CN115466330B - Coronavirus subunit vaccine based on virus-like particle presenting coronavirus receptor binding region - Google Patents

Abstract

The present application discloses a coronavirus subunit vaccine based on virus-like particles presenting coronavirus receptor binding domains. The application provides a recombinant virus-like particle, which is a chikungunya virus-like particle carrying specific proteins; the specific protein is S protein of SARS-CoV-2 or RBD segment of SARS-CoV-2S protein. The recombinant virus-like particles of the application are immunized on Balb/c mice, and the recombinant virus-like particles can be found to be capable of efficiently stimulating the mice to produce antibodies aiming at SARS-CoV-2RBD and inducing the production of neutralizing antibodies with high titer.

Description

Coronavirus subunit vaccine based on virus-like particle presenting coronavirus receptor binding region

Technical Field

The application relates to the technical field of biological medicine, in particular to a coronavirus subunit vaccine based on virus-like particles presenting a coronavirus receptor binding region.

Background

Coronaviruses are enveloped, sense RNA viruses belonging to the genus coronavirus of the family coronaviridae. Some coronaviruses are severely threatening human health, such as SARS-CoV, which causes severe respiratory syndrome (SARS) outbreaks, and MERS-CoV, which causes Middle East Respiratory Syndrome (MERS) outbreaks, and SARS-CoV-2, which causes new coronavirus pneumonia (COVID-19). In addition, there are also coronaviruses which cause less symptomatic diseases in humans, such as HCoV-NL63, HCoV-229E, HCoV-OC43, HCoV-HKU1, etc. Some coronaviruses can also infect animals and have an impact on pet health and livestock production, such as feline peritonitis virus (FIPV) can cause peritonitis and ascites in cats, with high mortality, and Porcine Epidemic Diarrhea Virus (PEDV) can cause diarrhea in live pigs, severely threatening production of live pigs. In addition, there are coronaviruses that can infect dogs, mice and cattle. The most effective way to combat viral infections is vaccination, which is now being tightly developed in all countries around the world against new coronaviruses and other coronaviruses.

In the early stages of a new coronal epidemic, scientists have demonstrated that the receptor taken by the virus into cells is the cell surface protein ACE2, and that the Receptor Binding Domain (RBD) on the virus surface spike protein (S) directly mediates interactions with ACE 2. Most neutralizing antibodies isolated from naturally infected patients are capable of binding RBD and preventing the interaction of RBD with ACE2, so RBD as an immunogen will be able to stimulate the body to produce neutralizing antibodies that inhibit the binding of virus to the receptor.

Current studies show that RBD monomeric or dimeric proteins or mRNA encoding RBD as vaccines can stimulate the body to produce neutralizing antibodies. However, the immunogenicity of RBD monomers or dimers is relatively weak, and no RNA vaccine is approved for marketing, and the long-term safety is still unknown, so that the development of RBD subunit vaccines with high safety and high immunogenicity is necessary.

Disclosure of Invention

It is an object of the present application to provide a coronavirus subunit vaccine based on virus-like particles presenting coronavirus receptor binding domains.

In a first aspect, the application claims a recombinant virus-like particle.

The recombinant virus-like particle claimed by the application is chikungunya virus-like particle carrying specific proteins; the specific protein is S protein of SARS-CoV-2 or RBD segment of SARS-CoV-2S protein. The specific protein is presented by virus-like particles.

The chikungunya virus-like particles are formed by self-assembly of the capsid protein, the E1 protein, the E2 protein, the E3 protein and the 6K protein of the chikungunya virus. The capsid protein, E1 protein, E2 protein, E3 protein and 6K protein of chikungunya virus are generated by cleavage of chikungunya virus multimeric protein capsid-E3-E2-6K-E1 (see figure 1) by host protease.

Further, the recombinant virus-like particle is formed by self-assembly of the capsid protein of chikungunya virus, the E1 protein of chikungunya virus, fusion protein a and the 6K protein of chikungunya virus. Each of the above proteins is produced by cleavage of the multimeric protein "capsid-fusion protein A-6K-E1" by the host protease. The fusion protein A contains E2 protein, E3 protein and the specific protein of chikungunya virus.

Wherein the specific protein replaces a Furin cleavage site between the chikungunya virus E3 protein and the E2 protein. The inventors tried some other locations nearby, but these attempts failed to detect VLP formation.

Further, the fusion protein A may also contain a tag protein for purification, such as a Flag tag.

Still further, the fusion protein A comprises, in order from N-terminus to C-terminus, the E3 protein of chikungunya virus, the specific protein, the N-terminal segment of the E2 protein of chikungunya virus, a Flag tag, and the C-terminal segment of the E2 protein of chikungunya virus.

Wherein the Flag tag is inserted between amino acids 204 and 205 of the E2 protein of chikungunya virus.

More specifically, the fusion protein A sequentially comprises an E3 protein of the chikungunya virus, a flexible peptide 1, the specific protein, a flexible peptide 2, an N-terminal segment of the E2 protein of the chikungunya virus, a flexible peptide 3, a Flag tag, a flexible peptide 4 and a C-terminal segment of the E2 protein of the chikungunya virus from the N end to the C end.

Wherein, four flexible peptides for connecting functional proteins or protein functional regions can be set according to actual conditions, including: if functional proteins or protein functional regions before and after one or more flexible peptides are directly connected without affecting the functions of related proteins, the corresponding flexible peptides can be omitted and a direct connection method can be adopted. Of course, a variety of flexible peptides may be used, such as GSGG, GGGS, SGS, SSSG, GGGGS, (GGGGS) n, etc., as long as the function of the protein of interest is not affected.

In a second aspect, the application claims any of the following biomaterials:

p1 and fusion protein B sequentially comprise, from the N end to the C end, a capsid protein of the chikungunya virus, an E1 protein of the chikungunya virus, the fusion protein A and a 6K protein of the chikungunya virus in the first aspect.

P2, nucleic acid molecule encoding the fusion protein B described in P1.

P3, an expression cassette, a recombinant vector, a recombinant cell or a recombinant bacterium comprising the nucleic acid molecule as described in P2.

The expression cassette refers to a DNA capable of expressing the fusion protein B in a host cell, and the DNA may include not only a promoter for initiating transcription of the gene encoding the fusion protein B, but also a terminator for terminating transcription of the gene encoding the fusion protein B. Further, the expression cassette may also include an enhancer sequence.

Further, the recombinant vector in P3 may be a eukaryotic expression vector containing the nucleic acid molecule.

Further, the recombinant vector is a recombinant plasmid obtained by cloning the nucleic acid molecule into a pcDNA3.1 (+) vector.

In a specific embodiment of the present application, the recombinant vector is specifically a recombinant plasmid obtained by substituting the nucleic acid molecule for a small fragment between KpnI and XhoI of pcDNA3.1 (+) vector.

In a third aspect, the application claims a method of preparing a recombinant virus-like particle as described in the first aspect above.

The method for preparing recombinant virus-like particles as claimed in the first aspect of the present application may comprise the steps of: introducing the recombinant vector described in the second aspect into mammalian cells, and then performing cell culture, thereby obtaining the recombinant virus-like particles.

In a specific embodiment of the application, the mammalian cell is a HEK293F cell.

In the method, HEK293F cells are cultured to a logarithmic phase by adopting a SMM293-TII culture medium, and then the recombinant vector is transfected; culturing for 24h after transfection, and then adding SMS 293-SUPI without serum and protein feed liquid for culturing for 48h; the recombinant virus-like particles are then obtained from the cell culture supernatant.

Further, the recombinant virus-like particles may be obtained from cell culture supernatants as follows: adding anti-Flag agarose beads into the cell culture supernatant, and shaking and uniformly mixing for 12 hours at 4 ℃; agarose beads were collected by centrifugation, transferred to a gravity flow empty column, washed off the contaminating proteins, and then competitively eluted with a buffer containing 200. Mu.g/ml 3 XFlag peptide (and then concentrated by a 100kD cutoff concentration tube) to give a CHIKV-RBD-Flag2-VLP solution (i.e., a solution containing the recombinant virus-like particles).

In a fourth aspect, the application claims any of the following applications:

use of Q1, a biological material according to the second aspect of the foregoing for the preparation of a recombinant virus-like particle according to the first aspect of the foregoing;

use of Q2, a recombinant virus-like particle as described in the first aspect hereinbefore or a biological material as described in the second aspect hereinbefore for the manufacture of a product (e.g. a vaccine or medicament) for the prophylaxis of a disease caused by infection with SARS-CoV-2.

In a fifth aspect, the application claims a vaccine for preventing diseases caused by SARS-CoV-2 infection.

The vaccine for preventing SARS-CoV-2 infection diseases has the active component of the recombinant virus-like particle.

Further, the vaccine also contains an adjuvant, such as aluminum adjuvant.

Still further, the aluminum adjuvant is an aluminum hydroxide adjuvant.

In a specific embodiment of the application, the adjuvant is specifically an Imject Alum (Thermo Scientific Corp, accession number 77161). Correspondingly, the vaccine is prepared from the recombinant virus-like particle and the Imject Alum according to 2 μg (in terms of protein content): 50 mu L of the components.

In the fourth and fifth aspects, the disease caused by SARS-CoV-2 infection can be specifically new coronavirus pneumonia (COVID-19).

In the above aspects, the amino acid sequence of the RBD segment of SARS-CoV-2S protein can be as indicated at positions 325-518 of SEQ ID No. 5; alternatively, a protein derived from SARS-CoV-2 and having more than 98% identity and the same function as the protein shown at positions 325-518 of SEQ ID No. 5; or the protein shown in 325-518 of SEQ ID No.5 is substituted and/or deleted and/or added by one or more amino acid residues to obtain the protein with the same function.

In the above aspects, the amino acid sequence of the capsid protein of chikungunya virus may be as shown in positions 1-261 of SEQ ID No. 5; or a protein derived from chikungunya virus and having 98% or more identity with the protein represented by positions 1 to 261 of SEQ ID No.5 and having the same function; or the protein shown in the 1 st to 261 st positions of SEQ ID No.5 is substituted and/or deleted and/or added by one or more amino acid residues to obtain the protein with the same function.

In the above aspects, the amino acid sequence of the E1 protein of the chikungunya virus can be shown in 1022-1460 of SEQ ID No. 5; or a protein derived from chikungunya virus and having 98% or more identity with the protein shown at positions 1022 to 1460 of SEQ ID No.5 and having the same function; or protein with the same function, which is obtained by substituting and/or deleting and/or adding one or more amino acid residues for the protein shown in 1022-1460 of SEQ ID No. 5.

In the above aspects, the amino acid sequence of the 6K protein of the chikungunya virus may be as shown in positions 960-1021 of SEQ ID No. 5; or a protein derived from chikungunya virus and having 98% or more identity with the protein represented by positions 960 to 1021 of SEQ ID No.5 and having the same function; or the protein shown in 960-1021 of SEQ ID No.5 is substituted and/or deleted and/or added by one or more amino acid residues to obtain the protein with the same function.

In the above aspects, the amino acid sequence of the E3 protein of the chikungunya virus may be as shown in positions 262-321 of SEQ ID No. 5; or a protein derived from chikungunya virus and having 98% or more identity with the protein represented at positions 262 to 321 of SEQ ID No.5 and having the same function; or the protein shown in 262-321 of SEQ ID No.5 is substituted and/or deleted and/or added by one or more amino acid residues to obtain the protein with the same function.

In the above aspects, the amino acid sequence of the E2 protein of the chikungunya virus may be as shown in positions 326-747 of SEQ ID No. 4; or a protein derived from chikungunya virus and having more than 98% identity with the protein shown at positions 326 to 747 of SEQ ID No.4 and having the same function; or a protein with the same function, which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein shown in 326-747 of SEQ ID No. 4.

In the above aspects, the amino acid sequence of the Flag tag may be as shown at positions 730-737 of SEQ ID No. 5.

In the above aspects, the amino acid sequence of the N-terminal segment of the E2 protein of the chikungunya virus can be shown as 523-725 of SEQ ID No. 5; or a protein derived from chikungunya virus and having more than 98% identity with the protein represented by positions 523 to 725 of SEQ ID No.5 and having the same function; or the protein shown in 523-725 of SEQ ID No.5 is substituted and/or deleted and/or added by one or more amino acid residues to obtain the protein with the same function.

In the above aspects, the amino acid sequence of the C-terminal segment of the E2 protein of the chikungunya virus may be as shown in positions 742-959 of SEQ ID No. 5; or a protein derived from chikungunya virus and having 98% or more identity with the protein shown at positions 742 to 959 of SEQ ID No.5 and having the same function; or a protein having the same function obtained by substituting and/or deleting and/or adding one or more amino acid residues to the protein shown in positions 742-959 of SEQ ID No. 5.

In the above aspects, the amino acid sequence of the flexible peptide 1 may be as shown at positions 322-324 of SEQ ID No. 5.

In the above aspects, the amino acid sequence of the flexible peptide 2 may be as shown in positions 519-522 of SEQ ID No. 5.

In the above aspects, the amino acid sequence of the flexible peptide 3 may be as shown at positions 726-729 of SEQ ID No. 5.

In the above aspects, the amino acid sequence of the flexible peptide 4 may be as shown in positions 738-741 of SEQ ID No. 5.

Further, the amino acid sequence of the fusion protein A can be shown in positions 262-959 of SEQ ID No. 5; or a protein having 98% or more identity and the same function as the protein shown at positions 262 to 959 of SEQ ID No. 5; or a protein with the same function, which is obtained by substituting and/or deleting and/or adding one or more amino acid residues for the protein shown in 262-959 of SEQ ID No. 5.

Furthermore, the amino acid sequence of the fusion protein B can be the whole sequence shown in SEQ ID No. 5; or a protein having 98% or more identity and the same function as the protein shown in SEQ ID No. 5; or the protein shown in SEQ ID No.5 is obtained by substituting and/or deleting and/or adding one or more amino acid residues, and has the same function.

In the above aspects, in the nucleic acid molecule described in the foregoing P2, the nucleic acid molecule encoding the capsid protein of the chikungunya virus may be as shown in positions 1 to 783 of SEQ ID No. 2; or a DNA molecule which has 98% or more identity with positions 1 to 783 of SEQ ID No.2 and encodes the capsid protein of the chikungunya virus.

In the above aspects, in the nucleic acid molecule described in the foregoing P2, the nucleic acid molecule encoding the E1 protein of the chikungunya virus may be as shown in positions 3064-4383 of SEQ ID No. 2; or a DNA molecule which has 98% or more identity with the 3064-4383 th site of SEQ ID No.2 and encodes the E1 protein of the chikungunya virus.

In the above aspects, in the nucleic acid molecule described in the foregoing P2, the nucleic acid molecule encoding the fusion protein a may be as shown in positions 784 to 2877 of SEQ ID No. 2; or a DNA molecule which has more than 98% identity with positions 784-2877 of SEQ ID No.2 and which encodes said fusion protein A.

In the above aspects, in the nucleic acid molecule described in the foregoing P2, the nucleic acid molecule encoding the 6K protein of the chikungunya virus may be as shown in positions 2878-3063 of SEQ ID No. 2; or a DNA molecule which has more than 98% identity with the 2878-3063 th site of SEQ ID No.2 and encodes the 6K protein of the chikungunya virus.

Further, the nucleic acid molecule described in the foregoing P2 may be represented by the entire sequence of SEQ ID No. 2; or a DNA molecule which has more than 98% identity with SEQ ID No.2 and which encodes said fusion protein B.

The present application provides chikungunya virus-like particles (CHIKV-VLPs) having a coronavirus receptor binding domain, useful as subunit vaccines for the prevention of diseases caused by infection with novel coronaviruses (SARS-CoV-2). The application expresses the CHIKV-RBD-Flag2 fusion protein in 293F cells by transient transfection of recombinant plasmids. The method comprises the steps of inserting a Flag tag (sequence: DYKDDDDK) between amino acids 204 and 205 of a chiKNKNKNKNKNKNKYV E2 protein in a chiKNKNKNKNKNKYV-RBD-Flag 2 fusion protein, replacing a Furin cleavage site (sequence RQRR) between the chiKNKNKNKNKNKYV E3 protein and the E2 protein by SARS-CoV-2RBD (containing T333-G526) peptide fragments, adding 3 and 4 flexible amino acids respectively at the upper and lower sides of the RBD, adding 4 flexible amino acids (GSGGDYKDDDDKGGGS) respectively at the upper and lower sides of the Flag tag, and forming a cap-E3-RBD-E2-DYDKDKDDD (Flag tag) -E2-6K-E1 polyprotein after translational expression in cells, and forming spherical particles of the Flag-DKNKDK 2-DKDK-2 protein from the spherical particles. The surface of CHIKV-RBD-Flag 2-VLPs displays SARS-CoV-2RBD at high density to stimulate immune cells to produce immune responses against RBD. Balb/c mice were immunized and found to be highly potent in stimulating antibodies against SARS-CoV-2RBD by CHIKV-RBD-Flag2-VLP and induced to produce high titers of neutralizing antibodies.

Drawings

FIG. 1 is a schematic diagram showing the structure of a CHIKV multimeric protein and a CHIKV-RBD-Flag2 fusion protein.

FIG. 2 is an electron microscopic negative dye photograph of CHIKV-RBD-Flag2-VLP.

FIG. 3 is a chromatogram of molecular sieve purification of SARS-CoV-2 RBD.

FIG. 4 shows the results of detecting SARS-CoV-2 RBD-specific IgG antibody titer in serum.

FIG. 5 shows the results of detecting the titer of neutralizing antibodies in serum. In the figures, EC50 units are serum dilutions.

Detailed Description

The following detailed description of the application is provided in connection with the accompanying drawings that are presented to illustrate the application and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the application in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

The buffers in the following examples are as follows, unless otherwise indicated: contains 25mM Tris-HCl and 150mM NaCl, the balance being water, pH8.0. Unless otherwise indicated, the quantitative tests in the examples below were all performed in triplicate, and the results averaged.

Example 1 preparation of Virus-like particles

1. Construction of recombinant plasmids

The double-stranded DNA molecule shown in SEQ ID No.1 is used for replacing a small fragment between KpnI and XhoI of the pcDNA3.1 (+) vector, and the recombinant plasmid pcDNA3.1-chiKV-VLP is obtained after sequencing and verification.

The 1 st to 783 rd positions of SEQ ID No.1 are the coding region of the capsid protein; the 784-963 position is the coding region of the E3 protein; the 964-975 position is the coding region of Furin enzyme cutting site; the 976-2241 position is the coding region of E2 protein; positions 2242-2427 are the 6K protein coding region; the 2428-3747 position is the E1 protein coding region.

The double-stranded DNA molecule shown in SEQ ID No.1 encodes the CHIKV polyprotein shown in SEQ ID No.4 (the schematic domain is shown in FIG. 1).

Positions 1-261 of SEQ ID No.4 are the capsid protein; e3 protein at 262-321; the 322 th to 325 th sites are Furin enzyme cutting sites; the 326-747 position is E2 protein; the 748-809 position is 6K protein; e1 protein at positions 810-1248.

CHIKV polyprotein is cleaved by host proteases to produce the capsid, E1, E2, E3, 6K proteins, which self-assemble to form virus-like particles, i.e., CHIKV-VLPs.

The double-stranded DNA molecule shown in SEQ ID No.2 is used for replacing a small fragment between KpnI and XhoI of the pcDNA3.1 (+) vector, and the recombinant plasmid pcDNA3.1-chiKV-RBD-Flag2 is obtained after sequencing and verification.

The 1 st to 783 rd positions of SEQ ID No.2 are the coding region of the capsid protein; the 784-963 position is the E3 protein coding region; positions 964-972 are flexible peptide 1 coding regions; bits 973-1554 are RBD coding region; flexible peptide 2 coding region at positions 1555-1566; positions 1567-2175 are coding regions of the N-terminal region of the E2 protein; 2176 to 2187 are flexible peptide 3 coding regions; bits 2188-2211 are Flag tag coding region; the 2212-2223 positions are flexible peptide 4 coding region; the 2224-2877 th bit is the coding region of the C-terminal region of the E2 protein; the 2878-3063 position is a 6K protein coding region; the E1 protein coding region is positioned 3064-4383.

The double-stranded DNA molecule shown in SEQ ID No.2 encodes the CHIKV-RBD-Flag2 fusion protein shown in SEQ ID No.5 (the schematic domain is shown in FIG. 1).

Positions 1-261 of SEQ ID No.5 are the capsid protein; e3 protein at 262-321; flexible peptide 1 at positions 322-324; bits 325-518 are RBD; flexible peptide 2 at positions 519-522; the 523 th to 725 th sites are N-terminal sections of the E2 protein; flexible peptide 3 at positions 726-729; bits 730-737 are Flag tags; flexible peptide 4 at positions 738-741; positions 742-959 are the C-terminal segment of E2 protein; 6K protein at 960-1021; e1 protein at 1022-1460.

The difference between CHIKV-RBD-Flag2 fusion proteins compared to CHIKV polyprotein is only that a Flag tag was inserted into the E2 protein (flexible linker "GSGG" was introduced upstream of the Flag tag, flexible linker "GGGS" was introduced downstream), and Furin cleavage site was replaced by SARS-CoV-2RBD (flexible linker "SGS" was introduced upstream of the insert, and flexible linker "SSSG" was introduced downstream). Cleavage of the CHIKV-RBD-Flag2 fusion protein by the host protease yields a capsid protein, an E1 protein, an E3-RBD-E2-Flag-E2 fusion protein, a 6K protein, which self-assemble to form virus-like particles, i.e., CHIKV-RBD-Flag 2-VLPs. The E3-RBD-E2-Flag-E2 fusion protein comprises the following components from the N end to the C end in sequence: e3 protein, SARS-CoV-2RBD, E2 protein N segment, flag tag, E2 protein C segment.

2. Preparation of CHIKV-VLPs

1. Culturing HEK293F cells to logarithmic phase Using SMM293-TII Medium(density reaches 1.5X10) ⁶ -2.0×10 ⁶ Individual cells/ml culture system), then the recombinant plasmid pcDNA3.1-chiKV-VLP (2. Mu.g recombinant plasmid/ml culture system) was transfected with PEI MAX (Polysciences, 24765-2), then the culture was continued for 24h, then the serum-free protein-free feed solution SMS 293-SUPI (35 ml/L culture system) was added, and then the culture was continued for 48h. SMM293-TII medium and serum-free protein-free feed supplement SMS 293-SUPI (also known as SMS 293-SUPI medium additive solution) are both manufactured by Yinqiao China Biotechnology Co.

2. After completion of step 1, 1000g was centrifuged for 10min, and the supernatant was collected.

3. Taking the supernatant obtained in the step 2, centrifuging 4500g for 1h, and collecting the supernatant.

4. The supernatant obtained in step 3 was taken, PEG6000 (to make the concentration thereof in the system 7g/100 mL) and NaCl (to make the concentration thereof in the system 0.5M) were added, stirred at 4℃for 12 hours, and then centrifuged at 4500g for 1 hour, and the precipitate was collected.

5. Taking the precipitate obtained in the step 4, carefully cleaning the precipitate with a buffer solution, and then re-suspending the precipitate with the buffer solution to obtain a precipitate re-suspension. Sucrose was used as a solute and buffer was used as a solvent to give a sucrose concentration of 24g/100ml, i.e. 24% sucrose solution. 2ml of 24% sucrose solution was added to the corresponding ultracentrifuge tube of the Beckman SW41Ti rotor, then about 7.5ml of the precipitate heavy suspension was carefully spread over the sucrose solution, and then centrifuged at 32000rpm for 2 hours at 4℃using an ultracentrifuge, and the precipitate at the bottom of the centrifuge tube was collected.

6. Taking the precipitate obtained in the step 5, and re-suspending the precipitate by using a buffer solution to obtain a precipitate suspension. Preparing 20% -60% continuous density gradient sucrose (sucrose is used as solute, buffer is used as solvent, concentration% of sucrose means g/100 ml) in a transparent overspeed centrifuge tube corresponding to Beckman SW41Ti rotor, then spreading 1ml of sediment suspension on top of the continuous density gradient sucrose, centrifuging for 15h at 4 ℃ using the overspeed centrifuge, and puncturing to extract a target strip at the upper middle part of the density gradient.

7. And (3) taking the extract obtained in the step (6), and replacing the buffer system with a buffer solution by adopting a 100kD cutoff type concentration tube, namely the CHIKV-VLP solution.

8. And (3) taking the solution obtained in the step (7), detecting the protein concentration by adopting nanodrop, subpackaging, and then quickly freezing with liquid nitrogen and storing at-80 ℃.

3. Preparation of CHIKV-RBD-Flag2-VLP

1. HEK293F cells were cultured to logarithmic growth phase (density 1.5X10) using SMM293-TII medium ⁶ -2.0×10 ⁶ Individual cells/ml culture system), then the recombinant plasmid pcDNA3.1-chiKV-RBD-Flag2 (2. Mu.g recombinant plasmid/ml culture system) was transfected with PEI MAX (Polysciences, 24765-2), then the culture was continued for 24 hours, then the serum-free protein-free feed solution SMS 293-SUPI (35 ml/L culture system) was added, and then the culture was continued for 48 hours. SMM293-TII medium and serum-free protein-free feed supplement SMS 293-SUPI (also known as SMS 293-SUPI medium additive solution) are both manufactured by Yinqiao China Biotechnology Co.

2. After completion of step 1, 1000g was centrifuged for 10min, and the supernatant was collected.

3. Taking the supernatant obtained in the step 2, centrifuging 4500g for 1h, and collecting the supernatant.

4. And (3) adding anti-Flag beads (1 ml beads per liter of supernatant) into the supernatant obtained in the step (3), and shaking and uniformly mixing at 4 ℃ for 12 hours. anti-Flag tags: jinsri biotechnology Co.

5. After the completion of step 4, 100g was centrifuged for 10min, and the beads were collected.

6. The beads collected in step 5 were transferred to a gravity flow empty column (BIO-RAD company, model Econo-Pac), the foreign proteins were washed out sufficiently with a buffer, then the target proteins were competitively eluted with a buffer containing 200. Mu.g/ml 3 XFlag peptides, and then concentrated and replaced with a buffer through a 100kD cutoff concentration tube to obtain a CHIKV-RBD-Flag2-VLP solution. 3×flag peptide: strong living things.

7. And (3) taking the solution obtained in the step (6), detecting the protein concentration by adopting nanodrop, subpackaging, and then quickly freezing with liquid nitrogen and storing at-80 ℃.

8. And (3) taking the CHIKV-RBD-Flag2-VLP solution obtained in the step (6) and carrying out electron microscope observation. An electron microscope counterstain photograph of the CHIKV-RBD-Flag2-VLP is shown in fig. 2. Exhibiting an irregular spherical shape.

EXAMPLE 2 evaluation of immune Effect

Aluminium gel adjuvant (image aluminium, aluminium hydroxide adjuvant): thermo Scientific Corp, product number 77161.

The protein concentration was adjusted with PBS buffer pH7.4 using the chiKV-VLP solution or the chiKV-RBD-Flag2-VLP solution prepared in example 1.

1. Group immunization

Balb/c females at 4-6 weeks of age were randomly divided into three groups of 3 females. Immunization process: primary immunization was performed on trial day 1 and booster immunization was performed on trial day 15. The immunization modes are as follows: intramuscular injection of the right hind limb. A single mouse was immunized with 100. Mu.l of the immunized material. CHIKV-VLP group: mu.l of the immunomer was emulsified from 50. Mu.l of CHIKV-VLP solution (protein content 2. Mu.g) and 50. Mu.l of aluminium gel adjuvant. CHIKV-RBD-Flag2-VLP group: each 100. Mu.l of the immunomer was emulsified from 50. Mu.l of CHIKV-RBD-Flag2-VLP solution (protein content 2. Mu.g) and 50. Mu.l of aluminium gel adjuvant. PBS control group: the immunomer was PBS buffer.

Mandibular blood was collected on day 21, day 35, and day 56 after primary immunization, respectively. Blood was taken, allowed to stand at 4℃for about 1 hour, and then centrifuged at 1500g for 15min to collect serum.

2. Serum was assayed for SARS-CoV-2 RBD-specific IgG antibody titer.

Serum from day 21 after primary immunization and serum from day 35 after primary immunization were taken and the titer of IgG antibodies that specifically bound to SARS-CoV-2RBD was detected by ELISA.

1. Preparation of SARS-CoV-2RBD

(1) The DNA molecule shown in SEQ ID No.3 (which is SARS-CoV-2RBD (319R-541F) -strep-Flag sequence with codon optimized and codes the amino acid sequence shown in SEQ ID No. 6) is inserted between NheI and BamHI cleavage sites of pcDNA3.1 (+) plasmid to obtain recombinant plasmid, and the recombinant plasmid is named as recombinant plasmid pcDNA3.1-RBD-Flag after sequencing verification.

(2) HEK293F cells were cultured to logarithmic growth phase (density 1.5X10) using SMM293-TII medium ⁶ -2.0×10 ⁶ Individual cells/ml culture system), then the recombinant plasmid pcDNA3.1-RBD-Flag (2. Mu.g recombinant plasmid/ml culture system) was transfected with PEI MAX (Polysciences, 24765-2), cultivation was continued for 24h, then the serum-free and protein-free feed supplement SMS 293-SUPI (35 ml/L culture system) was added, and cultivation was continuedAnd (5) culturing for 48 hours. SMM293-TII medium and serum-free protein-free feed supplement SMS 293-SUPI (also known as SMS 293-SUPI medium additive solution) are both manufactured by Yinqiao China Biotechnology Co.

(3) After completion of step 1, 1000g was centrifuged for 10min, and the supernatant was collected.

(4) Taking the supernatant obtained in the step (3), centrifuging 4500g for 1h, collecting the supernatant, filtering with a 0.45 μm filter membrane, and collecting filtrate.

(5) And (3) taking the filtrate obtained in the step (4), and purifying by adopting anti-Flag beads affinity chromatography.

Specific parameters are as follows: an anti-Flag beads (Kirschner Co., ltd.) was packed in a gravity flow column (BIO-RAD, model Econo-Pac), 1ml beads was mixed per liter of filtrate, the filtrate was passed through the beads 3 times in the gravity flow column, and then the foreign proteins were washed out sufficiently with a buffer, and then the target proteins were competitively eluted with a buffer containing 200. Mu.g/ml 3 XFlag peptides, and then concentrated by a 10kD cutoff concentration tube.

(6) And (3) taking the solution obtained in the step (5), and purifying through a molecular sieve.

Specific parameters are as follows: pre-packed column Superdex 200Increase 10/300GL (molecular sieves) using GE gel filtration; the sample was eluted at a loading volume of 0.2ml using 25ml buffer, and the chromatogram showed 2 peaks (corresponding to a retention volume of 13.3ml and 14.9ml, respectively) (see FIG. 3) corresponding to the dimer and monomer of SARS-CoV-2RBD, respectively (corresponding to 13.3ml of the pre-packed column used with a molecular weight of about 65kDa, and corresponding to 14.9ml with a molecular weight of about 32kDa. RBD-Flag protein with a molecular weight of 30.7kDa. Two peaks, respectively, were dimer and monomer according to molecular weight size), and the monomer peak was collected (corresponding to a retention volume of 14.9ml, which was the monomer peak), i.e., SARS-CoV-2RBD solution.

2. Detection of SARS-CoV-2RBD specific IgG antibody titre in serum

(1) Coating: taking an ELISA plate, adding 100 mu L of SARS-CoV-2RBD solution prepared in the step 1 (the coating amount of the SARS-CoV-2RBD is 100 ng/hole, adjusting the protein concentration by using a buffer solution) into each hole, incubating overnight at 4 ℃, and discarding the supernatant.

(2) B3T was blocked and incubated at 37℃for 1 hour, and the supernatant was discarded.

(3) 100. Mu.L of serum dilution was added to each well and incubated at 37℃for 1 hour. The supernatant was discarded and washed with PBST.

( Serum dilution: serum obtained in the step one is taken and diluted with a B3T gradient. At least 2 duplicate wells were set per dilution. )

(4) The B3T diluted secondary enzyme-labeled antibody was added, incubated at 37℃for 1 hour at 100. Mu.L per well, and the supernatant was discarded, followed by PBST washing.

( The enzyme-labeled secondary antibody is anti-mouse IgG (working concentration is 1:4000). Anti-mouse IgG: abcam corporation, cat No. ab6789. )

(5) mu.L TMB dye was added to each well and incubated at room temperature.

(6) mu.L of stop solution was added to each well.

(7) The absorbance at 450nm was read with a microplate reader.

PBS (1L formulation, pH 7.4): monopotassium phosphate (KH) ₂ PO ₄ ) 0.27g, disodium hydrogen phosphate (Na ₂ HPO ₄ ) 1.42g of sodium chloride (NaCl) 8g, 0.2g of potassium chloride (KCl) and about 800mL of deionized water are added, the mixture is fully stirred and dissolved, then concentrated hydrochloric acid is added to adjust the pH to 7.4, and finally the volume is fixed to 1L.

PBST:1LPBS was added with 0.5ml of Tween-20.

B3T: PBST containing 3% (3 g/100 ml) BSA.

The results are shown in FIG. 4.CHIKV-RBD-Flag 2-VLPs stimulated mice to produce high titers of antibodies that bind SARS-CoV-2 RBD.

3. Detection of serum neutralizing antibody titres

Serum from day 35 after primary immunization and serum from day 56 after primary immunization of animals of the CHIKV-RBD-Flag2-VLP group were used to detect neutralizing antibody titers in serum using SARS-CoV-2Surrogate Virus Neutralization Test Kit (GenScript, cat.No.:l 00847), for specific methods, see kit instructions.

The results are shown in FIG. 5. It can be seen that CHIKV-RBD-Flag2 stimulated mice produced high titers of neutralizing antibodies.

The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

<110> university of Qinghua

<120> a coronavirus subunit vaccine presenting coronavirus receptor binding region based on virus-like particles

<130> CGGNQALN216021

<160> 6

<170> PatentIn version 3.5

<210> 1

<211> 3747

<212> DNA

<213> Artificial sequence

<400> 1

atggagttca tcccgacgca aactttctat aacagaaggt accaaccccg accctgggcc 60

ccacgcccta caattcaagt aattagacct agaccacgtc cacagaggca ggctgggcaa 120

ctcgcccagc tgatctccgc agtcaacaaa ttgaccatgc gcgcggtacc tcaacagaag 180

cctcgcagaa atcggaaaaa caagaagcaa aggcagaaga agcaggcgcc gcaaaacgac 240

ccaaagcaaa agaagcaacc accacaaaag aagccggctc aaaagaagaa gaaaccaggc 300

cgtagggaga gaatgtgcat gaaaattgaa aatgattgca tcttcgaagt caagcatgaa 360

ggcaaagtga tgggctacgc atgcctggtg ggggataaag taatgaaacc agcacatgtg 420

aagggaacta tcgacaatgc cgatctggct aaactggcct ttaagcggtc gtctaaatac 480

gatcttgaat gtgcacagat accggtgcac atgaagtctg atgcctcgaa gtttacccac 540

gagaaacccg aggggtacta taactggcat cacggagcag tgcagtattc aggaggccgg 600

ttcactatcc cgacgggtgc aggcaagccg ggagacagcg gcagaccgat cttcgacaac 660

aaaggacggg tggtggccat cgtcctagga ggggccaacg aaggtgcccg cacggccctc 720

tccgtggtga cgtggaacaa agacatcgtc acaaaaatta cccctgaggg agccgaagag 780

tggagcctcg ccctcccggt cttgtgcctg ttggcaaaca ctacattccc ctgctctcag 840

ccgccttgca caccctgctg ctacgaaaag gaaccggaaa gcaccttgcg catgcttgag 900

gacaacgtga tgagacccgg atactaccag ctactaaaag catcgctgac ttgctctccc 960

caccgccaaa gacgcagtac taaggacaat tttaatgtct ataaagccac aagaccatat 1020

ctagctcatt gtcctgactg cggagaaggg cattcgtgcc acagccctat cgcattggag 1080

cgcatcagaa atgaagcaac ggacggaacg ctgaaaatcc aggtctcttt gcagatcggg 1140

ataaagacag atgacagcca cgattggacc aagctgcgct atatggatag ccatacgcca 1200

gcggacgcgg agcgagccgg attgcttgta aggacttcag caccgtgcac gatcaccggg 1260

accatgggac actttattct cgcccgatgc ccgaaaggag agacgctgac agtgggattt 1320

acggacagca gaaagatcag ccacacatgc acacacccgt tccatcatga accacctgtg 1380

ataggtaggg agaggttcca ctctcgacca caacatggta aagagttacc ttgcagcacg 1440

tacgtgcaga gcaccgctgc cactgctgag gagatagagg tgcatatgcc cccagatact 1500

cctgaccgca cgctgatgac gcagcagtct ggcaacgtga agatcacagt taatgggcag 1560

acggtgcggt acaagtgcaa ctgcggtggc tcaaacgagg gactgacaac cacagacaaa 1620

gtgatcaata actgcaaaat tgatcagtgc catgctgcag tcactaatca caagaattgg 1680

caatacaact cccctttagt cccgcgcaac gctgaactcg gggaccgtaa aggaaagatc 1740

cacatcccat tcccattggc aaacgtgact tgcagagtgc caaaagcaag aaaccctaca 1800

gtaacttacg gaaaaaacca agtcaccatg ctgctgtatc ctgaccatcc gacactcttg 1860

tcttaccgta acatgggaca ggaaccaaat taccacgagg agtgggtgac acacaagaag 1920

gaggttacct tgaccgtgcc tactgagggt ctggaggtca cttggggcaa caacgaacca 1980

tacaagtact ggccgcagat gtctacgaac ggtactgctc atggtcaccc acatgagata 2040

atcttgtact attatgagct gtaccccact atgactgtag tcattgtgtc ggtggcctcg 2100

ttcgtgcttc tgtcgatggt gggcacagca gtgggaatgt gtgtgtgcgc acggcgcaga 2160

tgcattacac catatgaatt aacaccagga gccactgttc ccttcctgct cagcctgcta 2220

tgctgcgtca gaacgaccaa ggcggccaca tattacgagg ctgcggcata tctatggaac 2280

gaacagcagc ccctgttctg gttgcaggct cttatcccgc tggccgcctt gatcgtcctg 2340

tgcaactgtc tgaaactctt gccatgctgc tgtaagaccc tggctttttt agccgtaatg 2400

agcatcggtg cccacactgt gagcgcgtac gaacacgtaa cagtgatccc gaacacggtg 2460

ggagtaccgt ataagactct tgtcaacaga ccgggttaca gccccatggt gttggagatg 2520

gagctacaat cagtcacctt ggaaccaaca ctgtcacttg actacatcac gtgcgagtac 2580

aaaactgtca tcccctcccc gtacgtgaag tgctgtggta cagcagagtg caaggacaag 2640

agcctaccag actacagctg caaggtcttt actggagtct acccatttat gtggggcggc 2700

gcctactgct tttgcgacgc cgaaaatacg caattgagcg aggcacatgt agagaaatct 2760

gaatcttgca aaacagagtt tgcatcggcc tacagagccc acaccgcatc ggcgtcggcg 2820

aagctccgcg tcctttacca aggaaacaac attaccgtag ctgcctacgc taacggtgac 2880

catgccgtca cagtaaagga cgccaagttt gtcgtgggcc caatgtcctc cgcctggaca 2940

ccttttgaca acaaaatcgt ggtgtacaaa ggcgacgtct acaacatgga ctacccacct 3000

tttggcgcag gaagaccagg acaatttggt gacattcaaa gtcgtacacc ggaaagtaaa 3060

gacgtttatg ccaacactca gttggtacta cagaggccag cagcaggcac ggtacatgta 3120

ccatactctc aggcaccatc tggcttcaag tattggctga aggaacgagg agcatcgcta 3180

cagcacacgg caccgttcgg ttgccagatt gcgacaaacc cggtaagagc tgtaaattgc 3240

gctgtgggga acataccaat ttccatcgac ataccggatg cggcctttac tagggttgtc 3300

gatgcaccct ctgtaacgga catgtcatgc gaagtaccag cctgcactca ctcctccgac 3360

tttgggggcg tcgccatcat caaatacaca gctagcaaga aaggtaaatg tgcagtacat 3420

tcgatgacca acgccgttac cattcgagaa gccgacgtag aagtagaggg gaactcccag 3480

ctgcaaatat ccttctcaac agccctggca agcgccgagt ttcgcgtgca agtgtgctcc 3540

acacaagtac actgcgcagc cgcatgccac cctccaaagg accacatagt caattaccca 3600

gcatcacaca ccacccttgg ggtccaggat atatccacaa cggcaatgtc ttgggtgcag 3660

aagattacgg gaggagtagg attaattgtt gctgttgctg ccttaatttt aattgtggtg 3720

ctatgcgtgt cgtttagcag gcactaa 3747

<210> 2

<211> 4383

<212> DNA

<213> Artificial sequence

<400> 2

atggagttca tcccgacgca aactttctat aacagaaggt accaaccccg accctgggcc 60

ccacgcccta caattcaagt aattagacct agaccacgtc cacagaggca ggctgggcaa 120

ctcgcccagc tgatctccgc agtcaacaaa ttgaccatgc gcgcggtacc tcaacagaag 180

cctcgcagaa atcggaaaaa caagaagcaa aggcagaaga agcaggcgcc gcaaaacgac 240

ccaaagcaaa agaagcaacc accacaaaag aagccggctc aaaagaagaa gaaaccaggc 300

cgtagggaga gaatgtgcat gaaaattgaa aatgattgca tcttcgaagt caagcatgaa 360

ggcaaagtga tgggctacgc atgcctggtg ggggataaag taatgaaacc agcacatgtg 420

aagggaacta tcgacaatgc cgatctggct aaactggcct ttaagcggtc gtctaaatac 480

gatcttgaat gtgcacagat accggtgcac atgaagtctg atgcctcgaa gtttacccac 540

gagaaacccg aggggtacta taactggcat cacggagcag tgcagtattc aggaggccgg 600

ttcactatcc cgacgggtgc aggcaagccg ggagacagcg gcagaccgat cttcgacaac 660

aaaggacggg tggtggccat cgtcctagga ggggccaacg aaggtgcccg cacggccctc 720

tccgtggtga cgtggaacaa agacatcgtc acaaaaatta cccctgaggg agccgaagag 780

tggagcctcg ccctcccggt cttgtgcctg ttggcaaaca ctacattccc ctgctctcag 840

ccgccttgca caccctgctg ctacgaaaag gaaccggaaa gcaccttgcg catgcttgag 900

gacaacgtga tgagacccgg atactaccag ctactaaaag catcgctgac ttgctctccc 960

cacagcggca gcaccaatct gtgccctttc ggcgaggtgt tcaacgccac cagattcgcc 1020

tctgtgtacg cctggaaccg gaagcggatc agcaattgcg tggccgacta cagcgtgctg 1080

tacaacagcg ccagcttcag caccttcaag tgctacggcg tgtcccctac caagctgaac 1140

gacctgtgct tcaccaacgt gtacgccgac agcttcgtga tcagaggcga cgaagtgcgg 1200

cagattgccc ctggacagac aggcaagatc gccgattaca actacaagct gcccgacgac 1260

ttcaccggct gtgtgattgc ctggaacagc aacaacctgg acagcaaagt cggcggcaac 1320

tacaactacc tgtaccggct gttccggaag tccaacctga agcctttcga gcgggacatc 1380

agcaccgaga tctatcaggc cggcagcacc ccttgcaatg gcgtggaagg cttcaactgc 1440

tacttcccac tgcagtccta cggcttccag cctacaaacg gcgtgggcta ccagccttac 1500

agagtggtgg tgctgagctt cgagctgctg catgctcctg ccacagtgtg cggcagctcc 1560

agcggcagta ctaaggacaa ttttaatgtc tataaagcca caagaccata tctagctcat 1620

tgtcctgact gcggagaagg gcattcgtgc cacagcccta tcgcattgga gcgcatcaga 1680

aatgaagcaa cggacggaac gctgaaaatc caggtctctt tgcagatcgg gataaagaca 1740

gatgacagcc acgattggac caagctgcgc tatatggata gccatacgcc agcggacgcg 1800

gagcgagccg gattgcttgt aaggacttca gcaccgtgca cgatcaccgg gaccatggga 1860

cactttattc tcgcccgatg cccgaaagga gagacgctga cagtgggatt tacggacagc 1920

agaaagatca gccacacatg cacacacccg ttccatcatg aaccacctgt gataggtagg 1980

gagaggttcc actctcgacc acaacatggt aaagagttac cttgcagcac gtacgtgcag 2040

agcaccgctg ccactgctga ggagatagag gtgcatatgc ccccagatac tcctgaccgc 2100

acgctgatga cgcagcagtc tggcaacgtg aagatcacag ttaatgggca gacggtgcgg 2160

tacaagtgca actgcggtag cggcggcgac tacaaagacg atgacgacaa gggaggcgga 2220

agcggctcaa acgagggact gacaaccaca gacaaagtga tcaataactg caaaattgat 2280

cagtgccatg ctgcagtcac taatcacaag aattggcaat acaactcccc tttagtcccg 2340

cgcaacgctg aactcgggga ccgtaaagga aagatccaca tcccattccc attggcaaac 2400

gtgacttgca gagtgccaaa agcaagaaac cctacagtaa cttacggaaa aaaccaagtc 2460

accatgctgc tgtatcctga ccatccgaca ctcttgtctt accgtaacat gggacaggaa 2520

ccaaattacc acgaggagtg ggtgacacac aagaaggagg ttaccttgac cgtgcctact 2580

gagggtctgg aggtcacttg gggcaacaac gaaccataca agtactggcc gcagatgtct 2640

acgaacggta ctgctcatgg tcacccacat gagataatct tgtactatta tgagctgtac 2700

cccactatga ctgtagtcat tgtgtcggtg gcctcgttcg tgcttctgtc gatggtgggc 2760

acagcagtgg gaatgtgtgt gtgcgcacgg cgcagatgca ttacaccata tgaattaaca 2820

ccaggagcca ctgttccctt cctgctcagc ctgctatgct gcgtcagaac gaccaaggcg 2880

gccacatatt acgaggctgc ggcatatcta tggaacgaac agcagcccct gttctggttg 2940

caggctctta tcccgctggc cgccttgatc gtcctgtgca actgtctgaa actcttgcca 3000

tgctgctgta agaccctggc ttttttagcc gtaatgagca tcggtgccca cactgtgagc 3060

gcgtacgaac acgtaacagt gatcccgaac acggtgggag taccgtataa gactcttgtc 3120

aacagaccgg gttacagccc catggtgttg gagatggagc tacaatcagt caccttggaa 3180

ccaacactgt cacttgacta catcacgtgc gagtacaaaa ctgtcatccc ctccccgtac 3240

gtgaagtgct gtggtacagc agagtgcaag gacaagagcc taccagacta cagctgcaag 3300

gtctttactg gagtctaccc atttatgtgg ggcggcgcct actgcttttg cgacgccgaa 3360

aatacgcaat tgagcgaggc acatgtagag aaatctgaat cttgcaaaac agagtttgca 3420

tcggcctaca gagcccacac cgcatcggcg tcggcgaagc tccgcgtcct ttaccaagga 3480

aacaacatta ccgtagctgc ctacgctaac ggtgaccatg ccgtcacagt aaaggacgcc 3540

aagtttgtcg tgggcccaat gtcctccgcc tggacacctt ttgacaacaa aatcgtggtg 3600

tacaaaggcg acgtctacaa catggactac ccaccttttg gcgcaggaag accaggacaa 3660

tttggtgaca ttcaaagtcg tacaccggaa agtaaagacg tttatgccaa cactcagttg 3720

gtactacaga ggccagcagc aggcacggta catgtaccat actctcaggc accatctggc 3780

ttcaagtatt ggctgaagga acgaggagca tcgctacagc acacggcacc gttcggttgc 3840

cagattgcga caaacccggt aagagctgta aattgcgctg tggggaacat accaatttcc 3900

atcgacatac cggatgcggc ctttactagg gttgtcgatg caccctctgt aacggacatg 3960

tcatgcgaag taccagcctg cactcactcc tccgactttg ggggcgtcgc catcatcaaa 4020

tacacagcta gcaagaaagg taaatgtgca gtacattcga tgaccaacgc cgttaccatt 4080

cgagaagccg acgtagaagt agaggggaac tcccagctgc aaatatcctt ctcaacagcc 4140

ctggcaagcg ccgagtttcg cgtgcaagtg tgctccacac aagtacactg cgcagccgca 4200

tgccaccctc caaaggacca catagtcaat tacccagcat cacacaccac ccttggggtc 4260

caggatatat ccacaacggc aatgtcttgg gtgcagaaga ttacgggagg agtaggatta 4320

attgttgctg ttgctgcctt aattttaatt gtggtgctat gcgtgtcgtt tagcaggcac 4380

taa 4383

<210> 3

<211> 819

<212> DNA

<213> Artificial sequence

<400> 3

atgctgcgcg gactgtgctg cgtgctgcta ctgtgcggcg ccgtgttcgt gagccccagc 60

caggagatcc acgcccgatt caggagagga gccagaggac gcgtgcagcc caccgagagc 120

atcgtgcgct tccccaacat caccaacctg tgccccttcg gcgaggtgtt caacgccacc 180

cgcttcgcca gcgtgtacgc ctggaaccgc aagcgcatca gcaactgcgt ggccgactac 240

agcgtgctgt acaacagcgc cagcttcagc accttcaagt gctacggcgt gagccccacc 300

aagctgaacg acctgtgctt caccaacgtg tacgccgaca gcttcgtgat ccgcggcgac 360

gaggtgcgcc agatcgcccc cggccagacc ggcaagatcg ccgactacaa ctacaagctg 420

cccgacgact tcaccggctg cgtgatcgcc tggaacagca acaacctgga cagcaaggtg 480

ggcggcaact acaactacct gtaccgcctg ttccgcaaga gcaacctgaa gcccttcgag 540

cgcgacatca gcaccgagat ctaccaggcc ggcagcaccc cctgcaacgg cgtggagggc 600

ttcaactgct acttccccct gcagagctac ggcttccagc ccaccaacgg cgtgggctac 660

cagccctacc gcgtggtggt gctgagcttc gagctgctgc acgcccccgc caccgtgtgc 720

ggccccaaga agagcaccaa cctggtgaag aacaagtgcg tgaacttctg gagccacccc 780

cagttcgaga aggactacaa ggacgacgac gacaagtaa 819

<210> 4

<211> 1248

<212> PRT

<213> Artificial sequence

<400> 4

Met Glu Phe Ile Pro Thr Gln Thr Phe Tyr Asn Arg Arg Tyr Gln Pro

1 5 10 15

Arg Pro Trp Ala Pro Arg Pro Thr Ile Gln Val Ile Arg Pro Arg Pro

20 25 30

Arg Pro Gln Arg Gln Ala Gly Gln Leu Ala Gln Leu Ile Ser Ala Val

35 40 45

Asn Lys Leu Thr Met Arg Ala Val Pro Gln Gln Lys Pro Arg Arg Asn

50 55 60

Arg Lys Asn Lys Lys Gln Arg Gln Lys Lys Gln Ala Pro Gln Asn Asp

65 70 75 80

Pro Lys Gln Lys Lys Gln Pro Pro Gln Lys Lys Pro Ala Gln Lys Lys

85 90 95

Lys Lys Pro Gly Arg Arg Glu Arg Met Cys Met Lys Ile Glu Asn Asp

100 105 110

Cys Ile Phe Glu Val Lys His Glu Gly Lys Val Met Gly Tyr Ala Cys

115 120 125

Leu Val Gly Asp Lys Val Met Lys Pro Ala His Val Lys Gly Thr Ile

130 135 140

Asp Asn Ala Asp Leu Ala Lys Leu Ala Phe Lys Arg Ser Ser Lys Tyr

145 150 155 160

Asp Leu Glu Cys Ala Gln Ile Pro Val His Met Lys Ser Asp Ala Ser

165 170 175

Lys Phe Thr His Glu Lys Pro Glu Gly Tyr Tyr Asn Trp His His Gly

180 185 190

Ala Val Gln Tyr Ser Gly Gly Arg Phe Thr Ile Pro Thr Gly Ala Gly

195 200 205

Lys Pro Gly Asp Ser Gly Arg Pro Ile Phe Asp Asn Lys Gly Arg Val

210 215 220

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

225 230 235 240

Ser Val Val Thr Trp Asn Lys Asp Ile Val Thr Lys Ile Thr Pro Glu

245 250 255

Gly Ala Glu Glu Trp Ser Leu Ala Leu Pro Val Leu Cys Leu Leu Ala

260 265 270

Asn Thr Thr Phe Pro Cys Ser Gln Pro Pro Cys Thr Pro Cys Cys Tyr

275 280 285

Glu Lys Glu Pro Glu Ser Thr Leu Arg Met Leu Glu Asp Asn Val Met

290 295 300

Arg Pro Gly Tyr Tyr Gln Leu Leu Lys Ala Ser Leu Thr Cys Ser Pro

305 310 315 320

His Arg Gln Arg Arg Ser Thr Lys Asp Asn Phe Asn Val Tyr Lys Ala

325 330 335

Thr Arg Pro Tyr Leu Ala His Cys Pro Asp Cys Gly Glu Gly His Ser

340 345 350

Cys His Ser Pro Ile Ala Leu Glu Arg Ile Arg Asn Glu Ala Thr Asp

355 360 365

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

370 375 380

Asp Ser His Asp Trp Thr Lys Leu Arg Tyr Met Asp Ser His Thr Pro

385 390 395 400

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

405 410 415

Thr Ile Thr Gly Thr Met Gly His Phe Ile Leu Ala Arg Cys Pro Lys

420 425 430

Gly Glu Thr Leu Thr Val Gly Phe Thr Asp Ser Arg Lys Ile Ser His

435 440 445

Thr Cys Thr His Pro Phe His His Glu Pro Pro Val Ile Gly Arg Glu

450 455 460

Arg Phe His Ser Arg Pro Gln His Gly Lys Glu Leu Pro Cys Ser Thr

465 470 475 480

Tyr Val Gln Ser Thr Ala Ala Thr Ala Glu Glu Ile Glu Val His Met

485 490 495

Pro Pro Asp Thr Pro Asp Arg Thr Leu Met Thr Gln Gln Ser Gly Asn

500 505 510

Val Lys Ile Thr Val Asn Gly Gln Thr Val Arg Tyr Lys Cys Asn Cys

515 520 525

Gly Gly Ser Asn Glu Gly Leu Thr Thr Thr Asp Lys Val Ile Asn Asn

530 535 540

Cys Lys Ile Asp Gln Cys His Ala Ala Val Thr Asn His Lys Asn Trp

545 550 555 560

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

565 570 575

Lys Gly Lys Ile His Ile Pro Phe Pro Leu Ala Asn Val Thr Cys Arg

580 585 590

Val Pro Lys Ala Arg Asn Pro Thr Val Thr Tyr Gly Lys Asn Gln Val

595 600 605

Thr Met Leu Leu Tyr Pro Asp His Pro Thr Leu Leu Ser Tyr Arg Asn

610 615 620

Met Gly Gln Glu Pro Asn Tyr His Glu Glu Trp Val Thr His Lys Lys

625 630 635 640

Glu Val Thr Leu Thr Val Pro Thr Glu Gly Leu Glu Val Thr Trp Gly

645 650 655

Asn Asn Glu Pro Tyr Lys Tyr Trp Pro Gln Met Ser Thr Asn Gly Thr

660 665 670

Ala His Gly His Pro His Glu Ile Ile Leu Tyr Tyr Tyr Glu Leu Tyr

675 680 685

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

690 695 700

Ser Met Val Gly Thr Ala Val Gly Met Cys Val Cys Ala Arg Arg Arg

705 710 715 720

Cys Ile Thr Pro Tyr Glu Leu Thr Pro Gly Ala Thr Val Pro Phe Leu

725 730 735

Leu Ser Leu Leu Cys Cys Val Arg Thr Thr Lys Ala Ala Thr Tyr Tyr

740 745 750

Glu Ala Ala Ala Tyr Leu Trp Asn Glu Gln Gln Pro Leu Phe Trp Leu

755 760 765

Gln Ala Leu Ile Pro Leu Ala Ala Leu Ile Val Leu Cys Asn Cys Leu

770 775 780

Lys Leu Leu Pro Cys Cys Cys Lys Thr Leu Ala Phe Leu Ala Val Met

785 790 795 800

Ser Ile Gly Ala His Thr Val Ser Ala Tyr Glu His Val Thr Val Ile

805 810 815

Pro Asn Thr Val Gly Val Pro Tyr Lys Thr Leu Val Asn Arg Pro Gly

820 825 830

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

835 840 845

Pro Thr Leu Ser Leu Asp Tyr Ile Thr Cys Glu Tyr Lys Thr Val Ile

850 855 860

Pro Ser Pro Tyr Val Lys Cys Cys Gly Thr Ala Glu Cys Lys Asp Lys

865 870 875 880

Ser Leu Pro Asp Tyr Ser Cys Lys Val Phe Thr Gly Val Tyr Pro Phe

885 890 895

Met Trp Gly Gly Ala Tyr Cys Phe Cys Asp Ala Glu Asn Thr Gln Leu

900 905 910

Ser Glu Ala His Val Glu Lys Ser Glu Ser Cys Lys Thr Glu Phe Ala

915 920 925

Ser Ala Tyr Arg Ala His Thr Ala Ser Ala Ser Ala Lys Leu Arg Val

930 935 940

Leu Tyr Gln Gly Asn Asn Ile Thr Val Ala Ala Tyr Ala Asn Gly Asp

945 950 955 960

His Ala Val Thr Val Lys Asp Ala Lys Phe Val Val Gly Pro Met Ser

965 970 975

Ser Ala Trp Thr Pro Phe Asp Asn Lys Ile Val Val Tyr Lys Gly Asp

980 985 990

Val Tyr Asn Met Asp Tyr Pro Pro Phe Gly Ala Gly Arg Pro Gly Gln

995 1000 1005

Phe Gly Asp Ile Gln Ser Arg Thr Pro Glu Ser Lys Asp Val Tyr

1010 1015 1020

Ala Asn Thr Gln Leu Val Leu Gln Arg Pro Ala Ala Gly Thr Val

1025 1030 1035

His Val Pro Tyr Ser Gln Ala Pro Ser Gly Phe Lys Tyr Trp Leu

1040 1045 1050

Lys Glu Arg Gly Ala Ser Leu Gln His Thr Ala Pro Phe Gly Cys

1055 1060 1065

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

1070 1075 1080

Asn Ile Pro Ile Ser Ile Asp Ile Pro Asp Ala Ala Phe Thr Arg

1085 1090 1095

Val Val Asp Ala Pro Ser Val Thr Asp Met Ser Cys Glu Val Pro

1100 1105 1110

Ala Cys Thr His Ser Ser Asp Phe Gly Gly Val Ala Ile Ile Lys

1115 1120 1125

Tyr Thr Ala Ser Lys Lys Gly Lys Cys Ala Val His Ser Met Thr

1130 1135 1140

Asn Ala Val Thr Ile Arg Glu Ala Asp Val Glu Val Glu Gly Asn

1145 1150 1155

Ser Gln Leu Gln Ile Ser Phe Ser Thr Ala Leu Ala Ser Ala Glu

1160 1165 1170

Phe Arg Val Gln Val Cys Ser Thr Gln Val His Cys Ala Ala Ala

1175 1180 1185

Cys His Pro Pro Lys Asp His Ile Val Asn Tyr Pro Ala Ser His

1190 1195 1200

Thr Thr Leu Gly Val Gln Asp Ile Ser Thr Thr Ala Met Ser Trp

1205 1210 1215

Val Gln Lys Ile Thr Gly Gly Val Gly Leu Ile Val Ala Val Ala

1220 1225 1230

Ala Leu Ile Leu Ile Val Val Leu Cys Val Ser Phe Ser Arg His

1235 1240 1245

<210> 5

<211> 1460

<212> PRT

<213> Artificial sequence

<400> 5

Met Glu Phe Ile Pro Thr Gln Thr Phe Tyr Asn Arg Arg Tyr Gln Pro

1 5 10 15

Arg Pro Trp Ala Pro Arg Pro Thr Ile Gln Val Ile Arg Pro Arg Pro

20 25 30

Arg Pro Gln Arg Gln Ala Gly Gln Leu Ala Gln Leu Ile Ser Ala Val

35 40 45

Asn Lys Leu Thr Met Arg Ala Val Pro Gln Gln Lys Pro Arg Arg Asn

50 55 60

Arg Lys Asn Lys Lys Gln Arg Gln Lys Lys Gln Ala Pro Gln Asn Asp

65 70 75 80

Pro Lys Gln Lys Lys Gln Pro Pro Gln Lys Lys Pro Ala Gln Lys Lys

85 90 95

Lys Lys Pro Gly Arg Arg Glu Arg Met Cys Met Lys Ile Glu Asn Asp

100 105 110

Cys Ile Phe Glu Val Lys His Glu Gly Lys Val Met Gly Tyr Ala Cys

115 120 125

Leu Val Gly Asp Lys Val Met Lys Pro Ala His Val Lys Gly Thr Ile

130 135 140

Asp Asn Ala Asp Leu Ala Lys Leu Ala Phe Lys Arg Ser Ser Lys Tyr

145 150 155 160

Asp Leu Glu Cys Ala Gln Ile Pro Val His Met Lys Ser Asp Ala Ser

165 170 175

Lys Phe Thr His Glu Lys Pro Glu Gly Tyr Tyr Asn Trp His His Gly

180 185 190

Ala Val Gln Tyr Ser Gly Gly Arg Phe Thr Ile Pro Thr Gly Ala Gly

195 200 205

Lys Pro Gly Asp Ser Gly Arg Pro Ile Phe Asp Asn Lys Gly Arg Val

210 215 220

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

225 230 235 240

Ser Val Val Thr Trp Asn Lys Asp Ile Val Thr Lys Ile Thr Pro Glu

245 250 255

Gly Ala Glu Glu Trp Ser Leu Ala Leu Pro Val Leu Cys Leu Leu Ala

260 265 270

Asn Thr Thr Phe Pro Cys Ser Gln Pro Pro Cys Thr Pro Cys Cys Tyr

275 280 285

Glu Lys Glu Pro Glu Ser Thr Leu Arg Met Leu Glu Asp Asn Val Met

290 295 300

Arg Pro Gly Tyr Tyr Gln Leu Leu Lys Ala Ser Leu Thr Cys Ser Pro

305 310 315 320

His Ser Gly Ser Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala

325 330 335

Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn

340 345 350

Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr

355 360 365

Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe

370 375 380

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

385 390 395 400

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

405 410 415

Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn

420 425 430

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

435 440 445

Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile

450 455 460

Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys

465 470 475 480

Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly

485 490 495

Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala

500 505 510

Pro Ala Thr Val Cys Gly Ser Ser Ser Gly Ser Thr Lys Asp Asn Phe

515 520 525

Asn Val Tyr Lys Ala Thr Arg Pro Tyr Leu Ala His Cys Pro Asp Cys

530 535 540

Gly Glu Gly His Ser Cys His Ser Pro Ile Ala Leu Glu Arg Ile Arg

545 550 555 560

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

565 570 575

Gly Ile Lys Thr Asp Asp Ser His Asp Trp Thr Lys Leu Arg Tyr Met

580 585 590

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

595 600 605

Thr Ser Ala Pro Cys Thr Ile Thr Gly Thr Met Gly His Phe Ile Leu

610 615 620

Ala Arg Cys Pro Lys Gly Glu Thr Leu Thr Val Gly Phe Thr Asp Ser

625 630 635 640

Arg Lys Ile Ser His Thr Cys Thr His Pro Phe His His Glu Pro Pro

645 650 655

Val Ile Gly Arg Glu Arg Phe His Ser Arg Pro Gln His Gly Lys Glu

660 665 670

Leu Pro Cys Ser Thr Tyr Val Gln Ser Thr Ala Ala Thr Ala Glu Glu

675 680 685

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

690 695 700

Gln Gln Ser Gly Asn Val Lys Ile Thr Val Asn Gly Gln Thr Val Arg

705 710 715 720

Tyr Lys Cys Asn Cys Gly Ser Gly Gly Asp Tyr Lys Asp Asp Asp Asp

725 730 735

Lys Gly Gly Gly Ser Gly Ser Asn Glu Gly Leu Thr Thr Thr Asp Lys

740 745 750

Val Ile Asn Asn Cys Lys Ile Asp Gln Cys His Ala Ala Val Thr Asn

755 760 765

His Lys Asn Trp Gln Tyr Asn Ser Pro Leu Val Pro Arg Asn Ala Glu

770 775 780

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

785 790 795 800

Val Thr Cys Arg Val Pro Lys Ala Arg Asn Pro Thr Val Thr Tyr Gly

805 810 815

Lys Asn Gln Val Thr Met Leu Leu Tyr Pro Asp His Pro Thr Leu Leu

820 825 830

Ser Tyr Arg Asn Met Gly Gln Glu Pro Asn Tyr His Glu Glu Trp Val

835 840 845

Thr His Lys Lys Glu Val Thr Leu Thr Val Pro Thr Glu Gly Leu Glu

850 855 860

Val Thr Trp Gly Asn Asn Glu Pro Tyr Lys Tyr Trp Pro Gln Met Ser

865 870 875 880

Thr Asn Gly Thr Ala His Gly His Pro His Glu Ile Ile Leu Tyr Tyr

885 890 895

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

900 905 910

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

915 920 925

Ala Arg Arg Arg Cys Ile Thr Pro Tyr Glu Leu Thr Pro Gly Ala Thr

930 935 940

Val Pro Phe Leu Leu Ser Leu Leu Cys Cys Val Arg Thr Thr Lys Ala

945 950 955 960

Ala Thr Tyr Tyr Glu Ala Ala Ala Tyr Leu Trp Asn Glu Gln Gln Pro

965 970 975

Leu Phe Trp Leu Gln Ala Leu Ile Pro Leu Ala Ala Leu Ile Val Leu

980 985 990

Cys Asn Cys Leu Lys Leu Leu Pro Cys Cys Cys Lys Thr Leu Ala Phe

995 1000 1005

Leu Ala Val Met Ser Ile Gly Ala His Thr Val Ser Ala Tyr Glu

1010 1015 1020

His Val Thr Val Ile Pro Asn Thr Val Gly Val Pro Tyr Lys Thr

1025 1030 1035

Leu Val Asn Arg Pro Gly Tyr Ser Pro Met Val Leu Glu Met Glu

1040 1045 1050

Leu Gln Ser Val Thr Leu Glu Pro Thr Leu Ser Leu Asp Tyr Ile

1055 1060 1065

Thr Cys Glu Tyr Lys Thr Val Ile Pro Ser Pro Tyr Val Lys Cys

1070 1075 1080

Cys Gly Thr Ala Glu Cys Lys Asp Lys Ser Leu Pro Asp Tyr Ser

1085 1090 1095

Cys Lys Val Phe Thr Gly Val Tyr Pro Phe Met Trp Gly Gly Ala

1100 1105 1110

Tyr Cys Phe Cys Asp Ala Glu Asn Thr Gln Leu Ser Glu Ala His

1115 1120 1125

Val Glu Lys Ser Glu Ser Cys Lys Thr Glu Phe Ala Ser Ala Tyr

1130 1135 1140

Arg Ala His Thr Ala Ser Ala Ser Ala Lys Leu Arg Val Leu Tyr

1145 1150 1155

Gln Gly Asn Asn Ile Thr Val Ala Ala Tyr Ala Asn Gly Asp His

1160 1165 1170

Ala Val Thr Val Lys Asp Ala Lys Phe Val Val Gly Pro Met Ser

1175 1180 1185

Ser Ala Trp Thr Pro Phe Asp Asn Lys Ile Val Val Tyr Lys Gly

1190 1195 1200

Asp Val Tyr Asn Met Asp Tyr Pro Pro Phe Gly Ala Gly Arg Pro

1205 1210 1215

Gly Gln Phe Gly Asp Ile Gln Ser Arg Thr Pro Glu Ser Lys Asp

1220 1225 1230

Val Tyr Ala Asn Thr Gln Leu Val Leu Gln Arg Pro Ala Ala Gly

1235 1240 1245

Thr Val His Val Pro Tyr Ser Gln Ala Pro Ser Gly Phe Lys Tyr

1250 1255 1260

Trp Leu Lys Glu Arg Gly Ala Ser Leu Gln His Thr Ala Pro Phe

1265 1270 1275

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

1280 1285 1290

Val Gly Asn Ile Pro Ile Ser Ile Asp Ile Pro Asp Ala Ala Phe

1295 1300 1305

Thr Arg Val Val Asp Ala Pro Ser Val Thr Asp Met Ser Cys Glu

1310 1315 1320

Val Pro Ala Cys Thr His Ser Ser Asp Phe Gly Gly Val Ala Ile

1325 1330 1335

Ile Lys Tyr Thr Ala Ser Lys Lys Gly Lys Cys Ala Val His Ser

1340 1345 1350

Met Thr Asn Ala Val Thr Ile Arg Glu Ala Asp Val Glu Val Glu

1355 1360 1365

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

1370 1375 1380

Ala Glu Phe Arg Val Gln Val Cys Ser Thr Gln Val His Cys Ala

1385 1390 1395

Ala Ala Cys His Pro Pro Lys Asp His Ile Val Asn Tyr Pro Ala

1400 1405 1410

Ser His Thr Thr Leu Gly Val Gln Asp Ile Ser Thr Thr Ala Met

1415 1420 1425

Ser Trp Val Gln Lys Ile Thr Gly Gly Val Gly Leu Ile Val Ala

1430 1435 1440

Val Ala Ala Leu Ile Leu Ile Val Val Leu Cys Val Ser Phe Ser

1445 1450 1455

Arg His

1460

<210> 6

<211> 272

<212> PRT

<213> Artificial sequence

<400> 6

Met Leu Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly Ala Val Phe

1 5 10 15

Val Ser Pro Ser Gln Glu Ile His Ala Arg Phe Arg Arg Gly Ala Arg

20 25 30

Gly Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr

35 40 45

Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser

50 55 60

Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr

65 70 75 80

Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly

85 90 95

Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu

165 170 175

Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser

180 185 190

Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln

195 200 205

Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg

210 215 220

Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys

225 230 235 240

Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe

245 250 255

Trp Ser His Pro Gln Phe Glu Lys Asp Tyr Lys Asp Asp Asp Asp Lys

260 265 270

Claims (20)

1. A recombinant virus-like particle, characterized in that: the recombinant virus-like particle is chikungunya virus-like particle carrying specific proteins; the specific protein is RBD section of SARS-CoV-2S protein;

the recombinant virus-like particle is formed by self-assembly of a capsid protein of the chikungunya virus, an E1 protein of the chikungunya virus, a fusion protein A and a 6K protein of the chikungunya virus;

the fusion protein A sequentially comprises an E3 protein of the chikungunya virus, a flexible peptide 1, the specific protein, a flexible peptide 2, an N-terminal section of the E2 protein of the chikungunya virus, a flexible peptide 3, a Flag tag, a flexible peptide 4 and a C-terminal section of the E2 protein of the chikungunya virus from N end to C end;

the amino acid sequence of RBD section of SARS-CoV-2S protein is shown in 325-518 th position of SEQ ID No. 5;

the amino acid sequence of the capsid protein of the chikungunya virus is shown in positions 1-261 of SEQ ID No. 5;

the amino acid sequence of the E1 protein of the chikungunya virus is shown in 1022-1460 positions of SEQ ID No. 5;

the amino acid sequence of the 6K protein of the chikungunya virus is shown in 960-1021 th site of SEQ ID No. 5;

the amino acid sequence of the E3 protein of the chikungunya virus is shown in positions 262-321 of SEQ ID No. 5;

the amino acid sequence of the E2 protein of the chikungunya virus is shown in positions 326-747 of SEQ ID No. 4;

the amino acid sequence of the Flag tag is shown in the 730 th-737 th positions of SEQ ID No. 5;

the amino acid sequence of the N-terminal section of the E2 protein of the chikungunya virus is shown in 523-725 of SEQ ID No. 5;

the amino acid sequence of the C-terminal section of the E2 protein of the chikungunya virus is shown in 742-959 positions of SEQ ID No. 5;

the amino acid sequence of the flexible peptide 1 is shown in 322-324 of SEQ ID No. 5;

the amino acid sequence of the flexible peptide 2 is shown in 519-522 positions of SEQ ID No. 5;

the amino acid sequence of the flexible peptide 3 is shown in 726-729 th positions of SEQ ID No. 5;

the amino acid sequence of the flexible peptide 4 is shown in 738-741 th position of SEQ ID No. 5.

2. The recombinant virus-like particle of claim 1, wherein: the amino acid sequence of the fusion protein A is shown in 262-959 of SEQ ID No. 5.

3. Fusion protein B consisting of, in order from the N-terminus to the C-terminus, the capsid protein of the chikungunya virus described in claim 1, the E1 protein of the chikungunya virus described in claim 1, the fusion protein a of claim 1 or 2, and the 6K protein of the chikungunya virus described in claim 1.

4. A fusion protein B according to claim 3, characterized in that: the amino acid sequence of the fusion protein B is shown as SEQ ID No. 5.

5. A nucleic acid molecule encoding the fusion protein B of claim 3.

6. The nucleic acid molecule of claim 5, wherein: in the nucleic acid molecule, the nucleic acid sequence of the capsid protein of the chikungunya virus is shown in positions 1-783 of SEQ ID No. 2.

7. The nucleic acid molecule of claim 5, wherein: in the nucleic acid molecule, the nucleic acid sequence of the E1 protein of the chikungunya virus is shown in the 3064-4383 th positions of SEQ ID No. 2.

8. The nucleic acid molecule of claim 5, wherein: in the nucleic acid molecule, the nucleic acid sequence encoding the fusion protein A is shown in positions 784-2877 of SEQ ID No. 2.

9. The nucleic acid molecule of claim 5, wherein: in the nucleic acid molecule, the nucleic acid sequence of the 6K protein of the chikungunya virus is shown in 2878-3063 of SEQ ID No. 2.

10. The nucleic acid molecule of claim 5, wherein: the nucleic acid molecule is shown as SEQ ID No. 2.

11. An expression cassette, recombinant vector, recombinant cell or recombinant bacterium comprising the nucleic acid molecule of any one of claims 5-10.

12. The recombinant vector according to claim 11, wherein: the recombinant vector is a eukaryotic expression vector containing the nucleic acid molecule.

13. The recombinant vector according to claim 12, wherein: the recombinant vector is a recombinant plasmid obtained by cloning the nucleic acid molecule into a pcDNA3.1 (+) vector.

14. A method of preparing a recombinant virus-like particle according to claim 1 or 2, comprising the steps of: the recombinant vector according to claim 12 or 13 is introduced into mammalian cells, followed by cell culture, thereby obtaining the recombinant virus-like particle.

15. Use of the fusion protein B of claim 3 or 4 or the nucleic acid molecule of any one of claims 5 to 10 or the expression cassette, recombinant vector, recombinant cell or recombinant bacterium of claim 11 for the preparation of the recombinant virus-like particle of claim 1 or 2.

16. Use of a recombinant virus-like particle according to claim 1 or 2 or a fusion protein B according to claim 3 or 4 or a nucleic acid molecule according to any one of claims 5 to 10 or an expression cassette, recombinant vector, recombinant cell or recombinant bacterium according to claim 11 for the preparation of a product for the prevention of diseases caused by infection with SARS-CoV-2.

17. A vaccine for preventing diseases caused by SARS-CoV-2 infection, comprising the recombinant virus-like particle of claim 1 or 2 as an active ingredient.

18. The vaccine of claim 17, wherein: the vaccine also contains an adjuvant.

19. The vaccine of claim 18, wherein: the adjuvant is aluminum adjuvant.

20. The vaccine of claim 19, wherein: the aluminum adjuvant is an aluminum hydroxide adjuvant.