CN114606218B - Coronavirus neutralizing effector protein and application thereof - Google Patents

Abstract

The application discloses coronavirus neutralizing effector protein and application thereof. The application provides a protein, which is a protein with the same function and more than 80% of identity through substitution and/or deletion and/or addition of one or more amino acid residues or through connection of protein labels on the 1 st-740 th site of an amino acid sequence shown as SEQ ID No. 1. The C4-2 protein provided by the application can be combined with the coronavirus S protein and inhibit the combination of the coronavirus S protein and the ACE2receptor of human cells, so that the coronavirus can be effectively neutralized, and the application is proved at the cellular level. Meanwhile, the corresponding DNA sequence is favorable for soluble expression in a human cell line, and provides a new direction for the treatment of coronaviruses.

Description

Coronavirus neutralizing effector protein and application thereof

Technical Field

The application relates to the field of biotechnology, in particular to coronavirus neutralizing effector protein and application thereof.

Background

Since the 21 st century, humans experienced three times atypical pneumonia (SARS), middle East Respiratory Syndrome (MERS), new coronavirus pneumonia (covd-19) causing severe respiratory symptoms and high mortality coronavirus pandemics. Among them, new coronavirus pneumonia in the late 2019 outbreak was caused by new coronavirus (SARS-CoV-2,severe acute respiratory syndrome-related coronavirus), and existing research evidence has shown that SARS-CoV-2 is the easiest of the three coronaviruses to spread.

The S protein is a structural protein on the surface of a coronavirus membrane. The main body of the S protein is exposed outside the virus phospholipid bilayer membrane structure, a trimer structure is formed on the surface of the mature virus membrane structure, and similar to other coronaviruses, the S protein of SARS-CoV-2 can mediate the initial process of virus invasion into cells through interaction with host cell surface receptors. After binding to the receptor protein of the host cell, the S protein can be recognized and cleaved by furin and transmembrane protease serine 2 (Transmembrane protease serine, TMPRSS2) to expose the membrane fusion domain, thereby effecting fusion of the viral membrane structure and the host cell membrane. On the other hand, the S protein is capable of mediating endocytosis by binding to cellular receptors, and the cells are encapsulated in vesicles to release genetic material in the cytoplasm.

Angiotensin converting enzyme 2 (ACE 2) is a human cell receptor membrane protein of several coronaviruses and is considered as a potential target for coronavirus therapy. Taking a novel coronavirus SARS-CoV-2 as an example, the Receptor Binding Domain (RBD) of the spike (S) protein of the virus and ACE2 are mutually identified and combined, so that virus particles are anchored on the surface of cells, and the processes of cell membrane fusion and the like are carried out to invade the cells. Thus, ACE2 can be an important target for coronavirus therapy, including SARS and new coronaviruses, and it is important to find new therapeutic approaches for coronavirus therapy by finding inhibitors of coronavirus S protein and ACE2 binding.

For the biological process of coronavirus cell invasion, inhibitors currently reported or under investigation fall roughly into two categories: the first is a small molecule. Including protease inhibitors such as boceprevir and acetylneuraminic acid. These small molecules prevent membrane fusion of the virus and cells by inhibiting the proteolytic cleavage of the S protein by proteases on the cell surface. The second class is monoclonal antibodies. Such as monoclonal antibody cocktail REGEN-COV from Zymomonas, bamlanivimab and Ecesevelimab from Lily, S309, cilgavimab and VHH-72, etc.

Both classes of coronavirus inhibitors have their respective drawbacks: small molecule inhibitors are less specific and therefore inhibit other similar physiological activities when inhibiting coronavirus targets. Excessive use of large-scale monoclonal antibodies can lead to a tendency of the virus to generate more immune escape, possibly failing to work with new mutant strains.

Disclosure of Invention

The application aims to provide a coronavirus neutralizing effector protein and application thereof.

In a first aspect, the application claims a protein.

The protein claimed by the application is named as C4-2 and is any one of the following:

(A1) A protein with an amino acid sequence shown in positions 1-740 of SEQ ID No. 1;

(A2) A protein having the same function by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence defined in (A1);

(A3) A protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity with the amino acid sequence defined in (A1) or (A2) and having the same function;

(A4) A protein obtained by ligating the N-terminal and/or C-terminal of the protein defined in any one of (A1) to (A3) with a protein tag.

Among the above proteins, the protein tag (protein-tag) refers to a polypeptide or protein that is fusion expressed together with a target protein by using a DNA in vitro recombination technique, so as to facilitate the expression, detection, tracing and/or purification of the target protein. The protein tag may be a Flag tag, his tag, MBP tag, HA tag, myc tag, GST tag, and/or SUMO tag, etc.

In the above proteins, the identity refers to the identity of amino acid sequences. The identity of amino acid sequences can be determined using homology search sites on the internet, such as BLAST web pages of the NCBI homepage website. For example, in advanced BLAST2.1, the identity of a pair of amino acid sequences can be searched for by using blastp as a program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as Matrix, setting Gap existence cost, per residue gap cost and Lambda ratio to 11,1 and 0.85 (default values), respectively, and calculating, and then obtaining the value (%) of the identity.

In the above protein, the homology of 95% or more may be at least 96%, 97% or 98% identical. The 90% or more homology may be at least 91%, 92%, 93%, 94% identical. The 85% or more homology may be at least 86%, 87%, 88%, 89% identical. The 80% or more homology may be at least 81%, 82%, 83%, 84% identical.

In a second aspect, the application claims a fusion protein.

The fusion protein claimed in the present application is formed by fusing the protein described in the first aspect and an Fc segment of an antibody.

Wherein the antibody Fc fragment fusion may be fused at the C-terminus or the N-terminus of the protein described in the first aspect.

Further, the antibody Fc fragment may be an Fc fragment of an IgG antibody.

Further, the antibody may be a human antibody or a mouse antibody.

In a specific embodiment of the application, the Fc segment of the antibody is the Fc segment of a mouse IgG antibody, and the amino acid sequence of the Fc segment is shown in 741-970 of SEQ ID No. 1.

More specifically, the amino acid sequence of the fusion protein is shown as SEQ ID No. 1.

In a third aspect, the application claims nucleic acid molecules encoding the proteins described in the first aspect above or the fusion proteins described in the second aspect above.

Further, the nucleic acid molecule encoding the protein described in the first aspect of the foregoing may be any one of the following:

(B1) A DNA molecule shown in positions 1-2220 of SEQ ID No. 2;

(B2) A DNA molecule which hybridizes under stringent conditions to a DNA molecule as defined in (B1) and which encodes a protein as described in the first aspect hereinbefore;

(B3) A DNA molecule having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology to the DNA sequence defined in (B1) or (B2) and encoding the protein described in the first aspect above.

Further, in the nucleic acid molecule encoding the fusion protein, the nucleic acid molecule encoding the Fc segment of the mouse IgG antibody may be the DNA molecule shown at positions 2221-2910 of SEQ ID No. 2.

More specifically, the nucleic acid molecule encoding the fusion protein may be any one of the following:

(C1) A DNA molecule shown in SEQ ID No. 2;

(C2) A DNA molecule which hybridizes under stringent conditions to a DNA molecule as defined in (C1) and which encodes a fusion protein as described in the second aspect hereinbefore;

(C3) A DNA molecule having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology to the DNA sequence defined in (C1) or (C2) and encoding the fusion protein described in the second aspect of the foregoing.

In the above nucleic acid molecule, the stringent conditions may be as follows: 50℃in 7% Sodium Dodecyl Sulfate (SDS), 0.5M Na ₃ PO ₄ Hybridization with 1mM EDTA, rinsing in 2 XSSC, 0.1% SDS at 50 ℃; the method can also be as follows: 50℃in 7% SDS, 0.5M Na ₃ PO ₄ Hybridization with 1mM EDTA, rinsing in 1 XSSC, 0.1% SDS at 50 ℃; the method can also be as follows: 50℃in 7% SDS, 0.5M Na ₃ PO ₄ Hybridization with 1mM EDTA, rinsing in 0.5 XSSC, 0.1% SDS at 50 ℃; the method can also be as follows: 50℃in 7% SDS, 0.5M Na ₃ PO ₄ Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 50 ℃; the method can also be as follows: 50℃in 7% SDS, 0.5M Na ₃ PO ₄ Hybridization with 1mM EDTA in a mixed solution at 65℃in 0.1 XSSC, 0.Rinsing in 1% SDS; the method can also be as follows: hybridization was performed in a solution of 6 XSSC, 0.5% SDS at 65℃and then washed once with 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS.

In the above nucleic acid molecules, homology refers to the identity of nucleotide sequences. The identity of nucleotide sequences can be determined using homology search sites on the internet, such as BLAST web pages of the NCBI homepage website. For example, in advanced BLAST2.1, by using blastp as a program, the Expect value is set to 10, all filters are set to OFF, BLOSUM62 is used as Matrix, gap existence cost, per residue gap cost and Lambda ratio are set to 11,1 and 0.85 (default values), respectively, and identity of a pair of nucleotide sequences is searched for and calculated, and then the value (%) of identity can be obtained.

In the nucleic acid molecule, the homology of 95% or more may be at least 96%, 97% or 98% identical. The 90% or more homology may be at least 91%, 92%, 93%, 94% identical. The 85% or more homology may be at least 86%, 87%, 88%, 89% identical. The 80% or more homology may be at least 81%, 82%, 83%, 84% identical.

In a fourth aspect, the application claims an expression cassette or recombinant vector or recombinant bacterium or transgenic cell line comprising a nucleic acid molecule as described in the third aspect above.

In a specific embodiment of the present application, the recombinant vector is specifically a recombinant vector obtained by inserting the nucleic acid molecule (SEQ ID No. 2) into the multiple cloning site (e.g., nheI and XbaI) of pcDNA3.1.

In a specific embodiment of the application, the transgenic cell line is in particular obtained after introduction of a nucleic acid molecule (SEQ ID No. 2) into 293F cells. Further, it is introduced in the form of the recombinant vector.

In a fifth aspect, the application claims an anti-coronavirus agent.

The anti-coronavirus agent as claimed in the present application comprises as an active ingredient the protein as described in the first aspect above or the fusion protein as described in the second aspect above.

The medicine may further contain pharmaceutically acceptable excipients, diluents or carriers, etc. as required.

In a sixth aspect, the application claims a method of preparing a fusion protein as described in the second aspect of the foregoing.

The method of preparing a fusion protein as claimed in the second aspect of the present application may comprise the steps of: cloning the nucleic acid molecule encoding the fusion protein to a pcDNA3.1 vector to obtain a recombinant vector; introducing the recombinant vector into 293F cells to obtain a transgenic cell line; culturing the transgenic cell line for 48 hours, centrifugally collecting cell culture supernatant, and performing affinity chromatography on the supernatant to obtain the fusion protein in the second aspect.

In a seventh aspect, the application claims any of the following applications:

(D1) Use of a protein as described in the first aspect hereinbefore or a fusion protein as described in the second aspect hereinbefore or a nucleic acid molecule as described in the third aspect hereinbefore or an expression cassette or recombinant vector or recombinant bacterium or transgenic cell line as described in the fourth aspect hereinbefore for the manufacture of an anti-coronavirus medicament;

(D2) Use of a protein as described in the first aspect hereinbefore or a fusion protein as described in the second aspect hereinbefore or a nucleic acid molecule as described in the third aspect hereinbefore or an expression cassette or recombinant vector or recombinant bacterium or transgenic cell line as described in the fourth aspect hereinbefore or a medicament as described in the fifth aspect hereinbefore for the preparation of a product capable of neutralising coronavirus;

(D3) Use of a protein as described in the first aspect hereinbefore or a fusion protein as described in the second aspect hereinbefore or a nucleic acid molecule as described in the third aspect hereinbefore or an expression cassette or recombinant vector or recombinant bacterium or transgenic cell line as described in the fourth aspect hereinbefore in the manufacture of a reagent for detecting coronavirus S protein;

(D4) Use of a protein as described in the first aspect hereinbefore or a fusion protein as described in the second aspect hereinbefore or a nucleic acid molecule as described in the third aspect hereinbefore or an expression cassette or recombinant vector or recombinant bacterium or transgenic cell line as described in the fourth aspect hereinbefore for the preparation of a detection reagent capable of binding to the RBD domain of the coronavirus S protein.

In the present application, the coronavirus may be SARS-CoV-2.

In the present application, the coronavirus S protein may be an S protein derived from any of SARS-CoV-2: wild type SARS-CoV-2, alpha epidemic strain of SARS-CoV-2, beta epidemic strain of SARS-CoV-2, gamma epidemic strain of SARS-CoV-2, delta epidemic strain of SARS-CoV-2.

The application carries out the combination of a plurality of point mutations and the mouse IgG-Fc modification of the protein C end on the angiotensin converting enzyme 2 (ACE 2) to obtain the protein mutant individual C4-2 with stronger affinity with the novel coronavirus S protein RBD, which has stable molecular structure, molecular weight of about 114kDa, can carry out soluble expression in a human cell line and has higher expression quantity. The application has the beneficial effects that: the C4-2 protein provided by the application can be combined with the coronavirus S protein and inhibit the combination of the coronavirus S protein and the ACE2receptor of human cells, so that the coronavirus is effectively neutralized and proved on the cellular level. While its corresponding DNA sequence facilitates soluble expression in human cell lines. Overcomes the problem of poor specificity of small molecule inhibitors of coronaviruses and provides a new direction for the treatment of coronaviruses.

Drawings

FIG. 1 shows a vector map of pCDNA3.1.

FIG. 2 is a diagram showing SDS-PAGE results of C4-2 Protein elution purification after Protein G purification. Lane 2 is the C4-2 protein, M is marker, and the rest are irrelevant plots.

FIG. 3 is a graph showing the results of in vitro activity measurement of C4-2 protein;

FIG. 4 is a graph showing SPR results for the C4-2 protein and the SARS-CoV-2S protein RBD.

FIG. 5 is a graph showing the experimental results of the C4-2 protein in HEK293T-ACE2 cells and various epidemic strains of SARS-CoV-2.

Detailed Description

The following detailed description of the application is provided in connection with the accompanying drawings that are presented to illustrate the application and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the application in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

Example 1 preparation of ACE2 mutant protein C4-2

1. Construction of recombinant expression vector pCDNA3.1/C4-2

The application obtains ACE2 mutant protein C4-2 by carrying out point mutation on the 30 th, 79 th, 322 th and 475 th sites of an angiotensin converting enzyme 2 (ACE 2) amino acid sequence and then fusing the ACE2 mutant protein with an Fc segment of a mouse IgG antibody, and the amino acid sequence of the ACE2 mutant protein C4-2 is shown as SEQ ID No. 1.

According to the amino acid sequence of C4-2 protein, designing and artificially synthesizing eukaryotic expression DNA sequence (SEQ ID No. 2), respectively adding NheI (GCTAGC) and XbaI (TCTAGA) double-restriction sites and homology arms at the front end and the rear end of the overlapping PCR on the basis of the DNA fragment shown in SEQ ID No.2, connecting to a pCDNA3.1 vector of double-restriction (NheI and XbaI) through Gibson Assembly, transferring into DH5 alpha thallus to amplify and extract plasmid, and obtaining recombinant expression vector pCDNA3.1/C4-2 after sequencing verification. FIG. 1 shows the framework sequence of pCDNA3.1.

The recombinant expression vector pCDNA3.1/C4-2 is described as: the recombinant plasmid obtained by inserting the DNA fragment shown in SEQ ID No.2 between the cleavage sites NheI and XbaI of the pCDNA3.1 vector.

2. Transfection of Large Scale culture/1L shake flask

293F cells (ThermoFisher) were cultured in suspension according to standard operating manual, typically 250ml cell culture shake flasks were started to culture volumes of 30ml to 100 ml. According to 0.5X10 ⁶ cell/ml inoculum size cells were inoculated into 300ml medium in 1L shake flasks. Incubating in a shaking incubator at 37℃and 120rpm with 5% carbon dioxide concentration until the cell density reached 1X 10 ⁶ cells/ml. Mu.g of DNA (i.e.the recombinant expression vector pCDNA3.1/C4-2 obtained in step one) was pipetted into 30ml of PBS and 1.2ml of filter sterilized PEI solution (0.5 mg/m)l) adding the mixture into PBS/DNA, standing for 20min, and adding cells. After transfection, incubation was performed for 48h in a shaker incubator. The cell culture supernatant was centrifuged at 3000g for 5min and the cell pellet was harvested.

3. Purification of protein complexes extracted from cell culture supernatants

The culture supernatant, which had been cultured for 48 hours after the transfection in the second step, was filtered through a 0.45 μm filter. The filtered supernatant was incubated with 1.25ml Protein G column (Cytiva) per liter, eluted with 10mM citric acid/200 mM NaCl buffer, then separated by Q-sepharose ion exchange column (Cytiva), loaded with 10mM phosphate buffer/200 mM NaCl, then purified by Sephacryal 200 gel chromatography column (Cytiva), loaded with 10mM phosphate buffer/150 mM NaCl buffer, and the eluate was the C4-2 stock solution.

10 μl of the concentrated protein sample was taken and added to a2 Xprotein loading buffer for electrophoresis detection. The protein was filtered through a 0.22 μm filter. FIG. 2 shows the C4-2 protein of interest extracted from 293F medium supernatant of transient transfection of 2L (8X 250 ml). As is clear from the SDS-PAGE electrophoresis of FIG. 2, the target protein was expressed at about 135kDa in the supernatant of the cell expressed by the C4-2 strain, the molecular weight of the target protein was close to the theoretical value (113 KD) (the actual molecular weight was slightly higher than the theoretical value due to the glycosylation process of the protein), and the purified target protein was substantially free of bands. Freezing the constructed expression strain to a refrigerator at-80 ℃ for standby. The purified protein yield of 1L medium was estimated to be approximately 1mg.

Example 2 measurement of in vitro Activity of ACE2 mutant protein C4-2

1. Construction of cell lines expressing 5S protein mutants of coronavirus by lentivirus transfection

The S proteins of the 5 SARS-CoV-2 are derived from: wild type SARS-CoV-2, alpha, beta, gamma of SARS-CoV-2 and delta epidemic strains. The amino acid sequence of the S protein from wild type SARS-CoV-2 (i.e., wuhan-Hu-1 strain) is identical to NCBI Reference Sequence:YP_009724390.1, the corresponding coding gene sequence is NCBI Reference Sequence:NC_045512.2 (21563..25384), NCBIGeneID is 43740568; the amino acid sequence of the S protein from the alpha-epidemic strain was changed as compared to YP_009724390.1 as follows: the amino acid residues 69-70 are deleted, the amino acid residue 144 is deleted, N501Y, A570D, D614G, P681H, T716I, S982A, D1118H, the corresponding coding genes are changed as follows compared with NC_045512.2 (21563..25384): nucleotide 205-210, nucleotide 430-432, tat from 1501-1503, gat from 1708-1710, ggt from 1840-1842, cat from 2041-2043, atc from 2146-2148, gcc from 2944-2946, and cac from 3352-3354; the amino acid sequence of the S protein from the beta-pop strain was changed as compared with YP_009724390.1 as follows: the amino acid residues 242-244 of L18F, D80A, D215G, K417N, E484K, N501Y, D614G, A701V, the corresponding coding gene changed as compared to NC_045512.2 (21563..25384): the 52 th to 54 th mutations are ttc, the 238 th to 240 th mutations are gcc, the 643 th to 645 th mutations are ggc, the 724 th to 732 th nucleotides are deleted, the 1249 th to 1251 th mutations are aat, the 1450 th to 1452 th mutations are aag, the 1501 th to 1503 th mutations are tat, the 1840 th to 1842 th mutations are ggt, and the 2101 th to 2103 th mutations are gta; the amino acid sequence of the S protein from the gamma-pop strain was changed as compared with YP_009724390.1 as follows: L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, T1027I, V1176F, the corresponding coding gene was altered as compared to NC_045512.2 (21563..25384): the 52 th to 54 th mutation is ttc, the 58 th to 60 th mutation is aac, the 76 th to 78 th mutation is tct, the 412 th to 414 th mutation is tac, the 568 th to 570 th mutation is agc, the 1249 th to 1251 th mutation is agc, the 1450 th to 1452 th mutation is aag, the 1501 th to 1503 th mutation is tat, the 1840 th to 1842 th mutation is ggt, the 1963 th to 1965 th mutation is tat, the 3079 th to 3081 th mutation is atc, and the 3526 th to 3528 th mutation is ttc; the amino acid sequence of the S protein from the delta epidemic was changed compared to YP_009724390.1 as follows: the deletion of amino acid residues 156-157 from T19R, G142D, R158G, L452R, T478K, D614G, P681R, D950N, the following changes occurred in the corresponding coding gene compared to nc_045512.2 (21563..25384): 55-57 is mutated to aga, 424-426 is mutated to gag, 466-471 is deleted, 472-474 is mutated to ggc, 1364-1356 is mutated to aga, 1432-1434 is mutated to aag, 1840-1842 is mutated to ggc, 2041-2043 is mutated to aga, 2848-2850 is mutated to aac.

Lentiviral packaging plasmids pCMV-dR8.91 (4. Mu.g, addgene: vector-database/2221 /), pCMV-VSV-G (1. Mu.g, addgene: 8454) were mixed with 5 lentiviral vector plasmids pWSLV03-SARS-CoV-2-Spike-mCherry (4. Mu.g) expressing SARS-CoV-Spike proteins (S-WT, S-alpha, S-beta, S-gamma and S-delta), respectively (the lentiviral vector plasmids were all pWSLV03 frameworks, the pLV 03 plasmid was harbored with mCherry sequence, the pLV 03 plasmid was shown as SEQ ID No.3, from Beijing Shang Lide Biotech Co., ltd., pWS03 was inserted into each strain S protein coding gene sequence by double cleavage at NotI and EcoRI sites, and was confirmed to be correct by sequencing) and transfection reagents PEI (30. Mu.l) were mixed in 3ml Glibco-OPLV MEM for 20min. The mixture was transferred to a pre-prepared 10cm dish 293T cell line (containing 7ml of medium with cell density controlled between 70% and 90%). After 6h, the medium was discarded and 10ml of fresh DMEM medium was added. After 48h, mCherry positive cells were collected using flow sorting.

2. Binding of ACE2 mutant protein C4-2 to SARS-CoV-2S protein expressing cell line

The mCherry positive cells collected in step one were cultured in an incubator at 37℃for 24 hours, the cells were digested with 0.25% of trypsin/EDTA, centrifuged at 1000rpm for 3 minutes, and the cells were resuspended in DMEM containing 1% FBS and added to 96-well plates. The mFc-labeled ACE2 mutant protein (i.e., C4-2 prepared in example 1) was formulated into 7 concentration gradients, up to 100nM, diluted 3-fold per gradient, and three parallel experiments were performed. In the experiment, the 96-well plate was centrifuged at 1000rmp for 3min to remove the medium, and the prepared protein was added to each well at a concentration of 100. Mu.l per well, and incubated in an incubator at 37℃for one hour. After the incubation was completed, the supernatant was removed by centrifugation at 1000rmp for 3min, rinsed once with 100. Mu.l PBS, and incubated in an incubator at 37℃for one hour with 100. Mu.l FITC-labeled anti-mouse IgG antibody (Biolegend, 1000 Xdilution) in the absence of light. Centrifugation at 1000rmp for 3min to remove supernatant, re-suspension with PBS followed by precipitation using LSRFortessa ^TM Cell Analyzer (BD) analyzed Cell fluorescence, calculated FITC mean fluorescence intensity (MFU) of mCherry positive cells,the results shown in FIG. 3 were obtained, and IC50 of C4-2 against different S protein expressing cell lines was obtained by the Reed-Muench method.

FIG. 3 shows the binding curves of ACE2 mutant protein C4-2 and different mutants of SARS-CoV-2, respectively, showing that the protein has obvious binding to cells, and this also demonstrates that C4-2 has strong binding capacity to S protein of various mutants.

Example 3 affinity assay of ACE2 mutant proteins C4-2 and SARS-CoV-2S protein RBD

For typical affinity determination, a Biacore 2000 instrument is used in the experiment, and the detection principle is based on the Surface Plasmon Resonance (SPR) technology, and can sensitively reflect the refractive index change of the chip surface. To study the interactions between molecules, one of the molecules is immobilized on the chip surface and the other molecule flows across the surface in solution. The response of SPR is proportional to the mass concentration change near the chip.

In a typical test, a CM5 chip was selected to measure the protein-protein interaction between soluble ACE2 protein and SARS-CoV-2 Spike-RBD. CM5 chips have moderate capacity and versatile properties based on a strategy of covalent coupling. The running buffer was HBS-EP (Cytiva). RBD-his (Yinqiao Shenzhou: 40592-V08H 105) (20 ng/. Mu.l) was dissolved in sodium acetate buffer (pH 4.5) and immobilized as a ligand on a chip at a flow rate of 5. Mu.l/min, stopped after about 30s, and the immobilization amount was about 60RU. The analyte was ACE2 mutant protein C4-2 at a concentration of from 100 to 1.56nM (2-fold dilution between each gradient) and the diluent was running buffer. The flow rate was set at 45 μl/min, the binding time was set at 180 seconds, and the dissociation time was set at 1800 seconds. After each cycle, the glycine solution was regenerated for 30 seconds at a flow rate of 30. Mu.l/min using a pH of 1.5.

The signal profile during the reaction is shown in FIG. 4. For binding of two proteins, to analyze their kinetic characteristics, it can be assumed that both fit a simple interaction model and that [ ACE2] (concentration of analyte) is constant, so the rate equation for the binding process is:

in the dissociation process, the rate equation is as follows:

we fit the binding curves for all concentration gradients to the data of table 1.

TABLE 1 molecular interaction parameters of C4-2 with Spike-RBD

Sample of	ka(1/Ms)	kd(1/s)	KD(nM)
				C4-2	1.01×10 5	4.62×10 -5	0.46

The ACE2 mutant protein C4-2 shows strong RBD affinity in the test, and can be seen that the protein has a relatively fast binding rate, and a dissociation curve shows that the binding of the protein is extremely stable. Furthermore, considering the combination and dissociation processes together, we can calculate the dissociation constant KD, which is much smaller than the nM magnitude and much higher than wild-type ACE2 (about 20nM. Refer to "Wrapp D, wang N, corbett KS, et al Cryo-EM structure of the 2019-nCoV spike in the prefusion Condition.science 2020;367 (6483): eabb2507.Glasgow A, glasgow J, limanta D, et al engineered ACE2receptor traps potently neutralize SARS-CoV-2.Proceedings of the National Academy of Sciences.2020;117 (45): 28046-28055." A.) which makes it a great advantage as a therapeutic drug.

This example demonstrates the strong affinity of the C4-2 protein for the Spike protein RBD, which exceeds the affinity of wild-type ACE2 for the Spike protein RBD in terms of KD.

EXAMPLE 4 ACE2 mutant proteins C4-2 and SARS-CoV-2 pseudovirus neutralization assay

The pseudovirus neutralization experiment uses Vesicular Stomatitis Virus (VSV) as a basic skeleton, and replaces the receptor binding protein G protein of the Vesicular Stomatitis Virus (VSV) with the Spike protein of SARS-CoV-2, so that the process of entering cells and being inhibited by drugs is simulated. Compared with in vitro experiments, the pseudo virus simulates the process of virus infection cells, and the experimental result is more real and reliable. The ACE2 mutant protein C4-2 is excellent in vitro experiments, and thus further experiments are required to confirm the therapeutic effect.

1. Preparation of pseudoviruses

Construction of SARS-CoV-2 pseudotype virus and mutant strain. The method comprises the following steps: the day before transfection, 293T cells were digested and adjusted to 5X 10 ⁵ To 7X 10 ⁵ concentration of cells/mL. Then, the cells in 15ml of medium were transferred to T75 cell medium and treated with 5% CO at 37 ℃ ₂ The cells were cultured overnight in an incubator. When the cells reached 70% -90% coverage, the supernatant was discarded and used at a concentration of 7X 10 ⁴ TCID ₅₀ A15 mL VSV-. DELTA.G-luciferase plasmid expression vector system (Kerafast: EH 1008) per mL was infected. Meanwhile, 30 μg of S protein expression plasmids of different epidemic strains (recombinant plasmids obtained by taking pCDNA3.1 of figure 1 as a vector and inserting all the several S protein coding genes between cleavage sites NheI and XbaI of the pCDNA3.1 vector) are respectively transferred into cells. Cells were incubated at 37℃with 5% CO ₂ Is cultured in an incubator of (a). After 6-8 hours, the cell supernatant was discarded and the cells were gently rinsed twice with PBS+1% FBS and 15mL fresh DMEM was added to the T75 cell culture flask. At 37℃and 5% CO ₂ After culturing in an incubator for 24 hours, collecting the culture containing the pseudotyped virusThe supernatant is filtered and split-packed to obtain VSV pseudotyped virus with S-WT, S-alpha, S-beta, S-gamma or S-delta on the surface of the virus (respectively representing wild type SARS-CoV-2, alpha, beta, gamma of SARS-CoV-2 and S protein of delta epidemic strain), and the VSV pseudotyped virus is frozen and stored at-70 ℃ for subsequent use.

2. Pseudovirus inhibition assay

After RT-PCR quantitative analysis of the pseudoviruses collected in the step one, each pseudotype virus was diluted to a titer of 2X 10 ⁵ TCID ₅₀ /mL, and 100. Mu.L was added to the 96-well cell culture plate. Wild-type ACE2 (Yinqiao China: 10108-H08H) and C4-2 protein samples were each subjected to a gradient dilution (3-fold dilution starting at 100nM, total of 8 gradients) in 96-well plates and added to corresponding wells to which the virus solution had been added. 8 control groups to which only the virus solution was added and 8 control groups to which only the cells were added were set on the well plate. Incubation at 37℃for 1 hour, wild-type ACE2 overexpressing cells (next holy bioscience (Shanghai): 41107ES 03) were digested with trypsin and incubated at 2X 10 ⁴ A cell concentration of 100. Mu.L was added to each well of the 96-well plate. After incubation for 24 hours in a 37 ℃ incubator containing 5% carbon dioxide, changes in luciferase gene expression were examined to assess the neutralization effect of wild-type ACE2 protein and C4-2 protein samples, respectively, on pseudovirus-infected ACE2 overexpressing cells. To each well 100 μl of luciferase substrate (perkinemer) was added, incubated for 2 minutes at room temperature, then transferred to a detection whiteboard, and measured using a photometer (perkinemer). Each group contained two replicates. EC was calculated for each sample using Reed-Muench method ₅₀ Values.

3. Results and analysis

As can be seen from FIG. 5, the ACE2 mutant protein C4-2 has a very strong inhibitory effect on all mutant pseudoviruses in the experiment, and EC is shown in Table 2 ₅₀ As a result of calculation, the inhibition effect of C4-2 on wild-type S protein pseudovirus is improved by more than 200 times compared with that of non-mutated ACE2 protein (EC ₅₀ Value comparison).

TABLE 2 results of Virus inhibition of wild type ACE2 and C4-2 in pseudoviruses of different novel coronastrains

EC 50 (ng/mL)	Wild type ACE2	C4-2
			Wild strain	>10000	205
alpha	780	103
			beta	2221	61
gamma	1010	39
			delta	1009	41

The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

<110> university of Beijing

<120> coronavirus neutralizing effector protein and use thereof

<130> GNCLN220643

<160> 3

<170> PatentIn version 3.5

<210> 1

<211> 970

<212> PRT

<213> Artificial sequence

<400> 1

Met Ser Ser Ser Ser Trp Leu Leu Leu Ser Leu Val Ala Val Thr Ala

1 5 10 15

Ala Gln Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Ile Lys Phe

20 25 30

Asn His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp

35 40 45

Asn Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn

50 55 60

Ala Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Trp Ala

65 70 75 80

Gln Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln

85 90 95

Leu Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys

100 105 110

Ser Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser

115 120 125

Thr Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu

130 135 140

Glu Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu

145 150 155 160

Arg Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu

165 170 175

Arg Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg

180 185 190

Ala Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu

195 200 205

Val Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu

210 215 220

Asp Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu

225 230 235 240

His Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile

245 250 255

Ser Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly

260 265 270

Arg Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys

275 280 285

Pro Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala

290 295 300

Gln Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu

305 310 315 320

Pro Val Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro

325 330 335

Gly Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly

340 345 350

Lys Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp

355 360 365

Phe Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala

370 375 380

Tyr Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe

385 390 395 400

His Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys

405 410 415

His Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn

420 425 430

Glu Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly

435 440 445

Thr Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe

450 455 460

Lys Gly Glu Ile Pro Lys Asp Gln Trp Met Phe Lys Trp Trp Glu Met

465 470 475 480

Lys Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr

485 490 495

Tyr Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe

500 505 510

Ile Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala

515 520 525

Leu Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile

530 535 540

Ser Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu

545 550 555 560

Gly Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala

565 570 575

Lys Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe

580 585 590

Thr Trp Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr

595 600 605

Asp Trp Ser Pro Tyr Ala Asp Gln Ser Ile Lys Val Arg Ile Ser Leu

610 615 620

Lys Ser Ala Leu Gly Asp Lys Ala Tyr Glu Trp Asn Asp Asn Glu Met

625 630 635 640

Tyr Leu Phe Arg Ser Ser Val Ala Tyr Ala Met Arg Gln Tyr Phe Leu

645 650 655

Lys Val Lys Asn Gln Met Ile Leu Phe Gly Glu Glu Asp Val Arg Val

660 665 670

Ala Asn Leu Lys Pro Arg Ile Ser Phe Asn Phe Phe Val Thr Ala Pro

675 680 685

Lys Asn Val Ser Asp Ile Ile Pro Arg Thr Glu Val Glu Lys Ala Ile

690 695 700

Arg Met Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asn Asp Asn

705 710 715 720

Ser Leu Glu Phe Leu Gly Ile Gln Pro Thr Leu Gly Pro Pro Asn Gln

725 730 735

Pro Pro Val Ser Met Asp Pro Lys Ser Ser Asp Lys Thr His Thr Cys

740 745 750

Pro Pro Cys Pro Ala Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro

755 760 765

Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys

770 775 780

Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp

785 790 795 800

Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu

805 810 815

Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met

820 825 830

His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser

835 840 845

Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly

850 855 860

Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln

865 870 875 880

Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe

885 890 895

Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu

900 905 910

Asn Tyr Lys Asn Thr Gln Pro Ile Met Asn Thr Asn Gly Ser Tyr Phe

915 920 925

Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn

930 935 940

Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr

945 950 955 960

Glu Lys Ser Leu Ser His Ser Pro Gly Lys

965 970

<210> 2

<211> 2910

<212> DNA

<213> Artificial sequence

<400> 2

atgtcaagct cttcctggct ccttctcagc cttgttgctg taactgctgc tcagtccacc 60

attgaggaac aggccaagac atttttgatt aagtttaacc acgaagccga agacctgttc 120

tatcaaagtt cacttgcttc ttggaattat aacaccaata ttactgaaga gaatgtccaa 180

aacatgaata atgctgggga caaatggtct gcctttttaa aggaacagtc cacatgggcc 240

caaatgtatc cactacaaga aattcagaat ctcacagtca agcttcagct gcaggctctt 300

cagcaaaatg ggtcttcagt gctctcagaa gacaagagca aacggttgaa cacaattcta 360

aatacaatga gcaccatcta cagtactgga aaagtttgta acccagataa tccacaagaa 420

tgcttattac ttgaaccagg tttgaatgaa ataatggcaa acagtttaga ctacaatgag 480

aggctctggg cttgggaaag ctggagatct gaggtcggca agcagctgag gccattatat 540

gaagagtatg tggtcttgaa aaatgagatg gcaagagcaa atcattatga ggactatggg 600

gattattgga gaggagacta tgaagtaaat ggggtagatg gctatgacta cagccgcggc 660

cagttgattg aagatgtgga acataccttt gaagagatta aaccattata tgaacatctt 720

catgcctatg tgagggcaaa gttgatgaat gcctatcctt cctatatcag tccaattgga 780

tgcctccctg ctcatttgct tggtgatatg tggggtagat tttggacaaa tctgtactct 840

ttgacagttc cctttggaca gaaaccaaac atagatgtta ctgatgcaat ggtggaccag 900

gcctgggatg cacagagaat attcaaggag gccgagaagt tctttgtatc tgttggtctt 960

cctgtaatga ctcaaggatt ctgggaaaat tccatgctaa cggacccagg aaatgttcag 1020

aaagcagtct gccatcccac agcttgggac ctggggaagg gcgacttcag gatccttatg 1080

tgcacaaagg tgacaatgga cgacttcctg acagctcatc atgagatggg gcatatccag 1140

tatgatatgg catatgctgc acaacctttt ctgctaagaa atggagctaa tgaaggattc 1200

catgaagctg ttggggaaat catgtcactt tctgcagcca cacctaagca tttaaaatcc 1260

attggtcttc tgtcacccga ttttcaagaa gacaatgaaa cagaaataaa cttcctgctc 1320

aaacaagcac tcacgattgt tgggactctg ccatttactt acatgttaga gaagtggagg 1380

tggatggtct ttaaagggga aattcccaaa gaccagtgga tgtttaagtg gtgggagatg 1440

aagcgagaga tagttggggt ggtggaacct gtgccccatg atgaaacata ctgtgacccc 1500

gcatctctgt tccatgtttc taatgattac tcattcattc gatattacac aaggaccctt 1560

taccaattcc agtttcaaga agcactttgt caagcagcta aacatgaagg ccctctgcac 1620

aaatgtgaca tctcaaactc tacagaagct ggacagaaac tgttcaatat gctgaggctt 1680

ggaaaatcag aaccctggac cctagcattg gaaaatgttg taggagcaaa gaacatgaat 1740

gtaaggccac tgctcaacta ctttgagccc ttatttacct ggctgaaaga ccagaacaag 1800

aattcttttg tgggatggag taccgactgg agtccatatg cagaccaaag catcaaagtg 1860

aggataagcc taaaatcagc tcttggagat aaagcatatg aatggaacga caatgaaatg 1920

tacctgttcc gatcatctgt tgcatatgct atgaggcagt actttttaaa agtaaaaaat 1980

cagatgattc tttttgggga ggaggatgtg cgagtggcta atttgaaacc aagaatctcc 2040

tttaatttct ttgtcactgc acctaaaaat gtgtctgata tcattcctag aactgaagtt 2100

gaaaaggcca tcaggatgtc ccggagccgt atcaatgatg ctttccgtct gaatgacaac 2160

agcctagagt ttctggggat acagccaaca cttggacctc ctaaccagcc ccctgtttcc 2220

atggatccga aatcctctga caaaactcac acatgcccac cgtgcccagc tccggaagta 2280

tcatctgtct tcatcttccc cccaaagccc aaggatgtgc tcaccattac tctgactcct 2340

aaggtcacgt gtgttgtggt agacatcagc aaggatgatc ccgaggtcca gttcagctgg 2400

tttgtagatg atgtggaggt gcacacagct cagacgcaac cccgggagga gcagttcaac 2460

agcactttcc gctcagtcag tgaacttccc atcatgcacc aggactggct caatggcaag 2520

gagttcaaat gcagggtcaa cagtgcagct ttccctgccc ccatcgagaa aaccatctcc 2580

aaaaccaaag gcagaccgaa ggctccacag gtgtacacca ttccacctcc caaggagcag 2640

atggccaagg ataaagtcag tctgacctgc atgataacag acttcttccc tgaagacatt 2700

actgtggagt ggcagtggaa tgggcagcca gcggagaact acaagaacac tcagcccatc 2760

atgaacacga atggctctta cttcgtctac agcaagctca atgtgcagaa gagcaactgg 2820

gaggcaggaa atactttcac ctgctctgtg ttacatgagg gcctgcacaa ccaccatact 2880

gagaagagcc tctcccactc tcctgggaaa 2910

<210> 3

<211> 7978

<212> DNA

<213> Artificial sequence

<400> 3

gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc 60

ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg 120

tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc 180

gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc cgtgtgtggt 240

tcccgcgggc ctggcctctt tacgggttat ggcccttgcg tgccttgaat tacttccacc 300

tggctgcagt acgtgattct tgatcccgag cttcgggttg gaagtgggtg ggagagttcg 360

aggccttgcg cttaaggagc cccttcgcct cgtgcttgag ttgaggcctg gcctgggcgc 420

tggggccgcc gcgtgcgaat ctggtggcac cttcgcgcct gtctcgctgc tttcgataag 480

tctctagcca tttaaaattt ttgatgacct gctgcgacgc tttttttctg gcaagatagt 540

cttgtaaatg cgggccaaga tctgcacact ggtatttcgg tttttggggc cgcgggcggc 600

gacggggccc gtgcgtccca gcgcacatgt tcggcgaggc ggggcctgcg agcgcggcca 660

ccgagaatcg gacgggggta gtctcaagct ggccggcctg ctctggtgcc tggcctcgcg 720

ccgccgtgta tcgccccgcc ctgggcggca aggctggccc ggtcggcacc agttgcgtga 780

gcggaaagat ggccgcttcc cggccctgct gcagggagct caaaatggag gacgcggcgc 840

tcgggagagc gggcgggtga gtcacccaca caaaggaaaa gggcctttcc gtcctcagcc 900

gtcgcttcat gtgactccac ggagtaccgg gcgccgtcca ggcacctcga ttagttctcg 960

agcttttgga gtacgtcgtc tttaggttgg ggggaggggt tttatgcgat ggagtttccc 1020

cacactgagt gggtggagac tgaagttagg ccagcttggc acttgatgta attctccttg 1080

gaatttgccc tttttgagtt tggatcttgg ttcattctca agcctcagac agtggttcaa 1140

agtttttttc ttccatttca ggtgtcgtga ggtgtcgtga gcatgcgatg actgatcatg 1200

accctcgagg tcgacggtat cgataagctc gcttcacgag attccagcag gtcgagggac 1260

ctaataactt cgtatagcat acattatacg aagttatatt aagggttcca agcttaagcg 1320

gccgcgtgga taaccgtatt accgccatgc atctacctag ggatggatcc ggaagcgaat 1380

tcacgcgtga gggcagagga agtctactaa catgcggtga cgtggaggag aatcccggcc 1440

ctgacatgtt gatggccatc atcaaggagt tcatgcgctt caaggtgcac atggagggct 1500

ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc tacgagggca 1560

cccagaccgc caagctgaag gtgaccaagg gtggccccct gcccttcgcc tgggacatcc 1620

tgtcccctca gttcatgtac ggctccaagg cctacgtgaa gcaccccgcc gacatccccg 1680

actacttgaa gctgtccttc cccgagggct tcaagtggga gcgcgtgatg aacttcgagg 1740

acggcggcgt ggtgaccgtg acccaggact cctccctcca ggacggcgag ttcatctaca 1800

aggtgaagct gcgcggcacc aacttcccct ccgacggccc cgtaatgcag aagaaaacca 1860

tgggctggga ggcctcctcc gagcggatgt accccgagga cggcgccctg aagggcgaga 1920

tcaagcagag gctgaagctg aaggacggcg gccactacga cgctgaggtc aagaccacct 1980

acaaggccaa gaagcccgtg cagctgcccg gcgcctacaa cgtcaacatc aagttggaca 2040

tcacctccca caacgaggac tacaccatcg tggaacagta cgaacgcgcc gagggccgcc 2100

actccaccgg cggcatggac gagctgtaca agtagtaaca attcgtcgag ggacctaata 2160

acttcgtata gcatacatta tacgaagtta tacatgttta agggttccgg ttccactagg 2220

tacaattcga tatcaagctt atcgataatc aacctctgga ttacaaaatt tgtgaaagat 2280

tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc 2340

ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct 2400

ggttgctgtc tctttatgag gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca 2460

ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc caccacctgt cagctccttt 2520

ccgggacttt cgctttcccc ctccctattg ccacggcgga actcatcgcc gcctgccttg 2580

cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga 2640

aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac ctggattctg cgcgggacgt 2700

ccttctgcta cgtcccttcg gccctcaatc cagcggacct tccttcccgc ggcctgctgc 2760

cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt 2820

gggccgcctc cccgcatcga taccgtcgac ctcgatcgag acctagaaaa acatggagca 2880

atcacaagta gcaatacagc agctaccaat gctgattgtg cctggctaga agcacaagag 2940

gaggaggagg tgggttttcc agtcacacct caggtacctt taagaccaat gacttacaag 3000

gcagctgtag atcttagcca ctttttaaaa gaaaaggggg gactggaagg gctaattcac 3060

tcccaacgaa gacaagatat ccttgatctg tggatctacc acacacaagg ctacttccct 3120

gattggcaga actacacacc agggccaggg atcagatatc cactgacctt tggatggtgc 3180

tacaagctag taccagttga gcaagagaag gtagaagaag ccaatgaagg agagaacacc 3240

cgcttgttac accctgtgag cctgcatggg atggatgacc cggagagaga agtattagag 3300

tggaggtttg acagccgcct agcatttcat cacatggccc gagagctgca tccggactgt 3360

actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac 3420

ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg 3480

ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct 3540

agcagcatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 3600

gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 3660

aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 3720

gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 3780

ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 3840

cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 3900

ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 3960

actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 4020

tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca 4080

gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 4140

ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 4200

cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 4260

ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt 4320

tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc 4380

agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc 4440

gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata 4500

ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg 4560

gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc 4620

cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct 4680

gcaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa 4740

cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt 4800

cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca 4860

ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac 4920

tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca 4980

atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt 5040

tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc 5100

actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca 5160

aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata 5220

ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc 5280

ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc 5340

cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc cgatccccta tggtgcactc 5400

tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct gcttgtgtgt 5460

tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg 5520

acaattgcat gaagaatctg cttagggtta ggcgttttgc gctgcttcgc gatgtacggg 5580

ccagatatac gcgttgacat tgattattga ctagttatta atagtaatca attacggggt 5640

cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc 5700

ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag 5760

taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc 5820

acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg 5880

gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc 5940

agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca 6000

atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca 6060

atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg 6120

ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagcgcgt 6180

tttgcctgta ctgggtctct ctggttagac cagatctgag cctgggagct ctctggctaa 6240

ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgtgt 6300

gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta gtcagtgtgg 6360

aaaatctcta gcagtggcgc ccgaacaggg acttgaaagc gaaagggaaa ccagaggagc 6420

tctctcgacg caggactcgg cttgctgaag cgcgcacggc aagaggcgag gggcggcgac 6480

tggtgagtac gccaaaaatt ttgactagcg gaggctagaa agagagagat gggtgcgaga 6540

gcgtcagtat taagcggggg agaattagat cgcgatggga aaaattcggt taaggccagg 6600

gggaaagaaa aaatataaat taaaacatat agtatgggca agcagggagc tagaacgatt 6660

cgcagttaat cctggcctgt tagaaacatc agaaggctgt agacaaatac tgggacagct 6720

acaaccatcc cttcagacag gatcagaaga acttagatca ttatataata cagtagcaac 6780

cctctattgt gtgcatcaaa ggatagagat aaaagacacc aaggaagctt tagacaagat 6840

agaggaagag caaaacaaaa gtaagaccac cgcacagcaa gcggccggcc gctgatcttc 6900

agacctggag gaggagatat gagggacaat tggagaagtg aattatataa atataaagta 6960

gtaaaaattg aaccattagg agtagcaccc accaaggcaa agagaagagt ggtgcagaga 7020

gaaaaaagag cagtgggaat aggagctttg ttccttgggt tcttgggagc agcaggaagc 7080

actatgggcg cagcgtcaat gacgctgacg gtacaggcca gacaattatt gtctggtata 7140

gtgcagcagc agaacaattt gctgagggct attgaggcgc aacagcatct gttgcaactc 7200

acagtctggg gcatcaagca gctccaggca agaatcctgg ctgtggaaag atacctaaag 7260

gatcaacagc tcctggggat ttggggttgc tctggaaaac tcatttgcac cactgctgtg 7320

ccttggaatg ctagttggag taataaatct ctggaacaga tttggaatca cacgacctgg 7380

atggagtggg acagagaaat taacaattac acaagcttaa tacactcctt aattgaagaa 7440

tcgcaaaacc agcaagaaaa gaatgaacaa gaattattgg aattagataa atgggcaagt 7500

ttgtggaatt ggtttaacat aacaaattgg ctgtggtata taaaattatt cataatgata 7560

gtaggaggct tggtaggttt aagaatagtt tttgctgtac tttctatagt gaatagagtt 7620

aggcagggat attcaccatt atcgtttcag acccacctcc caaccccgag gggacccgac 7680

aggcccgaag gaatagaaga agaaggtgga gagagagaca gagacagatc cattcgatta 7740

gtgaacggat cggcactgcg tgcgccaatt ctgcagacaa atggcagtat tcatccacaa 7800

ttttaaaaga aaagggggga ttggggggta cagtgcaggg gaaagaatag tagacataat 7860

agcaacagac atacaaacta aagaattaca aaaacaaatt acaaaaattc aaaattttcg 7920

ggtttattac agggacagca gagatccagt ttggttagta ccgggcccgc tctagagt 7978

Claims (16)

1. Protein, which is any one of the following:

(A1) A protein with an amino acid sequence shown in positions 1-740 of SEQ ID No. 1;

(A2) A protein obtained by ligating the N-terminal and/or C-terminal of the protein defined in (A1) with a protein tag.

2. A fusion protein comprising the protein of claim 1 fused to an Fc fragment of an antibody.

3. The fusion protein of claim 2, wherein: the antibody Fc fragment is fused to the C-terminus or N-terminus of the protein of claim 1.

4. The fusion protein of claim 2, wherein: the antibody Fc segment is the Fc segment of an IgG antibody.

5. The fusion protein of claim 2, wherein: the antibody is a human antibody or a mouse antibody.

6. The fusion protein of claim 5, wherein: the Fc segment of the antibody is the Fc segment of a mouse IgG antibody, and the amino acid sequence of the Fc segment is shown in 741-970 of SEQ ID No. 1.

7. The fusion protein of any one of claims 2-6, wherein: the amino acid sequence of the fusion protein is shown as SEQ ID No. 1.

8. A nucleic acid molecule encoding the protein of claim 1 or the fusion protein of any one of claims 2-7.

9. The nucleic acid molecule of claim 8, wherein: the nucleic acid molecule encoding the protein of claim 1 is a DNA molecule shown at positions 1-2220 of SEQ ID No. 2.

10. The nucleic acid molecule of claim 8, wherein: among the nucleic acid molecules encoding the fusion protein, the nucleic acid molecule encoding the Fc segment of the mouse IgG antibody is the DNA molecule shown in positions 2221-2910 of SEQ ID No. 2.

11. The nucleic acid molecule of claim 10, wherein: the nucleic acid molecule encoding the fusion protein is a DNA molecule shown as SEQ ID No. 2.

12. An expression cassette or recombinant vector or recombinant bacterium or transgenic cell line comprising the nucleic acid molecule of any one of claims 8-11.

13. An anti-coronavirus agent comprising the protein of claim 1 or the fusion protein of any one of claims 2 to 7 as an active ingredient;

the coronavirus is SARS-CoV-2.

14. A method of preparing the fusion protein of any one of claims 2-7, comprising the steps of: cloning the nucleic acid molecule encoding the fusion protein to a pcDNA3.1 vector to obtain a recombinant vector; introducing the recombinant vector into 293F cells to obtain a transgenic cell line; culturing the transgenic cell line for 48 hours, centrifugally collecting cell culture supernatant, and performing affinity chromatography on the supernatant to obtain the fusion protein of any one of claims 2-7.

15. Any of the following applications:

(D1) Use of a protein according to claim 1 or a fusion protein according to any one of claims 2 to 7 or a nucleic acid molecule according to any one of claims 8 to 11 or an expression cassette or a recombinant vector or a recombinant bacterium or a transgenic cell line according to claim 12 for the preparation of an anti-coronavirus drug;

(D2) Use of a protein according to claim 1 or a fusion protein according to any one of claims 2 to 7 or a nucleic acid molecule according to any one of claims 8 to 11 or an expression cassette or recombinant vector or recombinant bacterium or transgenic cell line or a medicament according to claim 12 for the preparation of a product capable of neutralizing coronaviruses;

(D3) Use of a protein according to claim 1 or a fusion protein according to any one of claims 2 to 7 or a nucleic acid molecule according to any one of claims 8 to 11 or an expression cassette or recombinant vector or recombinant bacterium or transgenic cell line according to claim 12 for the preparation of a reagent for detecting coronavirus S protein;

(D4) Use of a protein according to claim 1 or a fusion protein according to any one of claims 2 to 7 or a nucleic acid molecule according to any one of claims 8 to 11 or an expression cassette or recombinant vector or recombinant bacterium or transgenic cell line according to claim 12 for the preparation of a detection reagent capable of binding to the RBD domain of the coronavirus S protein;

the coronavirus is SARS-CoV-2.

16. The use according to claim 15, characterized in that:

the coronavirus S protein is S protein from any one of the following SARS-CoV-2: wild type SARS-CoV-2, alpha epidemic strain of SARS-CoV-2, beta epidemic strain of SARS-CoV-2, gamma epidemic strain of SARS-CoV-2, delta epidemic strain of SARS-CoV-2.