Application of biological nanopore protein and mutant thereof in detection

Abstract

The invention belongs to the technical field of methods for characterizing target polynucleotides, and provides application of a biological nanopore protein and a mutant thereof in detection. The biological nanopore protein mutant is obtained by mutating wild type PANX1 nanopore protein, the amino acid sequence of the wild type PANX1 nanopore protein is shown as SEQ ID NO: 1, and the mutation means that one or more amino acids of the wild type PANX1 nanopore protein are mutated into common amino acids except original amino acids, or carrying out truncation or truncation substitution on the C region of the wild type PANX1 nanopore protein to obtain the PANX1 nanopore protein mutant. The wild type PANX1 nanopore protein can be normally expressed after mutation, compared with the wild type PANX1 nanopore protein, the biological nanopore protein mutant is stable in property, the C region is truncated, so that the pore diameter of the contraction region of the protein mutant is enlarged, and the pore channel is prevented from being blocked.

Description

Technical Field

[0001] The present invention belongs to the technical field of methods for characterizing target polynucleotides, and particularly relates to the application of a biological nanopore protein and its mutants in detection. Background Art

[0002] Nanopore single-molecule sensing technology has shown significant advantages in the fields of small molecule detection, chemical reaction monitoring, DNA sequencing, and protein identification. This technology originated from the particle size measurement method based on the continuous pulse principle. Its classic representative, the Coulter counter, accurately estimates the cell number and size by monitoring the current pulse displacement generated when blood cells pass through pores with diameters ranging from 20 μm to 2 mm. However, when the analyte size is much smaller than the pore diameter, the resulting current change amplitude will be too small to detect. Therefore, pore proteins with appropriate pore diameters are one of the key technologies affecting nanopore sequencing. The geometric characteristics of the constriction region (i.e., the core region for signal reading) of pore proteins directly determine the quality and resolution accuracy of the original current signal.

[0003] Currently, the types of pore proteins are single. Taking the commonly used mutant of Escherichia coli CsgG as an example, its constriction region can only accommodate 4 - 5 bases, resulting in the current signal recorded each time being generated by the combined action of multiple bases, forming superposition interference, which significantly increases the complexity of subsequent signal analysis and makes it difficult to achieve the sequencing accuracy level of second-generation sequencing technology. In addition, existing mainstream pore proteins such as CsgG are mostly non-axisymmetric nine-mer structures, and such complex oligomeric forms make the protein cloning, expression, and purification processes more cumbersome. Therefore, it is necessary to discover pore proteins with novel constriction regions, which can, on the one hand, increase the available types of nanopores, and on the other hand, reduce the difficulty of current signal analysis and further improve the sequencing accuracy. Summary of the Invention

[0004] Aiming at the deficiencies of the prior art, the present invention provides the application of a biological nanopore protein and its mutants in detection, aiming to solve the problems mentioned in the background art.

[0005] In the first aspect, the present invention provides a nanopore protein mutant, which is obtained by mutating the wild-type PANX1 nanopore protein. The amino acid sequence of the wild-type PANX1 nanopore protein is shown as SEQ ID NO:1. The mutation means that one or more amino acids of the wild-type PANX1 nanopore protein are mutated into common amino acids other than the original amino acids, or the C region of the wild-type PANX1 nanopore protein is truncated or truncated and replaced; 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] Further, it is any one selected from the following A, B, and C: A: One or more of the amino acids W74, R75, S73, and F72 in the wild-type PANX1 nanopore protein are mutated to common amino acids other than the original amino acids; B: One or more of the amino acids I58, T21, E22, and P23 in the wild-type PANX1 nanopore protein are mutated to common amino acids other than the original amino acids; C: The C region of the wild-type PANX1 nanopore protein is truncated or truncated and replaced.

[0007] Further, in A: The amino acid W74 in the wild-type PANX1 nanopore protein is mutated to a common amino acid other than the original amino acid; the amino acid W74 in the wild-type PANX1 nanopore protein is mutated to W74M, W74P, W74L, W74G, W74I, W74Q, W74E, W74C, W74R, W74H, W74K, W74T, W74A, W74S, W74Y, W74N, W74V, or W74D; preferably, the amino acid W74 in the wild-type PANX1 nanopore protein is mutated to W74T, W74L, or W74G; The amino acid R75 in the wild-type PANX1 nanopore protein is mutated to a common amino acid other than the original amino acid; the amino acid R75 in the wild-type PANX1 nanopore protein is mutated to R75N or R75S; The amino acid S73 in the wild-type PANX1 nanopore protein is mutated to a common amino acid other than the original amino acid; the amino acid S73 in the wild-type PANX1 nanopore protein is mutated to S73N, S73K, S73H, S73D, S73W, S73R, S73F, S73E, S73P, S73L, S73A, S73V, S73G, or S73M; preferably, the amino acid S73 in the wild-type PANX1 nanopore protein is mutated to S73N or S73K; The amino acid F72 in the wild-type PANX1 nanopore protein is mutated to a common amino acid other than the original amino acid; the amino acid F72 in the wild-type PANX1 nanopore protein is mutated to F72N, F72H, F72Q, F72S, F72L, F72D, F72C, F72I, F72P, F72T, F72G, F72M, F72W, or F72R; preferably, the amino acid F72 in the wild-type PANX1 nanopore protein is mutated to F72I.

[0008] Furthermore, in B: the amino acid I58 of the wild-type PANX1 nanopore protein is mutated to a common amino acid other than the original amino acid; the amino acid I58 of the wild-type PANX1 nanopore protein is mutated to I58N; the amino acid T21 of the wild-type PANX1 nanopore protein is mutated to a common amino acid other than the original amino acid; the amino acid T21 of the wild-type PANX1 nanopore protein is mutated to T21N, T21L; the amino acid E22 of the wild-type PANX1 nanopore protein is mutated to a common amino acid other than the original amino acid; the amino acid E22 of the wild-type PANX1 nanopore protein is mutated to E22T, E22L; the amino acid P23 of the wild-type PANX1 nanopore protein is mutated to a common amino acid other than the original amino acid; the amino acid P23 of the wild-type PANX1 nanopore protein is mutated to P23N.

[0009] Furthermore, in C: the truncation is a 1 - 10 amino acid truncation from G371 towards the N-terminus; preferably, a 2 amino acid truncation from G371 towards the N-terminus, i.e., truncating G371-L370; a 4 amino acid truncation from G371 towards the N-terminus, i.e., truncating G371-T368; a 6 amino acid truncation from G371 towards the N-terminus, i.e., truncating G371-L366; an 8 amino acid truncation from G371 towards the N-terminus, i.e., truncating G371-M364.

[0010] Furthermore, in C: the truncation substitution is replacing with a short amino acid sequence from G371 towards the N-terminus, 3 amino acids replacing 8, 9, 10, or 11 amino acids; preferably, replacing 11 amino acids with GSG, i.e., truncation substitution of G371-I361.

[0011] Furthermore, the amino acid sequence of the biological nanopore protein mutant is as shown in SEQ ID NO:2 - SEQ ID NO:6; preferably, the amino acid sequence of the biological nanopore protein mutant is as shown in SEQ ID NO:5.

[0012] In a second aspect, the present invention provides a coding gene of a biological nanopore protein mutant, selected from: D: a nucleic acid sequence encoding a biological nanopore protein mutant; E: a nucleic acid having at least 85% homology with the nucleic acid sequence in D and encoding a biological nanopore protein mutant; F: a nucleic acid complementary to D and E; wherein the homology is between 85% - 99%.

[0013] In a third aspect, the present invention provides a biological nanopore protein, which is composed of seven identical subunits, the overall channel is funnel-shaped, and the amino acid sequence is as shown in SEQ ID NO: 1.

[0014] In a fourth aspect, the present invention provides a method for preparing a biological nanopore protein and its mutants, comprising the following steps: Step S1: Construct a vector of the biological nanopore protein or its mutant. Step S2: Express and purify the biological nanopore protein or its mutant.

[0015] In a fifth aspect, the present invention provides a membrane layer embedded with the biological nanopore protein and its mutants, and the membrane layer is a lipid bilayer.

[0016] In a sixth aspect, the present invention provides the use of the biological nanopore protein and its mutants in the sequencing or detection of one or more of nucleic acids, oligopeptides, oligosaccharides, and macromolecular drugs.

[0017] The present invention has the following beneficial effects: (1) The wild-type PANX1 nanopore protein is a unique nanopore protein different from MspA and CsgG, with a heptameric structure. Each subunit contains 4 transmembrane domains TM1-TM4. The N-terminus and C-terminus of TM1 and TM4 are respectively anchored intracellularly, and TM2 and TM3 form the lateral support of the channel pore. The overall channel is funnel-shaped. Compared with the existing nanopore proteins, its cloning expression and purification processes are simple, it is easy to assemble the protein of the biological nanopore, and it has a wide range of applications.

[0018] (2) Mutagenesis experiments found that when one or more amino acids of the wild-type PANX1 nanopore protein are mutated to common amino acids other than the original amino acids, or the C region of the wild-type PANX1 nanopore protein is truncated or truncated and replaced, it can be normally expressed. Compared with the wild-type PANX1 nanopore protein, the PANX1 nanopore protein mutant (biological nanopore protein mutant) has stable properties, and the truncation of the C region enlarges the pore diameter of the contraction region of the protein mutant, avoiding blocking the pore. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] By referring to the following drawings, the exemplary embodiments of the present invention can be more fully understood: Figure 1 It is a top view of the surface structure model of the wild-type PANX1 nanopore protein channel of the present invention.

[0020] Figure 2 It is a side view of the surface structure model of the wild-type PANX1 nanopore protein channel of the present invention.

[0021] Figure 3Top view of the ribbon structure model of the wild-type PANX1 nanopore protein channel of the present invention.

[0022] Figure 4 Side view of the ribbon structure model of the wild-type PANX1 nanopore protein channel of the present invention.

[0023] Figure 5 Purification electrophoresis diagram of the PANX1 nanopore protein mutant after truncating 10 amino acids at the C-terminus and mutating amino acid F72 to F72I in Example 6 of the present invention; Lane 1: Protein Marker; Lane 2: Wash sample; Lane 3: Elution sample; Lane 4: Elution sample; Lane 5: Elution sample.

[0024] Figure 6 Detection result diagram of the open pore current of the wild-type PANX1 nanopore protein in Example 2 of the present invention.

[0025] Figure 7 Detection result diagram of the open pore current of the PANX1 nanopore protein mutant after truncating 10 amino acids at the C-terminus and mutating amino acid F72 to F72I in Example 7 of the present invention.

[0026] Figure 8 Detection result diagram of the current during single-stranded DNA perforation of the PANX1 nanopore protein mutant after truncating 10 amino acids at the C-terminus and mutating amino acid F72 to F72I in Example 8 of the present invention. Detailed implementation manners

[0027] In order to make the technical problems, technical solutions and beneficial effects to be solved by 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 only used to explain the present invention and are not used to limit the present invention.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs; the terms used herein are only for the purpose of describing specific embodiments and do not limit the present invention.

[0029] The embodiments of the present invention provide a biological nanopore protein mutant, which is obtained by mutating the wild-type PANX1 nanopore protein. The amino acid sequence of the wild-type PANX1 nanopore protein is shown in SEQ ID NO:1. The mutation means that one or more amino acids of the wild-type PANX1 nanopore protein are mutated into common amino acids other than the original amino acids, or the C region of the wild-type PANX1 nanopore protein is truncated or truncated and replaced; Among them, 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.

[0030] In some embodiments, any one selected from the following A, B, and C: A: One or more of the amino acids W74, R75, S73, and F72 of the wild-type PANX1 nanopore protein are mutated to common amino acids other than the original amino acids; B: One or more of the amino acids I58, T21, E22, and P23 of the wild-type PANX1 nanopore protein are mutated to common amino acids other than the original amino acids; C: The C region of the wild-type PANX1 nanopore protein is truncated or truncated and replaced.

[0031] In some embodiments, in A: The amino acid W74 of the wild-type PANX1 nanopore protein is mutated to a common amino acid other than the original amino acid; the amino acid W74 of the wild-type PANX1 nanopore protein is mutated to W74M, W74P, W74L, W74G, W74I, W74Q, W74E, W74C, W74R, W74H, W74K, W74T, W74A, W74S, W74Y, W74N, W74V, or W74D.

[0032] In some embodiments, the amino acid W74 of the wild-type PANX1 nanopore protein is mutated to W74T, W74L, or W74G.

[0033] In some embodiments, in A: The amino acid R75 of the wild-type PANX1 nanopore protein is mutated to a common amino acid other than the original amino acid; the amino acid R75 of the wild-type PANX1 nanopore protein is mutated to R75N or R75S.

[0034] In some embodiments, in A: The amino acid S73 of the wild-type PANX1 nanopore protein is mutated to a common amino acid other than the original amino acid; the amino acid S73 of the wild-type PANX1 nanopore protein is mutated to S73N, S73K, S73H, S73D, S73W, S73R, S73F, S73E, S73P, S73L, S73A, S73V, S73G, or S73M.

[0035] In some embodiments, the amino acid S73 of the wild-type PANX1 nanopore protein is mutated to S73N or S73K.

[0036] In some embodiments, in A: the amino acid F72 of the wild-type PANX1 nanopore protein is mutated to a common amino acid other than the original amino acid; the amino acid F72 of the wild-type PANX1 nanopore protein is mutated to F72N, F72H, F72Q, F72S, F72L, F72D, F72C, F72I, F72P, F72T, F72G, F72M, F72W or F72R.

[0037] In some embodiments, the amino acid F72 of the wild-type PANX1 nanopore protein is mutated to F72I.

[0038] In some embodiments, in B: the amino acid I58 of the wild-type PANX1 nanopore protein is mutated to a common amino acid other than the original amino acid; the amino acid I58 of the wild-type PANX1 nanopore protein is mutated to I58N.

[0039] In some embodiments, in B: the amino acid T21 of the wild-type PANX1 nanopore protein is mutated to a common amino acid other than the original amino acid; the amino acid T21 of the wild-type PANX1 nanopore protein is mutated to T21N, T21L.

[0040] In some embodiments, in B: the amino acid E22 of the wild-type PANX1 nanopore protein is mutated to a common amino acid other than the original amino acid; the amino acid E22 of the wild-type PANX1 nanopore protein is mutated to E22T, E22L.

[0041] In some embodiments, in B: the amino acid P23 of the wild-type PANX1 nanopore protein is mutated to a common amino acid other than the original amino acid; the amino acid P23 of the wild-type PANX1 nanopore protein is mutated to P23N.

[0042] In some embodiments, in C: the truncation is a 1- to 10-amino acid truncation from G371 towards the N-terminus; preferably, a 2-amino acid truncation from G371 towards the N-terminus, i.e., truncating G371-L370; a 4-amino acid truncation from G371 towards the N-terminus, i.e., truncating G371-T368; a 6-amino acid truncation from G371 towards the N-terminus, i.e., truncating G371-L366; an 8-amino acid truncation from G371 towards the N-terminus, i.e., truncating G371-M364.

[0043] In some embodiments, in C: the truncation substitution is a substitution from G371 towards the N-terminus with a short amino acid sequence, where 3 amino acids replace 8, 9, 10 or 11 amino acids; preferably, replacing 11 amino acids with GSG (glycyl-glycyl-glycine), i.e., truncation substitution of G371-I361.

[0044] In some embodiments, the amino acid sequences of the bio-nanopore protein mutants are as shown in SEQ ID NO:2 - SEQ ID NO:6, and the DNA sequences of their encoding genes are respectively as shown in SEQ ID NO:8 - SEQ ID NO:12.

[0045] In some embodiments, the amino acid sequence of the bio-nanopore protein mutant is as shown in SEQ ID NO:5.

[0046] In some embodiments, the present invention provides a coding gene for a bio-nanopore protein mutant, selected from: D: a nucleic acid sequence encoding a bio-nanopore protein mutant; E: a nucleic acid having at least 85% homology with the nucleic acid sequence in D and encoding a bio-nanopore protein mutant; F: a nucleic acid complementary to D and E; wherein the homology is between 85% and 99%.

[0047] In some embodiments, the present invention provides a bio-nanopore protein, which is composed of seven identical subunits, and the overall channel is funnel-shaped, and the amino acid sequence is as shown in SEQ ID NO:1.

[0048] In some embodiments, the present invention provides a method for preparing a bio-nanopore protein and its mutant, comprising the following steps: Step S1: Construct a bio-nanopore protein or bio-nanopore protein mutant vector; Step S2: Express and purify the bio-nanopore protein or bio-nanopore protein mutant.

[0049] In some embodiments, the present invention provides a membrane layer embedded with a bio-nanopore protein and its mutant, and the membrane layer is a lipid bilayer.

[0050] In some embodiments, the present invention provides the application of a bio-nanopore protein and its mutant in the sequencing or detection of one or more of nucleic acids, oligopeptides, oligosaccharides, and macromolecular drugs.

[0051] Example 1: Expression and purification of wild-type PANX1 nanopore protein 1. Recombinant Bacmid construction and virus packaging (1) Transformation and screening: Transform the pEG-PANX1 vector containing the PANX1 gene into E. coli DH10bac cells, and identify positive clones (containing recombinant Bacmid) by blue-white screening; (2) Transfect Sf9 cells: Extract the recombinant Bacmid and transfect adherently cultured Sf9 insect cells with Cellfectin reagent. After culturing for 4 days, observe the GFP fluorescence under a fluorescence microscope to confirm the successful packaging of the virus. (3) Virus amplification (P1 to P2 virus): Collect the virus supernatant (filtered through a 0.22 μm filter) and infect suspension-cultured Sf9 cells (density cells / mL). After culturing for 4 days, centrifuge (7000g) to collect the supernatant and obtain high-titer P2 virus, which is stored in the dark at 4°C.

[0052] 2. Infection of HEK293S cells and protein induction expression (1) Cell culture: Culture HEK293S GnTI⁻ cells in Freestyle 293 medium containing 1.5% FBS at 37°C until the density reaches cells / mL; (2) Virus infection and induction: Inoculate the P2 virus. After 12 hours, add sodium butyrate at a final concentration of 5 mM and lower the culture temperature to 32°C. Collect the cells after 60 hours of infection. (3) Cell lysis: Lyse the collected cells by sonication (35% amplitude, 15 minutes). Centrifuge the lysate at 100,000×g for 1 hour to enrich the cell membrane fraction in the precipitate. (4) Membrane protein solubilization: Solubilize the membrane fraction with a buffer containing 10 mM glycone ligand (sodium glycocholate), 25 mM Tris (tris(hydroxymethyl)aminomethane, pH = 8.0), and 100 mM KCl. Centrifuge again at 100,000×g for 1 hour and collect the supernatant.

[0053] 3. Affinity chromatography purification (nickel column purification) (1) Binding: Incubate the supernatant with Ni-NTA resin (pre-equilibrated binding buffer: 25 mM Tris, pH = 8.0; 100 mM KCl; 100 μM GDN) for 2 hours, and the target protein (containing an 8×His tag at the C-terminus) specifically binds to the resin. (2) Elution: Elute the target protein with an elution buffer containing 0.3 M imidazole (25 mM Tris, pH = 8.0; 100 mM KCl, 100 μM GDN, 0.3 M imidazole), and collect the eluate.

[0054] 4. Tag removal and size exclusion chromatography (1) Tag cleavage: Add TEV protease to the eluate to remove the C-terminal GFP and 8×His tags and obtain tag-free PANX1 protein. (2)Fine purification: The digested protein was purified by a size exclusion chromatography column. The buffer was 25 mM Tris (pH = 8.0), 100 mM KCl, 1% glycerol, and 50 μM GDN. The main peak fraction was collected.

[0055] The top view of the surface structure model of the wild-type PANX1 nanopore protein channel is as shown in Figure 1 ; The side view of the surface structure model of the wild-type PANX1 nanopore protein channel is as shown in Figure 2 ; The top view of the ribbon structure model of the wild-type PANX1 nanopore protein channel is as shown in Figure 3 ; The parts with the same color show one protein monomer. The side view of the ribbon structure model of the wild-type PANX1 nanopore protein channel is as shown in Figure 4 ; The parts with the same color show one protein monomer. The results show that the wild-type PANX1 nanopore protein is a heptamer structure and the overall channel is funnel-shaped.

[0056] Example 2: Characterization of wild-type PANX1 nanopore protein The wild-type PANX1 nanopore protein with the amino acid sequence shown in SEQ ID NO: 1 was obtained, and the DNA sequence of its encoding gene is shown in SEQ ID NO: 7.

[0057] On a polytetrafluoroethylene horizontal pore diameter of about 20 μm, a lipid bilayer was formed with 1,2-diphytanoyl-sn-glycero-3-phosphocholine. A buffer solution containing 1 M KCl and 20 mM HEPES / KOH (pH = 8.0 ± 0.05) was added to each 1 ml compartment on both sides of the bilayer. A transmembrane voltage of 100 mV was applied to the bilayer using an Axopatch 200B integrated patch clamp amplifier (the reverse side was positive and the cis chamber was grounded). The wild-type PANX1 nanopore protein with a concentration of 4.0 ng / ml was added to the grounded cis chamber. After detecting the insertion of a single pore, the compartment was immediately rinsed with the experimental buffer to avoid further insertion of free protein.

[0058] The experiment was carried out at room temperature (23 ± 1 °C). The analog signal was low-pass filtered with a 4-pole Bessel filter at 100 kHz and digitized at 500 kHz. Origin was used to control data acquisition. The detection result of the open pore current of the wild-type PANX1 nanopore protein is as shown in Figure 6 .

[0059] Example 3: Detection of single-stranded DNA using wild-type PANX1 nanopore protein On a polytetrafluoroethylene horizontal pore diameter of approximately 20 μm, a lipid bilayer was formed with 1,2-diphytanoyl-sn-glycero-3-phosphocholine. A buffer solution containing 1 M KCl and 20 mM HEPES / KOH (pH = 8.0 ± 0.05) was added to each 1 ml compartment on both sides of the bilayer. Using an Axopatch 200B integrated patch clamp amplifier, a transmembrane voltage of 100 mV was applied across the bilayer (the reverse side was positive and the cis chamber was grounded). The wild-type PANX1 nanopore protein with a concentration of 4.0 ng / ml was added to the grounded cis chamber. After detecting the insertion of a single pore, the compartment was immediately rinsed with the experimental buffer to avoid further insertion of free protein. Then, single-stranded DNA with a fluorescent label (SEQ ID NO: 13: 5’-ACGTACGTACGTCAG-3’, concentration 1 μM) was added to the cis chamber, a voltage of 100 mV was applied, and the current signal was recorded.

[0060] The experiment was carried out at room temperature (23 ± 1 °C). The analog signal was low-pass filtered with a 4-pole Bessel filter at 100 kHz and digitized at 500 kHz. Data acquisition was controlled by Origin. The current signal of single-stranded DNA passing through the wild-type PANX1 nanopore protein could be seen. The diameter of the constriction region of the wild-type PANX1 protein was too small, which might block the pore.

[0061] Example 4: Detection of single-stranded DNA using the PANX1 nanopore protein mutant (biological nanopore protein mutant) with amino acid P23 mutated to P23N On a polytetrafluoroethylene horizontal pore diameter of approximately 20 μm, a lipid bilayer was formed with 1,2-diphytanoyl-sn-glycero-3-phosphocholine. A buffer solution containing 1 M KCl and 20 mM HEPES / KOH (pH = 8.0 ± 0.05) was added to each 1 ml compartment on both sides of the bilayer. Using an Axopatch 200B integrated patch clamp amplifier, a transmembrane voltage of 100 mV was applied across the bilayer (the reverse side was positive and the cis chamber was grounded). The PANX1 nanopore protein mutant with amino acid P23 mutated to P23N and a concentration of 4.0 ng / ml was added to the grounded cis chamber. After detecting the insertion of a single pore, the compartment was immediately rinsed with the experimental buffer to avoid further insertion of free protein. Then, single-stranded DNA with a fluorescent label (SEQ ID NO: 13: 5’-ACGTACGTACGTCAG-3’, concentration 1 μM) was added to the cis chamber, a voltage of 100 mV was applied, and the current signal was recorded.

[0062] The experiment was carried out at room temperature (23±1 °C). The analog signal was low-pass filtered with a 4-pole Bessel filter at 100 kHz and digitized at 500 kHz. The data acquisition was controlled by Origin. The results showed that compared with the wild-type PANX1 nanopore protein, the PANX1 nanopore protein mutant with amino acid P23 mutated to P23N had a stable current property when detecting single-stranded DNA, a narrower current signal width, and fewer spikes.

[0063] Example 5: Detection of single-stranded DNA using a PANX1 nanopore protein mutant (biological nanopore protein mutant) with 10 amino acids truncated from the C-terminus On a polytetrafluoroethylene horizontal pore diameter of about 20 μm, a lipid bilayer was formed with 1,2-diphytanoyl-sn-glycero-3-phosphocholine. A buffer solution containing 1 M KCl and 20 mM HEPES / KOH (pH = 8.0±0.05) was added to each 1 ml compartment on both sides of the bilayer. Using an Axopatch 200B integrated patch clamp amplifier, a transmembrane voltage of 100 mV was applied to the bilayer (the reverse side was positive and the cis chamber was grounded). The PANX1 nanopore protein mutant with 10 amino acids truncated from the C-terminus at a concentration of 4.0 ng / ml was added to the grounded cis chamber. After detecting the insertion of a single pore, the compartment was immediately rinsed with the experimental buffer to avoid further insertion of free protein. Then, the single-stranded DNA with fluorescent label (SEQ ID NO: 13: 5’-ACGTACGTACGTCAG-3’, concentration 1 μM) was added to the cis chamber, and a voltage of 100 mV was applied to record the current signal.

[0064] The experiment was carried out at room temperature (23±1 °C). The analog signal was low-pass filtered with a 4-pole Bessel filter at 100 kHz and digitized at a sampling rate of 500 kHz. The data acquisition was controlled by Origin software. It can be seen that compared with the wild-type PANX1 nanopore protein, the PANX1 nanopore protein mutant with 10 amino acids truncated from the C-terminus had a stable current property when detecting single-stranded DNA, and the current signal noise decreased.

[0065] Example 6: Expression and purification of a PANX1 nanopore protein mutant (biological nanopore protein mutant) with 10 amino acids truncated from the C-terminus and amino acid F72 mutated to F72I 1. Recombinant Bacmid construction and virus packaging (1) Transformation and screening: The pEG-PANX1 vector containing the PANX1 gene with 10 amino acids truncated from the C-terminus and amino acid F72 mutated to F72I was transformed into E. coli DH10bac cells, and positive clones (containing recombinant Bacmid) were identified by blue-white screening; (2) Transfect Sf9 cells: Extract the recombinant Bacmid and transfect adherently cultured Sf9 insect cells with Cellfectin reagent. After culturing for 4 days, observe the GFP fluorescence under a fluorescence microscope to confirm the successful packaging of the virus. (3) Virus amplification (P1 to P2 virus): Collect the virus supernatant (filtered through a 0.22 μm filter) and infect suspension-cultured Sf9 cells (density cells / mL). After culturing for 4 days, centrifuge (7000g) to collect the supernatant and obtain high-titer P2 virus, which is stored in the dark at 4°C.

[0066] 2. Infection of HEK293S cells and protein induction expression (1) Cell culture: Culture HEK293S GnTI⁻ cells in Freestyle 293 medium containing 1.5% FBS at 37°C until the density reaches cells / mL; (2) Virus infection and induction: Inoculate the P2 virus. After 12 hours, add sodium butyrate at a final concentration of 5 mM and lower the culture temperature to 32°C. Collect the cells after 60 hours of infection. (3) Cell lysis: Lyse the collected cells by sonication (35% amplitude, 15 minutes). Centrifuge the lysate at 100,000×g for 1 hour to enrich the cell membrane fraction in the pellet. (4) Membrane protein solubilization: Solubilize the membrane fraction with a buffer containing 10 mM glycon ligand (sodium glycocholate), 25 mM Tris (tris(hydroxymethyl)aminomethane, pH = 8.0), and 100 mM KCl. Centrifuge again at 100,000×g for 1 hour and collect the supernatant.

[0067] 3. Affinity chromatography purification (nickel column purification) (1) Binding: Incubate the supernatant with Ni-NTA resin (pre-equilibrated binding buffer: 25 mM Tris pH = 8.0, 100 mM KCl, 100 μM GDN) for 2 hours. The target protein (containing an 8×His tag at the C-terminus) specifically binds to the resin. (2) Elution: Elute the target protein with an elution buffer containing 0.3 M imidazole (25 mM Tris pH = 8.0, 100 mM KCl, 100 μM GDN, 0.3 M imidazole) and collect the eluate.

[0068] 4. Tag removal and size exclusion chromatography (1) Tag cleavage: Add TEV protease to the eluate to remove the C-terminal GFP and 8×His tags and obtain tag-free PANX1 protein. (2)Fine purification: The digested protein was purified by a size-exclusion chromatography column with a buffer of 25 mM Tris pH = 8.0, 100 mM KCl, 1% glycerol and 0 μM GDN, and the main peak fraction was collected.

[0069] The purification electrophoresis results of the PANX1 nanopore protein mutant with 10 amino acids truncated at the C-terminus and amino acid F72 mutated to F72I are as Figure 6 shown.

[0070] Example 7: Characterization of the PANX1 nanopore protein mutant (biological nanopore protein mutant) with 10 amino acids truncated at the C-terminus and amino acid F72 mutated to F72I On a polytetrafluoroethylene horizontal pore diameter of about 20 μm, a lipid bilayer was formed with 1,2-diphytanoyl-sn-glycero-3-phosphocholine. A buffer containing 1 M KCl and 20 mM HEPES / KOH (pH = 8.0 ± 0.05) was added to each 1 ml compartment on both sides of the bilayer. A transmembrane voltage of 100 mV was applied to the bilayer using an Axopatch 200B integrated patch clamp amplifier (the reverse side was positive and the cis chamber was grounded). The PANX1 nanopore protein mutant with 10 amino acids truncated at the C-terminus and amino acid F72 mutated to F72I at a concentration of 4.0 ng / ml was added to the grounded cis chamber. After detecting the insertion of a single pore, the compartment was immediately rinsed with the experimental buffer to avoid further insertion of free protein.

[0071] The experiment was carried out at room temperature (23 ± 1 °C). The analog signal was low-pass filtered by a 100 kHz, 4-pole Bessel filter and digitized at a sampling rate of 500 kHz. The data acquisition was controlled by Origin software. The nanopore opening current detection results of the PANX1 nanopore protein mutant with 10 amino acids truncated at the C-terminus and amino acid F72 mutated to F72I are as Figure 7 shown.

[0072] Example 8: Detection of single-stranded DNA using the PANX1 nanopore protein mutant (biological nanopore protein mutant) with 10 amino acids truncated at the C-terminus and amino acid F72 mutated to F72I On a polytetrafluoroethylene horizontal pore with a diameter of about 20 μm, a lipid bilayer was formed with 1,2-diphytanoyl-sn-glycero-3-phosphocholine. A buffer solution containing 1 M KCl and 20 mM HEPES / KOH (pH = 8.0 ± 0.05) was added to each 1 ml compartment on both sides of the bilayer. Using an Axopatch 200B integrated patch clamp amplifier, a transmembrane voltage of 100 mV was applied to the bilayer membrane (the reverse side was positive and the cis chamber was grounded). The PANX1 nanopore protein with 10 amino acids truncated at the C-terminus and the amino acid F72 mutated to F72I at a concentration of 4.0 ng / ml was added to the grounded cis chamber. After a single pore insertion was detected, the compartment was immediately rinsed with the experimental buffer to avoid further insertion of free protein. Then, single-stranded DNA with a fluorescent label (SEQ ID NO: 13: 5'-ACGTACGTACGTCAG-3', concentration 1 μM) was added to the cis chamber, a voltage of 100 mV was applied, and the current signal was recorded.

[0073] The experiment was carried out at room temperature (23 ± 1 °C). The analog signal was low-pass filtered by a 100 kHz, 4-pole Bessel filter and digitized at a sampling rate of 500 kHz. The data acquisition was controlled by Origin software. The results of the current detection during the perforation of single-stranded DNA by the PANX1 nanopore protein mutant with 10 amino acids truncated at the C-terminus and the amino acid F72 mutated to F72I are as Figure 8 shown.

[0074] By comparison Figures 6 - 8 , it can be seen that compared with the wild-type PANX1 nanopore protein, the open pore current of the PANX1 nanopore protein mutant is significantly increased, and the current properties and current signals are significantly improved, which is suitable for the sequencing and / or detection of samples such as DNA.

[0075] The narrowest pore diameter of the wild-type PANX1 nanopore protein mutant is 1.2 nm, and the truncation of its C region expands the pore diameter of the protein mutant's constriction region. Therefore, the PANX1 nanopore protein mutant is more suitable for detecting macromolecular compounds such as nucleic acids, oligopeptides, oligosaccharides, and large molecule drugs (the diameter of the drug is smaller than the protein pore diameter).

[0076] In summary, the present invention discovers that the wild-type PANX1 nanopore protein is a unique nanopore protein different from MspA and CsgG, with a heptameric structure. Each subunit contains 4 transmembrane domains TM1-TM4. The N-terminus and C-terminus of TM1 and TM4 are respectively anchored intracellularly, and TM2 and TM3 form the lateral support of the channel pore. The overall channel is funnel-shaped. Compared with the existing nanopore proteins, its cloning expression and purification process are simple, it is easy to assemble proteins into biological nanopores, and it has a wide range of applications.

[0077] Mutation experiments found that when one or more amino acids of the wild-type PANX1 nanopore protein were mutated to common amino acids other than the original amino acids, or when the C region of the wild-type PANX1 nanopore protein was truncated or truncated and replaced, it could be normally expressed. Compared with the wild-type PANX1 nanopore protein, the PANX1 nanopore protein mutant (biological nanopore protein mutant) was stable in nature, and the truncation of the C region enlarged the pore diameter of the contraction region of the protein mutant, avoiding the blockage of the pore channel.

[0078] Amino acid sequence: SEQ ID NO:1: MAIAQLATEYVFSDFLLKEPTEPKFKGLRLELAVDKMVTCIAVGLPLLLISLAFAQEISIGTQISCFSPSSFSWRQAAFVDSYCWAAVQQKNSLQSESGNLPLWLHKFFPYILLLFAILLYLPPLFWRFAAAPHICSDLKFIMEELDKVYNRAIKAAKSARDLDMRDGACSVPGVTENLGQSLWEVSESHFKYPIVEQYLKTKKNSNNLIIKYISCRLLTLIIILLACIYLGYYFSLSSLSDEFVCSIKSGILRNDSTVPDQFQCKLIAVGIFQLLSVINLVVYVLLAPVVVYTLFVPFRQKTDVLKVYEILPTFDVLHFKSEGYNDLSLYNLFLEENISEVKSYKCLKVLENIKSSGQGIDPMLLLTNLG

[0079] SEQ ID NO:2: MAIAQLATEYVFSDFLLKEPTEPKFKGLRLELAVDKMVTCIAVGLPLLLISLAFAQEISIGTQISCFSPSSFSWRQAAFVDSYCWAAVQQKNSLQSESGNLPLWLHKFFPYILLLFAILLYLPPLFWRFAAAPHICSDLKFIMEELDKVYNRAIKAAKSARDLDMRDGACSVPGVTENLGQSLWEVSESHFKYPIVEQYLKTKKNSNNLIIKYISCRLLTLIIILLACIYLGYYFSLSSLSDEFVCSIKSGILRNDSTVPDQFQCKLIAVGIFQLLSVINLVVYVLLAPVVVYTLFVPFRQKTDVLKVYEILPTFDVLHFKSEGYNDLSLYNLFLEENISEVKSYKCLKVLENIKSSGQGIDPMLLLTN

[0080] SEQ ID NO:3: MAIAQLATEYVFSDFLLKEPTEPKFKGLRLELAVDKMVTCIAVGLPLLLISLAFAQEISIGTQISCFSPSSFSWRQAAFVDSYCWAAVQQKNSLQSESGNLPLWLHKFFPYILLLFAILLYLPPLFWRFAAAPHICSDLKFIMEELDKVYNRAIKAAKSARDLDMRDGACSVPGVTENLGQSLWEVSESHFKYPIVEQYLKTKKNSNNLIIKYISCRLLTLIIILLACIYLGYYFSLSSLSDEFVCSIKSGILRNDSTVPDQFQCKLIAVGIFQLLSVINLVVYVLLAPVVVYTLFVPFRQKTDVLKVYEILPTFDVLHFKSEGYNDLSLYNLFLEENISEVKSYKCLKVLENIKSSGQGIDPMLLL

[0081] SEQ ID NO:4: MAIAQLATEYVFSDFLLKEPTEPKFKGLRLELAVDKMVTCIAVGLPLLLISLAFAQEISIGTQISCFSPSSFSWRQAAFVDSYCWAAVQQKNSLQSESGNLPLWLHKFFPYILLLFAILLYLPPLFWRFAAAPHICSDLKFIMEELDKVYNRAIKAAKSARDLDMRDGACSVPGVTENLGQSLWEVSESHFKYPIVEQYLKTKKNSNNLIIKYISCRLLTLIIILLACIYLGYYFSLSSLSDEFVCSIKSGILRNDSTVPDQFQCKLIAVGIFQLLSVINLVVYVLLAPVVVYTLFVPFRQKTDVLKVYEILPTFDVLHFKSEGYNDLSLYNLFLEENISEVKSYKCLKVLENIKSSGQGIDPML

[0082] SEQ ID NO:5: MAIAQLATEYVFSDFLLKEPTEPKFKGLRLELAVDKMVTCIAVGLPLLLISLAFAQEISIGTQISCFSPSSFSWRQAAFVDSYCWAAVQQKNSLQSESGNLPLWLHKFFPYILLLFAILLYLPPLFWRFAAAPHICSDLKFIMEELDKVYNRAIKAAKSARDLDMRDGACSVPGVTENLGQSLWEVSESHFKYPIVEQYLKTKKNSNNLIIKYISCRLLTLIIILLACIYLGYYFSLSSLSDEFVCSIKSGILRNDSTVPDQFQCKLIAVGIFQLLSVINLVVYVLLAPVVVYTLFVPFRQKTDVLKVYEILPTFDVLHFKSEGYNDLSLYNLFLEENISEVKSYKCLKVLENIKSSGQGIDP

[0083] SEQ ID NO:6: MAIAQLATEYVFSDFLLKEPTEPKFKGLRLELAVDKMVTCIAVGLPLLLISLAFAQEISIGTQISCFSPSSFSWRQAAFVDSYCWAAVQQKNSLQSESGNLPLWLHKFFPYILLLFAILLYLPPLFWRFAAAPHICSDLKFIMEELDKVYNRAIKAAKSARDLDMRDGACSVPGVTENLGQSLWEVSESHFKYPIVEQYLKTKKNSNNLIIKYISCRLLTLIIILLACIYLGYYFSLSSLSDEFVCSIKSGILRNDSTVPDQFQCKLIAVGIFQLLSVINLVVYVLLAPVVVYTLFVPFRQKTDVLKVYEILPTFDVLHFKSEGYNDLSLYNLFLEENISEVKSYKCLKVLENIKSSGQGI

[0084] DNA sequence: SEQ ID NO:7:

[0085] SEQ ID NO:8:

[0086] SEQ ID NO:9:

[0087] SEQ ID NO:10:

[0088] SEQ ID NO:11:

[0089] SEQ ID NO:12:

[0090] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A biological nanopore protein mutant, characterized in that: The biological nanopore protein mutant is obtained by mutating the wild-type PANX1 nanopore protein. The amino acid sequence of the wild-type PANX1 nanopore protein is shown in SEQ ID NO:

1. The mutation means that one or more amino acids of the wild-type PANX1 nanopore protein are mutated into common amino acids other than the original amino acids, or the C region of the wild-type PANX1 nanopore protein is truncated or truncated and replaced; 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 bio-nanopore protein mutant according to claim 1, characterized in that: Any one selected from the following A, B and C: A: One or more of the amino acids W74, R75, S73, F72 of the wild-type PANX1 nanopore protein are mutated into common amino acids other than the original amino acids; B: One or more of the amino acids I58, T21, E22, P23 of the wild-type PANX1 nanopore protein are mutated into common amino acids other than the original amino acids; C: The C region of the wild-type PANX1 nanopore protein is truncated or truncated and replaced.

3. The biological nanopore protein mutant according to claim 2, wherein: In A: The amino acid W74 of the wild-type PANX1 nanopore protein is mutated into a common amino acid other than the original amino acid; the amino acid W74 of the wild-type PANX1 nanopore protein is mutated into W74M, W74P, W74L, W74G, W74I, W74Q, W74E, W74C, W74R, W74H, W74K, W74T, W74A, W74S, W74Y, W74N, W74V or W74D; The amino acid R75 of the wild-type PANX1 nanopore protein is mutated into a common amino acid other than the original amino acid; the amino acid R75 of the wild-type PANX1 nanopore protein is mutated into R75N or R75S; The amino acid S73 of the wild-type PANX1 nanopore protein is mutated into a common amino acid other than the original amino acid; the amino acid S73 of the wild-type PANX1 nanopore protein is mutated into S73N, S73K, S73H, S73D, S73W, S73R, S73F, S73E, S73P, S73L, S73A, S73V, S73G or S73M; The amino acid F72 of the wild-type PANX1 nanopore protein is mutated into a common amino acid other than the original amino acid; the amino acid F72 of the wild-type PANX1 nanopore protein is mutated into F72N, F72H, F72Q, F72S, F72L, F72D, F72C, F72I, F72P, F72T, F72G, F72M, F72W or F72R.

4. The bio-nanopore protein mutant according to claim 3, characterized in that: In B: The amino acid I58 of the wild-type PANX1 nanopore protein is mutated into a common amino acid other than the original amino acid; the amino acid I58 of the wild-type PANX1 nanopore protein is mutated into I58N; The amino acid T21 of the wild-type PANX1 nanopore protein is mutated to a common amino acid other than the original amino acid; the amino acid T21 of the wild-type PANX1 nanopore protein is mutated to T21N and T21L; The amino acid E22 of the wild-type PANX1 nanopore protein is mutated to a common amino acid other than the original amino acid; the amino acid E22 of the wild-type PANX1 nanopore protein is mutated to E22T and E22L; The amino acid P23 of the wild-type PANX1 nanopore protein is mutated to a common amino acid other than the original amino acid; the amino acid P23 of the wild-type PANX1 nanopore protein is mutated to P23N.

5. The biological nanopore protein mutant according to claim 4, wherein: In C: The truncation is a 1- to 10-amino acid truncation from G371 towards the N-terminus.

6. The coding gene of a nanopore protein mutant, characterized in that: Selected from: D: A nucleic acid sequence encoding a PANX1 nanopore protein mutant; E: A nucleic acid that has at least 85% homology with the nucleic acid sequence in D and encodes a nanopore protein mutant; F: A nucleic acid complementary to D and E; wherein the homology is between 85% and 99%.

7. A biological nanopore protein, characterized in that: The biological nanopore protein is composed of seven identical subunits, and the overall channel is funnel-shaped, and the amino acid sequence is as shown in SEQ ID NO:

1.

8. A method for preparing a biological nanopore protein and its mutants, characterized in that: Including the following steps: Step S1: Construct a biological nanopore protein or biological nanopore protein mutant vector; Step S2: Express and purify the biological nanopore protein or biological nanopore protein mutant.

9. A membrane layer embedded with biological nanopore proteins and their mutants, characterized in that: The membrane layer is a lipid bilayer.

10. Use of the biological nanopore protein and its mutants in the sequencing or detection of one or more of nucleic acids, oligopeptides, oligosaccharides, and macromolecular drugs.

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