Use of a biological nanopore protein and mutants thereof in detection

Abstract

The application belongs to the technical field of methods for characterizing target polynucleotides, and provides an application of a biological nanopore protein and a mutant thereof in detection. The biological nanopore protein mutant is obtained by mutating a wild-type PANX1 nanopore protein, and an amino acid sequence of the wild-type PANX1 nanopore protein is shown as SEQ ID NO:1. The mutation refers to that one or more amino acids of the wild-type PANX1 nanopore protein are mutated into common amino acids other than original amino acids, or a C region of the wild-type PANX1 nanopore protein is truncated or truncated and replaced to obtain the PANX1 nanopore protein mutant. After the wild-type PANX1 nanopore protein is mutated, the wild-type PANX1 nanopore protein can be normally expressed. Compared with the wild-type PANX1 nanopore protein, the biological nanopore protein mutant is stable in property, and the C region is truncated to expand the pore diameter of the contraction region of the protein mutant, so that the pore channel is avoided from being blocked.

Description

Technical Field

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

[0002] Nanopore single-molecule sensing technology has shown significant advantages in small molecule detection, chemical reaction monitoring, DNA sequencing, and protein identification. This technology originated from particle size measurement methods based on the continuous pulse principle. A classic example is the Coulter counter, which monitors the displacement of the current pulse generated when blood cells pass through pores with diameters ranging from 20 μm to 2 mm, enabling precise estimation of cell number and size. However, when the analyte size is much smaller than the pore size, the resulting current change is too small to be detected. Therefore, pore proteins with suitable pore size are one of the key technologies affecting nanopore sequencing. The geometric characteristics of the contraction region (i.e., the core region for signal readout) of the pore protein directly determine the quality and resolution accuracy of the original current signal.

[0003] Currently, the variety of pore proteins is limited. Taking the commonly used E. coli CsgG mutant as an example, its contractile region can only accommodate 4-5 bases, resulting in a single recorded current signal being generated by the combined action of multiple bases, creating superimposed interference. This significantly increases the complexity of subsequent signal analysis, making it difficult to achieve sequencing accuracy comparable to next-generation sequencing technologies. Furthermore, existing mainstream pore proteins, such as CsgG, are mostly nonamerical non-axisymmetric structures. This complex oligomeric morphology makes protein cloning, expression, and purification processes more cumbersome. Therefore, it is necessary to discover pore proteins with novel contractile regions. This would increase the variety of nanopores available and reduce the difficulty of current signal analysis, further improving sequencing accuracy. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides an application of biological nanoporous proteins and their mutants in detection, aiming to solve the problems mentioned in the background art.

[0005] In a first aspect, the present invention provides a nanoporin mutant, wherein the bio-nanoporin mutant is obtained by mutation of wild-type PANX1 nanoporin, the amino acid sequence of wild-type PANX1 nanoporin is shown in SEQ ID NO:1, and the mutation refers to: one or more amino acids of wild-type PANX1 nanoporin being mutated to common amino acids other than the original amino acids, or the C region of wild-type PANX1 nanoporin being truncated or truncated and replaced.

[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, choose any one of the following: A, B, and C:

[0008] A: One or more of the amino acids W74, R75, S73, and F72 in wild-type PANX1 nanoporin are mutated to common amino acids other than the original amino acids;

[0009] B: One or more of the amino acids I58, T21, E22, and P23 of wild-type PANX1 nanoporin are mutated to common amino acids other than the original amino acids;

[0010] C: The C region of wild-type PANX1 nanoporin is truncated or replaced.

[0011] Further, in A: the amino acid W74 of the wild-type PANX1 nanoporin is mutated to a common amino acid other than the original amino acid; the amino acid W74 of the wild-type PANX1 nanoporin 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 of the wild-type PANX1 nanoporin is mutated to W74T, W74L or W74G;

[0012] The amino acid R75 of wild-type PANX1 nanoporin is mutated to a common amino acid other than the original amino acid; the amino acid R75 of wild-type PANX1 nanoporin is mutated to R75N ​​or R75S.

[0013] The wild-type PANX1 nanoporin has an amino acid S73 mutation to a common amino acid other than the original amino acid; the wild-type PANX1 nanoporin has an amino acid S73 mutation to S73N, S73K, S73H, S73D, S73W, S73R, S73F, S73E, S73P, S73L, S73A, S73V, S73G or S73M; preferably, the wild-type PANX1 nanoporin has an amino acid S73 mutation to S73N or S73K;

[0014] The amino acid F72 of wild-type PANX1 nanoporin is mutated to a common amino acid other than the original amino acid; the amino acid F72 of wild-type PANX1 nanoporin is mutated to F72N, F72H, F72Q, F72S, F72L, F72D, F72C, F72I, F72P, F72T, F72G, F72M, F72W or F72R; preferably, the amino acid F72 of wild-type PANX1 nanoporin is mutated to F72I.

[0015] Furthermore, in B: the amino acid I58 of wild-type PANX1 nanoporin is mutated to a common amino acid other than the original amino acid; the amino acid I58 of wild-type PANX1 nanoporin is mutated to I58N;

[0016] The amino acid T21 of wild-type PANX1 nanoporin is mutated to common amino acids other than the original amino acid; the amino acid T21 of wild-type PANX1 nanoporin is mutated to T21N and T21L.

[0017] The amino acid E22 of wild-type PANX1 nanoporin is mutated to common amino acids other than the original amino acid; the amino acid E22 of wild-type PANX1 nanoporin is mutated to E22T and E22L.

[0018] The amino acid P23 of wild-type PANX1 nanoporin is mutated to a common amino acid other than the original amino acid; the amino acid P23 of wild-type PANX1 nanoporin is mutated to P23N.

[0019] Further, in C: the truncation is a truncation of 1-10 amino acids from G371 to the N-terminus; preferably, a truncation of 2 amino acids from G371 to the N-terminus, i.e., truncation of G371-L370; a truncation of 4 amino acids from G371 to the N-terminus, i.e., truncation of G371-T368; a truncation of 6 amino acids from G371 to the N-terminus, i.e., truncation of G371-L366; and a truncation of 8 amino acids from G371 to the N-terminus, i.e., truncation of G371-M364.

[0020] Further, in C: truncated substitution is the replacement of G371 to the N-terminus with a short amino acid sequence, where 3 amino acids replace 8, 9, 10, or 11 amino acids; preferably, GSG replaces 11 amino acids, i.e., truncated substitution of G371-I361.

[0021] Furthermore, the amino acid sequences of the bio-nanoporin mutant are shown in SEQ ID NO:2-SEQ ID NO:6; preferably, the amino acid sequence of the bio-nanoporin mutant is shown in SEQ ID NO:5.

[0022] Secondly, this invention provides a gene encoding a bio-nanoporin mutant, selected from:

[0023] D: Nucleic acid sequence encoding a mutant of a biological nanoporin;

[0024] E: Nucleic acid sequence with at least 85% homology to that in D, and encoding a bio-nanoporin mutant;

[0025] F: Nucleic acid complementary to D and E;

[0026] Among them, the homology is between 85% and 99%.

[0027] Thirdly, the present invention provides a bio-nanoporin composed of seven identical subunits, with the overall channel being funnel-shaped, and the amino acid sequence is shown in SEQ ID NO:1.

[0028] Fourthly, the present invention provides a method for preparing biological nanoporous proteins and their mutants, comprising the following steps:

[0029] Step S1: Construct bio-nanoporin or bio-nanoporin mutant vector;

[0030] Step S2: Expression and purification of bio-nanoporin or bio-nanoporin mutant.

[0031] Fifthly, the present invention provides a membrane layer embedded with biological nanoporous proteins and their mutants, wherein the membrane layer is a lipid bilayer.

[0032] In a sixth aspect, the present invention provides the application of biological nanoporous proteins and their mutants in the sequencing or detection of one or more of nucleic acids, oligopeptides, oligosaccharides, and macromolecular drugs.

[0033] The present invention has the following beneficial effects:

[0034] (1) Wild-type PANX1 nanoporous protein is a unique nanoporous protein, different from MspA and CsgG. It has a heptameric structure, with each subunit containing four transmembrane domains TM1-TM4. The N-terminus and C-terminus of TM1 and TM4 are anchored to the intracellular space, respectively. TM2 and TM3 provide lateral support for the channel, and the overall channel is funnel-shaped. Compared with existing nanoporous proteins, its cloning, expression, and purification processes are simple, it is easy to assemble into proteins for biological nanopores, and it has a wide range of applications.

[0035] (2) Mutation experiments revealed that one or more amino acids of wild-type PANX1 nanoporin were mutated to common amino acids other than the original amino acids, or the C region of wild-type PANX1 nanoporin was truncated or replaced, and it could be expressed normally. Compared with wild-type PANX1 nanoporin, the PANX1 nanoporin mutant (biological nanoporin mutant) is stable and the truncation of the C region makes the pore size of the protein mutant shrinkage region larger, avoiding pore blockage. Attached Figure Description

[0036] Exemplary embodiments of the present invention can be more fully understood by referring to the following figures:

[0037] Figure 1 This is a top view of the surface structure model of the wild-type PANX1 nanoporous protein channel of this invention.

[0038] Figure 2 This is a side view of the surface structure model of the wild-type PANX1 nanoporous protein channel of the present invention.

[0039] Figure 3 This is a top view of the wild-type PANX1 nanoporous protein channel ribbon structure model of the present invention.

[0040] Figure 4 This is a side view of the wild-type PANX1 nanoporous protein channel ribbon structure model of the present invention.

[0041] Figure 5 This is a purification electrophoresis image of the PANX1 nanoporous protein mutant after C-terminus truncation of 10 amino acids and mutation of amino acid F72 to F72I in Example 6 of the present invention; wherein lane 1: protein marker; lane 2: washing sample; lane 3: elution sample; lane 4: elution sample; lane 5: elution sample.

[0042] Figure 6 This is a graph showing the detection results of the pore opening current of wild-type PANX1 nanoporous protein in Example 2 of the present invention.

[0043] Figure 7 The image shows the nanopore opening current detection results of the PANX1 nanopore protein mutant after 10 amino acids were truncated at the C-terminus and amino acid F72 was mutated to F72I in Example 7 of the present invention.

[0044] Figure 8 The graph shows the current detection results during single-stranded DNA perforation of the PANX1 nanoporous protein mutant after 10 amino acids were truncated at the C-terminus and amino acid F72 was mutated to F72I in Example 8 of the present invention. Detailed Implementation

[0045] 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.

[0046] 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.

[0047] This invention provides a bio-nanoporin mutant, which is obtained by mutating wild-type PANX1 nanoporin. The amino acid sequence of wild-type PANX1 nanoporin is shown in SEQ ID NO:1. Mutation refers to: one or more amino acids of wild-type PANX1 nanoporin being mutated to common amino acids other than the original amino acids, or the C region of wild-type PANX1 nanoporin being truncated or truncated and replaced.

[0048] 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.

[0049] In some embodiments, any one of the following A, B, and C is selected:

[0050] A: One or more of the amino acids W74, R75, S73, and F72 in wild-type PANX1 nanoporin are mutated to common amino acids other than the original amino acids;

[0051] B: One or more of the amino acids I58, T21, E22, and P23 of wild-type PANX1 nanoporin are mutated to common amino acids other than the original amino acids;

[0052] C: The C region of wild-type PANX1 nanoporin is truncated or replaced.

[0053] In some embodiments, in A: the amino acid W74 of the wild-type PANX1 nanoporin is mutated to a common amino acid other than the original amino acid; the amino acid W74 of the wild-type PANX1 nanoporin is mutated to W74M, W74P, W74L, W74G, W74I, W74Q, W74E, W74C, W74R, W74H, W74K, W74T, W74A, W74S, W74Y, W74N, W74V or W74D.

[0054] In some embodiments, the amino acid W74 of wild-type PANX1 nanoporin is mutated to W74T, W74L, or W74G.

[0055] In some embodiments, in A: the amino acid R75 of wild-type PANX1 nanoporin is mutated to a common amino acid other than the original amino acid; the amino acid R75 of wild-type PANX1 nanoporin is mutated to R75N ​​or R75S.

[0056] In some embodiments, in A: the S73 amino acid of wild-type PANX1 nanoporin is mutated to a common amino acid other than the original amino acid; the S73 amino acid of wild-type PANX1 nanoporin is mutated to S73N, S73K, S73H, S73D, S73W, S73R, S73F, S73E, S73P, S73L, S73A, S73V, S73G or S73M.

[0057] In some embodiments, the amino acid S73 of wild-type PANX1 nanoporin is mutated to S73N or S73K.

[0058] In some embodiments, in A: the amino acid F72 of wild-type PANX1 nanoporin is mutated to a common amino acid other than the original amino acid; the amino acid F72 of wild-type PANX1 nanoporin is mutated to F72N, F72H, F72Q, F72S, F72L, F72D, F72C, F72I, F72P, F72T, F72G, F72M, F72W or F72R.

[0059] In some embodiments, the amino acid F72 of wild-type PANX1 nanoporous protein is mutated to F72I.

[0060] In some embodiments, in B: the amino acid I58 of wild-type PANX1 nanoporin is mutated to a common amino acid other than the original amino acid; the amino acid I58 of wild-type PANX1 nanoporin is mutated to I58N.

[0061] In some embodiments, in B: the amino acid T21 of wild-type PANX1 nanoporin is mutated to a common amino acid other than the original amino acid; the amino acid T21 of wild-type PANX1 nanoporin is mutated to T21N or T21L.

[0062] In some embodiments, in B: the amino acid E22 of wild-type PANX1 nanoporous protein is mutated to a common amino acid other than the original amino acid; the amino acid E22 of wild-type PANX1 nanoporous protein is mutated to E22T or E22L.

[0063] In some embodiments, in B: the amino acid P23 of wild-type PANX1 nanoporin is mutated to a common amino acid other than the original amino acid; the amino acid P23 of wild-type PANX1 nanoporin is mutated to P23N.

[0064] In some embodiments, in C: the truncation is a truncation of 1-10 amino acids from G371 to the N-terminus; preferably, G371 is truncated by 2 amino acids from the N-terminus, i.e., truncated G371-L370; G371 is truncated by 4 amino acids from the N-terminus, i.e., truncated G371-T368; G371 is truncated by 6 amino acids from the N-terminus, i.e., truncated G371-L366; G371 is truncated by 8 amino acids from the N-terminus, i.e., truncated G371-M364.

[0065] In some embodiments, in C: truncation substitution is the replacement of G371 to the N-terminus with a short amino acid sequence, where 3 amino acids replace 8, 9, 10, or 11 amino acids; preferably, 11 amino acids are replaced with GSG (glycyl-glycyl-glycine), i.e., truncation substitution of G371-I361.

[0066] In some embodiments, the amino acid sequences of the bio-nanoporin mutants are shown in SEQ ID NO:2-SEQ ID NO:6, and the DNA sequences of their encoding genes are shown in SEQ ID NO:8-SEQ ID NO:12, respectively.

[0067] In some embodiments, the amino acid sequence of the bio-nanoporin mutant is shown in SEQ ID NO:5.

[0068] In some embodiments, the present invention provides a gene encoding a bio-nanoporin mutant, selected from:

[0069] D: Nucleic acid sequence encoding a mutant of a biological nanoporin;

[0070] E: Nucleic acid sequence with at least 85% homology to that in D, and encoding a bio-nanoporin mutant;

[0071] F: Nucleic acid complementary to D and E;

[0072] Among them, the homology is between 85% and 99%.

[0073] In some embodiments, the present invention provides a bio-nanoporin composed of seven identical subunits, with the overall channel being funnel-shaped, and the amino acid sequence is shown in SEQ ID NO:1.

[0074] In some embodiments, the present invention provides a method for preparing biological nanoporous proteins and their mutants, comprising the following steps:

[0075] Step S1: Construct bio-nanoporin or bio-nanoporin mutant vector;

[0076] Step S2: Expression and purification of bio-nanoporin or bio-nanoporin mutant.

[0077] In some embodiments, the present invention provides a membrane layer in which a bioporous protein and its mutant are embedded, the membrane layer being a lipid bilayer.

[0078] In some embodiments, the present invention provides the application of bio-nanoporins and their mutants in the sequencing or detection of one or more of nucleic acids, oligopeptides, oligosaccharides, and macromolecular drugs.

[0079] Example 1: Expression and purification of wild-type PANX1 nanoporous protein

[0080] 1. Recombinant Bacmid Construction and Viral Packaging

[0081] (1) Transformation and screening: pEG-PANX1 vector containing the PANX1 gene was transformed into E. coli DH10bac cells, and positive clones (containing recombinant Bacmid) were identified by blue-white screening.

[0082] (2) Transfection of Sf9 cells: Recombinant Bacmid was extracted and transfected into adherent Sf9 insect cells with Cellfectin reagent. After 4 days of culture, GFP fluorescence was observed by fluorescence microscopy to confirm successful virus packaging.

[0083] (3) Viral amplification (P1 to P2 virus): Collect viral supernatant (filtered through a 0.22 μm filter) and infect suspension-cultured Sf9 cells (density) After culturing for 4 days, the supernatant was collected by centrifugation (7000g) to obtain high-titer P2 virus, which was then stored at 4°C in the dark.

[0084] 2. HEK293S cell infection and protein-induced expression

[0085] (1) Cell culture: HEK293S GnTI⁻ cells were cultured at 37°C in Freestyle 293 medium containing 1.5% FBS until the desired density was reached. cells / mL;

[0086] (2) Viral infection and induction: P2 virus was inoculated, and 5 mM sodium butyrate was added 12 hours later. The culture temperature was lowered to 32°C, and cells were collected 60 hours after infection.

[0087] (3) Cell disruption: The collected cells were disrupted by sonication (35% amplitude, 15 minutes), and the lysate was centrifuged at 100,000 × g for 1 hour to precipitate and enrich cell membrane components.

[0088] (4) Membrane protein dissolution: Dissolve membrane components in a buffer solution containing 10 mM glycoside (sodium glycocholate), 25 mM Tris (tris(hydroxymethyl)aminomethane, pH=8.0) and 100 mM KCl, centrifuge again at 100000×g for 1 hour, and collect the supernatant.

[0089] 3. Affinity chromatography purification (nickel column purification)

[0090] (1) Binding: The supernatant was incubated with Ni-NTA resin (pre-equilibration binding buffer: 25 mM Tris, pH=8.0; 100 mM KCl; 100 μM GDN) for 2 hours, and the target protein (containing the C-terminal 8×His tag) specifically bound to the resin.

[0091] (2) Elution: The target protein was eluted with elution buffer containing 0.3 M imidazole (25 mM Tris, pH=8.0; 100 mM KCl, 100 μM GDN, 0.3 M imidazole), and the eluent was collected.

[0092] 4. Label removal and size exclusion chromatography

[0093] (1) Tag cleavage: TEV protease was added to the elution buffer to remove the C-terminal GFP and 8×His tag, and tag-free PANX1 protein was obtained;

[0094] (2) Fine purification: The protein after enzyme digestion was purified by size exclusion chromatography column with buffer of 25 mM Tris (pH=8.0), 100 mM KCl, 1% glycerol and 50 μM MDN, and the main peak component was collected.

[0095] A top view of the surface structure model of wild-type PANX1 nanoporous protein channels is shown below. Figure 1 As shown; a side view of the surface structure model of wild-type PANX1 nanoporous protein channels is shown below. Figure 2 As shown; Top view of the wild-type PANX1 nanoporous protein channel ribbon structure model. Figure 3 As shown, the parts of the same color represent a single protein monomer; a side view of the wild-type PANX1 nanoporous protein channel ribbon structure model is shown below. Figure 4 As shown, the parts of the same color represent a single protein monomer. The results indicate that the wild-type PANX1 nanoporous protein has a heptamer structure, and the overall channel is funnel-shaped.

[0096] Example 2: Characterization of wild-type PANX1 nanoporous protein

[0097] Wild-type PANX1 nanoporous protein with an amino acid sequence as shown in SEQ ID NO:1 was obtained, and the DNA sequence of its encoding gene is shown in SEQ ID NO:7.

[0098] A lipid bilayer was formed on a polytetrafluoroethylene horizontal pore with a diameter of approximately 20 μm using 1,2-diphynoglycerol-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 100 mV transmembrane voltage (positive on the reverse side, cis compartment grounded) was applied to the bilayer using an Axopatch 200B integrated patch-clamp amplifier. Wild-type PANX1 nanoporous protein at a concentration of 4.0 ng / ml was added to the grounded cis compartment. Once a single pore insertion was detected, the compartment was immediately flushed with experimental buffer to prevent further insertion of free protein.

[0099] The experiment was conducted 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 using Origin. The pore opening current detection results of wild-type PANX1 nanoporous protein are as follows: Figure 6 As shown.

[0100] Example 3: Detection of single-stranded DNA using wild-type PANX1 nanoporous protein

[0101] A lipid bilayer was formed with 1,2-diphynoglycerol-3-phosphocholine on a polytetrafluoroethylene horizontal pore with a diameter of approximately 20 μm. 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 100 mV transmembrane voltage (positive on the reverse side, cis compartment grounded) was applied to the bilayer using an Axopatch 200B integrated patch-clamp amplifier. Wild-type PANX1 nanoporous protein at a concentration of 4.0 ng / ml was added to the grounded cis compartment. After detecting insertion of a single pore, the compartment was immediately flushed with experimental buffer to prevent further insertion of free protein. Then, fluorescently labeled single-stranded DNA (SEQ ID NO: 13: 5'-ACGTACGTACGTCAG-3', concentration 1 μM) was added to the cis compartment, and a 100 mV voltage was applied, recording the current signal.

[0102] The experiment was conducted 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 using Origin. The current signal of single-stranded DNA passing through wild-type PANX1 nanoporous protein can be observed. The diameter of the contraction region of wild-type PANX1 protein is too small, which may block the pores.

[0103] Example 4: Detection of single-stranded DNA using the PANX1 nanoporin mutant (biological nanoporin mutant) with amino acid P23 mutated to P23N.

[0104] A lipid bilayer was formed with 1,2-diphynoglycerol-3-phosphocholine on a polytetrafluoroethylene horizontal pore with a diameter of approximately 20 μm. 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 100 mV transmembrane voltage (positive on the reverse side, cis compartment grounded) was applied to the bilayer using an Axopatch 200B integrated patch-clamp amplifier. A PANX1 nanoporous protein mutant with amino acid P23 mutated to P23N at a concentration of 4.0 ng / ml was added to the grounded cis compartment. After detecting a single pore insertion, the compartment was immediately flushed with experimental buffer to prevent further insertion of free protein. Then, fluorescently labeled single-stranded DNA (SEQ ID NO: 13: 5'-ACGTACGTACGTCAG-3', concentration 1 μM) was added to the cis compartment, a 100 mV voltage was applied, and the current signal was recorded.

[0105] The experiment was conducted 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 using Origin. The results showed that compared with wild-type PANX1 nanoporous protein, the PANX1 nanoporous protein mutant with amino acid P23 mutated to P23N exhibited stable current properties, narrower current signal width, and fewer spikes when detecting single-stranded DNA.

[0106] Example 5: Detection of single-stranded DNA using a PANX1 nanoporin mutant with a 10-amino acid C-terminus truncated (biological nanoporin mutant).

[0107] A lipid bilayer was formed with 1,2-diphynoglycerol-3-phosphocholine on a polytetrafluoroethylene horizontal pore with a diameter of approximately 20 μm. 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 100 mV transmembrane voltage (positive on the reverse side, cis compartment grounded) was applied to the bilayer using an Axopatch 200B integrated patch-clamp amplifier. A 4.0 ng / ml mutant of PANX1 nanoporous protein with a 10-amino acid truncation at the C-terminus was added to the grounded cis compartment. After detecting a single pore insertion, the compartment was immediately flushed with experimental buffer to prevent further insertion of free protein. Then, fluorescently labeled single-stranded DNA (SEQ ID NO: 13: 5'-ACGTACGTACGTCAG-3', concentration 1 μM) was added to the cis compartment, a 100 mV voltage was applied, and the current signal was recorded.

[0108] The experiment was conducted at room temperature (23±1℃). The simulated signal was low-pass filtered at 100 kHz using a 4-pole Bessel filter and then digitized at a sampling rate of 500 kHz. Data acquisition was controlled by Origin software. It can be seen that compared to wild-type PANX1 nanoporous protein, the PANX1 nanoporous protein mutant with a 10-amino acid truncation at the C-terminus exhibits stable current properties and reduced current signal noise when detecting single-stranded DNA.

[0109] Example 6: Expression and purification of the PANX1 nanoporin mutant (biological nanoporin mutant) after C-terminus truncation of 10 amino acids and mutation of amino acid F72 to F72I.

[0110] 1. Recombinant Bacmid Construction and Viral Packaging

[0111] (1) Transformation and screening: The pEG-PANX1 vector containing the PANX1 gene with 10 amino acids truncated at the C-terminus and the 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.

[0112] (2) Transfection of Sf9 cells: Recombinant Bacmid was extracted and transfected into adherent Sf9 insect cells with Cellfectin reagent. After 4 days of culture, GFP fluorescence was observed by fluorescence microscopy to confirm successful virus packaging.

[0113] (3) Viral amplification (P1 to P2 virus): Collect viral supernatant (filtered through a 0.22 μm filter) and infect suspension-cultured Sf9 cells (density) After culturing for 4 days, the supernatant was collected by centrifugation (7000g) to obtain high-titer P2 virus, which was then stored at 4°C in the dark.

[0114] 2. HEK293S cell infection and protein-induced expression

[0115] (1) Cell culture: HEK293S GnTI⁻ cells were cultured at 37°C in Freestyle 293 medium containing 1.5% FBS until the desired density was reached. cells / mL;

[0116] (2) Viral infection and induction: P2 virus was inoculated, and 5 mM sodium butyrate was added 12 hours later. The culture temperature was lowered to 32°C, and cells were collected 60 hours after infection.

[0117] (3) Cell disruption: The collected cells were disrupted by sonication (35% amplitude, 15 minutes), and the lysate was centrifuged at 100,000 × g for 1 hour to precipitate and enrich cell membrane components.

[0118] (4) Membrane protein dissolution: Dissolve membrane components in a buffer containing 10 mM glycoside (sodium glycocholate), 25 mM Tris (tris(hydroxymethyl)aminomethane, pH=8.0) and 100 mM KCl, centrifuge again at 100000×g for 1 hour, and collect the supernatant.

[0119] 3. Affinity chromatography purification (nickel column purification)

[0120] (1) Binding: The supernatant was incubated 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 the C-terminal 8×His tag) specifically bound to the resin.

[0121] (2) Elution: The target protein was eluted with elution buffer containing 0.3 M imidazole (25 mM Tris pH=8.0, 100 mM KCl, 100 μM GDN, 0.3 M imidazole), and the eluent was collected.

[0122] 4. Label removal and size exclusion chromatography

[0123] (1) Tag cleavage: TEV protease was added to the elution buffer to remove the C-terminal GFP and 8×His tag, and tag-free PANX1 protein was obtained;

[0124] (2) Fine purification: The protein after enzyme digestion was purified by size exclusion chromatography column with buffer of 25 mM Tris pH=8.0, 100 mM KCl, 1% glycerol and 0 μM GDN, and the main peak component was collected.

[0125] The purification electrophoresis results of the PANX1 nanoporin mutant, which had 10 amino acids truncated at the C-terminus and amino acid F72 mutated to F72I, are as follows: Figure 6 As shown.

[0126] Example 7: Characterization of the PANX1 nanoporin mutant (biological nanoporin mutant) resulting from C-terminal truncation of 10 amino acids and mutation of amino acid F72 to F72I.

[0127] A lipid bilayer was formed with 1,2-diphynoglycerol-3-phosphocholine on a polytetrafluoroethylene horizontal pore with a diameter of approximately 20 μm. A buffer solution (pH = 8.0 ± 0.05) containing 1 M KCl and 20 mM HEPES / KOH was added to each 1 ml compartment on both sides of the bilayer. A transmembrane voltage of 100 mV (positive on the reverse side, cis compartment grounded) was applied to the bilayer using an Axopatch 200B integrated patch-clamp amplifier. A 4.0 ng / ml PANX1 nanoporous protein mutant, with 10 amino acids truncated at the C-terminus and amino acid F72 mutated to F72I, was added to the grounded cis compartment. Once a single pore insertion was detected, the compartment was immediately flushed with experimental buffer to prevent further insertion of free protein.

[0128] The experiment was conducted at room temperature (23±1℃). The analog signal was low-pass filtered by a 100 kHz, 4-pole Bessel filter, and then digitized at a sampling rate of 500 kHz. Data acquisition was controlled by Origin software. The nanopore opening current detection results of the PANX1 nanopore protein mutant, after truncating 10 amino acids at the C-terminus and mutating amino acid F72 to F72I, are as follows: Figure 7 As shown.

[0129] Example 8: Detection of single-stranded DNA using a PANX1 nanoporin mutant (biological nanoporin mutant) with 10 amino acids truncated at the C-terminus and amino acid F72 mutated to F72I.

[0130] A lipid bilayer was formed with 1,2-diphynoglycerol-3-phosphocholine on a polytetrafluoroethylene horizontal pore with a diameter of approximately 20 μm. A buffer solution (pH = 8.0 ± 0.05) containing 1 M KCl and 20 mM HEPES / KOH was added to each 1 ml compartment on both sides of the bilayer. A transmembrane voltage of 100 mV (positive on the reverse side, cis compartment grounded) was applied to the bilayer using an Axopatch 200B integrated patch-clamp amplifier. PANX1 nanoporous protein with a C-terminus cleaved by 10 amino acids and amino acid F72 mutated to F72I at a concentration of 4.0 ng / ml was added to the grounded cis compartment. After detecting a single pore insertion, the compartment was immediately flushed with experimental buffer to prevent further insertion of free protein. Then, fluorescently labeled single-stranded DNA (SEQ ID NO: 13: 5'-ACGTACGTACGTCAG-3', concentration 1 μM) was added to the cis compartment, a voltage of 100 mV was applied, and the current signal was recorded.

[0131] The experiment was conducted at room temperature (23±1℃). The analog signal was low-pass filtered at 100 kHz using a 4-pole Bessel filter and then digitized at a sampling rate of 500 kHz. Data acquisition was controlled by Origin software. The current detection results during single-stranded DNA perforation of the PANX1 nanoporin mutant (with 10 amino acids truncated at the C-terminus and amino acid F72 mutated to F72I) are as follows: Figure 8 As shown.

[0132] By comparison Figures 6-8 As can be seen, compared with wild-type PANX1 nanoporin, the opening current of PANX1 nanoporin mutant is significantly increased, and the current properties and current signal are significantly improved, making it suitable for sequencing and / or detection of samples such as DNA.

[0133] The wild-type PANX1 nanoporous protein mutant has a pore size of 1.2 nm at its narrowest point, and its truncated C region expands the pore size of the shrinkage region of the protein mutant. Therefore, the PANX1 nanoporous protein mutant is more suitable for detecting large molecular compounds such as nucleic acids, oligopeptides, oligosaccharides, and large molecular drugs (the diameter of the drug is smaller than the protein pore size).

[0134] In summary, this invention reveals that wild-type PANX1 nanoporous protein is a unique nanoporous protein, distinct from MspA and CsgG. It exhibits a heptameric structure, with each subunit containing four transmembrane domains TM1-TM4. The N-termini and C-termini of TM1 and TM4 are anchored intracellularly, respectively, while TM2 and TM3 provide lateral support for the channel, resulting in a funnel-shaped overall channel. Compared to existing nanoporous proteins, its cloning, expression, and purification processes are simpler, facilitating protein assembly into biological nanopores and broadening its applicability.

[0135] Mutation experiments revealed that one or more amino acids in wild-type PANX1 nanoporin were mutated to common amino acids other than the original amino acids, or that the C region of wild-type PANX1 nanoporin was truncated or replaced, and that it could be expressed normally. Compared with wild-type PANX1 nanoporin, the PANX1 nanoporin mutant (biological nanoporin mutant) is more stable and the truncation of the C region increases the pore size of the protein mutant's contraction zone, thus avoiding pore blockage.

[0136] amino acid sequence:

[0137] SEQ ID NO:1:

[0138] MAIAQLATEYVFSDFLLKEPTEPKFKGLRLELAVDKMVTCIAVGLPLLLISLAFAQEISIGTQISCFSPSSFSWRQAAFVDSYCWAAVQQKNSLQSESGNLPLWLHKFFPYILLLFAILLYLPPLFWRFAAAPHICSDLKFIMEELDKVYNRAIKAAKSARDLDMRDGACSVPGVTENLGQSLWE VSESHFKYPIVEQYLKTKKNSNNLIIKYISCRLLTLIIILLACIYLGYYFSLSSLSDEFVCSIKSGILRNDSTVPDQFQCKLIAVGIFQLLSVINLVVYVLLAPVVVYTLFVPFRQKTDVLKVYEILPTFDVLHFKSEGYNDLSLYNLFLEENISEVKSYKCLKVLENIKSSGQGIDPMLLLTNLG

[0139] SEQ ID NO:2:

[0140] MAIAQLATEYVFSDFLLKEPTEPKFKGLRLELAVDKMVTCIAVGLPLLLISLAFAQEISIGTQISCFSPSSFSWRQAAFVDSYCWAAVQQKNSLQSESGNLPLWLHKFFPYILLLFAILLYLPPLFWRFAAAPHICSDLKFIMEELDKVYNRAIKAAKSARDLDMRDGACSVPGVTENLGQSLWEVSESHFKYPIVEQYLKTKKNSNNLIIKYISCRLLTLIIILLACIYLGYYFSLSSLSDEFVCSIKSGILRNDSTVPDQFQCKLIAVGIFQLLSVINLVVYVLLAPVVVYTLFVPFRQKTDVLKVYEILPTFDVLHFKSEGYNDLSLYNLFLEENISEVKSYKCLKVLENIKSSGQGIDPMLLLTN

[0141] SEQ ID NO:3:

[0142] MAIAQLATEYVFSDFLLKEPTEPKFKGLRLELAVDKMVTCIAVGLPLLLISLAFAQEISIGTQISCFSPSSFSWRQAAFVDSYCWAAVQQKNSLQSESGNLPLWLHKFFPYILLLFAILLYLPPLFWRFAAAPHICSDLKFIMEELDKVYNRAIKAAKSARDLDMRDGACSVPGVTENLGQSLWEVSESHFKYPIVEQYLKTKKNSNNLIIKYISCRLLTLIIILLACIYLGYYFSLSSLSDEFVCSIKSGILRNDSTVPDQFQCKLIAVGIFQLLSVINLVVYVLLAPVVVYTLFVPFRQKTDVLKVYEILPTFDVLHFKSEGYNDLSLYNLFLEENISEVKSYKCLKVLENIKSSGQGIDPMLLL

[0143] SEQ ID NO:4:

[0144] MAIAQLATEYVFSDFLLKEPTEPKFKGLRLELAVDKMVTCIAVGLPLLLISLAFAQEISIGTQISCFSPSSFSWRQAAFVDSYCWAAVQQKNSLQSESGNLPLWLHKFFPYILLLFAILLYLPPLFWRFAAAPHICSDLKFIMEELDKVYNRAIKAAKSARDLDMRDGACSVPGVTENLGQSLWEVSESHFKYPIVEQYLKTKKNSNNLIIKYISCRLLTLIIILLACIYLGYYFSLSSLSDEFVCSIKSGILRNDSTVPDQFQCKLIAVGIFQLLSVINLVVYVLLAPVVVYTLFVPFRQKTDVLKVYEILPTFDVLHFKSEGYNDLSLYNLFLEENISEVKSYKCLKVLENIKSSGQGIDPML

[0145] SEQ ID NO:5:

[0146] MAIAQLATEYVFSDFLLKEPTEPKFKGLRLELAVDKMVTCIAVGLPLLLISLAFAQEISIGTQISCFSPSSFSWRQAAFVDSYCWAAVQQKNSLQSESGNLPLWLHKFFPYILLLFAILLYLPPLFWRFAAAPHICSDLKFIMEELDKVYNRAIKAAKSARDLDMRDGACSVPGVTENLGQSLWEVSESHFKYPIVEQYLKTKKNSNNLIIKYISCRLLTLIIILLACIYLGYYFSLSSLSDEFVCSIKSGILRNDSTVPDQFQCKLIAVGIFQLLSVINLVVYVLLAPVVVYTLFVPFRQKTDVLKVYEILPTFDVLHFKSEGYNDLSLYNLFLEENISEVKSYKCLKVLENIKSSGQGIDP

[0147] SEQ ID NO:6:

[0148] MAIAQLATEYVFSDFLLKEPTEPKFKGLRLELAVDKMVTCIAVGLPLLLISLAFAQEISIGTQISCFSPSSFSWRQAAFVDSYCWAAVQQKNSLQSESGNLPLWLHKFFPYILLLFAILLYLPPLFWRFAAAPHICSDLKFIMEELDKVYNRAIKAAKSARDLDMRDGACSVPGVTENLGQSLWEVSESHFKYPIVEQYLKTKKNSNNLIIKYISCRLLTLIIILLACIYLGYYFSLSSLSDEFVCSIKSGILRNDSTVPDQFQCKLIAVGIFQLLSVINLVVYVLLAPVVVYTLFVPFRQKTDVLKVYEILPTFDVLHFKSEGYNDLSLYNLFLEENISEVKSYKCLKVLENIKSSGQGI

[0149] DNA sequence:

[0150] SEQ ID NO:7:

[0151]

[0152] SEQ ID NO:8:

[0153]

[0154] SEQ ID NO:9:

[0155]

[0156] SEQ ID NO:10:

[0157]

[0158] SEQ ID NO:11:

[0159]

[0160] SEQ ID NO:12:

[0161]

[0162] 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 bio-nanoporin mutants in sequencing or detection of one or more of nucleic acids, oligopeptides, oligosaccharides, and macromolecular drugs, characterized by: The amino acid sequence of wild-type PANX1 nanoporin is shown in SEQ ID NO:1, and the biomolecular nanoporin mutant is selected from any one of the following A, B and C: A: The amino acid P23 of wild-type PANX1 nanoporin is mutated to P23N; B: The C region of wild-type PANX1 nanoporous protein is truncated, specifically by shortening it by 10 amino acids from G371 to the N-terminus; C: The amino acid F72 of wild-type PANX1 nanoporin is mutated to F72I, and the C region is truncated, specifically by 10 amino acids from G371 to the N-terminus.

2. The application as described in claim 1, characterized in that: The nucleic acid includes single-stranded DNA.

3. The application as described in claim 1, characterized in that: The method for preparing the bio-nanoporin mutant includes the following steps: Step S1: Construct a biomolecular nanoporous protein mutant vector; Step S2: Expression and purification of bio-nanoporin mutants.

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