A broad-spectrum antiviral fusion protein, its preparation method and application

Abstract

The present invention provides a broad-spectrum antiviral fusion protein, a preparation method and an application thereof, relating to the technical field of biomedicine. The present invention constructs a fusion protein NS3b-Apaf1 composed of the dsRNA binding domain of the NS3b protein of coronavirus HKU4-2 and the CARD signaling domain of human Apaf-1 protein. And the antiviral effect of the fusion protein NS3b-Apaf1 is studied. The fusion protein NS3b-Apaf1 constructed by the present invention has strong inhibitory, preventive and killing effects on viruses. The fusion protein can be used for preparing drugs or vaccines against highly pathogenic viruses, providing a new idea for antiviral treatment.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a broad-spectrum antiviral fusion protein and a preparation method and application thereof. Background Art

[0002] The virus has strong transmission ability and high mutation rate, posing a serious threat to human life and health.

[0003] Following viral infection, host cells produce large amounts of dsRNA. It is generally believed that dsRNA is primarily a product of RNA polymerase during RNA viral replication, or bidirectional transcription during DNA viral replication. It is also believed that viral infection can induce host cell stress, leading to the production of large amounts of endogenous dsRNA. Consequently, animal cells have evolved a series of receptors for virus-associated dsRNA that specifically recognize and sense intracellular virus-associated dsRNA signals and initiate corresponding innate immune responses. Retinoic acid-inducible gene I (RLR)-like receptors (RLRs) are a recently discovered class of viral dsRNA-dependent receptors that can initiate antiviral immune responses. RLRs are primarily composed of RIG-I and MDA5 receptors. They share a high degree of family homology and possess two functionally independent domains: the N-terminal CARD signaling domain (CARD) and the C-terminal RNA-binding domain (RBD). The RNA-binding domain determines the specificity of RLRs for recognizing abnormal viral RNA within cells, while the CARD signaling domain determines the downstream signaling pathways of RLR receptors. However, the continuous emergence of highly pathogenic viruses indicates that viruses have evolved to evade the natural immune response mediated by human RLRs, causing viral infection and widespread spread. Therefore, the development of new fusion proteins to reshape RLRs, enhance their ability to recognize viruses, trigger the body's antiviral immune response, and prevent viral immune evasion is a key issue that needs to be addressed in the development of viral drugs. Summary of the Invention

[0004] (1) Technical problems solved

[0005] In view of the deficiencies of the existing technology, the present invention provides a broad-spectrum antiviral fusion protein and its preparation method and application.

[0006] (2) Technical solution

[0007] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0008] In a first aspect, the present invention provides a fusion protein, which is a fusion protein NS3b-Apaf1 composed of the dsRNA binding domain (dsRBD) of the NS3b protein of coronavirus HKU4-2 and the CARD signaling domain of the human Apaf-1 protein.

[0009] In a second aspect, the present invention further provides a gene encoding a fusion protein NS3b-Apaf1. The structure of the fusion protein NS3b-Apaf1 is: 6*His - [BamHI] - PTD4– [XhoI] – RBD– [KpnI] – CARD- [HindIII]; the amino acid sequence of the fusion protein NS3b-Apaf1 is shown in SEQ ID NO. 1, specifically:

[0010] HHHHHHGSYARAAARQARASGLEYVSLLNQFWQKQIKSYKETPSQYHYLYPPRFFYKPVLGNLQHPTKWCCTIKFYEYSAQATECTKASAKQDAARLIC EQLGTMDAKARNCLLQHREALEKDIKTSYIMDHMISDGFLTISEEEKVRNEPTQQQRAAMLIKMILKKDNDSYVSFYNALLHEGYKDLAALLHDGIPVV

[0011] Specifically, the method for constructing the gene of the fusion protein NS3b-Apaf1 is characterized in that the PTD4 sequence as shown in SEQ ID NO.4, the RBD sequence of NS3b of the coronavirus HKU4-2 as shown in SEQ ID NO.6, and the CARD sequence of Apaf-1 as shown in SEQID NO.7 are double-enzyme-cut and then connected into a vector for integration.

[0012] The present invention also contains an expression vector of the gene of the fusion protein NS3b-Apaf1.

[0013] The present invention also provides a recombinant bacterium containing a vector for expressing the gene of the fusion protein NS3b-Apaf1.

[0014] In a third aspect, the present invention provides a method for preparing a fusion protein, wherein the fusion protein is prepared according to the following steps:

[0015] (1) The above-mentioned fusion protein NS3b-Apaf1 gene sequence was ligated into the pRSET-B vector by double enzyme digestion to obtain the pRSET-NS3b-Apaf1 recombinant plasmid;

[0016] (2) The recombinant plasmid obtained in step (1) was transformed into Rosetta gami (DE3) Escherichia coli, coated on an LB plate containing ampicillin and cultured overnight, a single colony on the LB plate was picked for amplification and culture, and sequencing was performed for identification. The matching of the sequencing sequence indicated that the NS3b-Apaf1 fusion protein expression vector was successfully constructed, and a recombinant bacterium expressing the NS3b-Apaf1 fusion protein was obtained;

[0017] (3) The recombinant bacteria in step (2) are amplified and cultured, and the expression is induced by IPTG. The bacteria are collected and crushed, and the precipitate is collected after centrifugation. The precipitate is washed, denatured, and renatured to obtain the NS3b-Apaf1 fusion protein;

[0018] Furthermore, step (1) specifically includes the following steps:

[0019] a. Obtain pRSET-PTD4 vector:

[0020] Synthesize two oligonucleotides with the following sequences:

[0021] CATGGATCCTACGCCCGTGCCGCCGCCCGTCAGGCCCGTGCCAGTGGT (SEQ ID NO. 2) and CGTACTCGAGACCACTGGCACGGGCCTG (SEQ ID NO. 3) were annealed and extended, and then digested with BamHI and XhoI restriction enzymes to obtain the cell-penetrating peptide domain (PTD4) cDNA, which encodes the amino acid sequence: YARAAARQARA (SEQ ID NO. 4). The PTD4 sequence was inserted between the BamHI and XhoI residues of the pRSET-B vector to generate the pRSET-PTD4 vector.

[0022] b. Obtain pRSET-PTD4-RBD vector:

[0023] The RBD of the NS3b-Apaf1 fusion protein is the RBD of A3EXA5 (Uncharacterized protein NS3b) of Bat coronavirus HKU4-2. The RBD fragment of NS3b was fully synthesized by the company, with an XhoI restriction site introduced before the sequence and a KpnI restriction site introduced after the sequence. The synthetic sequence is:

[0024] CTCGAGTACGTCTCTTTGCTTAACCAATTTTGGCAGAAGCAAATTAAGTCTTATAAAGAGACTCCTAGTCAGTATCATTACCTGTATCCACCCAGGTTTTTCTATAAACCTGTTTTGGGTAATTTA CAGCACCCTACCAAGTGGTGTTGTACTATTAAATTTTATGAGTATAGTGCTCAGGCTACTGAGTGTACTAAAGCATCAGCAAAACAAGATGCAGCTAGACTTATCTGTGAACAGTTAGGTACC (SEQ ID NO.5)

[0025] Its protein sequence is:

[0026] YVSLLNQF WQKQIKSYKE TPSQYHYLYP PRFFYKPVLG NLQHPTKWCC TIKFYEYSAQATECTKASAK QDAARLICEQ L (SEQ ID NO.6)

[0027] The NS3b RBD was digested with XhoI and KpnI restriction enzymes and inserted between XhoI and KpnI of the pRSET-PTD4 vector to obtain the pRSET-PTD4-RBD vector.

[0028] c. Obtain the pRSET-NS3b-Apaf1 recombinant plasmid:

[0029] The CARD of the NS3b-Apaf1 fusion protein is the CARD of human Apaf-1. A KpnI restriction site was introduced before the upstream primer, and a HindIII restriction site was introduced before the downstream primer. Human cell RNA was extracted and reverse transcribed into cDNA. The human Apaf-1 CARD was amplified using primers. The sequence is:

[0030] GGTACCATGGATGCAAAAGCTCGAAATTGTTTGCTTCAACATAGAGAAGCTCTGGAAAAGGACATCAAGACATCCTACATCATGGATCACATGATTAGTGATGGATTTTTAACAATATCAGAAGAGGAAAAAGTAAGAAATGAGCCCAC TCAACAGCAAAGAGCAGCTATGCTGATTAAAATGATACTTAAAAAAGATAATGATTCCTACGTATCATTCTACAATGCTCTACTACATGAAGGATATAAAGATCTTGCTGCCCTTCTCCATGATGGCATTCCTGTTGTCTAAGCTT (SEQ ID NO.7)

[0031] The human Apaf-1 CARD was obtained by digestion with KpnI and HindIII restriction enzymes, and then inserted between KpnI and HindIII of the pRSET-PTD4-RBD vector to obtain the pRSET-NS3b-Apaf1 recombinant plasmid.

[0032] Furthermore, step (3) specifically includes the following steps:

[0033] NS3b-Apaf1 recombinant bacteria were inoculated into 20ml of Amp+LB medium and cultured at 37°C to an OD600 of approximately 0.5. 3ml of the culture was transferred into 250ml of Amp+LB medium and cultured at 37°C with shaking overnight until mid-logarithmic phase (OD600 = 0.5-1.0). Protein expression was induced by adding IPTG. After overnight induction, cells were harvested by centrifugation at 4,000 rpm for 30 minutes at 4°C. 10g of wet cells were resuspended in 50ml of Lysis Buffer and centrifuged at 8,000 rpm for 10 minutes. The supernatant was discarded. 10g of wet cells were resuspended in 100ml of 2M urea-Lysis Buffer and sonicated at 200W power, working for 2 seconds and stopping for 5 seconds for a total of 20 minutes. After sonication, the sample was stirred at 4°C for 1-2 hours and then centrifuged at 12,000 rpm at 4°C. After 10 minutes, discard the supernatant and resuspend the pellet in 8M urea-Lysis Buffer with an equal volume of 0.01% NP-40 and 2M urea-Lysis Buffer. Stir at 4°C overnight (more than 15 hours). The next day, centrifuge at 12,000 rpm for 20 minutes at 4°C. Remove the supernatant, filter through a 0.45μm filter, and use it for affinity chromatography to purify the protein.

[0034] Furthermore, step (3) further includes the following steps: agarose affinity purification of the recombinant fusion protein NS3b-Apaf1 and on-column renaturation, specifically the following steps:

[0035] 1) Column Packing: Load 5 ml of Ni-NTA filler into the column, remove bubbles and compact the filler. Turn on the UV detection of the purification system and rinse with 50 ml of deionized water at a flow rate of 2 ml / min.

[0036] 2) Column equilibration: Equilibrate the column with 30 ml of Lysis Buffer; then equilibrate the column with 20 ml of 8 M urea-Lysis Buffer at a flow rate of 2 ml / min.

[0037] 3) Protein loading: Add an equal volume of 8 M urea-Lysis Buffer to the protein solution to dilute it, then slowly pass the protein solution through the chromatography column at a flow rate of 0.5 ml / min;

[0038] 4) On-column renaturation: rinse with 50 ml of 8 M, 6 M, 4 M, 2 M, and 1 M urea-Lysis Buffer, respectively, followed by 200 ml of Lysis Buffer at a flow rate of 1 ml / min.

[0039] 5) Rinse: Rinse with 100 ml of Wash Buffer until the A280 curve is flat, flow rate 1 ml / min;

[0040] 6) Elution: Add Elution Buffer and collect the protein at a flow rate of 1 ml / min until the A280 curve flattens;

[0041] 7) Column cleaning: Rinse with 100 ml of distilled water, then rinse with 30 ml of 20% ethanol and immerse the column in it.

[0042] 8) Dialysis desalination: Prepare three cylinders of 2 L Lysis Buffer each and pre-cool them. Load the eluted protein into dialysis tape and dialyze at 4°C, changing the external buffer every 6 hours.

[0043] 9) Ultrafiltration Concentration: Add the dialyzed protein solution to the inner tube of an ultrafiltration tube with a cutoff of 3 kD. Centrifuge at 12,000 rpm for 30 minutes at 4°C. Discard the waste liquid in the tube, add PBS, and centrifuge again to collect the protein in the inner tube.

[0044] 10) Add 10% glycerol to the protein and filter the protein with a 0.2 μm filter. Determine the protein concentration using the BCA assay and store at -70°C.

[0045] In a fourth aspect, the present invention further provides an application of a fusion protein, wherein the fusion protein NS3b-Apaf1 is used to prepare antiviral drugs and / or vaccines against highly pathogenic viruses.

[0046] (3) Beneficial effects

[0047] The present invention constructs a fusion protein, NS3b-Apaf1, composed of the dsRNA binding domain of the NS3b protein of coronavirus HKU4-2 and the CARD signaling domain of the human Apaf-1 protein. The antiviral effects of this fusion protein, NS3b-Apaf1, have been studied. The fusion protein NS3b-Apaf1 constructed in the present invention has strong inhibitory, preventive, and killing effects on viruses. This fusion protein can be used to prepare drugs or vaccines against highly pathogenic viruses, providing new ideas for antiviral treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 Schematic diagram of the structure of the fusion protein NS3b-Apaf1.

[0049] Figure 2 This is the sequencing result of the fusion protein NS3b-Apaf1.

[0050] Figure 3 Figure 3 shows the SDS-PAGE electrophoresis analysis (A) and Western blot identification (B) results of the fusion protein NS3b-Apaf1, where channel 3 represents the target protein.

[0051] Figure 4 The following are the results of cytotoxicity assay of the fusion protein NS3b-Apaf1, where (A) is the cell survival rate under treatment with different concentrations of NS3b-Apaf1; (B) is the stable existence time of 200 nM NS3b-Apaf1 protein added to 293T cells.

[0052] Figure 5 Figure 3 shows the inhibitory effect of the fusion protein NS3b-Apaf1 on AdV, where (A) is the fluorescence image of cells infected with AdV-GFP under treatment with different concentrations of NS3b-Apaf1; (B) is the FFU value of cells infected with AdV-GFP under treatment with different concentrations of NS3b-Apaf1; (C) is the AdV-GFP content results of cells infected with AdV-GFP under treatment with different concentrations of NS3b-Apaf1.

[0053] Figure 6The figures show the preventive effect of the fusion protein NS3b-Apaf1 against AdV, where (A) is the fluorescence image of cells infected with AdV-GFP under treatment with different concentrations of NS3b-Apaf1; (B) is the FFU value of cells infected with AdV-GFP under treatment with different concentrations of NS3b-Apaf1.

[0054] Figure 7 Figure 3 shows the killing effect of the fusion protein NS3b-Apaf1 on AdV, where (A) is the fluorescence image of cells infected with AdV-GFP under treatment with different concentrations of NS3b-Apaf1; (B) is the FFU value of cells infected with AdV-GFP under treatment with different concentrations of NS3b-Apaf1.

[0055] Figure 8 The figures show the inhibitory effect of the fusion protein NS3b-Apaf1 on VTT, where (A) is the fluorescence image of cells infected with VTT-GFP under treatment with different concentrations of NS3b-Apaf1; (B) is the FFU value of cells infected with VTT-GFP under treatment with different concentrations of NS3b-Apaf1. DETAILED DESCRIPTION

[0056] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention. Example

[0057] 1. Preparation of NS3b-Apaf1 fusion protein

[0058] 1.1 Construction of pRSET-NS3b-Apaf1 recombinant plasmid

[0059] a. Obtain pRSET-PTD4 vector:

[0060] Synthesize two oligonucleotides with the following sequences:

[0061] CATGGATCCTACGCCCGTGCCGCCGCCCGTCAGGCCCGTGCCAGTGGT (SEQ ID NO. 2) and CGTACTCGAGACCACTGGCACGGGCCTG (SEQ ID NO. 3) were annealed and extended, and then digested with BamHI and XhoI restriction enzymes to obtain the cell-penetrating peptide domain (PTD4) cDNA, which encodes the amino acid sequence: YARAAARQARA (SEQ ID NO. 4). The PTD4 sequence was inserted between the BamHI and XhoI residues of the pRSET-B vector to generate the pRSET-PTD4 vector.

[0062] b. Obtain pRSET-PTD4-RBD vector:

[0063] The RBD of the NS3b-Apaf1 fusion protein is the RBD of A3EXA5 (Uncharacterized protein NS3b) of Bat coronavirus HKU4-2. The RBD fragment of NS3b was fully synthesized by the company, with an XhoI restriction site introduced before the sequence and a KpnI restriction site introduced after the sequence. The synthetic sequence is:

[0064] CTCGAGTACGTCTCTTTGCTTAACCAATTTTGGCAGAAGCAAATTAAGTCTTATAAAGAGACTCCTAGTCAGTATCATTACCTGTATCCACCCAGGTTTTTCTATAAACCTGTTTTGGGTAATTTA CAGCACCCTACCAAGTGGTGTTGTACTATTAAATTTTATGAGTATAGTGCTCAGGCTACTGAGTGTACTAAAGCATCAGCAAAACAAGATGCAGCTAGACTTATCTGTGAACAGTTAGGTACC (SEQ ID NO.5)

[0065] Its protein sequence is:

[0066] YVSLLNQF WQKQIKSYKE TPSQYHYLYP PRFFYKPVLG NLQHPTKWCC TIKFYEYSAQATECTKASAK QDAARLICEQ L (SEQ ID NO.6)

[0067] The NS3b RBD was digested with XhoI and KpnI restriction enzymes and inserted between XhoI and KpnI of the pRSET-PTD4 vector to obtain the pRSET-PTD4-RBD vector.

[0068] c. Obtain the pRSET-NS3b-Apaf1 recombinant plasmid:

[0069] The CARD of the NS3b-Apaf1 fusion protein is the CARD of human Apaf-1. A KpnI restriction site was introduced before the upstream primer, and a HindIII restriction site was introduced before the downstream primer. Human cell RNA was extracted and reverse transcribed into cDNA. The human Apaf-1 CARD was amplified using primers. The sequence is:

[0070] GGTACCATGGATGCAAAAGCTCGAAATTGTTTGCTTCAACATAGAGAAGCTCTGGAAAAGGACATCAAGACATCCTACATCATGGATCACATGATTAGTGATGGATTTTTAACAATATCAGAAGAGGAAAAAGTAAGAAATGAGCCCAC TCAACAGCAAAGAGCAGCTATGCTGATTAAAATGATACTTAAAAAAGATAATGATTCCTACGTATCATTCTACAATGCTCTACTACATGAAGGATATAAAGATCTTGCTGCCCTTCTCCATGATGGCATTCCTGTTGTCTAAGCTT (SEQ ID NO.7)

[0071] The Apaf-1 CARD was obtained by digestion with KpnI and HindIII restriction enzymes, and then inserted between KpnI and HindIII of the pRSET-PTD4-RBD vector to obtain the pRSET-NS3b-Apaf1 recombinant plasmid.

[0072] 1.2 Construction of NS3b-Apaf1 fusion protein expression vector

[0073] The pRSET-NS3b-Apaf1 recombinant plasmid obtained in 1.1 was transformed into Rosetta gami (DE3) Escherichia coli, coated on an LB plate containing ampicillin and cultured overnight, a single colony on the LB plate was picked for amplification and identification by sequencing. A match in the sequencing sequence indicated that the NS3b-Apaf1 fusion protein expression vector was successfully constructed, and a recombinant bacterium expressing the NS3b-Apaf1 fusion protein was obtained; the structure of the fusion protein NS3b-Apaf1 is: 6*His - [BamHI] - PTD4 - [XhoI] - RBD - [KpnI] - CARD - [HindIII], as shown in the structural diagram. Figure 1 The amino acid sequence of the fusion protein NS3b-Apaf1 is shown in SEQ ID NO. 1, specifically:

[0074] HHHHHHGSYARAAARQARASGLEYVSLLNQFWQKQIKSYKETPSQYHYLYPPRFFYKPVLGNLQHPTKWCCTIKFYEYSAQATECTKASAKQDAARLIC EQLGTMDAKARNCLLQHREALEKDIKTSYIMDHMISDGFLTISEEEKVRNEPTQQQRAAMLIKMILKKDNDSYVSFYNALLHEGYKDLAALLHDGIPVV

[0075] The obtained fusion protein NS3b-Apaf1 was sequenced and identified. Figure 2 As shown, after comparing the sequencing results with the designed sequence, the sequencing sequence was consistent with the designed sequence, indicating that the fusion protein NS3b-Apaf1 expression vector was successfully constructed.

[0076] The fusion protein NS3b-Apaf1 was identified by Western blot. The NS3b-Apaf1 recombinant bacteria were selected and amplified in LB medium containing ampicillin. When the OD600 value was 0.6-0.8, IPTG was added to induce expression for 6 hours at a final concentration of 1.0mM. The bacteria were collected and disrupted. After centrifugation, the supernatant and precipitate were collected separately. The supernatant and precipitate were analyzed by SDS-PAGE electrophoresis. The results are as follows: Figure 3 As shown in A. After 6 hours of IPTG induction, the bacterial protein can be seen at 23.4kD expression bands, and the recombinant protein is expressed in inclusion bodies. Western blot analysis showed that the bacterial protein after 6 hours of IPTG induction can react specifically with anti-6*His monoclonal antibody, and a specific band appears at around 23.4kD with high specificity. Channel 3 is the target protein. The results are shown in Figure 3 As shown in B.

[0077] The above sequencing results showed that the fusion protein NS3b-Apaf1 expression vector was successfully constructed.

[0078] 1.3 Expression, purification, and refolding of NS3b-Apaf1 fusion protein

[0079] The recombinant bacteria in 1.2 are cultured and induced for expression with IPTG, and then the cells are collected. The collected cells are crushed, centrifuged, and the precipitate is collected. The precipitate is washed, denatured, and renatured to obtain the NS3b-Apaf1 fusion protein. The specific steps are as follows:

[0080] (1) Pick NS3b-Apaf1 recombinant bacteria and inoculate them into 20ml Amp+LB culture medium, and culture them at 37ºC until OD600 is about 0.5; take 3ml of culture and transfer them into 250ml Amp+LB culture medium, culture them at 37ºC with shaking overnight, until the mid-logarithmic phase (OD600=0.5-1.0), add IPTG to induce protein expression; after induction overnight, centrifuge at 4,000rpm for 30min at 4ºC to collect cells; resuspend every 10g of wet bacteria with 50ml Lysis Buffer, centrifuge again at 8,000rpm for 10min, and discard the supernatant; resuspend every 10g of wet bacteria with 100ml 2M urea-Lysis Buffer, sonicate at 200W power, work for 2s and stop for 5s, for a total of 20min; after sonication, place the sample at 4ºC and stir for 1-2h; centrifuge at 4ºC at 12,000rpm After 10 minutes, the supernatant was discarded and the pellet was resuspended in 8M urea-Lysis Buffer with an equal volume of 0.01% NP-40 and 2M urea-Lysis Buffer. The pellet was stirred at 4°C overnight (more than 15 hours). The next day, the pellet was centrifuged at 12,000 rpm for 20 minutes at 4°C. The supernatant was collected and filtered through a 0.45 μm filter membrane for protein purification by affinity chromatography.

[0081] (2) Agarose affinity purification of the recombinant fusion protein NS3b-Apaf1 and on-column renaturation, the specific steps are as follows:

[0082] 1) Column Packing: Load 5 ml of Ni-NTA filler into the column, remove bubbles and compact the filler. Turn on the UV detection of the purification system and rinse with 50 ml of deionized water at a flow rate of 2 ml / min.

[0083] 2) Column equilibration: Equilibrate the column with 30 ml of Lysis Buffer; then equilibrate the column with 20 ml of 8 M urea-Lysis Buffer at a flow rate of 2 ml / min.

[0084] 3) Protein loading: Add an equal volume of 8M urea-Lysis Buffer to the protein solution to dilute it, then slowly pass the protein solution through the chromatography column at a flow rate of 0.5 ml / min;

[0085] 4) On-column renaturation: rinse with 50 ml of 8 M, 6 M, 4 M, 2 M, and 1 M urea-Lysis Buffer, respectively, followed by 200 ml of Lysis Buffer at a flow rate of 1 ml / min.

[0086] 5) Rinse: Rinse with 100 ml of Wash Buffer until the A280 curve is flat, flow rate 1 ml / min;

[0087] 6) Elution: Add Elution Buffer and collect the protein at a flow rate of 1 ml / min until the A280 curve flattens;

[0088] 7) Column cleaning: Rinse with 100 ml of distilled water, then rinse with 30 ml of 20% ethanol and immerse the column in it.

[0089] 8) Dialysis desalination: Prepare three cylinders of 2 L Lysis Buffer each and pre-cool them. Load the eluted protein into dialysis tape and dialyze at 4°C, changing the external buffer every 6 hours.

[0090] 9) Ultrafiltration Concentration: Add the dialyzed protein solution to the inner tube of an ultrafiltration tube with a cutoff of 3 kD. Centrifuge at 12,000 rpm for 30 min at 4°C. Discard the waste liquid in the tube, add PBS, and centrifuge again to collect the protein in the inner tube.

[0091] 10) Add 10% glycerol to the protein and filter the protein with a 0.2 μm filter. Determine the protein concentration using the BCA assay and store at -70°C.

[0092] 2 Cytotoxicity assay of fusion protein NS3b-Apaf1

[0093] The cytotoxicity of NS3b-Apaf1 protein was determined using a CCK8 assay. Specific methods: 293T cells were seeded into 96-well plates. When cells reached 60% confluence, the culture medium was discarded and NS3b-Apaf1 protein was introduced at concentrations of 10, 25, 50, 100, 150, 200, 400, and 800 nM. Ten replicate wells were plated for each concentration. Cell culture medium without cells or NS3b-Apaf1 protein served as a blank control, and cells without any NS3b-Apaf1 protein (0 nM) served as a control. The 96-well plates were removed at 0, 24, 48, and 72 hours, and CCK8 assay solution was added to each well. After incubation at 37°C for 2 hours, the absorbance of each well was measured using a microplate reader. Cell viability was then calculated.

[0094] Cell viability = (OD450 of experimental group - OD450 of blank group) / (OD450 of control group - OD450 of blank group) 100%.

[0095] from Figure 4 A (original Figure 1 As shown in B), compared with the control group, there was no significant difference in the survival rate of cells treated with 10, 20, 50, 100, 150, and 200 nM NS3b-Apaf1 (P > 0.05), and the cell viability of the 400 and 800 nM NS3b-Apaf1 treatment groups decreased, and the difference was significant (P < 0.05); Based on the above results, 200 nM NS3b-Apaf1 was used as the maximum safe concentration that affects cell activity. NS3b-Apaf1 protein with a final concentration of 200 nM was then added to 293T cells, and the cells were collected at 2h, 12h, D1, D2, D3, and D4, and western blot was performed to determine the efficiency of NS3b-Apaf1 protein entering the cells, and the histidine tag antibody was produced after the cells were lysed. The results showed that NS3b-Apaf1 protein could enter the cells within 2 hours and stably exist in 293T cells for at least 3 days, as shown in the following figure. Figure 4 As shown in B.

[0096] 3. Antiviral effect of fusion protein NS3b-Apaf1 on AdV infection

[0097] Adenovirus (AdV) is a common double-stranded DNA virus that produces a large amount of double-stranded RNA during replication. We investigated the antiviral effect of the fusion protein NS3b-Apaf1 against AdV infection.

[0098] 3.1 Inhibitory effect of fusion protein NS3b-Apaf1 on AdV

[0099] First, AdV was added to the cells, followed by the addition of NS3b-Apaf1 protein. Specifically, 293T cells were robustly infected with AdV-GFP for 2 hours. NS3b-Apaf1 protein was then added at final concentrations of 0 nM, 10 nM, 25 nM, 50 nM, 100 nM, and 200 nM for incubation. AdV-GFP-infected cells were observed and photographed daily under a fluorescence microscope.

[0100] The results are as follows Figure 5 As shown in Figure 2, the number of cells infected with AdV-GFP decreased with increasing concentrations of NS3b-Apaf1 protein. At a concentration of 200 nM, no significant GFP fluorescence was observed, and the infected cells clearly lost their normal morphology (e.g., Figure 5 A). The FFU of cells in different groups were quantified using the Spearman-Karber method, and it was found that NS3b-Apaf1 protein reduced the titer of AdV-GFP in a dose-dependent manner (e.g. Figure 5 B). Cells were collected and lysed for Western blotting, and the level of AdV-GFP was detected using GFP antibody; the results are shown in Figure 2. Figure 5 As shown in Figure C, the GFP in the cells decreased with the increase of NS3b-Apaf1 protein concentration. The above results show that the fusion protein NS3b-Apaf1 provided by the present invention has a strong inhibitory effect on AdV.

[0101] 3.2 Preventive effect of fusion protein NS3b-Apaf1 against AdV

[0102] First, NS3b-Apaf1 protein was added to the cells, and then AdV was added. The specific method was: before AdV-GFP infection of 293T cells, NS3b-Apaf1 was added at final concentrations of 0nM, 10nM, 25nM, 50nM, 100nM, and 200nM. The cells infected with AdV-GFP were observed under a fluorescence microscope and photographed every day. The results are shown in Figure 2. Figure 6 As shown in Figure 3, the protective effect of NS3b-Apaf1 protein on AdV-infected 293T cells is related to its concentration. As the concentration of NS3b-Apaf1 protein increases, the number of cells infected with AdV-GFP decreases ( Figure 6 A). Quantification of FFU of cells in different groups showed that NS3b-Apaf1 protein reduced the titer of AdV-GFP in a dose-dependent manner ( Figure 6 B). The above results indicate that the fusion protein NS3b-Apaf1 provided by the present invention has a strong preventive effect on AdV.

[0103] 3.3 Killing effect of fusion protein NS3b-Apaf1 on AdV

[0104] NS3b-Apaf1 protein and AdV were added to the cells at the same time. The specific method was: NS3b-Apaf1 and AdV-GFP were added to A549 cells at the final concentrations of 0nM, 10nM, 25nM, 50nM, 100nM and 200nM. The results are shown in Figure 2. Figure 7 As shown in the figure, compared with the AdV-GFP control group (0nM NS3b-Apaf1), different concentrations of NS3b-Apaf1 protein showed a certain killing effect on AdV-GFP, and there was a certain dose-effect relationship ( Figure 7 A). FFU quantification of cells in different groups showed that NS3b-Apaf1 protein reduced the titer of AdV-GFP in a dose-dependent manner (( Figure 7 B). The above results show that the fusion protein NS3b-Apaf1 provided by the present invention has a significant killing effect on AdV.

[0105] 4. Antiviral effect of fusion protein NS3b-Apaf1 on VTT infection

[0106] We also studied the inhibitory effect of the fusion protein NS3b-Apaf1 on VTT virus. The specific method was as follows: A549 cells were infected with VTT-GFP for 2 hours and then incubated with NS3b-Apaf1 protein at final concentrations of 0 nM, 10 nM, 25 nM, 50 nM, 100 nM, and 200 nM. VTT-GFP infected cells were observed under a fluorescence microscope and photographed every day. The results are shown in Figure 2. Figure 8 As shown in Figure 2, as the concentration of NS3b-Apaf1 protein increased, the number of cells infected with VTT-GFP decreased. At a concentration of 200 nM, no obvious GFP fluorescence was observed, and the infected cells clearly lost their normal morphology ( Figure 8 A). Cell suspension was collected for FFU quantification, and it was found that NS3b-Apaf1 protein reduced the VTT-GFP titer in a dose-dependent manner ( Figure 8 B) The above results show that the fusion protein NS3b-Apaf1 provided by the present invention has a significant inhibitory effect on VTT virus.

[0107] 5 Discussions

[0108] The innate immune system constitutes the initial line of defense against viral infection. It recognizes viral pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs), thereby initiating an innate immune response. RLRs are a crucial class of PRRs, and the antiviral innate immune response they mediate is the most important cytoplasmic antiviral mechanism. RLRs have a high degree of family homology and possess two functionally independent domains: an N-terminal CARD signaling domain and a C-terminal RNA-binding domain. They are primarily composed of RIG-I and MDA5 receptors. After recognizing viral RNA through the RNA-binding domain, RLR receptors utilize CARD-CARD interactions to recruit IPS-1, which contains the CARD signaling domain, to activate the downstream NF-κB pathway and exert a potent antiviral effect.

[0109] Based on the structure and function of the RLR domain, we hypothesized that its dsRNA-binding domain and CARD signaling domain are functionally independent. Since dsRNA is a common feature of viral infection, and long dsRNA can be detected in the cytoplasm of nearly all viruses, with the exception of ssRNA viruses, remodeling the RLR receptor, particularly its dsRNA-binding domain, may improve RLR expression. RLR receptors can recognize viruses and trigger antiviral immune responses, thereby preventing viral immune evasion. We previously successfully engineered an RLR receptor (PKR-dsCARE) with antiviral properties by utilizing the dsRNA-binding domain of human PACT and the CARD signaling domain of Apaf-1. However, the dsRNA-binding proteins of some viruses can interact with human PACT, competitively binding to RNA and evading the natural immune response. The MERS-CoV 4A protein can interact with PACT, competitively binding to RNA, interfering with PACT function, inhibiting MDA5 activity, and evading the immune response. Both the Ebola virus VP35 protein and the herpes simplex virus Us11 protein are RNA-binding proteins that can evade the body's natural antiviral response by preventing RIG-I activation. To enhance the sensitivity and specificity of RLR proteins in blocking viral immune escape, the present invention constructed and expressed a fusion protein, NS3b-Apaf1, consisting of the dsRNA-binding domain of the NS3b protein of the bat coronavirus HKU4-2, linked to the signaling domain of human Apaf-1, which is composed of the CARD.

[0110] To validate the antiviral efficacy of NS3b-Apaf1, we first tested AdV-GFP, a virus widely used in the laboratory that produces large amounts of dsRNA during replication. We investigated the virulence-inhibiting, antiviral, and antiviral activities of NS3b-Apaf1 based on the order of addition of NS3b-Apaf1 and virus. The results showed that the number of cells infected with AdV-GFP decreased with increasing NS3b-Apaf1 protein concentration. FFU quantification also revealed that NS3b-Apaf1 reduced AdV-GFP titers in a dose-dependent manner. Western blot analysis of intracellular GFP expression revealed that GFP expression decreased with increasing NS3b-Apaf1 protein concentration. These results confirm that NS3b-Apaf1 has potent inhibitory, preventive, and antiviral activities against AdV-GFP. Furthermore, we also tested the inhibitory effect of NS3b-Apaf1 against VTT virus and found that increasing NS3b-Apaf1 protein concentration reduced the number of cells infected with VTT-GFP. Cell suspensions were collected and FFU quantified, and it was found that NS3b-Apaf1 protein reduced the titer of VTT-GFP in a dose-dependent manner, confirming the inhibitory effect of NS3b-Apaf1 protein on VTT virus. These results confirm that NS3b-Apaf1 protein has antiviral effects.

[0111] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A fusion protein, characterized in that, The fusion protein comprises the dsRNA binding domain of the NS3b protein of coronavirus HKU4-2 and the structural domain of the CARD signal of human Apaf-1 protein; the structure of the fusion protein NS3b-Apaf is: 6*His-【BamHI】-PTD4–【XhoI】–RBD–【KpnI】–CARD-【HindIII】; the amino acid sequence of the fusion protein NS3b-Apaf1 is shown in SEQ ID NO.

1.

2. A gene encoding the fusion protein according to claim 1, characterized in that, The amino acid sequence of the fusion protein is shown in SEQ ID NO.

1.

3. The method for constructing the gene of the fusion protein NS3b-Apaf1 according to claim 1, characterized in that, The PTD4 sequence shown in SEQ ID NO.4, the RBD sequence of NS3b of coronavirus HKU4-2 shown in SEQ ID NO.6, and the CARD sequence of human Apaf-1 shown in SEQ ID NO.7 are integrated into the vector by double digestion and ligation.

4. An expression vector containing the gene according to claim 2.

5. A recombinant bacterium containing the expression vector according to claim 4.

6. The preparation method of a fusion protein according to claim 1, characterized in that, The fusion protein is prepared according to the following steps: (1) The gene sequence encoding the fusion protein NS3b-Apaf1 of claim 1 is digested with double enzymes and then ligated into the pRSET-B vector to obtain the pRSET-NS3b-Apaf1 recombinant plasmid. (2) The recombinant plasmid obtained in step (1) is transformed into Rosetta gami(DE3) Escherichia coli, spread on an LB plate containing ampicillin and cultured overnight, a single colony on the LB plate is picked for large-scale culture, and sequencing identification is carried out. A matching sequencing sequence indicates that the NS3b-Apaf1 fusion protein expression vector is successfully constructed, and a recombinant bacterium expressing the NS3b-Apaf1 fusion protein is obtained. (3) The recombinant bacterium in step (2) is cultured on a large scale, induced to express by IPTG, the bacterial cells are collected and lysed, the precipitate is collected after centrifugation, and the precipitate is washed, denatured, and renatured to obtain the NS3b-Apaf1 fusion protein.

7. Use of a fusion protein NS3b-Apaf1 as described in claim 1 for the preparation of an antiviral drug against highly pathogenic viruses, characterized in that, The highly pathogenic virus is adenovirus or vaccinia virus.

8. Use of a fusion protein NS3b-Apaf1 as described in claim 1 for the preparation of a vaccine against highly pathogenic viruses, characterized in that, The highly pathogenic virus is adenovirus or vaccinia virus.

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