CN109266666A

CN109266666A - A kind of Hybrid virus like particles of duck tembusu virus E protein and its application

Info

Publication number: CN109266666A
Application number: CN201810997202.6A
Authority: CN
Inventors: 李林林; 孙敏华; 董嘉文; 张俊勤; 张春红
Original assignee: Institute of Animal Health of Guangdong Academy of Agricultural Sciences
Current assignee: Institute of Animal Health of Guangdong Academy of Agricultural Sciences
Priority date: 2018-08-29
Filing date: 2018-08-29
Publication date: 2019-01-25
Anticipated expiration: 2038-08-29
Also published as: CN109266666B

Abstract

The invention discloses a kind of Hybrid virus like particles of duck tembusu virus E protein and its applications.Virus-like particle fusion as shown in SEQ ID NO.15 and avian influenza virus H3N2 M1 gene co-expressing obtain.The present invention develops to obtain the Hybrid virus like particles of expression duck tembusu virus (DTMUV) E protein using baculoviral/insect cell expression system, with significant immune effect, theory and practice basis has been established for exploitation duck tembusu virus new generation vaccine and polyvaccine.Meanwhile the virus-like particle that the present invention is prepared does not contain viral nucleic acid, because that duck tembusu virus will not be caused to infect, the risk that vaccine uses can be farthest reduced, have a good application prospect without replication capacity.

Description

A kind of Hybrid virus like particles of duck tembusu virus E protein and its application

Technical field

The present invention relates to duck tembusu virus technical fields, more particularly, to a kind of the embedding of duck tembusu virus E protein Close virus-like particle and its application.

Background technique

Duck tembusu virus disease is newly to break out and pandemic communicable disease in China in recent years, can cause infected duck Group's feed intake and egg production dramatic decrease, and other poultry can be infected, huge economic losses are caused to aviculture.Currently, the disease Prevention and control have a commercialization inactivated vaccine, but still there is duck tembusu virus disease to break out often in producing, also in the epidemic disease of development phase The problems such as it is not high that seedling has that there are virus titers, and antigenic content is insufficient.Therefore, urgently new generation vaccine is ground for effective prevention and control of the disease Hair.

Virus-like particle (VLPs) refers to the hollow bead containing one or several virulent structural proteins.Currently, one The VLPs of a little viruses has been used as vaccine to be successfully applied to clinic, such as: human papilloma virus (HPV) tetravalence VLPs vaccine, B-mode liver Scorching virus (HBV) etc..With containing the chimeric VLPs from two kinds of viral structural proteins to VLPs increasingly in-depth study Become VLPs and applies a upper extremely important and promising direction.It there is no duck tembusu virus disease chimeric at present The research of VLPs vaccine is reported.

The influenza virus of a variety of hypotypes, including H5N1, H3N2, H1N1, H9N2 etc. have been used for the packaging of VL Ps, and take It must succeed.It recent studies have shown that single HA albumen and M1 albumen can successfully pack out influenza VLPs.It is sick with A type influenza is belonged to The transmembrane region (TM) of other all 17 kinds of subtype influenza virus HA albumen of poison compares, H3 subtype influenza virus HA albumen across Film area has unique structure, facilitates HA albumen and forms intermolecular disulfide bond, improves the stability of HA albumen and exempting from for albumen Epidemic focus.This research is packed out using influenza virus H3N2VLPs as skeleton, and surface display duck tembusu virus principal immune is protected The chimeric VLPs of albumen E is protected, and purified chimeric VLPs is subjected to animal immune to evaluate its immune protective effect.

Summary of the invention

The purpose of the invention is to overcome the deficiencies of the prior art and provide a kind of being fitted into for duck tembusu virus E protein The preparation and application of virus-like particle.

The first purpose of the invention is to provide a kind of fusions.

A second object of the present invention is to provide a kind of fusion proteins.

Third object of the present invention is to provide a kind of carriers.

Fourth object of the present invention is to provide a kind of carrier combination.

Fifth object of the present invention is to provide a kind of baculovirals.

Sixth object of the present invention is to provide a kind of Hybrid virus like particles for expressing duck tembusu virus E protein.

7th purpose of the invention is to provide fusion described above, fusion protein described above, described above carries The Hybrid virus like particles of body, carrier combination described above or baculoviral described above in preparation duck tembusu virus E protein In application.

8th purpose of the invention is to provide fusion described above, fusion protein described above, described above carries Body, carrier combination described above, baculoviral described above or Hybrid virus like particles described above are in preparation duck Tan Busu disease Application in malicious vaccine.

9th purpose of the invention is to provide a kind of duck tembusu virus vaccine.

To achieve the goals above, the present invention is achieved by the following technical programs:

The present invention is prepared for a kind of Hybrid virus like particles for expressing duck tembusu virus E protein, smooth with the duck that it is prepared Cloth Soviet Union viral vaccine has good immune protective effect, therefore claimed the following contents:

A kind of fusion, the nucleotide sequence of the fusion is as shown in SEQ ID NO.15.

A kind of fusion protein, the amino acid sequence of the fusion protein is as shown in SEQ ID NO.16.

A kind of recombinant vector, the carrier contain the M1 gene of fusion and avian influenza virus H3N2 described above, and It is located at two open reading frame of carrier.

One recombinant vector combination, including recombinant vector 1 and recombinant vector 2, recombinant vector 1 contain fusion base described above Cause, M1 gene of the recombinant vector 2 containing avian influenza virus H3N2.

Preferably, the carrier is pFastBacDual.

The M1 gene of avian influenza virus H3N2, nucleotide sequence is as shown in SEQ ID NO.17；The ammonia of the albumen of coding Base acid sequence is as shown in SEQ ID NO.18.

The baculoviral of one recombination, the baculoviral contain fusion and avian influenza virus H3N2's described above M1 gene.

A kind of Hybrid virus like particles for expressing duck tembusu virus E protein, the virus is by using the above Fusion, fusion protein described above, carrier described above, carrier combination described above or baculoviral system described above Standby.

Preferably, the Hybrid virus like particles are isolated and purified from the culture solution of above-mentioned cell.

A kind of preparation method of Hybrid virus like particles that expressing duck tembusu virus E protein, comprising the following steps:

S1. nucleotide sequence fusion as shown in SEQ ID NO.15 and influenza virus H3N2M1 gene are obtained；

S2. above-mentioned two gene is separately connected two open reading frame into expressing viral transfer vector, converts large intestine Bacillus competent cell, screening obtain recombinant transfer vector, convert DH10Bac competent cell, and screening obtains recombination shuttle matter Grain, and transfect cell；

S3. the cell being transfected is cultivated, cell conditioned medium is collected, obtain recombinant virus and is passed on；

S4. recombinant virus infection cell is obtained, simultaneously purified virus sample particle is obtained.

Preferably, in step S2, expressing viral transfer vector is baculovirus expression transfer vector.

It is highly preferred that expressing viral transfer vector is pFastBacDual baculovirus expression transfer vector in step S2.

Preferably, in step S2, transfection insect cell.

It is highly preferred that in step S2, transfection insect cell Sf9.

Preferably, in step S3, the time for the cell being transfected is cultivated are as follows: when typical cells lesion occurs in cell or 25 ~30 DEG C, cultivate 70~75h.

Cell becomes larger showing themselves in that when typical cells lesion occurs in cell, is rounded, and nucleus increases, and refractivity intracellular is strong Substance significantly increase, cell slow growth even stops, and more suspension cell occurs in culture medium.

Preferably, in step S3, the time for cultivating the cell being transfected is 27 DEG C, cultivates 72h.

Preferably, in step S4, purified virus sample particle method particularly includes: take cell supernatant, 8,000~12, 000rpm is centrifuged 8~12min, takes supernatant；35,000~45,000rpm are centrifuged 100~140min, take precipitating with PBS (pH7.4) Buffer is resuspended；Re-suspension liquid is centrifuged using 20%~60% saccharose gradient liquid for having formed gradient, 25,000~30, 000rpm swing bucket rotor is centrifuged 100~140min；Isolated viral sample granule strip band, after PBS buffer solution is resuspended, 35,000~45, 000rpm 100~140min of ultracentrifugation abandons supernatant；Appropriate PBS is resuspended precipitating and saves.

Most preferably, the preparation method of the Hybrid virus like particles of duck tembusu virus E protein, including following step are expressed It is rapid:

S2. above-mentioned two gene is separately connected into pFastBacDual baculovirus expression transfer vector, conversion DH10Bac competent cell obtains recombinant baculovirus shuttle vector through screening, and recombinant baculovirus shuttle vector transfects insect Cell Sf9；

S3.27 DEG C is cultivated the cell 72h being transfected, and collects cell conditioned medium, is obtained recombinant virus and is passed on；

S4. recombinant virus is obtained, 5 infection cells are equal to MOI, takes cell supernatant, 10,000rpm centrifugation 10min take Supernatant；40,000rpm centrifugation 2h, take precipitating to be resuspended with PBS buffer solution；Re-suspension liquid is utilized and has formed the 20%~60% of gradient Saccharose gradient liquid, 27,000rpm swing bucket rotors be centrifuged 2h；Isolated viral sample granule strip band, after PBS buffer solution is resuspended, 40, 000rpm ultracentrifugation 2h abandons supernatant；Appropriate PBS is resuspended precipitating and saves.

Fusion described above, fusion protein described above, carrier described above, carrier combination described above or more Application of the baculoviral in the Hybrid virus like particles of preparation duck tembusu virus E gene.

Fusion described above, fusion protein described above, carrier described above, carrier combination described above, more than The application of the baculoviral or Hybrid virus like particles described above in preparation duck tembusu virus vaccine.

A kind of duck tembusu virus vaccine, the embedded virus sample including expression duck tembusu virus E gene described above Grain.

A kind of preparation method of above-mentioned duck tembusu virus vaccine, comprising the following steps:

S1. water phase is configured with the Hybrid virus like particles and twen-80 of expression duck tembusu virus E protein described above；

S2. mineral oil, Si Ben -80 and aluminum stearate configure oily phase, sterilizing；

S2. water phase and oil mutually according to 1:1.5~1:2 ratio prepare and it is fully emulsified.

Preferably, the volume ratio of the Hybrid virus like particles and twen-80 of E protein is 90~95:5~10.

It is highly preferred that the Hybrid virus like particles of E protein and the volume ratio of twen-80 are 93:7.

Preferably, the volume ratio of mineral oil, Si Ben -80 and aluminum stearate is 91~95:4~6:1~3.

It is highly preferred that the volume ratio of mineral oil, Si Ben -80 and aluminum stearate is 93:5:2.

Preferably, the mineral oil is white oil.

Duck tembusu virus vaccine prepared by the above method, also should be within protection scope of the present invention.

Compared with prior art, the invention has the following beneficial effects:

The present invention utilizes the chimeric disease of baculoviral/insect cell expression system development expression duck tembusu virus E protein Malicious sample particle, and find it with good immune effect.To develop duck tembusu virus new generation vaccine and multivalence epidemic disease in next step Seedling establishes theory and practice basis.Meanwhile the virus-like particle being prepared of the invention be free of virulent nucleic acid, because without With replication capacity, duck tembusu virus will not be caused to infect, can farthest reduce the risk that vaccine uses.

Detailed description of the invention

Fig. 1 is the building of fusion protein sp/E/HA2.

Fig. 2 is the PCR amplification of E segment.

Fig. 3 is the PCR amplification of H3N2 HA gene HA2 segment.

Fig. 4 is the PCR amplification of H3N2 HA gene signal peptide fragment.

Fig. 5 is the PCR amplification for merging segment sp/E/HA2.

Fig. 6 is the PCR amplification of H3N2 M1 gene.

Fig. 7 is the digestion qualification result of pFastBacDual-sp/E/HA2-M1 plasmid；M:DNA Marker DL5,000； 1: plasmid single endonuclease digestion；2: being directed to sp/E/HA2 segment double digestion；3: being directed to M1 segment double digestion.

Fig. 8 is restructuring rod granule rBacmid-sp/E/HA2-M1 bacterium solution PCR identification；M:DNA Marker DL5,000；1,2: Recombinant plasmid rBacmid-sp/E/HA2-M1 bacterium solution.

Fig. 9 is that recombinant baculovirus expression albumen western-blot detects (H3N2 primary antibody)；1:r-sp/E/HA2-M1 sense Contaminate Sf9 product of cell lysis；2:r-sp/E/HA2-M1 infects Sf9 supernatant；3: infecting the Sf9 cell pair of wild-type baculovirus According to.

Figure 10 is that recombinant baculovirus expression albumen western-blot detects (DTMUV primary antibody).

Figure 11 is r-sp/E/HA2-M1 plaque purification figure.

Figure 12 is the expression and positioning of indirect immunofluorescene assay recombinant protein sp/E/HA2 and M1.

Figure 13 is that SDS-PAGE electrophoresis estimates E protein content.

The Electronic Speculum that Figure 14 is sp/E/HA2-M1VLP observes result.

Figure 15 is antibody level variation in different immune group mice serums.

Figure 16 is the variation of different immune group duckling Serum Antibody levels.

Specific embodiment

The present invention is made with specific embodiment with reference to the accompanying drawings of the specification and further being elaborated, the embodiment It is served only for explaining the present invention, be not intended to limit the scope of the present invention.Test method as used in the following examples is such as without spy Different explanation, is conventional method；Used material, reagent etc., unless otherwise specified, for the reagent commercially obtained And material.

1 construction of fusion protein sp/E/HA2 gene of embodiment and pFastBacDual-sp/E/HA2-M1 recombinant plasmid

One, fusion protein sp/E/HA2 building and PCR amplification

Fusion protein successively includes signal peptide (H3N2HA-sp), DTMUV E gene and the H3N2- of influenza virus H3N2-HA Entire HA2 segment (transmembrane region containing influenza virus H3N2HA gene and intracellular region (TM+CT), the H3N2HA- of HA gene HA2), antigen-4 fusion protein gene building structure is as shown in Figure 1.

1, experimental implementation

Design 4 pairs of primers of synthesis, (contain DTMUV E base with this experimental construction and the plasmid pFastBacDual-E of preservation Cause) carry out target gene fragment DTMUV E gene PCR amplification；(contain influenza virus with plasmid H3N2HA-T vector H3N2HA gene is controlled by Zhongshan University Life Science College harmful organism and is given with utilization of resources National Key Laboratory) be Template carries out the PCR amplification of target gene fragment H3N2HA-sp and H3N2HA-HA2.With expand obtain H3N2HA-sp, E, H3N2-HA-HA2 is that template carries out overlapping PCR amplification acquisition fusion segment H3N2HAsp-E-H3N2-HA- HA2 is used to expand the nucleotide sequence of the primer sequence of H3N2HA-sp as shown in NO.7~8 SEQ ID；

For expanding nucleotide sequence such as NO.9~10 SEQ ID of the primer sequence of DTMUV E (except signal peptide sequence) It is shown；

For expand H3N2HA-HA2 primer sequence nucleotide sequence as shown in NO.11~12 SEQ ID.

Template is blended together as with the above amplified production and carries out overlapping PCR amplification, obtains fusion protein Genetic fragment, the nucleotide sequence of primer sequence is as shown in NO.13~14 SEQ ID.

2, experimental result

The clip size and be expected consistent (Fig. 2 to Fig. 4) that PCR amplification obtains.

Obtained H3N2HA-sp is expanded, 180bp nucleotide sequence is as shown in SEQ ID NO.1；60 amino acid are encoded, Amino acid sequence is as shown in SEQ ID NO.2.Expand obtained DTMUV E (removing signal peptide and transmembrane region), 1047bp nucleotide Sequence is as shown in SEQ ID NO.3；349 amino acid sequences are encoded as shown in SEQ ID NO.4.

Obtained H3N2HA-HA2 is expanded, 666bp nucleotide sequence is as shown in SEQ ID NO.5；Encode 221 amino acid Sequence is as shown in SEQ ID NO.6.

Overlapping PCR is obtained target fragment sp/E/HA2 (Fig. 5): 1893bp nucleotide sequence such as SEQ ID Shown in NO.15；631 amino acid are encoded, amino acid sequence is as shown in SEQ ID NO.16.

Two, target fragment is connected into the digestion qualification result of positive plasmid after pFastBacDual carrier

1, experimental implementation

The sp/E/HA2 segment of acquisition is inserted into the baculovirus expression transfer vector of the gene containing H3N2M1 PFastBacDual-M1 is (by the control of Zhongshan University Life Science College harmful organism and utilization of resources National Key Laboratory favour Give) another expression cassette in.Building pFastBacDual-sp/E/HA2-M1 recombinant plasmid and carry out bacterium solution PCR verifying and Digestion identification.Wherein, the nucleotide sequence of H3N2M1 gene is as shown in SEQ ID NO.17；The amino acid sequence of the albumen of coding As shown in SEQ ID NO.18.

2, experimental result

H3N2M1 genetic fragment is as shown in Figure 6 in pFastBacDual-M1.

By being accredited as positive clone, plasmid is extracted after expanding culture, the positive is accredited as with double digestion, send Ying Jungong Department's sequencing, it is right-on through being sequenced, -80 DEG C are stored in, pFastBacDual-sp/E/HA2-M1 is named as.Digestion identification knot Fruit such as Fig. 7.

2 recombinant baculovirus shuttle plasmid rBacmid-sp/E/HA2-M1 transfection insect cell Sf9 of embodiment

1, experimental implementation

Identified correct recombinant plasmid pFastBacDual-sp/E/HA2-M1 is converted into DH10Bac^TMCompetent cell, It is screened through blue hickie and triple antibiotic (gentamicin, kanamycins and tetracycline) drug resistance selects, trained in 37 DEG C of incubators After supporting 48h, picking hickie is simultaneously inoculated into new containing there are three types of in the LB plate of antibiotic, culture 48h picking again with hatched manner White single colonie is inoculated with containing there are three types of in the fresh liquid LB culture medium of antibiotic, after 6h, takes out 1 μ l bacterium solution as template Bacterium solution PCR identification is carried out, screening obtains the positive restructuring containing recombinant plasmid pFastBacDual-sp/E/HA2-M1 DH10Bac, and extract recombinant plasmid.Choosing identification, correctly recombination rBacmid-sp/E/HA2-M1 rod granule transfects Sf9 cell. When typical cells lesion occurs in cell (or after infection 72h), virus liquid and cell are collected.The specific manifestation of cytopathy are as follows: Cell becomes larger, and is rounded, and nucleus increases, and the strong substance of refractivity intracellular significantly increases, and cell slow growth even stops, culture Occurs more suspension cell in base.Gained supernatant contains recombinant virus, and as P1 for virus stock solution used, -80 DEG C are saved backup.Detection Whether there is or not destination protein expression for gained precipitating.

2, experimental result

The bacterium solution PCR qualification result of restructuring rod granule rBacmid-sp/E/HA2-M1 is as shown in figure 8, obtained containing purposeful The positive bacterium colony of gene.The expression of destination protein is detected after positive restructuring rod granule transfection Sf9 cell.

The detection of albumen after 3 recombinate shape virus infection Sf9 cell of embodiment

1, experimental implementation

(1) 72h collects cell conditioned medium, as first generation recombinant baculovirus virus (label after restructuring rod granule transfection cell For P1), it is named as r-sp/E/HA2-M1.Continue the Sf9 cell of infection logarithmic growth phase with P1 generation poison, is collected on cell after 72h Clearly, it is labeled as P2.Infected cell is collected, PBS is washed three times, protease inhibitors is added, is put in -80 DEG C of multigelations three It is secondary, it is crushed with sonicator, 12,000 × g, 4 DEG C of centrifugation 1min.

(2) it takes supernatant sample-loading buffer is added to boil and carries out SDS-PAGE electrophoresis after sample, after electrophoresis, polypropylene segment amide is solidifying Glue carries out normal dyeing with coomassie brilliant blue R_250 decoration method.

(3) polyacrylamide gel of rest part carries out electricity turn, and the Protein transfer in gel is enterprising to pvdf membrane Row Westernblot detection.

2, experimental result

Western-blot testing result such as Fig. 9,10, fusion protein sp/E/HA2 size are about 70KD, and M1 size is about 28KD is consistent with expected expression albumen size.

4 recombinant baculovirus plaque purification of embodiment and virus titer calculate

Selection pass through Western blot detection prove can express express target protein P1 for virus stock solution used infect Sf9 it is thin Born of the same parents after 27 DEG C of incubation 72h, collect and contain virulent supernatant, are denoted as P2 generation virus.The above method is repeated to upload in Sf9 cell Generation to P3, P4 and P5 generation.Referring to the method for Invitrogen company operation instructions, recombination disease is measured using plaque test technology The titre (PFU/mL) of poison

1, experimental implementation

It (1) is 1 × 10 by concentration⁶The Sf9 cell suspension inoculation of a/ml is put into 28 DEG C of cell incubators in 12 orifice plates In be incubated overnight and keep cell adherent.

(2) third generation recombinant baculovirus liquid (P3) is cannotd be used up into full culture medium and does gradient dilution, 10^-1-10^-8。

The first and second generation poison of baculoviral is saved generally as kind of a poison, often selects stable third generation virus to carry out subsequent Experiment.

(3) culture medium in 12 orifice plates is discarded, 3 cells is washed with PBS, is sequentially added into the virus liquid diluted, Every 400 μ l of hole, each dilution set three multiple holes, and using normal cell as control, 12 orifice plates are put into incubator, stand 1 hour Make virus absorption onto cell.

(4) agarose and high pressure sterilization of configuration 2% are spare.

(5) after viruses adsorption 1 hour, the virus liquid in hole is discarded, is cleaned cell 3 times with PBS.Slowly spread agarose (configuration method: the agarose of dual anti-(penicillin+the streptomysin)+14ml 2% of+300 μ l of 14ml 2 × Grace ' s culture medium+ 3ml FBS uses preceding configuration).

12 orifice plates are put into 28 DEG C of incubators after agarose solidifies completely and cultivate 7- by (6) 4 DEG C of standing 10min 10d。

(7) plaque test can be observed under light to naked eyes.The neutral red solution of 1ml 0.03% is added in every hole, puts back to 28 DEG C incubator culture 2 hours, dyeing liquor is sucked out, observes plaque.

(8) single plaque is chosen to the Sf9 cell in logarithmic growth phase with fresh culture culture with 10 μ l pipette tips Middle culture.After 72 hours, cell supernatant is collected.1ml PBS is added and dispels cell on plate, and is washed three times with PBS, ultrasonic wave is broken Broken instrument smudge cells detect protein expression situation.

(9) Virus plaque formational situation (PFU) is calculated

Virus titer=(plaque number × viral dilution multiple)/every hole virus inoculation amount

2, experimental result

Figure 11 is r-sp/E/HA2-M1 plaque purification figure.By virus titer=5.2 × 10 are calculated⁶。

5 indirect immunofluorescence of embodiment (IFA) detects the expression of sp/E/HA2 and M1 albumen in chimeric VLPs

Sf9 cell inoculation recombinant baculovirus supernatant, after infecting 72h, using indirect immunofluorescence observation recombinant protein Expression.

1, experimental implementation

It is 1 × 10 by concentration⁶The Sf9 cell suspension inoculation of a/ml is put into mistake in 28 DEG C of cell incubators in 6 orifice plates Night culture keeps cell adherent.Sf9 cell (MOI=5) is infected with recombinant baculovirus r-sp/E/HA2-M1, is trained in 28 DEG C of cells It supports and is cultivated 48 hours in case.Overnight with 4 DEG C of 3%BSA closings.With the 1%BSA dilution how anti-mouse serum of TMUV, (1:200 is dilute for addition Release), it is stored at room temperature 2 hours.PBS clean 3 times after be added with 1%BSA dilution the more anti-rabbit serum of influenza virus H3N2 (1:50-1: 200 dilutions), it is stored at room temperature 2 hours.PBS is added fluorescent marker FITC after cleaning 3 times and marks anti-mouse secondary antibody (1:1000 dilution), Room temperature is protected from light standing 1 hour.Fluorescent marker CY3 label anti-rabbit secondary antibody (1:1000 dilution) is added after cleaning, room temperature is protected from light standing 1 Hour.It is washed cell 3 times, each 5min with PH7.4PBST, DAPI is added and contaminates core, is protected from light 15min, washes cell 3 with PH7.4PBST It is secondary, each 5min.With glycerol mounting, 4 DEG C are kept in dark place or are directly observed with laser co-focusing.

2, experimental result

Recombinant baculovirus r-sp/E/HA2-M1 is infected into Sf9 cell, Figure 12 is recombinant protein sp/E/HA2 and M1 Expression.As can be seen, green fluorescence shows the expression of recombinant protein sp/E/HA2, and is primarily targeted for infection cell Surface.Red fluorescence shows the expression of M1 albumen, and M1 albumen is distributed in entire cell, is primarily targeted for cytoplasm.

6 transmission electron microscope observing of embodiment is fitted into the form of VLPs (v-sp/E/HA2)

1, experimental implementation

(1) preparation and purification of VLPs

The purified recombinant baculovirus r-sp/E/HA2-M1 that transfection insect cell is obtained is equal to 5 infection with MOI Insect cell, after cultivating 72h, harvests supernatant, -80 DEG C of preservations by 27 DEG C.By the supernatant of harvest, 10,000rpm centrifugation 10min Remove cell fragment；Supernatant 40000rpm ultracentrifugation 2h is concentrated, and 2~4ml PBS (PH7.4) buffer weight is precipitated It is outstanding；Concentrate is carefully slowly splined on the saccharose gradient liquid level for having formed the 20%-60% of gradient, 27000rpm is horizontal Rotary head is centrifuged 2h；Virus band is slowly drawn to concentration tube, 40000rpm ultracentrifugation 2h desugar after PBS buffer solution is filled it up with. It is finally resuspended and is precipitated with appropriate PBS, -80 DEG C save or carry out next step identification.

(2) albumen quantifies in VLPs

E protein content in virus-like particle is measured using PAGE gel electrophoresis gray scale scanning method: by ox blood Pure albumen (BSA) standard items (1mg/ml) and VLPs make gradient dilution with PBS respectively；Take 20 μ L BSA or virus-like particle dilute It releases liquid and after 5 × electrophoresis sample-loading buffer boils, 10 μ L mixed liquors is taken to carry out 10% SDS-PAGE electrophoresis, Coomassie brilliant blue dye After color, according to band thickness, the depth, gray scale scanning analysis is carried out using gel imaging system, estimates the about concentration model of VLPs It encloses and (is calculated with the content of sp/E/HA2 albumen).

(3) form of transmission electron microscope observing VLPs

Chimeric VLPs is purified by sucrose density gradient, uses transmission electron microscope observing after carrying out negative staining with phosphotungstic acid.It will purifying VLPs be placed on 2min on copper mesh, then dye 1min with the phosphotungstic acid of pH 6.5, DTMUV virus-like observed under transmission electron microscope The form of particle.

2, experimental result

With software according to the relative amount (Figure 13) of the thickness of band on SDS-PAGE glue and each destination protein of gray count, The concentration of sp/E/HA2 albumen is about 0.052mg/mL.

As Figure 14 electromicroscopic photograph can be seen that, chimeric VLPs form is similar to protovirus, and diameter is about 50nm.Explanation VLPs particle is expressed in Sf9 cell and packed out to sp/E/HA2 albumen, is discharged into cells and supernatant.

The immunogenicity determining of embodiment 7VLPs

1, experimental implementation

(1) preparation of VLPs oil seepage

The VLPs and twen-80 purified with embodiment 6 configures water phase, and wherein the volume ratio of VLPs and twen-80 is 93:7, With 93 parts, Si Ben -805 parts and 2 parts of aluminum stearate of mineral oil (white oil), oily phase is configured.By water phase and oil mutually according to 1:1.5~1: 2 volume ratio preparation is simultaneously fully emulsified.After the completion of oil seepage preparation, packing is stored in 4 DEG C, and carries out appearance, stability and sterile Detection.

(2) immune detection of VLPs vaccine

40 health Balb/c mouse and the susceptible duckling of health are respectively taken, are respectively divided into 4 groups, every group 10.4 groups of difference neck skins Lower injection v-sp/E/HA2 oil seepage (oil seepage group), v-sp/E/HA2 albumen (protein groups), inactivated vaccine (commodity inactivated vaccine) and PBS.Wherein, the injection dosage of v-sp/E/HA2 oil seepage and v-sp/E/HA2 albumen is 2 μ of sp/E/HA2 protein content g/.

The adjuvant added when wherein, to mouse immune is Freund's adjuvant, and the adjuvant to the immune addition of duckling is white-oil adjuvant. Secondary immunity is carried out altogether, and 7 age in days head of duckling exempts from, and head exempts from rear 14d progress two and exempts from.

Before immune, head exempts from rear 7d, 14d, and two exempt from rear 7d, 14d, 21d, 28d blood sampling, anti-with ELISA measurement DTMUV Body；Meanwhile exempting from rear 14d in two and carrying out protest test with DTMUV is virulent, observe the clinical symptoms and the dead feelings of duck of duckling Condition is at least observed 15 days.

All data carry out statistical analysis and significance test (paired-samples with SPSS software in experiment Ttest), experimental result is indicated with average value ± standard error (mean ± SE).

2, experimental result

As the result is shown: after immune mouse, oil seepage group and the growth of protein groups antibody level of serum are approached with inactivated vaccine group, all 21 days after exempting from two, antibody level peaks, and protein groups antibody level is substantially less than oil seepage group and inactivated vaccine group (figure 15)。

After immune duckling, 21 days antibody level of serum peak after each immune group is also exempted from two, but inactivated vaccine group and Oil seepage group is apparently higher than protein groups (Figure 16).

Duckling attacks poison protection the result shows that the protecting effect of (table 1), inactivated vaccine and oil seepage group is up to 90% after immune, The protecting effect of protein groups is 70%.Immune duckling can obtain good exempt from after illustrating sp/E/HA2 virus-like particle production oil seepage Epidemic disease protecting effect.

Challenge viral dosage result after duckling is immunized in table 1:

Comparative example 1

1, experimental implementation

Embodiment 7 is arrived with embodiment 1, the length of the signal peptide of H3N2-HA is only changed to 30aa, then its nucleotide sequence is such as Shown in SEQ ID NO.19；Amino acid sequence is as shown in SEQ ID NO.20, the nucleotide sequence of amplimer such as SEQ ID Shown in NO.21~22.

2, experimental result

Obtain target fragment 30sp/E/HA2: its nucleotide sequence is as shown in SEQ ID NO.23, amino acid sequence such as SEQ Shown in ID NO.24.

Comparative example 2

1, experimental implementation

Embodiment 7 is arrived with embodiment 1, the length of the signal peptide of H3N2-HA is only changed to 45aa, then its nucleotide sequence is such as Shown in SEQ ID NO.25；Amino acid sequence is as shown in SEQ ID NO.26, the nucleotide sequence of amplimer such as SEQ ID Shown in NO.27~28.

2, experimental result

Obtain target fragment 45sp/E/HA2: its nucleotide sequence is as shown in SEQ ID NO.29, amino acid sequence such as SEQ Shown in ID NO.30.

It is shown according to experimental result, secretory volume of the Hybrid virus like particles in cell conditioned medium is recombinated in the solution of the present invention It is apparently higher than comparative example 1 and 2.

The nucleotide sequence of the signal peptide of SEQ ID NO.1:H3N2-HA

atgaagactatcattgctttgagctacattttctgtctggttttcgcccaagaccttccaggaaatga caacagcacagcaactctttgcctgggacatcatgcggtgccaaacggaacgctagtgaaaacaatcacgaatgat cagattgaagtgactaatgctactgaactggttcag

The amino acid sequence of the signal peptide of SEQ ID NO.2:H3N2-HA

MKTIIALSYIFCLVFAQDLPGNDNSTATLCLGHHAVPNGTLVKTITNDQIEVTNATELVQ

The nucleotide sequence of SEQ ID NO.3:DTMUV E gene

gaattagcggttgtgagatcttactgctatgagccgaaagtgtcggacgtgacgacagaatccagatg cccaaccatgggagaggctcataatcccaaggcaacttatgctgaatacatatgcaaaaaagattttgtggacagg ggttggggcaatggctgtggcttgtttggaaaggggagcata cagacatgtgccaagtttgactgcacaaagaaa gcagaaggcaggatcgtgcagaaggagaacgtccagtttgaagttgcagttttcatacatggttccacggaagcga gcacctaccacaattattcagcccagcagtcgctgaaacatgccgccagattcgttataacgcccaaaagtcccgt ctacaccgctgagatggaggattatggtaccatcacactcgaatgtgaaccccgatctggggttgacatggggcaa ttctatgtctttaccatgaacacaaaaagctggcttgttaacagagactggtttcatgatctcaacttaccatgga cagggtcatcagcggggacgtggcaaaacaaagagtcattgatagaatttgaggaggcccacgccaccaaacaatc agtggtggctttggcatcacaagaaggagccctccatgcagcattggcgggagctattccagtgaagtactctgga agcaaattggaaatgacctcaggtcatcttaaatgtagggttaaaatgcagggtttgaagctgaaaggaatgacct acccgatgtgtagcaatacattttccctagtgaagaatcctaccgacactgggcatggcactgtcgtggtggaatt gtcttatgcaggtaccgatgggccctgtagagttcccatatccatgtcggcagatctgaatgacatgacaccagtt ggacgcttgataacagtcaacccatacgtgtcgacctcctccacgggtgccaagataatggtggaagtggaacctc cattcggggattcattcatcttagtaggaagtggaaaaggacagatcaggtaccagtggcatagaagt

The amino acid sequence of SEQ ID NO.4:DTMUV E gene

ELAVVRSYCYEPKVSDVTTESRCPTMGEAHNPKATYAEYICKKDFVDRGWGNGCGLFGKGSIQTCAKFD CTKKAEGRIVQKENVQFEVAVFIHGSTEASTYHNYSAQQSLKHAARFVITPKSPVYTAEMEDYGTITLECEPRSGVD MGQFYVFTMNTKSWLVNRDWFHDLNLPWTGSSAGTWQNKESLIEFEEAHATKQSVVALASQEGALHAALAGAIPVKY SGSKLEMTSGHLKCRVKMQGLKLKGMTYPMCSNTFSLVKNPTDTGHGTVVVELSYAGTDGPCRVPISMSADLNDMTP VGRLITVNPYVSTSSTGAKIMVEVEPPFGDSFILVGSGKGQIRYQWHRS

The nucleotide sequence of SEQ ID NO.5:H3HA2

ggcatattcggcgcaatagcaggtttcatagaaaatggttgggagggaatgataggcggttggtacgg tttcaggcatcaaaattctgagggcacaggacaagcagcagatcttaaaagcactcaagcagccatcgaccaaatc aatgggaaactgaatagggtaatcgagaagacgaacgagaaattccatcaaatcgaaaaggaattctcagaagtag aagggagaattcaggacctcgagaaatacgttgaagacactaaaatagatctctggtcttacaatgcggagcttct tgtcgctctggagaaccaacatacaattgatctgactgactcggaaatgaacaaactgtttgaaaaaacaaggagg caactgagggaaaatgctgaggacatgggcaatggttgcttcaaaatataccacaaatgtgacaatgcttgcatag ggtcaatcagaaatgggacttatgaccatgatgtatacagagacgaagcattaaacaaccggtttcagatcaaagg tgttgaactgaagtcaggatacaaagactggatcctgtggatttcctttgccatatcatgctttttgctttgtgtt gttttgctggggttcatcatgtgggcctgccagaaaggcaacattaggtgcaacatttgcatctga

The amino acid sequence of SEQ ID NO.6:H3HA2

GIFGAIAGFIENGWEGMIGGWYGFRHQNSEGTGQAADLKSTQAAIDQINGKLNRVIEKTNEKFHQIEKE FSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTRRQLRENAEDMGNGCFKIYHKCDN ACIGSIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDWILWISFAISCFLLCVVLLGFIMWACQKGNIRCNICI

SEQ ID NO.7

atgaagactatcattgctttg

SEQ ID NO.8

ctgaaccagttcagtagcat

SEQ ID NO.9

gaattagcggttgtgagatct

SEQ ID NO.10

acttctatgccactggtacct

SEQ ID NO.11

ggcatattcggcgcaa

SEQ ID NO.12

tcagatgcaaatgttgcacc

SEQ ID NO.13

ggactagtatgaagactatcattgcttt

SEQ ID NO.14

Ccaagctttcagatgcaaatgttgcacc

The nucleotide sequence of SEQ ID NO.15:sp/E/HA2

atgaagactatcattgctttgagctacattttctgtctggttttcgcccaagaccttccaggaaatga caacagcacagcaactctttgcctgggacatcatgcggtgccaaacggaacgctagtgaaaacaatcacgaatgat cagattgaagtgactaatgctactgaactggttcaggaattagcggttgtgagatcttactgctatgagccgaaag tgtcggacgtgacgacagaatccagatgcccaaccatgggagaggctcataatcccaaggcaacttatgctgaata catatgcaaaaaagattttgtggacaggggttggggcaatggctgtggcttgtttggaaaggggagcatacagaca tgtgccaagtttgactgcacaaagaaagcagaaggcaggatcgtgcagaaggagaacgtccagtttgaagttgcag ttttcatacatggttccacggaagcgagcacctaccacaattattcagcccagcagtcgctgaaacatgccgccag attcgttataacgcccaaaagtcccgtctacaccgctgagatggaggattatggtaccatcacactcgaatgtgaa ccccgatctggggttgacatggggcaattctatgtctttaccatgaacacaaaaagctggcttgttaacagagact ggtttcatgatctcaacttaccatggacagggtcatcagcggggacgtggcaaaacaaagagtcattgatagaatt tgaggaggcccacgccaccaaacaatcagtggtggctttggcatcacaagaaggagccctccatgcagcattggcg ggagctattccagtgaagtactctggaagcaaattggaaatgacctcaggtcatcttaaatgtagggttaaaatgc agggtttgaagctgaaaggaatgacctacccgatgtgtagcaatacattttccctagtgaagaatcctaccgacac tgggcatggcactgtcgtggtggaattgtcttatgcaggtaccgatgggccctgtagagttcccatatccatgtcg gcagatctgaatgacatgacaccagttggacgcttgataacagtcaacccatacgtgtcgacctcctccacgggtg ccaagataatggtggaagtggaacctccattcggggattcattcatcttagtaggaagtggaaaaggacagatcag gtaccagtggcatagaagtggcatattcggcgcaatagcaggtttcatagaaaatggttgggagggaatgataggc ggttggtacggtttcaggcatcaaaattctgagggcacaggacaagcagcagatcttaaaagcactcaagcagcca tcgaccaaatcaatgggaaactgaatagggtaatcgagaagacgaacgagaaattccatcaaatcgaaaaggaatt ctcagaagtagaagggagaattcaggacctcgagaaatacgttgaagacactaaaatagatctctggtcttacaat gcggagcttcttgtcgctctggagaaccaacatacaattgatctgactgactcggaaatgaacaaactgtttgaaa aaacaaggaggcaactgagggaaaatgctgaggacatgggcaatggttgcttcaaaatataccacaaatgtgacaa tgcttgcatagggtcaatcagaaatgggacttatgaccatgatgtatacagagacgaagcattaaacaaccggttt cagatcaaaggtgttgaactgaagtcaggatacaaagactggatcctgtggatttcctttgccatatcatgctttt tgctttgtgttgttttgctggggttcatcatgtgggcctgccagaaaggcaacattaggtgcaacatttgcatctga

The protein sequence of SEQ ID NO.16:sp/E/HA2

MKTIIALSYIFCLVFAQDLPGNDNSTATLCLGHHAVPNGTLVKTITNDQIEVTNATELVQELAVVRSY CYEPKVSDVTTESRCPTMGEAHNPKATYAEYICKKDFVD RGWGNGCGLFGKGSIQTCAKFDCTKKAEGRIVQKEN VQFEVAVFIHGSTEASTYHNYSAQQSLKHAARFVITPKSPVYTAEMEDYGTITLECEPRSGVDMGQFYVFTMNTKSW LVNRDWFHDLNLPWTGSSAGTWQNKESLIEFEEAHATKQSVVALASQEGALHAALAGAIPVKYSGSKLEMTSGHLKC RVKMQGLKLKGMTYPMCSNTFSLVKNPTDTGHGTVVVELSYAGTDGPCRVPISMSADLNDMTPVGRLITVNPYVSTS STGAKIMVEVEPPFGDSFILVGSGKGQIRYQWHRSGIFGAIAGFIENGWEGMIGGWYGFRHQNSEGTGQAADLKSTQ AAIDQINGKLNRVIEKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLF EKTRRQLRENAEDMGNGCFKIYHKCDNACIGSIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDWILWISFAISCF LLCVVLLGFIMWACQKGNIRCNICI

The nucleotide sequence of SEQ ID NO.17:H3N2M1 gene

atgagccttctaaccgaggtcgaaacgtatgttctctctatcgttccgtcaggccccctcaaagccga aatcgcgcagagacttgaagatgtctttgctggaaagaacacagatcttgaggctctcatggaatggctaaagaca agaccaatcctgtcacctctgactaaggggattttgggatttgtattcacgctcaccgtgcccagtgagcgaggac tgcagcgtagacgctttgtccaaaatgccctcaatgggaatggggatccaaataacatggacaaagcagttaaact gtacagaaaacttaagagggagataacattccatggggccaaagaaatagcactcagttattctgctggtgcactt gccagttgcatgggcctcatatacaacaggatgggggctgtgaccactgaagtggcctttggcctggtatgtgcaa cctgtgaacagattgctgactcccagcacaggtctcataggcaaatggtggcaacaaccaatccactaataagaca tgagaacagaatggttctggccagcactacagctaaggctatggagcaaatggctggatcaagtgagcaggcagca gaggccatggaggttgctagtcaggccaggcaaatggtacaggcaatgagagccattgggactcatcctagctcca gtgctggtctaaaagatgatcttcttgaaaatttgcaggcctatcagaagcgaatgggggtgcagatgcaacgatt caagtga

SEQ ID NO.18:H3N2M1 amino acid sequence

MSLLTEVETYVLSIVPSGPLKAEIAQRLEDVFAGKNTDLEALMEWLKTRPILSPLTKGILGFVFTLTVP SERGLQRRRFVQNALNGNGDPNNMDKAVKLYRKLKREITFHGAKEIALSYSAGALASCMGLIYNRMGAVTTEVAFGL VCATCEQIADSQHRSHRQMVATTNPLIRHENRMVLASTTAKAMEQMAGSSEQAAEAMEVASQARQMVQAMRAIGTHP SSSAGLKDDLLENLQAYQKRMGVQMQRFK

The nucleotide sequence of the signal peptide of SEQ ID NO.19:H3N2-HA

atgaagactatcattgctttgagctacattttctgtctggttttcgcccaagaccttccaggaaatga caacagcacagcaactctttgc

The amino acid sequence of the signal peptide of SEQ ID NO.20:H3N2-HA

MKTIIALSYIFCLVFAQDLPGNDNSTATLC

SEQ ID NO.21

atgaagactatcattgctttg

SEQ ID NO.22

gcaaagagttgctgtgct

SEQ ID NO.23 30sp/E/HA2 nucleotide sequence

atgaagactatcattgctttgagctacattttctgtctggttttcgcccaagaccttccaggaaatga caacagcacagcaactctttgcgaattagcggttgtgagatcttactgctatgagccgaaagtgtcggacgtgacg acagaatccagatgcccaaccatgggagaggctcataatcccaaggcaacttatgctgaatacatatgcaaaaaag attttgtggacaggggttggggcaatggctgtggcttgtttggaaaggggagcatacagacatgtgccaagtttga ctgcacaaagaaagcagaaggcaggatcgtgcagaaggagaacgtccagtttgaagttgcagttttcatacatggt tccacggaagcgagcacctaccacaattattcagcccagcagtcgctgaaacatgccgccagattcgttataacgc ccaaaagtcccgtctacaccgctgagatggaggattatggtaccatcacactcgaatgtgaaccccgatctggggt tgacatggggcaattctatgtctttaccatgaacacaaaaagctggcttgttaacagagactggtttcatgatctc aacttaccatggacagggtcatcagcggggacgtggcaaaacaaagagtcattgatagaatttgaggaggcccacg ccaccaaacaatcagtggtggctttggcatcacaagaaggagccctccatgcagcattggcgggagctattccagt gaagtactctggaagcaaattggaaatgacctcaggtcatcttaaatgtagggttaaaatgcagggtttgaagctg aaaggaatgacctacccgatgtgtagcaatacattttccctagtgaagaatcctaccgacactgggcatggcactg tcgtggtggaattgtcttatgcaggtaccgatgggccctgtagagttcccatatccatgtcggcagatctgaatga catgacaccagttggacgcttgataacagtcaacccatacgtgtcgacctcctccacgggtgccaagataatggtg gaagtggaacctccattcggggattcattcatcttagtaggaagtggaaaaggacagatcaggtaccagtggcata gaagtggcatattcggcgcaatagcaggtttcatagaaaatggttgggagggaatgataggcggttggtacggttt caggcatcaaaattctgagggcacaggacaagcagcagatcttaaaagcactcaagcagccatcgaccaaatcaat gggaaactgaatagggtaatcga gaagacgaacgagaaattccatcaaatcgaaaaggaattctcagaagtagaa gggagaattcaggacctcgagaaatacgttgaagacactaaaatagatctctggtcttacaatgcggagcttcttg tcgctctggagaaccaacatacaattgatctgactgactcggaaatgaacaaactgtttgaaaaaacaaggaggca actgagggaaaatgctgaggacatgggcaatggttgcttcaaaatataccacaaatgtgacaatgcttgcataggg tcaatcagaaatgggacttatgaccatgatgtatacagagacgaagcattaaacaaccggtttcagatcaaaggtg ttgaactgaagtcaggatacaaagactggatcctgtggatttcctttgccatatcatgctttttgctttgtgttgt tttgctggggttcatcatgtgggcctgccagaaaggcaacattaggtgcaacatttgcatctga

SEQ ID NO.24 30sp/E/HA2 amino acid sequence

MKTIIALSYIFCLVFAQDLPGNDNSTATLCELAVVRSYCYEPKVSDVTTESRCPTMGEAHNPKATYAEY ICKKDFVDRGWGNGCGLFGKGSIQTCAKFDCTKKAEGRIVQKENVQFEVAVFIHGSTEASTYHNYSAQQSLKHAARF VITPKSPVYTAEMEDYGTITLECEPRSGVDMGQFYVFTMNTKSWLVNRDWFHDLNLPWTGSSAGTWQNKESLIEFEE AHATKQSVVALASQEGALHAALAGAIPVKYSGSKLEMTSGHLKCRVKMQGLKLKGMTYPMCSNTFSLVKNPTDTGHG TVVVELSYAGTDGPCRVPISMSADLNDMTPVGRLITVNPYVSTSSTGAKIMVEVEPPFGDSFILVGSGKGQIRYQWH RSGIFGAIAGFIENGWEGMIGGWYGFRHQNSEGTGQAADLKSTQAAIDQINGKLNRVIEKTNEKFHQIEKEFSEVEG RIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIGSI RNGTYDHDVYRDEALNNRFQIKGVELKSGYKDWILWISFAISCFLLCVVLLGFIMWACQKGNIRCNICI

SEQ ID NO.25

atgaagactatcattgctttgagctacattttctgtctggttttcgcccaagaccttccaggaaatga caacagcacagcaactctttgcctgggacatcatgcggtgccaaacggaacgctagtgaaaacaatc

SEQ ID NO.26

MKTIIALSYIFCLVFAQDLPGNDNSTATLCLGHHAVPNGTLVKTI

SEQ ID NO.27

atgaagactatcattgctttg

SEQ ID NO.28

gattgttttcactagcgttc

SEQ ID NO.29 45sp/E/HA2 nucleotide sequence

atgaagactatcattgctttgagctacattttctgtctggttttcgcccaagaccttccaggaaatga caacagcacagcaactctttgcctgggacatcatgcggtgccaaacggaacgctagtgaaaacaatcgaattagcg gttgtgagatcttactgctatgagccgaaagtgtcggacgtgacgacagaatccagatgcccaaccatgggagagg ctcataatcccaaggcaacttatgctgaatacatatgcaaaaaagattttgtggacaggggttggggcaatggctg tggcttgtttggaaaggggagcatacagacatgtgccaagtttgactgcacaaagaaagcagaaggcaggatcgtg cagaaggagaacgtccagtttgaagttgcagttttcatacatggttccacggaagcgagcacctaccacaattatt cagcccagcagtcgctgaaacatgccgccagattcgttataacgcccaaaagtcccgtctacaccgctgagatgga ggattatggtaccatcacactcgaatgtgaaccccgatctggggttgacatggggcaattctatgtctttaccatg aacacaaaaagctggcttgttaacagagactggtttcatgatctcaacttaccatggacagggtcatcagcgggga cgtggcaaaacaaagagtcattgatagaatttgaggaggcccacgccaccaaacaatcagtggtggctttggcatc acaagaaggagccctccatgcagcattggcgggagctattccagtgaagtactctggaagcaaattggaaatgacc tcaggtcatcttaaatgtagggttaaaatgcagggtttgaagctgaaaggaatgacctacccgatgtgtagcaata cattttccctagtgaagaatcctaccgacactgggcatggcactgtcgtggtggaattgtcttatgcaggtaccga tgggccctgtagagttcccatatccatgtcggcagatctgaatgacatgacaccagttggacgcttgataacagtc aacccatacgtgtcgacctcctccacgggtgccaagataatggtggaagtggaacctccattcggggattcattca tcttagtaggaagtggaaaaggacagatcaggtaccagtggcatagaagtggcatattcggcgcaatagcaggttt catagaaaatggttgggagggaatgataggcggttggtacggtttcaggcatcaaaattctgagggcacaggacaa gcagcagatcttaaaagcactcaagcagccatcgaccaaatcaatgggaaactgaatagggtaatcgagaagacga acgagaaattccatcaaatcgaaaaggaattctcagaagtagaagggagaattcaggacctcgagaaatacgttga agacactaaaatagatctctggtcttacaatgcggagcttcttgtcgctctggagaaccaacatacaattgatctg actgactcggaaatgaacaaactgtttgaaaaaacaaggaggcaactgagggaaaatgctgaggacatgggcaatg gttgcttcaaaatataccacaaatgtgacaatgcttgcatagggtcaatcagaaatgggacttatgaccatgatgt atacagagacgaagcattaaacaaccggtttcagatcaaaggtgttgaactgaagtcaggatacaaagactggatc ctgtggatttcctttgccatatcatgctttttgctttgtgttgttttgctggggttcatcatgtgggcctgccaga aaggcaacattaggtgcaacatttgcatctga

SEQ ID NO.30 45sp/E/HA2 amino acid sequence

MKTIIALSYIFCLVFAQDLPGNDNSTATLCLGHHAVPNGTLVKTIELAVVRSYCYEPKVSDVTTESRCP TMGEAHNPKATYAEYICKKDFVDRGWGNGCGLFGKGSIQTCAKFDCTKKAEGRIVQKENVQFEVAVFIHGSTEASTY HNYSAQQSLKHAARFVITPKSPVYTAEMEDYGTITLECEPRSGVDMGQFYVFTMNTKSWLVNRDWFHDLNLPWTGSS AGTWQNKESLIEFEEAHATKQSVVALASQEGALHAALAGAIPVKYSGSKLEMTSGHLKCRVKMQGLKLKGMTYPMCS NTFSLVKNPTDTGHGTVVVELSYAGTDGPCRVPISMSADLNDMTPVGRLITVNPYVSTSSTGAKIMVEVEPPFGDSF ILVGSGKGQIRYQWHRSGIFGAIAGFIENGWEGMIGGWYGFRHQNSEGTGQAADLKSTQAAIDQINGKLNRVIEKTN EKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTRRQLRENAEDMGNGC FKIYHKCDNACIGSIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDWILWISFAISCFLLCVVLLGFIMWACQKGN IRCNICI

Claims

1. a kind of fusion, which is characterized in that the nucleotide sequence of the fusion is as shown in SEQ ID NO.15.

2. a kind of fusion protein, which is characterized in that the amino acid sequence of the fusion is as shown in SEQ ID NO.16.

3. a kind of recombinant vector, which is characterized in that the carrier contains fusion and avian influenza virus described in claim 1 The M1 gene of H3N2, and it is located at two open reading frame of carrier.

4. a kind of recombinant vector combination, which is characterized in that including recombinant vector 1 and recombinant vector 2, recombinant vector 1, which contains, has the right It is required that 1 fusion, M1 gene of the recombinant vector 2 containing avian influenza virus H3N2.

5. a kind of baculoviral of recombination, which is characterized in that the baculoviral contains fusion and fowl described in claim 1 The M1 gene of influenza virus H3N2.

6. fusion protein described in fusion, claim 2 described in claim 1, carrier, claim 4 described in claim 3 Baculoviral described in the carrier combination or claim 5 is in the Hybrid virus like particles of preparation duck tembusu virus E protein Application.

7. a kind of Hybrid virus like particles for expressing duck tembusu virus E protein, which is characterized in that the virus-like particle is logical It crosses using carrier, claim 4 described in fusion protein described in fusion, claim 2 described in claim 1, claim 3 Baculoviral described in the carrier combination or claim 5 is prepared.

8. fusion protein described in fusion, claim 2 described in claim 1, carrier, claim 4 described in claim 3 Hybrid virus like particles described in baculoviral described in the carrier combination, claim 5 or claim 7 are in preparation duck Tan Busu Application in viral vaccine.

9. a kind of duck tembusu virus vaccine, which is characterized in that including expressing duck tembusu virus E protein described in claim 7 Hybrid virus like particles.