KR20220002156A - Kit for screening for therapeutic agent for preventing or treating Corona-virus infection by using of esterase activity of Corona-19 virus spike protein and pharmaceutical composition comprising material selected therefrom - Google Patents

Abstract

The present invention relates to a discovery of an activity of esterase of a recombinant spike protein of the Coronavirus Disease 2019 (COVID-19) virus and to development of a treatment for the COVID-19 virus using same. Specifically, the present inventors confirmed for the first time that the S1 subunit and the S2 subunit of the spike protein of the COVID-19 virus each showed high esterase activity, and have conceived a method for developing a therapeutic agent for COVID-19 in such a way that a substance inhibiting the activity of the esterase is selected, on the basis of the activity of the esterase of the spike protein of the COVID-19 virus. In addition, the present invention provides a substance inhibiting esterase, selected through the above method, as a therapeutic agent for COVID-19. The selected substance inhibits cell infection by the virus and a release process of the virus from the cell by inhibiting the activity of the esterase of the spike protein of the COVID-19 virus, thereby inhibiting the proliferation of the virus in a body, and thus has an effect of treating COVID-19.

Description

A pharmaceutical composition for preventing or treating a corona virus infection comprising a screening kit for a therapeutic agent for corona virus infection using the esterase activity of the spike protein of the corona-19 virus and a material screened using the kit {Kit for screening for therapeutic agent for preventing or treating Corona-virus infection by using of esterase activity of Corona-19 virus spike protein and pharmaceutical composition comprising material selected therefrom}

The present invention relates to the discovery of the activity of an esterase of a recombinant spike protein of the Corona-19 virus (COVID-19, Coronavirus Disease 2019) and the development of a treatment for the Corona-19 virus using the same. In detail, the present inventors first confirmed that the spike protein of the Corona-19 virus exhibited high esterase activity, and using this, the activity of the esterase protein of the spike protein of the Corona-19 virus was used. Thus, a method for screening a therapeutic agent for the COVID-19 virus was developed. Substances that inhibit esterases selected through screening inhibit the activity of esterases of the COVID-19 virus spike protein, thereby inhibiting the cell infection or release process of the virus. Through this, it has the effect of suppressing the proliferation of the virus in the body and treating the infection of the Corona 19 virus. Accordingly, the present invention provides a method for screening a coronavirus therapeutic agent using the esterase activity of the spike protein of the Corona-19 virus, and a pharmaceutical composition for preventing or treating the Corona-19 virus using the screened material.

Corona virus is one of the three major viruses that cause colds in humans along with adenovirus and rhinovirus, and is an RNA virus with a gene size of 27 to 32 kb that can infect humans and various animals. When viewed under an electron microscope, the surface of the virus particle protrudes like a protrusion. It accounts for 10 to 30% of adult colds that occur mainly in the cold winter, and the main symptom is a nasal cold accompanied by a headache, sore throat, or cough. Since the coronavirus was first discovered in chickens in the 1930s, it was discovered in animals such as dogs, pigs, and birds, and in the 1960s, it was also found in humans, confirming that it is found in both animals and humans.

As the field of human activity expands, viruses that only spread among animals cause genetic mutations for survival and are passed on to humans, for example, SARS (bats and civets), MERS (bats and camels), Coronavirus Infectious Disease-19 (presumed to be a bat) is one such case. The coronaviruses discovered so far are classified into four genera: Alpha, Beta, Gamma, and Delta. Here, alpha is further divided into types 1a and 1b, and beta is divided into types 2a, 2b, 2c, and 2d. Of these, alpha and beta infect humans and animals, and gamma and delta infect animals. There are a total of 7 types of coronaviruses that have been identified so far, including HCoV 229E · HCoV NL63 · HCoV OC43 · HCoV HKU1 · SARS-CoV · MERS-CoV · SARS-CoV-2. Of these, four (229E, OC43, NL63, and HKU1) caused only mild symptoms similar to colds. However, SARS (Severe Acute Respiratory Syndrome), MERS (MERS-CoV), and Coronavirus Infectious Disease-19 (SARS-CoV-2, severe acute respiratory syndrome coronavirus 2) It can cause disease and cause many deaths.

Coronavirus (COVID-19) is a new type of coronavirus (SARS-CoV-2) that first emerged in Wuhan, China in December 2019 and has spread throughout China and around the world. The COVID-19 virus has a very high transmission rate, making it particularly contagious. After being infected with the COVID-19 virus, after an incubation period of about 2 to 14 days (estimated), the main symptoms include fever (37.5 degrees), respiratory symptoms such as cough or shortness of breath, and pneumonia, but asymptomatic infections are not uncommon. The spike protein of the COVID-19 virus (COVID-19) is a type 1 membrane glycoprotein comprising a very large ecto-domain region, a single transmembrane domain (TMD) and a short cytoplasmic tail. to be. The spike protein, like the spike (S) protein of other coronaviruses, exists as a trimer on the surface of the virus, and has a receptor binding domain necessary for the virus to invade the host cell and the virus membrane and organelles during cell invasion. It has a fusion peptide that induces fusion between membranes, and is known to play a role, such as inducing neutralized antibodies against the spike protein in a natural host. The corona spike protein exists as a trimer on the surface of the virus membrane.

When a virus invades a cell, it must bind to the cell's surface, and for this, it uses a variety of receptors. Corona-19 is known to recognize and bind to a protein called ACE present on the surface of human cells by a spike protein present on the surface of the virus, thereby recognizing target cells. Coronaviruses are classified into three types in terms of receptor binding. One type binds to a specific protein, a second type binds to a sugar, and another type binds to both a protein and a sugar. Specifically, NL63 and PRCV of the α-genus of the corona virus bind to respective protein receptors called ACE2 and aminopeptidase N (APN), and are of the same α-genus. TGEV binds both APN and sugar. MHV and BCoV of the corona virus β-genus bind to both carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) and sugar, respectively, whereas MERS-CoV and SARS are proteins Binds to dipeptidyl peptidase 4 (DPP4) and angiotensin-converting enzyme 2 (ACE2) (Garcia-Sastre, A. (2010) Influenza Virus Receptor Specificity. Disease and Transmission. Am J Pathol. 176: 1584-1585). However, MERS is known to bind to sialic acid in addition to DPP4 (Li et al., (2017) Identification of sialic acid-binding function for the Middle East respiratory syndrome coronavirus spike glycoprotein. Proc Natl Acad Sci US A. 114:E8508-E8517). Since MERS-CoV transmission is inhibited in cells from which sialic acid has been removed, it has been suggested that binding to sialic acid is very important for virus infection. IBV, a γ-genus, is known to bind to sugars.

In the case of viruses in which the surface protein of a virus binds to sugar residues such as sialic acid present on the surface of animal cells, when they enter the cell or when released from the cell, between the surface protein of the virus and the sugar residue on the surface of the animal cell binding should be removed. One of the methods for removing the bond with sialic acid is to cut the sialic acid residue from the sugar chain. Proteins having this activity are called receptor destroying enzymes (RDEs), and some genus of coronavirus, such as influenza C, are hemagglutinin-esterase (HE). is known to have such an activity. In particular, human coronavirus OC43 (HCoV-OC43) and bovine coronavirus (BCoV) are known to have hemagglutinin-esterase (HE). In the case of HCoV-OC43, it was reported that when the activity was inhibited using a neutralizing antibody or modification, the proliferation of the virus was significantly reduced, and when the activity was reinforced, the proliferation of the virus was increased again (Desforges et al.) al., (2013) The acetyl-esterase activity of the hemagglutinin-esterase protein of human coronavirus OC43 strongly enhances the production of infectious virus. J Virol). HE of this virus has the activity of acetyl esterase, and shows the activity of removing an acetyl group from O-acetylated sialic acid. And in the case of influenza virus, which is a different kind of virus, it has different kinds of sugar hydrolysing enzymes depending on the virus type. Types A and B have neuraminidase (NA), and type C has HEF. Thus, these influenza viruses use the activity of these proteins to cut sialic acid residues. It has been reported that the NA activity of influenza virus is important, especially when influenza virus is released from cells. It is known that SARS-CoV and SARS-CoV-2 (COVID-19) do not have independent HE genes. Therefore, it is not known whether these viruses require enzymatic activity such as esterases. In the present invention, it was revealed for the first time that the S protein of SARS-CoV-2 virus has an esterase activity that hydrolyzes p-nitrophenol acetate, and in particular, both S1 and S2 subunits The purpose of this study is to reveal the esterase activity, and to develop an inhibitor that can interfere with virus cell infection and proliferation by interfering with the esterase activity of the spike protein.

When a virus invades the human body, the proteins on the surface of the virus recognize the sugar chain of the protein on the surface of the target cell and use it as a receptor, or first recognize the sugar chain of the protein on the surface of the target cell and enter the target cell. After attachment, it subsequently binds to its main receptor, allowing the virus to infect. In addition, when the virus is released from the target cell after it has proliferated, the protein on the virus surface also binds to the sugar chain of the target cell's surface protein. Therefore, it is important to break the binding of these proteins to sugar chains in the virus target cell infection and release. Esterases play an important role in this process. Therefore, these esterases are becoming important targets for the development of antiviral agents. In particular, Tamiflu, a treatment for swine flu, has been developed as an activity inhibitor of neuraminidase involved in this process.

In the present invention, it was confirmed that the spike protein of corona virus has a strong esterase activity. In particular, it was confirmed that each subunit exhibited esterase activity even when divided into S1 and S2 subunits. Esterases are capable of cleaving ester bonds in various compounds, including various forms of sialic acid, as well as standard substrates such as para-nitrophenyl acetate (p-NPA).

In the present invention, the three types of S proteins (full length, S1 and S2) hydrolyze p-NPA after production and separation and purification of the trimer form of the full-length, S1 and S2 subunits of the spike protein. It was confirmed that it has the activity of a hydrolase, and a compound that inhibits this activity was screened. Various compounds have been reported to inhibit the activity of esterases. In particular, substances that inhibit the activity of some esterases are used as medicines such as anticancer drugs and antibiotics. First, it was confirmed whether the activity of the spike protein was inhibited using these. And it is intended to utilize these esterase activity inhibitors as antiviral agents. In the case of influenza virus, it is well known that Tamiflu, an NA inhibitor, inhibits the proliferation of H1N1 influenza. It is known that Tamiflu inhibits the activity of NA so that the virus particles are not released from the target cells, thereby preventing the proliferation of the virus. In the case of HCoV-OC43, by inhibiting HE activity, infection into target cells and release of virus from target cells were inhibited, thereby inhibiting virus proliferation overall. These results support the claim that by inhibiting the activity of the esterase of the spike protein identified in the present invention, the proliferation of the coronavirus can be inhibited. Therefore, in the present invention, it is intended to find a compound that inhibits the esterase activity of the spike protein, and to develop it as a drug that inhibits the proliferation of viruses.

Accordingly, it is an object of the present invention to provide a recombinant protein derived from the spike protein of coronavirus, which has esterase activity.

Another object of the present invention is a kit capable of screening a substance capable of preventing or treating a corona virus infection by checking whether the esterase activity of the spike protein of the corona virus is inhibited, and prevention or treatment of a corona virus infection using the same It is to provide a method for screening a substance for use.

Further, another object of the present invention is a pharmaceutical composition for preventing or treating coronavirus infection comprising an esterase activity inhibitor of the spike protein of coronavirus as an active ingredient and/or corona virus infection prevention comprising the same composition as an active ingredient Or to provide health functional food for improvement.

In order to solve the above problems, the present invention provides a recombinant protein comprising the S1 subunit or S2 subunit of the coronavirus spike protein, or the S1 subunit and the S2 subunit having esterase activity. Provided is a recombinant protein comprising a unit.

According to a preferred embodiment of the present invention, when the corona virus is SARS-Corona Virus-2, the S1 subunit comprises the amino acid of SEQ ID NO: 4, and the S2 subunit is SEQ ID NO: The recombinant protein comprising the amino acid sequence of 6, the recombinant protein comprising the S1 subunit and the S2 subunit may include the amino acid sequence of SEQ ID NO: 2.

According to another preferred embodiment of the present invention, the recombinant protein may be in a trimeric form.

According to another preferred embodiment of the present invention, the recombinant protein can be produced using a recombinant vector containing the gene construct of Figure 1 (a), (b) or (c).

In addition, the present invention provides a screening kit for a candidate substance for the prevention or treatment of a coronavirus infection, comprising:

Recombinant protein derived from the spike protein of coronavirus; and

A substance capable of measuring the esterase activity of the recombinant protein derived from the spike protein of the corona virus.

According to a preferred embodiment of the present invention, the corona virus is selected from the group consisting of SARS-Corona Virus, MERS-Corona Virus, and SARS-Corona Virus-2 may be any one of

According to another preferred embodiment of the present invention, the recombinant protein derived from the spike protein of the coronavirus is a recombinant protein comprising the S1 subunit, the S2 subunit, the S1 subunit or the S2 subunit of the spike protein of the Corona-19 virus. , and may be a recombinant protein comprising the S1 subunit and the S2 subunit.

According to another preferred embodiment of the present invention, when the corona virus is SARS-Corona Virus-2, the S1 subunit comprises the amino acid of SEQ ID NO: 4, and the S2 subunit is The recombinant protein comprising the amino acid sequence of SEQ ID NO: 6, the recombinant protein comprising the S1 subunit and the S2 subunit may include the amino acid sequence of SEQ ID NO: 2.

According to another preferred embodiment of the present invention, the recombinant protein derived from the spike protein of the corona virus may be in the form of a trimer.

According to another preferred embodiment of the present invention, the substance capable of measuring the esterase activity is a substrate of a general esterase; compounds comprising sialic acid; BCECF, AM(2',7'-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester) (BCECF, AM(2',7'-Bis- (2-Carboxyethyl)-5-(and-6)-Carboxyfluorescein, Acetoxymethyl Ester)); Calcein AM; Carboxyeosin diacetate, succinimidyl ester; Carboxyfluorescein diacetate, acetoxymethyl ester; Or it may be carboxyfluorescein diacetate, succinimidyl ester (Carboxyfluorescein diacetate, succinimidyl ester).

According to another preferred embodiment of the present invention, the substrate of the esterase is para nitropheny acetate (p-NPA), and the compound containing sialic acid is Neu5,9Ac2, 4-methyl It may be umbelliferyl acetate (4-methylumbelliferyl acetate) or 9Ac2-lactose (9Ac2-lactose).

In addition, the present invention provides a method for screening a candidate substance for the prevention or treatment of a coronavirus infection using the above-described kit.

According to a preferred embodiment of the present invention, the method for screening a candidate substance for the prevention or treatment of coronavirus infection may include the following steps:

(i) treating a candidate material with a recombinant protein derived from the spike protein of coronavirus;

(ii) measuring the esterase activity of the recombinant protein derived from the spike protein of the corona virus treated with the candidate substance; and

(iii) when the candidate material inhibits esterase activity by 30% or more, selecting the candidate material as a candidate material for prevention or treatment of coronavirus.

According to another preferred embodiment of the present invention, the recombinant protein derived from the spike protein of the coronavirus is a recombinant protein comprising the S1 subunit, the S2 subunit, the S1 subunit or the S2 subunit of the spike protein of the coronavirus, and It may be a recombinant protein comprising the S1 subunit and the S2 subunit.

Furthermore, the present invention provides a pharmaceutical composition for the prevention or treatment of corona virus infection, comprising the inhibitor of esterase activity of the recombinant protein derived from the spike protein of the corona virus described above.

According to a preferred embodiment of the present invention, the selected substance binds to the active site of the esterase and inhibits the activity of the esterase, or binds to a site other than the active site of the esterase and binds to an ester. It may be to inhibit the activity of the esterase by inducing a structural change of the hydrolase.

In addition, the present invention provides a pharmaceutical composition for preventing or treating COVID-19 virus infection, comprising sulfamethoxazole, a derivative, isomer, or pharmaceutically acceptable salt thereof, and sulfamethoxazole; It provides a health functional food composition for preventing or improving Corona-19 virus infection, including a derivative, isomer, or food pharmaceutically acceptable salt thereof.

The present invention is based on the esterase activity of the S1 and S2 subunits of the spike protein of the Corona-19 virus to develop an inhibitor that inhibits its activity, and uses this to develop a coronavirus therapeutic agent. It can directly inhibit the activity of esterase by binding to the active site of esterase, or indirectly by inducing a change in structure by binding to a site other than the active site of esterase of the spike protein. It may also inhibit the activity of esterases. These substances may be various compounds including chemical substances, nucleic acid-based substances such as aptamers, or small oligopeptides containing amino acids or mutated amino acids.

1 is a schematic diagram of a recombinant protein gene of three types (Sf, S1 and S2) of the spike protein of the Corona-19 virus used in the present invention.
Figure 2 is a recombinant protein of three types (Sf, S1 and S2) of the spike protein of the corona-19 virus of Figure 1 is produced in Nicotaiana benthamiana , and it is produced by protein A affinity column chromatography (Protein A It was confirmed that they were separated and purified through affinity column chromatography).
3 is a graph showing the activity of esterases of three types of spike recombinant proteins (full length, S1 and S2).
Figure 4 shows the activity of the esterase after treatment with sulfamethoxazole to three types of spike recombinant proteins (full length, S1 and S2).

Hereinafter, the present invention will be described in detail.

As described above, ester hydrolase is an important target for the development of antiviral agents, and in particular, Tamiflu, a treatment for H1N1 influenza, has been developed as an activity inhibitor of neuraminidase involved in this process. Under this background, the present inventors have developed a coronavirus spike protein such as SARS-CoV and SARS-CoV-2 (COVID-19), in particular, a recombinant protein in a form comprising the S1 subunit and/or the S2 subunit in the spike protein. It was first revealed that it has an esterase activity that hydrolyzes p-nitrophenol acetate, and an inhibitor that can interfere with virus cell infection and proliferation by interfering with the esterase activity of the spike protein of coronavirus developed.

Accordingly, the first aspect of the present invention is a recombinant protein comprising the S1 subunit or S2 subunit of the coronavirus spike protein, or the S1 subunit and the S2 subunit having esterase activity. It relates to a screening kit for a recombinant protein comprising the same and a candidate material for the prevention or treatment of coronavirus infection using the same.

Specifically, the kit of the present invention may comprise:

Recombinant protein derived from the spike protein of coronavirus; and

A substance capable of measuring the esterase activity of the recombinant protein derived from the spike protein of the corona virus.

In the kit of the present invention, the coronavirus is any one selected from the group consisting of SARS-Corona Virus, MERS-Corona Virus, and SARS-Corona Virus-2 can be

As used herein, the term “screening kit” refers to a kit used to select a substance exhibiting a specific effect from among a plurality of candidate substances. For the purpose of the present invention, the screening kit inhibits the esterase activity of the spike protein and/or the spike protein-derived recombinant protein of the coronavirus to effectively inhibit the cell infection or release process of the virus. It can be a kit used to screen for substances capable of inhibiting.

In the kit of the present invention, the recombinant protein derived from the coronavirus spike protein is a recombinant protein comprising the S1 subunit, the S2 subunit, the S1 subunit or the S2 subunit of the coronavirus spike protein, or the S1 subunit It may be a recombinant protein comprising a unit and the S2 subunit.

In SARS-Corona Virus-2, the spike protein plays a key role in receptor recognition and cell membrane fusion processes, and is composed of two subunits, the S1 subunit and the S2 subunit. The S1 subunit contains a receptor binding domain that recognizes and binds to the host receptor angiotensin converting enzyme 2, whereas the S2 subunit contains a six-helical bundle via a two-heptad repeat domain. bundle) to mediate viral cell membrane fusion.

In the kit of the present invention, when the corona virus is SARS-Corona Virus-2, the S1 subunit includes the amino acid sequence of SEQ ID NO: 4, and the S2 subunit includes the amino acid sequence of SEQ ID NO: 6 It includes an amino acid sequence, and the recombinant protein including the S1 subunit and the S2 subunit may include the amino acid sequence of SEQ ID NO:2.

The gene encoding the S1 subunit comprises the nucleotide sequence of SEQ ID NO: 3, or the nucleotide sequence of SEQ ID NO: 3 and at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably It may include a nucleotide sequence having 95% or more sequence homology.

The gene encoding the S2 subunit comprises the nucleotide sequence of SEQ ID NO: 5, or the nucleotide sequence of SEQ ID NO: 5 and at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably It may include a nucleotide sequence having 95% or more sequence homology.

The recombinant protein comprising the S1 subunit and the S2 subunit is 70% or more, more preferably 80% or more, even more preferably 90% or more of the nucleotide sequence of SEQ ID NO: 1 or the nucleotide sequence of SEQ ID NO: 1 , most preferably a nucleotide sequence having 95% or more sequence homology.

The "% sequence homology" for a polynucleotide is determined by comparing two optimally aligned sequences with a comparison region, wherein a portion of the polynucleotide sequence in the comparison region is a reference sequence (additions or deletions) to the optimal alignment of the two sequences. may include additions or deletions (ie, gaps) compared to (not including).

In the kit of the present invention, the recombinant protein derived from the spike protein of the coronavirus may be in a trimeric form.

Since the above-mentioned spike protein of corona virus exists as a trimer on the surface of the virus, in the present invention, in order to produce a recombinant protein derived from the spike protein of corona virus in a trimeric form, the protein of the trimeric motif region of Coronin 1 is encoded. genes were used.

The Coronin 1 may be mouse-derived Coronin 1 (mCor 1) (GenBank: EDL17419.1), and the protein of its trimeric motif region may include the amino acid sequence of SEQ ID NO: 8.

The gene encoding the protein of the trimeric motif region of Coronin 1 (mCor 1) may include the nucleotide sequence of SEQ ID NO: 7. Specifically, the gene includes a nucleotide sequence having a sequence homology of 70% or more, more preferably 80% or more, even more preferably 90% or more, and most preferably 95% or more to the nucleotide sequence of SEQ ID NO: 7. can

In addition, in order to accumulate the recombinant protein derived from the spike protein of the coronavirus at a high concentration in the ER (endoplasmic reticulum), BiP (chaperone binding protein) at the N- and C-terminus of the gene encoding the recombinant protein derived from the spike protein of the corona virus, respectively of the leader sequence and HDEL, which is an ER retention signal, can be added. The BiP-encoding gene may include the nucleotide sequence of SEQ ID NO: 9, and HDEL (His-Asp-Glu-Leu) may include the nucleotide sequence of SEQ ID NO: 10.

In a specific embodiment of the present invention, by using a construct comprising the following, 1) a full length ectodomain lacking from the transmembrane domain to the C-terminus in the spike protein of Corona-19 virus (full length ectodomain) ) comprising a recombinant protein (named full-length or Sf), 2) a recombinant protein comprising from the N-terminus to subdomain 2 (SD2) in the spike protein of Corona-19 virus (named S1), and a recombinant protein (designated as S2) comprising a region lacking from the transmembrane domain to the C-terminus in the S2 subunit of the spike protein of Corona-19 virus (named as S2) was prepared, respectively:

(i) a recombinant protein comprising a full length ectodomain lacking from the transmembrane domain to the C-terminus in the spike protein of the Corona-19 virus (named full-length or Sf), Corona-19 Recombinant protein containing from N-terminus to subdomain 2 (SD2) in the spike protein of the virus (designated as S1), or from the transmembrane domain to the C-terminus in the S2 subunit of the spike protein of the Corona-19 virus A gene encoding a recombinant protein (named S2) containing a region lacking until; (ii) Arabidopsis thaliana fused to the N-terminus of each recombinant protein to express the three recombinant proteins in the endoplasmic reticulum; A gene region encoding the leader sequence of BiP; (iii) a gene encoding a protein of a trimeric motif region of Coronin 1 (mCor 1) sequentially fused to the C-terminus of each recombinant protein, monomer A gene encoding human Fc and a gene encoding HDEL, which is an ER retention motif.

The expression of the genes encoding the three types of spike protein-derived recombinant proteins constructed as described above (BiP:Sf:mCor1:mhFC:HDEL, BiP:S1:mCor1:mhFC:HDEL and BiP:S2:mCor1:mhFC:HDEL) was It can be applied by selecting an appropriate promoter for this purpose. For example, 35S promoter derived from cauliflower mosaic virus, 19S RNA promoter derived from cauliflower mosaic virus, Mac promoter, plant actin protein promoter and ubiquitin protein It may further include any one promoter selected from the group consisting of promoters, preferably it may include a Mac promoter (Mac promoter), more preferably it may include a MacT promoter (MacT promoter), It is not limited to this.

The MacT promoter may be a promoter in which A, which is the 3' terminal base of the Mac promoter base sequence, is substituted with T, and the MacT promoter may include the base sequence of SEQ ID NO: 16, and specifically, the gene is of SEQ ID NO: 16 It may include a nucleotide sequence having a sequence homology of 70% or more, more preferably 80% or more, even more preferably 90% or more, and most preferably 95% or more to the nucleotide sequence.

In addition, the construct may further include an RD29B-t termination site, and the RD29B-t termination site gene may include the nucleotide sequence of SEQ ID NO: 17, and specifically, the gene includes the nucleotide sequence of SEQ ID NO: 17 and 70% or more, more preferably 80% or more, even more preferably 90% or more, and most preferably 95% or more.

According to a preferred embodiment of the present invention, the three types of recombinant spike proteins forming the above-mentioned trimer are obtained by using a recombinant vector containing the gene construct of FIG. 1 (a), (b) or (c). can be produced by

The construct encoding the three recombinant spike proteins forming the trimer designed in the present invention can be introduced into a host using an appropriate vector.

As used herein, the term "recombinant" refers to a cell in which the cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a peptide, a heterologous peptide, or a protein encoded by the heterologous nucleic acid. Recombinant cells can express genes or gene segments not found in the native form of the cell in either the sense or antisense form. Recombinant cells can also express genes found in cells in a natural state, but the genes are modified and re-introduced into cells by artificial means.

The term "recombinant expression vector" means a bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus or other vector. In general, any plasmid and vector can be used as long as it can be replicated and stabilized in a host. An important characteristic of the expression vector is that it has an origin of replication, a promoter, a marker gene and a translation control element. The recombinant expression vector and the expression vector containing appropriate transcriptional/translational control signals can be constructed by methods well known to those skilled in the art. The method includes in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology.

A preferred example of the recombinant vector of the present invention is a Ti-plasmid vector capable of transferring a part of itself, the so-called T-region, into a plant cell when present in a suitable host. Another type of Ti-plasmid vector is currently being used to transfer hybrid DNA sequences into plant cells, or protoplasts from which new plants can be produced that properly insert the hybrid DNA into the genome of the plant. A particularly preferred form of the Ti-plasmid vector is the so-called binary vector as claimed in EP 0 120 516 B1 and US Pat. No. 4,940,838. Other suitable vectors that can be used to introduce into plant hosts the constructs encoding the trimeric-forming recombinant spike proteins from the coronavirus designed in the present invention are double-stranded plant viruses (e.g., CaMV) and single-stranded. It can be selected from viral vectors such as those derived from viruses, geminiviruses, and the like, for example incomplete plant viral vectors. The use of such vectors can be advantageous, especially when it is difficult to adequately transform a plant host.

The method for delivering the above-described recombinant vector into a host cell may be carried out by a CaCl2 method, a Hanhan method, an electroporation method, or the like when the host cell is a prokaryotic cell. In addition, when the host cell is a eukaryotic cell, the vector may be injected into the host cell by microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, DEAE-dextran treatment, and gene bombardment. can

The host cell may be a prokaryote or a eukaryote, for example, yeast (Saccharomyce cerevisiae), fungi such as E. coli, insect cells, human cells (eg, CHO cell line (Chinese hamster ovary), W138, BHK, COS- 7, 293, HepG2, 3T3, RIN and MDCK cell lines) and plant cells may be used, preferably Agrobacterium. In the case of insect cells and human cells, a gene encoding a recombinant HA protein forming a trimer can be expressed using an expression vector required for expression of each type of cell.

In a specific embodiment of the present invention, a recombinant vector comprising a construct encoding three types of recombinant spike proteins is introduced into leaf cells of a plant using Agrobacterium-mediated infiltration, and then from these A recombinant protein encoded by the recombinant gene was produced. Then, the three recombinant spike proteins from the leaf extract of the plant were separated and purified using protein A affinity column chromatography.

In a specific embodiment of the present invention, in order to confirm whether the separated and purified three kinds of trimer spike recombinant proteins have esterase activity, these three kinds of trimer spike recombinant proteins are esters called p-NPA. It was cultured in vitro with a general substrate of the hydrolase. As a result, as shown in FIG. 3 , the three recombinant spike proteins (BiP:Sf:mCor1:mhFC:HDEL, BiP:S1:mCor1:mhFC:HDEL, BiP:S2:mCor1:mhFC:HDEL) were All were shown to have esterase activity. Therefore, it was confirmed that S1 and S2 each have independent esterase activity.

In the case of some coronaviruses, another protein called HE is known to show esterase activity, but so far, it has not been confirmed whether SARS-CoV or COVID-19 coronaviruses have esterase activity. none. Although the exact role of the activity of the esterases identified in the present invention is not known, viral esterases play a role in removing the binding of viral proteins to sugar residues such as sialic acid present on the surface of animal cells. it is known Therefore, the esterase activity of the spike protein of coronavirus is thought to play a similar role. Accordingly, interfering with the activity of esterases is expected to play an important role in virus infection and release from cells, and is expected to result in inhibition of virus production.

Accordingly, the kit of the present invention includes a material capable of measuring the esterase activity of a recombinant protein derived from the spike protein of coronavirus, and examples of such material include an esterase substrate; compounds comprising sialic acid; BCECF, AM(2',7'-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester) (BCECF, AM(2',7'-Bis- (2-Carboxyethyl)-5-(and-6)-Carboxyfluorescein, Acetoxymethyl Ester)); Calcein AM; Carboxyeosin diacetate, succinimidyl ester; Carboxyfluorescein diacetate, acetoxymethyl ester; Or carboxyfluorescein diacetate, succinimidyl ester (Carboxyfluorescein diacetate, succinimidyl ester), but is not limited thereto.

According to another preferred embodiment of the present invention, the substrate of the esterase may be a standard substrate such as para nitropheny acetate (p-NPA), and the compound containing sialic acid is Neu5 , 9Ac2, 4-methylumbelliferyl acetate (4-methylumbelliferyl acetate) or 9Ac2-lactose (9Ac2-lactose), but is not limited thereto.

A second aspect of the present invention relates to a method of screening a candidate for preventing or treating a coronavirus infection using the above-described screening kit.

Specifically, the screening method of the present invention may include the following steps:

(i) treating a candidate material with a recombinant protein derived from the spike protein of coronavirus;

(ii) measuring the esterase activity of the recombinant protein derived from the spike protein of the corona virus treated with the candidate substance; and

(iii) when the candidate material inhibits esterase activity by 30% or more, selecting the candidate material as a candidate material for prevention or treatment of coronavirus.

In the screening method of the present invention, the recombinant protein derived from the spike protein of the corona virus in step (i) includes the S1 subunit or the S2 subunit of the spike protein, or includes both the S1 subunit and the S2 subunit , it may be a trimeric form of recombinant spike protein.

As described above, in the present invention, since it was confirmed that the separated and purified recombinant proteins S1 and S2 each independently exhibit esterase activity, various types of recombinant proteins including the S1 and/or S2 were used to ester their esters. By confirming the hydrolase activity, it is possible to screen candidate substances for the prevention or treatment of coronavirus infection.

In the screening method of the present invention, the candidate substances include, without limitation, substances that inhibit esterases, for example, compounds, microbial cultures, microbial extracts, nucleic acids, antibodies, proteins, peptides, aptamers and natural substances. It may be any one selected from the group consisting of extracts, but is not limited thereto.

The nucleic acid may be selected from the group consisting of microRNA, siRNA, shRNA, antisense RNA, aptamer, LNA (locked nucleic acid), PNA (peptide nucleic acid), and morpholino (morpholino), but It is not limited.

As used herein, the term "micro RNA" refers to a short non-coding RNA consisting of about 22 nucleotide sequences. It is known to function as a post-transcriptional regulator in the process of gene expression. By complementary binding to a target mRNA having a complementary base sequence, degradation of the target mRNA or translation into a protein is inhibited.

As used herein, the term “small interfering RNA (siRNA)” refers to a short double-stranded RNA capable of inducing an RNA interference (RNAi) phenomenon through cleavage of a specific mRNA. It is composed of a sense RNA strand having a sequence homologous to the mRNA of a target gene and an antisense RNA strand having a sequence complementary thereto. Since siRNA can inhibit the expression of a target gene, it is provided as an efficient gene knock-down method or as a gene therapy method.

As used herein, the term “shRNA (small hairpin RNA)” refers to a single strand consisting of 50 to 60 nucleotides, and forms a stem-loop structure in vivo. That is, shRNA is an RNA sequence that creates a tight hairpin structure for suppressing gene expression through RNA interference. A long RNA of 15-30 nucleotides is complementary to both sides of a loop of 5-10 nucleotides to form a double-stranded stem. In order to always be expressed, shRNA is transfected into cells through a vector containing a U6 promoter, and is usually transferred to daughter cells so that gene expression suppression is inherited. The shRNA hairpin structure is cleaved by an intracellular mechanism to become siRNA and then binds to RISC (RNA-induced silencing complex). These RISCs bind to and cleave mRNA. shRNA is transcribed by RNA polymerase.

In the present invention, the term “aptamer” refers to single-stranded DNA (ssDNA) or RNA having high specificity and affinity for a specific substance. Aptamers have very high affinity for specific substances and are stable, can be synthesized by a relatively simple method, can be modified in various ways to increase binding strength, and can be targeted to cells, proteins, and even small organic substances. Its specificity and stability are very high compared to antibodies that have already been developed.

In the screening method of the present invention, the esterase activity in step (ii) can be measured using an esterase substrate or a compound containing sialic acid. For example, the esterase inhibitory activity of a candidate substance can be evaluated by incubating pNPA, a standard substrate for an esterase, with a recombinant spike protein treated with a candidate substance, and measuring the amount of paranitrophenol released therefrom. have. Alternatively, the esterase inhibitory activity of the candidate substance may be evaluated by incubating a compound containing sialic acid with a recombinant spike protein treated with a candidate substance, and measuring the amount of sialic acid released therefrom.

In the screening method of the present invention, since the candidate material selected in step (iii) exhibits esterase inhibitory activity of at least 30% or more, preferably 50% or more, effective for development as a therapeutic agent for coronavirus infection It can be used as an ingredient.

In a specific embodiment of the present invention, it was attempted to screen for substances that inhibit the activity of esterases present in S1 and S2 of the spike protein using three kinds of recombinant spike proteins isolated and purified from plant leaves. For this purpose, among the various types of compounds known as inhibitors of esterases, first four types of compounds, namely acetazoleamide, sulfanilaminde, sulfapyridin, and sulfamethoxazole, were prepared. By selection, it was determined whether these compounds inhibit the esterase activity of the spike protein. pNPA was used as a substrate for esterase, and 0.2 μg of pure isolated spike protein was treated with the above four compounds at a concentration of 0.5 mM, 1.0 mM, or 2.0 mM, respectively, and incubated at 37 ° C. Activity was measured. As a result, as shown in FIG. 4 , it was confirmed that among the four compounds, sulfamethoxazole inhibited the activity of the spike protein esterase by about 50% at a concentration of 1.0 mM.

It will be possible to synthesize a substance having better inhibitory activity through additional modification based on sulfamethoxazole, which is an esterase activity inhibitor of the spike protein identified in the present invention. In addition, the structure of the complex in which the spike protein and p-NPA, which is an esterase substrate, is combined, and the active site of the esterase by using it, is identified, and based on this, a better inhibitor of esterase activity is developed. it is possible to do

Accordingly, a third aspect of the present invention relates to a pharmaceutical composition for preventing or treating coronavirus infection, comprising an inhibitor of esterase activity of a recombinant protein derived from a spike protein of coronavirus as an active ingredient.

In the pharmaceutical composition of the present invention, the selected esterase activity inhibitor binds to the active site of the esterase to inhibit the activity of the esterase, or to a site other than the active site of the esterase. It may be to inhibit the activity of the esterase by inducing a structural change of the esterase by binding.

As described above, in a specific embodiment of the present invention, it was confirmed that sulfamethoxazole inhibited the esterase activity of the spike protein of Corona-19 virus by about 50%. Accordingly, the present invention provides a pharmaceutical composition for preventing or treating COVID-19 virus infection, comprising sulfamethoxazole, a derivative, isomer, or pharmaceutically acceptable salt thereof as an active ingredient.

In the present invention, the term "derivative" refers to a compound obtained by substituting a part of the structure of sulfamethoxazole with another atom or group of atoms. The "pharmaceutically acceptable salt" may be an acid addition salt formed using an organic acid or an inorganic acid, and the organic acid is, for example, formic acid, acetic acid, propionic acid, lactic acid, butyric acid, isobutyric acid, trifluoroacetic acid, malic acid, maleic acid Acid, malonic acid, fumaric acid, succinic acid, succinic acid monoamide, glutamic acid, tartaric acid, oxalic acid, citric acid, glycolic acid, glucuronic acid, ascorbic acid, benzoic acid, phthalic acid, salicylic acid, anthranilic acid, dichloroacetic acid, aminooxyacetic acid, benzenesulfonic acid, p-toluenesulfonic acid or methanesulfonic acid. Inorganic acids include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, carbonic acid or boric acid. The acid addition salt may preferably be in the form of hydrochloride or acetate, and more preferably in the form of hydrochloride.

In addition, additional salts available include gaba salt, gabapentin salt, pregabalin salt, nicotinate salt, adipate salt, hemimalonate, cysteine salt, acetylcysteine salt, methionine salt, arginine salt, lysine salt, ornithine salt or aspartate salt etc.

In addition, the pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may further include, for example, a carrier for oral administration or a carrier for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Carriers for parenteral administration may include water, suitable oils, saline, aqueous glucose and glycols, and the like. In addition, it may further include a stabilizer and a preservative. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives are benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. As other pharmaceutically acceptable carriers, reference may be made to those described in the following literature (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

In the present invention, the term "isomer" means a relationship between compounds having the same chemical formula but not identical, and the types of such isomers include structural isomers, geometric isomers, optical isomers and geometric isomers. By stereoisomer is meant a compound that has the same chemical constitution but differs in the arrangement of atoms or groups in space, and optical isomer (enantiomer) means two stereoisomers of a compound that have non-superimposable mirror images of each other, diastereomers An isomer means a stereoisomer that has two or more asymmetric centers and whose molecules are not mirror images of each other.

The pharmaceutical composition of the present invention may include one or more active ingredients exhibiting the same or similar function in addition to the above ingredients. The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable additive, wherein the pharmaceutically acceptable additive includes starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate. , lactose, mannitol, syrup, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, lead carnauba, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, Calcium stearate, sucrose, dextrose, sorbitol and talc and the like may be used. The pharmaceutically acceptable additive according to the present invention may be included in an amount of 0.1 to 90 parts by weight based on the composition, but is not limited thereto.

The pharmaceutical composition of the present invention may be administered in various oral and parenteral formulations during actual clinical administration, and when formulated, commonly used fillers, extenders, binders, wetting agents, disintegrants, diluents or excipients such as surfactants It can be prepared using Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in the extract of the present invention, for example, starch, calcium carbonate, sucrose (Sucrose), may be prepared by mixing lactose (Lactose) or gelatin. In addition to simple excipients, lubricants such as magnesium stearate talc may also be used. Liquid formulations for oral use include suspensions, solutions, emulsions, and syrups, and various excipients such as wetting agents, sweetening agents, fragrances, and preservatives in addition to commonly used simple diluents such as water and liquid paraffin may be included. . Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.

The pharmaceutical composition of the present invention may be administered orally or administered parenterally according to a desired method, and when administered parenterally, external skin or intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection You can choose the injection method. The dosage may vary depending on the patient's weight, age, sex, health status, diet, administration time, administration method, excretion rate, and severity of disease.

The dosage of the pharmaceutical composition of the present invention varies depending on the patient's weight, age, sex, health status, diet, administration time, administration method, excretion rate and severity of disease, and the daily dosage is the dosage of the present invention. It may be 0.0001 mg/kg to 100 mg/kg based on the amount of the extract, and specifically 0.001 mg/kg to 10 mg/kg, but is not limited thereto.

In the present invention, the terms “prevention”, “amelioration” and/or “treatment” refer to any action that inhibits or delays the onset of a disease or condition, any action that ameliorates or beneficially alters the disease or condition state, and the treatment of the disease or condition. means any action that delays, stops or reverses progress.

The pharmaceutical composition according to the present invention may be administered in combination with an existing therapeutic agent for coronavirus infection. The term "administered in combination" means that the pharmaceutical composition of the present invention is administered to a subject in need thereof together with an existing therapeutic agent for coronavirus infection. Administering each component together means that each component may be administered simultaneously, separately, or sequentially to obtain a desired effect.

Furthermore, a fourth aspect of the present invention relates to a health functional food composition for preventing or improving Corona-19 virus infection, comprising sulfamethoxazole, a derivative, isomer, or pharmaceutically acceptable salt thereof as an active ingredient. will be.

As used herein, the term "food" includes, for example, various foods, beverages, gum, tea, vitamin complexes, health functional foods, and the like, and includes all foods in a conventional sense. The food can be prepared in dosage forms such as tablets, granules, powders, capsules, liquid solutions and pills according to a known manufacturing method, and the content of the extract according to the present invention is 0.0001 based on the total weight of the food composition depending on the dosage form. It can be adjusted to 100% by weight to 100% by weight. There is no particular limitation on other ingredients other than including the compound of the present invention, and various conventional flavoring agents or natural carbohydrates may be included as additional ingredients.

Examples of the natural carbohydrate include monosaccharides such as glucose, fructose and the like; disaccharides such as maltose, sucrose and the like; and polysaccharides such as conventional sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (taumatine, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. have.

In the present invention, the term "health functional (sex) food" is the same term as food for special health use (FoSHU), and in addition to nutritional supply, it has medical and medical effects processed to efficiently exhibit bioregulatory functions. It means high food. Here, "function (sex)" means to obtain a useful effect for health purposes such as regulating nutrients or physiological action with respect to the structure and function of the human body. The food of the present invention can be prepared by a method commonly used in the art, and during the manufacture, it can be prepared by adding raw materials and components commonly added in the art. In addition, the formulation of the food may be prepared without limitation as long as it is a formulation recognized as a food. The food composition of the present invention can be prepared in various types of dosage forms, and unlike general drugs, there is no side effect that may occur during long-term administration of the drug using food as a raw material, and has excellent portability.

Specifically, the health functional food is a food prepared by adding the composition of the present invention to food materials such as beverages, teas, spices, gum, and confectionery, or encapsulating, powdering, suspension, etc., and when ingested, It means that it is effective, but unlike general drugs, it has the advantage that there are no side effects that may occur when taking the drug for a long time using food as a raw material.

The food composition may further include a physiologically acceptable carrier, the type of carrier is not particularly limited.

Hereinafter, preferred examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

Design of the gene encoding the recombinant spike protein of the COVID-19 virus

In the transmembrane domain (TMD) of the spike protein (GenBank No. MZ433938.1) to induce the expression of a spike (S) derived from the corona-19 virus surface protein in the ER lumen in a soluble form Spike protein ecto-domain (from the 16th amino acid to the 1213th amino acid, a total of 1198 residues; Sf), from the N-terminus to the SD2 domain (from the 16th amino acid) Up to amino acid 681, a total of 666 residues; S1), and a protein region from the transmembrane domain (TMD) to the C-terminus in the S2 subunit (from amino acid 682 to 1213, a total of 532 residues; S2) secured. The ER targeting signal obtained from BiP, an Arabidopsis protein, was fused to the 5'-end of the spike gene of the Corona-19 virus to enable ER targeting. Additionally, a motif inducing the formation of a homotrimer from a protein called mouse mCoronin1 was added to the spike to construct a construct. And human FC in a monomeric form was included as a tag for isolating and purifying these recombinant proteins, and then HDEL was included to allow the recombinant protein to accumulate in the ER. For the expression of these genes, macT was used, and the end of Rd29b was used as a transcription terminator (FIG. 1). The transcription terminator was confirmed to show high transcription efficiency as a result of previous studies. The nucleotide sequences used in the experiment are shown in Table 1 below.

sequence (5'-3') SEQ ID NO: Sf
base sequence gttaaccttaccaccagaactcagttacccccagcatacactaattctttcacacgtggtgtttactaccctgacaaagttttcagaagcagcgttttacacagcactcaggatttattcctacctttcttttccaacgtgacctggttccatgctatacatgtatctgggaccaatggtaccaagaggtttgataacccggtcctaccatttaatgatggagtctattttgcctccactgagaagtctaatataataagaggctggatttttggaactactcttgattcgaagacccagagtctacttattgttaataacgctacaaatgttgttatcaaagtatgtgaatttcaattctgtaatgatccattcttgggtgtttactaccacaaaaacaacaaaagttggatggaaagtgagtttcgggtttatagcagtgcgaataattgcacttttgagtacgtctcccaaccttttcttatggaccttgaaggaaagcagggaaatttcaagaatcttcgcgaatttgtgtttaagaatatcgatggttatttcaagatatattctaagcacacgcctattaatttagtgcgagatctccctcagggtttttcggcgctggaaccattggtagatttgccgataggaatcaatatcactaggttccagactttacttgctctgcatagaagttacttgacccctggagatagctcatcaggttggacagctggtgcggcagcttattacgtggggtatcttcagcctaggacgttcctattaaaatataatgaaaatggaaccattacagatgctgtagactgtgcacttgaccctctctcagaaacaaagtgtacgttgaaatccttcacggtagaaaaagggatctaccaaacgtctaacttcagagtccagccaacagaatctattgtgagatttcccaatattacaaacttgtgccctttcggagaagtttttaacgccaccaggtttgcat cggtttatgcttggaacaggaaaagaatcagcaactgtgttgctgattatagtgtcctatataactccgcatccttttccactttcaagtgttacggagtttctcctactaaattaaatgatctctgctttactaatgtctatgcagattcatttgtaatcagaggtgatgaggtcagacaaatcgctccagggcagactggaaagattgctgattataattataagcttcctgatgattttacaggctgcgttatagcatggaattctaataatcttgactctaaggtggggggaaattataattacctgtatagactgtttaggaagagcaatctcaagcctttcgagagagacatttcaactgagatctaccaggcgggaagcactccgtgtaatggtgttgagggttttaattgttactttcctttacagtcatacggtttccaacccacgaatggggttggttaccaaccgtaccgagtagtagtactttctttcgagcttctacatgccccagcaactgtttgtggacctaagaagtctactaatttggttaaaaataagtgtgtcaattttaatttcaatggacttacgggcacaggagttcttactgagtctaacaagaagtttctgcctttccagcagttcggcagagatattgctgacactactgatgctgtgcgtgatccacagacacttgaaattcttgacattacaccatgttcttttggtggcgtgagtgttataactcccggaacaaatacctccaaccaggtggctgttctgtatcaggacgtgaactgtacagaagtccctgttgcaattcatgcagatcagcttactcctacctggcgtgtttattctacgggttccaatgtttttcaaacacgtgcaggctgcttgataggggctgaacatgtcaacaactcatatgaatgcgacatacccataggtgcaggtatatgcgctagttatcagactcagaccaattctccgcg gcgggcacgaagtgtagctagtcaatccatcattgcctacactatgtcacttggtgcagaaaattcagttgcttactctaataactctattgccatacccacaaattttactattagtgttaccacagaaattctaccagtgtctatgaccaagacatcagttgactgtacaatgtatatttgcggggattcaactgagtgctcgaatctgttgttgcaatacggcagtttttgtacccaattgaaccgggctctgactggaatagctgtggaacaagataaaaacacccaagaagtttttgcacaagtcaaacaaatttataaaacaccaccaattaaagatttcggtggtttcaacttctcacaaatactgccagatccgagcaaaccaagcaagaggtcattcattgaagacctacttttcaacaaagtgacacttgcagatgctggcttcattaaacagtatggtgattgcttgggggatattgctgctagagacctcatttgtgcacaaaagtttaacgggctgacagtgttgccacctttgttgacagatgagatgattgctcagtacacttctgcactgctcgctggtacaatcacatctgggtggacctttggtgcaggtgctgccttacaaataccatttgctatgcagatggcttataggttcaatggtatcggagttacacagaacgttctctatgagaaccaaaaattgattgccaaccaattcaatagtgccattggcaagattcaggactcactttcaagcacagcgagtgcacttggaaagttgcaagatgtggtcaaccagaatgcacaagctttaaacacgcttgtgaaacaactcagctccaactttggggcaatttcaagtgttttgaatgatatcctttcacgtcttgataaagtggaagccgaggtgcaaattgacaggttgatcacaggccgacttcaaagtttgcagacttatgtgactcaacaattaattagggca gcagaaatccgcgcttcggctaatctggcggctactaaaatgtcagagtgtgtacttggacaatctaaacgagttgatttttgcggaaagggctatcatctcatgtccttccctcagtcagcgcctcacggtgtagtgttcttgcacgtgacttacgttcctgcacaagaaaagaatttcacaactgctccggccatttgtcatgatggaaaagcccactttccgcgtgaaggtgtctttgtttcgaatggcacacactggtttgtaacccaaaggaatttttatgagccacaaatcattacgacggacaacacttttgtgtctggtaattgtgatgttgtaatcggaatcgtcaacaacaccgtttacgatcctttgcagcctgagttagattctttcaaagaggagctggataagtatttcaagaatcatacatcacccgatgttgatctcggtgatatctctggaattaatgcttcagttgtgaacattcaaaaggagattgaccgcctcaatgaggttgccaagaatttgaatgaatcgctcatcgatctccaagaacttggaaagtatgagcagtatatcaagtggcca One Sf
amino acid sequence vnlttrtqlppaytnsftrgvyypdkvfrssvlhstqdlflpffsnvtwfhaihvsgtngtkrfdnpvlpfndgvyfasteksniirgwifgttldsktqsllivnnatnvvikvcefqfcndpflgvyyhknnkswmesefrvyssannctfeyvsqpflmdlegkqgnfknlrefvfknidgyfkiyskhtpinlvrdlpqgfsaleplvdlpiginitrfqtllalhrsyltpgdsssgwtagaaayyvgylqprtfllkynengtitdavdcaldplsetkctlksftvekgiyqtsnfrvqptesivrfpnitnlcpfgevfnatrfasvyawnrkrisncvadysvlynsasfstfkcygvsptklndlcftnvyadsfvirgdevrqiapgqtgkiadynyklpddftgcviawnsnnldskvggnynylyrlfrksnlkpferdisteiyqagstpcngvegfncyfplqsygfqptngvgyqpyrvvvlsfellhapatvcgpkkstnlvknkcvnfnfngltgtgvltesnkkflpfqqfgrdiadttdavrdpqtleilditpcsfggvsvitpgtntsnqvavlyqdvnctevpvaihadqltptwrvystgsnvfqtragcligaehvnnsyecdipigagicasyqtqtnsprrarsvasqsiiaytmslgaensvaysnnsiaiptnftisvtteilpvsmtktsvdctmyicgdstecsnlllqygsfctqlnraltgiaveqdkntqevfaqvkqiyktppikdfggfnfsqilpdpskpskrsfiedllfnkvtladagfikqygdclgdiaardlicaqkfngltvlpplltdemiaqytsallagtitsgwtfgagaalqipfamqmayrfngigvtqnvlyenqklianqfnsaigkiqdslsstasalgklqdvvnqnaqalntlvkqlssnfgaissvlndilsrldkveaevqidrlitgrlqslqtyvtqqlira aeirasanlaatkmsecvlgqskrvdfcgkgyhlmsfpqsaphgvvflhvtyvpaqeknfttapaichdgkahfpregvfvsngthwfvtqrnfyepqiittdntfvsgncdvvigivnntvydplqginpeldsfkeeldneskyfklqnkyveqniqniqkevdlqnhk 2 S1
base sequence gttaaccttaccaccagaactcagttacccccagcatacactaattctttcacacgtggtgtttactaccctgacaaagttttcagaagcagcgttttacacagcactcaggatttattcctacctttcttttccaacgtgacctggttccatgctatacatgtatctgggaccaatggtaccaagaggtttgataacccggtcctaccatttaatgatggagtctattttgcctccactgagaagtctaatataataagaggctggatttttggaactactcttgattcgaagacccagagtctacttattgttaataacgctacaaatgttgttatcaaagtatgtgaatttcaattctgtaatgatccattcttgggtgtttactaccacaaaaacaacaaaagttggatggaaagtgagtttcgggtttatagcagtgcgaataattgcacttttgagtacgtctcccaaccttttcttatggaccttgaaggaaagcagggaaatttcaagaatcttcgcgaatttgtgtttaagaatatcgatggttatttcaagatatattctaagcacacgcctattaatttagtgcgagatctccctcagggtttttcggcgctggaaccattggtagatttgccgataggaatcaatatcactaggttccagactttacttgctctgcatagaagttacttgacccctggagatagctcatcaggttggacagctggtgcggcagcttattacgtggggtatcttcagcctaggacgttcctattaaaatataatgaaaatggaaccattacagatgctgtagactgtgcacttgaccctctctcagaaacaaagtgtacgttgaaatccttcacggtagaaaaagggatctaccaaacgtctaacttcagagtccagccaacagaatctattgtgagatttcccaatattacaaacttgtgccctttcggagaagtttttaacgccaccaggtttgcat cggtttatgcttggaacaggaaaagaatcagcaactgtgttgctgattatagtgtcctatataactccgcatccttttccactttcaagtgttacggagtttctcctactaaattaaatgatctctgctttactaatgtctatgcagattcatttgtaatcagaggtgatgaggtcagacaaatcgctccagggcagactggaaagattgctgattataattataagcttcctgatgattttacaggctgcgttatagcatggaattctaataatcttgactctaaggtggggggaaattataattacctgtatagactgtttaggaagagcaatctcaagcctttcgagagagacatttcaactgagatctaccaggcgggaagcactccgtgtaatggtgttgagggttttaattgttactttcctttacagtcatacggtttccaacccacgaatggggttggttaccaaccgtaccgagtagtagtactttctttcgagcttctacatgccccagcaactgtttgtggacctaagaagtctactaatttggttaaaaataagtgtgtcaattttaatttcaatggacttacgggcacaggagttcttactgagtctaacaagaagtttctgcctttccagcagttcggcagagatattgctgacactactgatgctgtgcgtgatccacagacacttgaaattcttgacattacaccatgttcttttggtggcgtgagtgttataactcccggaacaaatacctccaaccaggtggctgttctgtatcaggacgtgaactgtacagaagtccctgttgcaattcatgcagatcagcttactcctacctggcgtgtttattctacgggttccaatgtttttcaaacacgtgcaggctgcttgataggggctgaacatgtcaacaactcatatgaatgcgacatacccataggtgcaggtatatgcgctagttatcagactcagaccaattctccg 3 S1
amino acid sequence vnlttrtqlppaytnsftrgvyypdkvfrssvlhstqdlflpffsnvtwfhaihvsgtngtkrfdnpvlpfndgvyfasteksniirgwifgttldsktqsllivnnatnvvikvcefqfcndpflgvyyhknnkswmesefrvyssannctfeyvsqpflmdlegkqgnfknlrefvfknidgyfkiyskhtpinlvrdlpqgfsaleplvdlpiginitrfqtllalhrsyltpgdsssgwtagaaayyvgylqprtfllkynengtitdavdcaldplsetkctlksftvekgiyqtsnfrvqptesivrfpnitnlcpfgevfnatrfasvyawnrkrisncvadysvlynsasfstfkcygvsptklndlcftnvyadsfvirgdevrqiapgqtgkiadynyklpddftgcviawnsnnldskvggnynylyrlfrksnlkpferdisteiyqagstpcngvegfncyfplqsygfqptngvgyqpyrvvvlsfellhapatvcgpkkstnlvknkcvnfnfngltgtgvltesnkkflpfqqfgrdiadttdavrdpqtleilditpcsfggvsvitpgtntsnqvavlyqdvnctevpvaihadqltptwrvystgsnvfqtragcligaehvnnsyecdipigagicasyqtqtnsp 4 S2
base sequence cggcgggcacgaagtgtagctagtcaatccatcattgcctacactatgtcacttggtgcagaaaattcagttgcttactctaataactctattgccatacccacaaattttactattagtgttaccacagaaattctaccagtgtctatgaccaagacatcagttgactgtacaatgtatatttgcggggattcaactgagtgctcgaatctgttgttgcaatacggcagtttttgtacccaattgaaccgggctctgactggaatagctgtggaacaagataaaaacacccaagaagtttttgcacaagtcaaacaaatttataaaacaccaccaattaaagatttcggtggtttcaacttctcacaaatactgccagatccgagcaaaccaagcaagaggtcattcattgaagacctacttttcaacaaagtgacacttgcagatgctggcttcattaaacagtatggtgattgcttgggggatattgctgctagagacctcatttgtgcacaaaagtttaacgggctgacagtgttgccacctttgttgacagatgagatgattgctcagtacacttctgcactgctcgctggtacaatcacatctgggtggacctttggtgcaggtgctgccttacaaataccatttgctatgcagatggcttataggttcaatggtatcggagttacacagaacgttctctatgagaaccaaaaattgattgccaaccaattcaatagtgccattggcaagattcaggactcactttcaagcacagcgagtgcacttggaaagttgcaagatgtggtcaaccagaatgcacaagctttaaacacgcttgtgaaacaactcagctccaactttggggcaatttcaagtgttttgaatgatatcctttcacgtcttgataaagtggaagccgaggtgcaaattgacaggttgatcacaggccgacttcaaagtttgcagacttatgtgactcaacaattaattaggg cagcagaaatccgcgcttcggctaatctggcggctactaaaatgtcagagtgtgtacttggacaatctaaacgagttgatttttgcggaaagggctatcatctcatgtccttccctcagtcagcgcctcacggtgtagtgttcttgcacgtgacttacgttcctgcacaagaaaagaatttcacaactgctccggccatttgtcatgatggaaaagcccactttccgcgtgaaggtgtctttgtttcgaatggcacacactggtttgtaacccaaaggaatttttatgagccacaaatcattacgacggacaacacttttgtgtctggtaattgtgatgttgtaatcggaatcgtcaacaacaccgtttacgatcctttgcagcctgagttagattctttcaaagaggagctggataagtatttcaagaatcatacatcacccgatgttgatctcggtgatatctctggaattaatgcttcagttgtgaacattcaaaaggagattgaccgcctcaatgaggttgccaagaatttgaatgaatcgctcatcgatctccaagaacttggaaagtatgagcagtatatcaagtggcca 5 S2
amino acid sequence RRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWP 6 mCOr1
base sequence gtgtctaggcttgaggaagatgttagaaatctcaacgcaattgtccagaaacttcaggaaaggttggataggctggaggaaactgttcaagctaag 7 mCor1
amino acid sequence vsrleedvrnlnaivqklqerldrleetvqak 8 BiP
base sequence atggctcgct cgtttggagc taacagtacc gttgtgttgg cgatcatctt cttcggtgag tgattttccg atcttcttct ccgatttaga tctcctctac attgttgctt aatctcagaa ccttttttcg ttgttcctgg atctgaatgt gtttgtttgc aatttcacga tcttaaaagg ttagatctcg attggtattg acgattggaa tctttacgat ttcaggatgt ttatttgcgt tgtcctctgc aatagaagag gctacgaagt ta 9 HDEL
base sequence cacgatgagctc 10 HDEL
amino acid sequence His-Asp-Glu-Leu 11 linker
base sequence gatgatgatgataagacccggggtggtggaagtggtggaagt 12 linker
amino acid sequence DDDDKTRGGGSGGS 13 monomer
human FC
base sequence gcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacacctcgcccccatcccgtgaggagatgaccaagaaccaggtcagcctgaggtgccacgtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgaagcccgtgctggactccgacggctccttcgagctcaagagcgcgctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtcccctggtaaa 14 monomer
human FC
amino acid
order APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTSPPSREEMTKNQVSLRPVLDPEVKSFYPSDIAVEKTSKSRDQPQENNYFSTHYKLSKPSKHKPSDIAVEKCS 15 MacT
base sequence agagatctcctttgccccagagatcacaatggacgacttcctctatctctacgatctagtcaggaagttcgacggagaaggtgacgataccatgttcaccactgataatgagaagattagccttttcaatttcagaaagaatgctaacccacagatggttagagaggcttacgcagcaggtctcatcaagacgatctacccgagcaataatctccaggagatcaaataccttcccaagaaggttaaagatgcagtcaaaagattcaggactaactgcatcaagaacacagagaaagatatatttctcaagatcagaagtactattccagtatggacgattcaaggcttgcttcacaaaccaaggcaagtaatagagattggagtctctaaaaaggtagttcccactgaatcaaaggccatggagtcaaagattcaaatagaggacctaacagaactcgccgtaaagactggcgaacagttcatacagagtctcttacgactcaatgacaagaagaaaatcttcgtcaacatggtggagcacgacacgcttgtctactccaaaaatatcaaagatacagtctcagaagaccaaagggcaattgagacttttcaacaaagggtaatatccggaaacctcctcggattccattgcccagctatctgtcactttattgtgaagatagtggaaaaggaaggtggctcctacaaatgccatcattgcgataaaggaaaggccatcgttgaagatgcctctgccgacagtggtcccaaagatggacccccacccacgaggagcatcgtggaaaaagaagacgttccaaccacgtcttcaaagcaagtggattgatgtgacgcaagacgtgacgtaagtatctgagctagtttttatttttctactaatttggtcgtttatttcggcgtgtaggacatggcaaccgggcctgaatttcgcgggtattctgtttctattccaactttttcttgatccgcagccattaac gacttttgaatagatacgctgacacgccaagcctcgctagtcaaaagtgtaccaaacaacgctttacagcaagaacggaatgcgcgtgacgctcgcggtgacgccatttcgccttttcagaaatggataaaatagcctttgctaagcctcgctagtcaaaagtgtaccaaacaacgctttacagcaagaacggaatgcgcgtgacgctcgt 16 RD29B
Warrior
terminator
base sequence aattttactcaaaatgttttggttgctatggtagggactatggggttttcggattccggtggaagtgagtggggaggcagtggcggaggtaagggagttcaagattctggaactgaagatttggggttttgcttttgaatgtttgcgtttttgtatgatgcctctgtttgtgaactttgatgtattttatctttgtgtgaaaaagagattgggttaataaaatatttgcttttttggataagaaactcttttagcggcccattaataaaggttacaaatgcaaaatcatgttagcgtcagatatttaattattcgaagatgattgtgatagatttaaaattatcctagtcaaaaagaaagagtaggttgagcagaaacagtgacatctgttgtttgtaccatacaaattagtttagattattggttaacatgttaaatggctatgcatgtgacatttagaccttatcggaattaatttgtagaattattaattaagatgttgattagttcaaacaaaaat 17

Separation and purification of recombinant spike protein of COVID-19 virus using protein A affinity column chromatography

Expression of the recombinant spike protein of the Corona-19 virus was induced through transient expression in 4-5 week-old Nicotiana benthamiana plant leaves using a vacuum infiltration method. The infiltrated leaves are harvested 5 days (dpi) after infiltration, and Nicotiana benthamiana leaf tissue expressing the trimeric S protein is placed in liquid nitrogen and ground to a powder, where the fresh weight of the leaf is obtained. A buffer solution corresponding to 5 times was added, and total soluble protein was extracted. And this was separated and purified using protein A affinity column chromatography. A 1 mg protein A affinity resin column was used for a total protein extract corresponding to 1 g of leaf fresh tissue. After the total aqueous extract was passed through the column, the column was washed with 10 volumes of washing buffer, the protein A resin was recovered from the column, and then the protein affinity resin using 500 μml of 200 mM glycine solution. The protein bound to was eluted. The pH was then immediately neutralized with 0.1 M NaOH. The separated and purified protein was verified using Coommassie brilliant blue and anti-human IgG antibody using SDS/PAGE (FIG. 2).

Esterase Activity of Recombinant Spike Proteins of Corona-19 Virus Forming Trimers

The esterase activity of the three recombinant proteins (Sf, S1 and S2) of the Corona 19 virus S protein to form a trimer was determined by para-nitrophenyl acetate (pNPA) paranitrophenol, pNP) was determined by measuring the release amount. To measure the activity, the substrate (p-NPA) was made to 1 mM in 0.5 ml of 50 mM sodium phosphate buffer (pNPA is pH 7.0), and the mixture was prepared to contain 200 ng of the isolated and purified S protein recombinant protein. Then, it was incubated at 37°C for 10 minutes. The reaction was then quenched by the addition of 1 ml of 1M sodium carbonate. Absorbance was measured at 405 nm. Steady-state kinetics measurements were performed at the optimum temperature and pH of pNPA while changing the substrate concentration (0.5 to 2 mM). Km and Vmax values were calculated from the original pNP free ratio. The esterase activity of the recombinant spike protein of Corona-19 virus to form a trimer was determined by measuring the amount of reducing sugars. One unit enzyme activity was defined as the amount of enzyme that produced 1 μmol of pNP or reducing sugar per minute. As the concentration of the S protein increased, it was confirmed that the esterase activity increased with time (FIG. 3).

Esterase activity inhibitor screening of recombinant spike proteins of Corona-19 virus that form trimers

Esterase activity was confirmed by treatment with sulfamethoxazole at 0.5 mM, 1.0 mM, and 2 mM, respectively, to the recombinant spike protein of Corona-19 virus forming a trimer. Esterase activity was determined by measuring the release amount of para-nitrophenol (pNP) in para-nitrophenyl acetate (pNPA). To measure the activity, the substrate (p-NPA) was made to 6 mM in 0.5 ml of 50 mM sodium phosphate buffer (pH 7.0), and a mixture was prepared to contain 200 ng of the isolated and purified S protein recombinant protein. Incubated at ℃ for 10 minutes. The reaction was quenched by the addition of 1 ml of 1M sodium carbonate. Absorbance was measured at 405 nm. One unit enzyme activity was defined as the amount of enzyme that produced 1 μmol of pNP or reducing sugar per minute. As a result, it was confirmed that the esterase activity of S-F, S1 and S2 decreased as the concentration of sulfamethoxazole increased (FIG. 4). Sulfamethoxazole inhibits the activity of the esterase of the spike protein of the Corona-19 virus, thereby inhibiting the cell infection or release process of the virus, thereby inhibiting the proliferation of the virus in the body Infection can be treated.

SEQUENCE LISTING <110> POSTECH ACADEMY-INDUSTRY FOUNDATION <120> Kit for screening for therapeutic agent for preventing or treating Corona-virus infection by using of esterase activity of Corona-19 virus spike protein and pharmaceutical composition comprising material selected therefrom <130> 1069405 <150> KR 10-2020-0080622 <151> 2020-06-30 <160> 17 <170> PatentIn version 3.2 <210> 1 <211> 3594 <212> DNA <213> Artificial <220> <223> Sf <400> 1 gttaacctta ccaccagaac tcagttaccc ccagcataca ctaattcttt cacacgtggt 60 gtttactacc ctgacaaagt tttcagaagc agcgttttac acagcactca ggatttattc 120 ctacctttct tttccaacgt gacctggttc catgctatac atgtatctgg gaccaatggt 180 accaagaggt ttgataaccc ggtcctacca tttaatgatg gagtctattt tgcctccact 240 gagaagtcta atataataag aggctggatt tttggaacta ctcttgattc gaagacccag 300 agtctactta ttgttaataa cgctacaaat gttgttatca aagtatgtga atttcaattc 360 tgtaatgatc cattcttggg tgtttactac cacaaaaaca acaaaagttg gatggaaagt 420 gagtttcggg tttatagcag tgcgaataat tgcacttttg agtacgtctc ccaacct ttt 480 cttatggacc ttgaaggaaa gcagggaaat ttcaagaatc ttcgcgaatt tgtgtttaag 540 aatatcgatg gttatttcaa gatatattct aagcacacgc ctattaattt agtgcgagat 600 ctccctcagg gtttttcggc gctggaacca ttggtagatt tgccgatagg aatcaatatc 660 actaggttcc agactttact tgctctgcat agaagttact tgacccctgg agatagctca 720 tcaggttgga cagctggtgc ggcagcttat tacgtggggt atcttcagcc taggacgttc 780 ctattaaaat ataatgaaaa tggaaccatt acagatgctg tagactgtgc acttgaccct 840 ctctcagaaa caaagtgtac gttgaaatcc ttcacggtag aaaaagggat ctaccaaacg 900 tctaacttca gagtccagcc aacagaatct attgtgagat ttcccaatat tacaaacttg 960 tgccctttcg gagaagtttt taacgccacc aggtttgcat cggtttatgc ttggaacagg 1020 aaaagaatca gcaactgtgt tgctgattat agtgtcctat ataactccgc atccttttcc 1080 actttcaagt gttacggagt ttctcctact aaattaaatg atctctgctt tactaatgtc 1140 tatgcagatt catttgtaat cagaggtgat gaggtcagac aaatcgctcc agggcagact 1200 ggaaagattg ctgattataa ttataagctt cctgatgatt ttacaggctg cgttatagca 1260 tggaattcta ataatcttga ctctaaggtg gggggaaatt ataattacct gtatagactg 1320 tttagg aaga gcaatctcaa gcctttcgag agagacattt caactgagat ctaccaggcg 1380 ggaagcactc cgtgtaatgg tgttgagggt tttaattgtt actttccttt acagtcatac 1440 ggtttccaac ccacgaatgg ggttggttac caaccgtacc gagtagtagt actttctttc 1500 gagcttctac atgccccagc aactgtttgt ggacctaaga agtctactaa tttggttaaa 1560 aataagtgtg tcaattttaa tttcaatgga cttacgggca caggagttct tactgagtct 1620 aacaagaagt ttctgccttt ccagcagttc ggcagagata ttgctgacac tactgatgct 1680 gtgcgtgatc cacagacact tgaaattctt gacattacac catgttcttt tggtggcgtg 1740 agtgttataa ctcccggaac aaatacctcc aaccaggtgg ctgttctgta tcaggacgtg 1800 aactgtacag aagtccctgt tgcaattcat gcagatcagc ttactcctac ctggcgtgtt 1860 tattctacgg gttccaatgt ttttcaaaca cgtgcaggct gcttgatagg ggctgaacat 1920 gtcaacaact catatgaatg cgacataccc ataggtgcag gtatatgcgc tagttatcag 1980 actcagacca attctccgcg gcgggcacga agtgtagcta gtcaatccat cattgcctac 2040 actatgtcac ttggtgcaga aaattcagtt gcttactcta ataactctat tgccataccc 2100 acaaatttta ctattagtgt taccacagaa attctaccag tgtctatgac caagacatca 2160 gttgactgta c aatgtatat ttgcggggat tcaactgagt gctcgaatct gttgttgcaa 2220 tacggcagtt tttgtaccca attgaaccgg gctctgactg gaatagctgt ggaacaagat 2280 aaaaacaccc aagaagtttt tgcacaagtc aaacaaattt ataaaacacc accaattaaa 2340 gatttcggtg gtttcaactt ctcacaaata ctgccagatc cgagcaaacc aagcaagagg 2400 tcattcattg aagacctact tttcaacaaa gtgacacttg cagatgctgg cttcattaaa 2460 cagtatggtg attgcttggg ggatattgct gctagagacc tcatttgtgc acaaaagttt 2520 aacgggctga cagtgttgcc acctttgttg acagatgaga tgattgctca gtacacttct 2580 gcactgctcg ctggtacaat cacatctggg tggacctttg gtgcaggtgc tgccttacaa 2640 ataccatttg ctatgcagat ggcttatagg ttcaatggta tcggagttac acagaacgtt 2700 ctctatgaga accaaaaatt gattgccaac caattcaata gtgccattgg caagattcag 2760 gactcacttt caagcacagc gagtgcactt ggaaagttgc aagatgtggt caaccagaat 2820 gcacaagctt taaacacgct tgtgaaacaa ctcagctcca actttggggc aatttcaagt 2880 gttttgaatg atatcctttc acgtcttgat aaagtggaag ccgaggtgca aattgacagg 2940 ttgatcacag gccgacttca aagtttgcag acttatgtga ctcaacaatt aattagggca 3000 gcagaaatcc gcgcttc ggc taatctggcg gctactaaaa tgtcagagtg tgtacttgga 3060 caatctaaac gagttgattt ttgcggaaag ggctatcatc tcatgtcctt ccctcagtca 3120 gcgcctcacg gtgtagtgtt cttgcacgtg acttacgttc ctgcacaaga aaagaatttc 3180 acaactgctc cggccatttg tcatgatgga aaagcccact ttccgcgtga aggtgtcttt 3240 gtttcgaatg gcacacactg gtttgtaacc caaaggaatt tttatgagcc acaaatcatt 3300 acgacggaca acacttttgt gtctggtaat tgtgatgttg taatcggaat cgtcaacaac 3360 accgtttacg atcctttgca gcctgagtta gattctttca aagaggagct ggataagtat 3420 ttcaagaatc atacatcacc Legatgttgat ctcggtgata tctctggaat taatgcttca 3480 gttgtgaaca ttcaaaagga gattgaccgc ctcaatgagg ttgccaagaa tttgaatgaa 3540 tcgctcatcg atctccaaga actt g223 3521194 <n f 210 Pu 210 Ptg Artificial <2 Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser 1 5 10 15 Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val 20 25 30 Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr 35 40 45 Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe 50 55 60 Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr 65 70 75 80 Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp 85 90 95 Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val 100 105 110 Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val 115 120 125 Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val 130 135 140 Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe 145 150 155 160 Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu 165 170 175 Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His 180 185 190 Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu 195 200 205 Glu Pro Leu V al Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln 210 215 220 Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser 225 230 235 240 Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln 245 250 255 Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp 260 265 270 Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu 275 280 285 Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg 290 295 300 Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu 305 310 315 320 Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr 325 330 335 Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val 340 345 350 L eu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser 355 360 365 Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser 370 375 380 Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr 385 390 395 400 Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly 405 410 415 Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly 420 425 430 Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro 435 440 445 Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro 450 455 460 Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr 465 470 475 480 Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val 485 490 495 Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro 500 505 510 Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe 515 520 525 Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe 530 535 540 Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala 545 550 555 560 Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser 565 570 575 Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln 580 585 590 Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala 595 600 605 Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly 610 615 620 Ser Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His 625 630 6 35 640 Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys 645 650 655 Ala Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg Ser Val 660 665 670 Ala Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn 675 680 685 Ser Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr 690 695 700 Ile Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser 705 710 715 720 Val Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn 725 730 735 Leu Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu 740 745 750 Thr Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala 755 760 765 Gln Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly 770 775 780 Phe Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg 785 790 795 800 Ser Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala 805 810 815 Gly Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg 820 825 830 Asp Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro 835 840 845 Leu Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala 850 855 860 Gly Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln 865 870 875 880 Ile Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val 885 890 895 Thr Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe 900 905 910 Asn Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser 915 920 925 Ala Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu 930 935 940 Asn Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser 945 950 955 960 Val Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val 965 970 975 Gln Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr 980 985 990 Val Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn 995 1000 1005 Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys 1010 1015 1020 Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro 1025 1030 1035 Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val 1040 1045 1050 Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His 1055 1060 1065 Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn 1070 1075 1080 Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln 1085 109 0 1095 Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val 1100 1105 1110 Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro 1115 1120 1125 Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn 1130 1135 1140 His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn 1145 1150 1155 Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu 1160 1165 1170 Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu 1175 1180 1185 Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro 1190 1195 <210> 3 <211> 1998 <212> DNA <213> Artificial <220> <223> S1 <400> 3 gttaacctta ccaccagaac tcagttaccc ccagcataca ctaattcttt cacacgtggt 60 gtttactacc ctgacaaagt tttcagaagc agcgttttac acagcactca ggatttattc 120 ctacctttct tttccaacgt gacctggttc catgctatac atgtatctgg gaccaatggt 180 accaagaggt ttgataaccc ggtcctacca tttaatgatg gagtctattt tgcctccact 240 gagaagtcta atataataag aggctggatt tttggaacta ctcttgattc gaagacccag 300 agtctactta ttgttaataa cgctacaaat gttgttatca aagtatgtga atttcaattc 360 tgtaa tgatc cattcttggg tgtttactac cacaaaaaca acaaaagttg gatggaaagt 420 gagtttcggg tttatagcag tgcgaataat tgcacttttg agtacgtctc ccaacctttt 480 cttatggacc ttgaaggaaa gcagggaaat ttcaagaatc ttcgcgaatt tgtgtttaag 540 aatatcgatg gttatttcaa gatatattct aagcacacgc ctattaattt agtgcgagat 600 ctccctcagg gtttttcggc gctggaacca ttggtagatt tgccgatagg aatcaatatc 660 actaggttcc agactttact tgctctgcat agaagttact tgacccctgg agatagctca 720 tcaggttgga cagctggtgc ggcagcttat tacgtggggt atcttcagcc taggacgttc 780 ctattaaaat ataatgaaaa tggaaccatt acagatgctg tagactgtgc acttgaccct 840 ctctcagaaa caaagtgtac gttgaaatcc ttcacggtag aaaaagggat ctaccaaacg 900 tctaacttca gagtccagcc aacagaatct attgtgagat ttcccaatat tacaaacttg 960 tgccctttcg gagaagtttt taacgccacc aggtttgcat cggtttatgc ttggaacagg 1020 aaaagaatca gcaactgtgt tgctgattat agtgtcctat ataactccgc atccttttcc 1080 actttcaagt gttacggagt ttctcctact aaattaaatg atctctgctt tactaatgtc 1140 tatgcagatt catttgtaat cagaggtgat gaggtcagac aaatcgctcc agggcagact 1200 ggaaagattg ctgattataa ttataagctt cctgatgatt ttacaggctg cgttatagca 1260 tggaattcta ataatcttga ctctaaggtg gggggaaatt ataattacct gtatagactg 1320 tttaggaaga gcaatctcaa gcctttcgag agagacattt caactgagat ctaccaggcg 1380 ggaagcactc cgtgtaatgg tgttgagggt tttaattgtt actttccttt acagtcatac 1440 ggtttccaac ccacgaatgg ggttggttac caaccgtacc gagtagtagt actttctttc 1500 gagcttctac atgccccagc aactgtttgt ggacctaaga agtctactaa tttggttaaa 1560 aataagtgtg tcaattttaa tttcaatgga cttacgggca caggagttct tactgagtct 1620 aacaagaagt ttctgccttt ccagcagttc ggcagagata ttgctgacac tactgatgct 1680 gtgcgtgatc cacagacact tgaaattctt gacattacac catgttcttt tggtggcgtg 1740 agtgttataa ctcccggaac aaatacctcc aaccaggtgg ctgttctgta tcaggacgtg 1800 aactgtacag aagtccctgt tgcaattcat gcagatcagc ttactcctac ctggcgtgtt 1860 tattctacgg gttccaatgt ttttcaaaca cgtgcaggct gcttgatagg ggctgaacat 1920 gtcaacaact catatgaatg cgacataccc ataggtgcag gtatatgcgc tagttatcag 1980 actcagacca attctccg 1998 <210> 4 <211> 666 <212> PRT <213> Artificial <220> <223> S1 <400> 4 Val A sn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser 1 5 10 15 Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val 20 25 30 Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr 35 40 45 Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe 50 55 60 Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr 65 70 75 80 Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp 85 90 95 Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val 100 105 110 Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val 115 120 125 Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val 130 135 140 Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe 145 150 155 160 Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu 165 170 175 Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His 180 185 190 Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu 195 200 205 Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln 210 215 220 Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser 225 230 235 240 Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln 245 250 255 Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp 260 265 270 Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu 275 280 285 Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg 290 295 300 Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu 305 310 3 15 320 Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr 325 330 335 Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val 340 345 350 Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser 355 360 365 Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser 370 375 380 Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr 385 390 395 400 Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly 405 410 415 Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly 420 425 430 Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro 435 440 445 Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro 450 455 460 Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr 465 470 475 480 Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val 485 490 495 Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro 500 505 510 Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe 515 520 525 Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe 530 535 540 Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala 545 550 555 560 Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser 565 570 575 Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln 580 585 590 Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala 595 600 605 Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly 610 615 620 Ser Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His 625 630 635 640 Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys 645 650 655 Ala Ser Tyr Gln Thr Gln Thr Asn Ser Pro 660 665 <210> 5 <211> 1596 <212> DNA <213> Artificial <220> <223> S2 <400> 5 cggcgggcac gaagtgtagc tagtcaatcc atcattgcct acactatgtc acttggtgca 60 gaaaattcag ttgcttactc taataactct attgccatac ccacaaattt tactattagt 120 gttaccacag aaattctacc agtgtctatg accaagacat cagttgactg tacaatgtat 180 atttgcgggg attcaactga gtgctcgaat ctgttgttgc aatacggcag tttttgtacc 240 caattgaacc gggctctgac tggaatagct gtggaacaag ataaaaacac ccaagaagtt 300 tttgcacaag tcaaacaaat ttataaaaca ccaccaatta aagatttcgg tggtttcaac 360 ttctcacaaa tactgccaga tccgagcaaa ccaagcaaga ggtcattcat tgaagaccta 420 cttttca aca aagtgacact tgcagatgct ggcttcatta aacagtatgg tgattgcttg 480 ggggatattg ctgctagaga cctcatttgt gcacaaaagt ttaacgggct gacagtgttg 540 ccacctttgt tgacagatga gatgattgct cagtacactt ctgcactgct cgctggtaca 600 atcacatctg ggtggacctt tggtgcaggt gctgccttac aaataccatt tgctatgcag 660 atggcttata ggttcaatgg tatcggagtt acacagaacg ttctctatga gaaccaaaaa 720 ttgattgcca accaattcaa tagtgccatt ggcaagattc aggactcact ttcaagcaca 780 gcgagtgcac ttggaaagtt gcaagatgtg gtcaaccaga atgcacaagc tttaaacacg 840 cttgtgaaac aactcagctc caactttggg gcaatttcaa gtgttttgaa tgatatcctt 900 tcacgtcttg ataaagtgga agccgaggtg caaattgaca ggttgatcac aggccgactt 960 caaagtttgc agacttatgt gactcaacaa ttaattaggg cagcagaaat ccgcgcttcg 1020 gctaatctgg cggctactaa aatgtcagag tgtgtacttg gacaatctaa acgagttgat 1080 ttttgcggaa agggctatca tctcatgtcc ttccctcagt cagcgcctca cggtgtagtg 1140 ttcttgcacg tgacttacgt tcctgcacaa gaaaagaatt tcacaactgc tccggccatt 1200 tgtcatgatg gaaaagccca ctttccgcgt gaaggtgtct ttgtttcgaa tggcacacac 1260 tggtttgtaa cccaaaggaatttttatgag ccacaaatca ttacgacgga caacactttt 1320 gtgtctggta attgtgatgt tgtaatcgga atcgtcaaca acaccgttta cgatcctttg 1380 cagcctgagt tagattcttt caaagaggag ctggataagt atttcaagaa tcatacatca 1440 cccgatgttg atctcggtga tatctctgga attaatgctt cagttgtgaa cattcaaaag 1500 gagattgacc gcctcaatga ggttgccaag aatttgaatg aatcgctcat cgatctccaa 1560 gaacttggaa agtatgagca gtatatcaag tggcca 1596 <210> 6 <211> 532 <212> PRT <213> Artificial <220> <223> S2 <400> 6 Arg Arg Ala Arg Ser Val Ala Ser Gln Ser Ile Ile Ala Tyr Thr Met 1 5 10 15 Ser Leu Gly Ala Glu Asn Ser Val Ala Tyr Ser Asn Asn Ser Ile Ala 20 25 30 Ile Pro Thr Asn Phe Thr Ile Ser Val Thr Thr Glu Ile Leu Pro Val 35 40 45 Ser Met Thr Lys Thr Ser Val Asp Cys Thr Met Tyr Ile Cys Gly Asp 50 55 60 Ser Thr Glu Cys Ser Asn Leu Leu Leu Gln Tyr Gly Ser Phe Cys Thr 65 70 75 80 Gln Leu Asn Arg Ala Leu Thr Gly Ile Ala Val Glu Gln Asp Lys Asn 85 90 95 Thr Gln Glu Val Phe Ala Gln Val Lys Gln Ile Tyr Lys Thr Pro 100 105 110 Ile Lys Asp Phe Gly Gly Phe Asn Phe Ser Gln Ile Leu Pro Asp Pro 115 120 125 Ser Lys Pro Ser Lys Arg Ser Phe Ile Glu Asp Leu Leu Phe Asn Lys 130 135 140 Val Thr Leu Ala Asp Ala Gly Phe Ile Lys Gln Tyr Gly Asp Cys Leu 145 150 155 160 Gly Asp Ile Ala Ala Arg Asp Leu Ile Cys Ala Gln Lys Phe Asn Gly 165 170 175 Leu Thr Val Leu Pro Pro Leu Leu Thr Asp Glu Met Ile Ala Gln Tyr 180 185 190 Thr Ser Ala Leu Leu Ala Gly Thr Ile Thr Ser Gly Trp Thr Phe Gly 195 200 205 Ala Gly Ala Ala Leu Gln Ile Pro Phe Ala Met Gln Met Ala Tyr Arg 210 215 220 Phe Asn Gly Ile Gly Val Thr Gln Asn Val Leu Tyr Glu Asn Gln Lys 225 230 235 240 Leu Ile Ala Asn Gln Phe Asn Ser Ala Ile Gly Lys Ile Gln Asp Ser 245 25 0 255 Leu Ser Ser Thr Ala Ser Ala Leu Gly Lys Leu Gln Asp Val Val Asn 260 265 270 Gln Asn Ala Gln Ala Leu Asn Thr Leu Val Lys Gln Leu Ser Ser Asn 275 280 285 Phe Gly Ala Ile Ser Ser Val Leu Asn Asp Ile Leu Ser Arg Leu Asp 290 295 300 Lys Val Glu Ala Glu Val Gln Ile Asp Arg Leu Ile Thr Gly Arg Leu 305 310 315 320 Gln Ser Leu Gln Thr Tyr Val Thr Gln Gln Leu Ile Arg Ala Ala Glu 325 330 335 Ile Arg Ala Ser Ala Asn Leu Ala Ala Thr Lys Met Ser Glu Cys Val 340 345 350 Leu Gly Gln Ser Lys Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu 355 360 365 Met Ser Phe Pro Gln Ser Ala Pro His Gly Val Val Phe Leu His Val 370 375 380 Thr Tyr Val Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile 385 390 395 400 Cys His Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser 405 410 415 Asn Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln 420 425 430 Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val Val 435 440 445 Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro Glu Leu 450 455 460 Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn His Thr Ser 465 470 475 480 Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn Ala Ser Val Val 485 490 495 Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu Val Ala Lys Asn Leu 500 505 510 Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu Gly Lys Tyr Glu Gln Tyr 515 520 525 Ile Lys Trp Pro 530 <210> 7 <211> 96 <212> DNA <213> Artificial <220> <223> mCOr1 <400> 7 gtgtctaggc ttgaggaaga tgttagaaat ctcaacgcaa ttgtccagaa acttcaggaa 60 aggttggata ggctggagga aactgttcaa gctaag 96 <210> 8 <211> 32 <212> PRT <213> Val Glug Leu Glue <220> <223> mCor Val Arg Asn Leu Asn Ala Ile Val Gln 1 5 10 15 Lys Leu Gln Glu Arg Leu Asp Arg Leu Glu Glu Thr Val Gln Ala Lys 20 25 30 <210> 9 <211> 272 <212> DNA <213> Artificial <220 > <223> BiP <400> 9 atggctcgct cgtttggagc taacagtacc gttgtgttgg cgatcatctt cttcggtgag 60 tgattttccg atcttcttct ccgatttaga tctcctctac attgttgctt aatctcagaa 120 ccttttttcg ttgttcctgg atctgaatgt gtttgtttgc aatttcacga tcttaaaagg 180 ttagatctcg attggtattg acgattggaa tctttacgat ttcaggatgt ttatttgcgt 240 tgtcctctgc aatagaagag gctacgaagt ta 272 <210> 10 <211> 12 <212> DNA <213> Artificial <220> <223> HDEL <400> 10 cacgatgagc tc 12 <210> 11 <211> 4 <212> PRT <213> Artificial <220> <223> HDEL <400> 11 His Asp Glu Leu 1 <210> 12 <211> 42 <212> DNA <213> Artificial <220> <223> Linker <400> 12 gatgatgatg a taagacccg gggtggtgga agtggtggaa gt 42 <210> 13 <211> 14 <212> PRT <213> Artificial <220> <223> Linker <400> 13 Asp Asp Asp Asp Lys Thr Arg Gly Gly Gly Ser Gly Gly Ser 1 5 10 < 210> 14 <211> 651 <212> DNA <213> Artificial <220> <223> Monomeric human Fc <400> 14 gcacctgaac tcctgggggg accgtcagtc ttcctcttcc ccccaaaacc caaggacacc 60 ctcatgatct cccggacccc tgaggtcaca tgcgtggtgg tggacgtgag ccacgaagac 120 cctgaggtca agttcaactg gtacgtggac ggcgtggagg tgcataatgc caagacaaag 180 ccgcgggagg agcagtacaa cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac 240 caggactggc tgaatggcaa ggagtacaag tgcaaggtct ccaacaaagc cctcccagcc 300 cccatcgaga aaaccatctc caaagccaaa gggcagcccc gagaaccaca ggtgtacacc 360 tcgcccccat cccgtgagga gatgaccaag aaccaggtca gcctgaggtg ccacgtcaaa 420 ggcttctatc ccagcgacat cgccgtggag tgggagagca atgggcagcc ggagaacaac 480 tacaagacca cgaagcccgt gctggactcc gacggctcct tcgagctcaa gagcgcgctc 540 accgtggaca agagcaggtg gcagcagggg aacgtcttct catgctccgt gatgcatgag 600 gctctgcaca accactacac gcaga agagc ctctccctgt cccctggtaa a 651 <210> 15 <211> 217 <212> PRT <213> Artificial <220> <223> Monomeric human Fc <400> 15 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 100 105 110 Pro Arg Glu Pro Gln Val Tyr Thr Ser Pro Pro Ser Arg Glu Glu Met 115 120 125 Thr Lys Asn Gln Val Ser Leu Arg Cys His Val Lys Gly Phe Tyr Pro 130 135 140 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 145 150 155 160 Tyr Lys Thr Thr Lys Pro Val Leu Asp Ser Asp Gly Ser Phe Glu Leu 165 170 175 Lys Ser Ala Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 180 185 190 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 195 200 205 Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 <210> 16 <211> 1220 <212> DNA <213> Artificial <220> <223> MacT <400> 16 agagatctcc tttgccccag agatcacaat ggacgacttc ctctatctct acgatctagt 60 caggaagttc gacggagaag gtgacgatac catgttcacc actgataatg agaagattag 120 ccttttcaat ttcagaaaga atgctaaccc acagatggtt agagaggctt acgcagcagg 180 tctcatcaag acgatctacc cgagcaataa tctccaggag atcaaatacc ttcccaagaa 240 ggttaaagat gcagtcaaaa gattcaggac taactgcatc aagaacacag agaaagatat 300 atttctcaag atcagaagta ctattccagt atggacgatt caaggcttgc ttcacaaacc 360 aaggcaagta atagagattg gagtctctaa aaaggtagtt cccactgaat caaaggccat 420 ggagtcaaag attcaaatag agga cctaac agaactcgcc gtaaagactg gcgaacagtt 480 catacagagt ctcttacgac tcaatgacaa gaagaaaatc ttcgtcaaca tggtggagca 540 cgacacgctt gtctactcca aaaatatcaa agatacagtc tcagaagacc aaagggcaat 600 tgagactttt caacaaaggg taatatccgg aaacctcctc ggattccatt gcccagctat 660 ctgtcacttt attgtgaaga tagtggaaaa ggaaggtggc tcctacaaat gccatcattg 720 cgataaagga aaggccatcg ttgaagatgc ctctgccgac agtggtccca aagatggacc 780 cccacccacg aggagcatcg tggaaaaaga agacgttcca accacgtctt caaagcaagt 840 ggattgatgt gacgcaagac gtgacgtaag tatctgagct agtttttatt tttctactaa 900 tttggtcgtt tatttcggcg tgtaggacat ggcaaccggg cctgaatttc gcgggtattc 960 tgtttctatt ccaacttttt cttgatccgc agccattaac gacttttgaa tagatacgct 1020 gacacgccaa gcctcgctag tcaaaagtgt accaaacaac gctttacagc aagaacggaa 1080 tgcgcgtgac gctcgcggtg acgccatttc gccttttcag aaatggataa atagccttgc 1140 ttcctattat atcttcccaa attaccaata cattacacta gcatctgaat ttcataacca 1200 atctcgatac accaaatcgt 1220 <210> 17 <211> 520 <212> DNA <213> Artificial <220> <223> RD29B <400> 17 aattttactc a aaatgtttt ggttgctatg gtagggacta tggggttttc ggattccggt 60 ggaagtgagt ggggaggcag tggcggaggt aagggagttc aagattctgg aactgaagat 120 ttggggtttt gcttttgaat gtttgcgttt ttgtatgatg cctctgtttg tgaactttga 180 tgtattttat ctttgtgtga aaaagagatt gggttaataa aatatttgct tttttggata 240 agaaactctt ttagcggccc attaataaag gttacaaatg caaaatcatg ttagcgtcag 300 atatttaatt attcgaagat gattgtgata gatttaaaat tatcctagtc aaaaagaaag 360 agtaggttga gcagaaacag tgacatctgt tgtttgtacc atacaaatta gtttagatta 420 ttggttaaca tgttaaatgg ctatgcatgt gacatttaga ccttatcgga attaatttgt 480agaattatta attaagatgt tgattagttc aaacaaaaat 520

Claims (17)

A recombinant protein comprising the S1 subunit or the S2 subunit of a coronavirus spike protein, or a recombinant protein comprising the S1 subunit and the S2 subunit having esterase activity. The method according to claim 1, wherein when the corona virus is SARS-Corona Virus-2, the S1 subunit comprises the amino acid of SEQ ID NO: 4, and the S2 subunit has the amino acid sequence of SEQ ID NO: 6 Including, wherein the recombinant protein comprising the S1 subunit and the S2 subunit comprises the amino acid sequence of SEQ ID NO: 2, the recombinant protein. The recombinant protein according to claim 1, wherein the recombinant protein is in a trimeric form. The recombinant protein according to any one of claims 1 to 3, which is produced using a recombinant vector comprising the gene construct of Figure 1 (a), (b) or (c). Recombinant protein derived from the spike protein of coronavirus; and
a substance capable of measuring the esterase activity of the recombinant protein derived from the spike protein of the corona virus;
A screening kit of candidate substances for the prevention or treatment of coronavirus infection, comprising a. According to claim 5, wherein the corona virus is any one selected from the group consisting of SARS virus (SARS-Corona Virus), MERS virus (MERS-Corona Virus) and Corona-19 virus (SARS-Corona Virus-2) , a screening kit for candidate substances for the prevention or treatment of coronavirus infection. The method according to claim 6, wherein the recombinant protein derived from the spike protein of the coronavirus is a recombinant protein comprising an S1 subunit, an S2 subunit, the S1 subunit or the S2 subunit of the spike protein of the Corona-19 virus, and the S1 subunit. A screening kit for a candidate substance for the prevention or treatment of a coronavirus infection, which is a recombinant protein comprising a unit and an S2 subunit. The method according to claim 7, wherein when the coronavirus is SARS-Corona Virus-2, the S1 subunit comprises the amino acid of SEQ ID NO: 4, and the S2 subunit has the amino acid sequence of SEQ ID NO: 6. Including, wherein the recombinant protein comprising the S1 subunit and the S2 subunit comprises the amino acid sequence of SEQ ID NO: 2, a screening kit for a candidate material for the prevention or treatment of coronavirus infection. The screening kit of claim 5, wherein the recombinant protein derived from the spike protein of the coronavirus is in a trimeric form. According to claim 5, wherein the substance capable of measuring the esterase activity is an esterase substrate; compounds comprising sialic acid; BCECF, AM(2',7'-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester) (BCECF, AM(2',7'-Bis- (2-Carboxyethyl)-5-(and-6)-Carboxyfluorescein, Acetoxymethyl Ester)); Calcein AM; Carboxyeosin diacetate, succinimidyl ester; Carboxyfluorescein diacetate, acetoxymethyl ester; Or carboxyfluorescein diacetate, succinimidyl ester (Carboxyfluorescein diacetate, succinimidyl ester), a screening kit for a candidate material for the prevention or treatment of coronavirus infection. The method according to claim 10, wherein the substrate of the esterase is para nitropheny acetate (p-NPA), and the compound containing sialic acid is Neu5,9Ac2, 4-methylumbelliferyl acetate ( 4-methylumbelliferyl acetate) or 9Ac2-lactose, a screening kit for a candidate for the prevention or treatment of coronavirus infection. Using the kit of any one of claims 5 to 11, a method of screening a candidate for preventing or treating a coronavirus infection. 13. The method of claim 12,
(i) treating a candidate material with a recombinant protein derived from the spike protein of coronavirus;
(ii) measuring the esterase activity of the recombinant protein derived from the spike protein of the corona virus treated with the candidate substance; and
(iii) when the candidate material inhibits esterase activity by 30% or more, selecting the candidate material as a candidate material for prevention or treatment of coronavirus; How to screen for candidates. A pharmaceutical composition for the prevention or treatment of corona virus infection, comprising an inhibitor of esterase activity of the spike protein of coronavirus. 15. The method of claim 14, wherein the esterase activity inhibitor binds to the active site of the esterase to inhibit the activity of the esterase, or binds to a site other than the active site of the esterase to form an esterase. A pharmaceutical composition for preventing or treating corona virus infection, which inhibits the activity of an esterase by inducing a structural change of the degrading enzyme. A pharmaceutical composition for preventing or treating COVID-19 virus infection, comprising sulfamethoxazole, a derivative, isomer, or pharmaceutically acceptable salt thereof. A health functional food composition for preventing or improving corona-19 virus infection, comprising sulfamethoxazole, a derivative, isomer, or food pharmaceutically acceptable salt thereof.

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