Nanopore protein as well as mutant and application thereof

Abstract

The invention relates to the field of nanopore sequencing, in particular to a novel nanopore protein BCP52 as well as a mutant and application thereof. The novel nanopore protein BCP52 is found from a deep sea metagenome, protein preparation and nanopore sequencing verification show that the nanopore protein BCP52 has the capability of being applied to nanopore sequencing, and optimization of a mutant of the nanopore protein BCP52 can improve the accuracy of nanopore sequencing.

Description

Nanopore protein and its mutants and applications Technical Field

[0001] The present invention relates to the field of nanopore sequencing, and in particular to nanopore proteins and mutants thereof and applications thereof. Background Art

[0002] Nanopore sequencing, a newly emerging third-generation sequencing technology, has revolutionized the gene sequencing industry thanks to its advantages, including long read lengths, high throughput, low cost, and portability. Nanopore sequencing technology has extensive applications in fundamental life science research and biomedical clinical practice.

[0003] Nanopore sequencing is an electrical signal-based sequencing technology, primarily developed by Oxford Nanopore Technologies (ONT) in the UK. This technology can be applied to DNA, RNA, and protein sequencing. It records the electrical signals generated by the continuous blockade of the nanopore protein as the analyte passes through it in real time, and then converts them into sequence information for sequencing. This technology offers advantages in sequencing speed, throughput, portability, and direct RNA sequencing, and has garnered widespread attention in recent years.

[0004] Currently, Oxford Nanopore Technologies in the UK has commercialized a series of nanopore sequencers, including the MinION, GridION, and PromethION, as well as the QNome-3841 nanopore gene sequencer. However, these instruments still have significant shortcomings in sequencing accuracy, throughput, and chip stability, failing to meet the ultimate requirements of molecular biology research. Therefore, there is an urgent need to develop a highly accurate, highly integrated, and highly stable single-molecule sequencer. A nanopore single-molecule sequencer is a highly integrated detection system that integrates multiple disciplines and technologies. Its development requires deep cross-disciplinary collaboration and innovation across multiple disciplines, including physics, biology, chemistry, semiconductors, and computer science, to build a high-precision nanopore single-molecule sequencing system from the ground up.

[0005] Nanopore sequencing requires that the sensing region within the pore protein be sufficiently sharp to achieve high spatial resolution in both the horizontal and vertical directions. Among the pore proteins being studied, three main categories have the potential for sequencing: pore-forming toxins produced by bacteria or other organisms that disrupt cell membrane permeability; transporter proteins that serve as channels for the transport of various biomacromolecules and small molecules inside and outside the cell; and viral connectors that provide genome transport channels for viruses when they infect their hosts. Currently, only a few naturally occurring proteins, such as Mycobacterium smegmatis porin A (MspA) and the curli-specific transporter (CsgG), meet these requirements. Identifying more excellent sequencing nanopore proteins through gene discovery remains an unresolved issue.

[0006] Natural nanopore proteins generally possess the ability to form pores, but in in vitro recombinant protein expression and purification systems, the pore stability of recombinant nanopore proteins may not meet the requirements of single-molecule detection instrumentation. Furthermore, the wide range of pore size distribution of natural nanopore proteins may not meet the requirements of single-molecule detection, resulting in insufficient sequencing accuracy. The properties of the amino acid residues within the pore walls of natural nanopore proteins, particularly their charge, may not necessarily meet the requirements of specific analytes.

[0007] Summary of the Invention

[0008] In view of this, the present invention provides nanopore proteins and mutants and applications thereof.

[0009] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0010] The present invention provides a nanopore protein having:

[0011] (I), the amino acid sequence shown in SEQ ID NO.1; or

[0012] (II) a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence as shown in (I); or

[0013] (III) An amino acid sequence having a homology of 70% or more to the amino acid sequence shown in (I) or (II).

[0014] In some specific embodiments of the present invention, amino acid sequences having 70%, 75%, 80%, 85%, 90% or more homology to the amino acid sequence shown in (I) or (II) are also provided.

[0015] The present invention also provides a mutant of the nanopore protein, comprising:

[0016] (I), mutations in the sensor region; and / or

[0017] (II), mutations in the transmembrane region; and / or

[0018] (III), mutations in the entry region; and / or

[0019] (IV) Mutations in the export zone.

[0020] In some specific embodiments of the present invention, the mutation in the sensor region includes mutation in any one or more of S75, G79 and / or F80; and / or

[0021] The transmembrane region mutation includes any one or more mutations in E166, R200, T204 or S220; and / or

[0022] The mutation of the entry region includes any one or more mutations of R107, E108, E116, R117, K118, R121, K124, D125, K127 or E131;

[0023] In some specific embodiments of the present invention, the mutations of S75, G79 or F80 in the sensor region include but are not limited to A, G, S, T, N or Q; and / or

[0024] The mutations of E166, R200, T204 or S220 in the transmembrane region include but are not limited to A, G, V, L, I, F, Y or W; and / or

[0025] Mutations of R107, E108, E116, R117, K118, R121, K124, D125, K127 or E131 in the entry region include, but are not limited to, K, R, N, A, G, S, T or Q.

[0026] In some specific embodiments of the present invention,

[0027] (I) Mutation of the sensor region:

[0028] The mutation of S75 includes but is not limited to G, A or T; and / or

[0029] The mutation of G79 includes but is not limited to A, S, T, N or Q; and / or

[0030] The mutation of F80 includes but is not limited to G, A, S, T, N or Q; and / or

[0031] (II) Mutation of the transmembrane region:

[0032] The mutation of E166 includes but is not limited to A, G, V, L, I, Y, F or W; and / or

[0033] The mutation of R200 includes but is not limited to A, G, V, L, I, Y, F or W; and / or

[0034] The mutation of T204 includes but is not limited to A, G, V, L, I, Y, F or W; and / or

[0035] The mutation of S220 includes but is not limited to A, G, V, L, I, Y, F or W; and / or

[0036] (III) Mutation of the entry region:

[0037] The mutation of R107 includes but is not limited to N, A, G, S, T or Q; and / or

[0038] The mutation of E108 includes but is not limited to K, R, N, A, G, S, T or Q; and / or

[0039] The mutation of E116 includes but is not limited to K, R, N, A, G, S, T or Q; and / or

[0040] The mutation of R117 includes but is not limited to N, A, G, S or T; and / or

[0041] The mutation of K118 includes but is not limited to N, A, G, S or Q; and / or

[0042] The mutation of R121 includes but is not limited to N, A, G, S, T or Q; and / or

[0043] The mutation of K124 includes but is not limited to N, A, G, S, T or Q; and / or

[0044] The mutation of D125 includes but is not limited to K, R, N, A, G, S, T or Q; and / or

[0045] The mutation of K127 includes but is not limited to N, A, G, S, T or Q; and / or

[0046] The mutation of E131 includes but is not limited to K, R, N, A, G, S, T or Q.

[0047] In some specific embodiments of the present invention, the mutation in the sensor region includes F80N in the sensor region.

[0048] In some specific embodiments of the present invention, the mutant has:

[0049] (I), the amino acid sequence shown in SEQ ID NO. 2; or

[0050] (II) a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence as shown in (I); or

[0051] (III) An amino acid sequence having a homology of 70% or more to the amino acid sequence shown in (I) or (II).

[0052] In some specific embodiments of the present invention, amino acid sequences having 70%, 75%, 80%, 85%, 90% or more homology to the amino acid sequence shown in (I) or (II) are also provided.

[0053] The present invention also provides a nucleic acid molecule encoding the nanopore protein or the mutant.

[0054] In some embodiments of the present invention, the nucleic acid molecule encoding the nanopore protein has:

[0055] (I), the nucleotide sequence shown in SEQ ID NO: 8; or

[0056] (II) a nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I) but differs from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

[0057] (III) a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and having the same or similar functions as the nucleotide sequence shown in (I) or (II); or

[0058] (IV) A nucleotide sequence having a nucleotide sequence homology of 70% or more with the nucleotide sequence of (I), (II) or (III).

[0059] In some specific embodiments of the present invention, nucleotide sequences having 70%, 75%, 80%, 85%, 90% or more homology to the amino acid sequence as shown in (I), (II) or (III) are also provided.

[0060] In some embodiments of the present invention, the nucleic acid molecule encoding the nanopore protein mutant has:

[0061] (I), the nucleotide sequence shown in SEQ ID NO: 9; or

[0062] (II) a nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I) but differs from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

[0063] (III) a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and having the same or similar functions as the nucleotide sequence shown in (I) or (II); or

[0064] (IV) A nucleotide sequence having a sequence homology of 70% or more with the nucleotide sequence of (I), (II) or (III).

[0065] In some specific embodiments of the present invention, nucleotide sequences having 70%, 75%, 80%, 85%, 90% or more homology to the amino acid sequence as shown in (I), (II) or (III) are also provided.

[0066] The present invention also provides an expression vector, which includes the nucleic acid molecule and a backbone vector.

[0067] The present invention also provides a host comprising the recombinant vector.

[0068] The present invention also provides a construct, which consists of 7 to 11 covalently linked or non-covalently polymerized nanopore proteins.

[0069] In some embodiments of the present invention, the construct consists of 9 covalently linked or non-covalently aggregated nanopore proteins.

[0070] In some specific embodiments of the present invention, the porin BCP52 is the nanoporin nonamer.

[0071] The present invention also provides a method for preparing the nanopore protein or the nanopore protein mutant, which comprises the following steps:

[0072] (1) constructing an expression vector using the nucleic acid molecule;

[0073] (II), transforming the expression vector into a host for expression to obtain an expression product;

[0074] (III) extracting and purifying the expression product, and heating it at 90-98° C. to obtain the nanopore protein or the nanopore protein mutant.

[0075] The present invention also provides a method for preparing the construct, which comprises the following steps:

[0076] (1) constructing an expression vector using the nucleic acid molecule;

[0077] (II), transforming the expression vector into a host for expression to obtain an expression product;

[0078] (III) extracting and purifying the expression product to obtain the construct.

[0079] The present invention also provides a biosensor comprising any of the following items and acceptable auxiliary agents or components:

[0080] (I), the nanopore protein; and / or

[0081] (II), the nanopore protein mutant; and / or

[0082] (III), the construct; and / or

[0083] (IV), the nanopore protein or nanopore protein mutant prepared by the preparation method; and / or

[0084] (V) The construct prepared by the preparation method.

[0085] The present invention also provides a kit comprising any of the following items and an acceptable adjuvant or carrier:

[0086] (I), the nanopore protein; and / or

[0087] (II), the nanopore protein mutant; and / or

[0088] (III), the construct; and / or

[0089] (IV), the nanopore protein or nanopore protein mutant prepared by the preparation method; and / or

[0090] (V), a construct obtained by the preparation method; and / or

[0091] (VI), the biosensor. The present invention also provides the use of any of the following in single-molecule sequencing:

[0092] (I), the nanopore protein BCP52; and / or

[0093] (II), the nanopore protein BCP52 mutant; and / or

[0094] (III), the nucleic acid molecule; and / or

[0095] (IV), the expression vector; and / or

[0096] (V), the host; and / or

[0097] (VI), the construct; and / or

[0098] (VII), the nanopore protein or nanopore protein mutant prepared by the preparation method; and / or

[0099] (VIII), the construct prepared by the preparation method; and / or

[0100] (IX), the biosensor; and / or

[0101] (X), the kit.

[0102] In some embodiments of the invention, the single molecule comprises protein, DNA and / or RNA.

[0103] The present invention also provides a single molecule sequencing method, which comprises the following steps:

[0104] (1) constructing a sequencing library;

[0105] (II) Insert any of the following into the phospholipid bilayer:

[0106] A: The nanopore protein according to claim 1; and / or

[0107] B: The nanopore protein mutant according to any one of claims 2 to 7; and / or

[0108] C: The construct according to claim 13 or 14; and / or

[0109] D: a nanopore protein or a nanopore protein mutant prepared by the preparation method according to claim 15; and / or

[0110] E: a construct prepared by the preparation method according to claim 16;

[0111] (III) applying an external voltage, recording the current value, and obtaining the singleton sequence information based on the current value.

[0112] In some embodiments of the present invention, constructing a sequencing library specifically includes annealing two partially complementary DNA strands (top strand and bottom strand) to form an adapter, ligating the double-stranded target fragment to be tested using T4 DNA ligase at room temperature, and purifying the resulting sequencing library. The sequencing library is then incubated with the helicase BCH105 at 25°C for 1 hour (at a molar ratio of 1:8). After cross-linking and purification, a sequencing library containing the BCH105 motor protein is formed.

[0113] In some specific embodiments of the present invention, the phospholipid bilayer is composed of diacylphosphatidylcholine (DPhPC, 1,2-diphytanoyl-sn-glycero-3-phosphocholine).

[0114] The present invention also provides a sequencing device, comprising the biosensor and an acceptable auxiliary agent or carrier.

[0115] The beneficial effects achieved by the present invention include but are not limited to:

[0116] The present invention discovered a novel nanopore protein BCP52 from the deep-sea metagenome. Protein preparation and nanopore sequencing verification showed that it has the ability to be applied to nanopore sequencing, and the optimization of its mutants can improve the accuracy of nanopore sequencing. BRIEF DESCRIPTION OF THE DRAWINGS

[0117] Figure 1 shows the three-dimensional structure of BCP52 predicted by Alphafold2 multimer (side view);

[0118] Figure 2 shows the three-dimensional structure of BCP52 predicted by Alphafold2 multimer (top view);

[0119] FIG3 shows a schematic diagram of the predicted structure of the alphafold2 multimer of key amino acids in the sensor region of the nanopore protein;

[0120] FIG4 is a schematic diagram showing the amino acid residues in the sensor region of the predicted structure of the nanopore protein;

[0121] FIG5 is a schematic diagram showing the charged and polar amino acids facing the membrane in the transmembrane region of the predicted structure of the nanopore protein;

[0122] Figure 6 shows some key amino acids in the entrance region of the predicted structure of BCP52;

[0123] Figure 7 shows the proteins obtained by nanopore protein purification; among them, E1 and E2 are undenatured samples after elution, and D1 and D2 are samples after denaturation at 100°C;

[0124] Figure 8 shows a schematic diagram of the sequencing library structure; wherein, a: top strand; b: bottom strand; c: double-stranded target fragment; d: helicase BCH105;

[0125] FIG9 shows the pore opening current of wild-type nanopore protein at different voltages in a phospholipid bilayer;

[0126] FIG10 shows the pore opening current of the nanopore mutant 1 protein at different voltages in a phospholipid bilayer;

[0127] FIG11 shows the current trace of the DNA to be tested passing through the nanopore protein mutant 1 protein;

[0128] FIG12 shows local details of the DNA sequencing trace of nanopore protein mutant 1. DETAILED DESCRIPTION

[0129] The present invention discloses nanopore proteins, their mutants, and applications. Those skilled in the art can draw upon the disclosure herein to appropriately modify process parameters. It is particularly important to note that all similar substitutions and modifications readily apparent to those skilled in the art are considered encompassed by the present invention. The methods and applications of the present invention have been described through preferred embodiments. It is apparent that those skilled in the art can modify, adapt, and combine the methods and applications described herein to implement and apply the technology of the present invention without departing from the content, spirit, and scope of the present invention.

[0130] This study, using gene mining and computer-assisted structure prediction, identified a nanopore protein from deep-sea metagenomes. This protein can be used as a detector protein in nanopore sequencing, including the detection of small molecules, DNA, RNA, and peptides. Protein preparation and nanopore sequencing validation demonstrated that the nanopore protein is capable of nanopore sequencing applications, and that optimizing its mutants can improve the accuracy of nanopore sequencing.

[0131] Table 1 Sequence information

[0132]

[0133]

[0134]

[0135]

[0136]

[0137] The sequence information involved in the present invention is shown in Table 1:

[0138] The novel nanopore protein and its mutants provided by the present invention, as well as the raw materials and reagents used in their applications, can all be purchased from the market.

[0139] The present invention will be further described below in conjunction with the embodiments:

[0140] Example 1 Predicted structure of the Alphafold2 multimer of wild-type nanopore protein

[0141] The alphafold2 multimer was used to predict the structure of the nonamer (porin BCP52) of the nanopore protein (SEQ ID No. 1). The prediction results are shown in Figures 1 and 2. Figure 1 shows the side view of the predicted nanopore protein structure, and Figure 2 shows the top view of the predicted nanopore protein structure.

[0142] FIG3 and FIG4 are side chain structures of important amino acids in the sensor region of the predicted structure of the nanopore protein, showing that the three amino acids in the amino acid side chains are S75, G79, and F80.

[0143] FIG5 shows four charged and polar amino acids facing the membrane in the transmembrane region of the nanopore protein, namely K166, R200, T204, and S220.

[0144] Figure 6 shows some important amino acids in the entrance region of the nanopore protein, especially the charged amino acids, which are R107, E108, E116, R117, K118, R121, K124, D125, K127, and E131.

[0145] Example 2 Construction of expression vectors for nanopore proteins and their mutants

[0146] Using an in-fusion method, the DNA sequence encoding the nanopore protein was digested with NdeI and XhoI and then inserted into the multiple cloning region of the pET24a vector. StrepII amino acids were added to the C-termini of the wild-type nanopore protein amino acid sequence (SEQ ID No. 1) and nanopore protein mutant 1 (F80N SEQ ID No. 2) as purification tags, with kanamycin as the screening tag. The constructed vectors were named pET24a-BCP52-wt and pET24a-BCP52-F80N. Using an Agilent site-directed mutagenesis kit and the nanopore protein expression vector as a template, the corresponding nanopore protein mutant expression vectors were constructed.

[0147] Example 3 Cultivation and induction of nanopore protein and its mutant strains

[0148] Transform the constructed nanopore protein or its mutant expression plasmid into the E. coli expression strain E. coli BL21 (DE3), spread the bacterial solution evenly on a plate containing 50 μg / mL kanamycin, and culture at 37°C overnight. The next day, pick a single colony and place it in 5 mL LB medium containing 50 μg / mL kanamycin, and culture it at 37°C, 200 rpm, overnight. Inoculate the above bacterial solution at a ratio of 1:100 into 50 mL LB containing 50 μg / mL kanamycin, and culture it at 37°C, 200 rpm for 4 hours. Inoculate the expanded cultured bacterial solution at a ratio of 1:100 into 2 L LB containing 50 μg / mL kanamycin and culture it at 37°C, 200 rpm. Wait for OD 600When the value reaches about 0.6-0.8, add IPTG to a final concentration of 0.5 mmol / L and culture at 16°C, 200 rpm for about 16-18 hours. Collect the bacterial solution by centrifugation at 8000 rpm and freeze at -20°C until use.

[0149] Example 4 Extraction and purification of recombinant nanopore protein and its mutant proteins

[0150] Purification Buffer preparation:

[0151] Buffer A: 20mmol / L Tris-HCl, 250mmol / L NaCl, 1% DDM, pH 8.0.

[0152] Buffer B: 20mmol / L Tris-HCl, 250mmol / L NaCl, 0.05% DDM, pH 8.0.

[0153] Buffer C: 20 mmol / L Tris-HCl, 250 mmol / L NaCl, 0.05% DDM, 5 mmol / L desthiobiotin, pH 8.0.

[0154] Purification steps:

[0155] Resuspend 1 g of cells in 10 mL of Buffer A. Disrupt the cells by sonication until the solution is clear. Rotate overnight at 4°C. The next day, centrifuge at 18,000 rpm for 1 hour at 4°C. Remove the supernatant, filter through a 0.22 μm filter, and store at 4°C until ready for use.

[0156] A Strep-Tactin beads (IBA Lifesciences) column was equilibrated with Buffer A for 5 column volumes (CV) using an AKTA pure chromatography instrument and loaded with sample at 2 mL / min. After loading, the column was washed with Buffer B for 20 CV and eluted with Buffer C to collect the target protein.

[0157] The protein obtained by Strep column affinity was concentrated to 1 mL, passed through a Superdex 6 increase 10 / 300GL (Cytiva) column and a SEC6 30 / 300 column equilibrated with buffer B, and the target protein was collected and subsequently stored at -80°C.

[0158] Figure 7 shows an SDS-PAGE image of the purified nanopore protein. E1 and E2 are samples that were not denatured after elution, while D1 and D2 are samples that were boiled at 95°C. The results show that the target protein is in an aggregated state before boiling and in a monomeric state after boiling.

[0159] Example 5 Library Construction

[0160] Two partially complementary DNA strands (top strand and bottom strand (SEQ ID No. 3, 4)) were annealed to form a linker, which was then ligated with the double-stranded target fragment (SEQ ID No. 7) using T4 DNA ligase at room temperature and purified to prepare a sequencing library. This sequencing library was then incubated with the helicase BCH105 (SEQ ID No. 6) at 25°C for 1 hour (molar concentration ratio of 1:8). After cross-linking and purification, a sequencing library containing the BCH105 motor protein was formed (as shown in Figure 8).

[0161] Example 6 Construction of Nanopore Biosensors Using Nanopore Proteins and Their Mutants

[0162] Single-channel nanopore current measurement is based on an amplifier in a digital device. Ag / AgCl electrodes are immersed in sequencing buffer and located in the cis and trans regions of the electrolytic cell, respectively. Reagents such as the sequencing library and nanopore are added to the cis region. After the nanopore protein is diluted 100-fold with 1× PBS buffer, a single nanopore is inserted into a phospholipid bilayer composed of diacylphosphatidylcholine (DPhPC) under an applied electric field of 0.05 V to form a nanopore biosensor. An applied voltage is applied to obtain the current amplitude of a single nanopore protein. Figure 9 shows the nanopore biosensor current of the nanopore protein when voltages of 0.02 V, 0.04 V, 0.10 V, 0.14 V, and 0.18 V are applied.

[0163] In this example, the pore opening currents of the wild-type nanopore protein (SEQ ID No. 1) and the nanopore protein mutant 1 (SEQ ID No. 2) after being inserted into a phospholipid membrane and applying different voltages were mainly tested.

[0164] In this embodiment, the nanopore protein mutant 1 is a mutant of the wild-type nanopore protein containing the mutation site F80N.

[0165] The experimental results are shown in Figures 9 and 10. It can be seen that the wild-type nanopore protein has a large pore opening noise, while the nanopore protein mutant 1 protein has a very small pore opening current noise, indicating that the nanopore protein mutant 1 in the sensor region can effectively reduce the pore opening current noise.

[0166] Example 7 Using Nanopore Protein and Its Mutants for DNA Sequencing

[0167] One microgram of the sequencing library obtained in Example 5 and a cholesterol-laden single-stranded DNA (SEQ ID No. 5) at a concentration five times that of the library were mixed with sequencing buffer (0.47M KCl, 25mM HEPES, 1mM EDTA, 5mM ATP, 25mM MgCl2, pH 7.6) and added to a nanopore biosensor. After applying an external voltage of 0.14V or 0.18V, DNA was observed to be captured by the nanopore, generating a characteristic retarded current amplitude. Furthermore, the current amplitude varied as the DNA moved through the nanopore. Different DNA sequences produced different retarded current amplitudes. The cholesterol-laden single-stranded DNA can bind to the phospholipid bilayer, facilitating nanopore capture of the sequencing library and reducing the amount of sequencing library loaded.

[0168] The wild-type nanopore protein (SEQ ID No. 1) cannot be used for DNA sequencing due to excessive pore current noise. The sequencing current changes of the nanopore protein mutant 1 (F80N, SEQ ID No. 2) were used for DNA sequencing.

[0169] Figure 11 shows the current trace of library DNA passing through nanopore mutant 1 (F80N, SEQ ID No. 2) at an applied voltage of 0.18 V. This indicates that nanopore mutant 1 can be used for DNA sequencing. The current trace oscillates with DNA penetration, with a sequencing amplitude of approximately 50 pA.

[0170] Figure 12 is a partial detail of the DNA sequencing trace of nanopore protein mutant 1. The novel nanopore protein BCP52, its mutants, and applications provided by the present invention are described in detail above. This article uses specific examples to illustrate the principles and implementation methods of the present invention. The description of the above examples is only intended to help understand the methods and core concepts of the present invention. It should be noted that for those skilled in the art, without departing from the principles of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

[0171]

[0172]

[0173]

[0174]

[0175]

Claims

1. A nanopore protein, characterized in that It has: (I), the amino acid sequence shown in SEQ ID NO.1; or (II) a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence as shown in (I); or (III) An amino acid sequence having a homology of 70% or more to the amino acid sequence shown in (I) or (II).

2. The mutant of the nanopore protein according to claim 1, characterized in that include: (I), mutations in the sensor region; and / or (II), mutations in the transmembrane region; and / or (III), mutations in the entry region; and / or (IV) Mutations in the export zone.

3. The mutant according to claim 2, characterized in that The mutation in the sensor region includes any one or more mutations in S75, G79 and / or F80; and / or The transmembrane region mutation includes any one or more mutations in E166, R200, T204 or S220; and / or The mutation in the entry region includes any one or more mutations in R107, E108, E116, R117, K118, R121, K124, D125, K127 or E131.

4. The mutant according to claim 3, characterized in that The mutation of S75, G79 or F80 in the sensor region includes but is not limited to A, G, S, T, N or Q; and / or The mutations of E166, R200, T204 or S220 in the transmembrane region include but are not limited to A, G, V, L, I, F, Y or W; and / or Mutations of R107, E108, E116, R117, K118, R121, K124, D125, K127 or E131 in the entry region include but are not limited to K, R, N, A, G, S, T or Q.

5. The mutant according to any one of claims 2 to 4, characterized in that (I) Mutation of the sensor region: The mutation of S75 includes but is not limited to G, A or T; and / or The mutation of G79 includes but is not limited to A, S, T, N or Q; and / or The mutation of F80 includes but is not limited to G, A, S, T, N or Q; and / or (II), mutation of the transmembrane region: The mutation of E166 includes but is not limited to A, G, V, L, I, Y, F or W; and / or The mutation of R200 includes but is not limited to A, G, V, L, I, Y, F or W; and / or The mutation of T204 includes but is not limited to A, G, V, L, I, Y, F or W; and / or The mutation of S220 includes but is not limited to A, G, V, L, I, Y, F or W; and / or (III) Mutation of the entry zone: The mutation of R107 includes but is not limited to N, A, G, S, T or Q; and / or The mutation of E108 includes but is not limited to K, R, N, A, G, S, T or Q; and / or The mutation of E116 includes but is not limited to K, R, N, A, G, S, T or Q; and / or The mutation of R117 includes but is not limited to N, A, G, S or T; and / or The mutation of K118 includes but is not limited to N, A, G, S or Q; and / or The mutation of R121 includes but is not limited to N, A, G, S, T or Q; and / or The mutation of K124 includes but is not limited to N, A, G, S, T or Q; and / or The mutation of D125 includes but is not limited to K, R, N, A, G, S, T or Q; and / or The mutation of K127 includes but is not limited to N, A, G, S, T or Q; and / or The mutation of E131 includes but is not limited to K, R, N, A, G, S, T or Q.

6. The mutant according to any one of claims 2 to 5, characterized in that The mutation in the sensor region includes F80N in the sensor region.

7. The mutant according to any one of claims 2 to 6, characterized in that It has: (I), the amino acid sequence shown in SEQ ID NO.2; or (II) a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence as shown in (I); or (III) An amino acid sequence having a homology of 70% or more to the amino acid sequence shown in (I) or (II).

8. A nucleic acid molecule encoding the nanopore protein according to claim 1 or the mutant according to any one of claims 2 to 7.

9. A nucleic acid molecule encoding the nanopore protein according to claim 1, characterized in that: have: (I), the nucleotide sequence shown in SEQ ID NO: 8; or (II) a nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I) but is different from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or (III) a nucleotide sequence obtained by replacing, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and having the same or similar function as the nucleotide sequence shown in (I) or (II); or (IV) A nucleotide sequence having a nucleotide sequence homology of 70% or more with the nucleotide sequence described in (I), (II) or (III).

10. A nucleic acid molecule encoding the nanopore protein mutant according to any one of claims 2 to 7, characterized in that: have: (I), the nucleotide sequence shown in SEQ ID NO:9; or (II) a nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I) but is different from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or (III) a nucleotide sequence obtained by replacing, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and having the same or similar function as the nucleotide sequence shown in (I) or (II); or (IV) A nucleotide sequence having a sequence homology of 70% or more with the nucleotide sequence described in (I), (II) or (III).

11. An expression vector, characterized in that It comprises the nucleic acid molecule and a backbone vector as described in any one of claims 8 to 10.

12. A host, characterized in that Comprising the recombinant vector as described in claim 11.

13. A construct, characterized in that The construct consists of 7 to 11 covalently linked or non-covalently polymerized nanopore proteins according to any one of claims 1 to 7.

14. The construct according to claim 13, wherein The construct consists of 9 covalently linked or non-covalently polymerized nanopore proteins according to any one of claims 1 to 7.

15. The method for preparing the nanopore protein according to claim 1 or the nanopore protein mutant according to any one of claims 2 to 7, characterized in that: The steps include: (I) constructing an expression vector using the nucleic acid molecule according to any one of claims 8 to 10; (II), taking the expression vector and transforming it into a host for expression to obtain an expression product; (III) extracting and purifying the expression product, and heating at 90-98° C. to obtain the nanopore protein or the nanopore protein mutant.

16. The method for preparing the construct according to claim 13 or 14, characterized in that: The steps include: (I) constructing an expression vector using the nucleic acid molecule according to any one of claims 8 to 10; (II), taking the expression vector and transforming it into a host for expression to obtain an expression product; (III) extracting and purifying the expression product to obtain the construct.

17. A biosensor, characterized in that Includes any of the following and acceptable additives or components: (I), the nanopore protein as claimed in claim 1; and / or (II), the nanopore protein mutant according to any one of claims 2 to 7; and / or (III), the construct according to claim 13 or 14; and / or (IV) A nanopore protein or a nanopore protein mutant prepared by the preparation method according to claim 15; and / or (V) A construct obtained by the preparation method according to claim 16.

18. A kit, characterized in that Includes any of the following items and acceptable adjuvants or carriers: (I), the nanopore protein as claimed in claim 1; and / or (II), the nanopore protein mutant according to any one of claims 2 to 7; and / or (III), the construct according to claim 13 or 14; and / or (IV) A nanopore protein or a nanopore protein mutant prepared by the preparation method according to claim 15; and / or (V) a construct obtained by the preparation method according to claim 16; and / or (VI) The biosensor according to claim 17.

19. Application of any of the following in single-molecule sequencing: (I), the nanopore protein as claimed in claim 1; and / or (II), the nanopore protein mutant according to any one of claims 2 to 7; and / or (III), a nucleic acid molecule according to any one of claims 8 to 10; and / or (IV), the expression vector according to claim 11; and / or (V), the host according to claim 12; and / or (VI), the construct of claim 13 or 14; and / or (VII) a nanopore protein or a nanopore protein mutant prepared by the preparation method according to claim 15; and / or (VIII) a construct obtained by the preparation method according to claim 16; and / or (IX), the biosensor of claim 17; and / or (X) A kit as described in claim 18.

20. A single molecule sequencing method, characterized in that: The following steps are involved: (I), constructing a sequencing library; (II) Insert any of the following into the phospholipid bilayer; A: The nanopore protein according to claim 1; and / or B: The nanopore protein mutant according to any one of claims 2 to 7; and / or C: The construct according to claim 13 or 14; and / or D: a nanopore protein or a nanopore protein mutant prepared by the preparation method according to claim 15; and / or E: A construct obtained by the preparation method as described in claim 16; (III), applying an external voltage, recording the current value, and obtaining the singleton sequence information based on the current value.

21. A sequencing device, characterized in that The biosensor comprises the biosensor as claimed in claim 17 and an acceptable auxiliary agent or carrier.

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