CN113880924A - SARS-CoV-2 pseudovirus - Google Patents

SARS-CoV-2 pseudovirus Download PDF

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CN113880924A
CN113880924A CN202010630831.2A CN202010630831A CN113880924A CN 113880924 A CN113880924 A CN 113880924A CN 202010630831 A CN202010630831 A CN 202010630831A CN 113880924 A CN113880924 A CN 113880924A
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王佑春
李倩倩
黄维金
聂建辉
吴佳静
张黎
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National Institutes for Food and Drug Control
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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    • C07—ORGANIC CHEMISTRY
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    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
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    • C07—ORGANIC CHEMISTRY
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    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/23—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a GST-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C07K2319/42—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a HA(hemagglutinin)-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C07K2319/43—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a FLAG-tag
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    • C12N2770/00011—Details
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    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Abstract

The present invention relates to the field of genetic engineering and molecular biology, in particular to truncated Spike proteins or variants thereof, fusion proteins, vectors, host cells and viroid-like particles comprising said proteins. The viroid particles can be used for screening of antibodies against SARS-CoV-2 and drugs (e.g., detecting the neutralizing activity of SARS-CoV-2 antibodies, or screening drugs that inhibit SARS-CoV-2 infection at the cellular level). The invention also relates to a pharmaceutical composition and a kit comprising the protein.

Description

SARS-CoV-2 pseudovirus
Technical Field
The present invention relates to the field of genetic engineering and molecular biology, in particular to truncated Spike proteins or variants thereof, fusion proteins and viroid particles comprising said proteins. The viroid particles can be used for screening of antibodies against SARS-CoV-2 and drugs (e.g., detecting the neutralizing activity of SARS-CoV-2 antibodies, or screening drugs that inhibit SARS-CoV-2 infection at the cellular level). The invention also relates to a preparation method and a kit containing the protein.
Background
Pseudoviruses (pseudoviruses) are infectious viroid particles similar to true viruses formed by wrapping a heterologous nucleic acid carrying a reporter gene by a viral capsid protein or an envelope protein, and the pseudoviruses only have one round of infectivity and are safer to operate because the wrapped nucleic acid does not have the whole nucleic acid sequence for replicating and forming the viruses.
The pseudovirus is mainly formed by transfecting 293T cells together with multiple plasmids, is automatically replicated and expressed in the cells and is obtained by methods such as collecting culture medium supernatant or cracking the cells. The pseudovirus has the same infectivity as the true virus, the process of entering the cell is the same as the true virus, and the pseudovirus does not have the capability of replicating to generate the virus after entering the cell, so the pseudovirus has no harm and can be more safely used for detecting neutralizing antibodies and antiviral drugs.
The virus with strong infectivity and pathogenicity, such as SARS-CoV-2, Ebola virus, rabies virus, H7N9, etc., has great danger for research of true virus, limited quantity and good safety of pseudovirus, and can be prepared in large scale by transfection technology, thus providing convenience for research of the virus with high danger.
The pseudovirus system is characterized by overcoming the defects of the traditional method, ensuring that the detection technology is safer, simpler, more convenient, faster and higher in flux, and ensuring that some viruses which are difficult to culture can also be detected by adopting a culture method.
However, the virus titer is often low in the construction of pseudoviruses which usually utilize viral membrane proteins directly, which limits the application of pseudoviruses. Therefore, there is a need to further increase the virus titer of pseudoviruses.
Disclosure of Invention
The inventors of the present application performed a large number of experiments and repeated investigations to truncate and transform the Spike protein, thereby obtaining SARS-CoV-2 pseudovirus with high titer. Based on this, further modification of vectors was carried out through a large number of experiments to obtain modified vectors and transfection conditions for the preparation of high-titer pseudoviruses, and thus the present invention was completed.
Thus, in a first aspect, the present invention provides a truncated Spike protein or variant thereof, wherein (1) the C-terminus of said truncated Spike protein is truncated by 13-23 amino acids, and/or (2) the amino acids of said truncated Spike protein at the following positions are deleted, as compared to the Spike protein of wild-type SARS-CoV-2: positions corresponding to positions 669 and 672 of SEQ ID NO: 1;
wherein the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions; e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid conservative substitutions) as compared to the truncated Spike protein.
In certain embodiments, the C-terminus of the truncated Spike protein is truncated by 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 amino acids compared to the Spike protein of wild-type SARS-CoV-2.
In certain embodiments, the truncated Spike protein also contains deletions compared to the Spike protein of wild SARS-CoV-2, i.e., the amino acids at the following positions are deleted: positions corresponding to positions 669 and 672 of SEQ ID NO: 1.
In certain embodiments, the amino acid that is deleted is a furin cleavage site.
In certain embodiments, the amino acid that is deleted is PRRA.
In certain embodiments, the truncated Spike protein has amino acids deleted at the following positions compared to the Spike protein of wild SARS-CoV-2: positions corresponding to positions 669 and 672 of SEQ ID NO: 1.
In certain embodiments, the C-terminus of the truncated Spike protein is also truncated by 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 amino acids compared to the Spike protein of wild-type SARS-CoV-2.
In certain embodiments, the amino acid that is deleted is a furin cleavage site.
In certain embodiments, the amino acid that is deleted is PRRA.
In certain embodiments, the truncated Spike protein or variant thereof has an amino acid sequence selected from the group consisting of:
(1)SEQ ID NO:2-SEQ ID NO:23；
(2) an amino acid sequence having one or more amino acid substitutions, deletions or additions (e.g.1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions; e.g.1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid conservative substitutions) compared to the sequence set forth in any of SEQ ID NO 2-23;
(3) an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a sequence as set forth in any one of SEQ ID No. 2-SEQ ID No. 23.
In the present invention, the above variants substantially retain the biological function of the protein from which they are derived. In the present invention, the biological function mainly refers to the biological function of the Spike protein of SARS-CoV-2, including, but not limited to, the activity of binding to the cell surface receptor (ACE2), the ability to undergo conformational changes (e.g., coil folding to form hairpin-like structures), self-assembly into viroid particles, and the like.
In a second aspect, the invention provides a fusion protein comprising a truncated Spike protein as described above or a variant thereof and a further polypeptide.
In certain embodiments, the additional polypeptide is linked to the N-terminus or C-terminus of the protein, optionally through a linker. Such linkers are well known in the art and examples include, but are not limited to, those comprising aA peptide linker of one or more (e.g., 1, 2, 3, 4, or 5) amino acids (e.g., Glu or Ser). In certain embodiments, the peptide linker is flexible. In certain embodiments, a flexible peptide linker may be advantageous, which is capable of linking two protein/polypeptide components and maintaining their respective activities and functions. Such peptide linkers include, but are not limited to, (GGGGS)3。
In certain embodiments, the additional polypeptide is selected from a tag, a signal peptide or a leader, a detectable label (e.g., luciferase (fluc), Green Fluorescent Protein (GFP)), or any combination thereof. In certain embodiments, a protein of the invention or a variant thereof may be linked to a tag sequence to facilitate expression, detection and/or purification of the protein of the invention. In certain embodiments, a protein of the invention or a variant thereof may be linked to a signal peptide or leader sequence to direct secretion of the protein of the invention. In certain embodiments, a protein of the invention or a variant thereof may be linked to a detectable marker sequence to facilitate detection or tracking of the protein of the invention.
In certain embodiments, the signal peptide is selected from the group consisting of Spike protein, CD5, CD14, IgG heavy chain, Immunoglobulin light chain (Immunoglobulin light chain), tissue plasminogen activator (tPA), SCGB1D1, Serum albumin preproprotein (Serum albumin preproprotein), Gluc, IL-2, and the native signal peptide of IFN α 2.
In certain embodiments, the fusion proteins of the invention comprise tags, such tags being well known to those skilled in the art, including but not limited to histidine tag (His), glutathione mercaptotransferase tag (GST), influenza virus Hemagglutinin (HA), Flag-tag. The skilled person knows how to select a suitable tag according to the desired purpose (e.g. expression, purification or detection of a protein).
In certain embodiments, the signal peptide has an amino acid sequence selected from the group consisting of: 24-33 of SEQ ID NO.
In certain embodiments, the tag has the amino acid sequence shown as SEQ ID NO 35.
The above-described fusion protein/additional polypeptide (e.g., tag or signal peptide) does not adversely affect the desired activity of the protein of the invention (e.g., activity of binding to cell surface receptor (ACE2), is capable of undergoing conformational changes (e.g., coil folding to form hairpin-like structures), self-assembly into viroid particles).
In a third aspect, the invention provides an isolated nucleic acid molecule encoding a truncated Spike protein or variant thereof as described above, or encoding a fusion protein as described above.
In a fourth aspect, the present invention provides a vector comprising a nucleic acid molecule as described above. The vector of the present invention may be a cloning vector or an expression vector. In certain embodiments, the vectors of the invention are, for example, plasmids, cosmids, phages, cosmids, and the like. In certain embodiments, the vector is capable of expressing a protein of the invention or a fusion protein as described above in a host cell (e.g., a mammalian cell, e.g., a human cell).
In certain embodiments, the vector is a eukaryotic expression plasmid comprising a nucleic acid molecule as described above.
In certain embodiments, the eukaryotic expression plasmid is selected from pcdna3.1+, pcag3.1, and pcmv3.1.
In a fifth aspect, the invention provides a host cell comprising a nucleic acid molecule as described above or a vector as described above.
In certain embodiments, the cell is a mammalian cell.
In certain embodiments, the mammalian cell is a human cell, e.g., a hematopoietic cell, an epithelial cell, a liver cell, a tumor cell, a neural cell.
In certain embodiments, the cell is a cell that expresses or overexpresses the human ACE2 protein (e.g., 293T-ACE 2).
In certain embodiments, the host cell further comprises a packaging plasmid.
In a sixth aspect, the present invention provides a viroid-like particle comprising a truncated Spike protein or variant thereof as described above or a fusion protein as described above; alternatively, the viroid consists of a truncated Spike protein as described above or a variant thereof or a fusion protein as described above.
In the present invention, the truncated Spike protein or variant thereof or the fusion protein self-assembles to form the outer shell of the viroid. In certain embodiments, the viroid further comprises a nucleic acid component. Such nucleic acid components are, for example, encapsulated within a shell formed by the self-assembly of the truncated Spike protein or variant thereof or the fusion protein. In certain embodiments, the nucleic acid component is selected from any one or more of:
(a) exogenous DNA, or exogenous DNA encoding a foreign protein or polypeptide, or exogenous DNA encoding RNA;
(b) a vector optionally comprising the exogenous DNA described in (a);
(c) exogenous RNA, or exogenous RNA encoding an exogenous protein or polypeptide;
(d) a vector, optionally comprising the exogenous RNA described in (c).
In the present invention, the nucleic acid component may be double-stranded, single-stranded, or any combination thereof. In the present invention, the nucleic acid component may be DNA, RNA, or any combination thereof.
In certain embodiments, the nucleic acid component is co-transfected into a host cell with a vector as described above and self-assembled, and the host cell expresses a protein that encapsulates the nucleic acid, forming a viroid.
In certain embodiments, the nucleic acid component is a packaging plasmid.
In a seventh aspect, the present invention provides a method of producing a SARS-CoV-2 pseudovirus, the method comprising expressing in a suitable host cell a truncated Spike protein as described above or a variant thereof or a fusion protein as described above. In certain embodiments, a vector as described above is transferred into a suitable host cell and expresses a truncated Spike protein or variant thereof as described above or a fusion protein as described above. In certain embodiments, the vector and the packaging plasmid, as described above, are co-transformed into a host cell.
In certain embodiments, the vectors and packaging plasmids described above are transfected into a host cell.
In certain embodiments, the mass ratio of vector used in the transfection process and the packaging plasmid is 1-3:1-4, e.g., 3: 1. 2: 1. 1: 1. 1: 2. 1: 3 or 1: 4. in certain embodiments, the mass ratio of vector used in the transfection process and the packaging plasmid is 1: 3.
in certain embodiments, the transfection reagent used in the transfection process is selected from the group consisting of: lipofectamine2000, lipofectamine3000, PEI, or any combination thereof.
In certain embodiments, the host cell is a human cell, e.g., a hematopoietic cell, an epithelial cell, a liver cell, a tumor cell, a neural cell.
In certain embodiments, the viroid-infected cell is a cell that overexpresses human ACE2 protein (e.g., 293T-ACE 2).
In an eighth aspect, the present invention provides a kit comprising one or more components selected from the group consisting of: the truncated Spike protein or variant thereof as described above, the fusion protein as described above, the isolated nucleic acid molecule as described above, the vector as described above, the host cell as described above, the viroid as described above.
In certain embodiments, the kit further comprises a packaging plasmid.
In certain embodiments, the kit further comprises a transfection reagent.
In certain embodiments, the transfection reagent is selected from the group consisting of: lipofectamine2000, lipofectamine3000, PEI, or any combination thereof.
In certain embodiments, the kits of the invention further comprise instructions for use. Various optimization schemes are discussed herein to increase the titer of pseudoviruses produced. For example, selection of packaging plasmid, ratio of expression plasmid to packaging plasmid, selection of transfection reagent, ratio of transfection reagent to plasmid, pseudovirus harvest time, and the like. The above protocol may be included in the instructions for use of the kit of the invention.
In a ninth aspect, the invention provides a method of detecting SARS-CoV-2 antibody neutralizing activity, the method comprising contacting a viroid particle as described above with a host cell before, simultaneously with or after contacting the viroid particle with the antibody or host cell.
Compared with a direct ELISA method and a single epitope competition ELISA method, the pseudovirus method can reflect the neutralizing capacity of the antibody better, can simulate the biological process that the antibody blocks the virus from entering cells, and can directly reflect the protective effect of the antibody. The methods generally involve contacting the test antibody with a host cell or viroid prior to contacting the pseudovirus with the host cell (i.e., prior to infecting the host cell with the pseudovirus), and then testing for the ability of the pseudovirus to infect the host cell. If the infection activity of the pseudovirus is reduced, the antibody has an inhibiting effect on pseudovirus infected cells and has a protective effect on the cells.
In a tenth aspect, the present invention provides a method of screening for a drug candidate capable of inhibiting SARS-CoV-2 infection of a cell, the method comprising contacting a viroid particle or a host cell as described above with the drug candidate before, simultaneously with or after contacting the viroid particle with the host cell.
In certain embodiments, the methods generally involve contacting the candidate agent to be tested with the host cell or viroid prior to contacting the pseudovirus with the host cell (i.e., prior to infecting the host cell with the pseudovirus), and then testing for the ability of the pseudovirus to infect the host cell. If the infection activity of the pseudovirus is reduced, the candidate drug has an inhibition effect on pseudovirus infected cells and has a protective effect on the cells.
In an eleventh aspect, the present invention provides the use of a truncated Spike protein or variant thereof as described above, a fusion protein as described above, an isolated nucleic acid molecule as described above, a vector as described above, a host cell as described above, a viroid particle as described above for detecting or screening an anti-SARS-CoV-2 antibody or a medicament.
Definition of terms
In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Also, the procedures of molecular genetics, nucleic acid chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics, and recombinant DNA, etc., used herein, are all conventional procedures widely used in the corresponding field. Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.
As used herein, the term "SARS-CoV-2" is an abbreviation for "Severe acute respiratory syndrome coronavirus 2(Severe acute respiratory syndrome coronavirus 2)" which is old called "novel coronavirus" or "2019-nCov", which belongs to the genus β -coronavirus, being an envelope-containing single-stranded positive-sense RNA virus. The genomic sequence of SARS-CoV-2 is known to those skilled in the art and can be found, for example, in GenBank: MN 908947. SARS-CoV-2 contains at least three membrane proteins, including surface spike protein (S), integral membrane protein (M) and membrane protein (E). Like SARS-CoV, the SARS-CoV-2 Receptor is formed by specific binding of Receptor Binding Domain (RBD) on S protein and angiotensin converting enzyme 2(ACE2) on host cell, then the virus undergoes membrane fusion and cell entry, and S protein plays a crucial role in the process of virus infection of cells.
As used herein, the terms "surface Spike protein", "Spike protein" and "S protein" refer to a membrane protein of SARS-CoV-2, which have the same meaning and are used interchangeably.
As used herein, the terms "novel coronavirus pneumonia" and "COVID-19" refer to pneumonia resulting from SARS-CoV-2 infection, which have the same meaning and are used interchangeably.
As used herein, the term "SARS-CoV" or "SARS-CoV-1" is short for "severe acute respiratory syndrome coronavirus 1(Severe acute respiratory syndrome coronavirus 1)" belonging to the genus β -coronavirus, which is an envelope-containing single-stranded positive-sense RNA virus. The genomic sequence of SARS-CoV-1 is known to those skilled in the art and can be found, for example, in GenBank: AAP 13567.1. The pneumonia caused by SARS-CoV-1 is called SARS.
As used herein, the terms "wild" or "native" are used interchangeably. When these terms are used to describe a nucleic acid molecule, polypeptide or protein, they mean that the nucleic acid molecule, polypeptide or protein exists in nature, is found in nature, and has not been artificially modified or processed in any way. As used herein, the Spike protein of SARS-CoV-2, which is wild, refers to a naturally occurring, biologically active Spike protein. The amino acid sequence of Spike protein can be readily obtained by those skilled in the art from various public databases (e.g., GenBank database). For example, the amino acid sequence of the Spike protein of wild SARS-CoV-2 can be as shown in SEQ ID NO:1 is shown.
As used herein, the term "C-terminally truncated by X amino acids" means that the most terminal consecutive X amino acids of the C-terminus are truncated.
As used herein, the term "corresponding position" refers to an amino acid position at an equivalent position in two sequences that are compared when the two sequences are optimally aligned, i.e., when the two sequences are aligned for the highest percent identity. For example, the expression "position corresponding to position 669 + 672 of SEQ ID NO: 1" means the amino acid position in the sequence which is at the equivalent position to position 669 + 672 of SEQ ID NO:1, which is compared when a sequence is optimally aligned with SEQ ID NO:1, i.e. when a sequence is aligned with SEQ ID NO:1 to obtain the highest percentage identity.
As used herein, the terms "pseudovirus" and "viroid" have the same meaning and are used interchangeably; it is meant that a virus-like particle, formed by self-assembly of viral proteins, does not encapsulate nucleic acids or encapsulates other nucleic acids, such that the pseudovirus or viroid, while able to infect a host cell, is not capable of autonomous replication. Therefore, it is highly biologically safe compared to the true virus. Pseudoviruses are typically packaged in two parts, a packaging component and an expression component. The packaging component is constructed from the viral (e.g., HIV-1) genome with the genetic information required for packaging, reverse transcription, and integration removed, providing the proteins necessary for pseudovirions; the expression component is complementary to the packaging component, contains the genetic information required for packaging, reverse transcription and integration, and also contains the exogenous gene of interest. The pseudovirions can be harvested in the cell supernatant by co-transfecting the host cells with the packaging component and the vector component.
As used herein, the terms "backbone plasmid" and "packaging plasmid" have the same meaning and are used interchangeably. As is generally understood by those skilled in the art, a viral vector system (particularly a lentiviral vector system) may be composed of two parts, i.e., a packaging component (e.g., a packaging plasmid or backbone plasmid) and a vector component (e.g., a recombinant expression vector carrying a gene of interest); among other things, the packaging components (e.g., packaging plasmid or backbone plasmid) can provide all of the helper proteins necessary for transcription and packaging of genetic material (e.g., RNA) into recombinant pseudoviral particles. Thus, high titer pseudovirions can be produced by: cells are co-transfected with the recombinant expression vector and the packaging plasmid, followed by packaging of the pseudovirus in the cells, followed by secretion of the packaged pseudovirus particles into extracellular medium. Such packaging or backbone plasmids are well known to those skilled in the art, for example, backbone plasmids constructed on the basis of HIV-1: including, but not limited to, pSG3.Δ env (Wei X et al, Antibody neutralization and escape by HIV-1.Nature 422:307-312,2003) and NL4-3.fluc.R-. E- (Connor RI, et al, Vpr is required for the implementation of human immunological virus type-1in monoclonal medicines.virology 206: 935-944, 1995), as well as pSG3.Δ env.fluc and pSG3.Δ env.vcfluc constructed in the examples of this application.
As used herein, the term "identity" is used to refer to the match of sequences between two polypeptides or between two nucleic acids. When a position in both of the sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 of 10 positions of two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 of the total 6 positions match). Typically, the comparison is made when the two sequences are aligned to yield maximum identity. Such alignments can be performed by using, for example, Needleman et al (1970) j.mol.biol.48: 443-453. The algorithm of E.Meyers and W.Miller (Compout.appl biosci., 4:11-17(1988)) which has been incorporated into the ALIGN program (version 2.0) can also be used to determine percent identity between two amino acid sequences using a PAM120 weight residue table (weight residue table), a gap length penalty of 12, and a gap penalty of 4. Furthermore, percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J MoI biol.48: 444-.
As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the biological activity of a protein/polypeptide comprising the amino acid sequence. For example, conservative substitutions may be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include those in which an amino acid residue is replaced with an amino acid residue having a similar side chain, e.g., a substitution with a residue that is physically or functionally similar to the corresponding amino acid residue (e.g., of similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine tryptophan, histidine). Thus, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative substitutions of amino acids are well known in the art (see, e.g., Brummell et al, biochem.32:1180-1187 (1993); Kobayashi et al Protein Eng.12(10):879-884 (1999); and Burks et al, Proc. Natl Acad. set USA94:412-417(1997), which are incorporated herein by reference).
As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction, or transfection, and the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes such as Yeast Artificial Chromosomes (YACs), Bacterial Artificial Chromosomes (BACs), or artificial chromosomes (PACs) derived from P1; bacteriophage such as lambda phage or M13 phage, animal virus, etc. Animal viruses that may be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, papilloma polyoma vacuolatum viruses (e.g., SV 40). A vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may contain a replication initiation site.
As used herein, the term "host cell" refers to a cell that can be used for introducing a vector, and includes, but is not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblast, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.
One skilled in the art will appreciate that the design of an expression vector may depend on factors such as the choice of host cell to be transformed, the level of expression desired, and the like. A vector may be introduced into a host cell to produce transcripts, proteins, or peptides therefrom, including by proteins, fusion proteins, isolated nucleic acid molecules, and the like, as described herein.
As used herein, the term "effective amount" refers to an amount effective to achieve the intended purpose. For example, a prophylactically or therapeutically effective amount of a disease (e.g., rotavirus infection) is an amount effective to prevent, prevent or delay the onset of a disease (e.g., rotavirus infection), or to alleviate, reduce or treat the severity of an existing disease (e.g., a disease caused by rotavirus infection). It is within the ability of those skilled in the art to determine such an effective amount. For example, an amount effective for therapeutic use will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient, e.g., age, weight and sex, the mode of administration of the drug, and other treatments administered concurrently, and the like.
As used herein, the term "neutralizing activity" means that the antibody or antibody fragment has a functional activity of binding to an antigenic protein on the virus, thereby preventing the virus from infecting cells and/or maturation of viral progeny and/or release of viral progeny, and the antibody or antibody fragment having neutralizing activity can prevent amplification of the virus, thereby inhibiting or eliminating infection by the virus.
Advantageous effects of the invention
Compared with the prior art, the pseudovirus titer of SARS-CoV-2 prepared by the application is improved by at least 10 times, and the titer is improved by 826 times at most. The development of pseudoviruses of the present application makes the study of SARS-CoV-2 not restricted to laboratories with higher biological safety conditions. In addition, the pseudovirus can be applied to the detection of the neutralizing capacity of the antibody, and provides a good technical support means for SARS-CoV-2 vaccine, drug evaluation and the research of virus pathogenic mechanism.
Embodiments of the present invention will be described in detail below with reference to the drawings and examples, but those skilled in the art will understand that the following drawings and examples are only for illustrating the present invention and do not limit the scope of the present invention. Various objects and advantageous aspects of the present invention will become apparent to those skilled in the art from the accompanying drawings and the following detailed description of the preferred embodiments.
Drawings
FIG. 1 shows fluorescence values (RLU) of pseudoviruses prepared from 22 truncated Spike proteins in example 1.
FIG. 2 shows fluorescence values (RLU) of pseudoviruses prepared by fusing spike proteins of 10 signal peptides.
FIG. 3 shows fluorescence values (RLU) of pseudoviruses prepared from the constructed 3 vectors.
FIG. 4 shows fluorescence values (RLU) of pseudoviruses prepared from 4 vectors constructed by engineering.
FIG. 5 shows fluorescence values (RLU) of pseudoviruses prepared from 3 backbone plasmids.
FIG. 6 shows fluorescence values (RLU) of pseudoviruses prepared using 3 different transfection reagents.
FIG. 7 shows fluorescence values (RLU) of pseudoviruses prepared with different mass ratios of recombinant vector to backbone plasmid.
Sequence information
Information on the partial sequences to which the present invention relates is provided in table 1 below.
Table 1: description of the sequences
Figure BDA0002568590850000141
Figure BDA0002568590850000151
Figure BDA0002568590850000161
Figure BDA0002568590850000171
Figure BDA0002568590850000181
Figure BDA0002568590850000191
Figure BDA0002568590850000201
Figure BDA0002568590850000211
Figure BDA0002568590850000221
Figure BDA0002568590850000231
Figure BDA0002568590850000241
Figure BDA0002568590850000251
Figure BDA0002568590850000261
Detailed Description
The invention will now be described with reference to the following examples, which are intended to illustrate the invention, but not to limit it.
Unless otherwise indicated, the experiments and procedures described in the examples were performed essentially according to conventional methods well known in the art and described in various references. For example, conventional techniques in immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA used in the present invention can be found in Sambrook (Sambrook), friesch (Fritsch), and manitis (manitis), molecular cloning: a LABORATORY Manual (Molecular CLONING: A Laboratory Manual), 2 nd edition (1989); a Current Manual of MOLECULAR BIOLOGY experiments (Current PROTOCOLS IN MOLECULAR BIOLOGY BIOLOGY) (edited by F.M. Otsubel et al, (1987)); METHODS IN ENZYMOLOGY (METHODS IN Enzymology) series (academic Press): PCR 2: practical methods (PCR 2: A PRACTICAL APPROACH) (M.J. Mefferson, B.D. Hemsh (B.D. Hames) and G.R. Taylor (edited by G.R. Taylor) (1995)), and animal cell CULTURE (ANIMAL CELL CURTURE) (edited by R.I. Fresherni (R.I. Freshney) (1987)).
In addition, those whose specific conditions are not specified in the examples are conducted under the conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available. The examples are given by way of illustration and are not intended to limit the scope of the invention as claimed. All publications and other references mentioned herein are incorporated by reference in their entirety.
Example 1 construction of recombinant vector pcDNA3.1-Spike
The nucleotide sequence encoding the SARS-CoV-2Spike protein (SEQ ID NO:1) and the nucleotide sequence encoding the natural signal peptide (SEQ ID NO:34) were ligated to pcDNA3.1+ plasmid according to the BamH1 and Xho1 cleavage sites on plasmid pcDNA3.1+ (purchased from Invitrogen, cat # V79020) to construct a recombinant vector pcDNA3.1-Spike (Genbank: MT 613044).
The specific process is as follows: the plasmid pcDNA3.1+ after double digestion is recovered, and is mixed with the target fragment evenly, added with Infusion enzyme, and placed on a PCR instrument to be connected for 15 minutes at 50 ℃. The ligation product was transferred to 50. mu.l DH 5. alpha. competent cells and kept on ice for 30 min. After taking out the competent cells, the cells were heat-shocked in a water bath at 42 ℃ for 45-60s and rapidly placed on ice for 2 min. Adding 500 mul of non-resistant LB culture medium, placing in a shaker at 37 ℃, and carrying out shaking culture at 120rpm for 1 hour; centrifuging the cultured competent cells at 2800rpm for 2min, discarding 500. mu.l of supernatant, mixing the remaining thallus, and smearing on a plate with ampicillin resistance (Amp); culturing overnight (14-16 hours) in an incubator at 37 ℃ in an inverted manner; monoclonal colonies were picked, inoculated into 5mL LB medium (100. mu.g/mL Amp), placed in a shaker at 37 ℃ for 14-16 hours with shaking at 220rpm, stored in glycerol, and sent to the sequencer for identification of the resulting expression plasmid.
Example 2 engineering of Spike proteins and titer testing
Engineering of Spike proteins
A key site difference between the Spike protein of SARS-CoV-2 and the Spike protein of SARS-CoV-1 is that four amino acids, PRRA, are added at the cleavage sites of S1 and S2. The addition of these four amino acids forms a new Furin enzyme cleavage site corresponding to amino acids 669-672 of SEQ ID NO: 1. Therefore, this example modifies Spike protein of SARS-CoV-2 and constructs recombinant vector containing modified Spike protein, which is as follows:
(1) truncating the C-terminal intracellular region of the Spike protein by 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 and 23 amino acids respectively, wherein the corresponding amino acid sequence of the truncated Spike protein is shown as SEQ ID NO. 2-12, and constructing recombinant vectors according to the steps described in example 1, wherein the recombinant vectors are named as pcDNA3.1-Spike13 to pcDNA3.1-Spike 23;
(2) deleting the enzyme cutting site of Furin enzyme of Spike protein, namely deleting the amino acid (namely PRRA) corresponding to 669-672 th site of SEQ ID NO. 1, wherein the amino acid sequence corresponding to the modified Spike protein is shown as SEQ ID NO. 13, and the correspondingly constructed recombinant vector is named pcDNA3.1-Furin;
(3) the C-terminal intracellular domain of the Spike protein is respectively truncated by 13, 14, 15, 16, 17, 19, 20, 21, 22 and 23 amino acids, on the basis, the Furin enzyme digestion site is deleted, namely the amino acid corresponding to the 669-672 th site of the SEQ ID NO. 1 is deleted (namely PRRA), and the amino acid sequence corresponding to the modified Spike protein is shown as the SEQ ID NO. 14-23. The recombinant vectors constructed accordingly were named pcDNA3.1-Spike13-furin to pcDNA3.1-Spike23-furin, respectively.
The specific process is as follows: combining the principle of circular PCR, and using the sequence of the vector pcDNA3.1-Spike (Genebank: MT613044) as a template to perform PCR sequence amplification; because the PCR product has template strand, in order to reduce the interference and improve the success rate of the subsequent experiment, Dpn I restriction endonuclease is used to remove the template strand and stay overnight at 37 ℃ (the principle is that the template DNA sequence comes from conventional escherichia coli, is subjected to dam methylation modification and is sensitive to Dpn I endonuclease, so that the DNA sequence can be cut up, and the DNA amplified in vitro is not subjected to methylation, so that the DNA sequence cannot be recognized by the Dpn I endonuclease). The PCR product was constructed into pcDNA3.1+ plasmid, transformed into DH 5. alpha. competent cells, plated and cultured as described in example 1. Approximately 5 monoclonal colonies were picked from each plate and transferred to 5mL LB liquid medium containing ampicillin, respectively, and cultured with shaking at 220rpm in a shaker at 37 ℃ for about 16 hours. And carrying out small-amount extraction on the plasmid of the bacterial liquid, carrying out agarose gel electrophoresis on the small-amount extracted plasmid, and carrying out primary identification according to the size of a band. And (5) sending the band with the correct primary identification result to sequencing, further determining whether mutation is successful, and carrying out the next pseudovirus construction.
2. Titre test
The same amount of 22 recombinant vectors constructed as described above (with vector pcDNA3.1-Spike as a control) were co-transfected with the backbone plasmid pSG3. delta. env. cmvFluc (which was the laboratory construct containing the luciferase gene (fluc) carrying the CMV promoter), the plasmid-related information and sequences disclosed in patent CN201510293955.5) into 293T cells according to the instructions of Lipofectamine3000 transfection reagent. Supernatants were harvested at approximately 48h, and the pseudoviruses collected were infected into 293T-hACE2 cells (purchased from Okayama), discarded after 48h, and chemiluminescence values (RLU) were detected and calculated using a Britelite plus reporter gene assay system (Perkinelmer, cat. No.6066769) luciferase assay kit.
The results are shown in FIG. 1. After 13-23 amino acids are truncated in the C-terminal intracellular region of the Spike protein, the virus titer is improved by about 10 times. After the furin enzyme cutting site is deleted, the virus titer is improved by about 40 times. After the two are deleted at the same time, the virus titer is further improved. Particularly, after the furin enzyme cutting site is deleted and 21 amino acids in the C-terminal intracellular region of the truncated Spike protein are simultaneously cut, the chemiluminescence value (RLU) reaches the highest value, and is calculated to be improved by 826 times compared with the control.
Example 3 engineering of recombinant vectors and titer testing
1. Replacement of Signal peptide
To investigate the effect of the substitution of different signal peptides on the titer of pseudoviruses, 10 signal peptides (i.e., signal peptides of CD5, CD14, IgGheavy chain, immunoglubulin light chain, tPA, SCGB1D1, Serum albubmin preproprotein, Gluc, IL-2, IFN. alpha.2) were substituted for the signal peptide of the SARS-CoV-2Spike protein itself (SEQ ID NO:34), and the amino acid sequences corresponding to the 10 signal peptides were shown in SEQ ID NO:24-33, respectively. And (3) respectively constructing modified vectors by referring to the experimental steps, packaging the modified vectors with pseudoviruses according to the steps, and testing the virus titer.
As shown in FIG. 2, the titer of the pseudovirus constructed from the vector containing the signal peptide CD5 and the Immunoglobulin light chain was significantly increased, and the titer was increased 3-5 times, as compared with the control vector pcDNA3.1-Spike (2019-nCoV).
2. Expression vector
In order to investigate the effect of different expression vectors on viral titer, 4 different vectors were constructed based on 3 plasmids, and the constructed vectors were pseudovirally packaged to test viral titer by referring to the above experimental procedure. The plasmids used were as follows: (1) pCAG3.1 plasmid, the specific information of which is disclosed in patent CN 201510293955.5; (2) pCMV3.1 plasmid, the specific information of which is disclosed in patent CN 201510293955.5; (3) pcDNA3.1 +. The nucleotide sequence encoding the Spike protein (SEQ ID NO:1) and the nucleotide sequence encoding the signal peptide thereof (SEQ ID NO:34) were inserted into the multiple cloning site of the above plasmid, and the following vectors were constructed: (1) pCAG3.1-Sipke vector, which expresses Sipke protein shown as SEQ ID NO. 1 and signal peptide shown as SEQ ID NO. 34; (2) a pCAG-Sipke-tag vector which expresses Sipke protein shown as SEQ ID NO. 1 and a signal peptide shown as SEQ ID NO. 34, and further expresses a tag shown as SEQ ID NO. 35 at the C-terminal of the Sipke protein; (3) pCMV3.1-Spike vector, which expresses Sipke protein shown as SEQ ID NO. 1 and signal peptide shown as SEQ ID NO. 34; (4) pcDNA3.1-Spike vector, which expresses Sipke protein shown as SEQ ID NO. 1 and signal peptide shown as SEQ ID NO. 34.
As shown in FIG. 3, the virus titer was significantly increased in the pseudovirus prepared using the pCAG-Sipke-tag vector.
3. Carrier engineering
The above experimental results were combined, and the vector was further modified based on 3 plasmids. The plasmids used were as follows: (1) pCAG3.1 plasmid; (2) pCMV3.1 plasmid; (3) pcDNA3.1 +. The following vectors are constructed by respectively inserting Sipke protein or modified Sipke protein into the multiple cloning sites of the plasmids: (1) a pCAG-Sipke-tag vector which expresses Sipke protein shown as SEQ ID NO. 1 and a signal peptide shown as SEQ ID NO. 34, and further expresses a tag shown as SEQ ID NO. 35 at the C-terminal of the Sipke protein; (2) pCAG-LC-furin-tag vector, which expresses a signal peptide shown as SEQ ID NO. 27, a Spike protein shown as SEQ ID NO. 13 with furin site deleted and a label shown as SEQ ID NO. 35; (3) pCAG-CD5-furin-tag vector, which expresses a signal peptide shown as SEQ ID NO. 24, a Spike protein shown as SEQ ID NO. 13 with furin site deleted and a tag shown as SEQ ID NO. 35; (4) pCAG-furin vector, which expresses Spike protein with furin site deleted as shown in SEQ ID NO. 13 and signal peptide as shown in SEQ ID NO. 34; (5) pcDNA3.1-Spike vector (i.e., 2019-S vector in FIG. 4).
As shown in FIG. 4, the pCAG-LC-furin-tag vector can package pseudoviruses with higher titer, which is 30-50 times higher than that of the control (2019-S vector).
Example 4 search for pseudovirion-like conditions
1. Replacement of backbone plasmids
With reference to the above procedure, the recombinant vector pcDNA3.1-Spike and the 3-backbone plasmid were co-transfected into 293T cells, respectively, according to the instructions of lipofectamine3000 transfection reagent. The supernatant was collected at about 48h, the collected pseudovirus was infected into 293T-hACE2 cells, and after 48h, the supernatant was discarded, and chemiluminescence was measured using a Britelite plus reporter gene assay system (Perkinelmer, cat. No.6066769) luciferase assay kit. The National Institute of Health (NIH) provides 2 HIV-1 based constructed backbone plasmids, namely, plasmid pSG3. delta env (Wei X, Decker JM, Wang S, Hui H, kappa JC, Wu X, Salazar-Gonzalez JF, Salazar MG, Kilby JM, Saag MS, Komarova NL, Nowak MA, Hahn BH, Kwong PD and Shaw GM. antibody mutagenesis and expression by HIV-1.Nature 422:307-312,2003.) and NL4-3. flu. R-. E- (Connor RI, Chen BK, Choe S, Vplauu NR. expression for reagent type of virus-935, 1995: 206. fig. 4. the plasmid containing the flu gene was introduced into HIV-1. the plasmid of the genus of virus type of virus strain of the genus Vibrio et al, Japan. On the basis, the Fluc gene is amplified to construct a pSG3. delta env. Fluc vector carrying the Fluc gene, and then a CMV promoter is added to the upstream of the Fluc gene on the basis of the pSG3. delta env. Fluc vector to construct the pSG3. delta env. cmvfluc vector (the specific information of the pSG3. delta env. Fluc and the pSG3. delta env. cmvfluc is disclosed in patent CN 201510293955.5). Thus, the 3 backbone plasmids used in this example were: (1) pNL4-3.fluc.r-. E-; (2) psg3.Δ env. fluc; (3) psg3.Δ env. cmvfluc.
As a result, as shown in FIG. 5, the titer of the pseudovirus prepared using pSG3. delta. env. cmvFluc backbone plasmid was significantly increased as compared with the rest.
2. Replacement of transfection reagents
Referring to the above procedure, the recombinant vector pcDNA3.1-Spike and the backbone plasmid pSG3. delta. env. cmvFluc were co-transfected into 293T cells using 3 transfection reagents, respectively. The supernatant was collected at about 48h, the collected pseudovirus was infected into 293T-hACE2 cells, and after 48h, the supernatant was discarded, and chemiluminescence was measured using a Britelite plus reporter gene assay system (Perkinelmer, cat. No.6066769) luciferase assay kit. The 3 transfection reagents were: lipofectamine2000, lipofectamine3000, and PEI.
Results are shown in FIG. 6, where higher pseudoviral titers were produced using lipofectamine3000 transfection reagent.
3. Ratio of recombinant vector to backbone plasmid
With reference to the above procedure, the recombinant vector pcDNA3.1-Spike and the backbone plasmid pSG3. delta. env. cmvFluc were co-transfected into 293T cells, respectively, at different mass ratios, according to the instructions of lipofectamine3000 transfection reagent. The supernatant was collected at about 48h, the collected pseudovirus was infected into 293T-hACE2 cells, and after 48h, the supernatant was discarded, and chemiluminescence was measured using a Britelite plus reporter gene assay system (Perkinelmer, cat. No.6066769) luciferase assay kit. Wherein the mass ratio of the recombinant vector pcDNA3.1-Spike to the skeleton plasmid pSG3. delta. env. cmvFluc is 3: 1. 2: 1. 1: 1. 1: 2. 1: 3 and 1: 4.
as shown in FIG. 7, the mass ratio of the recombinant vector pcDNA3.1-Spike to the backbone plasmid pSG3. delta. env. cmvFluc was 1: 3, the pseudovirus was prepared with higher titer.
While specific embodiments of the invention have been described in detail, those skilled in the art will understand that: various modifications and changes in detail can be made in light of the overall teachings of the disclosure, and such changes are intended to be within the scope of the present invention. A full appreciation of the invention is gained by taking the entire specification as a whole in the light of the appended claims and any equivalents thereof.
Sequence listing
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<120> a SARS-CoV-2 pseudovirus
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<223> SARS-CoV-2Spike protein
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Ser Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr
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Thr Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg
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Ser Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser
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Asn Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr
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Lys Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe
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Ala Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr
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Thr Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr
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Asn Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe
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Leu Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu
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Phe Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser
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Gln Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn
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Leu Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr
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Ser Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe
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Ser Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr
210 215 220
Arg Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly
225 230 235 240
Asp Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly
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Tyr Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr
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Ile Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys
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Cys Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser
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Asn Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile
305 310 315 320
Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala
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Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp
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Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr
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Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr
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Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro
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Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp
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Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys
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Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn
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Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly
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Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu
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Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr
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Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val
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Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn
515 520 525
Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn
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Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr
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Thr Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr
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Pro Cys Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr
580 585 590
Ser Asn Gln Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val
595 600 605
Pro Val Ala Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr
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Ser Thr Gly Ser Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly
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Ala Glu His Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala
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Gly Ile Cys Ala Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala
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Arg Ser Val Ala Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly
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Ala Glu Asn Ser Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr
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Asn Phe Thr Ile Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr
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Lys Thr Ser Val Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu
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755 760 765
Val Phe Ala Gln Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp
770 775 780
Phe Gly Gly Phe Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro
785 790 795 800
Ser Lys Arg Ser Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu
805 810 815
Ala Asp Ala Gly Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile
820 825 830
Ala Ala Arg Asp Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val
835 840 845
Leu Pro Pro Leu Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala
850 855 860
Leu Leu Ala Gly Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala
865 870 875 880
Ala Leu Gln Ile Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly
885 890 895
Ile Gly Val Thr Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala
900 905 910
Asn Gln Phe Asn Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser
915 920 925
Thr Ala Ser Ala Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala
930 935 940
Gln Ala Leu Asn Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala
945 950 955 960
Ile Ser Ser Val Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu
965 970 975
Ala Glu Val Gln Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu
980 985 990
Gln Thr Tyr Val Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala
995 1000 1005
Ser Ala Asn Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly
1010 1015 1020
Gln Ser Lys Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met
1025 1030 1035
Ser Phe Pro Gln Ser Ala Pro His Gly Val Val Phe Leu His Val
1040 1045 1050
Thr Tyr Val Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala
1055 1060 1065
Ile Cys His Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe
1070 1075 1080
Val Ser Asn Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr
1085 1090 1095
Glu Pro Gln Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn
1100 1105 1110
Cys Asp Val Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro
1115 1120 1125
Leu Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr
1130 1135 1140
Phe Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser
1145 1150 1155
Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg
1160 1165 1170
Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu
1175 1180 1185
Gln Glu Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr
1190 1195 1200
Ile Trp Leu Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val
1205 1210 1215
Thr Ile Met Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys
1220 1225 1230
Gly Cys Cys Ser Cys Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp
1235 1240 1245
Ser Glu Pro Val Leu Lys Gly Val Lys Leu His Tyr Thr
1250 1255 1260
<210> 2
<211> 1248
<212> PRT
<213> artificial
<220>
<223> 13 amino acid truncated Spike protein from C terminal
<400> 2
Ser Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr
1 5 10 15
Thr Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg
20 25 30
Ser Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser
35 40 45
Asn Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr
50 55 60
Lys Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe
65 70 75 80
Ala Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr
85 90 95
Thr Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr
100 105 110
Asn Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe
115 120 125
Leu Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu
130 135 140
Phe Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser
145 150 155 160
Gln Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn
165 170 175
Leu Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr
180 185 190
Ser Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe
195 200 205
Ser Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr
210 215 220
Arg Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly
225 230 235 240
Asp Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly
245 250 255
Tyr Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr
260 265 270
Ile Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys
275 280 285
Cys Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser
290 295 300
Asn Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile
305 310 315 320
Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala
325 330 335
Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp
340 345 350
Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr
355 360 365
Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr
370 375 380
Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro
385 390 395 400
Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp
405 410 415
Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys
420 425 430
Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn
435 440 445
Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly
450 455 460
Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu
465 470 475 480
Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr
485 490 495
Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val
500 505 510
Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn
515 520 525
Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn
530 535 540
Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr
545 550 555 560
Thr Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr
565 570 575
Pro Cys Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr
580 585 590
Ser Asn Gln Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val
595 600 605
Pro Val Ala Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr
610 615 620
Ser Thr Gly Ser Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly
625 630 635 640
Ala Glu His Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala
645 650 655
Gly Ile Cys Ala Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala
660 665 670
Arg Ser Val Ala Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly
675 680 685
Ala Glu Asn Ser Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr
690 695 700
Asn Phe Thr Ile Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr
705 710 715 720
Lys Thr Ser Val Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu
725 730 735
Cys Ser Asn Leu Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn
740 745 750
Arg Ala Leu Thr Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu
755 760 765
Val Phe Ala Gln Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp
770 775 780
Phe Gly Gly Phe Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro
785 790 795 800
Ser Lys Arg Ser Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu
805 810 815
Ala Asp Ala Gly Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile
820 825 830
Ala Ala Arg Asp Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val
835 840 845
Leu Pro Pro Leu Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala
850 855 860
Leu Leu Ala Gly Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala
865 870 875 880
Ala Leu Gln Ile Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly
885 890 895
Ile Gly Val Thr Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala
900 905 910
Asn Gln Phe Asn Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser
915 920 925
Thr Ala Ser Ala Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala
930 935 940
Gln Ala Leu Asn Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala
945 950 955 960
Ile Ser Ser Val Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu
965 970 975
Ala Glu Val Gln Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu
980 985 990
Gln Thr Tyr Val Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala
995 1000 1005
Ser Ala Asn Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly
1010 1015 1020
Gln Ser Lys Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met
1025 1030 1035
Ser Phe Pro Gln Ser Ala Pro His Gly Val Val Phe Leu His Val
1040 1045 1050
Thr Tyr Val Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala
1055 1060 1065
Ile Cys His Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe
1070 1075 1080
Val Ser Asn Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr
1085 1090 1095
Glu Pro Gln Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn
1100 1105 1110
Cys Asp Val Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro
1115 1120 1125
Leu Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr
1130 1135 1140
Phe Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser
1145 1150 1155
Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg
1160 1165 1170
Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu
1175 1180 1185
Gln Glu Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr
1190 1195 1200
Ile Trp Leu Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val
1205 1210 1215
Thr Ile Met Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys
1220 1225 1230
Gly Cys Cys Ser Cys Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp
1235 1240 1245
<210> 3
<211> 1247
<212> PRT
<213> artificial
<220>
<223> Spike protein with 14 amino acids truncated at C terminal
<400> 3
Ser Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr
1 5 10 15
Thr Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg
20 25 30
Ser Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser
35 40 45
Asn Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr
50 55 60
Lys Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe
65 70 75 80
Ala Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr
85 90 95
Thr Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr
100 105 110
Asn Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe
115 120 125
Leu Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu
130 135 140
Phe Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser
145 150 155 160
Gln Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn
165 170 175
Leu Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr
180 185 190
Ser Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe
195 200 205
Ser Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr
210 215 220
Arg Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly
225 230 235 240
Asp Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly
245 250 255
Tyr Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr
260 265 270
Ile Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys
275 280 285
Cys Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser
290 295 300
Asn Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile
305 310 315 320
Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala
325 330 335
Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp
340 345 350
Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr
355 360 365
Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr
370 375 380
Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro
385 390 395 400
Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp
405 410 415
Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys
420 425 430
Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn
435 440 445
Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly
450 455 460
Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu
465 470 475 480
Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr
485 490 495
Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val
500 505 510
Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn
515 520 525
Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn
530 535 540
Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr
545 550 555 560
Thr Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr
565 570 575
Pro Cys Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr
580 585 590
Ser Asn Gln Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val
595 600 605
Pro Val Ala Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr
610 615 620
Ser Thr Gly Ser Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly
625 630 635 640
Ala Glu His Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala
645 650 655
Gly Ile Cys Ala Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala
660 665 670
Arg Ser Val Ala Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly
675 680 685
Ala Glu Asn Ser Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr
690 695 700
Asn Phe Thr Ile Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr
705 710 715 720
Lys Thr Ser Val Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu
725 730 735
Cys Ser Asn Leu Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn
740 745 750
Arg Ala Leu Thr Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu
755 760 765
Val Phe Ala Gln Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp
770 775 780
Phe Gly Gly Phe Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro
785 790 795 800
Ser Lys Arg Ser Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu
805 810 815
Ala Asp Ala Gly Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile
820 825 830
Ala Ala Arg Asp Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val
835 840 845
Leu Pro Pro Leu Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala
850 855 860
Leu Leu Ala Gly Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala
865 870 875 880
Ala Leu Gln Ile Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly
885 890 895
Ile Gly Val Thr Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala
900 905 910
Asn Gln Phe Asn Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser
915 920 925
Thr Ala Ser Ala Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala
930 935 940
Gln Ala Leu Asn Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala
945 950 955 960
Ile Ser Ser Val Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu
965 970 975
Ala Glu Val Gln Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu
980 985 990
Gln Thr Tyr Val Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala
995 1000 1005
Ser Ala Asn Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly
1010 1015 1020
Gln Ser Lys Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met
1025 1030 1035
Ser Phe Pro Gln Ser Ala Pro His Gly Val Val Phe Leu His Val
1040 1045 1050
Thr Tyr Val Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala
1055 1060 1065
Ile Cys His Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe
1070 1075 1080
Val Ser Asn Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr
1085 1090 1095
Glu Pro Gln Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn
1100 1105 1110
Cys Asp Val Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro
1115 1120 1125
Leu Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr
1130 1135 1140
Phe Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser
1145 1150 1155
Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg
1160 1165 1170
Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu
1175 1180 1185
Gln Glu Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr
1190 1195 1200
Ile Trp Leu Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val
1205 1210 1215
Thr Ile Met Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys
1220 1225 1230
Gly Cys Cys Ser Cys Gly Ser Cys Cys Lys Phe Asp Glu Asp
1235 1240 1245
<210> 4
<211> 1246
<212> PRT
<213> artificial
<220>
<223> Spike protein with 15 amino acids truncated at C terminal
<400> 4
Ser Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr
1 5 10 15
Thr Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg
20 25 30
Ser Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser
35 40 45
Asn Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr
50 55 60
Lys Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe
65 70 75 80
Ala Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr
85 90 95
Thr Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr
100 105 110
Asn Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe
115 120 125
Leu Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu
130 135 140
Phe Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser
145 150 155 160
Gln Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn
165 170 175
Leu Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr
180 185 190
Ser Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe
195 200 205
Ser Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr
210 215 220
Arg Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly
225 230 235 240
Asp Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly
245 250 255
Tyr Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr
260 265 270
Ile Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys
275 280 285
Cys Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser
290 295 300
Asn Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile
305 310 315 320
Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala
325 330 335
Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp
340 345 350
Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr
355 360 365
Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr
370 375 380
Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro
385 390 395 400
Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp
405 410 415
Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys
420 425 430
Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn
435 440 445
Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly
450 455 460
Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu
465 470 475 480
Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr
485 490 495
Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val
500 505 510
Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn
515 520 525
Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn
530 535 540
Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr
545 550 555 560
Thr Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr
565 570 575
Pro Cys Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr
580 585 590
Ser Asn Gln Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val
595 600 605
Pro Val Ala Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr
610 615 620
Ser Thr Gly Ser Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly
625 630 635 640
Ala Glu His Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala
645 650 655
Gly Ile Cys Ala Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala
660 665 670
Arg Ser Val Ala Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly
675 680 685
Ala Glu Asn Ser Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr
690 695 700
Asn Phe Thr Ile Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr
705 710 715 720
Lys Thr Ser Val Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu
725 730 735
Cys Ser Asn Leu Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn
740 745 750
Arg Ala Leu Thr Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu
755 760 765
Val Phe Ala Gln Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp
770 775 780
Phe Gly Gly Phe Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro
785 790 795 800
Ser Lys Arg Ser Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu
805 810 815
Ala Asp Ala Gly Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile
820 825 830
Ala Ala Arg Asp Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val
835 840 845
Leu Pro Pro Leu Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala
850 855 860
Leu Leu Ala Gly Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala
865 870 875 880
Ala Leu Gln Ile Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly
885 890 895
Ile Gly Val Thr Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala
900 905 910
Asn Gln Phe Asn Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser
915 920 925
Thr Ala Ser Ala Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala
930 935 940
Gln Ala Leu Asn Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala
945 950 955 960
Ile Ser Ser Val Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu
965 970 975
Ala Glu Val Gln Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu
980 985 990
Gln Thr Tyr Val Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala
995 1000 1005
Ser Ala Asn Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly
1010 1015 1020
Gln Ser Lys Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met
1025 1030 1035
Ser Phe Pro Gln Ser Ala Pro His Gly Val Val Phe Leu His Val
1040 1045 1050
Thr Tyr Val Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala
1055 1060 1065
Ile Cys His Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe
1070 1075 1080
Val Ser Asn Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr
1085 1090 1095
Glu Pro Gln Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn
1100 1105 1110
Cys Asp Val Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro
1115 1120 1125
Leu Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr
1130 1135 1140
Phe Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser
1145 1150 1155
Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg
1160 1165 1170
Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu
1175 1180 1185
Gln Glu Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr
1190 1195 1200
Ile Trp Leu Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val
1205 1210 1215
Thr Ile Met Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys
1220 1225 1230
Gly Cys Cys Ser Cys Gly Ser Cys Cys Lys Phe Asp Glu
1235 1240 1245
<210> 5
<211> 1245
<212> PRT
<213> artificial
<220>
<223> Spike protein with 16 amino acids truncated at C terminal
<400> 5
Ser Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr
1 5 10 15
Thr Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg
20 25 30
Ser Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser
35 40 45
Asn Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr
50 55 60
Lys Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe
65 70 75 80
Ala Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr
85 90 95
Thr Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr
100 105 110
Asn Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe
115 120 125
Leu Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu
130 135 140
Phe Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser
145 150 155 160
Gln Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn
165 170 175
Leu Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr
180 185 190
Ser Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe
195 200 205
Ser Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr
210 215 220
Arg Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly
225 230 235 240
Asp Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly
245 250 255
Tyr Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr
260 265 270
Ile Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys
275 280 285
Cys Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser
290 295 300
Asn Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile
305 310 315 320
Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala
325 330 335
Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp
340 345 350
Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr
355 360 365
Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr
370 375 380
Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro
385 390 395 400
Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp
405 410 415
Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys
420 425 430
Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn
435 440 445
Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly
450 455 460
Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu
465 470 475 480
Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr
485 490 495
Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val
500 505 510
Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn
515 520 525
Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn
530 535 540
Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr
545 550 555 560
Thr Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr
565 570 575
Pro Cys Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr
580 585 590
Ser Asn Gln Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val
595 600 605
Pro Val Ala Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr
610 615 620
Ser Thr Gly Ser Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly
625 630 635 640
Ala Glu His Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala
645 650 655
Gly Ile Cys Ala Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala
660 665 670
Arg Ser Val Ala Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly
675 680 685
Ala Glu Asn Ser Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr
690 695 700
Asn Phe Thr Ile Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr
705 710 715 720
Lys Thr Ser Val Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu
725 730 735
Cys Ser Asn Leu Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn
740 745 750
Arg Ala Leu Thr Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu
755 760 765
Val Phe Ala Gln Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp
770 775 780
Phe Gly Gly Phe Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro
785 790 795 800
Ser Lys Arg Ser Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu
805 810 815
Ala Asp Ala Gly Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile
820 825 830
Ala Ala Arg Asp Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val
835 840 845
Leu Pro Pro Leu Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala
850 855 860
Leu Leu Ala Gly Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala
865 870 875 880
Ala Leu Gln Ile Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly
885 890 895
Ile Gly Val Thr Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala
900 905 910
Asn Gln Phe Asn Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser
915 920 925
Thr Ala Ser Ala Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala
930 935 940
Gln Ala Leu Asn Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala
945 950 955 960
Ile Ser Ser Val Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu
965 970 975
Ala Glu Val Gln Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu
980 985 990
Gln Thr Tyr Val Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala
995 1000 1005
Ser Ala Asn Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly
1010 1015 1020
Gln Ser Lys Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met
1025 1030 1035
Ser Phe Pro Gln Ser Ala Pro His Gly Val Val Phe Leu His Val
1040 1045 1050
Thr Tyr Val Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala
1055 1060 1065
Ile Cys His Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe
1070 1075 1080
Val Ser Asn Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr
1085 1090 1095
Glu Pro Gln Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn
1100 1105 1110
Cys Asp Val Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro
1115 1120 1125
Leu Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr
1130 1135 1140
Phe Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser
1145 1150 1155
Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg
1160 1165 1170
Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu
1175 1180 1185
Gln Glu Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr
1190 1195 1200
Ile Trp Leu Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val
1205 1210 1215
Thr Ile Met Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys
1220 1225 1230
Gly Cys Cys Ser Cys Gly Ser Cys Cys Lys Phe Asp
1235 1240 1245
<210> 6
<211> 1244
<212> PRT
<213> artificial
<220>
<223> Spike protein with 17 amino acids truncated at C terminal
<400> 6
Ser Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr
1 5 10 15
Thr Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg
20 25 30
Ser Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser
35 40 45
Asn Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr
50 55 60
Lys Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe
65 70 75 80
Ala Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr
85 90 95
Thr Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr
100 105 110
Asn Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe
115 120 125
Leu Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu
130 135 140
Phe Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser
145 150 155 160
Gln Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn
165 170 175
Leu Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr
180 185 190
Ser Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe
195 200 205
Ser Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr
210 215 220
Arg Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly
225 230 235 240
Asp Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly
245 250 255
Tyr Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr
260 265 270
Ile Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys
275 280 285
Cys Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser
290 295 300
Asn Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile
305 310 315 320
Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala
325 330 335
Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp
340 345 350
Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr
355 360 365
Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr
370 375 380
Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro
385 390 395 400
Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp
405 410 415
Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys
420 425 430
Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn
435 440 445
Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly
450 455 460
Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu
465 470 475 480
Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr
485 490 495
Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val
500 505 510
Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn
515 520 525
Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn
530 535 540
Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr
545 550 555 560
Thr Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr
565 570 575
Pro Cys Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr
580 585 590
Ser Asn Gln Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val
595 600 605
Pro Val Ala Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr
610 615 620
Ser Thr Gly Ser Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly
625 630 635 640
Ala Glu His Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala
645 650 655
Gly Ile Cys Ala Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala
660 665 670
Arg Ser Val Ala Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly
675 680 685
Ala Glu Asn Ser Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr
690 695 700
Asn Phe Thr Ile Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr
705 710 715 720
Lys Thr Ser Val Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu
725 730 735
Cys Ser Asn Leu Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn
740 745 750
Arg Ala Leu Thr Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu
755 760 765
Val Phe Ala Gln Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp
770 775 780
Phe Gly Gly Phe Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro
785 790 795 800
Ser Lys Arg Ser Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu
805 810 815
Ala Asp Ala Gly Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile
820 825 830
Ala Ala Arg Asp Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val
835 840 845
Leu Pro Pro Leu Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala
850 855 860
Leu Leu Ala Gly Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala
865 870 875 880
Ala Leu Gln Ile Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly
885 890 895
Ile Gly Val Thr Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala
900 905 910
Asn Gln Phe Asn Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser
915 920 925
Thr Ala Ser Ala Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala
930 935 940
Gln Ala Leu Asn Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala
945 950 955 960
Ile Ser Ser Val Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu
965 970 975
Ala Glu Val Gln Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu
980 985 990
Gln Thr Tyr Val Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala
995 1000 1005
Ser Ala Asn Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly
1010 1015 1020
Gln Ser Lys Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met
1025 1030 1035
Ser Phe Pro Gln Ser Ala Pro His Gly Val Val Phe Leu His Val
1040 1045 1050
Thr Tyr Val Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala
1055 1060 1065
Ile Cys His Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe
1070 1075 1080
Val Ser Asn Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr
1085 1090 1095
Glu Pro Gln Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn
1100 1105 1110
Cys Asp Val Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro
1115 1120 1125
Leu Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr
1130 1135 1140
Phe Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser
1145 1150 1155
Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg
1160 1165 1170
Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu
1175 1180 1185
Gln Glu Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr
1190 1195 1200
Ile Trp Leu Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val
1205 1210 1215
Thr Ile Met Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys
1220 1225 1230
Gly Cys Cys Ser Cys Gly Ser Cys Cys Lys Phe
1235 1240
<210> 7
<211> 1243
<212> PRT
<213> artificial
<220>
<223> C-terminal 18 amino acid truncated Spike protein
<400> 7
Ser Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr
1 5 10 15
Thr Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg
20 25 30
Ser Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser
35 40 45
Asn Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr
50 55 60
Lys Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe
65 70 75 80
Ala Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr
85 90 95
Thr Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr
100 105 110
Asn Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe
115 120 125
Leu Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu
130 135 140
Phe Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser
145 150 155 160
Gln Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn
165 170 175
Leu Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr
180 185 190
Ser Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe
195 200 205
Ser Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr
210 215 220
Arg Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly
225 230 235 240
Asp Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly
245 250 255
Tyr Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr
260 265 270
Ile Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys
275 280 285
Cys Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser
290 295 300
Asn Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile
305 310 315 320
Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala
325 330 335
Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp
340 345 350
Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr
355 360 365
Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr
370 375 380
Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro
385 390 395 400
Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp
405 410 415
Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys
420 425 430
Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn
435 440 445
Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly
450 455 460
Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu
465 470 475 480
Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr
485 490 495
Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val
500 505 510
Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn
515 520 525
Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn
530 535 540
Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr
545 550 555 560
Thr Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr
565 570 575
Pro Cys Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr
580 585 590
Ser Asn Gln Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val
595 600 605
Pro Val Ala Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr
610 615 620
Ser Thr Gly Ser Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly
625 630 635 640
Ala Glu His Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala
645 650 655
Gly Ile Cys Ala Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala
660 665 670
Arg Ser Val Ala Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly
675 680 685
Ala Glu Asn Ser Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr
690 695 700
Asn Phe Thr Ile Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr
705 710 715 720
Lys Thr Ser Val Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu
725 730 735
Cys Ser Asn Leu Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn
740 745 750
Arg Ala Leu Thr Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu
755 760 765
Val Phe Ala Gln Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp
770 775 780
Phe Gly Gly Phe Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro
785 790 795 800
Ser Lys Arg Ser Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu
805 810 815
Ala Asp Ala Gly Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile
820 825 830
Ala Ala Arg Asp Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val
835 840 845
Leu Pro Pro Leu Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala
850 855 860
Leu Leu Ala Gly Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala
865 870 875 880
Ala Leu Gln Ile Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly
885 890 895
Ile Gly Val Thr Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala
900 905 910
Asn Gln Phe Asn Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser
915 920 925
Thr Ala Ser Ala Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala
930 935 940
Gln Ala Leu Asn Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala
945 950 955 960
Ile Ser Ser Val Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu
965 970 975
Ala Glu Val Gln Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu
980 985 990
Gln Thr Tyr Val Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala
995 1000 1005
Ser Ala Asn Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly
1010 1015 1020
Gln Ser Lys Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met
1025 1030 1035
Ser Phe Pro Gln Ser Ala Pro His Gly Val Val Phe Leu His Val
1040 1045 1050
Thr Tyr Val Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala
1055 1060 1065
Ile Cys His Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe
1070 1075 1080
Val Ser Asn Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr
1085 1090 1095
Glu Pro Gln Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn
1100 1105 1110
Cys Asp Val Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro
1115 1120 1125
Leu Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr
1130 1135 1140
Phe Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser
1145 1150 1155
Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg
1160 1165 1170
Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu
1175 1180 1185
Gln Glu Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr
1190 1195 1200
Ile Trp Leu Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val
1205 1210 1215
Thr Ile Met Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys
1220 1225 1230
Gly Cys Cys Ser Cys Gly Ser Cys Cys Lys
1235 1240
<210> 8
<211> 1242
<212> PRT
<213> artificial
<220>
<223> Spike protein with 19 amino acids truncated at C terminal
<400> 8
Ser Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr
1 5 10 15
Thr Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg
20 25 30
Ser Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser
35 40 45
Asn Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr
50 55 60
Lys Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe
65 70 75 80
Ala Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr
85 90 95
Thr Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr
100 105 110
Asn Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe
115 120 125
Leu Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu
130 135 140
Phe Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser
145 150 155 160
Gln Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn
165 170 175
Leu Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr
180 185 190
Ser Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe
195 200 205
Ser Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr
210 215 220
Arg Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly
225 230 235 240
Asp Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly
245 250 255
Tyr Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr
260 265 270
Ile Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys
275 280 285
Cys Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser
290 295 300
Asn Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile
305 310 315 320
Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala
325 330 335
Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp
340 345 350
Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr
355 360 365
Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr
370 375 380
Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro
385 390 395 400
Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp
405 410 415
Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys
420 425 430
Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn
435 440 445
Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly
450 455 460
Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu
465 470 475 480
Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr
485 490 495
Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val
500 505 510
Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn
515 520 525
Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn
530 535 540
Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr
545 550 555 560
Thr Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr
565 570 575
Pro Cys Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr
580 585 590
Ser Asn Gln Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val
595 600 605
Pro Val Ala Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr
610 615 620
Ser Thr Gly Ser Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly
625 630 635 640
Ala Glu His Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala
645 650 655
Gly Ile Cys Ala Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala
660 665 670
Arg Ser Val Ala Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly
675 680 685
Ala Glu Asn Ser Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr
690 695 700
Asn Phe Thr Ile Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr
705 710 715 720
Lys Thr Ser Val Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu
725 730 735
Cys Ser Asn Leu Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn
740 745 750
Arg Ala Leu Thr Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu
755 760 765
Val Phe Ala Gln Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp
770 775 780
Phe Gly Gly Phe Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro
785 790 795 800
Ser Lys Arg Ser Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu
805 810 815
Ala Asp Ala Gly Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile
820 825 830
Ala Ala Arg Asp Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val
835 840 845
Leu Pro Pro Leu Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala
850 855 860
Leu Leu Ala Gly Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala
865 870 875 880
Ala Leu Gln Ile Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly
885 890 895
Ile Gly Val Thr Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala
900 905 910
Asn Gln Phe Asn Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser
915 920 925
Thr Ala Ser Ala Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala
930 935 940
Gln Ala Leu Asn Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala
945 950 955 960
Ile Ser Ser Val Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu
965 970 975
Ala Glu Val Gln Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu
980 985 990
Gln Thr Tyr Val Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala
995 1000 1005
Ser Ala Asn Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly
1010 1015 1020
Gln Ser Lys Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met
1025 1030 1035
Ser Phe Pro Gln Ser Ala Pro His Gly Val Val Phe Leu His Val
1040 1045 1050
Thr Tyr Val Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala
1055 1060 1065
Ile Cys His Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe
1070 1075 1080
Val Ser Asn Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr
1085 1090 1095
Glu Pro Gln Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn
1100 1105 1110
Cys Asp Val Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro
1115 1120 1125
Leu Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr
1130 1135 1140
Phe Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser
1145 1150 1155
Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg
1160 1165 1170
Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu
1175 1180 1185
Gln Glu Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr
1190 1195 1200
Ile Trp Leu Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val
1205 1210 1215
Thr Ile Met Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys
1220 1225 1230
Gly Cys Cys Ser Cys Gly Ser Cys Cys
1235 1240
<210> 9
<211> 1241
<212> PRT
<213> artificial
<220>
<223> Spike protein with 20 amino acids truncated at C terminal
<400> 9
Ser Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr
1 5 10 15
Thr Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg
20 25 30
Ser Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser
35 40 45
Asn Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr
50 55 60
Lys Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe
65 70 75 80
Ala Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr
85 90 95
Thr Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr
100 105 110
Asn Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe
115 120 125
Leu Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu
130 135 140
Phe Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser
145 150 155 160
Gln Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn
165 170 175
Leu Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr
180 185 190
Ser Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe
195 200 205
Ser Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr
210 215 220
Arg Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly
225 230 235 240
Asp Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly
245 250 255
Tyr Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr
260 265 270
Ile Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys
275 280 285
Cys Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser
290 295 300
Asn Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile
305 310 315 320
Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala
325 330 335
Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp
340 345 350
Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr
355 360 365
Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr
370 375 380
Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro
385 390 395 400
Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp
405 410 415
Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys
420 425 430
Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn
435 440 445
Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly
450 455 460
Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu
465 470 475 480
Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr
485 490 495
Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val
500 505 510
Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn
515 520 525
Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn
530 535 540
Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr
545 550 555 560
Thr Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr
565 570 575
Pro Cys Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr
580 585 590
Ser Asn Gln Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val
595 600 605
Pro Val Ala Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr
610 615 620
Ser Thr Gly Ser Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly
625 630 635 640
Ala Glu His Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala
645 650 655
Gly Ile Cys Ala Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala
660 665 670
Arg Ser Val Ala Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly
675 680 685
Ala Glu Asn Ser Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr
690 695 700
Asn Phe Thr Ile Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr
705 710 715 720
Lys Thr Ser Val Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu
725 730 735
Cys Ser Asn Leu Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn
740 745 750
Arg Ala Leu Thr Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu
755 760 765
Val Phe Ala Gln Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp
770 775 780
Phe Gly Gly Phe Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro
785 790 795 800
Ser Lys Arg Ser Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu
805 810 815
Ala Asp Ala Gly Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile
820 825 830
Ala Ala Arg Asp Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val
835 840 845
Leu Pro Pro Leu Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala
850 855 860
Leu Leu Ala Gly Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala
865 870 875 880
Ala Leu Gln Ile Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly
885 890 895
Ile Gly Val Thr Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala
900 905 910
Asn Gln Phe Asn Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser
915 920 925
Thr Ala Ser Ala Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala
930 935 940
Gln Ala Leu Asn Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala
945 950 955 960
Ile Ser Ser Val Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu
965 970 975
Ala Glu Val Gln Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu
980 985 990
Gln Thr Tyr Val Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala
995 1000 1005
Ser Ala Asn Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly
1010 1015 1020
Gln Ser Lys Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met
1025 1030 1035
Ser Phe Pro Gln Ser Ala Pro His Gly Val Val Phe Leu His Val
1040 1045 1050
Thr Tyr Val Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala
1055 1060 1065
Ile Cys His Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe
1070 1075 1080
Val Ser Asn Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr
1085 1090 1095
Glu Pro Gln Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn
1100 1105 1110
Cys Asp Val Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro
1115 1120 1125
Leu Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr
1130 1135 1140
Phe Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser
1145 1150 1155
Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg
1160 1165 1170
Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu
1175 1180 1185
Gln Glu Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr
1190 1195 1200
Ile Trp Leu Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val
1205 1210 1215
Thr Ile Met Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys
1220 1225 1230
Gly Cys Cys Ser Cys Gly Ser Cys
1235 1240
<210> 10
<211> 1240
<212> PRT
<213> artificial
<220>
<223> Spike protein with 21 amino acids truncated at C terminal
<400> 10
Ser Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr
1 5 10 15
Thr Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg
20 25 30
Ser Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser
35 40 45
Asn Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr
50 55 60
Lys Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe
65 70 75 80
Ala Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr
85 90 95
Thr Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr
100 105 110
Asn Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe
115 120 125
Leu Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu
130 135 140
Phe Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser
145 150 155 160
Gln Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn
165 170 175
Leu Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr
180 185 190
Ser Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe
195 200 205
Ser Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr
210 215 220
Arg Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly
225 230 235 240
Asp Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly
245 250 255
Tyr Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr
260 265 270
Ile Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys
275 280 285
Cys Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser
290 295 300
Asn Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile
305 310 315 320
Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala
325 330 335
Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp
340 345 350
Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr
355 360 365
Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr
370 375 380
Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro
385 390 395 400
Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp
405 410 415
Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys
420 425 430
Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn
435 440 445
Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly
450 455 460
Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu
465 470 475 480
Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr
485 490 495
Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val
500 505 510
Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn
515 520 525
Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn
530 535 540
Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr
545 550 555 560
Thr Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr
565 570 575
Pro Cys Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr
580 585 590
Ser Asn Gln Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val
595 600 605
Pro Val Ala Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr
610 615 620
Ser Thr Gly Ser Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly
625 630 635 640
Ala Glu His Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala
645 650 655
Gly Ile Cys Ala Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala
660 665 670
Arg Ser Val Ala Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly
675 680 685
Ala Glu Asn Ser Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr
690 695 700
Asn Phe Thr Ile Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr
705 710 715 720
Lys Thr Ser Val Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu
725 730 735
Cys Ser Asn Leu Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn
740 745 750
Arg Ala Leu Thr Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu
755 760 765
Val Phe Ala Gln Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp
770 775 780
Phe Gly Gly Phe Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro
785 790 795 800
Ser Lys Arg Ser Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu
805 810 815
Ala Asp Ala Gly Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile
820 825 830
Ala Ala Arg Asp Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val
835 840 845
Leu Pro Pro Leu Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala
850 855 860
Leu Leu Ala Gly Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala
865 870 875 880
Ala Leu Gln Ile Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly
885 890 895
Ile Gly Val Thr Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala
900 905 910
Asn Gln Phe Asn Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser
915 920 925
Thr Ala Ser Ala Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala
930 935 940
Gln Ala Leu Asn Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala
945 950 955 960
Ile Ser Ser Val Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu
965 970 975
Ala Glu Val Gln Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu
980 985 990
Gln Thr Tyr Val Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala
995 1000 1005
Ser Ala Asn Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly
1010 1015 1020
Gln Ser Lys Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met
1025 1030 1035
Ser Phe Pro Gln Ser Ala Pro His Gly Val Val Phe Leu His Val
1040 1045 1050
Thr Tyr Val Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala
1055 1060 1065
Ile Cys His Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe
1070 1075 1080
Val Ser Asn Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr
1085 1090 1095
Glu Pro Gln Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn
1100 1105 1110
Cys Asp Val Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro
1115 1120 1125
Leu Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr
1130 1135 1140
Phe Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser
1145 1150 1155
Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg
1160 1165 1170
Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu
1175 1180 1185
Gln Glu Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr
1190 1195 1200
Ile Trp Leu Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val
1205 1210 1215
Thr Ile Met Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys
1220 1225 1230
Gly Cys Cys Ser Cys Gly Ser
1235 1240
<210> 11
<211> 1239
<212> PRT
<213> artificial
<220>
<223> Spike protein with 22 amino acids truncated at C terminal
<400> 11
Ser Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr
1 5 10 15
Thr Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg
20 25 30
Ser Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser
35 40 45
Asn Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr
50 55 60
Lys Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe
65 70 75 80
Ala Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr
85 90 95
Thr Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr
100 105 110
Asn Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe
115 120 125
Leu Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu
130 135 140
Phe Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser
145 150 155 160
Gln Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn
165 170 175
Leu Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr
180 185 190
Ser Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe
195 200 205
Ser Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr
210 215 220
Arg Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly
225 230 235 240
Asp Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly
245 250 255
Tyr Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr
260 265 270
Ile Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys
275 280 285
Cys Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser
290 295 300
Asn Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile
305 310 315 320
Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala
325 330 335
Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp
340 345 350
Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr
355 360 365
Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr
370 375 380
Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro
385 390 395 400
Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp
405 410 415
Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys
420 425 430
Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn
435 440 445
Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly
450 455 460
Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu
465 470 475 480
Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr
485 490 495
Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val
500 505 510
Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn
515 520 525
Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn
530 535 540
Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr
545 550 555 560
Thr Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr
565 570 575
Pro Cys Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr
580 585 590
Ser Asn Gln Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val
595 600 605
Pro Val Ala Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr
610 615 620
Ser Thr Gly Ser Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly
625 630 635 640
Ala Glu His Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala
645 650 655
Gly Ile Cys Ala Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala
660 665 670
Arg Ser Val Ala Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly
675 680 685
Ala Glu Asn Ser Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr
690 695 700
Asn Phe Thr Ile Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr
705 710 715 720
Lys Thr Ser Val Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu
725 730 735
Cys Ser Asn Leu Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn
740 745 750
Arg Ala Leu Thr Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu
755 760 765
Val Phe Ala Gln Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp
770 775 780
Phe Gly Gly Phe Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro
785 790 795 800
Ser Lys Arg Ser Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu
805 810 815
Ala Asp Ala Gly Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile
820 825 830
Ala Ala Arg Asp Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val
835 840 845
Leu Pro Pro Leu Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala
850 855 860
Leu Leu Ala Gly Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala
865 870 875 880
Ala Leu Gln Ile Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly
885 890 895
Ile Gly Val Thr Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala
900 905 910
Asn Gln Phe Asn Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser
915 920 925
Thr Ala Ser Ala Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala
930 935 940
Gln Ala Leu Asn Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala
945 950 955 960
Ile Ser Ser Val Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu
965 970 975
Ala Glu Val Gln Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu
980 985 990
Gln Thr Tyr Val Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala
995 1000 1005
Ser Ala Asn Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly
1010 1015 1020
Gln Ser Lys Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met
1025 1030 1035
Ser Phe Pro Gln Ser Ala Pro His Gly Val Val Phe Leu His Val
1040 1045 1050
Thr Tyr Val Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala
1055 1060 1065
Ile Cys His Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe
1070 1075 1080
Val Ser Asn Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr
1085 1090 1095
Glu Pro Gln Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn
1100 1105 1110
Cys Asp Val Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro
1115 1120 1125
Leu Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr
1130 1135 1140
Phe Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser
1145 1150 1155
Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg
1160 1165 1170
Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu
1175 1180 1185
Gln Glu Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr
1190 1195 1200
Ile Trp Leu Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val
1205 1210 1215
Thr Ile Met Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys
1220 1225 1230
Gly Cys Cys Ser Cys Gly
1235
<210> 12
<211> 1238
<212> PRT
<213> artificial
<220>
<223> Spike protein with 23 amino acids truncated at C terminal
<400> 12
Ser Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr
1 5 10 15
Thr Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg
20 25 30
Ser Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser
35 40 45
Asn Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr
50 55 60
Lys Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe
65 70 75 80
Ala Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr
85 90 95
Thr Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr
100 105 110
Asn Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe
115 120 125
Leu Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu
130 135 140
Phe Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser
145 150 155 160
Gln Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn
165 170 175
Leu Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr
180 185 190
Ser Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe
195 200 205
Ser Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr
210 215 220
Arg Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly
225 230 235 240
Asp Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly
245 250 255
Tyr Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr
260 265 270
Ile Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys
275 280 285
Cys Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser
290 295 300
Asn Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile
305 310 315 320
Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala
325 330 335
Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp
340 345 350
Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr
355 360 365
Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr
370 375 380
Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro
385 390 395 400
Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp
405 410 415
Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys
420 425 430
Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn
435 440 445
Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly
450 455 460
Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu
465 470 475 480
Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr
485 490 495
Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val
500 505 510
Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn
515 520 525
Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn
530 535 540
Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr
545 550 555 560
Thr Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr
565 570 575
Pro Cys Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr
580 585 590
Ser Asn Gln Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val
595 600 605
Pro Val Ala Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr
610 615 620
Ser Thr Gly Ser Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly
625 630 635 640
Ala Glu His Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala
645 650 655
Gly Ile Cys Ala Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala
660 665 670
Arg Ser Val Ala Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly
675 680 685
Ala Glu Asn Ser Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr
690 695 700
Asn Phe Thr Ile Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr
705 710 715 720
Lys Thr Ser Val Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu
725 730 735
Cys Ser Asn Leu Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn
740 745 750
Arg Ala Leu Thr Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu
755 760 765
Val Phe Ala Gln Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp
770 775 780
Phe Gly Gly Phe Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro
785 790 795 800
Ser Lys Arg Ser Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu
805 810 815
Ala Asp Ala Gly Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile
820 825 830
Ala Ala Arg Asp Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val
835 840 845
Leu Pro Pro Leu Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala
850 855 860
Leu Leu Ala Gly Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala
865 870 875 880
Ala Leu Gln Ile Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly
885 890 895
Ile Gly Val Thr Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala
900 905 910
Asn Gln Phe Asn Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser
915 920 925
Thr Ala Ser Ala Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala
930 935 940
Gln Ala Leu Asn Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala
945 950 955 960
Ile Ser Ser Val Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu
965 970 975
Ala Glu Val Gln Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu
980 985 990
Gln Thr Tyr Val Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala
995 1000 1005
Ser Ala Asn Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly
1010 1015 1020
Gln Ser Lys Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met
1025 1030 1035
Ser Phe Pro Gln Ser Ala Pro His Gly Val Val Phe Leu His Val
1040 1045 1050
Thr Tyr Val Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala
1055 1060 1065
Ile Cys His Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe
1070 1075 1080
Val Ser Asn Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr
1085 1090 1095
Glu Pro Gln Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn
1100 1105 1110
Cys Asp Val Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro
1115 1120 1125
Leu Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr
1130 1135 1140
Phe Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser
1145 1150 1155
Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg
1160 1165 1170
Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu
1175 1180 1185
Gln Glu Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr
1190 1195 1200
Ile Trp Leu Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val
1205 1210 1215
Thr Ile Met Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys
1220 1225 1230
Gly Cys Cys Ser Cys
1235
<210> 13
<211> 1257
<212> PRT
<213> artificial
<220>
<223> Spike protein deleting furin site
<400> 13
Ser Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr
1 5 10 15
Thr Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg
20 25 30
Ser Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser
35 40 45
Asn Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr
50 55 60
Lys Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe
65 70 75 80
Ala Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr
85 90 95
Thr Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr
100 105 110
Asn Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe
115 120 125
Leu Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu
130 135 140
Phe Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser
145 150 155 160
Gln Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn
165 170 175
Leu Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr
180 185 190
Ser Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe
195 200 205
Ser Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr
210 215 220
Arg Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly
225 230 235 240
Asp Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly
245 250 255
Tyr Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr
260 265 270
Ile Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys
275 280 285
Cys Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser
290 295 300
Asn Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile
305 310 315 320
Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala
325 330 335
Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp
340 345 350
Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr
355 360 365
Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr
370 375 380
Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro
385 390 395 400
Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp
405 410 415
Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys
420 425 430
Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn
435 440 445
Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly
450 455 460
Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu
465 470 475 480
Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr
485 490 495
Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val
500 505 510
Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn
515 520 525
Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn
530 535 540
Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr
545 550 555 560
Thr Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr
565 570 575
Pro Cys Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr
580 585 590
Ser Asn Gln Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val
595 600 605
Pro Val Ala Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr
610 615 620
Ser Thr Gly Ser Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly
625 630 635 640
Ala Glu His Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala
645 650 655
Gly Ile Cys Ala Ser Tyr Gln Thr Gln Thr Asn Ser Arg Ser Val Ala
660 665 670
Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser
675 680 685
Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile
690 695 700
Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val
705 710 715 720
Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu
725 730 735
Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr
740 745 750
Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln
755 760 765
Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe
770 775 780
Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser
785 790 795 800
Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly
805 810 815
Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp
820 825 830
Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu
835 840 845
Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly
850 855 860
Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile
865 870 875 880
Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr
885 890 895
Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn
900 905 910
Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala
915 920 925
Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn
930 935 940
Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val
945 950 955 960
Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln
965 970 975
Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val
980 985 990
Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu
995 1000 1005
Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg
1010 1015 1020
Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln
1025 1030 1035
Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val Pro
1040 1045 1050
Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His Asp
1055 1060 1065
Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn Gly
1070 1075 1080
Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln Ile
1085 1090 1095
Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val Val
1100 1105 1110
Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro Glu
1115 1120 1125
Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn His
1130 1135 1140
Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn Ala
1145 1150 1155
Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu Val
1160 1165 1170
Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu Gly
1175 1180 1185
Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu Gly
1190 1195 1200
Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Met Leu
1205 1210 1215
Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys Ser
1220 1225 1230
Cys Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro Val
1235 1240 1245
Leu Lys Gly Val Lys Leu His Tyr Thr
1250 1255
<210> 14
<211> 1244
<212> PRT
<213> artificial
<220>
<223> Spike protein truncated by 13 amino acids at C-terminus and deleted in furin site
<400> 14
Ser Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr
1 5 10 15
Thr Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg
20 25 30
Ser Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser
35 40 45
Asn Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr
50 55 60
Lys Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe
65 70 75 80
Ala Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr
85 90 95
Thr Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr
100 105 110
Asn Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe
115 120 125
Leu Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu
130 135 140
Phe Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser
145 150 155 160
Gln Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn
165 170 175
Leu Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr
180 185 190
Ser Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe
195 200 205
Ser Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr
210 215 220
Arg Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly
225 230 235 240
Asp Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly
245 250 255
Tyr Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr
260 265 270
Ile Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys
275 280 285
Cys Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser
290 295 300
Asn Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile
305 310 315 320
Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala
325 330 335
Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp
340 345 350
Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr
355 360 365
Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr
370 375 380
Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro
385 390 395 400
Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp
405 410 415
Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys
420 425 430
Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn
435 440 445
Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly
450 455 460
Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu
465 470 475 480
Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr
485 490 495
Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val
500 505 510
Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn
515 520 525
Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn
530 535 540
Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr
545 550 555 560
Thr Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr
565 570 575
Pro Cys Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr
580 585 590
Ser Asn Gln Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val
595 600 605
Pro Val Ala Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr
610 615 620
Ser Thr Gly Ser Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly
625 630 635 640
Ala Glu His Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala
645 650 655
Gly Ile Cys Ala Ser Tyr Gln Thr Gln Thr Asn Ser Arg Ser Val Ala
660 665 670
Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser
675 680 685
Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile
690 695 700
Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val
705 710 715 720
Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu
725 730 735
Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr
740 745 750
Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln
755 760 765
Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe
770 775 780
Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser
785 790 795 800
Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly
805 810 815
Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp
820 825 830
Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu
835 840 845
Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly
850 855 860
Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile
865 870 875 880
Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr
885 890 895
Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn
900 905 910
Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala
915 920 925
Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn
930 935 940
Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val
945 950 955 960
Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln
965 970 975
Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val
980 985 990
Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu
995 1000 1005
Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg
1010 1015 1020
Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln
1025 1030 1035
Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val Pro
1040 1045 1050
Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His Asp
1055 1060 1065
Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn Gly
1070 1075 1080
Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln Ile
1085 1090 1095
Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val Val
1100 1105 1110
Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro Glu
1115 1120 1125
Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn His
1130 1135 1140
Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn Ala
1145 1150 1155
Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu Val
1160 1165 1170
Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu Gly
1175 1180 1185
Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu Gly
1190 1195 1200
Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Met Leu
1205 1210 1215
Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys Ser
1220 1225 1230
Cys Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp
1235 1240
<210> 15
<211> 1243
<212> PRT
<213> artificial
<220>
<223> Spike protein with 14 amino acids truncated at C-terminus and furin site deleted
<400> 15
Ser Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr
1 5 10 15
Thr Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg
20 25 30
Ser Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser
35 40 45
Asn Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr
50 55 60
Lys Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe
65 70 75 80
Ala Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr
85 90 95
Thr Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr
100 105 110
Asn Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe
115 120 125
Leu Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu
130 135 140
Phe Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser
145 150 155 160
Gln Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn
165 170 175
Leu Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr
180 185 190
Ser Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe
195 200 205
Ser Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr
210 215 220
Arg Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly
225 230 235 240
Asp Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly
245 250 255
Tyr Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr
260 265 270
Ile Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys
275 280 285
Cys Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser
290 295 300
Asn Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile
305 310 315 320
Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala
325 330 335
Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp
340 345 350
Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr
355 360 365
Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr
370 375 380
Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro
385 390 395 400
Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp
405 410 415
Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys
420 425 430
Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn
435 440 445
Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly
450 455 460
Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu
465 470 475 480
Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr
485 490 495
Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val
500 505 510
Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn
515 520 525
Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn
530 535 540
Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr
545 550 555 560
Thr Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr
565 570 575
Pro Cys Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr
580 585 590
Ser Asn Gln Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val
595 600 605
Pro Val Ala Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr
610 615 620
Ser Thr Gly Ser Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly
625 630 635 640
Ala Glu His Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala
645 650 655
Gly Ile Cys Ala Ser Tyr Gln Thr Gln Thr Asn Ser Arg Ser Val Ala
660 665 670
Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser
675 680 685
Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile
690 695 700
Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val
705 710 715 720
Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu
725 730 735
Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr
740 745 750
Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln
755 760 765
Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe
770 775 780
Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser
785 790 795 800
Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly
805 810 815
Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp
820 825 830
Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu
835 840 845
Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly
850 855 860
Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile
865 870 875 880
Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr
885 890 895
Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn
900 905 910
Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala
915 920 925
Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn
930 935 940
Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val
945 950 955 960
Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln
965 970 975
Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val
980 985 990
Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu
995 1000 1005
Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg
1010 1015 1020
Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln
1025 1030 1035
Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val Pro
1040 1045 1050
Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His Asp
1055 1060 1065
Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn Gly
1070 1075 1080
Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln Ile
1085 1090 1095
Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val Val
1100 1105 1110
Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro Glu
1115 1120 1125
Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn His
1130 1135 1140
Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn Ala
1145 1150 1155
Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu Val
1160 1165 1170
Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu Gly
1175 1180 1185
Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu Gly
1190 1195 1200
Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Met Leu
1205 1210 1215
Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys Ser
1220 1225 1230
Cys Gly Ser Cys Cys Lys Phe Asp Glu Asp
1235 1240
<210> 16
<211> 1242
<212> PRT
<213> artificial
<220>
<223> Spike protein truncated by 15 amino acids at C-terminus and having furin site deleted
<400> 16
Ser Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr
1 5 10 15
Thr Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg
20 25 30
Ser Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser
35 40 45
Asn Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr
50 55 60
Lys Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe
65 70 75 80
Ala Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr
85 90 95
Thr Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr
100 105 110
Asn Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe
115 120 125
Leu Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu
130 135 140
Phe Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser
145 150 155 160
Gln Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn
165 170 175
Leu Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr
180 185 190
Ser Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe
195 200 205
Ser Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr
210 215 220
Arg Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly
225 230 235 240
Asp Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly
245 250 255
Tyr Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr
260 265 270
Ile Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys
275 280 285
Cys Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser
290 295 300
Asn Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile
305 310 315 320
Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala
325 330 335
Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp
340 345 350
Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr
355 360 365
Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr
370 375 380
Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro
385 390 395 400
Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp
405 410 415
Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys
420 425 430
Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn
435 440 445
Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly
450 455 460
Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu
465 470 475 480
Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr
485 490 495
Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val
500 505 510
Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn
515 520 525
Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn
530 535 540
Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr
545 550 555 560
Thr Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr
565 570 575
Pro Cys Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr
580 585 590
Ser Asn Gln Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val
595 600 605
Pro Val Ala Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr
610 615 620
Ser Thr Gly Ser Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly
625 630 635 640
Ala Glu His Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala
645 650 655
Gly Ile Cys Ala Ser Tyr Gln Thr Gln Thr Asn Ser Arg Ser Val Ala
660 665 670
Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser
675 680 685
Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile
690 695 700
Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val
705 710 715 720
Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu
725 730 735
Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr
740 745 750
Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln
755 760 765
Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe
770 775 780
Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser
785 790 795 800
Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly
805 810 815
Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp
820 825 830
Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu
835 840 845
Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly
850 855 860
Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile
865 870 875 880
Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr
885 890 895
Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn
900 905 910
Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala
915 920 925
Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn
930 935 940
Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val
945 950 955 960
Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln
965 970 975
Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val
980 985 990
Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu
995 1000 1005
Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg
1010 1015 1020
Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln
1025 1030 1035
Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val Pro
1040 1045 1050
Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His Asp
1055 1060 1065
Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn Gly
1070 1075 1080
Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln Ile
1085 1090 1095
Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val Val
1100 1105 1110
Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro Glu
1115 1120 1125
Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn His
1130 1135 1140
Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn Ala
1145 1150 1155
Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu Val
1160 1165 1170
Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu Gly
1175 1180 1185
Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu Gly
1190 1195 1200
Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Met Leu
1205 1210 1215
Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys Ser
1220 1225 1230
Cys Gly Ser Cys Cys Lys Phe Asp Glu
1235 1240
<210> 17
<211> 1241
<212> PRT
<213> artificial
<220>
<223> Spike protein truncated by 16 amino acids at C-terminus and having furin site deleted
<400> 17
Ser Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr
1 5 10 15
Thr Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg
20 25 30
Ser Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser
35 40 45
Asn Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr
50 55 60
Lys Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe
65 70 75 80
Ala Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr
85 90 95
Thr Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr
100 105 110
Asn Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe
115 120 125
Leu Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu
130 135 140
Phe Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser
145 150 155 160
Gln Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn
165 170 175
Leu Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr
180 185 190
Ser Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe
195 200 205
Ser Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr
210 215 220
Arg Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly
225 230 235 240
Asp Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly
245 250 255
Tyr Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr
260 265 270
Ile Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys
275 280 285
Cys Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser
290 295 300
Asn Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile
305 310 315 320
Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala
325 330 335
Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp
340 345 350
Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr
355 360 365
Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr
370 375 380
Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro
385 390 395 400
Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp
405 410 415
Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys
420 425 430
Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn
435 440 445
Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly
450 455 460
Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu
465 470 475 480
Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr
485 490 495
Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val
500 505 510
Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn
515 520 525
Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn
530 535 540
Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr
545 550 555 560
Thr Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr
565 570 575
Pro Cys Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr
580 585 590
Ser Asn Gln Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val
595 600 605
Pro Val Ala Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr
610 615 620
Ser Thr Gly Ser Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly
625 630 635 640
Ala Glu His Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala
645 650 655
Gly Ile Cys Ala Ser Tyr Gln Thr Gln Thr Asn Ser Arg Ser Val Ala
660 665 670
Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser
675 680 685
Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile
690 695 700
Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val
705 710 715 720
Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu
725 730 735
Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr
740 745 750
Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln
755 760 765
Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe
770 775 780
Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser
785 790 795 800
Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly
805 810 815
Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp
820 825 830
Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu
835 840 845
Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly
850 855 860
Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile
865 870 875 880
Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr
885 890 895
Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn
900 905 910
Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala
915 920 925
Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn
930 935 940
Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val
945 950 955 960
Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln
965 970 975
Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val
980 985 990
Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu
995 1000 1005
Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg
1010 1015 1020
Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln
1025 1030 1035
Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val Pro
1040 1045 1050
Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His Asp
1055 1060 1065
Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn Gly
1070 1075 1080
Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln Ile
1085 1090 1095
Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val Val
1100 1105 1110
Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro Glu
1115 1120 1125
Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn His
1130 1135 1140
Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn Ala
1145 1150 1155
Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu Val
1160 1165 1170
Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu Gly
1175 1180 1185
Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu Gly
1190 1195 1200
Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Met Leu
1205 1210 1215
Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys Ser
1220 1225 1230
Cys Gly Ser Cys Cys Lys Phe Asp
1235 1240
<210> 18
<211> 1240
<212> PRT
<213> artificial
<220>
<223> Spike protein truncated by 17 amino acids at C-terminus and having furin site deleted
<400> 18
Ser Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr
1 5 10 15
Thr Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg
20 25 30
Ser Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser
35 40 45
Asn Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr
50 55 60
Lys Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe
65 70 75 80
Ala Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr
85 90 95
Thr Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr
100 105 110
Asn Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe
115 120 125
Leu Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu
130 135 140
Phe Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser
145 150 155 160
Gln Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn
165 170 175
Leu Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr
180 185 190
Ser Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe
195 200 205
Ser Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr
210 215 220
Arg Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly
225 230 235 240
Asp Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly
245 250 255
Tyr Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr
260 265 270
Ile Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys
275 280 285
Cys Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser
290 295 300
Asn Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile
305 310 315 320
Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala
325 330 335
Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp
340 345 350
Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr
355 360 365
Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr
370 375 380
Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro
385 390 395 400
Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp
405 410 415
Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys
420 425 430
Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn
435 440 445
Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly
450 455 460
Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu
465 470 475 480
Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr
485 490 495
Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val
500 505 510
Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn
515 520 525
Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn
530 535 540
Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr
545 550 555 560
Thr Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr
565 570 575
Pro Cys Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr
580 585 590
Ser Asn Gln Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val
595 600 605
Pro Val Ala Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr
610 615 620
Ser Thr Gly Ser Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly
625 630 635 640
Ala Glu His Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala
645 650 655
Gly Ile Cys Ala Ser Tyr Gln Thr Gln Thr Asn Ser Arg Ser Val Ala
660 665 670
Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser
675 680 685
Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile
690 695 700
Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val
705 710 715 720
Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu
725 730 735
Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr
740 745 750
Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln
755 760 765
Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe
770 775 780
Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser
785 790 795 800
Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly
805 810 815
Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp
820 825 830
Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu
835 840 845
Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly
850 855 860
Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile
865 870 875 880
Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr
885 890 895
Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn
900 905 910
Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala
915 920 925
Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn
930 935 940
Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val
945 950 955 960
Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln
965 970 975
Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val
980 985 990
Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu
995 1000 1005
Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg
1010 1015 1020
Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln
1025 1030 1035
Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val Pro
1040 1045 1050
Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His Asp
1055 1060 1065
Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn Gly
1070 1075 1080
Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln Ile
1085 1090 1095
Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val Val
1100 1105 1110
Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro Glu
1115 1120 1125
Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn His
1130 1135 1140
Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn Ala
1145 1150 1155
Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu Val
1160 1165 1170
Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu Gly
1175 1180 1185
Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu Gly
1190 1195 1200
Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Met Leu
1205 1210 1215
Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys Ser
1220 1225 1230
Cys Gly Ser Cys Cys Lys Phe
1235 1240
<210> 19
<211> 1238
<212> PRT
<213> artificial
<220>
<223> Spike protein with 19 amino acids truncated at C-terminus and furin site deleted
<400> 19
Ser Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr
1 5 10 15
Thr Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg
20 25 30
Ser Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser
35 40 45
Asn Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr
50 55 60
Lys Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe
65 70 75 80
Ala Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr
85 90 95
Thr Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr
100 105 110
Asn Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe
115 120 125
Leu Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu
130 135 140
Phe Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser
145 150 155 160
Gln Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn
165 170 175
Leu Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr
180 185 190
Ser Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe
195 200 205
Ser Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr
210 215 220
Arg Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly
225 230 235 240
Asp Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly
245 250 255
Tyr Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr
260 265 270
Ile Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys
275 280 285
Cys Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser
290 295 300
Asn Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile
305 310 315 320
Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala
325 330 335
Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp
340 345 350
Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr
355 360 365
Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr
370 375 380
Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro
385 390 395 400
Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp
405 410 415
Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys
420 425 430
Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn
435 440 445
Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly
450 455 460
Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu
465 470 475 480
Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr
485 490 495
Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val
500 505 510
Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn
515 520 525
Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn
530 535 540
Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr
545 550 555 560
Thr Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr
565 570 575
Pro Cys Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr
580 585 590
Ser Asn Gln Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val
595 600 605
Pro Val Ala Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr
610 615 620
Ser Thr Gly Ser Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly
625 630 635 640
Ala Glu His Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala
645 650 655
Gly Ile Cys Ala Ser Tyr Gln Thr Gln Thr Asn Ser Arg Ser Val Ala
660 665 670
Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser
675 680 685
Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile
690 695 700
Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val
705 710 715 720
Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu
725 730 735
Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr
740 745 750
Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln
755 760 765
Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe
770 775 780
Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser
785 790 795 800
Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly
805 810 815
Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp
820 825 830
Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu
835 840 845
Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly
850 855 860
Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile
865 870 875 880
Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr
885 890 895
Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn
900 905 910
Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala
915 920 925
Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn
930 935 940
Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val
945 950 955 960
Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln
965 970 975
Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val
980 985 990
Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu
995 1000 1005
Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg
1010 1015 1020
Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln
1025 1030 1035
Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val Pro
1040 1045 1050
Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His Asp
1055 1060 1065
Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn Gly
1070 1075 1080
Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln Ile
1085 1090 1095
Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val Val
1100 1105 1110
Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro Glu
1115 1120 1125
Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn His
1130 1135 1140
Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn Ala
1145 1150 1155
Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu Val
1160 1165 1170
Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu Gly
1175 1180 1185
Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu Gly
1190 1195 1200
Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Met Leu
1205 1210 1215
Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys Ser
1220 1225 1230
Cys Gly Ser Cys Cys
1235
<210> 20
<211> 1237
<212> PRT
<213> artificial
<220>
<223> Spike protein truncated by 20 amino acids at C-terminus and having furin site deleted
<400> 20
Ser Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr
1 5 10 15
Thr Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg
20 25 30
Ser Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser
35 40 45
Asn Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr
50 55 60
Lys Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe
65 70 75 80
Ala Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr
85 90 95
Thr Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr
100 105 110
Asn Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe
115 120 125
Leu Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu
130 135 140
Phe Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser
145 150 155 160
Gln Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn
165 170 175
Leu Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr
180 185 190
Ser Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe
195 200 205
Ser Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr
210 215 220
Arg Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly
225 230 235 240
Asp Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly
245 250 255
Tyr Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr
260 265 270
Ile Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys
275 280 285
Cys Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser
290 295 300
Asn Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile
305 310 315 320
Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala
325 330 335
Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp
340 345 350
Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr
355 360 365
Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr
370 375 380
Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro
385 390 395 400
Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp
405 410 415
Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys
420 425 430
Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn
435 440 445
Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly
450 455 460
Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu
465 470 475 480
Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr
485 490 495
Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val
500 505 510
Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn
515 520 525
Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn
530 535 540
Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr
545 550 555 560
Thr Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr
565 570 575
Pro Cys Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr
580 585 590
Ser Asn Gln Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val
595 600 605
Pro Val Ala Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr
610 615 620
Ser Thr Gly Ser Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly
625 630 635 640
Ala Glu His Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala
645 650 655
Gly Ile Cys Ala Ser Tyr Gln Thr Gln Thr Asn Ser Arg Ser Val Ala
660 665 670
Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser
675 680 685
Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile
690 695 700
Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val
705 710 715 720
Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu
725 730 735
Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr
740 745 750
Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln
755 760 765
Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe
770 775 780
Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser
785 790 795 800
Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly
805 810 815
Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp
820 825 830
Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu
835 840 845
Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly
850 855 860
Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile
865 870 875 880
Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr
885 890 895
Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn
900 905 910
Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala
915 920 925
Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn
930 935 940
Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val
945 950 955 960
Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln
965 970 975
Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val
980 985 990
Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu
995 1000 1005
Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg
1010 1015 1020
Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln
1025 1030 1035
Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val Pro
1040 1045 1050
Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His Asp
1055 1060 1065
Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn Gly
1070 1075 1080
Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln Ile
1085 1090 1095
Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val Val
1100 1105 1110
Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro Glu
1115 1120 1125
Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn His
1130 1135 1140
Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn Ala
1145 1150 1155
Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu Val
1160 1165 1170
Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu Gly
1175 1180 1185
Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu Gly
1190 1195 1200
Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Met Leu
1205 1210 1215
Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys Ser
1220 1225 1230
Cys Gly Ser Cys
1235
<210> 21
<211> 1236
<212> PRT
<213> artificial
<220>
<223> Spike protein truncated by 21 amino acids at C-terminus and having furin site deleted
<400> 21
Ser Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr
1 5 10 15
Thr Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg
20 25 30
Ser Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser
35 40 45
Asn Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr
50 55 60
Lys Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe
65 70 75 80
Ala Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr
85 90 95
Thr Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr
100 105 110
Asn Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe
115 120 125
Leu Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu
130 135 140
Phe Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser
145 150 155 160
Gln Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn
165 170 175
Leu Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr
180 185 190
Ser Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe
195 200 205
Ser Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr
210 215 220
Arg Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly
225 230 235 240
Asp Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly
245 250 255
Tyr Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr
260 265 270
Ile Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys
275 280 285
Cys Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser
290 295 300
Asn Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile
305 310 315 320
Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala
325 330 335
Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp
340 345 350
Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr
355 360 365
Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr
370 375 380
Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro
385 390 395 400
Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp
405 410 415
Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys
420 425 430
Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn
435 440 445
Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly
450 455 460
Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu
465 470 475 480
Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr
485 490 495
Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val
500 505 510
Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn
515 520 525
Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn
530 535 540
Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr
545 550 555 560
Thr Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr
565 570 575
Pro Cys Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr
580 585 590
Ser Asn Gln Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val
595 600 605
Pro Val Ala Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr
610 615 620
Ser Thr Gly Ser Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly
625 630 635 640
Ala Glu His Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala
645 650 655
Gly Ile Cys Ala Ser Tyr Gln Thr Gln Thr Asn Ser Arg Ser Val Ala
660 665 670
Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser
675 680 685
Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile
690 695 700
Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val
705 710 715 720
Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu
725 730 735
Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr
740 745 750
Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln
755 760 765
Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe
770 775 780
Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser
785 790 795 800
Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly
805 810 815
Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp
820 825 830
Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu
835 840 845
Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly
850 855 860
Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile
865 870 875 880
Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr
885 890 895
Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn
900 905 910
Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala
915 920 925
Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn
930 935 940
Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val
945 950 955 960
Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln
965 970 975
Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val
980 985 990
Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu
995 1000 1005
Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg
1010 1015 1020
Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln
1025 1030 1035
Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val Pro
1040 1045 1050
Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His Asp
1055 1060 1065
Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn Gly
1070 1075 1080
Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln Ile
1085 1090 1095
Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val Val
1100 1105 1110
Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro Glu
1115 1120 1125
Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn His
1130 1135 1140
Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn Ala
1145 1150 1155
Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu Val
1160 1165 1170
Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu Gly
1175 1180 1185
Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu Gly
1190 1195 1200
Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Met Leu
1205 1210 1215
Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys Ser
1220 1225 1230
Cys Gly Ser
1235
<210> 22
<211> 1235
<212> PRT
<213> artificial
<220>
<223> Spike protein truncated by 22 amino acids at C-terminus and having furin site deleted
<400> 22
Ser Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr
1 5 10 15
Thr Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg
20 25 30
Ser Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser
35 40 45
Asn Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr
50 55 60
Lys Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe
65 70 75 80
Ala Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr
85 90 95
Thr Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr
100 105 110
Asn Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe
115 120 125
Leu Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu
130 135 140
Phe Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser
145 150 155 160
Gln Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn
165 170 175
Leu Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr
180 185 190
Ser Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe
195 200 205
Ser Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr
210 215 220
Arg Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly
225 230 235 240
Asp Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly
245 250 255
Tyr Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr
260 265 270
Ile Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys
275 280 285
Cys Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser
290 295 300
Asn Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile
305 310 315 320
Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala
325 330 335
Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp
340 345 350
Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr
355 360 365
Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr
370 375 380
Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro
385 390 395 400
Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp
405 410 415
Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys
420 425 430
Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn
435 440 445
Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly
450 455 460
Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu
465 470 475 480
Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr
485 490 495
Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val
500 505 510
Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn
515 520 525
Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn
530 535 540
Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr
545 550 555 560
Thr Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr
565 570 575
Pro Cys Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr
580 585 590
Ser Asn Gln Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val
595 600 605
Pro Val Ala Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr
610 615 620
Ser Thr Gly Ser Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly
625 630 635 640
Ala Glu His Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala
645 650 655
Gly Ile Cys Ala Ser Tyr Gln Thr Gln Thr Asn Ser Arg Ser Val Ala
660 665 670
Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser
675 680 685
Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile
690 695 700
Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val
705 710 715 720
Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu
725 730 735
Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr
740 745 750
Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln
755 760 765
Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe
770 775 780
Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser
785 790 795 800
Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly
805 810 815
Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp
820 825 830
Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu
835 840 845
Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly
850 855 860
Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile
865 870 875 880
Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr
885 890 895
Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn
900 905 910
Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala
915 920 925
Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn
930 935 940
Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val
945 950 955 960
Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln
965 970 975
Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val
980 985 990
Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu
995 1000 1005
Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg
1010 1015 1020
Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln
1025 1030 1035
Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val Pro
1040 1045 1050
Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His Asp
1055 1060 1065
Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn Gly
1070 1075 1080
Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln Ile
1085 1090 1095
Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val Val
1100 1105 1110
Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro Glu
1115 1120 1125
Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn His
1130 1135 1140
Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn Ala
1145 1150 1155
Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu Val
1160 1165 1170
Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu Gly
1175 1180 1185
Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu Gly
1190 1195 1200
Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Met Leu
1205 1210 1215
Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys Ser
1220 1225 1230
Cys Gly
1235
<210> 23
<211> 1234
<212> PRT
<213> artificial
<220>
<223> Spike protein truncated by 23 amino acids at C-terminus and deleted at furin site
<400> 23
Ser Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr
1 5 10 15
Thr Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg
20 25 30
Ser Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser
35 40 45
Asn Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr
50 55 60
Lys Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe
65 70 75 80
Ala Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr
85 90 95
Thr Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr
100 105 110
Asn Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe
115 120 125
Leu Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu
130 135 140
Phe Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser
145 150 155 160
Gln Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn
165 170 175
Leu Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr
180 185 190
Ser Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe
195 200 205
Ser Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr
210 215 220
Arg Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly
225 230 235 240
Asp Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly
245 250 255
Tyr Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr
260 265 270
Ile Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys
275 280 285
Cys Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser
290 295 300
Asn Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile
305 310 315 320
Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala
325 330 335
Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp
340 345 350
Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr
355 360 365
Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr
370 375 380
Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro
385 390 395 400
Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp
405 410 415
Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys
420 425 430
Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn
435 440 445
Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly
450 455 460
Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu
465 470 475 480
Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr
485 490 495
Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val
500 505 510
Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn
515 520 525
Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn
530 535 540
Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr
545 550 555 560
Thr Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr
565 570 575
Pro Cys Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr
580 585 590
Ser Asn Gln Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val
595 600 605
Pro Val Ala Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr
610 615 620
Ser Thr Gly Ser Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly
625 630 635 640
Ala Glu His Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala
645 650 655
Gly Ile Cys Ala Ser Tyr Gln Thr Gln Thr Asn Ser Arg Ser Val Ala
660 665 670
Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser
675 680 685
Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile
690 695 700
Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val
705 710 715 720
Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu
725 730 735
Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr
740 745 750
Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln
755 760 765
Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe
770 775 780
Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser
785 790 795 800
Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly
805 810 815
Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp
820 825 830
Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu
835 840 845
Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly
850 855 860
Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile
865 870 875 880
Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr
885 890 895
Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn
900 905 910
Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala
915 920 925
Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn
930 935 940
Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val
945 950 955 960
Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln
965 970 975
Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val
980 985 990
Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu
995 1000 1005
Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg
1010 1015 1020
Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln
1025 1030 1035
Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val Pro
1040 1045 1050
Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His Asp
1055 1060 1065
Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn Gly
1070 1075 1080
Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln Ile
1085 1090 1095
Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val Val
1100 1105 1110
Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro Glu
1115 1120 1125
Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn His
1130 1135 1140
Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn Ala
1145 1150 1155
Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu Val
1160 1165 1170
Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu Gly
1175 1180 1185
Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu Gly
1190 1195 1200
Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Met Leu
1205 1210 1215
Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys Ser
1220 1225 1230
Cys
<210> 24
<211> 24
<212> PRT
<213> artificial
<220>
<223> Signal peptide of CD5
<400> 24
Met Pro Met Gly Ser Leu Gln Pro Leu Ala Thr Leu Tyr Leu Leu Gly
1 5 10 15
Met Leu Val Ala Ser Val Leu Ala
20
<210> 25
<211> 20
<212> PRT
<213> artificial
<220>
<223> Signal peptide of CD14
<400> 25
Met Glu Arg Ala Ser Cys Leu Leu Leu Leu Leu Leu Pro Leu Val His
1 5 10 15
Val Ser Ala Thr
20
<210> 26
<211> 19
<212> PRT
<213> artificial
<220>
<223> signal peptide of IgG heavy chain
<400> 26
Met Asp Trp Thr Trp Arg Val Phe Cys Leu Leu Ala Val Thr Pro Gly
1 5 10 15
Ala His Pro
<210> 27
<211> 19
<212> PRT
<213> artificial
<220>
<223> Signal peptide of Immunoglobulin light chain
<400> 27
Met Ala Trp Ser Pro Leu Phe Leu Thr Leu Ile Thr His Cys Ala Gly
1 5 10 15
Ser Trp Ala
<210> 28
<211> 22
<212> PRT
<213> artificial
<220>
<223> Signal peptide of tPA
<400> 28
Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly
1 5 10 15
Ala Val Phe Val Ser Pro
20
<210> 29
<211> 21
<212> PRT
<213> artificial
<220>
<223> signal peptide of SCGB1D1
<400> 29
Met Arg Leu Ser Val Cys Leu Leu Leu Leu Thr Leu Ala Leu Cys Cys
1 5 10 15
Tyr Arg Ala Asn Ala
20
<210> 30
<211> 18
<212> PRT
<213> artificial
<220>
<223> Signal peptide of Serum album preproprotein
<400> 30
Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala
1 5 10 15
Tyr Ser
<210> 31
<211> 17
<212> PRT
<213> artificial
<220>
<223> Gluc Signal peptide
<400> 31
Met Gly Val Lys Val Leu Phe Ala Leu Ile Cys Ile Ala Val Ala Glu
1 5 10 15
Ala
<210> 32
<211> 20
<212> PRT
<213> artificial
<220>
<223> Signal peptide of IL-2
<400> 32
Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu
1 5 10 15
Val Thr Asn Ser
20
<210> 33
<211> 23
<212> PRT
<213> artificial
<220>
<223> IFN alpha 2 signal peptide
<400> 33
Met Ala Leu Thr Phe Ala Leu Leu Val Ala Leu Leu Val Leu Ser Cys
1 5 10 15
Lys Ser Ser Cys Ser Val Gly
20
<210> 34
<211> 12
<212> PRT
<213> artificial
<220>
<223> Signal peptide of Spike protein
<400> 34
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser
1 5 10
<210> 35
<211> 11
<212> PRT
<213> artificial
<220>
<223> tag
<400> 35
Gly Gly Thr Glu Thr Ser Gln Val Ala Pro Ala
1 5 10

Claims (14)

1. A truncated Spike protein or variant thereof, wherein, (1) the C-terminus of said truncated Spike protein is truncated by 13-23 amino acids, and/or, (2) the amino acids of said truncated Spike protein are deleted at the following positions: positions corresponding to positions 669 and 672 of SEQ ID NO: 1;
wherein the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions; e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid conservative substitutions) as compared to the truncated Spike protein.
2. The truncated Spike protein or variant thereof of claim 1, wherein the C-terminus of the truncated Spike protein is truncated by 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 amino acids compared to the Spike protein of wild-type SARS-CoV-2;
preferably, the truncated Spike protein also contains deletions compared to the Spike protein of wild SARS-CoV-2, i.e., the amino acids at the following positions are deleted: positions corresponding to positions 669 and 672 of SEQ ID NO: 1;
preferably, the amino acid deleted is a furin cleavage site;
preferably, the amino acid that is deleted is PRRA.
3. The truncated Spike protein or variant thereof of claim 1 wherein the amino acids of said truncated Spike protein are deleted at the following positions compared to the Spike protein of wild SARS-CoV-2: positions corresponding to positions 669 and 672 of SEQ ID NO: 1;
preferably, the C-terminus of the truncated Spike protein is further truncated by 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 amino acids compared to the Spike protein of wild SARS-CoV-2;
preferably, the amino acid deleted is a furin cleavage site;
preferably, the amino acid that is deleted is PRRA.
4. The truncated Spike protein or variant thereof of any one of claims 1 to 3 having an amino acid sequence selected from the group consisting of:
(1)SEQ ID NO:2-SEQ ID NO:23；
(2) an amino acid sequence having one or more amino acid substitutions, deletions or additions (e.g.1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions; e.g.1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid conservative substitutions) compared to the sequence set forth in any of SEQ ID NO 2-23;
(3) an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a sequence as set forth in any one of SEQ ID No. 2-SEQ ID No. 23.
5. A fusion protein comprising the truncated Spike protein of any one of claims 1-4 or a variant thereof and an additional polypeptide;
preferably, the additional polypeptide is linked to the N-terminus or C-terminus of the protein, optionally via a linker;
preferably, the additional polypeptide is selected from a tag, a signal peptide or a leader, a detectable label (e.g., luciferase (fluc), Green Fluorescent Protein (GFP)), or any combination thereof;
preferably, the signal peptide is selected from the group consisting of Spike protein, CD5, CD14, IgG heavy chain, Immunoglobulin light chain (Immunoglobulin light chain), tissue plasminogen activator (tPA), SCGB1D1, Serum albumin preproprotein (Serum albumin), Gluc, IL-2, and the native signal peptide of IFN α 2;
preferably, the tag comprises a histidine tag (His), a glutathione mercaptotransferase tag (GST), influenza virus Hemagglutinin (HA), Flag-tag;
preferably, the signal peptide has an amino acid sequence selected from the group consisting of: 24-33 of SEQ ID NO;
preferably, the tag has an amino acid sequence as shown in SEQ ID NO. 35.
6. An isolated nucleic acid molecule encoding the truncated Spike protein or variant thereof of any one of claims 1 to 4, or encoding the fusion protein of claim 5.
7. A vector comprising the nucleic acid molecule of claim 6;
preferably, the vector is a eukaryotic expression plasmid comprising the nucleic acid molecule of claim 6;
preferably, the eukaryotic expression plasmid is selected from pcDNA3.1+, pCAG3.1 and pCMV3.1.
8. A host cell comprising the nucleic acid molecule of claim 6 or the vector of claim 7;
preferably, the cell is a mammalian cell;
preferably, the mammalian cell is a human cell, e.g., a hematopoietic cell, an epithelial cell, a liver cell, a tumor cell, a neural cell;
preferably, the cell is a cell that expresses or overexpresses human ACE2 protein (e.g., 293T-ACE 2);
preferably, the host cell further comprises a packaging plasmid.
9. A viroid particle comprising a truncated Spike protein according to any one of claims 1 to 4 or a variant thereof or comprising a fusion protein according to claim 5; alternatively, the viroid consists of Spike protein or variant thereof according to any of claims 1 to 4 or fusion protein according to claim 5;
preferably, the viroid particle further comprises a nucleic acid component selected from any one or more of the following:
(a) exogenous DNA, or exogenous DNA encoding a foreign protein or polypeptide, or exogenous DNA encoding RNA;
(b) a vector optionally comprising the exogenous DNA described in (a);
(c) exogenous RNA, or exogenous RNA encoding an exogenous protein or polypeptide;
(d) a vector, optionally comprising the exogenous RNA described in (c);
preferably, the nucleic acid component is a packaging plasmid.
10. A method for producing SARS-CoV-2 pseudovirus, the method comprising transferring the vector of claim 7 and a packaging plasmid into a host cell;
preferably, the vector of claim 7 and a packaging plasmid are transfected into a host cell;
preferably, the vector of claim 7 and the packaging plasmid are used in a transfection procedure in a mass ratio of 1-3:1-4, e.g., 3: 1. 2: 1. 1: 1. 1: 2. 1: 3 or 1: 4; preferably, the vector of claim 7 and the packaging plasmid used in the transfection process are present in a mass ratio of 1: 3;
preferably, the transfection reagent used in the transfection process is selected from the group consisting of: lipofectamine2000, lipofectamine3000, PEI, or any combination thereof;
preferably, the host cell is a human cell, e.g., a hematopoietic cell, an epithelial cell, a liver cell, a tumor cell, a neural cell;
preferably, the viroid-infected cell is a cell that overexpresses human ACE2 protein (e.g., 293T-ACE 2).
11. A kit comprising one or more components selected from the group consisting of: the truncated Spike protein or variant thereof according to any one of claims 1 to 4, the fusion protein according to claim 5, the isolated nucleic acid molecule according to claim 6, the vector according to claim 7, the host cell according to claim 8, the viroid according to claim 9;
preferably, the kit further comprises a packaging plasmid;
preferably, the kit further comprises a transfection reagent;
preferably, the transfection reagent is selected from: lipofectamine2000, lipofectamine3000, PEI, or any combination thereof.
12. A method of detecting SARS-CoV-2 antibody neutralizing activity, the method comprising contacting the viroid particle of claim 9 with the antibody or host cell prior to, simultaneously with, or after contacting the viroid particle with the host cell.
13. A method of screening for a drug candidate capable of inhibiting SARS-CoV-2 infected cells, the method comprising contacting the viroid particle or host cell of claim 9 with the drug candidate prior to, simultaneously with, or after contacting the viroid particle with the host cell.
14. Use of the truncated Spike protein or variant thereof according to any one of claims 1 to 4, the fusion protein according to claim 5, the isolated nucleic acid molecule according to claim 6, the vector according to claim 7, the host cell according to claim 8, the viroid particle according to claim 9 for the detection or screening of anti-SARS-CoV-2 antibodies or medicaments.
CN202010630831.2A 2020-07-03 2020-07-03 SARS-CoV-2 pseudovirus Pending CN113880924A (en)

Priority Applications (1)

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Publications (1)

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Country Link
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114560915A (en) * 2021-12-27 2022-05-31 中国食品药品检定研究院 Modified high-titer SARS-CoV-2 pseudovirus

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114560915A (en) * 2021-12-27 2022-05-31 中国食品药品检定研究院 Modified high-titer SARS-CoV-2 pseudovirus
CN114560915B (en) * 2021-12-27 2024-01-09 中国食品药品检定研究院 Modified high-titer SARS-CoV-2 pseudovirus

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