WO2024138565A1

WO2024138565A1 - Nanopore protein, and mutant and use thereof

Info

Publication number: WO2024138565A1
Application number: PCT/CN2022/143608
Authority: WO
Inventors: 王乐乐; 刘欢欢; 姜楠; 王子; 陈俊毅; 季州翔; 郭斐; 曾涛; 黎宇翔; 董宇亮; 章文蔚; 徐讯
Original assignee: 深圳华大生命科学研究院
Priority date: 2022-12-29
Filing date: 2022-12-29
Publication date: 2024-07-04

Abstract

The present invention relates to the field of nanopore sequencing, and in particular to a novel nanopore protein BCP52, and a mutant and use thereof. According to the present invention, a novel nanopore protein BCP52 is found in 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 the mutant of the nanopore protein BCP52 can improve the accuracy of nanopore sequencing.

Description

Nanopore protein and its mutants and applications

Technical Field

The present invention relates to the field of nanopore sequencing, and in particular to nanopore proteins and mutants thereof and applications thereof.

Background technique

Nanopore sequencing is a third-generation sequencing technology that has emerged in recent years. It has brought disruptive changes to the gene sequencing industry due to its advantages such as long read length, high throughput, low cost and portability. Nanopore sequencing technology has a wide range of applications in basic theoretical research in life sciences and clinical practice in biomedicine.

Nanopore sequencing technology is a sequencing technology based on electrical signals, with Oxford Nanopore Technologies (ONT) as the main representative. This technology can be applied to DNA, RNA and protein sequencing at the same time. It records the electrical signals generated by the continuous blockage when the analytes pass through the nanopore protein one by one in real time, and converts them into sequence information through analysis to achieve sequencing. This technology has high advantages in sequencing speed, throughput, portability, and direct RNA sequencing, and has received widespread attention in recent years.

At present, Oxford Nanopore Technologies in the UK has developed a series of nanopore sequencers such as MinION, GridION and PromethION, as well as commercial instruments such as the QNome-3841 nanopore gene sequencer. However, there are still major deficiencies in terms of sequencing accuracy, throughput and chip stability, which cannot meet the ultimate needs of molecular biology research. Therefore, it is urgent to develop a single-molecule sequencer with high accuracy, high integration and high stability. The nanopore single-molecule sequencer is a detection system that is highly integrated with multiple disciplines and technologies. The development of this instrument requires deep cross-disciplinary and collaborative innovation in physics, biology, chemistry, semiconductors, computers and other disciplines, and builds a high-precision nanopore single-molecule sequencing system from the underlying core modules.

Nanopore sequencing requires that the sensing region inside the pore protein is sharp enough to have high spatial resolution in both the horizontal and vertical directions. Among the pore proteins studied, there are three main types that can be potentially used for sequencing: pore-forming toxin proteins produced by bacteria or other organisms that destroy the permeability of cell membranes, transporter proteins that serve as transport channels for various biological macromolecules and small molecules inside and outside cells, and viral connectors that provide genome transport channels when viruses infect hosts. At present, only a few natural proteins such as Mycobacterium smegmatis pore protein A (MspA) and curli-specific transport channel (CsgG) meet the requirements. Finding more excellent sequencing nanopore proteins through gene mining methods is still an unresolved problem.

Natural nanopore proteins generally have the ability to form pores, but in the in vitro expression and purification system of recombinant proteins, the pore stability of recombinant nanopore proteins may not meet the needs of single-molecule detector-related instrument products. At the same time, the pore size distribution range of natural nanopore proteins is relatively wide, which may not meet the needs of single-molecule detection, resulting in insufficient sequencing accuracy. The properties of the amino acid residues on the inner wall of the natural nanopore protein pore, especially the charged properties, may not meet the properties of the specific analyte.

Summary of the invention

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

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

The present invention provides a nanopore protein having:

(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).

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.

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

(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.

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

The mutation of the transmembrane region includes any one or more mutations in E166, R200, T204 or S220; and/or

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

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

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.

In some specific embodiments of the present invention,

(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.

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

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

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

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

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

(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 functions 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).

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

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

(I), the nucleotide sequence shown in SEQ ID NO:9; 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).

The present invention also provides an expression vector, which comprises the nucleic acid molecule and a backbone vector.

The present invention also provides a host, which comprises the recombinant vector.

The present invention also provides a construct, which is composed of 7 to 11 covalently linked or non-covalently polymerized nanopore proteins.

In some specific embodiments of the present invention, the construct consists of 9 covalently linked or non-covalently polymerized nanopore proteins.

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

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

(I) constructing an expression vector using the nucleic acid molecule;

(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.

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

(I) constructing an expression vector using the nucleic acid molecule;

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

The present invention also provides a biosensor, which includes any of the following items and acceptable auxiliary agents or components:

(I), the nanopore protein; and/or

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

(III), the construct; and/or

(IV), the nanopore protein or nanopore protein mutant obtained by the preparation method; and/or

(V) A construct obtained by the preparation method.

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

(I), the nanopore protein; and/or

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

(III), the construct; and/or

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

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

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

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

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

(IV), the expression vector; and/or

(V), the host; and/or

(VI), the construct; and/or

(VII), the nanopore protein or nanopore protein mutant obtained by the preparation method; and/or

(VIII), a construct obtained by the preparation method; and/or

(IX), the biosensor; and/or

(X), the kit.

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

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

(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 prepared by the preparation method according to claim 16;

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

In some specific embodiments of the present invention, the construction of the sequencing library specifically includes: annealing two partially complementary DNA strands (top strand and bottom strand) to form a linker, connecting the double-stranded target fragment to be tested with T4 DNA ligase at room temperature and purifying to prepare a sequencing library. The sequencing library is then incubated with the helicase BCH105 at 25°C for 1 hour (molar concentration ratio 1:8), and after cross-linking and purification, a sequencing library containing the BCH105 motor protein is formed.

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

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

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

The present invention found a new type of nanopore protein BCP52 from the deep-sea metagenome. Through protein preparation and nanopore sequencing verification, it was shown 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

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

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

Figure 3 shows a schematic diagram of the predicted structure of the alphafold2 multimer of key amino acids in the sensor region of the nanopore protein;

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

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

Figure 6 shows some key amino acids in the entry region of the predicted structure of BCP52;

FIG7 shows the protein obtained by nanopore protein purification; wherein, E1 and E2 are samples without denaturation after elution, and D1 and D2 are samples after denaturation at 100° C.;

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;

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

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

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

FIG. 12 shows local details of the DNA sequencing trace of nanopore protein mutant 1.

Detailed ways

The present invention discloses nanopore proteins and their mutants and applications. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

The present invention uses a gene mining method of computer-aided structure prediction to mine a nanopore protein from deep-sea metagenomes, so that it can be used as a detection protein and applied to nanopore sequencing, including the detection of small molecules, DNA, RNA and polypeptides. The present invention is verified by protein preparation and nanopore sequencing, indicating that nanopore protein has the ability to be applied to nanopore sequencing, and the optimization of its mutants can improve the accuracy of nanopore sequencing.

Table 1 Sequence information

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

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

The present invention will be further described below in conjunction with embodiments:

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

The structure of the nonamer (porin BCP52) of nanopore protein (SEQ ID No. 1) was predicted using alphafold2 multimer. The prediction results are shown in Figures 1 and 2. Figure 1 is a sideview of the predicted structure of nanopore protein, and Figure 2 is a topview of the predicted structure of nanopore protein.

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 chain are S75, G79, and F80.

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.

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.

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

Through the In-fusion method, the DNA sequence encoding the nanopore protein was inserted into the multiple cloning region of the vector pET24a after digestion with NdeI and XhoI. StrepII amino acids were added to the C-terminus of the wild-type nanopore protein amino acid sequence (SEQ ID No.1) and the nanopore protein mutant 1 (F80N SEQ ID No.2) as purification tags, where the screening tag was kanamycin, and the constructed vectors were named pET24a-BCP52-wt and pET24a-BCP52-F80N. Through the site-directed mutagenesis method, the Agilent site-directed mutagenesis kit was used to construct the corresponding nanopore protein mutant expression vector with the nanopore protein expression vector as a template.

Example 3 Cultivation and induction of nanopore protein and its mutant strains

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 in 5mL LB medium containing 50μg/mL kanamycin, and culture it at 37°C, 200rpm, overnight. Inoculate the above bacterial solution at a ratio of 1:100 into 50mL LB containing 50μg/mL kanamycin, and culture it at 37°C, 200rpm for 4h. Inoculate the expanded cultured bacterial solution at a ratio of 1:100 into 2L LB containing 50μg/mL kanamycin, and culture it at 37°C, 200rpm. When the OD ₆₀₀ value reaches about 0.6-0.8, add IPTG with a final concentration of 0.5mmol/L, and culture it at 16°C, 200rpm for about 16-18h. The bacterial solution was collected by centrifugation at 8000 rpm and the bacteria were frozen at -20°C until use.

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

Purification Buffer Preparation:

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

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

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

Purification steps:

Resuspend the cells thoroughly at a ratio of 10 mL Buffer A per 1 g of cells, and disrupt the cells by ultrasound until the cell solution is clear. Rotate overnight at 4°C on a rotator. Centrifuge at 18,000 rpm at 4°C for 1 hour the next day, take the supernatant, filter with a 0.22 μm filter membrane, and store at 4°C for later use.

The Strep-Tactin beads (IBA Lifesciences) column was equilibrated with Buffer A for 5 column volumes (CV) using an AKTA pure chromatograph, and then sampled at 2 mL/min. After sample loading, Buffer B was used to wash for 20 CV, and Buffer C was used to elute and collect the target protein.

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 then stored at -80°C.

Figure 7 is an SDS-PAGE image of the purified nanopore protein, where E1 and E2 are samples that were not denatured at high temperature after elution, and D1 and D2 are samples that were boiled at 95°C. The results show that the target protein is in a polymer state before boiling and in a monomer state after boiling.

Example 5 Library construction

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

Example 6: Construction of nanopore biosensors using nanopore proteins and their mutants

Single-channel nanopore current measurement is based on an amplifier of a digital device. Ag/AgCl electrodes are immersed in sequencing buffer and the electrodes are located in the cis and trans regions of the electrolytic cell, respectively. Reagents such as sequencing library and nanopore are added to the cis region. After the nanopore protein is diluted 100 times with 1×PBS buffer, a single nanopore is inserted into a phospholipid bilayer composed of diacylphosphatidylcholine (DPhPC, 1,2-diphytanoyl-sn-glycero-3-phosphocholine) under an applied electric field force of 0.05V to form a nanopore biosensor. An applied voltage is applied to obtain the current amplitude value of a single pore protein. Figure 9 shows the nanopore biosensing current of the nanopore protein when 0.02V, 0.04V, 0.10V, 0.14V and 0.18V voltages are applied.

In this example, the pore opening current of the wild-type nanopore protein (SEQ ID No. 1) and the nanopore protein mutant 1 (SEQ ID No. 2) after being inserted into the phospholipid membrane and applying different voltages was mainly attempted.

In this example, the nanopore protein mutant 1 is a mutant of the wild-type nanopore protein containing a mutation site of F80N.

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

Example 7 Using nanopore proteins and their mutants for DNA sequencing

1 microgram of the sequencing library obtained in Example 5 and single-stranded DNA with cholesterol (SEQ ID No. 5) at 5 times the library concentration were mixed with sequencing buffer (0.47M KCl, 25mM HEPES, 1mM EDTA, 5mM ATP, 25mM MgCl ₂ , pH 7.6) and added to the nanopore biosensor; after applying an applied voltage of 0.14V or 0.18V, it was observed that the DNA was captured by the nanopore, generating a characteristic blocking current amplitude value. And as the DNA moves through the nanopore, the current amplitude value changes. Different DNA sequences generate different blocking current amplitude values. Single-stranded DNA with cholesterol can bind to the phospholipid bilayer, which helps the nanopore capture the sequencing library and reduces the amount of sequencing library loading.

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) are applied to DNA sequencing.

Under the action of an applied voltage of 0.18 V, the current trace of the library DNA passing through the nanopore protein mutant 1 (F80N, SEQ ID No. 2) protein is shown in Figure 11. It can be seen that the nanopore protein mutant 1 can be used for DNA sequencing. As the DNA perforates, the current trace oscillates, and the sequencing amplitude is about 50pA.

Figure 12 is a partial detail of the DNA sequencing trace of nanopore protein mutant 1. The novel nanopore protein BCP52 and its mutants and applications provided by the present invention are introduced in detail above. This article uses specific examples to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core idea. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, the present invention can also be improved and modified in a number of ways, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

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).
The mutant of the nanopore protein according to claim 1, characterized in that it comprises:

(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.
The mutant according to claim 2, characterized in that the mutation in the sensor region comprises any one or more mutations in S75, G79 and/or F80; and/or

The mutation of the transmembrane region 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.
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.
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.
The mutant according to any one of claims 2 to 5, characterized in that the mutation in the sensor region comprises F80N in the sensor region.
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 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).
A nucleic acid molecule encoding the nanopore protein according to claim 1 or the mutant according to any one of claims 2 to 7.
The nucleic acid molecule encoding the nanopore protein according to claim 1, characterized in that it has:

(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).
A nucleic acid molecule encoding a nanopore protein mutant according to any one of claims 2 to 7, characterized in that it has:

(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).
An expression vector, characterized in that it comprises the nucleic acid molecule and a backbone vector according to any one of claims 8 to 10.
A host, characterized in that it comprises the recombinant vector according to claim 11.
The construct is characterized in that the construct is composed of 7 to 11 covalently linked or non-covalently polymerized nanopore proteins according to any one of claims 1 to 7.
The construct according to claim 13, characterized in that the construct consists of 9 covalently linked or non-covalently polymerized nanopore proteins according to any one of claims 1 to 7.
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 it comprises the following steps:

(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.
The method for preparing the construct according to claim 13 or 14, characterized in that it comprises the following steps:

(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.
A biosensor, characterized in that it comprises any of the following items and acceptable auxiliary agents 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.
The kit is characterized by comprising any of the following items and an acceptable auxiliary agent or carrier:

(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.
Application of any of the following to 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.
The single molecule sequencing method comprises the following steps:

(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.
A sequencing device, characterized in that it comprises the biosensor as described in claim 17 and an acceptable auxiliary agent or carrier.