Search

CN-121975763-A - NTase protein, screening method thereof and application thereof in preparation of anti-phage signal system antagonist and/or antibacterial preparation

CN121975763ACN 121975763 ACN121975763 ACN 121975763ACN-121975763-A

Abstract

The invention belongs to the technical field of microorganisms, and discloses an NTase protein, a screening method thereof and application thereof in preparing an antagonist of an anti-phage signal system and/or an antibacterial preparation. The NTase protein provided by the invention specifically comprises an amino acid fragment with a sequence shown as SEQ ID NO.1 or a variant fragment with at least 70% sequence identity with the sequence shown as SEQ ID NO.1, has excellent antagonism on CBASS systems of prokaryotes, can improve the infection efficiency of viruses on prokaryotes by inhibiting CBASS systems, thereby realizing better antibacterial effect and having very excellent application prospects in the research and development of novel antibacterial drugs.

Inventors

  • DONG XIYANG
  • LIU XINYUE
  • GUO XIAOMEI
  • LU ZIJIAN
  • CHENG RUI

Assignees

  • 自然资源部第三海洋研究所

Dates

Publication Date
20260505
Application Date
20251208

Claims (10)

  1. 1. An NTase protein, characterized in that it comprises an amino acid fragment having a sequence as shown in SEQ ID No. 1 or a variant fragment having at least 70% sequence identity to the sequence as shown in SEQ ID No. 1.
  2. 2. An expression cassette comprising a nucleic acid fragment encoding the NTase protein of claim 1.
  3. 3. The expression cassette of claim 2, wherein the expression cassette comprises a nucleotide sequence as set forth in SEQ ID No. 2 or a variant fragment having at least 70% sequence identity to the sequence set forth in SEQ ID No. 2.
  4. 4. The expression cassette of claim 2, wherein the expression cassette is selected from one or more of a plasmid, linear DNA, mRNA, and circRNA.
  5. 5. An expression strain, wherein the expression strain expresses the NTase protein of claim 1.
  6. 6. The method for screening the NTase protein according to claim 1 is characterized by comprising the steps of S1, screening a deep sea cold spring gene contig with a length of more than 5kb to obtain a deep sea cold spring virus genome set, S2, carrying out protein coding gene prediction and sequence clustering on the deep sea cold spring virus genome set to obtain a cold spring virus genome set, S3, screening an anti-defense gene and comparing a NTase family complete sequence with a structural domain model on the cold spring virus genome set to obtain an NTase gene set, and S4, carrying out three-dimensional structure prediction and known NTase reference protein structure comparison on the NTase gene set to obtain the NTase protein.
  7. 7. The method of claim 6, wherein the method of screening comprises at least one of the following features: (1) In the step S1, the virus gene screening comprises one or more of geNomad, virSorter and VIBRANT of analysis tools adopted by the virus gene screening; (2) In the step S2, the analysis tool adopted by the protein coding gene prediction is Prodigal, and the analysis tool adopted by the sequence clustering is MMseqs; (3) In the step S3, the analysis tool adopted by the screening of the anti-defense genes is ANTIDEFENSEFINDER, the database for comparison in the comparison of the NTase family full sequence and the structural domain model is an APIS database, and the analysis tool adopted by the screening of the anti-defense genes is DIAMOND and HMMER; (4) In step S4, the analysis tool adopted for the three-dimensional structure prediction of the protein is AlphaFold3, and the known NTase reference protein for comparison in the known NTase reference protein structure comparison is derived from an APIS database, and the analysis tool adopted is Foldseek.
  8. 8. Use of the NTase protein of claim 1, the expression cassette of any one of claims 2 to 4 or the expression strain of claim 5 for the preparation of an antagonist of an anti-phage signaling system.
  9. 9. Use of the NTase protein of claim 1, the expression cassette of any one of claims 2 to 4 or the expression strain of claim 5 in the preparation of an antibacterial formulation.
  10. 10. An antimicrobial preparation comprising a bacteriophage and the NTase protein of claim 1 or the expression cassette of any one of claims 2 to 4.

Description

NTase protein, screening method thereof and application thereof in preparation of anti-phage signal system antagonist and/or antibacterial preparation Technical Field The invention belongs to the technical field of microorganisms, and particularly relates to an NTase protein, a screening method thereof and application thereof in preparation of an antagonist of an anti-phage signal system and/or an antibacterial preparation. Background In recent years, with the increasing severity of drug-resistant bacteria, viruses are widely focused due to the specific sterilization characteristics, and the viruses have wide application prospects in the fields of clinical treatment, food safety, agricultural production and the like. However, prokaryotes possess a complex defense system that can be used to combat viral infections. The Cyclic oligonucleotide mediated anti-phage signaling system (CBASS) is a widely distributed and highly diverse prokaryotic antiviral defense system. CBASS is started when the cells are infected by viruses, and the coded CD-NTase and other circularized oligonucleotide synthases synthesize circulating oligonucleotide second messenger molecules after sensing the invasion of the viruses, the signal molecules are recognized by effector proteins (usually containing STING, TIR or SAVED domain structures) and trigger programmed cell death through membrane destruction, DNA degradation, NAD + consumption and other modes, so that the transmission of the viruses is blocked before the viruses are replicated and released, and the infection of the viruses is blocked. Large-scale genome and function analysis shows that CBASS system has wide distribution in microbiota and obvious diversity in system composition, has various structural and functional variants, forms a huge and highly diversified antiviral defense system family, and severely restricts the antibacterial effect of viruses. Current studies indicate that to combat the CBASS system, viruses evolve a series of anti-defenses to increase the efficiency of infection of prokaryotic cells by the virus. Recent studies have found that viruses themselves can encode ntases, they can synthesize competitive cyclic dinucleotide (cyclic dinucleotide, CDN) ligands and inhibit TIR NADASE effector proteins activated by linked STING-type CDN sensing domains (TIR-STING), preventing natural signal-triggered cell death, and that viruses encode MazG-like nucleotide pyrophosphorohydrolases (e.g., atd 1), depleting cell starvation alert molecules (p) ppGpp, blocking cytotoxin initiation, to avoid suicide reactions and provide conditions for the virus to complete replication, enabling the virus to better achieve lysis or proliferation inhibition of host cells, thus achieving better infection. Therefore, the NTase protein capable of effectively inhibiting CBASS systems is obtained, so that the infection efficiency of viruses to drug-resistant bacteria is improved, and the method has important significance for developing novel antibacterial drugs. Disclosure of Invention The first object of the present invention is to provide an NTase protein, which solves the problems of significant decrease in virus infection effect and unsatisfactory virus antibacterial effect caused by the presence of an anti-phage signal system in bacterial cells. A second object of the present invention is to provide an expression cassette. It is a third object of the present invention to provide an expression strain. The fourth object of the present invention is to provide a method for screening the above-mentioned NTase protein. It is a fifth object of the present invention to provide the use of the above-described NTase protein, expression cassette or expression strain for the preparation of an antagonist of an anti-phage signaling system. A sixth object of the present invention is to provide an application of the above-mentioned NTase protein, expression cassette or expression strain in the preparation of an antibacterial agent. A seventh object of the present invention is to provide an antibacterial agent. Specifically, the NTase protein provided by the invention comprises an amino acid fragment with a sequence shown as SEQ ID NO. 1 or a variant fragment with at least 70% sequence identity with the sequence shown as SEQ ID NO. 1. The expression cassette provided by the invention comprises a nucleic acid fragment for encoding the NTase protein. Further, the expression cassette comprises a nucleotide sequence as set forth in SEQ ID NO. 2 or a variant fragment having at least 70% sequence identity to the sequence set forth in SEQ ID NO. 2. Further, the expression cassette is selected from one or more of plasmid, linear DNA, mRNA, and circRNA. The screening method of the NTase protein comprises the steps of S1, screening a deep sea cold spring gene contig with the length of more than 5kb to obtain a deep sea cold spring virus genome set, S2, carrying out protein coding gene prediction and sequence clust