WO2023043213A2

WO2023043213A2 - Angiotensin converting enzyme 2 receptor and use thereof

Info

Publication number: WO2023043213A2
Application number: PCT/KR2022/013772
Authority: WO
Inventors: 한장현; 서미영
Original assignee: 한장현; 서미영
Priority date: 2021-09-16
Filing date: 2022-09-15
Publication date: 2023-03-23
Also published as: WO2023043213A3

Abstract

The present invention relates to an ACE2 ectodomain-redesigned ACE2 variant comprising: i) an ACE variant, lacking the enzymatic activity of ACE2, for capturing virions of Sars CoV-2 or variants thereof and ii) an ACE2 receptor, having the enzymatic activity of ACE2, for detecting virions of Sars CoV-2 or variants thereof, and a method for detecting virions of Sars CoV-2 or its variants by using same. Compared to conventional antigen and antibody diagnostic kits that detect past viral infections by using non-structural proteins of viruses and cannot distinguish past inactive viral infections from currently active viral infections, the present invention excludes causative substances that may come from past infection and inactive infection history in light of the feature wherein it detects virions of Sars CoV-2 or its variants that are currently active. In addition, in the present invention, the ACE2 variants were polymerized to increase the affinity for the virions by up to thousands of times, compared to the conventional ACE2, and the number of ACE2 molecules bound per virion was significantly increased to amplify the diagnostic signal.

Description

Angiotensin-converting enzyme 2 receptor and uses thereof

The present invention relates to a method for detecting virions of Sars coronavirus-2 (Sars CoV-2) using an angiotensin converting enzyme 2 (ACE2) variant and an ACE2 multimer.

Sars CoV-2 is a single-stranded RNA coronavirus that is infectious to humans and causes coronavirus disease-19 (COVID-19).

Conventional detection of Sars CoV-2 used an antibody recognizing spike protein (S protein) or its receptor binding domain (RBD) region present on the surface of Sars CoV-2. However, in the case of the newly emerging Sars CoV-2 variant, it was not possible to diagnose whether or not the mutant virus was infected because it could not be recognized by an antibody that binds to the existing spike protein. In addition, traditional antibody and antigen diagnostic kits mainly detect past viral infections by using nonstructural proteins of viruses. Existing PCR or nucleic acid diagnostic kits are also methods of detecting nucleic acids, such as viral RNA, that have not been released from the body for several months after virus infection or have even been inserted into the genome of the host. There was a problem that could not be detected separately from infection.

Viruses that infect the human body produce active viral particles (virions) capable of infecting and reproducing within host cells and inactive viral particles (ineterfering particles) thousands to tens of thousands of times larger, and secrete both of them to the outside of the cell. Existing diagnostic kits using antigens and antibodies do not identify these two and measure them randomly, and virus tests using PCR only measure the amount of virus, but cannot measure the activity of the virus produced.

Accordingly, the present invention attempted to develop a method for specifically detecting viral particles of not only the currently active form of Sars CoV-2 but also variants thereof.

The inventors of the present invention point out that since Sars CoV-2 or its variants must recognize the ACE2 receptor present in the host cell in order to invade the host cell, the structure determinant of the spike protein necessary for recognizing the receptor must be maintained. it was cognition Despite the mutation of the spike protein, it binds to the ACE2 receptor through the structure determinant of the spike protein, so it is confirmed that the presence of Sars CoV-2 or its variant virions can be clearly diagnosed using the ACE2 receptor completed the present invention.

In order to achieve the above object, an object of the present invention is i) an ACE2 variant without enzymatic activity of ACE2 for capturing Sars CoV-2 or variant virions thereof and ii) for detecting Sars CoV-2 or variant virions thereof It is to provide ACE2 variants in which the ectodomain of ACE2 is redesigned, including ACE2 variants having an enzymatic activity of ACE2.

Another object of the present invention is to provide a composition for detecting Sars CoV-2 or variant virions thereof, including an ACE2 variant in which the ACE2 ectodomain is redesigned.

Another object of the present invention is to provide a diagnostic kit comprising the composition for detecting Sars CoV-2 or its variant virions.

Another object of the present invention is i) contacting a biological sample with the redesigned ACE2 variant comprising the capture ACE2 variant immobilized on a solid support; and ii) measuring the enzymatic activity of the ACE2 variant for detection bound to Sars CoV-2 or its variant virions captured by the ACE2 variant for capture immobilized on the solid support. It is to provide a method for detecting mutant virions.

Another object of the present invention is to i) contact a biological sample with the redesigned ACE2 variant comprising the capture ACE2 variant immobilized on a solid support; and ii) amplifying RNA of Sars CoV-2 or its variant virion bound to the ACE2 variant for capture and the ACE2 variant for detection immobilized on the solid support with a primer specific to the captured virion, Sars CoV It is to provide a method for detecting -2 or variant virions thereof.

The present invention can detect virions of active Sars CoV-2 or variants thereof using ACE2 variants.

Compared to conventional antigen and antibody diagnostic kits that used non-structural proteins of viruses to detect inactive viral infections in the past, the present invention detects virions of currently active Sars CoV-2 or variants thereof. However, it has the advantage of excluding possible causative agents from past and inactive infection histories.

In addition, by polymerizing the ACE2 variant, the binding force with virions is exponentially increased by tens, hundreds, and thousands of times, thereby increasing the number of molecules bound to virions and strongly increasing the enzymatic activity. . This significantly increases the affinity with virions and amplifies the diagnostic signal.

1 shows an overall schematic diagram of the present invention for detecting Sars CoV-2 virions, and Sars CoV-2 virions are detected through FRET ASSAY, RNA amplification, and LFT (Laterial flow assay) methods.

Figure 2 shows the FRET ASSAY method for detecting virions of Sars CoV-2.

Figure 3 shows a method for capturing Sars CoV-2 virions using heparin coated on a solid support.

4 shows a method for capturing Sars CoV-2 virions by using the binding of protein A or protein G coated on a solid support and an Fc fragment (IgG, IgA, IgM) bound to an ACE2 variant for capture. In this case, when it binds to the Fc fragment of IgA, it can polymerize up to a tetramer of the ACE2 variant for capture and a decamer of the ACE2 variant when it binds to the Fc fragment of IgM.

FIG. 5 shows a method for capturing Sars CoV-2 virions using a combination of streptavidin coated on a solid support and biotinylated Abitac coupled to an ACE2 variant for capture.

FIG. 6 shows a method for capturing Sars CoV-2 virions using the binding of streptactin-XT coated on a solid support and streptactin II coupled to the ACE2 variant for capture.

7 shows a process of detecting Sars CoV-2 virion RNA using RT-PCR using virion-specific primers.

8 shows the FRET ASSAY results.

9 shows the results of detecting Sars CoV-2 virion RNA using virion-specific primers.

10 shows FRET ASSAY for some using ACE2 variants for capture, ACE2 variants for detection, and fluorescently labeled substrates, and RT-PCR using virion-specific primers for the remaining samples after releasing RNA shows a result.

11 confirms the binding ability of ACE2 variants for capture to virions. Specifically, after attaching the ACE2 variant for capture to Protein A sepharose, the activity of binding to the diluted Omicron virus was confirmed by RT-PCR.

12 confirms the formation of multimers of ACE2 variants for capture.

Hereinafter, the present specification will be described in more detail.

A detailed description of this is as follows. Meanwhile, each description and embodiment disclosed in the present invention may also be applied to other descriptions and embodiments for each. That is, all combinations of the various elements disclosed herein fall within the scope of the present invention. In addition, it cannot be seen that the scope of the present invention is limited by the specific descriptions described below.

Expressions such as “comprising” used in this specification are understood as open-ended terms that include the possibility of including other embodiments, unless specifically stated otherwise in a phrase or sentence in which the expression is included. It should be.

The terms or words used in the description and claims of the present invention should not be construed as being limited to ordinary or dictionary meanings, and the inventors use the concept of terms appropriately in order to explain their invention in the best way. Based on the principle that it can be defined, it should be interpreted as meaning and concept consistent with the technical spirit of the present invention.

As one aspect for achieving the above object, the present invention provides i) an ACE2 variant in which the enzymatic activity of ACE2 is removed for capturing Sars CoV-2 or variant virions thereof, and ii) detection of Sars CoV-2 or variant virions thereof Provided are ACE2 variants in which the ACE2 ectodomain has been redesigned, including ACE2 variants with enzymatic activity of ACE2 for use.

In the present invention, the term "Sars CoV-2" is a single-stranded RNA coronavirus, which is contagious to humans and causes Coronavirus Disease-19 (COVID-19).

In the present invention, the term "the variant of Sars CoV-2" refers to variant viruses of Sars CoV-2, including all viral variants such as alpha, beta, gamma, delta, and omicron. The Sars CoV-2 enters into the target cell by receptor-mediated endocytosis by the interaction between the spike protein present on the surface of the virus and ACE2, a receptor of the host cell. Even after an effective vaccine is introduced, Sars-CoV-2 evades the host's immune system and continuously induces mutations to enable breakthrough infection, making diagnosis impossible by traditional diagnostic methods using antibodies.

In the receptor binding site (RBD) of the spike protein of Sars CoV-2, there are separate epitopes that antibodies can recognize and structural determinants that ACE2 receptors can recognize. Viruses maintain their survival by changing the former (epithelial determinant) to evade host neutralization and conserving the latter (structural determinant). According to this principle, diagnosis using receptors can capture and recognize variants of all viruses. Therefore, even in the case of a Sars CoV-2 mutant having a mutation in the spike protein, the structure determinant required for receptor recognition to invade the host cell remains the same, and therefore, it does not lose its ability to bind to the ACE2 receptor. Therefore, focusing on the fact that the structural determinant that the ACE2 receptor can recognize is conserved in Sars CoV-2 or its variant virions, the C-terminal region is reduced to capture Sars CoV-2 or its variants, and ACE2 An ACE2 variant in which the ectodomain of ACE2 is redesigned, including an ACE2 variant in which the enzymatic function of ACE2 has been removed and a C-terminal region is reduced to detect the Sars CoV-2 or its variant virion, and an ACE2 variant with enzymatic activity of ACE2 is included. was used to detect active Sars CoV-2 or its mutant virions in an individual infected with Sars CoV-2. In addition, according to the present invention, not only existing Sars CoV-2 or its variants but also future variants can be detected without limitation.

In the present invention, the term "ACE2" refers to a host cell receptor to which the spike protein present on the surface of Sars CoV-2 binds, and in the present invention, ACE2 or ACE2 receptors may be used interchangeably and are interpreted in the same meaning.

The term “virion” refers to an intact viral particle capable of infecting.

The present invention can detect all variants of Sars CoV-2, such as alpha, beta, gamma, delta, and omicron, and can detect not only Sars CoV-2 but also Sars CoV, and coronavirus-NL63 (CoV-NL63), a common cold virus. can also be applied to

The ACE2 receptor polypeptide is an ACE2 protein derived from Homo sapiens listed in NCBI GenBank (NCBI GenBank: NP_068576.1, its gene sequence_NCBI GenBank: AF241254.1) and has SEQ ID NO: 30 (amino acid sequence from 1 to 805). .

The ACE2 variants in which the ACE2 ectodomain is redesigned include an ACE2 variant for capture and an ACE2 variant for detection, and the ACE2 ectodomain is C in ACE2 (NCBI GenBank: AAF78220.1) (SEQ ID NO: 30). -The terminal region is reduced, the 18-740th amino acid sequence (723 pieces) (SEQ ID NO: 1), the 18-615th amino acid sequence (598 pieces) (SEQ ID NO: 2), or the 18-357th amino acid sequence (340 pieces) Corresponds to (SEQ ID NO: 3).

The ACE2 variant for detection is a reduction of the C-terminal region in the ACE2 amino acid sequence (SEQ ID NO: 30), and has enzymatic activity for the ACE2 substrate, so Sars CoV-2 or its variant virions can be detected. And, it may include a sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 3. Specifically, in the amino acid sequence of SEQ ID NO: 30, the 18-740th amino acid sequence (723) (SEQ ID NO: 1), the 18-615th amino acid sequence (598) (SEQ ID NO: 2) and the 18-357th amino acid sequence (340 dog) (SEQ ID NO: 3) may include a sequence selected from the group consisting of.

In the present invention, the gene encoding the ACE2 variant for detection may be selected from the group consisting of the nucleotide sequences of SEQ ID NOs: 4 to 6.

The ACE2 variant for capture is a polypeptide into which a mutation has been introduced to remove the enzymatic activity, and there is no enzymatic activity for the ACE2 substrate by artificially inducing mutation, but Sars CoV-2 or its variant Spike protein of virion or its variant Can be combined with to capture Sars CoV-2 or its mutant virions. In the ACE2 variant for capture, histidine (H) at positions 357 and 361 in the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 is substituted with asparagine (N), or at position 256 in the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 Arginine (R), histidine at position 361 (H) and glutamic acid at position 385 (E) may be substituted with alanine (A).

Specifically, the ACE2 variant for capture may be selected from the group consisting of the amino acid sequences of SEQ ID NOs: 7 to 10, and the gene encoding the ACE2 variant for capture may be selected from the group consisting of the nucleotide sequences of SEQ ID NOs: 11 to 14 can

The ACE2 variant for detection and the ACE2 variant for capture are obtained by reducing the C-terminal region in the amino acid sequence of ACE2 (SEQ ID NO: 30), and when the ACE2 variant for detection includes the amino acid sequence of SEQ ID NO: 1, the capture The ACE2 variant comprises the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 10. In addition, when the ACE2 variant for detection includes the amino acid sequence of SEQ ID NO: 2, the ACE2 variant for capture includes the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 9. Accordingly, in the amino acid sequence of SEQ ID NO: 30, either of the ACE2 variants for detection or capture does not include the C-terminal region (amino acid residues 616 to 740). The C-terminal region (616-740 amino acid residues) of ACE2 is a region capable of dimer binding between ACE2s. Due to the reduction of the C-terminal region of ACE2, the binding between the ACE2 variant for capture and the ACE2 variant for detection or multimers thereof is blocked in the present invention. That is, by reducing the C-terminal region (amino acid residues 616-740) of either the ACE2 variant for capture or ACE2 for detection, dimer binding between the ACE2 variant for capture and ACE2 variant for detection can be blocked. Through this, the ACE2 variant for detection that is not bound to virion does not form a dimer with the ACE2 variant for capture, and as described later, it is washed and removed in the detection step, leaving only the ACE2 variant for detection that is bound to virion. do.

Noninfectious inactive virions and infectiously active virions coexist in biological samples collected after viral infection. Only the latter (active virions) retain intact ACE2 receptor binding moieties. Due to these properties, the present invention using the ACE2 receptor can detect or diagnose infectiously active virions existing in vivo.

In addition, the ACE2 variant for capture or the ACE2 variant for detection may include an alanine (A)-alanine (A) sequence at the N-terminus.

Due to the alanine-alanine dipeptide bond, the amount of the ACE2 variant for capture or detection of the ACE2 variant for capture or detection and the amount of production and extracellular secretion of the ACE2 variant for capture or detection can be increased. Specifically, a recombinant plasmid is prepared using a nucleotide sequence complementary to the ACE2 variant for capture or ACE2 variant for detection, and then introduced into cells for transformation to prepare an ACE2 variant for capture or ACE2 variant for detection. , The amount of the ACE2 variant for capture or the ACE2 variant for detection produced in the transformed cell and secreted out of the cell by the binding of the alanine-alanine dipeptide is greatly increased.

The ACE2 variant for capture or the ACE2 variant for detection may be a receptor that binds to spike protein or a variant thereof.

The ACE2 variant for capture or the ACE2 variant for detection may be a dimer, a trimer, a tetramer, a pentamer, a hexamer, a heptamer, an octamer, a decamer, or more multimers, and Sars CoV- 2 or a variant thereof, the number of capturing virions is significantly increased, and stronger binding becomes possible. The ACE2 variants for capture or ACE2 variants for detection include i) a synthetic peptide of foldon or three helix bundle (3HB), ii) an Fc fragment of immunoglobulin IgG, IgA, or IgM, iii) streptavidin, Abs. Dean (avidin), neutravidin (NeutrAvidin), or derivatives thereof and iv) AviTag™, biotinylated Abitak, Strep-tag (Strep-tag II ^® ) or derivatives thereof selected from the group consisting of It can be polymerized by covalent bonding using one or more linkers, thereby making it possible to prepare in the form of a multimer.

The foldon is the C-terminus of T4 fibritin and has the amino acid sequence of GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 25). The three helix bundle (3HB) has the amino acid sequence of GEIAAIKQEIAAIKKEIAAIKWEIAAIKQGYG (SEQ ID NO: 26). The streptavidin has an amino acid sequence of SEQ ID NO: 27. The Avitag™ may have the GLNDIFEAQKIEWHE amino acid sequence (SEQ ID NO: 28), and specifically, biotinylated Avitag may be used. The Streptag II ^® may have the WSHPQFEK amino acid sequence (SEQ ID NO: 29).

In another aspect, the present invention provides a composition for detecting Sars CoV-2 or variant virions thereof, including an ACE2 variant in which the ACE2 ectodomain is redesigned.

The redesigned ACE2 variants include ACE2 variants for capture and ACE2 multimers for detection. In the present invention, the term "redesigned ACE2 variant", "ACE2 ectodomain", "ACE2 variant for capture", "ACE2 variant for detection", "multimer", "Sars CoV-2", "variant of Sars CoV-2" ", and the description of "virion" is as described above.

The composition for detection may include, in addition to the ACE2 variant for capture and the ACE2 variant for detection, a substrate for the ACE2 variant for detection. Specifically, the ACE2 variant for detection is cleaved by the ACE2 enzyme activity to emit fluorescence or Contains a substrate that causes color development. The ACE2 variant for capture has no enzymatic activity for a substrate, and the ACE2 variant or multimer for detection may have an enzymatic activity for a substrate. The fluorescence detection method using the enzyme activity of ACE2 can use FRET (Fluorescent Resonance Energy Transfer) assay, and each part of the 5' and 3' of the substrate is labeled with a fluorescent dye and a scavenger (or vice versa), and the It is the principle that a fluorescent signal is emitted by enzyme activity. The specific method used may vary depending on the detection equipment, the specific fluorescent dye and scavenger used, and the peptide sequence.

The term "fluorescent dye" (fluorophore, fluorochrome) used in the present invention refers to a material capable of absorbing energy of a specific first wavelength and emitting energy of a different second wavelength. For example, fluorescein or a derivative thereof such as fluorescein isothiocyanate, carboxy fluorescein such as FAM, fluorescein amadite, cyanine or derivative thereof such as Cy3, Cy5, Cy7, TAMRA (tetramethylrhodamine) and Rhodamine or derivatives thereof, coumarin or derivatives thereof, and the like, but are not limited thereto.

The "quencher" is a fluorophore quencher or a dark quencher that accepts excitation energy generated from a fluorescent dye. In the fluorescent quencher, the transferred energy is emitted as light of a specific wavelength, and the non-fluorescent quencher can be identified using heat or the like. The fluorescent dye and the quencher may be appropriately selected in consideration of the above-described fluorescence energy transfer method, wavelength of each material, and detection equipment.

In the present invention, the substrate peptide of the ACE2 variant for detection can be labeled with a fluorescent dye at 5' and a scavenger at 3', and can be used in a FRET method.

The substrate is a substrate of ACE2 labeled with a fluorescent dye (fluoropore) and a quencher, and may be selected from the group consisting of Mca-YVADAPK (Dnp), Mca-RPPGFSAFK (Dnp), and Mca-APK (Dnp) And, Mca means methoxycoumarin as a fluorescent dye (fluoropore), and Dnp means dinitrophenol as a quencher.

By preparing and using the ACE2 variant having enzymatic activity in the form of a multimer, the number of ACE2 molecules bound to virions can be increased, thereby amplifying the signal in FRET analysis.

As described above, the ACE2 variant for capture has no enzymatic activity for a substrate, and the ACE2 variant for detection has an enzymatic activity for a substrate. That is, the ACE2 variant for capture has no enzymatic activity for the substrate by removing the enzymatic function of ACE2 through amino acid sequence modification, and serves only to capture Sars CoV-2 or its mutant virions.

Since the ACE2 variant or multimer for detection has enzymatic activity for the substrate, it not only binds to Sars CoV-2 or its variant virion captured by the ACE2 variant for capture, but also cleaves the substrate of ACE2 as an enzyme Since it emits fluorescence, Sars CoV-2 or its variant virions are detected by the FRET method described above.

As another aspect, the present invention provides a diagnostic kit including the composition for detecting Sars CoV-2 or its variant virions.

In the present invention, the term "redesigned ACE2 variant", "ACE2 ectodomain", "ACE2 variant for capture", "ACE2 variant for detection", "multimer", "Sars CoV-2", "variant of Sars CoV-2" ", "virion", and "detection composition" are as described above.

The diagnostic kit includes a composition for detecting Sars CoV-2 or variant virions thereof, and, as described above and below, a capture ACE2 variant immobilized on a solid support and an ACE2 variant for detection having enzymatic activity or a multimer thereof Sars CoV-2 or its variant virions can be detected by the FRET method using. In addition, it is possible to detect Sars CoV-2 or its variant virions by lateral flow assay using an ACE2 variant for capture immobilized on a solid support, an ACE2 variant for detection having enzymatic activity, or a multimer thereof. can The lateral flow assay can be applied using a method known in the art.

In another aspect, the present invention provides a method comprising: i) contacting a biological sample with the redesigned ACE2 variant including the capture ACE2 variant immobilized on a solid support; and ii) measuring the enzymatic activity of the ACE2 variant for detection bound to Sars CoV-2 or its variant virions captured by the ACE2 variant for capture immobilized on the solid support. A method for detecting its mutant virion is provided.

In the present invention, the term “redesigned ACE2 variant”, “capture ACE2 variant”, “detection ACE2 variant”, “multimer”, “FRET assay”, “Sars CoV-2”, “sars CoV-2 variant” , The description of "virion" is as described above.

The detection method of the present invention includes i) contacting a biological sample with the redesigned ACE2 variant including the capture ACE2 variant immobilized on a solid support.

The "biological sample" means nasal or nasopharyngeal secretions, saliva, urine, feces, whole blood, serum, plasma, tissue, cells, tissue autopsy samples (brain, skin, lymph node, spinal cord, etc.), cell culture supernatant, ruptured eukaryotic cells Samples isolated from the human body may be used, but the present invention is not limited thereto.

In the contacting step, the ACE2 variant for capture serves to capture and concentrate Sars CoV-2 or its variant virions present in the biological sample in a state immobilized on a solid support as described above, and the redesigned ACE2 The ACE2 variant for detection included in the variant binds to Sars CoV-2 or its mutant virion captured by the ACE2 variant for capture immobilized on a solid support.

The detection method of the present invention includes ii) measuring the enzyme activity of the ACE2 variant for detection.

A method of using, as an enzyme, an ACE2 variant for detection bound to Sars CoV-2 or its variant virion captured in a capture ACE2 variant without enzymatic activity immobilized on the solid support, wherein the ACE2 variant for detection cleaves the substrate Sars CoV-2 or its mutant virions can be detected by measuring the enzymatic activity of ACE2 by the FRET assay described above.

In addition, after step i), the ACE2 variant for detection that does not form a bond between the ACE2 variant for capture-Sars CoV-2 or its variant virion-detection ACE2 variant immobilized on the solid support is removed by washing Further steps may be included.

By the washing process, all but the virion captured by the ACE2 variant for capture and the ACE2 variant for detection bound thereto are removed. That is, the washing process removes the ACE2 variants for detection that do not bind to virions, and only the ACE2 variants for detection that bind to infectiously active virions having complete ACE receptor binding residues exist. Enzymatic activity is measured to allow detection of infectiously active virions.

1 shows an overall schematic diagram of the present invention for detecting Sars CoV-2 virions, and FIG. 2 shows a FRET ASSAY method for detecting Sars CoV-2 virions.

Specifically, an ACE2 variant for capture without enzymatic activity immobilized on a solid support captures Sars CoV-2 or a variant virion thereof, and an ACE2 variant for detection with enzymatic activity captures Sars CoV-2 or a mutant virion thereof. 2 or its variant After binding to virions, virions are detected by cleavage of the substrate through enzymatic activity and detecting fluorescence that appears through the FRET method. Sars CoV-2 or its variant virions can be detected by a lateral flow assay method using an ACE2 variant for capture immobilized on a solid support, an ACE2 variant for detection having enzymatic activity, or a multimer thereof. there is. The lateral flow assay can be applied using a method known in the art.

3 to 6 show methods for capturing Sars CoV-2 and variants thereof using a coating material immobilized on a solid support and a linker coupled to the ACE2 variant for capture. Figure 3 shows a method for capturing Sars CoV-2 virions using heparin coated on a solid support. 4 shows a method for capturing Sars CoV-2 virions by using the binding of protein A or protein G coated on a solid support and an Fc fragment (IgG, IgA, IgM) bound to an ACE2 variant for capture. 5 shows a method for capturing virions of Sars CoV-2 using a combination of streptavidin coated on a solid support and biotinylated AviTag™ coupled to an ACE2 variant for capture. . Figure 6 captures virions of Sars CoV-2 using the combination of StrepTactin-XT coated on a solid support and Strep-tag II ^® bound to a variant of ACE2 for capture It shows how to do it.

As described above, the linker bound to the ACE2 variant for capture is bound to the coating material of the solid support, thereby fixing the variant of ACE2 for capture to the solid support. The immobilized ACE2 variant for capture functions as a capture receptor that captures Sars CoV-2 or a variant thereof present in a biological sample. Since a small amount of biological sample may be flowed by the capture as described above, there is an effect of concentrating the virus.

The solid support is used to immobilize the ACE2 variant for capture, and the virion formed by the binding of the ACE2 variant for capture-virion-ACE2 variant for detection immobilized on the solid support is not removed by washing, and the ACE2 for capture It is fixed to the solid support through the deformable body.

The solid support includes, for example, a support in the form of a surface, particle, porous matrix, etc., and may be an inert support or carrier that is essentially water-insoluble and useful in immunoassay assays. Examples of commonly used supports include small sheets, sephadex, polyvinyl chloride, polyacrylamide, plastic beads, magnetic beads, gold particles, filter strips, and 96-well microtiter plates, polyethylene, polypropylene particulate materials such as filter paper, agarose, cross-linked dextran, and other polysaccharides, as well as assay plates or test tubes, tubes made from , polystyrene, and the like.

The solid support may be coated with a coating material selected from the group consisting of i) heparin or a derivative thereof, ii) protein A or protein G, and iii) streptavidin, avidin, neutravidin, streptactin, or a derivative thereof. there is.

The ACE2 variant for capture is i) a synthetic peptide of foldon or three helix bundle (3HB), ii) an Fc fragment of immunoglobulin IgG, IgA, or IgM, iii) streptavidin, avidin, neutravidin, or any of these derivatives and iv) at least one linker selected from the group consisting of Abitac, biotinylated Abitac, Streptac, or derivatives thereof, and the ACE2 variant for capture is polymerized using the linker to obtain the ACE2 variant. Can form multimers. In addition, the ACE2 variant for capture can strongly bind and capture virions in a multimeric state. The coating material of the solid support and the linker bound to the ACE2 variant for capture may be bonded to the solid support to immobilize the ACE2 variant for capture as shown in FIGS. 3 to 6 .

Sars CoV-2 and its variant virions are captured by the ACE2 variant for capture immobilized on the solid support by binding of the linker to the coating material immobilized on the solid support and the ACE2 variant for capture. As a result, the virions captured by the ACE2 variant for capture are strongly immobilized without being separated from the solid support. In this way, the ACE2 variant for capture is strongly bound to the coating material coated on the solid support through the linker attached to the ACE2 variant for capture, thereby strongly immobilizing the ACE2 variant for capture on the solid support, thereby capturing the ACE2 variant for capture immobilized on the solid support It is possible to measure the enzymatic activity of ACE2 variants and multimers for detection bound to Sars CoV-2 or its mutant virions.

In another aspect, the present invention provides a method comprising: i) contacting a biological sample with the redesigned ACE2 variant including the capture ACE2 variant immobilized on a solid support; and amplifying RNA of Sars CoV-2 or its mutant virion bound to the ACE2 variant for capture and the ACE2 variant for detection immobilized on the solid support with a primer specific to the captured virion, Sars CoV-2 Or it provides a method for detecting a variant virion thereof.

In the present invention, the terms "biological sample", "capture ACE2 variant immobilized on a solid support", "redesigned ACE2 variant", "capture ACE2 variant", "detection ACE2 variant", "multimer", "Sars CoV -2", "variant of Sars CoV-2", and "virion" are described above.

This method releases RNA by applying heat or a detergent to Sars CoV-2 or its variant virions bound to the ACE2 variant for capture and the ACE2 variant for detection immobilized on a solid support, and the captured RNA Sars CoV-2 or its variant virions are detected by amplification with virion-specific primers.

The virion-specific primers consist of a primer pair consisting of SEQ ID NO: 23 and SEQ ID NO: 24 or a primer pair consisting of SEQ ID NO: 25 and SEQ ID NO: 26.

The primer pair consisting of SEQ ID NO: 23 and SEQ ID NO: 24 and the primer pair consisting of SEQ ID NO: 25 and SEQ ID NO: 26 correspond to two types of primer pairs targeting the N gene of Sars CoV-2 or its mutant virion. Since an active virion can be captured with the ACE2 variant for capture and RNA in the virion to which the ACE2 variant for detection is bound can be specifically detected, it is superior in specificity to a method using only enzymatic activity.

7 shows a process of detecting Sars CoV-2 virion RNA using the RT-PCR method using the virion-specific primers as described above.

8 shows a partial FRET ASSAY process using ACE2 variants for capture, ACE2 variants for detection, and fluorescently labeled substrates.

9 shows the result of amplifying Sars CoV-2 virion RNA using virion-specific primers. Since the virus-specific RNA can be detected using a virion-specific primer, it is possible to detect Sars CoV-2 or mutant virions thereof by this method.

When the FRET ASSAY detection method using the ACE2 variant for capture, the ACE2 variant for detection, and a fluorescently labeled substrate is combined with the viral real-time gene amplification method (RT-PCR), the activity associated with the ACE2 variant for capture and the ACE2 variant for detection Viral RNA from infectious viral pathogens can be detected.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings so that those skilled in the art can easily carry out the present invention. However, the present invention may be embodied in many different forms and is not limited to the embodiments described herein.

실시예 1. 포획용 ACE2 변형체와 검출용 ACE2 변형체의 유전자 재조합 클로닝Example 1. Recombinant cloning of ACE2 variants for capture and ACE2 variants for detection

1-1. 포획용 ACE2 변형체의 유전자 재조합 클로닝1-1. Genetic recombination cloning of ACE2 variants for capture

The nucleotide sequence of the IL2 signal peptide and the nucleotide sequence of Ala-Ala (alanine dipeptide) are the N-terminal of the NN variant ACE2 nucleotide sequence of SEQ ID NO: 12 (corresponding to the NN variant ACE2 of amino acid residues 18 to 615 of SEQ ID NO: 8) , and a ACE2 variant for capture consisting of the nucleotide sequence of SEQ ID NO: 18 linked at the C-terminal with the nucleotide sequence corresponding to the Fc fragment of IgG1 was prepared. Plasmid DNA expressing the ACE2 variant for capture of SEQ ID NO: 18 (corresponding to the amino acid sequence of SEQ ID NO: 16) in mammalian cells was prepared through genetic recombination cloning.

1-2. 검출용 ACE2 변형체의 유전자 재조합 클로닝1-2. Genetic recombination cloning of ACE2 variants for detection

The nucleotide sequence of IL2 signal peptide and the nucleotide sequence of Ala-Ala (alanine dipeptide) of SEQ ID NO: 17 are located at the N-terminal of the ACE2 nucleotide sequence of SEQ ID NO: 4 (corresponding to 18-740 amino acid residues of ACE2 of SEQ ID NO: 1) A base sequence (corresponding to the amino acid sequence of SEQ ID NO: 15) was designed. A plasmid DNA expressing a nucleotide sequence identical to the wild-type ACE2 gene sequence without the Fc fragment in mammalian animal cells through genetic recombination cloning was prepared so as to maintain enzyme activity.

실시예 2. 포획용 ACE2 변형체와 검출용 ACE2 변형체 단백질의 준비Example 2. Preparation of ACE2 variants for capture and ACE2 variant proteins for detection

In order to transfect the plasmid DNA prepared in Example 1, 293 cells were seeded in a T25 flask at a cell number of 3X10 ⁶ one day before transfection. About 5 μg of ACE2 variant plasmid DNA for capture or detection with enzyme activity remaining was transfected using liposomes. After 2 to 4 days, ACE2 variants for capture or detection secreted out of cells and their multimeric proteins were collected from the culture medium. In the case of a multimer, it was prepared in the form of an octamer as shown in FIG. 12 . As shown in FIG. 12, it was confirmed that the dimer could be multimerized into an octamer.

실시예 3. Sars CoV-2와 Mers CoV의 pseudovirus의 준비Example 3. Preparation of pseudoviruses of Sars CoV-2 and Mers CoV

Sars CoV-2 pseudovirus is suppressed by neutralizing antibodies against the spike protein of Sars CoV-2 (SINO biological, #40592-MM57, China), so Sars CoV-2 pseudovirus mimics the outer membrane of Sars CoV-2. ( Nature Communications volume 11, Article number: 2070, 2020).

Therefore, in this experiment, a pseudovirus with the spike protein of Sars CoV-2 as the outer membrane was used. These pseudoviruses have the same characteristics as the outer membrane of the SARS CoV-2 virus and can penetrate cells, so they are useful in laboratories. Sars CoV-2 pseudovirus is based on Lentivirus, 1) expression plasmid: pLVX-firefly luciferase, 2) packaging plasmid: pCMV dr8.2 dvpr, 3) envelope plasmid: pCMV Sars CoV-2 spike prepared by transfection.

In addition, Mers-CoV was prepared as a pseudovirus using Mers-CoV's unique spike protein as an outer membrane protein. Three types of plasmid DNA required for pseudovirus production were co-transfected into 293 cells, and pseudovirus was obtained by collecting the culture medium on

day

2 or 3 of transfection.

실시예 4. RT-PCR 진행Example 4. RT-PCR progress

Sars CoV-2 pseudovirus was shed to the ACE2 variant for capture and the ACE2 variant for detection including the NN mutant prepared in Example 1. The Sars CoV-2 pseudovirus captured by the ACE2 variant for capture and bound to the ACE2 variant for detection was heated in a PCR machine at 95° C. for 10 minutes to induce RNA release naturally from the virus. After mixing 5 μl of the RNA thus released, virion-specific primers (SEQ ID NO: 19 and SEQ ID NO: 20), and RT-PCR mix (Enzynomics' TOPscript ^TM One-step RT PCR kit), RT according to the following conditions - PCR was performed. After reverse transcription at 50°C for 30 minutes, denaturation and PCR cycles (95°C for 30 seconds and 60°C for 30 seconds) were performed 35 times at 95°C for 10 minutes. DNA was electrophoresed to confirm virus-specific PCR products, and the results are shown in FIG. 9 .

9 shows the result of detecting the RNA of the virion of Sars CoV-2 using a primer for the Luciferase reporter gene included in the pseudovirus. Since the pseudovirus of the present invention contains an exogenous luciferase gene, pseudovirus-specific primers were used in this example. As a result, as shown in FIG. 9, PCR amplification occurred only in the sample (lane 6) and the positive control (lane 7) of the pseudovirus of Sars CoV-2 that was captured by the ACE2 variant for capture and bound to the ACE2 variant for detection. .

실시예 5. FRET 분석법Example 5. FRET analysis

For the purpose of capturing Sars CoV-2 or its variants using the ACE2 variant for capturing the NN mutant prepared in Example 1, an ACE2 variant (ACE2, enzyme inactive) for capturing the NN mutant with enzymatic activity removed It was attached to Protein A beads fixed on a solid support. In addition, the ACE2 variant (ACE2, enzyme active) for detection of intact enzymatic activity prepared in Example 1 searches for viruses bound to the NN-type ACE2 variant (ACE2, enzyme inactive) for capture, and It remains attached even during the PBS washing process. Therefore, the activity of the ACE2 variant for detection with enzymatic activity was measured by fluorescence by FRET assay. The composition of the buffer used at this time is as follows.

[Composition of buffer used for FRET analysis]

50 mM MES (pH 6.5)

300 mM NaCl

10 µM ZnCl2

0.01% Triton X-100

20 μM Mca-APK(Dnp)-Substrate

Mca-APK (Dnp) was used as a substrate for FRET analysis. The total reaction volume was 100 μl, the reaction time was up to 1 hour, and fluorescence was measured at 5-minute intervals. Fluostar Omega was used as the instrument used, and fluorescence results were analyzed with the Mars program. 8 shows the FRET ASSAY results. As confirmed in FIG. 8, only the pseudovirus (Spp) with the wild-type spike protein of Sars CoV-2 and the pseudovirus (SApp) with the African mutant spike protein as the outer membrane were detected as significant fluorescence signals.

실시예 6. FRET 분석과 NAT (nucleic acid amplification testing)의 결합Example 6. Combination of FRET analysis and nucleic acid amplification testing (NAT)

As in Example 5, RT-PCR was performed on the remaining samples after being used for FRET analysis in the same manner as in Example 4. The NN-type ACE2 variant (ACE2, enzyme inactive) for capture for virus capture was bound to Protein A beads immobilized on a solid support through an Fc linker, and captured Sars CoV-2 pseudovirus. The spike protein present in the outer membrane of the captured Sars CoV-2 pseudovirus is recognized and bound by the ACE2 variant for detection and shows fluorescence through FRET analysis.

10 shows that FRET ASSAY is performed on some of the samples using ACE2 variants for capture, ACE2 variants for detection, and fluorescently labeled substrates, and RNA is released for the remaining samples, followed by virion-specific primers (SEQ ID NOs: 21 and 22). ) shows the results of performing RT-PCR using. Since the pseudovirus used in this experiment contains an exogenous luciferase gene, pseudovirus-specific primers were used in this example.

By performing RT-PCR using the remaining samples, RNA from the Sars CoV-2 pseudovirus intactly captured and bound by the ACE2 variants for capture and ACE2 variants for detection can be amplified. As a result, unlike the pseudovirus (lane 5) with Mers CoV spike protein as outer membrane, pseudovirus (Spp) (lane 3) with wild-type spike protein of Sars CoV-2 as outer membrane and pseudovirus with African mutant spike protein as outer membrane (lane 3) It was confirmed that the target RNA was detected in SApp) (lane 4).

The RT-PCR method used in this experiment is as follows.

After using FRET ASSAY, a 5 μl volume of the remaining sample was transferred to a PCR tube, denatured at 95 ° C for 10 minutes, and one step RT-PCR was performed.

[RT-PCR Composition]

one step RT-PCR mix (Topscript, enzynomics) 5 μl

5 μl of remaining sample

Virion specific primer (10 μM) 0.5 μl + 0.5 μl

9 μl distilled water

Total volume 20 μl

[RT-PCR cycle (cycle #35)]

Reverse transcription 50℃, 30 minutes

Initial denaturation 95℃, 10 minutes

Denaturation 95℃, 3 seconds

Annealing/elongation 60℃, 30 seconds

실시예 7. 포획용 ACE2 변형체의 비리온에 대한 결합능 시험Example 7. Virion binding ability test of ACE2 variants for capture

2.5X10 ⁴ , 5X10 ³ , 1X10 ³ , 2X10 ² pfu (plaque forming units) as shown in FIG. The binding ability of the ACE2 variant for capture (Capture) prepared in Example 1 was confirmed for virions having .

Using Protein A sepharose resin, the NN-type ACE2 variant for capture prepared in Example 1-1 was bound to a solid support, and then 2.5X10 ⁴ , 5X10 ³ , 1X10 ³ , 2X10 ² pfu of virions were mixed to After being captured by the NN-type ACE2 transformant for capture, RT-PCR was performed in the same manner as in Example 4 using pseudovirus-specific primers (SEQ ID NO: 21 and SEQ ID NO: 22).

11 shows the activity of capturing the diluted Omicron virus by RT-PCR after attaching the NN-type ACE2 variant for capture to Protein A sepharose. As confirmed in lanes 2 to lane 5 of FIG. 11, it was confirmed that the Sarc CoV-2 omicron virus was captured up to 2.5X10 ⁴ ~ 2X10 ² pfu, and the NN-type ACE2 variant for capture captures and detects very low concentrations of the virus. confirmed that there is

Example 8 . Test of multimer formation ability of ACE2 variants for capture

After designing an amino acid sequence capable of forming a multimer attached to the C-term end of the NN-type ACE2 variant for capture of SEQ ID NO: 12 (corresponding to the NN variant ACE2 of SEQ ID NO: 8), A multimer of the NN-type ACE2 variant for capture was prepared to form a sieve, and the results are shown in FIG. 12 .

As a result, a band of 254 kDa, which is the expected molecular weight of the Fc dimer, was confirmed in box A of FIG. It was confirmed that the band position was shifted by This result was confirmed in the non-denaturing protein gel (No reducing agent, No heat). The A box is a dimer structure (Fc-dimer) in which the Fc linker of the ACE2 variant for capture is bound, and the B box is the Fc linker. This means that a molecular weight increase to the size of the Fc-octamer has occurred, resulting in the formation of a multimer.

Accordingly, it was found that by attaching an amino acid linker capable of forming a multimer, the ACE2 variant for capture could be multimerized to the size of an octamer (B box).

Claims

i) an ACE2 variant in which the enzymatic activity of ACE2 is removed for capturing Sars CoV-2 or its variant virions; and

ii) ACE2 variants with redesigned ACE2 ectodomains, including ACE2 variants having enzymatic activity of ACE2 for detection of Sars CoV-2 or variant virions thereof.
According to claim 1,

The ACE2 variant for detection with the enzymatic activity comprises a sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 3, wherein the ACE2 ectodomain is redesigned ACE2 variant.
According to claim 1,

The gene encoding the ACE2 variant for detection is selected from the group consisting of the nucleotide sequences of SEQ ID NOs: 4 to 6, an ACE2 variant with a redesigned ACE2 ectodomain.
According to claim 1,

In the ACE2 variant for capture with the enzymatic activity removed, histidine (H) at positions 357 and 361 in the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 is substituted with asparagine (N), or SEQ ID NO: 1 or SEQ ID NO: 2 Arginine (R) at position 256, histidine (H) at position 361, and glutamic acid (E) at position 385 are substituted with alanine (A) in the amino acid sequence of ACE2 variant with redesigned ACE2 ectodomain.
According to claim 1,

The ACE2 variant for capture is selected from the group consisting of the amino acid sequences of SEQ ID NOs: 7 to 10, an ACE2 variant with a redesigned ACE2 ectodomain.
According to claim 1,

An ACE2 variant with a redesigned ACE2 ectodomain, wherein the gene encoding the ACE2 variant for capture is selected from the group consisting of the nucleotide sequences of SEQ ID NOs: 11 to 14.
According to claim 1,

The ACE2 variant for capture or the ACE2 variant for detection includes an alanine (A)-alanine (A) sequence at the N-terminus, wherein the ACE2 ectodomain is redesigned ACE2 variant.
According to claim 1,

The ACE2 variant for capture or the ACE2 variant for detection is a receptor that binds to a spike protein or a variant thereof, an ACE2 variant with a redesigned ACE2 ectodomain.
According to claim 1,

The ACE2 variant for capture or ACE2 variant for detection is i) a synthetic peptide of foldon or three helix bundle (3HB), ii) an Fc fragment of immunoglobulin IgG, IgA, or IgM, iii) streptavidin, avidin, neutra An ACE2 variant having a redesigned ACE2 ectodomain, which is polymerized using at least one linker selected from the group consisting of Vidin, or a derivative thereof, and iv) AbITAC, biotinylated AbITAC, StrepTAC, or derivatives thereof.
A composition for detecting Sars CoV-2 or variant virions thereof, comprising an ACE2 variant in which the ACE2 ectodomain of claim 1 is redesigned.
According to claim 10,

Wherein the composition for detection comprises a substrate for detection of ACE2 variants, a composition for detecting Sars CoV-2 or variant virions thereof.
According to claim 10,

The ACE2 variant for capture has no enzymatic activity for the substrate,

The composition for detecting Sars CoV-2 or variant virions thereof, wherein the ACE2 variant for detection has an enzymatic activity for a substrate.
According to claim 12,

The substrate is selected from the group consisting of Mca-YVADAPK (Dnp), Mca-RPPGFSAFK (Dnp), and Mca-APK (Dnp), the composition for detecting Sars CoV-2 or variant virions thereof.
A diagnostic kit comprising the composition for detecting Sars CoV-2 or its variant virions according to claim 10.
i) contacting the biological sample with the redesigned ACE2 variant of claim 1 comprising the capture ACE2 variant immobilized on a solid support; and

ii) measuring the enzymatic activity of the ACE2 variant for detection bound to Sars CoV-2 or its variant virions captured in the ACE2 variant for capture immobilized on the solid support,

A method for detecting Sars CoV-2 or variant virions thereof.
According to claim 15,

Further comprising the step of washing and removing the ACE2 variant for detection that does not form a bond with the ACE2 variant for capture-Sars CoV-2 or its variant virion-detection ACE2 receptor immobilized on the solid support after step i) A method for detecting Sars CoV-2 or variant virions thereof.
According to claim 15,

The detection method uses, as an enzyme, the ACE2 receptor for detection bound to Sars CoV-2 or its variant virion in a state captured by the capture ACE2 variant immobilized on the solid support, Sars CoV-2 or its variant virion How to detect.
According to claim 15,

The solid support is coated with a coating material selected from the group consisting of i) heparin or a derivative thereof, ii) protein A or protein G, and iii) streptavidin, avidin, neutravidin, streptactin, or a derivative thereof. A method for detecting phosphorus, Sars CoV-2 or variant virions thereof.
According to claim 15,

The ACE2 variant for capture is i) a synthetic peptide of foldon or three helix bundle (3HB), ii) an Fc fragment of immunoglobulin IgG, IgA, or IgM, iii) streptavidin, avidin, neutravidin, or any of these and iv) a method for detecting Sars CoV-2 or a variant virion thereof coupled with at least one linker selected from the group consisting of Abitac, biotinylated Abitac, Streptag, or derivatives thereof.
According to claim 15,

Sars CoV-2 and its variant virions are captured by the ACE2 variant for capture immobilized on the solid support by binding of the linker to the coating material immobilized on the solid support and the ACE2 variant for capture Sars CoV-2 Or a method for detecting variant virions thereof.
i) contacting the biological sample with the redesigned ACE2 variant of claim 1 comprising the capture ACE2 variant immobilized on a solid support; and

ii) amplifying RNA of Sars CoV-2 or its variant virion bound to the ACE2 variant for capture and the ACE2 variant for detection immobilized on the solid support with a primer specific to the captured virion,

A method for detecting Sars CoV-2 or variant virions thereof.
According to claim 21,

The virion-specific primer is a primer pair consisting of SEQ ID NO: 21 and SEQ ID NO: 22 or a primer pair consisting of SEQ ID NO: 23 and SEQ ID NO: 24, Sars CoV-2 or a method for detecting a variant virion thereof.