Application of the two-component toxin nanopore protein xaxab after site-directed mutagenesis

Abstract

The application belongs to the technical field of biochemistry and molecular biology, and provides application of a two-component toxin nanopore protein XaxAB after site-directed mutation modification. The two-component toxin nanopore protein XaxAB comprises equal numbers of XaxA subunits and XaxB subunits, the amino acid sequence of the XaxA subunit is shown as SEQ ID NO: 1, and the amino acid sequence of the XaxB subunit is shown as SEQ ID NO: 2. The two-component toxin nanopore protein XaxAB is subjected to site-directed mutation modification, T290C mutation is introduced into the XaxA subunit and I30C mutation is introduced into the XaxB subunit, the two-component toxin nanopore protein XaxAB after site-directed mutation modification improves the current signal quality of pore opening, sequencing or detection, the current property is stable, the current signal width is narrower, and burrs are less.

Description

Technical Field

[0001] This invention belongs to the field of biochemistry and molecular biology, and in particular relates to the application of the two-component toxin nanoporous protein XaxAB modified by site-directed mutagenesis. Background Technology

[0002] Nanopore technology, as a revolutionary tool in the field of single-molecule detection, enables ultra-high resolution analysis of nucleic acid sequences, protein conformations, and small molecule metabolites by capturing the characteristic changes in ion currents caused by biomolecules passing through nanopores. It has irreplaceable application value in fields such as precision medicine, environmental monitoring, and biosafety.

[0003] However, existing bio-nanopore systems still face multiple technical bottlenecks in dynamic pore size regulation, severely limiting their broad applicability: First, structural rigidity limits the regulation range. The pore structure of traditional nanopores (such as α-hemolysin and MspA) is formed by the assembly of fixed oligomers, and their pore size is strictly constrained by the spatial stacking of amino acid side chains. This contradiction between the inherent stability of the structure and the regulation requirements makes traditional nanopores unable to meet the cross-scale detection needs from single nucleotides (0.3 nm in diameter) to complete antibody molecules (10 nm in diameter). Second, it is difficult to balance regulation precision and dynamic response. Existing regulation methods have significant limitations: chemical modification methods can achieve fine-tuning of 0.2-0.3 nm by modifying functional groups on the inner wall of the pore, but uneven modification efficiency leads to increased dispersion in pore size distribution (coefficient of variation > 15%); pH or voltage-responsive pores (such as CsgG) can achieve dynamic regulation, but the response amplitude is usually < 1 nm, and the regulation precision decays after multiple cycles due to protein conformational fatigue, making them unable to meet the simultaneous analysis of multiple targets of different sizes in complex samples. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides the application of a two-component toxin nanoporin XaxAB modified by site-directed mutagenesis, with the aim of solving the problems mentioned in the background art.

[0005] This invention discovers that the two-component toxin XaxAB, derived from *Xenorhabdus nematophila*, possesses unique potential to solve the problems mentioned in the background art. XaxAB, a two-component toxin derived from *Xenorhabdus nematophila*, is encoded by the XaxA and XaxB genes, and its structural and functional characteristics offer new directions for nanopore technology. The funnel-shaped channels formed by the XaxAB oligomers exhibit tunable pore size characteristics. In terms of membrane interactions, XaxAB can stably insert into DPhPC-cholesterol composite membranes (cholesterol molar ratio 10%), and its insertion mechanism is independent of specific lipid modifications, making it suitable for various membrane systems. Based on the structural biology data of XaxAB (such as the amino acid sequence homology of XaxA and XaxB, and the three-dimensional structure of the oligomers), this invention develops it into a tunable pore size nanopore sensor. Through membrane composition optimization and innovative detection methods, it overcomes the technical bottlenecks of traditional nanopores in terms of dynamic range, charge adaptability, and stability, providing a systematic solution for high-resolution analysis of complex biological samples.

[0006] This invention provides the application of a site-directed mutagenesis-modified two-component toxin nanoporin XaxAB in the sequencing or detection of target analytes. The two-component toxin nanoporin XaxAB comprises equal numbers of XaxA and XaxB subunits. The amino acid sequence of the XaxA subunit is shown in SEQ ID NO: 1, and the amino acid sequence of the XaxB subunit is shown in SEQ ID NO: 2.

[0007] The two-component toxin nanoporin XaxAB was subjected to site-directed mutagenesis. Specifically, the amino acid T290 of the XaxA subunit was mutated to T290C, and the amino acid I30 of the XaxB subunit was mutated to I30C, thus obtaining the site-directed mutagenesis-modified two-component toxin nanoporin XaxAB.

[0008] Furthermore, the target analytes include one or more of the following: metal ions, inorganic salts, polymers, amino acids, peptides, polypeptides, proteins, nucleotides, oligonucleotides, polynucleotides, dyes, bleaching agents, drugs, diagnostic agents, narcotics, explosive pollutants, and environmental pollutants.

[0009] Furthermore, the two-component toxin nanoporin XaxAB has a conical structure, including a cis-open portion, a lumen, and a trans-constriction portion, with the lumen connecting the cis-open portion and the trans-constriction portion;

[0010] The outer diameter of the cis-type opening at the connection with the lumen is ≥6nm, the depth of the lumen is ≥6nm, and the diameter of the inverse contraction is ≥0.5nm.

[0011] Furthermore, the site-directed mutagenesis-modified two-component toxin nanoporin XaxAB regulates the pore size by adjusting the number of XaxA and XaxB subunits, enabling sequencing or detection of target analytes with different diameters.

[0012] Furthermore, the total number of XaxA and XaxB subunits is 8-40.

[0013] Furthermore, the gene encoding the two-component toxin nanoporous protein XaxAB is selected from:

[0014] A: Nucleic acid sequences encoding the XaxA and XaxB subunits of the two-component toxin nanoporin XaxAB;

[0015] B: Nucleic acid sequence having at least 85% homology with the nucleic acid sequence in A, and encoding the XaxA and XaxB subunits of the two-component toxin nanoporin XaxAB;

[0016] C: Nucleic acid complementary to A and B;

[0017] Among them, the homology is between 85% and 99%.

[0018] Furthermore, the site-directed mutagenesis-modified two-component toxin nanoporin XaxAB is inserted into the membrane layer, and the membrane layer comprises: 1,2-diphydanyl-sn-glycero-3-phosphate choline, 1,2-dipalmitoyl-sn-glycero-3-phosphate choline, 1,2-dipalmitoyl-sn-glycero-3-phosphate ethanolamine, 1,2-dipalmitoyl-sn-glycero-3-phosphate glycerol, 1,2-dipalmitoyl-sn-glycero-3-phosphate-L-serine, 1,2-adiacyl-sn-glycero-3-phosphate choline, and 1,2-dilauroyl-sn... -Glyceryl-3-phosphocholine, 1,2-Dimyristico-sn-glyceryl-3-phosphocholine, 1,2-Dimyristico-sn-glyceryl-3-phosphoethanolamine, 1,2-Dimyristico-sn-glyceryl-3-phosphoglyceride, 1,2-Dimyristico-sn-glyceryl-3-phospho-L-serine, 1,2-Dioleoyl-sn-glyceryl-3-phospho-L-serine, 1,2-Dioleoyl-sn-glyceryl-3-phosphoethanolamine, 1,2-Dioleoyl-sn-glyceryl-3-phosphoglyceride, 1,2-distearatel-sn-glyceryl-3-phosphocholine, 1-palmitoyl-2-oleoyl-S At least one of the following: N-glycero-3-phosphocholine, mycolic acid, campesterol, sitosterol, stigmasterol, cholesterol, ergosterol, cardiolipin, sphingomyelin, 1-palmitoyl-2-cholesterolylhemisuccino-sn-glycero-3-phosphocholine, 1-oleoyl-2-cholesterolylhemisuccino-sn-glycero-3-phosphocholine, 1-palmitoyl-2-cholesterolylcarbonyl-sn-glycero-3-phosphocholine, 1,2-dicholesterolylhemisuccino-sn-glycero-3-phosphocholine, 10,12-pentadecanodiyne, 10,12-triadecanodiyne, 5,7-hexadecadecanodiyne, or 9,12-octadecadecanodiyne.

[0019] Furthermore, the preparation method of the site-directed mutagenesis-modified two-component toxin nanoporous protein XaxAB includes the following steps:

[0020] Step S1: Construct a recombinant vector encoding the XaxA and XaxB subunits of the two-component toxin nanoporous protein XaxAB after site-directed mutagenesis modification, wherein the amino acid T290 of the XaxA subunit is mutated to T290C and the amino acid I30 of the XaxB subunit is mutated to I30C.

[0021] Step S2: Expression and purification of the site-directed mutagenesis-modified two-component toxin nanoporous protein XaxAB.

[0022] The present invention has the following beneficial effects:

[0023] (1) The funnel-shaped structure of the two-component toxin nanoporin XaxAB increases the ion density in the narrow portion. By adjusting the number of subunits, XaxAB precisely controls the pore size of the XaxAB nanopores, adapting to a wide range of analytes and overcoming the limitations of the detection range caused by the fixed pore size of traditional biological nanopores, thus improving detection flexibility. Therefore, XaxAB can detect analytes from small molecules to ultra-large molecules without changing the nanopore system. XaxAB can be purified into multiple polymers in a single step, allowing for selective screening of nanopores with the desired pore size without multiple experiments, simplifying the operation process. The multiple polymeric forms of XaxAB endow the nanopore system with a wide range, high flexibility, and high sensitivity through structural diversity, enabling sensitive detection of fine structural and kinetic changes in analytes, providing key advantages for single-molecule detection and drug screening.

[0024] (2) The two-component toxin nanoporin XaxAB was modified by site-directed mutagenesis. The T290C mutation was introduced into the XaxA subunit and the I30C mutation was introduced into the XaxB subunit, resulting in covalent cross-linking. Therefore, a "structural anchoring network" was constructed between the XaxA and XaxB subunits, which significantly enhanced the anti-depolymerization ability of the nanopores. The site-directed mutagenesis-modified two-component toxin nanoporin XaxAB improved the quality of current signals in pore opening, sequencing, or detection. The current properties were stable, the current signal width was narrower, and there were fewer spikes. Attached Figure Description

[0025] Exemplary embodiments of the present invention can be more fully understood by referring to the following figures:

[0026] Figure 1 This is a top view of the two-component toxin nanoporous protein XaxAB channel ribbon structure model of Example 1 of the present invention, where the parts of the same color show a protein monomer.

[0027] Figure 2 This is a bottom view of the two-component toxin nanoporous protein XaxAB channel ribbon structure model of Example 1 of the present invention, where the parts of the same color show a protein monomer.

[0028] Figure 3 This is a side view of the two-component toxin nanoporous protein XaxAB channel ribbon structure model of Example 1 of the present invention, wherein the parts of the same color show a protein monomer.

[0029] Figure 4 This is a top view of the surface structure model of the two-component toxin nanoporous protein XaxAB channel in Example 1 of the present invention.

[0030] Figure 5This is a bottom view of the surface structure model of the two-component toxin nanoporous protein XaxAB channel in Example 1 of the present invention.

[0031] Figure 6 This is a side view of the surface structure model of the two-component toxin nanoporous protein XaxAB channel in Example 1 of the present invention.

[0032] Figure 7 The image shows the detection results of the opening current of the two-component toxin nanoporous protein XaxAB nanopores in Example 2 of this invention.

[0033] Figure 8 This is a graph showing the current detection results of the polypeptide chain passing through the two-component toxin nanoporous protein XaxAB in Example 3 of the present invention.

[0034] Figure 9 This is a purification electrophoresis diagram of the XaxA subunit of the present invention with amino acid T290 mutated to T290C in Example 4 of the present invention; lane 1: protein marker; lane 2: elution sample; lane 3: elution sample.

[0035] Figure 10 This is an electrophoresis diagram of the purification of the XaxB subunit of the present invention, in Example 4, where the amino acid I30 is mutated to I30C; lane 1: protein marker; lane 2: elution sample; lane 3: elution sample; lane 4: elution sample.

[0036] Figure 11 The graph shows the detection results of the nanopore opening current of the two-component toxin nanoporous protein XaxAB after site-directed mutagenesis modification in Example 5 of the present invention.

[0037] Figure 12 This is a graph showing the current detection results of the polypeptide chain crossing the two-component toxin nanoporous protein XaxAB modified by site-directed mutagenesis in Example 6 of the present invention.

[0038] Figure 13 The graph shows the current detection results of dextran passing through the two-component toxin nanoporous protein XaxAB modified by site-directed mutagenesis in Example 7 of the present invention.

[0039] Figure 14 This is a graph showing the current detection results of λDNA passing through the two-component toxin nanoporous protein XaxAB modified by site-directed mutagenesis in Example 8 of the present invention. Detailed Implementation

[0040] 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.

[0041] 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.

[0042] This invention provides the application of a site-directed mutagenesis-modified two-component toxin nanoporin XaxAB in the sequencing or detection of target analytes. The two-component toxin nanoporin XaxAB includes equal numbers of XaxA and XaxB subunits. The amino acid sequence of the XaxA subunit is shown in SEQ ID NO: 1, and the amino acid sequence of the XaxB subunit is shown in SEQ ID NO: 2.

[0043] The two-component toxin nanoporin XaxAB was modified by site-directed mutagenesis. Specifically, the amino acid T290 of the XaxA subunit was mutated to T290C, and the amino acid I30 of the XaxB subunit was mutated to I30C, thus obtaining the site-directed mutagenesis-modified two-component toxin nanoporin XaxAB.

[0044] In some embodiments, the target analyte includes one or more of the following: metal ions, inorganic salts, polymers, amino acids, peptides, polypeptides, proteins, nucleotides, oligonucleotides, polynucleotides, dyes, bleaching agents, drugs, diagnostic agents, narcotics, explosive pollutants, and environmental pollutants.

[0045] In some embodiments, the two-component toxin nanoporin XaxAB has a conical structure including a cis-open portion, a lumen, and a trans-constriction portion, wherein the lumen connects the cis-open portion and the trans-constriction portion;

[0046] The outer diameter of the cis-type opening at the connection with the lumen is ≥6nm, the depth of the lumen is ≥6nm, and the diameter of the inverse contraction is ≥0.5nm.

[0047] In some embodiments, the site-directed mutagenesis-modified two-component toxin nanoporin XaxAB regulates the pore size by adjusting the number of XaxA and XaxB subunits, enabling sequencing or detection of target analytes of different diameters.

[0048] In some embodiments, the total number of XaxA and XaxB subunits is 8-40.

[0049] In some embodiments, the gene encoding the two-component toxin nanoporin XaxAB is selected from:

[0050] A: Nucleic acid sequences encoding the XaxA and XaxB subunits of the two-component toxin nanoporin XaxAB;

[0051] B: Nucleic acid sequence with at least 85% homology to the nucleic acid sequence in A, and encoding the XaxA and XaxB subunits of the two-component toxin nanoporin XaxAB;

[0052] C: Nucleic acid complementary to A and B;

[0053] Among them, the homology is between 85% and 99%.

[0054] In some embodiments, the site-directed mutagenesis-modified two-component toxin nanoporin XaxAB is inserted into the membrane layer, and the membrane layer comprises: 1,2-diphydanoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol, 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine, 1,2-adiacyl-sn-glycero-3-phosphocholine, and 1,2-dilauroyl-sn... -Glyceryl-3-phosphocholine, 1,2-Dimyristico-sn-glyceryl-3-phosphocholine, 1,2-Dimyristico-sn-glyceryl-3-phosphoethanolamine, 1,2-Dimyristico-sn-glyceryl-3-phosphoglyceride, 1,2-Dimyristico-sn-glyceryl-3-phospho-L-serine, 1,2-Dioleoyl-sn-glyceryl-3-phospho-L-serine, 1,2-Dioleoyl-sn-glyceryl-3-phosphoethanolamine, 1,2-Dioleoyl-sn-glyceryl-3-phosphoglyceride, 1,2-distearatel-sn-glyceryl-3-phosphocholine, 1-palmitoyl-2-oleoyl-S At least one of the following: N-glycero-3-phosphocholine, mycolic acid, campesterol, sitosterol, stigmasterol, cholesterol, ergosterol, cardiolipin, sphingomyelin, 1-palmitoyl-2-cholesterolylhemisuccino-sn-glycero-3-phosphocholine, 1-oleoyl-2-cholesterolylhemisuccino-sn-glycero-3-phosphocholine, 1-palmitoyl-2-cholesterolylcarbonyl-sn-glycero-3-phosphocholine, 1,2-dicholesterolylhemisuccino-sn-glycero-3-phosphocholine, 10,12-pentadecanodiyne, 10,12-triadecanodiyne, 5,7-hexadecadecanodiyne, or 9,12-octadecadecanodiyne.

[0055] In some embodiments, the preparation method of the site-directed mutagenesis-modified two-component toxin nanoporous protein XaxAB includes the following steps:

[0056] Step S1: Construct a recombinant vector encoding the XaxA and XaxB subunits of the two-component toxin nanoporous protein XaxAB after site-directed mutagenesis modification, wherein the amino acid T290 of the XaxA subunit is mutated to T290C and the amino acid I30 of the XaxB subunit is mutated to I30C.

[0057] Step S2: Expression and purification of the site-directed mutagenesis-modified two-component toxin nanoporous protein XaxAB.

[0058] Example 1: Preparation of two-component toxin nanoporous protein XaxAB

[0059] (1) The amino acid sequence of the XaxA subunit is shown in SEQ ID NO: 1, and the amino acid sequence of the XaxB subunit is shown in SEQ ID NO: 2. The DNA fragments of the XaxA subunit and the XaxB subunit are synthesized, and the DNA fragments are cloned into the pET-30a(+) vector to construct a recombinant expression plasmid.

[0060] (2) The recombinant expression plasmid was transformed into E. coli Dh5α competent cells, and the plasmid was amplified by plate culture and liquid culture;

[0061] (3) The recombinant plasmids were transformed into Escherichia coli BL21(DE3)RIPL strain, inoculated into 2L of LB medium (containing 125mg / ml ampicillin), cultured at 37℃ until OD600=0.5-0.8, 0.4mM IPTG was added to induce expression, and cultured at 20℃ for 20 hours.

[0062] (4) Collect bacterial cells and resuspend them in a buffer consisting of 50 mM Tris-HCl (pH=8.5), 300 mM NaCl, 5 mM imidazole, 1 mM PMSF and 0.1% Triton X-100 (containing 10% glycerol). The cells are then sonicated to disrupt their structure. After centrifugation (38000 rpm, 4℃), the supernatant is passed through a Ni-NTA affinity chromatography column and eluted with a linear gradient of a buffer consisting of 50 mM Tris-HCl (pH=8.5), 300 mM NaCl and 500 mM imidazole. The eluent is then subjected to PreScission protease to remove the His tag and further purified by a Superdex200 size-exclusion column. The peak fraction is collected.

[0063] (5) Prepare a stock solution of 10 mg / ml 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine and brain extract polar lipids in a buffer containing 50 mM HEPES (pH=7.6), 250 mM NaCl and 5% n-octyl-bD-glucopyranoside (w / v); mix 10 mM XaxA subunit and XaxB subunit at a 1:1 molar ratio to a final lipid concentration of 2 mg / ml, and incubate at room temperature for 30 minutes to form oligomers (12-15 polymers, of which one polymer is one XaxA subunit and one XaxB subunit).

[0064] (6) Dialyze the oligomers with a buffer solution containing 50 mM HEPES (pH=7.6), 250 mM NaCl, and 0.002% amphipol A8-35;

[0065] (7) Load the sample onto a Superose 6 10 / 300 column. The mobile phase is 50 mM HEPES (pH=7.6), 250 mM NaCl, and 0.002% amphipol A8-35. The flow rate is 0.5 mL / min. Collect the elution peak. Verify the oligomer type by blue natural PAGE (BN-PAGE) and separate the two-component toxin nanoporous protein XaxAB (12-mer, 13-mer, 14-mer, and 15-mer).

[0066] Top view of the two-component toxin nanoporous protein XaxAB channel ribbon structure model as shown below Figure 1 As shown; Top view of the two-component toxin nanoporous protein XaxAB channel ribbon structure model. Figure 2 As shown; a side view of the two-component toxin nanoporous protein XaxAB channel ribbon structure model is shown. Figure 3 As shown; Top view of the surface structure model of the two-component toxin nanoporous protein XaxAB channel. Figure 4 As shown; Top view of the surface structure model of the two-component toxin nanoporous protein XaxAB channel. Figure 5 As shown; a side view of the surface structure model of the two-component toxin nanoporous protein XaxAB channel is shown. Figure 6 As shown.

[0067] Example 2: Characterization of the two-component toxin nanoporous protein XaxAB

[0068] (1) To construct a single two-component toxin nanoporous protein XaxAB pore in a lipid bilayer, first construct an artificial lipid bilayer on both sides of the pore of a polytetrafluoroethylene membrane, and then inject 1.2 mL of detection buffer (20 mM HEPES at pH=7.5 and 0.5 M KCl) into both chambers, with a volume of 1.2 mL / side, to ensure that the liquid levels on both sides of the membrane are balanced.

[0069] (2) Add 8 ng / mL of two-component toxin nanoporous protein XaxAB solution to the cis cavity and blank buffer to the trans cavity; apply a trans membrane voltage of 160 mV (Axopatch 200B amplifier) ​​and monitor the current change in real time. When the protein is inserted to form a single well, record the characteristic current step signal; after the protein well is formed, rinse the cis cavity three 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.

[0070] The detection results of the nanopore opening current of the two-component toxin nanoporous protein XaxAB are as follows: Figure 7 As shown.

[0071] Example 3: Two-component toxin nanoporin XaxAB detection of polypeptide chains

[0072] (1) To construct a single two-component toxin nanoporous protein XaxAB pore in a lipid bilayer, first construct an artificial lipid bilayer on both sides of the pore of a polytetrafluoroethylene membrane, and then inject 1.2 mL of detection buffer (20 mM HEPES at pH=7.5 and 0.5 M KCl) into both chambers, with a volume of 1.2 mL / side, to ensure that the liquid levels on both sides of the membrane are balanced.

[0073] (2) Add 8 ng / mL of two-component toxin nanoporous protein XaxAB solution to the cis cavity and blank buffer to the trans cavity; apply a trans membrane voltage of 160 mV (Axopatch 200B amplifier) ​​and monitor the current change in real time. When the protein is inserted to form a single well, record the characteristic current step signal. After the protein well is formed, rinse the cis cavity three times with 50 μL buffer to remove unbound protein and avoid interference from multiple pores.

[0074] (3) Add the polypeptide chain (SEQ ID NO:3: ALRHQVSSLSLRASSSSG, unmodified 10 nM), 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 °C) using Origin to control data acquisition.

[0075] Current detection results of polypeptide chains passing through the two-component toxin nanoporous protein XaxAB are as follows: Figure 8 As shown in the figure, the results indicate that the current signal baseline is wide and has many spikes.

[0076] Example 4: Preparation of the two-component toxin nanoporous protein XaxAB modified by site-directed mutagenesis

[0077] (1) The DNA fragments that mutate the amino acid T290 of the XaxA subunit to T290C and the amino acid I30 of the XaxB subunit to I30C were cloned into the pET-30a(+) vector to construct a recombinant expression plasmid.

[0078] (2) The recombinant expression plasmid was transformed into E. coli Dh5α competent cells, and the plasmid was amplified by plate culture and liquid culture;

[0079] (3) The recombinant plasmids were transformed into Escherichia coli BL21(DE3)RIPL strain, inoculated into 2L of LB medium (containing 125mg / ml ampicillin), cultured at 37℃ until OD600=0.5-0.8, 0.4mM IPTG was added to induce expression, and cultured at 20℃ for 20 hours.

[0080] (4) Collect bacterial cells and resuspend them in a buffer consisting of 50 mM Tris-HCl (pH=8.5), 300 mM NaCl, 5 mM imidazole, 1 mM PMSF and 0.1% Triton X-100 (containing 10% glycerol). The cells are then sonicated to disrupt their structure. After centrifugation (38000 rpm, 4℃), the supernatant is passed through a Ni-NTA affinity chromatography column and eluted with a linear gradient of a buffer consisting of 50 mM Tris-HCl (pH=8.5), 300 mM NaCl and 500 mM imidazole. The eluent is then subjected to PreScission protease to remove the His tag and further purified by a Superdex200 size-exclusion chromatography column. The peak fraction is collected.

[0081] (5) Prepare a stock solution of 10 mg / ml 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine and brain extract polar lipids in a buffer containing 50 mM HEPES (pH=7.6), 250 mM NaCl and 5% n-octyl-bD-glucopyranoside (w / v); mix 10 mM XaxA subunit and XaxB subunit at a 1:1 molar ratio to a final lipid concentration of 2 mg / ml, and incubate at room temperature for 30 minutes to form oligomers (12-15 polymers);

[0082] (6) Dialyze the oligomers with a buffer solution containing 50 mM HEPES (pH=7.6), 250 mM NaCl, and 0.002% amphipol A8-35;

[0083] (7) Load the sample onto a Superose 6 10 / 300 column. The mobile phase is 50 mM HEPES (pH=7.6), 250 mM NaCl, and 0.002% amphipol A8-35. The flow rate is 0.5 mL / min. Collect the elution peak. Verify the oligomer type by blue natural PAGE (BN-PAGE) and separate the site-directed mutagenesis-modified two-component toxin nanoporous protein XaxAB (12-mer, 13-mer, 14-mer, and 15-mer).

[0084] The purified electrophoresis image of the XaxA subunit with amino acid T290 mutated to T290C is shown below. Figure 9 As shown; the purified electrophoresis diagram of the XaxB subunit with amino acid I30 mutated to I30C is shown below. Figure 10 As shown.

[0085] Example 5: Characterization of the two-component toxin nanoporous protein XaxAB modified by site-directed mutagenesis

[0086] (1) A single site-directed mutagenesis-modified two-component toxin nanoporous protein XaxAB pore (XaxA T290C, XaxB I30C) was constructed in a lipid bilayer. First, an artificial lipid bilayer was constructed on both sides of the pore of the polytetrafluoroethylene membrane. Then, 1.2 mL of detection buffer (20 mM HEPES at pH 7.5 and 0.5 M KCl) 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.

[0087] (2) 8 ng / mL of the site-directed mutagenesis-modified two-component toxin nanoporous protein XaxAB solution was added to the cis-cavity, and the trans-cavity was filled with blank buffer. 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. 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.

[0088] The results of the site-directed mutagenesis-modified two-component toxin nanoporous protein XaxAB nanopore opening current detection are as follows: Figure 11 As shown, the results indicate that the quality of the pore current signal of the two-component toxin nanoporous protein XaxAB after site-directed mutagenesis modification is improved, the current properties are stable, the current signal width is narrower, and there are fewer spikes.

[0089] Example 6: Detection of polypeptide chains by XaxAB, a two-component toxin nanoporous protein modified by site-directed mutagenesis.

[0090] (1) A single site-directed mutagenesis-modified two-component toxin nanoporous protein XaxAB pore (XaxA T290C, XaxB I30C) was constructed in a lipid bilayer. First, an artificial lipid bilayer was constructed on both sides of the pore of the polytetrafluoroethylene membrane. Then, 1.2 mL of detection buffer (20 mM HEPES at pH=7.5 and 0.5 M KCl) 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.

[0091] (2) Add 8 ng / mL of the site-directed mutagenesis-modified two-component toxin nanoporous protein XaxAB solution to the cis-cavity, and use blank buffer for 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 well, record the characteristic current step signal. After the protein well is formed, rinse the cis-cavity three times with 50 μL buffer to remove unbound protein and avoid interference from multiple pores.

[0092] (3) Add the polypeptide chain (SEQ ID NO:3: ALRHQVSSLSLRASSSSG, unmodified 10 nM), 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 °C) using Origin to control data acquisition.

[0093] The current detection results of the polypeptide chain crossing the site-directed mutagenesis-modified two-component toxin nanoporous protein XaxAB are as follows: Figure 12 As shown, the results indicate that after site-directed mutation, covalent crosslinking significantly improves the stability of the current signal, resulting in smaller baseline fluctuations and fewer spikes during continuous detection.

[0094] Example 7: Detection of dextran by XaxAB, a two-component toxin nanoporous protein modified by site-directed mutagenesis.

[0095] (1) A single site-directed mutagenesis-modified two-component toxin nanoporous protein XaxAB pore (XaxA T290C, XaxB I30C) was constructed in a lipid bilayer. First, an artificial lipid bilayer was constructed on both sides of the pore of the polytetrafluoroethylene membrane. Then, 1.2 mL of detection buffer (20 mM HEPES at pH=7.5 and 0.5 M KCl) 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.

[0096] (2) Add 8 ng / mL of the site-directed mutagenesis-modified two-component toxin nanoporous protein XaxAB solution to the cis-cavity, and use blank buffer for 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 well, record the characteristic current step signal. After the protein well is formed, rinse the cis-cavity three times with 50 μL buffer to remove unbound protein and avoid interference from multiple pores.

[0097] (3) Add 10 kDa of dextran (10 nM), 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 °C) using Origin to control data acquisition.

[0098] Current detection results of dextran passing through the site-directed mutagenesis-modified two-component toxin nanoporous protein XaxAB are as follows: Figure 13 As shown, the results indicate that after site-directed mutation, covalent crosslinking significantly improves the stability of the current signal, resulting in smaller baseline fluctuations and fewer spikes during continuous detection.

[0099] Example 8: Detection of λDNA by XaxAB, a two-component toxin nanoporin modified with site-directed mutagenesis

[0100] (1) A single site-directed mutagenesis-modified two-component toxin nanoporous protein XaxAB pore (XaxA T290C, XaxB I30C) was constructed in a lipid bilayer. First, an artificial lipid bilayer was constructed on both sides of the pore of the polytetrafluoroethylene membrane. Then, 1.2 mL of detection buffer (20 mM HEPES at pH=7.5 and 0.5 M KCl) 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 the site-directed mutagenesis-modified two-component toxin nanoporous protein XaxAB solution to the cis-cavity, and use blank buffer for 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 well, record the characteristic current step signal. After the protein well is formed, rinse the cis-cavity three times with 50 μL buffer to remove unbound protein and avoid interference from multiple pores.

[0102] (3) Add λDNA (10 nM), 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 °C). Data acquisition is controlled by Origin.

[0103] The current detection results of λDNA passing through the site-directed mutagenesis-modified two-component toxin nanoporin XaxAB are as follows: Figure 14 As shown, the results indicate that after site-directed mutation, covalent crosslinking significantly improves the stability of the current signal, resulting in smaller baseline fluctuations and fewer spikes during continuous detection.

[0104] SEQ ID NO:1:

[0105] MGIFTKGDLINIKLYVKHSLELPFTLEGVKEYIGYNDIDIDGLKPAKMATLFKEIHDHALSWSGVESKVQQQSIDLENAGKQITLTGDEIISVIDQMPIIERVKNKLGDLTDKQLAEITYTNDDKEIAVELGNILESMKKDIKKQQENTQKVKTAVSDFKLKLIGGELSDGTIAQGLQPQISSK KKLMDDNNLSTIKDLQSKIDEKNKEIDQLQKDYNKYVGLAFSGMVGGIISWAITGGIFGDKAEKARKQKNKLIDEVKDLQSQVKDKSALQTSVQNLSLSFAGIHTSMVDAEEALNHLDFMWNTMLTQITTSRDKFDDINDALKLTSFVIAFKQVIEPWRDVQGSAAQLIQTFDEALAEYKKLYH

[0106] SEQ ID NO:2:

[0107] MYPEINIKAMNQAVNTIWLLAQRQTSGIEIINDKVKRISLYSREFDEMMRDSLAQLAPVLKQLTSDAAFQTIAQIDEALADPSLSKDDREALTLERNNLLQNLSKDIDNVIVSFTGRTNKLTNKISDISDMVIAERLQDLVTQAESQKTELQSDIDPKTEKRNKLDADR EKIIESQDVIRQNNIADMFKDFIPSAKDIDGLDFTQPKKEAIKQAIKQGAEIARKILGKVSEGLKYIDLADARMKLSDQIDQLITETDELKAKIREVELRLSGLKDVMQIDTERTTLLTEAVKIEQVWISFAEQLHKLSNDEINQQDLSNLINGQLDFLNNLTLQYNKLK

[0108] 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. The application of mutant two-component toxin nanoporin XaxAB in nanopore detection, characterized by: The mutant two-component toxin nanoporin XaxAB is obtained by site-directed mutagenesis of wild-type two-component toxin nanoporin XaxAB; the wild-type two-component toxin nanoporin XaxAB includes an equal number of wild-type XaxA subunits and wild-type XaxB subunits, the amino acid sequence of the wild-type XaxA subunit is shown in SEQ ID NO: 1, and the amino acid sequence of the wild-type XaxB subunit is shown in SEQ ID NO: 2; The site-directed mutagenesis modification specifically involves: the amino acid T290 of the wild-type XaxA subunit being mutated to T290C, and the amino acid I30 of the wild-type XaxB subunit being mutated to I30C.

2. The application as described in claim 1, characterized in that: The target analytes for nanopore detection include one or more of peptides, proteins, dextran, and nucleotides.

3. The application as described in claim 2, characterized in that: The target analyte is a polypeptide or DNA.

4. The application as described in claim 2, characterized in that: The mutant two-component toxin nanoporin XaxAB regulates the pore size by adjusting the number of XaxA and XaxB subunits, enabling sequencing or detection of target analytes with different diameters.

5. The application as described in claim 4, characterized in that: The total number of XaxA and XaxB subunits is 8-40.

6. The application as described in claim 5, characterized in that: The preparation method of the mutant two-component toxin nanoporin XaxAB includes the following steps: Step S1: Construct a recombinant vector encoding the genes of the XaxA and XaxB subunits of the mutant two-component toxin nanoporin XaxAB; Step S2: Expression and purification of the mutant two-component toxin nanoporous protein XaxAB.

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