An escrt-iii-based anti-phage system and uses thereof

Abstract

This invention discloses an anti-phage system based on ESCRT-III and its applications, relating to the field of biotechnology. This invention utilizes the coding genes for two structural proteins of the Hoda_Snf7_1 and Hoda_Snf7_2 from the Hoda archaea ESCRT-III. Hoda_ snf7_1 and Hoda_snf7_2 This invention is applied to antiphage systems. Compared with existing technologies, the main advantages of the antiphage system of this invention are: (1) it consists of only two genes, making system construction simple and quick; (2) it is composed of structural proteins, making it less prone to losing antiphage activity due to gene mutations; (3) it has a broad-spectrum antiphage effect; and (4) it does not affect the normal growth of host bacteria. This system expands the understanding of antiphage systems and provides new ideas and technical means for preventing and controlling phage contamination in industrial microbial production processes.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to an antiphage system based on ESCRT-III and its applications. Background Technology

[0002] Bacteriophages are viruses that infect bacteria. In industrial microbial fermentation systems, bacteriophages can infect and lyse core fermentation strains (such as E. coli), leading to reduced yields or even fermentation failure, resulting in significant economic losses. To combat bacteriophage contamination, measures such as equipment and pipeline sterilization, fermentation environment disinfection, and strain rotation are commonly employed. However, these measures are often complex, time-consuming, costly, and their effectiveness is inconsistent, making it difficult to fundamentally improve the fermentation strain's tolerance to bacteriophage contamination. Therefore, obtaining strains with stable resistance to bacteriophages is crucial for preventing bacteriophage contamination in the fermentation industry and ensuring production stability.

[0003] Currently, identifying and developing systems resistant to bacteriophage infection from biological genetic resources, and then genetically modifying bacteria to resist bacteriophages, is one of the important ways to prevent bacteriophage infection in microbial production and develop related biotechnologies. However, existing anti-phage systems have the following limitations: (1) Some systems are complex in composition, usually consisting of multiple genes, making it difficult to modify the host bacterial genes; (2) Some systems resist bacteriophages by modifying target genes or proteins through catalytic activity, and enzyme activity is easily lost due to mutations in the bacterial host genes, resulting in a decrease in the resistance of the bacterial host to bacteriophages in the later stages of passage; (3) Most systems only produce resistance to specific bacteriophages or a few types of bacteriophages, making it difficult to cope with the diversity and rapid evolution of bacteriophages in the production environment; (4) Some systems affect the normal growth or protein expression of the bacterial host, reducing industrial production efficiency.

[0004] Therefore, there is an urgent need to develop a novel antiphage system that is simplified in composition, easier to implement in engineering, has less impact on host growth, and can provide protection against a variety of bacteriophages. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide an antiphage system based on ESCRT-III and its application, aiming to solve the problems of existing antiphage systems being complex in composition, having low resistance efficiency, affecting host growth, and having insufficient types of antiphages.

[0006] The technical solution of the present invention is as follows: Firstly, providing Walk_snf7_1 Genes and Hoda_snf7_2 The application of genes in the preparation of antiphage systems, wherein, Walk_snf7_1The gene encodes the Hoda_Snf7_1 protein, whose amino acid sequence is shown in SEQ ID NO.1. Hoda_snf7_2 The gene encodes the Hoda_Snf7_2 protein, whose amino acid sequence is shown in SEQ ID NO.2.

[0007] Secondly, an anti-phage system based on ESCRT-III is provided, wherein the anti-phage system comprises... Walk_ snf7_1 Genes and Hoda_snf7_2 Genome composition; Among them, the Walk_snf7_1 The gene encodes the Hoda_Snf7_1 protein, whose amino acid sequence is shown in SEQ ID NO.1. Hoda_snf7_2 The gene encodes the Hoda_Snf7_2 protein, whose amino acid sequence is shown in SEQ ID NO.2.

[0008] The preferred technical solution, the Walk_snf7_1 The nucleotide sequence of the gene is shown in SEQ ID NO.3. Hoda_snf7_2 The nucleotide sequence of the gene is shown in SEQ ID NO.4.

[0009] Thirdly, an expression vector is provided that expresses the antiphage system described in the second aspect.

[0010] In a preferred technical solution, the expression vector delivers the... Walk_snf7_1 Genes and Hoda_snf7_2 The gene was obtained by recombination into a plasmid.

[0011] In a further preferred embodiment, the plasmid is selected from one of the following: pBAD plasmid, pET plasmid, pBR plasmid, and pUC plasmid.

[0012] Fourthly, a host cell is provided, said host cell containing the expression vector described in the third aspect.

[0013] In a preferred embodiment, the host cell is Escherichia coli.

[0014] In a further preferred embodiment, the *E. coli* strain is TOP10 (araBADC). - ).

[0015] Fifthly, the application of the antiphage system described in the second aspect, the expression vector described in the third aspect, or the host cell described in the fourth aspect in the preparation of antiphage products is provided.

[0016] Beneficial effects: This invention provides an ESCRT-III-based antiphage system and its application, which encodes the genes corresponding to the two structural proteins Hoda_Snf7_1 and Hoda_Snf7_2 of the Hoda archaea ESCRT-III. Walk_snf7_1 and Hoda_snf7_2 This invention is applied to antiphage systems. Compared with existing technologies, the main advantages of the antiphage system of this invention are: (1) it consists of only two genes, making system construction simple and quick; (2) it is composed of structural proteins, making it less prone to losing antiphage activity due to gene mutations; (3) it has a broad-spectrum antiphage effect; and (4) it does not affect the normal growth of host bacteria. This system expands the understanding of antiphage systems and provides new ideas and technical means for preventing and controlling phage contamination in industrial microbial production processes. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the recombinant pBAD plasmid containing the archaea ESCRT-III constructed in Example 2 of the present invention.

[0018] Figure 2 This is the growth curve of Escherichia coli TOP10 (araBADC-) expressing and not expressing the recombinant pBAD plasmid, as detected in Example 3 of this invention.

[0019] Figure 3 This is a graph showing the results of the resistance experiments of Escherichia coli TOP10 (araBADC-) expressing and not expressing the recombinant pBAD plasmid against 16 phages, as detected in Example 4 of this invention. Detailed Implementation

[0020] This invention provides an antiphage system based on ESCRT-III and its application. To make the purpose, technical solution and effects of this invention clearer and more explicit, the invention is further described in detail below.

[0021] To develop more novel anti-phage systems, researchers are continuously expanding and deepening their exploration of microbial resources. Currently, archaea, as prokaryotic microorganisms distinct from bacteria, are widely distributed in the natural environment and even the human body, and their biological resources urgently need development and utilization. Archaeoviral defense systems, represented by CRISPR / Cas and RM systems, exhibit anti-phage activity in *Escherichia coli*, suggesting the application value of archaeoviral defense systems in combating bacterial phages. The Endosomal Sorting Complex Required for Transport (ESCRT) is a unique protein complex discovered in the *Asgard archaea* group since 2015, possibly related to the process of cell membrane cleavage. This complex can be subdivided into subcomplexes such as ESCRT-I, ESCRT-II, and ESCRT-III based on its composition and function.

[0022] Among them, ESCRT-III is composed of Snf7_1 (encoding gene) snf7_1 ) and Snf7_2 (encoding gene) snf7_2 The archaic ESCRT-III system consists of two structural proteins (not catalytically active enzymes) (DOI: 10.1128 / mbio.00417-20). Snf7_1 (also known as Vps2 / 24 / 46) and Snf7_2 (also known as Vps20 / 32 / 60) can bind and alter membrane lipid structure (DOI: 10.1038 / s44318-024-00346-4). Notably, the human genome-encoded ESCRT is closely associated with viral infection (DOI: 10.1038 / s41580-019-0177-4), suggesting that archaic ESCRT may be involved in viral infection. These characteristics indicate that the archaic ESCRT-III system, composed of a few structural proteins, has unique potential in developing novel antiphage tools. However, to date, no archaic ESCRT-III system has been reported as an antiphage element.

[0023] Based on this, the embodiments of the present invention provide Walk_snf7_1 Genes and Hoda_snf7_2 The application of genes in the preparation of antiphage systems, wherein, Walk_snf7_1 The gene encodes the Hoda_Snf7_1 protein, whose amino acid sequence is shown in SEQ ID NO.1. Hoda_snf7_2 The gene encodes the Hoda_Snf7_2 protein, whose amino acid sequence is shown in SEQ ID NO.2.

[0024] Specifically, Asgardian archaea is a collective term for a group of archaea at the phylum level, encompassing multiple archaea species (such as Hodarchaeota, also known as Hodarchaeales), and this group continues to expand. The ESCRT encoded by the Asgardian archaea genome is similar in composition and mechanism of action to the ESCRT in eukaryotes, and the two can be considered homologous proteins closely related in evolution and function. The Asgardian ESCRT-III contains at least two structural proteins, Snf7_1 and Snf7_2. Both of these structural proteins contain an ESCRT-core domain at their amino terminus similar to that of the eukaryotic ESCRT. Furthermore, the genes encoding Snf7_1 and Snf7_2... snf7_1 and snf7_2 These proteins are typically located adjacent to each other on the genome and form gene clusters close to other ESCRT protein-coding genes. Furthermore, other archaea genomes are known to encode ESCRT homologs; for example, the cell division system encoded by some TACK archaea genomes contains a homolog of ESCRT-III (CdvB), which is also an archaea ESCRT protein.

[0025] This invention uses *Hoda* archaea as an example to explore the antiphage activity of archaea ESCRT-III. *Hoda_Snf7_1* and *Hoda_Snf7_2* are two structural proteins of *Hoda* archaea ESCRT-III, and this invention encodes their genes. Walk_snf7_1 and Hoda_snf7_2 It is used in antiphage systems.

[0026] The following conditions can enable these two genes to exert their antiphage effect in E. coli, including: (1) Two genes can be cloned into an E. coli vector and regulated by an operon, and exert antiphage effects by expressing proteins through gene induction; there are no restrictions on the position of the two genes, the operon is a common E. coli operon, and there are no specific requirements for the E. coli vector.

[0027] (2) Two genes can be adjacent to each other and recombined into the Escherichia coli genome under the regulation of one operon, and exert antiphage effect by expressing protein through gene induction; there is no restriction on the position of the two genes before or after, and the operon is a common operon in Escherichia coli.

[0028] (3) The two genes can be separated and cloned into an E. coli vector under the regulation of the same operon. The protein is expressed through gene induction to exert antiphage effect. The operon is a common E. coli operon, and there are no specific requirements for the E. coli vector.

[0029] (4) The two genes can be separated and recombined into the Escherichia coli genome under the regulation of the same operon, and exert antiphage effect by expressing protein through gene induction; among them, the operon is a common operon in Escherichia coli.

[0030] Since the expression of the two genes alone inhibits the growth of E. coli, if the two genes are regulated by different operons and cloned into vectors or recombined into the E. coli genome, the expression levels of the two genes may differ, thereby inhibiting the growth of E. coli.

[0031] Based on the same inventive concept, embodiments of the present invention provide an antiphage system based on ESCRT-III, wherein the antiphage system comprises... Walk_snf7_1 Genes and Hoda_snf7_2 Genome composition; Among them, the Walk_snf7_1 The gene encodes the Hoda_Snf7_1 protein, whose amino acid sequence is shown in SEQ ID NO.1. Hoda_snf7_2 The gene encodes the Hoda_Snf7_2 protein, whose amino acid sequence is shown in SEQ ID NO.2.

[0032] Specifically, the antiphage system of this invention involves the encoding genes of two structural proteins, Hoda_Snf7_1 and Hoda_Snf7_2, of the Hoddle archaea ESCRT-III. This invention introduces Hoddle archaea ESCRT-III into *E. coli* via the pBAD plasmid. Walk_snf7_1 Genes and Hoda_snf7_2 The system induces the simultaneous expression of two genes. Growth curve analysis using liquid culture showed that, compared to the control group, this system did not affect the growth of *E. coli* under phage-free conditions and effectively protected *E. coli* against infection by 16 phages. Furthermore, expressing either gene alone caused growth defects in *E. coli*, effectively reducing the probability of gene mutations leading to loss of phage resistance.

[0033] Compared with existing technologies, the main advantages of the antiphage system of this invention are: (1) it consists of only two genes, making system construction simple and quick; (2) it is composed of structural proteins, making it less prone to losing antiphage activity due to gene mutations; (3) it has a broad-spectrum antiphage effect; and (4) it does not affect the normal growth of host bacteria. This system expands the understanding of antiphage systems and provides new ideas and technical means for preventing and controlling phage contamination in industrial microbial production processes.

[0034] In some embodiments, the Walk_snf7_1 The nucleotide sequence of the gene is shown in SEQ ID NO.3. Hoda_snf7_2The nucleotide sequence of the gene is shown in SEQ ID NO.4, but is not limited thereto.

[0035] This invention also provides an expression vector that expresses the antiphage system described above.

[0036] In some embodiments, the expression vector transmits the... Walk_snf7_1 Genes and Hoda_snf7_2 Genes can be obtained by recombination into plasmids, but are not limited to this.

[0037] In some more specific embodiments, the plasmid is selected from one of the pBAD plasmid, pET plasmid, pBR plasmid, and pUC plasmid, but is not limited thereto.

[0038] This invention also provides a host cell containing the expression vector described above.

[0039] In some embodiments, the host cell is Escherichia coli, but it is not limited thereto.

[0040] In some more specific embodiments, the *E. coli* is TOP10 (araBADC). - (but not limited to this).

[0041] The present invention also provides the use of the antiphage system, the expression vector, or the host cell as described above in the preparation of antiphage products.

[0042] The present invention will be further described below through specific embodiments.

[0043] In the examples, unless otherwise stated, all reagents used were commercially available products. The culture medium components used are as follows: LB solid medium: prepared with water, comprising 1% (w / v) tryptone, 0.5% (w / v) yeast extract, 1% (w / v) sodium chloride and 1.5% agar.

[0044] LB liquid medium: prepared with water, including 1% (w / v) tryptone, 0.5% (w / v) yeast extract and 1% (w / v) sodium chloride.

[0045] Example 1: Identification of the antiphage gene cluster of the archaea ESCRT This embodiment identified two structural proteins, Hoda_Snf7_1 and Hoda_Snf7_2, and their corresponding coding genes in Hoda archaea ESCRT-III, as detailed below: The amino acid sequence of the Hoda_Snf7_1 protein is shown in SEQ ID NO.1, and the amino acid sequence of the Hoda_Snf7_2 protein is shown in SEQ ID NO.2. Walk_snf7_1 The nucleotide sequence of the gene is shown in SEQ ID NO.3. Hoda_snf7_ 2 The nucleotide sequence of the gene is shown in SEQ ID NO.4.

[0046] Example 2: Construction of recombinant pBAD plasmid containing archaea ESCRT-III Using the PrimeSTAR®Max high-fidelity enzyme system from TaKaRa, as described in Example 1 Walk_snf7_1 Using a gene (already inserted into the pET28a(+) plasmid, a gene synthesis service product customized by General Biotechnology (Anhui) Co., Ltd.) as a DNA template, polymerase chain reaction (PCR) amplification was performed using forward primer 1 and reverse primer 1 to obtain DNA fragment 1 (SEQ ID NO. 5) with a length of 649 base pairs. The same method was used in Example 1... Hoda_snf7_2 Using a gene (already inserted into the pET28a(+) plasmid, a gene synthesis service product customized by General Biotechnology (Anhui) Co., Ltd.) as a DNA template, PCR amplification was performed using forward and reverse primers 2 to obtain DNA fragment 2 (SEQ ID NO. 6) with a length of 699 base pairs. Subsequently, using pBAD as a template, PCR amplification was performed using forward and reverse primers 3 to obtain DNA fragment 3 (SEQ ID NO. 7) with a length of 4102 base pairs. DNA fragments 1, 2, and 3 were purified and recovered separately using the TaKaRa MiniBEST gel extraction kit, and the DNA concentration was determined using NanoDrop. According to the TaKaRa In-Fusion seamless cloning system instructions, the TaKaRa seamless cloning system was prepared at a concentration ratio of DNA fragment 1: DNA fragment 2: DNA fragment 3 = 1:3:1 and reacted at 55°C for 10 minutes. The mixture was then transformed into competent E. coli cells. Next, the cells were plated on LB agar containing 100 mg / L ampicillin. Finally, colony PCR was performed using forward and reverse primers 4 to detect whether a 1493-base-pair DNA fragment 4 (SEQ ID NO. 8) was inserted into the pBAD plasmid. The inserted fragment region was sent to BGI Genomics Co., Ltd. in Shenzhen for gene sequencing verification. The final result was an archaea containing ESCRT-III (…). Walk_snf7_1 Genes and Hoda_snf7_2 Recombinant pBAD plasmid (gene) Figure 1 ).

[0047] The specific information about the primers is shown in Table 1.

[0048] Table 1. Specific information on primers used in this embodiment

[0049] Example 3: Effect of recombinant pBAD plasmid expressing archaea ESCRT-III on the growth of Escherichia coli Using the Omega Plasmid Mini Kit, plasmids containing the archaea ESCRT-III prepared in Example 2 were extracted. Walk_snf7_1 Genes and Hoda_snf7_2 The recombinant pBAD plasmid (containing the gene) was transformed into *E. coli* TOP10 (araBADC-) competent cells via chemical transformation. Simultaneously, the empty vector pBAD plasmid was transformed into *E. coli* TOP10 (araBADC-) competent cells using the same method. - Cells were used as controls. After transformation, single colonies were picked and sent to BGI Genomics Co., Ltd. in Shenzhen for sequencing verification of the inserted fragment. Clones with correct sequencing results were screened and inoculated into LB liquid medium containing 100 mg / L ampicillin, and cultured overnight at 37°C with shaking. The absorbance of the culture was measured the next day, and the overnight culture was diluted to OD using LB liquid medium containing 0.3% (w / v) L-arabinose and 100 mg / L ampicillin. 600 =0.01. The diluted bacterial culture was aliquoted into 200 μL wells of a clear 96-well plate. The 96-well plates were then sealed and placed in an automated microbial growth curve analyzer manufactured by Hainan MicroKrypton Biotechnology Co., Ltd., where the OD values ​​of each well were continuously monitored and recorded under incubation conditions at 37°C. 600 Growth curves were obtained by varying the growth over time. During cultivation, reciprocating oscillation was performed at 30 s intervals, a frequency of 20 Hz, and an amplitude of 2 mm to evaluate the effect of the expressed archaea ESCRT-III on the growth of host bacteria. Experimental results are as follows: Figure 2 The results showed that, under 0.3% (w / v) L-arabinose induction conditions, *E. coli* carrying the archaea ESCRT-III did not exhibit significant growth inhibition compared to the control strain carrying the empty vector pBAD, and the growth rate showed no significant difference. This indicates that expression of the archaea ESCRT-III protein has an inhibitory effect on the host *E. coli* TOP10 (araBADC). - The growth of ) was not significantly affected.

[0050] Example 4: Recombinant pBAD plasmid expressing archaea ESCRT-III confers phage resistance in Escherichia coli. To evaluate whether the expression of the archaea ESCRT-III enhances host cell resistance to bacteriophages, 16 bacteriophages were selected to infect *E. coli*. Bacteriophages T1 and T4 were commercially available products, while the remaining bacteriophages were isolated and preserved in the laboratory. All listed bacteriophages are capable of lysing *E. coli*. The experimental group consisted of *E. coli* strain TOP10 (araBADC-) carrying the recombinant pBAD plasmid containing the archaea ESCRT-III, as described in Example 3; the control group consisted of *E. coli* strain TOP10 (araBADC-) carrying the empty pBAD plasmid. Both the experimental and control groups were inoculated into LB liquid medium containing 100 mg / L ampicillin and cultured overnight at 37°C with shaking. The next day, the overnight cultures were diluted to OD using LB liquid medium containing 0.3% (w / v) L-arabinose and 100 mg / L ampicillin. 600 =0.01, and dispensed into transparent 96-well plates, 200 μL per well. The 96-well plates were sealed and placed in a fully automated microbial growth curve analyzer manufactured by Hainan MicroKrypton Biotechnology Co., Ltd., where the OD of each well was continuously monitored and recorded under incubation conditions at 37℃. 600 Growth curves were obtained by varying the growth over time; during cultivation, reciprocating oscillation was performed for mixing, with each oscillation lasting 30 seconds, a frequency of 20 times / second, and an amplitude of 2 mm. When OD... 600 When the culture volume reaches approximately 0.3-0.35 (incubation time approximately 4-4.5 hours), add the corresponding phages according to MOI values ​​of 0.01, 0.1, and 1, and continuously monitor the OD of the culture system. 600 The changes over time are used to reflect the host lysis process caused by bacteriophage infection. Experimental results are as follows: Figure 3 As shown, the results indicate that, compared with the control group, *Escherichia coli* expressing the archaea ESCRT-III exhibited a significant phage-resistant phenotype under all 16 tested phage infection conditions. Specifically, this was manifested in the OD of the culture system after phage infection. 600 The decline has slowed significantly, OD 600 The lowest value increases, or OD 600 The levels remained relatively stable with minimal decline. These results indicate that the recombinant pBAD plasmid expressing the archaea ESCRT-III can inhibit host lysis induced by various bacteriophage infections, thereby conferring resistance to multiple bacteriophages on Escherichia coli TOP10 (araBADC-).

[0051] To quantify the anti-phage effect, under MOI=1, the time point of phage addition was defined as t0 (t0=270 min). Within 600 min post-infection (270 min–870 min), the growth curve was analyzed using the trapezoidal method, and the area under the curve (AUC) was calculated. The AUC ratio was further calculated as AUC(expressed) / AUC(unexpressed), used to characterize the overall growth level of the culture system after infection (Table 2). The results showed that compared with the empty vector control, the AUC of the ESCRT-III expression group increased, and the AUC ratio ranged from 2.31 to 6.87, suggesting that ESCRT-III expression can inhibit host death caused by phage infection.

[0052] Table 2. AUC (OD) values ​​of different phages at 270-870 min post-infection under MOI=1 conditions. 600 (min) and AUC ratio

[0053] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

[0054] The specific nucleotide and amino acid sequences involved in this invention are as follows: SEQ ID NO.1: MVFDKFGNWLKGGSNSQNTRKWIVRLRMVEKRMVRQRNKLIKEERQMLKEVEHAIEQGDMSTARLLARDVAKNRSMARGCQQMASRVKAIKFKLEQAAATQA IGKDLKGLVRTLHTMNAQLKIPQLEGVLQQMEIETERIDIATEAMDEGFEMMTTSEEEDEVVDKIIGELSASKAATADFGLPSPDIRSQELQKELEKIKKG.

[0055] SEQ ID NO.2: MKRFKGFLTGKKPSSSTETKAKLKRTINQMEVKIKNYNRQADEQYELARNLLKSGNKTGAQQALKRRELYLTQITNTHAKIANLQRTIDTLDVSADNVSLTQTLGEAQVAIDQNIASASPEKTEEIMMALEDSIEQVSDMEEALTDTTITEIGMDVEVDDRISAQMAELEAEIASGSLPSTGVRTSEPPIPVSSDTPEKREAEKKKIDEMRKQIEEGLRS。

[0056] SEQ ID NO.3: ATGGTTTTTGATAAATTCGGCAATTGGCTGAAAGGTGGTAGTAATAGTCAGAATACCCGCAAATGGATTGTGCGTCTGCGTATGGTTGAAAAACGCATGGTGCGTCAGCGTAATAAACTGATTAAAGAAGAACGTCAGATGCTGAAAGAAGTGGAACATGCAATTGAACAGGGTGATATGAGTACCGCCCGTCTGCTGGCACGTGATGTTGCCAAAAATCGTAGTATGGCACGTGGTTGTCAGCAGATGGCCAGTCGTGTTAAAGCAATTAAATTTAAACTGGAGCAGGCAGCAGCCACCCAGGCAATTGGTAAAGATCTGAAAGGTCTGGTGCGTACCCTGCATACCATGAATGCCCAGCTGAAAATTCCGCAGCTGGAAGGTGTTCTGCAGCAGATGGAAATTGAAACCGAACGCATTGATATTGCCACCGAAGCAATGGATGAAGGTTTTGAAATGATGACCACCAGTGAAGAAGAAGATGAAGTTGTTGATAAAATCATCGGCGAACTGAGTGCAAGTAAAGCAGCCACCGCAGATTTTGGCCTGCCGAGTCCGGATATTCGTAGCCAGGAACTGCAGAAAGAACTGGAAAAAATTAAAAAGGGCTAA。

[0057] SEQ ID NO.4: ATGAAACGTTTTAAAGGTTTTCTGACCGGCAAAAAACCGAGCAGTAGCACCGAAACCAAAGCAAAACTGAAACGCACCATTAATCAGATGGAAGTTAAAATTAAGAACTACAACCGCCAGGCCGATGAACAGTATGAACTGGCCCGCAATCTGCTGAAAAGTGGTAATAAAACCGGTGCCCAGCAGGCACTGAAACGTCGTGAACTGTATCTGACCCAGATTACCAATACCCATGCCAAAATTGCAAATCTGCAGCGTACCATTGATACCCTGGATGTGAGTGCAGATAATGTTAGCCTGACCCAGACCCTGGGCGAAGCACAGGTGGCCATTGATCAGAATATTGCCAGCGCAAGTCCGGAAAAAACCGAAGAAATTATGATGGCCCTGGAAGATAGTATTGAACAGGTTAGTGATATGGAAGAAGCACTGACCGATACCACCATTACCGAAATTGGTATGGATGTTGAAGTTGATGATCGTATTAGCGCACAGATGGCCGAACTGGAAGCCGAAATTGCCAGTGGCAGTCTGCCGAGCACCGGTGTGCGTACCAGTGAACCGCCGATTCCGGTTAGTAGCGATACACCTGAAAAACGTGAAGCAGAAAAAAAAAAAATCGACGAAATGCGCAAACAGATTGAAGAAGGTCTGCGCAGTTAA。

[0058] SEQ ID NO.5: CTAACAGGAGGAATTAACCATGGTTTTTGATAAATTCGGCAATTGGCTGAAAGGTGGTAGTAATAGTCAGAATACCCGCAAATGGATTGTGCGTCTGCGTATGGTTGAAAAACGCATGGTGCGTCAGCGTAATAAACTGATTAAAGAAGAACGTCAGATGCTGAAAGAAGTGGAACATGCAATTGAACAGGGTGATATGAGTACCGCCCGTCTGCTGGCACGTGATGTTGCCAAAAATCGTAGTATGGCACGTGGTTGTCAGCAGATGGCCAGTCGTGTTAAAGCAATTAAATTTAAACTGGAGCAGGCAGCAGCCACCCAGGCAATTGGTAAAGATCTGAAAGGTCTGGTGCGTACCCTGCATACCATGAATGCCCAGCTGAAAATTCCGCAGCTGGAAGGTGTTCTGCAGCAGATGGAAATTGAAACCGAACGCATTGATATTGCCACCGAAGCAATGGATGAAGGTTTTGAAATGATGACCACCAGTGAAGAAGAAGATGAAGTTGTTGATAAAATCATCGGCGAACTGAGTGCAAGTAAAGCAGCCACCGCAGATTTTGGCCTGCCGAGTCCGGATATTCGTAGCCAGGAACTGCAGAAAGAACTGGAAAAAATTAAAAAGGGCTAAATGAAACGTTTTAAAGGT。

[0059] SEQ ID NO.6: AAAATTAAAAAGGGCTAAATGAAACGTTTTAAAGGTTTTCTGACCGGCAAAAAACCGAGCAGTAGCACCGAAACCAAAGCAAAACTGAAACGCACCATTAATCAGATGGAAGTTAAAATTAAGAACTACAACCGCCAGGCCGATGAACAGTATGAACTGGCCCGCAATCTGCTGAAAAGTGGTAATAAAACCGGTGCCCAGCAGGCACTGAAACGTCGTGAACTGTATCTGACCCAGATTACCAATACCCATGCCAAAATTGCAAATCTGCAGCGTACCATTGATACCCTGGATGTGAGTGCAGATAATGTTAGCCTGACCCAGACCCTGGGCGAAGCACAGGTGGCCATTGATCAGAATATTGCCAGCGCAAGTCCGGAAAAAACCGAAGAAATTATGATGGCCCTGGAAGATAGTATTGAACAGGTTAGTGATATGGAAGAAGCACTGACCGATACCACCATTACCGAAATTGGTATGGATGTTGAAGTTGATGATCGTATTAGCGCACAGATGGCCGAACTGGAAGCCGAAATTGCCAGTGGCAGTCTGCCGAGCACCGGTGTGCGTACCAGTGAACCGCCGATTCCGGTTAGTAGCGATACACCTGAAAAACGTGAAGCAGAAAAAAAAAAAATCGACGAAATGCGCAAACAGATTGAAGAAGGTCTGCGCAGTTAAATGGGGGGTTCTCATCAT。

[0060] SEQ ID NO.7:

[0061] SEQ ID NO.8:

Claims

1. Hoda_snf7_1 Genes and Hoda_snf7_2 The application of genes in the preparation of antiphage systems is characterized by, The Hoda_snf7_1 The gene encodes the Hoda_Snf7_1 protein, whose amino acid sequence is shown in SEQ ID NO.

1. Hoda_ snf7_2 The gene encodes the Hoda_Snf7_2 protein, whose amino acid sequence is shown in SEQ ID NO.

2.

2. An anti-phage system based on ESCRT-III, characterized in that, The antiphage system consists of Hoda_snf7_ 1 Genes and Hoda_snf7_2 Genome composition; Among them, the Hoda_snf7_1 The gene encodes the Hoda_Snf7_1 protein, whose amino acid sequence is shown in SEQ ID NO.

1. Hoda_snf7_2 The gene encodes the Hoda_Snf7_2 protein, whose amino acid sequence is shown in SEQ ID NO.

2.

3. The antiphage system according to claim 2, characterized in that, The Hoda_snf7_1 The nucleotide sequence of the gene is shown in SEQ ID NO.

3. Hoda_snf7_2 The nucleotide sequence of the gene is shown in SEQ ID NO.

4.

4. An expression carrier, characterized in that, The expression vector expresses the antiphage system according to any one of claims 2-3.

5. The expression vector according to claim 4, characterized in that, The expression vector will use the Hoda_snf7_ 1 Genes and Hoda_snf7_2 The gene was obtained by recombination into a plasmid.

6. The expression vector according to claim 5, characterized in that, The plasmid is selected from one of the following: pBAD plasmid, pET plasmid, pBR plasmid, and pUC plasmid.

7. A host cell, characterized in that, The host cell contains the expression vector according to any one of claims 4-6.

8. The host cell according to claim 7, characterized in that, The host cell is Escherichia coli.

9. The host cell according to claim 8, characterized in that, The E. coli strain is TOP10 (araBADC) - ).

10. The use of the antiphage system according to any one of claims 2-3, the expression vector according to any one of claims 4-6, or the host cell according to any one of claims 7-9 in the preparation of antiphage products.

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