CN114606219A

CN114606219A - Coronavirus neutralizing effect protein and application thereof

Info

Publication number: CN114606219A
Application number: CN202210338777.3A
Authority: CN
Inventors: 席建忠; 王博仑; 赵俊轩
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2022-04-01
Filing date: 2022-04-01
Publication date: 2022-06-10
Anticipated expiration: 2042-04-01
Also published as: CN114606219B

Abstract

The invention discloses a coronavirus neutralizing effect protein and application thereof. The invention provides a protein, which is a protein with the amino acid sequence as the 1 st-740 th position of SEQ ID No.1 or through the substitution and/or deletion and/or addition of one or more amino acid residues or with more than 80% of identity and the same function, or a protein obtained by connecting a protein label on the basis. The C4-1 protein provided by the invention can be combined with coronavirus S protein and inhibit the combination of coronavirus S protein and ACE2 receptor of human cells, thereby effectively neutralizing coronaviruses and being proved on a cellular level. Meanwhile, the corresponding DNA sequence is favorable for soluble expression in a human cell line, and a new direction is provided for the treatment of the coronavirus.

Description

Coronavirus neutralizing effect protein and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a coronavirus neutralizing effect protein and application thereof.

Background

Since the 21 st century, humans have experienced a coronavirus pandemic with severe respiratory symptoms and high mortality three times as a result of atypical pneumonia (SARS), Middle East Respiratory Syndrome (MERS), novel coronavirus pneumonia (COVID-19). Among them, the novel coronavirus pneumonia which is outbreak at the end of 2019 is caused by novel coronavirus (SARS-CoV-2), and the existing research evidence indicates that SARS-CoV-2 is most easily transmitted among the three coronaviruses. The World Health Organization (WHO) announced in 3 months 2020 that COVID-19 has become a global pandemic, and by now more than 200 countries and regions with emerging new coronary confirmed cases. According to statistical data issued by the WHO, more than 2 hundred million cases of confirmed diagnosis of new coronavirus infection and 460 ten thousand cases of death are accumulated in the world. Researchers predict new coronavirus or will coexist with humans for long periods.

The S protein is a structural protein at the membrane surface of coronaviruses. The S protein body is exposed outside a virus phospholipid bimolecular membrane structure to form a trimer structure on the surface of a mature virus membrane structure, and similar to other coronaviruses, the S protein of SARS-CoV-2 can mediate the initial process of virus invading cells through the interaction with host cell surface receptors. After binding with the receptor protein of the host cell, the S protein can be identified and cut by furin protease (furin) and Transmembrane protease serine 2 (TMPRSS 2), so that the membrane fusion domain is exposed, and the fusion of the virus membrane structure and the host cell membrane is carried out. On the other hand, the S protein mediates endocytosis of cells through a binding process with cell receptors, and the cells release genetic material in the cytoplasm after being encapsulated into vesicles.

Angiotensin converting enzyme 2(ACE2) is a human cell receptor membrane protein of several coronaviruses and is considered as a potential target for coronavirus therapy. Taking the novel coronavirus SARS-CoV-2 as an example, the Receptor Binding Domain (RBD) of the spinous process (S) protein of the virus and ACE2 recognize and bind to each other, and the viral particle is anchored on the cell surface, thereby invading the cell by a process such as cell membrane fusion. Therefore, ACE2 can be an important target for coronavirus therapy including SARS and new coronavirus, and it is important to find a new therapeutic approach for coronavirus therapy by finding coronavirus s protein and ACE2 binding inhibitor.

In view of the biological process of coronavirus cell invasion, the inhibitors reported or under investigation are roughly divided into two categories: first, small molecules. Including protease inhibitors such as boceprevir, acetylneuraminic acid and the like. These small molecules prevent the fusion of the viral and cellular membranes by inhibiting the enzymatic cleavage of the S protein by proteases on the cell surface. The second, monoclonal antibodies. Such as REGEN-COV, BamHI cocktail of Biogenes, BamHI and Eteseveimab of Lily, S309, cilgavimab and VHH-72, etc.

Both these classes of coronavirus inhibitors have their own disadvantages: small molecule inhibitors are less specific and therefore inhibit other similar physiological activities when inhibiting coronavirus targets. The extensive use of monoclonal antibodies will result in a virus that tends to generate more immune escape, possibly disabling new mutant strains.

Disclosure of Invention

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

In a first aspect, the invention claims a protein.

The protein claimed by the invention is named C4-1 and is any one of the following:

(A1) protein with amino acid sequence shown as 1-740 th position of SEQ ID No. 1;

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

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

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

In the above protein, the protein tag (protein-tag) refers to a polypeptide or protein that is expressed by fusion with a target protein using in vitro recombinant DNA technology, so as to facilitate expression, detection, tracking and/or purification of the target protein. The protein tag may be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag, and/or a SUMO tag, among others.

In the above proteins, identity refers to the identity of amino acid sequences. The identity of the amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST web pages of the NCBI home website. For example, in the advanced BLAST2.1, by using blastp as a program, setting the value of Expect to 10, setting all filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residual Gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively, and performing a calculation by searching for the identity of a pair of amino acid sequences, a value (%) of identity can be obtained.

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

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

The fusion protein claimed in the present invention is obtained by fusing the protein described in the first aspect and the Fc region of the antibody.

Wherein the antibody Fc fragment fusion may be fused to the C-terminus or to the N-terminus of the protein of 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 present invention, the Fc fragment of the antibody is the Fc fragment of a mouse IgG antibody, and the amino acid sequence thereof is shown in the 741-970 position 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 invention claims a nucleic acid molecule encoding a protein as described in the first aspect above or a fusion protein as described in the second aspect above.

Further, the nucleic acid molecule encoding the protein of the first aspect may be any of:

(B1) a DNA molecule represented by positions 1-2220 of SEQ ID No. 2;

(B2) a DNA molecule which hybridizes under stringent conditions to the DNA molecule 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 as defined in the first aspect above.

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

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

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

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

(C3) a DNA molecule having more than 99%, more than 95%, more than 90%, more than 85% or more than 80% homology with the DNA sequence defined in (C1) or (C2) and encoding the fusion protein as described in the second aspect above.

In the above nucleic acid molecule, the stringent conditions may be as follows: at 50 ℃ in 7% twelveSodium alkyl sulfate (SDS), 0.5M Na₃PO₄Hybridization with 1mM EDTA, rinsing in 2 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M Na₃PO₄Hybridization with 1mM EDTA, rinsing at 50 ℃ in 1 XSSC, 0.1% SDS; also can be: 50 ℃ in 7% SDS, 0.5M Na₃PO₄Hybridization with 1mM EDTA, rinsing in 0.5 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M Na₃PO₄Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M Na₃PO₄Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 65 ℃; can also be: in a solution of 6 XSSC, 0.5% SDS at 65 ℃ and then washed once with each of 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS.

In the above nucleic acid molecules, homology means the identity of nucleotide sequences. The identity of the nucleotide sequences can be determined using homology search sites on the Internet, such as the BLAST web page of the NCBI home website. For example, in the advanced BLAST2.1, by using blastp as a program, setting the value of Expect to 10, setting all filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residual Gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively, and performing a calculation by searching for the identity of a pair of nucleotide sequences, a value (%) of identity can be obtained.

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

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

In a specific embodiment of the present invention, 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 particular embodiment of the invention, the transgenic cell line is obtained by introducing 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 invention claims an anti-coronavirus drug.

The invention claims an anti-coronavirus drug, the active ingredient of which is the protein described in the first aspect or the fusion protein described in the second aspect.

The medicine can also contain a pharmaceutically acceptable carrier according to requirements.

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

The method of preparing the fusion protein of the second aspect, as claimed herein, may comprise the steps of: cloning the nucleic acid molecule for encoding the fusion protein to pcDNA3.1 vector to obtain recombinant vector; introducing the recombinant vector into 293F cells to obtain a transgenic cell line; culturing the transgenic cell line for 48h, centrifuging and 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 invention claims any of the following applications:

(D1) use of a protein as described in the first aspect or a fusion protein as described in the second aspect or a nucleic acid molecule as described in the third aspect or an expression cassette or a recombinant vector or a recombinant bacterium or a transgenic cell line as described in the fourth aspect for the preparation 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 in the manufacture of a product capable of neutralising coronavirus;

(D3) use of a protein as described in the first aspect or a fusion protein as described in the second aspect or a nucleic acid molecule as described in the third aspect or an expression cassette or a recombinant vector or a recombinant bacterium or a transgenic cell line as described in the fourth aspect for the preparation of an agent for the detection of a coronavirus S protein;

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

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

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

The invention carries out combination of a plurality of point mutations and mouse IgG-Fc modification at the C end of protein on angiotensin converting enzyme 2(ACE2), obtains protein mutation individual C4-1 with stronger affinity with new coronavirus S protein RBD, has stable molecular structure and molecular weight of about 114kDa, can carry out soluble expression in a human cell line and has higher expression quantity. The invention has the beneficial effects that: the C4-1 protein provided by the invention can be combined with coronavirus S protein and inhibit the combination of coronavirus S protein and ACE2 receptor of human cells, thereby effectively neutralizing coronaviruses and being proved on a cellular level. And simultaneously, the corresponding DNA sequence is favorable for soluble expression in a human cell line. Overcomes the problem of poor specificity of the coronavirus small molecule inhibitor, and provides a new direction for the treatment of the coronavirus.

Drawings

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

FIG. 2 is a SDS-PAGE result of elution purification of C4-1 Protein after Protein G purification. Lane 1 is the C4-1 protein, M is marker, and the remainder are unrelated.

FIG. 3 is a diagram showing the results of in vitro activity assay of C4-1 protein;

FIG. 4 is a graph showing the SPR results of the C4-1 protein and the SARS-CoV-2S protein RBD. The black line represents the raw data and the grey is the fitted data.

FIG. 5 is a graph showing the results of experiments on neutralization of SARS-CoV-2 epidemic strains by C4-1 protein in HEK293T-ACE2 cells.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 preparation of ACE2 mutant protein C4-1

Firstly, construction of recombinant expression vector pCDNA3.1/C4-1

The invention carries out point mutation on the 30 th, 78 th, 91 th and 474 th positions of the amino acid sequence of angiotensin converting enzyme 2(ACE2), and then the point mutation is fused with the Fc segment of a mouse IgG antibody to obtain the ACE2 mutant protein C4-1, wherein the amino acid sequence of the ACE2 mutant protein C4-1 is shown as SEQ ID No. 1.

Designing and artificially synthesizing a eukaryotic expression DNA sequence (SEQ ID No.2) according to an amino acid sequence of the C4-1 protein, respectively adding a NheI (GCTAGC) and an XbaI (TCTAGA) double enzyme cutting site and a homology arm to the front end and the rear end of a DNA fragment shown in the SEQ ID No.2 through overlapping PCR, connecting the double enzyme cutting site and the homology arm to a pCDNA3.1 vector subjected to double enzyme cutting (NheI and XbaI) through Gibson Assembly, transferring the pCDNA3.1 vector to a5 alpha thallus for amplification and plasmid extraction, and obtaining a recombinant expression vector pCDNA3.1/C4-1 after the correctness of sequencing verification. FIG. 1 shows the backbone sequence of pCDNA3.1.

The recombinant expression vector pCDNA3.1/C4-1 structure is described as: the DNA fragment shown in SEQ ID No.2 is inserted between the enzyme cutting sites NheI and XbaI of the pCDNA3.1 vector to obtain the recombinant plasmid.

Second, Large Scale culture/transfection of 1L Shake flasks

293F cells (ThermoFisher) are cultured in suspension according to standard operating manual, typically 250ml cell culture flasks are started in a volume of 30ml to 100 ml. According to 0.5X 10⁶Inoculation of cells/ml cells were inoculated into 300ml of medium in a 1L shake flask. Incubated at 37 ℃ in a shaker incubator at 120rpm with 5% carbon dioxide concentration until a cell density of 1X 10 was reached⁶cells/ml. Mu.g of DNA (i.e., the recombinant expression vector pCDNA3.1/C4-1 obtained in step one) was pipetted into 30ml of PBS, 1.2ml of filter sterilized PEI solution (0.5mg/ml) was added to the PBS/DNA mixture, and the mixture was allowed to stand for 20min and added to the cells. After transfection, the cells were incubated in a shaker incubator for 48 h. Centrifuging at 3000g for 5min to separate cell culture medium supernatant from cell precipitate, and collecting culture medium supernatant.

Thirdly, purification of protein complex extracted from cell culture medium supernatant

The supernatant of the medium cultured for 48 hours after transfection in step two was filtered through a 0.45 μm filter. The filtered supernatant was incubated with 1.25ml of Protein G column (Cytiva) per liter, eluted with 10mM citric acid/200 mM NaCl buffer, and then separated by Q-sepharose ion exchange column (Cytiva) with 10mM phosphate buffer/200 mM NaCl buffer, followed by purification with Sephacryal 200 gel chromatography column (Cytiva), and eluted with 10mM phosphate buffer/150 mM NaCl buffer after loading to the column, as the C4-1 stock solution.

A10. mu.l sample of the concentrated protein was added to a2 Xprotein loading buffer for electrophoretic detection. The protein was filtered through a 0.22 μm filter. FIG. 2 shows C4-1 protein of interest extracted from the supernatant of 293F medium transiently transfected at 2L (8X 250 ml). As can be seen from the SDS-PAGE electrophoresis of FIG. 2, the target protein was expressed at about 135kDa molecular weight in the supernatant of cells expressed by C4-1 cells, and the target protein had a molecular weight close to the theoretical value (113KD) (the actual molecular weight was slightly greater than the theoretical value due to the presence of glycosylation process of the protein), and was purified to have substantially no bands. The constructed expression strain is frozen and stored in a refrigerator at the temperature of minus 80 ℃ for later use. The yield of purified protein was estimated to be approximately 1mg for 1L of medium.

Example 2 in vitro Activity assay of ACE2 mutant protein C4-1

Cell line expressed by 5S protein mutants of coronavirus constructed by using lentivirus transfection method

The S proteins of 5 SARS-CoV-2 are respectively from: wild type SARS-CoV-2, alpha, beta, gamma and delta circulating strains of SARS-CoV-2. The amino acid Sequence of the S protein from wild-type SARS-CoV-2 (i.e., strain Wuhan-Hu-1) is identical to NCBI Reference Sequence YP-009724390.1, the corresponding coding gene Sequence is NCBI Reference Sequence NC-045512.2 (21563..25384), and NCBIGeneID is 43740568; the amino acid sequence of the S protein from an epidemic strain of alpha was changed from YP-009724390.1 as follows: amino acid residues 69-70, 144, N501Y, a570D, D614G, P681H, T716I, S982A, D1118H, corresponding to the following changes in the encoded gene compared to NC _045512.2(21563.. 25384): the nucleotide deletion at the 210 th site from 205-; the amino acid sequence of the S protein from the beta epidemic strain was changed from YP _009724390.1 as follows: L18F, D80A, D215G, deletion of amino acid residue 242-244, K417N, E484K, N501Y, D614G, A701V, corresponding to the following changes in the encoded gene compared to NC 045512.2(21563.. 25384): the 52-54 th mutation is ttc, the 238 th and 240 th mutations are gcc, the 643 st and 645 th mutations are ggc, the 724 th and 732 th nucleotide deletions, the 1249 th and 1251 th mutations are aat, the 1450 nd and 1452 th mutations are aag, the 1501 th and 1503 th mutations are tat, the 1840 th and 1842 th mutations are ggt, and the 2101 nd and 2103 th mutations are gta; the amino acid sequence of the S protein from an epidemic strain of gamma was changed from YP _009724390.1 as follows: L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, T1027I, V1176F, the corresponding coding gene has the following changes compared with NC _045512.2(21563.. 25384): the 52-54 th mutation is ttc, the 58-60 th mutation is aac, the 76-78 th mutation is tct, the 412-414 th mutation is tac, the 568-570 th mutation is agc, the 1249-1251 th mutation is agc, the 1450-1452 th mutation is aag, the 1501-1503-gt, the 1963-1503-1965 th mutation is tat, the 3079-3081 th mutation is atc, and the 3526-3528 th mutation is ttc; the amino acid sequence of the S protein from delta epidemic strains was changed from YP _009724390.1 as follows: T19R, G142D, deletion of amino acid residue 157 at position 156-157, R158G, L452R, T478K, D614G, P681R, D950N, corresponding to the following changes in the encoded gene compared to NC-045512.2 (21563.. 25384): the 55-57 mutation is aga, the 424-426 mutation is gag, the 466-471 nucleotide deletion, the 472-474 mutation is ggc, the 1364-1356 mutation is aga, the 1432-1434 mutation is aag, the 1840-1842 mutation is ggc, the 2041-2043 mutation is aga, and the 2848-2850 mutation is aac.

The lentiviral packaging plasmid pCMV-dR8.91 (4. mu.g, Addgene: vector-database/2221/), pCMV-VSV-G (1. mu.g, Addge: 8454) was 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 (lentiviral vector plasmids were all pWSLV03 backbone, pWSLV03 plasmid carries mCherry sequence itself, pWSLV03 plasmid complete sequence is shown in SEQ ID No.3, from Beijing has only been limited bioscience, pWSLV03 plasmid was inserted into S protein coding gene sequence of each strain by nick and EcoRI site double digestion and verified to be correct by sequencing) and transfection reagent (30. mu.l) in 3ml of Glibco OpMEM, and left standing for 20 min. The mixture was transferred to a 293T cell line (containing 7ml of medium, cell density controlled at 70% to 90%) in a pre-prepared 10cm dish. After 6h, the medium was discarded and 10ml of fresh DMEM medium was added. After 48h, mCherry positive cells were collected by flow sorting.

II, ACE2 mutant protein C4-1 is combined with SARS-CoV-2S protein expressed cell line

Culturing the mCherry positive cells collected in the first step in an incubator at 37 ℃ for 24h, digesting the cells with 0.25% of trypsin/EDTA, centrifuging at 1000rpm for 3min, resuspending the cells with DMEM containing 1% FBS, and adding the cells into a 96-well plate. Mixing the mFcThe labeled ACE2 mutant protein (i.e., C4-1 prepared in example 1) was formulated in 7 concentration gradients with a maximum concentration of 100nM, each gradient diluted 3-fold, and three parallel experiments were performed. In the experiment, a 96-well plate is centrifuged at 1000rmp for 3min to remove the culture medium, the prepared protein is added to each well according to the concentration, 100. mu.l of the protein is added to each well, and the wells are incubated for one hour in an incubator at 37 ℃. After completion of the incubation, the supernatant was removed by centrifugation at 1000rmp for 3min, rinsed once with 100. mu.l PBS and incubated for one hour at 37 ℃ in an incubator protected from light with the addition of 100. mu.l FITC-labeled anti-mouse IgG antibody (Biolegend, 1000 Xdilution). Centrifuging at 1000rmp for 3min to remove supernatant, resuspending with PBS, precipitating, and performing LSRFortessa^TMCell Analyzer (BD) analyzes Cell fluorescence, calculates the FITC mean fluorescence intensity (MFU) of mCherry positive cells, and obtains the results shown in FIG. 3, and obtains IC50 of C4-1 for different S protein expression Cell lines by the Reed-Muench method.

FIG. 3 shows the binding curves of the ACE2 mutant protein C4-1 and different mutants of SARS-CoV-2, respectively, which shows that the protein has obvious binding with cells, and this also proves that C4-1 has strong binding ability to S protein of various mutants.

Example 3 affinity assay for ACE2 mutant protein C4-1 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 Surface Plasmon Resonance (SPR) technology, and the refractive index change of the chip surface can be reflected sensitively. To study the interaction between molecules, one molecule is immobilized on the chip surface and the other molecule flows through the surface in the form of a solution. The response value of SPR is proportional to the change in mass concentration near the chip.

In a typical assay, a CM5 chip was chosen to determine the protein-protein interaction between soluble ACE2 protein and SARS-CoV-2 Spike-RBD. CM5 chips are based on a strategy of covalent coupling, with medium capacity and universal characteristics. The running buffer was HBS-EP (Cytiva). RBD-his (Cassia, Chinese, supra: 40592-V08H105) (20 ng/. mu.l) was dissolved in sodium acetate buffer (pH 4.5) and immobilized as a ligand on the chip at a flow rate of 5. mu.l/min, stopping after about 30s, at an immobilization rate of about 60 RU. The analyte was ACE2 mutant protein C4-1 at a concentration of 100 to 1.56nM (2-fold dilution between each gradient) and the diluent was running buffer. The flow rate was set at 45. mu.l/min, the binding time was set at 180 seconds, and the dissociation time was 1800 seconds. At the end of each cycle, the regeneration was carried out for 30 seconds using a glycine solution at pH1.5 at a flow rate of 30. mu.l/min.

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

in the dissociation process, the rate equation is:

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

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

Sample(s)	ka(1/Ms)	kd(1/s)	KD(nM)
				C4-1	1.05×10⁵	4.68×10^-5	0.44

The ACE2 mutant protein C4-1 shows strong RBD affinity in the test, and as can be seen, the protein has a faster binding rate, and the dissociation curve shows that the protein is extremely stable in binding. Furthermore, considering the binding and dissociation processes together, we can calculate the dissociation constant KD, which is of a magnitude less than nM and much higher than wild-type ACE2 (about 20 nM. in reference to "Wrapp D, Wang N, Corbett KS, et al, Cryo-EM structure of the 2019-nCoV spike in the prefusion compatibility. science.2020; 367(6483): ear 2507.Glasgow A, Glasgow J, limo D, et al, Engined ACE2 receptors trap porosity of the National Academy of sciences.2020; 117(45): 46 + 280280280280so that it shows great advantage as a therapeutic drug.

This example shows the strong affinity of the C4-1 protein to the Spike protein RBD, which is superior to that of wild-type ACE2 from the KD value.

Example 4 ACE2 mutant protein C4-1 and SARS-CoV-2 pseudovirus neutralization assay

The pseudovirus neutralization experiment uses Vesicular Stomatitis Virus (VSV) as a basic framework, and replaces the receptor binding protein G protein with the Spike protein of SARS-CoV-2, thereby simulating the process of entering cells and being inhibited by drugs. Compared with in vitro experiments, the pseudovirus simulates the process of infecting cells by the virus, and the experimental result is more real and reliable. The ACE2 mutant protein C4-1 is excellent in vitro experiment, and further experiments are needed to confirm the treatment effect.

Preparation of Mono-and pseudotype viruses

Pseudotyped virus and mutant of SARS-CoV-2 were constructed. The method comprises the following specific steps: the day before transfection, 293T cells were digested and adjusted to 5X 10⁵To 7X 10⁵cell/mL. Then, the cells in 15ml of the medium were transferred to T75 cell medium, andusing 5% CO at 37 deg.C₂Incubated overnight in an incubator. When the cells reached 70% -90% coverage, the supernatant was discarded and used at a concentration of 7X 10⁴TCID₅₀Infection was carried out with 15mL of VSV-. DELTA.G-luciferase plasmid expression vector system (Kerafast: EH 1008). Meanwhile, 30. mu.g of S protein expression plasmids of different epidemic strains (recombinant plasmids obtained by using pCDNA3.1 of FIG. 1 as a vector and inserting all of several S protein coding genes between the enzyme cutting sites NheI and XbaI of the pCDNA3.1 vector) were respectively transferred into cells. Cells were incubated at 37 ℃ with 5% CO₂Cultured in an incubator. Cell supernatants were discarded after 6-8 hours and cells were gently washed twice with PBS + 1% FBS and 15mL of fresh DMEM was added to T75 cell culture flasks. At 37 ℃ and 5% CO₂After culturing in the incubator of (1) for 24 hours, the culture supernatant containing pseudotype virus is collected, filtered and split-packaged to obtain VSV pseudotype virus with S-WT, S-alpha, S-beta, S-gamma or S-delta (respectively representing S proteins of alpha, beta, gamma and delta epidemic strains of wild type SARS-CoV-2 and SARS-CoV-2) on the virus surface, and the VSV pseudotype virus is frozen and stored at-70 ℃ for subsequent use.

Second, pseudo type virus inhibition test

Performing RT-PCR quantitative analysis on the pseudoviruses collected in the step one, and diluting all the pseudoviruses to the titer of 2 multiplied by 10⁵TCID₅₀mL, and 100. mu.L was added to a 96-well cell culture plate. Wild-type ACE2 (Ajingshan: 10108-H08H) and C4-1 protein samples were each diluted in a gradient (3-fold dilution starting at 100nM, 8 gradients) in 96-well plates and added to the corresponding wells to which the virus solution had been added. On the well plate, 8 control groups to which only the virus solution was added and 8 control groups to which only cells were added were set. After incubation at 37 ℃ for 1 hour, wild-type ACE2 overexpressing cells (Saint biotechnologies (Shanghai): 41107ES03) were trypsinized and incubated at 2X 10⁴Cell concentration of 100. mu.L was added to each well of the 96-well plate. After 24 hours of incubation in an incubator at 37 ℃ containing 5% carbon dioxide, changes in luciferase gene expression were examined to assess the neutralizing effect of the wild-type ACE2 protein and C4-1 protein samples on, respectively, pseudovirus-infected ACE 2-overexpressing cells. Add 100. mu.L luciferase substrate (P) to each wellerkinlemer), incubated at room temperature for 2 minutes, then transferred to a test whiteboard and measured using a photometer (Perkinlemer). Each group contained two replicates. EC for each sample was calculated using the Reed-Muench method₅₀The value is obtained.

Third, results and analysis

As can be seen from FIG. 5, the ACE2 mutant protein C4-1 has a very strong inhibitory effect on all mutant pseudoviruses in the experiment, and EC in Table 2₅₀The calculation result shows that the inhibition effect of the C4-1 on wild type S protein pseudovirus is improved by more than 200 times (EC) compared with the inhibition effect of the non-mutated ACE2 protein₅₀Value comparison).

TABLE 2 results of viral suppression of wild-type ACE2 and C4-1 in different new corona strain pseudoviruses

EC₅₀(ng/mL)	Wild type ACE2	C4-1
			Wild strain	>10000	61
alpha	780	55
			beta	2221	35
gamma	1010	27
			delta	1009	35

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

<110> Beijing university

<120> coronavirus neutralizing effector protein and application thereof

<130> GNCLN220642

<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 Asn 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 Asn 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 ctcaatgtca 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

cctaatatga 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

1. A protein, which is any one of:

2. A fusion protein obtained by fusing the protein of claim 1 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;

and/or

The Fc segment of the antibody is the Fc segment of an IgG antibody;

and/or

The antibody is a human antibody or a mouse antibody;

furthermore, 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 as the 741-970 position of SEQ ID No. 1.

4. The fusion protein of claim 3, wherein: the amino acid sequence of the fusion protein is shown as SEQ ID No. 1.

5. A nucleic acid molecule encoding the protein of claim 1 or the fusion protein of any one of claims 2-4.

6. The nucleic acid molecule of claim 5, wherein: the nucleic acid molecule encoding the protein of claim 1 is any one of:

(B1) a DNA molecule represented by positions 1-2220 of SEQ ID No. 2;

(B2) a DNA molecule which hybridizes under stringent conditions to the DNA molecule defined in (B1) and which encodes the protein of claim 1;

(B3) a DNA molecule which has 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 which encodes the protein of claim 1;

and/or

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 the 2221-2910 position of SEQ ID No. 2;

further, the nucleic acid molecule encoding the fusion protein is any one of:

(C1) a DNA molecule shown as SEQ ID No. 2;

(C2) a DNA molecule which hybridizes under stringent conditions to the DNA molecule defined in (C1) and which encodes the fusion protein of any one of claims 2 to 4;

(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 of any one of claims 2 to 4.

7. An expression cassette or recombinant vector or recombinant bacterium or transgenic cell line comprising the nucleic acid molecule of claim 5 or 6;

or

An anti-coronavirus drug, wherein the active ingredient of the anti-coronavirus drug is the protein as defined in claim 1 or the fusion protein as defined in any one of claims 2 to 4.

8. A method of making the fusion protein of any one of claims 2-4, comprising the steps of: cloning the nucleic acid molecule for encoding the fusion protein to pcDNA3.1 vector to obtain recombinant vector; introducing the recombinant vector into 293F cells to obtain a transgenic cell line; culturing the transgenic cell line for 48h, centrifuging and collecting cell culture supernatant, and performing affinity chromatography on the supernatant to obtain the fusion protein of any one of claims 2-4.

9. Any one 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 4 or a nucleic acid molecule according to claim 5 or 6 or an expression cassette or a recombinant vector or a recombinant bacterium or a transgenic cell line according to claim 7 for the preparation of an anti-coronavirus medicament;

(D2) use of a protein according to claim 1 or a fusion protein according to any one of claims 2 to 4 or a nucleic acid molecule according to claim 5 or 6 or an expression cassette or a recombinant vector or a recombinant bacterium or a transgenic cell line according to claim 7 or a pharmaceutical agent 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 4 or a nucleic acid molecule according to any one of claims 5 to 7 or an expression cassette or a recombinant vector or a recombinant bacterium or a transgenic cell line according to claim 8 for the preparation of a reagent for the detection of coronavirus S protein;

(D4) use of a protein according to claim 1 or a fusion protein according to any one of claims 2 to 4 or a nucleic acid molecule according to claim 5 or 6 or an expression cassette or a recombinant vector or a recombinant bacterium or a transgenic cell line according to claim 7 for the preparation of a detection reagent capable of binding to the RBD domain of the coronavirus S protein.

10. The medicament according to claim 7 or the use according to claim 9, characterized in that: the coronavirus is SARS-CoV-2; and/or

The coronavirus S protein is an S protein from any one of SARS-CoV-2 as follows: wild SARS-CoV-2, alpha epidemic strain of SARS-CoV-2, beta epidemic strain of SARS-CoV-2, gamma epidemic strain of SARS-CoV-2, and delta epidemic strain of SARS-CoV-2.