CN116874607B

CN116874607B - A recombinant chimeric vaccine of H9 subtype avian influenza and its preparation method

Info

Publication number: CN116874607B
Application number: CN202310682630.0A
Authority: CN
Inventors: 仝舟; 崔庆为; 杨佳; 邢潇; 刘志达; 田文霞; 张航川
Original assignee: Shanxi Institute Of Higher Innovation; Shanxi Agricultural University
Current assignee: Shanxi Institute Of Higher Innovation; Shanxi Agricultural University
Priority date: 2023-06-09
Filing date: 2023-06-09
Publication date: 2024-12-10
Anticipated expiration: 2043-06-09
Also published as: CN116874607A

Abstract

本发明属于免疫学领域，具体公开一种H9亚型禽流感重组嵌合疫苗及其制备方法。在保留H9亚型禽流感主要保护性抗原HA骨架中的头部结构的同时，将疫苗H9的颈部区替换为具有广谱免疫保护的流感HA颈部区，从而构建重组嵌合HA。本发明的重组嵌合HA与天然H9三聚体蛋白在结构、分子量上保持一致，通过打破头部结构的优势，增强针对保守茎部区域的免疫反应，将免疫应答集中至免疫亚优势的茎部区域，共同作用激发产生更多的保护性抗体。本发明提供获得高纯度、高表达、有活性的H9嵌合H1蛋白的方法，成功实现了对禽流感病毒H9N2亚型毒株入侵的保护性。具有表达产量高，成本简单，工艺安全，能有效应对H9N2流感亚型毒株的传播。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

H9 subtype avian influenza recombinant chimeric vaccine and preparation method thereof

Technical Field

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

Background

The H9N2 subtype avian influenza virus (Avian influenza virus, AIV) belongs to low-pathogenicity AIV according to pathogenicity, but has the advantages of wide host range, high transmission speed and long epidemic time, is extremely easy to cooperatively infect poultry with other bacteria or viruses to cause the reduction of production performance, and seriously affects the economic value brought by poultry. Avian Influenza Virus (AIV) subtype H9N2 is widely distributed worldwide and is generally divided into two major lineages, north american and euryalism. Specifically, the European sub-spectrum is further propagated 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 transmitted in quails, has geographic advantages in the southern region, while BJ/94-like and F/98-like, which are popular in chicken flocks, are dominant in the northern and eastern regions, respectively. Recent epidemiological studies have also suggested that the S genotype is highest. The G57 genotype (equivalent to the S genotype) is more contagious than other genotypes, and has been dominant in china since 2010, causing serious damage to poultry farming. AIVs contained in wild birds tend to be highly pathogenic in transmission across species. The research finds that 6 internal genes of H7N9, H10N8 and H5N6 subtype AIV which are newly appeared in China and cause human infection are all from H9N2 subtype AIV, and the research also provides a powerful environment for the explosion of HPAIV. Therefore, we need to pay 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 guarantee for the public health safety of us.

H9N2 AIV is monitored in many countries, particularly in china and other asian countries. Since 2016 years, most research teams in China sample tens of thousands of strains of HxNy subtype in 37 cities of 23 provinces, cities and national autonomous regions in China, and the strain is identified by second generation sequencing (NGS) to find that H9N2 is the dominant subtype. Serological investigation reports also show that the positive rate of the H9N2 virus antibody in the general population is 1.3% -1.4%, and the positive rate in retail poultry workers is more than 15%, so that the human infection of the H9N2 virus is proved, and the Live Poultry Market (LPM) is found to be an important exposure risk factor for the human infection of the avian influenza virus.

The latest samples collected from 2013 to 2018, combining phylogenetic and antigenic analysis of the H9N2 isolates, found that the most recent isolates were mainly concentrated in subgroup II and subgroup III, far from the vaccine strain (mainly located at subgroup I). The homology of the HA proteins of the different antigen clusters was analyzed to evaluate amino acid residues that may lead to antigen drift. It was found that 11 amino acid residues are conserved in vaccine cluster viruses, but mutations occur in viruses of the subgroup II and subgroup III lineages, whereas 10 mutated amino acid residues are in the HA protein between subgroup II and subgroup III, which mutated amino acids could potentially affect the protective efficacy of the H9N2 AIV inactivated vaccine.

At present, the avian influenza whole virus inactivated vaccine is the vaccine which is most widely applied in the world, and the vaccination is also the main measure for preventing the avian influenza. Shao Hua et al (Shao Hua, wen Guoyuan, luo Ling, etc.. Recombinant Newcastle disease heat-resistant vaccine strain expressing truncated HA protein of H9 subtype avian influenza virus and preparation method thereof, 2016.) select HA1 region (1-1041 nt) with strong immunogenicity in HA protein, insert into Newcastle disease virus heat-resistant vector, can pertinently enhance immune protection effect of vaccine, greatly reduce dependence on cold chain system, can be used for preparing H9 avian influenza, newcastle disease bigeminal heat-resistant live vaccine, also measure pathogenicity of recombinant virus, keep low toxicity characteristic of parent strain, and insertion of exogenous gene does not change pathogenicity of recombinant virus. Krammer and (Krammer F,et al.Chimeric hemagglutinin influenza virus vaccine constructs elicit broadly protective stalk-specific antibodies.J Virol.2013Jun;87(12):6542-50.;Krammer F,et al.H3stalk-based chimeric hemagglutinin influenza virus constructs protect mice from H7N9 challenge.J Virol.2014Feb;88(4):2340-3.) are chimeric by using H1 stems of different epidemic season A type H1N1 influenza viruses, and then cHAs vaccines are constructed by using H5, H6 and H9 influenza virus heads and the chimeric stems respectively. The study shows that the survival rate of mice continuously immunized by the vaccine is 100% after being challenged by various viruses such as H5N1, H6N1, H7N9 and the like, the mice are obviously higher than that of each control group, and high-titer stem reactive antibodies are generated, the groups of mice are sequentially immunized by cHA with H9, H5 and H6 on the heads, and the serum reactivity of the mice to pandemic H1N1 viruses in the bodies of the ferrets is respectively improved by 4 times and 8 times compared with the former 1 after the cHA with H5 and H6 on the heads is inoculated.

Common avian influenza vaccines include whole virus inactivated vaccines, attenuated live vaccines, genetically engineered subunit vaccines, gene recombinant live vector vaccines, nucleic acid vaccines, RNA replicon vaccines, universal influenza vaccines, transgenic plant vaccines and the like, but the avian influenza inactivated vaccines are still used for preventing the infection of poultry by the H9 subtype avian influenza virus in the current farms, but the H9 subtype avian influenza virus is continuously mutated and popular due to antigen drift caused by the immune pressure of the avian influenza virus. The H9N2 subtype avian influenza virus is actively involved in gene rearrangement in other subtype influenza viruses, and the generation of novel influenza viruses such as H5N2, H6N1, H7N7, H7N9, H10N8 and the like threatens human health. The outbreak of H7N9 influenza in 2013 is the result of mixing in chickens, with its external genes from the H7 subtype influenza virus and its internal genes from the H9N2 subtype influenza virus.

In addition, the inactivated vaccine has the problems of large chick embryo quantity, high chick embryo early death rate, small average single embryo harvest quantity, unstable potency, high cost and the like in the production process. The application of inactivated vaccine cannot distinguish naturally infected chickens from vaccinated chickens, so that the monitoring of avian influenza epidemic situation and the investigation of epidemiology are interfered, attenuated live vaccines can be rearranged with other influenza viruses to obtain reassortant viruses with recovered virulence, cold adaptation attenuated live vaccines are found to have pathogenic risks in immunodeficiency persons, genetic engineering vaccines are found to have the defects of short antibody duration, high cost and the like, the efficacy of the recombinant live vector vaccines on immunized chickens can only last for a short time, the use of the chickens which are vaccinated or infected with viral vectors is limited to a certain extent, researchers find that the carrier of the nucleic acid vaccines is provided with antibiotic genes, the prevention and treatment of bacterial diseases can be difficult, and the internal expression efficiency of the nucleic acid vaccines is not high. The ideal avian influenza virus vaccine has the characteristics of high safety for poultry, capability of distinguishing the natural infection of the avian influenza virus and the immunization of the avian by the vaccine, good production safety, long duration and the like, and no vaccine has the characteristics at the same time. The general influenza vaccine is designed to have effects on various influenza subtype viruses in the pandemic stage of avian influenza or in the transitional variation stage of viruses.

In addition, the continuous vaccine immunization can also cause the antigen variation to generate immune escape to different degrees, and the clinical situation that the replacement speed of the inactivated vaccine strain cannot catch up with the pace of the virus antigen variation can also occur, so that the continuous epidemic and variation of the antigen escape strain can be caused, and thus, the avian influenza virus is not effectively controlled, and the continuous epidemic and the spreading range of the virus are also tended to be expanded.

Therefore, the development and evaluation of vaccines with cross-immunity protection effect on antigen variant strains or different subtype avian influenza viruses are very necessary for the prevention and control of H9 subtype viruses.

Disclosure of Invention

The invention constructs a novel recombinant protein vaccine based on the trimeric mammal expression form protein of influenza surface key antigen HA. The recombinant chimeric HA (chimeric HA) is constructed by retaining the head structure in the HA skeleton of the main protective antigen of the H9 subtype avian influenza and simultaneously replacing the neck region of the vaccine H9 with the influenza HA neck region with broad-spectrum immune protection after structural biological design modification. The recombinant chimeric HA is consistent with the natural H9 trimeric protein in structure and molecular weight. The chimeric vaccine breaks the advantages of the head structure, enhances the immune response aiming at the conserved stem region, concentrates the immune response to the immunodominant stem region, and stimulates the generation of more protective antibodies under the combined action.

The present invention thus provides a recombinant chimeric vaccine of H9 subtype avian influenza comprising a head region in the HA backbone of the H9 subtype avian influenza antigen and an influenza HA neck region with broad spectrum immunoprotection. Preferably, from ATG, positions 1-921 are HA head regions, positions 922-1548 are HA neck regions, and positions 922-957 are neck region active peptide structures.

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

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

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

It is also preferred that the amino acid sequence of the influenza HA neck region with broad spectrum immunoprotection 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 structural region in the HA skeleton of the H9 subtype avian influenza antigen is 1-921 th site of the HA nucleotide sequence, and the influenza HA neck region is 922-1548 th site of the HA nucleotide sequence.

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

Also 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 invention thus also provides an expression vector, preferably a PCAGGS vector, comprising said nucleic acid.

The invention further provides a preparation method of the H9 subtype avian influenza recombinant chimeric vaccine, which comprises the following steps:

The first step is to link the nucleic acid with cohesive end synthesized by the complete sequence (specifically, the cohesive end is EcoRI and XhoI) and NA gene nucleic acid fragment (preferably, the two fused different purification tag peptides) into the vector (specifically, the starting vector is PCAGGS vector) after double enzyme cutting (corresponding EcoRI and XhoI) respectively, so as to construct the recombinant expression vector.

And secondly, co-transfecting the plasmids HA and NA into 293T mammalian cells according to the ratio of 1:1, carrying out secretory expression, collecting supernatant, and purifying by an affinity chromatography method according to a purification tag to obtain the target HA protein.

Thirdly, emulsifying the HA protein and the adjuvant according to a proportion (preferably 1:1 by volume) to prepare the recombinant chimeric vaccine.

The invention finally provides, inter alia, a recombinant chimeric vaccine of H9 subtype avian influenza comprising said chimeric vaccine antigen protein, optionally together with pharmaceutically acceptable adjuvants.

The invention designs a set of method for obtaining 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 avian influenza virus H9N2 subtype strains. The H9 chimeric H1 protein has high expression yield, simple cost and safe process, can effectively cope with the transmission of avian influenza H9N2 subtype strains, and provides support for preparing high-efficiency universal avian influenza subunit vaccine.

Drawings

FIG. 1 shows a typical molecular sieve and SDS-PAGE gel of H9 chimeric H1 protein, and the arrow indicates the protein marker 72KD.

FIG. 2H 1N1 strain (A) and H3N2 strain (B) were tested for hemagglutination inhibition for different groups.

FIGS. 3A and 3B show HI antibody detection results. The test group shown in FIG. 3A comprises 1 non-immune group serum, 2 positive control group (inactivated avian influenza virus H9 subtype A/Chicken/Hebei/G/2012 (H9N 2 strain)) serum, 3 test group H9 chimeric H1 ISA 71 vaccine group serum and 4 single-adjuvant control group serum. FIG. 3B test panel set-up of 1, non-immunized panel serum, 2, positive control panel (commercial plakoch H9 inactivated vaccine 032101001A) serum, 3, test panel H9 chimeric H1 white oil vaccine panel serum;

FIGS. 4A and 4B ELISA detect specific antibodies in chicken serum. Wherein, FIG. 4A shows the immune response of the positive control group (H9 + newcastle disease bigeminal vaccine) serum and the test group H9 chimeric H1 Freund vaccine group serum to different influenza HA, and the plate-coated proteins are H1 full-length protein, H5ORI protein and H9ORI protein. FIG. 4B shows the immune response of Shan Zuoji control group serum (group 1), positive control (H9 + newcastle disease bigeminal) group serum (group 2), test group H9 chimeric H1 Freund's vaccine group serum (group 3) to the plate protein H9 chimeric H1 protein, H1 stem protein, H9ORI protein.

FIG. 5 shows the results of the blood inhibition test.

Figure 6 results of antibody titer statistics. Wherein, 1, a single adjuvant control group serum, 2, a positive control (H9 + newcastle disease bigeminal vaccine) group serum, 3, an experimental group H9 chimeric H1 Freund vaccine group serum.

Detailed Description

The following examples further illustrate the invention but are not to be construed as limiting the invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention.

The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated.

Example H9 subtype HA Gene and NA Gene plasmid construction to aid HA expression

The designed nucleic acid sequence of HA or NA was sent to the company for full sequence synthesis of DNA fragments with cohesive ends (cohesive ends were EcoRI and XhoI cleavage sites). The vector is PCAGGS vector with ampicillin resistance, double enzyme cutting is carried out on the vector, DNA fragment synthesized by complete sequence is connected into the vector, the following HA gene and NA gene nucleic acid fragments (fused with different purification tag peptides) with cohesive ends (specifically, the cohesive ends are EcoRI and XhoI) synthesized by complete sequence are respectively connected into PCAGGS vector after double enzyme cutting (corresponding EcoRI and XhoI), and recombinant expression vector is constructed.

The HA gene sequence structure is EcoRI enzyme cutting site, kozak sequence, signal peptide sequence, HA head region sequence, HA neck region sequence, thrombin enzyme cutting site sequence, trimer tag sequence, histidine tag sequence and XhoI enzyme cutting site.

EcoRI cleavage site GAATTC;

kozak sequence GCCACC;

Signal peptide amino acid sequence METVSLITILLVVTVSNA

ATGGAGACAGTATCACTAATAACTATACTACTAGTAGTAACAGTAAGCAAT GCA signal peptide nucleotide sequence;

Thrombin cleavage site amino acid sequence LVPRGS

The nucleotide sequence of thrombin cleavage site is CTGGTGCCAAGAGGCTCT;

A trimer tag sequence CCTGGCAGCGGCTATATTCCTGAGGCTCCCAGAGATGGCCAGGCCTACGTTAGA AAGGATGGCGAGTGGGTGCTGCTGAGCACCTTTCTGGGA;

A histidine tag amino acid sequence HHHHHH;

a histidine tag nucleotide sequence CACCACCACCATCACCAC;

XhoI cleavage site, CTCGAG.

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

HA head region nucleotide sequence (SEQ ID NO：3)：ATGGAGACAGTATCACTAATAACTATACTACTAGTAG TAACAGTAAGCAATGCAGATAAAATCTGCATCGGCTATCAATCAACAAACTCCACAGAAACTGTAGACACACTAACAGAAAACAACGTCCCTGTGACACATGCCAAAGAATTGCTCCACACAGAGCATAATGGGATGCTGTGTGCAACAAGCTTGGGACACCCTCTTATTCTAGACACCTGTACCATTGAAGGACTAATCTATGGCAATCCTTCTTGTGATCTATTGTTGGGAGGAAGAGAATGGTCCTATATCGTCGAGAGACCATCAGCTGTTAACGGATTGTGTTATCCCGGGAATGTAGAAAATCTAGAAGAGCTAAGGTCACTTTTTAGTTCTGCTAGGTCTTATCAAAGGATCCAGATTTTCCCAGACACAATCTGGAATGTGTCTTACAGTGGGACAAGCAAAGCATGTTCAGATTCATTCTACAGAAGCATGAGATGGTTGACTCAAAAGAACAATGCTTACCCTATTCAAGACGCCCAATACACAAATAATCAAGAAAAGAACATTCTTTTCATGTGGGGCATAAATCACCCACCCACCGATACTGCGCAGACAAATCTGTACACAAGAACCGACACAACAACGAGTGTGGCAACAGAAGAAATAAATAGGACCTTCAAACCATTGATAGGACCAAGGCCTCTTGTCAACGGTTTGCAGGGAAGAATTGATTATTATTGGTCGGTATTGAAACCGGGTCAAACACTGCGAATAAGATCTAATGGGAATCTAATAGCTCCATGGTATGGACACATTCTTTCAGGAGAGAGCCACGGAAGAATCCTGAAGACTGATTTAAAAAGGGGTAGCTGCACAGTGCAATGTCAGACAGAAAAAGGTGGATTAAACACAACATTGCCATTCCAAAACGTAAGT

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

KYAIGDCPKYVKQNTLKLATGLRNIPSIQSRGLFGAIAGFTEGGWTGMVDGLYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQYTAVGKEFNKSERRMENLNKKVDDGKIDLWSYNAELLVALENQHTIDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKR

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

AAGTATGCCATCGGCGACTGCCCCAAATACGTGAAGCAGAATACCCTGAAGCTGGCCACCGGCCTGAGAAACATCCCCAGCATCCAGAGCAGAGGCCTGTTCGGAGCCATTGCCGGCTTTACTGAAGGCGGCTGGACAGGCATGGTGGATGGCCTGTATGGCTATCACCACCAGAATGAGCAAGGCAGCGGATACGCCGCTGACCAGAAGTCTACCCAGAACGCTATCAATGGCATCACCAACAAAGTGAACTCCGTGATCGAGAAGATGAACACCCAGTACACCGCCGTGGGCAAAGAGTTCAACAAGAGCGAGCGGCGGATGGAAAACCTGAACAAGAAGGTGGACGACGGCAAGATCGACCTGTGGTCCTACAATGCCGAACTGCTGGTGGCCCTGGAAAACCAGCACACCATCGACTTCCACGACAGCAACGTGAAGAACCTGTACGAGAAAGTGAAGTCCCAGCTGAAGAACAACGCCAAAGAGATCGGCAACGGCTGCTTCGAGTTCTACCACAAGTGCAACGACGAGTGCATGGAAAGCGTGAAGAATGGCACCTACGACTACCCCAAGTACAGCGAGGAATCCAAGCTGAACCGCGAGAAAATCGACGGCGTGAAGAGA.

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

ATGGCCAGGCCTACGTTAGAAAGGATGGCGAGTGGGTGCTGCTGAGCACCTTTCTGGGACACCACCACCATCACCACTGACTCGAG。

The NA gene sequence structure is EcoRI enzyme cutting site, signal peptide sequence, flag tag sequence, tetramer tag sequence, thrombin enzyme cutting site sequence, 09NA sequence and XhoI enzyme cutting site.

EcoRI cleavage site GAATTC;

The amino acid sequence of the signal peptide is MGAGATGRAMDGPRLLLLLLLGVSLGGA;

ATGGGGGCAGGTGCCACCGGCCGCGCCATGGACGGGCCGCGCCTGCTGCTG TTGCTGCTTCTGGGGGTGTCCCTTGGAGGTGCC signal peptide nucleotide sequence;

flag tag amino acid sequence DYKDDDDK;

the Flag tag nucleotide sequence is GATTATAAGGATGATGATGATAAG;

the amino acid sequence of the tetramer tag is SSSDYSDLQRVKQELLEEVKKELQKVKEEIIEAFVQELRKRGS;

Tetramer tag nucleotide sequence ：AGCTCCAGTGATTACTCGGACCTACAGAGGGTGAAACAGGAGCTTCTG GAAGAGGTGAAGAAGGAATTGCAGAAAGTGAAAGAGGAAATCATTGAAGCCTTCGTCCAGGAGCTG AGGAAGCGGGGTTCT;

The thrombin cleavage site amino acid sequence is LVPRGS;

The nucleotide sequence of thrombin cleavage site is CTGGTACCACGAGGTAGT;

09NA amino acid sequence (SEQ ID NO：6)：PSRSVKLAGNSSLCPVSGWAIYSKDNSVRIGSKGDVFVIRE PFISCSPLECRTFFLTQGALLNDKHSNGTIKDRSPYRTLMSCPIGEVPSPYNSRFESVAWSASACHDGINWLTIGISGPDNGAVAVLKYNGIITDTIKSWRNNILRTQESECACVNGSCFTVMTDGPSNGQASYKIFRIEKGKIVKSVEMNAPNYHYEECSCYPDSSEITCVCRDNWHGSNRPWVSFNQNLEYQIGYICSGIFGDNPRPNDKTGSCGPVSSNGANGVKGFSFKYGNGVWIGRTKSISSRNGFEMIWDPNGWTGTDNNFSIKQDIVGINEWSGYSGSFVQHPELTGLDCIRPCFWVELIRGRPKENTIWTSGSSISFCGVNSDTVGWSWPDGAELPFTIDK;

09NA nucleotide sequence (SEQ ID NO：7)：CCATCACGATCAGTGAAATTAGCGGGCAATTCCTCTCTCT GCCCTGTTAGTGGATGGGCTATATACAGTAAAGACAACAGTGTAAGAATCGGTTCCAAGGGGGATGTGTTTGTCATAAGGGAACCATTCATATCATGCTCCCCCTTGGAATGCAGAACCTTCTTCTTGACTCAAGGGGCCTTGCTAAATGACAAACATTCCAATGGAACCATTAAAGACAGGAGCCCATATCGAACCCTAATGAGCTGTCCTATTGGTGAAGTTCCCTCTCCATACAACTCAAGATTTGAGTCAGTCGCTTGGTCAGCAAGTGCTTGTCATGATGGCATCAATTGGCTAACAATTGGAATTTCTGGCCCAGACAATGGGGCAGTGGCTGTGTTAAAGTACAACGGCATAATAACAGACACTATCAAGAGTTGGAGAAACAATATATTGAGAACACAAGAGTCTGAATGTGCATGTGTAAATGGTTCTTGCTTTACTGTAATGACCGATGGACCAAGTAATGGACAGGCCTCATACAAGATCTTCAGAATAGAAAAGGGAAAGATAGTCAAATCAGTCGAAATGAATGCCCCTAATTATCACTATGAGGAATGCTCCTGTTATCCTGATTCTAGTGAAATCACATGTGTGTGCAGGGATAACTGGCATGGCTCGAATCGACCGTGGGTGTCTTTCAACCAGAATCTGGAATATCAGATAGGATACATATGCAGTGGGATTTTCGGAGACAATCCACGCCCTAATGATAAGACAGGCAGTTGTGGTCCAGTATCGTCTAATGGAGCAAATGGAGTAAAAGGGTTTTCATTCAAATACGGCAATGGTGTTTGGATAGGGAGAACTAAAAGCATTAGTTCAAGAAACGGTTTTGAGATGATTTGGGATCCGAACGGATGGACTGGGACAGACAATAACTTCTCAATAAAGCAAGATATCGTAGGAATAAATGAGTGGTCAGGATATAGCGGGAGTTTTGTTCAGCATCCAGAACTAACAGGGCTGGATTGTATAAGACCTTGCTTCTGGGTTGAACTAATCAGAGGGCGACCCAAAGAGAACACAATCTGGACTAGCGGGAGCAGCATATCCTTTTGTGGTGTAAACAGTGACACTGTGGGTTGGTCTTGGCCAGACGGTGCTGAGTTGCCATTTACCATTGACAAGTAA;

XhoI cleavage site, CTCGAG.

EXAMPLE II expression purification of subtype H9 HA protein

Top10 competent cells were transformed with the recombinant expression vector plasmid, an appropriate amount of bacterial liquid was pipetted and streaked onto solid LB plates (LB plate formulation: 1% NaCl,1% tryptophan Tryptone,0.5% yeast extract, 1.5% agar powder; ampicillin use 1:1000) with ampicillin resistance, and cultured overnight at 37 ℃. The monoclonal, 5ml of mini-shake (ampicillin resistance) was picked, followed by shaking in 300ml overnight, and influenza virus antigen plasmid was obtained using endotoxinfree plasmid large extraction kit (TIANGEN, DP 117).

293T cells were cultured in DMEM containing 10% FBS at 37℃in a 5% CO ₂ incubator. When 293T cells reached about 70% confluence, influenza virus antigen plasmids (HA: na=20ug: 20ug/disc) were transfected into 293T cells with PEI transfection reagent, and when 4-6 hours post-transfection, cell culture supernatants were collected after 3 days of continued culture with serum-free DMEM exchange, supplemented with serum-free DMEM for 4 days of continued culture, and cell culture supernatants were collected. Expression of neuraminidase NA is also performed because HA is expressed in mammalian cells, to which sialic acid will adhere, resulting in no binding capacity to the receptor. NA was only responsible for the expression of the HA protein, and the 09NA gene was selected and FLAG tagged for detection. Because the purification tags of HA and NA are different, we choose the affinity column with His tag to purify HA protein, so that the finally purified protein is only HA protein, we verify that the HA+NA co-transfection is more than the HA protein obtained by HA single transfection through western blot, and in order to purify a large amount of HA protein, the designed HA is secreted and expressed by adopting the mode of co-transfection with NA.

The collected cell culture supernatant was filtered through a 0.22 μm filter and combined with HisTrap ^TM excel (GE) overnight at 4 ℃ to prepare protein elution buffer a (20mM Tris,150mM NaCl,pH 8.0) and buffer B (20mM Tris,150mM NaCl,pH 8.0,1M imidazole). Washing His column with buffer solution A to remove non-specific binding protein, removing impurity protein with buffer solution 2% B solution (20mM Tris,150mM NaCl,pH 8.0,20mM imidazole), eluting target protein from His column with 30% B solution (20mM Tris,150mM NaCl,pH 8.0,300mM imidazole), changing solution with 30 KD-trapped (30 KD cutoff) protein concentration tube with buffer solution A (20mM Tris,150mM NaCl,pH 8.0) to remove imidazole concentration in protein solution, concentrating protein solution to 0.5ml, adding thrombin (1 mg protein plus 5ul thrombin), and enzyme cutting at 4deg.C overnight. The digested protein solution was further purified using molecular sieves, column equilibrated with buffer solution A (20mM Tris,150mM NaCl,pH 8.0) using AKTA-pulser (GE) and Column Hiload 16/60superdex 200PG molecular sieves (GE), 1ml loop loaded, and UV absorbance at 280nm was monitored to collect the protein of interest, and protein purity was identified by SDS-PAGE. Typical molecular sieve patterns and SDS-PAGE analysis of the target protein are shown in FIG. 1.

The peak position of the protein is 62.5ml in a Column Hiload 16/60superdex 200PG molecular sieve, and because a trimer tag is added after a thrombin cleavage site (LVPR ∈, an arrow indicates an amino acid capable of recognizing and cleaving the site) in construction, the main peak of the protein is in a trimer form, the trimer tag falls off from HA after thrombin cleavage, and the monomer size is about 70KD. SDS-PAGE gel lanes correspond to trimer and monomer of H9 chimeric H1 protein, respectively, in the molecular sieve plot.

Example three preparation of vaccine

The H9 chimeric H1 protein obtained by purification is mixed with different adjuvants to prepare a vaccine, and the vaccine is emulsified according to the volume ratio of 1:1 until the water is in a non-bath state for animal immunization. H1 The stem protein is a neck region protein of influenza H1 subtype, HAs a broad-spectrum immunoprotection effect, and on the basis of the broad-spectrum immunoprotection effect, the H9 chimeric H1 protein is designed, and HAs an immune response effect on different influenza HA, and the detail is shown in a sixth example. The specific vaccine preparation method is shown in the third embodiment, the influenza HA neck region (H1 stem) function verification with broad-spectrum immune protection is shown by an immune mouse experiment, the specific embodiment is shown in the fourth embodiment, the prepared H9 chimeric H1 vaccine immune SPF chicken experiment is shown in the fifth embodiment, and the prepared H9 chimeric H1 vaccine immune sea blue white layer chicken experiment is shown in the sixth embodiment.

1. Preparation of H1stem vaccine

H1 The concentration of the stent protein is 10mg/ml, PBS is a diluent, and the immunological adjuvant is MF59. Test group H1 stem protein was mixed with an equal volume of MF59 adjuvant and shaken until completely emulsified in the bath for immunization of the animals.

The amino acid sequence of H1stem vaccine (SEQ ID NO: 8) wherein positions 194-262 are conserved peptide segments ：MYRMQLLSCIALSLALVTNSTYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSKQSQGLFGAIAGFTEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQYTAIGCEYNKSERCMKQIEDKIEEIESKIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQIEGRHHHHHHH

The nucleotide sequence of an H1stem vaccine (SEQ ID NO: 9), wherein positions 580-786 are conserved peptide segments:

ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACGAATTCAACCTACGCCGACACCATTTGCATCGGCTACCACGCCAACAACAGCACCGACACCGTGGACACCGTGCTGGAGAAGAACGTGACCGTGACCCACAGCGTGAATCTGCTGGAGAACGGAGGAGGCGGCAAATACGTCTGCAGCGCCAAACTGAGGATGGTGACCGGACTGAGGAACAAGCCCAGCAAGCAGAGCCAGGGACTGTTCGGAGCCATTGCCGGATTCACCGAGGGAGGTTGGACAGGAATGGTGGACGGTTGGTACGGCTACCACCACCAGAACGAGCAGGGAAGCGGATACGCCGCCGATCAGAAAAGCACCCAGAACGCCATCAACGGCATCACCAACAAGGTCAACAGCGTGATCGAGAAGATGAACACCCAGTACACCGCCATCGGTTGCGAGTACAACAAGAGCGAGCGCTGCATGAAGCAGATCGAGGACAAGATCGAGGAGATCGAGAGCAAGATCTGGTGCTACAACGCCGAACTGCTGGTGCTGCTGGAGAACGAGAGGACCCTGGACTTCCACGACAGCAACGTGAAGAACCTGTACGAGAAGGTCAAGAGCCAGCTGAAGAACAACGCCAAGGAGATCGGCAACGGCTGCTTCGAGTTCTACCACAAGTGCAACGACGAGTGCATGGAGAGCGTGAAGAACGGCACCTACGACTACCCCAAGTACAGCGAGGAGAGCAAGCTGAACCGGGAGAAGATCGACGGCGTGAAGCTGGAGAGCATGGGCGTGTACCAGATCGAGGGCAGACATCACCACCACCACCATCATTAG.

2. Preparation of H9 chimeric H1ISA71 vaccine (Montanide ^TM ISA71 VG as adjuvant)

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

The adjuvant and the aqueous antigen medium are prepared into vaccine according to the mass ratio of 7:3, the aqueous antigen medium is added into Montanide ^TM ISA 71VG at room temperature or below, and mixed under strong stirring to obtain a stable preparation, animal inoculation is carried out, labeling is carried out, and the storage is carried out at the temperature of 2-8 ℃.

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

The concentration of H9 chimeric H1 protein is 10mg/ml, PBS is a diluent, and the immunoadjuvant is mineral white oil. The oil phase is prepared by heating 94 parts of white oil for injection and 2 parts of aluminum stearate to 80 ℃, adding 80-80 parts of span until the temperature reaches 116 ℃, maintaining for 30min, and cooling for later use. Preparing water phase, namely taking 4 parts of Tween-80, sterilizing, cooling, adding the Tween-80 into 96 parts of inactivated antigen liquid, and stirring while adding until the Tween-80 is completely dissolved. And (3) emulsifying, namely taking 2 parts of oil phase, starting a motor to stir slowly, then slowly adding 1 part of water phase, and emulsifying for 30min at 2800-3200 r/min after the water phase is added. Quantitatively split charging, capping, sealing, labeling, and storing at 2-8 ℃.

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

The concentration of H9 chimeric H1 protein is 10mg/ml, PBS is a diluent, and the immunological adjuvant is Freund's adjuvant. Completely emulsifying the H9 chimeric H1 protein and the Freund complete adjuvant with the same volume until the bath water is not changed for animal inoculation, wherein Freund complete adjuvant is adopted for the first immunization, and Freund incomplete adjuvant is adopted for the second immunization.

Example four animal experiments 1 influenza HA neck region (H1 stem) functional verification with broad-spectrum immunoprotection

1. Test animals, 4-6 week old BALB/c female mice, 6 animals per group, were immunized by intramuscular injection.

2. Group settings ① negative control (adjuvant MF 59), ② positive control (influenza virus split vaccine TIV), ③ test group H1stem vaccine.

3. The test steps are as follows:

3.1 immunization of animals:

preparation of vaccine procedure reference is made to the preparation of the 1.H1 stem vaccine of example three. The immunization dose of the positive group was 100. Mu.L each, the immunization dose of each animal of the test group was 20. Mu.g/100. Mu.L/each animal, the proteins were diluted with PBS, 6 animals of each group were immunized 3 times, and the immunization interval was 14 days.

3.2 Serum separation:

Mice of the above immunization group were subjected to eyeball blood collection to prepare serum, the mouse serum was treated with RED, and 4 volumes of RDE were added according to 1 volume of serum, 37 ℃ water bath for 18h, and 56 ℃ inactivated for 30min for subsequent experiments.

3.3H1N1 strain and H3N2 strain TCID ₅₀ assay

A, diluting the virus stock by adopting a 10-time serial dilution mode.

B, 96-well plates were plated 18-24h in advance, and 100. Mu.L (2X 10 ⁵/ml) of MDCK cells were added to each well. Culturing was performed in a 37℃and 5% CO ₂ incubator.

C, virus inoculation, washing the cells 1 time with PBS, 150 mu L of each well, adding the diluted virus solution according to the first step into a 96-well plate, inoculating one longitudinal row of each dilution, 100 mu L of each well, setting a dilution control in 11 columns and 12 columns, and culturing in a 37 ℃ incubator.

And d, observing and recording the results day by day, and calculating the results after 72 hours according to the Reed-Muench method.

3.4 Neutralization experiment:

a, virus dilution, namely, diluting the virus culture solution to 200TCID ₅₀/50 mu L.

B, seronegative control (cell maintenance solution only)

Serum dilution the initial dilution factor of the mouse serum is 1:40, and neutralization test is carried out according to dilution ratio of 2 times.

And C, incubating the viruses, namely adding 50 mu l of diluted viruses (200 TCID ₅₀) into 50 mu l of serum, uniformly mixing, standing at 37 ℃ and incubating for 1h.

And d, adsorbing MDCK cells, namely washing 150 mu L of MDCK cells once by using PBS, discarding, adding 100 mu L of virus-serum mixed solution into each hole, and culturing in a cell culture box for 72 hours in parallel three holes.

E, results were obtained by observation and calculation using the blood inhibition test, and the results are shown in FIG. 2.

The results show that the positive vaccine TIV has complete neutralization effect on influenza virus H1N1 under the dilution of 1:40, has complete neutralization effect on influenza virus H3N2 under the dilution of 1:160, and the serum of the control group MF59 can not prevent the damage of influenza virus H1N1 and H3N2 to erythrocytes, the serum of the H1stem group has complete neutralization effect on H1N1 and H3N2 under the dilution of 1:40, and partial blood cells still remain intact under the dilution of serum of 1:1280, so that the antibody under the high dilution of serum can still prevent the damage of influenza virus H1N1 and H3N2 to erythrocytes, and the H1stem can generate specific antibodies aiming at influenza virus H1N1 and H3N2, thereby providing reference data for research and development of general vaccines. EXAMPLE five animal test 2 immunoprotection test of H9 chimeric H1 vaccine against SPF chickens

1. Test animals were 3 week old SPF chickens immunized by subcutaneous injection in the neck.

The test group A comprises 1, a non-immune group, 2, a positive control group (inactivated avian influenza virus H9 subtype A/Chicken/Hebei/G/2012 (H9N 2 strain)), 3, a test group H9 chimeric H1ISA71 vaccine group, 4, a single adjuvant control group, 10 groups each, a test group B comprises 1, a non-immune group, 2, a positive control group (commercial Protect H9 inactivated vaccine 032101001A), and 3, a test group H9 chimeric H1 white oil vaccine group.

3. The test steps are as follows:

3.1 immunization of animals:

A test shows that the preparation process of the vaccine is detailed in the preparation of the 2.H9 chimeric H1ISA71 vaccine in the third embodiment (the adjuvant is Montanide ^TM ISA71 VG). The immunization dose of the control group is used according to the specification, the injection dose of each animal of the test group is 20 ug/feather, the immunization amount is 10 feather parts of each group, the immunization is carried out for 2 times, the immunization interval is 14 days, the heart is used for blood sampling after 3 weeks of the 2 nd immunization, and serum is separated for the subsequent experiment.

B test, the vaccine preparation process is detailed in the preparation of the 3.H9 chimeric H1 white oil vaccine in the third embodiment (the adjuvant is mineral white oil). The immunization dose of the control group is used according to the specification, the injection dose of each animal of the test group is 30 ug/feather, the immunization quantity of each animal is 10 feather parts, the immunization mode is neck subcutaneous injection, the immunization is performed for 2 times, the immunization interval is 3 weeks, the heart is used for blood sampling after the 3 th immunization for 3 weeks, and serum is separated for subsequent experiments.

3.2 Serum separation:

after 3 weeks of second-time, heart blood is collected, and the blood is centrifuged at low temperature, serum is collected and sub-packaged for freezing storage at-80 ℃.

3.3HI antibody titer detection:

25. Mu.L of PBS was added to 1-12 wells of 96-well microplates, 25. Mu.L of serum after 3 weeks of immunization was added to 1-well, diluted to 10-well with a 25. Mu.L pipette, 25. Mu.L of 4HAU (four unit virus liquid) was added, PBS and a hemagglutinin control were used, and the mixture was allowed to act at 37℃for 10min, 25. Mu.L of 1% SPF chicken erythrocytes were added per air, and after mixing, the mixture was allowed to act at room temperature for 30min, and then the SPF chicken HI antibody titers of the immunized and non-immunized groups were calculated.

The results are shown in FIG. 3A, wherein the test results of A are shown in FIG. 3A, the test results of HI antibody detection titers of the non-immunized group (group 1) and Shan Zuoji control group (group 4) are 0, the HI antibody titer of the positive vaccine group (group 2) is about 11log2, the HI antibody titer of the test group H9 chimeric H1 ISA 71 vaccine group (group 3) is about 11log2, and the average value of the antibody titers of the chimeric vaccine of the invention is slightly higher than that of the positive vaccine group (group 2), so that the chimeric vaccine designed by the invention can induce more antibodies to neutralize H9N2 strain as effectively as commercial vaccine. FIG. 3B shows the results of B experiments, wherein the HI antibody titer after the second immunization is higher than that of the first immunization, the titer of the commercial H9 vaccine (group 2) is very high, the HI antibody titer of the H9 chimeric H1 white oil vaccine (group 3) after the second immunization is remarkably increased, and compared with the commercial H9 vaccine, the HI antibody titer is not remarkably different, and the H9N2 subtype avian influenza virus infection can be well neutralized.

3.4 Challenge after immunization of test animals

The strain is H9 subtype WD strain. After the SPF chickens are subjected to post-immune virus challenge, 5 commercial H9 vaccine control groups, 5 non-immune groups and 5 test groups (H9 chimeric H1 white oil vaccine) are respectively, 1:10 diluted avian influenza H9 subtype strains (0.5 ml/chicken) are intravenously injected into the wings of each SPF chicken, cloaca and throat swabs of each chicken are collected on the 5 th day after the virus challenge, virus separation is carried out, and the virus separation positive numbers of the immune group and the non-immune group chicken are compared.

Mixing cloaca and tracheal swabs of the same chicken to obtain 1 sample, inoculating 5 SPF chick embryos of 9-11 days old into each sample through an allantoic cavity, incubating and observing 0.2ml of each embryo for 5 days, and determining the HA titer of the chick embryo liquid embryo by embryo. The virus isolation positive can be judged as long as the HA titer of 1 chick embryo liquid in 5 chick embryos inoculated in each sample is more than or equal to 1:16 (micro method). Samples negative for virus isolation should be blinded 1 time before being judged. The immune group should have at least 4 chicken virus isolation negative, and the control group should have at least 4 chicken virus isolation positive.

The results show that the Proteus Ke Yimiao and H9 chimeric H1 white oil vaccine groups can protect immunized chickens by 100% after the virus is challenged by H9 subtype WD strains according to the regulations, and the challenged and non-immune groups meet the requirements.

TABLE 1 results after challenge in immunized and non-immunized groups

EXAMPLE six animal test 3 test of immunoprotection of H9 chimeric H1 vaccine against Heilanthan white layer

1. The test animals are 7-day old sea-blue white laying hens, and the immunization mode is neck subcutaneous injection.

2. The groups are 1, a single adjuvant control group, 2, a positive control (H9 + newcastle disease bigeminal vaccine) group, 3, a test group H9 chimeric H1 Freund vaccine group, and 7 groups.

3. The experimental steps are as follows:

3.1 immunization of animals:

the preparation of the vaccine is described in detail in the preparation of 4.H9 chimeric H1 Freund's vaccine of example III (Freund's adjuvant). Shan Zuoji control group immunity doses of 200ul each, test group animals have immunity doses of 60ug/200 ul/feather, PBS is used for diluting protein, 7 feather parts of each group are subcutaneously injected into the neck, the immunization is carried out for 2 times, each immunization is separated by 2 weeks, heart blood is collected after 3 weeks of the second immunization, and serum is separated for subsequent experiments.

3.2 Serum separation:

3.3ELISA detection of specific antibodies in hen serum:

a, coating the plate at 4 ℃ overnight according to the amount of 200ng per hole protein, wherein the coating plate protein is H1 full-length protein, H1 stem protein, H5ORI protein, H9ORI protein and H9 chimeric H1 protein;

b, washing, namely washing the coated plate by using PBST containing 0.05% Tween-20 for 4 times each time for 5min the next day;

c, sealing, namely adding 200ul of sealing liquid (5% skimmed milk powder, prepared by PBST) and sealing for 3 hours at room temperature;

Washing, namely washing the board to be detected by PBST for 4 times, wherein each time is 5min;

e, primary antibody incubation, namely diluting serum to be detected by PBST (1:50 and 1:100), adding 100ul of serum diluent to be detected into each hole, and incubating at 37 ℃ for 1h;

f, washing, namely washing the coated plate by PBST for 5 times, and 5 minutes each time;

g, secondary antibody incubation, namely diluting the HRP-marked goat anti-chicken secondary antibody with PBST (diluted 1:30000), adding 100ul of secondary antibody per well, and incubating for 1h at 37 ℃;

washing, namely washing the coated plate by PBST for 5 times, and 5 minutes each time;

i, developing, namely adding TMB substrate buffer solution for developing, 50 ul/hole, and after developing for 3min at 37 ℃ in a dark place, adding ELISA stopping solution for stopping reaction, and reading at OD _450nm at 50 ul/hole.

As a result, as shown in FIGS. 4A and 4B, the H9 chimeric H1 protein differs from the H9ORI protein in that the head region is the same and the neck region is different, and H1stem is a neck region protein of influenza H1 subtype, the conserved sequence of which is the same as that of the H9 chimeric H1 protein. Fig. 4A shows the results of C experiments, showing that the serum of the H9 chimeric H1 furs vaccine group immunoreacts with influenza H1, H5, H9 subtypes, compared with the control group, demonstrating that the serum produced by the H9 chimeric H1 furs vaccine after immunization elicits antibodies against different influenza HA in the serum produced by the body, providing theoretical support for the realization of the design of the universal influenza vaccine. FIG. 4B shows the results of the D test, wherein when the plate coating protein is H1stem, the H9 chimeric H1 Freund vaccine group serum (3 rd group) can excite more cervical region antibodies than the positive control group serum (H9 + Newcastle disease bigeminal vaccine) (2 nd group), so that the inertial superiority of the head region binding antibodies is broken, and the comparison of the H9 chimeric H1 Freund vaccine group serum (3 rd group) to the plate coating protein H9 chimeric H1 protein and the H1stem protein shows that the H9 chimeric H1 Freund vaccine group serum (3 rd group) can excite more head region and cervical region antibodies to improve the immunity of the organism.

3.4 Hemagglutination assay

A, 25ul of PBS was added to each well of the platelets. The first well was added with 25ul of inactivated H9N2 subtype avian influenza virus, serial dilutions were performed 2-fold in sequence, and negative control wells were established.

B, adding 25ul of 1% chicken erythrocyte suspension into each hole, oscillating on a horizontal oscillator for 1-2 min, uniformly mixing, standing at 37 ℃ for 30min, and judging the result. Results: virus most aggregated at 100% ((+ +++)) the large dilution is the hemagglutination value of the virus, namely an agglutination unit, and the hemagglutination titer of the H9N2 subtype avian influenza virus is 1:2 ⁷.

3.5 Hemagglutination inhibition assay

Preparing four units of virus liquid according to the result in 3.4;

a, adding 25ul of PBS into each hole of a blood coagulation plate, adding 25ul of serum to be detected (original serum) into a first hole, sequentially carrying out 2-time gradient dilution, and simultaneously setting a negative control hole (PBS);

b, adding 25ul of four-unit virus liquid into each hole except the negative control hole, placing the mixture on a horizontal oscillator for 12min, and standing at 37 ℃ for 15min;

And C, adding 25ul of 1% chicken erythrocyte suspension into each hole, oscillating on a horizontal oscillator for 1-2 min, uniformly mixing, standing at 37 ℃ for 30min, and judging the result.

Results are shown in FIG. 5, wherein the H9 chimeric H1 Freund's vaccine (group 3) has about 5log2 of blood inhibitory potency, can excite more antibodies to neutralize H9N2 subtype avian influenza virus compared with Shan Zuoji control group (group 1), has no obvious difference compared with commercial H9+ newcastle disease bigeminy vaccine (group 2), and provides theoretical support for the research and development of novel recombinant protein vaccine.

3.5 Challenge after immunization of test animals

After 3 weeks of the second immunization, the nasal drops attack A/chicken/Shanxi/1.23TGRL003-O/2019H9N2 with 300ul (4 HAU) of toxin per layer. Sampling the mouth and anus of each chicken on day 7 after virus challenge by using a cotton swab, storing in 1ml of virus preservation solution, inoculating 10-day-old SPF chick embryo after shaking, and collecting allantoic fluid after 72 hours to detect the toxin expelling condition of the chick embryo.

Viral isolation was performed on the pharyngeal and anal swabs collected on day 7 post challenge. And inoculating the separated virus liquid of 6 laying hen samples into 10-day-old SPF chick embryos, setting 3 repeated groups for each group, incubating and observing for 72 hours, collecting allantoic fluid for HA determination, determining the allantoic fluid hemagglutination value of the chick embryo no matter dead embryo or living embryo, and comparing the virus separation positive numbers of the immunized group and the control group. The virus isolation positive can be judged as long as the HA titer of 1 chick embryo liquid in 3 chick embryos inoculated in each sample is more than or equal to 1:16 (micro method). Samples negative for virus isolation should be blinded 1 time before being judged. The results are shown in Table 2.

TABLE 2 results after different groups of challenge

The result shows that Shan Zuoji control groups show that samples can detect H9N2 viruses, and compared with the control groups, the test vaccine groups can inhibit the propagation of H9N2 viruses in the laying hen sample, and the effect is consistent with that of a positive vaccine group, so that the vaccine designed by the invention can well resist the invasion of H9N2 viruses.

Claims

1. An H9 subtype avian influenza recombinant chimeric vaccine antigen protein is characterized in that the neck region is replaced by an influenza HA neck region with broad-spectrum immune protection on the basis of retaining a head structure region in an HA skeleton of the H9 subtype avian influenza antigen;

The amino acid sequence of the head structure region in the HA skeleton of the H9 subtype avian influenza antigen is shown as SEQ ID NO. 1, and the amino acid sequence of the neck region of the influenza HA is shown as SEQ ID NO. 2;

The amino acid sequence of the H9 subtype avian influenza recombinant chimeric vaccine antigen protein is SEQ ID NO. 1 and SEQ ID NO. 2 in sequence from the N end to the C end.

2. A nucleic acid encoding the chimeric vaccine antigen protein of claim 1.

3. The nucleic acid of claim 2, wherein the nucleotide sequence of the head structural region in the HA skeleton of the H9 subtype avian influenza antigen 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 of claim 3, wherein the full length nucleotide sequence is set forth in SEQ ID NO. 5.

5. An expression vector comprising the nucleic acid of any one of claims 2 to 4.

6. The expression vector of claim 5, wherein the starting vector is PCAGGS.

7. The preparation method of the H9 subtype avian influenza recombinant chimeric vaccine is characterized by comprising the following steps:

The first step, the nucleic acid fragment of the nucleic acid with the sticky end and the NA gene synthesized by the complete sequence is respectively connected into a vector after double enzyme digestion to construct a recombinant expression vector;

Secondly, co-transfecting 293T mammalian cells with plasmids HA and NA, carrying out secretory expression, collecting supernatant, and purifying by an affinity chromatography method according to a purification tag to obtain a target HA protein;

And thirdly, emulsifying the HA protein and an adjuvant to prepare the recombinant chimeric vaccine.

8. The method of claim 7, wherein the mass ratio of the plasmids HA and NA in the second step is 1:1 and the volume ratio of HA protein to adjuvant in the third step is 1:1.

9. The method of claim 7, wherein the vector is PCAGGS vector and the cohesive ends are EcoR I and Xho I.

10. An H9 subtype avian influenza recombinant chimeric vaccine comprising the chimeric vaccine antigen protein of claim 1, and a pharmaceutically acceptable adjuvant.

11. The recombinant chimeric H9 subtype avian influenza vaccine according to claim 10, wherein the adjuvant is Montanide ^TM ISA 71 VG, mineral white oil or freund's adjuvant.