Use of enterococcus pore-forming toxin mutant proteins in nanopore detection

Abstract

The application belongs to the technical field of characterizing target samples, and provides application of enterococcus pore-forming toxin mutant protein in nanopore detection. The enterococcus pore-forming toxin mutant protein is an EPX1 nanopore mutant protein, which is obtained by mutation of wild-type EPX1 nanopore protein, and the amino acid sequence of the wild-type EPX1 nanopore protein is shown as SEQ ID NO: 1. The mutation refers to that 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 EPX1 protein (wild type) of the application is a kind of pore-forming toxin protein, which widely exists in various organisms in nature. The mutant thereof exhibits excellent pore-forming ability after modification (after amino acid mutation). The EPX1 nanopore mutant protein realizes significant improvement in structural stability, molecular recognition efficiency and detection performance.

Description

Technical Field

[0001] This invention belongs to the field of target sample characterization technology, and particularly relates to the application of enterococcal pore-forming toxin mutant protein in nanopore detection. Background Technology

[0002] Nanopore sequencing technology, as the core of third-generation sequencing, has shown great potential in biomedical detection and genomic analysis due to its advantages such as real-time operation, label-free operation, and ultra-long reads. Traditional biochemical detection methods (such as ELISA, Western blotting, and mass spectrometry) have been applied to scenarios such as cancer biomarker detection, but they suffer from significant drawbacks such as being time-consuming and labor-intensive, having cumbersome sample preparation, and low sensitivity. In contrast, nanopore-based single-molecule sensing technology uses electromotive force to drive the analyte through the nanopore, utilizing changes in ion current to achieve highly sensitive detection, providing a new approach to overcome the shortcomings of traditional methods.

[0003] However, the structural limitations, insufficient detection accuracy, and complex preparation processes of existing nanoporous proteins severely restrict the widespread application of this technology. Most reported bio-nanopores (such as α-hemolysin, MspA, and CsgG) are formed by the polymerization of homogeneous monomers into multimeric structures, resulting in complex assembly processes and poor stability. For example, MspA and α-HL require complex recombinant expression and purification processes, which are costly and difficult to mass-produce. In summary, developing novel nanoporous proteins with simple structures, efficient preparation, and high sequencing accuracy, as well as optimizing analyte capture and rate control mechanisms, are key breakthroughs for advancing nanoporous technology towards industrial applications. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides the application of enterococcal pore-forming toxin mutant protein in nanopore detection, aiming to solve the problems mentioned in the background art.

[0005] In a first aspect, the present invention provides the application of enterococcal pore-forming toxin mutant protein in nanopore detection, wherein the enterococcal pore-forming toxin mutant protein is EPX1 nanopore mutant protein, which is obtained by mutation of wild-type EPX1 nanopore protein. The amino acid sequence of wild-type EPX1 nanopore protein is shown in SEQ ID NO:1. The mutation refers to: one or more amino acids of wild-type EPX1 nanopore protein being mutated to common amino acids other than the original amino acids.

[0006] The common amino acids mentioned include glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, and histidine.

[0007] Furthermore, the EPX1 nanopore mutant protein is selected from A, B, C, or D:

[0008] A: One or more of the amino acids V11, L12, and G13 of wild-type EPX1 nanoporin are mutated to common amino acids other than the original amino acids;

[0009] B: One or more of the amino acids N33, Q34, and Y35 of wild-type EPX1 nanoporin are mutated to common amino acids other than the original amino acids;

[0010] C: One or more of the amino acids K139, Q140, and V141 in wild-type EPX1 nanoporin are mutated to common amino acids other than the original amino acids;

[0011] D: One or more of the amino acids T174, T175, and Y176 in wild-type EPX1 nanoporin are mutated to common amino acids other than the original amino acids.

[0012] Further, in A: the V11 amino acid of the wild-type EPX1 nanoporous protein is mutated to V11P, V11L, V11G, V11R, V11Q, V11E, V11C, V11W, V11H, V11K, V11F, V11A, V11S, V11Y, V11N, or V11D; preferably, the V11 amino acid of the wild-type EPX1 nanoporous protein is mutated to V11A, V11L, or V11N;

[0013] The amino acid L12 of wild-type EPX1 nanoporin is mutated to L12A or L12V.

[0014] The amino acid G13 mutation of wild-type EPX1 nanoporin is G13N, G13K, G13S, G13D, G13W, G13R, G13F, G13E, G13P, G13L, G13A, G13V or G13M; preferably, the amino acid G13 mutation of wild-type EPX1 nanoporin is G13N or G13S.

[0015] Further, in B: the amino acid N33 mutation of wild-type EPX1 nanoporous protein is N33A, N33H, N33Q, N33S, N33L, N33D, N33C, N33I, N33P, N33T, N33G, N33M, N33W or N33R; preferably, the amino acid N33 mutation of wild-type EPX1 nanoporous protein is N33A;

[0016] The amino acid Q34 of wild-type EPX1 nanoporin is mutated to Q34N or Q34L;

[0017] The amino acid Y35 mutation of wild-type EPX1 nanoporin is Y35P, Y35L, Y35G, Y35I, Y35Q, Y35E, Y35C, Y35R, Y35H, Y35K, Y35T, Y35A, Y35S, Y35W, Y35N, Y35V or Y35D; preferably, the amino acid Y35 mutation of wild-type EPX1 nanoporin is Y35R, Y35L or Y35G.

[0018] Further, in C: the amino acid K139 of the wild-type EPX1 nanoporin 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 nanoporin is mutated to K139N, K139R or K139G;

[0019] The amino acid Q140 of wild-type EPX1 nanoporin is mutated to Q140R or Q140S;

[0020] The wild-type EPX1 nanoporin has an amino acid V141 mutation that is V141N, V141K, V141H, V141D, V141W, V141R, V141F, V141E, V141P, V141L, V141A, V141G, or V141M; preferably, the wild-type EPX1 nanoporin has an amino acid V141 mutation that is V141N or V141K.

[0021] Further, in D: the amino acid T174 mutation of wild-type EPX1 nanoporin is T174P, T174L, T174G, T174I, T174Q, T174E, T174C, T174R, T174H, T174K, T174A, T174S, T174Y, T174N, T174V or T174D; preferably, the amino acid T174 mutation of wild-type EPX1 nanoporin is T174L or T174G;

[0022] The amino acid T175 mutation of wild-type EPX1 nanoporin is T175P, T175L, T175G, T175I, T175Q, T175E, T175C, T175R, T175H, T175K, T175A, T175S, T175Y, T175N, T175V or T175D; preferably, the amino acid T175 mutation of wild-type EPX1 nanoporin is T175R, T175L or T175G.

[0023] The amino acid Y176 of wild-type EPX1 nanoporin is mutated to Y176N or Y176V.

[0024] Furthermore, the gene encoding the enterococcal pore-forming toxin mutant protein is selected from the following E, F, or G:

[0025] E: Nucleic acid encoding the EPX1 nanopore mutant protein;

[0026] F: Nucleic acid with 85-99% homology to the nucleic acid sequence in E, and encoding the EPX1 nanopore mutant protein;

[0027] G: Nucleic acid that is complementary to the nucleic acid of E or F.

[0028] Furthermore, it can detect one or more of the following: metal ions, inorganic salts, polymers, amino acids, peptides, polypeptides, proteins, nucleotides, oligonucleotides, polynucleotides, dyes, bleaching agents, drugs, diagnostic reagents, narcotics, explosive pollutants, or environmental pollutants.

[0029] This invention offers the following advantages: EPX1 protein (wild type) is a type of pore-forming toxin protein widely found in various organisms in nature. Its mutants, after modification (amino acid mutation), exhibit excellent pore-forming ability. Not only does it possess a simple expression and purification process, allowing for rapid preparation using conventional molecular biology methods, but it can also stably construct single nanopores in a lipid membrane environment, providing an ideal functional interface for single-molecule detection as a nanoporous protein.

[0030] The EPX1 protein assembles as a homooctamer, forming a barrel-shaped pore structure with a total length of 123 Å and a maximum outer diameter of 97 Å. This structure is precisely arranged along an octet of symmetry, consisting of an N-terminal β-barrel, a central vestibule formed by a cap domain, and a transmembrane β-barrel. This structure ensures the mechanical stability of the pores and creates a specific recognition space for molecular permeation events. The octamer symmetry structure endows the pores with excellent membrane intercalation capabilities. Furthermore, the central vestibule region of the EPX1 protein has a diameter of 68 Å, providing ample space for conformational changes of target molecules. Combined with precise mutational regulation of the contraction region (14.8-20.9 Å in diameter), gradient regulation of the sample permeation rate can be achieved. Moreover, through amino acid mutations, the EPX1 nanopore mutant protein exhibits significant improvements in structural stability, molecular recognition efficiency, and detection performance. Attached Figure Description

[0031] Exemplary embodiments of the present invention can be more fully understood by referring to the following figures:

[0032] Figure 1This is an electrophoresis diagram of the purification of wild-type EPX1 nanoporous protein in Example 1 of the present invention. In the diagram, lane 1: protein marker; lane 2: elution sample; lane 3: elution sample; lane 4: elution sample; lane 5: elution sample.

[0033] Figure 2 This is a graph showing the pore opening current detection results of wild-type EPX1 nanoporous protein in Example 2 of the present invention.

[0034] Figure 3 This is a current signal diagram of single-stranded DNA passing through wild-type EPX1 nanoporous protein in Example 3 of the present invention.

[0035] Figure 4 This is a current signal diagram of miRNA passing through EPX1 V11A nanoporous protein in Example 4 of the present invention.

[0036] Figure 5 This is a current signal diagram of miRNA passing through the EPX1 G13N nanoporous protein in Example 4 of the present invention.

[0037] Figure 6 This is a current signal diagram of aspartic acid passing through EPX1 T175R nanoporous protein in Example 5 of the present invention.

[0038] Figure 7 This is a current signal diagram of aspartic acid passing through the EPX1 Y176V nanoporous protein in Example 5 of the present invention.

[0039] Figure 8 This is a current signal diagram of ATP passing through the EPX1 N33A nanoporous protein in Example 6 of the present invention.

[0040] Figure 9 This is a current signal diagram of ATP passing through the EPX1 Q34N nanoporous protein in Example 6 of the present invention.

[0041] Figure 10 This is a current signal diagram of Hg²⁺ passing through EPX1 K139P nanoporous protein in Example 7 of the present invention.

[0042] Figure 11 This is a current signal diagram of Hg²⁺ passing through the EPX1 V141H nanoporous protein in Example 7 of the present invention. Detailed Implementation

[0043] To make the technical problems to be solved, the technical solutions, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.

[0044] 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 pertains; the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0045] This invention provides the application of enterococcal pore-forming toxin mutant protein in nanopore detection. The enterococcal pore-forming toxin mutant protein is EPX1 nanopore mutant protein, which is obtained by mutation of wild-type EPX1 nanopore protein. The amino acid sequence of wild-type EPX1 nanopore protein is shown in SEQ ID NO:1. Mutation refers to one or more amino acids of wild-type EPX1 nanopore protein being mutated to common amino acids other than the original amino acids.

[0046] 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.

[0047] In some embodiments, the EPX1 nanopore mutant protein is selected from A, B, C, or D:

[0048] A: One or more of the amino acids V11, L12, and G13 of wild-type EPX1 nanoporin are mutated to common amino acids other than the original amino acids;

[0049] B: One or more of the amino acids N33, Q34, and Y35 of wild-type EPX1 nanoporin are mutated to common amino acids other than the original amino acids;

[0050] C: One or more of the amino acids K139, Q140, and V141 in wild-type EPX1 nanoporin are mutated to common amino acids other than the original amino acids;

[0051] D: One or more of the amino acids T174, T175, and Y176 in wild-type EPX1 nanoporin are mutated to common amino acids other than the original amino acids.

[0052] In some embodiments, in A: the amino acid V11 of the wild-type EPX1 nanoporin is mutated to V11P, V11L, V11G, V11R, V11Q, V11E, V11C, V11W, V11H, V11K, V11F, V11A, V11S, V11Y, V11N, or V11D;

[0053] The amino acid L12 of wild-type EPX1 nanoporin is mutated to L12A or L12V.

[0054] The amino acid G13 mutation of wild-type EPX1 nanoporin is G13N, G13K, G13S, G13D, G13W, G13R, G13F, G13E, G13P, G13L, G13A, G13V or G13M.

[0055] In some embodiments, the amino acid V11 of the wild-type EPX1 nanoporin is mutated to V11A, V11L, or V11N.

[0056] In some embodiments, the amino acid G13 of the wild-type EPX1 nanoporin is mutated to G13N or G13S.

[0057] In some embodiments, in B: the amino acid N33 mutation of wild-type EPX1 nanoporin is N33A, N33H, N33Q, N33S, N33L, N33D, N33C, N33I, N33P, N33T, N33G, N33M, N33W or N33R.

[0058] The amino acid Q34 of wild-type EPX1 nanoporin is mutated to Q34N or Q34L;

[0059] The amino acid Y35 mutation of wild-type EPX1 nanoporin is Y35P, Y35L, Y35G, Y35I, Y35Q, Y35E, Y35C, Y35R, Y35H, Y35K, Y35T, Y35A, Y35S, Y35W, Y35N, Y35V or Y35D.

[0060] In some embodiments, the amino acid N33 of the wild-type EPX1 nanoporin is mutated to N33A.

[0061] In some embodiments, the amino acid Y35 of the wild-type EPX1 nanoporin is mutated to Y35R, Y35L, or Y35G.

[0062] In some embodiments, in C: the amino acid K139 mutation of wild-type EPX1 nanoporin is K139M, K139P, K139L, K139G, K139I, K139Q, K139E, K139C, K139R, K139H, K139W, K139T, K139A, K139S, K139Y, K139N, K139V or K139D;

[0063] The amino acid Q140 of wild-type EPX1 nanoporin is mutated to Q140R or Q140S;

[0064] The amino acid V141 mutation of wild-type EPX1 nanoporin is V141N, V141K, V141H, V141D, V141W, V141R, V141F, V141E, V141P, V141L, V141A, V141G, or V141M.

[0065] In some embodiments, the amino acid K139 of the wild-type EPX1 nanoporin is mutated to K139N, K139R, or K139G.

[0066] In some embodiments, the amino acid V141 of the wild-type EPX1 nanoporin is mutated to V141N or V141K.

[0067] In some embodiments, in D: the amino acid T174 mutation of wild-type EPX1 nanoporin is T174P, T174L, T174G, T174I, T174Q, T174E, T174C, T174R, T174H, T174K, T174A, T174S, T174Y, T174N, T174V or T174D;

[0068] The amino acid T175 mutation of wild-type EPX1 nanoporin can be T175P, T175L, T175G, T175I, T175Q, T175E, T175C, T175R, T175H, T175K, T175A, T175S, T175Y, T175N, T175V or T175D.

[0069] The amino acid Y176 of wild-type EPX1 nanoporin is mutated to Y176N or Y176V.

[0070] In some embodiments, the amino acid T174 of the wild-type EPX1 nanoporin is mutated to T174L or T174G.

[0071] In some embodiments, the amino acid T175 of the wild-type EPX1 nanoporin is mutated to T175R, T175L, or T175G.

[0072] In some embodiments, the preparation method of the EPX1 nanopore mutant protein includes the following steps:

[0073] Step S1: Construct the EPX1 nanopore mutant protein vector;

[0074] Step S2: Expression and purification of EPX1 nanopore mutant protein.

[0075] In some embodiments, the gene encoding the enterococcal pore-forming toxin mutant protein is selected from the following E, F, or G:

[0076] E: Nucleic acid encoding the EPX1 nanopore mutant protein;

[0077] F: Nucleic acid with 85-99% homology to the nucleic acid sequence in E, and encoding the EPX1 nanopore mutant protein;

[0078] G: Nucleic acid that is complementary to the nucleic acid of E or F.

[0079] In some embodiments, one or more of the following are detected: metal ions, inorganic salts, polymers, amino acids, peptides, polypeptides, proteins, nucleotides, oligonucleotides, polynucleotides, dyes, bleaching agents, drugs, diagnostic agents, narcotics, explosive pollutants, or environmental pollutants.

[0080] Example 1: Expression and purification of wild-type EPX1 nanoporous protein

[0081] (1) Gene construction and vector design: The wild-type EPX1 nanoporous protein DNA fragment was synthesized, and the DNA sequence is shown in SEQ ID NO:2. The fragment was cloned into the pET-30a(+) vector.

[0082] (2) The plasmid was transformed into E. coli Dh5α competent cells and amplified by plate culture and liquid culture;

[0083] (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 tested in a small volume of 50 ml LB medium to determine the optimal parameters.

[0084] (4) Large-scale culture and induction: Inoculation and culture: Inoculate 1 / 50 volume of overnight pre-culture (containing multiple single colonies) into 5LB medium (containing 100 μg / ml kanamycin and 1% glucose) and culture at 37℃ with shaking at 180 rpm until OD. 600 =0.6–0.8, then cool to 20°C, add 0.5 mM IPTG, and continue induction culture for 8–12 hours;

[0085] (5) Collection of bacterial cells: Centrifuge at 3400g for 60 minutes at 4℃, collect the bacterial cell precipitate, and freeze the precipitate at -20℃ for purification;

[0086] (6) Cell disruption: Resuspend the cells in lysis buffer (50mM Tris, pH=8.0, 500mM NaCl, 10mM imidazole, 1% Triton X-100), add lysozyme (0.2mg / ml) and protease inhibitor, lyse by high pressure homogenization, add benzamide enzyme, stir on ice for 30 minutes, and then perform high pressure homogenization twice to ensure complete lysis;

[0087] (7) Membrane component collection: The supernatant was collected by ultracentrifugation;

[0088] (8) The supernatant was loaded onto a Ni column for affinity chromatography purification;

[0089] (9) Washing off contaminating proteins: 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).

[0090] (10) Target protein elution: Elute with Buffer A containing 250 mM imidazole in 5 column volumes and collect the elution peak containing oligomers;

[0091] (11) Refolding treatment: The target protein component was transferred to dialysis buffer (20 mM Tris, pH=8.0, 150 mM NaCl, 20 mM sodium cholate) and dialyzed overnight at 4°C to remove small molecule impurities, and finally wild-type EPX1 nanoporous protein was obtained.

[0092] The amino acid sequence of wild-type EPX1 nanoporin is shown in SEQ ID NO:1, and the nucleic acid sequence encoding wild-type EPX1 nanoporin is shown in SEQ ID NO:2.

[0093] The purified electrophoresis image of wild-type EPX1 nanoporous protein is shown below. Figure 1 As shown.

[0094] Structural analysis of wild-type EPX1 nanoporous protein: Wild-type EPX1 nanoporous protein assembles as homooctamers, forming a barrel-shaped pore structure with a total length of 123 Å and a maximum outer diameter of 97 Å. This structure is precisely arranged along an octet of symmetry, consisting of an N-terminal β-barrel, a central vestibule formed by a cap domain, and a transmembrane β-barrel. This structure ensures the mechanical stability of the pores and creates a specific recognition space for molecular transpore events. The octamer symmetry structure endows the pores with excellent membrane intercalation capabilities. Furthermore, the central vestibule region of the EPX1 protein has a diameter of 68 Å, providing ample space for conformational changes of target molecules.

[0095] Example 2: Characterization of wild-type EPX1 nanoporous protein

[0096] (1) After obtaining wild-type EPX1 nanoporous protein, a single protein pore was constructed in a lipid bilayer. First, an artificial lipid bilayer was 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 both chambers, with a volume of 1.2 mL / side, to ensure that the liquid levels on both sides of the membrane were balanced.

[0097] (2) 8 ng / mL of wild-type EPX1 nanoporous protein solution was added to the cis-cavity, and blank buffer was used for the trans-cavity. A transmembrane voltage of 160 mV (Axopatch 200B amplifier) ​​was applied, and the current change was monitored in real time. When the protein was inserted to form a single well, the characteristic current step signal was recorded. After the protein well was formed, the cis-cavity was rinsed 3 times with 50 μL buffer to remove unbound protein. All experiments were performed at room temperature (23±1℃). 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.

[0098] The results of wild-type EPX1 nanoporous protein pore opening current detection are as follows: Figure 2 As shown.

[0099] Example 3: Detection of DNA using wild-type EPX1 nanoporous protein

[0100] (1) After obtaining wild-type EPX1 nanoporous protein, a single protein pore was constructed in a lipid bilayer. First, an artificial lipid bilayer was 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 both chambers, with a volume of 1.2 mL / side, to ensure that the liquid levels on both sides of the membrane were balanced.

[0101] (2) Add 8 ng / mL of wild-type EPX1 nanoporous protein solution to the cis-cavity and blank buffer to the trans-cavity. Apply a transmembrane voltage of 160 mV (Axopatch 200B amplifier) ​​and monitor the current change in real time. When the protein is inserted to form a single pore, record the characteristic current step signal. After the protein pore is formed, rinse the cis-cavity 3 times with 50 μL buffer to remove unbound protein and avoid interference from multiple pores.

[0102] (3) Add 10 nM single-stranded DNA (SEQ ID NO:3: 5'-ATCGATCGATCGATCGATCGATCGATCGATCGATCGATCG-3'), apply a voltage of 160 mV (optimize the voltage to reduce the influence of chain folding), sample at 50 kHz, use low-pass filtering (2 kHz), and complete all experiments at room temperature (23 ± 1 ℃). Data acquisition is controlled by Origin.

[0103] The electrical signal of single-stranded DNA passing through wild-type EPX1 nanoporous protein, such as Figure 3 As shown.

[0104] Example 4: Detection of miRNA using EPX1 nanopore mutant protein with mutation sites V11, L12, and G13.

[0105] (1) Following the preparation method of wild-type EPX1 nanoporous protein in Example 1, EPX1 nanoporous 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, G1) were obtained. After constructing artificial lipid bilayers (G13F, G13E, G13P, G13L, G13A, G13V, G13M), a single protein pore was created in the lipid bilayer. First, artificial lipid bilayers were formed on both sides of the pores 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 both chambers, with a volume of 1.2 mL / side, to ensure that the liquid levels on both sides of the membrane were balanced.

[0106] (2) Add 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 to the cis-chamber, and use blank buffer for the trans-chamber. Apply a transmembrane voltage of 160 mV (Axopatch). A 200B amplifier was used to monitor current changes in real time. When a protein was inserted to form a single well, a characteristic current step signal was recorded. After the protein well was formed, the cis-cavity was rinsed three times with 50 μL buffer to remove unbound protein and avoid interference from multiple wells.

[0107] (3) Add miRNA (SEQ ID NO:4: 5'-UAGCUUAUCAGUCUGAUGUUGA-3', 22nt, unmodified 10nM), apply a voltage of 160mV (optimize the voltage to reduce the influence of chain folding), sample at 50kHz, and use low-pass filtering (2kHz). All experiments were performed at room temperature (23±1℃), and data acquisition was controlled using Origin.

[0108] The current signal of miRNA passing through the EPX1 V11A nanoporin, such as Figure 4As shown, it can be seen that after the amino acid V11 in the contraction region of wild-type EPX1 nanoporous protein is mutated to V11A, the quality of the current signal for detecting miRNA is significantly improved, the current properties are stable, the current signal width is narrower, and there are fewer spikes. 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.

[0109] The current signal of miRNA passing through the EPX1 G13N nanoporin, such as Figure 5 As shown, it can be seen that after the amino acid G13 in the contraction region of wild-type EPX1 nanoporous protein is mutated to G13N, the quality of the current signal for detecting miRNA is significantly improved, the current properties are stable, the current signal width is narrower, and there are fewer spikes. This is because the G13N mutation replaces glycine with asparagine (containing a polar amide group), changing the polarity and hydrogen bonding interaction. Mutation type: polarity and hydrogen bonding regulation.

[0110] The current signals of other EPX1 nanopore mutant proteins are shown in Table 1.

[0111] Table 1. Current signals of other EPX1 nanopore mutant proteins in Example 4

[0112]

[0113] Example 5: Detection of aspartic acid using EPX1 nanopore mutant protein with mutation sites T174, T175, and Y176.

[0114] (1) Following the preparation method of wild-type EPX1 nanoporous protein in Example 1, EPX1 nanoporous 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, T174G, T174Q, T174E, T174C, T174R, T174G, T174Q ...Q, T174G, T174Q, T174Q, T174Q, T174Q, T174Q, T174Q, T174Q, T174Q, T174Q, T174Q, T174Q, T174Q, T174Q, T174Q, T174Q, T174Q, T174Q, T174Q, T17 After establishing the protein pores (5H, T175K, T175A, T175S, T175Y, T175N, T175V, T175D, Y176N, Y176V), a single protein pore was constructed in the lipid bilayer. First, an artificial lipid bilayer was 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 both chambers, with a volume of 1.2 mL / side, to ensure liquid level equilibrium on both sides of the membrane.

[0115] (2) Add 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 to the cis-cavity, and use blank buffer for the trans-cavity. A transmembrane voltage of 160 mV (Axopatch 200B amplifier) ​​was applied, and the current change was monitored in real time. When the protein was inserted to form a single well, the characteristic current step signal was recorded. After the protein well was formed, the cis-cavity was rinsed three times with 50 μL buffer to remove unbound protein and avoid interference from multiple wells.

[0116] (3) Add aspartic acid sample (10 nM), apply 160 mV voltage, sampling rate 50 kHz, low-pass filter (2 kHz), all experiments were completed at room temperature (23 ± 1 ℃), and data acquisition was controlled by Origin.

[0117] The current signal of aspartic acid passing through the EPX1 T175R nanoporous protein is as follows: Figure 6 As shown, it can be seen that after the amino acid T175 in the contraction region of wild-type EPX1 nanoporous protein is mutated to T175R, the quality of the current signal for detecting aspartic acid is significantly improved, the current properties are stable, the current signal width is narrower, and there are fewer spikes. This is because the T175R mutation introduces positively charged arginine residues. Mutation type: charge distribution regulation.

[0118] The current signal of aspartic acid passing through the EPX1 Y176V nanoporous protein is as follows: Figure 7 As shown, it can be seen that after the amino acid Y176 in the contraction region of wild-type EPX1 nanoporous protein is mutated to Y176V, the quality of the current signal for detecting aspartic acid is significantly improved, the current properties are stable, the current signal width is narrower, and there are fewer spikes. This is because Y176V eliminates the synergistic effect of tyrosine hydroxyl groups through mutation. Mutation type: steric hindrance and flexibility regulation.

[0119] The current signals of other EPX1 nanopore mutant proteins are shown in Table 2.

[0120] Table 2 Current signals of other EPX1 nanopore mutant proteins in Example 5

[0121]

[0122] Example 6: Detection of ATP using EPX1 nanopore mutant protein with mutation sites N33, Q34, Y35

[0123] (1) Following the preparation method of wild-type EPX1 nanoporous protein in Example 1, EPX1 nanoporous 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 ... After using 5A, Y35S, Y35W, Y35N, Y35V, Y35D, Q34N, and Q34L, a single protein pore was constructed in the lipid bilayer. First, an artificial lipid bilayer was 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, and 0.1 mM EDTA) was injected into both chambers, with a volume of 1.2 mL / side, to ensure that the liquid levels on both sides of the membrane were balanced.

[0124] (2) Add 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 to the cis chamber, and use blank buffer for the trans chamber. A transmembrane voltage of 160 mV (Axopatch 200B amplifier) ​​was applied, and the current change was monitored in real time. When the protein was inserted to form a single well, the characteristic current step signal was recorded. After the protein well was formed, the cis-cavity was rinsed three times with 50 μL buffer to remove unbound protein and avoid interference from multiple wells.

[0125] (3) Add ATP sample (10 nM), apply 160 mV voltage, sampling rate 50 kHz, low-pass filter (2 kHz), all experiments were performed at room temperature (23 ± 1 ℃), and data acquisition was controlled by Origin.

[0126] The current signal of ATP passing through the EPX1 N33A nanoporous protein is as follows: Figure 8As shown, it can be seen that after the amino acid N33 in the contraction region of wild-type EPX1 nanoporous protein is mutated to N33A, the quality of the current signal for detecting ATP is significantly improved, the current properties are stable, the current signal width is narrower, and there are fewer spikes. This is because N33A eliminates charge interference through mutation. Mutation type: charge distribution regulation.

[0127] The current signal of ATP passing through the EPX1 Q34N nanoporous protein is as follows: Figure 9 As shown, it can be seen that after the amino acid Q34 in the contraction region of wild-type EPX1 nanoporin is mutated to Q34N, the quality of the current signal for ATP detection is significantly improved, the current properties are stable, the current signal width is narrower, and there are fewer spikes. This is because Q34N introduces a more polar amide group through mutation. The mutation type is: polarity and hydrogen bond regulation.

[0128] The current signals of other EPX1 nanopore mutant proteins are shown in Table 3.

[0129] Table 3 Current signals of other EPX1 nanopore mutant proteins in Example 6

[0130]

[0131] Example 7: Detection of Hg²⁺ using EPX1 nanopore mutant protein with mutation sites K139, Q140, and V141.

[0132] (1) Following the preparation method of wild-type EPX1 nanoporous protein in Example 1, EPX1 nanoporous 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, ...) were obtained. After using V141W, V141R, V141F, V141E, V141P, V141L, V141A, V141G, and V141M, a single protein pore was created in a lipid bilayer. First, an artificial lipid bilayer was 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, and 0.1 mM EDTA) was injected into both chambers, with a volume of 1.2 mL / side, to ensure that the liquid levels on both sides of the membrane were balanced.

[0133] (2) Add 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 to the cis chamber, and use blank buffer for the trans chamber. A transmembrane voltage of 160 mV (Axopatch 200B amplifier) ​​was applied, and the current change was monitored in real time. When the protein was inserted to form a single well, the characteristic current step signal was recorded. After the protein well was formed, the cis-cavity was rinsed three times with 50 μL buffer to remove unbound protein and avoid interference from multiple wells.

[0134] (3) Add Hg²⁺ sample (10 nM), apply 160 mV voltage, sampling rate 50 kHz, low-pass filter (2 kHz), all experiments were completed at room temperature (23 ± 1 ℃), and data acquisition was controlled by Origin.

[0135] The current signal of Hg²⁺ passing through the EPX1 K139P nanoporous protein is as follows: Figure 10 As shown, it can be seen that after the amino acid K139 in the contraction region of wild-type EPX1 nanoporous protein is mutated to K139P, the quality of the current signal for detecting Hg²⁺ is significantly improved, the current properties are stable, the current signal width is narrower, and there are fewer spikes. This is because K139P introduces the rigid structure of proline to compress the space through mutation. Mutation type: steric hindrance and flexibility regulation.

[0136] The current signal of Hg²⁺ passing through the EPX1 V141H nanoporous protein is as follows: Figure 11 As shown, it can be seen that after the amino acid V141 in the contraction region of wild-type EPX1 nanoporous protein is mutated to V141H, the quality of the current signal for detecting Hg²⁺ is significantly improved, the current properties are stable, the current signal width is narrower, and there are fewer spikes. This is because V141H reduces random charge interference through mutation. The mutation type is charge distribution regulation.

[0137] The current signals of other EPX1 nanopore mutant proteins are shown in Table 4.

[0138] Table 4 Current signals of other EPX1 nanopore mutant proteins in Example 7

[0139]

[0140] Amino acid sequence: SEQ ID NO:1:

[0141] mdvqvfadnpvlgtevtfekdengrivkiisknqyritiynsvdagdtpndasvsldvdfiddknsgemga vasintfipsglryvegykykgvtnpiyknlsagmlwpkkyrvevvnipidqatkiitatpnnnikekqvsdtisygfggsvsadgkkpggsieanlaytktttydqpdyetsqikkttkeavwdtsfvetrdgytpnswnpvygnqmfmrgrysnvspidnikkggevsslisggfspkmgvvlaspngtkksqfvvrvsrmsdmyimrwsgtewggeneinqnvpkey

[0142] Nucleic acid sequence: SEQ ID NO:2:

[0143] atggatgtgcaggtgtttgcggataacccggtgctgggcaccgaagtgacctttgaaaaagatgaaaacggccgcattgtgaaaattattagcaaaaaccagtatcgcattaccatttataacagcgtggatgcgggcgataccccgaacgatgcgagcgtgagcctggatgtggattttattgatgataaaaacagcggcgaaatgggcgcggtggcgagcattaacacctttattccgagcggcctgcgctatgtggaaggctataaatataaaggcgtgaccaacccgatttataaaaacctgagcgcgggcatgctgtggccgaaaaaatatcgcgtggaagtggtgaacattccgattgatcaggcgaccaaaattattaccgcgaccccgaacaacaacattaaagaaaaacaggtgagcgataccattagctatggctttggcggcagcgtgagcgcggatggcaaaaaaccgggcggcagcattgaagcgaacctggcgtataccaaaaccaccacctatgatcagccggattatgaaaccagccagattaaaaaaaccaccaaagaagcggtgtgggataccagctttgtggaaacccgcgatggctataccccgaacagctggaacccggtgtatggcaaccagatgtttatgcgcggccgctatagcaacgtgagcccgattgataacattaaaaaaggcggcgaagtgagcagcctgattagcggcggctttagcccgaaaatgggcgtggtgctggcgagcccgaacggcaccaaaaaaagccagtttgtggtgcgcgtgagccgcatgagcgatatgtatattatgcgctggagcggcaccgaatggggcggcgaaaacgaaattaaccagaacgtgccgaaagaatataacgcgctgatgtatgaagatgtgaaatttgaaattgattgggaacagcgcaccgtgcgcaccattctggaa

[0144] 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 within the protection scope of the present invention.

Claims

1. Use of an Enterococcus pore-forming toxin mutant protein in nanopore detection, characterized in that: The Enterococcus 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 EPX1 nanopore mutant protein is selected from the following A, B, C or D: A: the amino acid V11 of the wild-type EPX1 nanopore protein is mutated into V11P, V11L, V11G, V11R, V11Q, V11E, V11C, V11W, V11H, V11K, V11F, V11A, V11S, V11Y, V11N or V11D; or, the amino acid L12 of the wild-type EPX1 nanopore protein is mutated into L12A or L12V; or, the amino acid G13 of the wild-type EPX1 nanopore protein is mutated into G13N, G13K, G13S, G13D, G13W, G13R, G13F, G13E, G13P, G13L, G13A, G13V or G13M; B: the amino acid N33 of the wild-type EPX1 nanopore protein is mutated into N33A, N33H, N33Q, N33S, N33L, N33D, N33C, N33I, N33P, N33T, N33G, N33M, N33W or N33R; or, the amino acid Q34 of the wild-type EPX1 nanopore protein is mutated into Q34N or Q34L; or, the amino acid Y35 of the wild-type EPX1 nanopore protein is mutated into Y35P, Y35L, Y35G, Y35I, Y35Q, Y35E, Y35C, Y35R, Y35H, Y35K, Y35T, Y35A, Y35S, Y35W, Y35N, Y35V or Y35D; C: the amino acid K139 of the wild-type EPX1 nanopore protein is mutated into K139M, K139P, K139L, K139G, K139I, K139Q, K139E, K139R, K139H, K139W, K139T, K139A, K139S, K139Y, K139N, K139V or K139D; or, the amino acid Q140 of the wild-type EPX1 nanopore protein is mutated into Q140R or Q140S; or, the amino acid V141 of the wild-type EPX1 nanopore protein is mutated into V141N, V141K, V141H, V141D, V141W, V141R, V141F, V141E, V141P, V141L, V141A, V141G or V141M; D: the amino acid T174 of the wild-type EPX1 nanopore protein is mutated into T174P, T174L, T174G, T174I, T174Q, T174E, T174C, T174H, T174K, T174A, T174S, T174Y, T174N, T174V or T174D; or amino acid Y176 of the wild-type EPX1 nanopore protein is mutated to Y176N or Y176V. A: amino acid V11 of the wild-type EPX1 nanopore protein is mutated to V11A, V11L, or V11N; or, amino acid G13 of the wild-type EPX1 nanopore protein is mutated to G13N or G13S.

2. Use according to claim 1, characterized in that: B: amino acid N33 of the wild-type EPX1 nanopore protein is mutated to N33A; or, amino acid Y35 of the wild-type EPX1 nanopore protein is mutated to Y35R, Y35L, or Y35G.

3. Use according to claim 1, characterized in that: C: amino acid K139 of the wild-type EPX1 nanopore protein is mutated to K139N, K139R, or K139G; or, amino acid V141 of the wild-type EPX1 nanopore protein is mutated to V141N or V141K.

4. The use according to claim 1, characterized in that: D: amino acid T174 of the wild-type EPX1 nanopore protein is mutated to T174L or T174G; or, amino acid T175 of the wild-type EPX1 nanopore protein is mutated to T175R, T175L, or T175G.

5. The use according to claim 1, characterized in that: The nanopore detection includes detecting one or more of a metal ion, an inorganic salt, a peptide, a protein, or a nucleotide.

6. Use according to any one of claims 1 to 5, characterized in that: ​

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