KR102412231B1 - FRET biosensor for targeting of coronaviridae-derived spike protein-ACE2 interaction and use thereof - Google Patents

Abstract

The present invention relates to a FRET biosensor that targets the binding of a coronavirus family-derived spike protein to ACE2, and a method for screening a spike protein and ACE2 binding inhibitor using the biosensor. The RBD of the biosensor according to the present invention specifically binds to ACE2 in the cell, and whether or not binding and the degree of binding can be measured through a change in the ratio of fluorescence intensity based on the FRET phenomenon, which inhibits RBD-ACE2 binding. It may be usefully used as a means for screening substances, furthermore, substances for preventing or treating coronavirus infection and verifying their efficacy.

Description

FRET biosensor for targeting of coronaviridae-derived spike protein and ACE2 binding target and use thereof {FRET biosensor for targeting of coronaviridae-derived spike protein-ACE2 interaction and use thereof}

The present invention relates to a FRET biosensor that targets the binding of a spike protein and ACE2 derived from Coronaviridae, and a screening method for a spike protein and an inhibitor of binding of ACE2 using the biosensor.

The first COVID-19 infection (COVID-19), which first occurred in Wuhan, China in December 2019, is caused by infection with a novel virus, Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), and mainly causes symptoms such as fever, cough and fatigue. causes It is known that SARS-CoV-2 binds the spike glycoprotein expressed on the surface with the ACE2 (Angiotensin-Converting Enzyme 2) protein of the host cell to advance the initial introduction of the virus. In particular, it is known that the initiation and key of viral infection that the receptor binding domain (RBD) of the spike glycoprotein forms a hydrophobic bond with ACE2 (Nature. 2020 May; 581(7807):215-220).

Because SARS-CoV-2 spreads rapidly between humans, the World Health Organization (WHO) has declared the COVID-19 outbreak a global pandemic. Currently, the number of confirmed cases and deaths due to viral infections and the control of human and material activities, including concerns about infection, are taking a heavy blow to the national/global economy.

Accordingly, numerous pharmaceutical companies around the world have started developing a COVID-19 vaccine and conducted clinical trials, but there is also a need for a treatment to be administered to patients already infected with COVID-19 or infected with a mutant virus that is outside the vaccine's immune range. Accordingly, many researchers are researching the development of new drugs for the treatment of COVID-19 infection and the potential therapeutic effects of drugs approved by the FDA. to be. Moreover, as the ‘era of virus storm’ is expected to arrive in the future, in which various infectious strain viruses spread all over the world, the establishment of a response system to be performed by researchers/medical staff is required when a new virus emerges in the future.

In the conventional drug development method, the efficacy of the drug was measured by administering a new drug to a living pathogenic organism model for a certain period of time, then sacrificing the biological model and analyzing the extracted genes and proteins. The gene of the sample is mainly analyzed by reverse transcription polymerase chain reaction (RT-PCR), and the protein is analyzed by an experimental technique called Western blot. However, the problems of the prior art are: 1) damage to the sample and the possibility that various chemicals may transform biological material in the course of the experiment, 2) that the activity of a new drug in a living sample cannot be analyzed spatio-temporally Point, 3) The time required for the experiment is very long, and the labor force of a skilled researcher is required for every hour, so the more various experimental conditions are formed, the more the human/material resource required increases exponentially.

Therefore, under the background of the above, the present inventors target the binding of RBD and ACE2 of the spike protein derived from coronavirus to overcome the limitations of the conventional drug verification system and to manufacture a biosensor that can efficiently measure the binding with an optical signal Thus, the present invention was completed.

Accordingly, the present invention provides a fluorescent donor; Receptor binding domain of the coronavirus family-derived spike protein; And it aims to provide a fluorescence resonance energy transfer (FRET) biosensor, including a fluorescent receptor.

Another object of the present invention is to provide a composition for screening a binding inhibitor of a coronavirus family-derived spike protein and ACE2, including the biosensor.

In addition, it is another object of the present invention to provide a screening method for an inhibitor of the binding of a spike protein and ACE2 derived from Coronaviridae.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention as described above, the present invention is a fluorescent donor (fluorescence donor); Receptor binding domain of a spike protein derived from Coronaviridae; and a fluorescence acceptor. It provides a fluorescence resonance energy transfer (FRET) biosensor.

In one embodiment of the present invention, the biosensor may be for measuring the interaction between the receptor binding domain (RBD) of the spike protein and Angiotensin-Converting Enzyme 2 (ACE2).

In another embodiment of the present invention, the biosensor may further include a fusion protein composed of an ACE2 protein and a fluorescent protein.

In another embodiment of the present invention, the coronavirus family may be SARS-CoV-2.

In another embodiment of the present invention, the fluorescent donor and the fluorescent acceptor may be cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), respectively.

In another embodiment of the present invention, the cyan fluorescent protein may be any one selected from the group consisting of Turquoise2-GL, mTurquoise, mTFP, ECFP, CyPet and ECGFP.

In another embodiment of the present invention, the yellow fluorescent protein is selected from the group consisting of YPet, mEYFP, SEYFP, phiYFP, Citrine, mCitrine, Venus, mVenus, iq-mVenus, cp50Venus, cp157Venus, cp172Venus, cp195Venus, and cp229Venus. It may be either one.

In another embodiment of the present invention, the fluorescent protein may be any one selected from the group consisting of mScarlet, mScarlet-i, mcherry, DsRed, mRFP, TagRFP, mGrape1, mKate, mRuby and FusionRed.

In another embodiment of the present invention, the biosensor may be composed of the amino acid sequence of SEQ ID NO: 1.

In another embodiment of the present invention, the fusion protein may consist of the amino acid sequence of SEQ ID NO: 3.

In addition, the present invention provides a composition for screening a binding inhibitor of a coronavirus family-derived spike protein and ACE2, including the biosensor.

In addition, the present invention provides a method for screening a binding inhibitor of the coronavirus family-derived spike protein and ACE2, comprising the following steps.

(a) transfecting the vector expressing the biosensor into cells expressing Angiotensin-Converting Enzyme 2 (ACE2);

(b) treating the transfected cells with a test substance; and

(c) measuring the ratio of the emission intensity of the fluorescent receptor to the emission intensity of the fluorescent donor in the cells treated with the test substance.

In one embodiment of the present invention, the cell may be one that expresses ACE2 endogenously, or is transfected with the fusion protein expression vector to express ACE2 protein.

In another embodiment of the present invention, in the method, the ratio value in cells treated with the test substance is compared with the ratio value of the emission intensity of the fluorescent receptor to the emission intensity of the fluorescent donor of the control group not treated with the test substance. If it decreases, it may further comprise the step of selecting the test substance as a binding inhibitor.

In the FRET biosensor according to the present invention, RBD and ACE2 of the spike protein constituting the biosensor specifically bind to each other in the cell, and when the binding is weakened and separated, the luminescence intensity versus fluorescence of the fluorescent receptor according to the structural change of the biosensor By changing the ratio of the luminescence intensity of the donor, it is possible to measure whether and the degree of binding to the RBD-ACE2. In addition, since the biosensor can conduct an experiment while the sample is alive, the possibility of transformation of the biological material can be excluded, and the degree of activity of the test material can be analyzed spatio-temporally based on real-time imaging data, and the time required and labor force. Furthermore, since there is no need to directly deal with live coronaviruses, infectious disease treatment research becomes possible even in research facilities that do not have a Biological Safety Level-3 (BSL-3) environment. Therefore, the FRET biosensor according to the present invention can be usefully used as a means for measuring RBD-ACE2 binding to screen for substances that inhibit the binding, furthermore, substances for preventing or treating coronavirus infection, and verifying the efficacy. There will be.

1 shows the structures of an ACE2 biosensor targeting RBD-ACE2 binding and an ACE2 fusion protein according to the present invention.
2 shows the operating principle of the biosensor according to the present invention.
Figure 3 shows that the wild-type or mutant (WT, E484P, F486L) of the biosensor and the wild-type or mutant (ACE2-WT, ACE2-M82K) of the biosensor were expressed in HEK293T cells that do not endogenously express the ACE2 protein. FRET/CFP ratio values were measured and displayed (error bars: SEM, ****p<0.0001).
4 is a result of measuring the change in FRET/CFP ratio in the HEK293T cells of FIG. 3 after treatment with hydroxychloroquine (HCQ), FIG. 4a shows real-time imaging results, and FIG. 4b is HCQ treatment and time-course graph measuring the change in FRET/CFP ratio over time (error bars: SEM, ***p<0.001).
Figure 5 is the result of measuring the change in FRET / CFP ratio in the cells when HCQ is treated after expressing ACE2 biosensor-WT in SH-SY5Y and HepG2 cells that endogenously express ACE2 protein, Figure 5a is real-time imaging The results are shown, and FIG. 5b is a time-course graph result of HCQ treatment and measurement of the FRET/CFP ratio change over time (error bars: SEM, ****p<0.0001).
6 is a result showing the comparison of the FRET / CFP ratio changed when HEK293T cells expressing both ACE2 biosensor-WT and ACE2-WT were treated with HCQ at various concentrations (20, 50, 100 uM) for 1 hour (error bars) : SEM, **p<0.01, ****p<0.0001).
7 shows the amino acid sequences of WT (SEQ ID NOs: 11 and 12), E484P (SEQ ID NOs: 13 and 14) and F486L (SEQ ID NOs: 15 and 16) of SARS-CoV-2 RBD, which is a component of the ACE2 biosensor according to the present invention; It shows the nucleotide sequence.
8 shows the amino acid sequences and base sequences of ACE2-WT (SEQ ID NOs: 17 and 18) and ACE2-M82K (SEQ ID NOs: 19 and 20) according to the present invention.
9 shows a plasmid vector map of the category of ACE2 biosensors constructed through gene cloning, respectively, of ACE2 Biosensor-WT (FIG. 9A), ACE2 Biosensor-E484P (FIG. 9B) and ACE2 Biosensor-F486L (FIG. 9C). A vector map is shown.
10 shows a plasmid vector map of the mScarlet-ACE2 category constructed through gene cloning, and shows a vector map of the ACE2-WT fusion protein (FIG. 10A) and ACE2-M82K fusion protein (FIG. 10B), respectively.

The present invention relates to a biosensor that can overcome the limitations of conventional drug screening and validation systems, and more specifically, by targeting the binding of RBD-ACE2 of the coronavirus spike glycoprotein, it is possible to select and verify the binding inhibitory substance. It relates to a biosensor and its use.

Accordingly, the present invention is a fluorescent donor (fluorescence donor);

Receptor binding domain of a spike protein derived from coronaviridae; and

Provided is a fluorescence resonance energy transfer (FRET) biosensor comprising a fluorescence acceptor.

As used herein, the term “fluorescence resonance energy transfer (FRET)” is a mechanism related to energy transfer between two chromophores that are close to each other. non-radiative energy transfer. This is a phenomenon in which the excitation level energy of the fluorescent donor in an excited state is transferred to the fluorescent acceptor, and emission from the fluorescent acceptor is observed or fluorescence quenching of the fluorescent donor is observed.

The fluorescent donor refers to a fluorescent material that acts as a donor in the FRET phenomenon, and the fluorescent acceptor refers to a fluorescent material that acts as an acceptor in the FRET phenomenon. More specifically, the fluorescent donor may be one selected from the group consisting of a fluorescent protein, a fluorescent dye, a bioluminescent protein, and a quantum dot, and the fluorescent acceptor is a fluorescent protein, It may be one selected from the group consisting of a quencher and gold nanoparticles (Au-nano particles).

In the present invention, the fluorescent donor and the fluorescent acceptor may be cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), respectively.

The cyan fluorescent protein may be any one selected from the group consisting of Turquoise2-GL, mTurquoise, mTFP, ECFP, CyPet and ECGFP, preferably Turquoise2-GL, but is not limited thereto.

The yellow fluorescent protein may be one selected from the group consisting of YPet, mEYFP, SEYFP, phiYFP, Citrine, mCitrine, Venus, mVenus, iq-mVenus, cp50Venus, cp157Venus, cp172Venus, cp195Venus, and cp229Venus, preferably any one selected from the group consisting of may be YPet, but is not limited thereto.

In a specific example of the present invention, a FRET biosensor was prepared for measuring the interaction between RBD of SARS-CoV-2 derived spike protein and ACE2 expressed in cells, and selecting and verifying substances that inhibit the interaction ( see Example 1).

According to an embodiment of the present invention, when the biosensor having the structure of FIG. 1 is expressed as a protein by transfecting a cell with the vector for biosensor expression, the RBD protein of the biosensor binds to ACE2 expressed in the cell. As can be seen in FIG. 2 , before RBD binds to ACE2, the distance between cyan fluorescent protein as a fluorescent donor and yellow fluorescent protein as a fluorescent acceptor is long, so stimulation of 440 nm wavelength light causes 475 nm wavelength light emission (CFP fluorescence emission intensity). However, after the binding of RBD and ACE2, the distance between the two fluorescent proteins becomes close, so that stimulation of light with a wavelength of 440 nm can induce emission of light with a wavelength of 530 nm (FRET fluorescence emission intensity). Therefore, in this state, the ratio value (FRET/CFP ratio) of the emission intensity of the yellow fluorescent protein that is the acceptor fluorescent protein to the emission intensity of the cyan fluorescent protein that is the donor fluorescent protein increases. However, when the RBD-ACE2 binding inhibitor candidate is treated and the RBD-ACE2 binding is weakened and separated from each other, the distance between the two fluorescent proteins increases again due to the structural change of the biosensor according to the present invention, and the ratio value is lowered. efficacy can be verified.

However, if the target cell for verification of a candidate substance does not introspectly express ACE2, a vector expressing a fusion protein composed of ACE2 protein and a fluorescent protein is introduced into the cell to induce exogenous ACE2 protein expression, and then the candidate substance can be used to verify the efficacy of

In the present invention, the fluorescent protein constituting the fusion protein is not particularly limited, but preferably has a different emission wavelength from the fluorescent donor and fluorescent receptor constituting the FRET biosensor, more preferably a red fluorescent protein may be, and more preferably mScarlet, mScarlet-i, mcherry, DsRed, mRFP, TagRFP, mGrape1, mKate, mRuby and FusionRed may be any one selected from the group consisting of, but is not limited thereto.

Accordingly, the biosensor according to the present invention can be used to measure the interaction between RBD and ACE2 protein of a spike protein derived from coronavirus family (coronaviridae), preferably SARS-CoV-2.

In the present invention, the biosensor may consist of the amino acid sequence of SEQ ID NO: 1, and the gene sequence encoding the biosensor protein may consist of the nucleotide sequence of SEQ ID NO: 2.

In addition, in the present invention, ACE2 Biosensor-E484P and ACE2 Biosensor-F486L, which are mutants of the ACE2 biosensor, may consist of the amino acid sequences of SEQ ID NOs: 5 and 7, and the nucleotide sequences of SEQ ID NOs: 6 and 8, respectively.

In the present invention, the fusion protein (ACE2-WT fusion protein) may consist of the amino acid sequence of SEQ ID NO: 3, and the gene sequence encoding the fusion protein may consist of the nucleotide sequence of SEQ ID NO: 4.

In addition, in the present invention, the variant (ACE2-M82K fusion protein) of the fusion protein may be composed of the amino acid sequence of SEQ ID NO: 9 and the nucleotide sequence of SEQ ID NO: 10.

In one embodiment of the present invention, it was attempted to verify the performance of the FRET biosensor according to the present invention for cells that endogenously express ACE2 and cells that do not express ACE2, respectively. At this time, a mutant type in which amino acid residues were substituted for the wild-type RBD constituting the biosensor and the ACE2 protein for exogenous expression in cells that do not express ACE2 was prepared, and the experiment was conducted under the same conditions. Specifically, cells were transfected with the expression vector of the biosensor to express the biosensor protein, and then treated with hydroxychloroquine (HCQ), which is known to inhibit the binding of RBD and ACE2 of SARS-CoV-2 derived spike protein. and real-time bioimaging and analysis of changes in FRET/CFP ratio. As a result, the performance of the biosensor for RBD-ACE2 binding inhibitory material selection and efficacy verification of the biosensor was confirmed through a decrease in the FRET/CFP ratio value when HCQ was treated. In addition, it was found that the biosensor according to the present invention specifically forms RBD-ACE2 binding by using the mutant type (see Examples 2 to 4).

In another embodiment of the present invention, as a result of analyzing the difference in the FRET/CFP ratio of the biosensor according to the present invention according to the treatment concentration of HCQ, the higher the treatment concentration of HCQ, the weaker the binding of RBD-ACE2, so the FRET/CFP ratio was detected at a lower level (see Example 5).

From the results of the above examples, it was found that the FRET biosensor according to the present invention can be easily used to measure the interaction between RBD and ACE2 of a spike protein derived from coronavirus.

Accordingly, as another aspect of the present invention, the present invention provides a composition for screening a binding inhibitor of a spike protein and ACE2 derived from coronaviridae, comprising the biosensor.

As another aspect of the present invention, the present invention provides a method for screening a spike protein derived from coronaviridae and an inhibitor of binding of ACE2.

(a) transfecting the vector expressing the biosensor into cells expressing Angiotensin-Converting Enzyme 2 (ACE2);

(b) treating the transfected cells with a test substance; and

(c) measuring the ratio of the emission intensity of the fluorescent receptor to the emission intensity of the fluorescent donor in the cells treated with the test substance.

In the present invention, the cell may be one that expresses ACE2 endogenously, or one that expresses the exogenous ACE2 protein by transfection with a fusion protein expression vector composed of an ACE2 protein and a fluorescent protein.

In the present invention, the test substance is not particularly limited, and specifically, it is selected from the group consisting of nucleic acids, compounds, microbial cultures or extracts, natural extracts, peptides, substrate analogues, aptamers, and antibodies. it could be

In the present invention, in the screening method, after step (c), the test substance-treated cells are compared with the value of the ratio of the emission intensity of the fluorescent receptor to the emission intensity of the fluorescent donor of the control group not treated with the test substance. When the ratio value decreases, the method may further include selecting the test substance as a binding inhibitor.

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

[Example]

Example 1. ACE2 biosensor fabrication and working principle

SARS-CoV-2 virus combines surface spike glycoprotein with ACE2 (Angiotensin-Converting Enzyme 2) protein of the host cell to initiate the initial introduction of the virus. In particular, binding of the receptor binding domain (RBD) of the spike glycoprotein to ACE2 in a hydrophobic network is the start and key of viral infection.

The present inventors tried to develop a biosensor for measuring the interaction between the two proteins as an optical signal by targeting the binding of RBD and ACE2 of the SARS-CoV-2 derived spike protein. To this end, specifically, as shown in FIG. 1, the biosensor was manufactured to include two categories.

More specifically, the ACE2 biosensor includes integral ACE2 Biosensor-WT (Wild Type), ACE2 Biosensor-E484P (mutant) and ACE2 Biosensor F486L (mutant). At this time, the ACE2 Biosensor-E484P was prepared to be used as a positive control and ACE2 Biosensor F486L as a negative control. When using cells that endogenously express ACE2, only the biosensor can be applied alone. However, mScarlet-ACE2 fusion protein was prepared in a different category for the case of using cells that are not resistant to ACE2 expression. mScarlet-ACE2 includes ACE2-WT and ACE2-M82K (mutant), and the ACE2-M82K (mutant) was prepared for use as a negative control.

The present inventors designed the biosensor protein to be finally translated in the form of FIG. 1 as described above, and after obtaining the gene sequence encoding the biosensor protein through various routes, gene cloning was performed. More specifically, the gene sequence of the RBD domain of SARS-CoV-2 virus was obtained, and polymerase chain reaction (PCR) was performed using it as a template to amplify the gene sequence. Through gene cloning, the gene sequence encoding the biosensors was made in the form of a plasmid by inserting the amplified gene fragment into the DNA carrier (vector) designed by the present inventors. Primer sequence information used in the site-specific mutagenesis technique for gene cloning and production of the mutant type in this Example is summarized in Table 1 below.

Genes 5'-SEQUENCE-3' SEQ ID NO: RBD_Forward CAGGCTCGAGACTAATCTTTGTCCGTTCG 21 RBD_Reverse AAATGCGGCCGCTCTTTTTGGGCCC 22 E484P_Forward ACGCCATGTAACGGAGTACCGGGATTTAATTGC 23 E484P_Reverse GCAATTAAATCCCGGTACTCCGTTACATGGCGT 24 F486L_Forward AACGGAGTAGAGGGACTTAATTGCTACTTTCCT 25 F486L_Reverse AGGAAAGTAGCAATTAAGTCCCCTCTACTCCGTT 26 ACE2-WT_Forward CATGTGTACAAGTCCACCATTGAGGAACAG 27 ACE2-WT_Reverse GTACTTCGAAAAAGGAGGTCTGAACATC 28 ACE2-M82K_Forward CTTGCCCAAAAGTATCCACTA 29 ACE2-M82K_Reverse TAGTGGATACTTTTGGGCAAG 30

When the gene sequence (plasmid) encoding the biosensor produced in the present invention is transduced into a cell, the introduced gene sequence is translated into a protein after 1 to 2 days in the cell to operate as a biosensor inside and outside the cell. For example, the biosensor may be used to verify the efficacy of a candidate substance for inhibiting the binding of RBD to ACE2 in cells expressing ACE2. Specifically, as shown in FIG. 2 , before the candidate material was treated, RBD-ACE2 was bound in the cell and the FRET fluorescence emission intensity/CFP fluorescence emission intensity (FRET/CFP ratio) value emitted by the biosensor was relatively high. situation. However, when the binding of RBD-ACE2 is weakened by the action of the candidate substance and separated from each other, the distance between the cyan fluorescent protein and the yellow fluorescent protein increases due to the conformational change of the ACE2 biosensor, and the As a result, the emission of light with a wavelength of 530 nm is lowered. Therefore, the intensity of the light emitted by each fluorescent protein using a fluorescence microscope or a spectrophotometer, specifically, the emission intensity of the yellow fluorescent protein as a fluorescent acceptor to the emission intensity of the cyan fluorescent protein as a fluorescent donor. By analyzing the (FRET/CFP ratio), it becomes possible to verify the efficacy of the drug.

Example 2. RBD-ACE2 specific binding verification of ACE2 biosensor using mutant biosensor

In order to verify RBD-ACE binding through the integrated ACE-2 biosensor, the present inventors have used the ACE2 biosensor prepared in the present invention by a site-directed mutagenesis method and binding to the amino acid residues of the ACE2 protein. A portion that plays an important role was substituted with another residue. Specifically, in ACE2-Biosensor-E484P, residue 484 of the RBD domain is substituted with proline (P) from glutamic acid (E), and this mutation is known to further enhance the bond between RBD-ACE2. It was used as a positive control in this verification. In another mutant, ACE2-Biosensor-F486L, residue 486 of the RBD domain is substituted from phenylalanine (F) to leucine (L), and this mutation is known to weaken the bond between RBD-ACE2. It was used as a negative control in the verification. In addition, in ACE-M82K, residue 82 of the ACE protein is substituted from methionine (M) to lysine (K), and the mutation is known to weaken the binding between RBD-ACE2. It was used as a negative control.

To verify the RBD-ACE2-specific binding of the integrated biosensor, wild-type ACE biosensor (WT), mutant ACE2 biosensor (E484P or F486) expression vector was introduced, and expression vectors of wild-type fusion protein (ACE2-WT) and mutant-type fusion protein (ACE2-M82K) were introduced as ACE2 protein constructs, respectively. In addition, the pmScarlet-I vector was introduced as a control.

Before treatment with a drug that inhibits RBD-ACE2 binding, as can be seen from the results showing the FRET/CFP ratio value in FIG. 3, if the bond formation between RBD-ACE2 is possible, that is, WT+ACE2-WT and E484P+ACE2 It was confirmed that -WT showed a relatively high FRET/CFP ratio value. On the other hand, when the mutant type, which is a negative control, was included among the constituents of RBD-ACE2 binding, binding between RBD-ACE2 was not made, and it was confirmed that the FRET/CFP ratio value was relatively low.

Example 3. Real-time imaging of ACE2 biosensors in cells that do not endogenously express ACE2

The present inventors treated the experimental conditions of Example 2, that is, hydroxychloroquine (HCQ), which is known to inhibit RBD-ACE2 binding to HEK293T cells into which the wild-type and mutant ACE2 biosensors and ACE2 protein were introduced, Changes in FRET/CFP ratio were measured in cells. For reference, the more red in the image, the higher the relative FRET/CFP ratio value, and each scale bar represents 10 μm.

As a result of the experiment, as shown in FIG. 4a, in the experimental group (WT+ACE2-WT and E484P+ACE2-WT) in FIG. 3 where binding between RBD-ACE2 was possible and showed a high FRET/CFP ratio value, when HCQ was treated It was found that the binding between RBD-ACE2 was dissociated and the value of the FRET/CFP ratio decreased. On the other hand, in the experimental group (WT-Control, WT+ACE2-M82K, E484P+ACE2-M82K and F486L+ACE2-WT) that showed a low FRET/CFP ratio value, RBD-ACE2 binding to be inhibited by HCQ was not formed, so FRET It was confirmed that there was no change in /CFP ratio.

In addition, as a result of analyzing the change in the FRET/CFP ratio over time by representing the imaging result as a time-course graph, in the upper graph of FIG. 4b , WT-Control and WT+ACE2- in which binding between RBD-ACE2 was not formed Unlike the M82K group, it was confirmed that the high FRET/CFP ratio value was gradually decreased by HCQ treatment only in the case of WT+ACE2-WT in which RBD-ACE2 bonding was formed. Even through the lower graph of FIG. 4b, high FRET/CFP only in the case of E484P+ACE2-WT in which the RBD-ACE2 bond was formed, unlike the E484P+ACE2-M82K and F486L+ACE2-WT groups in which the RBD-ACE2 bond was not formed. It was confirmed that the ratio value gradually decreased by HCQ treatment.

Example 4. Real-time Imaging of ACE2 Biosensors in Cells Endogenously Expressing ACE2

In addition to Example 3, the present inventors tried to verify the performance of the ACE2 biosensor in cells endogenously expressing ACE2. To this end, when only ACE2-biosensor was transduced into each of SH-SY5Y and HepG2 and treated with HCQ, the response of the biosensor was imaged in real time.

As a result, as can be seen in Figure 5a, the RBD domain of ACE2-biosensor binds to the ACE2 protein that is endogenously expressed in cells, and when HCQ is treated, the binding is separated, thereby reducing the FRET/CFP ratio. In addition, as a result of analyzing the change in the FRET/CFP ratio over time by representing the imaging result as a time-course graph, ACE2-biosensor-expressed SH-SY5Y (top) and HepG2 (bottom) as shown in FIG. 5b It was confirmed that the FRET/CFP ratio value in the cells gradually decreased over time.

Example 5. Confirmation of change in FRET/CFP ratio of ACE2 biosensor-WT according to hydroxycloroquine (HCQ) concentration

In addition to the results of the above examples, it was attempted to investigate the difference in the FRET/CFP ratio of the ACE2 biosensor according to the present invention according to the treatment concentration of HCQ. To this end, the FRET/CFP ratio changed when HEK293T cells expressing both ACE2 biosensor-WT and ACE2-WT were treated with HCQ at various concentrations (20, 50, 100 uM) for 1 hour were analyzed.

As a result, as shown in FIG. 6 , it was confirmed that the higher the concentration of HCQ, the weaker the binding of RBD-ACE2, the lower the FRET/CFP ratio was detected.

The description of the present invention stated above is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. There will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

<110> Pusan National University Industry-University Cooperation Foundation <120> FRET biosensor for targeting of coronaviridae-derived spike protein-ACE2 interaction and use thereof <130> PD20-367 <160> 30 <170> KoPatentIn 3.0 <210> 1 < 211> 993 <212> PRT <213> Artificial Sequence <220> <223> ACE2 Biosensor-WT <400> 1 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 1 5 10 15 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 20 25 30 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Leu 35 40 45 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60 Leu Gly Tyr Gly Leu Gln Cys Phe Ala Arg Tyr Pro Asp His Met Lys 65 70 75 80 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85 90 95 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105 110 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125 Ile Asp P he Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140 Asn Tyr Asn Ser His Asn Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn 145 150 155 160 Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Gly 165 170 175 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 180 185 190 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Tyr Gln Ser Ala Leu 195 200 205 Phe Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 210 215 220 Leu Thr Ala Ala Gly Ile Thr Glu Gly Met Asn Glu Leu Tyr Leu Glu 225 230 235 240 Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala 245 250 255 Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp 260 265 2 70 Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr 275 280 285 Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr 290 295 300 Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro 305 310 315 320 Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp 325 330 335 Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys 340 345 350 Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn 355 360 365 Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly 370 375 380 Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu 385 390 395 400 Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr 405 410 415 Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val 420 425 430 Cys Gly Pro Lys Lys Ser Gly Arg Met Val Ser Lys Gly Glu Glu Leu 435 440 445 Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn 450 455 460 Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr 465 470 475 480 Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val 485 490 495 Pro Trp Pro Thr Leu Val Thr Thr Leu Ser Trp Gly Val Gln Cys Phe 500 505 510 Ala Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala 515 520 525 Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp 530 535 540 Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu 545 550 555 560 Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn 565 570 575 Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Phe Ser Gly Asn Val Tyr 580 585 590 Ile Thr Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile 595 600 605 Arg His Asn Ile Glu Asp Gly Gly Val Gln Leu Ala Asp His Tyr Gln 610 615 620 Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His 625 630 635 640 Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg 645 650 655 Asp His Met Val Leu Leu Glu Phe Leu Thr Ala Ala Gly Gly Thr Lys 660 665 670 Val Tyr Asp Pro Glu Gln Arg Lys Arg Met Ile Thr Gly Pro Gln Trp 675 680 685 Trp Ala Arg Cys Lys Gln Met Asn Val Leu Asp Ser Phe Ile Asn Tyr 690 695 700 Tyr Asp Ser Glu Lys His Ala Glu Asn Ala Val Ile Phe Leu His Gly 705 710 715 720 Asn Ala Thr Ser Ser Tyr Leu Trp Arg His Val Val Pro His Ile Glu 725 730 735 Pro Val Ala Arg Cys Ile Ile Pro Asp Leu Ile Gly Met Gly Lys Ser 740 745 750 Gly Lys Ser Gly Asn Gly Ser Tyr Arg Leu Leu Asp His Tyr Lys Tyr 755 760 765 Leu Thr Ala Trp Phe Glu Leu Leu Asn Leu Pro Lys Lys Ile Ile Phe 770 775 780 Val Gly His Asp Trp Gly Ala Ala Leu Ala Phe His Tyr Ala Tyr Glu 785 790 795 800 His Gln Asp Arg Ile Lys Ala Ile Val His Met Glu Ser Val Val Asp 805 810 815 Val Ile Glu Ser Trp Asp Glu Trp Pro Asp Ile Glu Glu Asp Ile Ala 820 825 830 Leu Ile Lys Ser Glu Glu Gly Glu Lys Met Val Leu Glu Asn Asn Phe 835 840 845 Phe Val Glu Thr Val Leu Pro Ser Lys Ile Met Arg Lys Leu Glu Pro 850 855 860 Glu Glu Phe Ala Ala Tyr Leu Glu Pro Phe Lys Glu Lys Gly Glu Val 865 870 875 880 Arg Arg Pro Thr Leu Ser Trp Pro Arg Glu Ile Pro Leu Val Lys Gly 885 890 895 Gly Lys Pro Asp Val Val Gln Ile Val Arg Asn Tyr Asn Ala Tyr Leu 900 905 910 Arg Ala Ser Asp Asp Leu Pro Lys Leu Phe Ile Glu Gly Asp Pro Gly 915 920 925 Phe Phe Ser Asn Ala Ile Val Glu Gly Ala Lys Lys Phe Pro Asn Thr 930 935 940 Glu Phe Val Lys Val Lys Gly Leu His Phe Leu Gln Glu Asp Ala Pro 945 950 955 960 Asp Glu Met Gly Lys Tyr Ile Lys Ser Phe Val Glu Arg Val Leu Lys 965 970 975 Asn Glu Gln Ser Arg Leu Gln Leu Pro Leu Glu Arg Leu Thr Leu 9 80 985 990 Asp <210> 2 <211> 7713 <212> DNA <213> Artificial Sequence <220> <223> ACE2 Biosensor-WT <400> 2 ggtcgatcga cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcatacttac 60 tcatagccca tatagccgtgt ggtcatacttac 60 tcatagccca tatatggagt cc gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc 180 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc 240 agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg 300 gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat 360 ctacgtatta gtcatcgcta ttaccatggt cgaggtgagc cccacgttct gcttcactct 420 ccccatctcc cccccctccc cacccccaat tttgtattta tttatttttt aattattttg 480 tgcagcgatg ggggcggggg gggggggggg gcgcgccagg cggggcgggg cggggcgagg 540 ggcggggcgg ggcgaggcgg agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa 600 agtttccttt tatggcgagg cggcggcggc ggcggcccta taaaaagcga agcgcgcggc 660 gggcgggagt cgctgcc gcctgctgctgcc gcc accgcgttac tcccacaggt gagcgggcgg gacggccctt 780 ctcctccggg ctgtaattag cgcttggttt aatgacggct tgtttctttt ctgtggctgc 840 gtgaaagcct tgaggggctc cgggagggcc ctttgtgcgg ggggagcggc tcggggggtg 900 cgtgcgtgtg tgtgtgcgtg gggagcgccg cgtgcggctc cgcgctgccc ggcggctgtg 960 agcgctgcgg gcgcggcgcg gggctttgtg cgctccgcag tgtgcgcgag gggagcgcgg 1020 ccgggggcgg tgccccgcgg tgcggggggg gctgcgaggg gaacaaaggc tgcgtgcggg 1080 gtgtgtgcgt gggggggtga gcagggggtg tgggcgcgtc ggtcgggctg caaccccccc 1140 tgcacccccc tccccgagtt gctgagcacg gcccggcttc gggtgcgggg ctccgtacgg 1200 ggcgtggcgc ggggctcgcc gtgccgggcg gggggtggcg gcaggtgggg gtgccgggcg 1260 gggcggggcc gcctcgggcc ggggagggct cgggggaggg gcgcggcggc ccccggagcg 1320 ccggcggctg tcgaggcgcg gcgagccgca gccattgcct tttatggtaa tcgtgcgaga 1380 gggcgcaggg acttcctttg tcccaaatct gtgcggagcc gaaatctggg aggcgccgcc 1440 gcaccccctc tagcgggcgc ggggcgaagc ggtgcggcgc cggcaggaag gaaatgggcg 1500 gggagggcct tcgtgcgtcg ccgcgccgcc gtccccttct ccctctccag cctcggggct 1560 gtccgcgggg ggacggctgc cttcggggg g gacggggcag ggcggggttc ggcttctggc 1620 gtgtgaccgg cggctctagc tagagcctct gctaaccatg ttcatgcctt cttctttttc 1680 ctacagctcc tgggcaacgt gctggttatt gtgctgtctc atcattttgg caaagaattc 1740 ggcatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg 1800 gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc 1860 tacggcaagc tgaccctgaa gcttctatgc accaccggca agctgcccgt gccctggccc 1920 accctcgtga ccaccctggg ctacggcctg cagtgcttcg cccgctaccc cgaccacatg 1980 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc 2040 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc 2100 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg 2160 cacaagctgg agtacaacta caacagccac aacgtctata tcaccgccga caagcagaag 2220 aacggcatca aggccaactt caagatccgc cacaacatcg aggacggcgg cgtgcagctc 2280 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac 2340 cactacctga gctaccagtc cgccctgttc aaagacccca acgagaagcg cgatcacatg 2400 gtcctgctgg agttcctgac cgccgccggg atca ctgagg gcatgaacga gctgtacctc 2460 gagactaatc tttgtccgtt cggtgaggtt tttaacgcga caaggttcgc tagtgtatat 2520 gcttggaacc gaaagagaat ctccaattgc gtagctgatt actccgttct ctataacagt 2580 gcgtcctttt caacctttaa gtgttacggc gtttctccaa cgaagctgaa tgatctctgt 2640 tttacgaacg tgtatgctga ctctttcgtt atacgggggg acgaagtgag acagatagca 2700 ccaggtcaga ctgggaagat agcggattac aactataagt tgcccgatga ttttacgggg 2760 tgcgtaatcg catggaactc aaacaacctc gactccaaag taggtggtaa ttataattac 2820 ttgtatcgcc tgtttcgaaa gagcaatttg aagccttttg agcgggatat ttcaaccgaa 2880 atttaccaag caggcagtac gccatgtaac ggagtagagg gatttaattg ctactttcct 2940 cttcaatctt atggctttca accaacaaac ggagtggggt atcaacctta tagagtggta 3000 gtattgtcct ttgagctcct ccacgccccg gctacagttt gtgggcccaa aaagagcggc 3060 cgcatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg 3120 gacggcgacg tcaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc 3180 tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc 3240 accctcgtga ccaccctgtc ctggggcgtg cagtgcttcg cccgctaccc cgaccacatg 3300 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc 3360 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc 3420 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg 3480 cacaagctgg agtacaacta cttcagcggg aacgtctata tcaccgccga caagcagaag 3540 aacggcatca aggccaactt caagatccgc cacaacatcg aggacggcgg cgtgcagctc 3600 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac 3660 cactacctga gcacccagtc cgccttaagc aaagacccca acgagaagcg cgatcacatg 3720 gtcctgctgg agttcttgac cgccgccggg ggtaccaagg tgtacgaccc cgagcagagg 3780 aagaggatga tcaccggccc ccagtggtgg gccaggtgca agcagatgaa cgtgctggac 3840 agcttcatca actactacga cagcgagaag cacgccgaga acgccgtgat cttcctgcac 3900 ggcaacgcca ctagcagcta cctgtggagg cacgtggtgc cccacatcga gcccgtggcc 3960 aggtgcatca tccccgatct gatcggcatg ggcaagagcg gcaagagcgg caacggcagc 4020 tacaggctgc tggaccacta caagtacctg accgcctggt tcgagcttct gaacctgccc 4080 aagaagatca tcttcgtggg ccacgactgg ggcgccgccc tggcc ttcca ctacgcctac 4140 gagcaccagg acaggatcaa ggccatcgtg cacatggaga gcgtggtgga cgtgatcgag 4200 agctgggacg agtggccaga catcgaggag gacatcgccc tgatcaagag cgaggagggc 4260 gagaagatgg tgctggagaa caacttcttc gtggagaccg tgctgcccag caagatcatg 4320 agaaagctgg agcccgagga gttcgccgcc tacctggagc ccttcaagga gaagggcgag 4380 gtgagaagac ccaccctgag ctggcccaga gagatccccc tggtgaaggg cggcaagccc 4440 gacgtggtgc agatcgtgag aaactacaac gcctacctga gagccagcga cgacctgccc 4500 aagctgttca tcgagggcga ccccggcttc ttcagcaacg ccatcgtgga gggcgccaag 4560 aagttcccca acaccgagtt cgtgaaggtg aagggcctgc acttcctcca ggaggacgcc 4620 cccgacgaga tgggcaagta catcaagagc ttcgtggaga gagtgctgaa gaacgagcag 4680 tctagactgc agctgcctcc tctggaacgc ctgactctgg attaagtcga cggatctttt 4740 tccctctgcc aaaaattatg gggacatcat gaagcccctt gagcatctga cttctggcta 4800 ataaaggaaa tttattttca ttgcaatagt gtgttggaat tttttgtgtc tctcactcgg 4860 aaggacatat gggagggcaa atcatttaaa acatcagaat gagtatttgg tttagagttt 4920 ggcaacatat gcccatatgc tggctgccat gaacaaaggt tggctataaa gaggtcatca 4980 gtatatgaaa cagccccctg ctgtccattc cttattccat agaaaagcct tgacttgagg 5040 ttagattttt tttatatttt gttttgtgtt atttttttct ttaacatccc taaaattttc 5100 cttacatgtt ttactagcca gatttttcct cctctcctga ctactcccag tcatagctgt 5160 ccctcttctc ttatggagat ccctcgacct gcagcccaag cttggcgtaa tcatggtcat 5220 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa 5280 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc 5340 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gcggatccgc atctcaatta 5400 gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc 5460 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc 5520 ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg 5580 caaaaagcta acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca 5640 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc 5700 aatgtatctt atcatgtctg gatccgctgc attaatgaat cggccaacgc gcggggagag 5760 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcg gtcg 5820 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat 5880 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta 5940 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa 6000 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc 6060 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt 6120 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca 6180 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg 6240 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat 6300 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta 6360 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct 6420 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac 6480 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa 6540 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa 6600 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt 6 660 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca 6720 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca 6780 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc 6840 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa 6900 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc 6960 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca 7020 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat 7080 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag 7140 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac 7200 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt 7260 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt 7320 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc 7380 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat 7440 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca 7500 gc gtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga 7560 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg 7620 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg 7680 ttccgcgcac atttccccga aaagtgccac ctg 7713 <210> 3 <211> 1019 <212> PRT <213> Artificial Sequence <220> <223> ACE2-WT fusion protein <400> 3 Met Val Ser Lys Gly Glu Ala Val Ile Lys Glu Phe Met Arg Phe Lys 1 5 10 15 Val His Met Glu Gly Ser Met Asn Gly His Glu Phe Glu Ile Glu Gly 20 25 30 Glu Gly Glu Gly Arg Pro Tyr Glu Gly Thr Gln Thr Ala Lys Leu Lys 35 40 45 Val Thr Lys Gly Gly Pro Leu Pro Phe Ser Trp Asp Ile Leu Ser Pro 50 55 60 Gln Phe Met Tyr Gly Ser Arg Ala Phe Ile Lys His Pro Ala Asp Ile 65 70 75 80 Pro Asp Tyr Tyr Lys Gln Ser Phe Pro Glu Gly Phe Lys Trp Glu Arg 85 90 95 Val Met Asn Phe Glu Asp Gly Gly Ala Val Thr Val Thr Gln Asp Thr 100 105 110 Ser Leu Glu Asp Gly Thr Leu Ile Tyr Lys Val Lys Leu Arg Gly Thr 115 120 125 Asn Phe Pro Pro Asp Gly Pro Val Met Gln Lys Lys Thr Met Gly Trp 130 135 140 Glu Ala Ser Thr Glu Arg Leu Tyr Pro Glu Asp Gly Val Leu Lys Gly 145 150 155 160 Asp Ile Lys Met Ala Leu Arg Leu Lys Asp Gly Gly Arg Tyr Leu Ala 165 170 175 Asp Phe Lys Thr Thr Tyr Lys Ala Lys Lys Pro Val Gln Met Pro Gly 180 185 190 Ala Tyr Asn Val Asp Arg Lys Leu Asp Ile Thr Ser His Asn Glu Asp 195 200 205 Tyr Thr Val Val Glu Gln Tyr Glu Arg Ser Glu Gly Arg His Ser Thr 210 215 220 Gly Gly Met Asp Glu Leu Tyr Lys Ser Thr Ile Glu Glu Gln Ala Lys 225 230 235 240 Thr Phe Leu Asp Lys Phe Asn His Glu Ala Glu Asp Leu Phe Tyr Gln 245 250 255 Ser Ser Leu Ala Ser Trp Asn Tyr Asn Thr Asn Ile Thr Glu Glu Asn 260 265 270 Val Gln Asn Met Asn Asn Ala Gly Asp Lys Trp Ser Ala Phe Leu Lys 275 280 285 Glu Gln Ser Thr Leu Ala Gln Met Tyr Pro Leu Gln Glu Ile Gln Asn 290 295 300 Le u Thr Val Lys Leu Gln Leu Gln Ala Leu Gln Gln Asn Gly Ser Ser 305 310 315 320 Val Leu Ser Glu Asp Lys Ser Lys Arg Leu Asn Thr Ile Leu Asn Thr 325 330 335 Met Ser Thr Ile Tyr Ser Thr Gly Lys Val Cys Asn Pro Asp Asn Pro 340 345 350 Gln Glu Cys Leu Leu Leu Glu Pro Gly Leu Asn Glu Ile Met Ala Asn 355 360 365 Ser Leu Asp Tyr Asn Glu Arg Leu Trp Ala Trp Glu Ser Trp Arg Ser 370 375 380 Glu Val Gly Lys Gln Leu Arg Pro Leu Tyr Glu Glu Tyr Val Val Leu 385 390 395 400 Lys Asn Glu Met Ala Arg Ala Asn His Tyr Glu Asp Tyr Gly Asp Tyr 405 410 415 Trp Arg Gly Asp Tyr Glu Val Asn Gly Val Asp Gly Tyr Asp Tyr Ser 420 425 430 Arg Gly Gln Leu Ile Glu Asp Val Glu His Thr Phe Glu Glu Ile Lys 435 440 445 Pro Leu Tyr Glu His Leu His Ala Tyr Val Arg Ala Lys Leu Met Asn 450 455 460 Ala Tyr Pro Ser Tyr Ile Ser Pro Ile Gly Cys Leu Pro Ala His Leu 465 470 475 480 Leu Gly Asp Met Trp Gly Arg Phe Trp Thr Asn Leu Tyr Ser Leu Thr 485 490 495 Val Pro Phe Gly Gln Lys Pro Asn Ile Asp Val Thr Asp Ala Met Val 500 505 510 Asp Gln Ala Trp Asp Ala Gln Arg Ile Phe Lys Glu Ala Glu Lys Phe 515 520 525 Phe Val Ser Val Gly Leu Pro Asn Met Thr Gln Gly Phe Trp Glu Asn 530 535 540 Ser Met Leu Thr Asp Pro Gly Asn Val Gln Lys Ala Val Cys His Pro 545 550 555 560 Thr Ala Trp Asp Leu Gly Lys Gly Asp Phe Arg Ile Leu Met Cys Thr 565 570 575 Lys Val Thr Met Asp Asp Phe Leu Thr Ala His His Glu Met Gly His 580 58 5 590 Ile Gln Tyr Asp Met Ala Tyr Ala Ala Gln Pro Phe Leu Leu Arg Asn 595 600 605 Gly Ala Asn Glu Gly Phe His Glu Ala Val Gly Glu Ile Met Ser Leu 610 615 620 Ser Ala Ala Thr Pro Lys His Leu Lys Ser Ile Gly Leu Leu Ser Pro 625 630 635 640 Asp Phe Gln Glu Asp Asn Glu Thr Glu Ile Asn Phe Leu Leu Lys Gln 645 650 655 Ala Leu Thr Ile Val Gly Thr Leu Pro Phe Thr Tyr Met Leu Glu Lys 660 665 670 Trp Arg Trp Met Val Phe Lys Gly Glu Ile Pro Lys Asp Gln Trp Met 675 680 685 Lys Lys Trp Trp Glu Met Lys Arg Glu Ile Val Gly Val Val Glu Pro 690 695 700 Val Pro His Asp Glu Thr Tyr Cys Asp Pro Ala Ser Leu Phe His Val 705 710 715 720 Ser Asn Asp Tyr Ser Phe Ile Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln 725 730 735 Phe Gln Phe Gln Glu Ala Leu Cys Gln Ala Ala Lys His Glu Gly Pro 740 745 750 Leu His Lys Cys Asp Ile Ser Asn Ser Thr Glu Ala Gly Gln Lys Leu 755 760 765 Phe Asn Met Leu Arg Leu Gly Lys Ser Glu Pro Trp Thr Leu Ala Leu 770 775 780 Glu Asn Val Val Gly Ala Lys Asn Met Asn Val Arg Pro Leu Leu Asn 785 790 795 800 Tyr Phe Glu Pro Leu Phe Thr Trp Leu Lys Asp Gln Asn Lys Asn Ser 805 810 815 Phe Val Gly Trp Ser Thr Asp Trp Ser Pro Tyr Ala Asp Gln Ser Ile 820 825 830 Lys Val Arg Ile Ser Leu Lys Ser Ala Leu Gly Asp Lys Ala Tyr Glu 835 840 845 Trp Asn Asp Asn Glu Met Tyr Leu Phe Arg Ser Ser Val Ala Tyr Ala 850 855 860 Met Arg Gln Tyr Phe Leu Lys Val Lys Asn Gln Met Ile Leu Phe Gly 865 870 875 880 Glu Glu Asp Val Arg Val Ala Asn Leu Lys Pro Arg Ile Ser Phe Asn 885 890 895 Phe Phe Val Thr Ala Pro Lys Asn Val Ser Asp Ile Ile Pro Arg Thr 900 905 910 Glu Val Glu Lys Ala Ile Arg Met Ser Arg Ser Arg Ile Asn Asp Ala 915 920 925 Phe Arg Leu Asn Asp Asn Ser Leu Glu Phe Leu Gly Ile Gln Pro Thr 930 935 940 Leu Gly Pro Pro Asn Gln Pro Pro Val Ser Ile Trp Leu Ile Val Phe 945 950 955 960 Gly Val Val Met Gly Val Ile Val Val Gly Ile Val Ile Leu Ile Phe 965 970 975 Thr Gly Ile Arg Asp Arg Lys Lys Lys Asn Lys Ala Arg Ser Gly Glu 980 985 990 Asn Pro Tyr Ala Ser Ile Asp Ile Ser Lys Gly Glu Asn Asn Pro Gly 995 1000 1005 Phe Gln Asn Thr Asp Asp Val Gln Thr Ser Phe 1010 1015 <210> 4 <211> 7064 <212> DNA <213> Artificial Sequence <220> <223> ACE2-WT fusion protein <400> 4 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg 60 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 120 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 180 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 240 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 300 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 360 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 420 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 480 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 540 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta 600 ccggtcgcca ccatggtgag caagggcgag gcagtgatca aggagttcat gcggttcaag 660 gtgcacatgg agggctccat gaacggccac gagttcgaga tcgagggcga gggcgagggc 720 cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc 780 ttctcctggg acatcctgtc ccctcagttc atgtac ggct ccagggcctt catcaagcac 840 cccgccgaca tccccgacta ctataagcag tccttccccg agggcttcaa gtgggagcgc 900 gtgatgaact tcgaggacgg cggcgccgtg accgtgaccc aggacacctc cctggaggac 960 ggcaccctga tctacaaggt gaagctccgc ggcaccaact tccctcctga cggccccgta 1020 atgcagaaga agacaatggg ctgggaagcg tccaccgagc ggttgtaccc cgaggacggc 1080 gtgctgaagg gcgacattaa gatggccctg cgcctgaagg acggcggccg ctacctggcg 1140 gacttcaaga ccacctacaa ggccaagaag cccgtgcaga tgcccggcgc ctacaacgtc 1200 gaccgcaagt tggacatcac ctcccacaac gaggactaca ccgtggtgga acagtacgaa 1260 cgctccgagg gccgccactc caccggcggc atggacgagc tgtacaagtc caccattgag 1320 gaacaggcca agacattttt ggacaagttt aaccacgaag ccgaagacct gttctatcaa 1380 agttcacttg cttcttggaa ttataacacc aatattactg aagagaatgt ccaaaacatg 1440 aataatgctg gggacaaatg gtctgccttt ttaaaggaac agtccacact tgcccaaatg 1500 tatccactac aagaaattca gaatctcaca gtcaagcttc agctgcaggc tcttcagcaa 1560 aatgggtctt cagtgctctc agaagacaag agcaaacggt tgaacacaat tctaaataca 1620 atgagcacca tctacagtac tggaaaagtt tgtaacccag ataa tccaca agaatgctta 1680 ttacttgaac caggtttgaa tgaaataatg gcaaacagtt tagactacaa tgagaggctc 1740 tgggcttggg aaagctggag atctgaggtc ggcaagcagc tgaggccatt atatgaagag 1800 tatgtggtct tgaaaaatga gatggcaaga gcaaatcatt atgaggacta tggggattat 1860 tggagaggag actatgaagt aaatggggta gatggctatg actacagccg cggccagttg 1920 attgaagatg tggaacatac ctttgaagag attaaaccat tatatgaaca tcttcatgcc 1980 tatgtgaggg caaagttgat gaatgcctat ccttcctata tcagtccaat tggatgcctc 2040 cctgctcatt tgcttggtga tatgtggggt agattttgga caaatctgta ctctttgaca 2100 gttccctttg gacagaaacc aaacatagat gttactgatg caatggtgga ccaggcctgg 2160 gatgcacaga gaatattcaa ggaggccgag aagttctttg tatctgttgg tcttcctaat 2220 atgactcaag gattctggga aaattccatg ctaacggacc caggaaatgt tcagaaagca 2280 gtctgccatc ccacagcttg ggacctgggg aagggcgact tcaggatcct tatgtgcaca 2340 aaggtgacaa tggacgactt cctgacagct catcatgaga tggggcatat ccagtatgat 2400 atggcatatg ctgcacaacc ttttctgcta agaaatggag ctaatgaagg attccatgaa 2460 gctgttgggg aaatcatgtc actttctgca gccacaccta agcatttaaa atccattggt 2520 cttctgtcac ccgattttca agaagacaat gaaacagaaa taaacttcct gctcaaacaa 2580 gcactcacga ttgttgggac tctgccattt acttacatgt tagagaagtg gaggtggatg 2640 gtctttaaag gggaaattcc caaagaccag tggatgaaaa agtggtggga gatgaagcga 2700 gagatagttg gggtggtgga acctgtgccc catgatgaaa catactgtga ccccgcatct 2760 ctgttccatg tttctaatga ttactcattc attcgatatt acacaaggac cctttaccaa 2820 ttccagtttc aagaagcact ttgtcaagca gctaaacatg aaggccctct gcacaaatgt 2880 gacatctcaa actctacaga agctggacag aaactgttca atatgctgag gcttggaaaa 2940 tcagaaccct ggaccctagc attggaaaat gttgtaggag caaagaacat gaatgtaagg 3000 ccactgctca actactttga gcccttattt acctggctga aagaccagaa caagaattct 3060 tttgtgggat ggagtaccga ctggagtcca tatgcagacc aaagcatcaa agtgaggata 3120 agcctaaaat cagctcttgg agataaagca tatgaatgga acgacaatga aatgtacctg 3180 ttccgatcat ctgttgcata tgctatgagg cagtactttt taaaagtaaa aaatcagatg 3240 attctttttg gggaggagga tgtgcgagtg gctaatttga aaccaagaat ctcctttaat 3300 ttctttgtca ctgcacctaa aaatgtgtct gatatcattc ctagaactga agttg aaaag 3360 gccatcagga tgtcccggag ccgtatcaat gatgctttcc gtctgaatga caacagccta 3420 gagtttctgg ggatacagcc aacacttgga cctcctaacc agccccctgt ttccatatgg 3480 ctgattgttt ttggagttgt gatgggagtg atagtggttg gcattgtcat cctgatcttc 3540 actgggatca gagatcggaa gaagaaaaat aaagcaagaa gtggagaaaa tccttatgcc 3600 tccatcgata ttagcaaagg agaaaataat ccaggattcc aaaacactga tgatgttcag 3660 acctcctttt agtaactcga gctcaagctt cgaattctgc agtcgacggt accgcgggcc 3720 cgggatccac cggatctaga taactgatca taatcagcca taccacattt gtagaggttt 3780 tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa 3840 ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca 3900 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca 3960 tcaatgtatc ttaacgcgta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat 4020 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa 4080 tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta 4140 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca 4200 ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat 4260 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg 4320 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc 4380 acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcaggt 4440 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca 4500 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg 4560 aagagtcctg aggcggaaag aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt 4620 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca 4680 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt 4740 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt 4800 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg 4860 cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt 4920 gcaaagatcg atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg 4980 cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag 5040 a caatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt 5100 tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aagacgaggc agcgcggcta 5160 tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg 5220 ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt 5280 gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat 5340 ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg 5400 atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca 5460 gccgaactgt tcgccaggct caaggcgagc atgcccgacg gcgaggatct cgtcgtgacc 5520 catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc 5580 gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat 5640 attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc 5700 gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga 5760 ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt 5820 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga 5880 tgatcct cca gcgcggggat ctcatgctgg agttcttcgc ccaccctagg gggaggctaa 5940 ctgaaacacg gaaggagaca ataccggaag gaacccgcgc tatgacggca ataaaaagac 6000 agaataaaac gcacggtgtt gggtcgtttg ttcataaacg cggggttcgg tcccagggct 6060 ggcactctgt cgatacccca ccgagacccc attggggcca atacgcccgc gtttcttcct 6120 tttccccacc ccacccccca agttcgggtg aaggcccagg gctcgcagcc aacgtcgggg 6180 cggcaggccc tgccatagcc tcaggttact catatatact ttagattgat ttaaaacttc 6240 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc 6300 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt 6360 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac 6420 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct 6480 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact 6540 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg 6600 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata 6660 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga 6720 cctacaccga ac tgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag 6780 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg 6840 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac 6900 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca 6960 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg 7020 cgttatcccc tgattctgtg gataaccgta ttaccgccat gcat 7064 <210> 5 <211> 993 <212 > PRT <213> Artificial Sequence <220> <223> ACE2-Biosensor-E484P <400> 5 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Pro Ile Leu 1 5 10 15 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 20 25 30 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Leu 35 40 45 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60 Leu Gly Tyr Gly Leu Gln Cys Phe Ala Arg Tyr Pro Asp His Met Lys 65 70 75 80 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85 90 95 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr A rg Ala Glu 100 105 110 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140 Asn Tyr Asn Ser His Asn Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn 145 150 155 160 Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Gly 165 170 175 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 180 185 190 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Tyr Gln Ser Ala Leu 195 200 205 Phe Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 210 215 220 Leu Thr Ala Ala Gly Ile Thr Glu Gly Met Asn Glu Leu Tyr Leu Glu 225 230 235 240 Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala 245 250 255 Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp 260 265 270 Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr 275 280 285 Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr 290 295 300 Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro 305 310 315 320 Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp 325 330 335 Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys 340 345 350 Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn 355 360 365 Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly 370 375 380 Ser Thr Pro Cys Asn Gly Val Pro Gly Phe Asn Cys Tyr Phe Pro Leu 385 390 395 400 Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr 405 410 415 Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val 420 425 430 Cys Gly Pro Lys Lys Ser Gly Arg Met Val Ser Lys Gly Glu Glu Leu 435 440 445 Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn 450 455 460 Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr 465 470 475 480 Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val 485 490 495 Pro Trp Pro Thr Leu Val Thr Thr Leu Ser Trp Gly Val Gln Cys Phe 500 505 510 Ala Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala 515 520 525 Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp 530 535 540 Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu 545 550 555 560 Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn 565 570 575 Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Phe Ser Gly Asn Val Tyr 580 585 590 Ile Thr Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile 595 600 605 Arg His Asn Ile Glu Asp Gly Gly Val Gln Leu Ala Asp His Tyr Gln 610 615 620 Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His 625 630 635 640 Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg 645 650 655 Asp His Met Val Leu Leu Glu Phe Leu Thr Ala Ala Gly Gly Thr Lys 660 665 670 Val Tyr Asp Pro Glu Gln Arg Lys Arg Met Ile Thr Gly Pro Gln Trp 675 680 685 Trp Ala Arg Cys Lys Gln Met Asn Val Leu Asp Ser Phe Ile Asn Tyr 690 695 700 Tyr Asp Ser Glu Lys His Ala Glu Asn Ala Val Ile Phe Leu His Gly 705 710 715 720 Asn Ala Thr Ser Ser Tyr Leu Trp Arg His Val Val Pro His Ile Glu 725 730 735 Pro Val Ala Arg Cys Ile Ile Pro Asp Leu Ile Gly Met Gly Lys Ser 740 745 750 Gly Lys Ser Gly Asn Gly Ser Tyr Arg Leu Leu Asp His Tyr Lys Tyr 755 760 765 Leu Thr Ala Trp Phe Glu Leu Leu Asn Leu Pro Lys Lys Ile Ile Phe 770 775 780 Val Gly His Asp Trp Gly Ala Ala Leu Ala Phe His Tyr Ala Tyr Glu 785 790 795 800 His Gln Asp Arg Ile Lys Ala Ile Val His Met Glu Ser Val Val Asp 805 810 815 Val Ile Glu Ser Trp Asp Glu Trp Pro Asp Ile Glu Glu Asp Ile Ala 820 825 830 Leu Ile Lys Ser Glu Glu Gly Glu Lys Met Val Leu Glu Asn Asn Phe 835 840 845 Phe Val Glu Thr Val Leu Pro Ser Lys Ile Met Arg Lys Leu Glu Pro 850 855 860 Glu Glu Phe Ala Ala Tyr Leu Glu Pro Phe Lys Glu Lys Gly Glu Val 865 870 875 880 Arg Arg Pro Thr Leu Ser Trp Pro Arg Glu Ile Pro Leu Val Lys Gly 885 890 895 Gly Lys Pro Asp Val Val Gln Ile Val Arg Asn Tyr Asn Ala Tyr Leu 900 905 910 Arg Ala Ser Asp Asp Leu Pro Lys Leu Phe Ile Glu Gly Asp Pro Gly 915 920 925 Phe Phe Ser Asn Ala Ile Val Glu Gly Ala Lys Lys Phe Pro Asn Thr 930 935 940 Glu Phe Val Lys Val Lys Gly Leu His Phe Leu Gln Glu Asp Ala Pro 945 950 955 960 Asp Glu Met Gly Lys Tyr Ile Lys Ser Phe Val Glu Arg Val Leu Lys 965 970 975 Asn Glu Gln Ser Arg Leu Gln Leu Pro Pro Leu Glu Arg Leu Thr Leu 980 985 990 Asp <210> 6 <211> 7713 <212> DNA <213> Artificial Sequen ce <220> <223> ACE2-Biosensor-E484P <400> 6 ggtcgatcga cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt 60 tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg 120 accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc 180 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc 240 agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg 300 gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat 360 ctacgtatta gtcatcgcta ttaccatggt cgaggtgagc cccacgttct gcttcactct 420 ccccatctcc cccccctccc cacccccaat tttgtattta tttatttttt aattattttg 480 tgcagcgatg ggggcggggg gggggggggg gcgcgccagg cggggcgggg cggggcgagg 540 ggcggggcgg ggcgaggcgg agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa 600 agtttccttt tatggcgagg cggcggcggc ggcggcccta taaaaagcga agcgcgcggc 660 gggcgggagt cgctgcgcgc tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc 720 gcccgccccg gctctgactg accgcgttac tcccacaggt gagcgggcgg gacggccctt 780 ctcctccggg ctgtaattag cgcttggttt aatgacggct tgt ttctttt ctgtggctgc 840 gtgaaagcct tgaggggctc cgggagggcc ctttgtgcgg ggggagcggc tcggggggtg 900 cgtgcgtgtg tgtgtgcgtg gggagcgccg cgtgcggctc cgcgctgccc ggcggctgtg 960 agcgctgcgg gcgcggcgcg gggctttgtg cgctccgcag tgtgcgcgag gggagcgcgg 1020 ccgggggcgg tgccccgcgg tgcggggggg gctgcgaggg gaacaaaggc tgcgtgcggg 1080 gtgtgtgcgt gggggggtga gcagggggtg tgggcgcgtc ggtcgggctg caaccccccc 1140 tgcacccccc tccccgagtt gctgagcacg gcccggcttc gggtgcgggg ctccgtacgg 1200 ggcgtggcgc ggggctcgcc gtgccgggcg gggggtggcg gcaggtgggg gtgccgggcg 1260 gggcggggcc gcctcgggcc ggggagggct cgggggaggg gcgcggcggc ccccggagcg 1320 ccggcggctg tcgaggcgcg gcgagccgca gccattgcct tttatggtaa tcgtgcgaga 1380 gggcgcaggg acttcctttg tcccaaatct gtgcggagcc gaaatctggg aggcgccgcc 1440 gcaccccctc tagcgggcgc ggggcgaagc ggtgcggcgc cggcaggaag gaaatgggcg 1500 gggagggcct tcgtgcgtcg ccgcgccgcc gtccccttct ccctctccag cctcggggct 1560 gtccgcgggg ggacggctgc cttcgggggg gacggggcag ggcggggttc ggcttctggc 1620 gtgtgaccgg cggctctagc tagagcctct gctaaccatg ttcatgcctt c ttctttttc 1680 ctacagctcc tgggcaacgt gctggttatt gtgctgtctc atcattttgg caaagaattc 1740 ggcatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg 1800 gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc 1860 tacggcaagc tgaccctgaa gcttctatgc accaccggca agctgcccgt gccctggccc 1920 accctcgtga ccaccctggg ctacggcctg cagtgcttcg cccgctaccc cgaccacatg 1980 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc 2040 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc 2100 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg 2160 cacaagctgg agtacaacta caacagccac aacgtctata tcaccgccga caagcagaag 2220 aacggcatca aggccaactt caagatccgc cacaacatcg aggacggcgg cgtgcagctc 2280 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac 2340 cactacctga gctaccagtc cgccctgttc aaagacccca acgagaagcg cgatcacatg 2400 gtcctgctgg agttcctgac cgccgccggg atcactgagg gcatgaacga gctgtacctc 2460 gagactaatc tttgtccgtt cggtgaggtt tttaacgcga caaggttcgc tagtgta tat 2520 gcttggaacc gaaagagaat ctccaattgc gtagctgatt actccgttct ctataacagt 2580 gcgtcctttt caacctttaa gtgttacggc gtttctccaa cgaagctgaa tgatctctgt 2640 tttacgaacg tgtatgctga ctctttcgtt atacgggggg acgaagtgag acagatagca 2700 ccaggtcaga ctgggaagat agcggattac aactataagt tgcccgatga ttttacgggg 2760 tgcgtaatcg catggaactc aaacaacctc gactccaaag taggtggtaa ttataattac 2820 ttgtatcgcc tgtttcgaaa gagcaatttg aagccttttg agcgggatat ttcaaccgaa 2880 atttaccaag caggcagtac gccatgtaac ggagtaccgg gatttaattg ctactttcct 2940 cttcaatctt atggctttca accaacaaac ggagtggggt atcaacctta tagagtggta 3000 gtattgtcct ttgagctcct ccacgccccg gctacagttt gtgggcccaa aaagagcggc 3060 cgcatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg 3120 gacggcgacg tcaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc 3180 tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc 3240 accctcgtga ccaccctgtc ctggggcgtg cagtgcttcg cccgctaccc cgaccacatg 3300 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc 33 60 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc 3420 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg 3480 cacaagctgg agtacaacta cttcagcggg aacgtctata tcaccgccga caagcagaag 3540 aacggcatca aggccaactt caagatccgc cacaacatcg aggacggcgg cgtgcagctc 3600 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac 3660 cactacctga gcacccagtc cgccttaagc aaagacccca acgagaagcg cgatcacatg 3720 gtcctgctgg agttcttgac cgccgccggg ggtaccaagg tgtacgaccc cgagcagagg 3780 aagaggatga tcaccggccc ccagtggtgg gccaggtgca agcagatgaa cgtgctggac 3840 agcttcatca actactacga cagcgagaag cacgccgaga acgccgtgat cttcctgcac 3900 ggcaacgcca ctagcagcta cctgtggagg cacgtggtgc cccacatcga gcccgtggcc 3960 aggtgcatca tccccgatct gatcggcatg ggcaagagcg gcaagagcgg caacggcagc 4020 tacaggctgc tggaccacta caagtacctg accgcctggt tcgagcttct gaacctgccc 4080 aagaagatca tcttcgtggg ccacgactgg ggcgccgccc tggccttcca ctacgcctac 4140 gagcaccagg acaggatcaa ggccatcgtg cacatggaga gcgtggtgga cgtgatcgag 4200 agc tgggacg agtggccaga catcgaggag gacatcgccc tgatcaagag cgaggagggc 4260 gagaagatgg tgctggagaa caacttcttc gtggagaccg tgctgcccag caagatcatg 4320 agaaagctgg agcccgagga gttcgccgcc tacctggagc ccttcaagga gaagggcgag 4380 gtgagaagac ccaccctgag ctggcccaga gagatccccc tggtgaaggg cggcaagccc 4440 gacgtggtgc agatcgtgag aaactacaac gcctacctga gagccagcga cgacctgccc 4500 aagctgttca tcgagggcga ccccggcttc ttcagcaacg ccatcgtgga gggcgccaag 4560 aagttcccca acaccgagtt cgtgaaggtg aagggcctgc acttcctcca ggaggacgcc 4620 cccgacgaga tgggcaagta catcaagagc ttcgtggaga gagtgctgaa gaacgagcag 4680 tctagactgc agctgcctcc tctggaacgc ctgactctgg attaagtcga cggatctttt 4740 tccctctgcc aaaaattatg gggacatcat gaagcccctt gagcatctga cttctggcta 4800 ataaaggaaa tttattttca ttgcaatagt gtgttggaat tttttgtgtc tctcactcgg 4860 aaggacatat gggagggcaa atcatttaaa acatcagaat gagtatttgg tttagagttt 4920 ggcaacatat gcccatatgc tggctgccat gaacaaaggt tggctataaa gaggtcatca 4980 gtatatgaaa cagccccctg ctgtccattc cttattccat agaaaagcct tgacttgagg 5040 ttagatttt t tttatatttt gttttgtgtt atttttttct ttaacatccc taaaattttc 5100 cttacatgtt ttactagcca gatttttcct cctctcctga ctactcccag tcatagctgt 5160 ccctcttctc ttatggagat ccctcgacct gcagcccaag cttggcgtaa tcatggtcat 5220 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa 5280 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc 5340 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gcggatccgc atctcaatta 5400 gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc 5460 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc 5520 ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg 5580 caaaaagcta acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca 5640 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc 5700 aatgtatctt atcatgtctg gatccgctgc attaatgaat cggccaacgc gcggggagag 5760 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg 5820 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat 5880 caggggataa cgca ggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta 5940 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa 6000 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc 6060 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt 6120 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca 6180 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg 6240 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat 6300 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta 6360 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct 6420 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac 6480 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa 6540 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa 6600 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt 6660 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca 6720 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca 6780 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc 6840 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa 6900 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc 6960 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca 7020 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat 7080 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag 7140 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac 7200 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt 7260 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt 7320 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc 7380 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat 7440 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca 7500 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga 7560 cacggaaatg ttgaatactc atact Fcttcc tttttcaata ttattgaagc atttatcagg 7620 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg 7680 ttccgcgcgcac attttccccga aaagtgccac ctg < 213> 86 Mettccccac ctg < 213> 86 Mettccccac ctg < 213> <212 Meteor RT> 7223> Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 1 5 10 15 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 20 25 30 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Leu 35 40 45 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60 Leu Gly Tyr Gly Leu Gln Cys Phe Ala Arg Tyr Pro Asp His Met Lys 65 70 75 80 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85 90 95 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105 110 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140 Asn Tyr Asn Ser His Asn Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn 145 150 155 160 Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Gly 165 170 175 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 180 185 190 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Tyr Gln Ser Ala Leu 195 200 205 Phe Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 210 215 220 Leu Thr Ala Ala Gly Ile Thr Glu Gly Met Asn Glu Leu Tyr Leu Glu 225 230 235 240 Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala 245 250 255 Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp 260 265 270 Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr 275 280 285 Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr 290 295 300 Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro 305 310 315 320 Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp 325 330 335 Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys 340 345 350 Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn 355 360 365 Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly 370 375 380 Ser Thr Pro Cys Asn Gly Val Glu Gly Leu Asn Cys Tyr Phe Pro Leu 385 390 395 400 Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr 405 410 415 Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val 420 425 430 Cys Gly Pro Lys Lys Ser Gly Arg Met Val Ser Lys Gly Glu Glu Leu 435 440 445 Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn 450 455 460 Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr 465 470 475 480 Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val 485 490 495 Pro Trp Pro Thr Leu Val Thr Thr Leu Ser Trp Gly Val Gln Cys Phe 500 505 510 Ala Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala 515 520 525 Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp 530 535 540 Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu 545 550 555 560 Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn 565 570 575 Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Phe Ser Gly Asn Val Tyr 580 585 590 Ile Thr Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile 595 600 605 Arg His Asn Ile Glu Asp Gly Gly Val Gln Leu Ala Asp His Tyr Gln 610 615 620 Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His 625 630 635 640 Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg 645 650 655 Asp His Met Val Leu Leu Leu Glu Phe Leu Thr Ala Ala Gly Gly Thr Lys 660 665 670 Val Tyr Asp Pro Glu Gln Arg Lys Arg Met Ile Thr Gly Pro Gln Trp 675 680 685 Trp Ala Arg Cys Lys Gln Met Asn Val Leu Asp Ser Phe Ile Asn Tyr 690 695 700 Tyr Asp Ser Glu Lys His Ala Glu Asn Ala Val Ile Phe Leu His Gly 705 710 715 720 Asn Ala Thr Ser Ser Tyr Leu Trp Arg His Val Val Pro His Ile Glu 725 730 735 Pro Val Ala Arg Cys Ile Ile Pro Asp Leu Ile Gly Met Gly Lys Ser 740 745 750 Gly Lys Ser Gly Asn Gly Ser Tyr Arg Leu Leu Asp His Tyr Lys Tyr 755 760 765 Leu Thr Ala Trp Phe Glu Leu Leu Asn Leu Pro Lys Lys Ile Ile Phe 770 775 780 Val Gly His Asp Trp Gly Ala Ala Leu Ala Phe His Tyr Ala Tyr Glu 785 790 795 800 His Gln Asp Arg Ile Lys Ala Ile Val His Met Glu Ser Val Val Asp 805 810 815 Val Ile Glu Ser Trp Asp Glu Trp Pro Asp Ile Glu Glu Asp Ile Ala 820 825 830 Leu Ile Lys Ser Glu Glu Gly Glu Lys Met Val Leu Glu Asn Asn Phe 835 840 845 Phe Val Glu Thr Val Leu Pro Ser Lys Ile Met Arg Lys Leu Glu Pro 850 855 860 Glu Glu Phe Ala Ala Tyr Leu Glu Pro Phe Lys Glu Lys Gly Glu Val 865 870 875 880 Arg Arg Pro Thr Leu Ser Trp Pro Arg Glu Ile Pro Leu Val Lys Gly 885 890 895 Gly Lys Pro Asp Val Val Gln Ile Val Arg Asn Tyr Asn Ala Tyr Leu 900 905 910 Arg Ala Ser Asp Asp Leu Pro Lys Leu Phe Ile Glu Gly Asp Pro Gly 915 920 925 Phe Phe Ser Asn Ala Ile Val Glu Gly Ala Lys Lys Phe Pro Asn Thr 930 935 940 Glu Phe Val Lys Val Lys Gly Leu His Phe Leu Gln Glu Asp Ala Pro 945 950 955 960 Asp Glu Met Gly Lys Tyr Ile Lys Ser Phe Val Glu Arg Val Leu Lys 965 Glu 970 975 Asn Glu Gln Ser Arg Leu Gln Leu Pro Pro Leu Glu Arg Leu Thr Leu 980 985 990 Asp <210> 8 <211> 7713 <212> DNA <213> Artificial Sequence <220> <223> ACE2-Biosensor-F486L <400> 8 ggtcgatcga cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt 60 tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg tattgcccaac ggtccctcc aataatgacgtc aataatgacgtc a tagtaacgcc 180 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc 240 agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg 300 gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat 360 ctacgtatta gtcatcgcta ttaccatggt cgaggtgagc cccacgttct gcttcactct 420 ccccatctcc cccccctccc cacccccaat tttgtattta tttatttttt aattattttg 480 tgcagcgatg ggggcggggg gggggggggg gcgcgccagg cggggcgggg cggggcgagg 540 ggcggggcgg ggcgaggcgg agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa 600 agtttccttt tatggcgagg cggcggcggc ggcggcccta taaaaagcga agcgcgcggc 660 gggcgggagt cgctgcgcgc tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc 720 gcccgccccg gctctgactg accgcgttac tcccacaggt gagcgggcgg gacggccctt 780 ctcctccggg ctgtaattag cgcttggttt aatgacggct tgtttctttt ctgtggctgc 840 gtgaaagcct tgaggggctc cgggagggcc ctttgtgcgg ggggagcggc tcggggggtg 900 cgtgcgtgtg tgtgtgcgtg gggagcgccg cgtgcggctc cgcgctgccc ggcggctgtg 960 agcgctgcgg gcgcggcgcg gggctttgtg cgctccgcag tgtgcgcgag gggagcgcgg 1020cc gggggcgg tgccccgcgg tgcggggggg gctgcgaggg gaacaaaggc tgcgtgcggg 1080 gtgtgtgcgt gggggggtga gcagggggtg tgggcgcgtc ggtcgggctg caaccccccc 1140 tgcacccccc tccccgagtt gctgagcacg gcccggcttc gggtgcgggg ctccgtacgg 1200 ggcgtggcgc ggggctcgcc gtgccgggcg gggggtggcg gcaggtgggg gtgccgggcg 1260 gggcggggcc gcctcgggcc ggggagggct cgggggaggg gcgcggcggc ccccggagcg 1320 ccggcggctg tcgaggcgcg gcgagccgca gccattgcct tttatggtaa tcgtgcgaga 1380 gggcgcaggg acttcctttg tcccaaatct gtgcggagcc gaaatctggg aggcgccgcc 1440 gcaccccctc tagcgggcgc ggggcgaagc ggtgcggcgc cggcaggaag gaaatgggcg 1500 gggagggcct tcgtgcgtcg ccgcgccgcc gtccccttct ccctctccag cctcggggct 1560 gtccgcgggg ggacggctgc cttcgggggg gacggggcag ggcggggttc ggcttctggc 1620 gtgtgaccgg cggctctagc tagagcctct gctaaccatg ttcatgcctt cttctttttc 1680 ctacagctcc tgggcaacgt gctggttatt gtgctgtctc atcattttgg caaagaattc 1740 ggcatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg 1800 gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc 1860 tacggcaa gc tgaccctgaa gcttctatgc accaccggca agctgcccgt gccctggccc 1920 accctcgtga ccaccctggg ctacggcctg cagtgcttcg cccgctaccc cgaccacatg 1980 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc 2040 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc 2100 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg 2160 cacaagctgg agtacaacta caacagccac aacgtctata tcaccgccga caagcagaag 2220 aacggcatca aggccaactt caagatccgc cacaacatcg aggacggcgg cgtgcagctc 2280 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac 2340 cactacctga gctaccagtc cgccctgttc aaagacccca acgagaagcg cgatcacatg 2400 gtcctgctgg agttcctgac cgccgccggg atcactgagg gcatgaacga gctgtacctc 2460 gagactaatc tttgtccgtt cggtgaggtt tttaacgcga caaggttcgc tagtgtatat 2520 gcttggaacc gaaagagaat ctccaattgc gtagctgatt actccgttct ctataacagt 2580 gcgtcctttt caacctttaa gtgttacggc gtttctccaa cgaagctgaa tgatctctgt 2640 tttacgaacg tgtatgctga ctctttcgtt atacgggggg acgaagtgag acagatagca 2700 ccaggtcaga ctg ggaagat agcggattac aactataagt tgcccgatga ttttacgggg 2760 tgcgtaatcg catggaactc aaacaacctc gactccaaag taggtggtaa ttataattac 2820 ttgtatcgcc tgtttcgaaa gagcaatttg aagccttttg agcgggatat ttcaaccgaa 2880 atttaccaag caggcagtac gccatgtaac ggagtagagg gacttaattg ctactttcct 2940 cttcaatctt atggctttca accaacaaac ggagtggggt atcaacctta tagagtggta 3000 gtattgtcct ttgagctcct ccacgccccg gctacagttt gtgggcccaa aaagagcggc 3060 cgcatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg 3120 gacggcgacg tcaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc 3180 tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc 3240 accctcgtga ccaccctgtc ctggggcgtg cagtgcttcg cccgctaccc cgaccacatg 3300 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc 3360 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc 3420 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg 3480 cacaagctgg agtacaacta cttcagcggg aacgtctata tcaccgccga caagcagaag 3540 aacggcatca aggccaact t caagatccgc cacaacatcg aggacggcgg cgtgcagctc 3600 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac 3660 cactacctga gcacccagtc cgccttaagc aaagacccca acgagaagcg cgatcacatg 3720 gtcctgctgg agttcttgac cgccgccggg ggtaccaagg tgtacgaccc cgagcagagg 3780 aagaggatga tcaccggccc ccagtggtgg gccaggtgca agcagatgaa cgtgctggac 3840 agcttcatca actactacga cagcgagaag cacgccgaga acgccgtgat cttcctgcac 3900 ggcaacgcca ctagcagcta cctgtggagg cacgtggtgc cccacatcga gcccgtggcc 3960 aggtgcatca tccccgatct gatcggcatg ggcaagagcg gcaagagcgg caacggcagc 4020 tacaggctgc tggaccacta caagtacctg accgcctggt tcgagcttct gaacctgccc 4080 aagaagatca tcttcgtggg ccacgactgg ggcgccgccc tggccttcca ctacgcctac 4140 gagcaccagg acaggatcaa ggccatcgtg cacatggaga gcgtggtgga cgtgatcgag 4200 agctgggacg agtggccaga catcgaggag gacatcgccc tgatcaagag cgaggagggc 4260 gagaagatgg tgctggagaa caacttcttc gtggagaccg tgctgcccag caagatcatg 4320 agaaagctgg agcccgagga gttcgccgcc tacctggagc ccttcaagga gaagggcgag 4380 gtgagaagac ccaccctgag ctgg cccaga gagatccccc tggtgaaggg cggcaagccc 4440 gacgtggtgc agatcgtgag aaactacaac gcctacctga gagccagcga cgacctgccc 4500 aagctgttca tcgagggcga ccccggcttc ttcagcaacg ccatcgtgga gggcgccaag 4560 aagttcccca acaccgagtt cgtgaaggtg aagggcctgc acttcctcca ggaggacgcc 4620 cccgacgaga tgggcaagta catcaagagc ttcgtggaga gagtgctgaa gaacgagcag 4680 tctagactgc agctgcctcc tctggaacgc ctgactctgg attaagtcga cggatctttt 4740 tccctctgcc aaaaattatg gggacatcat gaagcccctt gagcatctga cttctggcta 4800 ataaaggaaa tttattttca ttgcaatagt gtgttggaat tttttgtgtc tctcactcgg 4860 aaggacatat gggagggcaa atcatttaaa acatcagaat gagtatttgg tttagagttt 4920 ggcaacatat gcccatatgc tggctgccat gaacaaaggt tggctataaa gaggtcatca 4980 gtatatgaaa cagccccctg ctgtccattc cttattccat agaaaagcct tgacttgagg 5040 ttagattttt tttatatttt gttttgtgtt atttttttct ttaacatccc taaaattttc 5100 cttacatgtt ttactagcca gatttttcct cctctcctga ctactcccag tcatagctgt 5160 ccctcttctc ttatggagat ccctcgacct gcagcccaag cttggcgtaa tcatggtcat 5220 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa 5280 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc 5340 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gcggatccgc atctcaatta 5400 gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc 5460 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc 5520 ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg 5580 caaaaagcta acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca 5640 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc 5700 aatgtatctt atcatgtctg gatccgctgc attaatgaat cggccaacgc gcggggagag 5760 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg 5820 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat 5880 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta 5940 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa 6000 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc 6060 cccctggaag ctccctcgtg cgctctcctg ttccg accct gccgcttacc ggatacctgt 6120 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca 6180 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg 6240 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat 6300 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta 6360 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct 6420 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac 6480 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa 6540 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa 6600 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt 6660 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca 6720 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca 6780 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc 6840 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa 6900 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc 6960 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca 7020 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat 7080 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag 7140 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac 7200 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt 7260 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt 7320 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc 7380 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat 7440 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca 7500 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga 7560 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg 7620 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg 7680 ttccgcgcac atttccccga aaagtgccac ctg 7713 <210> 9 <211> 1019 <212> PRT <213> Artificial Sequence <220> <223> ACE2-M82K fu sion protein <400> 9 Met Val Ser Lys Gly Glu Ala Val Ile Lys Glu Phe Met Arg Phe Lys 1 5 10 15 Val His Met Glu Gly Ser Met Asn Gly His Glu Phe Glu Ile Glu Gly 20 25 30 Glu Gly Glu Gly Arg Pro Tyr Glu Gly Thr Gln Thr Ala Lys Leu Lys 35 40 45 Val Thr Lys Gly Gly Pro Leu Pro Phe Ser Trp Asp Ile Leu Ser Pro 50 55 60 Gln Phe Met Tyr Gly Ser Arg Ala Phe Ile Lys His Pro Ala Asp Ile 65 70 75 80 Pro Asp Tyr Tyr Lys Gln Ser Phe Pro Glu Gly Phe Lys Trp Glu Arg 85 90 95 Val Met Asn Phe Glu Asp Gly Gly Ala Val Thr Val Thr Gln Asp Thr 100 105 110 Ser Leu Glu Asp Gly Thr Leu Ile Tyr Lys Val Lys Leu Arg Gly Thr 115 120 125 Asn Phe Pro Pro Asp Gly Pro Val Met Gln Lys Lys Thr Met Gly Trp 130 135 140 Glu Ala Ser Thr Glu Arg Leu Tyr Pro Glu Asp Gly Val Leu Lys Gly 145 150 155 160 Asp Ile Lys Met Ala Leu Arg Leu Lys Asp Gly Gly Arg Tyr Leu Ala 165 170 175 Asp Phe Lys Thr Thr Tyr Lys Ala Lys Lys Pro Val Gln Met Pro Gly 180 185 190 Ala Tyr Asn Val Asp Arg Lys Leu Asp Ile Thr Ser His Asn Glu Asp 195 200 205 Tyr Thr Val Val Glu Gln Tyr Glu Arg Ser Glu Gly Arg His Ser Thr 210 215 220 Gly Gly Met Asp Glu Leu Tyr Lys Ser Thr Ile Glu Glu Gln Ala Lys 225 230 235 240 Thr Phe Leu Asp Lys Phe Asn His Glu Ala Glu Asp Leu Phe Tyr Gln 245 250 255 Ser Ser Leu Ala Ser Trp Asn Tyr Asn Thr Asn Ile Thr Glu Glu Asn 260 265 270 Val Gln Asn Met Asn Asn Ala Gly Asp Lys Trp Ser Ala Phe Leu Lys 275 280 285 Glu Gln Ser Thr Leu Ala Gln Lys Tyr Pro Leu Gln Glu Ile Gln Asn 290 295 300 Leu Thr Val Lys Leu Gln Leu Gln Ala Leu Gln Gln Asn Gly Ser Ser 305 310 315 320 Val Leu Ser Glu Asp Lys Ser Lys Arg Leu Asn Thr Ile Leu Asn Thr 325 330 335 Met Ser Thr Ile Tyr Ser Thr Gly Lys Val Cys Asn Pro Asp Asn Pro 340 345 350 Gln Glu Cys Leu Leu Leu Glu Pro Gly Leu Asn Glu Ile Met Ala Asn 355 360 365 Ser Leu Asp Tyr Asn Glu Arg Leu Trp Ala Trp Glu Ser Trp Arg Ser 370 375 380 Glu Val Gly Lys Gln Leu Arg Pro Leu Tyr Glu Glu Tyr Val Val Leu 385 390 395 400 Lys Asn Glu Met Ala Arg Ala Asn His Tyr Glu Asp Tyr Gly Asp Tyr 405 410 415 Trp Arg Gly Asp Tyr Glu Val Asn Gly Val Asp Gly Tyr Asp Tyr Ser 420 425 430 Arg Gly Gln Leu Ile Glu Asp Val Glu His Thr Phe Glu Glu Ile Lys 435 440 445 Pro Leu Tyr Glu His Leu His Ala Tyr Val Arg Ala Lys Leu Met Asn 450 455 460 Ala Tyr Pro Ser Tyr Ile Ser Pro Ile Gly Cys Leu Pro Ala His Leu 465 470 475 480 Leu Gly Asp Met Trp Gly Arg Phe Trp Thr Asn Leu Tyr Ser Leu Thr 485 490 495 Val Pro Phe Gly Gln Lys Pro Asn Ile Asp Val Thr Asp Ala Met Val 500 505 510 Asp Gln Ala Trp Asp Ala Gln Arg Ile Phe Lys Glu Ala Glu Lys Phe 515 520 525 Phe Val Ser Val Gly Leu Pro Asn Met Thr Gln Gly Phe Trp Glu Asn 530 535 540 Ser Met Leu Thr Asp Pro Gly Asn Val Gln Lys Ala Val Cys His Pro 545 550 555 560 Thr Ala Trp Asp Leu Gly Lys Gly Asp Phe Arg Ile Leu Met Cys Thr 565 570 575 Lys Val Thr Met Asp Asp Phe Leu Thr Ala His His Glu Met Gly His 580 585 590 Ile Gln Tyr Asp Met Ala Tyr Ala Ala Gln Pro Phe Leu Leu Arg Asn 595 600 605 Gly Ala Asn Glu Gly Phe His Glu Ala Val Gly Glu Ile Met Ser Leu 610 615 620 Ser Ala Ala Thr Pro Lys His Leu Lys Ser Ile Gly Leu Leu Ser Pro 625 630 635 640 Asp Phe Gln Glu Asp Asn Glu Thr Glu Ile Asn Phe Leu Leu Lys Gln 645 650 655 Ala Leu Thr Ile Val Gly Thr Leu Pro Phe Thr Tyr Met Leu Glu Lys 660 665 670 Trp Arg Trp Met Val Phe Lys Gly Glu Ile Pro Lys Asp Gln Trp Met 675 680 685 Lys Lys Trp Trp Glu Met Lys Arg Glu Ile Val Gly Val Val Glu Pro 690 695 700 Val Pro His Asp Glu Thr Tyr Cys Asp Pro Ala Ser Leu Phe His Val 705 710 715 720 Ser Asn Asp Tyr Ser Phe Ile Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln 725 730 735 Phe Gln Phe Gln Glu Ala Leu Cys Gln Ala Ala Lys His Glu Gly Pro 740 745 750 Leu His Lys Cys Asp Ile Ser Asn Ser Thr Glu Ala Gly Gln Lys Leu 755 760 765 Phe Asn Met Leu Arg Leu Gly Lys Ser Glu Pro Trp Thr Leu Ala Leu 770 775 780 Glu Asn Val Val Gly Ala Lys Asn Met Asn Val Arg Pro Leu Leu Asn 785 790 795 800 Tyr Phe Glu Pro Leu Phe Thr Trp Leu Lys Asp Gln Asn Lys Asn Ser 805 810 815 Phe Val Gly Trp Ser Thr Asp Trp Ser Pro Tyr Ala Asp Gln Ser Ile 820 825 830 Lys Val Arg Ile Ser Leu Lys Ser Ala Leu Gly Asp Lys Ala Tyr Glu 835 840 845 Trp Asn Asp Asn Glu Met Tyr Leu Phe Arg Ser Ser Val Ala Tyr Ala 850 855 860 Met Arg Gln Tyr Phe Leu Lys Val Lys Asn Gln Met Ile Leu Phe Gly 865 870 875 880 Glu Glu Asp Val Arg Val Ala Asn Leu Lys Pro Arg Ile Ser Phe Asn 885 890 895 Phe Phe Val Thr Ala Pro Lys Asn Val Ser Asp Ile Ile Pro Arg Thr 900 905 910 Glu Val Glu Lys Ala Ile Arg Met Ser Arg Ser Arg Ile Asn Asp Ala 915 920 925 Phe Arg Leu Asn Asp Asn Ser Leu Glu Phe Leu Gly Ile Gln Pro Thr 930 935 940 Leu Gly Pro Pro Asn Gln Pro Val Ser Ile Trp Leu Ile Val Phe 945 950 955 960 Gly Val Val Met Gly Val Ile Val Val Gly Ile Val Ile Leu Ile Phe 965 970 975 Thr Gly Ile Arg Asp Arg Lys Lys Lys Asn Lys Ala Arg Ser Gly Glu 980 985 990 Asn Pro Tyr Ala Ser Ile Asp Ile Ser Lys Gly Glu Asn Asn Pro Gly 995 1000 1005 Phe Gln Asn Thr Asp Asp Val Gln Thr Ser Phe 1010 1015 <210> 10 <211> 7064 <212> DNA <213> Artificial Sequence <220> <223> ACE2-M82K fusion protein <400> 10 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg 60 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 120 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 180 atgggtgg atag tatttacggt aaactgccca cttgg 240 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 300 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 360 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 420 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 480 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 540 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta 600 ccggtcgcca ccatggtgag caagggcgag gcagtgatca aggagttcat gcggttcaag 660 gtgcacatgg agggctccat gaacggccac gagttcgaga tcgagggcga gggcgagggc 720 cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc 780 ttctcctggg acatcctgtc ccctcagttc atgtacggct ccagggcctt catcaagcac 840 cccgccgaca tccccgacta ctataagcag tccttccccg agggcttcaa gtgggagcgc 900 gtgatgaact tcgaggacgg cggcgccgtg accgtgaccc aggacacctc cctggaggac 960 ggcaccctga tctacaaggt gaagctccgc ggcaccaact tccctcctga cggccccgta 1020 atgcagaaga agacaatggg ctgggaagcg tccaccgagc ggttgtaccc cgaggacggc 1080 gtgctgaagg gc gacattaa gatggccctg cgcctgaagg acggcggccg ctacctggcg 1140 gacttcaaga ccacctacaa ggccaagaag cccgtgcaga tgcccggcgc ctacaacgtc 1200 gaccgcaagt tggacatcac ctcccacaac gaggactaca ccgtggtgga acagtacgaa 1260 cgctccgagg gccgccactc caccggcggc atggacgagc tgtacaagtc caccattgag 1320 gaacaggcca agacattttt ggacaagttt aaccacgaag ccgaagacct gttctatcaa 1380 agttcacttg cttcttggaa ttataacacc aatattactg aagagaatgt ccaaaacatg 1440 aataatgctg gggacaaatg gtctgccttt ttaaaggaac agtccacact tgcccaaaag 1500 tatccactac aagaaattca gaatctcaca gtcaagcttc agctgcaggc tcttcagcaa 1560 aatgggtctt cagtgctctc agaagacaag agcaaacggt tgaacacaat tctaaataca 1620 atgagcacca tctacagtac tggaaaagtt tgtaacccag ataatccaca agaatgctta 1680 ttacttgaac caggtttgaa tgaaataatg gcaaacagtt tagactacaa tgagaggctc 1740 tgggcttggg aaagctggag atctgaggtc ggcaagcagc tgaggccatt atatgaagag 1800 tatgtggtct tgaaaaatga gatggcaaga gcaaatcatt atgaggacta tggggattat 1860 tggagaggag actatgaagt aaatggggta gatggctatg actacagccg cggccagttg 1920 attgaagatg tggaacat ac ctttgaagag attaaaccat tatatgaaca tcttcatgcc 1980 tatgtgaggg caaagttgat gaatgcctat ccttcctata tcagtccaat tggatgcctc 2040 cctgctcatt tgcttggtga tatgtggggt agattttgga caaatctgta ctctttgaca 2100 gttccctttg gacagaaacc aaacatagat gttactgatg caatggtgga ccaggcctgg 2160 gatgcacaga gaatattcaa ggaggccgag aagttctttg tatctgttgg tcttcctaat 2220 atgactcaag gattctggga aaattccatg ctaacggacc caggaaatgt tcagaaagca 2280 gtctgccatc ccacagcttg ggacctgggg aagggcgact tcaggatcct tatgtgcaca 2340 aaggtgacaa tggacgactt cctgacagct catcatgaga tggggcatat ccagtatgat 2400 atggcatatg ctgcacaacc ttttctgcta agaaatggag ctaatgaagg attccatgaa 2460 gctgttgggg aaatcatgtc actttctgca gccacaccta agcatttaaa atccattggt 2520 cttctgtcac ccgattttca agaagacaat gaaacagaaa taaacttcct gctcaaacaa 2580 gcactcacga ttgttgggac tctgccattt acttacatgt tagagaagtg gaggtggatg 2640 gtctttaaag gggaaattcc caaagaccag tggatgaaaa agtggtggga gatgaagcga 2700 gagatagttg gggtggtgga acctgtgccc catgatgaaa catactgtga ccccgcatct 2760 ctgttccatg tttctaatga tta ctcattc attcgatatt acacaaggac cctttaccaa 2820 ttccagtttc aagaagcact ttgtcaagca gctaaacatg aaggccctct gcacaaatgt 2880 gacatctcaa actctacaga agctggacag aaactgttca atatgctgag gcttggaaaa 2940 tcagaaccct ggaccctagc attggaaaat gttgtaggag caaagaacat gaatgtaagg 3000 ccactgctca actactttga gcccttattt acctggctga aagaccagaa caagaattct 3060 tttgtgggat ggagtaccga ctggagtcca tatgcagacc aaagcatcaa agtgaggata 3120 agcctaaaat cagctcttgg agataaagca tatgaatgga acgacaatga aatgtacctg 3180 ttccgatcat ctgttgcata tgctatgagg cagtactttt taaaagtaaa aaatcagatg 3240 attctttttg gggaggagga tgtgcgagtg gctaatttga aaccaagaat ctcctttaat 3300 ttctttgtca ctgcacctaa aaatgtgtct gatatcattc ctagaactga agttgaaaag 3360 gccatcagga tgtcccggag ccgtatcaat gatgctttcc gtctgaatga caacagccta 3420 gagtttctgg ggatacagcc aacacttgga cctcctaacc agccccctgt ttccatatgg 3480 ctgattgttt ttggagttgt gatgggagtg atagtggttg gcattgtcat cctgatcttc 3540 actgggatca gagatcggaa gaagaaaaat aaagcaagaa gtggagaaaa tccttatgcc 3600 tccatcgata ttagcaaagg agaaaataa t ccaggattcc aaaacactga tgatgttcag 3660 acctcctttt agtaactcga gctcaagctt cgaattctgc agtcgacggt accgcgggcc 3720 cgggatccac cggatctaga taactgatca taatcagcca taccacattt gtagaggttt 3780 tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa 3840 ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca 3900 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca 3960 tcaatgtatc ttaacgcgta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat 4020 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa 4080 tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta 4140 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca 4200 ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat 4260 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg 4320 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc 4380 acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcaggt 4440 ggcacttttc ggggaaatgt gcgcggaacc ccta tttgtt tatttttcta aatacattca 4500 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg 4560 aagagtcctg aggcggaaag aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt 4620 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca 4680 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt 4740 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt 4800 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg 4860 cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt 4920 gcaaagatcg atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg 4980 cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag 5040 acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt 5100 tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aagacgaggc agcgcggcta 5160 tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg 5220 ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt 5280 gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat 5340 ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg 5400 atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca 5460 gccgaactgt tcgccaggct caaggcgagc atgcccgacg gcgaggatct cgtcgtgacc 5520 catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc 5580 gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat 5640 attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc 5700 gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga 5760 ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt 5820 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga 5880 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccctagg gggaggctaa 5940 ctgaaacacg gaaggagaca ataccggaag gaacccgcgc tatgacggca ataaaaagac 6000 agaataaaac gcacggtgtt gggtcgtttg ttcataaacg cggggttcgg tcccagggct 6060 ggcactctgt cgatacccca ccgagacccc attggggcca atacgcccgc gtttcttcct 6120 tttccccacc ccacccccca agttcgggtg aaggcccagg gctcg cagcc aacgtcgggg 6180 cggcaggccc tgccatagcc tcaggttact catatatact ttagattgat ttaaaacttc 6240 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc 6300 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt 6360 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac 6420 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct 6480 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact 6540 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg 6600 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata 6660 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga 6720 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag 6780 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg 6840 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac 6900 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca 6960 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg 7020 cgttatcccc tgattctgtg gataaccgta ttaccgccat gcat 7064 <210> 11 <211> 197 <212> PRT <213> Artificial Sequence <220> <223> RBD_SARS-CoV-2-WT <400> 11 Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala 1 5 10 15 Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp 20 25 30 Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr 35 40 45 Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr 50 55 60 Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro 65 70 75 80 Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp 85 90 95 Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys 100 105 110 Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn 115 120 125 Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly 130 135 140 Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu 145 150 155 160 Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr 165 170 175 Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val 180 185 190 Cys Gly Pro Lys Lys 195 <210> 12 <211> 591 <212> DNA <213> Artificial Sequence <220> <223> RBD_SARS-CoV-2-WT <400> 12 actaatcttt gtccgttcgg tgaggttttt aacgcgacaa ggtttcgctag tgtatatgct 60 tttaccattact tattagtgctc tttaccattact tattagtagctc tatta tagtgctc 120 tattaatcttt ttacggcgtt tctccaacga agctgaatga tctctgtttt 180 acgaacgtgt atgctgactc tttcgttata cggggggacg aagtgagaca gatagcacca 240 ggtcagactg ggaagatagc ggattacaac tataagttgc ccgatgattt tacggggtgc 300 gtaatcgcat ggaactcaaa caacctcgac tccaaagtag gtggtaatta taattacttg 360 tatcgcctgt ttcgaaagag caatttgaag ccttttgagc gggatatttc aaccgaaatt 420 taccaagcag gcagtacgcc atgtaacgga gtagagggat ttaattgcta ctttcctctt 480 caatcttatg gctttcaacc aacaaacgga gt ggggtatc aaccttatag agtggtagta 540 ttgtccttttg agctcctcca cgccccggct acagtttgtg ggcccaaaaa g 591 <210> 13 <211> 197 <212> PRT <213> Artificial Sequence <220> <223> RBD_SARS-CoV-2-E484P <400 Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala 1 5 10 15 Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp 20 25 30 Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr 35 40 45 Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr 50 55 60 Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro 65 70 75 80 Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp 85 90 95 Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys 100 105 110 Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn 115 120 125 Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly 130 135 140 Ser Thr Pro Cys Asn Gly Val Pro Gly Phe Asn Cys Tyr Phe Pro Leu 145 150 155 160 Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr 165 170 175 Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val 180 185 190 Cys Gly Pro Lys Lys 195 <210> 14 <211> 591 <212> DNA <213> Artificial Sequence <220> <223> RBD_SARS-CoV-2-E484P <400> 14 actaatcttt gtccgttcgg tgaggttttt aacgcgacaa ggttcgctag 60 tgtg agagaatctc caattgcgta gctgattact ccgttctcta taacagtgcg 120 tccttttcaa cctttaagtg ttacggcgtt tctccaacga agctgaatga tctctgtttt 180 acgaacgtgt atgctgactc tttcgttata cggggggacg aagtgagaca gatagcacca 240 ggtcagactg ggaagatagc ggattacaac tataagttgc ccgatgattt tacggggtgc 300 gtaatcgcat ggaactcaaa caacctcgac tccaaagtag gtggtaatta taattacttg 360 tatcgcctgt ttcgaaagag caatttgaag ccttttgagc gggatatttc aaccgaaatt 420 taccaagcag gcagtacgcc atgtaacgga gtaccgggat ttaattgcta ctttcctctt 480 caatcttatg gctttcaacc aacaaacgga gtggggtatc aaccttatag agtggtagta 540 ttgtcctttg agctcctcca cgccccggct acagtttgtg ggcccaaaaa g 591 <210> 15 <211> 197 <212> PRT <15> Artificial Sequence <220> <223> RBD_SARS-CoV-2-F486L <223> RBD_SARS-CoV Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala 1 5 10 15 Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp 20 25 30 Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr 35 40 45 Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr 50 55 60 Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro 65 70 75 80 Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp 85 90 95 Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys 100 105 110 Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn 115 120 125 Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly 130 135 140 Ser Thr Pro Cys Asn Gly Val Glu Gly Leu Asn Cys Tyr Phe Pro Leu 145 150 155 160 Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr 165 170 175 Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val 180 185 190 Cys Gly Pro Lys Lys 195 <210> 16 <211> 591 <212> DNA <213> Artificial Sequence <220> <223> RBD_SARS-CoV-2-F486L <400> 16 actaatcttt gtccgttcgg tgaggttttt aacgcgacaa ggttcgctag tgtatatgct 60 tggaaccgaa agagaatctc caattgcgta gctgattact ccgttctcta taacagtgcg 120 tccttttcaa cctttaagtg ttacggcgtt tctccaacga agctgaatga tctctgtttt 180 acgaacgtgt atgctgactc tttcgttata cggggggacg aagtgagaca gatagcacca 240 ggtcagactg ggaagatagc ggattacaac tataagttgc ccgatgattt tacggggtgc 300 gtaatcgcat ggaactcaaa caacctcgac tccaaagtag gtggtaatta taattacttg 360 tatcgcctgt ttcgaaagag caatttgaag ccttttgagc gggatatttc aaccgaaatt 420 taccaagcag gcagtacgcc atgtaacgga gtagagggac ttaattgcta ctttcctctt 480 caatcttatg gctttcaacc aacaaacgga gtggggtatc aaccttatag agtggtagta 540 ttgtccttttg agct cctcca 210 cgccccggct acagtta <213> WT> 212 Serial 400 220 cgccccggct acagtttt 211 <213 <ggcc> Artificial 400 Thr Ile Glu Glu Gln Ala Lys Thr P he Leu Asp Lys Phe Asn His 1 5 10 15 Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp Asn Tyr 20 25 30 Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn Ala Gly 35 40 45 Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala Gln Met 50 55 60 Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln Leu Gln 65 70 75 80 Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys Ser Lys 85 90 95 Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser Thr Gly 100 105 110 Lys Val Cys Asn Pro Asp Asn Pro 115 120 <210> 18 <211> 360 <212> DNA <213 > Artificial Sequence <220> <223> ACE2-WT <400> 18 tccaccattg aggaacaggc caagacattt ttggacaagt ttaaccacga agccgaagac 60 ctgttctatc aaagttcact tgcttcttgg aattataaca ccaatattac tgaagagaat 120 gtccaaaaca tgaataatgc tggggacaaa tggtctgcct ttttaaagga acagtccaca 180 cttgcccaaa tgtatccact acaagaaatt cagaatctca cagtcaagct tcagctgcag 240 gctcttcagc aaaatgggtc ttcagtgctc tcagaagaca agagcaaacg gttgaacaca 300 attctaaata caatgagcac catctacagt actggaaaag tttgtaaccc agataatcca 360 360 <210> 19 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> ACE2-M82K <400> 19 Ser Thr Ile Glu GluThr Pheln Ala Lys Leu Asp Lys Phe Asn His 1 5 10 15 Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp Asn Tyr 20 25 30 Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn Ala Gly 35 40 45 Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala Gln Lys 50 55 60 Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln Leu Gln 65 70 75 80 Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys Ser Lys 85 90 95 Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser Thr Gly 100 105 110 Lys Val Cys Asn Pro Asp Asn Pro 115 120 <210> 20 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> ACE2-M82K <400> 20 tccaccattg aggaacaggc caagacattt ttggacaagt ttaaccacga agccgaagac 60 ctgttctatc aaagttcact tgcttcttgg aattataaca ccaat attac tgaagagaat 120 gtccaaaaca tgaataatgc tggggacaaa tggtctgcct ttttaaagga acagtccaca 180 cttgcccaaa agtatccact acaagaaatt cagaatctca cagtcaagct tcagctgcag 240 gctcttcagc aaaatgggtc ttcagtgctc tcagaagaca agagcaaacg gttgaacaca 300 attctaaata caatgagcac catctacagt actggaaaag tttgtaaccc agataatcca 360 360 <210> 21 <211> 29 <212> DNA <213> Artificial Sequence < 220> <223> RBD_Forward <400> 21 caggctcgag actaatcttt gtccgttcg 29 <210> 22 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> RBD_Reverse <400> 22 aaatgcggcc gctctttttg ggccc 25 <210> 23 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> E484P_Forward <400> 23 acgccatgta acggagtacc gggatttaat tgc 33 <210> 24 <211> 33 <212> DNA <213> Artificial Sequence <220> < 223> E484P_Reverse <400> 24 gcaattaaat cccggtactc cgttacatgg cgt 33 <210> 25 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> F486L_Forward <400> 25 aacggagtag agggacttaa ttgctacttt cct 33 <210> 26 < 211> 33 <212> DNA <213> Artificial Sequence <220> <223> F4 86L_Reverse <400> 26 aggaaagtag caattaagtc cctctactcc gtt 33 <210> 27 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> ACE2-WT_Forward <400> 27 catgtgtaca agtccaccat tgaggaacag 30 <210> 28 <211 > 28 <212> DNA <213> Artificial Sequence <220> <223> ACE2-WT_Reverse <400> 28 gtacttcgaa aaaggaggtc tgaacatc 28 <210> 29 <211> 21 <212> DNA <213> Artificial Sequence <220> <223 > ACE2-M82K_Forward <400> 29 cttgcccaaa agtatccact a 21 <210> 30 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> ACE2-M82K_Reverse<400> 30 tagtggatac ttttgggcaa g 21

Claims (15)

fluorescence donors;
Receptor binding domain of a spike protein derived from coronaviridae; and
A fluorescence resonance energy transfer (FRET) biosensor comprising a fluorescence acceptor, comprising:
The biosensor is characterized in that consisting of the amino acid sequence of SEQ ID NO: 1, the biosensor.
The method of claim 1,
The biosensor is characterized in that for measuring the interaction between the receptor binding domain (RBD) of the spike protein and ACE2 (Angiotensin-Converting Enzyme 2), the biosensor.
The method of claim 1,
The biosensor is characterized in that it further comprises a fusion protein composed of an ACE2 protein and a fluorescent protein.
The method of claim 1,
The coronavirus family SARS-CoV-2, characterized in that the biosensor.
The method of claim 1,
The fluorescent donor and the fluorescent acceptor are each cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), characterized in that the biosensor.
6. The method of claim 5,
The cyan fluorescent protein is Turquoise2-GL, mTurquoise, mTFP, ECFP, CyPet, characterized in that any one selected from the group consisting of ECGFP, biosensor.
6. The method of claim 5,
The yellow fluorescent protein is any one selected from the group consisting of YPet, mEYFP, SEYFP, phiYFP, Citrine, mCitrine, Venus, mVenus, iq-mVenus, cp50Venus, cp157Venus, cp172Venus, cp195Venus, and cp229Venus. sensor.
4. The method of claim 3,
The fluorescent protein is characterized in that any one selected from the group consisting of mScarlet, mScarlet-i, mcherry, DsRed, mRFP, TagRFP, mGrape1, mKate, mRuby and FusionRed, the biosensor.
delete 4. The method of claim 3,
The fusion protein is characterized in that consisting of the amino acid sequence of SEQ ID NO: 3, biosensor.
A composition for screening a binding inhibitor of a spike protein and ACE2 derived from the coronaviridae, comprising the biosensor of any one of claims 1 to 8 and 10.
A method for screening a binding inhibitor of a spike protein and ACE2 derived from coronaviridae, comprising the steps of:
(a) transfecting the vector expressing the biosensor of any one of claims 1 to 8 and 10 into cells expressing Angiotensin-Converting Enzyme 2 (ACE2);
(b) treating the transfected cells with a test substance; and
(c) measuring the ratio of the emission intensity of the fluorescent receptor to the emission intensity of the fluorescent donor in the cells treated with the test substance.
13. The method of claim 12,
The screening method, characterized in that the coronavirus family SARS-CoV-2.
13. The method of claim 12,
The cell is characterized in that to express ACE2 endogenously, or transfected with the fusion protein expression vector of claim 3 to express the ACE2 protein, the screening method.
13. The method of claim 12,
In the method, when the ratio value is decreased in the cells treated with the test substance compared to the ratio value of the emission intensity of the fluorescent receptor to the emission intensity of the fluorescent donor in the control group not treated with the test substance, the test substance is Screening method, characterized in that it further comprises the step of selecting with a binding inhibitor.

Images