CN110042088A - A kind of bird flu H9N2 virus-like particle subunit vaccine - Google Patents

Abstract

The present invention provides a kind of virus-like particle of avian influenza virus, is using after recombinate shape virus infection insect cell, acquisition is collected in culture.Wherein recombinant baculovirus includes the nucleotide fragments for encoding HA, NA and M1 albumen；HA, NA and M1 albumen, amino acid sequence are respectively SEQ ID NO:8；SEQ ID NO:10；SEQ ID NO:6.Virus-like particle prepared by the present invention is used to prepare vaccine.The present invention is screened and is analyzed by the cDNA complete sequence to avian influenza virus HA, NA and M1 albumen, have chosen wherein epitope it is relatively abundant, can in insect cell high efficient expression cDNA sequence.After optimizing transformation to HA, NA and M albumen, using insect cell-baculovirus expression system preparation VLP, virus protein yield is significantly higher than the virus of SPF embryo culture, and production cost is substantially reduced.

Description

A kind of bird flu H9N2 virus-like particle subunit vaccine

Technical field

The invention belongs to recombinant vaccine technical fields, and in particular to a kind of bird flu H9N2 virus-like particle subunit Vaccine.

Background technique

Bird flu is one of birds deadly infectious disease, is by influenza A (Avian Influenza Virus, AIV) It is caused.Poultry infection H9 subtype avian influenza virus, especially when with mixed infection, can lead to and cause high mortality and Laying rate degradation.Acute, the hyperinfection high in one of animals and humans infectiousness, the death rate as Major Epidemic Property disease, bird flu betide extensively all over the world, to the mankind and aviculture harm it is very big.

Avian influenza virus belongs to the member of orthomyxovirus section, and influenza virus particles include following various albumen: blood cell is solidifying Collect element HA, nerve amines neuraminidase NA, matrix prote m1, ionophorous protein M2, nucleoprotein NP, varial polymerases PB1, virus The protein such as polymerase PB2, varial polymerases PA and non-structural protein NS2.Wherein HA protein and NA protein are film sugar Albumen is responsible for inside the adherent cell of virus and the penetrating cell of virion, is to neutralize and protect immunity for virus The source of principal immune epitope.HA protein and NA protein be considered as preventative influenza vaccines it is main at Point.

Baculoviral is a kind of large-scale rod-shaped togavirus, and genome is circular double stranded DNA, and size is about 80- 180kbp.Baculoviral parasitizes arthropod as pathogenic microorganism, and the host specificity with height, host mainly has Lepidoptera Diptera and hymenopteran, not yet the baculoviral host other than discovery arthropod.In the numerous of baculoviral In member, studying and utilize at most at present is more embedding nuclear polyhedrosis virus (AcMNPV) of autographa california. Double-stranded DNA of the AcMNPV viral genome for virus covalently closed circular supercoil, about 130kb, genome sequence have been measured at present. In recent years, rhabdovirus expression vector accounts for leading status with the advantage of itself on expression vector.Baculoviral table Up to system compared with other expression systems, baculovirus expression system have it is easy to operate, highly-safe, big mesh can be accommodated Gene, expression foreign protein effect is high, plays the role of posttranslational modification, expresses the immunogenicity, bioactivity and day of albumen The advantages that right albumen is similar (Anderson et al., 1995；Wang et al.,2001；Ribeiro et al., 2001)。

Existing avian influenza vaccine is mostly traditional vaccine, is to use complete pathogen as antigen for vaccine, there are one Fixed security risk.And the use of totivirus can cause selection pressure to virus, accelerate the variation of virus.

Summary of the invention

The present invention provides a kind of virus-like particle of avian influenza virus, and prepares vaccine using the virus-like particle, from And make up the deficiencies in the prior art.

Present invention firstly provides a kind of recombinant baculovirus, wherein including the nucleotide piece for encoding HA, NA and M1 albumen Section；HA, NA and M1 albumen, amino acid sequence are respectively SEQ ID NO:8；SEQ ID NO:10；SEQ ID NO: 6；

The nucleotide fragments, sequence are SEQ ID NO:7；SEQ ID NO:9；SEQ ID NO:11；

Recombinant baculovirus constructed by the present invention is used to prepare the virus-like particle of avian influenza virus in insect cell；

Another aspect of the present invention provides a kind of virus-like particle of avian influenza virus, is using the above-mentioned rod-shaped disease of recombination After malicious infected insect cell, acquisition is collected in culture.

The insect cell is Insect cells Sf9；

Virus-like particle prepared by the present invention is used to prepare vaccine；

Include antigen and vaccine adjuvant the present invention also provides a kind of avian influenza virus subunit vaccine, used in it is anti- It originally was virus-like particle prepared by the present invention.

The present invention is screened and is analyzed by the cDNA complete sequence to avian influenza virus HA, NA and M1 albumen, is had chosen Wherein epitope it is relatively abundant, can in insect cell high efficient expression cDNA sequence.HA, NA and M albumen are optimized After transformation, using insect cell-baculovirus expression system preparation VLP, virus protein yield is significantly higher than SPF embryo culture Virus, production cost are substantially reduced.

Specific embodiment

The recombinant baculovirus for being used to express antigen protein that the present invention constructs, can successfully give expression in insect cell Soluble HA, NA, M1 albumen, three albumen automatic assembling assembly virus-like particle (VLPS) in vivo.VLP is on space conformation More similar and natural viral can stimulate body to generate cellular immunity and humoral immunity simultaneously, good immune effect, and not due to VLP Containing viral nucleic acid, potential viral pathogenesis gene is not present, safety is higher, it is possible to reduce bird flu whole virus vaccine is immune To virus variation bring pressure, the variation of virus is reduced.Therefore, developing AIV-VLP vaccine using genetic engineering means is AIV The hot spot of new generation vaccine research and direction.

AIV-VLP subunit vaccine can be produced using of the invention, there is business use value

The present invention is described in detail combined with specific embodiments below.

The screening of embodiment 1, avian influenza virus HA protein, NA albumen, M1 albumen

Applicant in 2017 isolates one plant of H9 subtype avian influenza virus from the chicken group in Shandong chicken farm.Pathological material of disease is pressed 1:5 (w/v) ratio is added to containing in dual anti-physiological saline, and suspension is made in grinding, and 1000r/min is centrifuged 5 minutes, and supernatant is taken to urinate Blister cavities is inoculated with 10~11 age in days SPF chicken embryos, and every embryo 0.2ml, 36 DEG C~37 DEG C hatch 3~4, and every sunshine embryo 2 times takes 24 hours Dead chicken embryo afterwards sets 2~8 DEG C of coolings 8~16 hours, harvests chicken embryo liquid, carries out HA and HI test.The virus liquid of harvest is through pure The analysis detection of the virus characteristic of viral level, immunogenicity, specificity and pure property etc. has been carried out after change, the results showed that Isolated strain is H9N2 type avian influenza virus, no bacterium, mycoplasma and exogenous virus pollution；Strain after purification is named as YBF1701。

Sequence analysis is carried out to HA, NA, M1 gene of the YBF1701 strain of screening and identification, wherein HA full length gene 1683bp (nucleotides sequence is classified as SEQ ID NO:1) encodes 562 amino acid (amino acid sequence is SEQ ID NO:2)；NA base Because of overall length 1563bp (amino acid sequence be SEQ ID NO:3), encode 522 amino acid (amino acid sequence is SEQ ID NO: 4)；M1 full length gene 759bp (amino acid sequence is SEQ ID NO:5), encodes 253 amino acid (amino acid sequence SEQ ID NO:6).It is subjected to Phylogenetic tree and nucleotide sequences homologous with HA, NA, M1 gene order included in GenBank Property analysis, find YBF1701 plant HA amino acid compared with the nearest APY18098.1 of homology in GenBank, the 217th (I change M), the 239th (D become N), the 254th (R becomes K), the 320th (I becomes V), the 420th (N becomes D), the 422nd (I becomes V), the 473 (K becomes R) are made a variation, and homology is 97.5~98.7%；Homology is nearest in NA amino acid and GenBank AKA60605.1 is compared, the 108th (G becomes A), the 222nd (T becomes I), the 355th (N becomes I), the 379th (G becomes A), homology About 96.5~98.9%；M1 amino acid is compared with the nearest AIQ82571.1 of homology in GenBank, the 20th (L become K), the 62 (F becomes R), the 138th (V becomes G), the 171st (P becomes T), the 233rd (L becomes P), homology about 96.5~98.9%.It is anti- Former analysis of protein is the result shows that HA, NA, M1 albumen of screening virus and HA, NA and M1 gene of reported H9N2 virus are deposited In amino acid of differences.

Embodiment 2: the building of the recombinant baculovirus of expression NA, HA and M1 gene

1, the shearing, optimization of HA, NA and M1 gene

The transmembrane region of the signal peptide of 18 amino acid before the N-terminal structural domain of HA gene, 10 amino acid of C-terminal is sheared Fall；337 S are sported into K, it is easier to the formation of space structure；N-terminal is added into initiation codon ATG；Simultaneously to codon It optimizes, the nucleotides sequence after optimization is classified as SEQ ID NO:7；The nucleotide sequence contains the digestion position at 5 ' ends and 3 ' Point, the amino acid sequence for removing the albumen translated after the nucleotide of the two restriction enzyme sites is SEQ ID NO:8.

29AA before NA gene N-terminal is cut off, and initiation codon ATG, C-terminal one section of termination sequence of addition, while into The optimization of row codon, the nucleotides sequence after optimization are classified as SEQ ID NO:9, since there is terminator codon in inside, so only 438 amino acid can be translated, sequence is SEQ ID NO:10.

The N-terminal of M1 gene is added to the nucleotide sequence of one section of promoter, while carrying out codon optimization, after optimization Nucleotide is SEQ ID NO:11.The sequence sheared, optimized and the sequence without Shearing Optimization are by upper marine growth work Journey Co., Ltd is synthesized, and is connected into cloning vector.

2, the building of HA positive plasmid

2.2.1 endonuclease reaction

2.2.1.1 label needs well the 1.5mL EP used to manage, and sample-adding is carried out according to the following table in 1.5mL EP pipe, mixes Even: reaction system is 50 μ L, and sample-adding is as shown in the table:

2.2.1.2 the 1.5mL EP pipe in step 2.2.1.1 is placed in 37 DEG C of thermostat water baths, water-bath 2-3h.

2.2.1.3 double enzyme digestion product glue recycles

Above-mentioned double digestion system is taken out, carries out agarose gel electrophoresis to recycle DNA fragmentation therein.

(1) sample collection EP pipe, adsorption column and collecting pipe have been marked.

(2) the empty EP pipe weight marked is weighed, and records numerical value.

(3) single target DNA band is carefully cut from Ago-Gel with scalpel on bale cutting instrument be put into it is dry In net 1.5mL centrifuge tube.

(4) 600 μ L PC buffer50 DEG C water-baths are added in the 1.5mL centrifuge tube in step (3) and place 5min or so, Centrifuge tube is mildly constantly spun upside down therebetween, to ensure that blob of viscose sufficiently dissolves.

(5) column equilibration: into adsorption column CB2,500 μ L equilibrium liquid BL, centrifugation is added in (adsorption column is placed in advance in collecting pipe) 12,000rpm, 1min outwell the waste liquid in collecting pipe, adsorption column are placed back in collecting pipe.

(6) step (5) acquired solution is added in adsorption column CB2, stand 2min, 10,000rpm, be centrifuged 30s, outwell receipts Waste liquid in collector, then adsorption column CB2 is put into collecting pipe.

(7) 600 μ L rinsing liquid PW buffer are added into adsorption column, stand 3min, is centrifuged 10,000rpm, 30s, outwells Adsorption column CB2 is put into collecting pipe by the waste liquid in collecting pipe.

(8) step (7) are repeated.

(9) suction attached column is centrifuged, 12,000rpm, 2min, as far as possible removing rinsing liquid.Adsorption column is placed in and is placed at room temperature for 10min thoroughly dries.

(10) adsorption column CB2 is put into collecting pipe, 50 μ L is vacantly added dropwise to adsorbed film middle position Elutionbuffer (65 DEG C of preheatings), stands 3min, is centrifuged 12,000rpm, 2min.

(11) from centrifuge tube in step (10) is taken out in centrifuge, intermediate adsorption column CB2 is abandoned, centrifuge tube lid is covered Son retains the DNA sample in centrifuge tube.

(12) DNA sample in step 11 is placed in 4 DEG C of preservations, prepares agarose gel electrophoresis identification glue and recycles DNA piece Section.

2.2.3 connection reaction

(1) label needs the 0.2mL centrifuge tube used.

(2) it is loaded in marking complete 0.2mL pipe according to 20 μ L reaction systems of following table:

(3) it after completing sample-adding, is gently blown and beaten with pipettor and mixes each component several times.

(4) 0.2mL centrifuge tube is placed in 37 DEG C of reaction 30min, to after the reaction was completed, reaction tube is placed in ice-water bath immediately Middle cooling 5min.

(5) reaction product of step (4) can directly carry out transformation experiment, can also be stored in -20 DEG C, thaw turn when needed Change.

2.2.4 conversion reaction

(1) 10 μ L connection reaction solutions are rapidly joined in 100 μ L competent cells, and blows and beats mixing, ice bath 30min.

(2) after the completion of step (1), sample cell is taken out, is placed in 42 DEG C of water-bath 100s, immediately after ice bath 2min.

(3) it after the completion of step (2), takes out sample cell and 600 μ L liquid LB is added into sample cell in superclean bench Culture medium, is then placed in 37 DEG C of constant-temperature tables for sample cell, and 220rpm cultivates 1h.

(4) preparation conversion plate, according to plasmid resistance preparation conversion LB resistant panel.

(5) coated plate: taking out sample cell in step (3), and room temperature is centrifuged 8,000rpm, 2min, removes 600 μ L supernatant fluids, The thallus of bottom of the tube is resuspended in remaining supernatant, and the bacterium solution of resuspension is put into corresponding conversion plate center, will be converted with bacteria stick is applied The bacterium solution of plate center is uniformly spread out.

(6) step (5) plate is just being placed in biochemical constant incubator, after 37 DEG C of culture 1h, will conversion plate be inverted into Row culture 15h.

(7) conversion results are observed and recorded.

2.2.5 plasmid extraction and PCR are identified

2.2.5.1 plasmid extraction

(1) with 10 μ L liquid transfer gun heads from conversion plate in picking monoclonal to the 5ml resistance of benzyl containing ammonia LB liquid medium In, 37 DEG C, 220rpm shakes bacterium and stays overnight.

(2) bacterium solution is drawn into 1.5mL EP pipe, and room temperature centrifugation, 12,000rpm, 2min abandon supernatant.

(3) 250 μ L plasmids are added in the EP pipe in step (2) and extract reagent P1buffer, thorough suspension thalline.

(4) 250 μ L P2buffer are added into step (3) solution, immediately mildly 5-10 mixing of reverse centrifuge tube.Room Temperature stands 2-4min.

(5) 350 μ L P3 buffer are added into step (4) solution, immediately mildly 5-10 mixing of reverse centrifuge tube.Room Temperature stands 2-4min.

(6) by step (5) solution, room temperature centrifugation, 14,000rpm, 10min.

(7) supernatant solution in step (6) is moved into adsorption column center, room temperature centrifugation, 12,000rpm, 30s outwell collection Liquid in pipe.

(8) 500 μ L Buffer DW1 are added to adsorption column center, room temperature centrifugation, 12,000rpm, 30s outwell collecting pipe Middle liquid.

(9) 500 μ L wash solution are added to adsorption column center, room temperature centrifugation, 12,000rpm, 30s outwell receipts Liquid in collector.It is repeated once.

(10) suction attached column, room temperature centrifugation, 12,000rpm, 2min.

(11) adsorption column is put into a clean 1.5ml centrifuge tube, 30 μ L is added to absorption center membrane Elutionbuffer is stored at room temperature 5min, and room temperature is centrifuged, 12,000rpm, 2min, DNA solution in 4 DEG C of preservation pipes.

2.2.5.2 PCR is identified

(1) PCR pipe for needing to use well is marked, sample-adding is carried out according to the following table, mixes, reaction system is 25 μ L:

(2) PCR amplification program:

(3) it is sequenced: sending sequencing company to be sequenced the positive plasmid of pcr identification.

The building of 2.3 HA-NA positive plasmids

Correct positive plasmid will be sequenced and carry out digestion (BamHI/EcoRI), connection, conversion, method is same as above, PCR is reflected Fixed positive plasmid send sequencing company to be sequenced.

The building of 2.4 HA-NA-M1 positive plasmids

Correct positive plasmid will be sequenced and carry out digestion (Not I/Xba I), connection, conversion, method is same as above, PCR is reflected Fixed positive plasmid send sequencing company to be sequenced.

2.5 conversion

The positive plasmid of sequencing is converted into DH10bac competent cell, the same 2.2.4. of method

2.6 picking rod granules and PCR are identified

2.6.1 picking rod granule

(1) from 2.5 conversion plate in, with 10 μ L liquid transfer gun heads from conversion plate in picking white monoclonal colonies to 5ml containing kalamycin resistance, tetracyclin resistance, gentamicin resistance LB liquid medium in, 37 DEG C, 220rpm shakes bacterium mistake Night.

(2) bacterium solution is drawn into 1.5mL EP pipe, and room temperature centrifugation, 12,000rpm, 2min abandon supernatant.

(3) 250 μ L plasmids are added in the EP pipe in step (2) and extract reagent P1buffer, thorough suspension thalline.

(4) 250 μ L P2buffer are added into step (3) solution, immediately mildly 5-10 mixing of reverse centrifuge tube.Room Temperature stands 2-4min.

(5) 350 μ L P3 buffer are added into step (4) solution, immediately mildly 5-10 mixing of reverse centrifuge tube.Room Temperature stands 2-4min.

(6) by step (5) solution, room temperature centrifugation, 14,000rpm, 10min.

(7) supernatant solution in step (6) is moved into adsorption column center, room temperature centrifugation, 12,000rpm, 30s outwell collection Liquid in pipe.

(8) 500 μ L Buffer DW1 are added to adsorption column center, room temperature centrifugation, 12,000rpm, 30s outwell collecting pipe Middle liquid.

(9) 500 μ L wash solution are added to adsorption column center, room temperature centrifugation, 12,000rpm, 30s outwell receipts Liquid in collector.It is repeated once.

(10) suction attached column, room temperature centrifugation, 12,000rpm, 2min.

(11) adsorption column is put into a clean 1.5ml centrifuge tube, 30 μ L is added to absorption center membrane Elutionbuffer is stored at room temperature 5min, and room temperature is centrifuged, 12,000rpm, 2min, DNA solution in 4 DEG C of preservation pipes.

2.6.2 the DNA that 2.6.1 is extracted in PCR identification carries out PCR identification, and the plasmid of the result positive is carried out SF9 cell Transfection.

3 SF9 cell transfecting of embodiment

(1) prepare: Biohazard Safety Equipment ultraviolet sterilization 30min；TNM-FH culture solution is placed in 27 DEG C of water-baths and is preheated to 27 DEG C.

(2) recombinant DNA prepared by 2 μ g embodiments 2 is added in 100 μ l serum-frees and dual anti-TNM-FH culture solution, It mixes.9 μ l Cellfectin Reagent are added in 100 μ l serum-frees and dual anti-TNM-FH culture solution, are mixed.It will Liposome is mixed with recombinant DNA, the static 40min of room temperature.

(3) 6 orifice plates cell is taken out from 27 DEG C of incubators, is discarded supernatant culture medium, is washed with the TNM-FH culture solution of pre-temperature Cell three times, and discards TNM-FH culture solution.

(4) the TNM-FH culture solution of 10% fetal calf serum of 2ml is added in each cell hole.

(5) mixture of recombinant DNA and liposome is gently added in the cell of every hole, is mixed gently, in 27 DEG C of conditions 5~6h of lower static gas wave refrigerator.

(6) liquid in hole is discarded, is added the complete TNM-FH culture solution of 2ml (containing dual anti-and 10% serum), 27 DEG C Under the conditions of static gas wave refrigerator 5~6 days.

(7) to cellular swelling, volume becomes larger, and after falling off, collects supernatant, labeled as P1 for recombinant baculovirus, name For AIV-VLP-P1.

(8) the Sf9 cell newly cultivated is infected with AIV-VLP-P1, improves recombinant baculovirus content, was inoculated with for 2 generations repeatedly Afterwards, cell supernatant is collected, 4 DEG C or -80 DEG C save backup.

4 protein purification of embodiment and detection

Expression and identification of the 4.1 recombination HA-NA-M1 albumen in Insect cells Sf9

By recombinant baculovirus AIV-VLP-P1 infected insect cell Sf9,27 DEG C of culture 72h, while with uninfecting virus 27 DEG C of culture 48h of normal Insect cells Sf9 as control, harvest cell, culture supernatant frozen spare.Cell pH7.4 PBS washing after, be added 1 × SDS-PAGE sample loading buffer [50mM Tris-HCl (pH6.8), 100mM Dithiothreitol (DTT), 2%SDS, 0.05%Bromophemol blue, 10%Glycerol], 5min is boiled, is used 12% separation gel, 5% concentration glue carry out polyacrylamide gel electrophoresis, 100 volts of about 2.5h, and coomassie brilliant blue R250 dyeing is seen The specificity for whether occurring avian influenza virus HA, NA, M1 albumen in the Insect cells Sf9 lysate of recombinate shape virus infection examined Band.As a result the not optimized rod granule of sequence does not have the expression of any external source band, and the rod granule of sequence optimisation has external source mesh Band expression.

Recombinant baculovirus AIV-VLP will be inoculated with Insect cells Sf9 by 4.2 HA Blood coagulation tests, and 27 DEG C are cultivated 72 hours Afterwards, cell precipitation is collected by centrifugation, is added appropriate physiological saline in cell precipitation, cell precipitation is resuspended, through ultrasonic treatment cell, With 4 DEG C of centrifugation 10min of 1000r/min, supernatant is collected, carries out HA detection.HA > 15log2 as the result is shown, and the full disease separated Malicious HA only has 8log2.

Recombinant baculovirus AIV-VLP is inoculated with Insect cells Sf9,27 DEG C of trainings by the purifying of 4.3 recombination HA-NA-M1 albumen After supporting 72 hours, cell precipitation is collected by centrifugation, appropriate physiological saline is added in cell precipitation, cell precipitation is resuspended, through ultrasonic wave Lytic cell is collected supernatant, is purified with 35% ammonium sulfate precipitation, weighed with 4 DEG C of centrifugation 10min of 1000r/min Group HA-NA-M1 prion sample particle.

4.4 Western blot by the totivirus of acquisition and 4.3 obtain recombination HA-NA-M1 prion sample particle into Protein band is transferred to pvdf membrane by row SDS-PAGE, and using 20 volts of transfer 30min of semidry method, transfer film is closed with confining liquid Overnight, PBST is washed 3 times, and 1: 500 37 DEG C of diluted avian influenza virus positive serum effect 1.5h, PBST is washed 3 times, with 1: 2000 37 DEG C of goat-anti chicken enzyme labelled antibody the effect 1.5h, PBST washing 3 times of diluted HRP label, substrate solution acts on 5min, ChemiDOC develops the color.As a result totivirus band is very weak, and the nucleotides sequence after optimizing is classified as SEQ ID NO:7, SEQ ID NO:9, the protein band that the gene of SEQ ID NO:11 is expressed in Insect cells Sf9 are very bright, illustrate HA, NA, M1 egg of expression White immunogenicity is good.

5 transmission electron microscope observing of embodiment

The influenza virus YBF1701 of screening is inoculated with SPF chicken embryo, collects allantoic fluid after 72 hours, carries out 100 times of concentrations. Electronic Speculum is observed after the P3 of the totivirus liquid of concentration and AIV-VLP are carried out negative staining for expression product simultaneously.As a result AIV-VLP is assembled At many virus-like particles, size 100nm, and the totivirus liquid for being concentrated 100 times only has micro particle.

The preparation and effect detection of 6 vaccine of embodiment

1, the preparation of vaccine

1) oil is mutually prepared: 95 parts of white oil for animals are taken, 1 part of aluminum stearate, is placed in oily phase preparation tank after being heated to 80 DEG C, then 5 parts of Jia Siben -80, until 30min is maintained when temperature reaches 115 DEG C, it is spare after cooling.

2) prepared by water phase: the recombination HA-NA-M1 prion sample particle that 4.3 steps of embodiment 4 are purified and sterile life The mixing of salt water proper proportion is managed, is made in every 0.2ml water phase containing protein content not less than 20 μ g/ml (HA >=9log2).After taking sterilizing 5 parts of Tween-80, be added Agitation Tank in, while add 95 parts of hybrid antigen liquid, start stirring motor stir 20~30min, make Tween-80 is completely dissolved.

3) emulsification takes 1.5 parts of oily phase to be put in high-speed shearing machine, starts the stirring of motor slow rotation, while water being added slowly It 1 part of phase, with 10000r/min, emulsifies 5 minutes.After emulsification, 10ml is taken, with 3000r/min centrifugation 15 minutes, the water that tube bottom is precipitated Accordingly it is no more than 0.5ml.

2, the efficacy test of vaccine

2.1 serological methods:

With 30~60 age in days SPF chickens 25,10 each subcutaneously or intramuscularly injection whole virus vaccine 0.2ml, 10 each subcutaneous Or intramuscular injection AIV-VLP vaccine 0.2ml, another 5 not immune to compare.21~28 days after inoculation, every chicken is taken a blood sample respectively, point From serum, the measurement of HI antibody titer is carried out by existing " Chinese veterinary pharmacopoeia ".The geometrical mean of immune group HI antibody titer should not Lower than 1:180 (micromethod), the geometrical mean of non-immunized controls group HI antibody titer should be not higher than 1:4 (micromethod), as a result It is shown in Table 1.

2.2 Immunization methods:

With 30~60 age in days SPF chickens 25,10 each subcutaneously or intramuscularly injection whole virus vaccine 0.2ml, 10 each subcutaneous Or intramuscular injection AIV-VLP vaccine 0.2ml, another 5 not immune to compare.21~28 days after inoculation, every chicken wings intravenous injection 10 times of diluted YBF1701 plants of venom, every 0.2ml acquire cloacal swab, after processing, allantoic cavity 5 days after attacking poison 10~11 age in days SPF chicken embryos 5 are inoculated with, are incubated for observation 5 days, no matter dead germ, embryo living should all measure chicken embryo liquid erythrocyte agglutination valence, As long as having the agglutination titer of 1 chicken embryo liquid not less than 1:16 (micromethod) in 5 chicken embryos of each swab samples inoculation, can be judged to Virus purification is positive.To the sample of virus purification feminine gender, determined again after answering blind passage primary.Immune group should at least 9 chicken diseases Poison is separated into feminine gender；Control group should 4 chicken virus purifications be at least the positive, the results are shown in Table 1.

Acquisition cloacal swab inoculated into chick embryo isolated viral on the 3rd after poison is attacked, measures allantoic fluid HA, again without coagulation blastochyle Blind passage is primary, using HA feminine gender as protection criterion.

1 vaccine potency inspection result of table

Note: 1, antibody is the geometric average value for antibody of this group of chicken.

The result shows that after 35 age in days SPF chicken of vaccine immunity prepared by the present invention, take a blood sample within 21st, separate serum, by it is existing " in State's veterinary drug allusion quotation " carry out the measurement of HI antibody titer.The geometrical mean of totivirus immune group HI antibody titer is that 1:548.7 is (micro Method), the geometrical mean of subunit vaccine group HI antibody titer is 1:724.1 (micromethod), non-immunized controls group HI antibody effect The geometrical mean of valence should be not higher than 1:, 2 (micromethods).Illustrate to recombinate HA-NA-M1 prion sample particle dilution 2⁸After times, Totivirus effect of the immune effect than former times is good.

Every chicken wings is injected intravenously 10 times of diluted YBF1701 plants of venom simultaneously, and every 0.2ml is adopted 5 days after attacking poison Collect cloacal swab, after processing, allantoic cavity is inoculated with 10 age in days SPF chicken embryos 5, is incubated for observation 5 days, and no matter dead germ, embryo living are equal Chicken embryo liquid erythrocyte agglutination valence should be measured, as long as having the agglutination titer of 1 chicken embryo liquid not in 5 chicken embryos of each swab samples inoculation Lower than 1:16 (micromethod), the virus purification positive can be judged to.To the sample of virus purification feminine gender, carried out again after answering blind passage primary Determine.As a result 9 chicken virus purifications of totivirus immune group are feminine gender；Recombination HA-NA-M1 prion sample particle immune group 10 Chicken virus purification is feminine gender, and 10 chicken virus purifications of control group are the positive.

The above results show the effect of subunit vaccine prepared by the present invention better than whole virus vaccine, the virus-like particle system Standby vaccine can prevent the infection of H9N2 well, and the antibody titer that subunit vaccine generates is apparently higher than whole virus vaccine, So having a good application prospect.Moreover, compared to commercially available other bird flu H9N2 viral vaccines, it is provided by the present invention Subunit vaccine is best to the immune effect of YBF1701 plants of venom, shows the antibody of subunit vaccine generation of the present invention to generation The bird flu H9N2 Strain of variation has better neutralization.

To sum up, the present invention is by the HA-NA-M1 albumen being not optimised and the HA-NA-M1 albumen of optimization while building is used for table Up to the rod granule carrier of antigen protein, as a result not optimized overall length HA-NA-M1 gene cannot express in insect cell, and excellent The HA-NA-M1 albumen of change successfully gives expression to soluble recombinant protein in insect cell.100 times of YBF1701 will be concentrated Strain, expression HA-NA-M1 supernatant carry out Electronic Speculum observation simultaneously, 100 times of totivirus is as a result concentrated and there was only a small amount of VLP, and The vp2 supernatant of not diluted expression automatic assembling assembly virus-like particle (VLPS) in vivo, and there are many virion amount.VLP exists More similar and natural viral on Space Idea can stimulate body to generate cellular immunity and humoral immunity simultaneously, good immune effect, and Since VLP does not contain viral nucleic acid, potential viral pathogenesis gene is not present, safety is higher.

Sequence table

<110>YEBIO Bioengineering Co., Ltd of Qingdao

<120>a kind of bird flu H9N2 virus-like particle subunit vaccine

<160> 11

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1683

<212> DNA

<213>artificial sequence (Artificial Sequence)

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gagaagaaca ttcttttcat gtggggcata aatcacccac ccaccgatac tacgcagaca 600

aatctgtaca caagaaccga cacaacaacg agtgtggcaa cagaagaaat aaataggatc 660

ttcaaaccat tgataggacc aaggcctctt gtcaacggtt tgatgggaag aattgattat 720

tattggtcgg tattgaaacc gggtcaaaca ctgcgaataa gatctgatgg gaatctaata 780

gctccatggt atggacacat tctttcagga gagagccacg gaagaatcct gaagactgat 840

ttaaaaaggg gtagctgcac agtgcaatgt cagacagaga aaggtggatt aaacacaaca 900

ttgccattcc aaaatgtaag taagtatgca tttggaaact gctcgaaata cattggcata 960

aagagtctca aacttgcagt tggtctgagg aatgtgcctt ctagatctag tagaggacta 1020

ttcggggcca tagcaggatt tatagaggga ggttggtcag gactagttgc tggttggtat 1080

ggattccagc attcaaatga ccaaggggtt ggtatggcag cagatagaga ctcaacccaa 1140

aaggcaattg ataaaataac atccaaagtg aataacatag tcgacaaaat gaacaagcag 1200

tatgaaatta ttgatcatga attcagtgag gtagaaacta gacttaacat gatcaataat 1260

aagatcgatg atcaaatcca ggatatatgg gcatataatg cagaattgct agttctgctt 1320

gaaaaccaga aaacactcga tgagcatgac gcaaatgtaa acaatctata taataaagtg 1380

aagagggcgt tgggttccaa tgcggtggaa gatgggaaag gatgtttcga gctataccac 1440

aaatgtgatg accagtgcat ggagacaatt cggaacggga cctacaacag aaggaagtat 1500

caagaggagt caaaattaga aagacagaaa atagaggggg tcaagctgga atctgaagga 1560

acttacaaaa tcctcaccat ttattcgact gtcgcctcat ctcttgtgat tgcaatgggg 1620

tttgctgcct ttttgttctg ggctatgtcc aatgggtctt gcagatgcaa catttgtata 1680

taa 1683

<210> 2

<211> 560

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 2

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

1 5 10 15

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

20 25 30

Thr Val Asp Thr Leu Thr Glu Asn Asn Val Pro Val Thr His Val Lys

35 40 45

Glu Leu Leu His Thr Glu His Asn Gly Met Leu Cys Ala Thr Ser Leu

50 55 60

Gly His Pro Leu Ile Leu Asp Thr Cys Thr Ile Glu Gly Leu Ile Tyr

65 70 75 80

Gly Asn Pro Ser Cys Asp Leu Leu Leu Gly Gly Arg Glu Trp Ser Tyr

85 90 95

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

100 105 110

Val Glu Asn Leu Glu Glu Leu Arg Ser Leu Phe Ser Ser Ala Arg Ser

115 120 125

Tyr Gln Arg Ile Gln Ile Phe Pro Asp Thr Ile Trp Asn Val Ser Tyr

130 135 140

Ser Gly Thr Ser Lys Ala Cys Ser Asp Ser Phe Tyr Arg Ser Met Arg

145 150 155 160

Trp Leu Thr Gln Lys Asn Asn Ala Tyr Pro Ile Gln Asp Ala Gln Tyr

165 170 175

Thr Asn Asn Gln Glu Lys Asn Ile Leu Phe Met Trp Gly Ile Asn His

180 185 190

Pro Pro Thr Asp Thr Thr Gln Thr Asn Leu Tyr Thr Arg Thr Asp Thr

195 200 205

Thr Thr Ser Val Ala Thr Glu Glu Met Asn Arg Ile Phe Lys Pro Leu

210 215 220

Ile Gly Pro Arg Pro Leu Val Asn Gly Leu Met Gly Arg Ile Asn Tyr

225 230 235 240

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

245 250 255

Gly Asn Leu Ile Ala Pro Trp Tyr Gly His Ile Leu Ser Gly Glu Ser

260 265 270

His Gly Arg Ile Leu Lys Thr Asp Leu Lys Arg Gly Ser Cys Thr Val

275 280 285

Gln Cys Gln Thr Glu Lys Gly Gly Leu Asn Thr Thr Leu Pro Phe Gln

290 295 300

Asn Val Ser Lys Tyr Ala Phe Gly Asn Cys Ser Lys Tyr Ile Gly Val

305 310 315 320

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

325 330 335

Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp

340 345 350

Ser Gly Leu Val Ala Gly Trp Tyr Gly Phe Gln His Ser Asn Asp Gln

355 360 365

Gly Val Gly Met Ala Ala Asp Arg Asp Ser Thr Gln Lys Ala Ile Asp

370 375 380

Lys Ile Thr Ser Lys Val Asn Asn Ile Val Asp Lys Met Asn Lys Gln

385 390 395 400

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

405 410 415

Met Ile Asn Asp Lys Val Asp Asp Gln Ile Gln Asp Ile Trp Ala Tyr

420 425 430

Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Gln Lys Thr Leu Asp Glu

435 440 445

His Asp Ala Asn Val Asn Asn Leu Tyr Asn Lys Val Lys Arg Ala Leu

450 455 460

Gly Ser Asn Ala Val Glu Asp Gly Arg Gly Cys Phe Glu Leu Tyr His

465 470 475 480

Lys Cys Asp Asp Gln Cys Met Glu Thr Ile Arg Asn Gly Thr Tyr Asn

485 490 495

Arg Arg Lys Tyr Gln Glu Glu Ser Lys Leu Glu Arg Gln Lys Ile Glu

500 505 510

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

515 520 525

Ser Thr Val Ala Ser Ser Leu Val Ile Ala Met Gly Phe Ala Ala Phe

530 535 540

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

545 550 555 560

<210> 3

<211> 1401

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 3

atgaatccaa atcagaagat aatagcaatt ggctctgttt ctctaaccat tgcgataata 60

tgttttctca tgcagattgc catcttaaca acaaccatga cactacattt caggcagaat 120

gaatgcagca atccatcgaa taatcaagtg gtgccatgtg aacctatcat aatagagaag 180

aacacagtgc atttgaatag tactaccata gagagggaaa tttgtcctaa agtggcagaa 240

tacaagaatt ggtcaaaacc acaatgtcaa attacagggt tcgctccttt ctcaaaggac 300

aactcaatta ggctttctgc agctggagat atctggttga caagagaacc ttatgtatcg 360

tgcagtcttg gcaaatgtta tcaatttgca cttgggcagg gaaccactct gaaaaacaag 420

cactcaaatg gcactacaca tgatagaatc cctcacagaa ctctcttaat gaatgagtta 480

ggtgttccat ttcatttggg aaccaaacaa gtgtgcatag catggtctag ttcaagctgc 540

catgatggga aagcatggtt acatatttgt gtgactgggg atgataaaaa tgctactgct 600

agtattattt atgatgggat gctggctgac agtattggtt catggtccaa aaacatccta 660

agaatccagg agtcagaatg cgtttgcatc aatggaactt gtgcagtagt aatgactgat 720

ggaagtgcgt caggaaaggc tgacactaga atattattca taagagaggg gaaaattata 780

agtattagcc cattgtcagg aagtgctcag catgtggagg aatgttcctg ttacccccga 840

tatcctgaag ttaggtgtgt ttgcagagac aactggaagg gctccaatag gcccgttcta 900

tatataaata tggcagatta tagtattgag tccagttatg tatgctcagg acttgttggc 960

gacacaccaa gggatgatga tagcaccagc agcagcaact gtagagaccc taataacgaa 1020

agaggggccc caggagtgaa agggtgggcc tttgacgatg ggattgacat ttggatggga 1080

cgaacaatca aaagtgattc acgctcaggt tatgagactt ttagggtcgt taatgcttgg 1140

accacggcta attccaagtc acaggtaaat aggcaagtca tagttgacag tgacagttgg 1200

tctgggtatt ctggtatctt ctctgttgaa ggcaagaact gcatcaacag gtgtttttat 1260

gtggagttga taagagggag accacaggag accagagtgt ggtggacttc aaacagcatc 1320

attgtattct gtggaacctc aggtacatat ggaacaggct catggcctga tggagcgaat 1380

atcgacttca tgcctatata a 1401

<210> 4

<211> 466

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 4

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

1 5 10 15

Ile Ala Ile Ile Cys Phe Leu Met Gln Ile Ala Ile Leu Thr Thr Thr

20 25 30

Met Thr Leu His Phe Arg Gln Asn Glu Cys Ser Asn Pro Ser Asn Asn

35 40 45

Gln Val Val Pro Cys Glu Pro Ile Ile Ile Glu Lys Asn Thr Val His

50 55 60

Leu Asn Ser Thr Thr Ile Glu Arg Glu Ile Cys Pro Lys Val Ala Glu

65 70 75 80

Tyr Lys Asn Trp Ser Lys Pro Gln Cys Gln Ile Thr Gly Phe Ala Pro

85 90 95

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

100 105 110

Leu Thr Arg Glu Pro Tyr Val Ser Cys Ser Leu Gly Lys Cys Tyr Gln

115 120 125

Phe Ala Leu Gly Gln Gly Thr Thr Leu Lys Asn Lys His Ser Asn Gly

130 135 140

Thr Thr His Asp Arg Ile Pro His Arg Thr Leu Leu Met Asn Glu Leu

145 150 155 160

Gly Val Pro Phe His Leu Gly Thr Lys Gln Val Cys Ile Ala Trp Ser

165 170 175

Ser Ser Ser Cys His Asp Gly Lys Ala Trp Leu His Ile Cys Val Thr

180 185 190

Gly Asp Asp Lys Asn Ala Thr Ala Ser Ile Ile Tyr Asp Gly Met Leu

195 200 205

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

210 215 220

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

225 230 235 240

Gly Ser Ala Ser Gly Lys Ala Asp Thr Arg Ile Leu Phe Ile Arg Glu

245 250 255

Gly Lys Ile Ile Ser Ile Ser Pro Leu Ser Gly Ser Ala Gln His Val

260 265 270

Glu Glu Cys Ser Cys Tyr Pro Arg Tyr Pro Glu Val Arg Cys Val Cys

275 280 285

Arg Asp Asn Trp Lys Gly Ser Asn Arg Pro Val Leu Tyr Ile Asn Met

290 295 300

Ala Asp Tyr Ser Ile Glu Ser Ser Tyr Val Cys Ser Gly Leu Val Gly

305 310 315 320

Asp Thr Pro Arg Asp Asp Asp Ser Thr Ser Ser Ser Asn Cys Arg Asp

325 330 335

Pro Asn Asn Glu Arg Gly Ala Pro Gly Val Lys Gly Trp Ala Phe Asp

340 345 350

Asp Gly Ile Asp Ile Trp Met Gly Arg Thr Ile Lys Ser Asp Ser Arg

355 360 365

Ser Gly Tyr Glu Thr Phe Arg Val Val Asn Ala Trp Thr Thr Ala Asn

370 375 380

Ser Lys Ser Gln Val Asn Arg Gln Val Ile Val Asp Ser Asp Ser Trp

385 390 395 400

Ser Gly Tyr Ser Gly Ile Phe Ser Val Glu Gly Lys Asn Cys Ile Asn

405 410 415

Arg Cys Phe Tyr Val Glu Leu Ile Arg Gly Arg Pro Gln Glu Thr Arg

420 425 430

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

435 440 445

Thr Tyr Gly Thr Gly Ser Trp Pro Asp Gly Ala Asn Ile Asp Phe Met

450 455 460

Pro Ile

465

<210> 5

<211> 759

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 5

atgagtcttc taaccgaggt cgaaacgtac gttctctcta tcattccatc aggccccaag 60

aaagccgaga tcgcgcagag acttgaggat gtttttgcag ggaagaacac agatctcgag 120

gctctcatgg agtggataaa gacaagacca atcctgtcac ctctgactaa ggggatttta 180

gggcgtgtgt tcacgctcac cgtgcccagt gagcgaggac tgcagcgtag acggtttgtc 240

caaaacgccc taaatgggaa tggagaccca aacaacatgg acaaggcagt taaattatac 300

aagaagctga agagggaaat gacatttcat ggagcaaagg aagttgcact cagttactca 360

actggtgcgc ttgccagctg catgggtctc atatacaaca ggatgggaac aggtaccgca 420

gaaggggctc ttggactagt atgtgccact tgtgagcaga ttgctgacgc acaacatcgg 480

tcccacaggc agatggcgac tactaccaac accctaatta ggcatgagaa tagaatggta 540

ctagccagca ctacggctaa ggctatggag cagatggctg gatcaagtga acaggcagcg 600

gaagccatgg aagtcgcaag tcaggctagg caaatggtgc aggctatgag aacagtcggg 660

actcacccga actccagtac aggtctaaaa gatgatcccc ttgaaaattt gcaggcttac 720

cagaaccgga tgggagtgca actgcagcgg ttcaagtga 759

<210> 6

<211> 252

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 6

Met Ser Leu Leu Thr Glu Val Glu Thr Tyr Val Leu Ser Ile Ile Pro

1 5 10 15

Ser Gly Pro Lys Lys Ala Glu Ile Ala Gln Arg Leu Glu Asp Val Phe

20 25 30

Ala Gly Lys Asn Thr Asp Leu Glu Ala Leu Met Glu Trp Ile Lys Thr

35 40 45

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

50 55 60

Thr Leu Thr Val Pro Ser Glu Arg Gly Leu Gln Arg Arg Arg Phe Val

65 70 75 80

Gln Asn Ala Leu Asn Gly Asn Gly Asp Pro Asn Asn Met Asp Lys Ala

85 90 95

Val Lys Leu Tyr Lys Lys Leu Lys Arg Glu Met Thr Phe His Gly Ala

100 105 110

Lys Glu Val Ala Leu Ser Tyr Ser Thr Gly Ala Leu Ala Ser Cys Met

115 120 125

Gly Leu Ile Tyr Asn Arg Met Gly Thr Gly Thr Ala Glu Gly Ala Leu

130 135 140

Gly Leu Val Cys Ala Thr Cys Glu Gln Ile Ala Asp Ala Gln His Arg

145 150 155 160

Ser His Arg Gln Met Ala Thr Thr Thr Asn Thr Leu Ile Arg His Glu

165 170 175

Asn Arg Met Val Leu Ala Ser Thr Thr Ala Lys Ala Met Glu Gln Met

180 185 190

Ala Gly Ser Ser Glu Gln Ala Ala Glu Ala Met Glu Val Ala Ser Gln

195 200 205

Ala Arg Gln Met Val Gln Ala Met Arg Thr Val Gly Thr His Pro Asn

210 215 220

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

225 230 235 240

Gln Asn Arg Met Gly Val Gln Leu Gln Arg Phe Lys

245 250

<210> 7

<211> 1614

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 7

cccgggatgg acaaaatttg catcgggtac caaagtacaa actccactga gactgttgac 60

actttaacgg agaataatgt accggtgacc cacgtaaaag agcttctgca tacggagcat 120

aacggaatgc tgtgtgccac gagtctgggg caccccttaa tcttggacac ttgtacgatc 180

gaaggtttga tctatggaaa cccgagttgt gatctgcttc tggggggccg cgaatggtcg 240

tatattgtag aacgcccaag tgccgtgaac ggactttgct atccggggaa tgtggagaac 300

ctggaagaat tacgttctct gtttagctca gcgcgcagtt atcagcgcat tcagatcttc 360

ccggacacaa tttggaacgt gagttattcc ggtacgtcaa aggcgtgctc ggacagtttt 420

taccgctcaa tgcgctggtt aacccaaaag aacaacgcat atcccattca ggacgcgcag 480

tacactaaca accaagagaa gaatattctg ttcatgtggg gcattaatca tccgccgaca 540

gatacaactc agactaactt gtatacccgt actgatacga cgacatccgt tgccacagaa 600

gaaatcaatc gtatcttcaa accactgatt gggcctcgtc ctttagtcaa tgggttgatg 660

ggtcgtatcg attactactg gagtgtgctg aaaccgggcc aaacgttacg cattcgctcc 720

gacggcaatc ttattgcccc atggtatggc catattcttt ccggggagag ccacggccgc 780

attcttaaaa ccgaccttaa acgtgggtcc tgtacagtac aatgccagac agagaaaggc 840

gggttgaata ccaccctgcc gtttcagaat gtgtcgaagt acgcattcgg aaattgctcg 900

aaatacattg gcattaaaag tctgaagttg gctgttggac ttcgcaatgt gccgagccgt 960

agtaagcgtg gcctttttgg ggctattgcc ggttttatcg aggggggctg gagtggactt 1020

gtcgcgggat ggtatggatt ccaacattcg aatgaccaag gtgtcggaat ggctgcggac 1080

cgcgacagca cgcagaaggc tatcgataaa atcacttcaa aagtgaacaa cattgtggat 1140

aagatgaata agcaatacga aattattgat catgagtttt ctgaggtgga aacgcgctta 1200

aatatgatca acaacaagat tgatgatcag attcaagaca tttgggcgta caacgccgaa 1260

ttattggttc ttctggaaaa ccaaaagacc ttagacgaac acgatgcaaa cgtcaataat 1320

ctttataata aagtaaaacg tgccttgggt agcaatgctg tagaagatgg aaaggggtgt 1380

ttcgaattgt atcacaaatg cgatgaccaa tgtatggaga cgattcgcaa tggtacatac 1440

aatcgtcgca aataccaaga agaatctaag cttgagcgtc aaaaaattga aggcgtgaag 1500

ctggaaagtg aaggaactta taaaattttg acgatttact cgaccgtagc gtcatcactg 1560

gtgattgcaa tgggatttgc tgcttttctg ttctgggcga tgtcttaact cgag 1614

<210> 8

<211> 533

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 8

Met Asp Lys Ile Cys Ile Gly Tyr Gln Ser Thr Asn Ser Thr Glu Thr

1 5 10 15

Val Asp Thr Leu Thr Glu Asn Asn Val Pro Val Thr His Val Lys Glu

20 25 30

Leu Leu His Thr Glu His Asn Gly Met Leu Cys Ala Thr Ser Leu Gly

35 40 45

His Pro Leu Ile Leu Asp Thr Cys Thr Ile Glu Gly Leu Ile Tyr Gly

50 55 60

Asn Pro Ser Cys Asp Leu Leu Leu Gly Gly Arg Glu Trp Ser Tyr Ile

65 70 75 80

Val Glu Arg Pro Ser Ala Val Asn Gly Leu Cys Tyr Pro Gly Asn Val

85 90 95

Glu Asn Leu Glu Glu Leu Arg Ser Leu Phe Ser Ser Ala Arg Ser Tyr

100 105 110

Gln Arg Ile Gln Ile Phe Pro Asp Thr Ile Trp Asn Val Ser Tyr Ser

115 120 125

Gly Thr Ser Lys Ala Cys Ser Asp Ser Phe Tyr Arg Ser Met Arg Trp

130 135 140

Leu Thr Gln Lys Asn Asn Ala Tyr Pro Ile Gln Asp Ala Gln Tyr Thr

145 150 155 160

Asn Asn Gln Glu Lys Asn Ile Leu Phe Met Trp Gly Ile Asn His Pro

165 170 175

Pro Thr Asp Thr Thr Gln Thr Asn Leu Tyr Thr Arg Thr Asp Thr Thr

180 185 190

Thr Ser Val Ala Thr Glu Glu Ile Asn Arg Ile Phe Lys Pro Leu Ile

195 200 205

Gly Pro Arg Pro Leu Val Asn Gly Leu Met Gly Arg Ile Asp Tyr Tyr

210 215 220

Trp Ser Val Leu Lys Pro Gly Gln Thr Leu Arg Ile Arg Ser Asp Gly

225 230 235 240

Asn Leu Ile Ala Pro Trp Tyr Gly His Ile Leu Ser Gly Glu Ser His

245 250 255

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

260 265 270

Cys Gln Thr Glu Lys Gly Gly Leu Asn Thr Thr Leu Pro Phe Gln Asn

275 280 285

Val Ser Lys Tyr Ala Phe Gly Asn Cys Ser Lys Tyr Ile Gly Ile Lys

290 295 300

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

305 310 315 320

Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Ser

325 330 335

Gly Leu Val Ala Gly Trp Tyr Gly Phe Gln His Ser Asn Asp Gln Gly

340 345 350

Val Gly Met Ala Ala Asp Arg Asp Ser Thr Gln Lys Ala Ile Asp Lys

355 360 365

Ile Thr Ser Lys Val Asn Asn Ile Val Asp Lys Met Asn Lys Gln Tyr

370 375 380

Glu Ile Ile Asp His Glu Phe Ser Glu Val Glu Thr Arg Leu Asn Met

385 390 395 400

Ile Asn Asn Lys Ile Asp Asp Gln Ile Gln Asp Ile Trp Ala Tyr Asn

405 410 415

Ala Glu Leu Leu Val Leu Leu Glu Asn Gln Lys Thr Leu Asp Glu His

420 425 430

Asp Ala Asn Val Asn Asn Leu Tyr Asn Lys Val Lys Arg Ala Leu Gly

435 440 445

Ser Asn Ala Val Glu Asp Gly Lys Gly Cys Phe Glu Leu Tyr His Lys

450 455 460

Cys Asp Asp Gln Cys Met Glu Thr Ile Arg Asn Gly Thr Tyr Asn Arg

465 470 475 480

Arg Lys Tyr Gln Glu Glu Ser Lys Leu Glu Arg Gln Lys Ile Glu Gly

485 490 495

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

500 505 510

Thr Val Ala Ser Ser Leu Val Ile Ala Met Gly Phe Ala Ala Phe Leu

515 520 525

Phe Trp Ala Met Ser

530

<210> 9

<211> 1455

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 9

ggatccatga caactacaat gacgcttcac tttcgccaga acgagtgtag caatccatca 60

aacaaccaag ttgtgccttg tgaaccaatt attattgaga agaacacagt tcacttgaac 120

agtactacta tcgaaagaga gatctgccct aaggttgcgg agtacaagaa ctggtccaag 180

ccgcaatgcc aaataaccgg tttcgcgcca ttcagcaagg acaattcgat ccgcctttct 240

gcggcaggcg atatatggtt gacgcgtgaa ccatacgtat cttgctccct gggaaagtgt 300

tatcagttcg cgcttggtca gggtacaact ctgaaaaaca aacactctaa cggcacaacc 360

cacgacagga taccgcatcg cacactgttg atgaacgagc ttggagtacc ctttcatctt 420

ggcactaagc aagtatgtat agcttggtcg tcctcatcct gccacgacgg taaggcgtgg 480

cttcatatct gcgttacggg cgacgataag aacgccaccg cgagtatcat atacgacggt 540

atgctggccg actcaatcgg tagttggtct aaaaatatac tcaggataca ggagtccgag 600

tgcgtctgca taaatggcac atgcgcagtg gttatgactg atggcagtgc cagtggcaaa 660

gcagatacgc gcatactgtt tattagggag ggtaaaatca tcagtatttc gccattgtcc 720

ggctcggctc agcatgtaga agaatgctcc tgctacccga gataccccga agtaaggtgt 780

gtatgtcgcg ataactggaa aggttccaat aggccagtcc tctatataaa tatggcagat 840

tatagtattg aatcaagtta tgtttgttct ggacttgtag gagacacacc aagagacgat 900

gacagcacgt ccagctcgaa ctgcagagac ccaaataacg aacgtggtgc ccccggagtt 960

aagggttggg cattcgatga tggcattgac atctggatgg gaaggaccat aaagtcagat 1020

agtcgctcgg gttatgaaac atttagggtg gtcaacgcat ggactacggc aaactcgaag 1080

tcccaagtaa atcgccaggt cattgtggat tccgatagtt ggagtggata ctctggaatc 1140

ttctcggttg agggaaagaa ttgtattaat agatgttttt acgttgagct cattaggggc 1200

cgccctcaag aaacccgcgt ctggtggact agtaactcta taatcgtgtt ttgtggaacc 1260

agtggcacgt acggtactgg ctcttggcca gacggagcca acatagactt catgcctatc 1320

taagatcata atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc 1380

acacctcccc ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat 1440

tgctggagtg aattc 1455

<210> 10

<211> 438

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 10

Met Thr Thr Thr Met Thr Leu His Phe Arg Gln Asn Glu Cys Ser Asn

1 5 10 15

Pro Ser Asn Asn Gln Val Val Pro Cys Glu Pro Ile Ile Ile Glu Lys

20 25 30

Asn Thr Val His Leu Asn Ser Thr Thr Ile Glu Arg Glu Ile Cys Pro

35 40 45

Lys Val Ala Glu Tyr Lys Asn Trp Ser Lys Pro Gln Cys Gln Ile Thr

50 55 60

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

65 70 75 80

Gly Asp Ile Trp Leu Thr Arg Glu Pro Tyr Val Ser Cys Ser Leu Gly

85 90 95

Lys Cys Tyr Gln Phe Ala Leu Gly Gln Gly Thr Thr Leu Lys Asn Lys

100 105 110

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

115 120 125

Met Asn Glu Leu Gly Val Pro Phe His Leu Gly Thr Lys Gln Val Cys

130 135 140

Ile Ala Trp Ser Ser Ser Ser Cys His Asp Gly Lys Ala Trp Leu His

145 150 155 160

Ile Cys Val Thr Gly Asp Asp Lys Asn Ala Thr Ala Ser Ile Ile Tyr

165 170 175

Asp Gly Met Leu Ala Asp Ser Ile Gly Ser Trp Ser Lys Asn Ile Leu

180 185 190

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

195 200 205

Val Met Thr Asp Gly Ser Ala Ser Gly Lys Ala Asp Thr Arg Ile Leu

210 215 220

Phe Ile Arg Glu Gly Lys Ile Ile Ser Ile Ser Pro Leu Ser Gly Ser

225 230 235 240

Ala Gln His Val Glu Glu Cys Ser Cys Tyr Pro Arg Tyr Pro Glu Val

245 250 255

Arg Cys Val Cys Arg Asp Asn Trp Lys Gly Ser Asn Arg Pro Val Leu

260 265 270

Tyr Ile Asn Met Ala Asp Tyr Ser Ile Glu Ser Ser Tyr Val Cys Ser

275 280 285

Gly Leu Val Gly Asp Thr Pro Arg Asp Asp Asp Ser Thr Ser Ser Ser

290 295 300

Asn Cys Arg Asp Pro Asn Asn Glu Arg Gly Ala Pro Gly Val Lys Gly

305 310 315 320

Trp Ala Phe Asp Asp Gly Ile Asp Ile Trp Met Gly Arg Thr Ile Lys

325 330 335

Ser Asp Ser Arg Ser Gly Tyr Glu Thr Phe Arg Val Val Asn Ala Trp

340 345 350

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

355 360 365

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

370 375 380

Asn Cys Ile Asn Arg Cys Phe Tyr Val Glu Leu Ile Arg Gly Arg Pro

385 390 395 400

Gln Glu Thr Arg Val Trp Trp Thr Ser Asn Ser Ile Ile Val Phe Cys

405 410 415

Gly Thr Ser Gly Thr Tyr Gly Thr Gly Ser Trp Pro Asp Gly Ala Asn

420 425 430

Ile Asp Phe Met Pro Ile

435

<210> 11

<211> 890

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 11

gcggccgcac tcccgaatac taataaatta tggagatcat taagatgatc accatcagtc 60

agatcaacaa gtatttcact gtcttcgtga ctgttctcta gtaaaagaat ctttgaattt 120

ttaggatgtc gctgcttact gaagtagaga cttacgtgtt gtccatcata cctagcggac 180

caaagaaggc cgaaattgcg cagcgcctcg aagatgtatt cgcgggaaag aacacagact 240

tggaggccct catggagtgg attaaaacca gaccgattct ctctcccctg accaagggca 300

tactgggccg cgtctttaca ctgacggtgc cgtcagagcg tggtctgcaa aggcgcagat 360

ttgtccagaa cgcgcttaac ggaaatggcg atcctaataa catggataaa gcggtaaagt 420

tgtataaaaa actgaaacgc gagatgactt ttcacggagc aaaagaagta gccttgagtt 480

actcgacagg cgctcttgca tcttgtatgg gtcttatata taaccgtatg ggcaccggaa 540

ctgctgaggg tgcgttgggc ctcgtgtgcg caacttgcga gcagatagct gacgcgcagc 600

accgttctca tcgtcaaatg gcgactacga cgaacacttt gattcgccac gagaatagaa 660

tggtgctggc aagtacaact gcaaaagcca tggaacaaat ggcaggttca tcagaacagg 720

ctgcagaggc catggaggtg gcatcgcaag ccaggcaaat ggtacaggcg atgcgtactg 780

tcggaacgca cccgaactcg tcaaccggtt tgaaagacga cccccttgag aatctgcaag 840

catatcaaaa ccgtatggga gtccaactcc agaggtttaa gtgatctaga 890

Claims (10)

1. a kind of recombinant baculovirus, which is characterized in that include coding HA, NA and M1 albumen in the recombinant baculovirus Nucleotide fragments；HA, NA and M1 albumen, amino acid sequence are respectively SEQ ID NO:8；SEQ ID NO:10 and SEQ ID NO:6。

2. recombinant baculovirus as described in claim 1, which is characterized in that the nucleosides of coding HA, NA and M1 albumen Acid fragment, sequence are respectively SEQ ID NO:7；SEQ ID NO:9；SEQ ID NO:11.

3. recombinant baculovirus of any of claims 1 or 2 is prepared in insect cell in the virus-like particle of avian influenza virus Application.

4. application as claimed in claim 3, which is characterized in that the insect cell is Insect cells Sf9.

5. a kind of method for the virus-like particle for preparing avian influenza virus, which is characterized in that the method is wanted using right After recombinate shape virus infection insect cell described in asking 1 or 2, the virus-like particle of standby avian influenza virus is collected in culture.

6. method as claimed in claim 5, which is characterized in that the insect cell is Insect cells Sf9.

7. a kind of virus-like particle of avian influenza virus, which is characterized in that the virus-like particle be using claim 5 or The preparation of method described in 6.

8. virus-like particle as claimed in claim 7 is preparing the application in vaccine.

9. application as claimed in claim 8, which is characterized in that the vaccine is subunit vaccine.

10. a kind of avian influenza virus subunit vaccine, which is characterized in that the vaccine includes antigen and vaccine adjuvant, Middle antigen is virus-like particle as claimed in claim 7.