CN109731100A

CN109731100A - Avian influenza vaccine and preparation and application based on MultiBac baculovirus expression system

Info

Publication number: CN109731100A
Application number: CN201910117092.4A
Authority: CN
Inventors: 樊惠英; 孔德鑫; 王亚贞; 胡朝升
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2019-02-15
Filing date: 2019-02-15
Publication date: 2019-05-10

Abstract

The present invention relates to bioengineering and viral vaccine technical fields, and in particular to a kind of avian influenza vaccine and preparation and application based on MultiBac baculovirus expression system.The present invention selects the HA gene of H7N9 and/or H5N1 subtype avian influenza virus prevalence strain as target antigen, the HA albumen of H7N9 and H5N1 subtype avian influenza virus is rapidly and efficiently expressed using MultiBac baculovirus expression system, avian influenza vaccine is further prepared, the vaccine, which overcomes existing vaccinum influenzae inactivatum, there are problems that dissipating malicious risk in production and inactivation process, provide the candidate vaccine of a kind of prevention and control H7N9 and the propagation of H5N1 avian influenza virus prevalence.

Description

Avian influenza vaccine and preparation and application based on MultiBac baculovirus expression system

Technical field

The present invention relates to bioengineering and viral vaccine technical fields, and in particular to one kind is based on MultiBac baculoviral The avian influenza vaccine of expression system and preparation and application.

Background technique

Avian influenza virus prevention and control at present mainly aim at prevention, and currently used is most widely polyvalent inactivation bird flu epidemic disease Seedling.Current inactivated avian influenza vaccine be easy to cause viral expansion during production and inactivation of viruses based on mainly producing with chicken embryo Dissipate, cause bio-safety problem, therefore develop it is a kind of it is safer, quickly and effectively candidate vaccine is extremely urgent.Genetic engineering Vaccine becomes the hot spot of exploitation bird flu candidate vaccine because it is safe, quickly and effectively characteristic attracts people's attention.Fowl stream The HA albumen of hemagglutinin (hemagglutinin, HA) gene expression of Influenza Virus is its most important target antigen, and HA gene It is higher in the avian influenza virus nucleotide homology of same hypotype, so the exploitation of most of recombinant vaccines is mainly with HA base Because based on, specific avian influenza vaccine is produced.

Rod string design is the hot topic of recombinant vaccine exploitation, as a kind of eukaryotic expression system, The exogenous genes products and natural products of expression have similar biological activity, have high application value.MultiBac The characteristic that various exogenous genes are inserted into Baculovirus Gene group simultaneously can be caused people because of it by baculovirus expression system Attention, pass through the system simultaneously express various exogenous genes, for exploitation multivalent avian influenza vaccines have significant application value. And it is good by the foreign protein bioactivity of the system expression, there are a variety of application values.

Therefore the present invention is based on existing influenza vaccines there are the drawbacks of, it is intended to develop a kind of based on rhabdovirus expression vector The avian influenza genes engineered vaccine of system, with its safety, quickly and effectively characteristic prevention and control H7N9 and H5N1 subtype avian influenza virus Prevalence.

Summary of the invention

For overcome the deficiencies in the prior art and disadvantage, the primary purpose of the present invention is that providing a kind of based on MultiBac The avian influenza vaccine of baculovirus expression system.

Another object of the present invention is to provide the above-mentioned avian influenza vaccines based on MultiBac baculovirus expression system Preparation method, this method selects the HA gene of H7N9 and/or H5N1 subtype avian influenza virus prevalence strain as target antigen, makes It expresses the HA albumen of H7N9 and H5N1 subtype avian influenza virus respectively with MultiBac baculovirus expression system, further prepares Obtain avian influenza vaccine.

A further object of the present invention is to provide the above-mentioned avian influenza vaccines based on MultiBac baculovirus expression system Application.

The purpose of the invention is achieved by the following technical solution:

A kind of avian influenza vaccine based on MultiBac baculovirus expression system includes solubility HA albumen (hemagglutinin, HA), solubility HA albumen is by that can express the rod-shaped disease of the recombinant modified of avian influenza virus HA protein Poison expression；

The avian influenza virus is at least one of H7N9 subtype avian influenza virus and H5N1 subtype avian influenza virus；

The amino acid sequence of the HA albumen of the H7N9 subtype avian influenza virus is as shown in SEQ ID NO.1；

The amino acid sequence of the HA albumen of the H5N1 subtype avian influenza virus is as shown in SEQ ID NO.2；

Encode the nucleotide sequence such as SEQ ID NO.3 institute of the gene of the HA albumen of above-mentioned H7N9 subtype avian influenza virus Show；

Encode the nucleotide sequence such as SEQ ID NO.4 institute of the gene of the HA albumen of above-mentioned H5N1 subtype avian influenza virus Show；

V-cath gene and chiA gene are destroyed and lose function in the genome of the baculoviral of the recombinant modified It acts on and inserts single promoter gene box in genome；

Single promoter gene box is from upstream to downstream including such as lower component: polyhedrin (Polyhedron, PH) the gene of the HA albumen of promoter and coding avian influenza virus；

The carrier that sets out for constructing the baculoviral of above-mentioned recombinant modified is pACEBac1；

The soluble HA albumen is expressed by MultiBac baculovirus expression system；

The baculoviral is preferably autographa california nuclear polyhedrosis virus (AcMNPV)；

The preparation method of the baculoviral of the recombinant modified, preferably comprises following steps:

The HA gene of avian influenza virus is inserted into transmission plasmid and passes through the genome of swivel base and shuttle vector Bacmid Homologous recombination is carried out, recombinant baculovirus genomic DNA is obtained, it is then that recombinant baculovirus genomic DNA transfection insect is thin Born of the same parents obtain the baculoviral of recombinant modified in insect cell inner packing；

The preparation method of the avian influenza vaccine based on MultiBac baculovirus expression system includes following step It is rapid:

(1) genome for extracting avian influenza virus, is reversed to cDNA；Design primer is with avian influenza virus cDNA Template, amplification obtain HA gene；HA gene is inserted into behind the PH promoter of transmission plasmid pACEBac1, obtains recombination transmission Plasmid；Recombinant transfer plasmid is converted into DH10MultiBac Escherichia coli, is recombinated by swivel base, recombinant baculovirus matter is obtained Grain；Finally recombinant baculovirus is obtained respectively by recombinant baculovirus plasmid transfection sf9 insect cell with liposome method；

(2) P1 is infected for recombinant baculovirus (first generation recombinant baculovirus collected after transfection) with MOI=0.1 thin Born of the same parents' density is 1.5 × 10⁶The sf9 cell of cell/ml, collecting cells and supernatant after 27 DEG C of suspensions culture 72h is P2 generation weight Group baculoviral, by P2 for recombinant baculovirus with MOI=1 infection cell density be 2.5 × 10⁶The sf9 cell of cell/ml, Cell is collected after 27 DEG C of suspension culture 96h, through ultrasonication, obtains soluble HA albumen；

(3) soluble HA albumen is mixed with adjuvant, obtains the bird flu based on MultiBac baculovirus expression system Vaccine；

Adjuvant described in step (3) is preferably SEPPIC MONTANIDETM ISA 201VG；

When soluble HA albumen described in step (3) comes from H7N9 subtype avian influenza virus, the blood of soluble HA albumen Solidifying potency is 2¹⁰~2¹⁴, mixed with adjuvant according to the ratio of volume ratio 1:1；

When soluble HA albumen described in step (3) comes from H5N1 subtype avian influenza virus, the blood of soluble HA albumen Solidifying potency is preferably 2¹⁰~2¹⁴, mixed with adjuvant according to the ratio of volume ratio 1:1；

The avian influenza vaccine based on MultiBac baculovirus expression system is in preparing pre- avian influenza-prevention product Application；

The present invention has the following advantages and effects with respect to the prior art:

(1) the present invention provides a kind of avian influenza vaccines based on MultiBac baculovirus expression system, wherein MultiBac baculovirus expression system includes a variety of transmission plasmids, can facilitate the multiple foreign genes of insertion, there is preparation multivalence stream The potentiality of influenza vaccine；And the function that v-cath and chiA gene is destroyed in the Baculovirus Gene group that the system includes is made With being more advantageous to the great expression of foreign gene.Since foreign gene is eukaryotic expression, life similar with natural products is maintained Object activity, has high application value.

(2) preparation method of the avian influenza vaccine provided by the invention based on MultiBac baculovirus expression system and existing There is technology different, is to mix the HA albumen of suitable hemagglutinative titer with adjuvant, this allows for vaccine and is quickly prepared, and has province The advantages of Shi Shengli.

(3) present invention quickly expresses the HA albumen of bird flu prevalence strain using MultiBac baculovirus expression system, And avian influenza genes engineered vaccine is prepared using the high bioactivity HA albumen of expression, it is assessed in SPF chicken immune challenge viral dosage The Vaccine effectiveness of the vaccine is conducive to the popular of prevention and control of fowl influenza virus and propagates.

(4) HA albumen, shaking flask culture are expressed in the embodiment of the present invention in the way of flask suspension culture insect cell 20ml cell concentration can obtain potency >=2 HA¹⁶Protein liquid, gained HA albumen has the spy that expression quantity is high and bioactivity is good Point can greatly reduce vaccine cost in the future using the modes great expression HA albumen such as fermentor.

(5) avian influenza vaccine provided by the invention based on MultiBac baculovirus expression system has immunogenicity good With safe and reliable feature.Experiments prove that the specific advantage of the avian influenza vaccine is as follows: 1. the influenza vaccines safely may be used Lean on, after SPF chicken is immunized in subcutaneous injection mode, vaccine absorb it is fast, and injection site it is disease-free cash as.2. with HA potency for 2¹⁴ With 2¹⁰Protein liquid be prepared into the avian influenza vaccine of high low dose group, the experimental results showed that high low dose group vaccine obtain it is similar Immune effect, low dose group avian influenza vaccine can Protection chicken from homologous avian influenza virus attack.3. SPF chicken immune Afterwards challenge viral dosage the result shows that: after immune H7N9 avian influenza vaccine, experimental chicken is almost without toxin expelling, and without any morbidity in 14 days Phenomenon, weight gain is obvious, and the H7N9 vaccine of the high low dosage of single immunization obtains 100% protective rate；Immune H5N1 fowl stream After influenza vaccine, though experimental chicken has several days toxin expellings, without morbidity phenomenon in 14 days, weight gain is obvious, the high low dosage of single immunization H5N1 vaccine obtain 89% protective rate.

Detailed description of the invention

Fig. 1 is the qualification result figure of recombinant plasmid pACE-HA7；Wherein, M:DNA Marker (DL5000Marker), 1: Using plasmid pACE-HA7 as template amplification HA segment, 2: with ddH₂O is the negative control of template, 3:saII, Not I double digestion matter The DNA fragmentation that grain pACE-HA7 is obtained.

Fig. 2 is the qualification result figure of recombinant plasmid pACE-HA5；Wherein, M:DNA Marker (DL5000Marker), 1: Using plasmid pACE-HA5 as template amplification HA segment, 2: with ddH₂O is the negative control of template, 3:saII, Xba I double digestion matter The DNA fragmentation that grain pACE-HA7 is obtained.

Fig. 3 is the qualification result figure of recombinant baculovirus Bacmid-HA7 and Bacmid-HA5；Wherein, A:M13 primer reflects Determine Bacmid-HA7 plasmid, B:M13 primer identifies Bacmid-HA5 plasmid.

Fig. 4 is H7N9 subtype avian influenza HA albumen through Western Blot experimental identification result figure；Wherein, M:protein Maker (size 170Kda), 1,3:P2 for supernatant after the processing of Bv-HA7 infection cell, and 2,4:P2 for the processing of Bv-HA7 infection cell After precipitate, 5: compareing (mock) after full cell processing.

Fig. 5 is H5N1 subtype avian influenza HA albumen through Western Blot experimental identification result figure；Wherein, M:protein Maker (size 170Kda), 1,3:P2 for supernatant after the processing of Bv-HA5 infection cell, and 2,4:P2 for the processing of Bv-HA5 infection cell After precipitate, 5 for full cell processing after compare (mock).

Fig. 6 is that the horizontal result analysis chart of HI after SPF chicken is immunized in H7N9 vaccine group.

Fig. 7 is that the horizontal result analysis chart of HI after SPF chicken is immunized in H5N1 vaccine group.

Fig. 8 is the survival results analysis chart of chicken after attacking poison.

Specific embodiment

Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited In this.

Test material in following embodiments is derived from commercial sources unless otherwise noted.The test side Method is unless otherwise noted conventional methods.

The building and identification of 1 recombinant baculovirus plasmid (Bacmid) of embodiment

(1) extraction of H7N9 and H5N1 subtype avian influenza virus geneome RNA:

H7 subtype avian influenza virus H7N9 (E157) and H5 subtype avian influenza virus H5N1 used in this experiment and by Its positive serum prepared is provided by the local joint project laboratory of Amphixenosis's anti-control agent country, Agricultural University Of South China. Chick embryo allantoic liquid 12000r/min containing avian influenza virus is centrifuged 5min, takes 500 μ L to DEPC of supernatant processed RNA is extracted in Eppendorf pipe；

1. 500 μ L Trizol LS Reagent is added, it acutely is placed at room temperature for 10min after concussion, 200 μ L chloroforms are added, It is sufficiently mixed, after solution to be emulsified is in milky white shape, is stored at room temperature after 10min and is centrifuged (13000rpm, 15min, 4 DEG C)；

2. 600 μ L upper strata aqueous phases is taken to move to the processed 1.5mL sterilizing Eppendorf pipe of another new DEPC；

3. isometric isopropanol is added into supernatant, after the centrifuge tube that turns upside down mixes well, 10min is stood at room temperature It is centrifuged (13000rpm, 15min, 4 DEG C)；

4. discarding supernatant liquid, it is slowly added to the ethanol solution that the 1mL volume fraction of pre-cooling is 75% along centrifugation tube wall, slowly It is mixed by inversion, is centrifuged (8000rpm, 5min, 4 DEG C), discards supernatant liquid；

5. after air-drying in superclean bench, suitable DEPC processing water dissolution RNA, wait be completely dissolved, -70 DEG C of guarantors is added Deposit or be directly used in reverse transcription；

6. taking a small amount of RNA solution detectable concentration.

(2) RNA reverse transcription synthesizes cDNA

RT reaction system (20 μ L) is as follows: 10 μ L templates and 2 μ L primers, 72 DEG C of water-bath 10min are added to 0.2mL centrifuge tube Afterwards, reverse transcription is carried out according to the specification of TaKaRa company Reverse Transcriptase Reagents kit, i.e., is added in the PCR system of 20 μ L following Each component: 45 × Buffer of μ L, 2 μ L dNTPMix, 1 μ L M-MLVRT, 0.5 μ L Recombinant RNase, 42 DEG C of water-baths 1h。

According to influenza virus H7N9 (E157) and H5N1 hemagglutinin HA nucleotide sequence, two pairs of primers, P1, P2 are separately designed With P3, P4.Sal I, Not I restriction enzyme site wherein are introduced in P1, P2 upstream and downstream, and introduces 6 × HIS mark in downstream primer Label, primer sequence are as follows:

P1:5 '-ACGCGTCGACATGAACACTCAAATCCTGGTATTCGCTC-3 '；

P2:5 '-ATAAGAATGCGGCCGCTTAGTGATGGTGATGGTGATGTATACAA ATAGTGCACCGCATGTT- 3'；

The amplification condition of primer P1/P2 are as follows: loop parameter are as follows: 98 DEG C, 10s, 60 DEG C, 5s, 72 DEG C, 2min；30 circulations Extend 2min eventually for 72 DEG C afterwards.After PCR product is recycled and uses Sal I and Not I double digestion, it is inserted into transferring plasmid pACEBac1 Sal I and Not I site, convert escherichia coli DH5a competent cell, obtain carrier pACE-HA7.With PCR amplification purpose Gene and the mode of digestion identification identify recombinant plasmid, and the plasmid is sequenced, as a result correct (Fig. 1).

And primer P3, P4 introduce Sal I, Xba I restriction enzyme site in upstream and downstream, and downstream primer introduces 6 × HIS mark Label, primer sequence are as follows:

P3:5 '-ACGCGTCGAC ATGGAGAAAATAGTGCTTCTCTTTG-3 '；

P4:5 '-GCTCTAGATTAGTGATGGTGATGGTGATGAATGCAAATTCTGCA CTGCAAC-3 '；

The amplification condition of primer P3/P4 are as follows: loop parameter are as follows: 98 DEG C, 10s, 60 DEG C, 5s, 72 DEG C, 2min；30 circulations Extend 2min eventually for 72 DEG C afterwards.After PCR product is recycled and uses Sal I and Xba I double digestion, it is inserted into transferring plasmid pACEBac1 Sal I and Xba I site, convert escherichia coli DH5a competent cell, obtain carrier pACE-HA5.With PCR amplification purpose Gene and the mode of digestion identification identify recombinant plasmid, and the plasmid is sequenced, as a result correct (Fig. 2).

The amino acid sequence of the HA albumen of H7N9 (E157) subtype avian influenza virus:

MNTQILVFALIAIIPTNADKICLGHHAVSNGIKVNTLTEKGVEVVNATETVERTNTPRICSKGKRTVD LGQCGLLGTITGPPQCDQFLEFSADLIIERREGSDVCYPGKFVNEEALRQILRESGGIDKESMGLTYNGIRTNGVT SACRRSGSSFYAEMKWLLSNTDNAAFPQMTKSYKNTRESPAIIVWGIHHSVSTAEQTKLYGSGNKLVTVGSSNYQQ SFVPSPGARPQVNGQSGRIDFHWLILNPNDTVTFSFNGAFIAPDRASFLRGKSMGIQSGVQVDANCEGDCYHSGGT IISNLPFQNIDSRAVGKCPRYVKQRSLLLATGMKNVPEVPKGKRTARGLFGAIAGFIENGWEGLIDGWYGFRHQNA QGEGTAADYKSTQSAIDQITGKLNRLIAKTNQQFKLIDNEFNEVEKQIGNVINWTRDSITEVWSYNAELLVAMENQ HTIDLADSEMDKLYERVKRQLRENAEEDGTGCFEIFHKCDDDCMASIRNNTYDHRKYREEAMQNRIQIDPVKLSSG YKDVILWFSFGASCFILLAIVMGLVFICVKNGNMRCTICI.

Encode the nucleotide sequence of the gene of the HA albumen of H7N9 (E157) subtype avian influenza virus:

ATGAACACTCAAATCCTGGTATTCGCTCTGATTGCGATCATTCCAACAAATGCAGACAAAATCTGCCTC GGACATCATGCCGTGTCAAACGGAATCAAAGTAAACACATTAACTGAAAAAGGAGTGGAAGTCGTCAATGCAACTGA AACAGTGGAACGAACAAACACCCCCAGGATCTGCTCAAAAGGGAAAAGGACAGTTGATCTCGGTCAATGTGGACTCC TGGGGACAATCACTGGACCACCTCAATGTGACCAATTCCTAGAATTTTCGGCCGATTTAATTATTGAGAGGCGAGAA GGAAGTGATGTCTGTTATCCTGGAAAATTCGTGAATGAAGAAGCTCTGAGGCAAATTCTCAGAGAATCAGGCGGAAT TGACAAGGAATCCATGGGACTCACATACAATGGAATAAGAACTAATGGGGTGACCAGTGCATGTAGGAGATCAGGAT CTTCATTCTATGCAGAAATGAAATGGCTCCTGTCAAACACAGATAATGCTGCATTCCCGCAGATGACTAAGTCATAT AAAAATACAAGAGAAAGCCCAGCTATAATAGTATGGGGGATCCATCATTCCGTTTCAACTGCAGAGCAAACCAAGCT ATATGGGAGTGGAAACAAACTGGTGACAGTTGGGAGTTCTAATTATCAACAATCTTTCGTACCGAGTCCAGGAGCAA GACCACAAGTTAATGGTCAATCTGGAAGAATTGACTTTCATTGGCTAATACTAAATCCCAATGATACAGTCACTTTC AGTTTCAATGGGGCTTTCATAGCTCCAGACCGTGCAAGCTTCCTGAGAGGAAAATCTATGGGAATCCAGAGTGGAGT ACAGGTTGATGCCAATTGTGAAGGGGACTGCTATCATAGTGGAGGGACAATAATAAGTAACTTGCCATTTCAGAACA TAGATAGCAGGGCAGTTGGAAAATGTCCGAGATATGTTAAGCAAAGGAGTCTTCTGCTGGCAACAGGGATGAAGAAT GTTCCTGAGGTTCCAAAGGGAAAACGGACTGCGAGAGGCCTATTTGGTGCTATAGCGGGTTTCATTGAAAATGGATG GGAAGGCCTAATTGATGGTTGGTATGGTTTCAGACACCAGAATGCACAGGGAGAGGGAACTGCTGCAGATTACAAAA GCACTCAATCGGCAATTGATCAAATAACAGGGAAATTAAATCGGCTTATAGCAAAAACCAACCAACAATTTAAGTTG ATAGACAATGAATTCAATGAGGTAGAGAAGCAAATCGGTAATGTGATAAATTGGACCAGAGATTCTATAACAGAAGT ATGGTCATACAATGCTGAACTCTTGGTGGCAATGGAGAACCAGCATACAATTGATCTGGCTGATTCAGAAATGGACA AACTGTACGAACGAGTGAAAAGACAGCTGAGAGAGAATGCTGAAGAAGATGGCACGGGTTGCTTTGAAATATTTCAC AAGTGTGATGATGACTGTATGGCCAGTATTAGAAATAACACCTATGATCACAGAAAATACAGAGAGGAGGCAATGCA AAATAGAATACAGATTGACCCAGTCAAACTAAGCAGCGGCTACAAAGATGTGATACTTTGGTTTAGCTTCGGGGCAT CATGTTTCATACTTCTAGCCATTGTAATGGGCCTTGTCTTCATATGTGTGAAGAATGGAAACATGCGGTGCACTATT TGTATATAA

The amino acid sequence of the HA albumen of H5N1 subtype avian influenza virus:

MEKIVLLFATISLAKSDHICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLNGVKPLIL KDCSVAGWLLGNPWCDEFINVPEWSYIVEKANPVNGLCYPGNFNDYEELKHLLSRINHFEKIQIIPKDSWSDHEAS KGVSAACSYQGKSSFFRNVVWLIKKNDTYPTIKKDYNNTNREDLLVLWGIHHPNDKAEQITLYQNPTTYISIGTST LNQRLVPKIATRSKINGQSGRIDFFWTILKPNDAIHFESNGNFIAPEYAYKIVKKGDSTIMRSEVEYGNCNTRCQT PVGAINSSMPFHNIHPLTIGECPKYVKSNKLVLATGLRNSPQRERRRKRGLFGAIAGFIEGGWQGMVDGWYGYHHS NEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLME NERTLDFHDSNVKNLYDKVRLQLKDNAKELGNGCFEFYHKCNNECMESVRNGTYDYPQYSEEARLKREEISGVKLE SIGVYQILSIYSTVASSLVLAIMMAGLSLWMCSNGSLQCRICI.

Encode the nucleotide sequence of the HA albumen of H5N1 subtype avian influenza virus:

ATGGAGAAAATAGTGCTTCTCTTTGCAACAATCAGTCTTGCTAAAAGCGATCATATTTGCATTGGATAT CATGCAAATAACTCGACAGAGCAGGTTGACACAATAATGGAAAAGAACGTTACTGTTACACATGCCCAAGACATACT GGAAAAGACACACAACGGGAAGCTTTGCGATCTAAATGGGGTGAAGCCTCTGATTTTAAAAGATTGTAGTGTAGCAG GATGGCTACTCGGAAACCCATGGTGTGACGAATTCATCAATGTACCAGAATGGTCTTACATAGTAGAGAAGGCCAAT CCAGTCAATGGCCTCTGTTACCCAGGGAATTTCAACGATTATGAAGAATTGAAACACCTATTGAGCAGGATAAACCA TTTTGAGAAAATACAGATCATCCCCAAAGATTCTTGGTCAGATCACGAAGCCTCAAAGGGGGTGAGCGCAGCATGTT CATACCAGGGAAAGTCCTCCTTCTTCAGAAATGTAGTATGGCTTATCAAAAAGAACGATACATATCCAACAATAAAG AAAGATTACAATAATACCAACCGAGAAGATCTCTTGGTACTTTGGGGGATCCACCATCCTAATGATAAAGCAGAGCA AATAACGCTCTATCAAAACCCAACCACCTATATTTCCATTGGGACGTCAACACTAAACCAGAGATTGGTACCAAAAA TAGCCACTAGATCCAAAATAAACGGGCAAAGTGGCAGGATAGATTTCTTCTGGACAATTTTAAAACCGAATGATGCA ATCCACTTCGAGAGTAATGGAAATTTCATTGCTCCAGAATATGCATACAAAATTGTCAAGAAAGGAGACTCCACAAT TATGAGAAGTGAAGTGGAATATGGTAACTGCAACACCAGGTGTCAGACTCCGGTAGGGGCGATAAACTCTAGTATGC CATTCCATAACATACACCCCCTCACCATCGGAGAATGTCCCAAATATGTGAAATCGAACAAATTAGTCCTTGCGACT GGACTCAGAAATAGTCCTCAAAGAGAGAGAAGAAGAAAAAGAGGACTATTTGGAGCTATAGCAGGTTTCATAGAGGG AGGATGGCAGGGAATGGTAGATGGTTGGTATGGGTACCACCACAGCAATGAGCAGGGGAGTGGATACGCTGCAGACA AAGAATCTACTCAAAAGGCAATAGACGGAGTCACCAATAAGGTCAACTCGATCATTGACAAAATGAACACTCAGTTT GAGGCCGTAGGAAGGGAATTTAATAACTTAGAGAGGAGAATAGAGAATTTAAACAAGAAGATGGAAGACGGATTCCT AGATGTCTGGACTTATAATGCTGAACTTCTGGTTCTCATGGAAAATGAAAGAACTTTAGACTTCCATGACTCAAATG TCAAGAACCTTTACGATAAGGTCAGACTACAGCTTAAGGATAATGCAAAAGAGCTGGGCAACGGTTGTTTCGAGTTC TATCACAAATGTAATAATGAATGTATGGAAAGTGTAAGAAACGGAACGTATGACTACCCGCAGTATTCAGAAGAAGC AAGATTAAAAAGAGAGGAAATAAGTGGAGTAAAATTGGAGTCAATAGGAGTCTACCAAATACTGTCAATTTATTCAA CAGTGGCGAGTTCCCTAGTGCTGGCAATCATGATGGCTGGTCTATCTTTATGGATGTGTTCCAACGGGTCGTTGCAG TGCAGAATTTGCATTTAA

(3) acquisition and identification of recombinant baculovirus plasmid Bacmid-HA5 and Bacmid-HA7

Donor plasmid pACE-HA5 and pACE-HA7 are converted into DH10MultiBac competent escherichia coli cell respectively, led to Swivel base recombination is crossed, recombinant baculovirus plasmid Bacmid-HA5 and Bacmid-HA7 is obtained respectively, according to Invitrogen company Insect baculovirus operation manual identifies it with M13 primer amplification segment, as a result correct (Fig. 3).

The acquisition of embodiment 2 recombinant baculovirus BV-HA5 and BV-HA7

Using liposome mediated transfection method, recombinant baculovirus plasmid Bacmid-HA5 and Bacmid-HA7 are transfected respectively Sf9 insect cell is cultivated in 27 DEG C；When culture is to 72h, there is lesion in cell, collects cells and supernatant, i.e., obtains the respectively Generation recombinant baculovirus (P1) BV-HA5 and BV-HA7.

The acquisition and identification of embodiment 3H5N1 and H7N9 subtype influenza virus HA albumen

The present invention expresses HA albumen, specifically with recombinate shape virus infection sf9 cell by way of flask suspension culture Operating procedure is as follows: (1) recombinant baculovirus passes on: cell is after the recovery of Kolle flask static gas wave refrigerator, with cell density after passage Not less than 2.5 × 10⁵The inoculum concentration of cell/ml is gradually amplified to 250ml shaking flask, and shaking bottling liquid product is 50ml, close to cell Degree is 1.5 × 10⁶When cell/ml or so, P1 is inoculated with sf9 cell for recombinant baculovirus with MOI=0.1, cultivation temperature is 27 DEG C, shaking speed is 100~120rpm/min, and culture medium is sf-900TM II SFM, collects cell after infection cell 72h Supernatant is simultaneously filtered with the filter of 0.22um, and gained filtrate is second generation recombinant baculovirus (P2 generation).

(2) recombinant protein is expressed, specific steps are as follows:

By P2 for recombinant baculovirus with MOI=1 inoculating cell density be 2.5 × 10⁶The sf9 cell of cell/ml, It is 20ml that 100ml, which shakes bottling liquid product, and cultivation temperature is 27 DEG C, and shaking speed is 100~120rpm/min, is received when cultivating 96h Cell is taken, gained cell precipitation is resuspended using PBS phosphate buffer, the cell suspension after resuspension is used at ultrasonic wave Reason, treatment conditions are as follows: working time 3s, intermittent time 5s, 100~200w of operating power are crushed 10~15min of time.Through super Cell suspension can become to clarify bright after sonicated, will treated cell suspension with 4 DEG C, 13000rpm/min centrifugation 8~ 10min takes supernatant, and treated, and HA albumen exists in the form of soluble protein in supernatant, -80 DEG C of protein liquid are saved.Through Blood coagulation tests identification: H7N9-HA protein liquid HA potency is 2¹⁶, H5N1-HA protein liquid HA potency is 2¹⁶.H7N9-HA albumen warp Western Blot experimental identification is correct (see Fig. 4), and H5N1-HA albumen is correct (see Fig. 5) through Western Blot experimental identification.

4 Analysis of Immunogenicity of embodiment

(1) preparation of H5N1 and H7N9 subtype avian influenza virus recombinant vaccine

It is real that the HA protein liquid of H7N9 and H5N1 subtype avian influenza virus after ultrasonication in embodiment 3 is subjected to blood clotting It tests, it is final to determine that high dose group protein liquid hemagglutinative titer is 2 according to blood coagulation tests result treatment HA protein liquid¹⁴, low dose group egg White liquor hemagglutinative titer is 2¹⁰, finally by high low dose group protein liquid respectively at SEPPIC MONTANIDETM ISA 201VG adjuvant It is mixed with the ratio of volume ratio 1:1, finally respectively obtains the avian influenza genes engineered vaccine of the high low dose group of H5 and H7 hypotype.

(2) immune programme

2 week old SPF chickens are randomly divided into 5 groups and mark, and every group 10, grouping is immunized: first group is H7N9 high dose group epidemic disease Seedling (2¹⁴), immunization ways are neck subcutaneous injection, and immunizing dose is 500 μ L/；Second group is H7N9 low dose group vaccine (2¹⁰), immunization ways are neck subcutaneous injection, and immunizing dose is 500 μ L/；Third group is H5N1 high dose group vaccine (2¹⁴), Immunization ways are neck subcutaneous injection, and immunizing dose is 500 μ L/；4th group is H5N1 low dose group vaccine (2¹⁰), side is immunized Formula is neck subcutaneous injection, and immunizing dose is 500 μ L/；Wing venous blood sampling detects HI antibody weekly in 3 weeks after immune.

(3) Serum HI antibody testing result

25 μ L PBS are added in blood-coagulation-board, take the 25 continuous doubling dilutions of μ L serum, and respectively equivalent first is added in H7N9 vaccine group 4 unit isogeneic H5N1 of equivalent formalin-inactivated are added in 4 unit isogeneic H7N9 (E157) of aldehyde inactivation, H5N1 vaccine group (separation of this laboratory saves), mixing is stored at room temperature 30 minutes, and equivalent 1% (percent by volume) chicken red blood cell is added, and room temperature is put Observation after setting 30 minutes is as a result, the serum highest dilution that can completely inhibit erythrocyte agglutination is the HI potency of tested serum.

H7N9 vaccine group has antibody generation in second week, and at third week, HI potency reaches 2⁶More than, H7N9 high dose Two weeks HI potency of vaccine group are higher than H7N9 Low dose vaccine group, but the difference between the two is not significant (see Fig. 6), illustrate H7N9 height Dosage group vaccine one can make SPF chicken generate HI potency not less than 2 after exempting from⁶Antibody level.

H5N1 vaccine group antibody level in second week is lower, and at third week, HI potency reaches 2⁴~2⁵, but H5N1 high agent It measures two weeks HI potency of vaccine group and is higher than H5N1 Low dose vaccine group, the two difference is more significant (see Fig. 7).

Embodiment 5 attacks malicious Protection research

(1) swab toxin expelling detects after attacking poison

It is immunized according to 4 immune programme of embodiment, 3 weeks after being immunized, PBS control group is randomly divided into 2 groups, every group each 5 SPF chicken；H7N9 high Low dose vaccine group and one group of PBS control group collunarium are attacked into poison 10⁶EID50/200μL H7N9(E157)； H5N1 high Low dose vaccine group and one group of PBS control group collunarium are attacked into poison 10⁶EID50/200μL H5N1；Attack 3 after poison, 5,7,9, 11 days acquisition oropharynxs and cloacal swab conventionally carry out the separation and measurement of virus.

After attacking poison in 3 days, H7N9 and H5N1 control group experimental chicken is dead.H7N9 high dose group on day 3 with the 5th day Respectively detect 1 chicken cutlet poison, H7N9 low dose group only detects 3 chicken cutlet poison on day 3；H5N1 high dose group is examined on day 3 It measures 7 chicken cutlet poison and the 5th day detects 4 chicken cutlet poison, a chicken cutlet poison was detected at the 7th day, and this chicken was at the 8th day It is dead；H5N1 low dose group detects 5 chicken cutlet poison on day 3 and detects within the 5th day 2 chicken cutlet poison, a 5th day dead chicken (being pecked extremely by same cage chicken), but oropharynx and cloacal swab detect toxin expelling, are judged to dying of illness herein.As a result such as table 1.

Toxin expelling situation counts after table 1SPF chicken attacks poison

(2) survival rate of chicken after poison is attacked

The death condition of continuous 14 days observation chickens after poison is attacked, H7N9 control group is all dead in 3 days after attacking poison, H7N9 high Low dose vaccine group is without chicken death, and protective rate 100%, H5N1 control group is all dead after attacking poison 1 day, H5N1 vaccine Each group only remains 9 SPF chickens when group attacks poison, attacks after poison each dead 1, protective rate 89%, as shown in Figure 8.

It is above-mentioned all the results showed that 1, H7N9 and H5N1 subtype gene engineered vaccine only be immunized can once generate The antibody of effect.2, homologous H7N9 avian flu is resisted in the protection that H7N9 high low dose group vaccine immunity can once provide 100% Homologous H5N1 avian influenza virus is resisted in the attack of poison, the protection that H5N1 high low dose group vaccine immunity can once provide 89% Attack.

The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention, It should be equivalent substitute mode, be included within the scope of the present invention.

SEQUENCE LISTING

<110>Agricultural University Of South China

<120>avian influenza vaccine and preparation and application based on MultiBac baculovirus expression system

<130> 1

<160> 8

<170> PatentIn version 3.3

<210> 1

<211> 564

<212> PRT

<213> Artificial

<220>

<223>H7N9(E157) subtype avian influenza virus HA albumen amino acid sequence

<400> 1

Met Asn Thr Gln Ile Leu Val Phe Ala Leu Ile Ala Ile Ile Pro Thr

1 5 10 15

Asn Ala Asp Lys Ile Cys Leu Gly His His Ala Val Ser Asn Gly Ile

20 25 30

Lys Val Asn Thr Leu Thr Glu Lys Gly Val Glu Val Val Asn Ala Thr

35 40 45

Glu Thr Val Glu Arg Thr Asn Thr Pro Arg Ile Cys Ser Lys Gly Lys

50 55 60

Arg Thr Val Asp Leu Gly Gln Cys Gly Leu Leu Gly Thr Ile Thr Gly

65 70 75 80

Pro Pro Gln Cys Asp Gln Phe Leu Glu Phe Ser Ala Asp Leu Ile Ile

85 90 95

Glu Arg Arg Glu Gly Ser Asp Val Cys Tyr Pro Gly Lys Phe Val Asn

100 105 110

Glu Glu Ala Leu Arg Gln Ile Leu Arg Glu Ser Gly Gly Ile Asp Lys

115 120 125

Glu Ser Met Gly Leu Thr Tyr Asn Gly Ile Arg Thr Asn Gly Val Thr

130 135 140

Ser Ala Cys Arg Arg Ser Gly Ser Ser Phe Tyr Ala Glu Met Lys Trp

145 150 155 160

Leu Leu Ser Asn Thr Asp Asn Ala Ala Phe Pro Gln Met Thr Lys Ser

165 170 175

Tyr Lys Asn Thr Arg Glu Ser Pro Ala Ile Ile Val Trp Gly Ile His

180 185 190

His Ser Val Ser Thr Ala Glu Gln Thr Lys Leu Tyr Gly Ser Gly Asn

195 200 205

Lys Leu Val Thr Val Gly Ser Ser Asn Tyr Gln Gln Ser Phe Val Pro

210 215 220

Ser Pro Gly Ala Arg Pro Gln Val Asn Gly Gln Ser Gly Arg Ile Asp

225 230 235 240

Phe His Trp Leu Ile Leu Asn Pro Asn Asp Thr Val Thr Phe Ser Phe

245 250 255

Asn Gly Ala Phe Ile Ala Pro Asp Arg Ala Ser Phe Leu Arg Gly Lys

260 265 270

Ser Met Gly Ile Gln Ser Gly Val Gln Val Asp Ala Asn Cys Glu Gly

275 280 285

Asp Cys Tyr His Ser Gly Gly Thr Ile Ile Ser Asn Leu Pro Phe Gln

290 295 300

Asn Ile Asp Ser Arg Ala Val Gly Lys Cys Pro Arg Tyr Val Lys Gln

305 310 315 320

Arg Ser Leu Leu Leu Ala Thr Gly Met Lys Asn Val Pro Glu Val Pro

325 330 335

Lys Gly Lys Arg Thr Ala Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe

340 345 350

Ile Glu Asn Gly Trp Glu Gly Leu Ile Asp Gly Trp Tyr Gly Phe Arg

355 360 365

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

370 375 380

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

385 390 395 400

Lys Thr Asn Gln Gln Phe Lys Leu Ile Asp Asn Glu Phe Asn Glu Val

405 410 415

Glu Lys Gln Ile Gly Asn Val Ile Asn Trp Thr Arg Asp Ser Ile Thr

420 425 430

Glu Val Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Met Glu Asn Gln

435 440 445

His Thr Ile Asp Leu Ala Asp Ser Glu Met Asp Lys Leu Tyr Glu Arg

450 455 460

Val Lys Arg Gln Leu Arg Glu Asn Ala Glu Glu Asp Gly Thr Gly Cys

465 470 475 480

Phe Glu Ile Phe His Lys Cys Asp Asp Asp Cys Met Ala Ser Ile Arg

485 490 495

Asn Asn Thr Tyr Asp His Arg Lys Tyr Arg Glu Glu Ala Met Gln Asn

500 505 510

Arg Ile Gln Ile Asp Pro Val Lys Leu Ser Ser Gly Tyr Lys Asp Val

515 520 525

Ile Leu Trp Phe Ser Phe Gly Ala Ser Cys Phe Ile Leu Leu Ala Ile

530 535 540

Val Met Gly Leu Val Phe Ile Cys Val Lys Asn Gly Asn Met Arg Cys

545 550 555 560

Thr Ile Cys Ile

<210> 2

<211> 567

<212> PRT

<213> Artificial

<220>

<223>amino acid sequence of the HA albumen of H5N1 subtype avian influenza virus

<400> 2

Met Glu Lys Ile Val Leu Leu Phe Ala Thr Ile Ser Leu Ala Lys Ser

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Arg Ile Asp Phe Phe Trp Thr Ile Leu Lys Pro Asn Asp Ala Ile His

245 250 255

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

260 265 270

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

275 280 285

Asn Cys Asn Thr Arg Cys Gln Thr Pro Val Gly Ala Ile Asn Ser Ser

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

Gly Phe Ile Glu Gly Gly Trp Gln Gly Met Val Asp Gly Trp Tyr Gly

355 360 365

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

370 375 380

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

385 390 395 400

Ile Asp Lys Met Asn Thr Gln Phe Glu Ala Val Gly Arg Glu Phe Asn

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

Asp Lys Val Arg Leu Gln Leu Lys Asp Asn Ala Lys Glu Leu Gly Asn

465 470 475 480

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

485 490 495

Val Arg Asn Gly Thr Tyr Asp Tyr Pro Gln Tyr Ser Glu Glu Ala Arg

500 505 510

Leu Lys Arg Glu Glu Ile Ser Gly Val Lys Leu Glu Ser Ile Gly Val

515 520 525

Tyr Gln Ile Leu Ser Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu

530 535 540

Ala Ile Met Met Ala Gly Leu Ser Leu Trp Met Cys Ser Asn Gly Ser

545 550 555 560

Leu Gln Cys Arg Ile Cys Ile

565

<210> 3

<211> 1695

<212> DNA

<213> Artificial

<220>

<223>encode H7N9(E157) subtype avian influenza virus HA albumen gene nucleotide sequence

<400> 3

atgaacactc aaatcctggt attcgctctg attgcgatca ttccaacaaa tgcagacaaa 60

atctgcctcg gacatcatgc cgtgtcaaac ggaatcaaag taaacacatt aactgaaaaa 120

ggagtggaag tcgtcaatgc aactgaaaca gtggaacgaa caaacacccc caggatctgc 180

tcaaaaggga aaaggacagt tgatctcggt caatgtggac tcctggggac aatcactgga 240

ccacctcaat gtgaccaatt cctagaattt tcggccgatt taattattga gaggcgagaa 300

ggaagtgatg tctgttatcc tggaaaattc gtgaatgaag aagctctgag gcaaattctc 360

agagaatcag gcggaattga caaggaatcc atgggactca catacaatgg aataagaact 420

aatggggtga ccagtgcatg taggagatca ggatcttcat tctatgcaga aatgaaatgg 480

ctcctgtcaa acacagataa tgctgcattc ccgcagatga ctaagtcata taaaaataca 540

agagaaagcc cagctataat agtatggggg atccatcatt ccgtttcaac tgcagagcaa 600

accaagctat atgggagtgg aaacaaactg gtgacagttg ggagttctaa ttatcaacaa 660

tctttcgtac cgagtccagg agcaagacca caagttaatg gtcaatctgg aagaattgac 720

tttcattggc taatactaaa tcccaatgat acagtcactt tcagtttcaa tggggctttc 780

atagctccag accgtgcaag cttcctgaga ggaaaatcta tgggaatcca gagtggagta 840

caggttgatg ccaattgtga aggggactgc tatcatagtg gagggacaat aataagtaac 900

ttgccatttc agaacataga tagcagggca gttggaaaat gtccgagata tgttaagcaa 960

aggagtcttc tgctggcaac agggatgaag aatgttcctg aggttccaaa gggaaaacgg 1020

actgcgagag gcctatttgg tgctatagcg ggtttcattg aaaatggatg ggaaggccta 1080

attgatggtt ggtatggttt cagacaccag aatgcacagg gagagggaac tgctgcagat 1140

tacaaaagca ctcaatcggc aattgatcaa ataacaggga aattaaatcg gcttatagca 1200

aaaaccaacc aacaatttaa gttgatagac aatgaattca atgaggtaga gaagcaaatc 1260

ggtaatgtga taaattggac cagagattct ataacagaag tatggtcata caatgctgaa 1320

ctcttggtgg caatggagaa ccagcataca attgatctgg ctgattcaga aatggacaaa 1380

ctgtacgaac gagtgaaaag acagctgaga gagaatgctg aagaagatgg cacgggttgc 1440

tttgaaatat ttcacaagtg tgatgatgac tgtatggcca gtattagaaa taacacctat 1500

gatcacagaa aatacagaga ggaggcaatg caaaatagaa tacagattga cccagtcaaa 1560

ctaagcagcg gctacaaaga tgtgatactt tggtttagct tcggggcatc atgtttcata 1620

cttctagcca ttgtaatggg ccttgtcttc atatgtgtga agaatggaaa catgcggtgc 1680

actatttgta tataa 1695

<210> 4

<211> 1704

<212> DNA

<213> Artificial

<220>

<223>nucleotide sequence of the HA albumen of H5N1 subtype avian influenza virus is encoded

<400> 4

atggagaaaa tagtgcttct ctttgcaaca atcagtcttg ctaaaagcga tcatatttgc 60

attggatatc atgcaaataa ctcgacagag caggttgaca caataatgga aaagaacgtt 120

actgttacac atgcccaaga catactggaa aagacacaca acgggaagct ttgcgatcta 180

aatggggtga agcctctgat tttaaaagat tgtagtgtag caggatggct actcggaaac 240

ccatggtgtg acgaattcat caatgtacca gaatggtctt acatagtaga gaaggccaat 300

ccagtcaatg gcctctgtta cccagggaat ttcaacgatt atgaagaatt gaaacaccta 360

ttgagcagga taaaccattt tgagaaaata cagatcatcc ccaaagattc ttggtcagat 420

cacgaagcct caaagggggt gagcgcagca tgttcatacc agggaaagtc ctccttcttc 480

agaaatgtag tatggcttat caaaaagaac gatacatatc caacaataaa gaaagattac 540

aataatacca accgagaaga tctcttggta ctttggggga tccaccatcc taatgataaa 600

gcagagcaaa taacgctcta tcaaaaccca accacctata tttccattgg gacgtcaaca 660

ctaaaccaga gattggtacc aaaaatagcc actagatcca aaataaacgg gcaaagtggc 720

aggatagatt tcttctggac aattttaaaa ccgaatgatg caatccactt cgagagtaat 780

ggaaatttca ttgctccaga atatgcatac aaaattgtca agaaaggaga ctccacaatt 840

atgagaagtg aagtggaata tggtaactgc aacaccaggt gtcagactcc ggtaggggcg 900

ataaactcta gtatgccatt ccataacata caccccctca ccatcggaga atgtcccaaa 960

tatgtgaaat cgaacaaatt agtccttgcg actggactca gaaatagtcc tcaaagagag 1020

agaagaagaa aaagaggact atttggagct atagcaggtt tcatagaggg aggatggcag 1080

ggaatggtag atggttggta tgggtaccac cacagcaatg agcaggggag tggatacgct 1140

gcagacaaag aatctactca aaaggcaata gacggagtca ccaataaggt caactcgatc 1200

attgacaaaa tgaacactca gtttgaggcc gtaggaaggg aatttaataa cttagagagg 1260

agaatagaga atttaaacaa gaagatggaa gacggattcc tagatgtctg gacttataat 1320

gctgaacttc tggttctcat ggaaaatgaa agaactttag acttccatga ctcaaatgtc 1380

aagaaccttt acgataaggt cagactacag cttaaggata atgcaaaaga gctgggcaac 1440

ggttgtttcg agttctatca caaatgtaat aatgaatgta tggaaagtgt aagaaacgga 1500

acgtatgact acccgcagta ttcagaagaa gcaagattaa aaagagagga aataagtgga 1560

gtaaaattgg agtcaatagg agtctaccaa atactgtcaa tttattcaac agtggcgagt 1620

tccctagtgc tggcaatcat gatggctggt ctatctttat ggatgtgttc caacgggtcg 1680

ttgcagtgca gaatttgcat ttaa 1704

<210> 5

<211> 38

<212> DNA

<213> Artificial

<220>

<223>primer P1

<400> 5

acgcgtcgac atgaacactc aaatcctggt attcgctc 38

<210> 6

<211> 61

<212> DNA

<213> Artificial

<220>

<223>primer P2

<400> 6

ataagaatgc ggccgcttag tgatggtgat ggtgatgtat acaaatagtg caccgcatgt 60

t 61

<210> 7

<211> 35

<212> DNA

<213> Artificial

<220>

<223>primer P3

<400> 7

acgcgtcgac atggagaaaa tagtgcttct ctttg 35

<210> 8

<211> 51

<212> DNA

<213> Artificial

<220>

<223>primer P4

<400> 8

gctctagatt agtgatggtg atggtgatga atgcaaattc tgcactgcaa c 51

Claims

1. a kind of avian influenza vaccine based on MultiBac baculovirus expression system, it is characterised in that include solubility HA egg White, solubility HA albumen is by that can express the baculovirus expression of the recombinant modified of avian influenza virus HA protein；

The soluble HA albumen is expressed by MultiBac baculovirus expression system.

2. according to right want 1 described in the avian influenza vaccine based on MultiBac baculovirus expression system, it is characterised in that:

The nucleotide sequence of the gene of the HA albumen of H7N9 subtype avian influenza virus is as shown in SEQ IDNO.3 on coding；

The nucleotide sequence of the gene of the HA albumen of H5N1 subtype avian influenza virus is encoded as shown in SEQ ID NO.4.

3. according to right want 1 described in the avian influenza vaccine based on MultiBac baculovirus expression system, it is characterised in that:

V-cath gene and chiA gene are destroyed and lose function in the genome of the baculoviral of the recombinant modified And single promoter gene box is inserted in genome.

4. the avian influenza vaccine according to claim 3 based on MultiBac baculovirus expression system, it is characterised in that:

Single promoter gene box is from upstream to downstream including such as lower component: polyhedrin promoter and coding bird flu The gene of the HA albumen of virus.

5. according to right want 1 described in the avian influenza vaccine based on MultiBac baculovirus expression system, it is characterised in that:

The carrier that sets out for constructing the baculoviral of recombinant modified is pACEBac1.

6. according to right want 1 described in the avian influenza vaccine based on MultiBac baculovirus expression system, it is characterised in that:

The baculoviral is Autographa californica multicapsid nucleopolyhedrosisvirus nuclear polyhedrosis virus.

7. according to right want 1 described in the avian influenza vaccine based on MultiBac baculovirus expression system, it is characterised in that:

The preparation method of the baculoviral of the recombinant modified, comprises the following steps:

The HA gene of avian influenza virus is inserted into transmission plasmid and is carried out by the genome of swivel base and shuttle vector Bacmid Homologous recombination obtains recombinant baculovirus genomic DNA, then by recombinant baculovirus genomic DNA transfection insect cell, Insect cell inner packing obtains the baculoviral of recombinant modified.

8. the preparation of the described in any item avian influenza vaccines based on MultiBac baculovirus expression system of claim 1~7 Method, characterized by comprising the steps of:

(1) genome for extracting avian influenza virus, is reversed to cDNA；Design primer, using avian influenza virus cDNA as template, Amplification obtains HA gene；HA gene is inserted into behind the PH promoter of transmission plasmid pACEBac1, obtains recombination transmission plasmid； Recombinant transfer plasmid is converted into DH10MultiBac Escherichia coli, is recombinated by swivel base, obtains recombinant baculovirus plasmid respectively； Finally recombinant baculovirus is obtained respectively by recombinant baculovirus plasmid transfection sf9 insect cell with liposome method；

(2) by P1 for recombinant baculovirus with MOI=0.1 infection cell density be 1.5 × 10⁶The sf9 cell of cell/ml, 27 DEG C Suspending and collecting supernatant after cultivating 72h is P2 for recombinant baculovirus, and P2 is infected carefully for recombinant baculovirus with MOI=1 Born of the same parents' density is 2.5 × 10⁶The sf9 cell of cell/ml, 27 DEG C of suspensions are collected cell after cultivating 96h, through ultrasonication, are obtained Soluble HA albumen；

(3) soluble HA albumen is mixed with adjuvant, obtains the avian influenza vaccine based on MultiBac baculovirus expression system.

9. the preparation method of the avian influenza vaccine according to claim 8 based on MultiBac baculovirus expression system, It is characterized by:

Adjuvant described in step (3) is SEPPIC MONTANIDETM ISA 201VG；

When soluble HA albumen described in step (3) comes from H7N9 subtype avian influenza virus, the blood clotting effect of soluble HA albumen Valence is 2¹⁰~2¹⁴, mixed with adjuvant according to the ratio of volume ratio 1:1；

When soluble HA albumen described in step (3) comes from H5N1 subtype avian influenza virus, the blood clotting effect of soluble HA albumen Valence is preferably 2¹⁰~2¹⁴, mixed with adjuvant according to the ratio of volume ratio 1:1.

10. prepared by the described in any item avian influenza vaccines based on MultiBac baculovirus expression system of claim 1~7 Application in pre- avian influenza-prevention product.