A monkeypox virus protein B4R nuclease domain protein, crystal, its preparation and application

Abstract

This invention discloses a monkeypox virus protein B4R nuclease domain protein, its crystal, and its preparation and application, belonging to the field of protein crystal culture technology. This invention efficiently expresses and purifies B4R-CTD in *E. coli*, and confirms its endonuclease activity through biochemical experiments. Protein crystals are cultured using a gas-phase diffusion method, and the crystal structure of the B4R-CTD protein is resolved. It was found that the wild-type B4R-CTD exists in solution as monomers and tetramers, and the polymerization morphology affects enzyme activity. The B4R-CTD protein prepared by this invention has high purity, stable properties, and excellent crystal quality, and can be widely used in the structural biology research of monkeypox virus, possessing significant scientific research and application value.

Description

Technical Field

[0001] This invention belongs to the field of protein crystal culture technology, specifically relating to a method for preparing and crystallizing the B4R nuclease domain (B4R-CTD) of monkeypox virus protein. Background Technology

[0002] Monkeypox virus (MPXV) is a DNA virus belonging to the genus Orthopoxviruses (OPXVs) of the family Poxviridae. The Poxviridae family is a large class of double-stranded DNA (dsDNA) viruses. MPXV is enveloped, brick-shaped or oval in form, and can reach a diameter of 350 nm. Its genome is approximately 197 kb long, encoding over 180 proteins. Compared to other viruses, its composition is extremely complex and its size is enormous. Unlike most DNA viruses that replicate within the host cell nucleus, MPXV replicates entirely in the cytoplasm of the infected cell. Monkeypox virus more readily triggers an immune response and requires a more comprehensive strategy to survive within the host.

[0003] Among them, the B4R protein (also known as OPG188) is the core effector molecule antagonizing the cGAS-STING pathway. Studies have found that the B4R protein has a unique dual-domain feature: its N-terminal Poxin domain acts as a highly efficient 2',3'-cGAMP degrading enzyme, directly blocking STING activation by hydrolyzing the second messenger cGAMP; while its C-terminal domain further inhibits interferon-mediated signal transduction by chelating the key transcription factor STAT2.

[0004] The B4R protein consists of 503 amino acid residues with a molecular weight of 118 kDa. It has two main domains from the N-terminus to the C-terminus: the cGAMP hydrolytic domain (poxin) and the endonuclease domain (SLFN). The three-dimensional structure of the poxin domain has been resolved, providing a molecular basis for understanding its mechanism of action. However, the function and structure of the C-terminal domain (B4R-CTD) of the B4R protein remain unclear. Sequence analysis shows that the B4R-CTD is homologous to the human antiviral protein Schlafen (SLFN) family and contains potential nucleic acid binding motifs and endonuclease activity regions, mediating the interaction between viral proteins and host nucleic acid substances. Recent research suggests that this domain may further inhibit the host's antiviral response by chelating signaling molecules (such as STAT2) or directly processing nucleic acid substrates.

[0005] Currently, the specific function of the SLFN domain of poxvirus is not fully understood, its functional consistency with human homologous proteins has not been confirmed, and its interaction mechanism with poxvirus poxin requires further investigation. To address these issues, there is an urgent need for a method to prepare high-resolution B4R-CTD crystals to elucidate the relationship between protein structure and function through protein crystal structure analysis. Summary of the Invention

[0006] In order to overcome the shortcomings and deficiencies of the prior art, the present invention aims to provide a monkeypox virus protein B4R nuclease domain protein, crystal, and its preparation and application.

[0007] This invention employs a truncated design, removing the N-terminus while retaining the core endonuclease domain B4R-CTD. A highly efficient purification and crystallization system was established to resolve the structure of monkeypox virus B4R-CTD, and biochemical experiments were conducted to study the endonuclease activity of B4R-CTD.

[0008] Due to the need for functional studies of B4R protein nuclease domains and the development of small molecule drugs, high-resolution crystal structures of B4R protein nuclease domains are required. Therefore, this invention provides a method for crystallizing B4R protein nuclease domains.

[0009] This invention efficiently expressed and purified B4R-CTD in *E. coli*, and confirmed its endonuclease activity through biochemical experiments. Protein crystals were cultured using gas-phase diffusion, and the crystal structure of B4R-CTD was determined. It was found that wild-type B4R-CTD exists in solution as monomers and tetramers, and the polymerization morphology affects enzyme activity. The B4R-CTD protein prepared by this invention exhibits high purity, stable properties, and excellent crystal quality, making it widely applicable in the structural biology research of monkeypox virus and possessing significant scientific and applied value.

[0010] The objective of this invention is achieved through the following technical solution:

[0011] A monkeypox virus B4R nuclease domain (B4R-CTD) protein, the amino acid sequence of which is shown as SEQ ID NO: 2, segments 10-320aa.

[0012] A mutant of monkeypox virus B4R nuclease domain (B4R-CTD) protein, the amino acid sequence of which is shown as SEQ ID NO: 4, segments 10-320aa.

[0013] The aforementioned biological materials related to the monkeypox virus B4R nuclease domain (B4R-CTD) protein or mutants are any one or more combinations of the following biological materials:

[0014] (a) A nucleic acid molecule encoding the above-mentioned monkeypox virus B4R nuclease domain protein or its mutants;

[0015] (b) An expression cassette containing the nucleic acid molecule described in (a);

[0016] (c) A recombinant expression vector containing the nucleic acid molecules described in (a);

[0017] (d) A recombinant expression vector containing the expression cassette described in (b);

[0018] (e) Recombinant bacteria containing the nucleic acid molecules described in (a);

[0019] (f) Recombinant bacteria containing the expression cassette described in (b);

[0020] (g) Recombinant bacteria containing the recombinant expression vector described in (c) or (d).

[0021] Furthermore, the nucleic acid molecule described in (a) is a gene sequence encoding the monkeypox virus B4R nuclease domain (B4R-CTD) protein, the nucleotide sequence of which is shown in SEQ ID NO: 1, 28-963bp.

[0022] The nucleic acid molecule described in (a) is a gene sequence encoding a mutant of the monkeypox virus B4R nuclease domain (B4R-CTD) protein, and its nucleotide sequence is shown in SEQ ID NO: 3, 28-963bp.

[0023] Furthermore, the starting vector for the recombinant expression vector described in (c) and (d) is a pET series vector, etc.; preferably, it is a pET-22b(+) vector.

[0024] Furthermore, the host bacteria corresponding to the recombinant bacteria mentioned in (e), (f), and (g) are selected from prokaryotes, yeast, or higher eukaryotic cells; the prokaryotes include bacteria such as Escherichia. More specifically, the prokaryotes are Escherichia, preferably Escherichia coli, specifically Escherichia coli BL21 (DE3).

[0025] The application of the above-mentioned biomaterials related to the monkeypox virus B4R nuclease domain (B4R-CTD) protein in the preparation of monkeypox virus B4R nuclease domain protein or its mutants.

[0026] A method for preparing a monkeypox virus B4R nuclease domain (B4R-CTD) protein or a mutant thereof includes the following steps: culturing the above-mentioned recombinant bacteria to obtain the monkeypox virus B4R nuclease domain protein or a mutant thereof from the recombinant bacteria.

[0027] Specifically, the steps include the following:

[0028] (1) Construction of recombinant expression vector: The target gene of the above-mentioned monkeypox virus B4R nuclease domain protein or its mutant was cloned into the expression vector pET-22b(+) to construct the recombinant expression vector;

[0029] (2) Expression of the target gene: The recombinant expression vector was transformed into Escherichia coli BL21(DE3) competent cells and expressed by IPTG; the bacterial culture induced by IPTG was collected, and the bacterial cells were collected after centrifugation.

[0030] (3) Affinity chromatography purification of target protein: After resuspending the bacterial body, the mixture is broken and centrifuged to prepare target protein supernatant; after equilibrating the Ni-affinity chromatography column with affinity chromatography buffer, the target protein supernatant is eluted in a gradient.

[0031] (4) Purification of target protein by ion exchange chromatography: The predicted isoelectric point of the target protein is 7.0. It is purified by anion exchange column. The Q column is equilibrated with low salt buffer to allow the target protein to adhere to the column. It is then eluted with high salt buffer. Impurities are removed by gradient elution, and the elution peak is collected.

[0032] (5) Purification of target protein by molecular sieve chromatography: After equilibrating the molecular sieve chromatography column with molecular sieve chromatography buffer, collect 300 mM imidazole eluent and further purify the target protein by molecular sieve chromatography according to the protein size.

[0033] In step (2), the induced expression is induced by 0.1 mM IPTG at 16°C and 150 rpm for 14 h.

[0034] In step (3), the affinity chromatography buffer is formulated as follows: 50 mM Tris pH 8.0, 500 mM NaCl, 1 mM DTT, and 5% glycerol.

[0035] In step (4), the low-salt buffer solution is formulated with 50 mM Tris pH 8.0 and 50 mM NaCl; the high-salt buffer solution is formulated with 50 mM Tris pH 8.0 and 1 M NaCl.

[0036] In step (5), the molecular sieve chromatography buffer is formulated with 50 mM Tris pH 8.0 and 250 mM NaCl.

[0037] The aforementioned monkeypox virus B4R nuclease domain (B4R-CTD) protein, mutants, and their biological materials possess endonuclease activity, enabling nucleic acid cleavage.

[0038] A crystal of monkeypox virus B4R-CTD protein has the following structural parameters: space group P 62 2 2, cell parameters a = b = 71.85 Å, c = 404.16 Å, α = β = 90°, γ = 120.0°, resolution range: 2.65 Å, and integrity: 99.7%.

[0039] A method for preparing monkeypox virus B4R-CTD protein crystals includes the following steps: preparing a protein solution of the above-mentioned monkeypox virus B4R-CTD protein with a buffer solution, then crystallizing it by a sitting drop gas phase diffusion method in the presence of a crystallization reagent, and then incubating it statically to obtain monkeypox virus B4R-CTD protein crystals.

[0040] Furthermore, the buffer solution is formulated with 50 mM Tris, 150 mM NaCl, and pH 8.0.

[0041] Furthermore, the concentration of the protein solution is 5–10 mg / mL;

[0042] Furthermore, the crystallization reagent is 0.1 M Bis-Tris, 1.8 M-2.0 M ammonium sulfate, 0.1 MPraseodymium(III) acetate hydrate, pH 6.5.

[0043] Furthermore, the volume ratio of the protein solution to the crystallization reagent is 1:1.

[0044] Furthermore, the static incubation temperature is 16°C.

[0045] Furthermore, the static incubation period is 3 to 5 days.

[0046] The above-mentioned monkeypox virus B4R-CTD protein crystals are used in studying the function of the monkeypox virus SLFN domain, its interaction mechanism with poxin, or in the preparation of anti-monkeypox virus drugs.

[0047] Furthermore, the above-mentioned monkeypox virus B4R-CTD protein crystals are used in the preparation of monkeypox virus vaccines.

[0048] The present invention has the following advantages and effects compared with the prior art:

[0049] (1) This invention is the first to obtain the high-resolution (2.65 Å) crystal structure of monkeypox virus B4R-CTD, providing a high-resolution model for the study of the functional mechanism of the SLFN family;

[0050] (2) The truncated expression-three-step purification-optimized crystallization system established in this invention has good reproducibility, is easy to operate, and has a protein purity of >95%, which can be prepared on a large scale;

[0051] (3) The present invention systematically elucidates that B4R-CTD exists in solution in monomer / tetramer form. The tetramer significantly enhances nucleic acid binding activity, while the monomer has the strongest cleavage activity, providing new evidence for the viral immune escape mechanism.

[0052] (4) The truncated design of this invention does not affect the degradation efficiency of N-terminal poxin on cGAMP, laying the foundation for subsequent research on the synergistic effect of dual domains and the screening of small molecule drugs against monkeypox. The presence of the SLFN domain can enhance the affinity of the protein for cGAMP.

[0053] (5) The B4R-CTD protein, crystal structure and activity detection method provided by the present invention can be widely used in poxvirus structural biology, vaccine design and drug development, and has important scientific research and application value. Attached Figure Description

[0054] Figure 1 This is an SDS-PAGE electrophoresis image of the B4R-CTD protein samples in each component during affinity chromatography in Example 1; where S is the supernatant, total protein in the supernatant after centrifugation; P is the precipitate, total protein in the precipitate after centrifugation; Ft is the flow-through buffer, total protein in the flow-through buffer; W is the 60 mM imidazole eluted sample, E1 is the 300 mM imidazole eluted sample, E2 is the 500 mM imidazole eluted sample, B is the sample taken from the eluted medium; and Marker is the protein standard marker.

[0055] Figure 2 The images show the anion exchange peak diagram (A) and the SDS-PAGE electrophoresis diagram (B) of the B4R-CTD protein sample in Example 1; lane 1 is for sampling after desalting, and lanes 2 and 3 are for sampling from the ion column peaks.

[0056] Figure 3 These are the molecular sieve chromatography (Superdex 200 increase 10 / 300GL) images of the protein sample from Example 1 and the SDS-PAGE electrophoresis image of the main peak of the molecular sieve in the protein sample; where A: B4R; B: B4R-NTD; C: B4R-CTD; D: B4R-CTD-tetramer.

[0057] Figure 4 The images shown are the crystal image (A) and crystal structure analysis diagram (B) of the B4R-CTD protein in Example 2; where the scale bar in A is 1000 μm.

[0058] Figure 5 These are the binding results of B4R-CTD-tetramer, B4R, and B4R-CTD to ssDNA in Example 3.

[0059] Figure 6These are the results of the binding experiments of B4R-CTD-tetramer, B4R, and B4R-CTD to ssRNA in Example 3.

[0060] Figure 7 The results are the nucleic acid cleavage experiments of B4R-CTD-tetramer, B4R, and B4R-CTD with ssDNA in Example 3; where 1 represents B4R-CTD (monomer), 2 represents B4R (disomer), and 4 represents B4R-CTD-tetramer (tetramer).

[0061] Figure 8 The results are the nucleic acid cleavage experiments of B4R-CTD-tetramer, B4R, and B4R-CTD with stem-loop RNA in Example 3; where 1 represents B4R-CTD (monomer), 2 represents B4R (disomer), and 4 represents B4R-CTD-tetramer (tetramer).

[0062] Figure 9 The results are the nucleic acid cleavage experiments of B4R-CTD-tetramer, B4R, and B4R-CTD with tRNA in Example 3; where 1 represents B4R-CTD (monomer), 2 represents B4R (diomer), and 4 represents B4R-CTD-tetramer (tetraomer).

[0063] Figure 10 This is a graph comparing the efficiency of B4R and B4R-NTD in degrading cGAMP in Example 4; the horizontal axis corresponds to the concentration gradient of the protein (such as B4R-NTD or full-length B4R) added to the incubation system, and the vertical axis corresponds to the cGAMP hydrolysis clearance rate. Detailed Implementation

[0064] The present invention will be further described in detail below with reference to embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto. Test methods in the following embodiments that do not specify specific experimental conditions are generally performed under conventional experimental conditions or according to the manufacturer's recommended experimental conditions. Unless otherwise specified, the materials and reagents used are commercially available.

[0065] Example 1: Expression and purification of B4R-CTD protein

[0066] Based on the genomic information of the Zaire strain of monkeypox virus (Mpox virus) in the NCBI database (GenBank: AF380138.1), the full-length sequence of the target gene was obtained. According to the protein domain prediction (the N-terminal poxin domain is amino acids 1-236, and the C-terminal is an SLFN-like endonuclease domain), the N-terminus was truncated, and the nucleotide sequence containing amino acids 237-503 was retained, thus obtaining the B4R-CTD target gene fragment.

[0067] Since the natural sequence is derived from a eukaryotic virus, in order to improve its expression efficiency in the prokaryotic system (E. coli BL21(DE3)), this study commissioned a professional biotechnology company (General Biotech Co., Ltd.) to optimize and synthesize the whole genome sequence.

[0068] According to the high-frequency codon usage table of E. coli, rare codons (such as AGA and AGG) were replaced with high-frequency synonymous codons (such as CTT and GGT) to avoid ribosome arrest during translation. At the same time, the GC content was adjusted to 40%-60%, and mRNA secondary structures (such as hairpins) were removed to improve transcription and translation efficiency. Tag sequences (such as 8×His-tag) were added for purification. Finally, the gene was cloned into the pET-22b vector.

[0069] The corresponding gene (SEQ ID NO: 1, containing an N-terminal 8×His tag) was synthesized according to the amino acid sequence of B4R-CTD (SEQ ID NO: 2), cloned into the pET-22b(+) vector, and transformed into E. coli BL21(DE3) competent cells. Single colonies were picked and inoculated into 1 L LB liquid medium and cultured at 37°C until OD600. 600 =0.6-0.8, add 0.1 mM IPTG, and induce at 16℃ and 150 rpm for 14 h. Centrifuge to collect bacterial cells, resuspend at 1 g bacterial cells: 5 mL buffer A (50 mM Tris pH 8.0, 500 mM NaCl, 5% glycerol, 1 mM DTT), autoclave at 4℃, and centrifuge to collect the supernatant.

[0070] The supernatant was passed through a Ni-affinity chromatography column (equilibration buffer same as buffer A), washed with 60 mM imidazole, and the target protein was eluted with 300 mM imidazole. Figure 1 As shown, SDS-PAGE revealed a clear target band in the supernatant (S), and the 300 mM imidazole elution peak (E1) exhibited high purity. The eluent was collected, diluted to a final NaCl concentration of 50 mM, and loaded onto a Q-type anion exchange column (Buffer A: 50 mM Tris pH 8.0, 50 mM NaCl; Buffer B: 50 mM Tris pH 8.0, 1 M NaCl), with gradient elution to collect the main peak. Figure 2 As shown, the purity of the peak tip component was further improved. After concentration, the sample was loaded onto a Superdex 200 Increase 10 / 300 GL molecular sieve column (buffer C: 25 mM Tris pH 8.0, 250 mM NaCl), as shown. Figure 3 As shown in C, the main peak is symmetrical, and SDS-PAGE shows a single band with a purity >95%. Concentrate to 15 mg / mL, aliquot, flash freeze in liquid nitrogen, and store at -80°C.

[0071] Following the method described in this embodiment, B4R-CTD-tetramer, B4R protein, and B4R-NTD protein were prepared. The amino acid sequence of B4R-CTD-tetramer is shown in SEQ ID NO: 4, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO: 3; the amino acid sequence of B4R protein is shown in SEQ ID NO: 6, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO: 5; the amino acid sequence of B4R-NTD protein is shown in SEQ ID NO: 8, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO: 7.

[0072] Example 2: Crystallization and structural analysis of B4R-CTD protein

[0073] The purified protein from Example 1 was diluted to 5 mg / mL and 10 mg / mL with buffer (50 mM Tris, 150 mM NaCl, pH 8.0). A seated drop gas-phase diffusion method (mosquito LCP robot, SwissCi 96-well plate) was used. The pooling solution was from a commercial kit (Index, PEG / Ionic, SaltRx, Wizard, etc.). The protein:pooling solution ratio was 200 nL:200 nL, and the mixture was incubated statically at 16°C.

[0074] Observations were conducted from day 2 to 9 during the initial screening. After rod-shaped crystals were identified, optimization was performed. The final optimized conditions were: 0.1 M Bis-Tris (bis(2-hydroxyethyl)amino(tris(hydroxymethyl)methane)) pH 6.5, 2.0 M Ammonia sulfate, and 0.1 M Praseodymium(III) acetate hydrate. Figure 4As shown, the optimized single crystal is rod-shaped, large in size, and exhibits good single crystallinity. The crystal was retrieved, treated with 20% glycerol antifreeze, and flash-frozen in liquid nitrogen. Diffraction data were collected at the BL19U1 beamline (wavelength 0.979183 Å) of the Shanghai Synchrotron Radiation Facility. Data statistics are shown in Table 1: space group P62 2 2, cell parameters a=b=71.85 Å, c=404.16 Å, α=β=90°, γ=120.0°, resolution 2.65 Å (highest shell 2.72-2.65 Å), integrity 99.7%, R-merge=0.091. The structure was resolved using molecular substitution, yielding a high-quality model. Figure 4 The right figure shows the overall folding and active center.

[0075] Table 1. Diffraction Data Collection

[0076]

[0077] X-ray diffraction data collection for the crystal was conducted at the BL19U1 beamline of the Shanghai Synchrotron Radiation Facility (SSRF). During the experiment, the crystal was kept cryogenically cooled in a nitrogen gas flow at 100 K. The X-ray wavelength used for data collection was 0.9792 Å, and the detector model was Pilatus3 6M. An oscillatory method was employed for data collection, with a rotation angle of 1° per frame, resulting in a total of 360 diffraction images acquired.

[0078] First, the acquired raw diffraction images were processed using HKL2000 or XDS software packages to obtain accurate unit cell parameters, space group information, and normalized amplitude data. In the structure resolution stage, the B4R-CTD structure predicted by AlphaFold 3 was used as the search model, and the initial phase was calculated using the Molecular Replacement module in the PHENIX or CCP4 packages. After obtaining the initial electron density map, PHENIX.refine was used for automated model construction and parameter refinement. Finally, COOT software was used to adjust and repair the chiral atomic model based on the differential electron density map until all geometric parameters of the model reached reasonable thresholds. Table 2 shows that, overall, the protein structure model is of good quality and highly reliable. Although the number of water molecules is relatively small, the model almost reaches perfection in terms of geometry (main chain and side chain conformations) (zero outliers), and the data fitting process is rigorous with no obvious signs of overfitting.

[0079] Table 2 B4R-CTD Diffraction Data Processing

[0080]

[0081] Example 3: Effect of B4R-CTD endonuclease activity and oligomerization state on activity

[0082] 1. EMSA was used to detect the binding affinity of B4R-CTD (monomer / tetramer form) and full-length B4R to nucleic acid substrates. For example... Figure 5 As shown, in the ssDNA binding experiment, the tetrameric form showed the most significant change in migration rate; Figure 6 The results showed that ssRNA binding was strongest in tetramers.

[0083] EMSA experimental procedures:

[0084] This study used electrophoretic migration assay (EMSA) to detect the interaction between proteins and nucleic acid probes. Nucleic acids with a 5′ fluorescently labeled group (FAM) were used as probes. The labeled probes were incubated with the target protein in a binding buffer system. If the protein specifically binds to the nucleic acid, a protein-probe complex is formed. The reaction mixture was then separated using non-denaturing polyacrylamide gel electrophoresis (Native-PAGE). Under an electric field, the larger complex with a lower negative charge density migrated significantly slower than the unbound free probe. Fluorescence signals were detected using a laser scanning imaging system. The presence of a distinct retarded / shifted band at the lag position in the gel lane confirmed a direct and specific interaction between the protein and the target nucleic acid probe.

[0085] 5'-FAM-labeled DNA / RNA probes (synthesized by General Biotech) were designed targeting the sequence. Single-stranded nucleic acids, double-stranded nucleic acids, or nucleic acids containing stem-loops (such as ssDNA, ssRNA, tRNA, stem-loop RNA) were prepared by annealing (95°C for 5 min, followed by natural cooling to room temperature).

[0086] (1) Binding reaction of nucleic acid probe and protein: Prepare a 20 μL binding reaction system on ice. First, add the prepared 10×EMSA binding buffer (final concentration: 50 mM Tris-HCl pH 8.0, 50 mM NaCl) to a centrifuge tube. Then, add the serially diluted target protein to a final concentration range of 80 nM to 5.12 μM (corresponding to a protein to nucleic acid molar ratio of 2:1 to 128:1), and mix gently. Finally, add 40 nM of 5′-FAM fluorescently labeled nucleic acid probe. Incubate the reaction mixture on ice for 30 minutes to reach binding equilibrium.

[0087] (2) Non-denaturing polyacrylamide gel electrophoresis (Native-PAGE): Prepare 15% and 20% non-denaturing polyacrylamide gels (Native-PAGE) using 1×TBE buffer. Fix the gel in the electrophoresis tank, add pre-cooled 1×TBE electrophoresis buffer, and pre-electrophoresis at 100 V for 30 minutes to remove impurity ions from the gel and pre-cool the electrophoresis buffer.

[0088] (3) Sample loading: After electrophoresis incubation, add 5×EMSA loading buffer and mix well. Take 6μL of sample and load it into the gel well. The entire electrophoresis process is carried out in an ice-water bath with the voltage set at 100V and the electrophoresis time is about 120 minutes (judged by the migration position of the bromophenol blue indicator, which usually reaches 2 / 3 of the gel).

[0089] (4) Imaging and data analysis: After electrophoresis, carefully remove the glass plate and transfer the gel to the Bio-RadChemiDoc™ imaging system. Use the FAM or Cy2 channel (excitation light 488 nm, emission light 520 nm) for exposure imaging.

[0090] 1. Nucleic acid cleavage activity assay (reaction at 37℃, detection by polyacrylamide gel electrophoresis): Figure 7 (ssDNA) Figure 8 (stem-loop RNA) Figure 9 (tRNA) results showed that B4R-CTD had the highest cleavage efficiency, followed by full-length B4R, while the tetrameric form showed weaker activity. Nuclease activity was preserved under EDTA treatment, indicating that B4R-CTD protein and its mutants are non-metal ion-dependent endonucleases, but Mg... 2+ and Ca 2+ Both can promote the activity of endonucleases.

[0091] 1) Reaction system preparation: The nuclease reaction was carried out in reaction buffer (10 mM Tris, pH 8.0; 20 mM KCl; 1% Glycerol; 1 mM DTT). The purified target protein (final concentration 2 μM) was mixed with different types of 5′-FAM fluorescently labeled nucleic acid substrates (including ssRNA, ssDNA, tRNA, and stem-loop RNA, final concentration 40 nM). EDTA and Mg were added to the reaction system respectively. + or Ca + ; among them, EDTA served as the negative control group, with a final concentration of 5 mM; Mg + Ca + The final concentration was 2 mM.

[0092] 2) Incubation: The system was incubated in a water bath at 37°C for 30 min to carry out the cleavage reaction. Subsequently, an appropriate amount of proteinase K was added and incubation was continued at 42°C for 1 h to completely digest the protein in the system and eliminate the influence of protein on nucleic acid migration rate.

[0093] 3) Denaturing Polyacrylamide Gel Electrophoresis (Urea-PAGE): The reaction products are separated by urea denaturing gel electrophoresis. Before electrophoresis, the gel is pre-treated to remove residual urea and unpolymerized monomers from the sample wells and to preheat the gel to the working temperature. The treated samples are added to the sample wells and electrophoresed at a constant voltage of 200 V for 30 min.

[0094] 4) Development and Imaging: Since the substrates all have 5′-FAM fluorescent groups, after electrophoresis, the gel is directly scanned using a multifunctional gel imaging system to record and analyze the cleavage of the nucleic acid substrates.

[0095] The sequences of ssDNA, ssRNA, stem-loop RNA, and tRNA used in this embodiment are as follows:

[0096] ssDNA: 5'-GGCTTTTGACCTTTATGCTTAGTGGTCAAAAGCCCATGATTTAACTTTCCGAAAGACGGT-3';

[0097] ssRNA: 5'-GGCUUUUGACCUUUAUGCUUAGUGGUCAAAAGCCCAUGAUUUAACUUUCCGAAGACGGU-3';

[0098] stem-loopRNA: 5'-GUCAGUGUACGUCGACAGGUAAACUCCCGUCGGUAGCAGUUCUCAGGUCGAUGUGAGCG-3';

[0099] tRNA: 5'-GUAGUCGUGGCCGAGUGGUUAAGGCGAUGGACUUGAAAUCCAUUGUGGUUUCCCCGCGCAGGUUCGAAUCCUGCCGACUACGCCA-3'.

[0100] Example 4: Effect of SLFN domain on poxin activity

[0101] This assay is based on the competitive binding of a limited amount of 2',3'-cGAMP polyclonal antiserum to a 2',3'-cGAMP tracer conjugated with horseradish peroxidase (HRP) (2',3'-cGAMP-HRP Tracer). With a fixed tracer concentration and varying natural 2',3'-cGAMP concentration, the tracer dose binding to the antiserum is inversely proportional to the concentration of free 2',3'-cGAMP in the sample. This antibody-2',3'-cGAMP complex is further bound to a pre-coated mouse anti-rabbit IgG plate. After washing to remove unbound reagents, a tetramethylbenzidine (TMB) substrate solution (HRP substrate) is added, and the enzymatic reaction produces a yellow product with strong absorbance at 450 nm. The reaction is terminated by adding HRP stop solution. Absorbance is directly proportional to the bound tracer dose and inversely proportional to the concentration of 2',3'-cGAMP in the sample. The main steps are as follows:

[0102] (1) Reagent preparation and calibrator preparation: Wash buffer preparation: Dilute the concentrated wash buffer with ultrapure water to the working concentration according to the instructions. Standard gradient dilution: Serially dilute the 2',3'-cGAMP standard stock solution with ELISA buffer to construct a concentration gradient covering the range of 0.1 ng / mL to 100 ng / mL. Tracer and antibody preparation: Bring the 2',3'-cGAMP-HRP tracer (Tracer) and specific polyclonal antibody (Antiserum) to room temperature for later use.

[0103] (2) Sample pretreatment and incubation - Sample inactivation (key step): Add a constant concentration of 2',3'-cGAMP (100 nM) to the reaction buffer, followed by serially diluted test protein (1000 nM - 7.8 nM). Incubate overnight at 37°C to ensure the enzymatic degradation reaction reaches its endpoint or equilibrium. To prevent phosphodiesterases such as ENPP1 in the sample from degrading cGAMP, cell lysates or tissue samples should be heated at 95°C for 5 min, centrifuged, and the supernatant collected for detection.

[0104] (3) Washing: Rinse five times with 300 μL washing buffer. After washing, use absorbent paper to dry the excess liquid in the wells.

[0105] (4) Sample addition: In a 96-well plate pre-coated with mouse anti-rabbit IgG, add 50 μL of standard or test sample to each well. Set up a negative control with only ultrapure water and protein, and a positive control with only cGAMP.

[0106] (5) Competition reaction initiation: Add 50 μL of 2',3'-cGAMP-HRP tracer and 50 μL of specific antibody to each well.

[0107] (6) Sealing and Incubation: Cover with sealing film to prevent liquid evaporation and incubate in a shaker at room temperature for 2 hours.

[0108] (7) Washing: Discard the liquid in the wells and wash 5 times with 300 μL of washing buffer. Ensure that the liquid completely covers the well walls with each wash. After the last wash, pat dry on clean absorbent paper to completely remove any unbound tracer.

[0109] (8) Colorimetric reaction (Development): Add 175 μL of TMB colorimetric substrate solution to each well, seal the plate, and place it on a horizontal shaker. Incubate for 30 min at room temperature under dark conditions. At this time, HRP catalyzes the color change of TMB from colorless to blue.

[0110] (9) Termination of reaction: Quickly add 75 μL of HRP stop solution to each well. The reaction solution immediately changes from blue to yellow, while the colorless wells remain colorless.

[0111] (10) Absorbance detection: The absorbance (OD value) of each well was measured at a wavelength of 450 nm using an ELISA reader.

[0112] cGAMP clearance rate (%) = (OD positive control - OD sample) / OD positive control × 100%.

[0113] In addition, such as Figure 10 As shown, the EC50 of B4R is 31.25 nM, while that of B4R-NTD is 100 nM. The full-length B4R is three times more efficient than B4R-NTD in degrading cGAMP, confirming that C-terminal truncation does not impair N-terminal poxin function, but the presence of SLFN enhances cGAMP hydrolysis activity. These experimental results collectively demonstrate that B4R-CTD exists in solution as a monomer / tetramer, with the monomer being the predominant active form, and the tetramer enhancing the protein's ability to bind nucleic acids. This provides direct evidence for subsequent structure-function studies.

[0114] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A monkeypox virus B4R nuclease domain protein or a mutant thereof, characterized in that, The amino acid sequence of the protein is shown as 10-320aa in SEQ ID NO: 2; the amino acid sequence of the protein mutant is shown as 10-320aa in SEQ ID NO:

4.

2. The biomaterials related to the monkeypox virus B4R nuclease domain protein or its mutants as described in claim 1, characterized in that, It can be any one or more combinations of the following biological materials: (a) A nucleic acid molecule encoding the monkeypox virus B4R nuclease domain protein of claim 1 or a mutant thereof; (b) An expression cassette containing the nucleic acid molecule described in (a); (c) A recombinant expression vector containing the nucleic acid molecule described in (a); (d) A recombinant expression vector containing the expression cassette described in (b); (e) Recombinant bacteria containing the nucleic acid molecules described in (a); (f) Recombinant bacteria containing the expression cassette described in (b); (g) Recombinant bacteria containing the recombinant expression vector described in (c) or (d).

3. The biomaterial according to claim 2, characterized in that: The nucleic acid molecule described in (a) is a gene sequence encoding the monkeypox virus B4R nuclease domain protein, and its nucleotide sequence is shown as 28-963bp in SEQ ID NO: 1; The nucleic acid molecule described in (a) is a gene sequence encoding a mutant monkeypox virus B4R nuclease domain protein, and its nucleotide sequence is shown as 28-963bp in SEQ ID NO: 3; And / or, the starting vector for the recombinant expression vectors described in (c) and (d) is a pET series vector; And / or, the host bacteria corresponding to the recombinant bacteria described in (e), (f), and (g) are selected from prokaryotes, yeast, or higher eukaryotic cells.

4. The use of the biomaterial according to any one of claims 2 to 3 in the preparation of monkeypox virus B4R nuclease domain protein or mutant thereof.

5. A method for preparing a monkeypox virus B4R nuclease domain protein or a mutant thereof, characterized in that, The method includes the following steps: culturing the recombinant bacteria according to any one of claims 2 to 3, and obtaining the monkeypox virus B4R nuclease domain protein or its mutant according to claim 1 from the recombinant bacteria.

6. The application of the monkeypox virus B4R nuclease domain protein or its mutant as described in claim 1, or the biological material described in any one of claims 2 to 3, in nucleic acid cleavage.

7. A crystal of a monkeypox virus B4R nuclease domain protein, characterized in that, Its structural parameters are: space group P62 2 2, cell parameters a = b = 71.85 Å, c = 404.16 Å, α = β = 90°, γ = 120.0°, resolution 2.65 Å, integrity: 99.7%.

8. The method for preparing the monkeypox virus B4R nuclease domain protein crystal according to claim 7, characterized in that, The steps include: preparing a protein solution of the monkeypox virus B4R nuclease domain protein according to claim 1 using a buffer solution, and then crystallizing it using a sitting drop gas phase diffusion method in the presence of a crystallization reagent, followed by static incubation to obtain monkeypox virus B4R nuclease domain protein crystals. The crystallization reagent is 0.1 M Bis-Tris, 1.8 M-2.0 M ammonium sulfate, and 0.1 M Praseodymium (III) acetate hydrate, pH 6.

5.

9. The crystallization method according to claim 8, characterized in that: The concentration of the protein solution is 5–10 mg / mL; And / or, the volume ratio of protein solution to crystallization reagent is 1:1; And / or, the temperature for static incubation is 16°C; And / or, the static incubation time is 3 to 5 days.

10. The application of the monkeypox virus B4R nuclease domain protein crystal according to claim 7, characterized in that: For any of the following applications: (A) Applications in studying the function of the SLFN domain of monkeypox virus, its interaction mechanism with poxin, or in the preparation of anti-monkeypox virus drugs; (B) Application in the preparation of monkeypox virus vaccines.

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