CN106190993A - Avian influenza vaccine and the diagnostic antigen preparation method planting poison used - Google Patents

Abstract

The present invention relates to veterinary biological product field, be used for preparing avian influenza vaccine and diagnostic antigen required kind poison.Present invention application Reverse Genetics, six, inside gene (PB2 with influenza virus cold adaptation strain, PB1, PA, NP, M, NS) it is genome skeleton, together with bird flu virus HA and NA gene, artificial constructed influenza virus, this virus is as preparing avian influenza vaccine and diagnostic antigen required kind poison, there is higher safety, and keep titer high, the advantages such as matching is strong, attenuated vaccine or the inactivated vaccine of preparation are safe and reliable, stronger immunity can be produced after immunity, can preventing bird flu effectively popular and propagating, reduce the economic loss that primary disease causes, reduce the threat to human health, there is important public health meaning, have broad application prospects.

Description

Avian influenza vaccine and the diagnostic antigen preparation method planting poison used

Technical field

The present invention relates to avian influenza vaccine and the diagnostic antigen preparation method planting poison used, belong to biological technical field.

Background technology

Bird flu (Avian influenza, AI) is a kind of poultry and the infectious disease of wild fowl caused by bird flu virus.Bird flu Virus, according to hemagglutinin (HA) and the difference of neuraminidase (NA), can be divided into 16 HA hypotypes and 9 NA hypotypes. High pathogenic avian influenza is the deadly infectious disease of the serious threat birds caused by some H5 or H7 subtype avian influenza virus.Some High pathogenic avian influenza virus can also cause the infection of people, morbidity, even dead.

Since 2003, the H5N1 subtype highly pathogenic avian influenza multiple countries and regions including China always are popular, Not only causing serious economic loss to aviculture, the health of the mankind in also serious threat.Except constantly strengthening and improving influenza Beyond monitoring, vaccine remains the important means of prevention and control AI.2005, it is extensive strong that China comes into effect for bird flu Property immunity policy processed, and achieve remarkable effect.Along with the continuous variation of virus, vaccine also updates Re-4 and Re-5 from Re-1, Re-6 and Re-7 up till now.All these vaccines are all six internal gene by PR8 (A/PR/8/34) and popular poison Two external gene restructuring of HA and NA of strain form.Due to the PR8 virus high-affinity to mammal, for safety Considering, these vaccines that all PR8 recombinate and come can only be made inactivated vaccine and use.Inactivated vaccine is mainly produced by induction of antibodies Protection body can be accelerated in antibody mediated immunity pressure lower variation speed from virus attack, virus from birth so that virus is more difficult to prevention And control.This patent provides a kind of new avian influenza vaccine and the diagnostic antigen preparation method planting poison used.Plant poison, as epidemic disease " seed " that Seedling produces, is the key point of production of vaccine.Supporting diagnostic antigen is that vaccine quality controls and vaccine using effect The necessary material evaluated, and planting poison is supporting hemagglutination test or the necessary material of hemagglutination inhibition test diagnostic antigen production.

Summary of the invention

This patent determine with first cold adaptation influenzae strain virus A/Ann Arbor/6/60 (AAca) six internal gene (PB2, PB1, PA, NP, M and NS) and the external gene (HA and NA) of epidemic isolates, by reverse genetics manipulation technology, Avian influenza vaccine and diagnostic antigen required kind poison.Reverse genetics manipulation technology refers to by certain or some artificial constructed plasmids, Transfect specific cell, produce a kind of technology of the desired virus obtained.Utilize cold adaptation influenzae strain virus A/Ann Arbor/6/60 (AAca) and Reverse Genetics prepare avian influenza vaccine and the required poison of planting of diagnostic antigen has higher safety, And keeping the advantages such as titer is high, matching is strong, attenuated vaccine or the inactivated vaccine of preparation are safe and reliable, can produce relatively after immunity Strong immunity, it is possible to the popular and propagation of preventing bird flu effectively, reduces the economic loss that primary disease causes, reduces human health Threat, there is important public health meaning, have broad application prospects.

This patent determines first prepares six internal gene sequences of required viral backbone during avian influenza vaccine with Reverse Genetics Row.

Detailed description of the invention

Below by embodiment, further illustrate technical scheme, but protection scope of the present invention is not limited to this.

The recombiant plasmid of expression A/Ann Arbor/6/60 (AAca) six internal gene of virus described in embodiment is moved by China Thing health preserves with epidemiology center construction, and 293T cell is purchased from QbioGene company；Liposome Lipofectamine2000 Purchased from Invitrogen company.

Embodiment 1

The construction method of restructuring H5 subtype avian influenza virus (rH5/AAca), comprises the steps:

1, synthetic and six internal gene of clone A/Ann Arbor/6/60 (AAca)

PB2, PB1, PA, NP, M, NS gene order (SEQ ID NO.1～NO.6) specified according to the present invention, adopts By the method for synthetic, insert it in bi-directional expression vector (can simultaneously transcribe generation viral RNA and mRNA), Prepare pH-PB2, pH-PB1, pH-PA, pH-NP, pH-M, pH-NS plasmid.

2, the clone of H5 subtype avian influenza virus surface gene

Amplification H5 subtype avian influenza virus HA and NA surface gene, and HA gene is carried out molecular modification, remove and H5 The multiple basic amino acids at HA protein cleavage site that subtype avian influenza virus pathogenicity is relevant so that it is obtain low pathogenicity fowl The characterization of molecules of influenza virus (will-PLRERRRKR-sport-PQRETR-), inserts it in bi-directional expression vector, Prepare pH-mHA and plasmid pH-NA.

3, the restructuring packaging of H5 subtype avian influenza virus and qualification

Express 6 internal gene recombinant expression plasmids of A/Ann Arbor/6/60 (AAca) virus, with expression H5 hypotype fowl Influenza virus HA and NA gene recombination plasmid, through liposome Lipofectamine 2000 cotransfection 293T cell.Will transfection Cell is inoculated in 10 age in days SPF Embryo Gallus domesticus, every embryo 0.1mL, two embryos of each sample inoculation through allantoic cavity, puts 35 DEG C and cultivates extremely 72h, collects allantoic fluid, and the standard recommended with International Office of Epizootics (OIE) carries out blood clotting (HA) and blood clotting suppression (HI) examination Test identifying virus.HI positive allantoic fluid, the 2nd generation allantoic fluid passed on Embryo Gallus domesticus extract RNA, RT-PCR amplification HA and NS genetic fragment checks order.Expand without the RNA PCR of reverse transcription.RT-PCR amplification and sequencing result show, save The virus rescued is restructuring H5 subtype avian influenza virus, without the RNA PCR of reverse transcription without amplified band.Obtain restructuring disease The named rH5/AAca of poison.

4, the enrichment procedure of virus needed for production of vaccine

With physiological saline solution 1: 10000 times dilution rH5/AAca virus, inoculate 10 age in days SPF Embryo Gallus domesticus, 0.1mL/ through allantoic cavity Embryo, the survival chick embryo allantoic liquid after results cultivation 72h, measure HA titer, virus allantoic fluid HA titer is 2⁹, and aseptic It is qualified to check.

5, the virus inactivating method needed for production of vaccine

By addition formalin in the allantoic fluid of results to final concentration 0.2%, 2-8 DEG C of inactivation 48h, shake up once every 2h.Go out Living after 48h, each virus sample 10 pieces of Embryo Gallus domesticus of inoculation, every embryo 0.1mL, mensuration HA titer after hatching 72h, without coagulation An allantoic fluid blind passage generation again.The second filial generation is still judged to virus complete inactivation without coagulation.Result virus allantoic fluid Embryo Gallus domesticus passes on 2 times All coagulation without producing, inactivation is completely.

6, the quality testing of inactivated vaccine

Prepared rH5/AAca strain oil emulsion inactivated vaccine by the method for " veterinary biological product code " and carried out the aseptic examination of vaccine Test, physical behavior, safety detection.Indices meets the regulation of " veterinary biological product code ".

7, the immuning effect test of vaccine

50 21 age in days SPF chickens are randomly divided into 5 groups, often group 10.1-4 group intramuscular inoculation quality testing is qualified RH5/AAca avian influenza vaccine, immunizing dose is respectively 0.05,0.1,0.3,0.5mL/ only, the 5th group is blank group, Isolated rearing.After immunity 2 weeks and 3 weeks, blood sampling measured H5 subtype avian influenza virus antibody horizontal.Within after immunity 3 weeks, use homology H5 subtype highly pathogenic avian influenza virus counteracting toxic substances, it is determined that the immune protective effect (table 1) of vaccine.

The result of the test that affects of immune efficacy is shown by the different immunizing doses of H5 subtype avian influenza virus inactivated vaccine, is developed Vaccine immunity dosage be that 0.1mL/ only just can make vaccinated flock within 3 weeks after immunity, be obtained with the protection viral to H5.From From the point of view of antibody generation level, the antibody horizontal of 0.3mL/ immunizing dose group chicken is apparently higher than 0.1mL group, and 0.3mL group resists Body remained stable rises.Multiple comparisons shows, 0.3 is not notable (P > 0.05) with 0.5mL group antibody titer change difference, Illustrate under similarity condition, to select 0.3mL/ dose inoculation can reach effect of preferably preventing epidemic.

Table 1 is recombinated the immuning effect test of H5 subtype avian influenza virus oil emulsion inactivated vaccine

Note: * denominator represents test chicken number, molecule represents dead chicken number.

8, H5 subtype avian influenza virus is as the method for diagnostic antigen

The enrichment procedure of H5 subtype avian influenza virus is with step 4.The enrichment procedure of H5 subtype avian influenza virus is with step 4, inactivation Method is with step 5.Carry out hemagglutination inhibition test with this inactivation of viruses and detect the immune effect of vaccine (HI antibody titer) matched, As shown in step 7.

SEQUENCE LISTING

<110>China Animal Health and Epidemiology Center

<120>avian influenza vaccine and the diagnostic antigen preparation method planting poison used

<160> 6

<170> PatentIn version 3.3

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<211> 2341

<212> DNA

<213> Influenza virus

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agcgaaagca ggtcaattat attcaatatg gaaagaataa aagaactacg gaatctgatg 60

tcgcagtctc gcactcgcga gatactaaca aaaaccacag tggaccatat ggccataatt 120

aagaagtaca catcagggag acaggaaaag aacccgtcac ttaggatgaa atggatgatg 180

gcaatgaaat atccgattac agccgacaag aggataacag aaatgattcc tgagagaaat 240

gagcaagggc aaactctatg gagtaaaatg agtgatgccg gatcggatcg tgtgatggta 300

tcacctctgg ctgtgacatg gtggaataga aatggaccaa tgacaagtac ggttcattat 360

ccaaaaatct acaaaactta ttttgagaaa gtcgaaaggt taaaacatgg aacctttggc 420

cctgttcatt ttagaaacca agtcaaaata cgccgaagag ttgacataaa tcctggtcat 480

gcagacctca gtgccaagga ggcacaggat gtaatcatgg aagttgtttt ccctaacgaa 540

gtgggggcca ggatactaac gtcggaatcg caattaacaa taaccaaaga gaaaaaagaa 600

gaactccagg attgcaaaat ttcacctttg atggttgcgt acatgttaga gagagaactt 660

gtccgaaaaa cgagatttct cccagttgct ggtggaacaa gcagtgtgta catcgaagtg 720

ttgcacttga ctcaaggaac atgctgggaa cagatgtaca ctccaggtgg agaagtgagg 780

aatgatgatg ttgatcaaag tctaattatt gcagccagga gcatagtgag aagagcagca 840

gtatcagcag atccactagc atctttattg gagatgtgcc acagcacaca gattggcggg 900

acaaggatgg tggacattct taggcagaac ccaacagaag agcaagctgt ggaaatatgc 960

aaagctgcaa tgggactgag gatcagctca tccttcagtt ttggcgggtt cacatttaag 1020

agaacaagcg gatcatcagt caagagagag gaagaagtgc ttacgggcaa tcttcaaaca 1080

ttgaaaataa gggtgcatga gggatacgag gagttcacaa tggttgggaa aagggcaaca 1140

gctatactca gaaaagcaac caggagattg attcagctga tagtgagtgg aagagacgaa 1200

cagtcgatag ccgaagcaat aattgtggcc atggtatttt cacaagaaga ttgtatgata 1260

aaagcagtta gaggtgatct gaatttcgtt aatagggcaa atcagcgatt gaatcccatg 1320

catcaacttt taagacattt tcagaaggat gctaaagtgc tttttcaaaa ttggggaatt 1380

gaacatatcg acaatgtgat gggaatgatt ggggtattac cagacatgac tccaagcaca 1440

gagatgtcaa tgagaggggt aagagtcagc aaaatgggcg tagatgaata ctccagcgcg 1500

gagagagtag tggtgagcat tgaccggttt ttgagagttc gagaccaacg aggaaatgta 1560

ctactatctc ctgaggaggt cagtgaaaca cagggaacag agaaactgac aataacttac 1620

tcatcgtcaa tgatgtggga gattaatggc cctgagtcag tgttggtcaa tacctatcag 1680

tggatcatca gaaactggga aactgttaaa attcagtggt ctcagaatcc tacaatgcta 1740

tacaataaaa tggaatttga gccatttcag tctttagttc ctaaggccat tagaggccaa 1800

tacagtgggt ttgttaggac tctattccaa caaatgaggg atgtacttgg gacatttgat 1860

accacccaga taataaaact tcttcccttt gcagccgccc caccaaagca aagtagaatg 1920

caattctctt cattgactgt gaatgtgagg ggatcaggaa tgagaatact tgtaaggggc 1980

aattctccta tattcaacta caacaagacc actaagagac taacaattct cggaaaggat 2040

gctggcactt taactgaaga cccagatgaa ggcacatctg gagtggagtc cgctgttctg 2100

agaggattcc tcattctggg caaagaagat aggagatatg gaccagcatt aagcatcaat 2160

gaactgagta accttgcgaa aggagaaaag gctaatgtac taattgggca aggagacgtg 2220

gtgttggtaa tgaaacgaaa acggaactct agcatactta ctgacagcca gacagcgacc 2280

aaaaggattc ggatggccat caattaatgt tgaatagttt aaaaacgacc ttgtttctac 2340

t 2341

<210> 2

<211> 2341

<212> DNA

<213> Influenza virus

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agcgaaagca ggcaaaccat ttgaatggat gtcaatccga ccttactttt cttgaaagtt 60

ccagcgcaaa atgccataag tactacattc ccttatactg gagatcctcc atacagccat 120

ggaacaggaa caggatacac catggacaca gtcaacagaa cacatcaata ttcagaaaag 180

gggaagtgga caacaaacac ggaaactgga gcgcaccaac ttaacccaat tgatggacca 240

ctacctgagg acaatgaacc aagtggatat gcacaaacag actgcgtcct ggaagcaatg 300

gctttccttg aagaatccca cccaggaatc tttgaaaact cgtgtcttga aacgatggaa 360

gttattcaac aaacaagagt ggacaaactg acccaaggtc gtcagaccta tgattggaca 420

ttgaacagaa atcagccggc tgcaactgcg ctagccaaca ctatagaggt cttcagatcg 480

aatggcctga cagctaatga atcgggaagg ctaatagatt tcctcaagga tgtgatagaa 540

tcaatggata aagaggagat ggaaatcaca acacacttcc aaagaaaaag aagagtaaga 600

gacaacatga ccaagaaaat ggtcacacaa cgaacaatag gaaagaagaa gcaaagattg 660

aacaagagaa gctatctaat aagagcactg acattgaaca caatgactaa agatgcagag 720

agaggtaaat taaagagaag agcaattgca acacccggta tgcagatcag agggttcgtg 780

tactttgtcg aaacactagc gagaagtatt tgtgagaagc ttgaacagtc tgggcttccg 840

gttggaggta atgaaaagaa ggctaaactg gcaaatgttg tgcgaaaaat gatgactaat 900

tcacaagaca cagagctctc tttcacaatt actggagaca ataccaaatg gaatgagaat 960

caaaatcctc ggatgttcct ggcgatgata acatacatca caagaaatca acctgaatgg 1020

tttagaaacg tcctgagcat cgcacctata atgttctcaa ataaaatggc aagactaggg 1080

aaaggataca tgttcaaaag caagagcatg aagctccgaa cacaaatacc agcagaaatg 1140

ctagcaagta ttgacctgaa atactttaat gaatcaacaa gaaagaaaat cgaggaaata 1200

aggcctctcc taatagatgg cacagtctca ttgagtcctg gaatgatgat gggcatgttc 1260

aacatgctaa gtacagtctt aggagtctca atcctgaatc ttggacaaaa gaagtacacc 1320

aaaacaacat actggtggga cggactccaa tcctctgatg acttcgccct catagtgaat 1380

gcaccaaatc atgagggaat acaagcaggg gtggatagat tctacagaac ctgcaagcta 1440

gtcggaatca atatgagcaa aaagaagtcc tacataaata ggacagggac atttgaattc 1500

acaagctttt tctatcgcta tggatttgta gccaatttta gcatggagct gcccagcttt 1560

ggagtgtctg gaattaatga atcggctgat atgagcattg gggtaacagt gataaagaac 1620

aacatgataa acaatgacct tgggccagca acagcccaac tggctcttca actattcatc 1680

aaagactaca gatatacgta ccggtgccac agaggagaca cacaaattca gacaaggaga 1740

tcattcgagc taaagaagct gtgggggcaa acccgctcaa aggcaggact tttggtttcg 1800

gatggaggac caaacttata caatatccgg aatctccaca ttccagaagt ctgcttgaag 1860

tgggagctaa tggatgaaga ctatcagggg aggctttgta atcccctgaa tccatttgtc 1920

agtcataagg agattgagtc tgtaaacaat gctgtggtaa tgccagctca cggtccagcc 1980

aagagcatgg aatatgatgc tgttactact acacactcct ggatccctaa gaggaaccgc 2040

tccattctca acacaagcca aaggggaatt cttgaagatg aacagatgta tcagaagtgt 2100

tgcaatctat tcgagaaatt cttccctagc agttcgtaca ggagaccagt tggaatttcc 2160

agcatggtgg aggccatggt gtctagggcc cggattgatg cacggattga cttcgagtct 2220

ggacggatta agaaagagga gttcgctgag atcatgaaga tctgttccac cattgaagag 2280

ctcagacggc aaaaatagtg aatttagctt gtccttcatg aaaaaatgcc ttgtttctac 2340

t 2341

<210> 3

<211> 2233

<212> DNA

<213> Influenza virus

<400> 3

agcgaaagca ggtactgatt cgaaatggaa gattttgtgc gacaatgctt caatccgatg 60

attgtcgagc ttgcggaaaa agcaatgaaa gagtatggag aggatctgaa aatcgaaaca 120

aacaaatttg cagcaatatg cactcacttg gaagtatgct tcatgtattc agattttcat 180

ttcatcaatg agcaaggcga gtcaataata gtagagcttg atgatccaaa tgcacttttg 240

aagcacagat ttgaaataat agagggaaga gatcgcacaa tggcctggac agtagtaaac 300

agtatttgca acactacagg agctgagaaa ccgaagtttc tgccagattt gtatgattac 360

aaggagaata gattcatcga gattggagtg acaaggaggg aagtccacat atactatctt 420

gaaaaggcca ataaaattaa atctgagaag acacacatcc acattttctc attcactggg 480

gaagaaatgg ccacaaaggc cgactacact ctcgatgagg aaagcagggc taggatcaaa 540

accagactat tcaccataag acaagaaatg gctagcagag gcctctggga ttcctttcat 600

cagtccgaaa gaggcgaaga aacaattgaa gaaagatttg aaatcacagg gacaatgcgc 660

aggctcgccg accaaagtct cccgccgaac ttctcctgcc ttgagaattt tagagcctat 720

gtggatggat tcgaaccgaa cggctacatt gagggcaagc tttctcaaat gtccaaagaa 780

gtaaatgcta aaattgaacc ttttctgaaa acaacaccaa gaccaattag acttccggat 840

gggcctcctt gttctcagcg gtccaaattc ctgctgatgg atgctttaaa attaagcatt 900

gaggacccaa gtcacgaagg agagggaata ccactatatg atgcgatcaa gtgtatgaga 960

acattctttg gatggaaaga accctatgtt gttaaaccac acgaaaaggg aataaatcca 1020

aattatctgc tgtcatggaa gcaagtactg gcagaactgc aggacattga gaatgaggag 1080

aagattccaa gaaccaaaaa catgaagaaa acgagtcagc taaagtgggc acttggtgag 1140

aacatggcac cagagaaggt agactttgac gactgtagag atgtaagcga tttgaagcaa 1200

tatgatagtg atgaacctga attaaggtca ctttcaagct ggatccagaa tgagttcaac 1260

aaggcatgcg agctgaccga ttcaatctgg atagagctcg atgagattgg agaagatgtg 1320

gctccaattg aacacattgc aagcatgaga aggaattact tcacagcaga ggtgtctcat 1380

tgcagagcca cagaatatat aatgaagggg gtatacatta atactgcctt gcttaatgca 1440

tcctgtgcag caatggacga tttccaacta attcccatga taagcaaatg tagaactaaa 1500

gagggaaggc gaaagaccaa tttatatggt ttcatcataa aaggaagatc tcacttaagg 1560

aatgacaccg acgtggtaaa ctttgtgagc atggagtttt ctctcactga cccaagactt 1620

gagccacaca aatgggagaa gtactgtgtt cttgagatag gagatatgct actaagaagt 1680

gccataggcc aggtgtcaag gcccatgttc ttgtatgtga ggacaaatgg aacatcaaag 1740

attaaaatga aatggggaat ggagatgagg cgttgcctcc ttcagtcact ccaacaaatc 1800

gagagtatga ttgaagccga gtcctctgtc aaggagaaag acatgaccaa agagtttttc 1860

aagaataaat cagaaacatg gcccattgga gagtccccca aaggagtgga agaaggttcc 1920

attgggaagg tctgcaggac tttattagcc aagtcggtat tcaatagcct gtatgcatct 1980

ccacaattag aaggattttc agctgaatca agaaaactgc ttcttgtcgt tcaggctctt 2040

agggacaatc ttgaacctgg gacctttgat cttggggggc tatatgaagc aattgaggag 2100

tgcctgatta atgatccctg ggttttgctt aatgcgtctt ggttcaactc cttcctaaca 2160

catgcattaa gatagttgtg gcaatgctac tatttgctat ccatactgtc caaaaaagta 2220

ccttgtttct act 2233

<210> 4

<211> 1566

<212> DNA

<213> Influenza virus

<400> 4

agcgaaagca gggtagataa tcactcactg agtgacatca aaatcatggc gtcccaaggc 60

accaaacggt cttatgaaca gatggaaact gatggggaac gccagaatgc aaatgaaatc 120

agagcatccg tcgggaagat gattggtgga attggacgat tctacatcca aatgtgcacc 180

gaacttaaac tcagtgatta tgaggggcgg ctgatccaga acagcttaac aatagagaga 240

atggtgctct ctgcttttga cgagaggagg aataaatatc tggaagaaca tcccagcgcg 300

gggaaggatc ctaagaaaac tggaggaccc atatacaaga gagtagatgg aaagtggatg 360

agggaactcg tcctttatga caaagaagaa ataaggcgaa tctggcgcca agctaataat 420

ggtgatgatg caacagctgg tctgactcac atgatgatct ggcattccaa tttgaatgat 480

acaacatacc agaggacaag agctcttgtt cgcaccggaa tggatcccag gatgtgctct 540

ttgatgcagg gttcgactct ccctaggagg tctggagccg caggcgctgc agtcaaagga 600

gttgggacaa tggtgatgga gttgataagg atgatcaaac gtgggatcaa tgatcggaac 660

ttctggagag gtgagaatgg gcggaaaaca aggaatgctt atgagagaat gtgcaacatt 720

ctcaaaggaa aatttcaaac agctgcacaa agagcaatga tggatcaagt gagagaaagc 780

cggaacccag gaaatgctga gatcgaagat ctcatctttc tggcacggtc tgcactcata 840

ttgagagggt cagttgctca caaatcttgt ctgcctgcct gtgtgtatgg acctgccgta 900

gccagtggct acgacttcga aaaagaggga tactctttag tagggataga ccctttcaaa 960

ctgcttcaaa acagccaagt atacagccta atcagaccga atgagaatcc agcacacaag 1020

agtcagctgg tgtggatggc atgcaattct gctgcatttg aagatctaag agtatcaagc 1080

ttcatcagag ggaccaaagt aatcccaagg gggaaacttt ccactagagg agtacaaatt 1140

gcttcaaatg aaaacatgga tactatggga tcaagtactc ttgaactgag aagcaggtac 1200

tgggccataa ggaccagaag tggaggaaac actaatcaac agagggcctc tgcaggtcaa 1260

atcagtgtac aacctacgtt ttctgtgcaa agaaacctcc catttgacaa accaaccatc 1320

atggcagcat tcactgggaa tgcagaggga agaacatcag acatgagggc agaaatcata 1380

aggatgatgg aaggtgcaaa accagaagaa gtgtccttcc aggggcgggg agtcttcgag 1440

ctctcggacg aaaaggcaac gaacccgatc gtgccctctt ttgacatgag taatgaagga 1500

tcttatttct tcggagacaa tgcagaggag tacgacaatt aaggaaaaaa tacccttgtt 1560

tctact 1566

<210> 5

<211> 1027

<212> DNA

<213> Influenza virus

<400> 5

agcgaaagca ggtagatatt gaaagatgag tcttctaacc gaggtcgaaa cgtacgttct 60

ctctatcatc ccgtcaggcc ccctcaaagc cgagatcgca cagagacttg aagatgtctt 120

tgctgggaaa aacaccgatc ttgaggctct catggaatgg ctaaagacaa gaccaatcct 180

gtcacctctg actaagggga ttttgggatt tgtattcacg ctcaccgtgc ccagtgagcg 240

aggactgcag cgtagacgct ttgtccaaaa tgccctcaat gggaatgggg atccaaataa 300

catggacaga gcagttaaac tgtatagaaa gcttaagagg gagataacat tccatggggc 360

caaagaaata gcgctcagtt attctgctgg tgcacttgcc agttgtatgg gcctcatata 420

caacaggatg ggggctgtga ccactgaagt ggtcttaggc ctggtatgtg caacctgtga 480

acagattgct gactcccagc ataggtctca taggcaaatg gtgacaacaa ccaatccact 540

aataagacat gagaacagaa tggttctggc cagcactaca gctaaggcta tggagcaaat 600

ggctggatcg agtgagcaag cagcagaggc catggaggtt gctagtcagg ccaggcaaat 660

ggtgcaggca atgagagtta ttgggactca tcctagctcc agtgctggtc taaaaaatga 720

tcttcttgaa aatttgcagg cctatcagaa acgaatgggg gtgcagatgc aacgattcaa 780

gtgaccctct tgttgttgcc gcgagtatca ttgggatctt gcacttgata ttgtggattc 840

ttgatcatct ttttttcaaa tgcatttatc gcttctttaa acacggtctg aaaagagggc 900

cttctacgga aggagtacca gagtctatga gggaagaata tcgaaaggaa cagcagagtg 960

ctgtggatgc tgacgatagt cattttgtca gcatagagct ggagtaaaaa actaccttgt 1020

ttctact 1027

<210> 6

<211> 890

<212> DNA

<213> Influenza virus

<400> 6

agcgaaagca gggtgacaaa gacataatgg atcctaacac tgtgtcaagc tttcaggtag 60

attgcttcct ttggcatgtc cgcaaacaag ttgcagacca agaactaggt gatgccccat 120

tccttgatcg gcttcgccga gatcagaagt ccctaagggg aagaggcagt actctcggtc 180

tgaacatcga aacagccacc cgtgttggaa agcagatagt ggagaggatt ctgaaggaag 240

aatccgatga ggcacttaaa atgaccatgg cctccgcacc tgcttcgcga tacctaactg 300

acatgactat tgaggaaatg tcaagggact ggttcatgct aatgcccaag cagaaagtgg 360

caggccctct ttgtatcaga atggaccagg caatcatgga taagaacatc atattgaaag 420

cgaatttcag tgtgattttt gaccggctag agaccctaat attactaagg gctttcaccg 480

aagcgggagc aattgttggc gaaatttcac cattgccttc tcttccagga catactaatg 540

aggatgtcaa aaatgcaatt ggggtcctca tcggaggact tgaatggaat gataacacag 600

ttcgagtctc taaaactcta cagagattcg cttggagaag cagtgatgag aatgggagac 660

ctccactcac tccaaaatag aaacggaaaa tggcgagaac aattaggtca aaagttcgaa 720

gaaataagat ggctgattga agaagtgaga cacaaattga agataacaga gaatagtttt 780

gagcaaataa catttatgca agccttacag ctactatttg aagtggaaca agagataaga 840

actttctcgt ttcagcttat ttaatgataa aaaacaccct tgtttctact 890

Claims (5)

1. with Reverse Genetics and cold adaptation influenzae strain viral backbone, artificial constructed influenza virus, the kind poison produced as avian influenza vaccine.

Plant poison the most as claimed in claim 1, it is characterized in that, described bird flu virus PB2, PB1, PA, NP, M, NS gene order and SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 similarity (similarity) are higher than 95%.

3. the kind poison described in claim 2, for avian influenza vaccine or the production of supporting diagnostic antigen.

4. the kind poison described in claim 2, for H5 subtype avian influenza vaccine or the production of supporting diagnostic reagent.

5. the kind poison described in claim 2, for H7 subtype avian influenza vaccine or the production of supporting diagnostic reagent.