A binding protein Bntifitin and its application in preparing medicine for treating coronavirus infection

Abstract

The present invention provides a binding protein, Bntifitin, and its use in the preparation of a medicament for treating coronavirus infection. The protein has an amino acid sequence as shown in SEQ ID NO: 1. The binding protein Bntifitin provided by the present invention has a binding affinity KD for RBD of 1.3 nM, indicating that Bntifitin-S10 binds to RBD with high affinity and can inhibit SARS-CoV-2 pseudovirus infection and SARS-CoV-2 live virus infection. The binding protein can be used for the treatment or diagnosis of coronavirus infection, especially for the treatment of coronavirus brain infection.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a binding protein Bntifitin and an application thereof in preparing a medicine for treating coronavirus infection. Background Art

[0002] The spike protein, also known as the S protein, is located on the surface of the coronavirus and is a key structure that mediates viral invasion and infection of host cells. After invading the human respiratory tract, the coronavirus primarily relies on the receptor binding domain (RBD) on the S protein to recognize the host cell receptor angiotensin-converting enzyme 2 (ACE2), bind to it, and infect the host cell, causing or exacerbating acute lung injury. In other words, any cell expressing ACE2 can be infected by the coronavirus.

[0003] In addition to being expressed in human airway epithelium, lung tissue cells, kidneys, small intestine, testicles, muscles and vascular endothelial cells, ACE2 is also widely expressed throughout the nervous system and on the surface of various cells such as neurons, astrocytes, microglia and oligodendrocytes. Therefore, the nervous system is also a potential target for coronavirus infection.

[0004] Paniz-Mondolfi et al. (J MedVirol, 2020, 92(7): 699-702) first confirmed the presence of SARS-CoV-2 in human brain tissue. After SARS-CoV-2 infects the central nervous system, patients often present with neurological symptoms such as stroke, dizziness, headache, epilepsy, impaired consciousness, acute cerebrovascular disease, cerebral hemorrhage, and confusion.

[0005] The blood-brain barrier (BBB) ​​is a crucial structure for the development and proper function of the nervous system. While protecting the brain from external intrusions, it also restricts the delivery and transport of a large number of valuable drugs. Coronavirus invasion and infection of the brain present significant challenges in its symptomatic treatment. Therefore, there is an urgent need to develop drugs that target the coronavirus spike protein and can cross the BBB. Summary of the Invention

[0006] In order to solve the technical problem, the purpose of the present invention is to provide a binding protein Bntifitin and its use in the preparation of a drug for treating coronavirus infection. The binding protein Bntifitin can cross the blood-brain barrier to recognize and bind to the S1 subunit and RBD, thereby inhibiting SARS-CoV-2 live virus infection.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] In the first aspect of the present invention, a binding protein Bntifitin is provided, which has an amino acid sequence as shown in SEQ ID NO: 1, or an amino acid sequence of a coronavirus spike protein that has at least 90% homology to the amino acid sequence shown in SEQ ID NO.1 and can cross the blood-brain barrier.

[0009] In the second aspect of the present invention, a nucleic acid molecule encoding the binding protein Bntifitin is provided.

[0010] Furthermore, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO.2.

[0011] In the third aspect of the present invention, a nucleic acid construct of the binding protein Bntifitin is provided, wherein the nucleic acid construct is capable of expressing the binding protein Bntifitin.

[0012] Furthermore, the nucleic acid construct is selected from an expression cassette or a vector.

[0013] In the fourth aspect of the present invention, a host cell containing the nucleic acid molecule or the nucleic acid construct is provided.

[0014] Furthermore, the host cells include prokaryotic host cells and eukaryotic host cells. The prokaryotic host cells include Escherichia coli, and the eukaryotic host cells include yeast cells, insect cells, etc.

[0015] In the fifth aspect of the present invention, provided is the use of the binding protein Bntifitin, the nucleic acid molecule, the nucleic acid construct, or the host cell in the preparation of a medicament for treating coronavirus infection.

[0016] Furthermore, the coronavirus includes SARS-CoV-2 and its variants.

[0017] The use of the binding protein Bntifitin or the nucleic acid molecule or the nucleic acid construct or the host cell in the preparation of a drug for treating coronavirus infection and in the preparation of a kit for diagnosing coronavirus infection, especially in coronavirus brain infection.

[0018] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0019] The present invention provides a binding protein Bntifitin and its use in the preparation of a medicament for treating coronavirus infection. The binding protein Bntifitin of the present invention can cross the blood-brain barrier of the coronavirus spike protein, and simultaneously achieves the treatment or diagnosis of crossing the blood-brain barrier and coronavirus infection, providing a new method, platform and carrier for the treatment and diagnosis of coronavirus brain infection.

[0020] The binding affinity KD of a binding protein Bntifitin provided by the present invention to RBD is 1.3 nM, indicating that Bntifitin-S10 binds to RBD with high affinity and can inhibit SARS-CoV-2 pseudovirus infection and SARS-CoV-2 live virus infection, thereby being able to prepare drugs for treating coronavirus infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following is a brief introduction to the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0022] Figure 1 This is the plasmid map of the binding protein Bntifitin expression vector pBntifitin.

[0023] Figure 2 This is the ELISA test result of the binding protein Bntifitin and S protein RBD.

[0024] Figure 3 This is a graph showing the results of the neutralization activity test of the binding protein Bntifitin against SARS-CoV-2 pseudovirus.

[0025] Figure 4 This is a graph showing the results of the inhibitory activity test of the binding protein Bntifitin against live SARS-CoV-2 virus infection.

[0026] Figure 5 This is a graph showing the results of permeability test of the binding protein Bntifitin in the blood-brain barrier in vitro model. DETAILED DESCRIPTION

[0027] The present invention will be described in detail below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific embodiments and examples are for illustrating the present invention, rather than for limiting the present invention.

[0028] Throughout this specification, unless otherwise specified, the terms used herein should be understood as having the same meaning as commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the event of any conflict, the present specification shall take precedence.

[0029] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0030] The effects of this application will be described in detail below with reference to examples and experimental data.

[0031] Example 1 Gene and primer sequence synthesis

[0032] The amino acid sequence of the binding protein according to the present invention, as shown in SEQ ID NO: 1, was artificially designed and converted to a nucleotide sequence. After codon optimization, the target gene sequence (as shown in SEQ ID NO: 2) was ligated into the vector pUC57. Primer sequences were designed using primer design software, with SapI and PciI endonuclease cleavage sites added to the upstream and downstream primers, respectively. The sequences are shown in Table 1.

[0033] Table 1

[0034]

[0035]

[0036] Example 2 Construction and identification of recombinant plasmids

[0037] Using pUC57 harboring the target gene as a template and primers pDJ-F and pDJ-R (sequences shown in Table 1), PCR amplification of the target gene was performed. The PCR amplification product was separated and recovered by agarose gel electrophoresis. Next, the pEQ-80L plasmid (#VT1289) was double-digested with SapI and PciI endonucleases in a 37°C water bath for 2 h. The double-digested plasmid product was separated and recovered by agarose gel electrophoresis. The recovered PCR amplification product was ligated with the double-digested product using the Beyotime Seamless Cloning Kit. DH5ɑ competent cells were thawed on ice, and the ligation product was added. Heat shock was performed at 42°C for 90 seconds, and 900 μL of LB medium was added. The cells were incubated at 37°C with shaking at 220 rpm for 45 minutes. The cells were centrifuged at 5000 rpm for 1 minute. The pellet was evenly spread on a solid agar plate using an L-shaped sterile glass rod and incubated at 37°C for 14 hours. A single colony was picked and added to 5 mL of ampicillin-resistant LB liquid medium. The culture was shaken at 37°C and 220 rpm for 14 h. The bacterial suspension, pHK-F primer, and pHK-R primer were collected for identification of the recombinant plasmid. The plasmid pBntifitin was extracted using the EZNATMPlasmid Mini Kit (plasmid extraction kit) from the bacterial suspension with the correct sequencing results. The plasmid structure is shown in [ 1 ]. Figure 1 .

[0038] Example 3: Transformation of recombinant plasmid into Escherichia coli and screening of protein expression conditions

[0039] Thaw BL21(DE3) competent cells on ice, add the recombinant plasmid pBntifitin, heat shock at 42°C for 90 seconds, add 900 μL of LB medium, shake incubate at 220 rpm at 37°C for 45 minutes, centrifuge at 5000 rpm for 1 minute, spread the precipitate evenly on the surface of a solid agar plate using an L-shaped sterile glass rod, and incubate at 37°C for 14 hours. Pick a single colony and add it to 5 mL of ampicillin-resistant LB liquid medium. Incubate it in a shaker at 37°C at 220 rpm for 14 hours. Then, add 5 mL of the bacterial solution to 250 mL of LB medium containing ampicillin and shake in a shaker at 37°C at 220 rpm for 4-5 hours. When the OD reaches 0.6-0.8, add the inducer IPTG to a final concentration of 0.2 mM. Continue shaking in a shaker at 18°C ​​at 220 rpm for 20 hours to induce protein expression in E. coli. The bacterial precipitate was collected by centrifugation and resuspended in an appropriate amount of buffer A (20 mM phosphate buffer, 300 mM NaCl, 5 mM imidazole, pH 8.0). The bacteria were disrupted by ultrasonication in an ice bath and centrifuged at 12000 rpm for 25 min at 4°C. The supernatant and precipitate were collected and identified by reducing sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) electrophoresis.

[0040] Example 4 Purification of target protein by affinity chromatography

[0041] The Ni-NTA affinity chromatography filler was slowly eluted with deionized water and pre-balanced with 10 times the column bed volume of buffer A. The sample was loaded and the supernatant collected by high-speed centrifugation of ultrasonically disrupted bacteria was filtered through a 0.45 μm filter membrane and injected into the Ni-NTA affinity chromatography column.

[0042] The column was then rinsed with 10 column volumes of buffer A, and the filtrate was collected. Elution was then started with 5 column volumes of buffer B (20 mM phosphate buffer, 300 mM NaCl, 10 mM imidazole, pH 8.0), and the eluate was collected.

[0043] Then, elution was performed sequentially with 5 column volumes of buffer C (20 mM phosphate buffer, 300 mM NaCl, 40 mM imidazole, pH 8.0), buffer D (20 mM phosphate buffer, 300 mM NaCl, 80 mM imidazole, pH 8.0), and buffer E (20 mM phosphate buffer, 300 mM NaCl, 200 mM imidazole, pH 8.0), and the eluates were collected. Subsequently, the target protein was eluted with buffer F (20 mM phosphate buffer, 300 mM NaCl, 300 mM imidazole, pH 8.0). The target protein was collected and subjected to reducing SDS-PAGE electrophoresis.

[0044] A single band of approximately 14 kDa was detected, indicating that an affinity-binding protein for the S protein was obtained and named Bntifitin. The amino acid sequence is shown in SEQ NO: 1.

[0045] Example 5 Preparation of pseudovirus

[0046] After optimizing the S protein DNA fragments of SARS-CoV-2 Alpha variant (B.1.1.7), Beta variant (B.1.351), Gamma variant (P.1), Delta variant (B.1.617.2), Omicron variant (BA.2.75), and Omicron variant (XBB.1.5), the S protein expression plasmids of 6 SARS-CoV-2 variants of interest were obtained by seamless cloning technology, referring to Example 2. Then, 5ug of each of the 6 S protein expression plasmids was added to 500uL of DMEM, and then 5ug of pNL4.3-Luc-RE-shuttle plasmid and 5ug of HIV-1 lentiviral packaging auxiliary plasmid psPAX2 (Shanghai Haijihaoge Biotechnology Co., Ltd.) were added respectively, mixed, and allowed to stand at room temperature for 5 minutes. 15 μg of polyethyleneimine [Mw ≈ 25,000, Sigma-Aldrich (408727)] was added to 500 μL of DMEM and allowed to stand at room temperature for 5 minutes. Subsequently, the plasmid and polyethyleneimine mixtures were mixed and allowed to stand at room temperature for 15 minutes. Then, HEK293T cells with good growth status were taken and inoculated into a 10 cm culture dish at a density of 6 × 106 / mL. They were cultured with DMEM medium containing 10% fetal bovine serum and cultured in a 37 ° C, 5% CO2 incubator for 12 h. Then, a mixture of plasmid and polyethyleneimine was added dropwise and cultured in a 37 ° C, 5% CO2 cell culture incubator for 12 h. The original culture medium was discarded and replaced with 10 mL of DMEM medium containing 2% fetal bovine serum. The cells were cultured in a 37 ° C, 5% CO2 cell culture incubator for 48 h. The culture supernatant containing SARS-CoV-2 pseudovirus was collected and centrifuged at 3000g for 10 min at 4 ° C. The supernatant was taken, filtered with a 0.45 μm filter, and stored at -80 ° C for later use.

[0047] Example 6 Binding affinity determination

[0048] Binding affinity was determined using a Biacore 8K SPR instrument (GE Healthcare) at 25°C. 4.25 μg / mL of S protein RBD was diluted with 10 mM sodium acetate (pH 4.5) and flowed over the chip surface at a flow rate of 10 μL / min. Unreacted RBD was then washed away with 1 M hydrochloric acid-containing aminoethanol (pH 8.5). Unreacted active sites on the chip were also blocked by reaction with the hydrochloric acid-containing aminoethanol. The protein Bntifitin was dissolved in the Biacore buffer provided at various concentrations. 80 μL of samples of different concentrations were continuously passed over the chip surface at a flow rate of 10 μL / min. The sample dissociation process was performed using the buffer provided by Biacore, also at a flow rate of 10 μL / min, to flush the chip. The chip surface was regenerated with 70 μL of 10 mM aminoacetic acid (pH 2.5) to elute the bound drug protein. The real-time response signal was fitted with Biacore evaluation software to obtain a binding affinity KD of 1.3 nM between the drug protein and RBD, indicating that Bntifitin-S10 binds to RBD with high affinity.

[0049] Example 7 Binding Protein Recognizes and Binds to S1 Subunit and RBD

[0050] 100 μL of protein Bntifitin (concentration of 2 μg / mL) diluted with PBS was added to a 96-well microtiter plate (Corning Company) and incubated at 4°C overnight. 2+ (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM CaCl2, 0.1% Tween-20) and washed twice, and 200 μL of 2% bovine serum albumin (BSA)-TBST-Ca 2+ , reacted at 37℃ for 2h. 2 + After washing twice, different concentrations of TBST-Ca 2+ Diluted biotin-labeled RBD protein was incubated at 37°C for 1 h, with BSA as the control, and then TBST-Ca was added. 2+ Diluted HRP-conjugated streptavidin (1:10,000, Biospa) was incubated at 37°C for 1 h. After washing the plate three times with TBST-Ca2+, 50 μL of 3,3',5,5'-tetramethylbenzidine (TMB, Thermo Fisher Scientific) color development solution was added. After 15-30 min, 50 μL of 2 M H2SO4 was added to terminate the reaction. The plate was measured on a microplate reader at OD 450 The absorbance value of each well was measured under the following conditions. The results are as follows Figure 2As shown, as the RBD protein concentration increases, the amount of binding to bntifitin increases in a dose-dependent manner. In the BSA control group, despite increasing RBD protein concentration, RBD protein binding to bntifitin is not dose-dependent. This indicates that the coronavirus S protein-binding protein bntifitin can recognize and bind to the RBD protein.

[0051] Example 8 Detection of the inhibitory activity of binding protein on pseudovirus infection

[0052] Serum-free DMEM medium was used to dilute Bntifitin and BSA in a 96-well round-bottom plate in a 10-fold gradient, with a starting concentration of 20 μM and a total of 6 dilutions. The drug volume per well was 100 μL, and 3 replicates were set for each gradient; 100 μL of SARS-CoV-2 pseudovirus solution (RLU: 50,000-100,000) was added to a 96-well plate (wells with drugs and viruses were recorded as drug wells). At the same time, a positive control group (with virus but no drug, i.e., virus wells) and a negative control group (with no virus and no drug, i.e., no drug wells) for virus infection were set up. The virus and drug were incubated at 37°C for 1 hour; and then added to a 96-well plate with HuH-7 cells. Culture at 37°C, 5% CO2 for 12 hours, then replace the culture medium with DMEM containing 10% FBS. After 48 hours, discard the culture supernatant and wash the cells once with PBS. Add 50 μL of cell lysis buffer (purchased from Promega, catalog number 0000389797) and lyse at room temperature for 1 hour. Pipette 40 μL of lysis supernatant into a 96-well assay plate, add 40 μL of substrate (purchased from Promega, catalog number 0000369690), and immediately measure the fluorescence value. Calculate the inhibition rate of Bntifitin on pseudovirus infection using the following formula: Inhibition rate = (virus wells - drug wells) / (virus wells - drug-free wells) × 100%. The results are shown in Figure 2. Figure 3 As shown, Bntifitin can inhibit SARS-CoV-2 pseudovirus infection.

[0053] Example 9 Detection of inhibitory activity against SARS-CoV-2 live virus infection

[0054] Bntifitin was diluted 10-fold in a 96-well round-bottom plate using serum-free DMEM. The starting concentration was 20 μM, and a total of 6 dilutions were performed. The drug volume per well was 50 μL, and 3 replicates were set for each gradient. 50 μL of 100 TCID50 SARS-CoV-2 live virus (preserved by the P3 laboratory of Wuhan University) was added to the 96-well plate (the drug-added and virus-added wells were marked as drug wells). At the same time, a positive control group (with virus but no drug, i.e., virus wells) and a negative control group (with virus) for virus infection were set. No virus and no drug, i.e., no drug wells), the virus and drug were incubated at 37°C for 1 hour; 100 μL of the virus and drug mixture was added to a 96-well plate plated with Vero-E6 cells (purchased from ATCC, catalog number CRL-1586TM), cultured at 37°C, 5% CO2 for 1 hour, and the culture medium was replaced with DMEM medium containing 10% FBS; after 48 hours, the supernatant was collected and TRIZOL was added, and the cells were fixed with 4% formaldehyde and immersed in 0.2% Triton X-100 in PBS solution; the culture supernatant was used for qRT-PCR viral RNA, and the detection kit was purchased from Takara; the primers used were purchased from GENEWIZ, and the probe was purchased from GENEWIZ, and its sequence was 5'-FAM-TTGCTGCTGCTTGACAGATT-TAMRA-3'. The cells were detected for viral nucleocapsid by indirect immunofluorescence. The primary antibody was SARS-CoV-2 nucleocapsid antibody (Sino Biological) at a dilution ratio of 1:200, and the secondary antibody was AlexaFluor 488 goat anti-mouse IgG (Thermo Fisher) at a dilution ratio of 1:100. The infection rate was calculated using the following formula: infection rate = (viral wells - drug wells) / (viral wells - drug-free wells) × 100%.

[0055] The results are as follows Figure 4 As shown, it is shown that the binding protein Bntifitin of the present invention can inhibit SARS-CoV-2 live virus infection.

[0056] Example 10 Crossing the blood-brain barrier

[0057] Eight 4- to 6-week-old female CBA / J mice were harvested and their cerebral cortices were cut into 2- to 3-mm fragments. The fragments were digested with DMEM containing 0.1% trypsin and 0.1% EDTA at 37°C for 30 minutes, followed by digestion with 0.2% collagenase (type II) for 15 minutes. After homogenization, the fragments were filtered through a 200-μm nylon membrane. 5 mL of FCS was added to the homogenate, and the fragments were centrifuged at 500 rpm for 5 minutes. The supernatant was discarded, and the residue was redissolved in 5 mL of DMEM and then mixed with 20 mL of 20% Percoll. Brain microvascular endothelial cells (BMVECs) were collected by centrifugation at 1500 rpm for 15 minutes. The BMVECs were washed twice with DMEM and suspended in complete culture medium (20% inactivated FCS, 100 μg / mL streptomycin / penicillin, 2 mM L-glutamine, 100 μg / mL endothelial cell growth factor, and 40 U / mL heparin). Then, culture in gelatin-coated T25 plastic tissue culture flasks for 24 hours, wash with DMEM, and remove non-adherent cells. Change the medium every two days. After one week, BMVEC colonies have formed. Using an inverted light microscope, manually remove non-endothelial cells surrounding the BMVECs. After topical trypsin / EDTA digestion, transfer the BMVECs to a new flask using a glass micropipette. Once the cells are cultured to confluency, passage them using trypsin / EDTA (1:3).

[0058] BMVECs (passage 8) were suspended in endothelial cell culture medium (DMEM 20% fetal bovine serum, 100,000 u / L penicillin, 100 mg / L streptomycin, 2 mmol / L L-glutamine, 100 mg / L ECGF, 20 mg / L heparin, 40 μg / L insulin) and seeded into 24-well cell inserts pre-coated with 2% (mass fraction) gelatin at a seeding density of 200,000 cells / cm 2 Culture in a 5% (volume fraction) CO2, 37°C incubator until confluence (3-5 days). Add cell culture medium to the donor and recipient reservoirs of the cell inserter, ensuring a >0.5 cm difference in the liquid level between the donor and recipient reservoirs. After 4 hours, if a significant difference in the liquid level is still maintained between the two reservoirs of the inserter, the BMVECs are considered fully confluent and the BBB is essentially formed.

[0059] Select BMVECs that have reached confluence for permeability experiments. Dissolve Bntifitin in experimental culture medium (endothelial-specific culture medium without phenol red and ECGS). Discard the original endothelial-specific culture medium, then add 460 μL of experimental culture medium containing 500 ng Bntifitin to the cell inserter donor pool, and add 1140 μL of experimental culture medium to the receptor pool, so that the liquid levels inside and outside the cell inserter are level. The cells were cultured in a 37°C 5% CO2 incubator for 24 hours, and 50 μL of samples were taken from the receptor pool at different time points (50 μL of experimental culture medium was added to the receptor pool at the same time), placed in a 96-well plate, and the absorbance (D value) was measured at 450 nm. The permeability of Bntifitin was calculated based on the standard curve. Permeability calculation formula: P% = (C 受池 ×V 受池 ) / (C 上池 ×V 上池 )×100%.

[0060] The results are as follows Figure 5 As shown, at 1 hour, the permeability of the binding protein Bntifitin of the present invention reaches 40%, and then the permeability increases slowly over time, reaching 70% at 24 hours.

[0061] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements, but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0062] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0063] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A binding protein Bntifitin, characterized in that The protein has an amino acid sequence as shown in SEQ ID NO:

1.

2. A nucleic acid molecule encoding the binding protein Bntifitin according to claim 1.

3. A nucleic acid construct binding protein Bntifitin, characterized in that, The nucleic acid construct is capable of expressing the binding protein Bntifitin according to claim 1.

4. A nucleic acid construct binding protein Bntifitin according to claim 3, characterized in that, The nucleic acid construct is selected from an expression cassette or a vector.

5. A host cell containing the nucleic acid molecule according to claim 2 or the nucleic acid construct according to claim 3 or 4.

6. The host cell according to claim 5, characterized in that The host cells include prokaryotic host cells and eukaryotic host cells.

7. Use of the binding protein Bntifitin according to claim 1, or the nucleic acid molecule according to claim 2, or the nucleic acid construct according to claim 3 or 4, or the host cell according to claim 5 or 6 in the preparation of a medicine for treating coronavirus infection, wherein the coronavirus is SARS-CoV-2.

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