Application of enterococcus pore-forming toxin mutant protein in nanopore detection

Abstract

The invention belongs to the technical field of target sample characterization, and provides application of enterococcus pore-forming toxin mutant protein in nanopore detection. The enterococcus pore-forming toxin mutant protein is EPX1 nanopore mutant protein and is obtained by mutating wild type EPX1 nanopore protein, the amino acid sequence of the wild type EPX1 nanopore protein is as shown in SEQ ID NO: 1, and the mutation means that one or more amino acids of the wild type EPX1 nanopore protein are mutated into common amino acids except original amino acids. The EPX1 protein (wild type) is a type of pore-forming toxin protein and widely exists in various living bodies in nature, and a mutant of the EPX1 protein shows excellent pore-forming capacity after being modified (after amino acid mutation). The structural stability, the molecular recognition efficiency and the detection performance of the EPX1 nanopore mutant protein are remarkably improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of characterizing target samples, and in particular relates to the application of enterococcal pore-forming toxin mutant proteins in nanopore detection. Background Art

[0002] Nanopore sequencing, the core of third-generation sequencing technology, demonstrates tremendous potential in biomedical testing, genomic analysis, and other fields due to its real-time, label-free, and ultra-long read length advantages. While traditional biochemical detection methods (such as ELISA, protein blotting, and mass spectrometry) have been applied in scenarios such as cancer marker detection, they suffer from significant drawbacks such as time-consuming and labor-intensive processes, cumbersome sample preparation, and low sensitivity. Nanopore-based single-molecule sensing, however, uses electromotive force to drive analytes through the nanopore and exploits changes in ionic current for highly sensitive detection, offering a new approach to overcoming the shortcomings of traditional methods.

[0003] However, the structural limitations of existing nanopore proteins, insufficient detection precision, and complex preparation processes have severely hampered the widespread application of this technology. Most reported biological nanopores (such as α-hemolysin, MspA, and CsgG) are constructed from homogeneous monomers that aggregate to form multimeric structures. This assembly process is complex and results in poor stability. For example, MspA and α-HL require complex recombinant expression and purification processes, which are costly and difficult to produce on a large scale. In summary, the development of novel nanopore proteins with simple structures, efficient preparation, and high sequencing accuracy, as well as the optimization of analyte capture and rate control mechanisms, are key breakthroughs in advancing nanopore technology towards industrial application. Summary of the Invention

[0004] In view of the deficiencies of the prior art, the present invention provides the use of a mutant protein of enterococcal pore-forming toxin in nanopore detection, which aims to solve the problems mentioned in the background art.

[0005] In a first aspect, the present invention provides a use of an enterococcal pore-forming toxin mutant protein in nanopore detection, wherein the enterococcal pore-forming toxin mutant protein is an EPX1 nanopore mutant protein, which is obtained by mutating a wild-type EPX1 nanopore protein, the amino acid sequence of which is shown in SEQ ID NO: 1, wherein the mutation refers to: one or more amino acids of the wild-type EPX1 nanopore protein are mutated to common amino acids other than the original amino acids; The common amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, and histidine.

[0006] Furthermore, the EPX1 nanopore mutant protein is selected from the following A, B, C or D: A: One or more of amino acids V11, L12, and G13 of the wild-type EPX1 nanopore protein are mutated to common amino acids other than the original amino acids; B: One or more of amino acids N33, Q34, and Y35 of the wild-type EPX1 nanopore protein are mutated to common amino acids other than the original amino acids; C: One or more of amino acids K139, Q140, and V141 of the wild-type EPX1 nanopore protein are mutated to common amino acids other than the original amino acids; D: One or more of amino acids T174, T175, and Y176 of the wild-type EPX1 nanopore protein are mutated to common amino acids other than the original amino acids.

[0007] Furthermore, in A: the amino acid V11 of the wild-type EPX1 nanopore protein is mutated to V11P, V11L, V11G, V11R, V11Q, V11E, V11C, V11W, V11H, V11K, V11F, V11A, V11S, V11Y, V11N or V11D; preferably, the amino acid V11 of the wild-type EPX1 nanopore protein is mutated to V11A, V11L or V11N; Amino acid L12 of the wild-type EPX1 nanopore protein was mutated to L12A or L12V; The amino acid G13 of the wild-type EPX1 nanopore protein is mutated to G13N, G13K, G13S, G13D, G13W, G13R, G13F, G13E, G13P, G13L, G13A, G13V or G13M; preferably, the amino acid G13 of the wild-type EPX1 nanopore protein is mutated to G13N or G13S.

[0008] Further, in B: the amino acid N33 of the wild-type EPX1 nanopore protein is mutated to N33A, N33H, N33Q, N33S, N33L, N33D, N33C, N33I, N33P, N33T, N33G, N33M, N33W or N33R; preferably, the amino acid N33 of the wild-type EPX1 nanopore protein is mutated to N33A; Amino acid Q34 of the wild-type EPX1 nanopore protein was mutated to Q34N or Q34L; The amino acid Y35 of the wild-type EPX1 nanopore protein is mutated to Y35P, Y35L, Y35G, Y35I, Y35Q, Y35E, Y35C, Y35R, Y35H, Y35K, Y35T, Y35A, Y35S, Y35W, Y35N, Y35V or Y35D; preferably, the amino acid Y35 of the wild-type EPX1 nanopore protein is mutated to Y35R, Y35L or Y35G.

[0009] Further, in C: the amino acid K139 of the wild-type EPX1 nanopore protein is mutated to K139M, K139P, K139L, K139G, K139I, K139Q, K139E, K139C, K139R, K139H, K139W, K139T, K139A, K139S, K139Y, K139N, K139V or K139D; preferably, the amino acid K139 of the wild-type EPX1 nanopore protein is mutated to K139N, K139R or K139G; Amino acid Q140 of the wild-type EPX1 nanopore protein was mutated to Q140R or Q140S; The amino acid V141 of the wild-type EPX1 nanopore protein is mutated to V141N, V141K, V141H, V141D, V141W, V141R, V141F, V141E, V141P, V141L, V141A, V141G or V141M; preferably, the amino acid V141 of the wild-type EPX1 nanopore protein is mutated to V141N or V141K.

[0010] Further, in D: the amino acid T174 of the wild-type EPX1 nanopore protein is mutated to T174P, T174L, T174G, T174I, T174Q, T174E, T174C, T174R, T174H, T174K, T174A, T174S, T174Y, T174N, T174V or T174D; preferably, the amino acid T174 of the wild-type EPX1 nanopore protein is mutated to T174L or T174G; The amino acid T175 of the wild-type EPX1 nanopore protein is mutated to T175P, T175L, T175G, T175I, T175Q, T175E, T175C, T175R, T175H, T175K, T175A, T175S, T175Y, T175N, T175V or T175D; preferably, the amino acid T175 of the wild-type EPX1 nanopore protein is mutated to T175R, T175L or T175G; Amino acid Y176 of the wild-type EPX1 nanopore protein is mutated to Y176N or Y176V.

[0011] Furthermore, the gene encoding the enterococcal pore-forming toxin mutant protein is selected from the following E, F or G: E: nucleic acid encoding EPX1 nanopore mutant protein; F: nucleic acid having 85-99% homology to the nucleic acid sequence in E and encoding an EPX1 nanopore mutant protein; G: Nucleic acid complementary to the nucleic acid of E or F.

[0012] Furthermore, one or more of metal ions, inorganic salts, polymers, amino acids, peptides, polypeptides, proteins, nucleotides, oligonucleotides, polynucleotides, dyes, bleaches, drugs, diagnostic agents, narcotics, explosive pollutants or environmental pollutants are detected.

[0013] The present invention has the following beneficial effects: EPX1 (wild-type) is a pore-forming toxin protein widely found in various organisms in nature. Its mutants, after modification (after amino acid mutation), exhibit excellent pore-forming ability. Not only does this method have a simple expression and purification process, allowing for rapid preparation using conventional molecular biology methods, but it can also stably construct single nanopores within a lipid membrane environment. As a nanopore protein, it provides an ideal functional interface for single-molecule detection.

[0014] The EPX1 protein assembles as a homologous octamer, forming a barrel-shaped pore structure with a total length of 123 angstroms and a maximum outer diameter of 97 angstroms. This structure, precisely arranged along an eight-fold axis of symmetry, consists of an N-terminal β-barrel, a central vestibule formed by the cap domain, and a transmembrane β-barrel. This structure not only ensures mechanical stability but also creates a specific recognition space for molecular transit events. The octameric symmetry endows the pore with excellent membrane embedding capabilities. Furthermore, the central vestibule region of the EPX1 protein, with a diameter of 68 angstroms, provides ample conformational space for target molecules. Combined with precise mutagenesis of the constriction region (diameter 14.8-20.9 angstroms), this allows for gradient regulation of sample transit speed. Furthermore, through amino acid mutations, EPX1 nanopore mutants have significantly improved structural stability, molecular recognition efficiency, and detection performance. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] A more complete understanding of exemplary embodiments of the present invention may be obtained by referring to the following drawings: Figure 1 This is an electrophoresis diagram of the purification of the wild-type EPX1 nanopore protein in Example 1 of the present invention, wherein lane 1: protein marker; lane 2: eluted sample; lane 3: eluted sample; lane 4: eluted sample; lane 5: eluted sample.

[0016] Figure 2 This is a graph showing the pore opening current detection results of the wild-type EPX1 nanopore protein in Example 2 of the present invention.

[0017] Figure 3 This is a current signal diagram of single-stranded DNA passing through the wild-type EPX1 nanopore protein in Example 3 of the present invention.

[0018] Figure 4 This is a current signal diagram of the miRNA of Example 4 of the present invention passing through the EPX1 V11A nanopore protein.

[0019] Figure 5 This is a current signal diagram of the miRNA of Example 4 of the present invention passing through the EPX1 G13N nanopore protein.

[0020] Figure 6 This is a current signal diagram of aspartic acid passing through the EPX1 T175R nanopore protein in Example 5 of the present invention.

[0021] Figure 7 This is a current signal diagram of aspartic acid passing through the EPX1 Y176V nanopore protein in Example 5 of the present invention.

[0022] Figure 8 This is a current signal diagram of ATP passing through the EPX1 N33A nanopore protein in Example 6 of the present invention.

[0023] Figure 9 This is a current signal diagram of ATP passing through the EPX1 Q34N nanopore protein in Example 6 of the present invention.

[0024] Figure 10 This is a current signal diagram of Hg²⁺ passing through the EPX1 K139P nanopore protein in Example 7 of the present invention.

[0025] Figure 11 This is a current signal diagram of Hg²⁺ passing through the EPX1 V141H nanopore protein in Example 7 of the present invention. DETAILED DESCRIPTION

[0026] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs; the terms used herein are only for the purpose of describing specific embodiments rather than limiting the present invention.

[0028] The present invention provides an embodiment of the present invention for use of a mutant protein of an enterococcal pore-forming toxin in nanopore detection. The mutant protein of the enterococcal pore-forming toxin is an EPX1 nanopore mutant protein, which is obtained by mutation of a wild-type EPX1 nanopore protein. The amino acid sequence of the wild-type EPX1 nanopore protein is shown in SEQ ID NO: 1. The mutation refers to: one or more amino acids of the wild-type EPX1 nanopore protein are mutated to common amino acids other than the original amino acids; Common amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, and histidine.

[0029] In some embodiments, the EPX1 nanopore mutant protein is selected from the following A, B, C, or D: A: One or more of amino acids V11, L12, and G13 of the wild-type EPX1 nanopore protein are mutated to common amino acids other than the original amino acids; B: One or more of amino acids N33, Q34, and Y35 of the wild-type EPX1 nanopore protein are mutated to common amino acids other than the original amino acids; C: One or more of amino acids K139, Q140, and V141 of the wild-type EPX1 nanopore protein are mutated to common amino acids other than the original amino acids; D: One or more of amino acids T174, T175, and Y176 of the wild-type EPX1 nanopore protein are mutated to common amino acids other than the original amino acids.

[0030] In some embodiments, in A: amino acid V11 of the wild-type EPX1 nanopore protein is mutated to V11P, V11L, V11G, V11R, V11Q, V11E, V11C, V11W, V11H, V11K, V11F, V11A, V11S, V11Y, V11N, or V11D; Amino acid L12 of the wild-type EPX1 nanopore protein was mutated to L12A or L12V; Amino acid G13 of the wild-type EPX1 nanopore protein is mutated to G13N, G13K, G13S, G13D, G13W, G13R, G13F, G13E, G13P, G13L, G13A, G13V, or G13M.

[0031] In some embodiments, amino acid V11 of the wild-type EPX1 nanopore protein is mutated to V11A, V11L, or V11N.

[0032] In some embodiments, amino acid G13 of the wild-type EPX1 nanopore protein is mutated to G13N or G13S.

[0033] In some embodiments, in B: amino acid N33 of the wild-type EPX1 nanopore protein is mutated to N33A, N33H, N33Q, N33S, N33L, N33D, N33C, N33I, N33P, N33T, N33G, N33M, N33W or N33R; Amino acid Q34 of the wild-type EPX1 nanopore protein was mutated to Q34N or Q34L; Amino acid Y35 of the wild-type EPX1 nanopore protein is mutated to Y35P, Y35L, Y35G, Y35I, Y35Q, Y35E, Y35C, Y35R, Y35H, Y35K, Y35T, Y35A, Y35S, Y35W, Y35N, Y35V or Y35D.

[0034] In some embodiments, amino acid N33 of the wild-type EPX1 nanopore protein is mutated to N33A.

[0035] In some embodiments, amino acid Y35 of the wild-type EPX1 nanopore protein is mutated to Y35R, Y35L, or Y35G.

[0036] In some embodiments, in C: amino acid K139 of the wild-type EPX1 nanopore protein is mutated to K139M, K139P, K139L, K139G, K139I, K139Q, K139E, K139C, K139R, K139H, K139W, K139T, K139A, K139S, K139Y, K139N, K139V, or K139D; Amino acid Q140 of the wild-type EPX1 nanopore protein was mutated to Q140R or Q140S; Amino acid V141 of the wild-type EPX1 nanopore protein is mutated to V141N, V141K, V141H, V141D, V141W, V141R, V141F, V141E, V141P, V141L, V141A, V141G, or V141M.

[0037] In some embodiments, amino acid K139 of the wild-type EPX1 nanopore protein is mutated to K139N, K139R, or K139G.

[0038] In some embodiments, amino acid V141 of the wild-type EPX1 nanopore protein is mutated to V141N or V141K.

[0039] In some embodiments, in D: amino acid T174 of the wild-type EPX1 nanopore protein is mutated to T174P, T174L, T174G, T174I, T174Q, T174E, T174C, T174R, T174H, T174K, T174A, T174S, T174Y, T174N, T174V, or T174D; amino acid T175 of the wild-type EPX1 nanopore protein is mutated to T175P, T175L, T175G, T175I, T175Q, T175E, T175C, T175R, T175H, T175K, T175A, T175S, T175Y, T175N, T175V, or T175D; Amino acid Y176 of the wild-type EPX1 nanopore protein is mutated to Y176N or Y176V.

[0040] In some embodiments, amino acid T174 of the wild-type EPX1 nanopore protein is mutated to T174L or T174G.

[0041] In some embodiments, amino acid T175 of the wild-type EPX1 nanopore protein is mutated to T175R, T175L, or T175G.

[0042] In some embodiments, a method for preparing an EPX1 nanopore mutant protein comprises the following steps: Step S1: constructing EPX1 nanopore mutant protein vector; Step S2: Expression and purification of EPX1 nanopore mutant proteins.

[0043] In some embodiments, the gene encoding the Enterococcus pore-forming toxin mutant protein is selected from the following E, F, or G: E: nucleic acid encoding EPX1 nanopore mutant protein; F: nucleic acid having 85-99% homology to the nucleic acid sequence in E and encoding an EPX1 nanopore mutant protein; G: Nucleic acid complementary to the nucleic acid of E or F.

[0044] In some embodiments, one or more of a metal ion, an inorganic salt, a polymer, an amino acid, a peptide, a polypeptide, a protein, a nucleotide, an oligonucleotide, a polynucleotide, a dye, a bleach, a drug, a diagnostic agent, a narcotic, an explosive pollutant, or an environmental pollutant is detected.

[0045] Example 1: Expression and purification of wild-type EPX1 nanopore protein (1) Gene construction and vector design: Synthesize the wild-type EPX1 nanopore protein DNA fragment, the DNA sequence of which is shown in SEQ ID NO: 2, and clone the fragment into the pET-30a(+) vector; (2) The plasmid was transformed into E. coli Dh5α competent cells and amplified by plate culture and liquid culture; (3) Transformation and condition optimization: The plasmid was transformed into E. coli BL21 competent cells. The expression conditions (including IPTG concentration, induction temperature, and time) were first tested in a small volume of 50 ml LB medium to determine the optimal parameters. (4) Large-scale culture and induction: Inoculation and culture: Inoculate 1 / 50 volume of overnight pre-culture (including multiple single colonies) into 5LB medium (containing 100 μg / ml kanamycin and 1% glucose), and culture at 37°C and 180 rpm with shaking until OD 600 = 0.6–0.8, then cooled to 20°C, 0.5 mM IPTG was added, and the induction culture was continued for 8–12 h; (5) Bacterial cell collection: centrifuge at 3400 g for 60 min at 4°C to collect the bacterial pellet, which was then frozen at -20°C for purification. (6) Cell disruption: Resuspend the cells in lysis buffer (50 mM Tris, pH = 8.0, 500 mM NaCl, 10 mM imidazole, 1% Triton X-100), add lysozyme (0.2 mg / ml) and protease inhibitors, and lyse the cells by a high-pressure homogenizer cycle. Add benzamidase, stir on ice for 30 minutes, and then perform two high-pressure homogenization cycles to ensure sufficient lysis. (7) Membrane fraction collection: collect the supernatant by ultracentrifugation; (8) The supernatant was loaded onto a Ni column for affinity chromatography purification; (9) Washing of impurities: First wash with 50 ml of lysis buffer containing 1% Triton X-100, then wash the column with 50 ml of Buffer A (50 mM Tris, pH = 8.0, 150 mM NaCl, 10 mM imidazole, 20–50 mM sodium cholate); (10) Target protein elution: Elute with Buffer A containing 250 mM imidazole for 5 column volumes, and collect the elution peak containing oligomers; (11) Renaturation treatment: The target protein component was transferred to dialysis buffer (20 mM Tris, pH = 8.0, 150 mM NaCl, 20 mM sodium cholate), dialyzed overnight at 4 °C to remove small molecule impurities, and finally the wild-type EPX1 nanopore protein was obtained.

[0046] The amino acid sequence of the wild-type EPX1 nanopore protein is shown in SEQ ID NO: 1, and the nucleic acid sequence encoding the wild-type EPX1 nanopore protein is shown in SEQ ID NO: 2.

[0047] The electrophoresis diagram of the purified wild-type EPX1 nanopore protein is shown in Figure 1 shown.

[0048] Structural analysis of the wild-type EPX1 nanopore: The wild-type EPX1 nanopore assembles as a homo-octamer, forming a barrel-shaped pore structure with a total length of 123 angstroms and a maximum outer diameter of 97 angstroms. This structure is precisely arranged along an eight-fold symmetry axis, consisting of an N-terminal β-barrel, a central vestibule formed by the cap domain, and a transmembrane β-barrel. This structure not only ensures the mechanical stability of the pore but also creates a specific recognition space for molecular pore-crossing events. The octameric symmetry gives the pore excellent membrane embedding ability. Furthermore, the central vestibule region of the EPX1 protein has a diameter of 68 angstroms, providing ample space for conformational changes for target molecules.

[0049] Example 2: Characterization of wild-type EPX1 nanopore protein (1) After obtaining the wild-type EPX1 nanopore protein, a single protein pore was established in the lipid bilayer. First, artificial lipid bilayers were formed on both sides of the pore of the polytetrafluoroethylene membrane. Then, 1.2 mL of detection buffer (0.5 M KCl, 10 mM HEPES-KOH, pH = 8.0, 2 mM MgCl2, 0.1 mM EDTA) was injected into the chambers on both sides, with a volume of 1.2 ml / side, to ensure that the liquid level on both sides of the membrane was balanced; (2) 8 ng / mL wild-type EPX1 nanopore protein solution was added to the cis chamber, and blank buffer was added to the trans chamber. A 160 mV transmembrane voltage was applied (Axopatch 200B amplifier), and the current changes were monitored in real time. When the protein inserted into a single pore, a characteristic current step signal was recorded. After the protein pore was formed, the cis chamber was rinsed three times with 50 μL buffer to remove unbound protein. All experiments were performed at room temperature (23 ± 1 ° C). The analog signal was low-pass filtered at 100 kHz using a 4-pole Bessel filter and digitized at 500 kHz. Data acquisition was controlled by Origin.

[0050] The results of the pore opening current detection of wild-type EPX1 nanopore protein are as follows Figure 2 shown.

[0051] Example 3: DNA detection using wild-type EPX1 nanopore protein (1) After obtaining the wild-type EPX1 nanopore protein, a single protein pore was established in the lipid bilayer. First, artificial lipid bilayers were formed on both sides of the pore of the polytetrafluoroethylene membrane. Then, 1.2 mL of detection buffer (0.5 M KCl, 10 mM HEPES-KOH, pH = 8.0, 2 mM MgCl2, 0.1 mM EDTA) was injected into the chambers on both sides, with a volume of 1.2 ml / side, to ensure that the liquid level on both sides of the membrane was balanced; (2) 8 ng / mL wild-type EPX1 nanopore protein solution was added to the cis chamber, and blank buffer was added to the trans chamber. A 160 mV transmembrane voltage was applied (Axopatch 200B amplifier), and the current changes were monitored in real time. When the protein inserted into a single pore, a characteristic current step signal was recorded. After the protein pore was formed, the cis chamber was rinsed three times with 50 μL buffer to remove unbound proteins and avoid multi-pore interference; (3) 10 nM single-stranded DNA (SEQ ID NO: 3: 5'-ATCGATCGATCGATCGATCGATCGATCGATCGATCGATCG-3') was added, and a voltage of 160 mV was applied (the voltage was optimized to reduce the effect of chain folding). The sampling rate was 50 kHz and low-pass filtering was performed at 2 kHz. All experiments were performed at room temperature (23 ± 1 °C) and data acquisition was controlled by Origin.

[0052] The current signal of single-stranded DNA passing through the wild-type EPX1 nanopore protein is as follows Figure 3 shown.

[0053] Example 4: Detection of miRNA using EPX1 nanopore mutant protein with mutation sites V11, L12, and G13 (1) Referring to the preparation method of the wild-type EPX1 nanopore protein in Example 1, EPX1 nanopore mutant proteins (V11P, V11L, V11G, V11R, V11Q, V11E, V11C, V11W, V11H, V11K, V11F, V11A, V11S, V11Y, V11N, V11D, L12A, L12V, G13N, G13K, G13S, G13D, G13W, G13R, G13D) were obtained. After the pores were prepared (e.g., G13F, G13E, G13P, G13L, G13A, G13V, G13M), a single protein pore was established in the lipid bilayer. First, artificial lipid bilayers were formed on both sides of the pore in the polytetrafluoroethylene membrane. Then, 1.2 mL of detection buffer (0.5 M KCl, 10 mM HEPES-KOH, pH = 8.0, 2 mM MgCl2, 0.1 mM EDTA) was injected into the chambers on both sides at a volume of 1.2 mL per side to ensure that the liquid levels on both sides of the membrane were balanced. (2) 8 ng / mL of EPX1 nanopore mutant protein (V11P, V11L, V11G, V11R, V11Q, V11E, V11C, V11W, V11H, V11K, V11F, V11A, V11S, V11Y, V11N, V11D, L12A, L12V, G13N, G13K, G13S, G13D, G13W, G13R, G13F, G13E, G13P, G13L, G13A, G13V, G13M) solution was added to the cis chamber, blank buffer was added to the trans chamber, and a 160 mV transmembrane voltage was applied (Axopatch 200B amplifier), monitor the current changes in real time. When the protein is inserted into a single pore, the characteristic current step signal is recorded. After the protein pore is formed, the cis cavity is rinsed three times with 50μL buffer to remove unbound proteins and avoid multi-pore interference; (3) miRNA (SEQ ID NO:4: 5'-UAGCUUAUCAGUCUGAUGUUGA-3', 22 nt, unmodified, 10 nM) was added, and a voltage of 160 mV (optimized to minimize the effects of strand folding) was applied. The sampling rate was 50 kHz and low-pass filtering was performed at 2 kHz. All experiments were performed at room temperature (23 ± 1°C) and data acquisition was controlled by Origin.

[0054] The current signal of miRNA passing through EPX1 V11A nanopore protein is as follows Figure 4 As shown, it can be seen that after the amino acid V11 in the contraction region of the wild-type EPX1 nanopore protein is mutated to V11A, the current signal quality for detecting miRNA is significantly improved, the current properties are stable, the current signal width is narrower, and there are fewer glitches. This is because the V11A mutation replaces valine with alanine to eliminate the hydrophobicity of the side chain. Mutation type: steric hindrance and flexibility regulation.

[0055] The current signal of miRNA passing through EPX1 G13N nanopore protein is as follows Figure 5 As shown, it can be seen that after the amino acid G13 in the contraction region of the wild-type EPX1 nanopore protein mutates to G13N, the current signal quality for detecting miRNA is significantly improved, the current properties are stable, the current signal width is narrower, and there are fewer glitches. This is because the G13N mutation replaces glycine with asparagine (containing a polar amide group), changing the polarity and hydrogen bond interaction. Mutation type: polarity and hydrogen bond regulation.

[0056] The current signals of other EPX1 nanopore mutant proteins are shown in Table 1.

[0057] Table 1 Current signals of other EPX1 nanopore mutant proteins in Example 4

[0058] Example 5: Detection of aspartic acid using EPX1 nanopore mutant protein with mutation sites T174, T175, and Y176 (1) Referring to the preparation method of the wild-type EPX1 nanopore protein in Example 1, EPX1 nanopore mutant proteins (T174P, T174L, T174G, T174I, T174Q, T174E, T174C, T174R, T174H, T174K, T174A, T174S, T174Y, T174N, T174V, T174D, T175P, T175L, T175G, T175I, T175Q, T175E, T175C, T175R, T174H, T174K, T174A, T174S, T174Y, T174N, T174V, T174D, T175 After the pores were detected by PCR (5H, T175K, T175A, T175S, T175Y, T175N, T175V, T175D, Y176N, Y176V), a single protein pore was established in the lipid bilayer. First, artificial lipid bilayers were formed on both sides of the pore of the polytetrafluoroethylene membrane. Then, 1.2 mL of detection buffer (0.5 M KCl, 10 mM HEPES-KOH, pH = 8.0, 2 mM MgCl2, 0.1 mM EDTA) was injected into the chambers on both sides at a volume of 1.2 ml per side to ensure that the liquid level on both sides of the membrane was balanced. (2) 8 ng / mL of EPX1 nanopore mutant protein (T174P, T174L, T174G, T174I, T174Q, T174E, T174C, T174R, T174H, T174K, T174A, T174S, T174Y, T174N, T174V, T174D, T175P, T175L, T175G, T175I, T175Q, T175E, T175C, T175R, T175H, T175K, T175A, T175S, T175Y, T175N, T175V, T175D, Y176N, Y176V) solution was added to the cis chamber, and the trans chamber was filled with blank buffer. A 160 mV transmembrane voltage (Axopatch 200B amplifier) ​​was applied, and current changes were monitored in real time. When a single pore was formed by protein insertion, a characteristic current step signal was recorded. After protein pore formation, the cis cavity was rinsed three times with 50 μL of buffer to remove unbound protein and avoid multi-pore interference. (3) Aspartic acid sample (10 nM) was added, a voltage of 160 mV was applied, the sampling rate was 50 kHz, and a low-pass filter was used (2 kHz). All experiments were performed at room temperature (23 ± 1 °C), and data acquisition was controlled by Origin.

[0059] The current signal of aspartate passing through the EPX1 T175R nanopore protein is as follows Figure 6As shown, it can be seen that after the amino acid T175 in the contraction region of the wild-type EPX1 nanopore protein mutates to T175R, the current signal quality for detecting aspartic acid is significantly improved, the current properties are stable, the current signal width is narrower, and there are fewer glitches. This is because the T175R mutation introduces a positively charged arginine residue. Mutation type: charge distribution regulation.

[0060] The current signal of aspartate passing through the EPX1 Y176V nanopore protein is as follows Figure 7 As shown, it can be seen that after the amino acid Y176 in the contraction region of the wild-type EPX1 nanopore protein mutates to Y176V, the current signal quality for detecting aspartic acid is significantly improved, the current properties are stable, the current signal width is narrower, and there are fewer glitches. This is because Y176V eliminates the synergistic effect of tyrosine hydroxyl group through mutation. Mutation type: steric hindrance and flexibility regulation.

[0061] The current signals of other EPX1 nanopore mutant proteins are shown in Table 2.

[0062] Table 2 Current signals of other EPX1 nanopore mutant proteins in Example 5

[0063] Example 6: Detection of ATP using EPX1 nanopore mutant protein with mutation sites N33, Q34, and Y35 (1) Referring to the preparation method of the wild-type EPX1 nanopore protein in Example 1, EPX1 nanopore mutant proteins (N33A, N33H, N33Q, N33S, N33L, N33D, N33C, N33I, N33P, N33T, N33G, N33M, N33W, N33R, Y35P, Y35L, Y35G, Y35I, Y35Q, Y35E, Y35C, Y35R, Y35H, Y35K, Y35T, Y35 After the protein pores (5A, Y35S, Y35W, Y35N, Y35V, Y35D, Q34N, Q34L) were generated, a single protein pore was established in the lipid bilayer. First, artificial lipid bilayers were formed on both sides of the pore in the polytetrafluoroethylene membrane. Then, 1.2 mL of detection buffer (0.5 M KCl, 10 mM HEPES-KOH, pH = 8.0, 2 mM MgCl2, 0.1 mM EDTA) was injected into the chambers on both sides at a volume of 1.2 mL per side to ensure that the liquid levels on both sides of the membrane were balanced. (2) 8 ng / mL of EPX1 nanopore mutant protein (N33A, N33H, N33Q, N33S, N33L, N33D, N33C, N33I, N33P, N33T, N33G, N33M, N33W, N33R, Y35P, Y35L, Y35G, Y35I, Y35Q, Y35E, Y35C, Y35R, Y35H, Y35K, Y35T, Y35A, Y35S, Y35W, Y35N, Y35V, Y35D, Q34N, Q34L) solution was added to the cis chamber, and the trans chamber was filled with blank buffer. A 160 mV transmembrane voltage (Axopatch 200B amplifier) ​​was applied, and current changes were monitored in real time. When a single pore was formed by protein insertion, a characteristic current step signal was recorded. After protein pore formation, the cis cavity was rinsed three times with 50 μL of buffer to remove unbound protein and avoid multi-pore interference. (3) ATP sample (10 nM) was added, a voltage of 160 mV was applied, the sampling rate was 50 kHz, and a low-pass filter was used (2 kHz). All experiments were performed at room temperature (23 ± 1 °C), and data acquisition was controlled by Origin.

[0064] The current signal of ATP passing through the EPX1 N33A nanopore protein is as follows Figure 8 As shown, it can be seen that after the amino acid N33 in the contraction region of the wild-type EPX1 nanopore protein mutates to N33A, the current signal quality for detecting ATP is significantly improved, the current properties are stable, the current signal width is narrower, and there are fewer glitches. This is because N33A eliminates charge interference through mutation. Mutation type: charge distribution regulation.

[0065] The current signal of ATP passing through EPX1 Q34N nanopore protein is as follows Figure 9 As shown, it can be seen that after the amino acid Q34 in the contraction region of the wild-type EPX1 nanopore protein mutates to Q34N, the current signal quality of ATP detection is significantly improved, the current properties are stable, the current signal width is narrower, and there are fewer glitches. This is because Q34N introduces a more polar amide group through mutation. Mutation type: polarity and hydrogen bond regulation.

[0066] The current signals of other EPX1 nanopore mutant proteins are shown in Table 3.

[0067] Table 3 Current signals of other EPX1 nanopore mutant proteins in Example 6

[0068] Example 7: Detection of Hg²⁺ using EPX1 nanopore mutant protein with mutation sites K139, Q140, and V141 (1) Referring to the preparation method of the wild-type EPX1 nanopore protein in Example 1, EPX1 nanopore mutant proteins (K139M, K139P, K139L, K139G, K139I, K139Q, K139E, K139C, K139R, K139H, K139W, K139T, K139A, K139S, K139Y, K139N, K139V, K139D, Q140R, Q140S, V141N, V141K, V141H, V141D, After the addition of 141W, 141R, 141F, 141E, 141P, 141L, 141A, 141G, and 141M proteins, a single protein pore was established in the lipid bilayer. First, artificial lipid bilayers were formed on both sides of the pore in the polytetrafluoroethylene membrane. Then, 1.2 mL of assay buffer (0.5 M KCl, 10 mM HEPES-KOH, pH 8.0, 2 mM MgCl2, and 0.1 mM EDTA) was injected into the chambers on both sides at a volume of 1.2 mL per side to ensure that the liquid levels on both sides of the membrane were balanced. (2) 8 ng / mL of EPX1 nanopore mutant protein (K139M, K139P, K139L, K139G, K139I, K139Q, K139E, K139C, K139R, K139H, K139W, K139T, K139A, K139S, K139Y, K139N, K139V, K139D, Q140R, Q140S, V141N, V141K, V141H, V141D, V141W, V141R, V141F, V141E, V141P, V141L, V141A, V141G, V141M) solution was added to the cis chamber, and the trans chamber was filled with blank buffer. A 160 mV transmembrane voltage (Axopatch 200B amplifier) ​​was applied, and current changes were monitored in real time. When a single pore was formed by protein insertion, a characteristic current step signal was recorded. After protein pore formation, the cis cavity was rinsed three times with 50 μL of buffer to remove unbound protein and avoid multi-pore interference. (3) Hg²⁺ sample (10 nM) was added, a voltage of 160 mV was applied, a sampling rate of 50 kHz, and a low-pass filter (2 kHz) was used. All experiments were performed at room temperature (23 ± 1°C), and data acquisition was controlled by Origin.

[0069] The current signal of Hg²⁺ passing through EPX1 K139P nanopore protein is shown in Figure 2. Figure 10As shown in the figure, it can be seen that after the amino acid K139 in the contraction zone of the wild-type EPX1 nanopore protein mutates to K139P, the current signal quality for detecting Hg²⁺ is significantly improved, the current properties are stable, the current signal width is narrower, and there are fewer glitches. This is because K139P introduces the rigid structure of proline through mutation to compress the space. The mutation type is steric hindrance and flexibility regulation.

[0070] The current signal of Hg²⁺ passing through EPX1 V141H nanopore protein is shown in the figure. Figure 11 As shown in the figure, it can be seen that after the amino acid V141 in the contraction region of the wild-type EPX1 nanopore protein mutates to V141H, the current signal quality for detecting Hg²⁺ is significantly improved. The current properties are stable, the current signal width is narrower, and there are fewer glitches. This is because V141H reduces random charge interference through mutation. Mutation type: charge distribution regulation.

[0071] The current signals of other EPX1 nanopore mutant proteins are shown in Table 4.

[0072] Table 4 Current signals of other EPX1 nanopore mutant proteins in Example 7

[0073] Amino acid sequence: SEQ ID NO: 1: mdvqvfadnpvlgtevtfekdengrivkiisknqyritiynsvdagdtpndasvsldvdfiddknsgemga vasintfipsglryvegykykgvtnpiyknlsagmlwpkkyrvevvnipidqatkiitatpnnnikekqvsdtisygfggsvsadgkkpggsieanlaytktttydqpdyetsqi kkttkeavwdtsfvetrdgytpnswnpvygnqmfmrgrysnvspidnikkggevsslisggfspkmgvvlaspngtkksqfvvrvsrmsdmyimrwsgtewggeneinqnvpkey

[0074] Nucleic acid sequence: SEQ ID NO: 2: atggatgtgcaggtgtttgcggataacccggtgctgggcaccgaagtgacctttgaaaaagatgaaaacggccgcattgtgaaaattattagcaaaaaccagtatcgcattaccatttataacagcgtggatgcgggcgataccccgaacgatgcgagcgtgagcctggatgtggattttattgatgataaaaacagcggcgaaatgggcgcggtggcgagcattaacacctttattccgagcggcctgcgctatgtggaaggctataaatataaaggcgtgaccaacccgatttataaaaacctgagcgcgggcatgctgtggccgaaaaaatatcgcgtggaagtggtgaacattccgattgatcaggcgaccaaaattattaccgcgaccccgaacaacaacattaaagaaaaacaggtgagcgataccattagctatggctttggcggcagcgtgagcgcggatggcaaaaaaccgggcggcagcattgaagcgaacctggcgtataccaaaaccaccacctatgatcagccggattatgaaaccagccagattaaaaaaaccaccaaagaagcggtgtgggataccagctttgtggaaacccgcgatggctataccccgaacagctggaacccggtgtatggcaaccagatgtttatgcgcggccgctatagcaacgtgagcccgattgataacattaaaaaaggcggcgaagtgagcagcctgattagcggcggctttagcccgaaaatgggcgtggtgctggcgagcccgaacggcaccaaaaaaagccagtttgtggtgcgcgtgagccgcatgagcgatatgtatattatgcgctggagcggcaccgaatggggcggcgaaaacgaaattaaccagaacgtgccgaaagaatataacgcgctgatgtatgaagatgtgaaatttgaaattgattgggaacagcgcaccgtgcgcaccattctggaa The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Application of a mutant protein of enterococcal pore-forming toxin in nanopore detection, characterized by: The enterococcal pore-forming toxin mutant protein is an EPX1 nanopore mutant protein, which is obtained by mutating a wild-type EPX1 nanopore protein. The amino acid sequence of the wild-type EPX1 nanopore protein is shown in SEQ ID NO:

1. The mutation refers to: one or more amino acids of the wild-type EPX1 nanopore protein are mutated into common amino acids other than the original amino acids; The common amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, and histidine.

2. The use according to claim 1, characterized in that: The EPX1 nanopore mutant protein is selected from the following A, B, C or D: A: One or more of amino acids V11, L12, and G13 of the wild-type EPX1 nanopore protein are mutated to common amino acids other than the original amino acids; B: One or more of amino acids N33, Q34, and Y35 of the wild-type EPX1 nanopore protein are mutated to common amino acids other than the original amino acids; C: One or more of amino acids K139, Q140, and V141 of the wild-type EPX1 nanopore protein are mutated to common amino acids other than the original amino acids; D: One or more of amino acids T174, T175, and Y176 of the wild-type EPX1 nanopore protein are mutated to common amino acids other than the original amino acids.

3. The use according to claim 2, characterized in that: In A: amino acid V11 of the wild-type EPX1 nanopore protein is mutated to V11P, V11L, V11G, V11R, V11Q, V11E, V11C, V11W, V11H, V11K, V11F, V11A, V11S, V11Y, V11N, or V11D; Amino acid L12 of the wild-type EPX1 nanopore protein was mutated to L12A or L12V; Amino acid G13 of the wild-type EPX1 nanopore protein is mutated to G13N, G13K, G13S, G13D, G13W, G13R, G13F, G13E, G13P, G13L, G13A, G13V, or G13M.

4. The use according to claim 2, wherein: In B: amino acid N33 of the wild-type EPX1 nanopore protein is mutated to N33A, N33H, N33Q, N33S, N33L, N33D, N33C, N33I, N33P, N33T, N33G, N33M, N33W, or N33R; Amino acid Q34 of the wild-type EPX1 nanopore protein was mutated to Q34N or Q34L; Amino acid Y35 of the wild-type EPX1 nanopore protein is mutated to Y35P, Y35L, Y35G, Y35I, Y35Q, Y35E, Y35C, Y35R, Y35H, Y35K, Y35T, Y35A, Y35S, Y35W, Y35N, Y35V or Y35D.

5. The use according to claim 2, characterized in that: In C: amino acid K139 of the wild-type EPX1 nanopore protein is mutated to K139M, K139P, K139L, K139G, K139I, K139Q, K139E, K139C, K139R, K139H, K139W, K139T, K139A, K139S, K139Y, K139N, K139V, or K139D; Amino acid Q140 of the wild-type EPX1 nanopore protein was mutated to Q140R or Q140S; Amino acid V141 of the wild-type EPX1 nanopore protein is mutated to V141N, V141K, V141H, V141D, V141W, V141R, V141F, V141E, V141P, V141L, V141A, V141G, or V141M.

6. The use according to claim 2, characterized in that: In D: amino acid T174 of the wild-type EPX1 nanopore protein is mutated to T174P, T174L, T174G, T174I, T174Q, T174E, T174C, T174R, T174H, T174K, T174A, T174S, T174Y, T174N, T174V, or T174D; amino acid T175 of the wild-type EPX1 nanopore protein is mutated to T175P, T175L, T175G, T175I, T175Q, T175E, T175C, T175R, T175H, T175K, T175A, T175S, T175Y, T175N, T175V, or T175D; Amino acid Y176 of the wild-type EPX1 nanopore protein is mutated to Y176N or Y176V.

7. The use according to any one of claims 1 to 6, characterized in that: The gene encoding the enterococcal pore-forming toxin mutant protein is selected from the following E, F or G: E: nucleic acid encoding EPX1 nanopore mutant protein; F: nucleic acid having 85-99% homology to the nucleic acid sequence in E and encoding an EPX1 nanopore mutant protein; G: Nucleic acid complementary to the nucleic acid of E or F.

8. The use according to any one of claims 1 to 6, characterized in that: Detect one or more of metal ions, inorganic salts, polymers, amino acids, peptides, polypeptides, proteins, nucleotides, oligonucleotides, polynucleotides, dyes, bleaches, drugs, diagnostic agents, narcotics, explosive pollutants or environmental pollutants.

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