A novel anti-phage system and its application

Abstract

The present invention discloses a novel anti-phage system NBASS and its application, belonging to the fields of biotechnology and genetic engineering. NBASS is composed of three proteins, which can synthesize deoxyinosine triphosphate (dITP) after sensing the deoxynucleoside monophosphate kinase of the phage, activate the effector protein, and thus exert the anti-phage function. Based on this system, the present invention has developed a broad-spectrum anti-phage tool, which can be used for phage prevention and control in the process of microbial fermentation; the present invention has also developed a dITP detection tool, which can be used for scientific research and disease analysis related to nucleotide metabolism.

Description

Technical Field

[0001] The invention discloses a novel anti-bacteriophage system NBASS and application thereof, belonging to the fields of biotechnology and genetic engineering. Background Art

[0002] Bacteria have evolved complex and diverse anti-phage defense systems in the long-term coexistence and "arms race" with phages. These systems resist the invasion of phages by cutting phage nucleic acids, inhibiting phage genome replication, and killing infected cells to block phage proliferation. The proteins encoded by these systems often have unique activities and are the source of the development of various molecular tool enzymes. For example, restriction endonucleases derived from restriction modification systems have contributed to the development of genetic engineering technology, while sequence-specific nucleases such as Cas9 and Cas12 from the CRISPR-Cas system have promoted the revolution in gene editing technology. In recent years, many anti-phage systems have been studied, but more unknown systems are still waiting to be discovered, and the development of molecular tools based on these systems is also worth exploring.

[0003] Atypical nucleotides represented by deoxyinosine triphosphate (dITP) can be naturally generated in cells. For example, dATP can be converted to dITP by deamination. dITP accumulated in cells can be mistakenly inserted into newly synthesized DNA during DNA replication, causing DNA damage, leading to various diseases such as cancer and childhood encephalopathy (Mazin A. Zamzami, Inosine Triphosphate Pyrophosphatase (ITPase): Functions, Mutations, Polymorphisms and Its Impact on Cancer Therapies. Cells, 2022, 11, 384.). At present, the only method for detecting atypical nucleotides is LC-MS (Henryk Straube et al., An inosine triphosphate pyrophosphatase safeguards plant nucleic acids from aberrant purinenucleotides. New Phytologist, 2023, 237: 1759-1775), which has the disadvantages of low sensitivity, large number of samples required, reliance on large instruments, and long time consumption. In order to more quickly detect diseases caused by dITP accumulation and conduct scientific research related to dITP metabolism, it is urgent to develop a dITP detection tool.

[0004] In the process of industrial fermentation, the infection of fermentation strains by phages can cause significant economic losses. Model strains used in scientific research are often contaminated by phages, which brings troubles to the smooth progress of scientific research. Studies have shown that the use of natural anti-phage systems to construct phage-resistant strains is an effective phage prevention and control strategy (Xuan Zou et al., Systematic strategies for developing phage resistant Escherichia colistrains, Nature Communications, 2022, 13 (1): 4491.). For example, CRISPR-Cas systems and sulfur modification systems have been successfully used to construct phage-resistant strains of Escherichia coli, but a single system cannot achieve comprehensive defense against a wide variety of phages, and more broad-spectrum and efficient anti-phage systems are urgently needed. Summary of the invention

[0005] To achieve the above object, the present invention adopts the following technical solution:

[0006] The present invention provides a composition, characterized in that the composition is composed of three proteins: ADA, HAM1, and SIR2, wherein the ADA protein is an adenosine deaminase containing an adenosine deaminase domain, the HAM1 protein is a pyrophosphatase containing a HAM1-like non-canonical purine NTP pyrophosphatase domain, and the SIR2 protein is an NAD containing a SIR2-like domain. + Hydrolase.

[0007] In some embodiments, the adenosine deaminase domain is numbered PF00962 in the pfam database, the HAM1-like non-canonical purine NTP pyrophosphatase domain is numbered PF01725 in the pfam database, and the SIR2-like domain is numbered PF13289 in the pfam database.

[0008] In some embodiments, the sequences of the above-mentioned ADA, HAM1 and SIR2 proteins are shown as SEQ ID NOs. 1 to 3, respectively.

[0009] In some embodiments, the above-mentioned ADA, HAM1 and SIR2 proteins also include the proteins listed in Table 1 of the specification.

[0010] The present invention also provides an expression vector, characterized in that the expression vector can express the above-mentioned protein composition.

[0011] In some embodiments, the above expression vector contains a nucleic acid molecule with a sequence shown in SEQ ID NO.4.

[0012] The present invention also provides a host cell, characterized in that the host cell can produce the above-mentioned protein composition or the above-mentioned expression vector, and the host cell is a bacterial cell.

[0013] The present invention also provides the use of the above protein composition, or the above expression vector, or the above host cell in anti-phage.

[0014] The present invention also provides a dITP detection method, characterized in that NAD with a fluorescent group + The HS complex is incubated with the sample to be detected. If a fluorescent signal can be detected after incubation, the sample contains dITP; otherwise, it does not contain dITP. The HS complex is composed of HAM1 and SIR2 proteins.

[0015] In some embodiments, the HS complex is composed of HAM1 and SIR2 proteins in a molar ratio of 1:1.

[0016] In some embodiments, the above method comprises the steps of:

[0017] (1) preparing a reaction system containing 50 mM HEPES pH 7.5, 50 mM KCl, 1 mM TCEP, 50 μM ε-NAD, 100 μM ZnSO4, and 50 nM HS complex, wherein the HS complex is composed of HAM1 and SIR2 proteins at a molar concentration ratio of 1:1;

[0018] (2) Incubate the above reaction system and the sample to be tested at 37°C for 30 min;

[0019] (3) detecting a fluorescence signal on a fluorescence detection device;

[0020] (4) If a fluorescent signal is detected, the sample to be tested contains at least 0.1 nM dITP; if no fluorescent signal is detected, the sample to be tested contains less than 0.1 nM dITP.

[0021] The beneficial effects of the present invention are as follows: compared with the prior art, the present invention creatively discovered the NBASS system, which can synthesize deoxyinosine triphosphate (dITP) after sensing the deoxynucleoside monophosphate kinase of the phage, activate the effector protein, and exert the anti-phage function. Based on the system, the present invention has developed a broad-spectrum anti-phage tool that can be used for phage prevention and control in microbial fermentation processes; the present invention has also developed a dITP detection tool that can be used for scientific research and disease analysis related to nucleotide metabolism. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Gene cluster characteristics of NBASS system. (A) NBASS systems and upstream and downstream adjacent genes of different bacteria. Gabijia, CBASS, Cytidine deaminase, RM, and SPARTA are all known defense systems. (B) NBASS system from Escherichia coli NCTC13216 strain, the three proteins it encodes are ADA, HAM1, and SIR2.

[0023] Figure 2 The domain characteristics of the three proteins in the NBASS system. The first column is the structure of the three proteins in the NBASS system predicted by AlphaFold3 (ADA, HAM1, SIR2 from top to bottom), the second column is the structure of the ADA family, HAM1 family, and SIR2 family proteins that have been studied, and the third column is the superposition of their structures.

[0024] Figure 3 Verification of the anti-phage function of the NBASS system. (A) Expression of the NBASS system in this example. (B) Comparison of plaque formation units (PFU) of different phages in strains without NBASS (No system) and with NBASS (With system).

[0025] Figure 4 Verification of the anti-phage function of NBASS system in E. coli BL21 (DE3) strain. Plaque formation experiment of different phages in strains without NBASS (No system) and with NBASS (With system).

[0026] Figure 5Phage DNK protein is a key factor in activating the NBASS system. (A) Analysis of Rao1 phage escape mutants. On the left is the plaque formation experiment of 5 escape mutants in strains without NBASS (No system) and with NBASS (With system); on the right is the sequencing analysis of escape mutants, and their mutation sites are concentrated in the internal or promoter region of the DNK encoding gene gp56. (B) Co-expression of Rao1-DNK or its mutants (DNK m Analysis of the effects of Rao1-DNK and NBASS system on cell survival. Expression of Rao1-DNK activated NBASS system and caused cell death, while expression of its mutant (DNK m :DNK-K15A) cannot activate NBASS system and cause cell death. (C) DNK expressing other three phages activate NBASS system and cause cell death.

[0027] Figure 6 Analysis of the interaction between ADA, HAM1, SIR2, and Rao1-DNK. (A) Mass spectrometry analysis of co-purification of ADA, HAM1, SIR2 (with or without 6× His tag), and Rao1-DNK. The horizontal axis is the abundance of the identified protein in the co-purified sample with 6× His tag, and the vertical axis is the enrichment fold in the co-purified sample with 6× His tag relative to the co-purified sample without 6× His tag. (B) SDS-PAGE analysis of the single purification of SIR2 with 6× His tag and the co-purification with HAM1.

[0028] Figure 7 Activity analysis of ADA.

[0029] Figure 8 Activity analysis of Rao1-DNK.

[0030] Fig. 9 ADA and E. coli NDK tandem reaction using dADP as substrate to synthesize dITP. (A) Schematic diagram of this experiment. (B) LC-MS analysis of the products of the tandem reaction (using dITP as a standard). (C) Secondary mass spectrometry analysis of the products of the tandem reaction (using dITP as a standard).

[0031] Fig.10 dITP activates HS to degrade NAD + .

[0032] Fig.11 Analysis of the ability of the NBASS system effector protein complex HS to detect dITP.

[0033] Fig.12 Analysis of the specificity of the NBASS system effector protein complex for HS detection of dITP.

[0034] Fig.13 Schematic diagram of the mechanism of action of the NBASS system in resisting bacteria (A) and the principle diagram of the system effector complex detecting dITP (B). DETAILED DESCRIPTION

[0035] In order to more clearly illustrate the technical solutions and embodiments of the present invention, the technical solutions of the present invention are clearly and completely described below in conjunction with the accompanying drawings and specific technical methods. The described embodiments are only part of the embodiments of the present invention, not all of the embodiments, and do not constitute a limitation of the present invention in any way.

[0036] The experimental methods in the following examples are conventional methods unless otherwise specified, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, instruments, etc. used in the following examples are all commercially available unless otherwise specified. The quantitative tests in the following examples were repeated three times, and the results were averaged.

[0037] Example

[0038] Example 1 Identification of NBASS System

[0039] Prokaryotes are the most diverse and diverse cellular organisms on earth, and the number and diversity of their bacteriophages are about ten times that of prokaryotes. There is a fierce relationship between bacteriophages and prokaryotes in terms of infection and defense. The long-term "arms race" between them has prompted them to evolve complex and diverse defense and anti-defense systems. Defense systems are usually clustered and distributed in bacterial genomes to form so-called "defense islands". At the same time, there is a phenomenon of "modular evolution" in defense systems, that is, components of different defense systems are often exchanged. In previous work, the inventors identified nucleases, transmembrane proteins, and SIR2 family proteins associated with the long-B pAgos defense system (Catalytically inactive long prokaryotic Argonaute systems employ distinct effectors to confer immunity via abortive infection. Nature Communications, 2023, 14 (1): 6970.). On this basis, the inventors intend to combine the "defense island" strategy and the "modular evolution" strategy to identify new anti-phage systems.

[0040] The inventors first used the AlphaFold2 tool to search for proteins with similar structures to nucleases, transmembrane proteins, and SIR2 proteins on a large scale, and screened for new anti-phage systems based on whether there were other anti-phage systems near the location of the protein-encoding gene on the genome. After screening, the inventors discovered a new system that often exists in the same genomic region as other anti-phage systems and encodes SIR2 protein ( Figure 1 The inventors named it the atypical nucleotide-mediated anti-phage signaling pathway system ( n on-canonical nucleotide- b ased a ntiphage s signaling s sysstems, NBASS). This system conservedly encodes three proteins in different species. Through structural prediction, it was found that these three proteins are adenosine deaminase containing adenosine deaminase domain (PF00962 in the pfam protein family database), pyrophosphatase containing HAM1-like non-canonical purine NTP pyrophosphatase domain (PF01725), NAD+ hydrolase containing SIR2-like domain (PF13289) and NAD+ hydrolase containing SIR2-like domain (PF13289). Figure 2 ). The present invention named these three proteins ADA, HAM1, and SIR2. ADA, HAM1, and SIR2 proteins are widely present in various bacterial species (Table 1). In the upstream and downstream positions of the genome encoding genes of these three proteins, there are known defense systems such as Gabijia, CBASS, Cytidine deaminase, RM, and SPARTA. The inventors selected the NBASS system from E. coli NCTC13216 ( Figure 1 B) Functional verification was performed, and the sequences of the three proteins in the strain were shown as SEQ ID NOs. 1 to 3.

[0041] Table 1 ADA, HAM1 and SIR2 protein information in NBASS systems from various sources

[0042]

[0043]

[0044]

[0045]

[0046] Example 2 Determination of the anti-phage function of the NBASS system

[0047] The present invention synthesized the NBASS system derived from E. coli NCTC13216 (containing a natural promoter, the sequence of which is shown in SEQ ID NO.4), and constructed it on the pET28a plasmid to obtain the pET28a-NBASS plasmid (such as Figure 3 A) and the plasmid was transferred into E. coli MG1655 strain. The anti-phage ability of NBASS system was analyzed by plaque formation experiment, with MG1655 strain containing pET28a empty vector as control. The specific operation is as follows:

[0048] 1) The present invention uses E. coli MG1655 as a host to enrich and culture various phages (T1, T2, T3, T4, T5, T6, T7, SSL-2009a and Rao1). The stored E. coli MG1655 was inoculated into MMB medium (LB medium supplemented with 0.1 mM MnCl2 and 5 mM MgCl2), cultured at 37°C overnight, and the overnight culture was transferred to 5 mL of fresh MMB medium at a ratio of 1:100, and cultured at 37°C until the logarithmic phase (OD 600 ~0.2), then add phage with a multiplicity of infection (MOI) of 0.01 to 1 mL of culture, let it stand for 5 minutes, and then put it in a 37°C shaker for 4 hours to completely lyse the host bacteria. Then centrifuge the culture at 12000 rpm / min for 10 minutes, and filter the supernatant with a 0.22μm filter membrane (Millipore, SLGPM33RS) to remove cells. Finally, store the obtained phage suspension at 4°C.

[0049] 2) The pET28a-NBASS and pET28a plasmids were transferred into E.coli MG1655 competent cells respectively, and the transformation products were spread on LB plates containing Kan (50μg / mL) antibiotics and cultured at 37℃ overnight. Then, single clones were picked and added to 5mL MMB medium containing Kan and cultured at 37℃ overnight. Take 200μL of the bacterial solution after overnight culture, add it to 10mL 50℃ preheated MMB medium (containing 0.5% Agar and Kan), mix well and spread it on the lower MMB plate (containing 1.5% Agar and Kan), and wait for the plate to solidify on the clean bench. Take 3μL of the above gradient dilution of phage and spot it on the bacterial layer of the plate. After the plate is blown dry on the clean bench, it is inverted in a 37℃ incubator for 6-8h. When the plaques are clearly visible, take pictures of the plate and count them.

[0050] The results are as follows Figure 3As shown in Figure B, the NBASS system can resist the invasion of multiple phages (T2, T3, T4, T6, T7, SSL-2009a and Rao1) in the E. coli model strain MG1655. Among them, the plaque formation unit of T2, T4, T6, SSL-2009a and Rao1 can be reduced by about 100-1000 times. Although the resistance to T3 and T7 phages is less than 100 times, it will cause their plaques to become significantly smaller, which also confirms that NBASS has a certain resistance to T3 and T7 phages.

[0051] This example demonstrates that the NBASS system can resist the invasion of multiple bacteriophages.

[0052] Example 3 Anti-phage function of NBASS system in E. coli BL21 (DE3) strain

[0053] E.coli BL21 (DE3) strain is widely used to express recombinant proteins, and often encounters phage contamination, causing significant losses. In order to verify whether the NBASS system can resist phage invasion in the BL21 (DE3) strain, the present invention transfers the above-mentioned pET28a plasmid containing the NBASS system into the BL21 (DE3) strain, and uses a plaque formation experiment to analyze the ability of the NBASS system to resist phage in the BL21 (DE3) strain using the MG1655 strain containing the pET28a empty vector as a control. The specific operation is referred to Example 2, except that the competent cells are replaced with E.coli BL21 (DE3), and the phages tested are T2, T3, T4, T6, SSL-2009a and Rao1.

[0054] The results are as follows Figure 4 As shown, the NBASS system can also resist the invasion of multiple phages (T3, SSL-2009a and Rao1) in the BL21 strain, reducing the plaque formation unit by about 100-1000 times. Unlike Example 2, the NBASS system can only reduce the T3 phage plaque formation unit by about 10 times in the MG1655 strain, while it can reach more than 1000 times in the BL21 (DE3) strain. At the same time, in the MG1655 strain, the NBASS system can effectively resist T2, T4 and T6 phages, while in BL21 it can only slightly reduce the plaque size. The above results show that the NBASS system can also play a role in the BL21 (DE3) strain, but compared with the MG1655 strain, the inhibitory effect on different phages is different.

[0055] Example 4 Identification of key factors in phage-activated NBASS system

[0056] In order to determine the mechanism of action of the NBASS system and the scope of its ability to resist phages, the inventors used the "phage evolution" strategy to analyze how the NBASS system senses phage invasion. The specific implementation method is as follows: a gradient-diluted Rao1 phage is spotted onto the bacterial layer of an E.coli MG1655 plate containing the NBASS system. After the plate is dried in a clean bench, it is inverted in an incubator at 37°C for 6-8 hours. After plaques are formed on the bacterial layer, 5 plaques are picked, which are the escape mutants of the evolved Rao1 phage. These escape mutants are enriched using the E.coli MG1655 strain, and then spotted onto the bacterial layer of an E.coli MG1655 plate containing or not containing the NBASS system to verify whether the NBASS system can resist these escape mutants. The results showed that these mutants were able to successfully infect strains containing the NBASS system ( Figure 5 A). The genomes of these mutants were subsequently sequenced and compared with the genome of the wild-type Rao1 phage. The results showed that all mutations occurred within the gene encoding deoxynucleotide monophosphate kinase (DNK) or in the promoter region, suggesting that DNK may be a key factor in activating the NBASS system.

[0057] The present invention further tested whether Rao1-DNK (amino acid sequence as shown in SEQ ID NO.5) can activate the NBASS system. The specific implementation method is: first, based on the pCDFduet-1 vector, an expression plasmid pCDFduet-1-NBASS of the NBASS system (containing its own promoter) was constructed. At the same time, using Rao1 phage genomic DNA as a template, the coding gene sequence of Rao1-DNK was amplified and inserted into the pBadHisA plasmid to obtain the pBad-R1DNK plasmid (araBAD promoter controls the expression of R1DNK). At the same time, a plasmid expressing the Rao1-DNK mutant (pBad-R1DNK D15A The plasmid assembly method used was based on the ClonExpress assembly kit (Vazyme, C112-02). The above plasmids were co-transfected into E. coli MG1655 competent cells in different combinations, and single clones were picked and cultured overnight at 37°C in 5 mL of MMB medium containing antibiotics. Subsequently, the culture after overnight culture was diluted 1:100 into 5 mL of MMB medium containing antibiotics and cultured at 37°C for about 2 hours. 600 After reaching 0.8, the culture was diluted 10-fold using MMB medium (10 -1 ~10 -6After the dilution is completed, 5 μL of the dilution is spotted on an MMB plate (1.5% agar) with appropriate antibiotics and 1% glucose (to inhibit DNK expression) or 0.05% L-ara (to induce DNK expression), and the plate is dried in a clean bench and incubated overnight at 37°C. After the incubation is completed, the plate is photographed.

[0058] The results are as follows Figure 5 As shown in B, in the absence of the system, cells grow normally. However, in the presence of the NBASS system, induced expression of Rao1-DNK can lead to cell death and inhibit culture growth, indicating that Rao1-DNK can activate the NBASS system. In addition, the conservative catalytic site mutation of Rao1-DNK eliminates cytotoxicity, which also indicates that the enzymatic activity of Rao1-DNK protein is a necessary condition for activating the NBASS system.

[0059] Based on the above results, the present invention further synthesized DNKs from phages such as Klebsiella phage vB_KpnS-VAC2, Vibriophage XZ1 and Escherichia phage KarlJaspers (these DNK sequences are numbered in the database as QZE50419.1, UOL51207.1, and QXV82329.1, respectively) and used a similar method to test whether they can activate the NBASS system. The results are as follows Figure 5 As shown in C, all the DNKs tested can activate the NBASS system and cause cell death, which suggests that the NBASS system may defend against a variety of phages encoding DNK proteins.

[0060] Example 5 Analysis of the interactions between the components of the NBASS system and with Rao1-DNK

[0061] Many defense systems sense phage invasion by directly interacting with phage proteins and activate effector factors through interactions between defense system components, thereby mediating immune responses. Since Rao1-DNK can activate the immune response of the NBASS system, the inventors speculate that Rao1-DNK may have direct interactions with components of the NBASS system. The present invention uses the "protein co-purification-mass spectrometry analysis" strategy to verify this hypothesis. The specific implementation method is as follows: first construct pRSF-R1DNK-ADA, pCDF-SIR2 m -HAM1 and pCDF-SIR2 m (tag-free)-HAM1 three plasmids. pRSF-R1DNK-ADA is used to co-express Rao1-DNK and ADA, pCDF-SIR2 m-HAM1 is used to co-express the SIR2-H146A mutant and HAM1 containing a 6× histidine tag, pCDF-SIR2 m (tag-free)-HAM1 was used to co-express the SIR2-H146A mutant and HAM1 without the 6×histidine tag. ADA, HAM1, and SIR2 were expressed by the T7 promoter regulated by the lactose operator, and Rao1-DNK was expressed by the araBAD promoter. m -HAM1, pRSF-R1DNK-ADA, and pCDF-SIR2 m (tag-free)-HAM1 was transformed into E.coli BL21 (DE3) cells. After the transformants were expanded and cultured, protein expression was induced using 0.4mM IPTG and 0.2% L-arabinose, and then the protein was purified by nickel ion affinity chromatography, and finally the obtained protein sample was analyzed by mass spectrometry. The mass spectrometry data were aligned to the proteome database of Escherichia coli (strains B / BL21-DE3) (Uniprot, UP000009074) and the amino acid sequences of the four proteins to be tested (ADA, HAM1, SIR2, R1DNK), the relative abundance of the detected proteins was calculated, and their enrichment in the purified samples containing the histidine tag was compared. The results are shown in Figure 2. Figure 6 As shown in A, only SIR2 and HAM1 were highly enriched in the sample containing the histidine tag purification, suggesting that SIR2 and HAM1 interact with each other but not with ADA and Rao1-DNK.

[0062] In order to further verify whether HAM1 and SIR2 form a stable complex, the present invention constructed a pCDF-SIR2-HAM1 plasmid for co-expression of wild-type SIR2 and HAM1 containing a 6× histidine tag. The target protein was purified by nickel ion affinity chromatography and molecular exclusion chromatography, and the purified sample was analyzed by SDS-PAGE. The results showed that HAM1 and SIR2 were co-purified to obtain ( Figure 6 B) confirmed that HAM1 and SIR2 can form a stable complex (hereinafter referred to as HS).

[0063] Example 6 Analysis of the mechanism of synthesizing signal molecules by NBASS system

[0064] Because ADA and Rao1-DNK do not interact with HS, the inventors speculate that ADA and Rao1-DNK may activate the immune response of the NBASS system by synthesizing signal molecules. In order to verify this idea, the present invention characterizes the biochemical activities of ADA and Rao1-DNK. At the same time, the inventors speculate that the nucleoside diphosphate kinases (NDKs) of Escherichia coli may also be involved in the synthesis of signal molecules, so the biochemical activity of the enzyme was also analyzed. The analysis process includes constructing an expression plasmid, expressing the target protein in the E. coli BL21 (DE3) strain, purifying the target protein by nickel ion affinity chromatography and molecular exclusion chromatography, and finally analyzing the activity of the target protein by a specific reaction system. Among them, plasmid construction, protein expression and purification are all conventional methods.

[0065] (1) Analysis of ADA biochemical activity

[0066] Typical ADA family proteins can catalyze the deamination of adenine to obtain hypoxanthine. In order to determine the substrate specificity of the ADA protein of the NBASS system, the present invention tests the deamination activity of ADA on substrates such as dAMP, dADP, dATP, AMP, ADP, and ATP. The specific implementation method is: prepare a 50μL reaction system, including 50mM HEPES pH 7.5, 50mM KCl, 1mM TCEP, 1mM nucleotide substrate and 5μM ADA. The reaction system was incubated at 37°C for 1 hour. After incubation at 90°C for 5 minutes to inactivate the ADA protein, 1.5μL FastAP Thermosensitive Alkaline Phosphatase was added, and then incubated at 37°C for 1 hour and 15 minutes. After centrifugation to remove the denatured protein, 350μL MilliQ H2O was added, and then the product was analyzed by Ultra high-performance liquid chromatography (UHPLC). The results show that ADA has the strongest deamination activity for dAMP, and also has obvious deamination activity for dADP, AMP and ADP ( Figure 7 ).

[0067] (2) Determination of the biochemical activity of Rao1-DNK

[0068] In order to reveal the molecular mechanism of phage DNK activating the NBASS system, the present invention tested the kinase activity of Rao1-DNK against different nucleotides. The specific implementation method is: prepare a 50μL reaction system, including 50mM HEPES pH 7.5, 50mMKCl, 10mM MgCl2, 1mM ATP, 1mM TCEP, 1mM nucleotide substrate (dAMP, dGMP, dCMP, dTMP, dIMP, AMP or IMP) and 1μM Rao1-DNK. The reaction system was incubated at 37°C for 1 hour. After incubation at 90°C for 5 minutes to inactivate the ADA protein, the denatured protein was removed by centrifugation, and 750μL 5mM Tris-HCl pH 8.0 buffer was added, and the product was analyzed by ion exchange chromatography. The results show that Rao1-DNK has strong kinase activity against dAMP, dGMP, and dCMP, and also has partial activity against dTMP, but the activity against dIMP is almost unobservable ( Figure 8 ). Based on the activity analysis of ADA and Rao1-DNK, the inventors concluded that ADA and Rao1-DNK can convert dAMP into dIDP when they coexist. The specific process is that Rao1-DNK phosphorylates dAMP to obtain dADP, and ADA converts dADP into dIDP through deamination.

[0069] (3) NDK participates in the synthesis of dITP

[0070] NDK and ATP in bacterial cells can quickly convert nucleoside diphosphates into nucleoside triphosphates. Therefore, the inventors speculate that the signal molecule synthesized by the NBASS system sensing the phage DNK is not dIDP, but dITP phosphorylated by NDK. In order to verify this hypothesis, the inventors designed a tandem reaction with dADP as substrate and ADA and NDK as catalysis, and analyzed whether the final product contained dITP ( Fig. 9 A). The specific implementation method is as follows: prepare a 50 μL reaction system, including 50 mM HEPES pH 7.5, 50 mM KCl, 1 mM TCEP, ADA (0 / 20 μM), 1 mM dADP, incubate the reaction system at 37°C for 60 minutes, then add 100 μM ZnSO4, 10 mM MgCl2, NDK (0 / 4 μM), 2 mM ATP, and then incubate the reaction system at 37°C for 15 minutes. After incubating at 90°C for 5 minutes to inactivate the protein, add 150 μL MilliQ H2O, centrifuge to remove the precipitated protein, and analyze the final product by liquid chromatography-mass spectrometry. The results are as follows Fig. 9 As shown, E. coli NDK protein and ADA are able to convert dADP into dITP.

[0071] Example 7 dITP activates NBASS system effector protein complex HS to degrade NAD + .

[0072] The above results suggest that after phage invasion, ADA of the NBASS system synthesizes dITP together with DNK of the phage and NDK of the host. dITP may function as a signaling molecule of the NBASS system. + Therefore, the inventors tested whether dITP could degrade the HS complex to degrade NAD + The specific implementation method is: prepare a 50 μL reaction system, including 50 mM HEPES pH 7.5, 50 mM KCl, 1 mM TCEP, 1 mM NAD + , 100μM ZnSO4, 100nM HS and dITP (0 / 1nM), where HS was co-expressed with SIR2 and HAM1 proteins (with 6×His tag) using E. coli BL21 (DE3) strain, and the target protein was purified by nickel ion affinity chromatography and molecular exclusion chromatography, and the concentration of the target protein was measured by BCA protein quantification kit. The molar concentration of HS was calculated based on the ratio of SIR2 and HAM1 in the complex of 1:1. The reaction system was incubated at 37°C for 30 minutes. After incubation at 90°C for 5 minutes to inactivate the HS complex, 450μL MilliQ H2O was added, and after centrifugation to remove the denatured protein, the product was analyzed by UHPLC, and the substrate NAD was analyzed at the same time. + , and possible products ADPR and NAM as standard products. The results showed that HS can convert NAD + Degraded to ADPR and NAM, but the activity is low. Adding 1nM dITP can significantly increase the degradation of NAD by HS + The product is also ADPR and NAM ( Fig.10 ), confirming that dITP can activate the NADase activity of HS.

[0073] Example 8 Establishment of dITP detection method based on HS

[0074] Degradation of NAD by activation of dITP in the HS complex + The characteristics of the ε-NAD (NAD +Analogs of dITP) as substrates, a dITP detection tool is developed, the principle of which is: if the sample to be tested contains dITP, the sample to be tested is incubated with HS complex and ε-NAD, and the activated HS complex can degrade ε-NAD to produce a fluorescent product (ε-ADPR); conversely, ε-NAD will not be degraded and no fluorescence will be produced. The fluorescence change can be measured by an enzyme marker to detect dITP in the sample. The specific implementation method is: prepare a 50μL reaction system containing 50mM HEPES pH 7.5, 50mMKCl, 1mM TCEP, 50μMε-NAD (Sigma, N2630), 100μM ZnSO4, 50nM HS complex and different concentration gradients of dITP (0, 0.1nM, 1nM, 10nM, 100nM). Then, the samples were immediately incubated at 37°C in a Spark microplate reader, and the fluorescence intensity was measured every 30 seconds at an excitation wavelength of 300 nm and an emission wavelength of 410 nm. Fig.11 As shown, 0.1 nM dITP can also activate the HS complex and produce an obvious fluorescence signal, which proves that this reaction system can detect dITP and the detection sensitivity can reach 0.1 nM dITP.

[0075] Example 9 Specificity analysis of HS complex detection of dITP

[0076] The present invention further tested the specificity of HS complex in detecting nucleotides. In a 50 μL reaction system, 50 mM HEPES pH 7.5, 50 mM KCl, 1 mM TCEP, 50 μM ε-NAD (Sigma, N2630), 100 μM ZnSO4, 50 nM HS complex and 100 nM different nucleotides were contained. The tested nucleotides included dNTP, NTP, dITP, dUTP and ITP. Then, the samples were immediately incubated at 37°C in a Spark microplate reader, and the fluorescence intensity was measured every 30 seconds at an excitation wavelength of 300 nm and an emission wavelength of 410 nm. The results are shown in FIG. Fig.12 As shown, only dITP can activate the HS complex to degrade ε-NAD and generate fluorescent signals, confirming that the HS complex has a high specificity in detecting dITP.

[0077] The NBASS system can be used as an anti-phage and dITP detection tool. The schematic diagram of the mechanism of action of the NBASS system against bacteria and the schematic diagram of the principle of the system effector complex to detect dITP are shown in Fig.13 .

[0078] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A composition, characterized in that The composition is composed of three proteins: ADA, HAM1 and SIR2. The sequences of the ADA, HAM1 and SIR2 proteins are shown in SEQ ID NOs. 1 to 3, respectively.

2. An expression vector, characterized in that: The expression vector is capable of expressing the composition of claim 1.

3. The expression vector according to claim 2, characterized in that The expression vector contains a nucleic acid molecule with a sequence shown in SEQ ID NO.

4.

4. A host cell, characterized in that The host cell can produce the composition according to claim 1 or contains the expression vector according to any one of claims 2 to 3, and the host cell is an Escherichia coli cell.

5. Use of the composition according to claim 1, or the expression vector according to any one of claims 2 to 3, or the host cell according to claim 4 in anti-phage.

6. A dITP detection method for non-disease diagnosis purposes, characterized in that: The NAD and HS complex with fluorescent groups is incubated with the sample to be tested. If a fluorescent signal can be detected after incubation, the sample contains dITP; otherwise, it does not contain dITP; the HS complex is composed of HAM1 and SIR2 proteins; the sequences of the HAM1 and SIR2 proteins are shown in SEQ ID NO.2 and SEQ ID NO.3, respectively.

7. The method according to claim 6, characterized in that The HS complex is composed of HAM1 and SIR2 proteins in a 1:1 molar ratio.

8. The method according to claim 7, characterized in that The method comprises the following steps: (1) preparing a reaction system containing 50 mM HEPES pH 7.5, 50 mM KCl, 1 mM TCEP, 50 μM ε-NAD, 100 μM ZnSO4 and 50 nM HS complex, wherein the HS complex is composed of HAM1 and SIR2 proteins at a molar concentration ratio of 1:1; (2) Incubate the above reaction system and the sample to be tested at 37°C for 30 min; (3) detecting a fluorescence signal on a fluorescence detection device; (4) If a fluorescent signal is detected, the sample to be tested contains at least 0.1 nM dITP; if no fluorescent signal is detected, the sample to be tested contains less than 0.1 nM dITP.

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