Identification and application of host protein brap peptide segment interacting with coronavirus

Abstract

The application discloses a host protein BRAP peptide segment and application thereof in coronavirus research and treatment, and specifically comprises the following steps: identifying a BRAP peptide segment with specific interaction with a SARS-CoV-2 CoV-Y domain, developing a method for identifying the interaction between the peptide segment and the coronavirus in vitro, preparing a drug for inhibiting SARS-CoV-2-induced host inflammatory response by using the peptide segment, constructing a model for screening anti-SARS-CoV-2 drugs, and establishing an in-vitro model for studying the SARS-CoV-2 infection mechanism, so as to provide a theoretical basis and technical support for in-depth understanding of the pathogenic mechanism of the coronavirus, development of new anti-virus drugs and treatment strategies. The peptide segment has interaction with the CoV-Y domain, and 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 the global novel coronavirus pneumonia (COVID-19) pandemic, with profound impacts on human health and socioeconomic development. SARS-CoV-2 is transmitted through the respiratory tract and, upon infection of host cells, activates inflammatory responses within the host cells, leading to the massive release of inflammatory cytokines and, in turn, triggering an inflammatory storm. This is one of the main reasons for the severe symptoms experienced by COVID-19 patients. However, the molecular mechanisms by which SARS-CoV-2 induces host inflammatory responses remain incompletely understood.

[0003] During viral infection, interactions between viral and host proteins underlie key steps such as viral replication, assembly, and release. Studying these interactions helps to shed light on viral pathogenic mechanisms and provides 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 a crucial role in the interaction between the virus and host cells. Previous studies have shown that the CoV-Y domain in the C-terminal region of Nsp3 can interact with host cell proteins, such as the BRAP protein, triggering an inflammatory response in host cells. However, the specific interacting peptides of this domain in Nsp3 and host proteins, as well as their affinity, have not yet been fully characterized.

[0004] In addition, as SARS-CoV-2 continues to mutate, existing antiviral drugs are facing the problem of drug resistance, making the search for new drug targets particularly urgent. 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 relatively low mutation frequency. Therefore, in-depth research on the interaction between the SARS-CoV-2 CoV-Y domain and host proteins, especially the interaction peptides with the host protein BRAP and their affinity, 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 specifically interacts with the SARS-CoV-2 CoV-Y domain, developing an in vitro method for identifying the interaction between the peptide segment and the coronavirus, using the peptide segment to prepare drugs that inhibit SARS-CoV-2-induced host inflammatory responses, 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 listing SEQ ID NO: 1; the peptide segment interacts with the SARS-CoV-2 CoV-Y domain, and the dissociation constant Kd of the interaction is 12.67 ± 0.2 μM, and the stoichiometric ratio is 1:1; the SARS-CoV-2 CoV-Y domain has an NCBI accession number of NC_045512.2.

[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 it clearly identifies for the first time the specific interaction between a specific peptide segment of the host protein BRAP (amino acid sequence as shown in SEQ ID NO: 1 in the sequence listing) and the SARS-CoV-2 CoV-Y domain, which not only reveals a key molecular mechanism in the process of SARS-CoV-2 infecting host cells, but also accurately measures the dissociation constant Kd of the interaction between the two to be 12.67 ± 0.2 μM and the stoichiometric ratio to be 1:1. A lower Kd value generally means a higher binding affinity, that is, the interaction between the two molecules is tighter, and each CoV-Y molecule binds to one BRAP molecule to form a stable dimer structure, providing 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 the coronavirus SARS-CoV-2, comprising the following steps:

[0010] Purify SARS-CoV-2 CoV-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-2 CoV-Y domain recombinant protein and BRAP peptide in vitro and then utilizes isothermal titration calorimetry (ITC), a highly precise and sensitive biophysical technique, to accurately measure the binding affinity and binding ratio between the CoV-Y domain and the BRAP peptide. This method not only provides direct and reliable experimental data for studying the interaction between coronaviruses and host proteins, but also offers 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, providing strong technical support for the development of new antiviral drugs and treatment strategies for coronaviruses, and 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-2 CoV-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] With the further technical scheme, the application has remarkable advantages in preparing the SARS-CoV-2 CoV-Y domain recombinant protein. First, the first pair of specific primers (CoV-Y-F and CoV-Y-R) are designed and synthesized, so that 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 (+), and the recombinant plasmid is constructed. This process utilizes the high expression characteristics of the pET-28a (+) vector, which can realize high-level expression of the CoV-Y protein. Thirdly, the recombinant plasmid is transformed into the BL21 expression strain. As a commonly used high-efficiency expression host, the BL21 strain can provide a good expression environment, so that the CoV-Y recombinant protein can be expressed in large quantities. Finally, through the steps of induction expression and protein purification, high-purity and high-activity SARS-CoV-2 CoV-Y domain recombinant protein can be obtained, which provides high-quality experimental materials for subsequent interaction research and drug development.

[0025] On the basis of the above technical scheme, the application can also be improved as follows:

[0026] Further:

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

[0028] The BRAP fragment is obtained by PCR amplification using the second primer pair;

[0029] The second primer pair is:

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

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

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

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

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

[0035] Using this advanced technical approach, a specially designed second primer pair (BRAP-F and BRAP-R) enables efficient and specific amplification of the BRAP fragment, 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 was inserted into the prokaryotic expression vector pGEX4T-1 to construct a recombinant plasmid. This process leverages the high-efficiency expression and fusion tag advantages of the pGEX4T-1 vector, enabling high-level expression of the BRAP peptide. The fused GST tag improves protein solubility and stability, facilitating subsequent protein purification and functional studies. Furthermore, the recombinant plasmid was transformed into BL21 competent cells. As a commonly used high-efficiency expression host, BL21 provides an excellent expression environment, enabling high-quality expression of the BRAP peptide. Finally, through induction expression and protein purification, highly pure and active recombinant BRAP peptide protein was obtained, providing high-quality experimental material for studying its interaction with the SARS-CoV-2CoV-Y domain and its application in antiviral drug development.

[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 in-depth research on the interaction mechanism between the SARS-CoV-2 CoV-Y domain and host proteins. It has a clear biological function and mechanism of action, and can specifically interfere with the interaction between the CoV-Y domain and host cells, thereby effectively inhibiting the release of virus-induced inflammatory factors and the occurrence of inflammatory responses. Secondly, the application of this peptide segment expands the research and development ideas of antiviral drugs, breaking 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 the virus.

[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] The interaction between the host protein BRAP peptide and the SARS-CoV-2 CoV-Y domain is used 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-2 CoV-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 host cell function.

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

[0048] This model, using host cells expressing the SARS-CoV-2 CoV-Y domain, successfully mimics the binding pattern between key viral protein domains and host proteins, providing an important experimental basis for studying the functions and mechanisms of action of viral proteins. The introduction of an expression vector containing the BRAP peptide fragment of the host protein enables researchers to directly observe and analyze the interaction between the CoV-Y domain and the BRAP peptide fragment in vitro, as well as the specific effects of this interaction on host cell function, thereby further revealing the molecular mechanism by which SARS-CoV-2 infection of host cells 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 following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

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

[0051] Figure 2This 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 Figure A shows the purification results of the BRAP fragment recombinant protein; Figure A shows the gel filtration chromatography results of the BRAP fragment recombinant protein, and Figure B shows the SDS-PAGE identification results of the BRAP fragment recombinant protein.

[0054] Figure 5 This is the ITC experiment result of SARS-CoV-2 CoV-Y recombinant protein and BRAP fragment recombinant protein.

[0055] Figure 6 This is the result of Co-IP screening of the interaction between different domains of BRAP and SARS-CoV-2 CoV-Y recombinant protein. DETAILED DESCRIPTION

[0056] The following describes the details in conjunction with specific embodiments.

[0057] 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, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making 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.

[0058] 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 all conventional methods in the art.

[0059] Example 1

[0060] This example is an example of a method for identifying a host protein BRAP peptide segment and its interaction with a coronavirus.

[0061] During 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-2 CoV-Y domain. Through these experiments, the inventors ruled out the possibility of some non-specific binding and ultimately 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 6 .

[0062] See also Figure 6 Significant differences in cell expression were observed between the individual BRAP domains and the control vector. BRAP-HA, BRAP-1-HA, BRAP-2-HA, and BRAP-3-HA displayed clear bands at the expected molecular weights (approximately 70 kDa, 31 kDa, 17 kDa, and 22 kDa, respectively), indicating successful cell expression of these proteins. The vector control group showed no bands, confirming the absence of nonspecific binding.

[0063] In the presence of Flag-CoV-Y, BRAP-2-HA coprecipitated with Flag-CoV-Y in Flag immunoprecipitations, 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 do not interact with CoV-Y. BRAP-HA (full-length BRAP) also showed some coprecipitation, but given that full-length BRAP contains all tested domains, this result likely reflects contributions from multiple domains.

[0064] Figure 6 The results of the Co-IP experiment clearly indicate that BRAP-2 (amino acids 257-394) is the key region for interaction with the SARS-CoV-2 CoV-Y domain. This finding excludes the possibility that other BRAP domains are the main interaction regions and provides a clear direction for subsequent research.

[0065] 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.

[0066] 1. Identification of host protein BRAP peptides

[0067] 1.1 ZDOCK online prediction of complex structure

[0068] Using ZDOCK online prediction technology, the BRAP peptide motif that interacts with the SARS-CoV-2 CoV-Y domain was preliminarily determined.

[0069] Steps:

[0070] Obtain the three-dimensional structure file of the SARS-CoV-2 CoV-Y domain (PDB format), the PDB accession number is 8ILC.

[0071] Obtain the three-dimensional structure file of human BRAP protein (PDB format), Uniprot accession number is Q7Z569.

[0072] Visit the ZDOCK official website (https: / / zdock.wenglab.org / ) and upload the PDB files of the CoV-Y domain and BRAP protein to the ZDOCK platform.

[0073] Set the ZDOCK parameters, adjust the docking mode and search range, and start the prediction program.

[0074] 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.

[0075] 1.2 In vitro purification of SARS-CoV-2 CoV-Y domain recombinant protein

[0076] High-purity SARS-CoV-2 CoV-Y domain recombinant protein was obtained for subsequent interaction verification experiments.

[0077] Steps:

[0078] The SARS-CoV-2 CoV-Y domain has an NCBI accession number of NC_045512.2.

[0079] The upstream primer CoV-YF (5'-ggaattccatatggatcagagtagctatatt-3') and the downstream primer CoV-YR (5'-ccgctcgagttagccacctttcagtgcaattttggtgg-3') were designed and synthesized according to the nucleotide sequence of the SARS-CoV-2 CoV-Y domain.

[0080] 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. The nucleotide sequence of the PCR amplification product is shown in SEQ ID NO: 2.

[0081] The PCR reaction system and program settings are as follows:

[0082] Reagent volume: 2 μL upstream primer, 2 μL downstream primer, 1 μL template, 25 μL 2×Mix PCR polymerase, 20 μL ddH2O, totaling 50 μL.

[0083] The reaction procedure was as follows: 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 a final extension at 72°C for 10 min. The plates were then stored at 4°C.

[0084] Purify the PCR product using a PCR purification kit (such as OMEGA Product No. D6492).

[0085] The pET-28a(+) prokaryotic plasmid vector and the CoV-Y target gene fragment were digested with the rapid 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.

[0086] 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, and transform DH5α competent cells. Pick a single clone for sequencing to complete the construction of the recombinant plasmid. The schematic diagram of the pET-28a (+) - CoV-Y recombinant plasmid is shown in Figure 2 shown.

[0087] 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 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. The culture was then centrifuged at 8 000 rpm for 10 min, the supernatant was discarded, and the bacteria were collected.

[0088] Resuspend the harvested cells in buffer A (20 mM Tris-HCl, pH 7.5, 10 mM imidazole, 500 mM NaCl) with 1 mM PMSF to prevent degradation of the target protein. Disrupt the cells using a high-pressure disruptor at 4°C. First, rinse the disruptor tubing with buffer A at 0 bar. Add the resuspended bacterial solution to the filter. Allow the solution to flow into the sample cup and circulate until no bubbles remain in the tubing. Then, pressurize the solution to 200 bar for 2 minutes to disrupt nucleic acids. Continue pressurizing the cells to 600-800 bar for 3-4 minutes. After disruption, reduce the pressure to 0 bar, collect the liquid in a centrifuge tube, and centrifuge at 18,000 rpm at 4°C for 45 minutes to remove cell debris.

[0089] The supernatant after centrifugation was collected and filtered, and the sample was slowly loaded onto a HisTrapFF prepacked column (Cytiva, Product No. 17525501) equilibrated with buffer A using a peristaltic pump to allow the target protein to bind tightly to the nickel column material.

[0090] After sample loading, the prepacked column bound to the target protein was installed on the AKTA column position. Contaminants were washed away with 15-20 mL of buffer A at a flow rate of 2.5 mL / min. Contaminants were then further removed with 5% buffer B (20 mM Tris-HCl, pH 8.0, 500 mM imidazole, 500 mM NaCl) until the UV absorbance peak of the effluent leveled off. Finally, the concentration of buffer B was increased over time until the target protein was eluted and collected in a 10 mL collection tube. Thrombin protease was added to the target protein eluate to cleave the His-tag. The entire protein solution was transferred to a dialysis bag and dialyzed overnight against buffer C (20 mM Tris-HCl, pH 7.5, 150 mM NaCl) at 4°C to remove the imidazole from the protein solution. The next day, the protein sample after overnight enzymatic digestion was loaded onto the HisTrap FF prepacked 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 the His-tag removed and higher purity.

[0091] Gel filtration chromatography is a method that uses porous mesh particles as a separation medium to separate the components of 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, buffer C is sonicated at 70% power for 3 minutes to remove bubbles, and then a Superdex 75 Increase 10 / 300 GL chromatography column (Cytiva, Product No. 29148721) is equilibrated with buffer C. 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 collected protein is identified by size and purity by SDS-PAGE to obtain a highly pure and uniform SARS-CoV-2 CoV-Y recombinant protein. Figure 3 shown.

[0092] 1.3 In vitro purification of BRAP peptide recombinant protein

[0093] High-purity BRAP peptide recombinant protein was obtained for subsequent interaction verification experiments.

[0094] Steps:

[0095] The upstream primer BRAP-F (5'-ccgcgtggatccccggaattcgaagaaaataagtgttttga-3') and the downstream primer BRAP-R (5'-gtcacgatgcggccgctcgagttatcgatgaacatagttatctcca-3') were designed and synthesized based on the nucleotide sequence of the human BRAP fragment (shown in SEQ ID NO: 1).

[0096] Using total cell cDNA as a template, the target BRAP fragment was amplified by PCR using the aforementioned primers BRAP-F / BRAP-R. The PCR reaction system and program settings were the same as for the CoV-Y fragment amplification. The nucleotide sequence of the PCR amplification product is shown in SEQ ID NO: 3.

[0097] Purify the PCR product using a PCR purification kit.

[0098] The pGEX4T-1 prokaryotic expression plasmid vector was digested with the rapid restriction endonucleases EcoRI (Thermo Fisher, Cat. No. FD0274) and XhoI at 37°C for 30 min, and the digested plasmid was purified using a PCR purification kit.

[0099] Use homologous recombinase (such as Vazyme Product No. C116-02) to ligate the pGEX4T-1 double-digested product with the BRAP fragment target gene fragment, transform it into DH5α competent cells, pick a single clone for sequencing, and complete the construction of the recombinant plasmid.

[0100] 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 When the 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. The cells were then centrifuged at 8 000 rpm for 10 min, and the supernatant was discarded to collect the bacteria.

[0101] The harvested cells were thoroughly resuspended in GST buffer A (20 mM Tris-HCl, pH 7.5, 500 mM NaCl) and disrupted by sonication on ice at 20% power for 3 minutes, with a frequency of 6 seconds on, 3 seconds off, repeated three times until the cells were completely disrupted. After disruption, the cells were transferred to a dedicated high-speed centrifuge tube and centrifuged at 18,000 rpm for 45 minutes at 4°C. The supernatant was aspirated and filtered through a 0.45 μm filter. The filtrate was incubated on ice for 90 minutes with GST column material equilibrated with GST buffer A. The supernatant was removed by centrifugation at 2,000 rpm for 10 minutes, and the column material was washed three times with GST buffer A to remove nonspecific binding. The target protein was then eluted with GST buffer B (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 10 mM GSH) and collected in a collection tube. The eluate containing the target protein was placed at 4°C and Thrombin protease was added to cleave 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 minutes at 4°C. At the same time, buffer C was sonicated at 70% power for 3 minutes to remove bubbles. The Superdex 75 Increase10 / 300 GL chromatography column was then equilibrated with buffer C. Finally, the 1 mL protein sample after centrifugation was added to the sample loop of the chromatography column, and the protein was eluted using buffer C as the mobile phase at a flow rate of 0.5 mL / min. The collected protein was identified by SDS-PAGE for size and purity, and a high-purity, uniform BRAP fragment recombinant peptide was obtained, such as Figure 4 shown.

[0102] 1.4 ITC affinity detection

[0103] 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.

[0104] Steps:

[0105] The purified SARS-CoV-2 CoV-Y protein was concentrated to 460 μM, and the purified BRAP fragment protein was concentrated to 45 μM.

[0106] At 20°C, ITC titration experiments were performed using BRAP fragment protein as substrate protein and CoV-Y protein as ligand protein. 2.5 μL of ligand protein CoV-Y was added to the sample pool containing 180 μL of substrate protein BRAP fragment every 150-200 s, for a total of 20 injections. The stirring speed of the stirrer was set at 125 rpm.

[0107] The original titration data set was processed in Nano Analyze data analysis software, and the interaction between the CoV-Y domain recombinant protein and the BRAP fragment recombinant peptide was determined to be an exothermic process with a dissociation constant (Kd) of 12.67 ± 0.2 μM. 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. Figure 5 shown.

[0108] Complex structure prediction results: ZDOCK online prediction results showed that the Yc subdomain of the SARS-CoV-2 CoV-Y domain can form a complex with the amino acid 313-375 region of the BRAP protein, and preliminarily determined the interaction motif of the BRAP peptide segment.

[0109] Protein purification results: Through SDS-PAGE analysis, SARS-CoV-2CoV-Y recombinant protein and BRAP fragment recombinant peptide were successfully obtained with high purity and uniformity, providing high-quality experimental materials for subsequent interaction verification experiments.

[0110] ITC test results: The ITC experimental results 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 ± 0.2 μM and a stoichiometric ratio of 1:1, confirming the binding affinity and binding mode between the two, providing important experimental basis for studying the SARS-CoV-2 infection mechanism and developing antiviral drugs.

[0111] The present embodiment successfully identifies the interaction between the host protein BRAP peptide segment and the SARS-CoV-2 CoV-Y domain, and verifies the binding characteristics of the two through in vitro experiments, laying a solid foundation for subsequent research and application.

[0112] Embodiment 2

[0113] The present embodiment is an example of the application of the host protein BRAP peptide segment in the preparation of drugs for inhibiting SARS-CoV-2-induced host inflammatory response.

[0114] Based on the method of embodiment 1, a high-purity host protein BRAP peptide segment is obtained, and the amino acid sequence is shown in SEQ ID NO: 1 of the sequence listing, which is used for drug preparation.

[0115] The BRAP peptide segment is combined with a pharmaceutically acceptable carrier or excipient to prepare a pharmaceutical preparation suitable for in vivo application.

[0116] Select an appropriate drug carrier, such as polyethylene glycol (PEG) or liposomes, to improve the stability and bioavailability of the BRAP peptide segment.

[0117] Dissolve the purified BRAP peptide segment in an appropriate amount of buffer solution, mix with PEG or liposomes, and fix the peptide segment on the surface of the carrier or encapsulate it inside the carrier by physical adsorption or chemical bonding.

[0118] Homogenize the mixture to form a uniformly dispersed drug preparation.

[0119] In vitro cell experiment pre-experiment:

[0120] Select human alveolar epithelial cells (A549 cells) as the experimental cell strain, inoculate them in culture plates, and culture them to 80% confluence.

[0121] Divide the cells into control, virus infection, and drug treatment groups. The control group only adds culture medium, the virus infection group adds SARS-CoV-2 virus suspension to infect the cells, and the drug treatment group adds different concentrations of BRAP peptide segment drug preparation before or after virus infection.

[0122] After 3 days of culture, collect the cell supernatant and use enzyme-linked immunosorbent assay (ELISA) to detect the content of inflammatory factors (such as TNF-α, IL-8, etc.) in the supernatant.

[0123] At the same time, extract total RNA from the cells and detect the expression level of inflammation-related genes by real-time quantitative PCR (qPCR).

[0124] 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-treated 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.

[0125] Host protein BRAP peptide drugs significantly inhibit virus-induced host inflammatory responses by blocking or regulating the interaction between the SARS-CoV-2 CoV-Y domain and host cells, and have good antiviral activity and therapeutic potential.

[0126] 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.

[0127] Example 3

[0128] This example is an example of screening antiviral drugs by using the interaction between the host protein BRAP peptide segment and the SARS-CoV-2 CoV-Y domain.

[0129] SARS-CoV-2 CoV-Y domain recombinant protein and BRAP peptide recombinant protein were prepared according to the method in Example 1. The purity and molecular weight of the protein were confirmed by SDS-PAGE and mass spectrometry analysis to ensure its good biological activity and stability.

[0130] 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.

[0131] Using surface plasmon resonance (SPR) or ITC techniques, the binding constants and kinetic parameters of the BRAP peptide and the CoV-Y domain are determined to optimize the conditions of the interaction model, such as pH, ionic strength, and temperature.

[0132] 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.

[0133] Compounds to be screened are added to the interaction system between the BRAP peptide and the CoV-Y domain at varying concentrations. Fluorescence polarization (FP) or enzyme-linked immunosorbent assay (ELISA) is used to assess the effects of the compounds on the binding of the BRAP peptide to the CoV-Y domain. Compounds that significantly inhibit or enhance the interaction are screened, and their effective concentration range and degree of inhibition or enhancement are recorded.

[0134] Compounds initially screened undergo secondary validation, employing high-precision techniques such as SPR or ITC to determine detailed binding constants and kinetic parameters for the interaction between the BRAP peptide and the CoV-Y domain. Compounds with high affinity and favorable kinetic properties are selected for further cellular antiviral activity testing, such as inhibition of viral replication and cytoprotection in SARS-CoV-2-infected cells. Based on the results of these cellular experiments, the compounds are structurally optimized and modified to enhance their antiviral activity, stability, and bioavailability.

[0135] Example 4

[0136] This example is an implementation of an in vitro model for studying the mechanism of SARS-CoV-2 infection.

[0137] Based on the nucleotide sequence of the SARS-CoV-2 CoV-Y domain, a eukaryotic expression vector containing the CoV-Y gene, such as pCMV-CoV-Y, is constructed.

[0138] Select an appropriate host cell line, such as human alveolar epithelial cells (A549 cells) or human embryonic kidney cells (HEK293 cells), inoculate them into culture plates, and culture them to 70%-80% confluence.

[0139] 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 and transfection time.

[0140] After transfection, the cells are cultured for 24-48 hours to allow the CoV-Y gene to be expressed in the host cells.

[0141] 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.

[0142] 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.

[0143] Insert the BRAP peptide cDNA 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.

[0144] The pEGFP-BRAP expression vector was transfected into host cells that stably expressed the CoV-Y domain and appropriate transfection conditions were set.

[0145] After transfection, the cells are cultured for 24-48 hours to allow the BRAP peptide to be expressed in the host cells.

[0146] The transfected host cells were collected and the total cell protein was extracted.

[0147] Using co-immunoprecipitation (Co-IP) technology, antibodies against CoV-Y protein were used as bait to capture the BRAP peptide that interacts with it.

[0148] The presence of BRAP peptide in the immunoprecipitated product was detected by Western blot to confirm the interaction between the CoV-Y domain and the BRAP peptide.

[0149] 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 the cells.

[0150] The biological function indicators such as host cell proliferation, apoptosis, and inflammatory factor secretion were detected, and the effects of the interaction between the CoV-Y domain and the BRAP peptide on host cell function were compared.

[0151] 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 host cell supernatant is detected by ELISA.

[0152] 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.

[0153] In the description of the present invention, it should be understood that "-" and "~" represent a range between two values, and the range is inclusive. 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.

[0154] In the description of the present invention, the term "and / or" 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.

[0155] In the description of the invention, the numerical values ​​of time, temperature, ratio and mass involved may be based on actual measurements, standard parameters of equipment, simplified rounding results, or within an acceptable error range, ensuring the practicality and repeatability of the invention.

[0156] 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.

[0157] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be construed as limiting the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. Persons skilled in the art will appreciate that improvements and modifications may be made without departing from the spirit and scope of the present invention, and such improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A host protein BRAP peptide segment, characterized in that The amino acid sequence of the peptide is shown in SEQ ID NO: 1 in the sequence listing; the peptide interacts with the SARS-CoV-2 CoV-Y domain, and the dissociation constant Kd of the interaction is 12.67±0.2 μM, and the stoichiometric ratio is 1:1; the SARS-CoV-2 CoV-Y domain has an NCBI accession number of NC_045512.

2.

2. A method for identifying the interaction between the host protein BRAP peptide segment and the coronavirus SARS-CoV-2 according to claim 1, not for the purpose of diagnosing or treating a disease, characterized in that: The following steps are involved: Purify in vitro the SARS-CoV-2 CoV-Y domain recombinant protein and BRAP peptide; the SARS-CoV-2 CoV-Y domain has an NCBI accession number of NC_045512.2; 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-2 CoV-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, the BRAP fragment was 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. A use of the host protein BRAP peptide segment according to claim 1, 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: Utilizing the interaction between the host protein BRAP peptide segment described in claim 1 and the SARS-CoV-2 CoV-Y domain described in claim 1 as a screening model; The effects of the screened compounds on the interaction between the BRAP peptide and the CoV-Y domain were tested to screen potential anti-SARS-CoV-2 viral drugs.

9. An in vitro model for studying the infection mechanism of SARS-CoV-2, characterized in that include: A host cell expressing the SARS-CoV-2 CoV-Y domain of claim 1; 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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