Identification and application of interaction between host protein BRAP peptide fragment and coronavirus

Abstract

The invention discloses a host protein BRAP peptide fragment and application of the host protein BRAP peptide fragment in research and treatment of coronavirus, and particularly relates to identification of the BRAP peptide fragment having specific interaction with an SARS-CoV-2CoV-Y structural domain, development of a method for in-vitro identification of interaction of the peptide fragment and the coronavirus, preparation of drugs for inhibiting SARS-CoV-2 induced host inflammatory response by using the peptide fragment, and application of the host protein BRAP peptide fragment in research and treatment of the coronavirus. According to the method, a model for screening anti-SARS-CoV-2 drugs is constructed, and an in-vitro model for researching an SARS-CoV-2 infection mechanism is established, so that a theoretical basis and technical support are provided for deeply understanding the pathogenic mechanism of coronavirus and developing new antiviral drugs and treatment strategies. The peptide fragment interacts with a CoV-Y structural domain, the dissociation constant Kd of the interaction is 12.67 + / -0.2 mu M, and the stoichiometric ratio is 1: 1.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to the identification of the interaction between host proteins and coronaviruses and its application in the development of antiviral drugs. Background Art

[0002] Since the end of 2019, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has triggered a global pandemic of novel coronavirus pneumonia (COVID-19), which has had a profound impact on human health and socio-economy. SARS-CoV-2 is transmitted through the respiratory tract. After infecting host cells, it can activate the inflammatory response in host cells, leading to the massive release of inflammatory factors, which in turn triggers an inflammatory storm, which is one of the main reasons for the severe symptoms of COVID-19 patients. However, the molecular mechanism of SARS-CoV-2-induced host inflammatory response is not fully understood.

[0003] During viral infection, the interaction between viral proteins and host proteins is the basis for key steps such as viral replication, assembly, and release. Studying these interactions helps to reveal the pathogenic mechanism of the virus and provide potential targets for the development of new antiviral strategies. The nonstructural protein 3 (Nsp3) of SARS-CoV-2 is a multifunctional protein, and its C-terminal conserved domain CoV-Y plays an important role in the interaction between the virus and host cells. Studies have shown that the CoV-Y domain in the C-terminal region of Nsp3 can interact with host cell proteins, such as BRAP protein, causing inflammatory responses in host cells. However, the specific interaction peptides of this domain in Nsp3 and host proteins and their affinity have not yet been identified in detail.

[0004] In addition, as SARS-CoV-2 continues to mutate, existing antiviral drugs are facing the problem of drug resistance, which makes it particularly urgent to find new drug targets. The C-terminal region of Nsp3 is considered a potential target for antiviral drug development due to its key role in the viral life cycle and its relatively low mutation frequency. Therefore, in-depth research on the interaction between the SARS-CoV-2CoV-Y domain and host proteins, especially the interaction peptides and their affinity with the host protein BRAP, is of great significance for the development of new antiviral drugs and treatment strategies. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a host protein BRAP peptide segment and its application in coronavirus research and treatment, specifically including identifying a BRAP peptide segment that has a specific interaction with the SARS-CoV-2CoV-Y domain, developing an in vitro method for identifying the interaction between the peptide segment and the coronavirus, using the peptide segment to prepare a drug that inhibits SARS-CoV-2-induced host inflammatory response, constructing a model for screening anti-SARS-CoV-2 drugs, and establishing an in vitro model for studying the SARS-CoV-2 infection mechanism, thereby providing a theoretical basis and technical support for a deep understanding of the pathogenic mechanism of coronavirus and the development of new antiviral drugs and treatment strategies.

[0006] Through long-term exploration and attempts, as well as multiple experiments and efforts, the inventors have continuously reformed and innovated to solve the above technical problems. The technical solution provided by the present invention is to provide a host protein BRAP peptide segment, the amino acid sequence of which is shown in the sequence table SEQ ID NO: 1; the peptide segment interacts with the SARS-CoV-2CoV-Y domain, and the dissociation constant Kd of the interaction is 12.67±0.2μM, and the stoichiometric ratio is 1:1.

[0007] Compared with the prior art, the present invention has the following beneficial effects:

[0008] Compared with the prior art, the beneficial effect of the present invention lies in that, for the first time, the specific interaction between a specific peptide segment of the host protein BRAP (the amino acid sequence is shown in SEQ ID NO: 1 in the sequence table) and the SARS-CoV-2CoV-Y domain is clearly identified, which not only reveals a key molecular mechanism in the process of SARS-CoV-2 infecting host cells, but also accurately determines the dissociation constant Kd of the interaction between the two as 12.67±0.2μM and the stoichiometric ratio as 1:1. A lower Kd value usually means a higher binding affinity, that is, the interaction between the two molecules is tighter, and each CoV-Y molecule binds to a BRAP molecule to form a stable dimer structure, which provides an important quantitative basis for the subsequent development of antiviral drugs based on this interaction.

[0009] The present invention also provides a method for identifying the interaction between a host protein BRAP peptide segment and a coronavirus, comprising the following steps:

[0010] Purify SARS-CoV-2CoV-Y domain recombinant protein and BRAP peptide in vitro;

[0011] Isothermal titration calorimetry (ITC) was used to detect the binding affinity and binding ratio between the CoV-Y domain and the BRAP peptide.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] This method purifies the SARS-CoV-2CoV-Y domain recombinant protein and BRAP peptide in vitro, and then uses isothermal titration calorimetry (ITC), a high-precision and high-sensitivity biophysical technique, to accurately measure the binding affinity and binding ratio between the CoV-Y domain and the BRAP peptide. This not only provides direct and reliable experimental data for studying the interaction between coronaviruses and host proteins, but also has higher quantitative accuracy and wider applicability compared to traditional interaction research methods. The establishment of this method enables researchers to more deeply analyze the interaction mechanism between viruses and host proteins, and provides strong technical support for the development of new antiviral drugs and treatment strategies for coronaviruses. It has important scientific research value and application prospects.

[0014] On the basis of the above technical solution, the present invention can also be improved as follows:

[0015] Further:

[0016] The preparation of the SARS-CoV-2CoV-Y domain recombinant protein comprises the following steps:

[0017] Using the first primer pair, PCR amplification was performed to obtain the CoV-Y target gene fragment;

[0018] The first primer pair is:

[0019] CoV-YF: 5'-ggaattccatatggatcagagtagctatatt-3';

[0020] CoV-YR: 5'-ccgctcgagttagccacctttcagtgcaattttggtgg-3';

[0021] The amplified CoV-Y target gene fragment was inserted into the prokaryotic expression vector pET-28a+ to construct a recombinant plasmid;

[0022] The recombinant plasmid was transformed into the BL21 expression strain for induced expression and protein purification.

[0023] Compared with the prior art, the beneficial effects of adopting the above further technical solution are:

[0024] By adopting the above further technical scheme, the present invention has significant advantages in preparing SARS-CoV-2CoV-Y domain recombinant protein. First, by designing and synthesizing the first pair of specific primers (CoV-YF and CoV-YR), the CoV-Y target gene fragment can be efficiently and specifically amplified, ensuring the accuracy and reliability of PCR amplification, and laying a solid foundation for subsequent gene cloning and protein expression. Secondly, the amplified CoV-Y target gene fragment is inserted into the prokaryotic expression vector pET-28a (+) to construct a recombinant plasmid. This process utilizes the efficient expression characteristics of the pET-28a (+) vector and can achieve high-level expression of CoV-Y protein. Furthermore, the recombinant plasmid is transformed into the BL21 expression strain. As a commonly used efficient expression host, the BL21 strain can provide a good expression environment, so that the CoV-Y recombinant protein is expressed in large quantities. Finally, through the induction expression and protein purification steps, a high-purity and highly active SARS-CoV-2CoV-Y domain recombinant protein can be obtained, providing high-quality experimental materials for subsequent interaction research and drug development.

[0025] On the basis of the above technical solution, the present invention can also be improved as follows:

[0026] Further:

[0027] The preparation of the BRAP peptide recombinant protein comprises the following steps:

[0028] Using the second primer pair, BRAP fragments were obtained by PCR amplification;

[0029] The second primer pair is:

[0030] BRAP-F: 5'-ccgcgtggatccccggaattcgaagaaaataagtgttttga-3';

[0031] BRAP-R: 5'-gtcacgatgcggccgctcgagttatcgatgaacatagttatctcca-3';

[0032] The amplified BRAP fragment was inserted into the prokaryotic expression vector pGEX4T-1 to construct a recombinant plasmid;

[0033] The recombinant plasmid was transformed into BL21 competent cells for induced expression and protein purification.

[0034] Compared with the prior art, the beneficial effects of adopting the above further technical solution are:

[0035] By adopting the above further technical scheme, the BRAP fragment can be amplified efficiently and specifically through the specially designed second primer pair (BRAP-F and BRAP-R), ensuring the accuracy and reliability of PCR amplification, and providing a solid foundation for subsequent gene cloning and protein expression. Secondly, the amplified BRAP fragment is inserted into the prokaryotic expression vector pGEX4T-1 to construct a recombinant plasmid. This process takes advantage of the high-efficiency expression and fusion tag advantages of the pGEX4T-1 vector, which can achieve high-level expression of the BRAP peptide segment, and the fused GST tag helps to improve the solubility and stability of the protein, which is convenient for subsequent protein purification and functional research. Furthermore, the recombinant plasmid is transformed into the BL21 competent state. As a commonly used high-efficiency expression host, the BL21 strain can provide a good expression environment, allowing the BRAP peptide segment to be expressed in large quantities. Finally, through the induced expression and protein purification steps, a high-purity and high-activity BRAP peptide segment recombinant protein can be obtained, which provides high-quality experimental materials for studying its interaction with the SARS-CoV-2CoV-Y domain and its application in the development of antiviral drugs.

[0036] The present invention also provides an application of a host protein BRAP peptide segment for preparing a drug for inhibiting SARS-CoV-2-induced host inflammatory response.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] The host protein BRAP peptide provided by the present invention is a key target screened out based on an in-depth study of the interaction mechanism between the SARS-CoV-2CoV-Y domain and the host protein. It has a clear biological function and mechanism of action, and can specifically interfere with the interaction between the CoV-Y domain and the host cell, thereby effectively inhibiting the release of virus-induced inflammatory factors and the occurrence of inflammatory response. Secondly, the application of this peptide segment expands the research and development ideas of antiviral drugs, breaks through the limitations of traditional drugs that mainly target direct targets such as viral replicase, and provides new directions and possibilities for the development of new antiviral drugs. Finally, the application of this peptide segment also provides new options for personalized treatment and precision medicine. According to the patient's host protein characteristics and viral infection conditions, targeted drug treatment plans can be designed to improve the treatment effect and patient prognosis. In summary, the present invention has important theoretical significance and broad application prospects in clarifying the mechanism of SARS-CoV-2-induced host inflammatory response and developing drugs to alleviate the host inflammatory response caused by viruses.

[0039] The present invention also provides a drug for inhibiting SARS-CoV-2-induced host inflammatory response, comprising the host protein BRAP peptide segment.

[0040] Preferably, it also includes a pharmaceutically acceptable carrier or excipient.

[0041] The present invention also provides a method for screening anti-SARS-CoV-2 drugs, comprising the following steps:

[0042] Using the interaction between the host protein BRAP peptide and the SARS-CoV-2CoV-Y domain as a screening model;

[0043] The effects of the compounds to be screened on the interaction between the BRAP peptide and the CoV-Y domain were tested to screen out potential antiviral drugs.

[0044] The present invention also provides an in vitro model for studying the infection mechanism of SARS-CoV-2, comprising:

[0045] Host cells expressing the SARS-CoV-2CoV-Y domain;

[0046] The expression vector containing the host protein BRAP peptide segment is used to study the interaction between the CoV-Y domain and the BRAP peptide segment and its effect on the host cell function.

[0047] Compared with the prior art, the present invention has the following beneficial effects:

[0048] This model successfully simulates the binding mode between the key viral protein domain and the host protein by expressing the host cells of the SARS-CoV-2CoV-Y domain, providing an important experimental basis for studying the function and mechanism of action of the viral protein. The introduction of the expression vector containing the host protein BRAP peptide enables researchers to directly observe and analyze the interaction between the CoV-Y domain and the BRAP peptide under in vitro conditions, as well as the specific effects of this interaction on the function of host cells, thereby deeply revealing the molecular mechanism by which SARS-CoV-2 infects host cells and induces host inflammatory responses. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0050] Figure 1 It is the predicted structural result of the interaction between the Yc subdomain of the SARS-CoV-2CoV-Y domain and the BRAP fragment complex.

[0051] Figure 2 It is a schematic diagram of the pET-28a(+)-CoV-Y recombinant plasmid.

[0052] Figure 3 Figure 1 is the purification result of CoV-Y recombinant protein; Figure A is the gel filtration chromatography result of CoV-Y recombinant protein, and Figure B is the SDS-PAGE identification result of CoV-Y recombinant protein.

[0053] Figure 4 It is a schematic diagram of the pGEX4T-1-BRAP fragment recombinant plasmid.

[0054] Figure 5 Figure 1 is the purification result of BRAP fragment recombinant protein; Figure A is the gel filtration chromatography result of BRAP fragment recombinant protein, and Figure B is the SDS-PAGE identification result of BRAP fragment recombinant protein.

[0055] Figure 6 This is the result of the ITC experiment of SARS-CoV-2CoV-Y recombinant protein and BRAP fragment recombinant protein.

[0056] Figure 7 This is the result of the Co-IP screening experiment on the interaction between different domains of BRAP and the recombinant protein of SARS-CoV-2CoV-Y. DETAILED DESCRIPTION

[0057] The following describes the invention in conjunction with specific embodiments.

[0058] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention.

[0059] In the present invention, unless otherwise specified, all equipment and raw materials can be purchased from the market or are commonly used in the industry. The methods in the following embodiments, unless otherwise specified, are conventional methods in the art.

[0060] Example 1

[0061] This example is an example of a method for identifying a host protein BRAP peptide segment and its interaction with a coronavirus.

[0062] In the experimental process of the present invention, the inventors conducted a series of Co-IP experiments to explore the interaction between different domains of the host protein BRAP and the SARS-CoV-2CoV-Y domain. Through these experiments, the inventors ruled out the possibility of some non-specific binding and finally identified BRAP-2 (amino acids 257-394) as the key region for interaction with the CoV-Y domain. The experimental results are shown in Figure 7 .

[0063] See also Figure 7 , the expression of each BRAP domain and the control vector (Vector) in cells was significantly different. BRAP-HA, BRAP-1-HA, BRAP-2-HA, BRAP-3-HA showed clear bands at the expected molecular weights (approximately 70kDa, 31kDa, 17kDa, and 22kDa, respectively), indicating that these proteins were successfully expressed in cells. The Vector control group showed no bands, verifying that there was no nonspecific binding in the experiment.

[0064] In the presence of Flag-CoV-Y, BRAP-2-HA showed coprecipitation with Flag-CoV-Y in Flag immunoprecipitation, indicating a specific interaction between BRAP-2 and CoV-Y. BRAP-1-HA and BRAP-3-HA showed no specific bands, indicating that these regions did not interact with CoV-Y. BRAP-HA (full-length BRAP) also showed some coprecipitation, but considering that full-length BRAP contains all tested domains, this result may include contributions from multiple domains.

[0065] Figure 7 The results of the Co-IP experiment clearly pointed out that BRAP-2 (amino acids 257-394) is the key region for interaction with the SARS-CoV-2CoV-Y domain. This finding excludes the possibility that other domains of BRAP are the main interaction regions, providing a clear direction for subsequent research.

[0066] Through a series of experimental results, the inventors identified the interaction domain between the host protein BRAP and the CoV-Y domain, and further tested the binding mode and affinity of the peptide segment (amino acids 313-375) within this domain that interacts with the CoV-Y domain, providing an important molecular target for the development of antiviral strategies against SARS-CoV-2.

[0067] 1. Identification of the host protein BRAP peptide

[0068] 1.1 ZDOCK online prediction of complex structure

[0069] Through the ZDOCK online prediction technology, the BRAP peptide motif that interacts with the SARS-CoV-2CoV-Y domain was preliminarily determined.

[0070] Steps:

[0071] Get the three-dimensional structure file of the SARS-CoV-2CoV-Y domain (PDB format), the PDB accession number is 8ILC.

[0072] Obtain the three-dimensional structure file of human BRAP protein (PDB format), the Uniprot accession number is Q7Z569.

[0073] Visit the official website of ZDOCK (https: / / zdock.wenglab.org / ) and upload the PDB files of CoV-Y domain and BRAP protein to the ZDOCK platform.

[0074] Set ZDOCK parameters, adjust docking mode and search range, and start the prediction program.

[0075] The ZDOCK prediction results were analyzed and the BRAP peptide motif (peptide (amino acids 313-375)) that binds to the CoV-Y domain was screened out. The results are as follows Figure 1 Its amino acid sequence is shown in SEQ ID NO:1.

[0076] 1.2 In vitro purification of SARS-CoV-2CoV-Y domain recombinant protein

[0077] Obtain highly pure SARS-CoV-2CoV-Y domain recombinant protein for subsequent interaction verification experiments.

[0078] Steps:

[0079] The accession number of the SARS-CoV-2CoV-Y domain on NCBI is NC_045512.2.

[0080] According to the nucleotide sequence of the SARS-CoV-2CoV-Y domain, the upstream primer CoV-YF (5'-ggaattccatatggatcagagtagctatatt-3') and the downstream primer CoV-YR (5'-ccgctcgagttagccacctttcagtgcaattttggtgg-3') were designed and synthesized.

[0081] Using the synthesized Y plasmid as a template, the above-mentioned primers CoV-YF and CoV-YR were used to amplify the CoV-Y target gene fragment by PCR, and the nucleotide of the PCR amplification product is shown in SEQ ID NO: 2.

[0082] The PCR reaction system and program settings are as follows:

[0083] Reagent dosage: upstream primer 2μL, downstream primer 2μL, template 1μL, 2×Mix PCR polymerase 25μL, ddH2O 20μL, total 50μL.

[0084] Reaction procedure: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 15 s, annealing at 55°C for 15 s, extension at 72°C for 30 s / kb, for a total of 30 cycles, and final extension at 72°C for 10 min, and storage at 4°C.

[0085] Purify the PCR product using a PCR purification kit (such as OMEGA product number D6492).

[0086] The pET-28a(+) prokaryotic plasmid vector and the CoV-Y target gene fragment were digested with the fast restriction endonucleases NdeⅠ (Thermo Fisher, Catalog No. FD0585) and XhoⅠ (Thermo Fisher, Catalog No. FD0695) at 37°C for 30 min, and the digested fragments were purified using a PCR purification kit.

[0087] Use T4 DNA ligase (Thermo Fisher Product No. EL0011) to connect the pET-28a(+) double-digested product with the CoV-Y target gene fragment according to the instructions, transform DH5α competent cells, pick a single clone for sequencing, and complete the construction of the recombinant plasmid. The schematic diagram of the pET-28a(+)-CoV-Y recombinant plasmid is shown in Figure 2 shown.

[0088] The constructed pET-28a(+)-CoV-Y recombinant plasmid was transformed into the BL21(DE3) expression strain. The next day, clones were picked from the LB plate and cultured in a 37°C shaker until the bacterial solution OD 600 value was 0.6-0.8, IPTG (final concentration was 0.5 mM) was added, and the cells were induced at 18°C ​​and 220 rpm for 16-18 h, followed by centrifugation at 8 000 rpm for 10 min, the supernatant was discarded, and the bacteria were collected.

[0089] The collected bacteria were resuspended in buffer A (20mM Tris-HCl pH7.5, 10mM imidazole, 500mM NaCl), and 1mM PMSF was added to prevent degradation of the target protein. At a low temperature of 4°C, the bacteria were broken using a high-pressure crusher. First, the high-pressure crusher pipeline was rinsed with buffer A at 0 bar, and then the resuspended bacterial solution was added to the filter. The bacterial solution flowed into the sample cup and circulated until there were no bubbles in the pipeline. Then, the pressure was increased to 200 bar and run for 2 minutes to break the nucleic acid. The pressure was continued to be increased to 600 bar-800 bar to break the bacteria for 3-4 minutes. After the crushing was completed, the pressure was reduced to 0 bar, and all the liquid was collected in a centrifuge tube, and centrifuged at 4°C, 18,000 rpm for 45 minutes to remove cell debris.

[0090] The supernatant after centrifugation was collected and filtered, and the sample was slowly loaded onto a HisTrapFF pre-packed column (Cytiva, catalog number 17525501) equilibrated with buffer A using a peristaltic pump to allow the target protein to bind tightly to the nickel column material.

[0091] After loading, the pre-packed column bound to the target protein was installed on the AKTA column position, and 15-20mL buffer A was used to wash the impurities at a flow rate of 2.5mL / min, followed by 5% buffer B (20mM Tris-HCl pH 8.0, 500mM imidazole, 500mM NaCl) to further remove impurities until the UV absorption peak of the effluent tended to be flat. Finally, the concentration of buffer B was set to increase with time gradient until the target protein was eluted and collected in a 10mL collection tube. Thrombin protease was added to the target protein eluate to cut the His-tag, and all protein solutions were transferred to a dialysis bag and dialyzed overnight in buffer C (20mM Tris-HCl pH 7.5, 150mM NaCl) at 4°C to remove imidazole in the protein solution. The next day, the protein sample after overnight digestion was loaded onto the HisTrap FF pre-packed column and affinity chromatography was performed again. The target protein that was completely digested would no longer bind to the column material, while the target protein whose His-tag was not removed would still bind to the column material. Therefore, the flow-through was collected and eluted with buffer A to obtain a protein sample with His-tag removed and higher purity.

[0092] Gel filtration chromatography is a method that uses porous mesh particles as separation media to separate components in a protein mixture according to the size and shape of the protein. Large molecules are blocked outside the pores by the gel and have a short flow path, while small molecules can pass through the pores and have a long flow path, so large molecules flow out first and small molecules flow out later. First, use buffer C to sonicate at 70% power for 3 minutes to remove bubbles, and then use buffer C to balance the Superdex 75Increase 10 / 300GL chromatography column (Cytiva, catalog number 29148721). At the same time, the obtained protein sample is concentrated to 1 mL and centrifuged at 4°C to remove protein precipitates and bubbles. After centrifugation, the supernatant is added to the sample loop on the chromatography column, and buffer C is used as the mobile phase to elute the protein at a flow rate of 0.5 mL / min. The size and purity of the collected protein are identified by SDS-PAGE to obtain a high-purity, uniform SARS-CoV-2CoV-Y recombinant protein, such as Figure 3 shown.

[0093] 1.3 In vitro purification of BRAP peptide recombinant protein

[0094] High-purity BRAP peptide recombinant protein was obtained for subsequent interaction verification experiments.

[0095] Steps:

[0096] According to the nucleotide sequence of the human BRAP fragment (as shown in SEQ ID NO: 1), the upstream primer BRAP-F (5'-ccgcgtggatccccggaattcgaagaaaataagtgttttga-3') and the downstream primer BRAP-R (5'-gtcacgatgcggccgctcgagttatcgatgaacatagttatctcca-3') were designed and synthesized.

[0097] The total cell cDNA was used as a template, and the above primers BRAP-F / BRAP-R were used to amplify the target BRAP fragment by PCR. The PCR reaction system and program settings were the same as those for CoV-Y fragment amplification. The nucleotides of the PCR amplification product are shown in SEQ ID NO: 3.

[0098] Purify the PCR product using a PCR purification kit.

[0099] The pGEX4T-1 prokaryotic expression plasmid vector was digested with rapid restriction endonucleases EcoI (Thermo Fisher, catalog number FD0274) and XhoI at 37°C for 30 min, and then the digested plasmid was purified using a PCR purification kit.

[0100] Use homologous recombinase (such as Vazyme product number C116-02) to connect the pGEX4T-1 double-digested product with the BRAP fragment target gene fragment, transform DH5α competent cells, pick a single clone for sequencing, and complete the construction of the recombinant plasmid. The schematic diagram of the pGEX4T-1-BRAP fragment recombinant plasmid is shown in the figure. Figure 4 shown.

[0101] The constructed pGEX4T-1-BRAP fragment recombinant plasmid was transformed into BL21 competent cells. The next day, clones were picked from the LB plate and cultured in a shaking incubator at 37°C until the bacterial solution OD 600 The value was 0.6-0.8, IPTG (final concentration was 0.5 mM) was added, and the culture was induced at 18°C ​​and 220 rpm for 16-18 h. Then the culture was centrifuged at 8 000 rpm for 10 min, the supernatant was discarded, and the bacteria were collected.

[0102] The collected bacteria were fully resuspended with GST buffer A (20mM Tris-HCl pH7.5, 500mM NaCl), and ultrasonically broken on ice, with the power set to 20%, the time to 3min, the frequency of ultrasonic 6s interval 3s, and repeated three times until the bacteria were completely broken. After the bacterial liquid was broken, it was transferred to a dedicated high-speed centrifuge tube, centrifuged at 4℃, 18,000rpm for 45min, and the supernatant was aspirated and filtered with a 0.45μm filter membrane. The filtrate was equilibrated with GST buffer A and incubated on ice for 90min with the GST column material. The bacterial supernatant was removed by centrifugation at 2,000rpm for 10min, and the column material was washed three times with GST buffer A to remove non-specific binding in the GST column material; then GST buffer B (20mM Tris-HCl pH 8.0, 500mM NaCl, 10Mm GSH) was added to elute the target protein and collect it in the collection tube. The eluate containing the target protein was placed at 4℃ and Thrombin protease was added to cut the GST-Tag. The next day, the protein sample after enzyme digestion was concentrated to 1 mL and centrifuged at 13,000 rpm for 10 min at 4°C. At the same time, buffer C was ultrasonicated at 70% power for 3 min to remove bubbles, and then the Superdex 75Increase 10 / 300GL chromatography column was equilibrated with buffer C. Finally, 1 mL of the protein sample after centrifugation was added to the sample loop of the chromatography column, and the protein was eluted with buffer C as the mobile phase at a flow rate of 0.5 mL / min. The size and purity of the collected protein were identified by SDS-PAGE, and the recombinant peptide of BRAP fragment with high purity and uniform state was obtained, such as Figure 5 shown.

[0103] 1.4 ITC affinity test

[0104] Isothermal titration calorimetry (ITC) was used to detect the binding affinity and binding ratio between the CoV-Y domain and the BRAP peptide to verify the interaction between the two.

[0105] Steps:

[0106] The concentration of the purified SARS-CoV-2CoV-Y protein was concentrated to 460 μM, and the concentration of the purified BRAP fragment protein was concentrated to 45 μM.

[0107] At 20°C, an ITC titration experiment was carried out using BRAP fragment protein as substrate protein and CoV-Y protein as ligand protein. 2.5 μL of ligand protein CoV-Y was dripped into the sample pool containing 180 μL of substrate protein BRAP fragment every 150-200 s. The injection was performed 20 times in total, and the stirring speed of the stirrer was set to 125 rpm.

[0108] The original titration data set was processed in Nano Analyze data analysis software, and it was finally determined that the interaction between the CoV-Y domain recombinant protein and the BRAP fragment recombinant peptide was an exothermic process, and the dissociation constant (Kd) was 12.67±0.2μM. The stoichiometric ratio of CoV-Y and BRAP binding was approximately 1, indicating that the molar ratio of the two proteins during the interaction was 1:1, as shown in Figure 6 shown.

[0109] Complex structure prediction results: ZDOCK online prediction results showed that the Yc subdomain of the SARS-CoV-2CoV-Y domain could form a complex with the amino acid 313-375 region of the BRAP protein, and the interaction motif of the BRAP peptide segment was preliminarily determined.

[0110] Protein purification results: Through SDS-PAGE analysis, we successfully obtained high-purity and uniform SARS-CoV-2CoV-Y recombinant protein and BRAP fragment recombinant peptide, providing high-quality experimental materials for subsequent interaction verification experiments.

[0111] ITC test results: The results of the ITC experiment showed that there was a specific interaction between the CoV-Y domain and the BRAP peptide segment, with a clear dissociation constant Kd of 12.67±0.2μM and a stoichiometric ratio of 1:1, which confirmed the binding affinity and binding mode of the two, and provided an important experimental basis for studying the infection mechanism of SARS-CoV-2 and developing antiviral drugs.

[0112] This example successfully identified the interaction between the host protein BRAP peptide segment and the SARS-CoV-2CoV-Y domain, and verified the binding characteristics of the two through in vitro experiments, laying a solid foundation for subsequent research and application.

[0113] Example 2

[0114] This example is an example of the use of the host protein BRAP peptide segment in a drug for inhibiting SARS-CoV-2-induced host inflammatory response.

[0115] Based on the method of Example 1, a high-purity host protein BRAP peptide segment is obtained, and the amino acid sequence is shown in the sequence table SEQ ID NO: 1, which is used for drug preparation.

[0116] The BRAP peptide segment is combined with a pharmaceutically acceptable carrier or excipient to prepare a pharmaceutical preparation suitable for in vivo use.

[0117] Select appropriate drug carriers, such as polyethylene glycol (PEG) or liposomes, to improve the stability and bioavailability of the BRAP peptide segment.

[0118] The purified BRAP peptide is dissolved in an appropriate amount of buffer solution, mixed with PEG or liposomes, and the peptide is fixed on the surface of the carrier or encapsulated inside the carrier by physical adsorption or chemical bonding.

[0119] The mixture is homogenized to form a uniformly dispersed drug preparation.

[0120] In vitro cell experiment pre-experiment:

[0121] Human alveolar epithelial cells (A549 cells) were selected as the experimental cell line, inoculated in a culture plate, and cultured to 80% confluence.

[0122] The cells were divided into a control group, a virus infection group, and a drug treatment group. The control group only added culture medium, the virus infection group added SARS-CoV-2 virus suspension to infect the cells with the virus, and the drug treatment group added different concentrations of BRAP peptide drug preparations before or after virus infection.

[0123] After culturing for 3 days, the cell supernatant was collected and the content of inflammatory factors (such as TNF-α, IL-8, etc.) in the supernatant was detected by enzyme-linked immunosorbent assay (ELISA).

[0124] At the same time, total cellular RNA was extracted, and the expression levels of inflammation-related genes were detected by real-time quantitative PCR (qPCR).

[0125] Compared with the virus-infected group, the levels of inflammatory factors TNF-α and IL-8 in the cell supernatant of the BRAP peptide drug treatment group were significantly reduced, and the expression levels of inflammation-related genes were also significantly downregulated, indicating that BRAP peptide drugs can effectively inhibit the host cell inflammatory response induced by SARS-CoV-2.

[0126] Host protein BRAP peptide drugs significantly inhibit the virus-induced host inflammatory response by blocking or regulating the interaction between the SARS-CoV-2CoV-Y domain and host cells, and have good antiviral activity and therapeutic potential.

[0127] Both technical theory and in vitro cell pre-experiments show that the host protein BRAP peptide fragment can be used to prepare drugs that inhibit SARS-CoV-2-induced host inflammatory response.

[0128] Example 3

[0129] This example is an example of using the interaction between the host protein BRAP peptide segment and the SARS-CoV-2CoV-Y domain to screen antiviral drugs.

[0130] According to the method in Example 1, SARS-CoV-2CoV-Y domain recombinant protein and BRAP peptide recombinant protein were prepared respectively. The purity and molecular weight of the protein were confirmed by SDS-PAGE and mass spectrometry analysis to ensure that it had good biological activity and stability.

[0131] The purified BRAP peptide recombinant protein is mixed with the CoV-Y domain recombinant protein and incubated in a suitable buffer solution to promote the interaction between the two.

[0132] Through surface plasmon resonance (SPR) or ITC and other technologies, the binding constants and kinetic parameters of the BRAP peptide segment and the CoV-Y domain are determined to optimize the conditions of the interaction model, such as pH, ionic strength, temperature, etc.

[0133] Collect a variety of compounds to be screened, including small molecule compounds, natural product extracts, peptide compounds, etc. Dissolve the compounds in appropriate solvents and prepare solutions of different concentrations to meet the needs of subsequent screening experiments.

[0134] The compounds to be screened are added to the interaction system of BRAP peptide and CoV-Y domain, and different concentration gradients are set. Fluorescence polarization (FP) or enzyme-linked immunosorbent assay (ELISA) and other methods are used to detect the effect of the compounds on the binding of BRAP peptide and CoV-Y domain. Compounds that can significantly inhibit or enhance the interaction between the two are screened, and their effective concentration range and degree of inhibition or enhancement are recorded.

[0135] The compounds initially screened were validated for a second time, and high-precision techniques such as SPR or ITC were used to determine in detail the binding constants and kinetic parameters for the interaction between the BRAP peptide and the CoV-Y domain. Compounds with high affinity and good kinetic properties were selected for further antiviral activity testing at the cellular level, such as testing their viral replication inhibition effects on SARS-CoV-2 infected cells and cytoprotective effects. Based on the results of the cell experiments, the compounds were structurally optimized and modified to improve their antiviral activity, stability, and bioavailability.

[0136] Example 4

[0137] This example is an implementation of an in vitro model for studying the mechanism of SARS-CoV-2 infection.

[0138] Based on the nucleotide sequence of the SARS-CoV-2CoV-Y domain, a eukaryotic expression vector containing the CoV-Y gene, such as pCMV-CoV-Y, is constructed.

[0139] Select appropriate host cell lines, such as human alveolar epithelial cells (A549 cells) or human embryonic kidney cells (HEK293 cells), inoculate them in a culture plate, and culture them until they are 70%-80% confluent.

[0140] Use the liposome-mediated transfection method to transfect the pCMV-CoV-Y eukaryotic expression vector into the host cells, and set appropriate transfection conditions, such as liposome concentration, transfection time, etc.

[0141] After transfection, the cells are cultured for 24-48 hours to allow the CoV-Y gene to be expressed in the host cells.

[0142] The expression of CoV-Y protein in host cells was detected by immunofluorescence or Western blot, and host cell lines that stably expressed the CoV-Y domain were screened.

[0143] According to the amino acid sequence of the BRAP peptide segment (as shown in SEQ ID NO: 1 in the sequence listing), the corresponding cDNA sequence was designed and synthesized.

[0144] The BRAP peptide cDNA is inserted into a eukaryotic expression vector, such as pEGFP-N1, to construct the pEGFP-BRAP expression vector, so that the BRAP peptide is fused with green fluorescent protein (GFP) for expression, facilitating subsequent detection and positioning.

[0145] Transfect the pEGFP-BRAP expression vector into host cells that have stably expressed the CoV-Y domain and set appropriate transfection conditions.

[0146] After transfection, the cells are cultured for 24-48 hours to allow the BRAP peptide to be expressed in the host cells.

[0147] The transfected host cells were collected and the total cell protein was extracted.

[0148] Using co-immunoprecipitation (Co-IP) technology, antibodies against CoV-Y protein were used as bait to capture the BRAP peptide fragments that interacted with it.

[0149] The presence of BRAP peptide in the immunoprecipitation product was detected by Western blot to confirm the interaction between the CoV-Y domain and the BRAP peptide.

[0150] Co-immunofluorescence staining technology was further used to observe the co-localization of the CoV-Y domain and the BRAP peptide in host cells and analyze their interaction areas in cells.

[0151] The biological function indicators such as host cell proliferation, apoptosis, and secretion of inflammatory factors were detected, and the effects of the interaction between the CoV-Y domain and the BRAP peptide on the host cell function were compared.

[0152] For example, the proliferation of host cells is detected by a cell counting kit (CCK-8); the apoptosis rate of host cells is detected by Annexin V-FITC / PI double staining; and the content of inflammatory factors (such as TNF-α, IL-8, etc.) in the supernatant of host cells is detected by ELISA.

[0153] Analyze the specific effects of the interaction between the CoV-Y domain and the BRAP peptide on host cell function and explore its mechanism of action during SARS-CoV-2 infection.

[0154] In the description of the present invention, it should be understood that "-" and "~" represent the range between two values, and the range includes the endpoints. For example: "AB" represents a range greater than or equal to A and less than or equal to B. "A~B" represents a range greater than or equal to A and less than or equal to B.

[0155] In the description of the present invention, the term "and / or" herein is merely a description of the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B may represent three situations: A exists alone, A and B exist at the same time, and B exists alone.

[0156] In the description of the invention, the numerical values ​​of time, temperature, ratio, mass, etc. involved may be based on actual measurements, standard parameters of equipment, simplified rounding results, or within an acceptable error range, ensuring the practicability and repeatability of the invention.

[0157] In the description of the present invention, the term "about" or "approximately" is used to express the approximate value of a numerical value or range, allowing a certain error to ensure the flexibility and practicality of the description while remaining within an acceptable error range, with the maximum error range not exceeding 10% of the corresponding numerical value or numerical range.

[0158] The above are only preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention, and the protection scope of the present invention should be based on the scope defined by the claims. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

[0159] This invention is funded by the National Key R&D Program project "Development of humanized animal models for major infectious diseases based on new targets, 2021YFF0702000".

Claims

1. A host protein BRAP peptide segment, characterized in that: The amino acid sequence of the peptide is shown in the sequence listing SEQ ID NO: 1; the peptide interacts with the SARS-CoV-2CoV-Y domain, and the dissociation constant Kd of the interaction is 12.67±0.2μM, and the stoichiometric ratio is 1:

1.

2. A method for identifying the interaction between a host protein BRAP peptide segment and a coronavirus, characterized in that: The following steps are involved: Purify SARS-CoV-2CoV-Y domain recombinant protein and BRAP peptide in vitro; Isothermal titration calorimetry (ITC) was used to detect the binding affinity and binding ratio between the CoV-Y domain and the BRAP peptide.

3. The method according to claim 2, characterized in that The preparation of the SARS-CoV-2CoV-Y domain recombinant protein comprises the following steps: Using the first primer pair, PCR amplification was performed to obtain the CoV-Y target gene fragment; The first primer pair is: CoV-YF: 5'-ggaattccatatggatcagagtagctatatt-3'; CoV-YR: 5'-ccgctcgagttagccacctttcagtgcaattttggtgg-3'; The amplified CoV-Y target gene fragment was inserted into the prokaryotic expression vector pET-28a+ to construct a recombinant plasmid; The recombinant plasmid was transformed into the BL21 expression strain for induced expression and protein purification.

4. The method according to claim 2, characterized in that: The preparation of the BRAP peptide recombinant protein comprises the following steps: Using the second primer pair, BRAP fragments were obtained by PCR amplification; The second primer pair is: BRAP-F: 5'-ccgcgtggatccccggaattcgaagaaaataagtgttttga-3'; BRAP-R: 5'-gtcacgatgcggccgctcgagttatcgatgaacatagttatctcca-3'; The amplified BRAP fragment was inserted into the prokaryotic expression vector pGEX4T-1 to construct a recombinant plasmid; The recombinant plasmid was transformed into BL21 competent cells for induced expression and protein purification.

5. An application of a host protein BRAP peptide segment, characterized in that: Used to prepare drugs that inhibit SARS-CoV-2-induced host inflammatory response.

6. A drug for inhibiting SARS-CoV-2-induced host inflammatory response, characterized in that: Comprising the host protein BRAP peptide segment according to claim 1.

7. The drug according to claim 6, characterized in that It also includes pharmaceutically acceptable carriers or excipients.

8. A method for screening anti-SARS-CoV-2 drugs, characterized in that: The following steps are involved: Using the interaction between the host protein BRAP peptide segment and the SARS-CoV-2CoV-Y domain as claimed in claim 1 as a screening model; The effects of the compounds to be screened on the interaction between the BRAP peptide and the CoV-Y domain were tested to screen out potential antiviral drugs.

9. An in vitro model for studying the infection mechanism of SARS-CoV-2, characterized in that: include: Host cells expressing the SARS-CoV-2CoV-Y domain; An expression vector containing the host protein BRAP peptide segment according to claim 1 is used to study the interaction between the CoV-Y domain and the BRAP peptide segment and its effect on host cell function.

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