Antiviral fusion protein and use thereof

Abstract

The present application discloses an antiviral fusion protein. Specifically, the present application provides an antiviral fusion protein, which comprises a cell death inducing domain (lethal domain), a domain inhibiting the activity of the lethal domain (inhibitory domain) and a protease recognition sequence capable of removing or destroying the function of the inhibitory domain; the fusion protein has the activity of inducing cell death after being cut by a specific viral protease, thereby killing the cells infected by the virus in a targeted manner. The present application also discloses a programmable method for inducing the death of virus-infected cells, which comprises transferring the above-mentioned fusion protein or nucleic acid into cells so that the cells die before replication and assembly after being infected by the virus, thereby achieving timely and effective elimination of the virus.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more specifically, to an antiviral fusion protein. Background Technology

[0002] Viruses are characterized by rapid transmission, high pathogenicity, and high mutation rates. To mitigate viral epidemics, better treatment methods are needed. Viral proteases play a crucial role in their replication and transcription processes, making them an attractive target for therapeutic drugs.

[0003] Currently, drug design for proteases mainly focuses on small molecule inhibitors of proteases. For example, for the main protease of the novel coronavirus (SARS-CoV-2 Mpro), there are three main drug development methods: (1) reuse of Mpro inhibitors from homologous viruses, such as studying the potential of SARS-CoV-1 Mpro inhibitors as anti-SARS-CoV-2 drugs; (2) high-throughput screening based on activity: using fluorescence technology to screen the activity of compounds from drug libraries such as marketed drugs, clinical drugs and natural products; (3) virtual screening: using the reported Mpro crystal structure to reasonably narrow the range of active screening compounds.

[0004] However, the above methods still suffer from long development cycles and insufficient effective time, and cannot withstand the rapid mutation of viruses. In addition, there is poor compatibility between the development of targeted drugs for different viral proteases, failing to provide a unified, programmable approach for viral protease drug development.

[0005] Therefore, there is an urgent need in this field to develop a programmable, targeted engineered protein that induces cell death in virus-infected cells, thereby enabling timely and effective clearance of the virus. Summary of the Invention

[0006] The purpose of this invention is to provide a programmable engineered protein that can induce the death of virus-infected cells.

[0007] In a first aspect of the invention, a fusion protein is provided, the fusion protein comprising a lethal domain element, a protease recognition sequence element, and a repressor domain element; the location of the protease recognition sequence element is selected from the group consisting of: between the repressor domain element and the lethal domain element, within the repressor domain element, within the lethal domain element, or at other locations within the fusion protein.

[0008] In another preferred embodiment, the fusion protein has the structure shown in Formula Ia or Formula Ib: A-L1-B-L2-C formula Ia or C-L1-B-L2-A Formula Ib in, A is a lethal structural domain element. B is a protease recognition sequence element. C represents the suppression domain element. L1 and L2 are either non-linked or linked peptide elements. "-" represents the peptide bond that connects the components.

[0009] In another preferred embodiment, the lethal domain can induce cell death.

[0010] In another preferred embodiment, the protease recognition sequence element is capable of being recognized and cleaved by a specific protease.

[0011] In another preferred embodiment, in the absence of the specific protease, the inhibitory domain can inhibit the activity of the lethal domain.

[0012] In another preferred embodiment, the fusion protein is an antiviral fusion protein.

[0013] In another preferred embodiment, the protease recognition sequence element is a viral protease recognition sequence element.

[0014] In another preferred embodiment, the antiviral fusion protein has a structure shown in formula IIa or IIb: A-L1-B-L2-C Formula IIa or C-L1-B-L2-A IIb in, A is a lethal structural domain element. B is a viral protease recognition sequence element. C represents the suppression domain element. L1 and L2 are either non-linked or linked peptide elements. "-" represents the peptide bond that connects the components.

[0015] In another preferred embodiment, the amino acid sequence of the fusion protein is as shown in any one of SEQ ID NO. 14-17.

[0016] In another preferred embodiment, the lethal domain can induce cell death.

[0017] In another preferred embodiment, the viral protease recognition sequence element is capable of being recognized and cleaved by a specific viral protease.

[0018] In another preferred embodiment, in the absence of the specific viral protease, the inhibitory domain can inhibit the activity of the lethal domain.

[0019] In another preferred embodiment, in the presence of the specific viral protease, the fusion protein can be cleaved and acquire cell-killing activity, thereby targeting and killing cells infected by the virus.

[0020] In another preferred embodiment, the viral protease recognition sequence can remove or disrupt the function of the inhibitory domain by enzymatic cleavage, so that the fusion protein can acquire cell-killing activity after being cleaved by a specific viral protease, thereby targeting and killing cells infected by the virus.

[0021] In another preferred embodiment, the lethal domain element includes (but is not limited to) the following group: N-terminal domains of GSDM family proteins, Fas-associated death domain proteins, large and small subunits of Caspase family proteins, defensins, perforin and fragments or variants thereof.

[0022] In another preferred embodiment, the lethal domain element is the N-terminal domain of a GSDM family protein, as well as fragments and variants thereof.

[0023] In another preferred embodiment, the lethal domain element is the N-terminal domain of the GSDMD protein and its fragments and variants.

[0024] In another preferred embodiment, the amino acid sequence of the N-terminal domain of the GSDMD protein is shown in SEQ ID NO.18.

[0025] In another preferred embodiment, the inhibitory domain element includes (but is not limited to) the following group: C-terminal domains of GSDM family proteins, c-FLIP proteins, BIR domains of apoptosis inhibitory factor family proteins, and fragments and variants thereof.

[0026] In another preferred embodiment, the repressive domain element is the C-terminal domain of a GSDM family protein, as well as fragments and variants thereof.

[0027] In another preferred embodiment, the lethal domain element is the C-terminal domain of the GSDMD protein and its fragments and variants.

[0028] In another preferred embodiment, the amino acid sequence of the C-terminal domain of the GSDMD protein is shown in SEQ ID NO.19.

[0029] In another preferred embodiment, the GSDM family protein includes wild-type or mutant GSDMD protein, preferably, the GSDMD protein includes its full length or a fragment thereof.

[0030] In another preferred embodiment, the amino acid sequence of the wild-type GSDMD protein is shown in SEQ ID NO.20, and the polynucleotide encoding it is shown in SEQ ID NO.1.

[0031] In another preferred embodiment, the virus includes (but is not limited to): coronavirus, hepatitis C virus, HIV, dengue virus, human T-cell troponinophil virus, and tobacco mosaic virus.

[0032] In another preferred embodiment, the viral protease recognition sequence element includes (but is not limited to): a coronavirus main protease recognition sequence, a hepatitis C virus protease recognition sequence, an HIV protease recognition sequence, a dengue virus protease recognition sequence, a human T-cell tropic virus protease recognition sequence, and a tobacco mosaic virus protease recognition sequence.

[0033] In another preferred embodiment, the amino acid sequence of the viral protease recognition sequence is as shown in any of SEQ ID NO.21-27.

[0034] In another preferred embodiment, the coronavirus main protease recognition sequence is the Mpro recognition sequence as shown in SEQ ID NO.21.

[0035] In another preferred embodiment, the tobacco mosaic virus protease recognition sequence is the TEVP recognition sequence as shown in SEQ ID NO.22 or 27.

[0036] In another preferred embodiment, the hepatitis C virus protease recognition sequence is the HCVP recognition sequence shown in SEQ ID NO.23.

[0037] In another preferred embodiment, the HIV protease recognition sequence is shown in SEQ ID NO.24.

[0038] In another preferred embodiment, the dengue virus protease recognition sequence is shown in SEQ ID NO.25.

[0039] In another preferred embodiment, the human T-cell viral protease recognition sequence is shown in SEQ ID NO.26.

[0040] In another preferred embodiment, the linker peptide element comprises (but is not limited to) 0-20 identical or different amino acid residues.

[0041] In another preferred embodiment, the linker peptide element is a (GS)n flexible sequence, where n is 1-10, preferably 3-5.

[0042] In a second aspect of the invention, a polynucleotide is provided, the polynucleotide sequence encoding a fusion protein as described in the first aspect of the invention.

[0043] In another preferred embodiment, the fusion protein further includes a modification thereof, which is formed by site-directed PEGylation of the fusion protein.

[0044] In another preferred embodiment, the site-directed PEGylation is performed by PEG modification at the C-terminus of the fusion protein.

[0045] In another preferred embodiment, the fusion protein modifier contains PEG with a molecular weight of 3-40 KD, preferably 5 KD.

[0046] In a third aspect of the invention, a carrier is provided, the carrier containing the polynucleotide as described in the second aspect of the invention.

[0047] In another preferred embodiment, the vector is selected from the group consisting of plasmids, liposomes, and viral vectors.

[0048] In another preferred embodiment, the vector is selected from adenovirus, adeno-associated virus, lentivirus, or a combination thereof.

[0049] In another preferred embodiment, the vector is adeno-associated virus.

[0050] In a fourth aspect of the invention, a host cell is provided, said host cell containing a vector as described in the third aspect of the invention or the genes of said host cell containing polynucleotides as described in the second aspect of the invention.

[0051] In a fifth aspect of the present invention, a method for screening protease inhibitors is provided, comprising the steps of: (a) Providing an engineered cell and delivering a specific protease or its encoded nucleic acid into the engineered cell; (b) Add the reagent to be tested to the cells in (a); (c) Delivering the fusion protein or its encoded nucleic acid as described in the first aspect of the invention into cells (b); (d) Detect cell viability to screen for protease inhibitors.

[0052] In another preferred embodiment, (c), the protein recognition sequence in the fusion protein is inserted between the lethal domain and the inhibitory domain of the cell death-inducing protein, and the protease recognition sequence can be recognized and cleaved by the specific protease, thereby releasing the lethal domain and killing the cell.

[0053] In another preferred embodiment, in (d), if the cell state is detected to be normal or there is essentially no cell death, it indicates that the protease activity is inhibited, preventing it from performing its cleavage function and thus preventing the release of the lethal domain, indicating that the cell state is normal; then the reagent to be tested can be used as a protease inhibitor.

[0054] In another preferred embodiment, in (d), if a large number of cell deaths are detected, it indicates that the protease activity has not been inhibited, allowing it to perform its cleavage function, release the lethal domain, and thus kill the cell; therefore, the reagent to be tested cannot be used as a protease inhibitor.

[0055] In another preferred embodiment, the method further includes step (e) observing cell viability over a certain period of time and quantitatively determining the inhibitory effect of the protease inhibitor on the protease.

[0056] In a sixth aspect of the invention, the use of the fusion protein as described in the first aspect of the invention is provided for the preparation of pharmaceutical compositions for treating tumors.

[0057] In a seventh aspect of the invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising a fusion protein as described in the first aspect of the invention, and a pharmaceutically acceptable carrier.

[0058] In an eighth aspect of the invention, a method for producing a fusion protein as described in the first aspect of the invention is provided, comprising the steps of: (i) Under suitable conditions, host cells as described in the fourth aspect of the present invention are cultured to obtain expression of the fusion protein.

[0059] In another preferred embodiment, the method may further include the steps of: (ii) The obtained fusion protein is isolated and purified.

[0060] In a ninth aspect of the present invention, a method for in vitro non-therapeutic induction of cell death by a virus is provided, comprising the steps of: adding a fusion protein as described in the first aspect of the present invention or a pharmaceutical composition as described in the seventh aspect of the present invention to a virus-infected cell culture, thereby inducing cell death by the virus-infected cells.

[0061] In another preferred embodiment, the virus includes (but is not limited to): coronavirus, hepatitis C virus, HIV, dengue virus, human T-cell troponinophil virus, or tobacco mosaic virus.

[0062] In a tenth aspect of the invention, a method for treating a viral infection is provided, comprising the steps of: administering a safe dose of a fusion protein as described in the first aspect of the invention or a pharmaceutical composition as described in the seventh aspect of the invention to a desired subject, thereby treating the viral infection.

[0063] In another preferred embodiment, the desired object is a mammal, including (but not limited to) a mouse, a rat, or a human, preferably a human.

[0064] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0065] Figure 1 This invention illustrates the mechanism of action of a programmable antiviral engineered protein and its encoded nucleic acid in an embodiment of the present invention. After viral infection of cells, a protease is translated from RNA, which recognizes and cleaves specific protein sequences, playing a crucial role in viral growth and proliferation. The designed antiviral fusion protein includes a lethal domain, an inhibitory domain, a viral protease recognition sequence, and linker peptides (the latter two are located between the lethal and inhibitory domains in the figure). After the fusion protein or its encoded nucleic acid is delivered into eukaryotic cells via liposomes, cells infected with the corresponding virus, containing the protease that cleaves the recognition sequence, can cleave the protein and release the lethal domain. This directly or indirectly leads to cell membrane perforation, an imbalance in the intracellular and extracellular ion concentration gradient and osmotic pressure, and the release of other cell death signals, ultimately achieving rapid cell death in the early stages of viral infection, thereby effectively blocking viral proliferation and transmission. Uninfected cells do not contain the corresponding protease and are therefore unaffected.

[0066] Figure 2 The image shows the cell morphology results observed under a bright field optical microscope after 293 cells were transfected with different plasmids in the embodiments of the present invention. In the image, Mpro represents the plasmid group containing the gene encoding the main protease of the SARS-CoV-2 virus; Mprot represents the plasmid group containing the gene encoding GSDMDN-Mprot-GSDMDC, where Mprot is the recognition sequence of Mpro; and N represents the plasmid group containing the N-terminal sequence (lethal domain) of GSDMD.

[0067] Figure 3 The diagram shows the cell viability results of 293 cells transfected with different plasmids in this embodiment of the invention, as detected by CCK-8 assay. In this diagram, GFP represents the plasmid group containing the gene encoding green fluorescent protein (irrelevant plasmids used to detect transfection rate and DNA toxicity); N represents the plasmid group containing the N-terminal sequence (lethal domain) of GSDMD; TEVP represents the plasmid group containing the gene encoding tobacco mosaic virus protease; TEVPt represents the plasmid group containing the gene encoding GSDMDN-TEVPt-GSDMDC, where TEVPt is the recognition sequence for TEVP; Mpro represents the plasmid group containing the gene encoding the main protease of SARS-CoV-2; and Mprot represents the plasmid group containing the gene encoding GSDMDN-Mprot-GSDMDC, where Mprot is the recognition sequence for Mpro.

[0068] Figure 4This image shows the Western blot results of GSDMD cleavage mediated by the recognition and cleavage sequence of the tobacco mosaic virus protease (TEV Protease) in an embodiment of the present invention. In the image, "-" indicates the control group transfected only with the plasmid encoding 2×Flag-HA-bct3 (a GSDMD fusion protein containing the TEV Protease recognition sequence); "+" indicates the experimental group transfected with both the 2×Flag-HA-bct3 plasmid and the TEV protease expression plasmid; 2×flag-HA-bct3 is the full-length fusion protein band; 2×flag-HA-N is the N-terminal lethal domain fragment released after TEV protease cleavage; and β-tubulin is the internal control for sample loading. The results showed that, compared with the control group, the full-length 2×flag-HA-bct3 protein disappeared and a 2×flag-HA-N cleavage fragment appeared in the experimental group, confirming that the TEV protease can effectively recognize and cleave the fusion protein inserted into the TEVPt sequence, release the N-terminal lethal domain of GSDMD, and thus induce pyroptosis. This result was corroborated by the cell viability test data, proving that the cell death mediated by GSDMD cleavage sequence recognized by the tobacco mosaic virus protease is indeed caused by cleavage by the tobacco mosaic virus protease. Detailed Implementation

[0069] Through extensive and in-depth research, the inventors have, for the first time, prepared an antiviral fusion protein. This fusion protein comprises a lethal domain, a repressor domain, and a viral protease recognition sequence. The lethal domain induces cell death, the repressor domain inhibits the activity of the lethal domain under normal conditions, and the viral protease recognition sequence can remove or disrupt the function of the repressor domain through enzymatic cleavage. This allows the fusion protein to acquire cell-killing activity upon cleavage by a specific viral protease, thereby targeting and killing cells infected by the virus. Based on this, the present invention was completed.

[0070] Specifically, this invention utilizes the high activity and high specificity of viral proteases to design antiviral proteins with inserted viral protease recognition sequences. Upon delivery to virus-infected cells, these proteins are rapidly cleaved by viral proteases, releasing lethal domains and directly causing rapid cell death in the early stages of viral infection. This achieves precise killing, thereby inhibiting further viral spread within the host and effectively treating viral diseases.

[0071] Overcoming the limitations of traditional viral protease-targeted drug design, this drug eliminates the need for large-scale screening compared to small molecule inhibitors, shortening the drug development cycle. It also overcomes the problem of rapid viral mutation by utilizing the conservation of viral protease recognition sequences, enabling highly efficient and sustainable treatment of viral diseases.

[0072] Furthermore, the method provided by this invention is highly programmable, and the recognition and cleavage sequences of different types of viral proteases can be inserted into the protein to form a targeted drug for the virus. It has a wide range of applications, is simple and easy to operate, and is convenient and efficient.

[0073] Fusion proteins and their preparation In this invention, the terms "recombinant fusion protein," "inventory protein," "inventory fusion protein," "fusion protein," and "engineered protein" are used interchangeably, referring to a fusion protein having the structure described in formula Ia or Ib of the first aspect of this invention, i.e., containing a fusion protein including a lethal domain element, a viral protease recognition sequence element, and an inhibitory domain element. The protein of this invention can be a monomer or an oligomer or polymer formed from monomers. Furthermore, it should be understood that the terms also include the active fragment and derivatives of the fusion protein.

[0074] As used herein, the term “comprising” or its variations such as “including” or “comprises” are understood to include the said element or component without excluding the presence of other elements or other components.

[0075] As used herein, the terms “lethal domain” and “lethal domain” are used interchangeably, and the lethal domain can induce cell death; the elements of the lethal domain include (but are not limited to): N-terminal domains of GSDM family proteins, DED domains of Fas-associated death domain proteins, large and small subunits of Caspase family proteins, defensins, perforin, etc., and fragments and variants thereof.

[0076] As used herein, the terms “inhibition domain” and “inhibition domain” are used interchangeably, and the inhibition domain can inhibit the activity of the lethal domain under normal conditions without viral infection; the inhibition domain elements include (but are not limited to): C-terminal domains of GSDM family proteins, DED domains of c-FLIP proteins, BIR domains of apoptosis inhibitory factor family proteins, etc., and fragments and variants thereof.

[0077] As used herein, the terms "viral protease recognition sequence" and "viral protease recognition site" are used interchangeably. The viral protease recognition sequence can remove or disrupt the function of the inhibitory domain by enzymatic cleavage, so that the fusion protein can acquire the activity of inducing cell death after being cleaved by a specific viral protease, thereby targeting and killing cells infected by the virus.

[0078] The viral protease recognition sequence elements include (but are not limited to): coronavirus main protease recognition sequence, hepatitis C virus protease recognition sequence, HIV protease recognition sequence, dengue virus protease recognition sequence, human T-cell tropic virus protease recognition sequence, and tobacco mosaic virus protease recognition sequence.

[0079] This invention also includes active fragments, derivatives, and analogs of fusion proteins according to the invention. As used herein, the terms “fragment,” “derivative,” and “analyte” refer to a protein or polypeptide that substantially retains its protein function or activity. The protein or polypeptide fragments, derivatives, or analogs of the invention may be (i) proteins or polypeptides with partial amino acid residue deletions but still functional, (ii) proteins or polypeptides with one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) substituted, (iii) proteins or polypeptides having substituted or modified groups in one or more amino acid residues, (iv) proteins or polypeptides formed by fusing a fusion protein with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol), (v) proteins or polypeptides formed by fusing an additional amino acid sequence to this protein sequence (e.g., fusion proteins formed by fusing with a leader sequence, secretion sequence, or tag sequence such as 6His), and (vi) proteins whose amino acid sequences are not highly similar to this protein sequence but form similar spatial structures. Based on the teachings herein, these fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0080] A preferred class of active derivatives refers to proteins or polypeptides formed by replacing one or more amino acids with similar or related properties compared to the amino acid sequence of Formula Ia or Formula Ib. These conserved variant polypeptides are preferably produced by amino acid substitutions according to Table 1.

[0081] Table 1 The present invention also provides analogs of the fusion proteins of the present invention. These analogs may differ from any of the proteins or peptides shown in SEQ ID NO. 14-17 in that they may differ in amino acid sequence, in the form of modifications that do not affect the sequence, or both. Analogs also include those having residues different from native L-amino acids (such as D-amino acids), and those having non-naturally occurring or synthetic amino acids (such as β-, γ-amino acids). It should be understood that the proteins or peptides of the present invention are not limited to the representative proteins or peptides exemplified above.

[0082] Modifications (typically without altering the primary structure) include chemically derived forms of proteins or peptides, such as acetylation or carboxylation, either in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications during peptide synthesis and processing or further processing steps. This modification can be accomplished by exposing the protein or peptide to glycosylating enzymes (such as mammalian glycosylation or deglycosylation enzymes). Modifications also include sequences containing phosphorylated amino acid residues (such as phosphotyrosine, phosphotyserine, phosphotythreonine). Modifications also include proteins or peptides modified to improve their resistance to protease hydrolysis or optimize their solubility.

[0083] The proteins or polypeptides of this invention can also be used in the form of salts derived from pharmaceutically or physiologically acceptable acids or bases. These salts include (but are not limited to) salts formed with the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, citric acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, succinic acid, oxalic acid, fumaric acid, maleic acid, oxaloacetic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, or hydroxyethanesulfonic acid. Other salts include salts formed with alkali metals or alkaline earth metals (such as sodium, potassium, calcium, or magnesium), and salts in the form of esters, carbamates, or other conventional "prodrugs."

[0084] The polynucleotides of this invention can be in DNA or RNA form. DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. DNA can be single-stranded or double-stranded. Single-stranded DNA can be a coding strand or a non-coding strand.

[0085] This invention also relates to variants of the aforementioned polynucleotides that encode protein fragments, analogs, and derivatives having the same amino acid sequence as those of this invention. These polynucleotide variants can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be the substitution, deletion, or insertion of one or more nucleotides, but does not substantially alter the function of the encoded polypeptide.

[0086] As used herein, the term "primer" refers to a general term for oligonucleotides that, when paired with a template, can be used by DNA polymerase to synthesize a DNA strand complementary to the template. Primers can be natural RNA, DNA, or any form of natural nucleotide. Primers can even be non-natural nucleotides such as LNA or ZNA. A primer is "probably" (or "substantially") complementary to a specific sequence on one strand of the template. A primer must be fully complementary to one strand of the template to begin elongation, but the primer sequence does not need to be perfectly complementary to the template sequence. For example, adding a non-complementary sequence to the 5' end of a primer that is complementary to the template at the 3' end will still result in a primer that is probably complementary to the template. As long as the primer is long enough to bind sufficiently to the template, even a partially complementary primer can form a primer-template complex with the template, thereby enabling amplification.

[0087] The full-length nucleotide sequence or fragment thereof of the fusion protein or its elements of the present invention can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis. For PCR amplification, primers can be designed based on publicly available nucleotide sequences, especially open reading frame sequences, and commercially available cDNA libraries or cDNA libraries prepared using conventional methods known to those skilled in the art are used as templates to amplify the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the fragments amplified from each amplification in the correct order.

[0088] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transferring it into cells, and then isolating the sequence from the proliferated host cells using conventional methods.

[0089] In addition, sequences can be synthesized artificially, especially when the fragment length is short. Typically, long sequences can be obtained by first synthesizing multiple small fragments and then joining them.

[0090] The method of amplifying DNA / RNA using PCR technology is preferred for obtaining the gene of the present invention. Primers used for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein and can be synthesized using conventional methods. The amplified DNA / RNA fragments can be separated and purified using conventional methods such as gel electrophoresis.

[0091] The present invention also relates to vectors containing the polynucleotides of the present invention, host cells genetically engineered using the vectors or fusion protein coding sequences of the present invention, and methods for generating the proteins of the present invention via recombinant technology.

[0092] Using conventional recombinant DNA techniques, the polynucleotide sequence of this invention can be used to express or produce recombinant proteins. Generally, the following steps are involved: (1) Transform or transduce suitable host cells using the polynucleotide (or variant) encoding the protein of the present invention, or using a recombinant expression vector containing the polynucleotide; (2) Host cells cultured in a suitable culture medium; (3) Isolate and purify proteins from culture media or cells.

[0093] Methods well known to those skilled in the art can be used to construct expression vectors containing the coding DNA sequence of the protein of this invention and suitable transcription / translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, etc. The DNA sequence can be efficiently ligated to an appropriate promoter in the expression vector to guide mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0094] In addition, the expression vector preferably contains one or more selective marker genes to provide phenotypic traits for selecting host cells for transformation, such as dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.

[0095] Vectors containing the appropriate DNA sequence and appropriate promoter or control sequence can be used to transform appropriate host cells so that they can express proteins.

[0096] The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: bacterial cells of Escherichia coli and Streptomyces; fungal cells such as yeast; plant cells; insect cells of Drosophila S2 or Sf9; and animal cells of CHO, NSO, COS7, or 293 cells.

[0097] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as *E. coli*, competent cells capable of uptake DNA can be harvested after the exponential growth phase and treated with CaCl2, the steps of which are well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, polyethyleneimine packaging, etc.

[0098] The obtained transformants can be cultured using conventional methods to express the protein or polypeptide encoded by the gene of this invention. Depending on the host cells used, the culture medium can be selected from various conventional media. Culture is carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature adjustment or chemical induction), and the cells are cultured for a further period.

[0099] The proteins described in the above methods may be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, proteins can be separated and purified using various separation methods based on their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0100] Lethal Domain This invention provides a fusion protein containing a lethal domain. A lethal domain is a protein element that can be released after enzymatic cleavage of the fusion protein and induce rapid cell death in monomeric or polymeric form. Its mechanisms of inducing cell death include (but are not limited to) directly or indirectly causing plasma membrane perforation, releasing inflammatory factors to attract the immune system, blocking essential cell growth pathways, and activating downstream cell death signaling pathways. Preferably, the N-terminal domain of a GSDM family protein can serve as a lethal domain. After enzymatic cleavage and release, it forms oligomers, perforates the plasma membrane, causes an imbalance in the ion gradient and osmotic pressure across the membrane, and releases other cell death signals, thereby inducing rapid cell death.

[0101] Suppression domain This invention provides a fusion protein containing a repressor domain. A repressor domain is a protein element that, under normal conditions, binds to a lethal domain and inhibits its function, thereby maintaining normal cellular function. When the lethal domain is removed by enzymatic cleavage or its function is disrupted, it is released and restores normal function. Preferably, the C-terminal domain of a GSDM family protein can serve as a repressor domain, binding to and inhibiting the function of the N-terminal domain (lethal domain); after the C-terminal domain is removed by enzymatic cleavage or disrupted, the N-terminal domain is released and rapidly induces cell death.

[0102] Viral protease recognition sequence This invention provides a fusion protein containing a viral protease recognition sequence (hereinafter referred to as the "recognition sequence"). The recognition sequence is a short peptide that viral proteases can efficiently recognize and cleave, typically 5-10 amino acids in length, and most often 7-8 amino acids. It is highly conserved and rarely mutates during viral mutation. It is also highly programmable, capable of being replaced with recognition sequences from various different viral proteases, thereby enabling the rapid preparation of multiple fusion proteins resistant to different viruses.

[0103] GSDMD GSDMD is a cell death-inducing protein. The wild-type GSDMD is 484 amino acids long and consists of two domains. Its nucleotide sequence is shown in SEQ ID NO.1, and its amino acid sequence is shown in SEQ ID NO.120. The 31 kDa N-terminal domain (1-275 aa, of which 1-241 aa is functional) and the 22 kDa C-terminal domain (276-484 aa, of which 284-484 aa is functional) are the preferred lethal and inhibitory domains, respectively. They are separated by a linker (242-283 aa containing a protease recognition sequence). Under normal conditions, the first flexible loop of the C-terminal domain is located between the N-terminal domain and the linker, extending outwards and inserting into the pocket of the N-terminal domain, stabilizing the full-length protein conformation and simultaneously inhibiting the N-terminus function. When the C-terminus is removed (the N-terminus and C-terminus can be separated after the linker is cleaved by a protease) or inactivated, the N-terminus is released and forms a large transmembrane pore composed of 31-34 subunits, driving rapid cell death.

[0104] SARS-CoV-2 main protease (Mpro) The main protease of SARS-CoV-2 is a cysteine ​​protease, also known as C30 endopeptidase, 3-chymotrypsin, etc. It is the main protease found in coronaviruses and plays an important role in the processing of coronavirus replication polyprotein (POC6U8). It can recognize and cleave coronavirus polyprotein at 11 conserved sites, with the most efficient recognition sequence being AVLQ|SGFR (SEQ ID NO.21), where "|" represents the cleaved peptide bond.

[0105] Tobacco mosaic virus protease (TEV Protease) Tobacco mosaic virus protease is a highly sequence-specific cysteine ​​protease derived from tobacco etching virus and a member of the coagulase-like protease PA family. Due to its high sequence specificity, it is commonly used for controlled cleavage of fusion proteins in vitro and in vivo. Its most efficient recognition sequence is ENLYFQ|G / S (where ENLYFQG is SEQ ID NO. 22 and ENLYFQS is SEQ ID NO. 27), where "|" represents the cleaved peptide bond.

[0106] Hepatitis C virus protease (HCVP) The hepatitis C virus protease used in this invention is a protein complex composed of NS3 serine protease and the cofactor NS4A peptide linked by non-covalent bonds. NS3 serine protease is a key enzyme in the processing and maturation of HCV non-structural proteins; it is inactive when alone, but undergoes conformational rearrangement upon binding to NS4A peptide, becoming a functional protease that plays a crucial role in the hydrolytic processing of viral polymers. Its most efficient recognition sequence is EVVT|STWV (SEQ ID NO. 23), where "|" represents a cleaved peptide bond.

[0107] Linking peptides This invention provides a fusion protein that optionally contains a linker peptide (peptide linker). The size and complexity of the linker peptide can affect the activity of the protein. Generally, the linker peptide should have sufficient length and flexibility to ensure that the two linked proteins have sufficient spatial freedom to perform their functions. At the same time, the formation of α-helices or β-sheets in the linker peptide should be avoided to prevent the formation of these structures that could negatively impact the stability of the fusion protein.

[0108] The length of linker peptides is generally 0-20 amino acids, with (GS)n being preferred, where n=1-10.

[0109] Pharmaceutical Composition and Administration The present invention also provides a composition comprising an effective amount of the fusion protein of the present invention and a pharmaceutically acceptable carrier. Typically, the fusion protein of the present invention can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5-8, preferably about 6-8.

[0110] As used herein, the term “effective amount” or “effective dose” means an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals, such as 0.001-99 wt%; preferably 0.01-95 wt%; more preferably 0.1-90 wt%.

[0111] As used herein, a "pharmaceuticalally acceptable" ingredient is a substance suitable for human and / or mammalian use without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a substance with a reasonable benefit / risk ratio. The term "pharmaceuticalally acceptable carrier" refers to a carrier used for the administration of therapeutic agents, including various excipients and diluents.

[0112] The pharmaceutical compositions of the present invention contain a safe and effective amount of the fusion protein of the present invention and a pharmaceutically acceptable carrier. Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. Generally, the pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared using conventional methods with physiological saline or an aqueous solution containing glucose and other excipients. The pharmaceutical compositions are preferably manufactured under aseptic conditions. The dosage of the active ingredient is a therapeutically effective amount. The pharmaceutical formulations of the present invention can also be formulated into sustained-release formulations.

[0113] The effective amount of the fusion protein of this invention can vary depending on the administration method and the severity of the disease to be treated. The preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: the pharmacokinetic parameters of the fusion protein of this invention, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration. For cancer patients, generally, satisfactory results are obtained when the fusion protein of this invention is administered daily at a dose of approximately 0.5 mg to 5 mg / kg animal body weight (preferably 2 mg to 4 mg / kg animal body weight). For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be reduced proportionally.

[0114] The main beneficial effects of this invention include: (1) This invention utilizes the high activity and high specificity of viral proteases to design antiviral proteins with inserted viral protease recognition sequences. After being delivered to virus-infected cells, these proteins are rapidly cleaved by viral proteases, releasing lethal domains and directly causing rapid cell death in the early stages of viral infection. This achieves precise killing, thereby inhibiting the further spread of the virus in the host and effectively treating viral diseases.

[0115] (2) This invention breaks through the limitations of the original viral protease targeted drug design. Compared with small molecule inhibitor drugs, it does not require large-scale screening, shortens the drug development cycle, and overcomes the problem of rapid viral mutation by utilizing the conservation of viral protease recognition sequence, thus achieving efficient and sustainable treatment of viral diseases.

[0116] (3) The method provided by the present invention is also highly programmable. The recognition and cleavage sequences of different types of viral proteases can be inserted into the protein to form the target drug of the virus. It has a wide range of applications, is simple and easy to operate, and is convenient and efficient.

[0117] (4) This invention can be applied to specific viral disease treatment scenarios in the future. For example, for a future unknown epidemic virus, as long as preliminary experiments reveal that it possesses a protease and a recognition sequence for the protease, the recognition sequence can be inserted between the lethal and inhibitory domains according to the method proposed in this invention to form a new antiviral fusion protein drug. This drug can be delivered to the lesion via liposome delivery or other targeted delivery methods (such as plasmids or AAV viral vectors), enabling rapid death of target cells in the early stages of viral infection, while simultaneously providing effective protection for normal cells and inhibiting viral replication and spread. Based on the high efficiency, conservation, and programmability of the method provided by this invention, rapid production of fusion protein drugs capable of resisting viral mutations can be achieved for various different viruses. The fusion protein of this invention can exert beneficial effects in the treatment of various viral infections.

[0118] (5) The fusion protein or nucleic acid provided by the present invention is transferred into the cell, so that the cell contains the fusion protein. The engineered protein is cleaved by the protease specifically expressed by the virus in the infected cell, thereby activating its lethal activity, so that the target cell dies before the virus replicates and assembles after infection, thus achieving timely and effective clearance of the virus.

[0119] (6) This invention can also be applied to the screening of protease inhibitors. For example, to test the efficiency of an inhibitor against a specific protease, the recognition sequence of the protease can be inserted between the lethal domain and the inhibitory domain to form a fusion protein. After delivering a protease (or its encoding nucleic acid) to a specific cell line (such as the 293 cell line), the inhibitor to be tested is added to the system. After sufficient interaction, the fusion protein (or its encoding nucleic acid) is delivered to the cells. If the protease activity is sufficiently inhibited by the inhibitor, it cannot perform its cleavage function, preventing the release of the lethal domain, and the cell remains in a normal state. Conversely, if the protease retains some activity, it can cleave its recognition sequence, releasing the lethal domain and causing cell death. By observing the cell viability over a certain period of time, the inhibitory effect on the protease can be quantitatively determined. This method is simple and efficient and can be widely applied to the screening of various protease inhibitors.

[0120] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0121] Example 1 This embodiment verifies at the cell experiment level that GSDMD containing the SARS-CoV-2 main protease (Mpro) recognition and cleavage sequence can be induced to undergo cell death by the SARS-CoV-2 main protease. The specific process includes the following steps: (1) Search for genes on NCBI Homo sapiens The coding exon sequence of gasdermin D (GSDMD) (SEQ ID NO.1) was used as the basis for PCR amplification of the gene from a human cDNA library using primers designed at both ends (SEQ ID NO.2 and SEQ ID NO.3). The human cDNA library was obtained by extracting total RNA from 293 cells and reverse transcribing it.

[0122] (2) For the pBR322-CMV plasmid vector, PCR amplification primers (SEQ ID NO.4 and SEQ ID NO.5) were designed to amplify the linear backbone of the vector. The F primer included the 18 bp homologous arm sequence at the 3' end of GSDMD and a segment of the plasmid vector sequence after the insertion position, and the R primer included the 18 bp homologous arm sequence at the 5' end of GSDMD and a segment of the plasmid vector sequence before the insertion position.

[0123] (3) After PCR is completed, electrophoresis is performed and the gene product is recovered by gel cutting.

[0124] (4) The GSDMD gene was integrated into the vector backbone by homologous recombination to form the recombinant plasmid pBR322-CMV-GSDMD. The homologous recombination system was as follows: 2 μL of vector linear backbone, 1.5 μL of GSDMD gene, 1 μL of CEⅡ buffer, and 0.5 μL of ExnaseⅡ. The mixture was incubated at 37℃ for 30 minutes.

[0125] (5) Mix 5 μL of the product from (3) with 50 μL of pre-thawed Escherichia coli glycerol culture, incubate on ice for 20 minutes, heat shock at 42°C for 45 seconds, incubate on ice for another 2 minutes, then add 500 μL of antibiotic-free LB liquid medium, shake on a shaker at 200 rpm for 30-60 minutes at 37°C, then take 100 μL of the culture and spread it evenly on an LB plate with 50 μg / mL kanamycin, and incubate at 37°C for 14-16 hours.

[0126] (6) Pick positive colonies and add them to centrifuge tubes containing 5-10 mL of LB-anti-broth medium. Incubate at 37°C with shaking for 14-16 h. Whether a colony is positive can be determined by colony PCR.

[0127] (7) Extract plasmids using a plasmid extraction kit. After sequencing confirms that the plasmids are correct, the GSDMD master plasmid is obtained.

[0128] (8) Design primers (SEQ ID NO.6 and SEQ ID NO.7) for circular PCR, replacing the bases (bases 799-840) of the Caspase-recognized cleavage sequence in the GSDMD protein with the bases corresponding to the recognition and cleavage sequence of the SARS-CoV-2 main protease. The designed F primer includes the base sequence corresponding to the protease recognition and cleavage sequence and the 21bp parent plasmid sequence after the deletion sequence. The R primer includes the base sequence corresponding to the protease recognition and cleavage sequence and the 21bp parent plasmid sequence before the deletion sequence. There is a 24bp complementary region between the two primers, which allows the DNA to spontaneously ligate into a circular shape after transformation. To maintain the normal structure of the GSDMD protein and the binding strength between the C-terminus and N-terminus, a certain length of (GS)n flexible sequence bases corresponding to the Mpro recognition sequence can also be added on both sides.

[0129] (9) After PCR amplification, the residual GSDMD master plasmid in the Dpn1 degradation system is used to improve the purity of the recombinant plasmid.

[0130] (10) The gel was cut, the gel was recovered, the transformation was plated, single clones were picked, and the plasmid was extracted again to obtain the GSDMD plasmid pBR322-CMV-GSDMDN-Mprot-GSDMDC with protease recognition and cleavage sequence recombination.

[0131] (11) Design primers (SEQ ID NO.8 and SEQ ID NO.9) to perform circular PCR and synthesize plasmid pBR322-CMV-GSDMDN containing only the N-terminus (lethal domain) of GSDMD but not the C-terminus (inhibitory domain) using methods similar to (8)-(10).

[0132] (12) The nucleotide sequence of the main protease Mpro of the novel coronavirus was synthesized by overlap PCR and integrated into the plasmid backbone by the methods described in (2)-(7) to become pBR322-CMV-Mpro plasmid.

[0133] Overlap PCR is a method for de novo synthesis of DNA fragments. The target sequence is split into primer fragments of approximately 60 bp in length, with about 10 bp overlap between each other. These fragments are then annealed and joined into a long strand through a PCR reaction. A second round of PCR is then performed using head and tail primers to ensure the correct strand length.

[0134] (13) The obtained plasmid is transformed and amplified again, then endotoxin is removed and plasmid is extracted to remove cytotoxic substances such as lipopolysaccharide. If there is concern about insufficient plasmid quantity, large-scale plasmid extraction can be performed. The plasmid is diluted to 100 ng / μL for subsequent operations.

[0135] (14) Cellular experimental verification. 293 cells were diluted with complete culture medium to 5×10⁻⁶.5 After adding the plasmid at a rate of 1 / mL to each well of a 96-well plate (120 μL per well), transient plasmid transfection was performed.

[0136] Transfection reagent A: 5 μL OPTI + 0.6 μL PEI Solution B: The experimental group consisted of 5 μL OPTI + 30 ng Mpro + 20 ng GSDMDN-Mprot-GSDMDC. Negative control group 1 consisted of 5 μL OPTI + 30 ng Mpro. Negative control group 2 consisted of 5 μL OPTI + 20 ng GSDMDDN-Mprot-GSDMDC. The positive control group consisted of 5 μL OPTI + 20 ng GSDMDDN.

[0137] Each group should have at least 3 parallel replicates.

[0138] Mix solutions A and B and incubate at room temperature for 20 minutes. Then, add the mixture to each well of a 96-well plate, gently stirring with a pipette tip to ensure thorough mixing. Gently drag the 96-well plate horizontally in all directions and observe under a microscope until the cells in each well are evenly distributed at the bottom. Place the 96-well plate in a 37°C cell culture incubator containing 5% CO2. After 24 hours, remove the 96-well plate and examine the cell death status of each group under an optical microscope. Cell viability is quantitatively determined using a CCK8 assay kit (Sangon Biotech, product number E606335).

[0139] Experimental results: Observation results under bright field microscopy ( Figure 2 The results showed that when only Mpro was transfected, the cells maintained a basically normal morphology; when only GSDMDN-Mprot-GSDMDC (Mprot) was transfected, the cell morphology and activity were affected to some extent but not significantly, indicating that the engineered protein was not highly cytotoxic; when Mpro and GSDMDN-Mprot-GSDMDC (Mpro+Mprot) were transfected simultaneously or when GSDMDN (lethal domain, N) was transfected, the cells showed very obvious morphological changes, including a significant decrease in cell density, separation or oligo-aggregation into cell clusters, swelling and enlargement of some cells and the production of many bubble-like protrusions, exhibiting obvious characteristics of pyroptosis. Therefore, Mpro has the ability to cleave the engineered protein GSDMDN-Mprot-GSDMDC and produces a significant cell death effect.

[0140] Detected by CCK-8 ( Figure 3Similar conclusions can be drawn from this study. Groups transfected with irrelevant plasmids (such as GFP) or Mpro exhibited high cell viability. The cell viability of the group transfected with GSDMDN-Mprot-GSDMDC (MproT) decreased slightly (possibly due to oligomerization caused by accidental proximity of a few proteins in the cytoplasm), but still maintained high activity. In contrast, the cell viability of the group simultaneously transfected with Mpro and GSDMDN-Mprot-GSDMDC (Mpro+MproT) was significantly reduced, remaining at only about 10%, almost identical to the positive control group. This indicates that Mpro, after expression in cells, can efficiently cleave the engineered protein GSDMDN-Mprot-GSDMDC and rapidly induce cell death.

[0141] Example 2 This embodiment verifies at the cell experiment level that GSDMD containing the tobacco mosaic virus protease (TEV Protease) recognition and cleavage sequence can be induced to undergo cell death by the tobacco mosaic virus protease. The specific process includes the following steps: Using the same operating procedure as in Example 1, in step (8), the primers for the sequence insertion step were replaced with SEQ ID NO.10 and SEQ ID NO.11 to obtain the recombinant plasmid pBR322-CMV-GSDMDN-TEVPt-GSDMDC.

[0142] Experimental results: Detected by CCK-8 ( Figure 3 The engineered protein GSDMDN-TEVPt-GSDMDC exhibited stronger cleavage-induced cell death ability and lower cytotoxicity than that in Example 1. The GSDMDN-TEVPt-GSDMDC transfected group (TEVPt) showed a smaller decrease in cell viability compared to the negative control group, maintaining relatively higher activity. Simultaneously, the group transfected with both TEVP and GSDMDN-TEVPt-GSDMDC (TEVP+TEVPt) also showed a more significant decrease in cell viability, even lower than the positive control group (GSDMDN (lethal domain) transfected group, GFP+N), demonstrating stronger drug-forming ability and virus clearance effect.

[0143] Example 3 This embodiment verifies at the biochemical level that GSDMD-mediated cell death, which incorporates a tobacco mosaic virus protease (TEV Protease) recognition and cleavage sequence, is specifically triggered by TEV Protease cleavage. The specific process includes the following steps: Using the same procedure as in Example 2, the bct3 (linker-optimized TEVPt, SEQ ID NO. 16) gene was cloned into a plasmid containing a 2×Flag-HA double tag, resulting in pBR322-CMV-2×Flag-HA-GSDMDN-TEVPt-GSDMDC. HEK293T cells were seeded in 6-well plates (2×10⁻⁶ cells / wells). 5 Cells were cultured for 24 hours until confluence reached 70-80%. Then, they were transfected with polyethyleneimine (PEI). In the control group, each well was transfected with 100 ng of 2×Flag-HA-bct3 plasmid alone. In the experimental group, each well was transfected with a co-transfectation of 100 ng of 2×Flag-HA-bct3 plasmid and an appropriate amount of TEV protease expression plasmid (PEI to total DNA mass ratio of 3:1). After 6 hours of transfection, the culture medium was replaced with complete medium and cultured for another 24-48 hours to allow for sufficient TEV protease expression and substrate cleavage. Cells were collected, washed with PBS, and lysed on ice for 30 minutes with lysis buffer containing 1% Triton X-100 and protease inhibitor. The cells were centrifuged at 12,000×g for 10 minutes, and the supernatant was collected and boiled at 95°C for 5 minutes with SDS-PAGE loading buffer. 20-30 μg of protein was subjected to 10% SDS-PAGE electrophoresis, transferred to a PVDF membrane, and blocked with 5% skim milk powder for 1 h. The membrane was then incubated successively with anti-Flag or anti-HA primary antibody (4°C overnight, 1:1000) and HRP-labeled secondary antibody (room temperature 1 h, 1:5000). After washing with TBST, the membrane was developed using ECL chemiluminescence. Finally, the membrane was peeled off, reblocked, and incubated with β-tubulin antibody (1:5000) as an internal control for further development. The cleavage efficiency of TEV protease on 2×Flag-HA-bct3 was analyzed.

[0144] Development results ( Figure 4 The results showed that, compared with the control group, after TEV cleavage, 2xflag-HA-bct3 (full-length protein) disappeared and 2xflag-HA-N fragment (N-terminal fragment after cleavage) appeared. At the same time, the cell viability test results were consistent with those in Example 2, proving that the GSDMD-mediated cell death mediated by the insertion of the tobacco mosaic virus protease recognition cleavage sequence was specifically caused by tobacco mosaic virus protease cleavage.

[0145] The sequence involved in this invention is as follows: SEQ ID NO.1: Full-length nucleotide sequence of GSDMD SEQ ID NO.2: GSDMD primer-F ATGGGGTCGGCCTTTGAGCG SEQ ID NO.3: GSDMD primer-R CTAGTGGGGCTCCTGGCTCAGTC SEQ ID NO.4: Circular PCR primer-F AGCCAGGAGCCCCACTAGTCTGGCGGCTCAAAAAGA SEQ ID NO.5: Circular PCR primer-R CTCAAAGGCCGACCCCATGGTGGCGGCTCTCCCTAT SEQ ID NO.6: Mpro replacement primer-F GCCGTGCTGCAGAGCGGCTTCAGAGGGGCGTTCACTGAAGACTTC SEQ ID NO.7: Mpro replacement primer-R TCTGAAGCCGCTCTGCAGCACGGCCATCATGGAGAGGCCAGAGGG SEQ ID NO.8: GSDMD N-terminal primer-F TAGTCTGGCGGCTCAAAAAGAACCGCC SEQ ID NO.9: GSDMD N-terminal primer-R TTTTGAGCCGCCAGACTATCTGAAGCCGCTCTGCAG SEQ ID NO.10: TEVP replacement primer-F ATGGAGAACTTGTACTTCCAAGGCGGGGCGTTCACTGAAGAC SEQ ID NO.11: TEVP replacement primer-R CCCGCCTTGGAAGTACAAGTTCTCCATCATGGAGAGGCCAGAGG SEQ ID NO.12: HCVP replacement primer-F GAGGTGGTGACCAGCACCTGGGTGGGGGCGTTCACTGAAGACTTC SEQ ID NO.13: HCVP Replacement Primer-R CACCCAGGTGCTGGTCACCACCTCCATCATGGAGAGGCCAGAGGG SEQ ID NO.14: Amino acid sequence of GSDMDN-Mprot-GSDMDC MGSAFERVVRRVVQELDHGGEFIPVTSLQSSTGFQPYCLVVRKPSSSWFWKPRYKCVNLSIKDILEPDAAEPDVQRGRSFHFYDAMDGQIQGSVELAAPGQAKIAGGAAVSDSSSTSMNVYSLSVDPNTWQTLLHERHLRQPEHKVLQQLRSRGDNVYVVTEVLQTQKEVEVTRTHKREGSGRFSLPGATCLQGEGQGHLSQKKTVTIPSGSTLAFRVAQLVIDSDLDVLLFPDKKQRTFQPPATGHKRSTSEGAWPQLPSGLSMMKTSAVLQSGFRKMEGAFTEDFQGLRAEVETISKELELLDRELCQLLLEGLEGVLRDQLALRALEEALEQGQSLGPVEPLDGPAGAVLECLVLSSGMLVPELAIPVVYLLGALTMLSETQHKLLAEALESQTLLGPLELVGSLLEQSAPWQERSTMSLPPGLLGNSWGEGAPAWVLLDECGLELGEDTPHVCWEPQAQGRMCALYASLALLSGLSQEPH SEQ ID NO.15: Amino acid sequence of GSDMDN-TEVPt-GSDMDC MGSAFERVVRRVVQELDHGGEFIPVTSLQSSTGFQPYCLVVRKPSSSWFWKPRYKCVNLSIKDILEPDAAEPDVQRGRSFHFYDAMDGQIQGSVELAAPGQAKIAGGAAVSDSSSTSMNVYSLSVDPNTWQTLLHERHLRQPEHKVLQQLRSRGDNVYVVTEVLQTQKEVEVTRTHKREGSGRFSLPGATCLQGEGQGHLSQKKTVTIPSGSTLAFRVAQLVIDSDLDVLLFPDKKQRTFQPPATGHKRSTSEGAWPQLPSGLSMMKTSENLYFQGKMEGAFTEDFQGLRAEVETISKELELLDRELCQLLLEGLEGVLRDQLALRALEEALEQGQSLGPVEPLDGPAGAVLECLVLSSGMLVPELAIPVVYLLGALTMLSETQHKLLAEALESQTLLGPLELVGSLLEQSAPWQERSTMSLPPGLLGNSWGEGAPAWVLLDECGLELGEDTPHVCWEPQAQGRMCALYASLALLSGLSQEPH SEQ ID NO.16: Amino acid sequence of GSDMDN-bct3-GSDMDC MGSAFERVVRRVVQELDHGGEFIPVTSLQSSTGFQPYCLVVRKPSSSWFWKPRYKCVNLSIKDILEPDAAEPDVQRGRSFHFYDAMDGQIQGSVELAAPGQAKIAGGAAVSDSSSTSMNVYSLSVDPNTWQTLLHERHLRQPEHKVLQQLRSRGDNVYVVTEVLQTQKEVEVTRTHKREGSGRFSLPGATCLQGEGQGHLSQKKTVTIPSGSTLAFRVAQLVIDSDLDVLLFPDKKQRTFQPPATGGGSGGSGGSENLYFQGGGSGGSGGSLPSGLSMMRCLHNFAAAAVPAEGAFTEDFQGLRAEVETISKELELLDRELCQLLLEGLEGVLRDQLALRALEEALEQGQSLGPVEPLDGPAGAVLECLVLSSGMLVPELAIPVVYLLGALTMLSETQHKLLAEALESQTLLGPLELVGSLLEQSAPWQERSTMSLPPGLLGNSWGEGAPAWVLLDECGLELGEDTPHVCWEPQAQGRMCALYASLALLSGLSQEPH SEQ ID NO.17: Amino acid sequence of GSDMDN-HCVPt-GSDMDC MGSAFERVVRRVVQELDHGGEFIPVTSLQSSTGFQPYCLVVRKPSSSWFWKPRYKCVNLSIKDILEPDAAEPDVQRGRSFHFYDAMDGQIQGSVELAAPGQAKIAGGAAVSDSSSTSMNVYSLSVDPNTWQTLLHERHLRQPEHKVLQQLRSRGDNVYVVTEVLQTQKEVEVTRTHKREGSGRFSLPGATCLQGEGQGHLSQKKTVTIPSGSTLAFRVAQLVIDSDLDVLLFPDKKQRTFQPPATGHKRSTSEGAWPQLPSGLSMMEVVTSTWVGAFTEDFQGLRAEVETISKELELLDRELCQLLLEGLEGVLRDQLALRALEEALEQGQSLGPVEPLDGPAGAVLECLVLSSGMLVPELAIPVVYLLGALTMLSETQHKLLAEALESQTLLGPLELVGSLLEQSAPWQERSTMSLPPGLLGNSWGEGAPAWVLLDECGLELGEDTPHVCWEPQAQGRMCALYASLALLSGLSQEPH SEQ ID NO.18: Amino acid sequence of the lethal domain GSDMD N MGSAFERVVRRVVQELDHGGEFIPVTSLQSSTGFQPYCLVVRKPSSSWFWKPRYKCVNLSIKDILEPDAAEPDVQRGRSFHFYDAMDGQIQGSVELAAPGQAKIAGGAAVSDSSSTSMNVYSLSVDPNTWQTLLHERHLRQPEHKVLQQLRSRGDNVYVVTEVLQTQKEVEVTRTHKREGSGRFSLPGATCLQGEGQGHLSQKKTVTIPSGSTLAFRVAQLVIDSDLDVLLFPDKKQRTFQPPATGHKRSTSEGAWPQLPSGLSMMRCLHNFLTD SEQ ID NO.19: Amino acid sequence of the inhibitory domain GSDMD C GVPAEGAFTEDFQGLRAEVETISKELELLDRELCQLLLEGLEGVLRDQLALRALEEALEQGQSLGPVEPLDGPAGAVLECLVLSSGMLVPELAIPVVYLLGALTMLSETQHKLLAEALESQTLLGPLELLVGSLLEQSAPWQERSTMSLPPGLLGNSWGEGAPAWVLLDECGLELGEDTPHVCWEPQAQGRMCALYASLALLSGLSQEPH SEQ ID NO.20: Amino acid sequence of wild-type GSDMD protein MGSAFERVVRRVVQELDHGGEFIPVTSLQSSTGFQPYCLVVRKPSSSWFWKPRYKCVNLSIKDILEPDAAEPDVQRGRSFHFYDAMDGQIQGSVELAAPGQAKIAGGAAVSDSSSTSMNVY SLSVDPNTWQTLLHERHLRQPEHKVLQQLRSRGDNVYVVTEVLQTQKEVEVTRTHKREGSGFSLPGATCLQGEGQGHLSQKKTVTIPSGSTLAFRVAQLVIDSDLDVLLFPDKKQRTFQP PATGHKRSTSEGAWPQLPSGLSMMRCLHNFLTDGVPAEGAFTEDFQGLRAEVETISKELELLDRELCQLLLEGLEGVLRDQLALRALEEALEQGQSLGPVEPLDGPAGAVLECLVLSSGML VPELAIPVVYLLGALTMLSETQHKLLAEALESQTLLGPLELVGSLLEQSAPWQERSTMSLPPGLLGNSWGEGAPAWVLLDECGLELGEDTPHVCWEPQAQGRMCALYASLALLSGLSQEPH SEQ ID NO.21: (Amino acid sequence of the Mprot viral protease recognition element) AVLQSGFR SEQ ID NO.22: (Amino acid sequence of the TEVPt viral protease recognition element) ENLYFQG SEQ ID NO.23: (Amino acid sequence of HCVPt viral protease recognition element) EVVTSTWV SEQ ID NO.24: (Amino acid sequence of HIV protease recognition element) ATIMMQRG SEQ ID NO.25: (Amino acid sequence of dengue virus protease recognition element) TGKRSGAL SEQ ID NO.26: (Amino acid sequence of human T-cell viral protease recognition element) PQVLPVMH SEQ ID NO.27: (Amino acid sequence of the TEVPt viral protease recognition element) ENLYFQS All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A fusion protein, characterized in that, The fusion protein comprises a lethal domain element, a protease recognition sequence element, and a repressor domain element; the location of the protease recognition sequence element is selected from the group consisting of: between the repressor domain element and the lethal domain element, within the repressor domain element, within the lethal domain element, or at other locations within the fusion protein.

2. The fusion protein as described in claim 1, characterized in that, The fusion protein is an antiviral fusion protein, and the antiviral fusion protein has a structure shown in formula IIa or formula IIb: A-L1-B-L2-C Formula IIa or C-L1-B-L2-A IIb in, A is a lethal structural domain element. B is a viral protease recognition sequence element. C represents the suppression domain element. L1 and L2 are either non-linked or linked peptide elements. "-" represents the peptide bond that connects the components.

3. The fusion protein as described in claim 1, characterized in that, The amino acid sequence of the fusion protein is shown in any one of SEQ ID NO. 14-17.

4. The fusion protein as described in claim 2, characterized in that, The viral protease recognition sequence can remove or disrupt the function of the inhibitory domain by enzymatic cleavage, enabling the fusion protein to acquire cell-killing activity after being cleaved by a specific viral protease, thereby targeting and killing cells infected by the virus.

5. The fusion protein as described in claim 2, characterized in that, The lethal domain element is the N-terminal domain of a GSDM family protein, its fragments, and variants; the repressive domain element is the C-terminal domain of a GSDM family protein, its fragments, and variants.

6. The fusion protein as described in claim 2, characterized in that, The viruses mentioned include: coronavirus, hepatitis C virus, HIV, dengue virus, human T-cell troponinogen virus, and tobacco mosaic virus.

7. The fusion protein as described in claim 2, characterized in that, The amino acid sequence of the viral protease recognition sequence is as shown in any of SEQ ID NO.21-27.

8. A polynucleotide, characterized in that, The polynucleotide sequence encodes the fusion protein as described in claim 1.

9. A carrier, characterized in that, The carrier contains the polynucleotide as described in claim 8.

10. A method for screening protease inhibitors, characterized in that, Including the following steps: (a) Providing an engineered cell and delivering a specific protease or its encoded nucleic acid into the engineered cell; (b) Add the reagent to be tested to the cells in (a); (c) Delivery into (b) cells The fusion protein of claim 1 or its encoded nucleic acid; (d) Detect cell viability to screen for protease inhibitors.

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