KR102694894B1 - Composition for detection of neutralizing antibody against sars-cov2 variants, dectection method and kit comprising thereof - Google Patents

Abstract

The present invention relates to a composition for detecting neutralizing antibodies to type 2 severe acute respiratory syndrome coronavirus and its variants, a method and kit for detecting neutralizing antibodies comprising the same, and a technology related to a method for providing information for diagnosing possession of neutralizing antibodies.
The composition for detecting neutralizing antibodies according to the present invention utilizes a stable form of protein structure and is effective in detecting neutralizing antibodies with excellent sensitivity in the serum of patients infected with SARS-CoV-2 and vaccinated patients, and is effective in evaluating neutralizing efficacy, including the risk of viral infection.

Description

Composition for detection of neutralizing antibodies against type 2 severe acute respiratory syndrome coronavirus variants and method and kit for detecting neutralizing antibodies comprising the same {COMPOSITION FOR DETECTION OF NEUTRALIZING ANTIBODY AGAINST SARS-COV2 VARIANTS, DECTECTION METHOD AND KIT COMPRISING THEREOF}

The present invention relates to a composition for detecting neutralizing antibodies to type 2 severe acute respiratory syndrome coronavirus and its variants (e.g., alpha (B.1.1.7), beta (B1.351), gamma (P1), delta (B.1.617.2), omicron (B.1.1.529)), a method and kit for detecting neutralizing antibodies comprising the same, and a method for providing information for diagnosing possession of neutralizing antibodies.

SARS-CoV-2 is a newly discovered single-stranded RNA betacoronavirus that was named SARS-CoV-2 by the International Committee on Taxonomy of Viruses in January 2020. The infection route of SARS-CoV-2 has not yet been clearly identified, but some studies have reported that it is transmitted from bats to humans.

The genome of SARS-CoV-2 is non-segmented, approximately 30 kb in size, and encodes 10 proteins. The 10 proteins consist of a replicative polyprotein (open reading frames 1ab), four structural proteins (spike, envelope, membrane, and nucleocapsid), and five nonstructural proteins (open reading frames 3a, 6, 7a, 8, and 10).

COVID-19, caused by infection with SARS-CoV-2, has caused many infections and deaths worldwide, and efforts are currently being made to suppress COVID-19 through the development of vaccines and treatments. The World Health Organization (WHO) has listed several COVID-19 vaccine candidates undergoing clinical evaluation worldwide, and from a global perspective, it is expected that there will be many more COVID-19 vaccine candidates in the preclinical development stage.

Immune responses to vaccination are commonly measured in blood (cellular immune response) and serum (humoral immune response). Cell-mediated immune responses are measured by quantifying the number of subsets of lymphocyte populations (e.g., flow cytometry for CD4 and CD8 levels) and functional assays (e.g., interferon-gamma release assay). Humoral immune responses are measured by immunoassays (e.g., quantifying IgM and IgG antibody levels or titers using ELISA) and functional assays (e.g., neutralizing antibody bioassays).

IgA, IgM, and IgG antibody ELISA assays using plasma or serum for immunodiagnosis of SARS-CoV-2 are helpful in identifying individuals with an adaptive immune response to SARS-CoV-2, indicating recent or previous infection. IgM antibodies are typically detected in the early phase of infection, approximately 5–7 days after symptom onset. IgG antibodies are detected during the active and later phases of infection, or during recurrent infections. A small percentage of antibodies bind to sites on the virus that interact with host proteins, thereby blocking the virus from entering the host. These are known as neutralizing antibodies. The primary target of neutralizing antibodies in coronaviruses is the spike (S) protein, a trimeric glycoprotein anchored to the viral membrane. Potent neutralizing antibodies often target the receptor-interacting site of S1 (the N-terminal domain and receptor-binding domain region), inhibiting host-receptor interactions and preventing virus entry into cells.

Recently, there has been interest worldwide in why the UK and South Africa variants are spreading so quickly and whether they can be neutralized by existing vaccines. The first analysis results are gradually coming out by detecting the virus variants and constituent mutations using cell and animal models of SARS-CoV-2, and then testing the response to vaccines and antibodies triggered by natural infection.

The most worrisome variant viruses worldwide are 'B.1.1.7' first discovered in the UK, 'B.1.351' first reported in South Africa, and 'P.1' discovered in Brazil and Korea.

A nationwide COVID-19 genomics study of the UK has identified a variant called B.1.1.7 as the culprit behind a surge in cases in the southeast of England and London, and it has now spread to other parts of the UK and been detected in other countries. Epidemiologists studying the spread of B.1.1.7 in the UK estimate that it is around 50% more transmissible than the original circulating virus.

Meanwhile, many researchers are focusing on the 'change in the spike protein' shared by both variants, 'B.1.1.7' and 'B.1.351', which is N501Y, and this mutation changes the receptor binding domain (RBD), which plays a role in binding to human proteins and allowing infection.

The main mutations of N501Y include alpha, beta, gamma, delta, and omicron, all of which have been confirmed to have changes in viral characteristics such as transmissibility, pathogenicity, and immune evasion due to amino acid substitutions in the receptor binding domain (RBD) of the spike protein (S protein) that binds to host cells. Of these, the delta mutation has been prevalent worldwide since it first occurred in India. Some analysis results have been published that the high transmissibility of this delta mutation is because the initial viral load of the infected person is higher than that of the existing COVID-19 virus infected person, and in particular, it is reported that virus transmission from asymptomatic infected people due to the characteristics of this delta mutation is the cause of the spread.

The recently prevalent Omicron variant virus was first reported to the WHO in South Africa on November 24, 2021. On November 26, 2021, the World Health Organization (WHO) evaluated the characteristics of the B.1.1.529 lineage COVID-19 variant virus through an emergency meeting of the Technical Advisory Group on Virus Evolution (TAG-VE), named it "Omicron", and classified it as a major variant virus (Variants of Concern (VOC). In South Africa, the number of confirmed cases increased rapidly for several weeks from an average of about 270 in the first and second weeks of November to 1,275 on November 24 and 2,465 on the 25th, and Omicron was first confirmed in a sample collected in South Africa on November 9. Based on the genetic information registered in the GISAID (Global Initiative on Sharing All Influenza Data), a database for sharing genetic information about the COVID-19 virus, the share of the South African Omicron variant virus increased by 50.1% in one week as of November 30, but Delta showed a pattern of increasing by 48.6% in seven weeks, so it is estimated that it will spread faster than the Delta variant virus. As of November 30, 203 cases of Omicron have been confirmed in 18 countries, including South Africa, Botswana, the Netherlands, Hong Kong, Portugal, the United Kingdom, and Australia, and are showing signs of spreading worldwide.

Omicron has many amino acid mutations, so to check for omicron mutations, whole genome analysis or targeted genome analysis targeting the S protein must be performed, and for sequencing, a sufficient amount of virus must be present in the sample to allow for analysis.

As the WHO has designated Omicron as an urgent major variant, countries around the world are also assessing that the risk of Omicron is no less than that of other variant viruses such as Delta. In addition, as Omicron is rapidly spreading around the world, there is a high possibility that it will be brought into the country from countries other than Africa.

The Omicron variant virus is of the GR type, B.1.1.529 lineage, and has more mutations than the existing mutant virus, especially about 32 amino acid mutations in the spike (S) protein. In particular, there are 15 amino acid mutations in the receptor binding domain (RBD) of the S protein, which is much more mutations than 1 in alpha, 3 in beta, 3 in gamma, and 2 in delta. This means that mutations in the S protein are more likely to change the protein structure and antigenicity than the existing mutant virus. In addition, the presence of K417N, T478K, E484A, N501Y, and D614G mutations in the same positions as the major amino acid mutation sites confirmed in alpha, beta, gamma, and delta is estimated to have increased transmissibility and the possibility of immune evasion.

The present researchers completed the present invention by using mutant sequences in the receptor binding domain (RBD) of the Spike region of a mutant virus of severe acute respiratory syndrome coronavirus type 2 to produce a novel RBD sequence and using the RBD sequence to produce a novel composition for evaluating neutralizing antibodies that can replace existing neutralizing antibody assay methods (PRNT method, CPE method).

The purpose of the present invention is to provide a composition for detecting a neutralizing antibody of type 2 severe acute respiratory syndrome coronavirus (SARS-CoV2), comprising (i) a receptor binding domain (RBD) of the spike protein of type 2 severe acute respiratory syndrome coronavirus (SARS-CoV2) conjugated with a label, or a variant thereof; and (ii) human angiotensin-converting enzyme 2 (ACE2) protein, human angiotensin-converting enzyme 2 (ACE2) protein extracellular domain, or a fragment that specifically binds to the receptor binding domain (RBD) of the spike protein of (i) or a variant thereof.

Another object of the present invention is to provide a kit for detecting a neutralizing antibody of type 2 severe acute respiratory syndrome coronavirus (SARS-CoV2), comprising: (i) a receptor binding domain (RBD) of the spike protein of type 2 severe acute respiratory syndrome coronavirus (SARS-CoV2) conjugated with a label, or a variant thereof; and (ii) human angiotensin-converting enzyme 2 (ACE2) protein, human angiotensin-converting enzyme 2 (ACE2) protein extracellular domain, or a fragment that specifically binds to the receptor binding domain (RBD) of the spike protein of (i) or a variant thereof.

Another object of the present invention is to provide a method for detecting a neutralizing antibody of type 2 severe acute respiratory syndrome coronavirus (SARS-CoV2), comprising the steps of: determining the level of interaction between (i) a receptor binding domain (RBD) of type 2 severe acute respiratory syndrome coronavirus (SARS-CoV2) spike protein or a variant thereof; and (ii) human angiotensin-converting enzyme 2 (ACE2) protein and (ii) human angiotensin-converting enzyme 2 (ACE2) protein, human angiotensin-converting enzyme 2 (ACE2) protein extracellular domain or spike protein-specific binding fragment for sample analysis.

Another object of the present invention is to provide a method for producing a mixture, comprising: a) mixing a sample with a receptor binding domain (RBD) of the spike protein of severe acute respiratory syndrome coronavirus (SARS-CoV2) conjugated to a labeling substance or a variant thereof to prepare a mixture, and incubating the mixture;

b) mixing the mixture of step a) with a fragment that specifically binds to human angiotensin converting enzyme 2 (ACE2) protein, human angiotensin converting enzyme 2 (ACE2) protein extracellular domain or receptor binding domain (RBD) or a variant thereof; and

c) a step of detecting a signal of a marker substance; provides a method for detecting a neutralizing antibody of type 2 severe acute respiratory syndrome coronavirus (SARS-CoV2).

Another object of the present invention is to provide a method for providing information for diagnosing the possession of neutralizing antibodies to type 2 severe acute respiratory syndrome coronavirus (SARS-CoV2) using the detection composition, kit or detection method.

In order to avoid confusion due to overlapping content, overlapping content will be omitted in the following description. In other words, the content of the invention is not limited to the content below, and the content of the invention should be interpreted according to the content of the entire invention.

The present invention aims to provide a technology relating to a composition for detecting neutralizing antibodies and neutralizing ability against infection by severe acute respiratory syndrome coronavirus type 2 and its variants (e.g., alpha (B.1.1.7), beta (B1.351), gamma (P1), delta (B.1.617.2), omicron (B.1.1.529), etc.), a method and kit for detecting neutralizing antibodies comprising the same, and a method for providing information for diagnosing possession of neutralizing antibodies.

As one embodiment for achieving the above purpose, a composition for detecting a neutralizing antibody of type 2 severe acute respiratory syndrome coronavirus (SARS-CoV2) is provided, comprising: (i) a receptor binding domain (RBD) of the spike protein of type 2 severe acute respiratory syndrome coronavirus (SARS-CoV2) or a variant thereof, conjugated to a label; and (ii) a fragment that specifically binds to human ACE2 protein, human ACE2 protein extracellular domain, or the receptor binding domain (RBD) of the spike protein of (i) or a variant thereof.

The term 'severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)' of the present invention is a single-stranded RNA coronavirus, which is infectious to humans and is the causative virus of COVID-19. The outer surface of the membrane of this virus has spike-shaped proteins (spike proteins) densely attached, and these spike proteins bind to the ACE2 receptor of the host cell and support the rapid penetration of the virus into the host cell. This spike protein determines the specificity, selectivity, and infectivity of the host.

The term 'RBD (Receptor-Binding Domain)' of the present invention refers to a region where a virus binds to a receptor of a host cell, and is present in the S1 subunit of the spike protein. In the present invention, the RBD (Receptor-Binding Domain) of the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was used as a portion that binds to the angiotensin-converting enzyme 2 receptor. The RBD, in turn, includes a receptor binding motif (RBM), which is an RBD region that contacts ACE2.

The term 'angiotensin converting enzyme 2 (ACE2)' of the present invention is a type 1 transmembrane protein homologous to angiotensin converting enzyme, a type of metallocarboxypeptidase, and can be found in eukaryotes and bacteria. Angiotensin converting enzyme 2 plays an important role in the renin-angiotensin-aldosterone system (RAAS), which regulates body water and blood pressure, and is present in large quantities in the membranes of human tongue, respiratory tract, and intestinal epithelial cells. ACE2 provides an entry point into cells for SARS-CoV-2 through interaction with the spike protein, particularly the RBD region selected in the present invention. That is, ACE2 can be viewed as a key cell entry receptor for SARS-CoV-2.

In the present invention, "specific binding" refers to an interaction that is selective for a given molecule and can be distinguished from non-specific binding. A polypeptide that specifically binds to a given molecule preferably binds to the molecule with greater affinity and/or with a longer duration than the polypeptide binds to other molecules to which it does not specifically bind.

A composition according to the present invention comprises a receptor binding domain (RBD) of the spike protein of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV2) or a variant thereof.

In the present invention, the structure of the RBD protein was analyzed through protein structure analysis based on the structural prediction of the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Through this, the amino acid sequence 323 to 532 of the full-length sequence of the spike protein was selected. This selected amino acid sequence of RBD was confirmed to be a sequence region that not only has excellent structural stability compared to other regions, but also exhibits an efficacy suitable for detecting neutralizing antibodies.

Accordingly, in the present invention, the modified pcDNA3.4 vector, which was produced by modifying the pcDNA3.4 vector, was cut using BamHI and XhoI restriction enzymes, and a fragment amplified by PCR having a base sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, or 12 was inserted to produce an expression vector loaded with a polynucleotide encoding the receptor expression domain of the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or a fragment thereof. Each amino acid sequence produced through the above recombinant vector is represented by SEQ ID NO: 3, 5, 7, 9, 11, or 13, respectively.

In addition, in the present invention, the modified pcDNA3.4 vector, which was produced by modifying the pcDNA3.4 vector, was cut using NheI and XhoI restriction enzymes, and a fragment amplified by PCR having the base sequence represented by SEQ ID NO: 15 was inserted to produce an expression vector loaded with a polynucleotide encoding hACE2 or a fragment thereof. The amino acid sequence produced through the above recombinant vector is shown in SEQ ID NO: 16.

The polypeptide of the present disclosure may further comprise additional amino acids or amino acid sequences. For example, the polypeptide may comprise an amino acid sequence(s) that facilitates expression, folding, trafficking, processing, or purification of the polypeptide. For example, the polypeptide may optionally comprise a sequence encoding a His, (e.g., 6xHis), Myc, GST, MBP, FLAG, HA, E, or biotin tag at the N- or C-terminus of the polypeptide.

The polypeptide of the present disclosure may additionally comprise a signal peptide (also known as a leader sequence or signal sequence). A signal peptide is typically comprised of a sequence of 5-30 hydrophobic amino acids that forms a single alpha helix. Secreted proteins and proteins expressed on the cell surface often comprise a signal peptide.

A signal peptide may be present at the N-terminus of a polypeptide and may be present in a newly synthesized polypeptide. A signal peptide provides efficient trafficking and secretion of the polypeptide. A signal peptide is often removed by cleavage and is therefore not included in the mature polypeptide secreted from the cell expressing the polypeptide.

Signal peptides are known for many proteins and are recorded in databases such as GenBank, UniProt, Swiss-Prot, TrEMBL, Protein Information Resource, Protein Data Bank, Ensembl, and InterPro, and can be identified/predicted using amino acids, for example.

Accordingly, in embodiments of the present invention, the sequences may additionally include the above signal sequence and a His-tag sequence, etc.

In particular, for a fragment that specifically binds to human ACE2 protein, human ACE2 protein extracellular domain or receptor binding domain (RBD) of the spike protein of (i) above or a variant thereof, a His-tag may be included at the C-term so as to be immobilized toward the plate and have an orientation in which the N-term is up. In this case, it may exhibit excellent effects in that the binding ability to RBD can be enhanced.

Polypeptides according to the present disclosure can be produced according to methods for producing polypeptides known to those skilled in the art.

The naming of residues according to the present invention is based on the full-length sequence of the spike protein of severe acute respiratory syndrome coronavirus type 2. The full-length sequence of the spike protein of severe acute respiratory syndrome coronavirus type 2 is shown in SEQ ID NO: 1.

In the receptor binding domain (RBD) of the spike protein of type 2 severe acute respiratory syndrome coronavirus (SARS-CoV2) or a variant thereof according to the present invention, the sequence may include, for example, amino acid sequences at positions 323 to 532 of the amino acid sequence of SEQ ID NO: 1. More specifically, it may be amino acids 280 to 570, more preferably 290 to 560, even more preferably 300 to 550, and even more preferably 310 to 540 of the amino acid sequence of SEQ ID NO: 1. According to a specific embodiment of the present invention, a fragment of the spike receptor binding domain protein of type 2 severe acute respiratory syndrome coronavirus has an amino acid sequence at positions 323 to 532, and this sequence is shown in SEQ ID NO: 18.

The above variants include additional mutations within the above sequence categories mentioned above.

In particular, it may contain characteristic amino acid mutations that appear in alpha (B.1.1.7), beta (B1.351), gamma (P1), delta (B.1.617.2), and omicron (B.1.1.529).

In addition, it may include all polypeptides having a sequence identity of 80 to 99%, 85 to 99%, preferably 90 to 99%, for example, a sequence identity of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, with a polypeptide in which the amino acid thereof is substituted by conservative substitutions. The same also applies to the corresponding polynucleotide.

"Percent (%) sequence identity" is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in a reference polypeptide, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid identity can be accomplished in a variety of ways within the skill in the art, for example using publicly available computer software programs, such as those described in Current Protocols in Molecular Biology (Ausubel et al., eds., 1987), and using BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm necessary to achieve maximum alignment over the full length of the sequences being compared.

Accordingly, the 'receptor binding domain variant of SARS-CoV-2 spike protein' of the present invention may be one in which asparagine at position 501 of the wild-type SARS-CoV-2 spike protein represented by SEQ ID NO: 1 is substituted with tyrosine. That is, it may essentially include the N501Y mutation and include the amino acid sequence at positions 323 to 532 of the amino acid sequence of SEQ ID NO: 1. More specifically, it may essentially include the N501Y mutation and include amino acids at positions 280 to 570, more preferably 290 to 560, even more preferably 300 to 550, and even more preferably 310 to 540 of the amino acid sequence of SEQ ID NO: 1. According to a specific embodiment of the present invention, it may be an amino acid sequence represented by SEQ ID NO: 20 including N501Y, which is a characteristic of the SARS-CoV-2 RBD alpha variant.

In some embodiments, the SARS-CoV-2 RBD alpha variant or fragment thereof comprises or consists of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 20.

Accordingly, the 'SARS-CoV-2 Spike protein receptor binding domain variant' of the present invention may be, preferably, one in which lysine at position 417 of the wild-type SARS-CoV-2 Spike protein represented by SEQ ID NO: 1 is substituted with asparagine, glutamic acid at position 484 with lysine, and asparagine at position 501 with tyrosine. That is, it may essentially include the K417N/E484K/N501Y mutation and include amino acid sequences at positions 323 to 532 of the amino acid sequence of SEQ ID NO: 1. More specifically, it may essentially include the K417N/E484K/N501Y mutation and include amino acids at positions 280 to 570, more preferably 290 to 560, even more preferably 300 to 550, and even more preferably 310 to 540 of the amino acid sequence of SEQ ID NO: 1. According to a specific embodiment of the present invention, it may be an amino acid sequence represented by SEQ ID NO: 22 including K417N/E484K/N501Y, which are characteristic of the SARS-CoV-2 RBD beta variant.

In some embodiments, the SARS-CoV-2 RBD beta variant or fragment thereof comprises or consists of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 22.

Accordingly, the 'SARS-CoV-2 Spike protein receptor binding domain variant' of the present invention may be, preferably, one in which lysine at position 417 of the wild-type SARS-CoV-2 Spike protein represented by SEQ ID NO: 1 is substituted with threonine, glutamic acid at position 484 with lysine, and asparagine at position 501 with tyrosine. That is, it may essentially include the K417T/E484K/N501Y mutation and include amino acid sequences at positions 323 to 532 of the amino acid sequence of SEQ ID NO: 1. More specifically, it may essentially include the K417T/E484K/N501Y mutation and include amino acids at positions 280 to 570, more preferably 290 to 560, even more preferably 300 to 550, and even more preferably 310 to 540 of the amino acid sequence of SEQ ID NO: 1. According to a specific embodiment of the present invention, it may be an amino acid sequence represented by SEQ ID NO: 24 including K417T/E484K/N501Y, which are characteristic of the SARS-CoV-2 RBD gamma variant.

In some embodiments, the SARS-CoV-2 RBD gamma variant or fragment thereof comprises or consists of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 24.

Accordingly, the 'SARS-CoV-2 Spike protein receptor binding domain variant' of the present invention may be, preferably, one in which leucine at position 452 of the wild-type SARS-CoV-2 Spike protein represented by SEQ ID NO: 1 is substituted with arginine, and threonine at position 478 is substituted with lysine. That is, it may essentially include the L452R/T478K mutation and may include amino acid sequences at positions 323 to 532 of the amino acid sequence of SEQ ID NO: 1. More specifically, it may essentially include the L452R/T478K mutation and may be amino acids at positions 280 to 570, more preferably 290 to 560, even more preferably 300 to 550, and even more preferably 310 to 540 of the amino acid sequence of SEQ ID NO: 1. According to a specific embodiment of the present invention, it may be an amino acid sequence represented by SEQ ID NO: 26 including L452R/T478K, which is a characteristic of the SARS-CoV-2 RBD delta variant.

In some embodiments, the SARS-CoV-2 RBD delta variant or fragment thereof comprises or consists of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 26.

Accordingly, the 'SARS-CoV-2 Spike protein receptor binding domain variant' of the present invention is, for example, a variant in which glycine at position 339 of the wild-type SARS-CoV-2 Spike protein represented by SEQ ID NO: 1 is changed to aspartic acid, serine at position 371 to leucine, serine at position 373 to proline, serine at position 375 to phenylalanine, lysine at position 417 to asparagine, asparagine at position 440 to lysine, glycine at position 446 to serine, serine at position 477 to asparagine, threonine at position 478 to lysine, glutamic acid at position 484 to alanine, glutamine at position 493 to lysine, glycine at position 496 to serine, and phenylalanine at position 498. It may be a substitution of glutamine for arginine, asparagine at position 501 for tyrosine, and tyrosine at position 505 for histidine.

That is, it may include amino acid sequences at positions 323 to 532 of the amino acid sequence of SEQ ID NO: 1, and essentially includes at least one mutation selected from the group consisting of G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493K, G496S, Q498R, N501Y, and Y505H.

More specifically, it essentially contains mutations G339D/S371L/S373P/S375F/K417N/N440K/G446S/S477N/T478K/E484A/Q493K/G496S/Q498R/N501Y/Y505H and may be amino acids 280 to 570, more preferably 290 to 560, even more preferably 300 to 550, even more preferably 310 to 540 of the amino acid sequence of SEQ ID NO: 1. According to a specific embodiment of the present invention, it may be an amino acid sequence represented by SEQ ID NO: 28, which includes G339D/S371L/S373P/S375F/K417N/N440K/G446S/S477N/T478K/E484A/Q493K/G496S/Q498R/N501Y/Y505H, which are characteristic of SARS-CoV-2 RBD omicron variants.

In some embodiments, the SARS-CoV-2 RBD omicron variant or fragment thereof comprises or consists of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 28.

The composition according to the present invention may be at least one selected from the group consisting of the above-mentioned SEQ ID NOs: 18, 20, 22, 24, 26 and 28 as the receptor binding domain (RBD) of the spike protein of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV2) or a variant thereof.

The sequence encoding the receptor binding domain (RBD) of the spike protein of the above-mentioned severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) or a variant thereof is represented by SEQ ID NOs: 17, 19, 21, 23, 25, and 27, respectively.

The receptor binding domain variant of the SARS-CoV-2 spike protein may comprise any one mutation selected from the group consisting of N501Y, K417N/E484K/N501Y, K417N/E484K/N501Y, L452R/T478K, and G339D/S371L/S373P/S375F/K417N/N440K/G446S/S477N/T478K/E484A/Q493K/G496S/Q498R/N501Y/Y505H with respect to the amino acid sequence of SEQ ID NO: 1, and may include amino acid sequences at positions 323 to 532 of the amino acid sequence of SEQ ID NO: 1.

The composition according to the present invention may be for detecting neutralizing antibodies against any one or more selected from the group consisting of wild type SARS-CoV-2, alpha variant B.1.1.7; beta variant B1.351; gamma variant P1; delta variant B.1.617.2; and omicron variant B.1.1.529.

A composition according to the present invention comprises a fragment that specifically binds to human ACE2 protein, human ACE2 protein extracellular domain or receptor binding domain (RBD) of the spike protein of (i) above or a variant thereof.

The full-length sequence of human angiotensin converting enzyme 2 (ACE2) protein is shown in SEQ ID NO: 14. The human angiotensin converting enzyme 2 (ACE2) protein according to the present invention may include amino acids 16 to 740 of the sequence of SEQ ID NO: 14. More specifically, it may be amino acids 1 to 800, more preferably 10 to 780, and even more preferably 15 to 770 of the amino acid sequence of SEQ ID NO: 14. According to a specific embodiment of the present invention, a fragment of human angiotensin converting enzyme 2 (ACE2) protein has amino acid sequences 16 to 740, and this sequence is shown in SEQ ID NO: 30. A sequence encoding the human angiotensin converting enzyme 2 (ACE2) protein is shown in SEQ ID NO: 29.

In some embodiments, the hACE2 protein or fragment comprises or consists of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 30.

It may further include a His-tag at the C-terminus for the above sequence number 30.

The RBD region can also bind to the extracellular domain of ACE2, so the composition according to the present invention may also comprise a fragment that specifically binds to the extracellular domain of human ACE2 protein or the receptor binding domain (RBD) of the spike protein of (i) above or a variant thereof.

The receptor binding domain (RBD) of the spike protein of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV2) or a variant thereof; may be conjugated to a labeling agent.

The labeling substance of the present invention may be used in a detection method selected from the group consisting of immunofluorescence, radiolabeling, immunoblotting, western blotting, enzyme-linked immunosorbent assay (ELISA), flow cytometry, immunoprecipitation, immunohistochemistry, biofilm test, pinning ring test, antibody array optical density test and chemiluminescence, more preferably, it may be used in sVNT (surrogate virus neutralization test), more preferably, ELISA.

sVNT is a method of quantifying neutralizing antibodies by measuring the extent to which the RBD portion of the viral spike protein and the human cell receptor ACE2 protein are mixed with patient serum and inhibiting their binding by neutralizing antibodies in the serum. While existing methods required cell experiments, which took a long time (3 days) or required measurement in a facility with a high biosafety level, sVNT has the advantage of being simple and rapid in a relatively short time (within 3 hours).

In the ELISA detection method according to the present invention, a preferred labeling substance may be at least one selected from biotin, digoxigenin, aptamer, peptide, fluorescent compound, oligonucleotide, and polysaccharides.

The substance capable of recognizing the label that can indirectly generate a signal may be, for example, one or more selected from the group consisting of avidin or an avidin analogue, an antibody (for example, an anti-biotin antibody, an anti-digoxigenin antibody), a receptor, and a lectin.

Color development, etc. can be achieved through an enzyme and its substrate that produce a detectable signal based on the above-mentioned labeling substance and its pair.

For example, the enzyme is horseradish peroxidase, alkaline phosphatase or β-galactosidase.

The substrates for the above enzymes can include, for example, 3,3',5,5'-tetramethylbenzidine (TMB), 2,2' -azino-di-[3-ethylbenzthiazoline-6-sulfonic acid] (ABTS), o-phenylenediamine dihydrochloride (OPD), 3,3'-diaminobenzidine (DAB), Luminol, etc. for horseradish peroxidase, p-Nitrophenyl Phosphate, Disodium Salt (PNPP), etc. for alkaline phosphatase, and Chlorophenol red-B-D galactopyrano (CPRG), O-Nitrophenyl-β-D-galactopyranoside (ONPG), 5-Bromo-4-Chloro-3-Indolyl-β-D-Galactoside (X-Gal), etc. for β-galactosidase.

Preferably, it may be a process of providing a substance capable of recognizing a label by treating with avidin or an avidin analogue, and treating with an enzyme and substrate that produce a detectable signal. For example, horseradish hydrogen peroxide capable of reacting with avidin or an avidin analogue may be treated, and a substrate applicable thereto, for example, 3,3',5,5'-tetramethylbenzidine (TMB), 2,2' -azino-di-[3-ethylbenzthiazoline-6-sulfonic acid] (ABTS), o-phenylenediamine dihydrochloride (OPD), 3,3'-diaminobenzidine (DAB), luminol, etc. may be used, and for alkaline phosphatase, it may be a process of treating with p-Nitrophenyl Phosphate, Disodium Salt (PNPP), etc.

More specifically, biotin can be used as a labeling agent, and the horseradish peroxide conjugate of avidin or an avidin analogue can be treated to provide color information and/or fluorescence change information through TMB (3,3',5,5'-tetramethylbenzidine). Here, the avidin analogue can be, for example, streptavidin, neutravidin, or captavidin.

That is, more specifically, streptavidin-horseradish hydrogen peroxide conjugate is treated for biotin as a labeling substance, and color information and/or fluorescence color change information is confirmed through TMB (3,3',5,5'-tetramethylbenzidine).

Through the above composition, the present invention can provide specific information on the presence or absence of neutralizing antibodies, the extent of SARS-CoV infection, the presence or absence of previous infection, etc. In particular, since information can be provided to individually confirm the presence or absence of neutralizing antibodies for various mutations, specific information on the presence or absence of antibodies for various mutations indicating epidemic potential, the risk of viral infection, etc. can be provided.

As described above, the present invention provides, as one embodiment for achieving the above object, a kit for detecting a neutralizing antibody of severe acute respiratory syndrome coronavirus (SARS-CoV2), comprising: (i) a receptor binding domain (RBD) of the spike protein of severe acute respiratory syndrome coronavirus (SARS-CoV2) conjugated with a labeling substance or a variant thereof; and (ii) a fragment that specifically binds to human angiotensin-converting enzyme 2 (ACE2) protein, human angiotensin-converting enzyme 2 (ACE2) protein extracellular domain, or the receptor binding domain (RBD) of the spike protein of (i) or a variant thereof.

Preferably, the kit of the present invention may further comprise one or more of a carrier, a washing buffer, a sample dilution solution, an enzyme substrate, a reaction stop solution, and an instruction manual teaching how to use it.

Additionally, a solid support may be additionally included as needed.

The solid support can be any solid support to which the polypeptide can be readily immobilized (e.g., by adsorption or conjugation) and which is suitable for the analysis of an antibody-containing sample, e.g., a blood-derived sample, such as a serum sample according to the present disclosure. Suitable solid supports for use in such kits and compositions are well known to those skilled in the art.

In some embodiments, the solid support can comprise or consist of polystyrene, polypropylene, polycarbonate, cyclo-olefin, glass, or quartz. In some embodiments, the solid support can be a microtiter (or “multiwell”) plate or a microarray plate. In some embodiments, the solid support can be a bead, for example a magnetic bead.

A polypeptide according to the present disclosure can be immobilized (or 'coated') on a solid support according to the present disclosure by methods well known to those skilled in the art. The polypeptide can be covalently or non-covalently immobilized to the solid support.

For example, the polypeptide can be immobilized on the solid support by applying a solution of the polypeptide in a buffer solution under suitable conditions and for a sufficient period of time to allow the polypeptide to adsorb to the surface of the solid support. Alternatively, the polypeptide can be conjugated to the solid support, for example, via a covalent bond.

Preferably, the subject can be a mammal. In some embodiments, the subject can be a human.

It may also be a human suspected of being infected with Sars-Cov2 or suspected of having a disease due to a Sars-Cov2 infection. It may also be a human suspected of having previously been infected with Sars-Cov2 or suspected of having previously had a disease due to a Sars-Cov2 infection.

The sample of the present invention may be blood, lymph, saliva or synovial fluid.

For the purpose of understanding the present invention, a schematic diagram of a neutralizing antibody reaction according to the present invention is shown in Figure 1.

For example, when treating RBD conjugated with a labeled substance to hACE2 fixed on a solid support, whether or not a signal is generated varies depending on the presence or absence of a neutralizing antibody. If a neutralizing antibody is present, binding between hACE2 and RBD is inhibited by the neutralizing antibody, so that signal production does not occur. On the other hand, if a neutralizing antibody is not present, binding between hACE2 and RBD occurs, and thus signal generation increases.

Detection of neutralizing antibodies in a sample may indicate an ongoing or previous infection with the virus. Detection of the presence of neutralizing antibodies in a sample may indicate an ongoing or previous immune response, particularly a humoral immune response. Thus, methods for determining whether a subject is or has been infected may be provided, and may also provide information to determine whether a subject is or has mounted an immune response (e.g., a humoral immune response). It may also be used for the diagnosis of disease caused by infection with COVID-19.

As described above, the present invention is one embodiment for achieving the above purpose, comprising the step of determining the level of interaction between (i) a receptor binding domain (RBD) of a type 2 severe acute respiratory syndrome coronavirus (SARS-CoV2) spike protein or a variant thereof; and (ii) a human angiotensin-converting enzyme 2 (ACE2) protein and (ii) a human angiotensin-converting enzyme 2 (ACE2) protein, a human angiotensin-converting enzyme 2 (ACE2) protein extracellular domain or a spike protein-specific binding fragment;

The present invention provides a method for detecting a neutralizing antibody of severe acute respiratory syndrome coronavirus (SARS-CoV2), wherein the receptor binding domain (RBD) or a variant thereof of (i) above is conjugated to a label, and the human angiotensin converting enzyme 2 (ACE2) protein, human angiotensin converting enzyme 2 (ACE2) protein extracellular domain or spike protein-specific binding fragment of (ii) above is immobilized on a solid support.

That is, specifically, the present invention adopted a system that mimics a host cell, and adopted a method of fixing hACE2, which is about three times larger than RBD in size, to a plate in order to induce favorable RBD binding.

As described above, the present invention provides one embodiment for achieving the above object, comprising: a) a step of mixing a sample with a receptor binding domain (RBD) of the spike protein of severe acute respiratory syndrome coronavirus (SARS-CoV2) conjugated to a labeling substance or a variant thereof to prepare a mixture, and incubating the mixture;

b) mixing the mixture of step a) with a fragment that specifically binds to human angiotensin converting enzyme 2 (ACE2) protein, human angiotensin converting enzyme 2 (ACE2) protein extracellular domain or receptor binding domain (RBD) or a variant thereof; and

c) a step of detecting a signal of a marker substance; provides a method for detecting a neutralizing antibody of type 2 severe acute respiratory syndrome coronavirus (SARS-CoV2).

Preferably, the type 2 severe acute respiratory syndrome coronavirus (SARS-CoV2) for detecting the neutralizing antibody of the present invention is type 2 severe acute respiratory syndrome coronavirus (SARS-CoV-2) or a variant thereof, and the type 2 severe acute respiratory syndrome coronavirus (SARS-CoV-2) variant may be at least one selected from the group consisting of alpha variant B.1.1.7; beta variant B1.351; gamma variant P1; delta variant B.1.617.2; and omicron variant B.1.1.529.

As described above, the present invention is one embodiment for achieving the above purpose, comprising the steps of: a) mixing a sample with a receptor binding domain (RBD) of the spike protein of severe acute respiratory syndrome coronavirus (SARS-CoV2) conjugated to a labeling substance or a variant thereof to prepare a mixture, and incubating the mixture;

b) mixing the mixture of step a) with human angiotensin converting enzyme 2 (ACE2) protein, human angiotensin converting enzyme 2 (ACE2) protein extracellular domain or a fragment that specifically binds to the receptor binding domain (RBD) or a variant thereof;

c) a step of detecting a signal of a labeling substance; and

d) a step of determining that the presence of a signal indicates the presence of a neutralizing antibody in the sample, and the absence of a signal indicates that the absence of a signal indicates that the neutralizing antibody does not exist in the sample; thereby providing a method for providing information for diagnosing the presence of a neutralizing antibody for type 2 severe acute respiratory syndrome coronavirus (SARS-CoV2).

Through one embodiment of the present invention, it was confirmed that the technology of the present invention has an advantage in that it can correct the analysis value using standard neutralizing antibodies (Nb20 and 4G6). In addition, it was confirmed that the kit of the present invention exhibits excellent effects in detecting not only mutant viruses but also neutralizing antibodies for each type of vaccine.

In addition, the human ACE2 protein and the receptor binding domain protein of the SARS-CoV-2 spike protein or a fragment thereof according to the present invention have a stable protein structure and are effective in detecting neutralizing antibodies in the serum of patients infected with SARS-CoV-2 and vaccinated patients with excellent sensitivity.

In addition, by optimizing the receptor binding domain protein sequences of the human ACE2 protein and the SARS-CoV-2 spike protein inserted into the vector, a kit for detecting neutralizing antibodies with a reduced production cost can be efficiently produced.

The present invention can easily perform the existing neutralization analysis that requires a lot of time and is difficult to conduct in mass testing, and considering the extreme infectivity of SARS-CoV-2 type 2 and its variants, such analysis must be performed in a BSL-3 facility with highly trained testers, but in order to overcome the shortcomings of the existing virus neutralization biological analysis, the neutralizing antibody detection kit test method using the present invention is capable of mass testing, is automated, and is easy to perform by general laboratory testers using a BSL-2 safety level environment, and is effective in evaluating the neutralizing efficacy against recently prevalent SARS-CoV-2 type 2 variants (alpha (B.1.1.7), beta (B1.351), gamma (P1), delta (B.1.617.2), Omicron (B.1.1.529)) infected patients and mutant viruses after vaccination, and in providing information for diagnosing the possession of neutralizing antibodies.

The present invention also provides a nucleic acid encoding the above-mentioned protein or a variant thereof according to the present disclosure.

The present invention also provides a vector comprising a nucleic acid according to the present disclosure.

The present invention also provides a cell comprising a nucleic acid or vector according to the present disclosure.

The present invention provides the use of the aforementioned kit, composition for detecting the presence of the aforementioned neutralizing antibody in a sample.

The composition for detecting neutralizing antibodies according to the present invention utilizes a stable form of protein structure, and is effective in detecting neutralizing antibodies with excellent sensitivity in the sera of patients infected with SARS-CoV-2 and vaccinated patients, and is effective in evaluating neutralizing efficacy, including the risk of viral infection. In addition, through an optimized manufacturing process (i.e., optimization of the receptor binding domain protein sequence of the human ACE2 protein and the SARS-CoV-2 spike protein inserted into the vector, etc.), a kit for detecting neutralizing antibodies can be efficiently produced at a reduced production cost.

Figure 1 is a schematic diagram showing a neutralizing antibody reaction according to the present invention.
Figure 2 is a diagram showing the animal cell expression vector pcDNA3.4-hACE2 for expression of human ACE2 protein.
Figure 3 is a diagram confirming the purified human ACE2 protein.
Figure 4A is a schematic diagram of a recombinant plasmid constructed to obtain receptor-binding variant domain proteins of the SARS-CoV-2 spike protein, and Figure 4B is a diagram confirming the purified receptor-binding wild-type and variant (alpha, beta, gamma, and delta) domain proteins of the SARS-CoV-2 spike protein.
Figure 5A shows the results of confirming the binding concentration for detecting neutralizing antibodies of variants using the human ACE2 protein and the receptor-binding alpha variant (N501Y) protein of the SARS-CoV-2 spike protein, and Figure 5B shows the results of confirming the binding of hACE2 receptor and RBD at various concentrations.
FIG. 6A shows the results of measuring the amount of antibodies in the serum of a non-infected patient and a patient who tested positive one month after recovery from SARS-CoV-2 infection, FIG. 6B shows the results of measuring the neutralizing antibody titer in the serum of a non-infected patient and a patient who tested positive one month after recovery from SARS-CoV-2 infection, FIG. 6C (1:100 dilution of serum) shows the neutralizing capacity value using the neutralizing antibody detection kit of the present invention, and FIG. 6D (1:10 dilution of serum) and FIG. 6E (1:100 dilution of serum) show the results of measuring the neutralizing capacity value using GeneScript, a prototype.
Figure 7 shows the results of evaluating the neutralizing ability of the diagnostic kit of the present invention and the prototype GeneScript diagnostic kit (A: high-concentration diagnostic kit of the present invention, B: medium-concentration diagnostic kit of the present invention, C: low-concentration diagnostic kit of the present invention, D: GeneScript diagnostic kit).
Figure 8 shows the results of measuring the neutralizing ability (A: using Nb20 neutralizing antibody, B: using 4G6 neutralizing antibody) and antibody amount (C: using Nb20 neutralizing antibody, D: using 4G6 neutralizing antibody) of the diagnostic kit of the present invention.
Figure 9 shows the results of confirming general antibodies binding to wild-type RBD in the serum of vaccinated individuals.
Figure 10 shows the results of detecting neutralizing antibodies in the serum of vaccinated people.

Hereinafter, preferred examples are presented to help understand the present invention. However, the following examples are provided only to help understand the present invention more easily, and the content of the present invention is not limited by the following examples.

When a nucleic acid sequence is disclosed in the present invention, its reverse complement is also expressly contemplated. Furthermore, when a polypeptide-encoding nucleic acid sequence is disclosed in the present invention, an equivalent polypeptide-encoding sequence is also expressly contemplated as a result of the degeneracy of the genetic code.

The singular includes plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as "about" one particular value and/or "about" another particular value. When such a range is expressed, other embodiments include the one particular value and/or the other particular value.

The methods disclosed herein may be performed or the products may be present in vitro, ex vivo, or in vivo. The term "in vitro" is intended to include experiments using materials, biological materials, cells, and/or tissues under laboratory conditions or in culture, while the term "in vivo" is intended to include experiments and procedures using intact multicellular organisms. In some embodiments, methods performed in vivo can be performed in a non-human animal. "Ex vivo" refers to existing or occurring outside an organism, e.g., outside the human or animal body, which may be in a tissue (e.g., a whole organ) or in cells taken from an organism.

Example 1. Preparation of human ACE2 receptor protein

The cell surface protrusion protein (725 amino acids) with the transmembrane region of the human ACE2 protein removed, used in the experiment, was produced using the hACE2 (angiotensin-converting enzyme 2, human) base sequence. Specifically, the base sequence for the 16 to 740 amino acid sequence (SEQ ID NO: 16) of the full-length ACE2 protein was codon-optimized for the CHO (Chinese hamster ovary) cell line and synthesized by Bioneer Co., Ltd. The base sequence of the fragment of the protrusion protein of human ACE2 selected for protein production, which was codon-optimized for the CHO cell line, is shown in SEQ ID NO: 15.

In particular, the sequence was designed to include a His-tag at the C-terminus so that the sequence can be fixed to a solid support in the future.

A fragment of human ACE2 represented by the above-mentioned base sequence number 15 was produced using a modified pcDNA3.4 vector. Specifically, a recombinant plasmid was produced by subcloning into the NheI and XhoI sites of the modified pcDNA3.4 vector. A schematic diagram of the produced recombinant plasmid is shown in Fig. 2.

The recombinant plasmid was transfected into CHO cells using an Expi CHO transfection kit. hACE2 protein was inoculated into CHO cells at 6.0Ⅹ10 ⁶ cells/㎖ and cultured for 12 days. The culture supernatant was purified by nickel-affinity chromatography after removing medium components using a semipermeable membrane. The protein was then purified once more using HiPrep 16/60 Superdex S-200 gel-filtration chromatography using 2X PBS buffer to obtain hACE2 protein. The results are shown in Fig. 3.

Example 2. Preparation of receptor-binding wild-type and mutant domain proteins of SARS-CoV-2 spike protein

To establish the sequence of the receptor binding protein (RBD) of the SARS-CoV-2 spike protein, which can be used as an antigen for the detection of SARS-CoV-2, the structure of the RBD protein was confirmed using a protein structure analysis server (XtalPred-RF, RaptorX) based on structure prediction.

Through the above structural analysis, the amino acid sequence of the RBD protein with a stable secondary structure was determined, and the following antigen was prepared based on this.

The (210 amino acids) used in the experiment was manufactured using the base sequence of SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2). Specifically, the base sequence (SEQ ID NO: 2) for the 323 to 532 amino acid sequence (SEQ ID NO: 1) of the RBD full-length protein was codon-optimized for the CHO cell line and synthesized by Bioneer Co., Ltd.

Using site-directed mutagenesis, a SARS-CoV-2 RBD mutant protein was produced by subcloning an amino acid at a specific site of the wild-type RBD protein of the above sequence number 1 into the BamHI and XhoI sites of a modified pcDNA3.4 vector and replacing it with a different amino acid. An exemplary schematic diagram of this recombinant vector is shown in Fig. 4A.

Specifically, the protein of N501Y, a SARS-CoV-2 RBD alpha mutant represented by SEQ ID NO: 20, in which asparagine at position 501 of the wild-type RBD protein of SEQ ID NO: 1 is substituted with tyrosine, and its base sequence (SEQ ID NO: 19) was produced by codon optimization for a CHO cell line.

In addition, a protein of K417N-E484K-N501Y, a SARS-CoV-2 RBD beta mutant represented by SEQ ID NO: 22, in which lysine at position 417 of the wild-type RBD protein of SEQ ID NO: 1 was substituted with asparagine, glutamic acid at position 484 with lysine, and asparagine at position 501 with tyrosine, and its base sequence (SEQ ID NO: 21) was produced by codon optimization for a CHO cell line.

In addition, a protein of K417T-E484K-N501Y, a SARS-CoV-2 RBD gamma mutant represented by SEQ ID NO: 24, in which lysine at position 417 of the wild-type RBD protein of SEQ ID NO: 1 was substituted with threonine, glutamic acid at position 484 with lysine, and asparagine at position 501 with tyrosine, and its base sequence (SEQ ID NO: 23) was produced by codon optimization for a CHO cell line.

In addition, a protein of L452R-T478K, a SARS-CoV-2 RBD delta mutant represented by SEQ ID NO: 26, in which leucine at position 452 of the wild-type RBD protein of SEQ ID NO: 1 is substituted with arginine and threonine at position 478 is substituted with lysine, and a base sequence thereof (SEQ ID NO: 25) was produced by codon optimization for a CHO cell line.

Similarly, amino acid sequences for SEQ ID NO: 28 and sequences for SEQ ID NO: 27 were prepared to correspond to the omicron mutation.

The specific sequences used for the above sequence synthesis and protein synthesis are specifically shown in SEQ ID NOs: 2-14.

Thereafter, the recombinant plasmid was transfected into the CHO cell line using the Expi CHO transfection kit. The RBD protein was inoculated into 6.0 X 10 ⁶ cells/㎖ of the CHO cell line and cultured for 12 days. The culture supernatant was purified primarily with Phenyl Sepharose 6 FF after removing the medium components using a semi-permeable membrane. Thereafter, the wild-type and mutant RBD proteins were obtained using hydroxyapatite chromatography and HiPrep 16/60 Superdex S-200 gel-filtration chromatography sequentially, and the results are shown in Fig. 4B.

Example 3. Confirmation of detection of neutralizing antibodies of alpha variants using human ACE2 protein and receptor-binding alpha variant proteins of SARS-CoV-2 spike protein

It was confirmed whether the human ACE2 protein and SARS-CoV-2 receptor binding domain alpha variant (N501Y) RBD protein antigens prepared in Examples 1 and 2 above effectively induce binding.

For this purpose, the reagent was prepared by the following protocol (reagent preparation step):

Specifically, all samples were vortexed immediately before use. Then, biotin-RBD was prepared and biotin-RBD was diluted 1:40,000 using PBST buffer (based on using 1 mg/㎕ concentration stock of biotin RBD). [Biotin-RBD washing solution (2.0 ng/100 ㎕, when mixed 1:1 with the sample during neutralization reaction, the final concentration is 1 ng/100 ㎕)]. Next, streptavidin-HRP was prepared and Streptavidin-HRP was diluted 1:20,000 using PBS buffer and used.

For the neutralization reaction, the experiment was conducted according to the following protocol (neutralization reaction step):

hACE2 protein was dispensed at 100㎕/well using PBS buffer so that it would be 250 ng/well and counted (coated). Thereafter, the solution inside the plate was removed and blocked by reaction at 25°C for 30 minutes under 1% BSA. Then, after washing once with PBST, PBST was completely removed. Next, the positive control, negative control, and sample were diluted at a ratio of 1:50. Thereafter, the diluted control and sample were mixed with Biotin-RBD at a ratio of 1:1 and reacted at 25°C for 30 minutes. Thereafter, 100㎕ of the mixture was added to each well prepared previously, the plate was covered, and the reaction was performed at 25°C for 10 minutes. Next, the contents of the plate were removed, washed twice with 200㎕ of PBST, and PBST was completely removed. Then, 100 ㎕ of Streptavidin-HRP was added, the lid was covered, and the reaction was performed at 25°C for 1 hour. Next, the plate contents were removed, washed 4 times with 200 ㎕ of PBST, and then PBST was completely removed.

In addition, for substrate reaction and absorbance measurement (inhibition calculation), the experiment was conducted according to the following protocol (inhibition measurement step):

Specifically, 100 ㎕ of TMB solution was added to the result of the neutralization reaction protocol, the plate lid was covered, and the reaction was performed at 25°C for 10 minutes. The timer was started from the moment the TMB solution was added to the first well. Next, 100 ㎕ of stop solution was added to stop the additional reaction. Then, the absorbance was immediately measured at 450 nm, and the degree of neutralization was calculated by the following formula.

inhibition(%) = (1- Sample O.D value/negative control O.D value)

As can be confirmed through the experimental results in Fig. 5, it was confirmed that the binding of the SARS-CoV-2 receptor binding domain alpha variant (N501Y) RBD according to the present invention and hACE2 changes in a concentration-dependent manner. In addition, it was confirmed that the binding was significantly inhibited by the N501Y neutralizing antibody.

Specifically, as can be confirmed through A of Fig. 5, when the RBD antiserum of SARS-CoV-2 WT was diluted 10-fold and 100-fold to confirm the inhibition of binding of hACE2 and HRP-conjugated alpha mutant RBD (N501Y), it was confirmed that the 100-fold dilution group did not inhibit the binding of hACE2 and HRP-conjugated alpha mutant RBD (N501Y) at all.

In addition, as can be confirmed through B of Fig. 5, after immobilizing hACE2 and hDPP4 (the cell infection receptor of MERS-CoV) on the plate respectively, the binding reaction of the RBD of SARS-CoV-2 WT conjugated with HRP was confirmed according to the concentration. As a result, it was confirmed that the RBD of SARS-CoV-2 WT bound to hACE2 (red graph), but the RBD of SARS-CoV-2 WT did not bind to hDPP4 (blue graph).

Example 4. Confirmation of detection of neutralizing antibodies using normal antibodies, human ACE2 protein, and wild-type protein binding to SARS-CoV-2 spike protein in the sera of patients infected with SARS-CoV-2

The formation of antibodies that bind to the receptor binding site of the spike protein in the sera of patients who recovered after actual SARS-CoV-2 infection (1 month after recovery, 1 negative (NC1), 11 positive (001, 002, 003, 004, 005, 014, 015, 016, 017, 018, 019)) was evaluated using ELISA by comparing the sera of recovered patients and uninfected (-) by diluting them at a ratio of 1:2,500. In addition, the SARS-CoV-2 (BetaCoV/South Korea/KUMC01/2020) virus isolated from a domestically infected patient was distributed, and the antiserum of a recovered patient was diluted at a certain ratio (1:10, 1:20, 1:40, 1:80, 1:160, 1:320, 1:640, 1:1,280), and the viruses were mixed with 100 TCID50 each, treated with Vero E6 cells for 1 hour, then removed, and the value of the dilution ratio just before the cytopathic effect (CPE) appeared after 72 hours in a new culture medium was read to evaluate the neutralizing ability. Then, the serum of a recovered patient was diluted to 1:100, and the neutralizing ability was detected by the method of Example 3, and a comparative evaluation was performed on the commercialized product (GeneScript) under two conditions of 1:10 and 1:100. The results are shown in Fig. 6.

As a result of the experiment, it was found that the general antibody and the neutralizing antibody are not completely identical, and in fact, it is necessary to set the neutralizing ability standard depending on the evaluation conditions (A and B of Fig. 6), and as a result of the comparative analysis of the neutralizing ability value according to the serum dilution ratio, it was found that the neutralizing antibody detection ability of the present invention (C of Fig. 6) exhibits superior sensitivity compared to existing products (D and E of Fig. 6).

Example 5. Confirmation and optimization of neutralizing antibody detection of variants using SARS-CoV-2 neutralizing antibodies

Nanoantibody Nb20, a neutralizing antibody for wild-type SARS-CoV-2 that is well known from existing research results rather than clinical samples ( Science . 2020 Dec 18; 370(6523): 1479-1484, Versatile and multivalent nanobodies efficiently neutralize SARS-CoV-2), was directly expressed in E. coli, and its neutralizing ability was confirmed in an actual virus. In addition, 4G6 antibody from GeneScript, a monoclonal antibody as a neutralizing antibody for SARS-CoV-2, was purchased and used to evaluate the neutralizing ability for the variant of the present invention. Nb20 and 4G6 neutralizing antibodies were treated at 0.39, 0.78, 1.56, 3.125, 6.25, 12.5, 25, and 50 ng per well, respectively, by the method of Example 3.

As shown in Fig. 7, the neutralizing antibodies could be detected by reacting with the concentrations of wild-type RBD of 2.5 ng at high concentration, 2 ng at medium concentration, and 1 ng at low concentration, respectively, and the commercialized product was able to detect neutralizing antibodies even though the same concentration of neutralizing antibodies had to be treated with 3 ng of wild-type RBD. Through this, it was confirmed that this technology has the advantage of being able to diagnose according to the concentration of neutralizing antibodies by controlling the amount of RBD, and that it was possible to precisely analyze by distinguishing between high and low levels of actual neutralizing antibodies.

In addition, the wild type and mutant were treated with 0.01, 0.0625, 0.125, 0.25, 0.5, 1, 5, 10, 20, 50, 10, and 200 ng of Nb20 and 4G6 neutralizing antibodies per well, respectively, using the method of Example 3, and the wild type and mutant RBD were reacted at a concentration of 2 ng to evaluate the neutralizing ability, and the results are shown in Fig. 8.

The experimental results confirmed that this technology has the advantage of always being able to correct the analysis value using standard neutralizing antibodies (Nb20 and 4G6). In addition, it was confirmed that Nb20 and 4G6, which are neutralizing antibodies of wild-type SARS-CoV-2, have neutralizing ability against wild-type, alpha, and delta mutants, but very low neutralizing ability against beta and gamma mutants.

Example 6. Confirmation of detection of neutralizing antibodies to variants in the serum of SARS-CoV-2 vaccinated individuals

Using 32 actual vaccinated subjects (sera 4 weeks after the second vaccination), general antibodies were measured using the method of Example 4 to confirm antibodies binding to wild-type RBD, and the results are shown in Fig. 9.

Additionally, the vaccinated serum was diluted 1:50 and neutralizing antibodies were detected using the method of Example 3, and the results are shown in Figure 10.

The patient samples provided in the above experiment were arbitrarily labeled as 3-34, 3-36 to 3-48, 3-50 to 3-52, 3-148, 3-150, 3-151, 3-155, 3-157, 3-159 to 3-165, 3-172, and 3-174.

The experimental results confirmed that the wild type and delta mutant RBD showed high neutralizing ability overall. On the other hand, alpha, beta, and gamma mutants showed 20-30% lower neutralizing ability.

In addition, it was shown that there was a difference in neutralizing ability between those vaccinated with adenovirus (vector vaccine (AstraZeneca AZ)/AZ), mRNA vaccine (Moderna (M)/M), and cross-vaccination vaccine (AZ/Pfizer (PZ)) (neutralizing antibodies were higher in the serum of those vaccinated with mRNA vaccine or cross-vaccination vaccine than in the vector vaccine), and it was confirmed that the kit of the present invention can detect not only mutant viruses but also neutralizing antibodies by vaccine type with excellent sensitivity.

<110> Korea Research Institute of Bioscience and Biotechnology <120> COMPOSITION FOR DETECTION OF NEUTRALIZING ANTIBODY AGAINST SARS-COV2 VARIANTS, DECTECTION METHOD AND KIT COMPRISING THEREOF <130> P22017-KRIBB-PA <150> KR 1020210027938 <151> 2021-03 -03 <160> 30 <170> KoPatentIn 3.0 <210> 1 <211> 1273 <212> PRT <213> Human coronavirus <400> 1 Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val 1 5 10 15 Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe 20 25 30 Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu 35 40 45 His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp 50 55 60 Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp 65 70 75 80 Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu 85 90 95 Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser 100 105 110 Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile 115 120 125 Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr 130 135 140 Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr 145 150 155 160 Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu 165 170 175 Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe 180 185 190 Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr 195 200 205 Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu 210 215 220 Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr 225 230 235 240 Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser 245 250 255 Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro 260 265 270 Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala 275 280 285 Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys 290 295 300 Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val 305 310 315 320 Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys 325 330 335 Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala 340 345 350 Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu 355 360 365 Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro 370 375 380 Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe 385 390 395 400 Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly 405 410 415 Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys 420 425 430 Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn 435 440 445 Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe 450 455 460 Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys 465 470 475 480 Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly 485 490 495 Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val 500 505 510 Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys 515 520 525 Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn 530 535 540 Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu 545 550 555 560 Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val 565 570 575 Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe 580 585 590 Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val 595 600 605 Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile 610 615 620 His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser 625 630 635 640 Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val 645 650 655 Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala 660 665 670 Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg Ser Val Ala 675 680 685 Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser 690 695 700 Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile 705 710 715 720 Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val 725 730 735 Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu 740 745 750 Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr 755 760 765 Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln 770 775 780 Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe 785 790 795 800 Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser 805 810 815 Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly 820 825 830 Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp 835 840 845 Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu 850 855 860 Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly 865 870 875 880 Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile 885 890 895 Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr 900 905 910 Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn 915 920 925 Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala 930 935 940 Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn 945 950 955 960 Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val 965 970 975 Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln 980 985 990 Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val 995 1000 1005 Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu 1010 1015 1020 Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Val 1025 1030 1035 1040 Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln Ser Ala 1045 1050 1055 Pro His Gly Val Val Phe Leu His Val Thr Tyr Val Pro Ala Gln Glu 1060 1065 1070 Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His Asp Gly Lys Ala His 1075 1080 1085 Phe Pro Arg Glu Gly Val Phe Val Ser Asn Gly Thr His Trp Phe Val 1090 1095 1100 Thr Gln Arg Asn Phe Tyr Glu Pro Gln Ile Ile Thr Asp Asn Thr 1105 1110 1115 1120 Phe Val Ser Gly Asn Cys Asp Val Val Ile Gly Ile Val Asn Asn Thr 1125 1130 1135 Val Tyr Asp Pro Leu Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu 1140 1145 1150 Asp Lys Tyr Phe Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp 1155 1160 1165 Ile Ser Gly Ile Asn Ala Ser Val Asn Ile Gln Lys Glu Ile Asp 1170 1175 1180 Arg Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu 1185 1190 1195 1200 Gln Glu Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile 1205 1210 1215 Trp Leu Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile 1220 1225 1230 Met Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys 1235 1240 1245 Ser Cys Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro Val 1250 1255 1260 Leu Lys Gly Val Lys Leu His Tyr Thr 1265 1270 <210> 2 <211> 708 <212> DNA < 213> Artificial Sequence <220> <223> codon optimized DNA sequence of fragment of RBD of SARS-CoV-2 WT comprising signal peptide and restriction enzyme site <220> <221> sig_peptide <222> (1)..(69) <220> <221> misc_binding <222> (70)..(75) <223> restriction enzyme site (BamHI) <400> 2 gccaccatgg agacagacac actcctgcta tgggtactgc tgctctgggt tccaggttcc 60 actggtgacg gatccaccga gtctatcgtg cggttcccca acatcaccaa cctgtgtcct 120 ttcggcgagg tgttcaacgc caccag attc gcctctgtgt acgcctggaa ccggaagcgg 180 atctctaact gcgtggccga ctactccgtg ctgtacaact ccgcctcctt cagcaccttc 240 aagtgctacg gcgtgtcccc taccaagctg aacgacctgt gcttcaccaa cgtgtacgcc 300 gactccttcg tgatcagagg cgacgaagtg cggcagatcg ctcctggaca gaccggcaag 360 atcgccgact acaa gctgcccgac gacttcaccg gctgtgtgat cgcttggaac 420 tccaacaacc tggactccaa agtcggcggc aactacaatt acctgtaccg gctgttccgg 480 aagtccaacc tgaagccttt cgagcgggac atctccaccg agatctacca ggctggcagc 540 accccttgta atggcgtgga aggcttcaac tgctacttcc cactgcagtc ctacggcttc 600 cagcctacaa acggcgtggg ctaccagcct tacagagtgg tggtgctgtc cttcgagctg 660 ctgcatgctc ctgctaccgt gtgcggccct aagaaatcta ccaactaa 708 <210> 3 <211> 235 <212> PRT <213> Artificial Sequence <220> <223> 323-532 amino acid of RBD of SARS-CoV-2 WT comprising signal peptide <220> <221> SIGNAL <222> (1)..(23) <400> 3 Ala Thr Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp 1 5 10 15 Val Pro Gly Ser Thr Gly Asp Gly Ser Thr Glu Ser Ile Val Arg Phe 20 25 30 Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr 35 40 45 Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys 50 55 60 Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe 65 70 75 80 Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr 85 90 95 Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln 100 105 110 Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu 115 120 125 Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu 130 135 140 Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg 145 150 155 160 Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr 165 170 175 Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr 180 185 190 Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr 195 200 205 Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro 210 215 220 Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn 225 230 235 <210> 4 <211> 708 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence of RBD of alpha variant N501Y comprising signal peptide and restriction enzyme site <220> <221> sig_peptide <222> (1)..(69) <220> <221> misc_binding <222> (70)..(75) <223> restriction enzyme site(BamHI) <400> 4 gccaccatgg agacagacac actcctgcta tgggtactgc tgctctgggt tccaggttcc 60 actggtgacg gatccaccga gtctatcgtg cggttcccca acatcaccaa cctgtgtcct 120 ttcggcgagg tgttcaacgc caccagattc gcctctgtgt acgcctggaa ccggaagcgg 180 atctctaact gcgtggccga ctactccgtg ctgtacaact ccgcctcctt cagcaccttc 240 aagtgctacg gc gtgtcccc taccaagctg aacgacctgt gcttcaccaa cgtgtacgcc 300 gactccttcg tgatcagagg cgacgaagtg cggcagatcg ctcctggaca gaccggcaag 360 atcgccgact acaactacaa gctgcccgac gacttcaccg gctgtgtgat cgcttggaac 420 tccaacaacc tggactccaa agtcggcggc aactacaatt acctgtaccg gctgttccgg 480 aagtccaacc tgaagccttt cgagcgggac atctccaccg agatctacca ggctggcagc 540 accccttgta atggcgtgga aggcttcaac tgctacttcc cactgcagtc ctacggctt c 600 cagcctacat acggcgtggg ctaccagcct tacagagtgg tggtgctgtc cttcgagctg 660 ctgcatgctc ctgctaccgt gtgcggccct aagaaatcta ccaactaa 708 <210> 5 <211> 235 <212> PRT <213> Artificial Sequence <220> <223> amino acid of RBD of alpha variant N501Y comprising signal peptide <220> <221> SIGNAL <222> (1)..(23) <400> 5 Ala Thr Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp 1 5 10 15 Val Pro Gly Ser Thr Gly Asp Gly Ser Thr Glu Ser Ile Val Arg Phe 20 25 30 Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr 35 40 45 Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys 50 55 60 Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe 65 70 75 80 Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr 85 90 95 Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln 100 105 110 Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu 115 120 125 Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu 130 135 140 Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg 145 150 155 160 Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr 165 170 175 Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr 180 185 190 Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Tyr Gly Val Gly Tyr 195 200 205 Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro 210 215 220 Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn 225 230 235 <210> 6 <211> 705 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence of RBD of beta variant K417N-E484K-N501Y comprising signal peptide and restriction enzyme site <220> <221> sig_peptide <222> (1)..(69) <220> <221> misc_binding <222> (70)..(75) <223> restriction enzyme site(BamHI) <400> 6 gccaccatgg agacagacac actcctgcta tgggtactgc tgctctgggt tccaggttcc 60 actggtgacg gatccaccga gtctatcgtg cggttcccca acatcaccaa cctgtgtcct 120 ttcggcgagg t gttcaacgc caccagattc gcctctgtgt acgcctggaa ccggaagcgg 180 atctctaact gcgtggccga ctactccgtg ctgtacaact ccgcctcctt cagcaccttc 240 aagtgctacg gcgtgtcccc taccaagctg aacgacctgt gcttcaccaa cgtgtacgcc 300 gactccttcg tgatcagagg cgacgaagtg cggcagatcg ctcctggaca gaccggcaac 360 atcgccgact acaactacaa gctgcccgac gacttcaccg gctgtgtgat cgcttggaac 420 tccaacaacc tggactccaa agtcggcggc aactacaatt acctgtaccg gctgtt ccgg 480 aagtccaacc tgaagccttt cgagcgggac atctccaccg agatctacca ggctggcagc 540 accccttgta atggcgtgaa aggcttcaac tgctacttcc cactgcagtc ctacggcttc 600 cagcctacat acggcgtggg ctaccagcct tacagagtgg tggtgctgtc cttcgagctg 660 ctgcatgctc ctgctaccgt gtgcggccct aagaaatcta ccaac 705 <210> 7 <211> 235 <212> PRT <213> Artificial Sequence <220> <223> amino acid of RBD of beta variant K417N-E484K-N501 Y comprising signal peptide <220> <221> SIGNAL <222> (1)..(23) <400> 7 Ala Thr Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp 1 5 10 15 Val Pro Gly Ser Thr Gly Asp Gly Ser Thr Glu Ser Ile Val Arg Phe 20 25 30 Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr 35 40 45 Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys 50 55 60 Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe 65 70 75 80 Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr 85 90 95 Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln 100 105 110 Ile Ala Pro Gly Gln Thr Gly Asn Ile Ala Asp Tyr Asn Tyr Lys Leu 115 120 125 Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu 130 135 140 Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg 145 150 155 160 Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr 165 170 175 Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Lys Gly Phe Asn Cys Tyr 180 185 190 Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Tyr Gly Val Gly Tyr 195 200 205 Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro 210 215 220 Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn 225 230 235 <210> 8 <211> 705 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence of RBD of gamma variant K417T-E484K-N501Y comprising signal peptide and restriction enzyme site <220> <221> sig_peptide <222> (1)..(69) <220> <221> misc_binding <222> (70)..(75) <223> enzyme restriction site(BamHI) <400> 8 gccaccatgg agacagacac actcctgcta tgggtactgc tgctctgggt tccaggttcc 60 actggtgacg gatccaccga gtctatcgtg cggt tcccc acatcaccaa cctgtgtcct 120 ttcggcgagg tgttcaacgc caccagattc gcctctgtgt acgcctggaa ccggaagcgg 180 atctctaact gcgtggccga ctactccgtg ctgtacaact ccgcctcctt cagcaccttc 240 aagtgctacg gcgtgtcccc taccaagctg aacgacctgt gcttcaccaa cgtgtacgcc 300 gactccttcg tgatcagagg cgacgaagtg cggcagatcg ctcctggaca gaccggcacc 360 atcgccgact acaactacaa gctgcccgac gacttcaccg gctgtgtgat cgcttggaac 420 tccaacaacc tggactccaa agtcggcggc aactacaatt acctgtaccg gctgttccgg 480 aagtccaacc tgaagccttt cgagcgggac atctccaccg agatctacca ggctggcagc 540 accccttgta atggcgtgaa aggcttcaac tgctacttcc cactgcagtc ctacggcttc 600 cagcctacat acggcgtggg ctaccagcct tacagagtgg tggtgctgtc cttcgagctg 660 ctgcatgctc ctgctaccgt gtgcggccct aagaaatcta ccaac 705 <210> 9 <211> 235 <212> PRT <213> Artificial Sequence <220> <223> amino acid of RBD of gamma variant K417T-E484K-N501Y comprising signal peptide <220> <221> SIGNAL <222> (1)..(23) <400> 9 Ala Thr Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp 1 5 10 15 Val Pro Gly Ser Thr Gly Asp Gly Ser Thr Glu Ser Ile Val Arg Phe 20 25 30 Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr 35 40 45 Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys 50 55 60 Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe 65 70 75 80 Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr 85 90 95 Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln 100 105 110 Ile Ala Pro Gly Gln Thr Gly Thr Ile Ala Asp Tyr Asn Tyr Lys Leu 115 120 125 Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu 130 135 140 Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg 145 150 155 160 Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr 165 170 175 Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Lys Gly Phe Asn Cys Tyr 180 185 190 Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Tyr Gly Val Gly Tyr 195 200 205 Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro 210 215 220 Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn 225 230 235 <210> 10 <211> 705 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence of RBD of delta variant L452R-T478K comprising signal peptide and restriction enzyme site <220> <221> sig_peptide <222 > (1)..(69) <220> <221> misc_binding <222> (70)..(75) <223> restriction enzyme site(BamHI) <400> 10 gccaccatgg agacagacac actcctgcta tgggtactgc tgctctgggt tccaggttcc 60 actggtgacg gatccaccga gtctatcgtg cggttcccca acatcaccaa cctgtgtcct 120 ttcggcgagg tgttcaacgc caccagattc gcctctgtgt acgcctggaa ccggaagcgg 180 atctctaact gcgtggccga ctactccgtg ctgtacaact ccgcctcctt cagcaccttc 240 aagtgctacg gcgtgtcccc taccaagctg aacgacctgt gcttcaccaa cgtgtacg cc 300 gactccttcg tgatcagagg cgacgaagtg cggcagatcg ctcctggaca gaccggcaag 360 atcgccgact acaactacaa gctgcccgac gacttcaccg gctgtgtgat cgcttggaac 420 tccaacaacc tggactccaa agtcggcggc aactacaatt accggtaccg gctgttccgg 480 aagtccaacc tgaagccttt cgagcgggac atctccaccg agatctacca ggctggcagc 540 aagccttgta atggcgtgga aggcttcaac tgctacttcc cactgcagtc ctacggcttc 600 cagcctacaa acggcgtggg ctaccagagcct tggtgctgtc cttcgagctg 660 ctgcatgctc ctgctaccgt gtgcggccct aagaaatcta ccaac 705 <210> 11 <211> 235 <212> PRT <213> Artificial Sequence <220> < 223> amino acid of RBD of delta variant L452R-T478K comprising signal peptide <220> <221> SIGNAL <222> (1)..(23) <400> 11 Ala Thr Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp 1 5 10 15 Val Pro Gly Ser Thr Gly Asp Gly Ser Thr Glu Ser Ile Val Arg Phe 20 25 30 Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr 35 40 45 Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys 50 55 60 Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe 65 70 75 80 Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr 85 90 95 Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln 100 105 110 Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu 115 120 125 Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu 130 135 140 Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Arg Tyr Arg Leu Phe Arg 145 150 155 160 Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr 165 170 175 Gln Ala Gly Ser Lys Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr 180 185 190 Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr 195 200 205 Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro 210 215 220 Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn 225 230 235 < 210> 12 <211> 705 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence of RBD of omicron variant G339D-S371L-S373P-S375F-K417N-N440K-G446S-S477N-T478K-E484A-Q493K -G496S-Q498R-N501Y-Y505H comprising signal peptide and restriction enzyme site <220> <221> sig_peptide <222> (1)..(69) <220> <221> misc_binding <222> (70)..(75) <223> restriction enzyme site (BamHI) <400> 12 gccaccatgg agacagacac actcctgcta tgggtactgc tgctctgggt tccaggttcc 60 actggtgacg gatccaccga gtctatcgtg cggttcccca acatcaccaa cctgtgtcct 120 ttcgacgagg tgttcaacgc caccagattc gcctctgtgt acgcctggaa ccggaagcgg 180 atctctaact gcgtggccga ctactccgtg ctgtacaacc tggccccttt cttcaccttc 240 aagtgctacg gcgtgtcccc taccaagctg aacgacctgt gcttcaccaa cgtgtacgcc 300 gactccttcg tgatcagagg cgacgaagtg cggcagatcg ctcctggaca gaccggcaac 360 atcgccgact acaactacaa gctgcccgac gacttca ccg gctgtgtgat cgcttggaac 420 tccaacaagc tggactccaa agtctccggc aactacaatt acctgtaccg gctgttccgg 480 aagtccaacc tgaagccttt cgagcgggac atctccaccg agatctacca ggctggcaat 540 aaaccttgta atggcgtggc tggcttcaac tgctacttcc cactgaagtc ctactccttc 600 cggcctacat acggcgtggg ccatcagcct tacagagtgg tggtgctgtc cttcgagctg 660 ctgcatgctc ctgctaccgt gtgcggccct aagaaatcta ccaac 705 <210> 13 <211> 235 <212> PRT <213> Artificial Sequence <220> <223> amino acid of RBD of omicron variant G339D- S371L-S373P-S375F-K417N-N440K-G446S-S477N-T478K-E484A-Q493K -G496S-Q498R-N501Y-Y505H comprising signal peptide <220> <221> SIGNAL <222> (1)..(23) <400> 13 Ala Thr Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp 1 5 10 15 Val Pro Gly Ser Thr Gly Asp Gly Ser Thr Glu Ser Ile Val Arg Phe 20 25 30 Pro Asn Ile Thr Asn Leu Cys Pro Phe Asp Glu Val Phe Asn Ala Thr 35 40 45 Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys 50 55 60 Val Ala Asp Tyr Ser Val Leu Tyr Asn Leu Ala Pro Phe Phe Thr Phe 65 70 75 80 Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr 85 90 95 Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln 100 105 110 Ile Ala Pro Gly Gln Thr Gly Asn Ile Ala Asp Tyr Asn Tyr Lys Leu 115 120 125 Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Lys Leu 130 135 140 Asp Ser Lys Val Ser Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg 145 150 155 160 Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr 165 170 175 Gln Ala Gly Asn Lys Pro Cys Asn Gly Val Ala Gly Phe Asn Cys Tyr 180 185 190 Phe Pro Leu Lys Ser Tyr Ser Phe Arg Pro Thr Tyr Gly Val Gly His 195 200 205 Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro 210 215 220 Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn 225 230 235 <210> 14 <211> 805 <212> PRT <213> Homo sapiens <400> 14 Met Ser Ser Ser Ser Trp Leu Leu Leu Ser Leu Val Ala Val Thr Ala 1 5 10 15 Ala Gln Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe 20 25 30 Asn His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp 35 40 45 Asn Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn 50 55 60 Ala Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala 65 70 75 80 Gln Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln 85 90 95 Leu Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys 100 105 110 Ser Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser 115 120 125 Thr Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu 130 135 140 Glu Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu 145 150 155 160 Arg Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu 165 170 175 Arg Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg 180 185 190 Ala Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu 195 200 205 Val Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu 210 215 220 Asp Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu 225 230 235 240 His Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile 245 250 255 Ser Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly 260 265 270 Arg Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys 275 280 285 Pro Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala 290 295 300 Gln Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu 305 310 315 320 Pro Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro 325 330 335 Gly Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly 340 345 350 Lys Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp 355 360 365 Phe Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala 370 375 380 Tyr Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe 385 390 395 400 His Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys 405 410 415 His Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn 420 425 430 Glu Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly 435 440 445 Thr Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe 450 455 460 Lys Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met 465 470 475 480 Lys Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr 485 490 495 Tyr Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe 500 505 510 Ile Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala 515 520 525 Leu Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile 530 535 540 Ser Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu 545 550 555 560 Gly Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala 565 570 575 Lys Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe 580 585 590 Thr Trp Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr 595 600 605 Asp Trp Ser Pro Tyr Ala Asp Gln Ser Ile Lys Val Arg Ile Ser Leu 610 615 620 Lys Ser Ala Leu Gly Asp Lys Ala Tyr Glu Trp Asn Asp Asn Glu Met 625 630 635 640 Tyr Leu Phe Arg Ser Ser Val Ala Tyr Ala Met Arg Gln Tyr Phe Leu 645 650 655 Lys Val Lys Asn Gln Met Ile Leu Phe Gly Glu Glu Asp Val Arg Val 660 665 670 Ala Asn Leu Lys Pro Arg Ile Ser Phe Asn Phe Phe Val Thr Ala Pro 675 680 685 Lys Asn Val Ser Asp Ile Ile Pro Arg Thr Glu Val Glu Lys Ala Ile 690 695 700 Arg Met Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asn Asp Asn 705 710 715 720 Ser Leu Glu Phe Leu Gly Ile Gln Pro Thr Leu Gly Pro Pro Asn Gln 725 730 735 Pro Pro Val Ser Ile Trp Leu Ile Val Phe Gly Val Val Met Gly Val 740 745 750 Ile Val Val Gly Ile Val Ile Leu Ile Phe Thr Gly Ile Arg Asp Arg 755 760 765 Lys Lys Lys Asn Lys Ala Arg Ser Gly Glu Asn Pro Tyr Ala Ser Ile 770 775 780 Asp Ile Ser Lys Gly Glu Asn Asn Pro Gly Phe Gln Asn Thr Asp Asp 785 790 795 800 Val Gln Thr Ser Phe 805 <210> 15 <211> 2283 <212> DNA <213> Artificial Sequence <220> <223> codon optimized DNA sequence of fragment of projecting protein of hACE2 comprising signal peptide and restriction enzyme site <220> <221> sig_peptide <222> (1)..(69) <220> <221> misc_binding <222> (76)..(81) <223> restriction enzyme site(NheI) <220> <221> misc_binding <222> (2257)..(2262) <223> restriction enzyme site(XhoI) <400> 15 gccaccatgg agacagacac actcctgcta tgggtactgc tgctctgggt tccaggttcc 60 actggtgacg gatccgctag cgctgctcag tccaccattg aggaacaggc caagacattt 120 ttggacaagt ttaaccacga agccgaagac ctgttctatc aaagttcact tgcttcttgg 180 aattataaca ccaatattac tgaagagaat gtccaaaaca tgaataatgc tggggacaaa 240 tggtctgcct ttttaaagga acagtccaca cttgcccaaa tgtatccact acaagaaatt 300 cagaatctca cagtcaagct tcagctgcag gctcttcagc aaaatgggtc ttcagtgctc 360 tcagaagaca agagcaaacg gttgaacaca attctaaata caatgagcac catctacagt 420 actggaaaag tttgtaaccc agataatcca caagaatgct tattacttga accaggtttg 480 aatgaaataa tggcaaacag tttagactac aatgagaggc tctgggcttg ggaaagctgg 540 agatctgagg tcggcaagca gctgaggcca ttatatgaag agtatgtggt cttgaaaaat 600 gagatggcaa gagcaaatca ttatgaggac tatggggatt attggagagg agactatgaa 660 gtaaatgggg tagatggcta c cgcggccagt tgattgaaga tgtggaacat 720 acctttgaag agattaaacc attatatgaa catcttcatg cctatgtgag ggcaaagttg 780 atgaatgcct atccttccta tatcagtcca attggatgcc tccctgctca tttgcttggt 840 gatatgtggg gtagattttg gacaaatctg tactctttga cagttccctt tggacagaaa 900 ccaaacatag atgttactga tgcaatggtg gaccaggcct gggatgcaca gagaatattc 960 aaggaggccg agaagttctt tgtatctgtt ggtcttccta atatgactca aggattctgg 1020 gaaaattcca ctaacgga cccaggaaat gttcagaaag cagtctgcca tcccacagct 1080 tgggacctgg ggaagggcga cttcaggatc cttatgtgca caaaggtgac aatggacgac 1140 ttcctgacag ctcatcatga gatggggcat atccagtatg atatggcata tgctgcacaa 1200 ccttttctgc taagaaatgg agctaatgaa ggattccatg aagctgttgg ggaaatcatg 1260 tcactttctg cagccacacc taagcattta aaatccattg gtcttctgtc acccgatttt 1320 caagaagaca atgaaacaga aataaacttc ctgctcaaac aagcactcac gattg ttggg 1380 actctgccat ttacttacat gttagagaag tggaggtgga tggtctttaa aggggaaatt 1440 cccaaagacc agtggatgaa aaagtggtgg gagatgaagc gagagatagt tggggtggtg 1500 gaacctgtgc cccatgatga aacatactgt gaccccgcat ctctgttcca tgtttctaat 1 560 gattactcat tcattcgata ttacacaagg accctttacc aattccagtt tcaagaagca 1620 ctttgtcaag cagctaaaca tgaaggccct ctgcacaaat gtgacatctc aaactctaca 1680 gaagctggac agaaactgtt caatatgctg aggcttggaa aatcagaacc ctggacccta 1740 gcattggaaa atgttgtagg agcaaagaac atgaatgtaa ggccactgct caactacttt 1800 gagcccttat ttacctggct gaaagaccag aacaagaatt cttttgtggg atggagtacc 1860 gactggagtc catatgcaga ccaaagcatc aaagtgagga taagcctaaa at cagctctt 1920 ggagataaag catatgaatg gaacgacaat gaaatgtacc tgttccgatc atctgttgca 1980 tatgctatga ggcagtactt tttaaaagta aaaaatcaga tgattctttt tggggagggag 2040 gatgtgcgag tggctaattt gaaaccaaga atctccttta atttctttgt cactgcacct 2100 aaaaatgtgt ctgatatcat tcctagaact gaagttgaaa aggccatcag gatgtcccgg 2160 agccgtatca atgatgcttt ccgtctgaat gacaacagcc tagagtttct ggggatacag 2220 ccaacacttg gacctcctaa ccagcc ccct gtttccctcg agcatcatca tcatcatcat 2280 tga 2283 <210> 16 <211> 760 <212> PRT <213> Artificial Sequence <220> <223> 16 -740 amino acid of projecting protein of hACE2 comprising signal peptide and his tag <220> <221> SIGNAL <222> (1)..(23) <220> <221> SITE <222> (755)..(760) <223> His_Tag <400> 16 Ala Thr Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp 1 5 10 15 Val Pro Gly Ser Thr Gly Asp Gly Ser Ala Ser Ala Ala Gln Ser Thr 20 25 30 Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe Asn His Glu Ala 35 40 45 Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp Asn Tyr Asn Thr 50 55 60 Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn Ala Gly Asp Lys 65 70 75 80 Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala Gln Met Tyr Pro 85 90 95 Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln Leu Gln Ala Leu 100 105 110 Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys Ser Lys Arg Leu 115 120 125 Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser Thr Gly Lys Val 130 135 140 Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu Glu Pro Gly Leu 145 150 155 160 Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu Arg Leu Trp Ala 165 170 175 Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu Arg Pro Leu Tyr 180 185 190 Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg Ala Asn His Tyr 195 200 205 Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu Val Asn Gly Val 210 215 220 Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu Asp Val Glu His 225 230 235 240 Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu His Ala Tyr Val 245 250 255 Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile Ser Pro Ile Gly 260 265 270 Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly Arg Phe Trp Thr 275 280 285 Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys Pro Asn Ile Asp 290 295 300 Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala Gln Arg Ile Phe 305 310 315 320 Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu Pro Asn Met Thr 325 330 335 Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro Gly Asn Val Gln 340 345 350 Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly Lys Gly Asp Phe 355 360 365 Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp Phe Leu Thr Ala 370 375 380 His His Glu Met Gly His Ile Gln Tyr Asp Met Ala Tyr Ala Ala Gln 385 390 395 400 Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His Glu Ala Val 405 410 415 Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys His Leu Lys Ser 420 425 430 Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn Glu Thr Glu Ile 435 440 445 Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly Thr Leu Pro Phe 450 455 460 Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe Lys Gly Glu Ile 465 470 475 480 Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met Lys Arg Glu Ile 485 490 495 Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr Tyr Cys Asp Pro 500 505 510 Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe Ile Arg Tyr Tyr 515 520 525 Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala Leu Cys Gln Ala 530 535 540 Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile Ser Asn Ser Thr 545 550 555 560 Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu Gly Lys Ser Glu 565 570 575 Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala Lys Asn Met Asn 580 585 590 Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe Thr Trp Leu Lys 595 600 605 Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr Asp Trp Ser Pro 610 615 620 Tyr Ala Asp Gln Ser Ile Lys Val Arg Ile Ser Leu Lys Ser Ala Leu 625 630 635 640 Gly Asp Lys Ala Tyr Glu Trp Asn Asp Asn Glu Met Tyr Leu Phe Arg 645 650 655 Ser Ser Val Ala Tyr Ala Met Arg Gln Tyr Phe Leu Lys Val Lys Asn 660 665 670 Gln Met Ile Leu Phe Gly Glu Glu Asp Val Arg Val Ala Asn Leu Lys 675 680 685 Pro Arg Ile Ser Phe Asn Phe Phe Val Thr Ala Pro Lys Asn Val Ser 690 695 700 Asp Ile Ile Pro Arg Thr Glu Val Glu Lys Ala Ile Arg Met Ser Arg 705 710 715 720 Ser Arg Ile Asn Asp Ala Phe Arg Leu Asn Asp Asn Ser Leu Glu Phe 725 730 735 Leu Gly Ile Gln Pro Thr Leu Gly Pro Pro Asn Gln Pro Pro Val Ser 740 745 750 Leu Glu His His His His His His 755 760 <210> 17 <211 > 633 <212> DNA <213> Artificial Sequence <220> <223> codon optimized DNA sequence of fragment of RBD of SARS-CoV-2 WT <400> 17 accgagtcta tcgtgcggtt ccccaacatc accaacctgt gtcctttcgg cgaggtgttc 60 aacgccacca gattcgcctc tgtgtacgcc tggaaccgga agcggatctc taactgcgtg 120 gccgactact ccgtgctgta ca actccgcc tccttcagca ccttcaagtg ctacggcgtg 180 tcccctacca agctgaacga cctgtgcttc accaacgtgt acgccgactc cttcgtgatc 240 agaggcgacg aagtgcggca gatcgctcct ggacagaccg gcaagatcgc cgactacaac 300 tacaagctgc ccgacgactt caccggctgt gtgatcgctt ggaactccaa caacctggac 360 tccaaagtcg gcggcaacta caattacctg taccggctgt tccggaagtc caacctgaag 420 cctttcgagc gggacatctc caccgagatc taccaggctg gcagcacccc ttgtaatggc 480 gtggaaggct tcaactgcta cttcccactg cagtcctacg gcttccagcc tacaaacggc 540 gtgggctacc agccttacag agtggtggtg ctgtccttcg agctgctgca tgctcctgct 600 accgtgtgcg gccctaagaa atctaccaac taa 633 <210> 18 <211> 210 <212> PRT <213> Artificial Sequence <220> <223> 323-532 amino acid of RBD of SARS-CoV-2 WT <400> 18 Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe 1 5 10 15 Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn 20 25 30 Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn 35 40 45 Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys 50 55 60 Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile 65 70 75 80 Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile 85 90 95 Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile 100 105 110 Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn 115 120 125 Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg 130 135 140 Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly 145 150 155 160 Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln 165 170 175 Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser 180 185 190 Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser 195 200 205 Thr Asn 210 <210> 19 <211> 633 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence of RBD of alpha variant N501Y <400> 19 accgagtcta tcgtgcggtt ccccaacatc accaacctgt gtcctttcgg cgaggtgttc 60 aacgccacca gattcgcctc tgtgtacgcc tggaaccgga ag cggatctc taactgcgtg 120 gccgactact ccgtgctgta caactccgcc tccttcagca ccttcaagtg ctacggcgtg 180 tcccctacca agctgaacga cctgtgcttc accaacgtgt acgccgactc cttcgtgatc 240 agaggcgacg aagtgcggca gatcgctcct ggacagaccg gcaagatcgc aac 300 tacaagctgc ccgacgactt caccggctgt gtgatcgctt ggaactccaa caacctggac 360 tccaaagtcg gcggcaacta caattacctg taccggctgt tccggaagtc caacctgaag 420 cctttcgagc gggacatctc caccgagatc taccaggctg gcagcacccc ttgtaatggc 480 gtggaaggct tcaactgcta cttcccactg cagtcctacg gcttccagcc tacatacggc 540 gtgggctacc agccttacag agtggtggtg ctgtccttcg agctgctgca tgctcctgct 600 accgtgtgcg gccctaagaa atct accaac taa 633 <210> 20 <211> 210 <212> PRT <213> Artificial Sequence <220> <223> amino acid of RBD of alpha variant N501Y <400> 20 Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe 1 5 10 15 Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn 20 25 30 Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn 35 40 45 Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys 50 55 60 Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile 65 70 75 80 Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile 85 90 95 Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile 100 105 110 Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn 115 120 125 Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg 130 135 140 Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly 145 150 155 160 Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln 165 170 175 Pro Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser 180 185 190 Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser 195 200 205 Thr Asn 210 < 210> 21 <211> 630 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence of RBD of beta variant K417N-E484K-N501Y <400> 21 accgagtcta tcgtgcggtt ccccaacatc accaacctgt gtcctttcgg cgaggtgttc 60 aacgccacca gattcgcctc tgtgtacgcc tggaaccgga agcggatctc taactgcgtg 120 gccgact act ccgtgctgta caactccgcc tccttcagca ccttcaagtg ctacggcgtg 180 tcccctacca agctgaacga cctgtgcttc accaacgtgt acgccgactc cttcgtgatc 240 agaggcgacg aagtgcggca gatcgctcct ggacagaccg gcaacatcgc aac 300 tacaagctgc ccgacgactt caccggctgt gtgatcgctt ggaactccaa caacctggac 360 tccaaagtcg gcggcaacta caattacctg taccggctgt tccggaagtc caacctgaag 420 cctttcgagc gggacatctc caccgagatc taccaggctg gcagcacccc ttgtaatggc 480 gtgaaaggct tcaactgcta cttcccactg cagtcctacg gcttccagcc tacatacggc 540 gtgggctacc agccttacag agtggtggtg ctgtccttcg agctgctgca tgctcctgct 600 accgtgtgcg gccctaagaa atctaccaac 630 <210> 22 <211> 210 <212> PRT <213> Artificial Sequence <220> <223> amino acid of RBD of beta variant K417N-E484K-N501Y <400> 22 Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe 1 5 10 15 Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn 20 25 30 Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn 35 40 45 Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys 50 55 60 Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile 65 70 75 80 Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Asn Ile 85 90 95 Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile 100 105 110 Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn 115 120 125 Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg 130 135 140 Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly 145 150 155 160 Val Lys Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln 165 170 175 Pro Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser 180 185 190 Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser 195 200 205 Thr Asn 210 <210> 23 <211> 630 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence of RBD of gamma variant K417T-E484K-N501Y <400> 23 accgagtcta tcgtgcggtt ccccaacatc accaacctgt gtcctttcgg cgaggtgttc 60 aacgccacca gattcgcct c tgtgtacgcc tggaaccgga agcggatctc taactgcgtg 120 gccgactact ccgtgctgta caactccgcc tccttcagca ccttcaagtg ctacggcgtg 180 tcccctacca agctgaacga cctgtgcttc accaacgtgt acgccgactc cttcgtgatc 240 agaggcgacg aagtgcggca gatcgctcct ggacagaccg gcaccatcgc cgactacaac 300 tacaagctgc ccgacgactt caccggctgt gtgatcgctt ggaactccaa caacctggac 360 tccaaagtcg gcggcaacta caattacctg taccggctgt tccggaagtc caacctgaag 420 cctttcgagc gggacatctc caccgagatc taccaggctg gcagcacccc ttgtaatggc 480 gtgaaaggct tcaactgcta cttcccactg cagtcctacg gcttccagcc tacatacggc 540 gtgggctacc agccttacag agtggtggtg ctgtccttcg agctgctgca tgctcctgct 600 accgtgt gcg gccctaagaa atctaccaac 630 <210> 24 <211> 210 <212> PRT <213> Artificial Sequence <220> <223> amino acid of RBD of gamma variant K417T-E484K-N501Y <400> 24 Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe 1 5 10 15 Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn 20 25 30 Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn 35 40 45 Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys 50 55 60 Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile 65 70 75 80 Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Thr Ile 85 90 95 Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile 100 105 110 Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn 115 120 125 Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg 130 135 140 Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly 145 150 155 160 Val Lys Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln 165 170 175 Pro Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser 180 185 190 Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser 195 200 205 Thr Asn 210 <210> 25 <211> 630 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence of RBD of delta variant L452R-T478K <400> 25 accgagtcta tcgtgcggtt ccccaacatc accaacctgt gtcctttcgg cgaggtgttc 60 aacgccacca gattcgcctc tgtgtacgcc tggaaccgga a gcggatctc taactgcgtg 120 gccgactact ccgtgctgta caactccgcc tccttcagca ccttcaagtg ctacggcgtg 180 tcccctacca agctgaacga cctgtgcttc accaacgtgt acgccgactc cttcgtgatc 240 agaggcgacg aagtgcggca gatcgctcct ggacagaccg gcaagatcgc cgactacaac 300 tacaagctgc ccgacgactt caccggctgt gtgatcgctt ggaactccaa caacctggac 360 tccaaagtcg gcggcaacta caattaccgg taccggctgt tccggaagtc caacctgaag 420 cctttcgagc gggacatctc caccgagatc taccaggctg gcagcaagcc ttgtaatggc 480 gtggaaggct tcaactgcta cttcccactg cagtcctacg gcttccagcc tacaaacggc 540 gtgggctacc agccttacag agtggtggtg ctgtccttcg agctgctgca tgctcctgct 600 accgtgtgcg gccctaagaa atctaccaac 630 <210> 26 <211> 210 <212> PRT <213> Artificial Sequence <220> <223> amino acid of RBD of delta variant L452R-T478K <400> 26 Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe 1 5 10 15 Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn 20 25 30 Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn 35 40 45 Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys 50 55 60 Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile 65 70 75 80 Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile 85 90 95 Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile 100 105 110 Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn 115 120 125 Tyr Arg Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg 130 135 140 Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Lys Pro Cys Asn Gly 145 150 155 160 Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln 165 170 175 Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser 180 185 190 Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser 195 200 205 Thr Asn 210 <210> 27 <211> 630 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence of RBD of omicron variant G339D-S371L-S373P-S375F-K417N-N440K-G446S-S477N-T478K-E484A-Q493K -G496S-Q498R-N501Y-Y505H <400> 27 accgagtcta tcgtgcggtt ccccaacat c accaacctgt gtcctttcga cgaggtgttc 60 aacgccacca gattcgcctc tgtgtacgcc tggaaccgga agcggatctc taactgcgtg 120 gccgactact ccgtgctgta caacctggcc cctttcttca ccttcaagtg ctacggcgtg 180 tcccctacca agctgaacga cctgtgcttc accaacgtgt acgccgactc cttcgtga tc 240 agaggcgacg aagtgcggca gatcgctcct ggacagaccg gcaacatcgc cgactacaac 300 tacaagctgc ccgacgactt caccggctgt gtgatcgctt ggaactccaa caagctggac 360 tccaaagtct ccggcaacta caattacctg taccggctgt tccggaagtc caacctgaag 420 cctttcgagc gggacatctc caccgagatc taccaggctg gcaataaacc ttgtaatggc 480 gtggctggct tcaactgcta cttcccactg aagtcctact ccttccggcc tacatacggc 540 gtgggccatc agccttacag agtggtggtg ctgtcc ttcg agctgctgca tgctcctgct 600 accgtgtgcg gccctaagaa atctaccaac 630 <210> 28 <211> 210 <212> PRT <213> Artificial Sequence <220> <223 > amino acid of RBD of omicron variant G339D-S371L-S373P-S375F-K417N-N440K-G446S-S477N-T478K-E484A-Q493K -G496S-Q498R-N501Y-Y505H <400> 28 Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro he 1 5 10 15 Asp Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn 20 25 30 Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn 35 40 45 Leu Ala Pro Phe Phe Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys 50 55 60 Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile 65 70 75 80 Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Asn Ile 85 90 95 Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile 100 105 110 Ala Trp Asn Ser Asn Lys Leu Asp Ser Lys Val Ser Gly Asn Tyr Asn 115 120 125 Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg 130 135 140 Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Asn Lys Pro Cys Asn Gly 145 150 155 160 Val Ala Gly Phe Asn Cys Tyr Phe Pro Leu Lys Ser Tyr Ser Phe Arg 165 170 175 Pro Thr Tyr Gly Val Gly His Gln Pro Tyr Arg Val Val Val Leu Ser 180 185 190 Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser 195 200 205 Thr Asn 210 <210> 29 <211> 2175 <212> DNA <213> Artificial Sequence <220> <223> codon optimized DNA sequence of fragment of projecting protein of hACE2 <400> 29 gctgctcagt ccaccatga ggaacaggcc aagacatttt tggacaagtt taaccacgaa 60 gccgaagacc tgttctatca aagt tcactt gcttcttgga attataacac caatattact 120 gaagagaatg tccaaaacat gaataatgct ggggacaaat ggtctgcctt tttaaaggaa 180 cagtccacac ttgcccaaat gtatccacta caagaaattc agaatctcac agtcaagctt 240 cagctgcagg ctcttcagca aaatgggtct tcagtgctct cagaagacaa gagcaaacgg 300 t tgaacacaa ttctaaatac aatgagcacc atctacagta ctggaaaaagt ttgtaaccca 360 gataatccac aagaatgctt attacttgaa ccaggtttga atgaaataat ggcaaacagt 420 ttagactaca atgagaggct ctgggcttgg gaaagctgga gatctgaggt cggcaagcag 480 ctgaggccat tatatgaaga gtatgtggtc ttgaaaaatg agatggcaag agcaaatcat 540 tatgaggact atggggatta ttggagagga gactatgaag taaatggggt agatggctat 600 gactacagcc gcggccagtt gattgaagat gtggaacata cctttgaaga gattaaacca 660 ac atcttcatgc ctatgtgagg gcaaagttga tgaatgccta tccttcctat 720 atcagtccaa ttggatgcct ccctgctcat ttgcttggtg atatgtgggg tagattttgg 780 acaaatctgt actctttgac agttcccttt ggacagaaac caaacataga tgttactgat 840 gcaatggtgg accaggcctg ggatgcacag agaatattca aggaggccga gaagttcttt 900 gtatctgttg gtcttcctaa tatgactcaa ggattctggg aaaattccat gctaacggac 960 ccaggaaatg ttcagaaagc agtctgccat cccacagctt gggacctggg gaag ggcgac 1020 ttcaggatcc ttatgtgcac aaaggtgaca atggacgact tcctgacagc tcatcatgag 1080 atggggcata tccagtatga tatggcatat gctgcacaac cttttctgct aagaaatgga 1140 gctaatgaag gattccatga agctgttggg gaaatcatgt cactttctgc agccacacct 1200 aagcatttaa aatccattgg tcttctgtca cccgattttc aagaagacaa tgaaacagaa 1260 ataaacttcc tgctcaaaca agcactcacg attgttggga ctctgccatt tacttacatg 1320 ttagagaagt ggaggtggat ggtctttaaa ggggaaattc ccaaagacca gtggatgaaa 1380 aagtggtggg agatgaagcg agagatagtt ggggtggtgg aacctgtgcc ccatgatgaa 1440 acatactgtg accccgcatc tctgttccat gtttctaatg attactcatt cattcgatat 1500 tacacaagga ccctttacca attccagttt caagaagcac tttgtcaagc agctaaacat 1560 gaaggccctc tgcacaaatg tgacatctca aactctacag aagctggaca gaaactgttc 1620 aatatgctga ggcttggaaa atcagaaccc tggaccctag cattggaaaa tgttgtagga 1680 gcaaagaaca tgaat gtaag gccactgctc aactactttg agcccttatt tacctggctg 1740 aaagaccaga acaagaattc ttttgtggga tggagtaccg actggagtcc atatgcagac 1800 caaagcatca aagtgaggat aagcctaaaa tcagctcttg gagataaagc atatgaatgg 1860 aacgacaatg aaatgtacct gttccgatca tctgttgcat atgctatgag gcagtacttt 1920 ttaaaagtaa aaaatcagat gattcttttt ggggaggagg atgtgcgagt ggctaatttg 1980 aaaccaagaa tctcctttaa tttctttgtc actgcaccta aaaatgtgtc tgatatcatt 2040 cctagaactg aagttgaaaa ggccatcagg atgtcccgga gccgtatcaa tgatgctttc 2100 cgtctgaatg acaacagcct agagtttctg gggatacagc caacacttgg acctcctaac 2160 cagccccctg tttcc 2175 <210> 30 <211> 72 5 <212> PRT <213> Artificial Sequence <220> <223> amino acid of projecting protein of hACE2 <400> 30 Ala Ala Gln Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys 1 5 10 15 Phe Asn His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser 20 25 30 Trp Asn Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn 35 40 45 Asn Ala Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu 50 55 60 Ala Gln Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu 65 70 75 80 Gln Leu Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp 85 90 95 Lys Ser Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr 100 105 110 Ser Thr Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu 115 120 125 Leu Glu Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn 130 135 140 Glu Arg Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln 145 150 155 160 Leu Arg Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala 165 170 175 Arg Ala Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr 180 185 190 Glu Val Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile 195 200 205 Glu Asp Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His 210 215 220 Leu His Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr 225 230 235 240 Ile Ser Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp 245 250 255 Gly Arg Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln 260 265 270 Lys Pro Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp 275 280 285 Ala Gln Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly 290 295 300 Leu Pro Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp 305 310 315 320 Pro Gly Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu 325 330 335 Gly Lys Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp 340 345 350 Asp Phe Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met 355 360 365 Ala Tyr Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly 370 375 380 Phe His Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro 385 390 395 400 Lys His Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp 405 410 415 Asn Glu Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val 420 425 430 Gly Thr Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val 435 440 445 Phe Lys Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu 450 455 460 Met Lys Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu 465 470 475 480 Thr Tyr Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser 485 490 495 Phe Ile Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu 500 505 510 Ala Leu Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp 515 520 525 Ile Ser Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg 530 535 540 Leu Gly Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly 545 550 555 560 Ala Lys Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu 565 570 575 Phe Thr Trp Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser 580 585 590 Thr Asp Trp Ser Pro Tyr Ala Asp Gln Ser Ile Lys Val Arg Ile Ser 595 600 605 Leu Lys Ser Ala Leu Gly Asp Lys Ala Tyr Glu Trp Asn Asp Asn Glu 610 615 620 Met Tyr Leu Phe Arg Ser Ser Val Ala Tyr Ala Met Arg Gln Tyr Phe 625 630 635 640 Leu Lys Val Lys Asn Gln Met Ile Leu Phe Gly Glu Glu Asp Val Arg 645 650 655 Val Ala Asn Leu Lys Pro Arg Ile Ser Phe Asn Phe Phe Val Thr Ala 660 665 670 Pro Lys Asn Val Ser Asp Ile Ile Pro Arg Thr Glu Val Glu Lys Ala 675 680 685 Ile Arg Met Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asn Asp 690 695 700 Asn Ser Leu Glu Phe Leu Gly Ile Gln Pro Thr Leu Gly Pro Pro Asn 705 710 715 720Gln Pro Pro Val Ser 725

Claims (22)

(i) a receptor binding domain (RBD) of the spike protein of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV2) or a variant thereof, conjugated with a marker substance, biotin; and (ii) a human angiotensin converting enzyme 2 (ACE2) protein having a His-tag linked to the C-terminus,
The receptor binding domain (RBD) of the spike protein of the above-mentioned type 2 severe acute respiratory syndrome coronavirus (SARS-CoV2) or a variant thereof is at least one selected from the group consisting of SEQ ID NOs: 18, 20, 22, 24, 26 and 28,
The above human angiotensin converting enzyme 2 (ACE2) protein consists of sequence number 30.
A composition for detecting neutralizing antibodies to severe acute respiratory syndrome coronavirus type 2 (SARS-CoV2). delete delete In the first paragraph,
A composition for detecting a neutralizing antibody against severe acute respiratory syndrome coronavirus (SARS-CoV2), wherein the composition is for detecting a neutralizing antibody against at least one selected from the group consisting of wild type SARS-CoV-2, alpha variant B.1.1.7; beta variant B1.351; gamma variant P1; delta variant B.1.617.2; and omicron variant B.1.1.529. delete delete In the first paragraph,
A composition for detecting a neutralizing antibody to severe acute respiratory syndrome coronavirus type 2 (SARS-CoV2), wherein the labeling substance is for use in a detection method selected from the group consisting of immunofluorescence, immunoblotting, western blotting, enzyme-linked immunosorbent assay (ELISA), flow cytometry, immunoprecipitation, immunohistochemistry, biofilm test, pinning ring test, antibody array optical density test, and chemiluminescence. delete delete In the first paragraph,
A composition for detecting a neutralizing antibody of type 2 severe acute respiratory syndrome coronavirus (SARS-CoV2), wherein the substance capable of recognizing a label substance capable of indirectly generating a signal is avidin or an avidin analogue. A composition for detecting a neutralizing antibody against type 2 severe acute respiratory syndrome coronavirus (SARS-CoV2), wherein the avidin or avidin analogue is streptavidin, neutravidin, or captavidin. A kit for detecting a neutralizing antibody to severe acute respiratory syndrome coronavirus (SARS-CoV2), comprising a composition for detecting a neutralizing antibody to severe acute respiratory syndrome coronavirus (SARS-CoV2) according to any one of claims 1, 4, 7, 10, and 11. A kit for detecting neutralizing antibodies to type 2 severe acute respiratory syndrome coronavirus (SARS-CoV2), wherein the kit further comprises a solid support in claim 12. In Article 12,
A kit for detecting neutralizing antibodies to type 2 severe acute respiratory syndrome coronavirus (SARS-CoV2), wherein the kit further comprises at least one of a carrier, a washing buffer, a sample dilution solution, an enzyme substrate, a reaction stop solution, and an instruction manual teaching how to use. For the analysis of a sample, (i) a receptor binding domain (RBD) of the spike protein of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV2) or a variant thereof; and (ii) a human angiotensin converting enzyme 2 (ACE2) protein having a His-tag linked to the C-terminus, and (ii) a step of determining the level of interaction between the human angiotensin converting enzyme 2 (ACE2) protein;
The receptor binding domain (RBD) of the spike protein of the above-mentioned type 2 severe acute respiratory syndrome coronavirus (SARS-CoV2) or a variant thereof is at least one selected from the group consisting of SEQ ID NOs: 18, 20, 22, 24, 26 and 28,
The above human angiotensin converting enzyme 2 (ACE2) protein consists of sequence number 30.
A method for detecting a neutralizing antibody of severe acute respiratory syndrome coronavirus (SARS-CoV2), wherein the receptor binding domain (RBD) or a variant thereof of (i) above is conjugated to biotin, which is a labeling substance, and the human angiotensin converting enzyme 2 (ACE2) protein of (ii) above is immobilized on a solid support. In Article 15,
A method for detecting neutralizing antibodies to type 2 severe acute respiratory syndrome coronavirus (SARS-CoV2), wherein the sample is blood, lymph, saliva or synovial fluid. In Article 15,
A method for detecting a neutralizing antibody of type 2 severe acute respiratory syndrome coronavirus (SARS-CoV-2), wherein the type 2 severe acute respiratory syndrome coronavirus (SARS-CoV-2) variant is at least one selected from the group consisting of alpha variant B.1.1.7; beta variant B1.351; gamma variant P1; delta variant B.1.617.2; and omicron variant B.1.1.529. delete a) A step of preparing a mixture by mixing a sample with a receptor binding domain (RBD) of the spike protein of type 2 severe acute respiratory syndrome coronavirus (SARS-CoV2) or a variant thereof conjugated with a labeling substance, biotin, and incubating the mixture, wherein the receptor binding domain (RBD) of the spike protein of type 2 severe acute respiratory syndrome coronavirus (SARS-CoV2) or a variant thereof is at least one selected from the group consisting of SEQ ID NOs: 18, 20, 22, 24, 26, and 28,
b) a step of mixing the mixture of step a) with a human angiotensin converting enzyme 2 (ACE2) protein having a His-tag linked to the C-terminus, wherein the human angiotensin converting enzyme 2 (ACE2) protein consists of the sequence number 30,
c) a step of detecting a signal of biotin, which is a labeling substance; a method for detecting a neutralizing antibody of type 2 severe acute respiratory syndrome coronavirus (SARS-CoV2). In Article 19,
A method for detecting neutralizing antibodies to type 2 severe acute respiratory syndrome coronavirus (SARS-CoV2), wherein the sample is blood, lymph, saliva or synovial fluid. In Article 19,
A method for detecting a neutralizing antibody of type 2 severe acute respiratory syndrome coronavirus (SARS-CoV-2), wherein the type 2 severe acute respiratory syndrome coronavirus (SARS-CoV-2) variant is at least one selected from the group consisting of alpha variant B.1.1.7; beta variant B1.351; gamma variant P1; delta variant B.1.617.2; and omicron variant B.1.1.529. delete