CN114276969A - SARS-CoV-2 bacteria-like particle and its application in vaccine - Google Patents
SARS-CoV-2 bacteria-like particle and its application in vaccine Download PDFInfo
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Abstract
The invention discloses SARS-CoV-2RBD bacteria-like particles and application thereof in vaccines. SARS-CoV-2RBD protein is used as SARS-CoV-2 vaccine antigen, GEM-PA surface display technology is utilized, linker is added into fusion protein to improve the binding activity of the fusion protein combined with GEM particles and the immunogenicity of antigen protein, an insect-baculovirus expression system is utilized to express the fusion protein, the expressed fusion protein and the GEM particles are mixed uniformly, and SARS-CoV-2 bacteria-like particles are prepared after centrifugation and washing. The results of a BALB/C mouse model infected with old people by SARS-CoV-2BMA8 and a C57BL/6N mouse model infected with old people by CMA14 show that the BLP vaccine induces a neutralizing antibody response against BMA8 and C57MA14, can 100 percent protect old BALB/C and C57BL/6N mice against the attack of mouse adaptive SARS-CoV-2, and provides a new idea for the development of new crown vaccines for old people.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to SARS-CoV-2RBD bacteria-like particles and application thereof in vaccines.
Background
The novel Coronavirus (SARS-CoV-2) is a positive-strand RNA virus with envelope, the genome is the largest in known RNA viruses and consists of about 30000 nucleotides, viral genome RNA and nucleoprotein form a spirally symmetrical nucleocapsid, a lipid bilayer virus envelope is arranged outside the nucleocapsid, and three proteins are embedded in the envelope: spike protein (Spike, S), membrane protein (M) and envelope protein (E).
In the virus particle structure of SARS-CoV-2, the S protein is a high glycosylation protein, forms a special crown structure on the virus particle surface in the form of trimer, is composed of 1160-1400 amino acids, contains 21-35N-terminal glycosylation sites, has receptor binding activity and membrane fusion activity, and is closely related to virus infection and pathogenesis. Under the action of host cell protease, S protein is cleaved into two parts, S1 and S2. The S protein is mainly bound to angiotensin converting enzyme 2 (ACE 2) Receptor on the surface of host cell via Receptor-binding Domain (RBD) located in S1 subunit, which is also the main reason for SARS-CoV-2 cross-species spread to human and occurs between humans. Whereas S2 primarily mediates fusion of the viral envelope with the cell or endosomal membranes to allow the viral nucleocapsid to enter the cytoplasm. Because the full length of the S protein possibly causes (ADE) effect, the RBD region of the S1 protein can induce the body to generate strong neutralizing antibody response.
Thus, the focus of SARS-CoV-2 vaccine design is primarily on the development of RBD proteins. SARS-CoV-2 infection in humans can cause new forms of coronavirus pneumonia (COVID-19), and there is currently no effective drug therapy, and the most effective way to combat this viral infection is vaccination.
At present, a plurality of vaccine production enterprises in the world are added into the research and development of SARS-CoV-2 vaccine, and as long as 6 months and 16 days, according to NYT real-time data, 50 vaccines are currently subjected to phase I clinical test, phase II clinical test (36) and phase III clinical test (31) in the research and development situation of new crown vaccines in the world. In addition, more than 8 vaccines have been used to a limited extent; the 8 vaccines enter the normal use range.
However, existing vaccines have not been studied in the elderly population, and aging of the immune system of elderly people can predispose the body to infection and impair its response to the vaccine, thereby increasing infection, transmission and mortality.
The technical route of the new corona vaccine comprises an inactivated vaccine, a recombinant subunit vaccine, a viral vector vaccine and a nucleic acid vaccine. The recombinant subunit vaccine comprises different forms such as pure recombinant protein or nano particles, the virus vector vaccine comprises adenovirus vectors, poxvirus vectors, influenza virus vectors and the like, and the nucleic acid vaccine comprises DNA vaccine, non-replicative mRNA vaccine, replicative mRNA vaccine and the like.
The GEM-PA surface display system is a novel Protein surface display system, and consists of Gram positive bacteria enhanced Matrix (GEM) and anchoring Protein molecules (PA). The antigen protein is displayed on the surface of the GEM particles through PA, so that the immune effect of the vaccine can be effectively improved. The display system has the following characteristics: firstly, the safety is high. The food-grade lactic acid bacteria do not contain any nucleic acid substances, so that the risk of the transmission of the recombinant DNA to the environment and other organisms is reduced to the maximum extent; the antigen display density is high, the GEM-PA can display the foreign protein at high density, the load capacity of the GEM-PA is far higher than that of the female parent live bacteria, and the GEM-PA can effectively deliver antigen substances to induce an organism to generate immune response; and thirdly, the self-adjuvant effect is achieved. The main component of GEM-PA is peptidoglycan, which is one of ligands of TLR2, and the GEM-PA induces the maturation of host dendritic cells by activating TLR2 signal path, highly expresses surface molecules such as CD80, CD40 and MHC-II, and the mature DC cells secrete inflammatory cytokines to balance Th1 and Th2 type immunity and stimulate more effective immune response reaction. Fourthly, the mucous membrane delivery efficiency is high. The GEM particle size is about 1 mu M, is an ideal size for the M cell on the surface of the mucous membrane to absorb exogenous antigen, can be effectively absorbed by the M cell positioned on nasopharyngeal epithelial tissues and intestinal tracts and is transported to an antigen presenting cell, can effectively induce the reaction of antigen specific Th cells, cytotoxic T lymphocytes and IgA secretory B cells, and starts the local mucous membrane and systemic immune response reaction.
Therefore, the invention provides a vaccine with higher safety, better immune performance and wider application range by utilizing GEM-PA surface display technology.
Disclosure of Invention
One of the objectives of the present invention is to provide SARS-CoV-2 bacteria-like particles prepared by displaying rBV-RBD-linker-PA3 fusion protein on the surface of GEM particles.
The protein sequence of the linker of the SARS-CoV-2 bacterium-like particle is (Gly-Gly-Ser-Gly)2。
The amino acid sequence of rBV-RBD-linker-PA3 fusion protein of the SARS-CoV-2 bacteria-like particle is shown in SEQ No 1.
The SARS-CoV-2 bacteria-like particle, rBV-RBD-linker-PA3 fusion protein, is expressed by using insect-baculovirus expression system.
The invention also provides the application of the SARS-CoV-2 bacteria-like particle in the preparation of SARS-CoV-2 vaccine.
The invention also provides a SARS-CoV-2 vaccine, comprising the SARS-CoV-2 bacteria-like particle.
The protein sequence of the linker of the SARS-CoV-2 vaccine is (Gly-Gly-Ser-Gly)2。
The amino acid sequence of SARS-CoV-2 bacteria-like particle of the SARS-CoV-2 vaccine is shown in SEQ No 1.
The SARS-CoV-2 vaccine, rBV-RBD-linker-PA3 fusion protein, is expressed by insect-baculovirus expression system.
The fourth purpose of the invention is to provide a preparation method of the SARS-CoV-2 vaccine, which utilizes an insect-baculovirus expression system to express rBV-RBD-linker-PA3 fusion protein, wherein the protein sequence of the linker is (Gly-Gly-Ser-Gly)2Mixing rBV-RBD-linker-PA3 fusion protein with GEM particle, centrifuging, washing to obtain SARS-CoV-2 bacteria-like particle, adding pharmaceutically acceptable adjuvant, and making into vaccine.
The GEM particle surface display system is used as a novel antigen protein display system and consists of a gram-positive bacterium enhancement matrix and anchoring protein. The GEM particles are peptidoglycan skeletons of lactococcus lactis subjected to acid boiling treatment. Lactococcus lactis is a probiotic with high safety recognized worldwide, and has been used in food for a long time. Relative to lactic acid milk ballsThe bacteria and GEM particles subjected to acid boiling treatment mainly contain cell wall peptidoglycan, do not contain other components of cell walls and intracellular substances, and have higher safety. GEM particles can display exoprotein molecules with high affinity and high density through PA protein (10)5～106Individual antigenic molecules/GEM particles). In addition, the GEM particles can be efficiently combined with the fusion protein in the culture solution at room temperature, so that the aim of purifying the exoprotein in one step is fulfilled.
Different target genes are connected through a proper nucleotide sequence, so that the target genes are expressed into a single peptide chain in a certain protein expression system, wherein the amino acid playing a role in connection is called linker. The linker as an important component of the recombinant fusion protein plays an important role in maintaining the stability and biological activity of the fusion protein. Whether the two components in the fusion protein can form correct spatial structures respectively and better exert biological activity is closely related to a linker connecting the two components in the fusion protein. Linker is generally divided into three categories: flexible linker, rigid linker and shearable linker. Flexible linker is often used when certain mobility and interaction is required between the linked fusion proteins. Flexible linkers often consist of small, polar (e.g., Gly) or nonpolar (e.g., Ser or Thr) amino acid molecules. These small amino acid fragments can provide some flexibility to the various regions of the fusion protein. The addition of Ser or Thr can maintain the stability of the linker in aqueous solution by forming hydrogen bonds with water molecules, thereby reducing the adverse interaction between the linker and the protein moiety. The most commonly used flexible linker comprises mainly Gly and Ser residues ("GS" linker).
In this protocol, where PA is LysM of the C-terminal peptidoglycan binding domain of AcmA, which is an autolysin from lactococcus, the present invention contemplates both ensuring that PA maintains optimal orientation during binding to GEM particles, and that the SARS-CoV-2RBD protein conformation is not affected, thereby ensuring its immunogenicity. Through multiple tests and screens, the invention selects a flexible linker sequence (Gly-Gly-Ser-Gly)2The PA is allowed to bind to the GEM particles in a non-covalent manner. The amount of LysM determines the binding activity of the fusion proteinOne of the important factors of sex. Under natural conditions, the LysM domains are linked together by a linker rich in serine, asparagine and threonine to form a multivalent domain, so that the mobility and flexibility of the LysM are improved, and the anchoring activity of the LysM is improved. In the invention, linker (Gly-Gly-Ser-Gly) is added into the fusion protein2The binding activity of the fusion protein combined with GEM particles and the immunogenicity of antigen protein are improved, an insect-baculovirus expression system is used for expressing the fusion protein, the expressed fusion protein and the GEM particles are uniformly mixed, and the SARS-CoV-2 bacterial-like particles are prepared after centrifugation and washing, and experiments prove that the fusion protein can be successfully displayed on the surfaces of the GEM particles.
In addition, the invention selects two old mice BALB/C and C57BL/6N to evaluate the immunoreactivity of SARS-CoV-2 bacterial-like particle vaccine, designs a BALB/C mouse model of SARS-CoV-2BMA8 infected old and a C57BL/6N mouse model of CMA14 infected old respectively, and the experimental result shows that the BLP vaccine induces the neutralizing antibody response against BMA8 and C57MA14, can 100 percent protect the old BALB/C and C57BL/6N mice against the attack of mouse adaptive SARS-CoV-2, and provides a new thought for the development of new crown vaccine for the old.
Drawings
In order to more clearly illustrate the embodiments of the present application or technical solutions in the prior art, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments described in the present invention, and other drawings can be obtained by those skilled in the art according to the drawings.
FIG. 1 provides a schematic diagram of a fusion protein according to example 1 of the present invention.
FIG. 2 shows lactococcus lactis before and after treatment as provided in example 2 of the present invention.
FIG. 3 shows SDS-PAGE results of bound particles at different spin speeds as provided in example 3 of the present invention.
FIG. 4 shows the SDS-PAGE results of the GEM particles after binding provided in example 3 of the present invention.
FIG. 5 shows the results of indirect immunofluorescence of GEM particles after binding, as provided in example 3 of the present invention.
FIG. 6 shows the thin-layer scan results of the GEM particles after binding provided in example 3 of the present invention.
FIG. 7 is a graph of the immunization schedule and challenge time for BALB/C and C57BL/6N mice provided in example 4 of the present invention.
FIG. 8 shows the body weight changes of mice after challenge in BALB/c group provided in example 4 of the present invention.
FIG. 9 shows the body weight changes of mice after challenge in group C57BL/6N provided in example 4 of the present invention.
FIG. 10 is the body temperature changes of mice after challenge in BALB/c group provided in example 4 of the present invention.
FIG. 11 is the body temperature changes of mice after challenge in group C57BL/6N provided in example 4 of the present invention.
FIG. 12 is a graph showing the change in the level of neutralizing antibodies in the serum of mice after challenge in BALB/c group provided in example 4 of the present invention.
FIG. 13 is a graph showing the change in the level of neutralizing antibodies in the serum of mice after challenge in group C57BL/6N, provided in example 4 of the present invention.
FIG. 14 is a graph showing the serum neutralizing antibody levels in mice challenged with BALB/c group provided in example 4 of the present invention.
FIG. 15 is a graph showing the serum neutralizing antibody levels in mice challenged with group C57BL/6N, provided in example 4 of the present invention.
Detailed Description
In order to make the technical solutions of the present invention better understood by those skilled in the art, the present invention will be further described in detail with reference to the following embodiments and the accompanying drawings. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. 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 experimental procedures in the following examples are conventional unless otherwise specified.
The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. SARS-CoV-2BMA and C57MA14 strains are selected from SARS-CoV-2 Wuhan01 strain (Betacov/Wuhan/AMMS012020) Obtained by continuous passage in Balb/C and C57BL/6N mice respectively, DH5 alpha E.coli competent cells purchased from Takara; spodoptera frugiperda 9(Sf9) insect cell, lactococcus lactis (Lactoccoucoccus MG1363), pFastBac1 vector, DH10BacTMColi competent cells were kept in the laboratories of animal virology and special animal epidemics at the institute of veterinary and research, cathartics, academy of agricultural sciences, china.
3000Reagent from Thermo Fisher; DMEM and fetal bovine serum were purchased from GIBCO; phusion ultra fidelity DNA polymerase and T4 DNA ligase were purchased from NEB; DNA gel recovery kits and plasmid miniprep kits were purchased from Axygen.
EXAMPLE 1 construction of recombinant baculovirus
1. Primer design
The designed primer sequences are shown in Table 1, and the RBD-linker-PA3 gene fragment is synthesized by taking plasmids pUC57-S and pUC57-PA as templates.
TABLE 1 primer names and sequences
Note: 1 italicized thickening is enzyme cutting site gene sequence
2 the gene sequence is drawn as Middle linker (Gly-Gly-Ser-Gly)2
The bold of the 3 gene sequence is His-tag
2. Construction of recombinant bacmids
SARS-CoV-2RBD is selected as a target gene for amplification, a linker sequence is designed to be connected with PA3, a specific pattern diagram is shown in figure 2, SARS-CoV-2RBD is selected as a target gene for amplification, a linker sequence is designed to be connected with PA3, and a specific pattern diagram is shown in figure 1.
The specific method comprises the following steps: the RBD-linker-PA3 gene fragment and the pFastbac1-HBM vector are subjected to double enzyme digestion by Xba I and Xho I respectively, gel is recovered and then connected overnight, DH5 alpha E.coli competent cells are transformed, recombinant transfer plasmids pFastbac1-RBD-linker-PA3 are constructed, plates are coated, the shake bacteria are subjected to bacteria liquid PCR identification, plasmids are extracted and subjected to enzyme digestion identification, and the plasmid is sent to Ku Mei Biotech limited company for sequencing.
After the recombinant transfer plasmid pFastbac1-RBD-linker-PA3 is sequenced correctly, DH10Bac is transformedTMColi competent cells, coating a three-resistant plate for blue-white screening, identifying after purification, and naming as rBacmid-RBD-linker-PA3 after correct identification.
3. Rescue of recombinant baculovirus
The recombinant bacmid-RBD-linker-PA3 was transfected into Sf9 cells using the transfection Reagent Lipofectamine 3000Reagent, and the specific steps were as follows: adding Sf9 cells cultured by adherence into a 6-hole cell plate, culturing overnight at 27 ℃, and performing transfection when the cells grow to 80-90% confluence; adding 2.5. mu.g of recombinant bacmid DNA and 5. mu.L of P3000 into 125. mu.L of Grace culture medium without double antibody and serum, and gently mixing uniformly; adding 5 mu L of Lipofectamine 3000Reagent transfection Reagent into 125 mu L of Grace culture medium without double antibody and serum, and gently mixing uniformly; gently mixing bacmid and the transfection reagent, and incubating for 20min at room temperature to form bacmid and liposome complexes; washing cells in a 6-well plate for three times by using a Grace culture medium without double antibodies and serum, adding 500 mu L of Grace culture medium, respectively dropwise adding the bacmid and liposome compound into the 6-well plate, and culturing for 5 hours at 27 ℃; replacing the culture medium with a complete culture medium (10% FBS + 1% double antibody + Grace culture medium), and continuing to culture at 27 ℃ until the cells are obviously diseased (about 4-5 days); the supernatant of the transfected cells was harvested to obtain the 1 st generation of recombinant baculovirus and named rBV-RBD-linker-PA 3.
4. Identification of recombinant baculovirus
Subculturing rBV-RBD-linker baculovirus, extracting genome DNA of the 3 rd generation virus, and performing PCR amplification by taking baculovirus wild virus as a control. Transferring adherent Sf9 cells into a 24-well cell plate, inoculating P3 generation rBV-RBD-linker-PA3 and baculovirus wild virus respectively according to the volume ratio of 3% when the density is 80-90% of confluence degree, and carrying out indirect immunofluorescence detection after continuously culturing for 48h at 27 ℃.
Example 2 preparation and characterization of GEM particles
The lactococcus lactis is inoculated into a GM17 culture medium and cultured for 12-16 h at the temperature of 30 ℃ and the rpm of 180. Centrifuging at 6000 rpm for 15min, discarding the supernatant, resuspending the precipitate with 10mM PBS solution, centrifuging at 6000 rpm for 15min, discarding the supernatant, and washing repeatedly. And (3) resuspending the precipitate with 10% trichloroacetic acid solution with the volume 0.2 times that of the culture solution, boiling for 30min, centrifuging at 6000 rpm for 15min, discarding the supernatant, fully shaking and uniformly mixing the precipitate with 10mM PBS solution for 3-5 min, and washing for 3 times. Finally, the mixture was thoroughly mixed with 10mM PBS solution and the particle density was adjusted to 2.5X 10/ml9Each GEM particle was 1U.
Centrifuging the particles before and after lactococcus lactis treatment at room temperature and 3000rpm for 10min, removing supernatant, freezing tissue slices, and observing the particle morphology before and after treatment by a transmission electron microscope.
The results show that: the lactococcus lactis before treatment has darker cytoplasm and more contents (on the left side of figure 2), and the lactococcus lactis after treatment has less cell contents and maintains the skeleton and shape size of bacteria (on the right side of figure 2), which indicates that the GEM particles are successfully prepared and stored at-80 ℃ for later use.
Example 3 binding and identification of fusion proteins to GEM particles
1. Binding of fusion proteins to GEM particles
1U GEM particles prepared in a laboratory are respectively taken and shaken with 10mL supernatant of suspension cultured P4 generation rBV-RBD-linker-PA3 at room temperature for combination for 60min, and after combination, 6000g is centrifuged for 10min at 4 ℃, and the mixture is washed for 3 times by 10mM PBS and named as SARS-CoV-2 BLP.
2. Identification of SARS-CoV-2 BLP
2.1, centrifuge speed
1U GEM particles prepared in a laboratory are taken to be respectively shake-combined with 10mL supernatant of suspension-cultured P4 generation rBV-RBD-linker-PA3 at room temperature for 60min, after combination, 2mL combined liquid is taken to be added into a 2mL EP tube, and is respectively centrifuged for 15min under the conditions of 500g, 1500 g, 2500 g, 3500 g and 4500 g at 4 ℃, washed for 1 time by 10mM PBS, the color shade of a target band is observed by SDSP-PAGE, and the amount of target protein is analyzed.
The experimental results show that the bound particles can be obtained by centrifuging the bound particles at 1500 g for 15 min. Indicating that GEM particles were mixed with culture supernatant at room temperature and centrifuged at low speed (fig. 3).
2.2 SDS-PAGE identification
To determine whether rBV-RBD-linker-PA3 fusion protein was displayed on the GEM particle surface, protein loading buffers were added to the prepared BLP and GEM particles, respectively, and identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
The SDS-PAGE results showed: the GEM particle lane after being combined with RBD-linker-PA3 has a specific band, the size is about 60KDa, and the size is consistent with the size of the target protein (figure 4).
2.3 Indirect immunofluorescence identification
To further determine whether rBV-RBD-linker-PA3 fusion protein was displayed on the surface of GEM particles, the study used mouse polyclonal antibody against SARS-CoV-2S protein as the primary antibody for indirect immunofluorescence and thin-layer scan analysis.
The results of the indirect immunofluorescence experiments show that: the granular green fluorescence signal can be seen under a fluorescence microscope by the group of GEM granules combined with RBD-linker-PA3 (FIG. 5).
Thin-layer scanning results show that the GEM particles combined by the RBD-linker-PA3 have a single peak compared with the GEM particles, and the other peaks are basically matched with each other (figure 6). The above experimental results show that the fusion protein can be combined on the surface of GEM particles, and the construction of SARS-CoV-2 BLP is successful.
EXAMPLE 4 evaluation of the protective Effect of SARS-CoV-2 bacterium-like particle vaccine on mice
Immunization programs and challenge times for BALB/C and C57BL/6N mice are shown in FIG. 7. 9-month-old female BALB/C mice and C57BL/6N mice, 10 mice per group, were immunized 3 times with 10ug BLP in combination with Freund's Incomplete Adjuvant (FIA) at 3-week intervals, PBS + FIA was used as negative control, sera were collected 14 days after the last immunization and 7 days after SARS-CoV-2 challenge, respectively, and virus-neutralizing antibodies in sera were detected. 2 after 3 rd immunization of mice1 day, respectively using 50LD50The SARS-CoV-2BMA8 strain and CMA14 strain infect two groups of mice by nasal drip, and the weight, body temperature change and survival condition of each group of mice are monitored every day after infection.
The results show that: the BALB/C mice were not abnormal in body weight and body temperature, but the C57BL/6N mice slightly declined in body weight 2-4 days after challenge, then returned to normal, the control group lost body weight (FIGS. 8 and 9), and the body temperature decreased and died (FIGS. 10 and 11). Mice immunized with SARS-CoV-BLP were 100% protective against both SARS-CoV-2 lethal viruses (FIGS. 12 and 13).
The serum of BALB/C and C57BL/6N mice was collected 2 weeks after the 3 rd booster immunization, and the level of the neutralizing antibody of SARS-CoV-2 was detected, and the detection results showed that: BALB/c mice had an average neutralizing antibody titer of 1: 830 (FIG. 14), C57BL/6N mice had an average neutralizing antibody titer of 1: 338 (fig. 15).
The experimental results show that the BLP vaccine induces a neutralizing antibody response against BMA8 and C57MA14, and can 100 percent protect aged BALB/C and C57BL/6N mice against the attack of mouse adaptive SARS-CoV-2.
While certain exemplary embodiments of the present invention have been described above by way of illustration only, it will be apparent to those of ordinary skill in the art that the described embodiments may be modified in various different ways without departing from the spirit and scope of the invention. Accordingly, the drawings and description are illustrative in nature and should not be construed as limiting the scope of the invention.
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Gly Thr Thr Cys Thr Ala Cys Cys Gly Cys Thr Ala Cys Thr Ala Ala
1170 1175 1180
Cys Ala Ala Cys Thr Cys Cys Ala Ala Cys Thr Cys Thr Ala Cys Thr
1185 1190 1195 1200
Thr Cys Cys Thr Cys Ala Ala Ala Cys Thr Cys Thr Ala Ala Cys Gly
1205 1210 1215
Cys Thr Thr Cys Ala Ala Thr Cys Cys Ala Cys Ala Ala Gly Gly Thr
1220 1225 1230
Gly Gly Thr Gly Ala Ala Gly Gly Gly Thr Gly Ala Cys Ala Cys Cys
1235 1240 1245
Thr Thr Gly Thr Gly Gly Gly Gly Thr Cys Thr Gly Thr Cys Ala Cys
1250 1255 1260
Ala Gly Ala Ala Gly Thr Cys Ala Gly Gly Thr Thr Cys Cys Cys Cys
1265 1270 1275 1280
Cys Ala Thr Cys Gly Cys Thr Thr Cys Thr Ala Thr Cys Ala Ala Gly
1285 1290 1295
Gly Cys Thr Thr Gly Gly Ala Ala Cys Cys Ala Cys Cys Thr Gly Thr
1300 1305 1310
Cys Thr Thr Cys Thr Gly Ala Cys Ala Cys Cys Ala Thr Cys Cys Thr
1315 1320 1325
Gly Ala Thr Cys Gly Gly Thr Cys Ala Ala Thr Ala Cys Cys Thr Gly
1330 1335 1340
Cys Gly Thr Ala Thr Cys Ala Ala Gly
1345 1350
<210> 2
<211> 45
<212> DNA
<213> Artificial sequence (SARS-CoV-2)
<400> 2
tgctctagac atcaccatca ccatcacaga gtccaaccaa cagaa 45
<210> 3
<211> 44
<212> DNA
<213> Artificial sequence (SARS-CoV-2)
<400> 3
accagaacca ccaccagaac cacctttgtt tttaaccaaa ttag 44
<210> 4
<211> 44
<212> DNA
<213> Artificial sequence (SARS-CoV-2)
<400> 4
accagaacca ccaccagaac cacctttgtt tttaaccaaa ttag 44
<210> 5
<211> 37
<212> DNA
<213> Artificial sequence (SARS-CoV-2)
<400> 5
ccgctcgagt tacttgatac gcaggtattg accgatc 37
Claims (10)
1. SARS-CoV-2 bacteria-like particle is prepared by displaying rBV-RBD-linker-PA3 fusion protein on GEM particle surface.
2. A SARS-CoV-2 bacterium-like particle as claimed in claim 1, wherein the protein sequence of the linker is (Gly-Gly-Ser-Gly)2。
3. SARS-CoV-2 bacteria-like particle according to claim 1, wherein the amino acid sequence of the rBV-RBD-linker-PA3 fusion protein is shown in SEQ No 1.
4. A SARS-CoV-2 bacteria-like particle according to claim 1, wherein the rBV-RBD-linker-PA3 fusion protein is expressed using an insect-baculovirus expression system.
5. Use of SARS-CoV-2 bacteria-like particle according to any of claims 1 to 4 for the preparation of a SARS-CoV-2 vaccine.
6. A SARS-CoV-2 vaccine comprising the SARS-CoV-2 bacteria-like particle of claim 1.
7. A SARS-CoV-2 vaccine as claimed in claim 6 wherein the protein sequence of the linker is (Gly-Gly-Ser-Gly)2。
8. SARS-CoV-2 vaccine according to claim 6, wherein the amino acid sequence of the SARS-CoV-2 bacteria-like particle is as shown in SEQ No 1.
9. A SARS-CoV-2 vaccine as claimed in claim 6 wherein the rBV-RBD-linker-PA3 fusion protein is expressed using an insect-baculovirus expression system.
10. The method of claim 6, wherein the insect-baculovirus expression system is used to express rBV-RBD-linker-PA3 fusion protein, the protein sequence of the linker is (Gly-Gly-Ser-Gly)2Mixing rBV-RBD-linker-PA3 fusion protein with GEM particle, centrifuging, washing to obtain SARS-CoV-2 bacteria-like particle, adding pharmaceutically acceptable adjuvant, and making into vaccine.
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