9ORI protein.

Figure 5 Blood inhibition test results.

Figure 6 Antibody titer statistical results. Among them: 1. Serum from the single adjuvant control group; 2. Serum from the positive control (H9+ Newcastle disease dual vaccine) group; 3. Serum from the experimental group H9 chimeric H1 Freund's vaccine group.

Detailed ways

The following examples further illustrate the content of the present invention, but should not be understood as limiting the present invention. Without departing from the spirit and essence of the present invention, any modifications or substitutions made to the method, steps or conditions of the present invention shall fall within the scope of the present invention.

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

Example 1 Construction of H9 subtype HA gene and NA gene plasmid to assist HA expression

Send the designed nucleic acid sequence of HA or NA to the company for complete sequence synthesis of DNA fragments with sticky ends (the sticky ends are the enzyme cleavage sites of EcoRI and XhoI). The vector is a PCAGGS vector with ampicillin resistance. The vector is double-digested, and the DNA fragment synthesized with the full sequence is ligated into the vector. The full sequence is synthesized with sticky ends (specifically, the sticky ends are EcoRⅠ and XhoⅠ) The following HA gene and NA gene nucleic acid fragments (the two are fused with different purification tag peptides) were respectively connected into the PCAGGS vector after double enzyme digestion (corresponding EcoRI and XhoI) to construct a recombinant expression vector.

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

EcoRI restriction site: GAATTC;

Kozak sequence: GCCACC;

Signal peptide amino acid sequence: METVSLITILLVVTVSNA

Signal peptide nucleotide sequence: ATGGAGACAGTATCACTAATAACTATACTACTAGTAGTAACAGTAAGCAATGCA;

Thrombin cleavage site amino acid sequence: LVPRGS

Nucleotide sequence of thrombin cleavage site: CTGGTGCCAAGAGGCTCT;

Trimer tag sequence: CCTGGCAGCGGCTATATTCCTGAGGCTCCCAGAGATGGCCAGGCCTACGTTAGAAAGGATGGCGAGTGGGTGCTGCTGAGCACCTTTCTGGGA;

Histidine tag amino acid sequence: HHHHHH;

Histidine tag nucleotide sequence: CACCACCACCATCACCAC;

XhoI restriction site: CTCGAG.

HA head region amino acid sequence (SEQ ID NO: 1): METVSLITILLVVTVSNADKICIGYQSTNSTETVDTLTE NNVPVTHAKELLHTEHNGMLCATSLGHPLILDTCTIEGLIYGNPSCDLLLGGREWSYIVERPSAVNGLCYPGNVENLEELRSLFSSARSYQRIQIFPDTIWNVSYSGTSKACSDSFYRSMRWLTQKNNAYPIQDAQYTNNQEKNILFMWGI NHPPTDTAQTNLYTRTDTTTSVATEEINRTFKPLIGPRPLVNGLQGRIDYYWSVLKPGQTLRIRSNGNLIAPWYGHILSGESHGRILKTDLKRGSCTVQCQTEKGGLNTTLPFQNVS

HA head region nucleotide sequence (SEQ ID NO: 3): ATGGAGACAGTATCACTAATAACTATACTACTAGTAGTAACAGTAAGCAATGCAGATAAAATCTGCATCGGCTATCAATCAACAAACTCCACAGAAACTGTAGACACACTAACAGAAAACAACGTCCCTGTGACACATGCCAAAGAATTGTCCCACACAGAGCATAATGGGATGCTGTGTGCAACAAGCTTGGGACACCCTCTTATTCTAGACACCTGTACCATTGAAGGACTA ATCTATGGCAATCCTTCTTGTGATCTATTGTTGGGAGGAAGAGAATGGTCCTATATCGTCGAGAGACCATCAGCTGTTAACGGATTGTGTTATCCCGGGAATGTAGAAAATCTAGAAGAGCTAAGGTCACTTTTTAGTTCTGCTAGGTCTTATCAAAGGATCCAGATTTTCCCAGACACAATCTGGAATGTGTCTTACAGTGGGACAAGCAAAGCATGTTCAGATTCATTCTACAGAAGCATGAGATGGTTGACTCAAAA GAACAATGCTTACCCTATTCAAGACGCCCAATACACAAATAATCAAGAAAAGAACATTCTTTTCATGTGGGGCATAAATCACCCACCCACCGATACTGCGCAGACAAATCTGTACACAAGAACCGACACAACAACGAGTGTGGCAACAGAAGAAATAAATAGGACCTTCAAACCATTGATAGGACCAAGGCCTCTTGTCAACGGTTTGCAGGGAAGAATTGATTATTGGTCGGTATTGAAACCGGGTCAAACACTGCGAATAAGATCTAATGGGAA TCTAATAGCTCCATGGTATGGACACATTCTTTCAGGAGAGAGCCACGGAAGAATCCTGAAGACTGATTTAAAAAGGGGTAGCTGCACAGTGCAATGTCAGACAGAAAAAGGTGGATTAAACACAACATTGCCATTCCAAAACGTAAGT

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

KYAIGDCPKYVKQNTLKLATGLRNIPSIQSRGLFGAIAGFTEGGWTGMVDGLYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQYTAVGKEFNKSERRMENLNKKVDDGKIDLWSYNAELLVALENQHTIDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESK LNREKIDGVKR

HA neck region nucleotide sequence (SEQ ID NO: 4):

AAGTATGCCATCGGCGACTGCCCCAAATACGTGAAGCAGAATACCCTGAAGCTGGCCACCGGCCTGAGAAACATCCCCAGCATCCAGAGCAGAGGCCTGTTCGGAGCCATTGCCGGCTTTACTGAAGGCGGCTTTGGACAGGCATGGTGGATGGCCTGTATGGCTATCACCACCAGAATGAGCAAGGCAGCGGATACGCCGCTGACCAGAAGTCTACCCAGAACGCTATCAATGGCATCACCAACAAAGTGAACTCCGTGATC GAGAAGATGAACACCCAGTACACCGCCGTGGGCAAAGAGTTCAACAAGAGCGAGCGGCGGATGGAAAACCTGAACAAGAAGGTGGACGACGGCAAGATCGACCTGTGGTCCTACAATGCCGAACTGCTGGTGGCCCTGGAAAACCAGCACACCATCGACTTCCACGACAGCAACGTGAAGAACCTGTACGAGAAAGTGAAGTCCCAGCTGAAGAACAACGCCAAAGAGATCGGCAACGGCTGCTTCGAGTTCTACCACAAGTGCAA CGACGAGTGCATGGAAAGCGTGAAGAATGGCACCTACGACTACCCCAAGTACAGCGAGGAATCCAAGCTGAACCGCGAGAAAATCGACGGCGTGAAGAGA.

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

GAATTCGCCACCATGGAGACAGTATCACTAATAACTATACTACTAGTAGTAACAGTAAGCAATGCA

GATAAAATCTGCATCGGCTATCAATCAACAAACTCCACAGAAACTGTAGACACACTAACAGAAAAC

AACGTCCCTGTGACACATGCCAAAGAATTGCTCCACACAGAGCATAATGGGATGCTGTGTGCAACAA

GCTTGGGACACCCTCTTATTCTAGACACCTGTACCATTGAAGGACTAATCTATGGCAATCCTTCTTGTG

ATCTATTGTTGGGAGGAAGAGAATGGTCCTATATCGTCGAGAGACCATCAGCTGTTAACGGATTGTGTT

ATCCCGGGAATGTAGAAAATCTAGAAGAGCTAAGGTCACTTTTTAGTTCTGCTAGGTCTTATCAAAGG

ATCCAGATTTTCCCAGACACAATCTGGAATGTGTCTTACAGTGGGACAAGCAAAGCATGTTCAGATTC

ATTCTACAGAAGCATGAGATGGTTGACTCAAAAGAACAATGCTTACCCTATTCAAGACGCCCAATACA

CAAATAATCAAGAAAAGAACATTCTTTTCATGTGGGGCATAAATCACCCACCCACCGATACTGCGCAG

ACAAATCTGTACACAAGAACCGACACAACAACGAGTGTGGCAACAGAAGAAATAAATAGGACCTTC

AAACCATTGATAGGACCAAGGCCTCTTGTCAACGGTTTGCAGGGAAGAATTGATTATTATTGGTCGGT

ATTGAAACCGGGTCAAACACTGCGAATAAGATCTAATGGGAATCTAATAGCTCCATGGTATGGACACA

TTCTTTCAGGAGAGAGCCACGGAAGAATCCTGAAGACTGATTTAAAAAGGGGTAGCTGCACAGTGCA

ATGTCAGACAGAAAAAGGTGGATTAAACACAACATTGCCATTCCAAAACGTAAGTAAGTATGCCATCG

GCGACTGCCCCAAATACGTGAAGCAGAATACCCTGAAGCTGGCCACCGGCCTGAGAAACATCCCCAG

CATCCAGAGCAGAGGCCTGTTCGGAGCCATTGCCGGCTTTACTGAAGGCGGCTGGACAGGCATGGTG

GATGGCCTGTATGGCTATCACCACCAGAATGAGCAAGGCAGCGGATACGCCGCTGACCAGAAGTCTA

CCCAGAACGCTATCAATGGCATCACCAACAAAGTGAACTCCGTGATCGAGAAGATGAACACCCAGTA

CACCGCCGTGGGCAAAGAGTTCAACAAGAGCGAGCGGCGGATGGAAAACCTGAACAAGAAGGTGG

ACGACGGCAAGATCGACCTGTGGTCCTACAATGCCGAACTGCTGGTGGCCCTGGAAAACCAGCACAC

CATCGACTTCCACGACAGCAACGTGAAGAACCTGTACGAGAAAGTGAAGTCCCAGCTGAAGAACAA

CGCCAAAGAGATCGGCAACGGCTGCTTCGAGTTCTACCACAAGTGCAACGACGAGTGCATGGAAAG

CGTGAAGAATGGCACCTACGACTACCCCAAGTACAGCGAGGAATCCAAGCTGAACCGCGAGAAAATC

GACGGCGTGAAGAGACTGGTGCCCAGAGGCTCTCCTGGCAGCGGCTATATTCCTGAGGCTCCCAGAG

ATGGCCAGCCTACGTTAGAAAGGATGGCGAGTGGGTGCTGCTGAGCACCTTTCTGGGACACCACCACCATCACCACTGACTCGAG.

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

EcoRI restriction site: GAATTC;

Signal peptide amino acid sequence: MGAGATGRAMDGPRLLLLLLLGVSLGGA;

Signal peptide nucleotide sequence: ATGGGGGCAGGTGCCACCGGCCGCGCCATGGACGGGCCGCGCCTGCTGCTGTTGCTGCTTCTGGGGGTGTCCCTTGGAGGTGCC;

Flag tag amino acid sequence: DYKDDDDK;

Flag tag nucleotide sequence: GATTATAAGGATGATGATGATAAG;

Tetramer tag amino acid sequence: SSSDYSDLQRVKQELLEEVKKELQKVKEEIIEAFVQELRKRGS;

Tetramer tag nucleotide sequence: AGCTCCAGTGATTACTCGGACCTACAGAGGGTGAAACAGGAGCTTCTGGAAGAGGTGAAGAAGGAATTGCAGAAAGTGAAAGAGGAAATCATTGAAGCCTTCGTCCAGGAGCTGAGGAAGCGGGGTTCT;

Thrombin cleavage site amino acid sequence: LVPRGS;

Nucleotide sequence of thrombin cleavage site: CTGGTACCACGAGGTAGT;

09NA amino acid sequence (SEQ ID NO: 6): PSRSVKLAGNSSLCPVSGWAIYSKDNSVRIGSKGDVFVIREPFISCSPLECRTFFLTQGALLNDKHSNGTIKDRSPYRTLMSCPIGEVPSPYNSRFESVAWSASACHDGINWLTIGISGPDNGAVAVLKYNGIITDTIKSWRNNILRTQESECACVNGSCFTVMTDGPSNGQASYKIFRIEKGKIVKSVEM NAPNYHYEECSCYPDSSEITCVCRDNWHGSNRPWVSFNQNLEYQIGYICSGIFGDNPRPNDKTGSCGPVSSNGVKGFSFKYGNGVWIGRTKSISSRNGFEMIWDPNGWTGTDNNFSIKQDIVGINEWSGYSGSFVQHPELTGLDCIRPCFWVELIRGRPKENTIWTSGSSISFCGVNSDTVGWSWPDGAELPFTIDK;

09NA nucleotide sequence (SEQ ID NO: 7): CCATCACGATCAGTGAAATTAGCGGGCAATTCCTCTCTCTGCCCTGTTAGTGGATGGGCTATATACAGTAAAGACACAGTGTAAGAATCGGTTCCAAGGGGGATGTGTTTGTCATAAGGGAACCATTCATATGCTCCCCCTTGGAATGCAGAACCTTCTCTTGACTCAAGGGGCCTTGCTAAATGACAAACATTCCAATGGAACCATTAAAGACAGGAGCCCATATCGA ACCCTAATGAGCTGTCCTATTGGTGAAGTTCCCTCCATACAACTCAAGATTTGAGTCAGTCGCTTGGTCAGCAAGTGCTTGTCATGATGGCATCAATTGGCTAACAATTGGAATTTCTGGCCCAGACAATGGGGCAGTGGCTGTGTTAAAGTACAACGGCATAATAACAGACACTATCAAGAGTTGGAGAAACAATATATTGAGAACACAAGAGTCTGAATGTGCATGTGTAAATGGTTCTTGCTTTACTGTAAACTGACCGAT GGACCAAGTAATGGACAGGCCTCATACAAGATCTTCAGAATAGAAAAGGGAAAGATAGTCAAATCAGTCGAAATGAATGCCCCTAATTATCACTATGAGGAATGCTCCTGTTATCCTGATTCTAGTGAAATCACATGTGTGTGCAGGGATAACTGGCATGGCTCGAATCGACCGTGGGTGTCTTTCAACCAGAATCTGGAATATCAGATAGGATACATATGCAGTGGGATTTTCGGAGACAATCCACGCCCTAATGATAAGACAGGCA GTTGTGGTCCAGTATCGTCTAATGGAGCAAATGGAGTAAAAGGGTTTTCATTCAAATACGGCAATGGTGTTTGGATAGGGAGAACTAAAAGCATTAGTTCAAGAAACGGTTTTGAGATGATTTGGGATCCGAACGGATGGACTGGGACAGACAATAACTTCTCAATAAAGCAAGATATCGTAGGAATAAATGAGTGGTCAGGATATAGCGGGAGTTTTGTTCAGCATCCAGAACTAACAGGGCTGGATTGTATAAGACCTT GCTTCTGGGTTGAACTAATCAGAGGGCGACCCAAAGAGAACACAATCTGGACTAGCGGGAGCAGCATATCCTTTTGTGGTGTAAACAGTGACACTGTGGGTTGGTCTTGGCCAGACGGTGCTGAGTTGCCATTTACCATTGACAAGTAA;

XhoI restriction site: CTCGAG.

Example 2 Expression and Purification of H9 Subtype HA Protein

Transform the recombinant expression vector plasmid into Top10 competent cells, draw an appropriate amount of bacterial liquid and apply it on a solid LB plate with resistance to ampicillin (ampicillin mother solution concentration: 100 mg/ml) by streaking method (LB plate formula: 1% NaCl, 1% Tryptone, 0.5% yeast extract, 1.5% agar powder; ampicillin (1:1000) was used and cultured at 37°C overnight. Single clones were picked, 5 ml of micro-shake culture (ampicillin-resistant) was shaken, and then 300 ml was shaken overnight, and the influenza virus antigen plasmid was obtained using the endotoxin-free plasmid maximal extraction kit (TIANGEN, DP117).

Culture 293T cells with DMEM containing 10% FBS in a 37°C, 5% _CO2 incubator. When the confluence of 293T cells reaches about 70%, use PEI transfection reagent to transfect the influenza virus antigen plasmid (HA:NA=20ug:20ug/plate) into 293T cells. After 4-6 hours of transfection, use The DMEM medium was replaced with serum and the culture was continued for 3 days before the cell culture supernatant was collected. Serum-free DMEM was added to continue culturing 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 inability to bind to the receptor, so neuraminidase NA was also expressed. NA only assists the expression of HA protein. The 09NA gene was selected and FLAG tag was added for easy detection. Since the purification tags of HA and NA are different, we use an affinity column with a His tag to purify the HA protein, so that the final protein we purify is only the HA protein. We use western blot to verify the ratio of HA+NA co-transfection. The amount of HA protein obtained by single transfection of HA is larger. In order to purify a large amount of HA protein, the HA designed later is secreted by co-transfection with NA.

Filter the collected cell culture supernatant through a 0.22 μm filter membrane and combine it with HisTrap ^TM excel (GE) overnight at 4°C to prepare protein elution buffer A (20mM Tris, 150mM NaCl, pH 8.0) and buffer B. solution (20mM Tris, 150mM NaCl, pH 8.0, 1M imidazole). Then wash the His column with buffer A to remove non-specifically bound proteins, and then use buffer 2% B (20mM Tris, 150mM NaCl, pH 8.0, 20mM imidazole) to remove impurity proteins. Finally, the target protein is washed with 30 % of solution B (20mM Tris, 150mM NaCl, pH 8.0, 300mM imidazole) was eluted from the His column, and the protein concentration tube with 30KD cutoff was filled with buffer A (20mM Tris, 150mM NaCl, pH 8.0). Change the liquid to remove the imidazole concentration in the protein solution. Finally, concentrate the protein solution to 0.5 ml, add thrombin (1 mg protein plus 5 ul thrombin) and digest overnight at 4°C. The digested protein solution was further purified with molecular sieves, using AKTA-purifier (GE) and Column Hiload 16/60superdex 200PG molecular sieves (GE). The molecular sieves were column equilibrated with buffer A (20mM Tris, 150mM NaCl, pH 8.0). , load the sample into a 1ml loop, monitor the UV absorption value at 280nm at the same time, collect the target protein, and identify the protein purity through SDS-PAGE. The typical molecular sieve pattern and SDS-PAGE analysis of the target protein are shown in Figure 1.

The peak position of this protein in Column Hiload 16/60superdex 200PG molecular sieve is 62.5ml, because a trimer is added after the thrombin cleavage site (LVPR↓GS, the arrow points to the amino acid that can recognize and cleave this site) during the construction. The label is in the form of a trimer of the main peak of the protein. The trimer label is shed from HA after thrombin digestion, and the monomer size is about 70KD. The lanes of the SDS-PAGE gel map respectively correspond to the trimers and monomers of the H9 chimeric H1 protein in the molecular sieve map.

Example 3. Vaccine preparation

The purified H9 chimeric H1 protein is mixed with different adjuvants to prepare the vaccine, and the vaccine is emulsified according to a volume ratio of 1:1 until it is still in bath water for animal immunization. H1 stem protein is the neck region protein of influenza H1 subtype and has a broad-spectrum immune protective effect. On this basis, we designed an H9 chimeric H1 protein that has immune response effects on different influenza HAs. See Example 6 for details. . The specific vaccine preparation method is detailed in Example 3; the functional verification of the influenza HA neck region (H1stem) with broad-spectrum immune protection is demonstrated by immunizing mice, as shown in Example 4; the prepared H9 chimera For details on the test of immunizing SPF chickens with H1 vaccine, see Example 5; for the test of immunizing Hyline white laying hens with the prepared H9 chimeric H1 vaccine, please see Example 6 for details.

1. Preparation of H1stem vaccine

The H1 stem protein concentration is 10 mg/ml, PBS is the diluent, and the immune adjuvant is MF59. Mix the H1 stem protein of the test group with an equal volume of MF59 adjuvant, and shake until it is completely emulsified until the bath water does not melt, and then used for animal immunization.

Amino acid sequence of H1stem vaccine (SEQ ID NO:8), of which positions 194-262 are conserved peptides: MYRMQLLSCIALSLALVTNSTYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSKQSQGLFGAIAGFTEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQYTAI GCEYNKSERCMKQIEDKIEEIESKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQIEEGRHHHHHHH

The nucleotide sequence of H1stem vaccine (SEQ ID NO:9), of which positions 580-786 are conserved peptides:

ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACGAATTCAACCTACGCCGACACCATTTGCATCGGCTACCACGCCAACAACAGCACCGACACCGTGGACACCGTGCTGGAAGAACGTGACCGTGACCCACAGCGTGAATCTGCTGGAGAACGGAGGAGGCGGCAAATACGTCTGCAGCGCCAAACTGAGGATGGTGACCGGACTGAGGAACAAGCCCAGCAAGCAGAGCCAGGGACTGTTCG GAGCCATTGCCGGATTCACCGAGGGAGGTTGGACAGGAATGGTGGACGGTTGGTACGGCTACCACCACCACCAGAACGAGCAGGGAAGCGGATACGCCGCCGATCAGAAAAGCACCCAGAACGCCATCAACGGCATCACCAACAAGGTCAACAGCGTGATCGAGAAGATGAACACCCAGTACACCGCCATCGGTTGCGAGTACAACAAGAGCGAGCGCTGCATGAAGCAGATCGAGGACAAGATCGAGGAGATCGAGAGCAAGATC TGGTGCTACAACGCCGAACTGCTGGTGCTGCTGGAGAACGAGGACCCTGGACTTCCACGACAGCAACGTGAAGAACCTGTACGAGAAGGTCAAGAGCCAGCTGAAGAACAACGCCAAGGAGATCGGCAACGGCTGCTTCGAGTTCTACCACAAGTGCAACGACGAGTGCATGGAGAGCGTGAAGAACGGCACCTACGACTACCCCAAGTACAGCGAGGAGAGCAAGCTGAACCGGGAGAAGATCGACGGCGTGAAG CTGGAGAGCATGGGCGTGTACCAGATCGAGGGCAGACATCACCACCACCACCATCATTAG.

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

The concentration of H9 chimeric H1 protein is 10 mg/ml, PBS is the diluent, and the immune adjuvant is Montanide ^TM ISA 71VG.

The vaccine is prepared using a mass ratio of 7:3 between the adjuvant and the aqueous antigen medium. Add the aqueous antigen medium to Montanide ^TM ISA 71VG at room temperature or below room temperature, and mix under vigorous stirring to obtain a stable preparation. Animals are inoculated, labeled, and stored at 2 to 8°C.

3. Preparation of H9 chimeric H1 white oil vaccine (the adjuvant is mineral white oil)

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

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

The concentration of H9 chimeric H1 protein was 10 mg/ml, PBS was the diluent, and the immune adjuvant was Freund's adjuvant. Completely emulsify the H9 chimeric H1 protein with an equal volume of Freund's complete adjuvant until the bath water does not melt and then use it for animal vaccination. Freund's complete adjuvant is used for the first immunization, and Freund's incomplete adjuvant is used for the second immunization. agent.

Example 4 Animal Test 1: Functional verification of influenza HA neck region (H1 stem) with broad-spectrum immune protection

1. Experimental animals: 4-6 weeks old BALB/c female mice, 6 in each group. The immunization method is intramuscular injection.

2. Group settings: ① negative control (adjuvant MF59); ② positive control (split influenza virus vaccine TIV); ③ test group H1stem vaccine.

3. Test steps:

3.1 Animal immunity:

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

3.2 Serum separation:

Blood was collected from the eyeballs of the mice in the above immune groups to prepare serum. The mouse serum was treated with RED. According to 1 volume of serum, 4 volumes of RDE were added, and the water was bathed at 37°C for 18 hours and inactivated at 56°C for 30 minutes for subsequent use. experiment.

3.3 TCID ₅₀ determination of H1N1 strains and H3N2 strains

a, Use 10-fold serial dilution to dilute the virus stock solution.

b. Lay out the 96-well plate 18-24 hours in advance, and add 100 μL (2×10 ⁵ cells/ml) MDCK cells to each well. Cultivate in a 37°C, 5% _CO2 incubator.

c, virus inoculation, wash the cells once with PBS, 150 μL per well, then add the diluted virus solution according to the first step into the 96-well plate, inoculate one vertical row for each dilution, 100 μL per well, in columns 11 and 12 Column set dilution control and culture in 37°C incubator.

d. Observe and record the results daily. The results after 72 hours are calculated according to the Reed-Muench method.

3.4 Neutralization experiment:

a. Virus dilution: Use virus culture medium to dilute to 200TCID ₅₀ /50μL.

b, Serum negative control (only add cell maintenance solution)

Serum dilution: The initial dilution factor of mouse serum is 1:40, and the neutralization test is performed at a 2-fold dilution ratio.

c. Virus incubation: Add 50 μl of diluted virus (200TCID ₅₀ ) to 50 μl of serum, mix evenly, place at 37°C, and incubate for 1 hour.

d. MDCK cell adsorption: MDCK cells were washed once with 150 μL of PBS, and then discarded. Add 100 μL of virus-serum mixture to each well, make three parallel wells, and culture in a cell culture incubator for 72 hours.

e. Use the blood inhibition experiment to observe and calculate the results. The results are shown in Figure 2 below.

The results showed that the positive vaccine TIV had a complete neutralizing effect on influenza virus H1N1 at a dilution of 1:40, and it had a complete neutralizing effect on influenza virus H3N2 at a dilution of 1:160. However, the serum of the control group MF59 could not prevent influenza. Viruses H1N1 and H3N2 damage red blood cells. The H1stem group serum has a complete neutralizing effect on H1N1 and H3N2 at a 1:40-fold dilution. When the serum dilution is 1:1280, some blood cells still remain intact, indicating that the serum is highly diluted. The antibodies 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

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

2.A test group settings: 1. Unimmunized group; 2. Positive control group (inactivated avian influenza virus H9 subtype A/Chicken/Hebei/G/2012 (H9N2 strain)); 3. Test group H9 embedded H1ISA71 vaccine group; 4, single adjuvant control group; 10 animals in each group; B test group settings: 1, non-immunized group; 2, positive control group (commercial Pleco H9 inactivated vaccine 032101001A); 3. Experimental group H9 chimeric H1 white oil vaccine group.

3. Test steps:

3.1 Animal immunity:

Test A: For details on the vaccine preparation process, see 2. Preparation of H9 chimeric H1 ISA71 vaccine in Example 3 (the adjuvant is Montanide ^TM ISA 71VG). The vaccination dose in the control group was used according to the specifications; the injection dose for each animal in the test group was 20ug/feather, and the number of immunizations was 10 pigeons per group. The immunization was 2 times, with an interval of 14 days between each immunization. Blood was collected from the heart 3 weeks after the second immunization. The serum was isolated and used for subsequent experiments.

Test B: For details on the vaccine preparation process, see 3. Preparation of H9 chimeric H1 white oil vaccine in Example 3 (the adjuvant is mineral white oil). The vaccination dose in the control group was used according to the specifications; the injection dose of each animal in the test group was 30ug/feather, and the number of immunizations was 10 pigeons per group. The immunization method was subcutaneous injection in the neck, and the immunization was 2 times, with an interval of 3 weeks between each time. Three weeks after the first immunization, blood was collected from the heart, and serum was separated for subsequent experiments.

3.2 Serum separation:

Collect blood from the heart 3 weeks after the second vaccination, centrifuge at low temperature, collect serum, aliquot and freeze at -80°C.

3.3HI antibody titer detection:

Follow the standard operations specified in the national standard GB/T18936-2020. Add 25 μL of PBS to wells 1-12 of the 96-well microhemagglutination plate, then add 25 μL of serum after 3 weeks of immunity to the first well, and pipette with 25 μL. Doubling dilution to the 10th well, then add 25 μL of 4HAU (four units of virus solution), and set up PBS and hemagglutinin controls; incubate at 37°C for 10 min, add 25 μL of 1% SPF chicken red blood cells to each hole, and mix Incubate at room temperature for 30 minutes, and then calculate the HI antibody titers of SPF chickens in the immunized group and the unimmunized group.

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

3.4 Challenge the experimental animals after immunization

The strain is: H9 subtype WD strain. SPF chickens were challenged with the virus after primary immunization. There were 5 commercial H9 vaccine control groups, 5 unimmunized groups, and 5 experimental groups (H9 chimeric H1 white oil vaccine). Each SPF chicken wing was intravenously injected with avian influenza H9 subtype diluted 1:10. strain (0.5ml/bird), collect the cloaca and throat swabs of each chicken on the 5th day after the virus challenge, conduct virus isolation, and compare the number of positive virus isolations in the immune group and the unimmunized chicken group.

The cloacal and tracheal swabs of the same chicken were mixed and used as one sample. Each sample was inoculated through the allantoic cavity with 5 9- to 11-day-old SPF chicken embryos, 0.2 ml per embryo. Incubate and observe for 5 days, and determine the chicken embryo by embryo. HA titer of embryonic fluid. As long as one of the five chicken embryos inoculated in each sample has an HA titer of embryonic fluid ≥1:16 (micromethod), it can be judged as positive for virus isolation. Samples with negative virus isolation should be blindly passed once before being judged. There should be at least 4 chickens in the immune group with negative virus isolation, and at least 4 chickens in the control group with positive virus isolation.

The results showed that after being challenged with the H9 subtype WD strain according to the protocol, both the Pleco vaccine and the H9 chimeric H1 white oil vaccine group could protect 100% of the immune chickens, and the unimmunized group met the requirements.

Table 1: Result after challenge in immune group and non-immune group

Example 6 Animal Test 3: Immune Protection Test of H9 Chimeric H1 Vaccine on Hy-line White Laying Chickens

1. Experimental animals: 7-day-old Hyline white laying hens, immunized by subcutaneous injection in the neck.

2. Group settings: 1. Single adjuvant control group; 2. Positive control (H9 + Newcastle disease dual vaccine) group; 3. Experimental group H9 chimeric H1 Freund's vaccine group; 7 animals in each of the above groups.

3. Experimental steps:

3.1 Animal immunity:

For details on the process of preparing the vaccine, see 4. Preparation of H9 chimeric H1 Freund's vaccine in Example 3 (the adjuvant is Freund's adjuvant). The immune dose of the single adjuvant control group was 200ul per animal; the immune dose of each animal in the test group was 60ug/200ul/feather, and the protein was diluted with PBS, with 7 pigeons in each group. The immunization method was subcutaneous injection in the neck, 2 times each time The immunization interval was 2 weeks, and blood was collected from the heart 3 weeks after the second immunization, and the serum was separated for subsequent experiments.

3.2 Serum separation:

Collect blood from the heart 3 weeks after the second vaccination, centrifuge at low temperature, collect serum, aliquot and freeze at -80°C.

3.3 ELISA detects specific antibodies in laying hen serum:

a, Pack the plate with 200ng of protein per well overnight at 4°C. The coating proteins are H1 full-length protein, H1 stem protein, H5ORI protein, H9ORI protein, and H9 chimeric H1 protein;

b. Washing: Wash the coated plate with PBST containing 0.05% Tween-20 the next day, wash 4 times, 5 minutes each time;

c. Blocking: Add 200ul of blocking solution (5% skimmed milk powder, prepared with PBST), and block at room temperature for 3 hours;

d. Washing: Wash the plate to be tested with PBST, 4 times, 5 minutes each time;

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 1 hour; PBST is used as a negative control;

f. Washing: Rinse the coated plate with PBST, wash 5 times, 5 minutes each time;

g, Secondary antibody incubation: dilute HRP-labeled goat anti-chicken secondary antibody with PBST (1:30000 dilution), add 100ul to each well, and incubate at 37°C for 1 hour;

h, washing: rinse the coated plate with PBST, wash 5 times, 5 minutes each time;

i. Color development: Add TMB substrate buffer for color development, 50ul/well, develop color for 3 minutes at 37°C in the dark, then add ELISA stop solution to terminate the reaction, 50ul/well, and read at OD _450nm .

The results are shown in Figure 4A and Figure 4B: The difference between the H9 chimeric H1 protein and the H9ORI protein is that the head region is the same but the neck region is different; H1stem is the neck region protein of the influenza H1 subtype, and its conserved sequence is the same as that of the H9 chimeric protein. The neck region of the H1 protein is the same. Figure 4A shows the results of test C, showing that compared with the control group, the serum of the H9 chimeric H1 Freund's vaccine group produced immune responses to influenza H1, H5, and H9 subtypes, indicating that the H9 chimeric H1 Freund's vaccine stimulated the body to produce There are antibodies targeting different influenza HAs in the serum, providing theoretical support for the design of universal influenza vaccines. Figure 4B shows the results of D test, showing that when the packaging protein is H1stem, the serum of the H9 chimeric H1 Freund's vaccine group (Group 3) can stimulate more stimulation than the serum of the positive control group (H9+ Newcastle disease dual vaccine) (Group 2) More neck region antibodies were produced, breaking the usual advantage of binding antibodies in the head region; Comparative findings of the H9 chimeric H1 Freund's vaccine group serum (Group 3) on the package plate protein H9 chimeric H1 protein and H1stem protein The H9 chimeric H1 Freund's vaccine group serum (Group 3) can stimulate more antibodies in the head and neck areas to improve the body's immunity.

3.4 Hemagglutination test

a. Add 25ul PBS to each well of the hemagglutination plate. Add 25ul of inactivated H9N2 subtype avian influenza virus to the first well, perform 2-fold serial dilutions, and set up negative control wells at the same time.

b. Add 25ul of 1% chicken red blood cell suspension to each well, mix on a horizontal oscillator for 1 to 2 minutes, and let stand at 37°C for 30 minutes before judging the result. Results: 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 the H9N2 subtype avian influenza virus is 1:2 ⁷ .

3.5 Hemagglutination inhibition test

Prepare four units of virus liquid based on the results in 3.4;

a. Add 25ul of PBS to each well of the hemagglutination plate, add 25ul of the serum to be tested (original serum) to the first well, and perform 2-fold gradient dilution in sequence, and set up a negative control well (PBS) at the same time;

b. Except for the negative control well, add 25ul of four units of virus solution to each well: place it on a horizontal oscillator for 12 minutes, then let it stand at 37°C for 15 minutes;

c. Add 25ul of 1% chicken red blood cell suspension to each well, mix on a horizontal oscillator for 1 to 2 minutes, and let stand at 37°C for 30 minutes before determining the result.

The results are shown in Figure 5: the H9 chimeric H1 Freund's vaccine (Group 3) has a blood suppression titer of about 5log2 after immunization, and can stimulate more antibodies to neutralize H9N2 compared with the single adjuvant control group (Group 1). Subtype avian influenza virus, and there is no significant difference compared with the commercial H9+ Newcastle disease two-combination vaccine (Group 2), which provides theoretical support for the development of new recombinant protein vaccines.

3.5 Challenge the test animals after immunization

Three weeks after the second vaccination, challenge A/chicken/Shanxi/1.23TGRL003-O/2019H9N2 intranasally, with a challenge dose of 300ul (4HAU) per laying hen. Use cotton swabs to sample the mouth and anus of each chick on the 7th day after the virus challenge, and store them in 1 ml of virus preservation solution. After shaking, inoculate 10-day-old SPF chicken embryos, and collect allantoic fluid 72 hours later to detect the detoxification of the chicken embryos.

Viruses were isolated from pharyngeal and anal swabs collected on the 7th day after virus challenge. In each group, 6 laying hens were selected to inoculate the separated virus liquid from 10-day-old SPF chicken embryos, and 3 replicate groups were set up for each. They were incubated and observed for 72 hours, and the allantoic fluid was collected for HA measurement. Both dead and live embryos should be The hemagglutination value of chicken embryo allantoic fluid was measured, and the positive number of virus isolation in chickens in the immune group and the control group was compared. As long as one of the three chicken embryos inoculated in each sample has an HA titer of embryonic fluid ≥1:16 (micromethod), it can be judged as positive for virus isolation. Samples with negative virus isolation should be blindly passed once before being judged. The results are shown in Table 2.

Table 2: Results after virus challenge in different groups

The results showed that the single adjuvant control group showed that the H9N2 virus could be detected in all samples, while the test vaccine group could inhibit the reproduction of H9N2 virus in 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 It can well resist H9N2 virus invasion.

Claims (10)

1. An H9 subtype avian influenza recombinant chimeric vaccine antigen protein, characterized in that, on the basis of retaining the head structural region in the H9 subtype avian influenza antigen HA skeleton, its neck region is replaced with a broad-spectrum Immune protection is obtained from the influenza HA neck area. 2. The chimeric vaccine antigen protein according to claim 1, wherein the head structure region in the H9 subtype avian influenza antigen HA skeleton is specifically from positions 1 to 307 of the protein amino acid sequence, and the influenza HA neck region Specifically, starting from ATG of the HA protein amino acid sequence, positions 308-516. 3. The chimeric vaccine antigen protein according to claim 1, wherein 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, which has broad-spectrum immune protection. The amino acid sequence of the influenza HA neck region is shown in SEQ ID NO: 2. 4. Nucleic acid encoding the chimeric vaccine antigen protein according to any one of claims 1 to 3. 5. The nucleic acid according to claim 4, wherein the nucleotide sequence of the head structure region in the H9 subtype avian influenza antigen HA skeleton is the 1-921st position of the HA nucleotide sequence; the influenza HA neck The region ranges from positions 922-1548 of the HA nucleotide sequence. 6. The nucleic acid according to claim 5, wherein 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, and the nucleotide sequence of the neck region of the influenza HA is The acid sequence is shown in SEQ ID NO:4. 7. The nucleic acid according to claim 6, wherein its full-length nucleotide sequence is shown in SEQ ID NO: 5 . 8. An expression vector containing the nucleic acid according to any one of claims 5 to 7, preferably the starting vector is a PCAGGS vector. 9. A method for preparing a recombinant chimeric vaccine for H9 subtype avian influenza, which is characterized by comprising the following steps: The first step: synthesize the nucleic acid according to any one of claims 5-7 and the NA gene nucleic acid fragment (preferably, The two are fused with different purification tag peptides) and are respectively connected into the vector after double enzyme digestion (corresponding EcoR Ⅰ and Xho Ⅰ) (specifically, the starting vector is the PCAGGS vector) to construct a recombinant expression vector; Step 2: Co-transfect plasmids HA and NA into 293T mammalian cells preferably at a ratio of 1:1 for secretory expression, collect the supernatant, and purify the target HA protein through affinity chromatography according to the purification tag; Step 3: Emulsify HA protein and adjuvant in proportion (preferably 1:1 in volume) to prepare a recombinant chimeric vaccine. 10. A recombinant chimeric vaccine for H9 subtype avian influenza, characterized in that it includes the chimeric vaccine antigen protein according to any one of claims 1 to 4, and optionally, its adjuvant is Montanide ^TM ISA 71 VG , mineral white oil or Freund's adjuvant, optionally also including pharmaceutically acceptable auxiliaries.