SARS-CoV-2 broad-spectrum vaccine and application thereof

Abstract

The invention provides an application of a fusion protein in preparation of an SARS-CoV-2 broad-spectrum vaccine, the fusion protein comprises an NTD structural domain and an RBD structural domain of an SARS-CoV-2 spike protein, provides more conservative epitopes, can effectively stimulate the generation of a neutralizing antibody, and can effectively inhibit the generation of the SARS-CoV-2 spike protein. The vaccine prepared by using the fusion protein can provide broad-spectrum and efficient protection for the SARS-CoV-2 wild type and various mutant strains; the invention also provides an application of the broad-spectrum vaccine as an immune enhancer, and also provides an immune kit under a corresponding immune strategy, the broad-spectrum vaccine can cooperate with other existing vaccines to enhance immunity, and can independently perform a complete immune procedure, and excellent immune protection can be provided in any way.

Description

Technical Field

[0001] This application belongs to the field of biomedical engineering, and particularly relates to a SARS-CoV-2 broad-spectrum vaccine and its application in boosting the immunity against SARS-CoV-2. Background Art

[0002] The novel coronavirus (SARS-CoV-2) has been pandemic globally for over two years. During the spread of the virus, continuous evolution has occurred, and currently, multiple mutant strains have emerged. Among them, the infectivity of the main mutant strains such as Delta and Omicron is much stronger than that of the wild-type strain, bringing new challenges to epidemic prevention and control.

[0003] The SARS-CoV-2 virus particle contains four structural proteins, namely spike protein (S), nucleocapsid protein (N), membrane protein (M), and envelope protein (E). Studies have found that only antibodies against the S protein have neutralizing activity. Therefore, the S protein plays an important role in the research and development of related vaccines. Among them, the receptor-binding domain (RBD) of the S protein is considered to be the most important antigen target region for inducing neutralizing antibodies in the body; RBD as a vaccine can make the neutralizing antibodies stimulated by the body more focused on the receptor binding of the virus, which can improve the immunogenicity and immune efficiency of the vaccine. The N-terminal domain (NTD) of the S protein is a sequence at the N-terminal of the viral S protein, which can bind to the protein or glycoprotein of the host cell and mediate the invasion of the virus into the host cell. Therefore, this region may contain epitopes that induce the production of neutralizing antibodies.

[0004] Most of the currently marketed or under-developed COVID-19 vaccines only target a single strain (mainly the wild-type strain) and cannot produce corresponding neutralizing antibodies against newly emerging mutant strains. Therefore, it is difficult to cope with the spread and diffusion of mutant strains. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides a fusion protein based on RBD and NTD, a SARS-CoV-2 broad-spectrum vaccine based on the fusion protein, and an immune set based on the broad-spectrum vaccine.

[0006] The technical solution of the present invention is as follows:

[0007] The present invention provides an application of a fusion protein in the preparation of a SARS-CoV-2 broad-spectrum vaccine, and the fusion protein comprises the following parts:

[0008] The N-terminal domain NTD of the SARS-CoV-2 spike protein or its functional fragment, and

[0009] The receptor domain RBD of the SARS-CoV-2 spike protein or its functional fragment;

[0010] The broad-spectrum vaccine provides protection against at least one of the WH01 strain and the following mutant strains:

[0011] D614G strain, Alpha strain, Beta strain, Gamma strain, Delta strain, Eta strain, Iota strain, Kappa strain, Lambda strain, Miu strain, Omicron strain, and Omicron-BA.2 strain.

[0012] Furthermore, the mutant strains include Beta strain, Delta strain, Omicron strain, and Omicron-BA.2 strain.

[0013] Furthermore, the NTD is derived from the SARS-CoV-2 wild type or any mutant; the RBD is derived from the SARS-CoV-2 wild type or any mutant.

[0014] Furthermore, the NTD has an amino acid mutation at at least one of the following sites: L18, T20, P13, P26, D80, D138, R190, R203, D215, G204, L242-244, and R246.

[0015] Furthermore, the NTD contains at least one of the following amino acid mutations: L18F, T20N, P13L, P26S, D80A, D138Y, R190S, R203M, D215G, G204R, L242-244del, and R246I.

[0016] In a preferred case, the NTD contains the following amino acid mutations: L18F, D80A, D215G, L242-244del, and R246I.

[0017] In a preferred case, the NTD contains the following amino acid mutations: L18F, D80A, D215G, L242-244del, and R190S.

[0018] In a preferred case, the NTD contains the following amino acid mutations: L18F, T20N, P26S, D138Y, and R246I.

[0019] Furthermore, the amino acid sequence of the NTD is at least 90% identical to any one of SEQ ID NO.1-8.

[0020] Furthermore, the RBD has an amino acid mutation at one or more of the following sites: K417, E484, N501, L452, and T478.

[0021] Furthermore, the RBD comprises at least one of the following amino acid mutations: K417T / N, E484K, N501Y, L452R, and T478K.

[0022] In a preferred case, the RBD comprises the following amino acid mutations: K417T, E484K, N501Y.

[0023] In a preferred case, the RBD comprises the following amino acid mutations: K417N, E484K, N501Y.

[0024] Furthermore, the amino acid sequence of the RBD is at least 90% identical to any one of SEQ ID NOs. 9-25.

[0025] Furthermore, the fusion protein further comprises at least one of the following polypeptides:

[0026] P2 or its functionally active fragment, foldon domain or its functionally active fragment, ferritin or its functionally active fragment, and hepatitis B surface antigen HSsAg or its functionally active fragment;

[0027] At least one of the RBD or its functionally active fragment and the NTD or its active functional fragment is directly or indirectly linked to the polypeptide.

[0028] Furthermore, the polypeptide is in-frame fused with the RBD or its functionally active fragment and the NTD or its functionally active fragment.

[0029] Furthermore, the polypeptide is the foldon domain or its functionally active fragment.

[0030] Furthermore, the foldon domain or its functionally active fragment comprises the amino acid residues at the C-terminus of phage T4 fibritin.

[0031] Furthermore, the N-terminus of the foldon domain or its functionally active fragment is linked to the C-terminus of the RBD or its active functional fragment, and the N-terminus of the RBD or its functionally active fragment is linked to the C-terminus of the NTD or its functionally active fragment.

[0032] Furthermore, the amino acid sequence of the foldon domain is at least 90% identical to any one of SEQ ID NOs. 26-28.

[0033] On the other hand, the present invention provides a SARS-CoV-2 broad-spectrum vaccine, comprising the fusion protein in any one of the above; the broad-spectrum vaccine provides protection against at least one of the WH01 strain and the following mutant strains:

[0034] D614 strain, Alpha strain, Beta strain, Gamma strain, Delta strain, Eta strain, Iota strain, Kappa strain, Lambda strain, Miu strain, Omicron strain, and Omicron-BA.2 strain.

[0035] Further, the mutant strains include Delta strain, Omicron strain, and Omicron-BA.2 strain.

[0036] Further, the vaccine contains 10 - 40 μg of the fusion protein per single-dose administration, specifically, it can be equal to or approximately equal to 10 μg, 15 μg, 20 μg, 25 μg, 30 μg, 35 μg, or 40 μg, or it can be any value between any two adjacent numbers above.

[0037] Preferably, the vaccine contains 20 μg of the fusion protein per single-dose administration.

[0038] Further, the broad-spectrum vaccine also contains an adjuvant, which includes at least one of aluminum adjuvant, squalene, Tween, tocopherol, Span, MPL, LPA, CpG, poly(I:C), and QS21.

[0039] Further, the adjuvant contains the following components:

[0040] Squalene 42.16 mg / mL, α-tocopherol 47.44 mg / mL, Tween 80 19.44 mg / mL.

[0041] The third aspect of the present invention provides the application of the broad-spectrum vaccine of any one of the above in as a SARS-CoV-2 immune booster, and the broad-spectrum vaccine is administered 3 - 6 months after two immunizations with any SARS-CoV-2 vaccine.

[0042] The fourth aspect of the present invention provides a SARS-CoV-2 immune set, including a first immunization preparation, a second immunization preparation administered 14 - 28 days after the first immunization preparation, and a third immunization preparation administered 3 - 6 months after the second immunization preparation. The first immunization preparation and the second immunization preparation are inactivated vaccines or recombinant vaccines, and the third immunization preparation is the broad-spectrum vaccine according to any one of claims 23 - 26.

[0043] Further, both the first immunization preparation and the second immunization preparation are the broad-spectrum vaccines.

[0044] The beneficial effects of the present invention are as follows:

[0045] The present invention provides an application of a fusion protein in the preparation of a SARS-CoV-2 broad-spectrum vaccine. The fusion protein contains the NTD domain and the RBD domain of the SARS-CoV-2 spike protein, provides more conserved epitopes, can effectively stimulate the production of neutralizing antibodies, and the vaccine prepared by applying the fusion protein can provide broad-spectrum and efficient protection against wild-type SARS-CoV-2 and various mutant strains; the present invention also provides an application of the broad-spectrum vaccine as an immune booster, and at the same time provides an immune set under the corresponding immune strategy, wherein the broad-spectrum vaccine can be used for booster immunization in combination with other existing vaccines or can be independently used for a complete immunization program, and excellent immune protection can be provided in either way. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 It is the immunoblotting diagram of the vaccine stock solution in Example 1 (the primary antibody is the RBD antibody); among them, lane 1 is Marker, lane 2 is blank, and lanes 3 to 7 are samples of different batches;

[0047] Figure 2 It is the immunoblotting diagram of the vaccine stock solution in Example 1 (the primary antibody is the NTD antibody); among them, lane 1 is Marker, lane 2 is blank, and lanes 3 to 7 are samples of different batches;

[0048] Figure 3 It is the antibody titers against different mutant strains after different booster immunization regimens in Example 26 (from left to right in each group are D614G, Delta, Omicron pseudovirus neutralizing antibodies).

[0049] Figure 4 It is the neutralizing antibody titers against different mutant strains after booster immunization with different vaccines in Example 30 (from left to right in each group are D614G, Delta, Omicron-BA.1 and Omicron-BA.2 pseudovirus neutralizing antibodies). DETAILED DESCRIPTION OF THE EMBODIMENTS

[0050] The following further describes the present application in detail with reference to the drawings and embodiments.

[0051] TERM DEFINITION

[0052] In the present application, the term "fusion protein" generally refers to a protein molecule with biological functional activity obtained by genetic engineering techniques. In the present application, the fusion protein may be a fusion protein composed of RBD or its functional active fragment and any one of the following sequences: P2 or its functional active fragment, foldon domain or its functional active fragment, ferritin or its functional active fragment, hepatitis B surface antigen HBsAg or its functional active fragment. In the present application, the fusion protein may be a fusion protein composed of NTD or its functional active fragment and any one of the following sequences: P2 or its functional active fragment, foldon domain or its functional active fragment, ferritin or its functional active fragment, hepatitis B surface antigen HBsAg or its functional active fragment. In the present application, the fusion protein may be a fusion protein composed of NTD or its functional active fragment and RBD or its functional active fragment. In the present application, the fusion protein may be a fusion protein composed of NTD or its functional active fragment, RBD or its functional active fragment and any one of the following fragments: P2 or its functional active fragment, foldon domain or its functional active fragment, ferritin or its functional active fragment, hepatitis B surface antigen HBsAg or its functional active fragment.

[0053] In the present application, the term "P2" generally refers to the epitope peptide of tetanus toxin. For example, P2 may be the peptide segment of the epitope peptide 830 to 844 of tetanus toxin or its mutant. For example, P2 may be the peptide segment of the epitope peptide 830 to 845 of tetanus toxin or its mutant. For example, P2 may be the peptide segment of the epitope peptide 829 to 844 of tetanus toxin or its mutant. For example, P2 may be the peptide segment obtained by truncating the peptide segment of the epitope peptide 829 to 844 of tetanus toxin or adding 1, 2, 3, 4, 5, or 6 or 10 amino acids at the N-terminus or C-terminus. For example, P2 may be the peptide segment obtained by truncating the peptide segment of the epitope peptide 830 to 845 of tetanus toxin or adding 1, 2, 3, 4, 5, or 6 or 10 amino acids at the N-terminus or C-terminus. For example, P2 may be the peptide segment obtained by truncating the peptide segment of the epitope peptide 830 to 844 of tetanus toxin or adding 1, 2, 3, 4, 5, or 6 or 10 amino acids at the N-terminus or C-terminus. In the present application, the "mutant" generally refers to a sequence that is different from the reference sequence due to the presence of one or more differences (mutations). The difference may be the substitution, deletion or insertion of one or more amino acids.

[0054] In the present application, the term "foldon domain" generally refers to the residues at the C-terminus of the bacteriophage T4 fibritin. In the present application, the foldon domain can be the 27 residues at the C-terminus of the bacteriophage T4 fibritin or a mutant. In the present application, the foldon domain can be a truncated or elongated form obtained by truncating the 27 residues at the C-terminus of the bacteriophage T4 fibritin and adding 1, 2, 3, 4, 5, or 6 or 10 amino acids at the N-terminus or C-terminus. In the present application, "mutant" generally refers to a sequence that is different from a reference sequence due to the presence of one or more differences (mutations). The difference can be a substitution, deletion, or insertion of one or more amino acids.

[0055] In the present application, the terms "ferritin" and "ferritin" generally refer to Trichoplusia ni ferritin or Helicobacter pylori ferritin. In the present application, Trichoplusia ni ferritin has a heavy chain and a light chain (ferritin LC and ferritin HC). In the present application, Helicobacter pylori ferritin has a single-chain structure. In the present application, ferritin can be a mutant of Trichoplusia ni or Helicobacter pylori ferritin. In the present application, "mutant" generally refers to a sequence that is different from a reference sequence due to the presence of one or more differences (mutations). The difference can be a substitution, deletion, or insertion of one or more amino acids. In the present application, "the light chain of Trichoplusia ni ferritin", "LC" can be used interchangeably with "ferritin LC". In the present application, "the heavy chain of Trichoplusia ni ferritin", "HC" can be used interchangeably with "ferritin HC".

[0056] In the present application, the term "hepatitis B surface antigen (HBsAg)" generally refers to an outer envelope protein in the outermost envelope of the hepatitis B virus. For example, the amino acid sequence of the hepatitis B surface antigen can be the sequence corresponding to the protein sequence accession numbers AAA45524, ANJ76941, CAA24234, or AAC34729 in NCBI, and can also be appropriately truncated or have 1, 2, 3, 4, 5, or 6 or 10 amino acids added at the N-terminus or C-terminus, or there can be mutations in the protein, such as deletions, substitutions, or insertions of one or more amino acids.

[0057] In the present application, the term "functionally active fragment" generally refers to a fragment having similar biological activity to RBD, NTD, P2, foldon domain, ferritin, or hepatitis B surface antigen HBsAg.

[0058] In the present application, the term "immunogenic composition" generally refers to a subunit composition. In the present application, a subunit composition is a composition in which the components have been separated and purified to at least 50%, at least 60%, 70%, 80%, 90% purity before being mixed to form an antigenic composition. For example, the subunit composition can be an aqueous solution of a water-soluble protein. For example, the subunit composition may contain a detergent. For example, the subunit composition may contain a non-ionic, zwitterionic or ionic detergent. For example, the subunit composition may contain lipids. In some cases, the immunogenic composition may include an RBD or a functionally active fragment thereof and an NTD or a functionally active fragment thereof. In some cases, the immunogenic composition may include an RBD or a functionally active fragment thereof and a fusion protein comprising an NTD or a functionally active fragment thereof. In some cases, the immunogenic composition may include a fusion protein comprising an RBD or a functionally active fragment thereof and an NTD or a functionally active fragment thereof. In some cases, the immunogenic composition may include a fusion protein comprising an RBD or a functionally active fragment thereof and a fusion protein comprising an NTD or a functionally active fragment thereof. In the present application, the immunogenic composition may further include an adjuvant. For example, the adjuvant may include aluminum salts (such as aluminum hydroxide gel (alum) or aluminum phosphate), but may also be calcium salts, iron salts or zinc salts, or may be an insoluble suspension of acylated tyrosine or acylated sugar, cationic or anionic derivatized polysaccharide or polyphosphazene. For example, the immunogenic composition may also be selected as a Th1-type response preferential inducer. For example, a Th1-type response preferential inducer may include monophosphoryl lipid A or a derivative thereof. For example, the adjuvant may be a combination of monophosphoryl lipid A (for example, 3-de-O-acylated monophosphoryl lipid A (3D-MPL)) and an aluminum salt. An adjuvant enhancement system may include a combination of monophosphoryl lipid A and a saponin derivative, particularly the combination of QS21 and 3D-MPL disclosed in WO94 / 00153, or a composition in which QS21 is quenched with cholesterol to make it less reactogenic as disclosed in WO96 / 33739. For example, the adjuvant may also be the adjuvant described in WO95 / 17210, which contains QS21, 3D-MPL and tocopherol in an oil-in-water emulsion. For example, the adjuvant may be a homogeneous droplet emulsion formed by mixing Tween 80, sorbitan trioleate and squalene and subjecting it to microfluidization under high pressure conditions. For example, the adjuvant may contain unmethylated CpG of oligonucleotide (WO96 / 02555).

[0059] In the present application, the term "RBD" refers to the receptor-binding domain of the SARS-CoV-2 spike protein (S protein). In the present application, the RBD may be a peptide segment between amino acids 310-560 of the SARS-CoV-2 spike protein (S protein) or a mutant thereof, and may also be truncated at the N-terminus or C-terminus by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 20, 25 or 30 amino acids. In the present application, the RBD may be a peptide segment between amino acids 319-541 of the SARS-CoV-2 spike protein (S protein) or a mutant thereof, and may also be appropriately truncated at the N-terminus or C-terminus by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 20, 25 or 30 amino acids. In the present application, the RBD may be a peptide segment between amino acids 331-524 of the SARS-CoV-2 spike protein (S protein) or a mutant thereof, and may also be appropriately truncated at the N-terminus or C-terminus by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 20, 25 or 30 amino acids. In the present application, a "mutant" generally refers to a sequence that is different from a reference sequence due to the presence of one or more differences (mutations). The difference may be the substitution, deletion or insertion of one or more amino acids.

[0060] In the present application, the term "NTD" refers to the domain at the N-terminus of the SARS-CoV-2 spike protein (S protein). In the present application, the NTD may be a peptide segment between amino acids 13-353 of the SARS-CoV-2 spike protein (S protein) or a mutant thereof, and may also be appropriately truncated at the N-terminus or C-terminus by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 20, 25 or 30 amino acids. For example, the NTD may be a peptide segment between amino acids 13-303 of the SARS-CoV-2 spike protein (S protein) or a mutant thereof. In the present application, the NTD may be a peptide segment between amino acids 14-304 of the SARS-CoV-2 spike protein (S protein) or a mutant thereof. In the present application, the NTD may be a peptide segment between amino acids 18-353 of the SARS-CoV-2 spike protein (S protein) or a mutant thereof. In the present application, a "mutant" generally refers to a sequence that is different from a reference sequence due to the presence of one or more differences (mutations). The difference may be the substitution, deletion or insertion of one or more amino acids.

[0061] In this application, the term "S protein", also known as "Spike protein" or "spike protein", generally refers to the capsid surface glycoprotein of coronaviruses. SARS-COV-2 binds to the ACE2 receptor through the S protein and invades cells. The S protein consists of 1,213 amino acids, contains a transmembrane region, and includes at least the fragment from the N-terminus or the 14th amino acid to the 1,213th amino acid of the capsid surface glycoprotein from coronaviruses, or the corresponding region from other SARS viruses. The S1 protein is subunit 1 of the S protein, mainly referring to the fragment from the N-terminus or the 14th amino acid to the 685th amino acid.

[0062] In this application, the mutant may include any one or several of the currently known SAR-CoV-2 mutant strains (mutants). For example, the Gamma mutant strain may refer to the amino acid sequence of the SARS-CoV-2 surface glycoprotein of lineage B.1.1.28 with GenBank accession number QRN46961.1.

[0063] The term "hemagglutinin / hemagglutinin protein / HA" used herein generally refers to an antibody or other substance that can agglutinate red blood cells, and can be found on the surfaces of influenza viruses, measles viruses (and many other bacteria and viruses), etc. It can attach to the red blood cells of different animals, causing the red blood cells to agglutinate, and can be lethal in severe cases. In this context, it refers to the surface glycoprotein hemagglutinin (HA) of influenza viruses.

[0064] In this application, the term "comprising" generally means including, encompassing, containing, or subsuming. In some cases, it also means "being" or "consisting of".

[0065] In this application, the term "about" generally refers to a variation within the range of 0.5% - 10% above or below the specified value. For example, it can vary within the range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified value.

[0066] In the present application, the term "vector" generally refers to a nucleic acid molecule capable of self-replicating in a suitable host, which transfers the inserted nucleic acid molecule into host cells and / or between host cells. The vector may include vectors mainly used for inserting DNA or RNA into cells, vectors mainly used for replicating DNA or RNA, and expression vectors mainly used for transcription and / or translation of DNA or RNA. The vector also includes vectors having multiple of the above functions. The vector may be a polynucleotide capable of transcribing and translating into a polypeptide when introduced into a suitable host cell. Generally, by culturing a suitable host cell containing the vector, the vector can produce a desired expression product.

[0067] In the present application, the term "host cell" generally refers to an individual cell, cell line, or cell culture that can or already contains a plasmid or vector including the nucleic acid molecule described in the present application, or is capable of expressing the antibody or antigen-binding fragment thereof described in the present application. The host cell may include the progeny of a single host cell. Due to natural, accidental, or intentional mutations, the progeny cells may not necessarily be identical morphologically or genomically to the original parental cells, but are capable of expressing the fusion protein described in the present application. The host cell can be obtained by in vitro transfection of cells using the vector described in the present application. The host cell can be a prokaryotic cell (such as Escherichia coli), or a eukaryotic cell (such as a yeast cell, such as a COS cell, Chinese hamster ovary (CHO) cell, HeLa cell, HEK293 cell, COS-1 cell, NS0 cell, or myeloma cell). In some embodiments, the host cell is a mammalian cell. For example, the mammalian cell can be a CHO cell.

[0068] Example 1 Preparation of wild-type NTD-RBD-foldon

[0069] Based on the NTD (SEQ ID NO: 1) and RBD (SEQ ID NO: 9) sequences of the WH01 strain, and using the foldon sequence of SEQ ID NO: 28, a signal peptide (SEQ ID NO: 29) was added to the N-terminus of the NTD. After codon optimization according to the host CHO cells, it was sent out for synthesis. After sequencing verification of the target gene, PCR amplification and gel purification were carried out to obtain the target gene fragment, which was cloned into the expression vector pcDNA3.1(+), resulting in a transient expression vector. The reaction system is shown in Table 1.

[0070] Table 1 Reaction system for vector construction

[0071]

[0072]

[0073] The above expression vector was transformed into E. coli Top10 competent cells. The transformants were picked and cultured in liquid, and then streaked on LB agar plates (containing 100 μg / mL ampicillin). A single clone was picked from the plate and inoculated into 300 mL of LB medium for large-scale culture. Plasmids were prepared in large quantities and the target gene was verified by sequencing.

[0074] Verification of the recombinant expression plasmid: (1) Prepare agar plates containing the corresponding resistance. (2) Take 100 μL of the bacterial solution and spread it evenly on the agar plate containing the corresponding resistance. Gently spread the bacteria on the surface of the plate with a sterile glass spreader. Place the plate in an incubator at 37 °C for 15 minutes. (3) Invert the plate and incubate it at 37 °C for 12 - 16 hours to obtain colonies. (4) Pick colonies from the plate, shake the bacteria at 250 rpm at 37 °C for 14 hours, identify the bacteria by PCR using the bacterial solution, and send the positive clone bacterial solution for sequencing.

[0075] Identification method of the cloned plasmid: The NTD-RBD-foldon fragment was amplified by PCR. The primer sequences for identification were synthesized by the in-house primer department of the company. The expected amplified fragment length was 1761 bp. The PCR reaction used a 20 μL system: 0.5 μL of primer, 2 μL of template bacterial solution, 0.5 μL of polymerase buffer, 3 μL of buffer, and 14 μL of ddH2O. Cycling parameters: Pre-denaturation at 96 °C for 3 min; 95 °C for 15 s, 58 °C for 15 s, 72 °C for 20 s, for 23 cycles, and final extension at 72 °C for 1 min. Positive clones were screened by colony PCR. The positive bacterial solution obtained was shaken at 37 °C to extract plasmids and sequenced. For the plasmid with correct sequencing alignment, double digestion was performed with BamHI - XhoI to obtain two fragments of 1761 bp and 5372 bp.

[0076] Extraction of the NTD-RBD-foldon recombinant expression plasmid. Inoculate 1% of E. coli cells (stbl3) containing the NTD-RBD-foldon recombinant expression plasmid into 2 mL of LB medium and shake culture overnight at 37 °C. After treating the cells, use a plasmid extraction kit to extract 100 μg of plasmid. For the plasmid with correct sequencing alignment, double digestion was performed with BamHI - XhoI to obtain two fragments of 1761 bp and 5372 bp.

[0077] The obtained plasmid was introduced into CHO cells by transient transfection. After culture, the protein was collected and purified. CHO cells (ExpiCHO from Thermo) were subcultured and amplified using ExpiCHO Expression Medium. During all cell operations, gently rotate and mix the host cells; avoid violent mixing / pipetting. Subculture and amplify CHO cells until the cell density reaches 4×10 6 ~6×10 6cells / mL. Day - 1: Cell expansion, expand the cultured cells to 3×10 6 ~4×10 6 cells / mL, and allow the cells to grow overnight. Day 0: Transfect the cells, measure the viable cell density and viability. The density of the cells should reach 7×10 6 ~10×10 6 cells / mL, and the viability reaches 95 - 99% for transfection. Fresh cell expression medium, preheated to 37°C, dilute the cells to a final density of 6×10 6 cells / mL, and culture in a 50 mm amplitude incubator at 90 rpm, 37°C, and 8% CO2. Prepare the transfection reagent and recombinant expression plasmid complex (at 4°C) using OPti - PRO SFM medium. For example, for 1 mL of cells, prepare 40 μL of OPti - PRO SFM, add 0.8 μg of NTD - RBD - foldon - 8*HIS recombinant expression plasmid, mix well, and let stand for 5 min; prepare 40 μL of OPti - PRO SFM, add 3 μL of Expi Fectamine CHO reagent, mix well, and let stand for 5 min at room temperature for 1 - 5 minutes, then slowly transfer the solution to the shake flask and shake gently. During the addition process, gently shake the shake flask. Culture the cells in a 50 mm amplitude incubator at 90 rpm, 37°C, and 8% CO2. 18 - 22 hours after transfection, add Enhancer and ExpiCHO Feed, and perform the standard experimental protocol. For example, for 1 mL of cells, add 6 μL of Enhancer and 0.24 mL of ExpiCHO Feed, and culture the cells in a 50 mm amplitude incubator at 90 rpm at 37°C, 8% CO2. Collect after 8 days of transfection, and obtain the trimeric protein with a purity ≥95% after a series of chromatography purifications, which is the required purified NTD - RBD - foldon protein, and the results are shown in Table 2.

[0078] Table 2 Concentration and volume of purified NTD - RBD - foldon protein

[0079]

[0080] Verify the obtained purified protein by Western Blot, and the results are as Figure 1 and 2 shown. The complete sequence of this protein is shown as SEQ ID NO:30.

[0081] Example 2 Preparation of Beta strain NTD - RBD - foldon

[0082] Based on the NTD (SEQ ID NO: 3) and RBD (SEQ ID NO: 11) sequences of the Beta strain, and using the foldon sequence of SEQ ID NO: 27, the Beta strain NTD-RBD-foldon recombinant protein was prepared according to the method provided in Example 1.

[0083] Example 3 Preparation of Gamma Strain NTD-RBD-foldon

[0084] Based on the NTD (SEQ ID NO: 4) and RBD (SEQ ID NO: 12) sequences of the Gamma strain, and using the foldon sequence of SEQ ID NO: 26, the Gamma strain NTD-RBD-foldon recombinant protein was prepared according to the method provided in Example 1.

[0085] Example 4 Preparation of Delta Strain NTD-RBD-foldon

[0086] Based on the NTD (SEQ ID NO: 7) and RBD (SEQ ID NO: 22) sequences of the Delta strain, and using the foldon sequence of SEQ ID NO: 28, the Delta strain NTD-RBD-foldon recombinant protein was prepared according to the method provided in Example 1.

[0087] Example 5 Preparation of Omicron Strain NTD-RBD-foldon

[0088] Based on the NTD (SEQ ID NO: 8) and RBD (SEQ ID NO: 13) sequences of the Omicron strain, and using the foldon sequence of SEQ ID NO: 28, the Omicron strain NTD-RBD-foldon recombinant protein was prepared according to the method provided in Example 1. The complete sequence of this protein is shown in SEQ ID NO: 31.

[0089] Example 6 Preparation of Vaccine Composition

[0090] 0.02 mg / mL of wild-type NTD-RBD-foldon purified protein, 2% to 15% (w / v) sucrose; 0.01% to 0.05% (w / v) Tween 80; 5 mM to 25 mM histidine buffer with pH 5.0 - 7.0, dispensed into 2 mL ampoules, 0.5 mL per tube, and placed in a freeze dryer for freeze-drying.

[0091] Example 7 Preparation of Vaccine Composition

[0092] 0.04 mg / mL wild-type NTD-RBD-foldon purified protein, 2% to 15% (w / v) sucrose; 0.01% to 0.05% (w / v) Tween 80; 5 mM to 25 mM histidine buffer at pH 5.0 - 7.0, aliquoted into 2 mL ampoules, 0.5 mL per tube, and placed in a freeze dryer for freeze-drying.

[0093] Preparation of the vaccine composition in Example 8

[0094] 0.08 mg / mL wild-type NTD-RBD-foldon purified protein, 2% to 15% (w / v) sucrose; 0.01% to 0.05% (w / v) Tween 80; 5 mM to 25 mM histidine buffer at pH 5.0 - 7.0, aliquoted into 2 mL ampoules, 0.5 mL per tube, and placed in a freeze dryer for freeze-drying.

[0095] Preparation of the vaccine composition in Example 9

[0096] 0.12 mg / mL wild-type NTD-RBD-foldon purified protein, 2% to 15% (w / v) sucrose; 0.01% to 0.05% (w / v) Tween 80; 5 mM to 25 mM histidine buffer at pH 5.0 - 7.0, aliquoted into 2 mL ampoules, 0.5 mL per tube, and placed in a freeze dryer for freeze-drying.

[0097] Preparation of the vaccine composition in Example 10

[0098] 0.16 mg / mL wild-type NTD-RBD-foldon purified protein, 2% to 15% (w / v) sucrose; 0.01% to 0.05% (w / v) Tween 80; 5 mM to 25 mM histidine buffer at pH 5.0 - 7.0, aliquoted into 2 mL ampoules, 0.5 mL per tube, and placed in a freeze dryer for freeze-drying.

[0099] Preparation of the vaccine composition in Example 11

[0100] The vaccine composition described in Example 7, further comprising 500 μg of aluminum hydroxide.

[0101] Preparation of the vaccine composition in Example 12

[0102] The vaccine composition described in Example 6, further comprising 10.690 mg of squalene, 11.860 mg of α-tocopherol, and 4.86 mg of Tween 80 (this adjuvant is hereinafter referred to as BFA03 adjuvant), and further comprising 1.768 mg of NaCl, 0.044 mg of KCl, 0.253 mg of Na2HPO4, 0.044 mg of KH2PO4, and water for injection.

[0103] Preparation of Vaccine Composition in Example 13

[0104] The vaccine composition described in Example 7 further comprises 10.690 mg of squalene, 11.860 mg of α-tocopherol, and 4.86 mg of Tween 80, and also comprises 1.768 mg of NaCl, 0.044 mg of KCl, 0.253 mg of Na2HPO4, 0.044 mg of KH2PO4 and water for injection.

[0105] Preparation of Vaccine Composition in Example 14

[0106] The vaccine composition described in Example 8 further comprises 10.690 mg of squalene, 11.860 mg of α-tocopherol, and 4.86 mg of Tween 80, and also comprises 1.768 mg of NaCl, 0.044 mg of KCl, 0.253 mg of Na2HPO4, 0.044 mg of KH2PO4 and water for injection.

[0107] Preparation of Vaccine Composition in Example 15

[0108] The vaccine composition described in Example 9 further comprises 10.690 mg of squalene, 11.860 mg of α-tocopherol, and 4.86 mg of Tween 80, and also comprises 1.768 mg of NaCl, 0.044 mg of KCl, 0.253 mg of Na2HPO4, 0.044 mg of KH2PO4 and water for injection.

[0109] Preparation of Vaccine Composition in Example 16

[0110] The vaccine composition described in Example 10 further comprises 10.690 mg of squalene, 11.860 mg of α-tocopherol, and 4.86 mg of Tween 80, and also comprises 1.768 mg of NaCl, 0.044 mg of KCl, 0.253 mg of Na2HPO4, 0.044 mg of KH2PO4 and water for injection.

[0111] Preparation of Vaccine Composition in Example 17

[0112] The vaccine composition described in Example 7 further comprises 0.05 mg of aluminum hydroxide adjuvant and 3 mg of CpG1018 adjuvant (CpG was synthesized by Shanghai Sangon Biotech Co., Ltd. according to the CpG1018 sequence).

[0113] Preparation of Vaccine Composition in Example 18

[0114] The vaccine composition described in Example 7 further comprises 9.75 mg of squalene, 1.175 mg of Span 85, and 1.175 mg of Tween 80 (this adjuvant is hereinafter referred to as BFA02 adjuvant), and also comprises 0.66 mg of trisodium citrate dihydrate, 0.04 mg of citric acid monohydrate, and water for injection.

[0115] Preparation of the vaccine composition of Example 19

[0116] The vaccine composition described in Example 7 further comprises 50 μg of MPL, 50 μg of QS-21, 1 mg of dioleoyl phosphatidylcholine (DOPC), and 0.25 mg of cholesterol (this adjuvant is hereinafter referred to as BFA01 adjuvant), and also comprises 0.15 mg of disodium hydrogen phosphate anhydrous, 0.54 mg of potassium dihydrogen phosphate, 4.385 mg of sodium chloride, and water for injection.

[0117] Preparation of the vaccine composition of Example 20

[0118] The vaccine composition described in Example 7 further comprises 50 μg of MPL and 500 μg of aluminum hydroxide (this adjuvant is hereinafter referred to as BFA04 adjuvant).

[0119] Construction of the pseudovirus of Example 21

[0120] Zhongke Guobang (Beijing) Inspection and Testing Co., Ltd. was commissioned to construct pseudoviruses of WH01, D614G, Alpha, Beta, Gamma, Delta, Lambda, Omicron, and Omicron-BA.2, and corresponding detections were carried out for subsequent in vitro neutralization tests.

[0121] Screening of antigen combinations in Example 22

[0122] Take 50 BALB / c mice (female, 6 - 8 weeks old, purchased from the Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences), randomly divide them into 5 groups with 10 mice in each group. Based on the WH01 strain sequence, the adjuvant described in Example 12 (hereinafter referred to as BFA03 adjuvant) and excipients are used to prepare vaccines according to the method in Table 3 and conduct grouped immunization. After collecting sera, in vitro neutralization experiments are carried out with wild-type authentic virus and various pseudoviruses to screen the best protein expression combination method.

[0123] Table 3 Design of the recombinant COVID-19 vaccine candidate antigen screening experiment in BALB / c mice

[0124]

[0125] The results are shown in Table 4. After co - immunizing BALB / c mice with the NTD - RBD - foldon antigen and the BFA03 adjuvant, it can induce the body to produce high - titer neutralizing antibodies (detected based on the SARS - CoV - 2 virus strain and the VSV pseudovirus detection system) and antigen - specific IgG antibodies. Among them, the neutralization effect against the WH01 strain, D614G, and Gamma is significantly better than that of other antigens.

[0126] Table 4 In vitro neutralization test results after immunization with different antigens

[0127]

[0128] Example 23 Screening of adjuvants

[0129] Take 60 BALB / c mice (female, 6 - 8 weeks old, purchased from the Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences), randomly divide them into 6 groups with 10 mice in each group, and immunize the mice in groups according to the method in Table 5 using 1 / 10 of the human - used dose of the above - mentioned vaccine. After collecting the serum, perform in vitro neutralization tests with wild - type true virus and D614G pseudovirus respectively to screen the best adjuvant combination method.

[0130] Table 5 General situation of the experimental design for screening candidate adjuvants of the recombinant COVID - 19 vaccine in BALB / c mice

[0131]

[0132] The results are shown in Table 6. When immunizing according to Example 13, it can induce mice to produce high - titer neutralizing antibodies (detected based on the SARS - CoV - 2 virus strain and the VSV pseudovirus detection system) and antigen - specific IgG antibodies, and all indicators are significantly higher than those of the other groups, indicating that the BFA03 adjuvant has the best immune effect and is significantly better than the traditional aluminum adjuvant and other composite adjuvant systems based on aluminum salts.

[0133] Table 6 Screening results of candidate adjuvants of the recombinant COVID - 19 vaccine in BALB / c mice

[0134]

[0135]

[0136] Example 24 Comparison of immune effects in different animals

[0137] The broad-spectrum vaccine provided in Example 14 was used to immunize BALB / c mice, Japanese white rabbits, and rhesus monkeys with two doses (with a 14-day interval). There were 10 mice, 6 white rabbits, and 4 rhesus monkeys. The immunization dose for mice was 1 / 10 of the human dose, and the doses for white rabbits and rhesus monkeys were both one human dose. Serum was collected and in vitro neutralization experiments were conducted using wild-type and various mutant pseudoviruses to measure antibody titers. The results are shown in Table 7. This broad-spectrum vaccine could induce the production of high-titer neutralizing antibodies in different animals and also had a good immune effect against various mutant strains.

[0138] Table 7 Results of neutralizing antibody titers produced in different species of animals

[0139]

[0140] Comparison of immunization effects at different doses in Example 25

[0141] (1) Eighteen Japanese white rabbits were randomly divided into 3 groups of 6 each, and were immunized with two doses (with a 21-day interval) using the broad-spectrum vaccines (one human dose) provided in Examples 14 - 16. Serum was collected 14 days after the second immunization for in vitro neutralization experiments to measure the neutralizing antibody titers against different pseudoviruses.

[0142] The results are shown in Table 8. Broad-spectrum vaccines at different doses could all produce high-titer neutralizing antibodies against the D614G strain and the Alpha strain, and also had a certain immune effect against the Beta strain, Gamma strain, and Delta strain. For the D614G strain and the Alpha strain, the antibody titers produced in Example 15 were the highest, while for the Gamma strain and the Delta strain, the effect of Example 14 was the best. However, the remaining data did not show a pattern of change according to the antigen dose, indicating that the antigen dose used during immunization may not have a decisive impact on the immunization effect.

[0143] Table 8 Neutralizing antibody titers of white rabbit serum against different pseudoviruses

[0144]

[0145] (2) Twenty-four Japanese white rabbits were randomly divided into 4 groups of 6 each, and were immunized with two doses (with a 14-day interval) using the broad-spectrum vaccines (one human dose) provided in Examples 12 - 14, 16. Serum was collected 21 days after the second immunization for in vitro neutralization experiments to measure the neutralizing antibody titers and Ig antibody titers against the true virus and the D614G pseudovirus.

[0146] The results are shown in Table 9. Broad-spectrum vaccines with different doses can induce the body to produce high-titer specific Ig antibodies. In terms of pseudovirus neutralizing antibodies, the antibody titers produced in Example 16 and Example 14 are relatively high. In terms of authentic virus neutralizing antibodies, the antibody titer produced in Example 16 is relatively high, but overall, none of them reach the ideal level. However, the remaining data do not show a pattern of change depending on the antigen dose, indicating that the antigen dose used during immunization may not have a decisive impact on the immunization effect.

[0147] Table 9 Serum antibody titers of white rabbits immunized with different doses

[0148]

[0149] Comparison of immunization effects of different booster immunization regimens in Example 26

[0150] Forty BALB / c mice (female, 6 - 8 weeks old, purchased from the Institute of Laboratory Animals, Chinese Academy of Medical Sciences) were grouped and immunized as shown in Table 10. They were immunized with an inactivated vaccine at 1 / 10 human dose or the broad-spectrum vaccine provided in Example 12, and immunized via the intramuscular route according to an immunization schedule with an interval of 21 days (0 and 21 days). Booster immunization was carried out 12 weeks (3 months) after the second immunization. Serum was collected after each immunization, and pseudovirus in vitro neutralization tests were performed using D614G, Delta, and Omicron pseudoviruses, and the neutralizing antibody titers were measured.

[0151] Table 10 Grouping table of immunization regimens

[0152]

[0153]

[0154] As Figure 3 shown, the neutralizing antibody titers in group A are significantly lower than the corresponding data in group B, indicating that the broad-spectrum vaccine has more excellent immunological activity than the inactivated vaccine, thus effectively improving the immunization effect. The neutralizing antibody titers in A3 are significantly higher than those in A1 and A2, and at the same time, the titers in B3 are significantly higher than those in B1 and B2, indicating that when booster immunization is carried out after routine immunization, the broad-spectrum vaccine can more effectively promote the production of neutralizing antibodies than the inactivated vaccine, thus greatly enhancing the immunization effect. In addition, in B3, the titers of neutralizing antibodies against Delta and Omicron are also several times higher than those in the previously highest A3 group. In particular, the Omicron antibody has directly increased by an order of magnitude and reached a relatively high level. It can be seen that this broad-spectrum vaccine has an excellent inhibitory effect on representative mutant strains, and can also play a good immunological role even against Omicron, which is difficult to deal with by existing vaccines.

[0155] In addition, inFigure 3 It can also be seen that the antibody titers of A3 (inactivated vaccine primary immunization + broad-spectrum vaccine booster immunization) are significantly higher than those of B2 (broad-spectrum vaccine primary immunization + inactivated vaccine booster immunization). Based on this, it can be considered that the broad-spectrum vaccine provided by this application may play an excellent role in booster immunization, and it can even be reasonably expected that this broad-spectrum vaccine can be used as an efficient "booster shot" in combination with other existing vaccines.

[0156] Preparation of the vaccine composition of Example 27

[0157] According to the method provided in Example 8, a vaccine composition was prepared using the Omicron strain NTD-RBD-foldon purified protein, and further 10.690 mg of squalene, 11.860 mg of α-tocopherol, 4.86 mg of Tween 80 (BFA03), 1.768 mg of NaCl, 0.044 mg of KCl, 0.253 mg of Na2HPO4, 0.044 mg of KH2PO4 and water for injection were added.

[0158] Preparation of the vaccine composition of Example 28

[0159] According to the method provided in Example 8, a vaccine composition was prepared using the Omicron strain NTD-RBD-foldon purified protein, and further 0.5 mg of aluminum hydroxide adjuvant and 3 mg of CpG1018 adjuvant were added.

[0160] Preparation of the bivalent vaccine composition of Example 29

[0161] A wild-type - Omicron bivalent vaccine composition was prepared by mixing 0.04 mg / ml of wild-type NTD-RBD-foldon purified protein and 0.04 mg / ml of Omicron strain NTD-RBD-foldon purified protein in equal amounts according to the method provided in Example 27.

[0162] Comparison of the booster immunization effects of different antigens in Example 30

[0163] Twenty BALB / c mice (female, 6 - 8 weeks old, purchased from the Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences) were taken, grouped and immunized as shown in Table 11, immunized by the intramuscular route with the corresponding vaccine at 1 / 10 human dose, injected according to the immunization schedule with an interval of 21 days (0 and 21 days), boosted 30 days after the second immunization, and sera were collected 14 days later. Pseudovirus in vitro neutralization tests were performed using D614G, Delta, Omicron-BA.1 and Omicron-BA.2 pseudoviruses respectively through a SARS-CoV-2 pseudovirus neutralization detection method based on the VSV system, and the neutralizing antibody titers were measured.

[0164] Table 11 Immunization Protocol Grouping Table

[0165]

[0166] The results are as Figure 4 shown. After different booster immunizations in each group, the body can be induced to produce high-titer D614G antibodies. Among them, the GMT of groups 1 and 3 is significantly higher than that of groups 2 and 4; the situation of Delta neutralizing antibodies is similar to that of D614G, and groups 1 and 3 are also significantly higher than the other two groups, indicating that Example 14 (wild-type NTD-RBD-foldon) and Example 29 (wild-type-Omicron combination) can both exert excellent booster immunization effects against D614 strain and Delta strain. In terms of Omicron, the neutralizing antibody titer of Omicron-BA.1 is significantly lower than the previous two strains. Among them, group 3 is the highest and group 1 is the second; the situation of Omicron-BA.2 mutant strain is similar, group 3 is also the highest and group 1 is the second, both are significantly higher than group 4 (inactivated vaccine), indicating that Example 29 (wild-type-Omicron combination) has a certain booster immunization effect against Omicron-BA.1 and its mutant strains.

[0167] Overall, the neutralizing antibody levels of groups 1 and 3 are significantly higher than those of groups 2 and 4, and there is no significant difference between groups 2 and 4, which may indicate that Omicron antigen is not suitable for single use in booster immunization, but is very suitable for combination with wild-type antigen, especially showing unexpected application potential in resisting Omicron.

[0168] In addition, the overall trend in this example is basically consistent with that in Example 26, but the neutralizing antibody levels of groups 1, 2, and 3 all fail to reach the level of group A3 in Example 26, which may be related to the earlier time of the third immunization, and may indicate that the vaccination timing of the "booster shot" will also affect the immune effect, but further exploration is needed.

[0169] The above-described embodiments only represent several implementation manners of the present invention, and their descriptions are relatively specific and detailed, but should not be construed as limiting the scope of the patent of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention should be subject to the appended claims. Sequence Listing <110> Jiangsu Provincial Center for Disease Control and Prevention (Jiangsu Provincial Institute of Public Health); Jiangsu Recbio Biotechnology Co., Ltd.; Beijing Anbaisheng Biotechnology Co., Ltd. <120> A SARS-CoV-2 Broad-spectrum Vaccine and Its Application <160> 31 <170> SIPO Sequence Listing 1.0 <210> 1 <211> 291 <212> PRT <213> Artificial Sequence <400> 1 Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr 1 5 10 15 Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser 20 25 30 Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn 35 40 45 Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys 50 55 60 Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala 65 70 75 80 Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr 85 90 95 Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn 100 105 110 Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu 115 120 125 Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe 130 135 140 Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln 145 150 155 160 Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu 165 170 175 Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser 180 185 190 Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser 195 200 205 Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg 210 215 220 Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp 225 230 235 240 Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr 245 250 255 Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile 260 265 270 Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys 275 280 285 Thr Leu Lys 290 <210> 2 <211> 336 <212> PRT <213> Artificial Sequence <400> 2 Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe Thr 1 5 10 15 Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu His 20 25 30 Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp Phe 35 40 45 His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp Asn 50 55 60 Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu Lys 65 70 75 80 Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser Lys 85 90 95 Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile Lys 100 105 110 Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr Tyr 115 120 125 His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr Ser 130 135 140 Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu Met 145 150 155 160 Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe Val 165 170 175 Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr Pro 180 185 190 Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu Pro 195 200 205 Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr Leu 210 215 220 Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser Gly 225 230 235 240 Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro Arg 245 250 255 Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala Val 260 265 270 Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys Ser 275 280 285 Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val Gln 290 295 300 Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro 305 310 315 320 Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp 325 330 335 <210> 3 <211> 288 <212> PRT <213> Artificial Sequence <400> 3 Gln Cys Val Asn Phe Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr 1 5 10 15 Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser 20 25 30 Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn 35 40 45 Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys 50 55 60 Arg Phe Ala Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala 65 70 75 80 Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr 85 90 95 Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn 100 105 110 Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu 115 120 125 Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe 130 135 140 Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln 145 150 155 160 Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu 165 170 175 Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser 180 185 190 Lys His Thr Pro Ile Asn Leu Val Arg Gly Leu Pro Gln Gly Phe Ser 195 200 205 Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg 210 215 220 Phe Gln Thr Leu His Ile Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser 225 230 235 240 Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro 245 250 255 Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala 260 265 270 Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys 275 280 285 <210> 4 <211> 291 <212> PRT <213> Artificial Sequence <400> 4 Gln Cys Val Asn Phe Thr Asn Arg Thr Gln Leu Pro Ser Ala Tyr Thr 1 5 10 15 Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser 20 25 30 Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn 35 40 45 Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys 50 55 60 Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala 65 70 75 80 Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr 85 90 95 Leucine, Aspartic acid, Serine, Lysine, Threonine, Glutamine, Serine, Leucine, Leucine, Isoleucine, Valine, Asparagine, Asparagine, Alanine, Threonine, Asparagine 100 105 110 Valine, Valine, Isoleucine, Lysine, Valine, Cysteine, Glutamic acid, Phenylalanine, Glutamine, Phenylalanine, Cysteine, Asparagine, Tyrosine, Proline, Phenylalanine, Leucine 115 120 125 Glycine, Valine, Tyrosine, Tyrosine, Histidine, Lysine, Asparagine, Asparagine, Lysine, Serine, Tryptophan, Methionine, Glutamic acid, Serine, Glutamic acid, Phenylalanine 130 135 140 Arginine, Valine, Tyrosine, Serine, Serine, Alanine, Asparagine, Asparagine, Cysteine, Threonine, Phenylalanine, Glutamic acid, Tyrosine, Valine, Serine, Glutamine 145 150 155 160 Proline, Phenylalanine, Leucine, Methionine, Aspartic acid, Leucine, Glutamic acid, Glycine, Lysine, Glutamine, Glycine, Asparagine, Phenylalanine, Lysine, Asparagine, Leucine 165 170 175 Serine, Glutamic acid, Phenylalanine, Valine, Phenylalanine, Lysine, Asparagine, Isoleucine, Aspartic acid, Glycine, Tyrosine, Phenylalanine, Lysine, Isoleucine, Tyrosine, Serine 180 185 190 Lysine, Histidine, Threonine, Proline, Isoleucine, Asparagine, Leucine, Valine, Arginine, Aspartic acid, Leucine, Proline, Glutamine, Glycine, Phenylalanine, Serine 195 200 205 Alanine, Leucine, Glutamic acid, Proline, Leucine, Valine, Aspartic acid, Leucine, Proline, Isoleucine, Glycine, Isoleucine, Asparagine, Isoleucine, Threonine, Arginine 210 215 220 Phenylalanine, Glutamine, Threonine, Leucine, Leucine, Alanine, Leucine, Histidine, Arginine, Serine, Tyrosine, Leucine, Threonine, Proline, Glycine, Aspartic acid 225 230 235 240 Serine, Serine, Serine, Glycine, Tryptophan, Threonine, Alanine, Glycine, Alanine, Alanine, Alanine, Tyrosine, Tyrosine, Valine, Glycine, Tyrosine 245 250 255 Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile 260 265 270 Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys 275 280 285 Thr Leu Lys 290 <210> 5 <211> 305 <212> PRT <213> Artificial Sequence <400> 5 Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr 1 5 10 15 Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser 20 25 30 Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn 35 40 45 Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys 50 55 60 Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala 65 70 75 80 Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr 85 90 95 Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn 100 105 110 Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu 115 120 125 Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe 130 135 140 Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln 145 150 155 160 Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu 165 170 175 Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser 180 185 190 Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser 195 200 205 Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg 210 215 220 Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp 225 230 235 240 Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr 245 250 255 Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile 260 265 270 Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys 275 280 285 Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn 290 295 300 Phe 305 <210> 6 <211> 302 <212> PRT <213> Artificial Sequence <400> 6 Gln Cys Val Asn Phe Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr 1 5 10 15 Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser 20 25 30 Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn 35 40 45 Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys 50 55 60 Arg Phe Ala Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala 65 70 75 80 Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr 85 90 95 Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn 100 105 110 Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu 115 120 125 Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe 130 135 140 Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln 145 150 155 160 Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu 165 170 175 Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser 180 185 190 Lys His Thr Pro Ile Asn Leu Val Arg Gly Leu Pro Gln Gly Phe Ser 195 200 205 Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg 210 215 220 Phe Gln Thr Leu His Ile Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser 225 230 235 240 Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro 245 250 255 Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala 260 265 270 Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys 275 280 285 Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe 290 295 300 <210> 7 <211> 303 <212> PRT <213> Artificial Sequence <400> 7 Gln Cys Val Asn Leu Arg Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr 1 5 10 15 Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser 20 25 30 Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn 35 40 45 Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys 50 55 60 Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala 65 70 75 80 Ser Ile Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr 85 90 95 Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn 100 105 110 Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu 115 120 125 Asp Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Gly Val 130 135 140 Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe 145 150 155 160 Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu 165 170 175 Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His 180 185 190 Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu 195 200 205 Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln 210 215 220 Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser 225 230 235 240 Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln 245 250 255 Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp 260 265 270 Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu 275 280 285 Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe 290 295 300 <210> 8 <211> 305 <212> PRT <213> Artificial Sequence <400> 8 Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr 1 5 10 15 Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser 20 25 30 Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn 35 40 45 Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys 50 55 60 Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala 65 70 75 80 Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr 85 90 95 Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn 100 105 110 Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu 115 120 125 Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe 130 135 140 Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln 145 150 155 160 Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu 165 170 175 Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser 180 185 190 Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser 195 200 205 Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg 210 215 220 Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp 225 230 235 240 Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr 245 250 255 Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile 260 265 270 Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys 275 280 285 Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn 290 295 300 Phe 305 <210> 9 <211> 223 <212> PRT <213> Artificial Sequence <400> 9 Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn 1 5 10 15 Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val 20 25 30 Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser 35 40 45 Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val 50 55 60 Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp 65 70 75 80 Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln 85 90 95 Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr 100 105 110 Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly 115 120 125 Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys 130 135 140 Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr 145 150 155 160 Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser 165 170 175 Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val 180 185 190 Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly 195 200 205 Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe 210 215 220 <210> 10 <211> 194 <212> PRT <213> Artificial Sequence <400> 10 Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg 1 5 10 15 Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val 20 25 30 Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys 35 40 45 Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn 50 55 60 Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile 65 70 75 80 Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro 85 90 95 Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp 100 105 110 Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys 115 120 125 Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln 130 135 140 Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe 145 150 155 160 Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln 165 170 175 Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala 180 185 190 Thr Val <210> 11 <211> 223 <212> PRT <213> Artificial Sequence <400> 11 Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn 1 5 10 15 Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val 20 25 30 Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser 35 40 45 Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val 50 55 60 Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp 65 70 75 80 Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln 85 90 95 Thr Gly Asn Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr 100 105 110 Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly 115 120 125 Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys 130 135 140 Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr 145 150 155 160 Pro Cys Asn Gly Val Lys Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser 165 170 175 Tyr Gly Phe Gln Pro Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val 180 185 190 Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly 195 200 205 Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe 210 215 220 <210> 12 <211> 223 <212> PRT <213> Artificial Sequence <400> 12 Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn 1 5 10 15 Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val 20 25 30 Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser 35 40 45 Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val 50 55 60 Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp 65 70 75 80 Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln 85 90 95 Thr Gly Thr Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr 100 105 110 Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly 115 120 125 Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys 130 135 140 Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr 145 150 155 160 Pro Cys Asn Gly Val Lys Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser 165 170 175 Tyr Gly Phe Gln Pro Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val 180 185 190 Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly 195 200 205 Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe 210 215 220 <210> 13 <211> 223 <212> PRT <213> Artificial Sequence <400> 13 Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn 1 5 10 15 Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val 20 25 30 Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser 35 40 45 Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val 50 55 60 Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp 65 70 75 80 Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln 85 90 95 Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr 100 105 110 Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly 115 120 125 Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys 130 135 140 Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr 145 150 155 160 Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser 165 170 175 Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val 180 185 190 Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly 195 200 205 Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe 210 215 220 <210> 14 <211> 219 <212> PRT <213> Artificial Sequence <400> 14 Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn 1 5 10 15 Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val 20 25 30 Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser 35 40 45 Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val 50 55 60 Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp 65 70 75 80 Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln 85 90 95 Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr 100 105 110 Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly 115 120 125 Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys 130 135 140 Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr 145 150 155 160 Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser 165 170 175 Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val 180 185 190 Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly 195 200 205 Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys 210 215 <210> 15 <211> 248 <212> PRT <213> Artificial Sequence <400> 15 Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys Ser 1 5 10 15 Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val Gln 20 25 30 Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro 35 40 45 Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp 50 55 60 Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr 65 70 75 80 Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr 85 90 95 Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val 100 105 110 Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys 115 120 125 Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val 130 135 140 Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr 145 150 155 160 Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu 165 170 175 Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn 180 185 190 Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe 195 200 205 Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu 210 215 220 Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys 225 230 235 240 Ser Thr Asn Leu Val Lys Asn Lys 245 <210> 16 <211> 274 <212> PRT <213> Artificial Sequence <400> 16 Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn 1 5 10 15 Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val 20 25 30 Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser 35 40 45 Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val 50 55 60 Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp 65 70 75 80 Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln 85 90 95 Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr 100 105 110 Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly 115 120 125 Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys 130 135 140 Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr 145 150 155 160 Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser 165 170 175 Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val 180 185 190 Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly 195 200 205 Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn 210 215 220 Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys 225 230 235 240 Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp 245 250 255 Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys 260 265 270 Ser Phe <210> 17 <211> 303 <212> PRT <213> Artificial Sequence <400> 17 Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys Ser 1 5 10 15 Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val Gln 20 25 30 Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro 35 40 45 Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp 50 55 60 Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr 65 70 75 80 Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr 85 90 95 Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val 100 105 110 Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys 115 120 125 Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val 130 135 140 Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr 145 150 155 160 Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu 165 170 175 Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn 180 185 190 Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe 195 200 205 Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu 210 215 220 Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys 225 230 235 240 Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn Gly 245 250 255 Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu Pro 260 265 270 Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val Arg 275 280 285 Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe 290 295 300 <210> 18 <211> 219 <212> PRT <213> Artificial Sequence <400> 18 Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn 1 5 10 15 Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val 20 25 30 Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser 35 40 45 Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val 50 55 60 Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp 65 70 75 80 Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln 85 90 95 Thr Gly Asn Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr 100 105 110 Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly 115 120 125 Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys 130 135 140 Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr 145 150 155 160 Pro Cys Asn Gly Val Lys Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser 165 170 175 Tyr Gly Phe Gln Pro Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val 180 185 190 Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly 195 200 205 Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys 210 215 <210> 19 <211> 248 <212> PRT <213> Artificial Sequence <400> 19 Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys Ser 1 5 10 15 Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val Gln 20 25 30 Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro 35 40 45 Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp 50 55 60 Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr 65 70 75 80 Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr 85 90 95 Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val 100 105 110 Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Asn 115 120 125 Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val 130 135 140 Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr 145 150 155 160 Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu 165 170 175 Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn 180 185 190 Gly Val Lys Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe 195 200 205 Gln Pro Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu 210 215 220 Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys 225 230 235 240 Ser Thr Asn Leu Val Lys Asn Lys 245 <210> 20 <211> 274 <212> PRT <213> Artificial Sequence <400> 20 Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn 1 5 10 15 Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val 20 25 30 Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser 35 40 45 Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val 50 55 60 Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp 65 70 75 80 Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln 85 90 95 Thr Gly Asn Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr 100 105 110 Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly 115 120 125 Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys 130 135 140 Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr 145 150 155 160 Pro Cys Asn Gly Val Lys Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser 165 170 175 Tyr Gly Phe Gln Pro Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val 180 185 190 Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly 195 200 205 Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn 210 215 220 Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys 225 230 235 240 Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp 245 250 255 Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys 260 265 270 Ser Phe <210> 21 <211> 303 <212> PRT <213> Artificial Sequence <400> 21 Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys Ser 1 5 10 15 Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val Gln 20 25 30 Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro 35 40 45 Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp 50 55 60 Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr 65 70 75 80 Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr 85 90 95 Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val 100 105 110 Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Asn 115 120 125 Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val 130 135 140 Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr 145 150 155 160 Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu 165 170 175 Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn 180 185 190 Gly Val Lys Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe 195 200 205 Gln Pro Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu 210 215 220 Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys 225 230 235 240 Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn Gly 245 250 255 Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu Pro 260 265 270 Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val Arg 275 280 285 Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe 290 295 300 <210> 22 <211> 219 <212> PRT <213> Artificial Sequence <400> 22 Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn 1 5 10 15 Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val 20 25 30 Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser 35 40 45 Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val 50 55 60 Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp 65 70 75 80 Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln 85 90 95 Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr 100 105 110 Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly 115 120 125 Gly Asn Tyr Asn Tyr Arg Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys 130 135 140 Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Lys 145 150 155 160 Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser 165 170 175 Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val 180 185 190 Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly 195 200 205 Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys 210 215 <210> 23 <211> 248 <212> PRT <213> Artificial Sequence <400> 23 Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys Ser 1 5 10 15 Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val Gln 20 25 30 Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro 35 40 45 Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp 50 55 60 Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr 65 70 75 80 Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr 85 90 95 Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val 100 105 110 Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys 115 120 125 Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val 130 135 140 Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr 145 150 155 160 Asn Tyr Arg Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu 165 170 175 Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Lys Pro Cys Asn 180 185 190 Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe 195 200 205 Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu 210 215 220 Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys 225 230 235 240 Ser Thr Asn Leu Val Lys Asn Lys 245 <210> 24 <211> 274 <212> PRT <213> Artificial Sequence <400> 24 Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn 1 5 10 15 Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val 20 25 30 Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser 35 40 45 Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val 50 55 60 Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp 65 70 75 80 Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln 85 90 95 Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr 100 105 110 Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly 115 120 125 Gly Asn Tyr Asn Tyr Arg Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys 130 135 140 Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Lys 145 150 155 160 Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser 165 170 175 Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val 180 185 190 Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly 195 200 205 Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn 210 215 220 Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys 225 230 235 240 Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp 245 250 255 Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys 260 265 270 Ser Phe <210> 25 <211> 303 <212> PRT <213> Artificial Sequence <400> 25 Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys Ser 1 5 10 15 Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val Gln 20 25 30 Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro 35 40 45 Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp 50 55 60 Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr 65 70 75 80 Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr 85 90 95 Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val 100 105 110 Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys 115 120 125 Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val 130 135 140 Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr 145 150 155 160 Asn Tyr Arg Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu 165 170 175 Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Lys Pro Cys Asn 180 185 190 Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe 195 200 205 Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu 210 215 220 Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys 225 230 235 240 Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn Gly 245 250 255 Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu Pro 260 265 270 Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val Arg 275 280 285 Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe 290 295 300 <210> 26 <211> 27 <212> PRT <213> Artificial Sequence <400> 26 Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys Asp Gly Glu 1 5 10 15 Trp Val Leu Leu Ser Thr Phe Leu Ser Pro Ala 20 25 <210> 27 <211> 27 <212> PRT <213> Artificial Sequence <400> 27 Ala Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg 1 5 10 15 Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe 20 25 <210> 28 <211> 27 <212> PRT <213> Artificial Sequence <400> 28 Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys Asp 1 5 10 15 Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Gly 20 25 <210> 29 <211> 12 <212> PRT <213> Artificial Sequence <400> 29 Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser 1 5 10 <210> 30 <211> 560 <212> PRT <213> Artificial Sequence <400> 30 Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr 1 5 10 15 Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser 20 25 30 Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn 35 40 45 Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys 50 55 60 Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala 65 70 75 80 Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr 85 90 95 Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn 100 105 110 Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu 115 120 125 Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe 130 135 140 Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln 145 150 155 160 Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu 165 170 175 Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser 180 185 190 Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser 195 200 205 Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg 210 215 220 Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp 225 230 235 240 Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr 245 250 255 Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile 260 265 270 Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys 275 280 285 Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn 290 295 300 Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr 305 310 315 320 Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser 325 330 335 Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr 340 345 350 Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly 355 360 365 Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala 370 375 380 Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly 385 390 395 400 Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe 405 410 415 Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val 420 425 430 Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu 435 440 445 Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser 450 455 460 Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln 465 470 475 480 Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg 485 490 495 Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys 500 505 510 Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe 515 520 525 Gly Ser Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala 530 535 540 Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Gly 545 550 555 560 <210> 31 <211> 557 <212> PRT <213> Artificial Sequence <400> 31 Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr 1 5 10 15 Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser 20 25 30 Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn 35 40 45 Val Thr Trp Phe His Val Ile Ser Gly Thr Asn Gly Thr Lys Arg Phe 50 55 60 Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Ile 65 70 75 80 Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp 85 90 95 Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val 100 105 110 Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Asp His 115 120 125 Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr Ser Ser 130 135 140 Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu Met Asp 145 150 155 160 Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe Val Phe 165 170 175 Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr Pro Ile 180 185 190 Ile Val Arg Glu Pro Glu Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu 195 200 205 Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr 210 215 220 Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser 225 230 235 240 Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro 245 250 255 Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala 260 265 270 Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys 275 280 285 Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val 290 295 300 Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys 305 310 315 320 Pro Phe Asp Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala 325 330 335 Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu 340 345 350 Tyr Asn Leu Ala Pro Phe Phe Thr Phe Lys Cys Tyr Gly Val Ser Pro 355 360 365 Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe 370 375 380 Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly 385 390 395 400 Asn Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys 405 410 415 Val Ile Ala Trp Asn Ser Asn Lys Leu Asp Ser Lys Val Ser Gly Asn 420 425 430 Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe 435 440 445 Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Asn Lys Pro Cys 450 455 460 Asn Gly Val Ala Gly Phe Asn Cys Tyr Phe Pro Leu Arg Ser Tyr Ser 465 470 475 480 Phe Arg Pro Thr Tyr Gly Val Gly His Gln Pro Tyr Arg Val Val Val 485 490 495 Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys 500 505 510 Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Gly Ser Gly 515 520 525 Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg 530 535 540 Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Gly 545 550 555

Claims

1. Use of a fusion protein in the preparation of a SARS-CoV-2 broad-spectrum vaccine, characterized in that, The fusion protein comprises the following parts: the N-terminal domain NTD of the SARS-CoV-2 spike protein or its functional fragment, and the receptor-binding domain RBD of the SARS-CoV-2 spike protein or its functional fragment; The broad-spectrum vaccine provides protection against at least one of the following mutant strains of the WH01 strain: D614G strain, Alpha strain, Beta strain, Gamma strain, Delta strain, Eta strain, Iota strain, Kappa strain, Lambda strain, Miu strain, Omicron strain, and Omicron-BA.2 strain.

2. The application according to claim 1, characterized in that, The mutant strains include Beta strain, Delta strain, Omicron strain, and Omicron-BA.2 strain.

3. The application according to claim 1, characterized in that The NTD is derived from the SARS-CoV-2 wild type or any mutant; the RBD is derived from the SARS-CoV-2 wild type or any mutant.

4. The application according to claim 3, wherein The NTD has an amino acid mutation at at least one of the following sites: L18, T20, P13, P26, D80, D138, R190, R203, D215, G204, L242-244, and R246.

5. The application according to claim 4, characterized in that The NTD comprises at least one of the following amino acid mutations: L18F, T20N, P13L, P26S, D80A, D138Y, R190S, R203M, D215G, G204R, L242-244del, and R246I.

6. The application according to claim 5, characterized in that, The NTD comprises the following amino acid mutations: L18F, D80A, D215G, L242-244del, and R246I.

7. The application according to claim 5, wherein The NTD comprises the following amino acid mutations: L18F, D80A, D215G, L242-244del, and R190S.

8. The application according to claim 5, characterized in that, The NTD comprises the following amino acid mutations: L18F, T20N, P26S, D138Y, and R246I.

9. The application according to claim 4, wherein The amino acid sequence of the NTD is at least 90% identical to any one of SEQ ID NOs. 1-8.

10. The application according to claim 3, characterized in that The RBD has an amino acid mutation at one or more of the following sites: K417, E484, N501, L452, and T478.

11. The application according to claim 10, characterized in that, The RBD comprises at least one of the following amino acid mutations: K417T / N, E484K, N501Y, L452R, and T478K.

12. The application according to claim 11, wherein The RBD comprises the following amino acid mutations: K417T, E484K, N501Y.

13. The application according to claim 11, characterized in that The RBD comprises the following amino acid mutations: K417N, E484K, N501Y.

14. The application according to claim 10, wherein The amino acid sequence of the RBD is at least 90% identical to any one of SEQ ID NOs. 9-25.

15. The application according to claim 1, characterized in that, The fusion protein further comprises at least one of the following polypeptides: P2 or its functionally active fragment, foldon domain or its functionally active fragment, ferritin or its functionally active fragment, and hepatitis B surface antigen HSsAg or its functionally active fragment; At least one of the RBD or its functionally active fragment and the NTD or its active functional fragment is directly or indirectly linked to the polypeptide.

16. The application according to claim 15, characterized in that, The polypeptide is in-frame fused with the NTD or its functionally active fragment and the RBD or its functionally active fragment.

17. The application according to claim 15, wherein The polypeptide is a foldon domain or its functionally active fragment.

18. The application according to claim 17, characterized in that, The foldon domain or its functionally active fragment comprises the amino acid residues at the C-terminus of phage T4 fibritin.

19. The application according to claim 18, characterized in that, The N-terminus of the foldon domain or its functionally active fragment is linked to the C-terminus of the RBD or its active functional fragment, and the N-terminus of the RBD or its functionally active fragment is linked to the C-terminus of the NTD or its functionally active fragment.

20. The application according to claim 17, wherein The amino acid sequence of the foldon domain is at least 90% identical to any one of SEQ ID NOs. 26-28.

21. A SARS-CoV-2 broad-spectrum vaccine, characterized in that, Comprising the fusion protein according to any one of claims 1-20; the broad-spectrum vaccine provides protection against at least one of the following mutant strains: D614G strain, Alpha strain, Beta strain, Gamma strain, Delta strain, Eta strain, Iota strain, Kappa strain, Lambda strain, Miu strain, Omicron strain, and Omicron-BA.2 strain.

22. The broad-spectrum vaccine according to claim 21, wherein, The mutant strains include Delta strain, Omicron strain, and Omicron-BA.2 strain.

23. The broad-spectrum vaccine according to claim 21, wherein The vaccine in a single-dose administration contains 10-40 μg of the fusion protein.

24. The broad-spectrum vaccine according to claim 23, wherein The vaccine in a single-dose administration contains 20 μg of the fusion protein.

25. The broad-spectrum vaccine according to claim 21, wherein It further contains an adjuvant, which comprises at least one of aluminum adjuvant, squalene, Tween, tocopherol, Span, MPL, LPA, CpG, poly(I:C), and QS21.

26. The broad-spectrum vaccine according to claim 25, wherein, The adjuvant comprises the following components: Squalene 42.16 mg / mL, α-tocopherol 47.44 mg / mL, Tween 80 19.44 mg / mL.

27. Use of the broad-spectrum vaccine according to any one of claims 23 to 26 as an immune booster for SARS-CoV-2, characterized in that, The broad-spectrum vaccine is administered 3-6 months after two immunizations with any SARS-CoV-2 vaccine.

28. A SARS-CoV-2 immune set, characterized in that, Comprising a first immunization preparation, a second immunization preparation administered 14-28 days after the first immunization preparation, and a third immunization preparation administered 3-6 months after the second immunization preparation, wherein the first immunization preparation and the second immunization preparation are inactivated vaccines or recombinant vaccines, and the third immunization preparation is the broad-spectrum vaccine according to any one of claims 23-26.

29. The SARS-CoV-2 immune set according to claim 28, characterized in that, Both the first immunization preparation and the second immunization preparation are the broad-spectrum vaccine.

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