CN110272473B

CN110272473B - Influenza A universal virus-like particle and preparation method and application thereof

Info

Publication number: CN110272473B
Application number: CN201910618919.XA
Authority: CN
Inventors: 高玉伟; 任志广; 夏咸柱; 张亚敏; 赵永坤; 王铁成; 李元果; 王化磊; 冯娜; 孙伟洋; 杨松涛; 陈明涛
Original assignee: Military Veterinary Research Institute Academy Of Military Medical Sciences
Current assignee: Military Veterinary Research Institute Academy Of Military Medical Sciences
Priority date: 2019-07-10
Filing date: 2019-07-10
Publication date: 2021-03-02
Anticipated expiration: 2039-07-10
Also published as: CN110272473A

Abstract

The invention provides a universal influenza A virus-like particle and a preparation method and application thereof, the virus-like particle is prepared by respectively embedding 3 fusion proteins on the surface of influenza virus M1, and the fusion proteins are respectively marked as sHA-VLPs, mHA-VLPs and cHA-VLPs, and are respectively formed by highly conserved sequences or partial recombination of influenza virus HA protein, M2e protein, NP protein and PBI protein; the virus-like particle can be used for preventing specific influenza, tiger influenza homologous virus, heterologous virus and the like; the preparation method of the virus-like particles is simple, low in cost, good in immunogenicity and cross protection of the virus-like particles, capable of meeting the preparation requirements of mass influenza vaccines, safe in biology and capable of stimulating the immunity of a matrix to various influenza viruses.

Description

Influenza A universal virus-like particle and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biological pharmacy, and particularly relates to a universal influenza A virus-like particle as well as a preparation method and application thereof.

Background

Seasonal influenza is epidemic in the coldest season of temperate regions, is epidemic all the year round in tropical and subtropical regions, about one billion people are infected with influenza virus all the year round, wherein 3-5 million people have serious complications and 30-50 million people die, and the influenza virus is continuously varied, when a novel influenza virus appears, most people have no immunity, especially infants, old people, pregnant women and susceptible groups with low immune systems, the novel influenza virus avoids the immune system of the organism, is easy to infect people and spread among people to form pandemic diseases, and influenza pandemics in the world occur for many times historically; in 1918 Spain H1N1 influenza pandemic caused 4000 ten thousand people affected, in 1933 pathogen of influenza was identified for the first time, in 1957H 2N2 Asian influenza, in 1968H 3N2 hong Kong influenza, in 2009H 1N1 caused influenza virus pandemic, in 2017-18 the US national center for disease prevention and control reported 3 thousand influenza attacks in the United states, 171 people died due to influenza, compared with 2003 + 2004, in 2017 + 2018 patients in each age group all suffered from aggravation, and incidence rate and death rate of influenza virus are greatly increased.

Therefore, the continuous improvement of the current influenza vaccine and the improvement of the protective power, the broad spectrum and the multi-season coverage rate of the vaccine are the key points of the current influenza vaccine research, and in order to deal with the large-scale influenza which is likely to appear at any time, the development of a universal influenza vaccine with one-needle multi-prevention and broad-spectrum protective activity is urgently needed.

In order to cope with the continuous change of influenza virus antigens, the world health organization establishes a global influenza monitoring and response system (GISRS), and the consulting committee of the world health organization predicts the antigen drift according to the data monitored by the global influenza monitoring and response system every year to determine whether candidate strains of the vaccine are updated; currently marketed influenza virus vaccines mainly include: the inactivated influenza vaccine is prepared by inactivating complete virus propagated from chicken embryos by using formalin or beta-propiolactone, further cracking and purifying the inactivated complete virus, and removing foreign antigens and non-specific antigens, wherein the inactivated influenza vaccine used in the current market has the defects of long response time, high dependence on the chicken embryos, easiness in mutation of introduced allergen virus, incapability of propagating highly pathogenic strains and the like, and the candidate strains of the current influenza vaccine are obtained by prediction and can not be matched with actual epidemic influenza strains to cause the problems of low vaccine efficacy and the like; the influenza vaccine prepared by the influenza virus-like particles expressed by the insect baculovirus expression system becomes a hotspot of current research and development, the insect baculovirus expression system can simultaneously express a plurality of structural proteins of influenza virus and complete assembly to form virus-like particles, the virus-like particles are assembled by specific proteins of the virus, but do not contain the nucleic acid component of the virus, and are similar to the body virus in the outer surface antigen conformation, so that the humoral and cellular immune stimulation is caused, and the influenza vaccine has the advantages of high biological safety, low cost and high yield.

The influenza structural protein components contained in the current influenza virus-like particles are all complete natural protein structures, such as virus-like particles co-expressed by HA protein and M1 protein or co-infected by HA-NA-M1, and the immunogenicity of the virus-like particles is high, but the generated immune protection is narrow-spectrum, and the virus-like particles cannot play a cross protection role on heterologous strains.

Disclosure of Invention

The invention aims to provide an influenza A universal virus-like particle, a preparation method and application thereof, so that the yield of the influenza A universal virus-like particle is increased, the production cost is reduced, the immunogenicity and the cross protection effect of the virus-like particle are improved by reasonably selecting fusion protein fragments, and the influenza A universal virus-like particle can be applied to the aspects of preventing specific influenza, tiger influenza homologous viruses and heterologous viruses and has strong adaptability.

The technical scheme adopted by the invention is that the influenza A universal virus-like particle is prepared by respectively embedding and displaying 3 fusion proteins on the surface of influenza virus M1 protein, and the fusion proteins are respectively marked as sHA-VLPs, mHA-VLPs and cHA-VLPs;

the 3 fusion proteins are the sHA protein, mHA protein and cHA protein, respectively;

the gene sequence of the sHA protein is shown in SEQ ID NO.1, and the amino acid sequence of the sHA protein is shown in SEQ ID NO. 6;

the gene sequence of the mHA protein is shown as SEQ ID NO.2, and the amino acid sequence of the mHA protein is shown as SEQ ID NO. 7;

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

The preparation method of the influenza A universal virus-like particle comprises the following steps:

step 1, obtaining an encoding gene of an influenza virus M1 protein by a PCR method, carrying out multiple cloning site analysis on the encoding gene of M1 protein and a gene of a pFastBacDual vector, selecting two restriction endonuclease sites which are provided on the pFastBacdual vector and are not provided on a target fragment, designing a primer of the target fragment, recovering the target fragment after target fragment amplification and double enzyme digestion, inserting the target fragment into a multiple cloning site behind a p10 promoter of the pFastBacdual vector, and transforming a colon bacillus DH5 alpha competent cell to obtain a pFastBacdual-M1 plasmid;

step 2, obtaining coding genes of sHA protein, mHA protein and cHA protein by a gene synthesis technology, carrying out multiple cloning site analysis on the coding genes of the sHA protein, mHA protein and cHA protein and a gene of a pFastBacdual-M1 plasmid, selecting two restriction endonuclease sites which are provided on the pFastBacdual-M1 plasmid and are not contained in a target fragment, recovering the target fragment after double digestion, inserting the target fragment into the multiple cloning site behind a PH promoter of the pFastBacdual-M1 plasmid, and then transforming escherichia coli DH5 alpha competent cells to obtain a pFastBacdual-M1-fusion protein shuttle plasmid;

step 3, transforming the pFastBacdual-M1-fusion protein shuttle plasmid into an escherichia coli DH10Bac competent cell to obtain a recombinant baculovirus plasmid Bacmid, transfecting an Sf9 insect cell by using the recombinant baculovirus plasmid Bacmid, and rescuing to obtain a recombinant baculovirus, wherein the confluence of the Sf9 insect cell during transfection is more than 80%;

and 4, inoculating the recombinant baculovirus into Sf9 insect cells according to MOI =3, and harvesting supernatant after three days to obtain the influenza A universal virus-like particles.

Furthermore, the enzyme cutting sites of the M1 protein and the pFastBactual vector are SmaI and NsiI, and the enzyme cutting sites of the coding genes of the fusion proteins sHA, mHA and cHA and the plasmid pFastBacual-M1 are EcoRI and NotI.

Further, the specific steps for obtaining the recombinant baculovirus in the step 3 are as follows: adding 10ng of pFastBacdual-M1-fusion protein shuttle plasmid into DH10Bac competent cells, carrying out heat shock at 42 ℃ for 45s after ice bath for 30min, then carrying out ice bath for 2min, adding an anti-LB-free culture medium, shaking for 4h at 37 ℃ and at the rotating speed of 200rpm, taking 100 mu L of shaking solution, coating the shaking solution on a three-anti-LB (lysogeny broth) plate containing X-gal and IPTG (isopropyl-beta-thiogalactoside), and culturing for 48h at 37 ℃, wherein white spots screened by blue-white spots are recombinant baculovirus plasmid Bacmid.

Further, the concentration of Sf9 insect cells at the time of inoculating the recombinant baculovirus in the step 4 is 2X 10⁶More than mL.

Further, a sucrose density gradient centrifugation method is selected for purification of the influenza A universal virus-like particles, and the specific process is as follows: culturing insect cells inoculated with recombinant baculovirus for 72h, collecting suspension cell culture fluid, centrifuging for 20min at the rotating speed of 4 ℃ and 5000rpm to obtain supernatant, ultracentrifuging the supernatant at the rotating speed of 4 ℃ and 30000rpm for 1h to concentrate, using PBS to resuspend the concentrated solution, dissolving overnight at the temperature of 4 ℃, finally centrifuging for 1h at the rotating speed of 30000rpm in 20% -30% -60% of sucrose, centrifuging for 1h at the rotating speed of 30000rpm of a 30% -60% gradient solution after a sucrose removal step, and precipitating and resuspending to obtain purified virus-like particles.

The influenza A universal virus-like particle is used for preparing vaccines for preventing specific influenza, tiger influenza homologous viruses and heterologous viruses.

The invention has the beneficial effects that: the invention uses the baculovirus insect expression system as an expression vector, uses cells as a bioreactor to produce the influenza A virus-like particles, has the advantages of high yield and low production cost, and the produced influenza A virus-like particles have good immunogenicity and good cross protection, can be used for preparing a large amount of influenza vaccines, have no problems of biological safety risk and the like, and can stimulate the immunity of a matrix to various influenza viruses.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a schematic diagram showing the gene constitutions of mHA protein, sHA protein and cHA protein.

FIG. 2: a is an immunofluorescence chart of VLPs (HA-M1) obtained by taking HA rabbit polyclonal antibody as a primary antibody, B is an immunofluorescence chart of sHA-VLPs obtained by taking HA rabbit polyclonal antibody as a primary antibody, C is an immunofluorescence chart of mHA-VLPs obtained by taking HA rabbit polyclonal antibody as a primary antibody, D is an immunofluorescence chart of cHA-VLPs obtained by taking HA rabbit polyclonal antibody as a primary antibody, E is an immunofluorescence chart of VLPs (HA-M1) obtained by taking M1 mouse monoclonal antibody as a primary antibody, F is an immunofluorescence chart of sHA-VLPs obtained by taking M1 mouse monoclonal antibody as a primary antibody, G is an immunofluorescence chart of mHA-VLPs obtained by taking M1 mouse monoclonal antibody as a primary antibody, and H is an immunofluorescence chart of cHA-VLPs obtained by taking M1 mouse monoclonal antibody as a primary antibody.

FIG. 3: a is an electron micrograph of VLPs (HA-M1), B is an electron micrograph of virus-like particles sHA-VLPs, C is an electron micrograph of virus-like particles mHA-VLPs, and D is an electron micrograph of virus-like particles cHA-VLPs.

FIG. 4: a is a Western blot of 4 VLPs using M1 murine mAb as a primary antibody, B is a Western blot of VLPs (HA-M1) and mHA-VLPs, C is a Western blot of sHA-VLPs and cHA-VLPs, D is a Western blot of 4 VLPs using NP and M2 murine mAb as a primary antibody, and E is a Western blot of 4 VLPs.

FIG. 5A is a graph showing the detection of the level of antibodies specific for H1N1 immunized.

Figure 5B is a graph of the detection of the level of antibodies specific for H3N2 immunized.

Figure 5C is a graph of the detection of the level of antibodies specific for H5N1 immunized.

Figure 5D is a graph of the detection of the level of antibodies specific for H7N7 immunized.

Fig. 5E is a detection map of IgG1 and IgG2a antibodies.

Fig. 6A is a graph of the change in body weight of H1N1 challenged mice.

Fig. 6B is a graph of the change in survival rate of H1N1 challenge group mice.

Fig. 6C is a graph of the body weight change of H3N2 challenged mice.

Fig. 6D is a graph of the change in survival rate of H3N2 challenge group mice.

Fig. 6E is a graph of the body weight changes of H5N1 challenged mice.

Fig. 6F is a graph of the change in survival rate of H5N1 challenge group mice.

Fig. 6G is a graph of the body weight change of H7N7 challenged mice.

Fig. 6H is a graph of the change in survival rate of H7N7 challenge group mice.

FIG. 7A is a graph showing IL-4 secretion under H5N1 stimulation.

FIG. 7B is a graph showing IFN-. gamma.secretion in the presence of H5N 1.

Figure 8 is an influenza virus titer assay.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The influenza A universal virus-like particle is prepared by respectively displaying 3 fusion proteins on the surface of influenza virus M1 protein in a chimeric way, and is respectively marked as sHA-VLPs, mHA-VLPs and cHA-VLPs, the fusion proteins are composed of the genes of sHA protein, mHA protein and cHA protein as shown in figure 1, and the fusion proteins are respectively formed by recombining all or part of highly conserved sequences of influenza virus HA protein, M2e protein, NP protein and PBI protein.

The sequence of fusion protein sHA protein from N-terminus to C-terminus is: two sections of conserved sequences of the bee venom signal peptide + NP protein, namely + 5M 2e proteins from different subtypes and different species and HA2 proteins are connected by connecting peptide, the gene sequence of the sHA protein is shown as SEQ ID NO.1, and the amino acid sequence of the sHA protein is shown as SEQ ID NO. 6.

The sequence of the fusion protein mHA protein from N-terminus to C-terminus is: the 3-segment conserved sequence of the bee venom signal peptide + HA protein comprises the following 3-end conserved sequences: the sequences of E14-H37 of HA1 protein, N286-N319 of HA1 protein and T41-S113 of HA2 protein are connected by connecting peptide, the gene sequence of mHA protein is shown as SEQ ID NO.2, and the amino acid sequence of mHA protein is shown as SEQ ID NO. 7.

The sequence of the fusion protein cHA protein from N-terminal to C-terminal is: bee venom signal peptide + conserved sequence between HA1 protein E14-H37 + 2 conserved sequences of NP protein +5 conserved sequences of M2E protein from different subtypes and different species + two conserved sequences of HA transmembrane region, the conserved sequences at two ends of HA protein are N286-N319 of HA1 protein and T41-S113 of HA2 protein, the sequences are connected by connecting peptide, the gene sequence of cHA protein is shown as SEQ ID NO.3, and the amino acid sequence of cHA protein is shown as SEQ ID NO. 8.

The invention refers to the conservative regions of HA proteins of different influenza viruses, the conservative region M2e of M2 protein and the conservative region of NP protein for serial expression, combines a transmembrane region and an intracellular region of the HA proteins to recombine to obtain fusion protein, displays the fusion protein on the surface of a virus-like particle to construct different influenza A universal virus-like particles, and compared with single short peptide, the virus-like particle HAs better immunogenicity and can better stimulate an organism to generate humoral immunity and cellular immunity.

step 1, constructing a recombinant pFastBacdual-M1 plasmid: obtaining an encoding gene of an influenza virus M1 protein by a PCR method, carrying out multiple cloning site analysis on the encoding gene of the M1 protein and a gene of a pFastBacDual vector, selecting two restriction enzyme sites which are provided on the pFastBacdual vector and are not provided on a target fragment, designing a primer of the target fragment, recovering the target fragment after target fragment amplification and double enzyme digestion, inserting the target fragment into the multiple cloning site behind a p10 promoter of the pFastBacdual vector, and transforming escherichia coli DH5 alpha competent cells to obtain pFastBacdial-M1 plasmid;

step 2, constructing a recombinant shuttle plasmid: obtaining coding genes of fusion proteins sHA, mHA and cHA by a gene synthesis technology, carrying out multiple cloning site analysis on the coding genes of the fusion proteins sHA, mHA and cHA and a gene of a pFastBacdual-M1 plasmid, selecting two restriction endonuclease sites which are provided on the pFastBacdual-M1 plasmid and are not provided on the fusion proteins, recovering a target fragment of the fusion proteins after double enzyme digestion, inserting the target fragment into the multiple cloning sites behind a PH promoter of the pFastBacdual-M1 plasmid, and transforming escherichia coli DH5 alpha competent cells to obtain the pFastBacdual-M1-fusion protein shuttle plasmid;

step 3, constructing recombinant baculovirus plasmids and rescuing the recombinant baculovirus: transforming Escherichia coli DH10Bac competent cells by pFastBacdual-M1-fusion protein shuttle plasmids to obtain recombinant baculovirus plasmids Bacmid, transfecting Sf9 insect cells by using the recombinant baculovirus plasmids Bacmid, and rescuing to obtain recombinant baculovirus, wherein the confluence of the Sf9 insect cells during transfection reaches over 80%;

step 4, preparing influenza A universal virus-like particles: inoculating Sf9 insect cells with recombinant baculovirus according to MOI =3, and harvesting supernatant after 3 days to obtain influenza A universal virus-like particles, wherein the concentration of Sf9 insect cells needs to reach 2 × 10 when the recombinant baculovirus is inoculated⁶/mL。

In the invention, the enzyme cutting sites of M1 protein and pFastBacDual carrier in step 1 are SmaI and NsiI, and the enzyme cutting sites of the coding genes of fusion protein sHA, mHA and cHA and pFastBacdual-M1 plasmid in step 2 are EcoRI and NotI.

The molecular cloning of pFastBacdual-M1-fusion protein shuttle plasmid in the step 3 is obtained by cloning conventionally transformed DH5 alpha competent cells; the specific steps for obtaining the recombinant baculovirus plasmid are as follows: adding 10ng of pFastBactual-M1-fusion protein shuttle plasmid into a DH10Bac competent cell, carrying out heat shock at 42 ℃ for 45s after ice bath for 30min, then carrying out ice bath for 2min, adding an anti-LB-free culture medium, shaking for 4h at 37 ℃ and at the rotating speed of 200rpm, taking 100 mu L of shaking solution, coating the shaking solution on a three-resistant LB (lysogeny) plate containing X-gal and IPTG, carrying out culture for 48h at 37 ℃, wherein the white spot screened by the blue-white spot is recombinant baculovirus plasmid Bacmid, and realizing DNA homologous recombination between the hollow white rod shuttle plasmid and the pFastBactual-M1-fusion protein shuttle plasmid of the DH10Bac competent cell through DNA transposition.

The purification method of the virus-like particles adopts a sucrose density gradient centrifugation method, and comprises the following specific steps: culturing insect cells inoculated with recombinant baculovirus for 72h, collecting suspension cell culture fluid, centrifuging for 20min at the rotating speed of 4 ℃ and 5000rpm to remove cell debris to obtain supernatant, ultracentrifuging the supernatant at the rotating speed of 4 ℃ and 30000rpm for 1h to concentrate, using PBS (phosphate buffer solution) to resuspend the concentrated solution, dissolving overnight at the temperature of 4 ℃, finally centrifuging for 1h at the rotating speed of 30000rpm in 20% -30% -60% of sucrose, centrifuging for 1h at the rotating speed of 30000rpm for 30% -60% of solution between gradients after the sucrose removal step, and precipitating and resuspending to obtain purified virus-like particles.

Example 1

The following materials were selected to verify the preparation and use of the virus-like particles of the present invention.

H1N1 influenza mouse adapted strain A/Changchun/01/2009(H1N1, group1), H3N2 influenza mouse adapted strain A/baikaleal/Shanghai/SH-89/2013 (H3N2, group2), H5N1 avian influenza virus A/meerkat/Shanghai/SH-1/2012(H5N1, clade2.3.2.1; group1), H7N7 influenza mouse adapted strain A/Lesser White-front goose/Hunan/412/2010(H7N7, group2), all provided by the military veterinary institute virology institute of military medical academy of military science; main reagents e.coli DH5 α and e.coli DH10Bac were stored in the laboratory, donor plasmid pfastbacadual vector was purchased from Invitrogen, fusion protein gene synthesis was provided by jingzhi, technical services such as recombinant plasmid gene sequencing and primer synthesis were provided by changcoumei, insect cell transfection kit was purchased from Invitrogen, endonuclease was purchased from Thermo, genome extraction kit was purchased from AXYGEN, Sf-900 ii SFM was purchased from Thermo, antibiotics and the like were purchased from solibao, HA rabbit polyclonal antibody was purchased from kyoto, M2 murine mab and NP murine mab were purchased from abcam, and M1 murine mab was purchased from jien.

The experimental method is as follows:

s1, carrying out reverse transcription on RNA extracted from H5N1 virus to cDNA, carrying out PCR amplification by using the cDNA as a template and using an HA protein primer of H5N1 and an M1 protein primer respectively to obtain HA protein and an M1 protein fragment, using SEQ ID NO.15 and SEQ ID NO.16 as amplification primers of the HA protein fragment, using SEQ ID NO.17 and SEQ ID NO.18 as amplification primers of the M1 protein, obtaining gene fragments of fusion proteins sHA, mHA and cHA by using gene synthesis, carrying out PCR amplification process of maintaining 95 ℃ for 5min, then maintaining 95 ℃ for 30S, turning to 56 ℃ for 90S, then maintaining 72 ℃ for 1min, and carrying out 30 cycles in total;

cloning the obtained M1 protein gene into a plasmid pFastBacDual by using SmaI and NsiI enzyme cleavage sites, transforming the plasmid pFastBacdual into an escherichia coli DH5 alpha competent cell, plating the cell overnight for culture, screening positive bacteria, and obtaining a correct pFastBacdual-M1 plasmid through plasmid PCR, sequencing and enzyme digestion identification;

s2, cloning the obtained HA protein gene into a pFastBacdual-M1 plasmid by using SalI and NotI enzyme cleavage sites to obtain a correct pFastBacdual-HA-M1 plasmid;

obtaining pFastBacdual-sHA-M1 plasmid, pFastBacdual-mHA-M1 plasmid and pFastBacdual-cHA-M1 plasmid by using EcoRI and NotI of restriction enzyme cutting sites, wherein SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO.14 are PCR upstream and downstream primers for identifying sHA protein, mHA protein and cHA protein fragments respectively;

s3, constructing a recombinant rod-shaped plasmid:

transforming DH10Bac competent cells by pFastBacdual-HA-M1 plasmid, selecting white spots from blue white spot colonies generated by bacterial growth, extracting plasmid, performing PCR identification to obtain recombinant rod-shaped plasmid HA-M1-Bacmid, and obtaining sHA-M1-Bacmid, mHA-M1-Bacmid and cHA-M1-Bacmid by the same method;

s4, rescue of recombinant baculovirus:

transferring 4 positive Bacmid plasmids into Sf9 cells by using a Cellreagent transfection kit, culturing for three days at 27 ℃, collecting cell culture supernatant P1, inoculating P1 into new Sf9 cells according to the volume ratio of 3%, continuously culturing to amplify virus virulence, culturing for three days, collecting P2 generation supernatant, inoculating P2 into new Sf9 cells according to the volume ratio of 3%, continuously culturing for three days, collecting P3 generation supernatant, extracting DNA in P3 generation supernatant, performing PCR verification, observing and recording the morphological change of P3 generation cells, performing expression identification on the P3 generation cells by using indirect immunofluorescence, and respectively showing a test chart of Sf9 cells infected by 4 viruses obtained by using the indirect immunofluorescence method by using HA rabbit polyclonal antibody, respectively showing a chart of rod-shaped 9 cells by using M1 mouse monoclonal antibody as a test chart of Sf9 cells infected by using the indirect immunofluorescence method, 2A-2H show that 4 baculovirus-infected Sf9 cells of P3 generation all have strong green fluorescence expression, which indicates that 4 virus-like particles have the expression of corresponding proteins;

s5, bulk expression and purification of virus-like particles:

the titer of baculovirus of P3 generation is determined by a rapid baculovirus titer detection kit of Takara company, a large amount of cultured suspended Sf9 cells are inoculated by the baculovirus of P3 generation according to MOI =3, cell culture solution is collected after 4 days, and purified virus-like particles are obtained after a series of steps of cell debris removal → centrifugal concentration → sucrose density gradient centrifugation → sucrose removal and the like;

(1) western blot was used to validate the 4 virus-like particles prepared in the examples;

western blot verification is carried out by using HA rabbit polyclonal antibody as a primary antibody, and VLPs (HA-M1) are found to have a band at about 70KD, mHA-VLPs have a band at about 23KD as shown in figure 4B, and sHA-VLPs and cHA-VLPs have bands at about 50KD as shown in figures 4C and 4E, so that the expression of the fusion protein of 4 VLPs is correct;

western blot verification is carried out by using M1 mouse monoclonal antibody as primary antibody, the verification result is shown in figure 4A, and the 4 VLPs are found to have bands at about 30KD, which indicates that M1 proteins of the 4 VLPs are correctly expressed;

western blot verification is carried out by using NP and M2 mouse monoclonal antibodies as primary antibodies, the verification result is shown in figure 4D, and only sHA-VLPs and cHA-VLPs in 4 VLPs are found to have bands at about 50KD, which indicates that the fusion proteins of the sHA-VLPs and the cHA-VLPs in 4 VLPs are correctly expressed;

(2) observing the 4 virus-like particles by using a transmission electron microscope;

VLPs (HA-M1) were observed under a transmission electron microscope, and as a result of the observation shown in FIG. 3A, spheroidal particles with a size of about 100nm were observed, and a knob structure was formed around the spheroids, indicating that the VLPs (HA-M1) were assembled correctly; the HA polyclonal antibody is taken as a primary antibody to carry out the detection of an immune electron microscope on 3 universal virus-like particles, namely sHA-VLPs, mHA-VLPs and cHA-VLPs, the detection results are shown in figures 3B to 3D, the 3 virus-like particles are observed to be particles with the size of about 100nm under the electron microscope, and the peripheries of the particles are marked by gold particles, so that the 3 influenza A universal virus-like particles are assembled.

Example 2

The following materials were selected to study the immunological performance of influenza A universal virus-like particles of the present invention, BCA protein detection kit was purchased from Thermo corporation, 4 inactivated influenza virus-like particles were self-purified, HPR-labeled goat anti-mouse IgG, IgG1, IgG2a were purchased from Southern Biotechnology corporation, ELISApot detection kits for mouse IFN-. gamma.and IL-4 were purchased from Mabtech corporation, TMB was purchased from Sigma corporation, and 6-8 week-old BALB/c female mice were purchased from Wintonia, Beijing;

1. the specific procedure of the experiment is as follows:

s1, immunization and challenge:

detecting the protein concentration of influenza virus-like particles with a kit, intramuscular injecting 0.1mL of a vaccine containing 10 μ g of virus-like particles to mice at 0 and 3 weeks, with VLPs (HA-M1) as an immunization control group, PBS group as a Mock group, anesthetizing the mice with isoflurane two weeks after the second immunization, attacking the mice with 10MLD50 homologous virus (A/meerkat/Shanghai/SH-1/2012, H5N1, clade2.3.2.1; group1) and heterologous virus (mouse-adapted A/Changchun/01/2009, H1N1, group1), (mouse-adapted A/balkanteal/Shanghai/SH-89/2013, H3N2, group BCA 2), (mouse-adapted A/LeonsWheats-particle White/Huonghae/HA-7377, VLP/Na-73742) group and HAs-SACK group (A/Changchunchbun/BCA/Changchun-2 5, experiment group), mHA-VLPs, cHA-VLPs), mice were challenged by nasal drops, all test conditions and procedures were in accordance with the ethical guidelines of the International society for pain research;

s2, specific IgG antibody detection:

collecting blood in orbit at 0 week, 3 weeks and 5 weeks after immunization of mouse, standing the collected blood at room temperature for 2h, centrifuging at 3000rpm for 10min, separating upper layer serum, and storing at-80 deg.C; detecting influenza virus specific IgG, IgG1 and IgG2a in serum by ELISA assay, incubating inactivated H1N1 influenza virus mouse adapted strain A/Changchun/01/2009(H1N1, group1), H3N2 influenza virus mouse adapted strain 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 adapted strain A/Lesser White-oneded goose frost/Hunan/412/2010 (H7N7, group 42) with viruses of 39393996 mL, incubating the cells with serum-labeled IgG 37 ℃ after washing with serum and adding anti-PBST 5 IgG 461H, washing the cells with PBS 37, and washing the serum sample overnight at 37 ℃ after incubating with PBS 37H 37, adding anti-PBST 462, then adding 50 muL of 0.5mol/L H2SO4 to terminate the reaction, and detecting the result at 450nm by using a spectrophotometer;

s3, toxicity attacking and protecting experiment: observing the weight change and survival rate change of the mice in each group within 15 days after the challenge, wherein the weight change and survival rate change of the mice in each group are shown as figures 6A-6H;

s4, using elispot to detect cytokines, the procedure is as follows:

detecting virus-activated antigen-specific T cells induced by virus-like particles with ELISApot, pre-coating 96-well plates with IL-4 monoclonal antibody and IFN-gamma monoclonal antibody, spreading 1 × 106 splenocytes separated on day 4 after challenge per well, 3 mice per group, 6 replicate wells per mouse (3 replicate wells plus stimulus, and the other 3 control wells), the stimulus being inactivated H5N1 virus-like particles, and the final concentration of the stimulus being 10 μ g/mL, in 1640 medium containing 10% FBS at 37 ℃ in 5% CO₂Culturing for 48h, discarding cells in the wells, performing enzyme-linked spot detection according to the steps of the ELISApot detection kit, and finally calculating a spot forming unit by using an ELISApot reading system;

s5, performing a viral titration of the lung using the following steps:

inoculating chick embryos after diluting the lung homogenate by 1:10, 1:102, … and 1:1010, inoculating 3 SPF-level chick embryos of 9 days old at each dilution degree, inoculating 100 mu L of lung homogenate to each egg, incubating for 48h at 37 ℃ after sealing adhesive sticker, detecting the blood coagulation result of allantoic fluid after 48h, mixing 50 mu L of allantoic fluid and 50 mu L1% of chick red blood cell suspension for each egg, observing and recording the result after 15min, and calculating the half number of chick embryo infection EID50 of each lung mouse grinding fluid by using a Reed-Muench method.

2. Results of the experiment

(1) Detecting the result of immune response of the influenza A universal virus-like particles in a mouse;

detecting the level of specific antibodies against homologous virus and heterologous virus in the serum of mice immunized at

weeks

0, 3 and 5, respectively, the detection results of homologous virus (A/meerkat/Shanghai/SH-1/2012, H5N1, clade) are shown in FIG. 5C, and the detection results of 3 heterologous viruses (mouse-adapted A/Changchun/01/2009, H1N1, group1), (mouse-adapted A/baikaleal/Shanghai/SH-89/2013, H3N2, group2), (mouse-adapted A/Lesser White-bound goose/Hunan/412/2010, H7N7, group2) are shown in FIG. 5A, FIG. 5B and FIG. 5D, respectively; the specific IgG antibody levels in fig. 5A to 5D increased with the increase of the number of immunization weeks, indicating that specific antibodies were produced, and compared with control group VLPs (HA-M1), the specific antibody levels of the sHA-VLPs against H1N1 and H7N7 were significantly increased as shown in fig. 5A and 5D, the specific antibody levels of the mHA-VLPs against H3N2 as shown in fig. 5B, the specific antibody levels of the cHA-VLPs against H5N1 as shown in fig. 5C, the specific antibodies against 4 influenza viruses produced by the body stimulated by the group of sHA-VLPs in 4 virus-like particles were all kept at a high level, and the sHA-VLPs had better broad-spectrum; the IgG1 and IgG2a antibody subtypes of the fifth week sera of all immunization groups were tested, as shown in FIG. 5E, the IgG1 titer against H5N1 antigen was higher than that of IgG2a in all immunization groups, indicating that the antibody producing the specific immune response of the virus is mainly IgG1, while in the evaluation of the immune effect of VLPs, the high IgG1/IgG2a level indicates that the antibody produces cellular immunity, and the VLPs mainly induce humoral immunity to play a protective role.

(2) The challenge protection research of influenza A universal virus-like particles;

the weight change rate and survival rate change curves of the H1N1 challenge group mice are shown in fig. 6A and 6B, the weight change rate and survival rate change curves of the H3N2 challenge group mice are shown in fig. 6C and 6D, the weight change rate and survival rate change curves of the H5N1 challenge group mice are shown in fig. 6E and 6F, the weight change rate and survival rate change curves of the H7N7 challenge group mice are shown in fig. 6G and 6H, and it can be seen from fig. 6A, 6C, 6E and 6G that the weight change rates of the mice in the challenge groups are less different, but the H3N2 challenge groups mHA-VLPs show less weight loss;

as shown in FIG. 6B, the H1N1 challenge group showed no survival except for the survival rates of sHA-VLPs and mHA-VLPs of 60% and 20%, respectively; FIG. 6D shows that the survival rate of mHA-VLPs in the H3N2 challenge group is 60%, the survival rates of VLPs (HA-M1) in the sHA-VLPs group and the control group are both 20%, and no survival occurs in the cHA-VLPs and Mock group; FIG. 6F shows that the survival rates of the VLPs (HA-M1) in the control group, the mHA-VLPs and the cHA-VLPs in the H5N1 challenge group are all 40%, the survival rate of the sHA-VLPs is 20%, and the Mock group does not survive; as shown in FIG. 6H, the survival rates of the VLPs (HA-M1) and the cHA-VLPs in the control group and the cHA-VLPs in the H7N7 challenge group are both 80%, the survival rates of the sHA-VLPs and the mHA-VLPs are respectively 60% and 50%, and the Mock group does not survive; according to the body weight rate and survival rate detection results, the broad-spectrum protection effect of two groups of sHA-VLPs and mHA-VLPs on homologous and heterologous influenza viruses is better.

(3) Influenza a universal virus-like particles activate specific T lymphocytes;

spleen lymphocytes are separated on the fourth day after mouse challenge, the spleen lymphocytes are stimulated by H5N1 inactivated antigen, then the secretion condition of specific IL-4 (Th 2 type immune response) is detected, as shown in figure 7A, under the stimulation of the inactivated virus, a cHA-VLPs group can generate a higher IL-4 level compared with a control group VLPs (HA-M1), the secretion condition of IFN-gamma (Th 1 type immune response) is detected as shown in figure 7B, a mHA-VLPs group can generate a higher IFN-gamma level compared with a control group VLPs (HA-M1), and compared with the expression quantity of IL-4 in all immune groups, the expression quantity of IFN-gamma is higher than that of IL-gamma, which indicates that T lymphocytes are mainly immunized by Th2 type cells.

(4) Influenza a universal virus-like particles can inhibit replication of influenza virus;

as shown in fig. 8, the lungs of the mice on the fourth day after challenge were taken, and virus titers of various influenza viruses were measured in SPF chick embryos aged 9 days, and the general trend of the decrease in virus titers of the experimental groups of the remaining 3 heterologous viruses compared to the control group was consistent with the challenge protection experiment, except for H5N1 virus; in the H5N1 virus titer measurement, the virus titer of the sHA-VLPs group and the cHA-VLPs group is remarkably reduced compared with that of the VLPs (HA-M1) of the control group, which indicates that the particles of the sHA-VLPs group and the cHA-VLPs group can inhibit the replication of influenza virus in the lung of a mouse.

In 3 types of influenza A universal virus-like particles, the comprehensive immune protection effect of the sHA-VLPs group is better, the sHA-VLPs group can generate high-level attacking protection rate and specific IgG antibodies aiming at various heterogeneous influenza viruses and inhibit the replication of the influenza viruses, and the immune protection effect of the universal virus-like particles is mainly humoral immunity.

All the embodiments in the present specification are described in a related manner, and the same and similar parts among the embodiments may be referred to each other, and each embodiment focuses on the differences from the other embodiments. In particular, for the system embodiment, since it is substantially similar to the method embodiment, the description is simple, and for the relevant points, reference may be made to the partial description of the method embodiment.

The above description is only for the preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention shall fall within the protection scope of the present invention.

Sequence listing

<110> military medical institute of military sciences institute of military veterinary research institute

<120> influenza A universal virus-like particle and preparation method and application thereof

<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

1. The preparation method of the influenza A universal virus-like particle is characterized by comprising the following steps:

the enzyme cutting sites of the M1 protein and the pFastBacdual carrier are SmaI and NsiI;

step 2, obtaining a coding gene of the sHA protein by a gene synthesis technology, carrying out multiple cloning site analysis on the coding gene of the sHA protein and a gene of a pFastBacdial-M1 plasmid, selecting two restriction endonuclease sites which are provided on the pFastBacdial-M1 plasmid and are not contained in a target fragment, recovering the target fragment after double digestion, inserting the target fragment into the multiple cloning site behind a PH promoter of the pFastBacdial-M1 plasmid, and then transforming escherichia coli DH5 alpha competent cells to obtain a pFastBacdial-M1-sHA protein shuttle plasmid;

the coding gene of the sHA protein and the restriction enzyme sites of pFastBacdual-M1 plasmid are EcoRI and NotI, the gene sequence of the sHA protein is shown as SEQ ID NO.1, and the amino acid sequence of the sHA protein is shown as SEQ ID NO. 6;

step 3, transforming Escherichia coli DH10Bac competent cells by pFastBacdual-M1-sHA protein shuttle plasmids to obtain recombinant baculovirus plasmids Bacmid, transfecting Sf9 insect cells by using the recombinant baculovirus plasmids Bacmid, and rescuing to obtain recombinant baculovirus, wherein the confluence degree of the Sf9 insect cells during transfection reaches more than 80%;

step 4, making the concentration of Sf9 insect cells 2X 10⁶above/mL, recombinant baculovirus was inoculated with Sf9 insect cells at MOI =3, and after three days the supernatant was harvested to obtain influenza a universal virus-like particles.

2. The method for preparing influenza a universal virus-like particle according to claim 1, wherein the specific steps of obtaining the recombinant baculovirus plasmid Bacmid in the step 3 are as follows: adding 10ng of pFastBacdual-M1-sHA protein shuttle plasmid into DH10Bac competent cells, carrying out heat shock at 42 ℃ for 45s after ice bath for 30min, then carrying out ice bath for 2min, adding an anti-LB-free culture medium, shaking for 4h in a shaking table at 37 ℃ and a rotating speed of 200rpm, taking 100 mu L of shaking solution, coating the shaking solution on a three-anti-LB plate containing X-gal and IPTG, and culturing for 48h at 37 ℃, wherein the white spot screened by the blue-white spot is the recombinant baculovirus plasmid Bacmid.

3. The method for preparing influenza A virus-like particles according to claim 1, wherein the purification of the influenza A virus-like particles is performed by sucrose density gradient centrifugation, and the method comprises the following steps: culturing insect cells inoculated with recombinant baculovirus for 72h, collecting suspension cell culture fluid, centrifuging for 20min at the rotating speed of 4 ℃ and 5000rpm to obtain supernatant, ultracentrifuging for 1h at the rotating speed of 4 ℃ and 30000rpm to concentrate the supernatant, using PBS to resuspend the concentrated solution, dissolving overnight at the temperature of 4 ℃, finally centrifuging for 1h at the rotating speed of 30000rpm in 20% -30% -60% of sucrose, centrifuging for 1h at the rotating speed of 30000rpm of a 30% -60% gradient solution after a sucrose removal step, and precipitating and resuspending to obtain purified influenza A universal virus-like particles.

4. The influenza A virus-like particle produced by the method for producing an influenza A virus-like particle according to any one of claims 1 to 3, which is used for producing a vaccine for preventing influenza A virus.