CN110272473A - General virus-like particle of Flu-A and its preparation method and application - Google Patents

Abstract

The present invention provides a kind of general virus-like particle of Flu-A and its preparation method and application, the virus-like particle is that 3 kinds of fusion proteins are entrenched in the surface influenza virus M1 respectively to be prepared into, it is denoted as sHA-VLPs, mHA-VLPs, cHA-VLPs respectively, fusion protein is recombinated by the well-conserved sequence or part of Influenza virus HA protein, M2e albumen, NP albumen and PBI albumen respectively；The virus-like particle can be used to prevent specific influenza, the homologous virus of brave influenza and heterologus virus etc.；The preparation method of virus-like particle of the present invention is simple, at low cost, and virus-like particle immunogenicity is good, intersecting protective is good, is able to satisfy the preparation demand of high-volume influenza vaccines, and biological safety can excite matrix that a variety of influenza viruses are immunized.

Description

General virus-like particle of Flu-A and its preparation method and application

Technical field

The invention belongs to biopharmaceutical technology, more particularly to a kind of general virus-like particle of Flu-A and its Preparation method and application.

Background technique

Seasonal influenza is popular in Temperate Region in China the coldest season, and annual popular in subtropical and tropical zones, the whole world is every Year, there are about 1,000,000,000 people's influenza virus infections, and wherein severe complication occurs in 3-5 million people, and ten thousand people of 30-50 is dead, while influenza disease Malicious constantly variation, when a kind of novel influenza occurs, most people does not have immunity, especially child, old man, Yun Fuhe The vulnerable groups of weakened immune system, novel influenza avoid body immune system, it is easy to make one infection and in person to person Between propagate formed being very popular property disease, in history once occur repeatedly worldwide flu outbreak；1918 Nian Xiban Tooth H1N1 flu outbreak once caused 40,000,000 people impacted, and first identified in 1933 goes out the cause of disease of human influenza, nineteen fifty-seven H2N2 Asia influenza, nineteen sixty-eight H3N2 Mao flu, H1N1 in 2009 once lead to influenza virus pandemic, state, the U.S. 2017-18 Vertical disease prevention and control center reports the U.S. up to 30,000 person-times of flu episodes, and 171 people are because of influenza death, with 2003-2004 year It compares, 2017-2018 year, being aggravated by sense degree occurred in the patient of each age bracket, the morbidity and mortality of influenza virus It is substantially increased.

Therefore current influenza vaccines are continuously improved, improves the protection, broad spectrum activity and Duo Ji coverage rate of vaccine, is current The emphasis of influenza vaccines research needs to develop a needle more now in order to cope with the extensive popular influenza that can occur at any time Prevent, there are the active universal influenza vaccines of broad spectrum protection.

In order to cope with the continuous variation of influenza antigen, the World Health Organization establishes Global influenza monitoring and reply system It unites (GISRS), annual Advisory Board, the World Health Organization monitors the data that reply system monitoring arrives according to Global influenza and predicts Antigenic drift out, determines whether the candidate strain of vaccine updates；The influenza virus vaccine ratified currently on the market specifically includes that inactivation Influenza virus cracking vaccine, recombinant flu vaccines and acclimatization to cold attenuated live vaccine, no matter which kind of vaccine includes 3/4 candidates Strain, that is, H1N1, the Victoria system or/and Yanagata system of H3N2, IBV, inactivated influenza vaccine be chicken embryo is bred it is complete Totivirus formalin or beta-propiolactone inactivation, further crack purifying for the complete virus after inactivation, remove exotic antigen And heterogenetic antigen, split vaccine is made, but inactivated influenza vaccine currently used in the market has response time length, to chicken Embryo dependence is high, introduces the disadvantages of allergy provirus is easily mutated, highly pathogenic strain can not be proliferated, and current influenza vaccines marquis It selects strain prediction to obtain, may be mismatched with practical popular influenza strain, lead to problems such as vaccine potency low；Utilize insect The influenza virus-like particles preparation influenza vaccines of baculovirus expression system expression become current research and development focus, insect baculovirus Expression system can several structural proteins of expression of influenza virus and completion is assembled to form virus-like particle simultaneously, virus-like particle by Virus differential protein assemble, but not contain the viral nucleic acid composition, in outer surface antigen conformation with this precursor virus phase Seemingly, thus cause humoral and cellular immune response stimulation, have the advantages that biological safety is high, at low cost, yield is high.

The influenza structural proteins composition for including in influenza virus-like particles at present is complete native protein structure, such as The virus-like particle of HA and M1 coexpression or HA-NA-M1 coinfection, such virus-like particle immunogenicity is high, but generate Immune protective efficiency is narrow spectrum, plays cross-protection to heterologous strain difference.

Summary of the invention

The purpose of the present invention is to provide general virus-like particle of a kind of Flu-A and its preparation method and application, with It improves the yield of the general virus-like particle of Flu-A, reduce production cost, by reasonably selecting fusion protein segment, improve The immunogenicity and cross-protection of virus-like particle, can be applied to the homologous virus of prevention specific influenza, brave influenza and It is adaptable in terms of heterologus virus.

The technical scheme adopted by the invention is that the general virus-like particle of Flu-A is to distinguish 3 kinds of fusion proteins It is chimeric to show made of the influenza virus M1 protein surface, it is denoted as sHA-VLPs, mHA-VLPs, cHA-VLPs respectively；

3 kinds of fusion proteins are sHA albumen, mHA albumen and cHA albumen respectively；

The sHA protein gene sequence is as shown in SEQ ID NO.1, the amino acid sequence of sHA albumen such as SEQ ID NO.6 institute Show；

The gene order of the mHA albumen is as shown in SEQ ID NO.2, the amino acid sequence of mHA albumen such as SEQ ID NO.7 institute Show；

The gene order of the cHA is as shown in SEQ ID NO.3, and the amino acid sequence of cHA is as shown in SEQ ID NO.8.

The preparation method of the general virus-like particle of Flu-A the following steps are included:

Step 1, the encoding gene that influenza virus M1 is obtained by PCR method, to the encoding gene and pFastBacdual of M1 albumen Gene carry out multiple cloning sites analysis, select to provide on pFastBacdual and do not have in target fragment two it is restricted in Enzyme cutting, the primer of purpose of design segment recycle target fragment after target fragment amplification, double digestion, target fragment are inserted into In multiple cloning sites after insect cell expression vector p10 promoter, bacillus coli DH 5 alpha competent cell is converted, is obtained PFastBacdual-M1 plasmid；

Step 2, by gene synthesis technology obtain sHA albumen, mHA albumen and cHA albumen encoding gene, by sHA albumen, The gene of the encoding gene of mHA albumen and cHA albumen and pFastBacdual-M1 plasmid carries out multiple cloning sites analysis, selection Two restriction enzymes for providing on pFastBacdual-M1 plasmid and not having in target fragment recycle fusion after double digestion The target fragment of albumen, the multiple cloning sites after target fragment to be inserted into pFastBacdual-M1 plasmid PH promoter, then turns Change bacillus coli DH 5 alpha competent cell, obtains pFastBacdual-M1- fusion protein shuttle plasmid；

Step 3, pFastBacdual-M1- fusion protein shuttle plasmid is converted into Escherichia coli DH10Bac competent cell, obtained To recombinant baculovirus plasmid Bacmid, using recombinant baculovirus plasmid Bacmid transfection Sf 9 insect cell, rescue is weighed Group baculoviral, the convergence degree of Sf9 insect cell reaches 80% or more when transfection；

Step 4, recombinant baculovirus is inoculated with Sf9 insect cell according to MOI=3, it is logical to obtain Flu-A for harvest supernatant after three days Use virus-like particle.

Further, the restriction enzyme site of the M1 albumen and pFastBacdual carrier is SmaI and NsiI, fusion protein The encoding gene of sHA, mHA and cHA and the restriction enzyme site of pFastBacdual-M1 plasmid are EcoRI and NotI.

Further, specific step is as follows for acquisition recombinant baculovirus in the step 3: by 10ng PFastBacdual-M1- fusion protein shuttle plasmid is added in DH10Bac competent cell, hot at 42 DEG C after ice bath 30min Swash 45s, then ice bath 2min, nonreactive LB culture medium shaking table at 37 DEG C, revolving speed 200rpm is added and vibrates 4h, 100 μ L is taken to vibrate In solution coating to the three anti-LB plates containing X-gal, IPTG, 48h is cultivated at 37 DEG C, the hickie that blue hickie screens is attached most importance to Group baculovirus plasmid Bacmid.

Further, the concentration of Sf9 insect cell is 2 × 106/mL or more when the step 4 inoculation recombinant baculovirus.

Further, sucrose density gradient centrifugation, tool are selected in the purifying of the general virus-like particle of the Flu-A Body process is as follows: after being inoculated with the Insect cellculture 72h of recombinant baculovirus, collect suspension cell culture liquid 4 DEG C, It is centrifuged 20min under 5000rpm revolving speed and obtains supernatant, by supernatant, ultracentrifugation 1h is concentrated under 4 DEG C, 30000rpm revolving speed, Be resuspended and concentrate and dissolved overnight at 4 DEG C using PBS, finally in the sucrose of 20%-30%-60% with revolving speed 30000rpm from Heart 1h takes the solution 30000rpm between 30%-60% gradient to be centrifuged 1h after going sucrose step, is the virus of purifying after precipitating resuspension Sample particle.

The general virus-like particle of Flu-A is used to prepare prevention specific influenza, the homologous virus of brave influenza and heterologous disease The vaccine of poison.

The beneficial effects of the present invention are: the present invention using Baculovirus insect expression system as expression vector, using thin Born of the same parents produce influenza A virus sample particle as bioreactor, have the advantages that yield is high, production cost is low, the A type of production Influenza virus-like particles immunogenicity is good, intersecting protective is good, can be used in largely preparing influenza vaccines, and inanimate object safety wind The problems such as dangerous, can excite matrix that a variety of influenza viruses are immunized.

Detailed description of the invention

In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this Some embodiments of invention for those of ordinary skill in the art without creative efforts, can be with It obtains other drawings based on these drawings.

Fig. 1 is that the gene of mHA, sHA, cHA constitute schematic diagram.

Fig. 2: A is to resist VLPs (HA-M1) the immunofluorescence figure obtained for primary antibody with HA rabbit more, and B is to be resisted with HA rabbit for one more The sHA-VLPs immunofluorescence figure of anti-acquisition, C be with HA rabbit more resist for primary antibody obtain mHA-VLPs immunofluorescence figure, D be with HA rabbit resists the cHA-VLPs immunofluorescence figure obtained for primary antibody more, and E is that the VLPs (HA-M1) obtained using M1 mouse monoclonal antibody as primary antibody exempts from Epidemic disease fluorescent figure, F are the sHA-VLPs immunofluorescence figures obtained using M1 mouse monoclonal antibody as primary antibody, and G is obtained by primary antibody of M1 mouse monoclonal antibody MHA-VLPs immunofluorescence figure, H is the cHA-VLPs immunofluorescence figure obtained using M1 mouse monoclonal antibody as primary antibody.

Fig. 3: A is the electron microscope of VLPs (HA-M1), and B is the Electronic Speculum detection figure of virus-like particle sHA-VLPs, and C is virus The Electronic Speculum of sample particle mHA-VLPs detects figure, and D is the Electronic Speculum detection figure of virus-like particle cHA-VLPs.

Fig. 4: A is the western blot figure for making 4 kinds of VLPs of primary antibody with M1 mouse monoclonal antibody, and B is VLPs (HA-M1) and mHA-VLPs Western blot figure, C are the western blot figures of sHA-VLPs and cHA-VLPs, and D is 4 kinds of VLPs when making primary antibody with NP, M2 mouse monoclonal antibody Western blot figure, E is the western blot figure of 4 kinds of VLPs.

Fig. 5 A is the immune specific antibody level for H1N1 of detection.

Fig. 5 B is the immune specific antibody level for H3N2 of detection.

Fig. 5 C is the immune specific antibody level for H5N1 of detection.

Fig. 5 D is the immune specific antibody level for H7N7 of detection.

Fig. 5 E is the detection figure of IgG1 and IgG2a antibody.

Fig. 6 A is the changes of weight figure that H1N1 attacks poison group mouse.

Fig. 6 B is the survival rate variation diagram that H1N1 attacks poison group mouse.

Fig. 6 C is the changes of weight figure that H3N2 attacks poison group mouse.

Fig. 6 D is the survival rate variation diagram that H3N2 attacks poison group mouse.

Fig. 6 E is the changes of weight figure that H5N1 attacks poison group mouse.

Fig. 6 F is the survival rate variation diagram that H5N1 attacks poison group mouse.

Fig. 6 G is the changes of weight figure that H7N7 attacks poison group mouse.

Fig. 6 H is the survival rate variation diagram that H7N7 attacks poison group mouse.

Fig. 7 A is the secretion situation map that H5N1 stimulates lower IL-4.

Fig. 7 B is the secretion situation map that H5N1 stimulates lower IFN-γ.

Fig. 8 is Influenza virus titer detection figure.

Specific embodiment

Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other Embodiment shall fall within the protection scope of the present invention.

The general virus-like particle of Flu-A is to be fitted into 3 kinds of fusion proteins respectively to show in influenza virus M1 albumen table Made of wheat flour is standby, it is denoted as sHA-VLPs, mHA-VLPs, cHA-VLPs, fusion protein sHA albumen, mHA albumen and cHA respectively The gene of albumen is constituted as shown in Figure 1, fusion protein is respectively by Influenza virus HA protein, M2e albumen, NP albumen, PBI albumen Well-conserved sequence completely or partially recombinates.

Sequence composition of the fusion protein sHA albumen from N-terminal to C-terminal are as follows: two sections of conserved sequences of bee venom signal peptide+NP albumen + 5 M2e albumen+HA2 albumen from different subtype, different genera are connected between sequence, the gene sequence of sHA albumen with link peptide Column are as shown in SEQ ID NO.1, and the amino acid sequence of sHA albumen is as shown in SEQ ID NO.6.

Sequence composition of the fusion protein mHA albumen from N-terminal to C-terminal are as follows: 3 sections of conserved sequences of bee venom signal peptide+HA albumen, 3 end conserved sequences are respectively: the T41-S113 of the N286-N319 and HA2 albumen of E14-H37, HA1 albumen of HA1 albumen, sequence Between connected with link peptide, the gene order of mHA albumen is as shown in SEQ ID NO.2, the amino acid sequence of mHA albumen such as SEQ ID Shown in NO.7.

Sequence composition of the fusion protein cHA albumen from N-terminal to C-terminal are as follows: the guarantor between bee venom signal peptide+HA1 albumen E14-H37 Keep 2 sections of conserved sequences+5 of sequence+NP albumen from different subtype, two sections of the M2e albumen+HA albumen of different genera it is conservative Sequence+HA transmembrane region, the both ends conserved sequence of HA albumen are the T41-S113 of the N286-N319 and HA2 albumen of HA1 albumen, sequence It is connected between column with link peptide, the gene order of cHA is as shown in SEQ ID NO.3, the amino acid sequence of cHA such as SEQ ID NO.8 It is shown.

The present invention is with reference to the conservative region of different Influenza virus HA proteins, conservative region M2e, NP conservative region of M2 albumen Albumen carries out expressing in series, and the transmembrane region of HA albumen and intracellular region recombination is combined to obtain fusion protein, and fusion protein is shown On virus-like particle surface, to construct the general virus-like particle of different Flu-As, the virus-like particle is compared to independent Small peptide has better immunogenicity, and body can preferably be stimulated to generate humoral immunity and cellular immunity.

The preparation method of the general virus-like particle of Flu-A, comprising the following steps:

Step 1, building recombination pFastBacdual-M1 plasmid: the encoding gene of influenza virus M1 is obtained by PCR method, to M1 The gene of the encoding gene of albumen and pFastBacdual carry out multiple cloning sites analysis, select to provide on pFastBacdual and Two restriction enzymes not having in target fragment, the primer of purpose of design segment, after target fragment amplification, double digestion Target fragment is recycled, in the multiple cloning sites after target fragment to be inserted into insect cell expression vector p10 promoter, converts large intestine Bacillus DH5 α competent cell, obtains pFastBacdual-M1 plasmid；

Step 2, it constructs recombinant shuttle plasmid: fusion protein sHA, mHA and cHA encoding gene is obtained by gene synthesis technology, The gene of the encoding gene of fusion protein sHA, mHA and cHA and pFastBacdual-M1 plasmid is subjected to multiple cloning sites point Analysis, select to provide on pFastBacdual-M1 plasmid and two restriction enzymes not having on fusion protein, return after double digestion In multiple cloning sites after receiving the PH promoter of the target fragment insertion pFastBacdual-M1 plasmid of fusion protein, conversion is big Enterobacteria DH5 α competent cell, obtains pFastBacdual-M1- fusion protein shuttle plasmid；

Step 3, building recombinant baculovirus plasmid and recombinant baculovirus are saved: pFastBacdual-M1- fusion protein is worn Shuttle plasmid converts Escherichia coli DH10Bac competent cell, obtains recombinant baculovirus plasmid Bacmid, uses the rod-shaped disease of recombination Toxin grain Bacmid transfection Sf 9 insect cell, rescue obtain recombinant baculovirus, and the convergence degree of Sf9 insect cell reaches when transfection To 80% or more；

Step 4, be made Flu-A Universal virus-like particle: by recombinant baculovirus by MOI=3 be inoculated with Sf9 insect cell, 3 days Harvest supernatant is afterwards to get the general virus-like particle of Flu-A, and the concentration of Sf9 insect cell needs when being inoculated with recombinant baculovirus Reach 2 × 106/mL.

The restriction enzyme site of the present invention M1 albumen and pFastBacdual carrier in step 1 is SmaI and NsiI, in step 2 The encoding gene of fusion protein sHA, mHA and cHA and the restriction enzyme site of pFastBacdual-M1 plasmid are EcoRI and NotI.

The molecular cloning of pFastBacdual-M1- fusion protein shuttle plasmid is by routine transformation DH5 α in step 3 of the present invention Competent cell clones to obtain；Obtaining recombinant baculovirus plasmid, specific step is as follows: by 10ng pFastBacdual-M1- Fusion protein shuttle plasmid is added in DH10Bac competent cell, after ice bath 30min at 42 DEG C heat shock 45s, then ice bath 2min is added nonreactive LB culture medium shaking table at 37 DEG C, revolving speed 200rpm and vibrates 4h, 100 μ L is taken to vibrate solution coating to containing X- On the anti-LB plate of the three of gal, IPTG, 48h is cultivated at 37 DEG C, the hickie that blue hickie screens is recombinant baculovirus plasmid Bacmid realizes blank rod granule and pFastBacdual-M1- fusion protein in DH10Bac competent cell by the swivel base of DNA DNA homologous recombination between shuttle plasmid.

The purification process of virus-like particle selects sucrose density gradient centrifugation, and detailed process is as follows: inoculation is recombinated bar After the Insect cellculture 72h of shape virus, collects suspension cell culture liquid and be centrifuged 20min removal under 4 DEG C, 5000rpm revolving speed Cell fragment obtains supernatant, and by supernatant, ultracentrifugation 1h is concentrated under 4 DEG C, 30000rpm revolving speed, is resuspended using PBS dense Contracting liquid simultaneously dissolves at 4 DEG C overnight, finally 1h is centrifuged in the sucrose of 20%-30%-60% with revolving speed 30000rpm, through removing sucrose It takes the solution 30000rpm between 30%-60% gradient to be centrifuged 1h after step, is the virus-like particle of purifying after precipitating resuspension.

Embodiment 1

Following material is selected to verify virus-like particle preparation method and application of the invention.

H1N1 influenza virus mouse adapted strain A/Changchun/01/2009 (H1N1, group1), H3N2 influenza virus mouse are suitable It answers a plant A/baikalteal/Shanghai/SH-89/2013 (H3N2, group2), H5N1 avian influenza virus A/meerkat/ Shanghai/SH-1/2012(H5N1,clade2.3.2.1;Group1), H7N7 influenza virus mouse adapted strain A/Lesser White-fronted goose/HuNan/412/2010 (H7N7, group2), the above material is by army, Military Medical Science Institute Military affairs veterinary institute Viral Laboratory, thing Medical Research Institute provides；Main agents E.coli DH5 α and E.coli DH10Bac is by this Laboratory saves, and donor plasmid pFastBacdual is purchased from Invitrogen company, and antigen-4 fusion protein gene synthesis is public by gold only intelligence Department provides, and the technological services such as recombinant plasmid gene sequencing and primer synthesis are provided by Changchun library U.S. biology, insect cell transfection examination Agent box is purchased from Invitrogen company, and endonuclease is purchased from Thermo company, and genome extraction kit is public purchased from AXYGEN Department, II SFM of Sf-900 are purchased from Thermo company, and antibiotic etc. is purchased from Suo Laibao company, and HA rabbit is mostly anti-to be purchased from the Divine Land Beijing Yi Qiao Biotech firm, M2 mouse monoclonal antibody, NP mouse monoclonal antibody are purchased from abcam company, and M1 mouse monoclonal antibody is purchased from Suzhou outstanding person benefactor department.

Experimental method is as follows:

The RNA reverse transcription extracted in H5N1 virus is cDNA by S1, using cDNA as template, respectively with HA the and M1 albumen of H5N1 Primer carry out PCR amplification and obtain HA and M1 segment, SEQ ID NO.15, SEQ ID NO.16 are the amplimer of HA segment, SEQ ID NO.17, SEQ ID NO.18 are the amplimer of M1, obtain fusion protein sHA, mHA, cHA using gene chemical synthesis Genetic fragment, PCR amplification process are 95 DEG C of maintenance 30s after 95 DEG C of maintenance 5min, turn 56 DEG C of maintenance 90s, 72 DEG C of maintenance 1min again, 30 circulations are carried out altogether；

Using SmaI and NsiI restriction enzyme site by obtained M1 gene cloning into plasmid pFastBacdual, be transformed into large intestine bar In bacterium DH5 α competent cell, positive bacteria is screened in plated overnight culture, proposes plasmid PCR, sequencing and digestion identification, obtains correct PFastBacdual-M1 plasmid；

S2, using SalI and NotI restriction enzyme site by obtained HA gene cloning into pFastBacdual-M1 plasmid, obtain just True pFastBacdual-HA-M1 plasmid；

With restriction enzyme site EcoRI and NotI obtain pFastBacdual-sHA-M1 plasmid, pFastBacdual-mHA-M1 plasmid, PFastBacdual-cHA-M1 plasmid, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14 point The PCR upstream and downstream primer of sHA, mHA, cHA segment Wei not be identified；

S3, building recombinate rod-shaped plasmid:

DH10Bac competent cell is converted using pFastBacdual-HA-M1 plasmid, the blue hickie bacterium generated from bacterial growth Hickie is selected in falling, and is extracted plasmid PCR identification correctly, is obtained recombinating rod-shaped plasmid HA-M1-Bacmid, be obtained with identical method To sHA-M1-Bacmid, mHA-M1-Bacmid, cHA-M1-Bacmid；

S4 saves recombinant baculovirus:

4 kinds of positive Bacmid plasmids of identification are transferred to Sf9 cell with Cellreagent transfection reagent box, 27 DEG C are cultivated three days Afterwards, cells and supernatant P1 is collected, P1 is inoculated into new Sf9 cell by 3% volume ratio, continues culture to expand virus Virulence cultivates three days collection P2 for supernatant, P2 is inoculated into new Sf9 cell by 3% volume ratio, after continuing culture three days P3 is collected for supernatant, P3 is extracted for the DNA in supernatant and carries out PCR verifying, and observes and records P3 and changes for cellular morphology, with indirect Immunofluorescence carries out expression identification for cell to P3, and Fig. 2A~Fig. 2 D is respectively resisted with HA rabbit glimmering using being immunized indirectly for primary antibody more The inspection figure of the Sf9 cell for 4 kinds of baculovirus infections that light method obtains, Fig. 2 E~Fig. 2 H are respectively to make using M1 mouse monoclonal antibody as primary antibody With the inspection figure of the Sf9 cell of 4 kinds of baculovirus infections of indirect immunofluorescence acquisition, 4 kinds of bars are found by Fig. 2A~Fig. 2 H The P3 of shape virus infection has strong green fluorescence to express for Sf9 cell, illustrates that 4 kinds of virus-like particles have the table of corresponding albumen It reaches；

S5, great expression and purified virus sample particle:

With the quick baculovirus titers detection kit measurement P3 of Takara company for baculovirus titers, with P3 for rod-shaped disease Poison is inoculated with the suspension Sf9 cell of mass propgation by MOI=3, is collected cell culture fluid after 4 days, is removed cell fragment → centrifugal concentrating → sucrose density gradient centrifugation → removes the virus-like particle purified after the series of steps such as sucrose；

(1) using 4 kinds of virus-like particles of Western blot verifying embodiment preparation；

It uses HA rabbit how anti-as primary antibody and carries out Western blot verifying, it is found that VLPs (HA-M1) has band in 70KD or so, MHA-VLPs has that band is as shown in Figure 4 B in 23KD or so, sHA-VLPs and cHA-VLPs 50KD or so have band such as Fig. 4 C, Shown in Fig. 4 E, illustrate that the expressing fusion protein of 4 kinds of VLPs is correct；

M1 mouse monoclonal antibody is used to carry out Western blot verifying as primary antibody, verification result is as shown in Figure 4 A, it is found that 4 kinds of VLPs exist 30KD or so has band, illustrates that the M1 protein expression of 4 kinds of VLPs is correct；

NP, M2 mouse monoclonal antibody is used to carry out Western blot verifying as primary antibody, verification result is as shown in Figure 4 D, finds 4 kinds of VLPs In only sHA-VLPs and cHA-VLPs in 50KD or so have band, illustrate melting for sHA-VLPs and cHA-VLPs in 4 kinds of VLPs Hop protein expression is correct；

(2) 4 kinds of virus-like particles of transmission electron microscope observing are used；

VLPs (HA-M1) is observed under transmission electron microscope, observation result is as shown in Figure 3A, observes that size about 100nm's is spherical Particle, and spherical surrounding has belemnoid structure, illustrates that VLPs (HA-M1) assembling is correct；Resisted with HA for primary antibody to 3 kinds of general viruses more Sample particle sHA-VLPs, mHA-VLPs, cHA-VLPs make immuno-electron microscope detection, and testing result is as shown in Fig. 3 B~Fig. 3 D, in electricity The particle for being under the microscope size 100nm or so to this 3 kinds of virus-like particles, and marked around particle by gold particle, illustrate group 3 kinds of general virus-like particles of Flu-A are dressed up.

Embodiment 2

Select the immune performance of following investigation of materials Flu-A Universal virus-like particle of the present invention, BCA protein detection kit Purchased from Thermo company, 4 kinds of inactivating influenza virus sample particles are voluntarily purified, and HPR marks goat anti-mouse IgG, IgG1, IgG2a Purchased from Southern Biotechnology company, the ELISApot detection kit of mouse IFN-γ and IL-4 are purchased from Mabtech company, TMB are purchased from Sigma company, and the BALB/c female mice of 6-8 week old ties up company, tonneau China purchased from Beijing；

1, detailed process is as follows for experiment:

S1 is immunized and attacks poison:

With the protein concentration of BCA detection kit detection influenza virus-like particles, 0.1mL was injected to mouse muscle at 0 week and 3 weeks Vaccine containing 10 μ g virus-like particles, VLPs (HA-M1) are immunized controls group, and PBS group is Mock group, at second immune two Zhou Houyong isoflurane anesthetized mice, with 10MLD50 it is homologous virus (A/meerkat/Shanghai/SH-1/2012, H5N1, clade2.3.2.1；Group1) and heterologus virus (mouse-adapted A/Changchun/01/2009, H1N1, Group1), (mouse-adapted A/baikal teal/Shanghai/SH-89/2013, H3N2, group2), (mouse- Adapted A/Lesser White-fronted goose/HuNan/412/2010, H7N7, group2) attack Mock group, right According to a group VLPs (HA-M1) and experimental group (sHA-VLPs, mHA-VLPs, cHA-VLPs), carries out mouse collunarium and attack poison, all examinations Test the code of ethics that condition and test procedure all defer to International Association for Pain Research；

S2, specific IgG antibodies detection:

0 week after mouse immune, 3 weeks, eye socket blood sampling in 5 weeks, the blood taken is centrifuged after being placed at room temperature for 2h through 3000rpm 10min isolates upper serum and stores in -80 DEG C；With influenza virus specific IgG, IgG1 in ELISA testing inspection serum And IgG2a, with the H1N1 influenza virus mouse adapted strain A/Changchun/01/2009 (H1N1, group1) of inactivation, H3N2 influenza Viral mouse adapted strain A/baikalteal/Shanghai/SH-89/2013 (H3N2, group2), H5N1 avian influenza virus A/ Meerkat/Shanghai/SH-1/2012 (H5N1, clade 2.3.2.1；Group1), H7N7 influenza virus mouse adapted strain A/ The virus of Lesser White-fronted goose/HuNan/412/2010 (H7N7, group2) is stayed overnight with 5 μ g/mL at 4 DEG C It is coated with 96 orifice plates, closes 2h with 5% skim milk room temperature, 37 DEG C of the blood serum sample incubations diluted are added in 96 orifice plates 1.5h, again with sheep anti-mouse igg, IgG1 and the IgG2a of HRP label in 37 DEG C of incubation 1h after being cleaned with PBST, after being cleaned with PBST TMB is added to terminate reaction in 25 DEG C of effect 30min, the H2SO4 that 50 μ L 0.5mol/L are added later, existed with spectrophotometer Testing result at 450nm；

S3 attacks malicious Protection: attacking and observes the variation of each group mouse weight and survival rate variation after poison in 15 days, tests each group mouse Changes of weight and survival rate situation of change as shown in Fig. 6 A~Fig. 6 H；

S4 detects cell factor using ELISApot, and detailed process is as follows:

The T cells with antigenic specificity of viral activation induce with ELISApot detection by virus-like particle, by IL-4 monoclonal antibody with 96 orifice plate of IFN-γ monoclonal antibody pre-coated, every hole spread 1 × 106 splenocyte for attacking separation in the 4th day after poison, every group of 3 mouse, and every 6 multiple holes of mouse (wherein 3 multiple holes add stimulant, and in addition 3 are control wells), stimulant is the H5N1 virus sample of inactivation Grain, the final concentration of 10 μ g/mL of stimulant cultivate 48h with 37 DEG C of 1640 culture medium containing 10%FBS, 5%CO2, discard thin in hole Born of the same parents carry out enzyme-linked spot detection according to the step of ELISApot detection kit, finally calculate spot with ELISApot reading system Point forms unit；

S5 carries out the titration of virus of lung using following steps:

Lung homogenate presses 1:10, inoculated into chick embryo after 1:102 ..., 1:1010 dilution, and each dilution is inoculated with SPF grades of chickens of 3 piece of 9 age in days Embryo, every piece of egg are inoculated with 100 μ L lung homogenates, seal up after adhesive sticker the detection allantoic fluid blood clotting after 37 DEG C of incubation 48h, 48h as a result, Every piece of egg takes 50 μ L allantoic fluids and the chicken erythrocyte suspension of 50 μ L1% to mix, and is observed and recorded after 15min as a result, and using Reed- Muench method calculates the chicken embryo median infective dose EID50 of every mouse lungs lapping liquid.

2, experimental result

(1) the immune response testing result that Flu-A Universal virus-like particle excites in Mice Body；

The specific antibody water for homologous virus and heterologus virus in the mice serum of immune 0th, 3,5 week of detection respectively It is flat, the testing result of homologous virus (A/meerkat/Shanghai/SH-1/2012, H5N1, clade) as shown in Figure 5 C, 3 kinds Heterologus virus (mouse-adapted A/Changchun/01/2009, H1N1, group1), (mouse-adapted A/ Baikalteal/Shanghai/SH-89/2013, H3N2, group2), (mouse-adapted A/Lesser White- Fronted goose/HuNan/412/2010, H7N7, group2) respectively as shown in Fig. 5 A, Fig. 5 B, Fig. 5 D；Fig. 5 A~Fig. 5 D Middle specific IgG antibodies level increases with the growth of immune all numbers, shows the generation for having specific antibody and control group VLPs (HA-M1) is compared, and the sHA-VLPs as shown in Fig. 5 A and Fig. 5 D has significantly for the specific antibody level of H1N1 and H7N7 Property increase, mHA-VLPs as shown in Figure 5 B has conspicuousness raising, cHA- as shown in Figure 5 C for the specific antibody level of H3N2 VLPs has conspicuousness raising for the specific antibody level of H5N1, and sHA-VLPs group stimulation body produces in 4 kinds of virus-like particles The raw specific antibody for 4 kinds of influenza viruses is held at high level, and sHA-VLPs has better broad spectrum activity；To all IgG1 the and IgG2a antibody subtype of 5th week serum of immune group is detected, and as shown in fig. 5e, all immune groups are directed to H5N1 The titre of the titre ratio IgG2a of the IgG1 of antigen is high, illustrates that virus generates the antibody of specific immune response based on IgG1, and In the immune effect assessment of VLPs, for the horizontal high explanation of IgG1/IgG2a compared with cellular immunity, the main induction body fluid of VLPs is immune Generation play a protective role.

(2) Flu-A Universal virus-like particle attacks malicious Protective strategy；

For the changes of weight rate and survival rate change curve that H1N1 attacks poison group mouse as shown in Fig. 6 A, Fig. 6 B, H3N2 attacks poison group mouse Changes of weight rate and survival rate change curve as shown in Fig. 6 C, Fig. 6 D, H5N1 attack poison group mouse changes of weight rate and existence For rate change curve as shown in Fig. 6 E, Fig. 6 F, H7N7 attacks the changes of weight rate and survival rate change curve such as Fig. 6 G, figure of poison group mouse Shown in 6H, the changes of weight rate difference that poison group mouse is respectively attacked known to Fig. 6 A, Fig. 6 C, Fig. 6 E, Fig. 6 G is smaller, but attacks poison in H3N2 Group mHA-VLPs shows lesser body weight loss；

The H1N1 known to Fig. 6 B attacks malicious group other than the survival rate of sHA-VLPs and mHA-VLPs is respectively 60% and 20%, remaining Each group is without survival；The survival rate that the H3N2 known to Fig. 6 D attacks poison group mHA-VLPs group is 60%, sHA-VLPs group and control group The survival rate of VLPs (HA-M1) is 20%, cHA-VLPs and Mock group without survival；The H5N1 known to Fig. 6 F, which is attacked in malicious group, to be compareed Group VLPs (HA-M1), mHA-VLPs group and cHA-VLPs group survival rate are that 40%, sHA-VLPs group survival rate is 20%, Mock Group is without survival；It is 80% that the H7N7 known to Fig. 6 H, which attacks control group VLPs (HA-M1) and cHA-VLPs group survival rate in malicious group, SHA-VLPs group and mHA-VLPs group survival rate are respectively 60% and 50%, and Mock group is without survival；It is examined by proportion of weight to height and survival rate Result is surveyed it is found that sHA-VLPs and two groups of the mHA-VLPs broad spectrum protection effects for homologous and heterologous influenza virus are preferable.

(3) the general virus-like particle of Flu-A can activate special T lymphocyte；

The 4th day separation splenic lymphocytes after mouse attacks poison, and stimulated with H5N1 inactivation antigen, then detect the IL- of specificity 4(Th2 type immune response) secretion situation testing result it is as shown in Figure 7 A, under the stimulation of inactivation of viruses, cHA-VLPs group phase Higher IL-4 level can be generated than control group VLPs (HA-M1), the secretion situation detection of IFN-γ (immune response of Th1 type) is such as Shown in Fig. 7 B, VLPs (HA-M1) can generate that higher IFN-γ is horizontal to mHA-VLPs group compared to the control group, in contrast IL-4 Expression quantity is higher than the expression quantity of IFN-γ in all immune groups, illustrates T lymphocyte based on Th2 type cellular immunity.

(4) Flu-A Universal virus-like particle can inhibit the duplication of influenza virus；

As shown in figure 8, take attack poison after the 4th day mouse lung, the virus of various influenza virus is carried out in 9 age in days SPF chicken embryos Titer determination, in addition to H5N1 virus, the experimental group of remaining the 3 kinds of heterologus virus general trend that virus titer declines compared to the control group Malicious Protection is consistent with attacking；In H5N1 virus titer determination, sHA-VLPs group and cHA-VLPs group virus titer are than control Group VLPs (HA-M1) is decreased significantly, and shows that sHA-VLPs group and cHA-VLPs group particle are able to suppress influenza virus in mouse Duplication in lung.

In 3 kinds of Flu-A Universal virus-like particles, the combined immune protective effect of sHA-VLPs group is preferable, sHA-VLPs Group can attack malicious protective rate and specific IgG antibodies for a variety of heterologous influenza viruses generations are high-caliber, inhibit influenza virus Duplication, the immanoprotection action of general virus-like particle is based on humoral immunity.

Each embodiment in this specification is all made of relevant mode and describes, same and similar portion between each embodiment Dividing may refer to each other, and each embodiment focuses on the differences from other embodiments.Especially for system reality For applying example, since it is substantially similar to the method embodiment, so being described relatively simple, related place is referring to embodiment of the method Part explanation.

The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the scope of the present invention.It is all Any modification, equivalent replacement, improvement and so within the spirit and principles in the present invention, are all contained in protection scope of the present invention It is interior.

Sequence table

<110>Academy of Military Sciences's military medical research institute military affairs veterinary institute

<120>general virus-like particle of Flu-A and its preparation method and application

<130> 2019.4.22

<160> 18

<170> PatentIn version 3.3

<210> 1

<211> 1400

<212> DNA

<213>artificial sequence (Artificial sequence)

<400> 1

ccggaattca tgaaattcct ggtcaacgtg gctctggtgt tcatggtcgt ctacatctcc 60

tatatctacg ccgctgctgg tggtggtggt tccgacctga tcttcctggc ccgcagcgct 120

ctgattctgc gtggttccgt ggctcacaag tcctgtgctg ccgctcctgg tatcgctgac 180

atcgaggacc tgaccctgct ggctcgctcc atggtggtgg tgcgtcctgc tgccgcttcc 240

ctgctgactg aagtcgaaac ccccatccgt aacgagtggg gcagccgctc caatgattcc 300

tccgatgctg ctgctagcct cctgactgag gtggaaaccc ccattcgcaa cgagtggggt 360

tgccgctgca acggttcctc cgatgctgct gctagcctgc tgaccgaagt ggaaacccct 420

actcgtagcg aatgggagtc ccgcagctcc gactccagcg atgccgctgc tagcctgctg 480

accgaagtcg agacccccac ccgcaacggt tgggagtgca agtgttccga cagcagcgat 540

gctgccgctt ccctgctgac cgaggtcgaa accctgaccc gcaatggttg gggttgccgt 600

tgcagcgact cctccgacgc tgccgctggc tgtaacaaca acaacgctgc tgccggttgt 660

aataataata acgctgccgc cggctgcaac aataacaacg gtggcggcgg ctccggtctg 720

ttcggtgcca ttgctggttt catcgaaggc ggctggcagg gtatggtgga cggctggtac 780

ggctatcacc actccaacga gcagggttcc ggctacgccg ctgacaagga gtccacccag 840

aaagctatcg acggcgtcac caacaaggtc aactccatca ttgacaagat gaacactcaa 900

ttcgaagccg tcggccgcga gttcaacaac ctggagcgcc gtatcgagaa cctgaacaag 960

aagatggagg atggcttcct cgatgtctgg acttacaacg ccgagctgct ggtgctgatg 1020

gagaatggtc gcaccctgga cttccacgac agcaacgtga agaacctgta cgataaggtc 1080

cgcctgcagc tgaaggacaa cgccaaagag ctgggtaacg gctgcttcga gttttaccac 1140

aagtgcaata acgagtgcat ggagtccgtc cgcaacggca cctatgacta tccccagtac 1200

tccgaagagg cccgcctgaa gcgcgaggaa atcagcggcg tgaagctgga gagcatcggc 1260

atctaccaga ttctgagcat ttactccact gtcgcctcca gcctggtcct ggccatcatg 1320

atggccggtc tgtccctgtg gatgtgctcc aacggcagcc tgcagtgccg catctgcatc 1380

taagcggccg ctaaactatt 1400

<210> 2

<211> 791

<212> DNA

<213>artificial sequence (Artificial sequence)

<400> 2

ccggaattca tgaagttttt agtgaacgtg gctttagtgt tcatggtggt gtacatctcc 60

tacatctacg ccgctgctgg tggtggtggt tccatgggtt cctcctcctc cggtctggtg 120

cctcgcggtt cccacatgga gcaagttgac accatcatgg agaagaacgt gaccgtgacc 180

cacgctcaag atattttaga gaagacccac ggctccgcca acagcagcat gcccttccac 240

aacatccacc ctttaaccat cggtgagtgc cccaagtacg tgaagagcaa caagctggtg 300

ctggccaccg gcctccgtaa tggttccgct ggctccgcca cccagaaggc tatcgacggt 360

gtgaccaaca aggtgaactc catcatcgac aagatgaaca cccagttcga ggctgtgggc 420

cgcgagttca acaatttaga gcgtcgcatc gagaatttaa acaagaagat ggaggacggc 480

ttcctcgacg tgtggaccta caacgccgag ctgctggtgc tgatggagaa cggccgcact 540

ttagacttcc acgactccca aggtaccggc tacatccccg aagctcctcg cgacggtcaa 600

gcctatgtgc gcaaggacgg cgagtgggtg ctgctgtcca cctttttagg cggtggtggc 660

agcattttaa gcatctacag cactgtggct agctctttag tcctcgctat tatgatggct 720

ggtttatctt tatggatgtg ctccaacggt tctttacagt gccgcatctg catctaagcg 780

gccgctataa a 791

<210> 3

<211> 1442

<212> DNA

<213>artificial sequence (Artificial sequence)

<400> 3

ccggaattca tgaagttttt agtgaacgtg gctttagtgt tcatggtggt gtacatctcc 60

tacatctacg ccgctgctgg tggtggtggt tccatgggtt cctcctcctc cggtctggtg 120

cctcgcggtt cccacatgga gcaagttgac accatcatgg agaagaacgt gaccgtgacc 180

cacgctcaag atattttaga gaagacccac ggcggtggcg gttctttact gatcgacggc 240

accgcttctt tatcccccgg tatgatgatg ggcatgttca acatgctgtc caccgtttta 300

ggtgtgtcca ttttaaattt aggtcaagct gctgccgatt taatcttttt agctcgttcc 360

gctctgattt tacgcggttc cgtggctcac aagtcttgtg ctgctgctcc cggtatcgct 420

gacatcgagg atttaacttt actggctcgc tccatggtgg tggtgcgtcc cgctgctgct 480

tctttattga ccgaggtgga gacccccatt cgcaacgagt ggggttcccg cagcaacgac 540

tcttccgacg ctgctgcctc tttattgacc gaagtggaga cccctatccg caacgaatgg 600

ggttgccgct gcaacggttc ttctgacgct gccgcttccc tcttaaccga agttgaaacc 660

cctacccgtt ccgagtggga gtcccgctct tccgattcct ccgatgctgc tgcttcttta 720

ttaaccgagg tcgagacccc cacccgcaac ggctgggagt gcaagtgttc cgattcttct 780

gatgccgctg cctctttatt aactgaagtg gaaactttaa cccgtaacgg ctggggctgt 840

cgttgctccg attccagcga cgccgccgcc aacagcagca tgcccttcca caacatccac 900

cctttaacca tcggtgagtg ccccaagtac gtgaagagca acaagctggt gctggccacc 960

ggcctccgta atggttccgc tggctccgcc acccagaagg ctatcgacgg tgtgaccaac 1020

aaggtgaact ccatcatcga caagatgaac acccagttcg aggctgtggg ccgcgagttc 1080

aacaatttag agcgtcgcat cgagaattta aacaagaaga tggaggacgg cttcctcgac 1140

gtgtggacct acaacgccga gctgctggtg ctgatggaga acggccgcac tttagacttc 1200

cacgactccc aaggtaccgg ctacatcccc gaagctcctc gcgacggtca agcctatgtg 1260

cgcaaggacg gcgagtgggt gctgctgtcc acctttttag gcggtggtgg cagcatttta 1320

agcatctaca gcactgtggc tagctcttta gtcctcgcta ttatgatggc tggtttatct 1380

ttatggatgt gctccaacgg ttctttacag tgccgcatct gcatctaagc ggccgctata 1440

aa 1442

<210> 4

<211> 1704

<212> DNA

<213>avian influenza virus (Avian influenza virus)

<400> 4

atggagaaaa tagttcttct ctttacaaca atcagccttg tcaaaagcga tcatatttgc 60

attggttatc atgcaaataa ctcgacagag caggttgaca caataatgga gaagaacgtt 120

actgttacac atgcccaaga catactggaa aagacacaca acgggaagct ctgcgatcta 180

aatggagtga agcctctgat tttaaaagat tgtagtgtag caggatggct cctcggaaat 240

ccattgtgtg acgaattcac caatgtgcca gaatggtctt acatagtaga gaaggccaat 300

ccagccaatg acctctgtta cccagggaat ttcaacgatt atgaggaatt gaaacaccta 360

ttgagcagga taaaccattt tgagaaaata cagatcatcc ccaaagattc ttggtcagat 420

catgaagcct cattgggggt gagcgcggca tgttcatacc agggaaattc ctccttcttc 480

agaaatgtgg tgtggcttat caaaaaggac aatgcatacc caacaataaa gaaaggctac 540

aataatacca accgagaaga tctcttgata ctgtggggga tccaccatcc taatgatgag 600

gcagagcaga caaggctcta tcaaaaccca actacctata tttccattgg gacttcaaca 660

ctaaaccaga gattggtacc aaaaatagcc actagatcca aaataaacgg gcaaagtggc 720

aggatagatt tcttctggac aattttaaaa ccgaatgacg caatccattt cgagagcaat 780

ggaaatttca ttgctccaga atatgcatac aaaattgtca agaaaggaga ctccacaatc 840

atgagaagtg aagtggaata tggtaactgc aacaccaggt gtcagactcc aataggggcg 900

ataaactcta gcatgccatt ccacaacata caccctctca ctatcggaga atgtcccaaa 960

tatgtgaaat caaacaaatt agtccttgca actgggctca gaaatagtcc tcaaagagag 1020

agaagaagaa aaagaggact gtttggagct atagcaggtt ttatagaggg aggatggcag 1080

ggaatggtag atggttggta tgggtaccac cacagcaatg aacaggggag tggttacgct 1140

gcagacaaag aatctactca aaaggcgata gacggagtca ccaataaggt caattcgatc 1200

attgacaaaa tgaacactca gtttgaggct gtaggaaggg aatttaataa cttagagagg 1260

agaatagaaa atttaaacaa gaagatggaa gacggattcc tagatgtctg gacttataat 1320

gctgaacttc tggttctcat ggagaatggg agaactctag acttccatga ctcaaatgtc 1380

aagaaccttt acgataaggt ccgactacag cttaaggata atgcaaaaga gctgggaaac 1440

ggttgtttcg agttctatca caaatgtaat aatgaatgta tggaaagtgt aagaaacggg 1500

acgtatgact acccgcagta ttcagaagaa gcaagattaa aaagagagga aataagtgga 1560

gtaaaactgg aatcaatagg aatctaccaa atactgtcaa tttattcaac agtggcgagt 1620

tccctagtgc tggcaatcat gatggctggt ctatctttat ggatgtgttc caacgggtcg 1680

ttacagtgca gaatttgcat ttaa 1704

<210> 5

<211> 759

<212> DNA

<213>avian influenza virus (Avian influenza virus)

<400> 5

atgagtcttc taaccgaggt cgaaacgtac gttctctcta tcatcccatc aggccccctc 60

aaagccgaga tcgcgcagaa acttgaggat gtatttgcag gaaagaacac tgatctcgag 120

gctctcatgg agtggctaaa gacaagacca atcctgtcac ctctgactaa agggatcttg 180

ggatttgtat tcacgctcac cgtgcccagt gagcgaggac tgcagcgtag acgttttgtc 240

cagaatgccc taaatggaaa tggagatcca aataacatgg atagggcagt taagctatat 300

aagaagctga aaagagaaat aacattccat ggagctaagg aggtcgcact cagttactca 360

accggtgcac ttgccagttg catgggtctc atatacaaca ggatgggaac ggtgactaca 420

gaagtggctt ttggcctagt gtgtgccact tgtgagcaga ttgcagattc acagcatcgg 480

tctcacagac agatggcaac catcaccaac ccactaatca ggcatgagaa cagaatggtg 540

ctggccagca ctacagctaa ggccatggag cagatggcgg gatcaagcga gcaggcagca 600

gaagccatgg aggtcgccaa tcaggctaga cagatggtgc aggcgatgag gacaattggg 660

actcatccta actctagtgc tggtctgaga gataatcttc ttgaaaattt gcaggcctac 720

cagaaacgaa tgggagtgca gatgcagcga ttcaagtga 759

<210> 6

<211> 465

<212> PRT

<213>artificial sequence (Artificial sequence)

<400> 6

Pro Glu Phe Met Lys Phe Leu Val Asn Val Ala Leu Val Phe Met Val

1 5 10 15

Val Tyr Ile Ser Tyr Ile Tyr Ala Ala Ala Gly Gly Gly Gly Ser Asp

20 25 30

Leu Ile Phe Leu Ala Arg Ser Ala Leu Ile Leu Arg Gly Ser Val Ala

35 40 45

His Lys Ser Cys Ala Ala Ala Pro Gly Ile Ala Asp Ile Glu Asp Leu

50 55 60

Thr Leu Leu Ala Arg Ser Met Val Val Val Arg Pro Ala Ala Ala Ser

65 70 75 80

Leu Leu Thr Glu Val Glu Thr Pro Ile Arg Asn Glu Trp Gly Ser Arg

85 90 95

Ser Asn Asp Ser Ser Asp Ala Ala Ala Ser Leu Leu Thr Glu Val Glu

100 105 110

Thr Pro Ile Arg Asn Glu Trp Gly Cys Arg Cys Asn Gly Ser Ser Asp

115 120 125

Ala Ala Ala Ser Leu Leu Thr Glu Val Glu Thr Pro Thr Arg Ser Glu

130 135 140

Trp Glu Ser Arg Ser Ser Asp Ser Ser Asp Ala Ala Ala Ser Leu Leu

145 150 155 160

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

165 170 175

Asp Ser Ser Asp Ala Ala Ala Ser Leu Leu Thr Glu Val Glu Thr Leu

180 185 190

Thr Arg Asn Gly Trp Gly Cys Arg Cys Ser Asp Ser Ser Asp Ala Ala

195 200 205

Ala Gly Cys Asn Asn Asn Asn Ala Ala Ala Gly Cys Asn Asn Asn Asn

210 215 220

Ala Ala Ala Gly Cys Asn Asn Asn Asn Gly Gly Gly Gly Ser Gly Leu

225 230 235 240

Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Gln Gly Met Val

245 250 255

Asp Gly Trp Tyr Gly Tyr His His Ser Asn Glu Gln Gly Ser Gly Tyr

260 265 270

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

275 280 285

Lys Val Asn Ser Ile Ile Asp Lys Met Asn Thr Gln Phe Glu Ala Val

290 295 300

Gly Arg Glu Phe Asn Asn Leu Glu Arg Arg Ile Glu Asn Leu Asn Lys

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

Lys Glu Leu Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asn Asn

370 375 380

Glu Cys Met Glu Ser Val Arg Asn Gly Thr Tyr Asp Tyr Pro Gln Tyr

385 390 395 400

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

405 410 415

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

420 425 430

Ser Ser Leu Val Leu Ala Ile Met Met Ala Gly Leu Ser Leu Trp Met

435 440 445

Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile Cys Ile Ala Ala Ala Lys

450 455 460

Leu

465

<210> 7

<211> 262

<212> PRT

<213>artificial sequence (Artificial sequence)

<400> 7

Pro Glu Phe Met Lys Phe Leu Val Asn Val Ala Leu Val Phe Met Val

1 5 10 15

Val Tyr Ile Ser Tyr Ile Tyr Ala Ala Ala Gly Gly Gly Gly Ser Met

20 25 30

Gly Ser Ser Ser Ser Gly Leu Val Pro Arg Gly Ser His Met Glu Gln

35 40 45

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

50 55 60

Ile Leu Glu Lys Thr His Gly Ser Ala Asn Ser Ser Met Pro Phe His

65 70 75 80

Asn Ile His Pro Leu Thr Ile Gly Glu Cys Pro Lys Tyr Val Lys Ser

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Asn Gly Arg Thr Leu Asp Phe His Asp Ser Gln Gly Thr Gly Tyr Ile

180 185 190

Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys Asp Gly Glu

195 200 205

Trp Val Leu Leu Ser Thr Phe Leu Gly Gly Gly Gly Ser Ile Leu Ser

210 215 220

Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Ala Ile Met Met Ala

225 230 235 240

Gly Leu Ser Leu Trp Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile

245 250 255

Cys Ile Ala Ala Ala Ile

260

<210> 8

<211> 479

<212> PRT

<213>artificial sequence (Artificial sequence)

<400> 8

Pro Glu Phe Met Lys Phe Leu Val Asn Val Ala Leu Val Phe Met Val

1 5 10 15

Val Tyr Ile Ser Tyr Ile Tyr Ala Ala Ala Gly Gly Gly Gly Ser Met

20 25 30

Gly Ser Ser Ser Ser Gly Leu Val Pro Arg Gly Ser His Met Glu Gln

35 40 45

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

50 55 60

Ile Leu Glu Lys Thr His Gly Gly Gly Gly Ser Leu Leu Ile Asp Gly

65 70 75 80

Thr Ala Ser Leu Ser Pro Gly Met Met Met Gly Met Phe Asn Met Leu

85 90 95

Ser Thr Val Leu Gly Val Ser Ile Leu Asn Leu Gly Gln Ala Ala Ala

100 105 110

Asp Leu Ile Phe Leu Ala Arg Ser Ala Leu Ile Leu Arg Gly Ser Val

115 120 125

Ala His Lys Ser Cys Ala Ala Ala Pro Gly Ile Ala Asp Ile Glu Asp

130 135 140

Leu Thr Leu Leu Ala Arg Ser Met Val Val Val Arg Pro Ala Ala Ala

145 150 155 160

Ser Leu Leu Thr Glu Val Glu Thr Pro Ile Arg Asn Glu Trp Gly Ser

165 170 175

Arg Ser Asn Asp Ser Ser Asp Ala Ala Ala Ser Leu Leu Thr Glu Val

180 185 190

Glu Thr Pro Ile Arg Asn Glu Trp Gly Cys Arg Cys Asn Gly Ser Ser

195 200 205

Asp Ala Ala Ala Ser Leu Leu Thr Glu Val Glu Thr Pro Thr Arg Ser

210 215 220

Glu Trp Glu Ser Arg Ser Ser Asp Ser Ser Asp Ala Ala Ala Ser Leu

225 230 235 240

Leu Thr Glu Val Glu Thr Pro Thr Arg Asn Gly Trp Glu Cys Lys Cys

245 250 255

Ser Asp Ser Ser Asp Ala Ala Ala Ser Leu Leu Thr Glu Val Glu Thr

260 265 270

Leu Thr Arg Asn Gly Trp Gly Cys Arg Cys Ser Asp Ser Ser Asp Ala

275 280 285

Ala Ala Asn Ser Ser Met Pro Phe His Asn Ile His Pro Leu Thr Ile

290 295 300

Gly Glu Cys Pro Lys Tyr Val Lys Ser Asn Lys Leu Val Leu Ala Thr

305 310 315 320

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

325 330 335

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

340 345 350

Phe Glu Ala Val Gly Arg Glu Phe Asn Asn Leu Glu Arg Arg Ile Glu

355 360 365

Asn Leu Asn Lys Lys Met Glu Asp Gly Phe Leu Asp Val Trp Thr Tyr

370 375 380

Asn Ala Glu Leu Leu Val Leu Met Glu Asn Gly Arg Thr Leu Asp Phe

385 390 395 400

His Asp Ser Gln Gly Thr Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly

405 410 415

Gln Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe

420 425 430

Leu Gly Gly Gly Gly Ser Ile Leu Ser Ile Tyr Ser Thr Val Ala Ser

435 440 445

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

450 455 460

Ser Asn Gly Ser Leu Gln Cys Arg Ile Cys Ile Ala Ala Ala Ile

465 470 475

<210> 9

<211> 567

<212> PRT

<213>avian influenza virus (Avian influenza virus)

<400> 9

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

Leu Gly Val Ser Ala Ala Cys Ser Tyr Gln Gly Asn Ser Ser Phe Phe

145 150 155 160

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

165 170 175

Lys Lys Gly Tyr Asn Asn Thr Asn Arg Glu Asp Leu Leu Ile Leu Trp

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

Leu Gln Cys Arg Ile Cys Ile

565

<210> 10

<211> 252

<212> PRT

<213>avian influenza virus (Avian influenza virus)

<400> 10

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

1 5 10 15

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

20 25 30

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

35 40 45

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

85 90 95

Val Lys Leu Tyr Lys Lys Leu Lys Arg Glu Ile 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 Val Thr Thr Glu Val Ala Phe

130 135 140

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

145 150 155 160

Ser His Arg Gln Met Ala Thr Ile Thr Asn Pro 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 Asn Gln

195 200 205

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

210 215 220

Ser Ser Ala Gly Leu Arg Asp Asn Leu Leu Glu Asn Leu Gln Ala Tyr

225 230 235 240

Gln Lys Arg Met Gly Val Gln Met Gln Arg Phe Lys

245 250

<210> 11

<211> 34

<212> DNA

<213>artificial sequence (Artificial sequence)

<400> 11

ccggaattca tgaaattcct ggtcaacgtg gctc 34

<210> 12

<211> 39

<212> DNA

<213>artificial sequence (Artificial sequence)

<400> 12

aaatatgcgg ccgcttagat gcagatgcgg cactgcagg 39

<210> 13

<211> 34

<212> DNA

<213>artificial sequence (Artificial sequence)

<400> 13

cgcggatcca tgaagttttt agtcaacgtc gctc 34

<210> 14

<211> 57

<212> DNA

<213>artificial sequence (Artificial sequence)

<400> 14

aaatatgcgg ccgcttagtg atgatggtgg tgatggatgc agatgcggca ctgtaaa 57

<210> 15

<211> 35

<212> DNA

<213>artificial sequence (Artificial sequence)

<400> 15

acgcgtcgac atggagaaaa tagttcttct cttta 35

<210> 16

<211> 39

<212> DNA

<213>artificial sequence (Artificial sequence)

<400> 16

aaatatgcgg ccgcttaaat gcaaattctg cactgtaac 39

<210> 17

<211> 34

<212> DNA

<213>artificial sequence (Artificial sequence)

<400> 17

tcccccggga tgagtcttct aaccgaggtc gaaa 34

<210> 18

<211> 34

<212> DNA

<213>artificial sequence (Artificial sequence)

<400> 18

tgcatgcatt cacttgaatc gctgcatctg cact 34

Claims (7)

1. the general virus-like particle of Flu-A, which is characterized in that virus-like particle is that 3 kinds of fusion proteins are fitted into exhibition respectively Show made of the influenza virus M1 protein surface, is denoted as sHA-VLPs, mHA-VLPs, cHA-VLPs respectively；

3 kinds of fusion proteins are sHA albumen, mHA albumen and cHA albumen respectively；

The sHA protein gene sequence is as shown in SEQ ID NO.1, the amino acid sequence of sHA albumen such as SEQ ID NO.6 institute Show；

The gene order of the mHA albumen is as shown in SEQ ID NO.2, the amino acid sequence of mHA albumen such as SEQ ID NO.7 institute Show；

The gene order of the cHA is as shown in SEQ ID NO.3, and the amino acid sequence of cHA is as shown in SEQ ID NO.8.

2. the preparation method of the general virus-like particle of Flu-A as described in claim 1, which is characterized in that including following Step:

Step 1, the encoding gene that influenza virus M1 is obtained by PCR method, to the encoding gene and pFastBacdual of M1 albumen Gene carry out multiple cloning sites analysis, select to provide on pFastBacdual and do not have in target fragment two it is restricted in Enzyme cutting, the primer of purpose of design segment recycle target fragment after target fragment amplification, double digestion, target fragment are inserted into In multiple cloning sites after insect cell expression vector p10 promoter, bacillus coli DH 5 alpha competent cell is converted, is obtained PFastBacdual-M1 plasmid；

Step 2, by gene synthesis technology obtain sHA albumen, mHA albumen and cHA albumen encoding gene, by sHA albumen, The gene of the encoding gene of mHA albumen and cHA albumen and pFastBacdual-M1 plasmid carries out multiple cloning sites analysis, selection Two restriction enzymes for providing on pFastBacdual-M1 plasmid and not having in target fragment recycle fusion after double digestion The target fragment of albumen, the multiple cloning sites after target fragment to be inserted into pFastBacdual-M1 plasmid PH promoter, then turns Change bacillus coli DH 5 alpha competent cell, obtains pFastBacdual-M1- fusion protein shuttle plasmid；

Step 3, pFastBacdual-M1- fusion protein shuttle plasmid is converted into Escherichia coli DH10Bac competent cell, obtained To recombinant baculovirus plasmid Bacmid, using recombinant baculovirus plasmid Bacmid transfection Sf 9 insect cell, rescue is weighed Group baculoviral, the convergence degree of Sf9 insect cell reaches 80% or more when transfection；

Step 4, recombinant baculovirus is inoculated with Sf9 insect cell according to MOI=3, it is logical to obtain Flu-A for harvest supernatant after three days Use virus-like particle.

3. the preparation method of the general virus-like particle of Flu-A according to claim 2, which is characterized in that the M1 The restriction enzyme site of albumen and pFastBacdual carrier is SmaI and NsiI, the encoding gene of fusion protein sHA, mHA and cHA with The restriction enzyme site of pFastBacdual-M1 plasmid is EcoRI and NotI.

4. the preparation method of the general virus-like particle of Flu-A according to claim 2, which is characterized in that the step Recombinant baculovirus is obtained in rapid 3, and specific step is as follows: 10ng pFastBacdual-M1- fusion protein shuttle plasmid is added Enter in DH10Bac competent cell, after ice bath 30min at 42 DEG C heat shock 45s, then ice bath 2min, nonreactive LB culture is added Base shaking table at 37 DEG C, revolving speed 200rpm vibrates 4h, and 100 μ L is taken to vibrate solution coating to the three anti-LB plates containing X-gal, IPTG On, 48h is cultivated at 37 DEG C, the hickie that blue hickie screens is recombinant baculovirus plasmid Bacmid.

5. the preparation method of the general virus-like particle of Flu-A according to claim 2, which is characterized in that the step The concentration of Sf9 insect cell is 2 × 106/mL or more when rapid 4 inoculation recombinant baculovirus.

6. the preparation method of the general virus-like particle of Flu-A according to claim 2, which is characterized in that the first Sucrose density gradient centrifugation is selected in the purifying of the general virus-like particle of type influenza, and detailed process is as follows: inoculation is recombinated bar After the Insect cellculture 72h of shape virus, collects suspension cell culture liquid and be centrifuged 20min acquisition under 4 DEG C, 5000rpm revolving speed Supernatant, by supernatant, ultracentrifugation 1h is concentrated under 4 DEG C, 30000rpm revolving speed, concentrate is resuspended using PBS and at 4 DEG C Dissolution overnight is finally centrifuged 1h in the sucrose of 20%-30%-60% with revolving speed 30000rpm, takes 30%- after being gone sucrose step Solution 30000rpm between 60% gradient is centrifuged 1h, is the virus-like particle of purifying after precipitating resuspension.

7. the general virus-like particle of Flu-A as described in claim 1, which is characterized in that be used to prepare prevention specificity The vaccine of influenza, the homologous virus of brave influenza and heterologus virus.