CN113150083B - Recombinant avian influenza subunit vaccine and preparation method thereof - Google Patents

Abstract

The invention relates to the technical field of vaccine preparation, in particular to a recombinant avian influenza subunit vaccine and a preparation method thereof. The invention reserves the head structure in the main protective antigen HA skeleton of the H5 subtype avian influenza, and replaces the neck region of the vaccine H5 with the influenza HA neck region with broad-spectrum immune protection after structural biological design and modification, and the neck region can protect mutant strains of different H5 and provide broad-spectrum protection for H7 in mouse experiments.

Description

Recombinant avian influenza subunit vaccine and preparation method thereof

Technical Field

The invention relates to the technical field of vaccine preparation, in particular to a recombinant avian influenza subunit vaccine and a preparation method thereof.

Background

Influenza viruses (Influenza viruses) belong to the orthomyxoviridae family and are single-stranded, negative-stranded, segmented RNA viruses. Influenza viruses can be classified into A, B, C, D four types according to the difference of antigenic determinants on Nucleoprotein (NP) and matrix protein (M1), wherein type a and type B can pose a threat to human health, especially type a, approximately 30 to 50 million people die each year due to global seasonal influenza and sporadic influenza, and the death rate is up to 60%. According to the influenza virus infected subjects, the viruses can be classified into groups such as human influenza virus, swine influenza virus, equine influenza virus, and avian influenza virus. Avian influenza is a kind of zoonosis infectious disease caused by avian influenza virus, belongs to influenza A virus, and wild poultry are natural hosts of the influenza A virus, and are divided into three categories of high pathogenicity, low pathogenicity and non-pathogenicity according to different pathogenicity of the influenza virus to the poultry. Avian Influenza Virus (AIV) was first reported in italy by perronicito (1878) in 1878 and was subsequently discovered in several countries and regions of the world.

Highly Pathogenic Avian Influenza (HPAIV) has been classified as a group A virulent infectious disease by the International Bureau of veterinary medicine. The avian influenza virus subtypes which can directly infect humans have been found to be: H5N1, H7N2, H7N3, H7N7, H9N2 and H7N9 subtypes. Since 1905, avian influenza pathogen was identified, high-virulence avian influenza (HPAIV) caused by H5 subtype and H7 subtype was developed many times in the world, and it was found that high-virulence strain is formed after HA gene mutation of low-virulence strain (LPAIV) widely prevalent in nature, and avian influenza virus generally HAs no pathogenicity to wild birds but often HAs high pathogenicity when spread across species. Thus, the occurrence of low pathogenic avian influenza, which is occasionally transmitted to poultry, is not negligible for any form of avian influenza, including the persistent presence in waterfowl and wildbird bodies.

At present, HA protein is widely applied to the research and development of subunit vaccine, and research shows that HA protein expressed in various expression systems such as bacteria, insect cells, mammal cells and the like HAs activity, and the development of genetic engineering vaccine by using HA protein plays an important role in the prevention and control of avian influenza. Researchers believe that the head globular structure of the HA protein is the main protective antigen for avian influenza. The HA antibody titer can reflect the resistance of the body to the avian influenza virus to some extent. With the rapid development of genetic engineering technology, researchers clone HA genes onto eukaryotic expression vectors, and transform the HA genes into eukaryotes or cells for expression to obtain influenza subunit vaccines, thereby further completing the research on animal immunity.

Fan Wenhui and the like express and purify H5HA protein by using a pichia pastoris expression system, and the immune efficacy of an animal is evaluated after different doses of H5HA and different adjuvants are combined for an immune test. Test results show that H5HA HAs better immunogenicity, can induce SPF chickens to generate higher-level IgG, and HAs the hemagglutination inhibition titer of 1:64, virus neutralizing antibody titer 1:218, the research result provides theoretical support for the development of subunit vaccine of the H5 subtype influenza virus. Zhou Chenchen and the like use H7-HA protein purified by an insect baculovirus expression system to immunize SPF chickens, then a virus attacking protection test is carried out, the immune protection effect of the protein on the SPF chickens is evaluated, and the result shows that HA protein immune group chickens only obtain 100% protection. Research shows that the H-HA and L-HA recombinant proteins expressed by Sf9 cells are prepared into oil emulsion vaccines to immunize SPF (specific pathogen free) chickens of 14 days old. After immunization, blood is collected by the veins under the wings at 14, 21 and 28 days, the titer of the antibody is detected by hemagglutination inhibition test (HI) and ELISA test, and the result shows that each group after immunization can stimulate the organism to generate higher antibody. The law of change of CD3+/CD4+ and CD3+/CD8+ T lymphocyte subpopulations and lymphocyte transformation assays were examined by FCAS on days 3, 5, 7, 10 after immunization. The results show that the subpopulation proportion of CD3+/CD4+ and CD3+/CD8+ T lymphocytes of the 2 recombinant protein group chickens is slightly higher than that of the inactivated vaccine group, and lymphocyte transformation experiments also show that the lymphocyte transformation efficiency of the 2 recombinant protein group chickens is slightly higher than that of the conventional inactivated vaccine group. The results show that the vaccine prepared from the recombinant protein can stimulate the organism to generate humoral immunity and cellular immunity, has better immunogenicity, and has a slightly better comprehensive effect than the prior H5 subtype avian influenza inactivated vaccine.

However, most of the vaccines in the prior art are directed against only one subtype, and cannot have broad spectrum. Generally, multivalent vaccines are prepared by inactivating multiple viruses or recombining multiple fragments separately. The production cost is high, and the production period is long. Therefore, the avian influenza vaccine which is further developed and produced with low cost and can simultaneously stimulate the organism to generate immunity aiming at various subtype viruses has important significance.

Disclosure of Invention

In view of the above, the technical problem to be solved by the present invention is to provide a recombinant avian influenza subunit vaccine and a preparation method thereof, wherein the vaccine has broad-spectrum immunity.

The present invention provides an HA subunit having an amino acid sequence as shown in SEQ ID NO 1.

The present invention provides a nucleic acid encoding an HA subunit having the sequence shown in SEQ ID NO 2.

The invention provides an HA recombinant antigen, which comprises the following components connected in sequence: signal peptide fragments, HA subunits, thrombin fragments, trimer-tags and screenable markers as described herein.

Wherein the amino acid sequence of the signal peptide fragment is MKVKLLVLLCTFTATYA.

The amino acid sequence of the HA subunit is shown in SEQ ID NO. 1.

DQICIGYSANNSTEQVDTIMEKNVTVTQAQDILEKKHNGKLCDLDGVKPLILRD CSVAGWLLGNPMCDEFINVPEWSYIVEKANPVNDLCYPGDFNDYEELKHLLSRINHFEK IQIIPKSSWSSHEASLGVSSACPYQGKSSFFRNVVWLIKKDSTYPTIKRSYNNTNQEDLLV LWGIHHPNDAAEQTKLYQNPTTYISVGTSTLNQRLVPRIATRSKVKGLSGRMEFFWTIL KPNDAINFESNGNFIAPEYAYKIVKKGDSTIMKSELEYGNCNTKCQTPMGAINSSMPFHN IHPLTIGECPKYVKSNRLVLAIGLRNSPQRETQGLFGAIAGFTEGGWTGMVDGLYGYHH QNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQYTAVGKEFNKSERRMENLNKK VDDGFIDIWTYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYH KCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKR

The amino acid sequence of the thrombin fragment is LVPRGS.

The amino acid sequence of the trimer tag is GYIPEAPRDGQAYVRKDGEWVLLSTFLG.

The screening tag is a 6 × His tag, HHHHHH.

In the invention, the amino acid sequence of the HA recombinant antigen is shown as SEQ ID NO. 3.

ATMKVKLLVLLCTFTATYADQICIGYSANNSTEQVDTIMEKNVTVTQAQDILEKKH NGKLCDLDGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPVNDLCYPGDF NDYEELKHLLSRINHFEKIQIIPKSSWSSHEASLGVSSACPYQGKSSFFRNVVWLIKKDST YPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQNPTTYISVGTSTLNQRLVPRIATR SKVKGLSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTIMKSELEYGNCNT KCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNRLVLAIGLRNSPQRETQGLFGAIAGFTE GGWTGMVDGLYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQYTAVG KEFNKSERRMENLNKKVDDGFIDIWTYNAELLVLLENERTLDFHDSNVKNLYEKVKSQ LKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKRLVPRG SPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHH*

Nucleic acid encoding a recombinant HA antigen having the sequence shown in SEQ ID NO. 4.

gccaccatgaaggtcaagctgctggtcctcctctgcaccttcaccgccacctacgccgaccagatttgcatcggctactccgccaac aacagcaccgagcaggtggacaccatcatggagaagaacgtgacagtgacccaagcccaggatatcctggagaagaagcacaacggc aagctctgcgacctggacggagtgaaacccctgatcctgagggattgtagcgtggcaggttggctgctgggaaaccccatgtgcgacgag ttcatcaacgtccccgagtggagctatatcgtggagaaggccaacccagtgaacgacctctgctacccaggcgacttcaacgactacgag gagctgaagcacctgctgagccggatcaaccacttcgagaagatccagatcatccccaagagctcttggagcagccacgaagcctctctg ggagtgtctagcgcctgtccttaccagggcaagagcagcttcttccggaacgtcgtctggctgatcaagaaggatagcacctaccccacca tcaagcggagctacaacaacaccaaccaggaggacctgctcgtgctgtggggcattcaccaccctaacgacgcagccgagcagaccaa gctgtaccagaaccctaccacctacatcagcgtgggcacctctaccctgaaccagagactggtgcccaggatcgccaccagaagcaaagt gaagggcctgagcggccggatggagttcttttggaccatcctgaagcccaacgacgccatcaacttcgagagcaacggcaacttcatcgc ccccgagtacgcctacaagatcgtgaagaagggcgacagcaccatcatgaagagcgagctggagtacggcaactgcaacaccaagtgc cagacccctatgggcgccatcaacagcagcatgcccttccacaacatccaccccctgaccattggcgagtgtcctaagtacgtgaagagca accgcctggtgctggctatcggcctgagaaacagcccccagagagagacacaaggcctgttcggagccattgccggattcacagagggc ggttggacaggcatggtggacggactgtacggataccaccaccagaacgagcagggatcaggatacgccgccgatcagaagagcacc cagaacgccatcaacggcatcaccaacaaggtcaacagcgtgatcgagaagatgaacacccagtacaccgccgtgggcaaggagttca acaagagcgagcggcggatggagaacctgaacaagaaggtggacgacggcttcatcgacatttggacctacaacgccgagctgctggt gctgctggaaaacgagaggaccctggacttccacgacagcaacgtgaagaacctgtacgagaaggtcaagagccagctgaagaacaac gccaaggagatcggcaacggctgcttcgagttctaccacaagtgcaacgacgagtgcatggagagcgtgaagaacggcacctacgacta ccccaagtacagcgaggagagcaagctgaaccgggagaagatcgacggcgtgaagagactggtgccaagaggctctccaggaagcg gctacatcccagaagcccctagagacggacaggcttacgtgcgaaaagacggcgagtgggtgctgctgagcacatttctgggacaccatc atcaccaccactag

Wherein the nucleic acid sequence encoding the signal peptide fragment is atgaaggtcaagctgctggtcctcctctgcacctt caccgccacctacgcc.

The nucleic acid sequence encoding the HA subunit is shown in SEQ ID NO. 2.

gaccagatttgcatcggctactccgccaacaacagcaccgagcaggtggacaccatcatggagaagaacgtgacagtgacccaa gcccaggatatcctggagaagaagcacaacggcaagctctgcgacctggacggagtgaaacccctgatcctgagggattgtagcgtggc aggttggctgctgggaaaccccatgtgcgacgagttcatcaacgtccccgagtggagctatatcgtggagaaggccaacccagtgaacga cctctgctacccaggcgacttcaacgactacgaggagctgaagcacctgctgagccggatcaaccacttcgagaagatccagatcatccc caagagctcttggagcagccacgaagcctctctgggagtgtctagcgcctgtccttaccagggcaagagcagcttcttccggaacgtcgtct ggctgatcaagaaggatagcacctaccccaccatcaagcggagctacaacaacaccaaccaggaggacctgctcgtgctgtggggcatt caccaccctaacgacgcagccgagcagaccaagctgtaccagaaccctaccacctacatcagcgtgggcacctctaccctgaaccagag actggtgcccaggatcgccaccagaagcaaagtgaagggcctgagcggccggatggagttcttttggaccatcctgaagcccaacgacg ccatcaacttcgagagcaacggcaacttcatcgcccccgagtacgcctacaagatcgtgaagaagggcgacagcaccatcatgaagagc gagctggagtacggcaactgcaacaccaagtgccagacccctatgggcgccatcaacagcagcatgcccttccacaacatccaccccct gaccattggcgagtgtcctaagtacgtgaagagcaaccgcctggtgctggctatcggcctgagaaacagcccccagagagagacacaag gcctgttcggagccattgccggattcacagagggcggttggacaggcatggtggacggactgtacggataccaccaccagaacgagcag ggatcaggatacgccgccgatcagaagagcacccagaacgccatcaacggcatcaccaacaaggtcaacagcgtgatcgagaagatga acacccagtacaccgccgtgggcaaggagttcaacaagagcgagcggcggatggagaacctgaacaagaaggtggacgacggcttca tcgacatttggacctacaacgccgagctgctggtgctgctggaaaacgagaggaccctggacttccacgacagcaacgtgaagaacctgt acgagaaggtcaagagccagctgaagaacaacgccaaggagatcggcaacggctgcttcgagttctaccacaagtgcaacgacgagtg catggagagcgtgaagaacggcacctacgactaccccaagtacagcgaggagagcaagctgaaccgggagaagatcgacggcgtgaa gaga

The nucleic acid sequence encoding the thrombin fragment is ctggtgccaagaggctct.

The nucleic acid sequence encoding the trimeric tag is ggctacatcccagaagcccctagagacg gacaggcttacgtgcgaaaagacggcgagtgggtgctgctgagcacatttctggga.

The screening marker is a 6 XHis tag, and the coding nucleic acid sequence of the screening marker is caccatcatcaccaccac.

The invention also provides an NA subunit having an amino acid sequence as shown in SEQ ID NO. 5.

PSRSVKLAGNSSLCPVSGWAIYSKDNSVRIGSKGDVFVIREPFISCSPLECRTFFLTQ GALLNDKHSNGTIKDRSPYRTLMSCPIGEVPSPYNSRFESVAWSASACHDGINWLTIGIS GPDNGAVAVLKYNGIITDTIKSWRNNILRTQESECACVNGSCFTVMTDGPSNGQASYKI FRIEKGKIVKSVEMNAPNYHYEECSCYPDSSEITCVCRDNWHGSNRPWVSFNQNLEYQI GYICSGIFGDNPRPNDKTGSCGPVSSNGANGVKGFSFKYGNGVWIGRTKSISSRNGFEMI WDPNGWTGTDNNFSIKQDIVGINEWSGYSGSFVQHPELTGLDCIRPCFWVELIRGRPKE NTIWTSGSSISFCGVNSDTVGWSWPDGAELPFTIDK

Nucleic acid encoding an NA subunit having the sequence shown in SEQ ID NO 6.

The invention also provides a NA recombinant antigen comprising, in sequential linkage: signal peptide fragments, screening tags, tetramer tags, thrombin fragments and NA subunits as described herein.

Wherein the amino acid sequence of the signal peptide fragment is MGAGATGRAMDGPRLLLLLLLGVSLGGA.

The screening tag is a Flag tag sequence, and the amino acid sequence is DYKDDDDK.

The amino acid sequence of the NA subunit is shown in SEQ ID NO. 5.

PSRSVKLAGNSSLCPVSGWAIYSKDNSVRIGSKGDVFVIREPFISCSPLECRTFFLTQ GALLNDKHSNGTIKDRSPYRTLMSCPIGEVPSPYNSRFESVAWSASACHDGINWLTIGIS GPDNGAVAVLKYNGIITDTIKSWRNNILRTQESECACVNGSCFTVMTDGPSNGQASYKI FRIEKGKIVKSVEMNAPNYHYEECSCYPDSSEITCVCRDNWHGSNRPWVSFNQNLEYQI GYICSGIFGDNPRPNDKTGSCGPVSSNGANGVKGFSFKYGNGVWIGRTKSISSRNGFEMI WDPNGWTGTDNNFSIKQDIVGINEWSGYSGSFVQHPELTGLDCIRPCFWVELIRGRPKE NTIWTSGSSISFCGVNSDTVGWSWPDGAELPFTIDK

The nucleic acid sequence of the tetramer tag is

agctccagtgattactcggacctacagagggtgaaacaggagcttctggaagaggtgaagaaggaattgcagaaagtgaaagaggaaat cattgaagccttcgtccaggagctgaggaagcggggttct。

The nucleic acid sequence of the thrombin fragment is ctggtgccaagaggctct.

In the invention, the amino acid sequence of the NA recombinant antigen is shown as SEQ ID NO. 7.

ATMGAGATGRAMDGPRLLLLLLLGVSLGGADYKDDDDKSSSDYSDLQRVKQELL EEVKKELQKVKEEIIEAFVQELRKRGSLVPRGSPSRSVKLAGNSSLCPVSGWAIYSKDNS VRIGSKGDVFVIREPFISCSPLECRTFFLTQGALLNDKHSNGTIKDRSPYRTLMSCPIGEVP SPYNSRFESVAWSASACHDGINWLTIGISGPDNGAVAVLKYNGIITDTIKSWRNNILRTQ ESECACVNGSCFTVMTDGPSNGQASYKIFRIEKGKIVKSVEMNAPNYHYEECSCYPDSS EITCVCRDNWHGSNRPWVSFNQNLEYQIGYICSGIFGDNPRPNDKTGSCGPVSSNGANG VKGFSFKYGNGVWIGRTKSISSRNGFEMIWDPNGWTGTDNNFSIKQDIVGINEWSGYSG SFVQHPELTGLDCIRPCFWVELIRGRPKENTIWTSGSSISFCGVNSDTVGWSWPDGAELP FTIDK*

The invention also provides a nucleic acid for encoding the NA recombinant antigen, which has a sequence shown as SEQ ID NO. 8.

gccaccatgggggcaggtgccaccggccgcgccatggacgggccgcgcctgctgctgttgctgcttctgggggtgtcccttgga ggtgccgattataaggatgatgatgataagagctccagtgattactcggacctacagagggtgaaacaggagcttctggaagaggtgaaga aggaattgcagaaagtgaaagaggaaatcattgaagccttcgtccaggagctgaggaagcggggttctctggtaccacgaggtagtccat cacgatcagtgaaattagcgggcaattcctctctctgccctgttagtggatgggctatatacagtaaagacaacagtgtaagaatcggttccaa gggggatgtgtttgtcataagggaaccattcatatcatgctcccccttggaatgcagaaccttcttcttgactcaaggggccttgctaaatgaca aacattccaatggaaccattaaagacaggagcccatatcgaaccctaatgagctgtcctattggtgaagttccctctccatacaactcaagatt tgagtcagtcgcttggtcagcaagtgcttgtcatgatggcatcaattggctaacaattggaatttctggcccagacaatggggcagtggctgt gttaaagtacaacggcataataacagacactatcaagagttggagaaacaatatattgagaacacaagagtctgaatgtgcatgtgtaaatgg ttcttgctttactgtaatgaccgatggaccaagtaatggacaggcctcatacaagatcttcagaatagaaaagggaaagatagtcaaatcagt cgaaatgaatgcccctaattatcactatgaggaatgctcctgttatcctgattctagtgaaatcacatgtgtgtgcagggataactggcatggct cgaatcgaccgtgggtgtctttcaaccagaatctggaatatcagataggatacatatgcagtgggattttcggagacaatccacgccctaatg ataagacaggcagttgtggtccagtatcgtctaatggagcaaatggagtaaaagggttttcattcaaatacggcaatggtgtttggataggga gaactaaaagcattagttcaagaaacggttttgagatgatttgggatccgaacggatggactgggacagacaataacttctcaataaagcaa gatatcgtaggaataaatgagtggtcaggatatagcgggagttttgttcagcatccagaactaacagggctggattgtataagaccttgcttct gggttgaactaatcagagggcgacccaaagagaacacaatctggactagcgggagcagcatatccttttgtggtgtaaacagtgacactgt gggttggtcttggccagacggtgctgagttgccatttaccattgacaagtaa

Wherein the nucleic acid sequence encoding the signal peptide fragment is atgggggcaggtgccaccggccgcg ccatggacgggccgcgcctgctgctgttgctgcttctgggggtgtcccttggaggtgcc.

The screening tag is a Flag tag sequence, the amino acid sequence is DYKDDDDK, and the nucleic acid sequence for coding the tag is gattataaggatgatgatgataag.

The nucleic acid sequence encoding the NA subunit is shown in SEQ ID NO 6.

ccatcacgatcagtgaaattagcgggcaattcctctctctgccctgttagtggatgggctatatacagtaaagacaacagtgtaagaat cggttccaagggggatgtgtttgtcataagggaaccattcatatcatgctcccccttggaatgcagaaccttcttcttgactcaaggggccttg ctaaatgacaaacattccaatggaaccattaaagacaggagcccatatcgaaccctaatgagctgtcctattggtgaagttccctctccataca actcaagatttgagtcagtcgcttggtcagcaagtgcttgtcatgatggcatcaattggctaacaattggaatttctggcccagacaatggggc agtggctgtgttaaagtacaacggcataataacagacactatcaagagttggagaaacaatatattgagaacacaagagtctgaatgtgcat gtgtaaatggttcttgctttactgtaatgaccgatggaccaagtaatggacaggcctcatacaagatcttcagaatagaaaagggaaagatag tcaaatcagtcgaaatgaatgcccctaattatcactatgaggaatgctcctgttatcctgattctagtgaaatcacatgtgtgtgcagggataact ggcatggctcgaatcgaccgtgggtgtctttcaaccagaatctggaatatcagataggatacatatgcagtgggattttcggagacaatccac gccctaatgataagacaggcagttgtggtccagtatcgtctaatggagcaaatggagtaaaagggttttcattcaaatacggcaatggtgtttg gatagggagaactaaaagcattagttcaagaaacggttttgagatgatttgggatccgaacggatggactgggacagacaataacttctcaa taaagcaagatatcgtaggaataaatgagtggtcaggatatagcgggagttttgttcagcatccagaactaacagggctggattgtataagac cttgcttctgggttgaactaatcagagggcgacccaaagagaacacaatctggactagcgggagcagcatatccttttgtggtgtaaacagt gacactgtgggttggtcttggccagacggtgctgagttgccatttaccattgacaagtaa

The nucleic acid sequence encoding the tetramer tag is ctggtaccacgaggtagt.

The nucleic acid sequence encoding the thrombin fragment is ctggtaccacgaggtagt.

The invention also provides recombinant vectors comprising nucleic acids encoding recombinant antigens of HA.

The skeleton vector of the recombinant vector is a PCAGGS vector, and the insertion sites of the nucleic acid fragment are EcoRI and Xho I.

The invention also provides recombinant vectors comprising nucleic acids encoding NA recombinant antigens.

The skeleton vector of the recombinant vector is a PCAGGS vector, and the insertion sites of the nucleic acid fragment are EcoRI and Xho I.

The invention also provides host cells transformed or transfected with a recombinant vector comprising a nucleic acid encoding a recombinant antigen of HA.

In the present invention, the host cell is also transformed or transfected with a recombinant vector comprising a nucleic acid encoding a NA recombinant antigen.

In some embodiments, the host cell is a 293T cell

The invention also provides a recombinant HA protein which is obtained by the expression of the host cell.

The recombinant HA protein is obtained by expressing nucleic acid encoding the recombinant HA antigen by host cells, and the nucleic acid segment carries a trimer tag, so that the formed protein HAs a trimer structure, and the trimer tag is separated from the HA protein after Thrombin enzyme digestion. The host expresses NA simultaneously and is used for assisting the expression and secretion of HA, thereby greatly improving the expression quantity of HA and improving the immune effect.

The invention also provides a vaccine comprising the recombinant HA protein of the invention.

The vaccine also comprises an adjuvant, wherein the adjuvant is Freund's complete adjuvant, freund's incomplete adjuvant or MF59 adjuvant.

In the vaccine, the concentration of the recombinant HA protein is 10mg/ml.

The invention also provides application of the vaccine in preparation of a preparation for preventing and treating avian influenza.

The invention also provides a method for preventing and treating avian influenza, which is to administer the vaccine provided by the invention.

The avian influenza comprises H1 subtype avian influenza, H5 subtype avian influenza or H9 subtype avian influenza.

The avian influenza is avian influenza of poultry. In some embodiments, the avian influenza is avian influenza of chickens.

The invention reserves the head structure in the main protective antigen HA skeleton of the H5 subtype avian influenza, and replaces the neck region of the vaccine H5 with the influenza HA neck region with broad-spectrum immune protection after structural biological design and modification, and the neck region is proved to be capable of protecting different subtypes of avian influenza viruses in animal experiments.

Drawings

FIG. 1 shows a PCAGGS no-load map;

FIG. 2 shows a molecular sieve map and SDS-PAGE gel map of typical H5/H1-LSQ protein (A) and H5/H1 full-length protein (B);

in FIG. 3, A, B is the binding kinetics of antibody FL6V3 to H5/H1-LSQ (A) and H5/H1 (B) protein surface plasmon resonance; C. d is the binding H5/H1-LSQ (C) and H5/H1 (D) protein surface plasmon resonance kinetic curves of antibody CR 6261. The abscissa is the binding dissociation time in seconds(s); the ordinate is the bonding Response strength of the chip surface in units of RU (i.e., response unit);

FIG. 4 shows the results of neutralization experiments of the immune group sera against influenza virus H5N 8;

FIG. 5 shows the results of neutralization experiments of the immune group sera against influenza virus H1N 1;

FIG. 6 shows ELISA detection of specific antibodies in chicken sera, including the envelope proteins H5/H1 full-length protein (A), H5/H1-LSQ chimeric protein (B), and H9 protein (C); wherein, the column numbers 1-7 are respectively 1: H5/H1-LSQ chimeric vaccine immune serum, 2: H5/H1 full-length immune serum, 3: blank serum, 4: h9+ newcastle disease dual vaccine immune serum, 5: h5+ H7 bivalent vaccine immune serum, 6: PBS immune serum, 7: an irrelevant antibody strep negative control;

FIG. 7 shows the operation of the virus hemagglutination assay and the H9 virus titer;

FIG. 8A shows the results of a blood suppression test;

FIG. 8B shows statistics of antibody titers, where columns 1-5 are 1: H5/H1-LSQ chimeric seedlings; 2, a H5/H1 vaccine; 3, H9+ Newcastle disease bigeminy vaccine; 4, H5, H7 combined seedlings; 5, PBS immunization group;

FIG. 9 shows the virus titers after H9 challenge in H5/H1-LSQ chimeric vaccine, H9+ Newcastle disease dual vaccine, H5/H1 vaccine, and H5+ H7 dual vaccine immunized chicks for 2 weeks, 4 days after H9 challenge, and 10 days after H9 challenge detected by hemagglutination assay.

Detailed Description

The invention provides a recombinant avian influenza subunit vaccine and a preparation method thereof, and a person skilled in the art can use the content of the vaccine for reference and appropriately improve process parameters for realization. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the method and application of the present invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the method and application described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of the present invention without departing from the spirit and scope of the invention.

The test materials adopted by the invention are all common commercial products and can be purchased in the market.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. With regard to the definitions and nomenclature in this field, the expert can refer in particular to Current protocols in Molecular Biology (Ausubel). Abbreviations for amino acid residues are standard 3-letter and/or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

The H5 subtype Avian Influenza Virus (AIV) is a serious harm to the development of poultry industry in China. The influenza virus has a structure of a capsule membrane, a protein shell and a nucleocapsid from outside to inside in sequence. The surface of the virion HAs three membrane proteins, hemagglutinin (HA), neuraminidase (NA) and Matrix2 (M2) proteins.

The HA presents a tightly coiled structure at the proximal end of the viral envelope as the HA neck region, and the domain at the end distal to the surface of the envelope presents a globular structure as the HA head region, which HAs a receptor binding site. The HA precursor protein molecule HA0 is split into HA1 and HA2 by host protease, the HA head region is composed of most HA1 and is the main antigen target, a small part of HA1 and HA2 is composed of the HA neck region, HA1 is less conservative than HA2, but most of the neutralizing antibody generated after natural infection or influenza vaccine inoculation is directed to the HA head region.

The HA subunit is designed according to the nucleic acid sequence of the H5 subtype avian influenza virus, and the immune organism with the fragment can generate broad-spectrum protection, thereby being effective to the H5 subtype influenza virus and having good protection effect on the H1 subtype and the H9 subtype.

The invention constructs a novel recombinant protein vaccine based on the independently developed trimeric mammal expression form protein of the influenza surface key antigen HA. The head structure in the main protective antigen HA skeleton of the H5 subtype avian influenza is reserved, and meanwhile, the neck region of the vaccine H5 is replaced by the influenza HA neck region which is subjected to structural biological design and modification and HAs broad-spectrum immune protection, and the neck region is proved to be capable of providing broad-spectrum protection for different subtypes of avian influenza viruses in animal experiments. The new recombinant protein vaccine is consistent with the natural H5 tripolymer protein in structure and molecular weight. The mammalian cell expression system can be continuously adopted to express and purify the recombinant trimeric protein, so that the production cost is reduced, the limitation of virus culture required by inactivated vaccine is broken, and the safety is ensured to a certain extent.

The invention is further illustrated by the following examples:

example 1

1. H5 subtype HA gene and NA gene plasmid construction:

a DNA fragment for encoding HA or NA is subjected to double enzyme digestion (EcoRI and XhoI cleavage sites) and is connected into a PCAGGS vector (a map is shown as a figure 1), a signal peptide sequence is added in front of the encoding fragment of the protein to facilitate protein secretion expression, and a coding sequence of 6 histidine tags (hexa-His-tag) and a translation termination codon are inserted behind the encoding fragment of the protein, so that a recombinant protein with the histidine tag at the C-terminal end is obtained, and subsequent protein purification is facilitated. The recombinant plasmid is verified to be completely correct by PCR, enzyme digestion identification and sequencing. When HA is expressed in mammalian cells, sialic acid is adhered to HA, so that the HA cannot be combined with a receptor, the expression of NA is carried out, and a gene of 09NA is selected and is added with a FLAG label for detection.

HA gene sequence (full length shown as SEQ ID NO: 4): ecoRI cleavage site- -Signal peptide sequence- -HA Gene sequence- -Thrombin site- -trimer tag- -His tag-XhoI

NA gene sequence (full length shown as SEQ ID NO: 8): ecoRI cleavage site- -Signal peptide sequence- -Flag tag DYKDDDD- -tetramer tag- -Thrombin cleavage site- -09NA sequence- -XhoI cleavage site

The HA gene sequence is as follows:

gaccagatttgcatcggctactccgccaacaacagcaccgagcaggtggacaccatcatggagaagaacgtgacagtgacccaagccca ggatatcctggagaagaagcacaacggcaagctctgcgacctggacggagtgaaacccctgatcctgagggattgtagcgtggcaggtt ggctgctgggaaaccccatgtgcgacgagttcatcaacgtccccgagtggagctatatcgtggagaaggccaacccagtgaacgacctct gctacccaggcgacttcaacgactacgaggagctgaagcacctgctgagccggatcaaccacttcgagaagatccagatcatccccaaga gctcttggagcagccacgaagcctctctgggagtgtctagcgcctgtccttaccagggcaagagcagcttcttccggaacgtcgtctggctg atcaagaaggatagcacctaccccaccatcaagcggagctacaacaacaccaaccaggaggacctgctcgtgctgtggggcattcacca ccctaacgacgcagccgagcagaccaagctgtaccagaaccctaccacctacatcagcgtgggcacctctaccctgaaccagagactgg tgcccaggatcgccaccagaagcaaagtgaagggcctgagcggccggatggagttcttttggaccatcctgaagcccaacgacgccatc aacttcgagagcaacggcaacttcatcgcccccgagtacgcctacaagatcgtgaagaagggcgacagcaccatcatgaagagcgagct ggagtacggcaactgcaacaccaagtgccagacccctatgggcgccatcaacagcagcatgcccttccacaacatccaccccctgaccat tggcgagtgtcctaagtacgtgaagagcaaccgcctggtgctggctatcggcctgagaaacagcccccagagagagacacaaggcctgt tcggagccattgccggattcacagagggcggttggacaggcatggtggacggactgtacggataccaccaccagaacgagcagggatc aggatacgccgccgatcagaagagcacccagaacgccatcaacggcatcaccaacaaggtcaacagcgtgatcgagaagatgaacacc cagtacaccgccgtgggcaaggagttcaacaagagcgagcggcggatggagaacctgaacaagaaggtggacgacggcttcatcgac atttggacctacaacgccgagctgctggtgctgctggaaaacgagaggaccctggacttccacgacagcaacgtgaagaacctgtacgag aaggtcaagagccagctgaagaacaacgccaaggagatcggcaacggctgcttcgagttctaccacaagtgcaacgacgagtgcatgga gagcgtgaagaacggcacctacgactaccccaagtacagcgaggagagcaagctgaaccgggagaagatcgacggcgtgaagaga

the 09NA sequence is: ccatcacgatcagtgaaattagcgggcaattcctctctctgccctgttagtggatgggctatatacagtaaag acaacagtgtaagaatcggttccaagggggatgtgtttgtcataagggaaccattcatatcatgctcccccttggaatgcagaaccttcttcttg actcaaggggccttgctaaatgacaaacattccaatggaaccattaaagacaggagcccatatcgaaccctaatgagctgtcctattggtga agttccctctccatacaactcaagatttgagtcagtcgcttggtcagcaagtgcttgtcatgatggcatcaattggctaacaattggaatttctgg cccagacaatggggcagtggctgtgttaaagtacaacggcataataacagacactatcaagagttggagaaacaatatattgagaacacaa gagtctgaatgtgcatgtgtaaatggttcttgctttactgtaatgaccgatggaccaagtaatggacaggcctcatacaagatcttcagaataga aaagggaaagatagtcaaatcagtcgaaatgaatgcccctaattatcactatgaggaatgctcctgttatcctgattctagtgaaatcacatgtg tgtgcagggataactggcatggctcgaatcgaccgtgggtgtctttcaaccagaatctggaatatcagataggatacatatgcagtgggatttt cggagacaatccacgccctaatgataagacaggcagttgtggtccagtatcgtctaatggagcaaatggagtaaaagggttttcattcaaata cggcaatggtgtttggatagggagaactaaaagcattagttcaagaaacggttttgagatgatttgggatccgaacggatggactgggacag acaataacttctcaataaagcaagatatcgtaggaataaatgagtggtcaggatatagcgggagttttgttcagcatccagaactaacagggc tggattgtataagaccttgcttctgggttgaactaatcagagggcgacccaaagagaacacaatctggactagcgggagcagcatatcctttt gtggtgtaaacagtgacactgtgggttggtcttggccagacggtgctgagttgccatttaccattgacaagtaa

2. Expression and purification of H5 subtype HA protein:

the recombinant plasmid was transformed into Top10 competent cells, and an appropriate amount of the resulting bacterial solution was streaked onto solid LB plates (LB plate formulation: 1% NaCl,1% tryptone,0.5% Yeast extract,1.5% agar powder; ampicillin use: 1: 1000) having ampicillin (ampicillin mother liquor concentration: 100 mg/ml) resistance, followed by overnight culture at 37 ℃. Single clones were picked, shaken in 5ml of a small amount (ampicillin resistance) and then further shaken in 300ml overnight to obtain influenza antigen plasmids using an endotoxin-free plasmid macroextraction kit (TIANGEN, DP 117).

By using DMEM containing 10% FBS at 37 ℃, 5% ₂ 293T cells were cultured in an incubator. When the confluence of 293T cells reached about 70%, influenza virus antigen plasmids (HA: NA =20 μ g:20 μ g/disc) were transfected into 293T cells using PEI transfection reagent, and after 4-6 hours of transfection, culture was continued for 3 days with serum-free DMEM exchange solution, cell culture supernatant was collected, culture was continued for 4 days with serum-free DMEM, and cell culture supernatant was collected.

The cell culture supernatant was incubated with HisTrap ^TM Excel (GE) was allowed to bind 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). Thereafter, the His column was washed with buffer A to remove non-specifically bound proteins, then 2% of buffer B solution (20mMTris, 150mMNaCl, pH 8.0,20mM imidazole) was used to remove foreign proteins, and finally the objective protein was eluted from the His column with 30% of buffer B solution (20mMTris, 150mMNaCl, pH 8.0, 300mM imidazole), and the buffer A solution (20mMTris, 150mMNaCl, pH 8.0) was used as a protein concentration tube for 30KD (30 KD) to exchange the protein concentration tube to remove imidazole concentration in the protein solution, and finally the protein solution was concentrated to 0.5ml, and Thrombin enzyme (1 mg of protein plus 5. Mu. LThrombinase) was added overnight at 4 ℃. The digested protein solution was further purified by using molecular sieves, column-equilibrated with buffer solution A (20mMTris, 150mMNaCl, pH 8.0), using AKTA-purifier (GE) and Column Hiload 16/60superdex200PG molecular sieves (GE), loading 1ml loop, while monitoring the ultraviolet absorbance at 280nm, collecting the objective protein, and identifying the protein purity by SDS-PAGE. A typical molecular sieve profile and SDS-PAGE analysis of the protein of interest is shown in FIG. 2: the peak screening position of the protein in Column high 16/60superdex200PG molecule is 62.5ml, because a tripolymer label is added after a Thrombin enzyme cutting site (LVPR ↓GS) in construction, the main peak of the protein is in a tripolymer form, the tripolymer label falls off HA after the Thrombin enzyme cutting, and the size of a monomer is about 70KD. Lanes of the SDS-PAGE gel correspond to trimers and monomers of the H5/H1-LSQ protein in the molecular sieve image, respectively.

3. Detection of affinity of H5/H1-LSQ and H5/H1 protein and antibodies CR6261 and FL6V3 by surface plasmon resonance technology

Surface plasmon resonance analysis was performed using Biacore8000 (Biacore inc.). The method comprises the following specific steps:

first, antigens H5/H1-LSQ and H5/H1 protein were immobilized on an SA chip (GE), and then antibodies CR6261 and FL6V3 at a concentration gradient of 6.25nM to 100nM were injected into the chip, respectively, and the analysis was performed at a constant temperature of 25 ℃ using HEPES-Tween buffer (10 mM HEPES, [ pH 7.4],150 mM NaCl,0.005% Tween-20) at a flow rate of 30. Mu.l/min. The regeneration of the chip surface was performed with 10mM NaOH, the binding curve is shown in FIG. 3, and the curves for different concentrations constitute the kinetic curves shown graphically. The calculation of binding kinetic constants was performed using BIAevaluation software version3.2 (Biacore, inc.). The results show that the affinity constant of the antibodies FL6V3 and H5/H1-LSQ protein is 165nM (A); the affinity constants of antibodies FL6V3 and H5/H1 protein were 2.5nM (B); the affinity constants of antibodies CR6261 and H5/H1-LSQ protein were 1.46. Mu.M (C); the affinity constants of the antibodies CR6261 and H5/H1-LSQ protein were 8.12nM (D).

EXAMPLE 2 H5/H1-LSQ vaccine preparation

The concentration of H5/H1-LSQ protein is 10mg/ml, the protein is diluted by PBS, and the protein is mixed with equal volume of Freund's complete adjuvant or Freund's incomplete adjuvant and completely emulsified until the bath water is not dissolved for animal inoculation.

Comparative example 1 preparation of H5/H1 vaccine

The concentration of H5/H1 protein is 10mg/ml, the protein is diluted by PBS, and the protein is mixed with equal volume of Freund's complete adjuvant or Freund's incomplete adjuvant and completely emulsified until the bath water is not dissolved for animal inoculation.

Example 3

1, experimental animals: BALB/c female mice of 4-6 weeks old, 6 mice per group, and the immunization mode is intramuscular injection.

2, group setting: (1) negative control (adjuvant MF 59); (2) positive control (influenza virus split vaccine TIV); (3) experimental group H5/H1-LSQ (vaccine prepared in example 2); (4) experimental group H5/H1 (vaccine prepared in comparative example 1).

3. The experimental steps are as follows:

3.1 animal immunization:

firstly, the preparation method comprises the following steps: the immunization dose of the negative group and the experimental group is 100 mu L each; the immunization dose of the positive group was 100. Mu.L each.

The immunization dose of each animal in the experimental group is 20 mug/100 mug/mouse, proteins are diluted by PBS, 6 animals in each group, different experimental histones are mixed with the same volume of MF59 adjuvant, and the mixture is shaken until the mixture is completely emulsified until bath water is not dissolved for animal immunization.

And (2) avoiding: (first immunization was performed 14 days later): the immunization dose of each of the negative group and the experimental group is 100 mu L; the immunization dose of the positive group was 100. Mu.L each.

The immunization dose of each animal in the experimental group is 20 mug/100 mug/mouse, proteins are diluted by PBS, 6 animals in each group, different experimental histones and MF59 adjuvant with the same volume are mixed and shaken until the different experimental histones are completely emulsified until bath water is not dissolved for animal immunization.

And (3) three-step (I): (three immunizations 14 days after two immunizations): the immunization dose of each of the negative group and the experimental group is 100 mu L;

the dose of each animal in the experimental group is 20 mug/100 mug/animal, the protein is diluted by PBS, 6 animals in each group, different experimental histones are mixed and shaken with equal volume of MF59 adjuvant until complete emulsification is carried out until bath water is not dissolved for animal immunization.

3.2 serum separation:

and (3) taking eyeball blood from the mice of the immune group, preparing serum, treating the mouse serum by RED, adding RDE with the volume of 4 according to the volume of 1, carrying out water bath at 37 ℃ for 18h, and inactivating at 56 ℃ for 30min for subsequent experiments.

3.3 TCID on H5N8 and H1N1 strains ₅₀ Measurement of

a, adopting a 10-fold serial dilution mode, namely 10 ^-1 、10 ^-2 ....10 ^-10 A1.5 EP tube, 900. Mu.L virus dilution + 100. Mu.L virus stock, can be used.

b, laying 96-well plates 18-24 h in advance, adding 100 μ L (2 × 10) per well ⁵ Pieces/ml) MDCK cells. 5% CO at 37 ℃ ₂ Culturing in an incubator.

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

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

3.4, neutralization experiment:

a, dilution of the Virus

Using virusesThe culture broth was diluted to 200 TCID ₅₀ /100μL。

Serum negative control (cell maintenance solution added only)

Serum dilution: the mouse serum was initially diluted 1 fold 20 and the neutralization assay was performed at 2 fold dilution.

c, incubation of the virus

Diluting the virus (100 TCID) ₅₀ ) Adding 50 μ l into 50 μ l of serum, mixing well, standing at 37 deg.C, and incubating for 1h.

d, MDCK cell adsorption

MDCK cells are washed once with 150. Mu.L of PBS and then discarded, 100. Mu.L of virus-serum mixture is added to each well, and the mixture is placed into a cell incubator for 72 hours in parallel with three wells.

e, the results of observation and calculation by the blood inhibition experiment are shown in FIGS. 4 to 5.

FIG. 4 shows: "I" indicates that the chicken red blood cells vertically flow and the cell structure is complete, which indicates that the serum can inhibit the invasion of influenza virus; "-" indicates that the chicken red blood cells begin to agglutinate and do not flow vertically, which indicates that the serum can neutralize part of influenza virus;

this indicates that the chicken red blood cells were agglutinated, that the serum concentration was low, and that the influenza virus was not neutralized. The control MF59 group serum had no protective effect on the influenza virus H5N8, while the H5/H1-LSQ group serum had a neutralizing effect on the influenza virus H5N8, indicating that the H5/H1-LSQ group serum had a specific antibody against the influenza virus H5N8, which was capable of neutralizing the influenza virus H5N8.

FIG. 5 shows: "I" indicates that the chicken red blood cells vertically flow and the cell structure is complete, which indicates that the serum can inhibit the invasion of influenza virus; "-" indicates that the chicken red blood cells begin to agglutinate and do not flow vertically, which indicates that the serum can neutralize part of influenza virus;

indicating that the chicken erythrocytes agglutinated, and that the influenza virus was not neutralized by the low serum concentration. MF59 group sera had no effect on influenza H1N1The protective effect is achieved, and the H5/H1-LSQ group serum and the TIV group serum have the neutralizing effect on the influenza virus H1N1, so that specific antibodies aiming at the influenza virus H1N1 exist in the H5/H1-LSQ group serum and the TIV group serum, and the influenza virus H1N1 can be neutralized.

Example 4

1. Experimental animals:

7-day-old chicks, 5 in each group, were immunized by cervical subcutaneous injection.

2. The experimental group was set up:

(1) blank control; (2) negative control (PBS); (3) positive control (H9 + newcastle disease dual); (4) positive control group (H5 + H7 bigeminy); (5) experimental group H5/H1-LSQ (vaccine prepared in example 2); (6) experimental group H5/H1 (vaccine prepared in comparative example 1).

3. The experimental steps are as follows:

3.1 animal immunization:

firstly, the method avoids: the immunization dose of the control group is 200 mu L each;

experimental groups each animal was immunized at a dose of 50 μ g/100 μ L/animal, the proteins were diluted in PBS, 5 per group, and the different experimental histones were mixed with an equal volume of complete freund's adjuvant (immunization volume per chicken 200 μ L) and completely emulsified to the bath water for animal vaccination.

And (2) avoiding: (first immunization was performed 14 days later): the immunization dose of the control group is 200 mu L each;

experimental groups the immunization dose per animal was 50. Mu.g/100. Mu.L/animal, the proteins were diluted with PBS, 5 per group, and the different experimental histones were mixed with an equal volume of incomplete Freund's adjuvant (immunization volume per chicken was 200. Mu.L) and completely emulsified to bath water for animal vaccination.

Counteracting toxic substances: (after 2 weeks of hyperimmunization): the dose of the medicine for treating the viral disease by dripping the nose into the mouth is 300 mu L (four units of virus liquid 1:2) ⁵ ). The mouth and anus of each chicken were sampled with a cotton swab for 4 days and 10 days after challenge, and stored in 1ml of virus storage solution. The proliferation of H9 virus was examined by hemagglutination assay.

3.2 serum separation:

collecting blood from heart after 2 weeks of secondary immunization, centrifuging at low temperature, collecting serum, packaging, and freezing at-80 deg.C.

3.3 ELISA detection of specific antibodies in chicken sera:

a, coating the plate at 4 ℃ overnight according to the amount of 200ng of protein in each hole;

b, washing: the next day the coated plate was washed 4 times for 5min with PBST containing 0.05% Tween-20;

c, sealing: adding 200 μ L of sealing solution (5% skimmed milk powder, prepared by PBST), sealing at room temperature for 2 hr;

d, washing: washing the plate to be detected with PBST for 5min for 4 times;

e, primary antibody incubation: diluting the serum to be detected by PBST, adding 100 mu L of serum diluent to be detected into each hole, and incubating for 1h at 37 ℃; irrelevant antibody (primary antibody to strep 1 diluted (murine) in 5000) was used as a negative control;

f, washing: PBST washing coated plate, washing 5 times, each time for 5min;

g, secondary antibody incubation: diluting HRP-labeled goat-anti-chicken secondary antibody with PBST (1; a secondary antibody of an irrelevant antibody (primary anti-strep 1 diluted (murine)) was used as a negative control (goat anti-mouse secondary antibody (1 diluted 5000);

h, washing: PBST washing coated plate, washing 5 times, each time for 5min;

i, color development: adding TMB substrate buffer solution for color development at 50. Mu.L/well, shading for 3min, stopping reaction with ELISA stop solution at 50. Mu.L/well, OD _450nm And (4) reading.

The results are shown in FIG. 3, which shows: after the H5/H1-LSQ chimeric vaccine is immunized, more antibodies can be generated compared with the blank group and the PBS immunized group, and higher antibodies can be generated compared with the H9+ Newcastle disease bivalent vaccine after immunization; compared with the PBS group, the H5/H1-LSQ chimeric vaccine and the H5/H1 vaccine can also stimulate more antibodies which are combined with HA of the H9 strain after being immunized.

3.4 hemagglutination assay

a, 25 μ LPBS per well in the hemagglutination plate. 25 mu L of inactivated H9 subtype avian influenza virus is added into the first hole, serial dilution is carried out by 2 times in sequence, and meanwhile, a negative control hole is established.

b, adding 25 mu L 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 then judging the result.

Note: 1% chicken red blood cell suspension preparation:

preparing: anticoagulation of fresh chicken, PBS, centrifugal tube and low-speed centrifuge

The method comprises the following steps: anticoagulation first centrifugation: centrifuging at 1500rpm/min for 5min, removing upper layer leukocyte plasma, adding PBS, and mixing; and (3) centrifuging for the second time: centrifuging at 1500rpm/min for 5min to remove leukocyte plasma; and (3) centrifuging for the third time: centrifuging at 1500rpm/min for 5min to remove leukocyte plasma; 99ml PBS +1ml erythrocyte is prepared into 1% chicken erythrocyte suspension.

The specific operation method is shown in figure 7, and the result shows that: the maximum dilution of 100% agglutinated (+++) was the virus hemagglutination titer, i.e., one agglutination unit, and as can be seen from fig. 6, the hemagglutination titer of H9 subtype avian influenza virus was 1 ⁷ )。

3.5 hemagglutination inhibition assay

Four units of virus solution were prepared according to the results in 3.4: the hemagglutination valence of inactivated H9 subtype avian influenza virus was divided by 4 as the dilution of four unit virus solution (i.e., 128/4=32 (2) ⁵ ))；

a, adding 25 mu L PBS into each hole in a blood coagulation plate, adding 25 mu L serum to be detected (after the primary serum 1:4 is diluted) into the first hole, sequentially carrying out 2-fold gradient dilution, and simultaneously setting up a negative control hole (PBS);

b, in addition to negative control wells, 25 μ L of four units of virus fluid per well: placing on a horizontal oscillator, oscillating for 12min, and standing at 37 deg.C for 15min;

c, adding 25 mu L of 1% chicken red blood cell suspension into each hole, oscillating on a horizontal oscillator for 1-2 min, uniformly mixing, standing at 37 ℃ for 30min, and judging the result.

The results are shown in fig. 8A and 8B, showing: "I" indicates that the chicken red blood cells vertically flow and the cell structure is complete, which indicates that the serum can inhibit the invasion of influenza virus; "·" indicates that the chicken red blood cells begin to agglutinate and do not vertically flow, which indicates that the serum can neutralize part of influenza virus;

this indicates that the chicken red blood cells were agglutinated, that the serum concentration was low, and that the influenza virus was not neutralized. Post-immunization H5/H1-LSQ chimeric vaccine blood suppression potency 1 512 (1:2 ⁹ ) The H5/H1 vaccine has a post-immunization blood suppression titer of about 1 ⁷ ) The blood inhibitory potency after PBS immunization was 1 (1:2 ⁴ ) The post-H9 + newcastle disease dual vaccine immunization titer was about 1 128 (1:2 ⁷ ) The antibody titer generated after the H5/H1-LSQ chimeric vaccine is immunized is higher than that of an H9+ Newcastle disease dual vaccine, an H5/H1 vaccine and an H5+ H7 dual vaccine, and is obviously higher than that of a PBS group, so that the H5/H1-LSQ chimeric vaccine can effectively block the invasion of an H9 strain.

3.6, hemagglutination assay for H9 virus:

a, 25 μ L PBS per well was added to the hemagglutination plate. 25 mu L of inactivated H9 subtype avian influenza virus is added into the first hole, serial dilution is carried out by 2 times in sequence, and meanwhile, a negative control hole is established.

b, adding 25 mu L of 1% chicken red blood cell suspension into each hole, oscillating on a horizontal oscillator for 1-2 min, uniformly mixing, standing at 37 ℃ for 30min, and then judging the result.

The results are shown in FIG. 9, which shows: after H9 virus attack, the immune effect of the H5/H1-LSQ chimeric vaccine can better inhibit the proliferation of the H9 virus compared with the H9+ Newcastle disease bivalent vaccine and the H5/H1 vaccine.

The above is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, a plurality of modifications and embellishments can be made without departing from the principle of the present invention, and these modifications and embellishments should also be regarded as the protection scope of the present invention.

Sequence listing

<110> institute of advanced innovation in Shanxi province

<120> recombinant avian influenza subunit vaccine and preparation method thereof

<130> MP2031561

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 504

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Asp Gln Ile Cys Ile Gly Tyr Ser Ala Asn Asn Ser Thr Glu Gln Val

1 5 10 15

Asp Thr Ile Met Glu Lys Asn Val Thr Val Thr Gln Ala Gln Asp Ile

20 25 30

Leu Glu Lys Lys His Asn Gly Lys Leu Cys Asp Leu Asp Gly Val Lys

35 40 45

Pro Leu Ile Leu Arg Asp Cys Ser Val Ala Gly Trp Leu Leu Gly Asn

50 55 60

Pro Met Cys Asp Glu Phe Ile Asn Val Pro Glu Trp Ser Tyr Ile Val

65 70 75 80

Glu Lys Ala Asn Pro Val Asn Asp Leu Cys Tyr Pro Gly Asp Phe Asn

85 90 95

Asp Tyr Glu Glu Leu Lys His Leu Leu Ser Arg Ile Asn His Phe Glu

100 105 110

Lys Ile Gln Ile Ile Pro Lys Ser Ser Trp Ser Ser His Glu Ala Ser

115 120 125

Leu Gly Val Ser Ser Ala Cys Pro Tyr Gln Gly Lys Ser Ser Phe Phe

130 135 140

Arg Asn Val Val Trp Leu Ile Lys Lys Asp Ser Thr Tyr Pro Thr Ile

145 150 155 160

Lys Arg Ser Tyr Asn Asn Thr Asn Gln Glu Asp Leu Leu Val Leu Trp

165 170 175

Gly Ile His His Pro Asn Asp Ala Ala Glu Gln Thr Lys Leu Tyr Gln

180 185 190

Asn Pro Thr Thr Tyr Ile Ser Val Gly Thr Ser Thr Leu Asn Gln Arg

195 200 205

Leu Val Pro Arg Ile Ala Thr Arg Ser Lys Val Lys Gly Leu Ser Gly

210 215 220

Arg Met Glu Phe Phe Trp Thr Ile Leu Lys Pro Asn Asp Ala Ile Asn

225 230 235 240

Phe Glu Ser Asn Gly Asn Phe Ile Ala Pro Glu Tyr Ala Tyr Lys Ile

245 250 255

Val Lys Lys Gly Asp Ser Thr Ile Met Lys Ser Glu Leu Glu Tyr Gly

260 265 270

Asn Cys Asn Thr Lys Cys Gln Thr Pro Met Gly Ala Ile Asn Ser Ser

275 280 285

Met Pro Phe His Asn Ile His Pro Leu Thr Ile Gly Glu Cys Pro Lys

290 295 300

Tyr Val Lys Ser Asn Arg Leu Val Leu Ala Ile Gly Leu Arg Asn Ser

305 310 315 320

Pro Gln Arg Glu Thr Gln Gly Leu Phe Gly Ala Ile Ala Gly Phe Thr

325 330 335

Glu Gly Gly Trp Thr Gly Met Val Asp Gly Leu Tyr Gly Tyr His His

340 345 350

Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Gln Lys Ser Thr Gln

355 360 365

Asn Ala Ile Asn Gly Ile Thr Asn Lys Val Asn Ser Val Ile Glu Lys

370 375 380

Met Asn Thr Gln Tyr Thr Ala Val Gly Lys Glu Phe Asn Lys Ser Glu

385 390 395 400

Arg Arg Met Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe Ile Asp

405 410 415

Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu Arg

420 425 430

Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys Val

435 440 445

Lys Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe

450 455 460

Glu Phe Tyr His Lys Cys Asn Asp Glu Cys Met Glu Ser Val Lys Asn

465 470 475 480

Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ser Lys Leu Asn Arg

485 490 495

Glu Lys Ile Asp Gly Val Lys Arg

500

<210> 2

<211> 1512

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

gaccagattt gcatcggcta ctccgccaac aacagcaccg agcaggtgga caccatcatg 60

gagaagaacg tgacagtgac ccaagcccag gatatcctgg agaagaagca caacggcaag 120

ctctgcgacc tggacggagt gaaacccctg atcctgaggg attgtagcgt ggcaggttgg 180

ctgctgggaa accccatgtg cgacgagttc atcaacgtcc ccgagtggag ctatatcgtg 240

gagaaggcca acccagtgaa cgacctctgc tacccaggcg acttcaacga ctacgaggag 300

ctgaagcacc tgctgagccg gatcaaccac ttcgagaaga tccagatcat ccccaagagc 360

tcttggagca gccacgaagc ctctctggga gtgtctagcg cctgtcctta ccagggcaag 420

agcagcttct tccggaacgt cgtctggctg atcaagaagg atagcaccta ccccaccatc 480

aagcggagct acaacaacac caaccaggag gacctgctcg tgctgtgggg cattcaccac 540

cctaacgacg cagccgagca gaccaagctg taccagaacc ctaccaccta catcagcgtg 600

ggcacctcta ccctgaacca gagactggtg cccaggatcg ccaccagaag caaagtgaag 660

ggcctgagcg gccggatgga gttcttttgg accatcctga agcccaacga cgccatcaac 720

ttcgagagca acggcaactt catcgccccc gagtacgcct acaagatcgt gaagaagggc 780

gacagcacca tcatgaagag cgagctggag tacggcaact gcaacaccaa gtgccagacc 840

cctatgggcg ccatcaacag cagcatgccc ttccacaaca tccaccccct gaccattggc 900

gagtgtccta agtacgtgaa gagcaaccgc ctggtgctgg ctatcggcct gagaaacagc 960

ccccagagag agacacaagg cctgttcgga gccattgccg gattcacaga gggcggttgg 1020

acaggcatgg tggacggact gtacggatac caccaccaga acgagcaggg atcaggatac 1080

gccgccgatc agaagagcac ccagaacgcc atcaacggca tcaccaacaa ggtcaacagc 1140

gtgatcgaga agatgaacac ccagtacacc gccgtgggca aggagttcaa caagagcgag 1200

cggcggatgg agaacctgaa caagaaggtg gacgacggct tcatcgacat ttggacctac 1260

aacgccgagc tgctggtgct gctggaaaac gagaggaccc tggacttcca cgacagcaac 1320

gtgaagaacc tgtacgagaa ggtcaagagc cagctgaaga acaacgccaa ggagatcggc 1380

aacggctgct tcgagttcta ccacaagtgc aacgacgagt gcatggagag cgtgaagaac 1440

ggcacctacg actaccccaa gtacagcgag gagagcaagc tgaaccggga gaagatcgac 1500

ggcgtgaaga ga 1512

<210> 3

<211> 566

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Ala Thr Met Lys Val Lys Leu Leu Val Leu Leu Cys Thr Phe Thr Ala

1 5 10 15

Thr Tyr Ala Asp Gln Ile Cys Ile Gly Tyr Ser Ala Asn Asn Ser Thr

20 25 30

Glu Gln Val Asp Thr Ile Met Glu Lys Asn Val Thr Val Thr Gln Ala

35 40 45

Gln Asp Ile Leu Glu Lys Lys His Asn Gly Lys Leu Cys Asp Leu Asp

50 55 60

Gly Val Lys Pro Leu Ile Leu Arg Asp Cys Ser Val Ala Gly Trp Leu

65 70 75 80

Leu Gly Asn Pro Met Cys Asp Glu Phe Ile Asn Val Pro Glu Trp Ser

85 90 95

Tyr Ile Val Glu Lys Ala Asn Pro Val Asn Asp Leu Cys Tyr Pro Gly

100 105 110

Asp Phe Asn Asp Tyr Glu Glu Leu Lys His Leu Leu Ser Arg Ile Asn

115 120 125

His Phe Glu Lys Ile Gln Ile Ile Pro Lys Ser Ser Trp Ser Ser His

130 135 140

Glu Ala Ser Leu Gly Val Ser Ser Ala Cys Pro Tyr Gln Gly Lys Ser

145 150 155 160

Ser Phe Phe Arg Asn Val Val Trp Leu Ile Lys Lys Asp Ser Thr Tyr

165 170 175

Pro Thr Ile Lys Arg Ser Tyr Asn Asn Thr Asn Gln Glu Asp Leu Leu

180 185 190

Val Leu Trp Gly Ile His His Pro Asn Asp Ala Ala Glu Gln Thr Lys

195 200 205

Leu Tyr Gln Asn Pro Thr Thr Tyr Ile Ser Val Gly Thr Ser Thr Leu

210 215 220

Asn Gln Arg Leu Val Pro Arg Ile Ala Thr Arg Ser Lys Val Lys Gly

225 230 235 240

Leu Ser Gly Arg Met Glu Phe Phe Trp Thr Ile Leu Lys Pro Asn Asp

245 250 255

Ala Ile Asn Phe Glu Ser Asn Gly Asn Phe Ile Ala Pro Glu Tyr Ala

260 265 270

Tyr Lys Ile Val Lys Lys Gly Asp Ser Thr Ile Met Lys Ser Glu Leu

275 280 285

Glu Tyr Gly Asn Cys Asn Thr Lys Cys Gln Thr Pro Met Gly Ala Ile

290 295 300

Asn Ser Ser Met Pro Phe His Asn Ile His Pro Leu Thr Ile Gly Glu

305 310 315 320

Cys Pro Lys Tyr Val Lys Ser Asn Arg Leu Val Leu Ala Ile Gly Leu

325 330 335

Arg Asn Ser Pro Gln Arg Glu Thr Gln Gly Leu Phe Gly Ala Ile Ala

340 345 350

Gly Phe Thr Glu Gly Gly Trp Thr Gly Met Val Asp Gly Leu Tyr Gly

355 360 365

Tyr His His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Gln Lys

370 375 380

Ser Thr Gln Asn Ala Ile Asn Gly Ile Thr Asn Lys Val Asn Ser Val

385 390 395 400

Ile Glu Lys Met Asn Thr Gln Tyr Thr Ala Val Gly Lys Glu Phe Asn

405 410 415

Lys Ser Glu Arg Arg Met Glu Asn Leu Asn Lys Lys Val Asp Asp Gly

420 425 430

Phe Ile Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu

435 440 445

Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr

450 455 460

Glu Lys Val Lys Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn

465 470 475 480

Gly Cys Phe Glu Phe Tyr His Lys Cys Asn Asp Glu Cys Met Glu Ser

485 490 495

Val Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ser Lys

500 505 510

Leu Asn Arg Glu Lys Ile Asp Gly Val Lys Arg Leu Val Pro Arg Gly

515 520 525

Ser Pro Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala

530 535 540

Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Gly

545 550 555 560

His His His His His His

565

<210> 4

<211> 1701

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gccaccatga aggtcaagct gctggtcctc ctctgcacct tcaccgccac ctacgccgac 60

cagatttgca tcggctactc cgccaacaac agcaccgagc aggtggacac catcatggag 120

aagaacgtga cagtgaccca agcccaggat atcctggaga agaagcacaa cggcaagctc 180

tgcgacctgg acggagtgaa acccctgatc ctgagggatt gtagcgtggc aggttggctg 240

ctgggaaacc ccatgtgcga cgagttcatc aacgtccccg agtggagcta tatcgtggag 300

aaggccaacc cagtgaacga cctctgctac ccaggcgact tcaacgacta cgaggagctg 360

aagcacctgc tgagccggat caaccacttc gagaagatcc agatcatccc caagagctct 420

tggagcagcc acgaagcctc tctgggagtg tctagcgcct gtccttacca gggcaagagc 480

agcttcttcc ggaacgtcgt ctggctgatc aagaaggata gcacctaccc caccatcaag 540

cggagctaca acaacaccaa ccaggaggac ctgctcgtgc tgtggggcat tcaccaccct 600

aacgacgcag ccgagcagac caagctgtac cagaacccta ccacctacat cagcgtgggc 660

acctctaccc tgaaccagag actggtgccc aggatcgcca ccagaagcaa agtgaagggc 720

ctgagcggcc ggatggagtt cttttggacc atcctgaagc ccaacgacgc catcaacttc 780

gagagcaacg gcaacttcat cgcccccgag tacgcctaca agatcgtgaa gaagggcgac 840

agcaccatca tgaagagcga gctggagtac ggcaactgca acaccaagtg ccagacccct 900

atgggcgcca tcaacagcag catgcccttc cacaacatcc accccctgac cattggcgag 960

tgtcctaagt acgtgaagag caaccgcctg gtgctggcta tcggcctgag aaacagcccc 1020

cagagagaga cacaaggcct gttcggagcc attgccggat tcacagaggg cggttggaca 1080

ggcatggtgg acggactgta cggataccac caccagaacg agcagggatc aggatacgcc 1140

gccgatcaga agagcaccca gaacgccatc aacggcatca ccaacaaggt caacagcgtg 1200

atcgagaaga tgaacaccca gtacaccgcc gtgggcaagg agttcaacaa gagcgagcgg 1260

cggatggaga acctgaacaa gaaggtggac gacggcttca tcgacatttg gacctacaac 1320

gccgagctgc tggtgctgct ggaaaacgag aggaccctgg acttccacga cagcaacgtg 1380

aagaacctgt acgagaaggt caagagccag ctgaagaaca acgccaagga gatcggcaac 1440

ggctgcttcg agttctacca caagtgcaac gacgagtgca tggagagcgt gaagaacggc 1500

acctacgact accccaagta cagcgaggag agcaagctga accgggagaa gatcgacggc 1560

gtgaagagac tggtgccaag aggctctcca ggaagcggct acatcccaga agcccctaga 1620

gacggacagg cttacgtgcg aaaagacggc gagtgggtgc tgctgagcac atttctggga 1680

caccatcatc accaccacta g 1701

<210> 5

<211> 391

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Pro Ser Arg Ser Val Lys Leu Ala Gly Asn Ser Ser Leu Cys Pro Val

1 5 10 15

Ser Gly Trp Ala Ile Tyr Ser Lys Asp Asn Ser Val Arg Ile Gly Ser

20 25 30

Lys Gly Asp Val Phe Val Ile Arg Glu Pro Phe Ile Ser Cys Ser Pro

35 40 45

Leu Glu Cys Arg Thr Phe Phe Leu Thr Gln Gly Ala Leu Leu Asn Asp

50 55 60

Lys His Ser Asn Gly Thr Ile Lys Asp Arg Ser Pro Tyr Arg Thr Leu

65 70 75 80

Met Ser Cys Pro Ile Gly Glu Val Pro Ser Pro Tyr Asn Ser Arg Phe

85 90 95

Glu Ser Val Ala Trp Ser Ala Ser Ala Cys His Asp Gly Ile Asn Trp

100 105 110

Leu Thr Ile Gly Ile Ser Gly Pro Asp Asn Gly Ala Val Ala Val Leu

115 120 125

Lys Tyr Asn Gly Ile Ile Thr Asp Thr Ile Lys Ser Trp Arg Asn Asn

130 135 140

Ile Leu Arg Thr Gln Glu Ser Glu Cys Ala Cys Val Asn Gly Ser Cys

145 150 155 160

Phe Thr Val Met Thr Asp Gly Pro Ser Asn Gly Gln Ala Ser Tyr Lys

165 170 175

Ile Phe Arg Ile Glu Lys Gly Lys Ile Val Lys Ser Val Glu Met Asn

180 185 190

Ala Pro Asn Tyr His Tyr Glu Glu Cys Ser Cys Tyr Pro Asp Ser Ser

195 200 205

Glu Ile Thr Cys Val Cys Arg Asp Asn Trp His Gly Ser Asn Arg Pro

210 215 220

Trp Val Ser Phe Asn Gln Asn Leu Glu Tyr Gln Ile Gly Tyr Ile Cys

225 230 235 240

Ser Gly Ile Phe Gly Asp Asn Pro Arg Pro Asn Asp Lys Thr Gly Ser

245 250 255

Cys Gly Pro Val Ser Ser Asn Gly Ala Asn Gly Val Lys Gly Phe Ser

260 265 270

Phe Lys Tyr Gly Asn Gly Val Trp Ile Gly Arg Thr Lys Ser Ile Ser

275 280 285

Ser Arg Asn Gly Phe Glu Met Ile Trp Asp Pro Asn Gly Trp Thr Gly

290 295 300

Thr Asp Asn Asn Phe Ser Ile Lys Gln Asp Ile Val Gly Ile Asn Glu

305 310 315 320

Trp Ser Gly Tyr Ser Gly Ser Phe Val Gln His Pro Glu Leu Thr Gly

325 330 335

Leu Asp Cys Ile Arg Pro Cys Phe Trp Val Glu Leu Ile Arg Gly Arg

340 345 350

Pro Lys Glu Asn Thr Ile Trp Thr Ser Gly Ser Ser Ile Ser Phe Cys

355 360 365

Gly Val Asn Ser Asp Thr Val Gly Trp Ser Trp Pro Asp Gly Ala Glu

370 375 380

Leu Pro Phe Thr Ile Asp Lys

385 390

<210> 6

<211> 1176

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ccatcacgat cagtgaaatt agcgggcaat tcctctctct gccctgttag tggatgggct 60

atatacagta aagacaacag tgtaagaatc ggttccaagg gggatgtgtt tgtcataagg 120

gaaccattca tatcatgctc ccccttggaa tgcagaacct tcttcttgac tcaaggggcc 180

ttgctaaatg acaaacattc caatggaacc attaaagaca ggagcccata tcgaacccta 240

atgagctgtc ctattggtga agttccctct ccatacaact caagatttga gtcagtcgct 300

tggtcagcaa gtgcttgtca tgatggcatc aattggctaa caattggaat ttctggccca 360

gacaatgggg cagtggctgt gttaaagtac aacggcataa taacagacac tatcaagagt 420

tggagaaaca atatattgag aacacaagag tctgaatgtg catgtgtaaa tggttcttgc 480

tttactgtaa tgaccgatgg accaagtaat ggacaggcct catacaagat cttcagaata 540

gaaaagggaa agatagtcaa atcagtcgaa atgaatgccc ctaattatca ctatgaggaa 600

tgctcctgtt atcctgattc tagtgaaatc acatgtgtgt gcagggataa ctggcatggc 660

tcgaatcgac cgtgggtgtc tttcaaccag aatctggaat atcagatagg atacatatgc 720

agtgggattt tcggagacaa tccacgccct aatgataaga caggcagttg tggtccagta 780

tcgtctaatg gagcaaatgg agtaaaaggg ttttcattca aatacggcaa tggtgtttgg 840

atagggagaa ctaaaagcat tagttcaaga aacggttttg agatgatttg ggatccgaac 900

ggatggactg ggacagacaa taacttctca ataaagcaag atatcgtagg aataaatgag 960

tggtcaggat atagcgggag ttttgttcag catccagaac taacagggct ggattgtata 1020

agaccttgct tctgggttga actaatcaga gggcgaccca aagagaacac aatctggact 1080

agcgggagca gcatatcctt ttgtggtgta aacagtgaca ctgtgggttg gtcttggcca 1140

gacggtgctg agttgccatt taccattgac aagtaa 1176

<210> 7

<211> 478

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

Ala Thr Met Gly Ala Gly Ala Thr Gly Arg Ala Met Asp Gly Pro Arg

1 5 10 15

Leu Leu Leu Leu Leu Leu Leu Gly Val Ser Leu Gly Gly Ala Asp Tyr

20 25 30

Lys Asp Asp Asp Asp Lys Ser Ser Ser Asp Tyr Ser Asp Leu Gln Arg

35 40 45

Val Lys Gln Glu Leu Leu Glu Glu Val Lys Lys Glu Leu Gln Lys Val

50 55 60

Lys Glu Glu Ile Ile Glu Ala Phe Val Gln Glu Leu Arg Lys Arg Gly

65 70 75 80

Ser Leu Val Pro Arg Gly Ser Pro Ser Arg Ser Val Lys Leu Ala Gly

85 90 95

Asn Ser Ser Leu Cys Pro Val Ser Gly Trp Ala Ile Tyr Ser Lys Asp

100 105 110

Asn Ser Val Arg Ile Gly Ser Lys Gly Asp Val Phe Val Ile Arg Glu

115 120 125

Pro Phe Ile Ser Cys Ser Pro Leu Glu Cys Arg Thr Phe Phe Leu Thr

130 135 140

Gln Gly Ala Leu Leu Asn Asp Lys His Ser Asn Gly Thr Ile Lys Asp

145 150 155 160

Arg Ser Pro Tyr Arg Thr Leu Met Ser Cys Pro Ile Gly Glu Val Pro

165 170 175

Ser Pro Tyr Asn Ser Arg Phe Glu Ser Val Ala Trp Ser Ala Ser Ala

180 185 190

Cys His Asp Gly Ile Asn Trp Leu Thr Ile Gly Ile Ser Gly Pro Asp

195 200 205

Asn Gly Ala Val Ala Val Leu Lys Tyr Asn Gly Ile Ile Thr Asp Thr

210 215 220

Ile Lys Ser Trp Arg Asn Asn Ile Leu Arg Thr Gln Glu Ser Glu Cys

225 230 235 240

Ala Cys Val Asn Gly Ser Cys Phe Thr Val Met Thr Asp Gly Pro Ser

245 250 255

Asn Gly Gln Ala Ser Tyr Lys Ile Phe Arg Ile Glu Lys Gly Lys Ile

260 265 270

Val Lys Ser Val Glu Met Asn Ala Pro Asn Tyr His Tyr Glu Glu Cys

275 280 285

Ser Cys Tyr Pro Asp Ser Ser Glu Ile Thr Cys Val Cys Arg Asp Asn

290 295 300

Trp His Gly Ser Asn Arg Pro Trp Val Ser Phe Asn Gln Asn Leu Glu

305 310 315 320

Tyr Gln Ile Gly Tyr Ile Cys Ser Gly Ile Phe Gly Asp Asn Pro Arg

325 330 335

Pro Asn Asp Lys Thr Gly Ser Cys Gly Pro Val Ser Ser Asn Gly Ala

340 345 350

Asn Gly Val Lys Gly Phe Ser Phe Lys Tyr Gly Asn Gly Val Trp Ile

355 360 365

Gly Arg Thr Lys Ser Ile Ser Ser Arg Asn Gly Phe Glu Met Ile Trp

370 375 380

Asp Pro Asn Gly Trp Thr Gly Thr Asp Asn Asn Phe Ser Ile Lys Gln

385 390 395 400

Asp Ile Val Gly Ile Asn Glu Trp Ser Gly Tyr Ser Gly Ser Phe Val

405 410 415

Gln His Pro Glu Leu Thr Gly Leu Asp Cys Ile Arg Pro Cys Phe Trp

420 425 430

Val Glu Leu Ile Arg Gly Arg Pro Lys Glu Asn Thr Ile Trp Thr Ser

435 440 445

Gly Ser Ser Ile Ser Phe Cys Gly Val Asn Ser Asp Thr Val Gly Trp

450 455 460

Ser Trp Pro Asp Gly Ala Glu Leu Pro Phe Thr Ile Asp Lys

465 470 475

<210> 8

<211> 1437

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

gccaccatgg gggcaggtgc caccggccgc gccatggacg ggccgcgcct gctgctgttg 60

ctgcttctgg gggtgtccct tggaggtgcc gattataagg atgatgatga taagagctcc 120

agtgattact cggacctaca gagggtgaaa caggagcttc tggaagaggt gaagaaggaa 180

ttgcagaaag tgaaagagga aatcattgaa gccttcgtcc aggagctgag gaagcggggt 240

tctctggtac cacgaggtag tccatcacga tcagtgaaat tagcgggcaa ttcctctctc 300

tgccctgtta gtggatgggc tatatacagt aaagacaaca gtgtaagaat cggttccaag 360

ggggatgtgt ttgtcataag ggaaccattc atatcatgct cccccttgga atgcagaacc 420

ttcttcttga ctcaaggggc cttgctaaat gacaaacatt ccaatggaac cattaaagac 480

aggagcccat atcgaaccct aatgagctgt cctattggtg aagttccctc tccatacaac 540

tcaagatttg agtcagtcgc ttggtcagca agtgcttgtc atgatggcat caattggcta 600

acaattggaa tttctggccc agacaatggg gcagtggctg tgttaaagta caacggcata 660

ataacagaca ctatcaagag ttggagaaac aatatattga gaacacaaga gtctgaatgt 720

gcatgtgtaa atggttcttg ctttactgta atgaccgatg gaccaagtaa tggacaggcc 780

tcatacaaga tcttcagaat agaaaaggga aagatagtca aatcagtcga aatgaatgcc 840

cctaattatc actatgagga atgctcctgt tatcctgatt ctagtgaaat cacatgtgtg 900

tgcagggata actggcatgg ctcgaatcga ccgtgggtgt ctttcaacca gaatctggaa 960

tatcagatag gatacatatg cagtgggatt ttcggagaca atccacgccc taatgataag 1020

acaggcagtt gtggtccagt atcgtctaat ggagcaaatg gagtaaaagg gttttcattc 1080

aaatacggca atggtgtttg gatagggaga actaaaagca ttagttcaag aaacggtttt 1140

gagatgattt gggatccgaa cggatggact gggacagaca ataacttctc aataaagcaa 1200

gatatcgtag gaataaatga gtggtcagga tatagcggga gttttgttca gcatccagaa 1260

ctaacagggc tggattgtat aagaccttgc ttctgggttg aactaatcag agggcgaccc 1320

aaagagaaca caatctggac tagcgggagc agcatatcct tttgtggtgt aaacagtgac 1380

actgtgggtt ggtcttggcc agacggtgct gagttgccat ttaccattga caagtaa 1437

Claims (11)

1. The amino acid sequence of the HA subunit is shown as SEQ ID NO. 1.
2. 2. The nucleic acid sequence of the nucleic acid for coding the HA subunit is shown as SEQ ID NO. 2.
3. An ha recombinant antigen consisting of, connected in order: a signal peptide fragment, the HA subunit of claim 1, a thrombin fragment, a trimer-tag and a screenable marker.
4. 4. The HA recombinant antigen according to claim 3, wherein the amino acid sequence is as shown in SEQ ID NO 3.
5. 5. The nucleic acid sequence of the HA recombinant antigen is shown as SEQ ID NO. 4.
6. 6. A recombinant vector comprising the nucleic acid of claim 5.
7. 7. A host cell transformed or transfected with the recombinant vector of claim 6.
8. 8. The host cell of claim 7, further transformed or transfected with an expression vector for the NA antigen; the nucleic acid sequence for coding the NA antigen is shown as SEQ ID NO. 8.
9. 9. A recombinant HA protein expressed by the host cell of claim 7 or 8.
10. 10. A vaccine comprising the recombinant HA protein of claim 9.
11. 11. Use of the vaccine of claim 10 for the preparation of a formulation for the prevention and treatment of avian influenza; the avian influenza is H1 subtype avian influenza, H5 subtype avian influenza or H9 subtype avian influenza.

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