CN102234637B - Preparation for reconstruction influenza A H1N1 virus inactivated vaccine strain (SC/PR8), and use thereof - Google Patents

Abstract

The invention relates to a reconstruction influenza A H1N1 virus inactivated vaccine strain. The reconstruction influenza A H1N1 virus inactivated vaccine strain is characterized by expressing HA protein and NA protein of the influenza A H1N1 virus. In particular, the reconstruction influenza A H1N1 virus inactivated vaccine strain is SC/PR8. The invention further discloses a method for preparing the reconstruction influenza A H1N1 virus inactivated vaccine strain (SC/PR8), and a use of the reconstruction influenza A H1N1 virus inactivated vaccine strain (SC/PR8) in prevention of animal influenza A H1N1 or human influenza A H1N1.

Description

Preparation and the application of restructuring H1N1virus inactivated vaccine strain (SC/PR8)

Technical field

The present invention relates to recombination engineered vaccine, particularly, the present invention relates to a kind of for preventing the recombination engineered vaccine of animal and human's Influenza A H1N1, more specifically, vaccine involved in the present invention is a kind of for preventing the restructuring H1N1virus inactivated vaccine of animal and human's Influenza A H1N1.

Background technology

A type influenza virus is the togavirus that the extensive infected poultry of a kind of energy and mammiferous segmented strand RNA form.In the whole world, influenza often occurs sporadicly to break out the assorted random pandemic form of holding concurrently, in the past few decades, various countries mainly select to utilize remains totivirus deactivation vaccine, and it mainly stimulates body to produce humoral immunization to resist the hemagglutinin surface glycoprotein of identical hypotype.

By in December, 2009, start from being very popular of Mexican Influenza A H1N1, in the whole world, caused and surpassed 10,000 people's death, and epidemic situation is still continuing expansion.For the development of corresponding epidemic situation, we have carried out the development of natural recombinant vaccine strain.

Desirable vaccine strain should possess the antigen matching good with epidemic strain, to conditions such as chicken embryo had no pathogenicity and the high titre growth property of chicken embryo.Yet the strain that is difficult to possess above-mentioned condition from nature separation is as vaccine strain.At home, because street strain's chicken embryo adaptability of terrain separation is poor, cause existing domestic Influenza A H1N1 vaccine to have the shortcomings such as cost is high, the vaccine immunity phase is short.

Influenza virus A/PR/8/34 (H1N1) (being called for short PR8) is laboratory chicken embryo adapted strain, and this virus can grow to very high titre in chicken embryo, and this chicken embryo adaptability is determined by 6 internal gene of virus.In addition, when two influenza viruses cell of common infection or chicken embryo, can exchange by producer, thereby produce new gene recombined virus.Therefore, by genetic recombination method, can be by the HA of 6 internal gene of influenza virus A/PR/8/34 (H1N1) and epidemic strain, recombinating of NA gene and obtain a new recombinant virus, this recombinant virus has the antigenicity consistent with HA and NA genetic donor strain, also has the high titre growth characteristics of chicken embryo of influenza virus A/PR/8/34 (H1N1) simultaneously.

Artificial nature's recombinant technology is a kind of simple and effective structure 2:6 recombinant viral vaccine strain method, the genome of influenza virus is comprised of the strand RNA fragment of 8 segmentations, when 2 common infected chicken embryos of virus or cell, its genomic fragment can exchange, thereby produce some new recombinant viruses (reassortant), by the processing of serum, can effectively screen the strain of 2:6 recombinant viral vaccine.(Hualan Chen，Vaccine 2003)

Summary of the invention

We utilize artificial recombination method, with the pandemic influenza A in the world in 2009, the first Influenza A H1N1 isolate A/SC/1/2009 (H1N1) in inland of China (being called for short SC) is surface antigen hemagglutinin (HA) and neuraminidase (NA) genetic donor, influenza virus A/the PR/8/34 (H1N1) (being called for short PR8) of take is 6 internal gene (PB2, PB1, PA, NP, M and NS) donor, naturally restructuring has obtained 1 pnca gene recombinant virus, called after SC/PR8 (H1N1) (referred to as SC/PR8).; the present invention selects 6 internal gene of the laboratory chicken embryo adapted strain that can grow to very high titre in chicken embryo of influenza virus A/PR/8/34 (H1N1) as skeleton gene; and select the HA of the domestic influenza A virus isolate A/SC/1/2009 of flu outbreak in 2009 (H1N1) and NA gene as surface gene; utilize nature recombination method, prepared recombinant virus SC/PR8.The vaccine strain of Influenza A H1N1 will be can be used as.

Experiment confirmation, SC/PR8 is consistent with China H1N1virus epidemic strain A/SC/1/2009 (H1N1) antigenicity, will produce good special protectiveness to H1N1virus epidemic strain A/SC/1/2009 (H1N1).6 internal gene that this virus contains the high titre adapted strain of chicken embryo influenza virus A/PR/8/34 (H1N1), therefore have good chicken embryo adaptability, and viral growth titre is compared with wild poison, can improve more than 10 times; As vaccine strain, produce vaccine, can greatly reduce production of vaccine cost, improve vaccine quality.

Therefore, by experiment, we have prepared a good virus strain, and this is the further development research of Influenza A H1N1 vaccine, has established solid basis.

Therefore, the invention provides the following:

1. a H1N1virus inactivated vaccine strain for restructuring, is characterized in that expressing surface antigen hemagglutinin and the neuraminic acid zymoprotein of H1N1virus.

2. according to the H1N1virus inactivated vaccine strain of the restructuring described in above 1, it also expresses 6 albumen that chicken embryo adapts to vaccine strain: polysaccharase PB2 subunit (PB2), polysaccharase PB1 subunit (PB1), polysaccharase PA subunit (PA), nucleoprotein (NP), stromatin (M) and Nonstructural Protein (NS).

3. according to the H1N1virus inactivated vaccine strain of above 2 restructuring, it is influenza virus A/PR/8/34 (H1N1) (being called for short PR8) that wherein said chicken embryo adapts to vaccine strain.

4. according to the H1N1virus inactivated vaccine strain of the restructuring of any one in above 1 to 3, the aminoacid sequence of wherein said HA albumen and NA albumen derives from Influenza A H1N1 isolate A/SC/1/2009 (H1N1).

5. produce any one the method for H1N1virus inactivated vaccine strain of restructuring according to above 1-4, the method comprises:

(1) with H1N1virus strain and chicken embryo, adapt to vaccine strain co-inoculation chick embryo allantoic cavity or cell carries out nature restructuring;

(2) with anti-described chicken embryo, adapt to the antibody screening of vaccine strain, remove surface gene and derive from the strain that described chicken embryo adapts to vaccine strain; With

(3) dilution purifying, removes the strain that internal gene derives from described H1N1virus strain.

6. according to the method described in above 5, it is influenza virus A/PR/8/34 (H1N1) (being called for short PR8) that wherein said chicken embryo adapts to vaccine strain.

7. according to the method described in above 5, wherein said H1N1virus is Influenza A H1N1 isolate A/SC/1/2009 (H1N1).

8. the H1N1virus inactivated vaccine strain of the restructuring obtaining according to the method described in above 5-7 any one.

Restructuring H1N1virus inactivated vaccine strain SC/PR8 (H1N1), its preserving number is CCTCC NO:V201006.

10. the application in the medicine of preparation prevention disease that H1N1virus causes according to the H1N1virus inactivated vaccine strain of the restructuring of any one in above 1-4,8 or 9.

11. according to the application described in above 10, and wherein said medicine is for preventing animal or human's H 1 N 1 influenza A virus infection.

Biomaterial preservation

H1N1 recombinant influenza (Influenza virus) inactivated vaccine strain SC/PR8 (H1N1) is preserved in the Chinese Typical Representative culture collection center of the Wuhan University that is positioned at Wuhan, China on March 1st, 2010, its preserving number is CCTCC NO:V201006.

Accompanying drawing explanation

Figure 1A. the PR8 strain cDNA of take is template, the amplification that utilizes the Auele Specific Primer of SC and PR8 strain PB2, PB1, PA, HA, NP, NA, M, NS gene to carry out, wherein the first to the 8th swimming lane for using PB2, PB1, PA, HA, NP, NA, the M of SC strain, the amplification of the Auele Specific Primer of NS gene to PR8 strain cDNA; M is DNA Marker 2000; The amplification that the Auele Specific Primer of PB2, the PB1 that the 9th to the 16 swimming lane is use PR8 strain, PA, HA, NP, NA, M, NS gene is template to PR8 strain cDNA;

Figure 1B. the SC strain cDNA of take is template, the amplification that utilizes the Auele Specific Primer of PR8 and SC strain PB2, PB1, PA, HA, NP, NA, M, NS gene to carry out, wherein the first to the 8th swimming lane is for being used PB2, PB1, PA, HA, NP, NA, the M of PR8 strain, the amplification that the Auele Specific Primer of NS gene is template to SC strain cDNA; M is DNA Marker 2000; The amplification that the Auele Specific Primer of PB2, the PB1 that the 9th to the 16 swimming lane is use SC strain, PA, HA, NP, NA, M, NS gene is template to SC strain cDNA;

The RT-PCR amplification of PB2, the PB1 of Fig. 2 SC/AAca recombinant virus, PA, HA, NA, NP, M and NS gene,

1～8 swimming lane: recombinant virus SC/PR8 utilizes the PCR product of 8 pairs of specificity diagnostic primers amplifications of PR8; M:DNA molecular mass standard; 10～17 swimming lanes: recombinant virus SC/PR8 utilizes the PCR product of 8 pairs of specificity diagnostic primers amplifications of SC;

Fig. 3 SC/PR8 virus and wild-type H1N1virus A/SC/1/2009 (H1N1) (abbreviation SC) growth curve in 35 ℃ of mensuration of optimum culturing temperature of recombinating.

Sequence explanation

SEQ ID No.1:SC/PR8 HA gene;

SEQ ID No.2:SC/PR8 NA gene;

SEQ ID No.3:SC/PR8 PB2 gene;

SEQ ID No.4:SC/PR8 PB1 gene;

SEQ ID No.5:SC/PR8 PA gene;

SEQ ID No.6:SC/PR8 NP gene;

SEQ ID No.7:SC/PR8 M gene; With

SEQ ID No.8:SC/PR8 NS gene.

Embodiment

Below with reference to embodiment, describe the present invention in detail, described embodiment is only intended as illustrative explanation the present invention, rather than intention limits the scope of the invention.Scope of the present invention is specifically limited by accompanying claim.

Experiment material

1. virus strain

A/SC/1/2009 (H1N1) (being called for short SC)

A/PR/8/34 (H1N1) (being called for short PR8)

All purchased from Harbin Veterinary Medicine Inst., China Academy of Agriculture's animal influenza center.

2.SPF chicken embryo and cell

The SPF chicken embryo of 9-11 age in days is purchased from Harbin Veterinary Medicine Inst., China Academy of Agriculture's SPF hatching chamber, mdck cell (Madin-Darby canine kidney(cell line) (MDCK)) is purchased from Wuhan Virology Institute,Chinan academy of Sciences, and low generation Vero cell (African green monkey kidney cell) is purchased from Wuhan Virology Institute,Chinan academy of Sciences.

3. main agents

DATP, dGTP, dCTP, dTTP and rTaq archaeal dna polymerase are purchased from Dalian precious biotechnology company limited; RNA extracts reagent TRIzol, and mouse source ThermoScript II (MLV) test kit, DTT, RNaseOUT, Pfx high-fidelity DNA polymerase and serum free medium Opti-MEM are purchased from Invitrogen company; Glue recovery test kit and in a small amount plasmid extraction kit are purchased from Shanghai Hua Shun biotechnology company limited; Sequencing reaction test kit (CEQTM DTCS Quick Start Kit) is purchased from BECKMAN company; Anti-A/PR/8/34 (H1N1) antiserum(antisera) is purchased from Harbin Veterinary Medicine Inst., China Academy of Agriculture's animal influenza center.

4. primer

Analyze relatively, design is synthetic can be amplified 8 specific fragments of Influenza A H1N1 isolate A/SC/1/2009 (H1N1) gene and can not amplify 16 diagnostic primerses of Influenza A H1N1 isolate A/SC/1/2009 (H1N1) of any fragment of influenza virus A/PR/8/34 (H1N1), and synthetic primer sequence is in Table 1.

The diagnostic primers sequence of 8 specific fragments of table 1 H1N1virus A/SC/1/2009 (H1N1)

In addition, design is synthetic can only be amplified 8 specific fragments of influenza virus A/PR/8/34 (H1N1) and can not amplify 16 diagnostic primerses of influenza virus A/PR/8/34 (H1N1) of any fragment of Influenza A H1N1 isolate A/SC/1/2009 (H1N1), and synthetic primer sequence is in Table 2.

The diagnostic primers sequence of 8 specific fragments of table 2 influenza virus A/PR/8/34 (H1N1)

Embodiment 1: the foundation of screening, evaluation recombinant virus method

Utilize the TrIzol agent method of fissioning by force, extract the RNA of influenza virus A/PR/8/34 (H1N1) and Influenza A H1N1 isolate A/SC/1/2009 (H1N1), carry out reverse transcription, then use the influenza virus A/PR/8/34 (H1N1) of above-mentioned design and the fragments specific diagnostic primers of Influenza A H1N1 isolate A/SC/1/2009 (H1N1) to do PCR reaction.According to the fragments specific diagnostic primers of the gene design of influenza virus A/PR/8/34 (H1N1), can only amplify 8 specific fragments of influenza virus A/PR/8/34 (H1N1) strain and can not amplify any fragment of Influenza A H1N1 isolate A/SC/1/2009 (H1N1) strain, according to the fragments specific diagnostic primers of the gene design of Influenza A H1N1 isolate A/SC/1/2009 (H1N1), can only amplify 8 specific fragments of Influenza A H1N1 isolate A/SC/1/2009 (H1N1) strain and can not amplify any fragment of influenza virus A/PR/8/34 (H1N1) strain.This is by the foundation of proof screening, evaluation recombinant virus method.

As shown in Figure 1A, the cDNA of influenza virus A/PR/8/34 (H1N1) of take is template, utilize 32 bar segment specific amplifications of synthetic influenza virus A/PR/8/34 (H1N1) and the domestic isolate A/SC/1/2009 of H1N1virus (H1N1) strain, only have the specificity diagnostic primers of A/PR/8/34 (H1N1) strain to amplify 8 specific fragments of influenza virus A/PR/8/34 (H1N1) strain, and any fragment that the domestic isolate A/SC/1/2009 of H1N1virus (H1N1) strain specific diagnostic primers does not amplify.

As shown in Figure 1B, the cDNA of the domestic isolate A/SC/1/2009 of the H1N1virus of take (H1N1) strain is template, utilize 32 bar segment specific amplifications of synthetic influenza virus A/PR/8/34 (H1N1) and the domestic isolate A/SC/1/2009 of H1N1virus (H1N1) strain, only have the specificity diagnostic primers of the domestic isolate A/SC/1/2009 of H1N1virus (H1N1) strain to amplify 8 specific fragments of the domestic isolate A/SC/1/2009 of H1N1virus (H1N1) strain, and the specificity diagnostic primers of influenza virus A/PR/8/34 (H1N1) strain does not amplify any fragment.

Proof has been set up screening method thus, and this system can identify that 8 genes of prepared recombinant virus derive from respectively influenza virus A/PR/8/34 (H1N1) or the domestic isolate A/SC/1/2009 of H1N1virus (H1N1).

Embodiment 2: preparation and the evaluation of restructuring H1N1virus

1. coinfection-two viral genome are recombinated:

Domestic isolate A/the SC/1/2009 of H1N1virus (H1N1) of 100 μ l and influenza virus A/PR/8/34 (H1N1) of 50 μ l (being called for short PR8) mix and add in 850 μ l PBS, and the SPF chick embryo allantoic cavity of co-inoculation 10 ages in days carries out nature restructuring.Cultivate 24 hours for 35 ℃, collect allantoic fluid.

2. antibody screening-the remove strain that surface gene derives from influenza virus A/PR/8/34 (H1N1):

Get allantoic fluid that 50 μ l gather in the crops under above-mentioned specific artificial condition and anti-A/PR/8/34 (H1N1) antiserum(antisera) of 600 μ l multiple proportions (4 times) dilution and mix, room temperature effect 30 minutes, then three pieces of chicken embryos of each extent of dilution inoculation.Then, the chicken embryo of inoculation is cultivated 48 hours in 35 ℃ of incubators, measure the chick embryo allantoic liquid of each extent of dilution inoculation blood clotting valency (referring to laboratory animal hemagglutination-inhibition test country mark accurate (GB) (GB/T 14926.54-2001)), collect and retain low dilution A/PR/8/34 (H1N1) antiserum(antisera) and process the allantoic fluid that the chicken embryo of rear inoculation has blood clotting valency.Then, same procedure is done secondary antiserum(antisera) and is processed, and collects equally and retains low dilution A/PR/8/34 (H1N1) antiserum(antisera) and process the allantoic fluid that the chicken embryo of rear inoculation has blood clotting valency.

3. dilution purifying-the remove strain that internal gene derives from H1N1 strains of influenza viruses:

Process according to the method described above the virus obtain on chicken embryo, carry out 3 generation limited clone purification, utilize the TrIzol agent method of fissioning by force, extract viral RNA, carry out reverse transcription, then with the influenza virus A/PR/8/34 (H1N1) of design and each 8 pairs of specificity diagnostic primerses (table 1 and 2) of the domestic isolate A/SC/1/2009 of H1N1virus (H1N1) virus, carry out RT-PCR amplification.Then, amplified production is carried out to nucleic acid gel electrophoresis, the recombinant virus HA that evaluation obtains and NA come from the domestic isolate A/SC/1/2009 of H1N1virus (H1N1), 6 internal gene PB2, PB1, PA, NP, M and NS from influenza virus A/PR/8/34 (H1N1), and called after SC/PR8 (seeing Fig. 2).

4. the Sequence Identification of recombinant virus

The RNA that extracts recombinant virus SC/PR8, RT-PCR amplifies respectively complete genomic 8 fragments of recombinant virus SC/PR8, and PCR product is carried out to nucleic acid gel electrophoresis, measures the particular sequence of each fragment after glue reclaims.Utilize DNAStar software ( dNAStar Lasergene V7.1), the sequence of measuring and the sequence of the domestic isolate A/SC/1/2009 of wild strain H1N1virus (H1N1) and influenza virus A/PR/8/34 (H1N1) are carried out to sequence comparison, find that the genome of recombinant virus SC/PR8, all without any the variation of Nucleotide, shows that the HA of recombinant virus and NA come from the domestic isolate A/SC/1/2009 of H1N1virus (H1N1); 6 internal gene PB2, PB1, PA, NP, M and NS are from influenza virus A/PR/8/34 (H1N1).

5. the mensuration of recombinant virus SC/PR8 growth curve

Parental virus H1N1virus epidemic strain A/SC/1/2009 (H1N1) (being called for short SC) and recombinant virus SC/PR8 are inoculated to 5 groups of SPF chicken embryos, under 35 ℃ of conditions, cultivate respectively 24,48,72 and 96 hours, the chicken embryo of getting above-mentioned cultivation different time carries out blood clotting mensuration, detects the growth curve of parent strain A/SC/1/2009 (H1N1) and recombinant virus SC/PR8.

Recombinant virus is under 35 ℃ of culture condition as can be seen from the test results, and in the time of 72 hours, the hemagglutinative titer of recombinant virus is far above the domestic isolate A/SC/1/2009 of wild strain H1N1virus (H1N1) (as shown in Figure 3).

Prepare avian influenza vaccine strain, not only require it to there is good immunogenicity, and requirement has good growth characteristics aborning, the growth curve of measuring under 35 ℃ of culture condition from restructuring SC/PR8 virus can be found out, recombinant virus growth titre is significantly improved compared with the domestic isolate A/SC/1/2009 of parent's H1N1virus (H1N1), and hemagglutinative titer exceeds 16 times (as shown in Figure 3).This illustrates that we have prepared the recombinant virus of a height growth titre, will bring huge economic interests for later batch production.

6. the antigenicity analysis of recombinant virus SC/PR8

According to the antigen of the domestic isolate A/SC/1/2009 of parent's strain H1N1virus (H1N1) and recombinant virus and serum intersect HI test (referring to laboratory animal hemagglutination-inhibition test country standard (GB) (GB/T 14926.54-2001)) antigenicity analysis data, antigenic difference is less than 2 times as can be seen from the test results, illustrates that, after restructuring, virus is still keeping the antigenicity (referring to table 3) of parent's strain.

The antigenicity analysis of table 3: parent's strain A/SC/1/2009 (H1N1) and recombinant virus SC/PR8

Reference

1.Hualan Chen，Kanta Subbarao，David Swayne，Qi Chen，Xiuhua Lu，Jacqueline Katz，Nancy Cox，Yumiko Matsuoka.Generation and evaluation of a high-growth reassortant H9N2 influenza A virus as apandemic vaccine candidate.Vaccine 21(2003)1974-1979

Sequence table

<110> Harbin Veterinary Medicine Inst., China Academy of Agriculture

Preparation and the application thereof of <120> restructuring H1N1virus inactivated vaccine strain (SC/PR8)

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ctaagagggg tagccccatt gcatttgggt aaatgtaaca ttgctggctg gatcctggga 240

aatccagagt gtgaatcact ctccacagca agctcatggt cctacattgt ggaaacatct 300

agttcagaca atggaacgtg ttacccagga gatttcatcg attatgagga gctaagagag 360

caattgagct cagtgtcatc atttgaaagg tttgagatat tccccaagac aagttcatgg 420

cccgatcatg actcgaacaa aggtgtaacg gcagcatgtc ctcatgctgg agcaaaaagc 480

ttctacaaaa atttaatatg gctagttaaa aaaggaaatt catacccaaa gctcagcaaa 540

tcctacatta atgataaagg gaaagaagtc ctcgtgctat ggggcattca ccatccatct 600

actagtgctg accaacaaag tctctatcag aatgcagatg catatgtttt tgtggggtca 660

tcaagataca gcaagaagtt caagccggaa atagcaataa gacccaaagt gagggatcga 720

gaagggagaa tgaactatta ctggacacta gtagagccgg gagacaaaat aacattcgaa 780

gcaactggaa atctagtagt accgagatat gcattcgcaa tggaaagaaa tgctggatct 840

ggtattatca tttcagatac accagtccac gattgcaata caacttgtca gacacccaag 900

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atagagaatt taaataaaaa agttgatgat ggtttcctgg acatttggac ttacaatgcc 1320

gaactgttgg ttctattgga aaatgaaaga actttggact accacgattc aaatgtgaag 1380

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ggggcagtgg ctgtgttaaa gtacaacggc ataataacag acactatcaa gagttggaga 660

aacaatatat tgagaacaca agagtctgaa tgtgcatgtg taaatggttc ttgctttact 720

gtaatgaccg atggaccaag taatggacag gcctcataca agatcttcag aatagaaaag 780

ggaaagatag tcaaatcagt cgaaatgaat gctcctaatt atcactatga ggaatgctcc 840

tgttatcctg attctagtga aatcacatgt gtgtgcaggg ataactggca tggctcgaat 900

cgaccgtggg tgtctttcaa ccagaatctg gaatatcaga taggatacat atgcagtggg 960

attttcggag acaatccacg ccctaatgat aagacaggca gttgtggtcc agtatcgtct 1020

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aagaacccag cacttaggat gaaatggatg atggcaatga aatatccaat tacagcagac 180

aagaggataa cggaaatgat tcctgagaga aatgagcaag gacaaacttt atggagtaaa 240

atgaatgatg ccggatcaga ccgagtgatg gtatcacctc tggctgtgac atggtggaat 300

aggaatggac caatgacaaa tacagttcat tatccaaaaa tctacaaaac ttattttgaa 360

agagtcgaaa ggctaaagca tggaaccttt ggccctgtcc attttagaaa ccaagtcaaa 420

atacgtcgga gagttgacat aaatcctggt catgcagatc tcagtgccaa ggaggcacag 480

gatgtaatca tggaagttgt tttccctaac gaagtgggag ccaggatact aacatcggaa 540

tcgcaactaa cgataaccaa agagaagaaa gaagaactcc aggattgcaa aatttctcct 600

ttgatggttg catacatgtt ggagagagaa ctggtccgca aaacgagatt cctcccagtg 660

gctggtggaa caagcagtgt gtacattgaa gtgttgcatt tgactcaagg aacatgctgg 720

gaacagatgt atactccagg aggggaagtg aagaatgatg atgttgatca aagcttgatt 780

attgctgcta ggaacatagt gagaagagct gcagtatcag cagacccact agcatcttta 840

ttggagatgt gccacagcac acagattggt ggaattagga tggtagacat ccttaagcag 900

aacccaacag aagagcaagc cgtgggtata tgcaaggctg caatgggact gagaattagc 960

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gaggaagagg tgcttacggg caatcttcaa acattgaaga taagagtgca tgagggatat 1080

gaagagttca caatggttgg gagaagagca acagccatac tcagaaaagc aaccaggaga 1140

ttgattcagc tgatagtgag tgggagagac gaacagtcga ttgccgaagc aataattgtg 1200

gccatggtat tttcacaaga ggattgtatg ataaaagcag ttagaggtga tctgaatttc 1260

gtcaataggg cgaatcagcg actgaatcct atgcatcaac ttttaagaca ttttcagaag 1320

gatgcgaaag tgctttttca aaattgggga gttgaaccta tcgacaatgt gatgggaatg 1380

attgggatat tgcccgacat gactccaagc atcgagatgt caatgagagg agtgagaatc 1440

agcaaaatgg gtgtagatga gtactccagc acggagaggg tagtggtgag cattgaccgg 1500

ttcttgagag tccgggacca acgaggaaat gtactactgt ctcccgagga ggtcagtgaa 1560

acacagggaa cagagaaact gacaataact tactcatcgt caatgatgtg ggagattaat 1620

ggtcctgaat cagtgttggt caatacctat caatggatca tcagaaactg ggaaactgtt 1680

aaaattcagt ggtcccagaa ccctacaatg ctatacaata aaatggaatt tgaaccattt 1740

cagtctttag tacctaaggc cattagaggc caatacagtg ggtttgtgag aactctgttc 1800

caacaaatga gggatgtgct tgggacattt gataccgcac agataataaa acttcttccc 1860

ttcgcagccg ctccaccaaa gcaaagtaga atgcagttct cctcatttac tgtgaatgtg 1920

aggggatcag gaatgagaat acttgtaagg ggcaattctc ctgtattcaa ctacaacaag 1980

gccacgaaga gactcacagt tctcggaaag gatgctggca ctttaaccga agacccagat 2040

gaaggcacag ctggagtgga gtccgctgtt ctgaggggat tcctcattct gggcaaagaa 2100

gacaggagat atgggccagc attaagcatc aatgaactga gcaaccttgc gaaaggagag 2160

aaggctaatg tgctaattgg gcaaggagac gtggtgttgg taatgaaacg aaaacgggac 2220

tctagcatac ttactgacag ccagacagcg accaaaagaa ttcggatggc catcaattag 2280

<210>4

<211>2274

<212>DNA

<213>Influenza virus

<400>4

atggatgtca atccgacctt acttttctta aaagtgccag cacaaaatgc tataagcaca 60

actttccctt ataccggaga ccctccttac agccatggga caggaacagg atacaccatg 120

gatactgtca acaggacaca tcagtactca gaaaaggcaa gatggacaac aaacaccgaa 180

actggagcac cgcaactcaa cccgattgat gggccactgc cagaagacaa tgaaccaagt 240

ggttatgccc aaacagattg tgtattggaa gcaatggctt tccttgagga atcccatcct 300

ggtatttttg aaaactcgtg tattgaaacg atggaggttg ttcagcaaac acgagtagac 360

aagctgacac aaggccgaca gacctatgac tggactttaa atagaaacca gcctgctgca 420

acagcattgg ccaacacaat agaagtgttc agatcaaatg gcctcacggc caatgagtct 480

ggaaggctca tagacttcct taaggatgta atggagtcaa tgaaaaaaga agaaatgggg 540

atcacaactc attttcagag aaagagacgg gtgagagaca atatgactaa gaaaatgata 600

acacagagaa caataggtaa aaggaaacag agattgaaca aaaggagtta tctaattaga 660

gcattgaccc tgaacacaat gaccaaagat gctgagagag ggaagctaaa acggagagca 720

attgcaaccc cagggatgca aataaggggg tttgtatact ttgttgagac actggcaagg 780

agtatatgtg agaaacttga acaatcaggg ttgccagttg gaggcaatga gaagaaagca 840

aagttggcaa atgttgtaag gaagatgatg accaattctc aggacaccga actttctttg 900

accatcactg gagataacac caaatggaac gaaaatcaga atcctcggat gtttttggcc 960

atgatcacat atatgaccag aaatcagccc gaatggttca gaaatgttct aagtattgct 1020

ccaataatgt tctcaaacaa aatggcgaga ctgggaaaag ggtatatgtt tgagagcaag 1080

agtatgaaac ttagaactca aatacctgca gaaatgctag caagcattga tttgaaatat 1140

ttcaatgatt caacaagaaa gaagattgaa aaaatccgac cgctcttaat agaggggact 1200

gcatcattga gccctggaat gatgatgggc atgttcaata tgttaagcac tgtattaggc 1260

gtctccatcc tgaatcttgg acaaaagaga tacaccaaga ctacttactg gtgggatggt 1320

cttcaatcct ctgacgattt tgctctgatt gtgaatgcac ccaatcatga agggattcaa 1380

gccggagtcg acaggtttta tcgaacctgt aagctacatg gaatcaatat gagcaagaaa 1440

aagtcttaca taaacagaac aggtacattt gaattcacaa gttttttcta tcgttatggg 1500

tttgttgcca atttcagcat ggagcttccc agttttggtg tgtctgggag caacgagtca 1560

gcggacatga gtattggagt tactgtcatc aaaaacaata tgataaacaa tgatcttggt 1620

ccagcaacag ctcaaatggc ccttcagttg ttcatcaaag attacaggta cacgtaccga 1680

tgccatagag gtgacacaca aatacaaacc cgaagatcat ttgaaataaa gaaactgtgg 1740

gagcaaaccc gttccaaagc tggactgctg gtctccgacg gaggcccaaa tttatacaac 1800

attagaaatc tccacattcc tgaagtctgc ctaaaatggg aattgatgga tgaggattac 1860

caggggcgtt tatgcaaccc actgaaccca tttgtcagcc ataaagaaat tgaatcaatg 1920

aacaatgcag tgatgatgcc agcacatggt ccagccaaaa acatggagta tgatgctgtt 1980

gcaacaacac actcctggat ccccaaaaga aatcgatcca tcttgaatac aagtcaaaga 2040

ggagtacttg aagatgaaca aatgtaccaa aggtgctgca atttatttga aaaattcttc 2100

cccagcagtt catacagaag accagtcggg atatccagta tggtggaggc tatggtttcc 2160

agagcccgaa ttgatgcacg gattgatttc gaatctggaa ggataaagaa agaagagttc 2220

actgagatca tgaagatctg ttccaccatt gaagagctca gacggcaaaa atag 2274

<210>5

<211>2151

<212>DNA

<213>Influenza virus

<400>5

atggaagatt ttgtgcgaca atgcttcaat ccgatgattg tcgagcttgc ggaaaaaaca 60

atgaaagagt atggggagga cctgaaaatc gaaacaaaca aatttgcagc aatatgcact 120

cacttggaag tatgcttcat gtattcagat ttccacttca tcaatgagca aggcgagtca 180

ataatcgtag aacttggtga tcctaatgca cttttgaagc acagatttga aataatcgag 240

ggaagagatc gcacaatggc ctggacagta gtaaacagta tttgcaacac tacaggggct 300

gagaaaccaa agtttctacc agatttgtat gattacaagg aaaatagatt catcgaaatt 360

ggagtaacaa ggagagaagt tcacatatac tatctggaaa aggccaataa aattaaatct 420

gagaaaacac acatccacat tttctcgttc actggggaag aaatggccac aaaggccgac 480

tacactctcg atgaagaaag cagggctagg atcaaaacca ggctattcac cataagacaa 540

gaaatggcca gcagaggcct ctgggattcc tttcgtcagt ccgagagagg agaagagaca 600

attgaagaaa ggtttgaaat cacaggaaca atgcgcaagc ttgccgacca aagtctcccg 660

ccgaacttct ccagccttga aaattttaga gcctatgtgg atggattcga accgaacggc 720

tacattgagg gcaagctgtc tcaaatgtcc aaagaagtaa atgctagaat tgaacctttt 780

ttgaaaacaa caccacgacc acttagactt ccgaatgggc ctccctgttc tcagcggtcc 840

aaattcctgc tgatggatgc cttaaaatta agcattgagg acccaagtca tgaaggagag 900

ggaataccgc tatatgatgc aatcaaatgc atgagaacat tctttggatg gaaggaaccc 960

aatgttgtta aaccacacga aaagggaata aatccaaatt atcttctgtc atggaagcaa 1020

gtactggcag aactgcagga cattgagaat gaggagaaaa ttccaaagac taaaaatatg 1080

aaaaaaacaa gtcagctaaa gtgggcactt ggtgagaaca tggcaccaga aaaggtagac 1140

tttgacgact gtaaagatgt aggtgatttg aagcaatatg atagtgatga accagaattg 1200

aggtcgcttg caagttggat tcagaatgag ttcaacaagg catgcgaact gacagattca 1260

agctggatag agcttgatga gattggagaa gatgtggctc caattgaaca cattgcaagc 1320

atgagaagga attatttcac atcagaggtg tctcactgca gagccacaga atacataatg 1380

aagggggtgt acatcaatac tgccttactt aatgcatctt gtgcagcaat ggatgatttc 1440

caattaattc caatgataag caagtgtaga actaaggagg gaaggcgaaa gaccaacttg 1500

tatggtttca tcataaaagg aagatcccac ttaaggaatg acaccgacgt ggtaaacttt 1560

gtgagcatgg agttttctct cactgaccca agacttgaac cacacaaatg ggagaagtac 1620

tgtgttcttg agataggaga tatgcttcta agaagtgcca taggccaggt ttcaaggccc 1680

atgttcttgt atgtgaggac aaatggaacc tcaaaaatta aaatgaaatg gggaatggag 1740

atgaggcgtt gtctcctcca gtcacttcaa caaattgaga gtatgattga agctgagtcc 1800

tctgtcaaag agaaagacat gaccaaagag ttctttgaga acaaatcaga aacatggccc 1860

attggagagt ctcccaaagg agtggaggaa agttccattg ggaaggtctg caggacttta 1920

ttagcaaagt cggtatttaa cagcttgtat gcatctccac aactagaagg attttcagct 1980

gaatcaagaa aactgcttct tatcgttcag gctcttaggg acaatctgga acctgggacc 2040

tttgatcttg gggggctata tgaagcaatt gaggagtgcc taattaatga tccctgggtt 2100

ttgcttaatg cttcttggtt caactccttc cttacacatg cattgagtta g 2151

<210>6

<211>1497

<212>DNA

<213>Influenza virus

<400>6

atggcgtccc aaggcaccaa acggtcttac gaacagatgg agactgatgg agaacgccag 60

aatgccactg aaatcagagc atccgtcgga aaaatgattg gtggaattgg acgattctac 120

atccaaatgt gcacagaact taaactcagt gattatgagg gacggttgat ccaaaacagc 180

ttaacaatag agagaatggt gctctctgct tttgacgaaa ggagaaataa atacctggaa 240

gaacatccca gtgcggggaa ggatcctaag aaaactggag gacctatata cagaagagta 300

aacggaaagt ggatgagaga actcatcctt tatgacaaag aagaaataag gcgaatctgg 360

cgccaagcta ataatggtga cgatgcaacg gctggtctga ctcacatgat gatctggcat 420

tccaatttga atgatgcaac ttatcagagg acaagggctc ttgttcgcac cggaatggat 480

cccaggatgt gctctctgat gcaaggttca actctcccta ggaggtctgg agccgcaggt 540

gctgcagtca aaggagttgg aacaatggtg atggaattgg tcaggatgat caaacgtggg 600

atcaatgatc ggaacttctg gaggggtgag aatggacgaa aaacaagaat tgcttatgaa 660

agaatgtgca acattctcaa agggaaattt caaactgctg cacaaaaagc aatgatggat 720

caagtgagag agagccggga cccagggaat gctgagttcg aagatctcac ttttctagca 780

cggtctgcac tcatattgag agggtcggtt gctcacaagt cctgcctgcc tgcctgtgtg 840

tatggacctg ccgtagccag tgggtacgac tttgaaagag agggatactc tctagtcgga 900

atagaccctt tcagactgct tcaaaacagc caagtgtaca gcctaatcag accaaatgag 960

aatccagcac acaagagtca actggtgtgg atggcatgcc attctgccgc atttgaagat 1020

ctaagagtat tgagcttcat caaagggacg aaggtggtcc caagagggaa gctttccact 1080

agaggagttc aaattgcttc caatgaaaat atggagacta tggaatcaag tacacttgaa 1140

ctgagaagca ggtactgggc cataaggacc agaagtggag gaaacaccaa tcaacagagg 1200

gcatctgcgg gccaaatcag catacaacct acgttctcag tacagagaaa tctccctttt 1260

gacagaacaa ccgttatggc agcattcact gggaatacag aggggagaac atctgacatg 1320

aggaccgaaa tcataaggat gatggaaagt gcaagaccag aagatgtgtc tttccagggg 1380

cggggagtct tcgagctctc ggacgaaaag gcagcgagcc cgatcgtgcc ttcctttgac 1440

atgagtaatg aaggatctta tttcttcgga gacaatgcag aggagtacga caattaa 1497

<210>7

<211>1027

<212>DNA

<213>Influenza virus

<400>7

agcgaaagca ggtagatatt gaaagatgag tcttctaacc gaggtcgaaa cgtacgttct 60

ctctatcatc ccgtcaggcc ccctcaaagc cgagatcgca cagagacttg aagatgtctt 120

tgcagggaag aacaccgatc ttgaggttct catggaatgg ctaaagacaa gaccaatcct 180

gtcacctctg actaagggga ttttaggatt tgtgttcacg ctcaccgtgc ccagtgagcg 240

aggactgcag cgtagacgct ttgtccaaaa tgcccttaat gggaacgggg atccaaataa 300

catggacaaa gcagttaaac tgtataggaa gctcaagagg gagataacat tccatggggc 360

caaagaaatc tcactcagtt attctgctgg tgcacttgcc agttgtatgg gcctcatata 420

caacaggatg ggggctgtga ccactgaagt ggcatttggc ctggtatgtg caacctgtga 480

acagattgct gactcccagc atcggtctca taggcaaatg gtgacaacaa ccaacccact 540

aatcagacat gagaacagaa tggttttagc cagcactaca gctaaggcta tggagcaaat 600

ggctggatcg agtgagcaag cagcagaggc catggaggtt gctagtcagg ctaggcaaat 660

ggtgcaagcg atgagaacca ttgggactca tcctagctcc agtgctggtc tgaaaaatga 720

tcttcttgaa aatttgcagg cctatcagaa acgaatgggg gtgcagatgc aacggttcaa 780

gtgatcctct cgctattgcc gcaaatatca ttgggatctt gcacttgata ttgtggattc 840

ttgatcgtct ttttttcaaa tgcatttacc gtcgctttaa atacggactg aaaggagggc 900

cttctacgga aggagtgcca aagtctatga gggaagaata tcgaaaggaa cagcagagtg 960

ctgtggatgc tgacgatggt cattttgtca gcatagagct ggagtaaaaa actaccttgt 1020

ttctact 1027

<210>8

<211>890

<212>DNA

<213>Influenza virus

<400>8

agcaaaagca gggtgacaaa gacataatgg atccaaacac tgtgtcaagc tttcaggtag 60

attgctttct ttggcatgtc cgcaaacgag ttgcagacca agaactaggt gatgccccat 120

tccttgatcg gcttcgccga gatcagaaat ccctaagagg aaggggcagc actcttggtc 180

tggacatcga gacagccaca cgtgctggaa agcagatagt ggagcggatt ctgaaagaag 240

aatccgatga ggcacttaaa atgaccatgg cctctgtacc tgcgtcgcgt tacctaaccg 300

acatgactct tgaggaaatg tcaagggaat ggtccatgct catacccaag cagaaagtgg 360

caggccctct ttgtatcaga atggaccagg cgatcatgga taaaaacatc atactgaaag 420

cgaacttcag tgtgattttt gaccggctgg agactctaat attgctaagg gctttcaccg 480

aagagggagc aattgttggc gaaatttcac cattgccttc tcttccagga catactgctg 540

aggatgtcaa aaatgcagtt ggagtcctca tcggaggact tgaatggaat gataacacag 600

ttcgagtctc tgaaactcta cagagattcg cttggagaag cagtaatgag aatgggagac 660

ctccactcac tccaaaacag aaacgagaaa tggcgggaac aattaggtca gaagtttgaa 720

gaaataagat ggttgattga agaagtgaga cacaaactga aggtaacaga gaatagtttt 780

gagcaaataa catttatgca agccttacat ctattgcttg aagtggagca agagataaga 840

actttctcat ttcagcttat ttaataataa aaaacaccct tgtttctact 890

Claims (3)

1. a restructuring H1N1virus inactivated vaccine strain, its preserving number is CCTCC NO:V201006.

2. the application in the medicine of preparation prevention disease that H1N1virus causes according to the H1N1virus inactivated vaccine strain of the restructuring of claim 1.

3. application according to claim 2, wherein said medicine is for preventing animal or human's H 1 N 1 influenza A virus infection.

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