KR20230123115A - Ace2 variants and use thereof - Google Patents

Abstract

The present invention relates to an ACE-2 variant and a use thereof. The ACE-2 variant of the present invention is water-soluble, forms a trimer in vivo, and has more increased binding power to a coronavirus spike protein than the ACE-2 wild type, so that the ACE-2 variant can be usefully used as a therapeutic agent that can treat coronavirus infection or an agent that can diagnose coronavirus.

Description

ACE-2 variants and uses thereof {ACE2 VARIANTS AND USE THEREOF}

The present invention relates to ACE-2 variants and uses thereof.

Severe acute respiratory syndrome coronavirus 2 (SARS-Cov2) is a single-stranded RNA virus belonging to the subfamily Corona virus, and the cause was unknown in Wuhan, China in December 2019. The causative virus was first discovered in patients with pneumonia. Not to be confused with severe acute respiratory syndrome infection, the World Health Organization (WHO) also calls it '2019 coronavirus infection virus' or '2019 coronavirus infection virus'.

From December 2019 to early July 2020, 11.57 million confirmed cases occurred in 214 countries around the world, of which 537,138 died, with a fatality rate of 4.64. The clinical symptoms of SARS-CoV-2 are mainly respiratory symptoms accompanied by fever, and the main vulnerable target is the elderly suffering from underlying diseases and immune diseases in their 60s or older. Unlike previous viruses, it showed strong contagiousness and rapid worsening of symptoms, and in March 2020, about 3 months after the first report, the WHO declared a global pandemic, a pandemic.

On the other hand, it is well known that the coronavirus CoV-2 infects the human body through the binding of the RBD region of the spike protein and the cell receptor ACE-2 protein. The binding between the RBD region of the spike protein and its cellular receptor, the ACE-2 protein, occurs in three different regions. In addition, substitution of specific amino acids in the ACE-2 protein serves to strengthen the binding between the RBD protein and the ACE-2 protein. ACE-2 protein consists of an extracellular ligand-binding receptor site and a transmembrane part, and the ACE-2 protein from which the transmembrane part is removed exists as a water-soluble protein outside the cell instead of being present on the cell surface. However, the soluble ACE-2 protein still maintains binding to the coronavirus spike RBD protein. It is known that the spike protein binds to the ACE-2 protein in the form of a triplex when the corona virus penetrates the cell. In response, the duplex or triplex structure of the ACE-2 protein is known to have a stronger binding force with the coronavirus spike protein.

To date, there has been no report that ACE-2 can diagnose or treat COVID-19, but using the strong binding force between ACE-2 and spike protein, it is possible to prevent the spike protein of coronavirus from binding to ACE-2. It is being proposed as a treatment strategy for the novel coronavirus that competitively inhibits it.

Accordingly, in order to make a small-sized water-soluble ACE-2 protein that maintains the binding force with the coronavirus spike RBD protein, 1) amino acids that include three regions that act on binding with the RBD protein and increase the binding force with the RBD protein The present invention was completed by including a substitution and combining a protein structure that self-forms a triplex structure with this partial water-soluble form of ACE-2 protein to derive a water-soluble ACE-2 mutant of the present invention.

Korean Patent Publication No. 10-2021-0043363 (published on 21.10.13)

An object of the present invention is to provide ACE-2 variants or various uses thereof.

Therefore, the present invention provides an ACE-1 variant with increased binding ability to the water-soluble coronavirus spike protein.

In another aspect, the present invention provides a nucleic acid encoding the ACE-2 variant.

In another aspect, the present invention provides a vector comprising the nucleic acid molecule.

In another aspect, a host cell containing the vector is provided.

In addition, in another aspect, the present invention provides a composition for detecting coronavirus, including an ACE-1 variant.

In another aspect, the present invention provides a pharmaceutical composition for the treatment or prevention of coronavirus, comprising an ACE-2 variant, a nucleic acid molecule encoding the variant, or a vector containing the nucleic acid molecule as an active ingredient.

In another aspect, the present invention provides a composition for diagnosis of coronavirus infection comprising an ACE-2 variant.

In another aspect of the present invention, a) contacting a biological sample isolated from a subject with the ACE-2 variant of claim 1;

b) checking the binding level of the ACE-2 variant and the coronavirus spike protein; and

c) providing an informational method for diagnosing coronavirus infection, comprising the step of comparing the binding level of the ACE-2 variant and the corona virus spike protein in a normal control sample.

The ACE-2 variant of the present invention is water-soluble, forms a trimer in vivo, and has an increased binding affinity to the coronavirus spike protein than ACE-2 wild type, thereby diagnosing coronavirus or a therapeutic agent capable of treating coronavirus infection It can be usefully used as a possible agent.

1 is a schematic diagram of a vector containing sACE2.
2 is a schematic diagram of a vector containing sACE2.
3 is a diagram confirming the infection of lentivirus expressing spike protein in ACE2 overexpressing cell lines.
4 is a diagram showing the results of infection of ACE2-overexpressing human kidney cell lines after pre-treatment with coronavirus spike-like lentivirus with sACE2.

Hereinafter, the present invention will be described in detail as an embodiment of the present invention with reference to the accompanying drawings. However, the following embodiments are presented as examples of the present invention, and if it is determined that detailed descriptions of well-known techniques or configurations may unnecessarily obscure the gist of the present invention, the detailed descriptions may be omitted. , the present invention is not limited thereby. Various modifications and applications of the present invention are possible within the scope of the claims described below and equivalents interpreted therefrom.

In addition, the terms used in this specification (terminology) are terms used to appropriately express preferred embodiments of the present invention, which may vary according to the intention of a user or operator or customs in the field to which the present invention belongs. Therefore, definitions of these terms will have to be made based on the content throughout this specification. Throughout the specification, when a certain component is said to "include", it means that it may further include other components without excluding other components unless otherwise stated.

All technical terms used in the present invention, unless defined otherwise, are used with the same meaning as commonly understood by one of ordinary skill in the art related to the present invention. In addition, although preferred methods or samples are described in this specification, those similar or equivalent thereto are also included in the scope of the present invention. The contents of all publications incorporated herein by reference are incorporated herein by reference.

Throughout this specification, conventional one-letter and three-letter codes for naturally occurring amino acids are used, as well as generally accepted for other amino acids such as Aib (α-aminoisobutyric acid), Sar (N-methylglycine), etc. A three-letter code is used. Amino acids also referred to by abbreviations in the present invention are described according to the IUPAC-IUB nomenclature as follows:

Alanine: A, Arginine: R, Asparagine: N, Aspartic acid: D, Cysteine: C, Glutamic acid: E, Glutamine: Q, Glycine: G, Histidine: H, Isoleucine: I, Leucine: L, Lysine: K, Methionine : M, phenylalanine: F, proline: P, serine: S, threonine: T, tryptophan: W, tyrosine: Y, and valine: V.

In one aspect, the present invention is wild type (Wild type) ACE-2 ((Angiotensin-converting enzyme 2) amino acid substitutions consisting of T11Y, L63T, N74Q and T76Q in the amino acid substitution, the binding force with the spike protein of the corona virus It relates to an enhanced ACE-2 variant (in the present invention, ACE2 or ACE-2 may be used in combination).

In one embodiment, the amino acid of wild-type ACE-2 may include the amino acid sequence of SEQ ID NO: 1, and the amino acid position may be based on the amino acid sequence of SEQ ID NO: 1.

In one embodiment, the ACE-2 variant of the present invention may have a signal peptide sequence removed.

Further, in one embodiment, the ACE-2 variant of the present invention may be a sequence further comprising the sequence represented by SEQ ID NO: 2 at the carboxy terminus to form a trimer in the wild-type ACE-2 amino acid sequence.

Also, in one embodiment, the variant may be a water-soluble protein.

Also, in one embodiment, the variant may be a sequence represented by SEQ ID NOs: 3 to 6.

Also, in one embodiment, the variant may be encoded by the sequences represented by SEQ ID NOs: 7 to 10.

Also, in one embodiment, the corona virus may be corona virus CoV-2.

The ACE-2 variant of the present invention refers to a wild-type ACE-2 protein (or peptide) in which some amino acid sequences are substituted, deleted, or added,

As used herein, the term "variant" refers to a corresponding amino acid sequence that contains at least one amino acid difference (substitution, insertion or deletion) when compared to a reference material. In certain embodiments a “variant” has high amino acid sequence homology and/or conservative amino acid substitutions, deletions and/or insertions as compared to a reference sequence. In addition, as used herein, the term "ACE-2 mutant" refers to an ACE-2 mutant protein mutated in one or more amino acids in order to regulate its binding activity with the spike protein of coronavirus.

Specifically, the ACE-2 variants of the present invention can be prepared by standard synthetic methods, recombinant expression systems, or any other method in the art. Thus, peptides according to the present invention can be synthesized in a number of ways, including, for example, methods comprising:

(a) synthesizing peptides stepwise or by fragment assembly by means of solid-phase or liquid-phase methods, and isolating and purifying the final peptide product; or

(b) expressing a nucleic acid construct encoding the peptide in a host cell and recovering the expression product from the host cell culture; or

(c) a method of performing cell-free in vitro expression of a nucleic acid construct encoding a peptide and recovering the expression product; or

A method of obtaining a peptide fragment by any combination of (a), (b) and (c), then linking the fragments to obtain a peptide, and recovering the peptide.

As a more specific example, a gene encoding the ACE-2 variant of the present invention can be prepared through genetic manipulation, transformed into a host cell, and then expressed to produce the ACE-2 variant of the present invention.

In one aspect, the present invention relates to a nucleic acid molecule encoding the ACE-2 variant of the present invention, a vector containing the same, and a host cell containing the vector.

As used herein, the term "nucleic acid molecule" refers to deoxyribonucleotides or ribonucleotides that exist in single-stranded or double-stranded form, and includes natural nucleic acid analogs unless otherwise specified (Scheit, Nucleotide Analogs, John Wiley , New York (1980); Uhlman and Peyman, Chemical Reviews, 90:543-584 (1990)).

As used herein, the term “vector” refers to any nucleic acid comprising a competent nucleotide sequence that is inserted into a host cell to recombine with and integrate into the host cell genome, or to spontaneously replicate as an episome. Such vectors include linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors, and the like.

As used herein, the term "host cell" refers to a eukaryotic or prokaryotic cell into which one or more DNAs or vectors are introduced, and should be understood to refer not only to a specific target cell, but also to its progeny or potential progeny. In fact, the progeny are not identical to the parent cell, as certain modifications may occur in subsequent generations due to mutation or environmental influences, but are still included within the scope of the term as used herein.

In one aspect, the present invention relates to a spike protein and ACE-2 binding inhibitor of corona virus, including the ACE-2 variant of the present invention, a nucleic acid molecule thereof, or a vector containing the same.

In one aspect, the present invention relates to a composition for detecting a corona virus or spike protein of coronavirus, comprising the ACE-2 variant of the present invention.

In one embodiment, the composition can detect and quantify the expression level of spike protein of corona virus.

In one embodiment, the ACE-2 variant may be labeled with one selected from the group consisting of a chromogenic enzyme, a radioactive isotope, a chromopore, a luminescent material, and a fluorescent material, and the fluorescent material is a Cy (cyanine) series, It may be a rhodamine-based, Alexa-based, BODIPY-based or ROX-based fluorescent material, and may be Nile Red, BODIPY, 4,4-difluoro-4-bora-3a,4a -diaza-s-indacene), cyanine, fluorescein, rhodamine, coumarine or Alexa.

Since the ACE-2 variant of the present invention can detect and quantify the amount of coronavirus, in particular COVID-19 virus, it can determine the presence or absence of coronavirus and the degree of coronavirus infection in coronavirus patients.

In one aspect, the present invention relates to a pharmaceutical composition for the treatment or prevention of coronavirus infection comprising the ACE-2 variant of the present invention, a nucleic acid molecule thereof, or a vector containing the same. The coronavirus may be the COVID-19 virus.

The pharmaceutical composition of the present invention can be used as a single therapy, but can also be used in conjunction with other conventional biological therapies, chemotherapy or radiation therapy, and when such a combination therapy is performed, coronavirus infection can be treated more effectively. there is.

In the present invention, the term "prevention" refers to any action that inhibits or delays the occurrence, spread, and recurrence of a coronavirus infection by administration of an ACE-2 variant or a composition containing the same according to the present invention.

A therapeutically effective amount of the composition of the present invention may vary depending on several factors, such as the method of administration, the target site, and the condition of the patient. Therefore, when used in the human body, the dosage should be determined in an appropriate amount considering both safety and efficiency. It is also possible to estimate the amount to be used in humans from the effective amount determined through animal experiments. These considerations in determining an effective amount can be found, for example, in Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; and E.W. Martin ed., Remington's Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. As used herein, the term "pharmaceutically effective amount" means an amount that is sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment and does not cause side effects, and the effective dose level is the patient's Health condition, type and severity of disease, activity of drug, sensitivity to drug, method of administration, time of administration, route of administration and excretion rate, duration of treatment, factors including drugs used in combination or concurrently, and other factors well known in the medical field can be determined according to The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or in multiple doses. Considering all of the above factors, it is important to administer the amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.

The pharmaceutical composition of the present invention may include a carrier, diluent, excipient, or a combination of two or more commonly used in biological preparations. As used herein, the term "pharmaceutically acceptable" means that it exhibits non-toxic properties to normal cells or humans exposed to the composition. The carrier is not particularly limited as long as it is suitable for in vivo delivery of the composition. For example, Merck Index, 13th ed., Merck & Co. Inc. , saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components may be mixed and used. Customary additives may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate formulations for injection, such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, or tablets. Furthermore, it can be preferably formulated according to each disease or component by using an appropriate method in the art or by using a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

In one embodiment, the pharmaceutical composition may be one or more formulations selected from the group consisting of oral formulations, external preparations, suppositories, sterile injection solutions, and sprays.

The composition of the present invention may also include a carrier, diluent, excipient or a combination of two or more commonly used in biological preparations. The pharmaceutically acceptable carrier is not particularly limited as long as it is suitable for in vivo delivery of the composition, for example, Merck Index, 13th ed., Merck & Co. Inc. , saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components may be mixed and used. Customary additives may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate formulations for injection, such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, or tablets. Furthermore, it can be preferably formulated according to each disease or component by using an appropriate method in the art or by using a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

In addition to the composition of the present invention, one or more active ingredients exhibiting the same or similar functions may be contained. The composition of the present invention includes 0.0001 to 10% by weight of the protein, preferably 0.001 to 1% by weight, based on the total weight of the composition.

The pharmaceutical composition of the present invention may further include pharmaceutically acceptable additives, wherein the pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, and calcium hydrogen phosphate. , Lactose, Mannitol, Taffy, Gum Arabic, Pregelatinized Starch, Corn Starch, Powdered Cellulose, Hydroxypropyl Cellulose, Opadry, Sodium Starch Glycolate, Carnauba Lead, Synthetic Aluminum Silicate, Stearic Acid, Magnesium Stearate, Aluminum Stearate, Calcium stearate, white sugar, dextrose, sorbitol and talc may be used. The pharmaceutically acceptable additive according to the present invention is preferably included in an amount of 0.1 part by weight to 90 parts by weight based on the composition, but is not limited thereto.

The composition of the present invention may be parenterally administered (for example, intravenously, subcutaneously, intraperitoneally, or topically applied) or orally, depending on the desired method, and the dosage may vary depending on the patient's weight, age, sex, health condition, The range varies according to diet, administration time, administration method, excretion rate, and severity of disease. The daily dosage of the composition according to the present invention is 0.0001 to 10 mg/ml, preferably 0.0001 to 5 mg/ml, and it is more preferable to divide the administration once or several times a day.

Liquid formulations for oral administration of the composition of the present invention include suspensions, internal solutions, emulsions, syrups, etc., and various excipients such as wetting agents, sweeteners, aromatics, and preservatives in addition to water and liquid paraffin, which are commonly used simple diluents etc. may be included. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, suppositories, and the like.

In one aspect, the present invention relates to a composition for diagnosing coronavirus infection comprising an ACE-2 variant of the present invention.

As used herein, the term "detection" or "measurement" means detecting or quantifying the concentration of a measured object.

In one aspect, the present invention relates to a coronavirus infection diagnosis kit comprising the composition for diagnosis of coronavirus infection of the present invention.

In one embodiment, the kit may further comprise tools and/or reagents for collecting a biological sample from a subject or patient as well as tools and/or reagents for preparing genomic DNA, cDNA, RNA or protein from the sample. there is.

In the present invention, the term "corona virus infection diagnosis kit" means a kit including the composition for diagnosing corona virus of the present invention. Therefore, the expression “coronavirus infection diagnostic kit” can be used interchangeably or interchangeably with “coronavirus diagnostic composition”. As used herein, the term "diagnosis" refers to determining a subject's susceptibility to a specific disease or disorder, determining whether a subject currently has a specific disease or disorder, or suffering from a specific disease or disorder determining a subject's prognosis, or therametrics (eg, monitoring a subject's condition to provide information about treatment efficacy).

The ACE-2 variant of the present invention competitively inhibits the binding of the corona virus spike protein to the ACE-2 protein in the human body, and can be used as a therapeutic agent and a diagnostic reagent capable of detecting the virus.

In one aspect, the present invention comprises the steps of contacting a biological sample isolated from a subject with an ACE-2 variant of the present invention; Checking the binding level of the ACE-2 variant and the spike protein; And it relates to an information providing method for diagnosing coronavirus infection, comprising the step of comparing the binding level of the ACE-2 variant and spike protein in a normal control sample.

In one embodiment, the test subject A step of determining that the coronavirus is infected may be further included.

As used herein, the term "sample (sample)" refers to a biological sample obtained from a subject or patient. The source of the biological sample may be solid tissue from a fresh, frozen and/or preserved organ or tissue sample or biopsy or aspirate; blood or any blood component; It may be a cell at any point in the subject's pregnancy or development.

In one aspect, the present invention relates to a method for specific detection of coronavirus, comprising the steps of contacting an ACE-2 variant with a sample and detecting the binding of the ACE-2 variant with a coronavirus spike protein.

In one aspect, the present invention comprises the steps of culturing a host cell containing a vector containing a nucleic acid molecule encoding the ACE-2 variant of the present invention; and recovering the ACE-2 mutant expressed by the host cell, and a method for producing an ACE-2 mutant with increased binding ability of coronavirus or coronavirus to a spike protein.

The present invention will be described in more detail through the following examples. However, the following examples are only for specifying the content of the present invention, and the present invention is not limited thereto.

[Example 1] Preparation of sACE-2 (sACE2) protein

1. Cloning the pET15b-sACE2 Plasmid

To clone sACE2, part of the sACE2 gene was amplified from mRNA of human cells using a primer set (SEQ ID NO: 21 and SEQ ID NO: 22), and both ends were cut using Nde I and Xho I restriction enzymes, After binding to the pET15b plasmid (see FIG. 1 ) previously digested with Nde I and Xho I, the cloned plasmid pET15b-sACE2-R7 was obtained by transforming E. coli DH5a competent cells. This was amplified again with another primer set (SEQ ID NO: 23 and SEQ ID NO: 24), digested with Nde I and Hind III restriction enzymes, and then ligated to pET15b-sACE2-R7 from which short fragments were removed by previously digesting with Nde I and Hind III. After that, E.coli DH5a competent cells were transformed and pET15b-sACE2-m7 having 4 amino acid mutations was cloned. To clone sACE2-m4 (sACE2 having the amino acid sequence of SEQ ID NO: 3), primer sets of SEQ ID NOs: 15 and 16 were used, and to clone sACE2-m5 (SEQ ID NO: 4), SEQ ID NOs: 17 and 18 were used. A primer set was used, and in order to clone sACE2-m6 (SEQ ID NO: 5), amplification was carried out in pET15b-sACE2-m7 using SEQ ID NOs: 19 and 20, and the resulting amplification product was subjected to Nde I and Xho I restriction enzymes, respectively. , and cloned into the pET15b plasmid previously digested with Nde I and Xho I. In order to insert the Foldon sequence into each cloned plasmid, in the case of sACE2-m4, first PCR was performed using primer sets of SEQ ID NOs: 25 and 26, and then second PCR was performed using primer sets of SEQ ID NOs: 27 and 28 was cut with Nde I and Xho I restriction enzymes, and then cloned by binding to pET15b cut with Nde I and Xho I. In addition, primer sets of SEQ ID NOs: 29 and 30 were used to insert the foldon sequence into sACE2-m5. After the first PCR was performed using SEQ ID NOs: 31 and 32, the second PCR was performed again using the primer set of SEQ ID NOs: 31 and 32, and pET15b digested with Nde I and Xho I restriction enzymes was then digested with Nde I and Xho I. Combined and cloned.

In addition, in order to insert the foldon sequence into sACE2-m6, first PCR was performed using SEQ ID NOs: 33 and 34, and then second PCR was performed using primer sets of SEQ ID NO: 35 and SEQ ID NO: 36, Nde I, After digestion with Xho I restriction enzyme, it was cloned by binding to pET15b digested with Nde I and Xho I.

In addition, in order to insert the foldon sequence into sACE2-m7, first PCR was performed using primer sets of SEQ ID NOs: 37 and 38, followed by second PCR using primer sets of SEQ ID NOs: 39 and 40, and Nde I , Xho I was digested with a restriction enzyme, and then cloned by binding to pET15b digested with Nde I and Xho I (see FIGS. 1 and 2).

2. Cultivation of E.Coli strains transformed with sACE2 plasmids

For protein expression, each cloned pET15b plasmid was transformed into E.coli BL21 (DE3) strain and cultured in Luria Bertani solid medium containing 1 ug/mL ampicillin to obtain individual colonies. Individual colonies were inoculated into 1 mL of terrific broth medium containing ampicillin, cultured for more than 12 hours, and then inoculated into 200 mL of terrific broth containing ampicillin, incubated for more than 8 hours at 23°C and 250 rpm, with an OD of 600 nm After confirming the state of 0.6 or higher, IPTG was added to a final concentration of 1 mM.

3. Isolation of sACE2 protein

에서 사용시까지 보관하였다Cell pellets were obtained by centrifuging the cultured E. coli at 7000 rpm for 10 minutes or more in a centrifuge. On ice, 5 mL of cell lysis solution containing 100mM NaH2PO4, 10mM Tris Cl, and 8M Urea was added to the cells and kept at pH 8 for 15 minutes or longer, followed by shaking and reacting at room temperature for 1 hour. This was centrifuged at 12000 rpm for 30 minutes in a centrifuge to remove cell debris. 1 mL of 50% Ni-NTA was added to 4 mL of the obtained cell lysate and mixed while shaking at room temperature for 60 minutes. And this was carefully poured into the empty column with the bottom lid closed. After removing the bottom lid again and allowing the solution to flow, the column was washed with 4mL of a washing solution containing 100mM NaH2PO4 and 10mM Tris and adjusted to pH 6.3 in a state where the solution in the column had all flowed out. After washing, 0.5mL each of elution solution 1 containing 100mM NaH2PO4, 10mM Tris-Cl, and 8M Urea and adjusted to pH 5.9 was added to elute the protein bound to the column four times. In order to completely elute the remaining proteins that were not eluted and still remained in the column, 0.5 mL of elution solution 2 containing 100 mM NaH2PO4, 10 mM Tris-Cl, and 8 M Urea and adjusted to pH 4.5 was added and eluted four times. . Each protein obtained was -80

stored until use

Example 2. Confirmation of spike protein binding ability of sACE2

In order to verify the binding of sACE2 and coronavirus spike protein, binding inhibitory ability was confirmed using a reporter human kidney cell line. A similar lentivirus artificially made to express green fluorescent protein while having a coronavirus spike protein on its surface infects a human kidney cell line overexpressing the Ace2 receptor and causes the infected cell line to express green fluorescence. However, if the same experiment is performed after pre-treating the similar lentivirus with neutralizing antibody for coronavirus, the surface spike protein of the similar lentivirus is incapacitated by the neutralizing antibody, so that it no longer has infectivity. In the same principle, when sACE has binding ability to spike protein, the infectivity of the similar lentivirus is weakened, so the binding ability of sACE to spike protein can be tested according to the degree of green fluorescence in the reporter cell line, using the above method. , The binding ability of sACE2 protein and spike protein was confirmed.

The results of this are shown in Figure 3 (confirmation of lentivirus infection expressing spike protein in ACE2-overexpressing cell line) and Figure 4 (coronavirus spike-like lentivirus pre-treated with saline, vaccinated serum or sACE2, ACE2-overexpressing human kidney cell line The result of infection with ) is shown in .

As shown in FIG. 3, normal human kidney cell lines are hardly infected with corona virus spike-like lentivirus, but human kidney cell lines made to overexpress ACE2 show an increased degree of infection.

As shown in Figure 4, compared to the case of pre-treatment with saline as a control, the green fluorescence was completely removed when the vaccine recipient serum was pre-treated, suggesting that neutralizing antibodies were present in the serum to completely block the infectivity of the similar lentivirus. In the case of sACE2, it was confirmed that green fluorescence significantly decreased compared to the saline-treated group, confirming that sACE2 binds to the spike protein.

<110> LEE <120> ACE2 VARIANTS AND USE THEREOF <130> P-1 <160> 40 <170> KoPatentIn 3.0 <210> 1 <211> 367 <212> PRT <213> Artificial Sequence <220> <223> sACE2-wild type <400> 1 Met Gln Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe 1 5 10 15 Asn His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp 20 25 30 Asn Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn 35 40 45 Ala Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala 50 55 60 Gln Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln 65 70 75 80 Leu Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys 85 90 95 Ser Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser 100 105 110 Thr Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu 115 120 125 Glu Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu 130 135 140 Arg Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu 145 150 155 160 Arg Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg 165 170 175 Ala Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu 180 185 190 Val Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu 195 200 205 Asp Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu 210 215 220 His Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile 225 230 235 240 Ser Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly 245 250 255 Arg Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys 260 265 270 Pro Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala 275 280 285 Gln Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu 290 295 300 Pro Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro 305 310 315 320 Gly Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly 325 330 335 Lys Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp 340 345 350 Phe Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met 355 360 365 < 210> 2 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> peptide for inducing ACE2 to form trimer <400> 2 Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val 1 5 10 15 Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu 20 25 <210> 3 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> sACE2-m4 <400> 3 Met Gln Ser Thr Ile Glu Glu Gln Ala Lys Tyr Phe Leu Asp Lys Phe 1 5 10 15 Asn His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp 20 25 30 Asn Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn 35 40 45 Ala Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Thr Ala 50 55 60 Gln Met Tyr Pro Leu Gln Glu Ile Gln Gln Leu Gln Val Lys Leu Gln 65 70 75 80 Leu Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Gly Ser Gly Tyr 85 90 95 Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys Asp Gly 100 105 110 Glu Trp Val Leu Leu Ser Thr Phe Leu 115 120 <210> 4 <211 > 129 <212> PRT <213> Artificial Sequence <220> <223> sACE2-m5 <400> 4 Met Gln Ser Thr Ile Glu Glu Gln Ala Lys Tyr Phe Leu Asp Lys Phe 1 5 10 15 Asn His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp 20 25 30 Asn Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn 35 40 45 Ala Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Thr Ala 50 55 60 Gln Met Tyr Pro Leu Gln Glu Ile Gln Gln Leu Gln Val Lys Leu Gln 65 70 75 80 Leu Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys 85 90 95 Ser Lys Arg Leu Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly 100 105 110 Gln Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe 115 120 125 Leu <210> 5 <211> 177 <212> PRT <213> Artificial Sequence <220> < 223> sACE2-m6 <400> 5 Met Gln Ser Thr Ile Glu Glu Gln Ala Lys Tyr Phe Leu Asp Lys Phe 1 5 10 15 Asn His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp 20 25 30 Asn Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn 35 40 45 Ala Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Thr Ala 50 55 60 Gln Met Tyr Pro Leu Gln Glu Ile Gln Gln Leu Gln Val Lys Leu Gln 65 70 75 80 Leu Gln Ala Leu Gln Gln Asn Gly Ser Ser Ser Val Leu Ser Glu Asp Lys 85 90 95 Ser Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser 100 105 110 Thr Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu 115 120 125 Glu Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu 130 135 140 Arg Leu Trp Ala Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly 145 150 155 160 Gln Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe 165 170 175 Leu <210> 6 <211> 396 <212> PRT <213> Artificial Sequence <220> <223> sACE2-m7 <400 > 6 Met Gln Ser Thr Ile Glu Glu Gln Ala Lys Tyr Phe Leu Asp Lys Phe 1 5 10 15 Asn His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp 20 25 30 Asn Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn 35 40 45 Ala Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Thr Ala 50 55 60 Gln Met Tyr Pro Leu Gln Glu Ile Gln Gln Leu Gln Val Lys Leu Gln 65 70 75 80 Leu Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys 85 90 95 Ser Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser 100 105 110 Thr Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu 115 120 125 Glu Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu 130 135 140 Arg Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu 145 150 155 160 Arg Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg 165 170 175 Ala Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu 180 185 190 Val Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu 195 200 205 Asp Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu 210 215 220 His Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile 225 230 235 240 Ser Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly 245 250 255 Arg Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys 260 265 270 Pro Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala 275 280 285 Gln Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu 290 295 300 Pro Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro 305 310 315 320 Gly Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly 325 330 335 Lys Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp 340 345 350 Phe Leu Thr Ala His Glu Met Gly His Ile Gln Tyr Asp Met Gly 355 360 365 Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg 370 375 380 Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu 385 390 395 <210> 7 <211> 366 <212> DNA <213> Artificial Sequence <220> <223 > DNA sequence coding for sACE2-m4 <400> 7 atgcagtcca ccattgagga acaggccaag tattttttgg acaagtttaa ccacgaagcc 60 gaagacctgt tctatcaaag ttcacttgct tcttggaatt ataacaccaa tattactgaa 120 gagaatgtcc aaaacatgaa taatgctggg gacaaatggt ctgccttttt aaaggaacag 180 tccacaaccg cccaaatgta tccactacaa gaaattcagc agctccaggt caagcttcag 240 ctgcaggctc ttcagcaaaa tgggtcttca gtgctcggca gcggctatat tccggaagcc 300 ccgcgcga tg gccaggccta tgtgcgcaaa gatggcgaat gggtgctgct gagcaccttt 360 ctgtaa 366 <210> 8 <211> 390 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence coding for sACE2-m5 <400> 8 atgcagtcca ccattgagga acaggccaag tattttttgg acaagt ttaa ccacgaagcc 60 gaagacctgt tctatcaaag ttcacttgct tcttggaatt ataacaccaa tattactgaa 120 gagaatgtcc aaaacatgaa taatgctggg gacaaatggt ctgccttttt aaaggaacag 180 tccacaaccg cccaaatgta tccactacaa gaaattcagc agctccaggt caagcttcag 240 ctgcaggctc ttcag caaaa tgggtcttca gtgctctcag aagacaagag caaacggttg 300 ggcagcggct atattccgga agccccgcgc gatggccagg cctatgtgcg caaagatggc 360 gaatgggtgc tgctgagcac ctttctgtaa 390 <210> 9 <211> 534 < 212> DNA <213 > Artificial Sequence <220> <223> DNA sequence coding for sACE2-m6 <400> 9 atgcagtcca ccattgagga acaggccaag tattttttgg acaagtttaa ccacgaagcc 60 gaagacctgt tctatcaaag ttcacttgct tcttggaatt ataacaccaa tattactgaa 120 gagaatgt cc aaaacatgaa taatgctggg gacaaatggt ctgccttttt aaaggaacag 180 tccacaaccg cccaaatgta tccactacaa gaaattcagc agctccaggt caagcttcag 240 ctgcaggctc ttcagcaaaa tgggtcttca gtgctctcag aagacaagag caaacggttg 300 aacacaattc taaatacaat gagcaccatc tacagtactg gaaaagtttg taacccagat 360 aatccacaag aatgcttatt acttgaacca ggtttgaatg aaataatggc aaacagttta 420 gactacaatg agaggctctg gg ctggcagc ggctatattc cggaagcccc gcgcgatggc 480 caggcctatg tgcgcaaaga tggcgaatgg gtgctgctga gcacctttct gtaa 534 <210> 10 <211> 1101 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence coding for sACE2-m7 <400> 10 atgcagtcca ccattgagga acaggccaag acatttttgg acaagtttaa ccacgaagcc 60 gaagacctgt tctatcaaag ttcacttgct tcttggaatt ataacaccaa tattactgaa 120 gagaatgtcc aaaacat gaa taatgctggg gacaaatggt ctgccttttt aaaggaacag 180 tccacacttg cccaaatgta tccactacaa gaaattcaga atctcacagt caagcttcag 240 ctgcaggctc ttcagcaaaa tgggtcttca gtgctctcag aagacaagag caaacggttg 300 aacacaattc taaatacaat gagcaccatc tacagtactg gaaaagtttg taacccagat 360 aatccacaag aatgcttatt acttgaacca ggtttgaatg aaataatggc aaacagttta 420 gactacaatg agaggctctg ggcttgggaa agctggagat ctgaggtcgg caagcagctg 480 aggccattat atgaagagta tgtggtcttg aaaaatgaga tggcaagagc aaatcattat 540 gaggactatg gggattattg gagaggagac tatgaagtaa atggggtaga tggctatgac 600 tacagccgcg gccagttgat tgaagatgtg gacacatacct ttgaagaga t taaaccatta 660 tatgaacatc ttcatgccta tgtgagggca aagttgatga atgcctatcc ttcctatatc 720 agtccaattg gatgcctccc tgctcatttg cttggtgata tgtggggtag attttggaca 780 aatctgtact ctttgacagt tccctttgga cagaaaccaa acatagatgt tactgatgca 840 atggtggacc aggcctggga tgcacagaga atattcaagg aggccgagaa gt tctttgta 900 tctgttggtc ttcctaatat gactcaagga ttctgggaaa attccatgct aacggaccca 960 ggaaatgttc agaaagcagt ctgccatccc acagcttggg acctggggaa gggcgacttc 1020 aggatcctta tgtgcacaaa ggtgacaatg gacgactt cc tgacagctca tcatgagatg 1080 gggcatatcc agtatgatat g 1101 <210> 11 <211> 6206 <212> DNA <213> Artificial Sequence <220> <223> pET15b-m4F <400> 11 agatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa 60 ttcccctcta gaaataattt tgtttaactt taagaaggag atataccat g ggcagcagcc 120 atcatcatca tcatcacagc agcggcctgg tgccgcgcgg cagccatatg cagtccacca 180 ttgaggaaca ggccaagtat tttttggaca agtttaacca cgaagccgaa gacctgttct 240 atcaaagttc acttgcttct tggaattata acaccaatat tactgaagag aatgtccaaa 300 acatgaataa tgctggggac aaatggtctg cctttttaaa ggaacagtcc acaaccgccc 360 aaatgtatcc actacaa attcagcagc tccaggtcaa gcttcagctg caggctcttc 420 agcaaaatgg gtcttcagtg ctcggcagcg gctatattcc ggaagccccg cgcgatggcc 480 aggcctatgt gcgcaaagat ggcgaatggg tgctgctgag cacctttctg taactcgagc 540 ct ggctagcc tgcggctcaa agccaagcag cacgcctctt tcagctaccc cgccgtgccc 600 gggccgccgc cggccgctaa ccttagtccc tgccagtacg ccgtggaacg gccagtgtga 660 accgcaggtc tgtggatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg 720 ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt 780 ttttgctgaa agg aggaact atatccggat atcccgcaag aggcccggca gtaccggcat 840 aaccaagcct atgcctacag catccagggt gacggtgccg aggatgacga tgagcgcatt 900 gttagatttc atacacggtg cctgactgcg ttagcaattt aactgtgata aactaccgca 960 ttaaagctta tcgatg ataa gctgtcaaac atgagaattc ttgaagacga aagggcctcg 1020 tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg 1080 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa 1140 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga 1200 agagtatgag tattcacat ttcc gtgtcg cccttattcc cttttttgcg gcattttgcc 1260 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg 1320 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc 138 0 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat 1440 tatcccgtgt tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg 1500 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag 1560 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa 1620 cgatcggagg accgaaggag ctaaccgctt ttttgcaca a catgggggat catgtaactc 1680 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca 1740 cgatgcctgc agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc 1800 tagcttcccg gcaacaatta atagactgga tg gaggcgga taaagttgca ggaccacttc 1860 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg 21 00 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc 2160 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa 2220 agatcaaagg atcttcttga gatccttttt ttctgcgcgt a atctgctgc ttgcaaacaa 2280 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc 2340 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt 2400 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc 2460 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gact caagac 2520 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca 2580 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg 2640 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggca gg gtcggaacag 2700 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt 2760 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat 2820 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc 2880 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtatta cc gcctttgagt 2940 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag 3000 cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca 3060 tatatggtgc actctcagta caatctgct c tgatgccgca tagttaagcc agtatacact 3120 ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac 3180 gcgccctgac gggcttgtct a gctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta 3420 agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg 3480 ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac t gatgatgaa 3540 catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac 3600 cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca 3660 cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac 3720 ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag 3780 gtcgcagacg tt ttgcagca gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc 3840 tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc 3900 atgcgcaccc gtggccagga cccaacgctg cccgagatgc gccgcgtgcg gctg ctggag 3960 atggcggacg cgatggatat gttctgccaa gggttggttt gcgcattcac agttctccgc 4020 aagaattgat tggctccaat tcttggagtg gtgaatccgt tagcgaggtg ccgccggctt 4080 ccattcaggt cgaggtggcc cggctccatg caccgcgacg caacgcgggg aggcagacaa 4140 ggtatagggc ggcgcctaca atccatgcca acccgttcca tgtgctcgcc gaggcggcat 4200 aaatcgccgt gacgatcagc ggtccagtga tcgaagttag gctggtaaga gccgcgagcg 4260 atccttgaag ctgtccctga tggtcgtcat ctacctgcct ggacagcatg gcctgcaacg 4320 cgggcatccc gatgccgccg gaagcgagaa gaatcataat ggggaagg at ccagcctc 4380 gcgtcgcgaa cgccagcaag acgtagccca gcgcgtcggc cgccatgccg gcgataatgg 4440 cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt 4500 gcaagattcc gaataccgca agcgacaggc cgatcat cgt cgcgctccag cgaaagcggt 4560 cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga 4620 agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg 4680 ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta 4740 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc 4800 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt 4860 tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag 4920 agttgcagca agcggtccac gctgg tttgc cccagcaggc gaaaatcctg tttgatggtg 4980 gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata 5040 tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga 5100 tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt 5160 tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg 52 20 cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg 5280 cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc 5340 gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga gacat caaga 5400 aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc 5460 ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta 5520 caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg 5580 gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg 5640 gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg 5700 taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg 5760 gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc t gcgacatcg 5820 tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat 5880 gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt 5940 atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc 6000 cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc 6060 caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc 6120 atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc 6180 cacgatgcgt ccggcgtaga ggatcg 6206 <210> 12 <211> 6230 <212> DNA <213> Artificial Sequence <220> <223> pET15b-m5F <400> 12 agatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa 60 ttcccctcta gaaataattt tgtttaactt taagaaggag atataccatg ggcagcagcc 120 atcatcatca tcatcacagc a gcggcctgg tgccgcgcgg cagccatatg cagtccacca 180 ttgaggaaca ggccaagtat tttttggaca agtttaacca cgaagccgaa gacctgttct 240 atcaaagttc acttgcttct tggaattata acaccaatat tactgaagag aatgtccaaa 300 acatgaataa tgctggggac aaatggtctg cctttttaaa ggaacagtcc acaaccgccc 360 aaatgtatcc actacaagaa attcagcagc tccaggtcaa gcttcagctg caggctcttc 420 agcaaaatgg gtcttcagtg ctctcagaag acaagagcaa acggttgggc agcggct ata 480 ttccggaagc cccgcgcgat ggccaggcct atgtgcgcaa agatggcgaa tgggtgctgc 540 tgagcacctt tctgtaactc gagcctggct agcctgcggc tcaaagccaa gcagcacgcc 600 tctttcagct accccgccgt gcccgggccg ccgccggccg ctaaccttag tccctgccag 660 tacgccgtgg aacggccagt gtgaaccgca ggtctgtgga tccggctgct aacaaagccc 720 gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg 780 cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggatatcccg 840 caagaggccc ggcagtaccg gcataaccaa gcctatgcct acagcatcca gggtgacggt 900 gccgaggatg acgatgagcg cattgttaga tttcatacac ggtgcctgac tgcgttagca 960 atttaactgt gataaactac cgcattaaag cttatcgatg ataagctgtc aaacatgaga 1020 attcttgaag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa 10 80 taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt 1140 gtttatttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa 1200 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta 1260 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag 13 20 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca 1380 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta 1440 aagttctgct atgtggcgcg gtattatccc gtgttgacgc cgggca agag caactcggtc 1500 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc 1560 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca 1620 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc 1680 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca 1740 taccaaacga cgagc gtgac accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac 1800 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg 1860 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg 1920 ataaatct gg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg 1980 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac 2040 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc 2100 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct 2160 aggtgaagat cctttttgat aatctcatga ccaaaatcc c ttaacgtgag ttttcgttcc 2220 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc 2280 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg 2340 atca agagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa 2400 atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc 2460 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt 2520 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa 2580 cggggggttc gtgcacacag cccagcttgg agc gaacgac ctacaccgaa ctgagatacc 2640 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc 2700 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct 2760 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat 2820 gctcgtcagg ggggcggagc ctatgggaaaa acgccagcaa cgcggccttt ttacggttcc agcg cctgat gcggtatttt ctccttacgc 3060 atctgtgcgg tatttcacac cgcatatatg gtgcactctc agtacaatct gctctgatgc 3120 cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc 3180 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct 3240 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca 3300 ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag 3360 atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg 3420 cttctgataa agcgggccat gttaagggcg gttttttcct gtt tggtcac tgatgcctcc 3480 gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc 3540 acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa 3600 ctggcggtat ggatgcggcg ggaccagaga aa aatcactc agggtcaatg ccagcgcttc 3660 gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg 3720 aacataatgg aa ccccgcca gcctagccgg 3900 gtcctcaacg acaggagcac gatcatgcgc acccgtggcc aggaccaac gctgcccgag 3960 atgcgccgcg tgcggctgct ggagatggcg gacgcgatgg atatgttctg ccaagggttg 4020 gtttgcgcat tcacagttct cc gcaagaat tgattggctc caattcttgg agtggtgaat 4080 ccgttagcga ggtgccgccg gcttccattc aggtcgaggt ggcccggctc catgcaccgc 4140 gacgcaacgc ggggaggcag acaaggtata gggcggcgcc tacaatccat gccaacccgt 4200 tccatgtgct cgccgaggcg gcataaatcg ccgtgacgat cagcggtcca gtgatcgaag 4260 ttaggctggt aagagccgcg agcgatcctt gaagctgtcc ctgatggtcg tcatctacct 4320 gcc tggacag catggcctgc aacgcgggca tcccgatgcc gccggaagcg agaagaatca 4380 taatggggaa ggccatccag cctcgcgtcg cgaacgccag caagacgtag cccagcgcgt 4440 cggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg gcggga ccag 4500 tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca 4560 tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct 4620 gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc 4680 gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg 4740 gtgcctaatg agtga gctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt 4800 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt 4860 tgcgtattgg gcgccagggt ggttttctt ttcaccagtg agacgggcaa cag ctgattg 4920 cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc 4980 aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta 5040 tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg 5100 cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc 5160 tcattcagca tt tgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt 5220 tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga 5280 cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgc g 5340 accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg 5400 ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca 5460 gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg 5520 agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc 5580 accacgctgg cacccagttg atcggcgcga gatttaat cg ccgcgacaat ttgcgacggc 5640 gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt 5700 tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc 5760 cgcgttt tcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag 5820 acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat 6000 gttgaggccg ttgagcaccg ccgcc gcaag gaatggtgca tgcaaggaga tggcgcccaa 6060 cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc 6120 gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc 6180 acctgtgg cg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg 6230 <210> 13 <211> 6374 <212> DNA <213> Artificial Sequence <220> <223> pET15b-m6F <400> 13 agatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa 60 ttcccctcta gaaataattt tgtttaactt taagaaggag atataccatg ggcagcagcc 120 at catcatca tcatcacagc agcggcctgg tgccgcgcgg cagccatatg cagtccacca 180 ttgaggaaca ggccaagtat tttttggaca agtttaacca cgaagccgaa gacctgttct 240 atcaaagttc acttgcttct tggaattata acaccaatat tactgaagag aatgtccaaa 300 acatgaataa tgctggggac aaatggtctg cctttttaaa ggaacagtcc acaaccgccc 360 aaatgtatcc actacaagaa attcagcagc tccaggtcaa gcttcagctg caggctcttc 420 agcaaaatgg gtcttcagtg ctctcagaag acaagagcaa acggttgaac acaattctaa 480 atacaatgag caccatctac agtactggaa aagtttgtaa cccagataat ccacaagaat 540 gcttattact tgaaccaggt ttgaatgaaa taatggcaaa cagtttagac tacaatgaga 60 0 ggctctgggc tggcagcggc tatattccgg aagccccgcg cgatggccag gcctatgtgc 660 gcaaagatgg cgaatgggtg ctgctgagca cctttctgta actcgagcct ggctagcctg 720 cggctcaaag ccaagcagca cgcctctttc agctaccccg ccgtgcccgg gccgccgccg 780 gccgctaacc ttagtccctg ccagtacgcc gtggaacggc cagtgtgaac cgcaggtctg 840 tggatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc 900 aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag 960 gaggaactat atccggatat cccgcaagag gcccggcagt accggcataa ccaagccta t 1020 gcctacagca tccagggtga cggtgccgag gatgacgatg agcgcattgt tagatttcat 1080 acacggtgcc tgactgcgtt agcaatttaa ctgtgataaa ctaccgcatt aaagcttatc 1140 gatgataagc tgtcaaacat gagaattctt gaagacgaaa gggcctcgtg atacgcctat 1200 ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc actttcggg 1260 gaaatggg cg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc 1320 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta 1380 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtt tttg 1440 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg 1500 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac 1560 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg 1620 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt 1680 actcacc agt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg 1740 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac 1800 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt 18 60 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgcag 1920 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc 1980 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc 2040 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta 2100 tcattgcagc actggggcca gatggta aga tcctttt tgataatctc atgaccaaaa 2340 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat 2400 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc 2460 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg 2520 gcttcagcag agcg cagata ccaaatactg tccttctagt gtagccgtag ttaggccacc 2580 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg 2640 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg 2700 ataaggcg ca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa 2760 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg 2820 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga 2880 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct 2940 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca 3000 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc 3060 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg 3120 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc 3180 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata tatggtgcac 3240 tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta 3300 cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg 3360 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg 3420 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca 3480 gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt 3540 ttctccagaa gcgttaatgt ctggcttctg ataaagcggg c catgttaag ggcggttttt 3600 tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata 3660 ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta 3720 ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc 3780 actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag 3840 cag catcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc 3900 agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt 3960 ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta 4020 aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat gcgcacccgt 4080 ggccaggacc caacgctgcc cgagatgcgc cgcgtgcggc tgctggagat ggcggacgcg 4140 atggatatgt tctgccaagg gttggtttgc gcattcacag ttctccgcaa gaattgattg 4200 gctccaattc ttggagtggt gaatccgtta gcgaggtgcc gccggcttcc attcaggtc g 4260 aggtggcccg gctccatgca ccgcgacgca acgcggggag gcagacaagg tatagggcgg 4320 cgcctacaat ccatgccaac ccgttccatg tgctcgccga ggcggcataa atcgccgtga 4380 cgatcagcgg tccagtgatc gaagttaggc tggtaagagc cg cgagcgat ccttgaagct 4440 gtccctgatg gtcgtcatct acctgcctgg acagcatggc ctgcaacgcg ggcatcccga 4500 tgccgccgga agcgagaaga atcataatgg ggaaggccat ccagcctcgc gtcgcgaacg 4560 ccagcaagac gtagcccagc gcgtcggccg ccatgccggc gataatggcc tgcttctcgc 4620 cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc aagattccga 4680 ataccgcaag c gacaggccg atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa 4740 tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag acagtcataa 4800 gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc 48 60 tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt 4920 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg 4980 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc 5040 agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag 5100 cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg 5160 atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg 5220 cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttgg caacc 5280 agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac 5340 atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat 5400 ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc 5460 gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca 5520 tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa taac ctggcaccca gttgatcggc gcgagattta 5760 atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc 5820 agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc 5880 gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc 5940 acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta ta acgttact 6000 ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga 6060 aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctccccttat gcgactcctg 6120 cattaggaag cagcccagta ggtaggag gccgttgag c accgccgccg caaggaatgg 6180 tgcatgcaag gagatggcgc ccaacagtcc cccggccacg gggcctgcca ccatacccac 6240 gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc 6300 ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc 6360 ggcgtagagg atcg 6374 <210> 14 <211> 7031 <212> DNA <213> Artificial Sequence <220> <223> pET15b-m7F <400> 14 agatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa 60 ttcccctcta gaaataattt tgtttaactt taagaaggag atataccatg ggcagcagcc 120 atcatcatca tcatcacagc agcggcctgg tgccgcgcgg cagccatatg cagtccacca 180 ttgaggaaca ggccaagtat tttttggaca agtttaacca cgaag ccgaa gacctgttct 240 atcaaagttc acttgcttct tggaattata acaccaatat tactgaagag aatgtccaaa 300 acatgaataa tgctggggac aaatggtctg cctttttaaa ggaacagtcc acaaccgccc 360 aaatgtatcc actacaagaa attcagcagc tccaggtcaa gctt cagctg caggctcttc 420 agcaaaatgg gtcttcagtg ctctcagaag acaagagcaa acggttgaac acaattctaa 480 atacaatgag caccatctac agtactggaa aagtttgtaa cccagataat ccacaagaat 540 gcttattact tgaaccaggt ttgaatgaaa taatggcaaa cagtttagac tacaatgaga 600 ggctctgggc ttgggaaagc tggagatctg aggtcggcaa gcagctgagg ccattata tg 660 aagagtatgt ggtcttgaaa aatgagatgg caagagcaaa tcattatgag gactatgggg 720 attattggag aggagactat gaagtaaatg gggtagatgg ctatgactac agccgcggcc 780 agttgattga agatgtggaa catacctttg aagagattaa accattatat gaacatcttc 840 atgcctatgt gagggcaaag ttgatgaatg cctatccttc ctatatcagt ccaattggat 900 gcctccctgc tcatttgctt ggtgatatgt ggggtagatt ttggacaaat ctgtactctt 960 tgacagttcc ctttggacag aaaccaaaca tagatgttac tgatgcaatg gtggaccagg 1020 cctgggatgc acagagaata ttcaaggagg ccgagaagtt ctttgtatct gt tggtcttc 1080 ctaatatgac tcaaggattc tgggaaaatt ccatgctaac ggacccagga aatgttcaga 1140 aagcagtctg ccatcccaca gcttgggacc tggggaaggg cgacttcagg atccttatgt 1200 gcacaaaggt gacaatggac gacttcctga cagctcatca tgagatgggg catatccagt 1260 atgatatggg cagcggctat attccggaag ccccgcgcga tggccaggcc tatgtgcgca 1320 aagatggcga atgggtgctg ctgagcacct ttctgtaact cgagcctggc tagcctgcgg 1380 ctcaaagcca agcagcacgc ctctttcagc taccccgccg tgcccgggcc gccgccggcc 1440 gctaacctta gtccctgcca gtacgccgtg gaacggccag tgtgaaccgc agg tctgtgg 1500 atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat 1560 aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag 1620 gaactatatc cggatatccc gcaagaggcc cggcagtacc ggcata acca agcctatgcc 1680 tacagcatcc agggtgacgg tgccgaggat gacgatgagc gcattgttag atttcataca 1740 cggtgcctga ctgcgttagc aatttaactg tgataaacta ccgcattaaa gcttatcgat 1800 gataagctgt caaacatgag aattcttgaa gacgaaaggg cctcgtgata cgcctatttt 1860 tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa 1920 atgtgcg cgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca 1980 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc 2040 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gttttt gctc 2100 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt 2160 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt 2220 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtgttgacg 2280 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact 2340 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg 2400 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga 2460 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg 2520 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgcagcaa 2580 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac 2640 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc 2700 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca 2760 ttgcagcact ggggccagat gg taagccct cccgtatcgt agttatctac acgacgggga 2820 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gatagtgcc tcactgatta 2880 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc 2940 atttttaatt taaaaggatc taggtga aga tcctttttga taatctcatg accaaaatcc 3000 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt 3060 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac 3120 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct 3180 tcagcagagc gcagatacca a atactgtcc ttctagtgta gccgtagtta ggccaccact 3240 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg 3300 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata 3360 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga 3420 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag 3480 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg 3540 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac 3600 ttgagcgtcg atttttgtga tgctcgtcag ggggg cggag cctatggaaa aacgccagca 3660 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg 3720 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc 3780 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga 3840 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatatat ggtgcactct 3900 cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt 3960 gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct 4020 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ct gcatgtgt 4080 cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg 4140 tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc 4200 tccagaagcg ttaatgtctg gcttctgata aagc gggcca tgttaagggc ggttttttcc 4260 tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg 4320 atgaaacgag agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg 4380 gaacgttgtg agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact 4440 cagggtcaat gccagcgctt cgttaataca gatgtaggtg ttccacaggg tagccagcag 4500 catcc tgcga tgcagatccg gaacataatg gtgcagggcg ctgacttccg cgtttccaga 4560 ctttacgaaa cacggaaacc gaagaccatt catgttgttg ctcaggtcgc agacgttttg 4620 cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt cattctgc ta accagtaagg 4680 caaccccgcc agcctagccg ggtcctcaac gacaggagca cgatcatgcg cacccgtggc 4740 caggacccaa cgctgcccga gatgcgccgc gtgcggctgc tggagatggc ggacgcgatg 4800 gatatgttct gccaagggtt ggtttgcgca ttcacagttc tccgcaagaa ttgattggct 4860 ccaattcttg gagtggtgaa tccgttagcg aggtgccgcc ggcttccatt caggt cgagg 4920 tggcccggct ccatgcaccg cgacgcaacg cggggaggca gacaaggtat agggcggcgc 4980 ctacaatcca tgccaacccg ttccatgtgc tcgccgaggc ggcataaatc gccgtgacga 5040 tcagcggtcc agtgatcgaa gttaggctgg taagagcc gc gagcgatcct tgaagctgtc 5100 cctgatggtc gtcatctacc tgcctggaca gcatggcctg caacgcgggc atcccgatgc 5160 cgccggaagc gagaagaatc ataatgggga aggccatcca gcctcgcgtc gcgaacgcca 5220 gcaagacgta gcccagcgcg tcggccgcca tgccggcgat aatggcctgc ttctcgccga 5280 aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata 5340 ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga 5400 cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg 5460 cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgg gttg aaggctctca 5520 agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc 5580 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc 5640 aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt 5700 gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg 5760 tccacgctgg tttgcc ccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata 5820 taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc 5880 agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc 5940 atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg 6000 gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta 6060 tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg 6120 atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg 6180 gagaaaataa tactgttgat gggtgtctgg t cagagacat caagaaataa cgccggaaca 6240 ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc 6300 agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg 6360 cttcgttcta ccatcg acac caccacgctg gcacccagtt gatcggcgcg agatttaatc 6420 gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc 6480 aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc 6540 atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg 6600 cgggaaacgg tctgataaga gacaccggca tactctg ggt tgaggcc gttgagcacc gccgccgcaa ggaatggtgc 6840 atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc 6900 gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc 6960 gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc 7020 gtagaggatc g 7031 <210> 15 <211> 65 <2 12> DNA <213> Artificial Sequence <220> <223> forward primer for m4 <400> 15 agaaagcata tgcagtccac cattgaggaa caggccaagt attttttgga caagtttaac 60 cacga 65 <210> 16 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for m4 <400> 16 ggaatatagc cgctgccgag cactgaagac ccattttg 38 <210> 17 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> forward primer for m5 <400> 17 agaaagcata tgcagtccac cattgaggaa caggccaagt attttttgga caagtttaac 60 cacga 65 <210> 18 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for m5 <400> 18 aaactcgaga ctagtttaca accgtttgct cttgtcttct 40 <210> 19 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> forward primer for m6 <400> 19 agaaagcata tgcagtccac cattgaggaa caggccaagt attttttgga caagtttaac 60 cacga 65 <210> 20 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for m6 <400> 20 aaaaactcga gactagttta agcccagagc ctctcattgt 40 <210> 21 < 211> 33 <212> DNA <213> Artificial Sequence <220> <223> forward primer for ACE-2 <400> 21 agaaagcata tgcagtccac cattgaggaa cag 33 <210> 22 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for ACE-2 <400> 22 aagagactcg agttacatat catactggat atgccccat 39 <210> 23 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> forward primer for m7 <400 > 23 agaaagcata tgcagtccac cattgaggaa caggccaagt attttttgga caagtttaac 60 cacga 65 <210> 24 <211> 80 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for m7 <400> 24 cagctgaagc ttgacctgga gct gctgaat ttcttgtagt ggatacattt gggcggttgt 60 ggactgttcc tttaaaaagg 80 <210> 25 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> forward primer for foldon sequence in M4 <400> 25 agaaagcata tgcagtccac cattgaggaa caggccaagt attttttgga caagtttaac 60 cacga 65 <210> 26 < 211> 38 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for foldon sequence in M4 <400> 26 ggaatatagc cgctgccgag cactgaagac ccattttg 38 <210> 27 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> second forward primer for foldon sequence in M4 <400> 27 agaaagcata tgcagtccac cattgaggaa caggccaagt attttttgga caagtttaac 60 cacga 65 <210> 28 <211> 99 <212> DNA <213> Artificial Sequence <220> <223 > second reverse primer for foldon sequence in M4 <400> 28 aaactcgagt tacagaaagg tgctcagcag cacccattcg ccatctttgc gcacataggc 60 ctggccatcg cgcggggctt ccggaatata gccgctgcc 99 <210> 29 <211> 65 <212> DNA <213> Artificial Sequence <2 20> <223> first forward primer for foldon sequence in M5 <400> 29 agaaagcata tgcagtccac cattgaggaa caggccaagt attttttgga caagtttaac 60 cacga 65 <210> 30 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> first reverse primer for foldon sequence in M5 <400> 30 ggaatatagc cgctgcccaa ccgtttgctc ttgtcttct 39 <210> 31 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> second forward primer for foldon sequence in M5 <400> 31 agaaagcata tgcagtccac cattgaggaa caggccaagt attttttgga caagtttaac 60 cacga 65 <210> 32 <211> 99 <212> DNA <213> Artificial Sequence <220> <223> second reverse primer for foldon sequence in M5 <400> 32 aaactcgagt tacagaaagg tgctcagcag cacccattcg ccatctttgc gcacataggc 60 ctggccatcg cgcggggct t ccggaatata gccgctgcc 99 <210> 33 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> first forward primer for foldon sequence in M6 <400> 33 agaaagcata tgcagtccac cattgaggaa caggccaagt attttttgga caagtttaac 60 cacga 65 <210> 34 <211 > 37 <212> DNA <213> Artificial Sequence <220> <223> first reverse primer for foldon sequence in M6 <400> 34 ggaatatagc cgctgccagc ccagagcctc tcattgt 37 <210> 35 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> second forward primer for foldon sequence in M6 <400> 35 agaaagcata tgcagtccac cattgaggaa caggccaagt attttttgga caagtttaac 60 cacga 65 <210> 36 <211> 99 <212> DNA <213> Artificial Sequence <220> <223 > second reverse primer for foldon sequence in M6 <400> 36 aaactcgagt tacagaaagg tgctcagcag cacccattcg ccatctttgc gcacataggc 60 ctggccatcg cgcggggctt ccggaatata gccgctgcc 99 <210> 37 <211> 65 <212> DNA <213> Artificial Sequence <2 20> <223> first forward primer for foldon sequence in m7 <400> 37 agaaagcata tgcagtccac cattgaggaa caggccaagt attttttgga caagtttaac 60 cacga 65 <210> 38 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> first reverse primer for foldon sequence in m7 <400> 38 ggaatatagc cgctgcccat atcatactgg atatgcccca 40 <210> 39 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> second forward primer for foldon sequence in m7 <400> 39 agaaagcata tgcagtccac cattgaggaa caggccaagt atttttt gga caagtttaac 60 cacga 65 <210> 40 <211> 99 <212> DNA <213> Artificial Sequence <220> <223> second reverse primer for foldon sequence in m7 <400> 40 aaactcgagt tacagaaagg tgctcagcag cacccattcg ccatctttgc gcacataggc 60ctggccatcg cgcggggctt ccggaatata gccgctgcc 99

Claims (17)

Wild type ACE-2 (((Angiotensin-converting enzyme 2) amino acid substitution of T11Y, L63T, N74Q and T76Q in the amino acid substitution of water-soluble coronavirus spike protein ACE-1 variant with increased binding ability . The ACE-2 variant according to claim 1, wherein the wild-type ACE-2 amino acid comprises the amino acid sequence of SEQ ID NO: 1. The ACE-2 mutant according to claim 1, wherein the signal peptide sequence is removed from the ACE-2 mutant. The ACE-2 variant according to claim 1, further comprising the sequence represented by SEQ ID NO: 2 at the carboxy-terminus of the wild-type ACE-2 amino acid sequence to form a trimer. The ACE-2 variant according to claim 1, wherein the ACE-2 variant is a sequence represented by any one of the sequences represented by SEQ ID NOs: 3 to 6. Nucleic acid encoding the ACE-2 variant of claim 1 The nucleic acid according to claim 6, wherein the nucleic acid is a sequence represented by any one of the sequences represented by SEQ ID NOs: 7 to 10. 7. The vector according to claim 6, comprising a nucleic acid molecule. A host cell comprising the vector according to claim 8. A composition for detecting coronavirus, comprising the ACE-1 variant of claim 1. The composition for detecting coronavirus according to claim 10, wherein the ACE-2 variant is labeled with one selected from the group consisting of chromogenic enzyme, radioactive isotope, chromopore, luminescent material and fluorescent material. A pharmaceutical composition for the treatment or prevention of coronavirus, comprising the ACE-2 variant of claim 1, the nucleic acid molecule of claim 6, or the vector of claim 8 as an active ingredient. The composition of claim 12, wherein the corona virus is the corona virus COV-2. A composition for diagnosis of coronavirus infection comprising the ACE-2 variant of claim 1. a) contacting the biological sample isolated from the subject with the ACE-2 variant of claim 1;
b) checking the binding level of the ACE-2 variant and the coronavirus spike protein; and
c) an information providing method for diagnosing coronavirus infection, comprising the step of comparing the binding level of the ACE-2 variant and the coronavirus spike protein in a normal control sample. A method for specific detection of coronavirus, comprising the steps of contacting a sample with the ACE-2 variant of claim 1 and detecting the binding of the ACE-2 variant with the spike protein of coronavirus. a) culturing a host cell containing a vector containing a nucleic acid molecule encoding the ACE-2 variant of claim 1; and
b) a method for producing an ACE-2 variant with increased binding force with the spike protein of coronavirus, comprising the step of recovering the ACE-2 variant expressed by the host cell.