Reagent derived from deep sea cold spring and used for enhancing virus infection resistance of prokaryotic microorganisms and application of reagent

Abstract

The invention belongs to an industrial biotechnology, and discloses a deep sea cold spring-sourced reagent for enhancing the virus infection resistance of prokaryotic microorganisms and application of the reagent. The nucleic acid molecules and / or protein molecules contained in the reagent for enhancing the virus infection resistance of the prokaryotic microorganisms are / is obtained by being separated from a cold spring sediment sample or obtained through artificial preparation based on biological information of the cold spring sediment sample; by introducing the nucleic acid molecule into prokaryotic microorganisms such as escherichia coli and / or enabling the prokaryotic microorganisms to express corresponding protein molecules, the anti-infection ability of the prokaryotic microorganisms to viruses such as T7 bacteriophage, T5 bacteriophage and T4 bacteriophage can be effectively enhanced; the method is of great significance to reveal antiviral strategies of life bodies in extreme environments and development of novel biotechnology tools.

Description

Technical Field

[0001] This invention belongs to industrial biotechnology, and particularly relates to reagents derived from deep-sea cold seeps for enhancing the antiviral infection capabilities of prokaryotic microorganisms and their applications. Background Technology

[0002] Deep-sea cold seeps are unique submarine geological phenomena formed by the upwelling of methane-rich fluids. They are rich in hydrogen sulfide, methane, and other hydrocarbons, and exhibit typical extreme characteristics such as high pressure, low temperature, and oxygen deficiency. As one of Earth's important extreme ecosystems, cold seep sediments contain a wealth of unique microbial and viral resources. Studies have shown that cold seep microbial communities differ significantly from those in terrestrial or surface marine environments. Through long-term adaptive evolution, they have developed rich genetic resources and unique defense mechanisms. These defense mechanisms not only affect the survival of individual microorganisms but also play a crucial role in maintaining the stability of the cold seep ecosystem and biogeochemical cycles (such as methane oxidation, sulfate reduction, and nitrogen fixation).

[0003] In nature, prokaryotes have evolved diverse antiviral defense systems, including restriction-modification (RM) systems, CRISPR-Cas systems, BREX, DISARM, Gabija, and CBASS. These systems prevent viral infection and replication by recognizing, cleaving, or interfering with viral nucleic acids, and by inducing host suicide, thus achieving antiviral effects. Microorganisms in deep-sea cold seep environments, subjected to extreme conditions of high pressure, low temperature, and hypoxia, may exhibit significant differences in gene composition, mechanism of action, and regulatory mechanisms compared to systems in conventional environments. These unique defense systems may not only contain novel antiviral elements but may also function through specific signal transduction pathways or metabolic coupling mechanisms, which is of great significance for expanding human understanding of life's adaptability and developing new biotechnological tools. However, most of the defense systems that have been systematically studied and experimentally verified so far are concentrated on model strains or human-related environments, such as Escherichia coli, soil prokaryotic microorganisms, or gut prokaryotic microbiota. For the large number of candidate defense systems existing in extreme environments such as deep-sea cold seeps, due to the difficulty of sampling and experimental conditions, the research on antiviral systems of prokaryotes in deep-sea cold seeps is still at the stage of genome prediction, lacking functional verification and mechanism analysis.

[0004] Therefore, obtaining sequences from deep-sea cold seep prokaryotes that truly possess antiviral defense capabilities is of great significance for revealing the antiviral strategies of organisms in extreme environments and for the development of novel biotechnological tools. Summary of the Invention

[0005] The primary objective of this invention is to provide a reagent for enhancing the antiviral infection resistance of prokaryotic microorganisms. The nucleic acid molecules and / or protein molecules included in this reagent are isolated from cold seep sediment samples or artificially prepared based on the biological information of cold seep sediment samples. Introducing the nucleic acid molecules into prokaryotic microorganisms such as Escherichia coli and / or causing the aforementioned prokaryotic microorganisms to express corresponding protein molecules can effectively enhance the anti-infection resistance of prokaryotic microorganisms against bacteriophages such as T7, T5, and T4. This is of great significance for revealing the antiviral strategies of organisms in extreme environments and for the development of novel biotechnological tools.

[0006] A second objective of this invention is to provide an expression box.

[0007] A third objective of this invention is to provide a method for enhancing the antiviral infection resistance of prokaryotic microorganisms for purposes other than disease treatment and disease diagnosis.

[0008] A fourth objective of this invention is to provide the application of the above-mentioned reagents or expression cassettes in industrial biotechnology.

[0009] The fifth objective of this invention is to provide an antibacterial preparation.

[0010] Specifically, the reagent provided by the present invention for enhancing the antiviral infection ability of prokaryotic microorganisms includes at least one of the following substances: (1) one or more nucleic acid molecules with sequences as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5; (2) one or more protein molecules with amino acid sequences as shown in SEQ ID NO:6 or having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:6, protein molecules with amino acid sequences as shown in SEQ ID NO:7 or having at least 95% sequence identity with the sequence shown in SEQ ID NO:7, protein molecules with amino acid sequences as shown in SEQ ID NO:8 or having at least 95% sequence identity with the sequence shown in SEQ ID NO:8, protein molecules with amino acid sequences as shown in SEQ ID NO:9 or having at least 95% sequence identity with the sequence shown in SEQ ID NO:9, and protein molecules with amino acid sequences as shown in SEQ ID NO:10 or having at least 95% sequence identity with the sequence shown in SEQ ID NO:10; (3) one or more protein molecules encoding amino acid sequences as shown in SEQ ID NO:6 and / or those with SEQ ID NO:5. (3) Nucleic acid molecules that encode protein molecules with at least 95% sequence identity as shown in SEQ ID NO:6; (4) Nucleic acid molecules that encode protein molecules with amino acid sequences as shown in SEQ ID NO:7 and / or with at least 95% sequence identity as shown in SEQ ID NO:7; (5) Nucleic acid molecules that encode protein molecules with amino acid sequences as shown in SEQ ID NO:8 and / or with at least 95% sequence identity as shown in SEQ ID NO:8; (6) Nucleic acid molecules that encode protein molecules with amino acid sequences as shown in SEQ ID NO:9 and / or with at least 95% sequence identity as shown in SEQ ID NO:9; (7) Nucleic acid molecules that encode protein molecules with amino acid sequences as shown in SEQ ID NO:10 and / or with at least 95% sequence identity as shown in SEQ ID NO:10.

[0011] Furthermore, the protein molecule with an amino acid sequence as shown in SEQ ID NO:6 or having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:6 has at least one of the following technical features: (1) the protein molecule contains the Virulence_RhuM functional domain; (2) the protein molecule has biological activity that inhibits viral replication.

[0012] Furthermore, a protein molecule with an amino acid sequence as shown in SEQ ID NO:7 or having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:7 has at least one of the following technical features: (1) the protein molecule contains a nucleic acid binding domain; (2) the protein molecule has biological activity that blocks viral nucleic acid synthesis.

[0013] Furthermore, a protein molecule with an amino acid sequence as shown in SEQ ID NO:8 or having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:8 has at least one of the following technical features: (1) the protein molecule contains ArsR-like_HTH and NT_KNTase_like functional domains; (2) the protein molecule has biological activity that regulates the transcription process.

[0014] Furthermore, a protein molecule with an amino acid sequence as shown in SEQ ID NO:9 or having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:9 has the following technical features: (1) the protein molecule contains a Hepn domain; (2) the protein molecule has biological activity that regulates the transcription process.

[0015] Furthermore, a protein molecule with an amino acid sequence as shown in SEQ ID NO:10 or having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:10 has at least one of the following technical features: (1) the protein molecule contains the AAA_21 functional domain; (2) the protein molecule has biological activity that regulates the ATP hydrolysis process.

[0016] Furthermore, the reagent has at least one of the following technical features: (1) the reagent is obtained from cold seep sediment samples; (2) the reagent is artificially prepared based on biological information of nucleic acid molecules and / or protein molecules.

[0017] The expression cassette provided by the present invention includes at least one of the following nucleic acid molecules: (1) one or more nucleic acid molecules with sequences as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5; (2) a nucleic acid molecule encoding a protein molecule with an amino acid sequence as shown in SEQ ID NO:6 and / or having at least 95% sequence identity with the sequence shown in SEQ ID NO:6; (3) a nucleic acid molecule encoding a protein molecule with an amino acid sequence as shown in SEQ ID NO:7 and / or having at least 95% sequence identity with the sequence shown in SEQ ID NO:7; (4) a nucleic acid molecule encoding a protein molecule with an amino acid sequence as shown in SEQ ID NO:8 and / or having at least 95% sequence identity with the sequence shown in SEQ ID NO:8; (5) a nucleic acid molecule encoding a protein molecule with an amino acid sequence as shown in SEQ ID NO:9 and / or having at least 95% sequence identity with the sequence shown in SEQ ID NO:9; and (6) a nucleic acid molecule encoding a protein molecule with an amino acid sequence as shown in SEQ ID NO:10 and / or having at least 95% sequence identity with the sequence shown in SEQ ID NO:10.

[0018] Furthermore, the expression cassette is selected from one or more of linear DNA, plasmid vectors, mRNA, and circular RNA.

[0019] The method for enhancing the antiviral infection ability of prokaryotic microorganisms for non-disease treatment and disease diagnosis purposes provided by the present invention includes: introducing the above-mentioned expression cassette into prokaryotic microorganisms to enhance their antiviral infection ability.

[0020] Furthermore, the method includes at least one of the following technical features: (1) the prokaryotic microorganism is Escherichia coli; (2) the virus is selected from one or more of T7 phage, T5 phage and T4 phage.

[0021] The above-mentioned reagents or expression cassettes provided by the present invention are applied in industrial biotechnology, which utilizes prokaryotic microorganisms to produce products.

[0022] Furthermore, the industrial biotechnology is selected from one or more of the following: food and fermentation industry, enzyme preparation and biocatalysis industry, pharmaceutical and fine chemical industry, bioenergy and bio-based materials industry, and environmental governance.

[0023] The antibacterial preparation provided by the present invention comprises at least one of the following technical features: (1) a substance for inhibiting the expression of a protein molecule with an amino acid sequence as shown in SEQ ID NO:6 and / or having at least 95% sequence identity with the sequence shown in SEQ ID NO:6; (2) a substance for inhibiting the biological activity of a protein molecule with an amino acid sequence as shown in SEQ ID NO:6 and / or having at least 95% sequence identity with the sequence shown in SEQ ID NO:6; (3) a substance for inhibiting the expression of a protein molecule with an amino acid sequence as shown in SEQ ID NO:7 and / or having at least 95% sequence identity with the sequence shown in SEQ ID NO:7; (4) a substance for inhibiting the biological activity of a protein molecule with an amino acid sequence as shown in SEQ ID NO:7 and / or having at least 95% sequence identity with the sequence shown in SEQ ID NO:7; (5) a substance for inhibiting the expression of a protein molecule with an amino acid sequence as shown in SEQ ID NO:8 and / or having at least 95% sequence identity with the sequence shown in SEQ ID NO:6; (6) a substance for inhibiting the expression of a protein molecule with an amino acid sequence as shown in SEQ ID NO:8 and / or having at least 95% sequence identity with the sequence shown in SEQ ID NO:6. (7) A substance for inhibiting the expression of a protein molecule whose amino acid sequence is at least 95% identical to that shown in SEQ ID NO:9; (8) A substance for inhibiting the expression of a protein molecule whose amino acid sequence is at least 95% identical to that shown in SEQ ID NO:9; (9) A substance for inhibiting the expression of a protein molecule whose amino acid sequence is at least 95% identical to that shown in SEQ ID NO:9; (10) A substance for inhibiting the expression of a protein molecule whose amino acid sequence is at least 95% identical to that shown in SEQ ID NO:10; Attached Figure Description

[0024] Figure 1 This is one of the plaque experiment results provided in Example 2 of the present invention (T7 phage); Figure 2 This is the second image of the plaque experiment results provided in Example 2 of this invention (T5 phage); Figure 3 The third image shows the plaque experiment results provided in Example 2 of this invention (T4 phage).

[0025] Figure 4 This is a diagram showing the results of PDC-S05 protein domain annotation, structure prediction, and alignment provided in Example 3 of this invention. Figure 5 This is a diagram showing the results of PDC-S70 protein domain annotation, structure prediction, and alignment provided in Example 4 of this invention. Figure 6 This is a diagram showing the results of domain annotation, structure prediction, and alignment of PDC-M05A and PDC-M05B proteins provided in Example 5 of this invention. Figure 7 The figure shows the results of HEC-01 protein domain annotation, structure prediction, and alignment provided in Example 6 of this invention. Detailed Implementation

[0026] The inventors of this invention, aiming to overcome the challenges of unproven candidate defense system functions and lack of application translation in existing cold seep defense systems, used deep-sea cold seep sediment samples as research objects. Employing a combination of metagenomics mining and bioinformatics analysis, they performed high-throughput sequencing, short sequence assembly, open reading frame (ORF) prediction, functional annotation, and classification on DNA samples extracted from deep-sea cold seep sediment samples to screen for candidate deep-sea cold seep defense genes with potential antiviral functions. Furthermore, they verified the bioactivity and predicted the structure and function of the obtained candidate deep-sea cold seep defense genes, ultimately creatively obtaining five deep-sea cold seep defense genes that indeed possess antiviral activity. Based on this, the technical solution of this invention was obtained.

[0027] Based on the antiviral infection activity of the deep-sea cold seep defense genes obtained through screening, this invention provides a reagent for enhancing the antiviral infection ability of prokaryotic microorganisms.

[0028] In this invention, the reagent uses deep-sea cold seep defense genes and their encoded proteins as the core substances for enhancing the antiviral infection ability of prokaryotic microorganisms, specifically including at least one of the following nucleic acid molecules and / or protein molecules: (1) The deep-sea cold seep defense gene PDC-S05, which includes the nucleic acid fragment shown in SEQ ID NO:1, and / or the PDC-S05 protein molecule encoded by the deep-sea cold seep defense gene PDC-S05, wherein the amino acid sequence of the PDC-S05 protein molecule is shown in SEQ ID NO:6, and the PDC-S05 protein molecule has a Virulence_RhuM functional domain, which can achieve anti-infection effect by inhibiting the replication of viruses in prokaryotic microbial cells; (2) A variant of the PDC-S05 protein molecule that has at least 95% sequence identity with the PDC-S05 protein molecule, wherein the PDC-S05 protein molecule variant has one or more amino acid residues added, deleted and / or substituted compared to the PDC-S05 protein molecule, and the addition, deletion and / or substitution of amino acid residues does not change the Virulence_RhuM functional domain of the original protein molecule, and has a slightly inferior, equivalent or superior biological activity of inhibiting viral replication to the PDC-S05 protein molecule; (3) The deep-sea cold seep defense gene PDC-S70, which includes the nucleic acid fragment shown in SEQ ID NO:2, and / or the PDC-S70 protein molecule encoded by the deep-sea cold seep defense gene PDC-S70, wherein the amino acid sequence of the PDC-S70 protein molecule is shown in SEQ ID NO:7, and the PDC-S70 protein molecule has a nucleic acid binding domain, which can achieve anti-infection effect by inhibiting the nucleic acid synthesis of the virus in prokaryotic microbial cells; (4) A variant of the PDC-S70 protein molecule that has at least 95% sequence identity with the PDC-S70 protein molecule, wherein the PDC-S70 protein molecule variant has one or more amino acid residues added, deleted and / or substituted compared to the PDC-S70 protein molecule, and the addition, deletion and / or substitution of amino acid residues does not change the nucleic acid binding domain of the original protein molecule, and has a biological activity of blocking viral nucleic acid synthesis that is slightly inferior to, equivalent to or superior to that of the PDC-S70 protein molecule; (5) The deep-sea cold seep defense system PDC-M05 (containing two genes, PDC-M05A and PDC-M05B), including the nucleic acid fragments shown in SEQ ID NO:3 and SEQ ID NO:4, and / or the PDC-M05A and PDC-M05B protein molecules encoded by the deep-sea cold seep defense genes PDC-M05A and PDC-M05B, wherein the amino acid sequence of the PDC-M05A protein molecule is shown in SEQ ID NO:8, and the amino acid sequence of the PDC-M05B protein molecule is shown in SEQ ID NO:9. The PDC-M05A protein molecule has ArsR-like_HTH and NT_KNTase_like functional domains, and the PDC-M05B protein molecule has a Hepn domain. The combined effect of the two can affect the cell state by regulating the transcription process in prokaryotic microbial cells to achieve an anti-infection effect. (6) PDC-M05A and PDC-M05B protein molecule variants that have at least 95% sequence identity with the PDC-M05A and PDC-M05B protein molecules, wherein the PDC-M05A and PDC-M05B protein molecule variants have one or more amino acid residues added, deleted and / or substituted compared to the PDC-M05A and PDC-M05B protein molecules, and the added, deleted and / or substituted amino acid residues do not change the functional domains of the original protein molecules, and have biological activities that are slightly inferior to, equivalent to or superior to those of the original protein molecules in regulating transcription. (7) The deep-sea cold seep defense gene HEC-01, which includes the nucleic acid fragment shown in SEQ ID NO:5, and / or the HEC-01 protein molecule encoded by the deep-sea cold seep defense gene HEC-01, wherein the amino acid sequence of the HEC-01 protein molecule is shown in SEQ ID NO:10, and the HEC-01 protein molecule has the AAA_21 functional domain, which can affect the cell state by regulating the ATP hydrolysis process in prokaryotic microbial cells to achieve anti-infection effect; (8) A variant of the PHEC-01 protein molecule that has at least 95% sequence identity with the HEC-01 protein molecule, wherein the HEC-01 protein molecule variant has one or more amino acid residues added, deleted and / or substituted compared to the HEC-01 protein molecule, and the addition, deletion and / or substitution of amino acid residues does not change the AAA_21 functional domain of the original protein molecule, and has biological activity that is slightly inferior to, equivalent to or superior to that of the HEC-01 protein molecule in regulating the ATP hydrolysis process.

[0029] In this invention, the reagent enhances the antiviral infection ability of prokaryotic microorganisms by introducing the aforementioned nucleic acid molecules into the prokaryotic microorganisms, thereby enabling the prokaryotic microorganisms to express corresponding protein molecules or protein variants, thus achieving the effect of enhanced antiviral infection ability. Since different prokaryotic microorganisms have different codon preferences, in order to enable the prokaryotic microorganisms to better express proteins and achieve a more ideal enhanced antiviral infection ability, the reagent may further include at least one of the following nucleic acid molecules: (1) A nucleic acid molecule encoding a PDC-S05 protein molecule and / or a variant of the PDC-S05 protein molecule, wherein the nucleotide sequence of the nucleic acid molecule is determined based on the amino acid sequence of the PDC-S05 protein molecule and / or the variant of the PDC-S05 protein molecule and the prokaryotic microorganism to be introduced, and the present invention does not particularly limit it. (2) A nucleic acid molecule encoding a PDC-S70 protein molecule and / or a variant of the PDC-S70 protein molecule, wherein the nucleotide sequence of the nucleic acid molecule is determined based on the amino acid sequence of the PDC-S70 protein molecule and / or the variant of the PDC-S70 protein molecule and the prokaryotic microorganism to be introduced, and the present invention does not impose any particular limitation thereon. (3) Nucleic acid molecules encoding PDC-M05A protein molecules and PDC-M05B protein molecules and / or variants of PDC-M05A protein molecules and variants of PDC-M05B protein molecules, wherein the nucleotide sequence of the nucleic acid molecules is determined based on the amino acid sequence of PDC-M05A protein molecules and PDC-M05B protein molecules and / or variants of PDC-M05A protein molecules and the prokaryotic microorganisms to be introduced, and the present invention does not impose any particular limitation thereon; (4) A nucleic acid molecule encoding the HEC-01 protein molecule and / or a variant of the HEC-01 protein molecule, wherein the nucleotide sequence of the nucleic acid molecule is determined based on the amino acid sequence of the HEC-01 protein molecule and / or the variant of the HEC-01 protein molecule and the prokaryotic microorganism to be introduced, and the present invention does not particularly limit it.

[0030] In some specific embodiments, the reagent enhances the antiviral infection ability of prokaryotic microorganisms by specifically enabling the prokaryotic microorganisms to express PDC-S05 protein molecules and / or PDC-S05 protein molecule variants, thereby enhancing the anti-infection ability by inhibiting viral replication within the prokaryotic microorganisms. In a specific experimental example, by genetically modifying prokaryotic microorganisms to express PDC-S05 protein molecules and / or PDC-S05 protein molecule variants, the effect achieved was that the plaque formation efficiency (EOP) of prokaryotic microorganisms against T7 and T5 bacteriophages was reduced by approximately 1000-fold, and the EOP against T4 bacteriophage was reduced by approximately 10-fold.

[0031] In some specific embodiments, the reagent enhances the antiviral infection ability of prokaryotic microorganisms by specifically enabling the prokaryotic microorganisms to express PDC-S70 protein molecules and / or PDC-S70 protein molecule variants. This inhibits viral replication within the prokaryotic microorganisms by blocking viral nucleic acid synthesis, thereby enhancing their anti-infection ability. In a specific experimental example, by genetically modifying prokaryotic microorganisms to express PDC-S70 protein molecules and / or PDC-S70 protein molecule variants, the results show that the EOP of prokaryotic microorganisms against T5 phage is reduced by approximately 1000-fold, and the EOP against T7 and T4 phages is reduced by approximately 10-fold.

[0032] In some specific embodiments, the reagent enhances the antiviral infection ability of prokaryotic microorganisms by simultaneously expressing PDC-M05A and PDC-M05B protein molecules and / or simultaneously expressing variants of PDC-M05A and PDC-M05B protein molecules. This alters intracellular protein expression by regulating the transcriptional process of the prokaryotic microorganisms, thereby fully mobilizing the expression of other defense genes within the prokaryotic microorganisms and adjusting them to enter a state with stronger antiviral infection resistance. In a specific experimental example, by genetically modifying prokaryotic microorganisms to express PDC-M05 protein molecules and / or PDC-M05 protein variants, the effect achieved was a reduction in the EOP of prokaryotic T4 bacteriophage by approximately 10-fold.

[0033] In some specific embodiments, the reagent enhances the antiviral infection ability of prokaryotic microorganisms by specifically enabling prokaryotic microorganisms to express HEC-01 protein molecules and / or HEC-01 protein molecule variants. This is achieved by regulating the intracellular ATP hydrolysis process of the prokaryotic microorganisms, thereby adjusting them to a state with stronger antiviral infection resistance. In a specific experimental example, by genetically modifying prokaryotic microorganisms to express HEC-01 protein molecules and / or HEC-01 protein molecule variants, the effect achieved was a approximately 10-fold reduction in the EOP (excessive occlusion) of prokaryotic microorganisms against T4 phage.

[0034] In some specific embodiments, when the reagent enhances the antiviral infection ability of prokaryotic microorganisms, it can specifically cause the prokaryotic microorganisms to simultaneously express "PDC-S05 protein molecules and / or PDC-S05 protein molecule variants" and "PDC-S70 protein molecules and / or PDC-S70 protein molecule variants". By blocking the viral nucleic acid synthesis process and inhibiting the synthesis of related substances during viral replication, the reagent works together to achieve a more ideal effect of enhancing the antiviral infection ability of prokaryotic microorganisms.

[0035] In some specific embodiments, when the reagent enhances the antiviral infection ability of prokaryotic microorganisms, it can specifically cause the prokaryotic microorganisms to simultaneously express "PDC-S05 protein molecules and / or PDC-S05 protein molecule variants" and "PDC-M05A protein molecules and PDC-M05B protein molecules and / or PDC-M05A protein molecule variants and PDC-M05B protein molecule variants". By inhibiting viral replication and changing the protein expression in prokaryotic microorganisms, the reagent works together to achieve a more ideal effect of enhancing the antiviral infection ability of prokaryotic microorganisms.

[0036] In some specific embodiments, when the reagent enhances the antiviral infection ability of prokaryotic microorganisms, it can specifically enable prokaryotic microorganisms to simultaneously express "PDC-S05 protein molecules and / or PDC-S05 protein molecule variants" and "HEC-01 protein molecules and / or HEC-01 protein molecule variants". By inhibiting viral replication and regulating the ATP hydrolysis process, the expression of other defense genes in the cell is fully mobilized, and they work together to achieve a more ideal effect of enhancing the antiviral infection ability of prokaryotic microorganisms.

[0037] In some specific embodiments, when the reagent enhances the antiviral infection ability of prokaryotic microorganisms, it can specifically cause the prokaryotic microorganisms to simultaneously express "PDC-S70 protein molecules and / or PDC-S70 protein molecule variants" and "PDC-M05A protein molecules and PDC-M05B protein molecules and / or PDC-M05A protein molecule variants and PDC-M05B protein molecule variants". By blocking viral nucleic acid synthesis to inhibit viral replication and changing the protein expression in prokaryotic microorganisms, the reagent works together to achieve a more ideal effect of enhancing the antiviral infection ability of prokaryotic microorganisms.

[0038] In some specific embodiments, when the reagent enhances the antiviral infection ability of prokaryotic microorganisms, it can specifically enable prokaryotic microorganisms to simultaneously express "PDC-S70 protein molecules and / or PDC-S70 protein molecule variants" and "HEC-01 protein molecules and / or HEC-01 protein molecule variants". By blocking viral nucleic acid synthesis to inhibit viral replication and regulating ATP hydrolysis to fully mobilize the expression of other defense genes in the cell, the reagent works together to achieve a more ideal effect of enhancing the antiviral infection ability of prokaryotic microorganisms.

[0039] In some specific embodiments, when the reagent enhances the antiviral infection ability of prokaryotic microorganisms, it can specifically cause the prokaryotic microorganisms to simultaneously express "PDC-M05A protein molecules and PDC-M05B protein molecules and / or PDC-M05A protein molecule variants and PDC-M05B protein molecule variants" and "HEC-01 protein molecules and / or HEC-01 protein molecule variants". By changing the protein expression status in the prokaryotic microorganisms and regulating the ATP hydrolysis process, the state of the prokaryotic microorganisms is changed to fully mobilize the expression of other defense genes in the cell, so as to achieve a more ideal effect of enhancing the antiviral infection ability of prokaryotic microorganisms.

[0040] In some specific embodiments, when the reagent enhances the antiviral infection ability of prokaryotic microorganisms, it can specifically cause the prokaryotic microorganisms to simultaneously express "PDC-S05 protein molecules and / or PDC-S05 protein molecule variants", "PDC-S70 protein molecules and / or PDC-S70 protein molecule variants" and "PDC-M05A protein molecules and PDC-M05B protein molecules and / or PDC-M05A protein molecule variants and PDC-M05B protein molecule variants". By blocking viral replication and fully mobilizing the expression of other defense genes in prokaryotic microbial cells, the reagent works together to achieve a more ideal effect of enhancing the antiviral infection ability of prokaryotic microorganisms.

[0041] In some specific embodiments, when the reagent enhances the antiviral infection ability of prokaryotic microorganisms, it can specifically cause the prokaryotic microorganisms to simultaneously express "PDC-S05 protein molecules and / or PDC-S05 protein molecule variants", "PDC-S70 protein molecules and / or PDC-S70 protein molecule variants" and "HEC-01 protein molecules and / or HEC-01 protein molecule variants". By blocking viral replication and fully mobilizing the expression of other defense genes in the prokaryotic microorganism cells, they work together to achieve a more ideal effect of enhancing the antiviral infection ability of prokaryotic microorganisms.

[0042] In some specific embodiments, when the reagent enhances the antiviral infection ability of prokaryotic microorganisms, it can specifically cause the prokaryotic microorganisms to simultaneously express "PDC-S70 protein molecules and / or PDC-S70 protein molecule variants", "PDC-M05A protein molecules and PDC-M05B protein molecules and / or PDC-M05A protein molecule variants and PDC-M05B protein molecule variants" and "HEC-01 protein molecules and / or HEC-01 protein molecule variants". By blocking viral replication and fully mobilizing the expression of other defense genes in prokaryotic microbial cells, the reagent works together to achieve a more ideal effect of enhancing the antiviral infection ability of prokaryotic microorganisms.

[0043] In some specific embodiments, when the reagent enhances the antiviral infection ability of prokaryotic microorganisms, it can specifically cause the prokaryotic microorganisms to simultaneously express "PDC-S05 protein molecules and / or PDC-S05 protein molecule variants", "PDC-S70 protein molecules and / or PDC-S70 protein molecule variants", "PDC-M05A protein molecules and PDC-M05B protein molecules and / or PDC-M05A protein molecule variants and PDC-M05B protein molecule variants" and "HEC-01 protein molecules and / or HEC-01 protein molecule variants". By blocking viral replication and fully mobilizing the expression of other defense genes in prokaryotic microbial cells, the reagent works together to achieve a more ideal effect of enhancing the antiviral infection ability of prokaryotic microorganisms.

[0044] In this invention, the reagent can be obtained directly from cold seep sediment samples, specifically including at least one of the following: (1) DNA was extracted from cold seep sediment samples to obtain target nucleic acid molecules. The extraction method is a conventional technique used in existing molecular biology. Those skilled in the art can make adaptive selections and adjustments according to actual needs. This invention does not impose any special limitations on it. (2) Proteins are extracted from cold seep sediment samples to obtain target protein molecules. The extraction method is a conventional technique used in existing molecular biology. Those skilled in the art can make adaptive selections and adjustments according to actual needs. This invention does not impose any special limitations on it.

[0045] In this invention, the reagent can be artificially prepared using biotechnological means based on the biological information of nucleic acid molecules and / or protein molecules, specifically including at least one of the following: (1) Based on the nucleotide sequence information of nucleic acid molecules, the target nucleic acid molecules are synthesized by chemical synthesis. Chemical synthesis is a conventional technique used in existing molecular biology. Those skilled in the art can make adaptive selections and adjustments according to actual needs. This invention does not impose any special limitations on it. (2) Based on the nucleotide sequence information of nucleic acid molecules, primer pairs are designed to amplify DNA samples extracted from cold seep sediment samples by PCR to obtain target nucleic acid molecules. Primer design, sample DNA extraction and PCR amplification are conventional techniques used in existing molecular biology. Those skilled in the art can make adaptive selections and adjustments according to actual needs. This invention does not impose any special limitations on them. (3) Based on the amino acid sequence information of the protein molecule, the target protein molecule is obtained by chemical synthesis. Chemical synthesis is a conventional technique used in existing molecular biology. Those skilled in the art can make adaptive selections and adjustments according to actual needs. This invention does not impose any special limitations on it. (4) Based on the amino acid sequence information of the protein molecule, the corresponding coding nucleic acid molecule is designed, the corresponding coding nucleic acid molecule is introduced into the host cell to construct the expression strain, the expression strain is used for expression, and the target protein molecule is obtained. The design of the coding nucleic acid molecule, the construction of the expression strain and the protein expression are conventional techniques used in existing molecular biology. Those skilled in the art can make adaptive selections and adjustments according to actual needs. This invention does not impose any special limitations on it.

[0046] In this invention, the nucleic acid molecules and their encoded proteins contained in the reagent achieve antiviral infection effects by inhibiting the synthesis of virus replication-related substances in prokaryotic microbial cells and altering cellular transcription and energy metabolism. This strategy is based on altering viral replication and the core physiological processes of prokaryotic microorganisms and has good adaptability to different prokaryotic microorganisms. The applicable prokaryotic microorganisms for which the reagent can enhance antiviral infection effects include, but are not limited to, one or more of Escherichia coli, Lactobacillus, Acetobacter, Bacillus subtilis, Streptomyces, Bacillus licheniformis, Pseudomonas, and Rhizobium; and applicable viruses include, but are not limited to, one or more of T7 phage, T5 phage, and T4 phage.

[0047] Based on the antiviral infection activity of the aforementioned deep-sea cold seep defense gene and with the aim of modifying target prokaryotic microorganisms, this invention also provides an expression cassette. This expression cassette includes at least one of the following nucleic acid molecules: (1) One or more nucleic acid molecules with sequences as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5; (2) A nucleic acid molecule that encodes a protein molecule with an amino acid sequence as shown in SEQ ID NO:6 and / or with at least 95% sequence identity with the sequence shown in SEQ ID NO:6, wherein the nucleotide sequence information of the nucleic acid molecule is determined based on the amino acid sequence information of the encoded protein molecule, and the design of nucleotide sequence information from amino acid sequence information is a conventional technique used in existing molecular biology. Those skilled in the art can make adaptive designs according to actual needs, and the present invention does not impose any particular limitations on it. (3) A nucleic acid molecule that encodes a protein molecule with an amino acid sequence as shown in SEQ ID NO:7 and / or has at least 95% sequence identity with the sequence shown in SEQ ID NO:7. The nucleotide sequence information of the nucleic acid molecule is determined based on the amino acid sequence information of the encoded protein molecule. The design of nucleotide sequence information from amino acid sequence information is a conventional technique used in existing molecular biology. Those skilled in the art can make adaptive designs according to actual needs. This invention does not impose any particular limitations on it. (4) A nucleic acid molecule that encodes a protein molecule with an amino acid sequence as shown in SEQ ID NO:8 and / or has at least 95% sequence identity with the sequence shown in SEQ ID NO:8. The nucleotide sequence information of the nucleic acid molecule is determined based on the amino acid sequence information of the encoded protein molecule. The design of nucleotide sequence information from amino acid sequence information is a conventional technique used in existing molecular biology. Those skilled in the art can make adaptive designs according to actual needs. This invention does not impose any particular limitations on it. (5) A nucleic acid molecule that encodes a protein molecule with an amino acid sequence as shown in SEQ ID NO:9 and / or has at least 95% sequence identity with the sequence shown in SEQ ID NO:9. The nucleotide sequence information of the nucleic acid molecule is determined based on the amino acid sequence information of the encoded protein molecule. The design of nucleotide sequence information from amino acid sequence information is a conventional technique used in existing molecular biology. Those skilled in the art can make adaptive designs according to actual needs. This invention does not impose any special limitations on it.

[0048] (6) A nucleic acid molecule that encodes a protein molecule with an amino acid sequence as shown in SEQ ID NO:10 and / or has at least 95% sequence identity with the sequence shown in SEQ ID NO:10. The nucleotide sequence information of the nucleic acid molecule is determined based on the amino acid sequence information of the encoded protein molecule. The design of nucleotide sequence information from amino acid sequence information is a conventional technique used in existing molecular biology. Those skilled in the art can make adaptive designs according to actual needs. This invention does not impose any particular limitations on it.

[0049] In this invention, the expression cassette is a functional nucleic acid fragment that enables the deep-sea cold seep defense gene to be transcribed and translated in prokaryotic microorganisms. Its structure includes at least a promoter, a deep-sea cold seep defense gene, and a terminator. Preferably, it may also include, but is not limited to, regulatory elements for regulating protein expression and / or other functional elements for improving the stability of the expression cassette.

[0050] In this invention, the expression cassette can be introduced into prokaryotic microorganisms in the form of DNA, utilizing the transcription and translation systems within the prokaryotic microorganisms to express the corresponding protein molecules; or it can be introduced into prokaryotic microorganisms in the form of RNA, utilizing the translation systems within the prokaryotic microorganisms to express the corresponding protein molecules. That is, specific examples of the form in which the expression cassette exists include, but are not limited to, one or more of linear DNA, plasmid vectors, mRNA, and circular RNA.

[0051] Based on the application potential of the aforementioned deep-sea cold seep defense genes in enhancing the antiviral infection capabilities of prokaryotic microorganisms, this invention also provides a method for enhancing the antiviral infection capabilities of prokaryotic microorganisms for non-disease treatment and disease diagnosis purposes. The method specifically includes: introducing the aforementioned expression cassette into prokaryotic microorganisms to enhance their antiviral infection capabilities.

[0052] In this invention, the method of introducing the expression cassette into prokaryotic microorganisms is a conventional technique used in existing molecular biology. Those skilled in the art can make adaptive designs according to actual needs, and this invention does not impose any particular limitations on it.

[0053] In this invention, the method is specifically applied to practical application scenarios that are not for disease treatment or disease diagnosis, and may be, but is not limited to, one or more of the following: (1) When the method is applied to the industrial fermentation process, the production strain is modified to reduce the risk of production stoppage caused by virus contamination and improve production stability and economic benefits. (2) When the method is applied to biosynthesis, the deep-sea cold seep defense gene serves as a programmable defense element, improving the biosafety of the engineered strain; (3) When the method is applied to pharmaceutical research and development, the deep-sea cold seep defense gene provides molecular resources for the exploration of targets for novel antigenic nucleus microbial infections and provides a new approach for the development of non-antibiotic antigenic nucleus microbial infection drugs.

[0054] In some specific embodiments, the method has an excellent enhancing effect on the ability of Escherichia coli to resist infection by one or more viruses, including T7 phage, T5 phage, and T4 phage.

[0055] Based on the enhanced antiviral activity of the aforementioned deep-sea cold seep defense genes in prokaryotic microorganisms, this invention also provides the application of the aforementioned reagents or expression cassettes in industrial biotechnology. Industrial biotechnology refers to a technological system that designs, modifies, or utilizes prokaryotic microorganisms or their generated enzymes and metabolic pathways to produce industrial raw materials, chemicals, food additives, enzyme preparations, bioenergy, pharmaceuticals, and other products on a large scale, or for use in industrial processes such as environmental remediation and biocatalysis.

[0056] In this invention, specific examples of the industrial biotechnology field include, but are not limited to, one or more of the following: food and fermentation industry, enzyme preparation and biocatalysis industry, pharmaceutical and fine chemical industry, bioenergy and bio-based materials industry, and environmental governance.

[0057] Based on the application potential of the aforementioned deep-sea cold seep defense genes in the development of non-antibiotic drugs for antigenic microorganism infections, this invention also provides an antibacterial agent. The antibacterial agent includes at least one of the following technical features: (1) A substance for inhibiting the expression of protein molecules with amino acid sequences as shown in SEQ ID NO:6 and / or sequences with at least 95% sequence identity as shown in SEQ ID NO:6, wherein the antibacterial agent weakens the defense of prokaryotic microorganisms against viruses by inhibiting the expression of protein molecules with the Virulence_RhuM functional domain in prokaryotic microbial cells, and works synergistically with viruses to achieve the killing effect on prokaryotic microorganisms. (2) A substance for inhibiting the biological activity of protein molecules with amino acid sequences as shown in SEQ ID NO:6 and / or sequences with at least 95% sequence identity as shown in SEQ ID NO:6, wherein the antimicrobial agent weakens the defense of prokaryotic microorganisms against viruses by inhibiting the activity of protein molecules containing the Virulence_RhuM functional domain in prokaryotic microbial cells, and can work synergistically with viruses to achieve the killing effect on prokaryotic microorganisms; (3) A substance for inhibiting the expression of protein molecules with amino acid sequences as shown in SEQ ID NO:7 and / or having at least 95% sequence identity with the sequence shown in SEQ ID NO:7, wherein the antibacterial agent weakens the defense of prokaryotic microorganisms against viruses by inhibiting the expression of protein molecules containing nucleic acid binding domains in prokaryotic microbial cells, and works synergistically with viruses to achieve the killing effect on prokaryotic microorganisms. (4) A substance for inhibiting the biological activity of protein molecules with amino acid sequences as shown in SEQ ID NO:7 and / or having at least 95% sequence identity with the sequence shown in SEQ ID NO:7, wherein the antimicrobial agent weakens the defense of prokaryotic microorganisms against viruses by inhibiting the activity of protein molecules containing nucleic acid binding domains in prokaryotic microbial cells, and works synergistically with viruses to achieve the killing effect on prokaryotic microorganisms. (5) A substance for inhibiting the expression of protein molecules with amino acid sequences as shown in SEQ ID NO:8 and / or sequences with at least 95% sequence identity as shown in SEQ ID NO:8, wherein the antibacterial agent weakens the defense of prokaryotic microorganisms against viruses by inhibiting the expression of protein molecules containing ArsR-like_HTH and NT_KNTase_like functional domains in prokaryotic microbial cells, and works in conjunction with SEQ ID NO:9 to kill prokaryotic microorganisms; (6) A substance for inhibiting the biological activity of protein molecules with amino acid sequences as shown in SEQ ID NO:8 and / or having at least 95% sequence identity with the sequence shown in SEQ ID NO:8, wherein the antimicrobial agent weakens the defense of prokaryotic microorganisms against viruses by inhibiting the activity of protein molecules containing ArsR-like_HTH and NT_KNTase_like functional domains in prokaryotic microbial cells, and works in conjunction with SEQ ID NO:9 to kill prokaryotic microorganisms; (7) A substance for inhibiting the expression of protein molecules with an amino acid sequence as shown in SEQ ID NO:9 and / or having at least 95% sequence identity with the sequence shown in SEQ ID NO:9, wherein the antibacterial agent inhibits phage replication or regulates metabolic balance by degrading host RNA, thereby weakening the defense of prokaryotic microorganisms against viruses, and works in conjunction with SEQ ID NO:8 to kill prokaryotic microorganisms. (8) A substance for inhibiting the biological activity of protein molecules with amino acid sequences as shown in SEQ ID NO:9 and / or having at least 95% sequence identity with the sequence shown in SEQ ID NO:9, wherein the antimicrobial agent inhibits phage replication or regulates metabolic balance by degrading host RNA, thereby weakening the defense of prokaryotic microorganisms against viruses, and works in conjunction with SEQ ID NO:8 to kill prokaryotic microorganisms. (9) A substance for inhibiting the expression of protein molecules with amino acid sequences as shown in SEQ ID NO:10 and / or having at least 95% sequence identity with the sequence shown in SEQ ID NO:10, wherein the antibacterial agent weakens the defense of prokaryotic microorganisms against viruses by inhibiting the expression of protein molecules containing the AAA_21 functional domain in prokaryotic microbial cells, and works synergistically with viruses to achieve the killing effect on prokaryotic microorganisms. (10) A substance for inhibiting the biological activity of protein molecules with amino acid sequences as shown in SEQ ID NO:10 and / or having at least 95% sequence identity with the sequence shown in SEQ ID NO:10, wherein the antimicrobial agent weakens the defense of prokaryotic microorganisms against viruses by inhibiting the activity of protein molecules containing the AAA_21 functional domain in prokaryotic microbial cells, and works synergistically with viruses to achieve the killing effect on prokaryotic microorganisms.

[0058] The embodiments of the present invention are described in detail below. These embodiments are intended to explain the present invention and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0059] Example 1 This embodiment illustrates the acquisition of candidate deep-sea cold seep defense genes, specifically including the following steps: (1) 173 cold seep sediment samples from 17 cold seep sites worldwide were obtained, and gene extraction and sequencing were performed to construct a deep-sea cold seep sediment genome database. The cold seep sites include: ODP site 1244 cold seep (ENP) in the Eastern North Pacific; Santa Monica Mounds cold seep (SMM); Western Gulf of Mexico oil and gas seep (WGM); Northwestern Gulf of Mexico oil and gas seep (NGM); Eastern Gulf of Mexico oil and gas seep (EGM); Scotia Basin oil and gas seep (SB); Mediterranean Sea Amon mud volcano cold seep (MS); Haakon Mosby mud volcano cold seep (HM); Laptev Sea cold seep (LS); and Okinawa Trough cold seep. The cold seep includes the Xisha Trough cold seep (XST), as well as the Haima cold seep, Haiyang4 cold seep (HY4), Site F cold seep (SF), Jiaolong cold seep (JL), Qiongdongnan Basin cold seep (QDN), and Shenhuarea cold seep (SH) in the South China Sea, covering the seabed oxygenated sediment-water interface to the anoxic layer (reaching a maximum depth of 68.55m below the seabed).

[0060] (2) The deep-sea cold seep sediment genome data were quality controlled by the software fastp (v0.23.2; default parameters) and the Read_QC module (v1.3.2; -skip-bmtagger) in MetaWRAP, low-quality data were filtered out and the sequence data were trimmed and corrected to obtain a high-quality sequence set.

[0061] (3) The short sequence fragments were assembled using MEGAHIT (v1.2.9; default parameters) software. The binning module (v1.3.2; parameters: -metabat2 -maxbin2 -concoct -universal), SemiBin (v1.4.0; default parameters) single_easy_bin mode, and Rosella (v0.4.1; default parameters; https: / / github.com / rhysnewell / rosella) in MetaWRAP were used to integrate and optimize the obtained binning. The integrity and contamination of the optimized bins were evaluated using CheckM (v1.2.1). The genomes of 3813 representative cold seep prokaryotes were then deduplicated using dRep (v3.4.0; parameters: -comp 50 -con 10) and the average nucleotide similarity (ANI) of 95% was used as the standard to obtain the genomes of 3813 species-level cold seep prokaryotes.

[0062] (4) Use Prodigal v2.6.3 (parameter: -p meta) to predict open reading frames (ORFs) and protein-coding sequences in representative cold seep prokaryotic genomes.

[0063] (5) DefenseFinder v1.2.1 (parameter: --db-type gembase) and PADLOC v2.0.0 (parameter: --fix-prodigal) were used to annotate the defense genes / systems in MAGs. Finally, 36,783 defense-related genes (belonging to 26,389 defense systems) were detected in 65% (n = 2,466) of cold seep prokaryotes. Candidate deep-sea cold seep defense genes were obtained by using a proportion of more than 1% in the cold seep environment and a quality score (integrity - 5 × contamination) ≥ 75 as screening criteria. The specific details are shown in Table 1 (only the genes that were finally verified to have a defensive effect are shown).

[0064] Table 1.

[0065] (5) Based on the nucleic acid sequence information of each candidate deep-sea cold seep defense gene, the amino acid sequence of the corresponding encoded protein molecule was obtained by converting the genetic code, as shown in Table 2.

[0066] Table 2.

[0067] Example 2 This embodiment illustrates the verification of the antiviral activity of the candidate deep-sea cold seep defense gene provided in Example 1. The specific tests include: 1. Preparation of candidate deep-sea cold seep defense genes Based on the amino acid sequences described in Table 2, codons for E. coli were optimized and sent to a commercial synthesis company for artificial synthesis. The synthesized gene sequences were then cloned into a pET28a vector containing an N-terminal 6×His tag using Gibson Assembly cloning technology to construct recombinant plasmids carrying various defense genes, as shown in Table 3.

[0068] 2. Construction of recombinant strains The recombinant plasmids shown in Table 3 were transformed into competent cells using a transfection kit (Vazyme Biotech, China, catalog number TL301-01) according to the manufacturer's instructions. E. coli strain B (ATCC) ® 11303™ was used to obtain recombinant strains, as shown in Table 3.

[0069] Table 3.

[0070] 3. Validation of the antiviral activity of candidate deep-sea cold seep defense genes (1) Pick a single colony of the recombinant strain and inoculate it into 5 mL of LB liquid medium containing 100 μg / mL ampicillin. After incubating overnight at 37°C and 220 rpm, inoculate it into 100 mL of LB liquid medium containing 50 μg / mL ampicillin at an inoculation rate of 1% (v / v). Incubate at 37°C and 220 rpm until OD. 600 The concentration was 0.6; IPTG was added at a final concentration of 0.2 mM, and the cultures were induced at 37°C and 220 rpm for 1 h to obtain the bacterial solutions to be tested.

[0071] (2) Take E. coli DH5α was inoculated at a rate of 1% (v / v) into 100 mL of LB liquid medium containing 50 μg / mL ampicillin and cultured at 37°C and 220 rpm until OD500. 600The concentration was 0.6, and the host bacterial suspension was obtained. The phage suspension and host bacterial suspension were mixed evenly at a 1:1 volume ratio, and incubated at 37°C for 10 minutes. The phage and host bacterial suspension were then analyzed using the double-layer plate method. E. coli The mixture of DH5α was cultured to obtain plaques of various phages (T7 phage, T4 phage and T5 phage).

[0072] (3) Pick a single phage plaque and inoculate it into a container containing E. coli B was cultured in LB liquid medium at 37°C and 220 rpm until OD. 600 The concentration was set to 0.4, and then the mixture was centrifuged at 12000 rpm for 4 min. The supernatant was then filtered through a 0.2 µm filter to obtain the phage suspensions. The T7 phage suspension was then subjected to 10... -1 Up to 10 -8 Serial dilutions were performed on T4 and T5 phages for 10... -1 Up to 10 -7 Gradient dilution.

[0073] (4) Take 500 mL of each test bacterial suspension and mix it evenly with 14.5 mL of LB liquid medium containing 0.5% agar. Then add LB solid medium containing 100 µg / mL ampicillin and 0.1 mM IPTG. Spot 4 µL of each phage suspension and its dilution into the medium and incubate overnight at 25°C. Observe the plaque formation the next day and take pictures to record the results. Use the recombinant blank strain as a control. Calculate the reduction rate of plaque formation efficiency (EOP) according to the following formula. The results are as follows: Figures 1-3 As shown in Table 4.

[0074] EOP reduction factor = Number of single plaques in blank bacterial strain / Number of single plaques in test bacterial solution Table 4.

[0075] As shown in Table 4, the introduction of PDC-S05, PDC-S70, PDC-M05A, and PDC-M05B genes or the HEC-01 gene can enhance... E. coli Strain B exhibits resistance to T7, T5, and T4 phages. Among these, the recombinant strain PDC-S05 shows an approximately 1000-fold reduction in the EOP (Effect Opportunity Optimum) against T7 and T5 phages, and an approximately 10-fold reduction in the EOP against T4 phage. The recombinant strain PDC-S70 shows an approximately 1000-fold reduction in the EOP against T5 phage, and an approximately 100-fold reduction in the EOP against T7 and T4 phages.

[0076] Example 3 This embodiment illustrates the deep-sea cold seep defense gene PDC-S05 and its encoded protein's predicted structural domains and inferred antibacterial mechanism, specifically including: Based on the amino acid sequence of the protein encoded by the deep-sea cold seep defense gene PDC-S05, InterProScan was used for domain annotation, Alphafold3 was used for structure prediction, and Foldseek was used to search for structures. The structures were then compared with known protein structures in the PDB database to find the most similar structures. The results are as follows: Figure 4 As shown.

[0077] Depend on Figure 4 The results show that PDC-S05 has a Virulence_RhuM domain, which enhances DNA binding capacity through dimerization. This domain may rapidly repair host genome breaks under extreme radiation or oxidative stress, block DNA damage pathways dependent on phage integration or replication, and has biological activity to inhibit viral replication.

[0078] Example 4 This embodiment illustrates the deep-sea cold seep defense gene PDC-S70 and its encoded protein's predicted structural domains and inferred antibacterial mechanisms, specifically including: Based on the amino acid sequence of the protein encoded by the deep-sea cold seep defense gene PDC-S70, InterProScan was used for domain annotation, Alphafold3 was used for structure prediction, and Foldseek was used to search for structures. The structures were then compared with known protein structures in the PDB database to find the most similar structures. The results are as follows: Figure 5 As shown.

[0079] Depend on Figure 5 The results show that PDC-S70 has a DUF3368 domain and a PIN domain. The function of the DUF3368 domain is unknown, while the PIN domain has nuclease activity. It can inhibit cell translation by cleaving tRNA or rRNA, triggering abortion infection mechanism in the toxin-antitoxin system, thereby inhibiting phage proliferation and exhibiting biological activity to block viral nucleic acid synthesis.

[0080] Example 5 This embodiment illustrates the predicted structural domains and inferred antibacterial mechanisms of the deep-sea cold seep defense genes PDC-M05A and PDC-M05B and their encoded proteins, specifically including: Based on the amino acid sequences of the proteins encoded by the deep-sea cold seep defense genes PDC-M05A and PDC-M05B, InterProScan was used for domain annotation, Alphafold3 was used for structure prediction, and Foldseek was used to search for structures. The structures were then compared with known protein structures in the PDB database to find the most similar structures. The results are as follows: Figure 6 As shown.

[0081] Depend on Figure 6 The results show that the PDC-M05A and PDC-M05B subunits together constitute the PDC-M05 defense system. PDC-M05A includes an ArsR-like HTH domain and an NT-KNTase-like domain, possessing DNA-binding transcription factor activity. It may exert a restriction modification system defense function by recognizing and cleaving specific conformations of exogenous invading DNA, and has biological activity regulating transcription. PDC-M05B includes *Archaeopterygium* (…). Archaeoglobus fulgidus The Hepn domain of the nucleotide may act as a ribonuclease toxin in the toxin-antitoxin system, inhibiting phage replication or regulating metabolic balance to cope with infection pressure by degrading host RNA, and has biological activity in regulating transcription processes.

[0082] Example 6 This embodiment illustrates the predicted domains and inferred antibacterial mechanisms of the deep-sea cold seep defense gene HEC-01 and its encoded protein, specifically including: Based on the amino acid sequence of the protein encoded by the deep-sea cold seep defense gene HEC-01, InterProScan was used for domain annotation, Alphafold3 was used for structure prediction, and Foldseek was used to search for structures. The structures were then compared with known protein structures in the PDB database to find the most similar structures. The results are as follows: Figure 7 As shown.

[0083] Depend on Figure 7 The results show that HEC-01 includes the AAA_21 domain, which matches the PtuAB complex of E. coli Septu and has ATP binding and hydrolysis activities. It may mediate the excretion of exogenous toxic substances (such as bacteriophage toxins) through membrane transport or secretion systems, or act as a component of the toxin-antitoxin system. By regulating the ATP hydrolysis process, it can adjust cell activity and enhance the survival probability of bacteria under stress, thereby achieving antiviral or anti-stress effects.

[0084] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention without departing from the principles and spirit of the present invention.

Claims

1. An agent for enhancing the ability of a prokaryotic microorganism to resist viral infection, characterized in that, The reagent comprises at least one of the following: (1) one or more of the nucleic acid molecules having the sequence as set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5; (2) one or more of the protein molecules having the amino acid sequence as set forth in SEQ ID NO: 6 or having at least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 6, the amino acid sequence as set forth in SEQ ID NO: 7 or having at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 7, the amino acid sequence as set forth in SEQ ID NO: 8 or having at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 8, the amino acid sequence as set forth in SEQ ID NO: 9 or having at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 9, and the amino acid sequence as set forth in SEQ ID NO: 10 or having at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 10; (3) a nucleic acid molecule encoding a protein molecule having the amino acid sequence as set forth in SEQ ID NO: 6 and / or having at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 6; (4) a nucleic acid molecule encoding a protein molecule having the amino acid sequence as set forth in SEQ ID NO: 7 and / or having at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 7; (5) a nucleic acid molecule encoding a protein molecule having the amino acid sequence as set forth in SEQ ID NO: 8 and / or having at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 8; (6) a nucleic acid molecule encoding a protein molecule having the amino acid sequence as set forth in SEQ ID NO: 9 and / or having at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 9; (7) a nucleic acid molecule encoding a protein molecule having the amino acid sequence as set forth in SEQ ID NO: 10 and / or having at least 95% sequence identity to the sequence as set forth in SEQ ID NO:

10.

2. The agent for enhancing the ability of a prokaryotic microorganism to resist a viral infection according to claim 1, characterized by, The protein molecule having the amino acid sequence as set forth in SEQ ID NO: 6 or having at least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 6 has at least one of the following technical features: (1) the protein molecule comprises a Virulence_RhuM functional domain; (2) the protein molecule has a biological activity of inhibiting viral replication; Optionally, the protein molecule having the amino acid sequence as set forth in SEQ ID NO: 7 or having at least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 7 has at least one of the following technical features: (1) the protein molecule comprises a nucleic acid binding domain; (2) the protein molecule has a biological activity of blocking viral nucleic acid synthesis; Optionally, the protein molecule having an amino acid sequence as set forth in SEQ ID NO: 8 or having at least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 8 has at least one of the following technical features: (1) the protein molecule comprises ArsR-like_HTH and NT_KNTase_like functional domains; (2) the protein molecule has a biological activity of regulating a transcription process. Optionally, the protein molecule having an amino acid sequence as set forth in SEQ ID NO: 9 or having at least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 9 has at least one of the following technical features: (1) the protein molecule comprises a Hepn domain; (2) the protein molecule has a biological activity of regulating a transcription process. Optionally, the protein molecule having an amino acid sequence as set forth in SEQ ID NO: 10 or having at least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 10 has at least one of the following technical features: (1) the protein molecule comprises a AAA_21 functional domain; (2) the protein molecule has a biological activity of regulating an ATP hydrolysis process.

3. The agent for enhancing the ability of a prokaryotic microorganism to resist a viral infection according to claim 1, wherein The reagent has at least one of the following technical features: (1) the reagent is obtained by isolation from a cold spring sediment sample; (2) the reagent is obtained by artificial preparation based on biological information of a nucleic acid molecule and / or a protein molecule.

4. An expression cassette comprising, The expression cassette comprises at least one of the following nucleic acid molecules: (1) one or more of the nucleic acid molecules having a sequence as set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5; (2) a nucleic acid molecule encoding a protein molecule having an amino acid sequence as set forth in SEQ ID NO: 6 and / or having at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 6; (3) a nucleic acid molecule encoding a protein molecule having an amino acid sequence as set forth in SEQ ID NO: 7 and / or having at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 7; (4) a nucleic acid molecule encoding a protein molecule having an amino acid sequence as set forth in SEQ ID NO: 8 and / or having at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 8; (5) a nucleic acid molecule encoding a protein molecule having an amino acid sequence as set forth in SEQ ID NO: 9 and / or having at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 9; (6) a nucleic acid molecule encoding a protein molecule having an amino acid sequence as set forth in SEQ ID NO: 10 and / or having at least 95% sequence identity to the sequence as set forth in SEQ ID NO:

10.

5. The expression cassette of claim 4, wherein, The expression cassette is selected from one or more of linear DNA, a plasmid vector, mRNA, and circular RNA.

6. A method for enhancing the ability of a prokaryotic microorganism to resist viral infection for non-disease treatment and disease diagnostic purposes, characterized by, The method comprises: introducing the expression cassette of claim 4 into a prokaryotic microorganism to enhance the ability of the prokaryotic microorganism to resist viral infection.

7. The method for enhancing the ability of a prokaryotic microorganism to resist a viral infection for non-disease treatment and disease diagnosis purposes according to claim 6, characterized in that, The method comprises at least one of the following technical features: (1) the prokaryotic microorganism is Escherichia coli; (2) the virus is selected from one or more of T7 phage, T5 phage and T4 phage.

8. Use of the agent of claim 1 or the expression cassette of claim 4 in industrial biotechnology, characterized in that, The industrial biotechnology utilizes prokaryotic microorganisms for production of products.

9. Use of the agent or expression cassette according to claim 8 in industrial biotechnology, characterized in that, The industrial biotechnology is selected from one or more of food and fermentation industry, enzyme preparation and biocatalysis industry, pharmaceutical and fine chemical industry, bioenergy and bio-based material industry and environmental governance.

10. An antibacterial preparation, characterized by, The antibacterial preparation comprises at least one of the following technical features: (1) a substance for inhibiting expression of a protein molecule with an amino acid sequence as set forth in SEQ ID NO: 6 and / or having at least 95% sequence identity to the sequence set forth in SEQ ID NO: 6; (2) a substance for inhibiting biological activity of a protein molecule with an amino acid sequence as set forth in SEQ ID NO: 6 and / or having at least 95% sequence identity to the sequence set forth in SEQ ID NO: 6; (3) a substance for inhibiting expression of a protein molecule with an amino acid sequence as set forth in SEQ ID NO: 7 and / or having at least 95% sequence identity to the sequence set forth in SEQ ID NO: 7; (4) a substance for inhibiting biological activity of a protein molecule with an amino acid sequence as set forth in SEQ ID NO: 7 and / or having at least 95% sequence identity to the sequence set forth in SEQ ID NO: 7; (5) a substance for inhibiting expression of a protein molecule with an amino acid sequence as set forth in SEQ ID NO: 8 and / or having at least 95% sequence identity to the sequence set forth in SEQ ID NO: 8; (6) a substance for inhibiting biological activity of a protein molecule with an amino acid sequence as set forth in SEQ ID NO: 8 and / or having at least 95% sequence identity to the sequence set forth in SEQ ID NO: 8; (7) a substance for inhibiting expression of a protein molecule with an amino acid sequence as set forth in SEQ ID NO: 9 and / or having at least 95% sequence identity to the sequence set forth in SEQ ID NO: 9; (8) a substance for inhibiting biological activity of a protein molecule with an amino acid sequence as set forth in SEQ ID NO: 9 and / or having at least 95% sequence identity to the sequence set forth in SEQ ID NO: 9; (9) a substance for inhibiting expression of a protein molecule with an amino acid sequence as set forth in SEQ ID NO: 10 and / or having at least 95% sequence identity to the sequence set forth in SEQ ID NO: 10; (10) a substance for inhibiting biological activity of a protein molecule with an amino acid sequence as set forth in SEQ ID NO: 10 and / or having at least 95% sequence identity to the sequence set forth in SEQ ID NO: 10.

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