WO2024138425A1

WO2024138425A1 - Novel nanopore protein and use thereof

Info

Publication number: WO2024138425A1
Application number: PCT/CN2022/142827
Authority: WO
Inventors: 董宇亮; 罗婉婷; 章佳文; 郭斐; 曾涛; 王乐乐; 季州翔; 黎宇翔; 章文蔚; 徐讯
Original assignee: 深圳华大生命科学研究院
Priority date: 2022-12-28
Filing date: 2022-12-28
Publication date: 2024-07-04

Abstract

Provided is a nanopore protein BCP22, which is (a), (b) or (c): a) a protein having an amino acid sequence as shown in SEQ ID NO: 1; b) a protein having more than 70% identity to SEQ ID NO: 1 and having the function of SEQ ID NO: 1; and c) a fusion protein obtained by adding a tag to the end of the protein sequence as shown in a) or b), and can be applied to nanopore sequencing for detecting small molecules, DNA, RNA, polypeptides and the like.

Description

A novel nanoporous protein and its application

Technical Field

The invention belongs to the field of biotechnology and relates to a novel nanopore protein and application 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.

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.

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 currently being 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.

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.

Finding more excellent sequencing nanopore proteins through gene mining methods remains an unresolved problem.

Invention Disclosure

An object of the present invention is to provide porins.

In a first aspect, the present invention provides a porin, which is (a), (b) or (c):

a) a protein having the amino acid sequence shown in SEQ ID NO: 1;

b) a protein that has more than 70% identity to SEQ ID NO:1 and has the function of SEQ ID NO:1;

c) A fusion protein obtained by adding a tag to the end of the protein sequence shown in a) or b).

Among the pore proteins above, the first protein shown in b) is a mutant of the protein shown in a), and compared with the amino acid sequence shown in SEQ ID NO.1, the mutant has mutations in the amino acid residues at one or more of the following sites: 66, 67, 71 and 72.

In the porin above, the mutations at each site are as follows:

The N at position 66 mutates to A, G or S (located at sensor);

The D at position 67 is mutated to A, G, S, T, N or Q (located at sensor);

The E at position 71 mutates to A, G, S, T, N or Q (located at sensor);

The Y at position 72 is mutated to A, G, S, T, N or Q (located at sensor).

Among the pore proteins mentioned above, the second protein shown in b) is a mutant of the protein shown in a), and compared with the amino acid sequence shown in SEQ ID NO.1, the mutant has mutations in the amino acid residues at one or more of the following sites: 102, 111, 112, 113, 116, 119, 120, 123, 129, 162, 170, 173, 175, 204, 212, 217, 218, 221 and 224.

Or, in the porin above, the porin is compared with the protein shown in the first b) above (i.e., compared with the amino acid sequence shown in SEQ ID NO.1, the mutant has mutations in the amino acid residues at one or more of the following sites: 66, 67, 71 and 72), and further has mutations in the amino acid residues at one or more of the following sites: 102, 111, 112, 113, 116, 119, 120, 123, 129, 162, 170, 173, 175, 204, 212, 217, 218, 221 and 224.

In the above porin, the mutations at each site are as follows:

The R at position 102 was mutated to A, G, S, T, N, or Q (entry);

E at position 111 mutated to R, K, A, G, S, T, N, or Q (entry);

The R at position 112 was mutated to A, G, S, T, N, or Q (entry);

The K at position 113 was mutated to A, G, S, T, N, or Q (entry point);

The R at position 116 was mutated to A, G, S, T, N, or Q (entry);

The R at position 119 was mutated to A, G, S, T, N, or Q (entry);

The mutation of D at position 120 is A, G, S, T, N, or Q (entry);

The K at position 123 was mutated to A, G, S, T, N, or Q (entry point);

The K at position 129 was mutated to A, G, S, T, N, or Q (entry);

The R at position 162 mutated to A, G, S, T, N, or Q (barrel lining);

The S at position 170 was mutated to A, G, V, L, I, Y, F, or W (transmembrane region);

The R at position 173 mutated to A, G, S, T, N, or Q (barrel lining);

The D at position 175 mutates to A, G, S, T, N, or Q (barrel lining);

The S at position 204 was mutated to A, G, V, L, I, Y, F, or W (transmembrane region);

The K at position 212 mutates to D, E, A, G, S, T, N, or Q (exit loop);

The D at position 217 is mutated to A, G, S, T, N, or Q (exit loop);

The K at position 218 mutates to D, E, A, G, S, T, N, or Q (exit loop);

E at position 221 mutated to A, G, S, T, N, or Q (barrel lining);

The T at position 224 was mutated to A, G, V, L, I, Y, F or W (transmembrane region).

The porins mentioned above exist in the form of polymers;

Further, the porin exists in the form of 9-mers;

The pores formed by the porins mentioned above have a diameter of 0.5-3 nm;

Furthermore, the pore size formed by the porin is 1-2 nm.

In a second aspect, the present invention provides a nucleic acid molecule encoding the porin of the first aspect.

In a third aspect, the present invention provides an expression cassette, a recombinant vector or a transgenic cell line comprising the nucleic acid molecule of the second aspect.

In a fourth aspect, the present invention provides a recombinant bacterium comprising the nucleic acid molecule described in the second aspect.

In a fifth aspect, the present invention provides use of the nucleic acid molecule described in the second aspect, the expression cassette, recombinant vector or transgenic cell line described in the third aspect, or the recombinant bacteria described in the fourth aspect in preparing porins.

In a sixth aspect, the present invention provides the use of the porin described in the first aspect in the preparation of a nanobiosensor.

In a seventh aspect, the present invention provides a nanobiosensor comprising the porin and lipid bilayer of the first aspect.

In an eighth aspect, the present invention provides the use of the pore protein described in the first aspect, the nucleic acid molecule described in the second aspect, the expression cassette, recombinant vector or transgenic cell line described in the third aspect, the recombinant bacteria described in the fourth aspect, or the nanobiosensor described in the seventh aspect in nucleic acid nanopore sequencing.

In a ninth aspect, the present invention provides a method for preparing and producing the porin described in the first aspect, comprising the steps of: culturing the recombinant bacteria described in the fourth aspect, and then inducing the bacteria to obtain the porin described in the first aspect.

In a tenth aspect, the present invention provides a kit comprising the porin described in the first aspect.

The kit described above further comprises at least one of the following: a linker containing a helicase recognition site, a helicase, and single-stranded DNA with cholesterol.

In an eleventh aspect, the present invention provides a method for nanopore sequencing of nucleic acids, comprising the following steps:

1) Preparation of sequencing library and nanopore biosensor containing helicase;

The nanopore biosensor is prepared according to the following method: inserting the porin described in the first aspect into a lipid bilayer to form a nanopore channel, thereby constructing a nanopore biosensor;

2) The sequencing library containing the helicase, the single-stranded DNA containing cholesterol and the sequencing buffer are mixed and added to the nanopore biosensor, and a voltage is applied to capture the sequencing library.

In the above method, the sequencing library containing the helicase is prepared according to the following method: the nucleic acid to be tested is connected to a connector to prepare a sequencing library, and then the sequencing library is combined with a helicase to obtain a sequencing library containing the helicase; wherein the connector is obtained by annealing two single-stranded DNA molecules, and the single-stranded DNA molecule contains a helicase binding site.

In an embodiment of the present invention, the above-mentioned linker or the linker containing the helicase recognition site is specifically formed by annealing the single-stranded DNA molecule shown in SEQ ID No. 2 and the single-stranded DNA molecule shown in SEQ ID No. 3;

In an embodiment of the present invention, the above-mentioned helicase is specifically BCH105 shown in SEQ ID No.5;

In an embodiment of the present invention, the nucleotide sequence of the single-stranded DNA with cholesterol is specifically SEQ ID NO: 4, and the cholesterol is connected to the 5' end of the DNA;

In an embodiment of the present invention, the above-mentioned sequencing buffer formula is specifically: 0.47M KCl, 25mM HEPES, 1mM EDTA, 5mM ATP, 25mM MgCl2, the balance is water, pH 7.6.

In the above method, the pore size of the nanopore channel is 0.5-3 nm.

Furthermore, the pore size formed by the porin is 1-2 nm.

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

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is the three-dimensional structure of BCP22 predicted by Alphafold2 multimer (side view).

Figure 2 is the three-dimensional structure of BCP22 predicted by Alphafold2 multimer (top view).

Figure 3 is a schematic diagram of the alphafold2 multimer predicted structure of key amino acids in the BCP22 sensor region.

FIG4 is a schematic diagram of the key amino acids in the sensor region of the predicted structure of BCP22.

FIG5 is a schematic diagram of the key amino acids in the transmembrane region of the predicted structure of BCP22.

Figure 6 is a schematic diagram of the key amino acids in the entry region of the predicted structure of BCP22

Figure 7 is a schematic diagram of the key amino acids in the exit region and the inner wall of the barrel in the predicted structure of BCP22.

FIG. 8 shows the purified protein of BCP22.

Figure 9 is a schematic diagram of the sequencing library structure (a: top strand; b: bottom strand; c: double-stranded target fragment; d: helicase BCH105; e: chol-ssDNA); Figure 9A: Schematic diagram of the sequencing library; Figure 9B: Schematic diagram of the binding mode of chol-ssDNA and sequencing library.

FIG. 10 shows the pore opening current of BCP22 at different voltages in the phospholipid bilayer.

FIG. 11 is a current trace of the DNA to be tested passing through the nanopore BCP22.

Best Mode for Carrying Out the Invention

Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

Example 1. Discovery of wild-type BCP22 protein and predicted structure of Alphafold2 multimer

The inventors mined a BCP22 protein from the deep-sea metagenome, whose amino acid sequence is SEQ ID No.1, and the nucleotide sequence of the gene encoding the protein is SEQ ID No.7.

The alphafold2 multimer was used to predict the nonamer structure of BCP22 (SEQ ID No.1).

The prediction results are shown in Figures 1 and 2. Figure 1 is the side view of the predicted structure of BCP22, and Figure 2 is the top view of the predicted structure of BCP22. It can be seen from Figures 1 and 2 that the BCP22 protein is in the form of a nonameric structure with a pore size of 1-2 nm.

Based on common sense, the composition of the amino acids in the sensor region plays a decisive role in the generation of current signals. Figures 3 and 4 show the side chain structures of the important amino acids in the sensor region of the predicted structure of BCP22, showing that the four amino acids in the amino acid side chains are N66, D67, E71, and Y72. The middle number is the distance between the three amino acid side chains of the two protein monomers. The unit is angstrom.

Based on common sense, the amino acids in the transmembrane region of porins that face the membrane prefer hydrophobic amino acids, while charged amino acids and polar amino acids may affect the efficiency of pore insertion. Figure 5 shows three charged or polar amino acids facing the membrane in the transmembrane region of BCP22, namely S170, S204, and T224.

Based on common sense, since nucleic acids are negatively charged, if the amino acids at the entrance of the porin are positively charged, the library capture rate of nanopore sequencing can be increased, otherwise, the library capture rate can be reduced. At the same time, the strength of porin binding will also have a certain impact on the sequencing speed and sequencing current signal. Figure 6 shows some important amino acids in the entrance region of the BCP22 monomer, which are; R102, E111, R112, K113, R116, R119, D120, K123, K129.

At the same time, some amino acids on the inner wall and outlet of the barrel have an impact on the smooth penetration and exit of the analyte from the nanopore. Figure 7 shows some amino acids on the inner wall and outlet of the barrel of the BCP22 monomer that may affect the smooth penetration of the analyte, especially the charged amino acids, which are R162, R173, D175, K212, D217, K218, and E221.

Therefore, the wild-type BCP22 protein can be mutated at the above-mentioned key amino acid residues to obtain a mutant, which is predicted to have the function of the wild-type BCP22 protein.

The mutants may be mutated at one or more of the following amino acid residues based on the protein shown in SEQ ID No. 1: 66, 67, 71, 72, 102, 111, 112, 113, 116, 119, 120, 123, 129, 162, 170, 173, 175, 204, 212, 217, 218, 221 and 224;

The mutant can also be a mutant protein obtained by mutating the amino acid residues at one or more of the following sites based on the protein shown in SEQ ID No.1: 66, 67, 71, 72, and further mutating the amino acid residues at one or more of the following sites based on the mutant protein: 102, 111, 112, 113, 116, 119, 120, 123, 129, 162, 170, 173, 175, 204, 212, 217, 218, 221 and 224.

Example 2: Obtaining wild-type BCP22 protein

1. Plasmid expressing wild-type BCP22 protein

The plasmid expressing wild-type BCP22 protein is a vector obtained by inserting the coding gene of wild-type BCP22 protein (SEQ ID No.7) between the NdeI and XhoI sites of the pET24a vector. The vector expresses a fusion protein of wild-type BCP22 fused with a StrepII tag at the C-terminus. The StrepII tag is used for protein purification.

The corresponding mutants were constructed by site-directed mutagenesis using the Agilent site-directed mutagenesis kit and the plasmid expressing the wild-type BCP22 protein as a template.

2. Cultivation and induction of porin BCP22 strain

The plasmid expressing the wild-type BCP22 protein constructed above was transformed into the E. coli expression strain E. coli BL21 (DE3) to obtain the transformed bacterial solution. The transformed bacterial solution was then evenly spread on a plate containing 50 μg/mL kanamycin and cultured at 37°C overnight. The next day, a single colony was picked and cultured in 5 ml LB medium containing 50 μg/mL kanamycin at 37°C, 200 rpm, overnight. The obtained bacterial solution was inoculated into 50 ml LB containing 50 μg/mL kanamycin at a ratio of 1:100, and cultured at 37°C, 200 rpm for 4 hours. The expanded cultured bacterial solution was inoculated into 2L LB containing 50 μg/mL kanamycin at a ratio of 1:100, and cultured at 37°C, 200 rpm. When the OD600 value reached about 0.6-0.8, IPTG was added at a final concentration of 0.5 mM, and cultured at 16°C, 200 rpm for about 18 hours. The bacterial liquid was centrifuged at 8000 rpm for 10 minutes to collect the bacterial cells, and frozen at -20°C until use, thereby obtaining the porin BCP22 strain.

3. Extraction and purification of recombinant porin BCP22

1. Preparation of purification buffer

Buffer A: 20 mM Tris-HCl, 250 mM NaCl, 1% DDM, the remainder is water, pH 8.0.

Buffer B: 20 mM Tris-HCl, 250 mM NaCl, 0.05% DDM, balance water, pH 8.0.

Buffer C: 20 mM Tris-HCl, 250 mM NaCl, 0.05% DDM, 5 mM desthiobiotin, balance water, pH 8.0.

2. Purification

Resuspend the collected cells in the ratio of 1g porin BCP22 strain cells to 10ml Buffer A, and ultrasonically disrupt the cells until the cell solution is clear. Rotate overnight at 4℃ on a rotator. Centrifuge at 18000rpm at 4℃ for 1h the next day, take the supernatant, filter with a 0.22μm filter membrane, and store at 4℃ for later use.

After equilibration of the Strep-Tactin beads (IBA Lifesciences) column with Buffer A for 5 CV using AKTA pure, the sample was loaded at 2 ml/min. After loading, it was rinsed with Buffer B for 20 CV, eluted with Buffer C, and the eluate (containing the target protein) was collected.

The eluate (containing the target protein) after the above-mentioned Strep column affinity chromatography was concentrated to 1 ml, passed through a Superdex 6 increase 10/300GL (Cytiva) column equilibrated with buffer B, the target protein was collected, and then stored at -80°C to obtain a homozygous target protein solution.

The target protein solution obtained after purification was subjected to SDS-PAGE electrophoresis.

The results are shown in Figure 8. Lanes 2 and 3 are the gel bands of the target protein solution before and after boiling at 95°C, respectively. The results show that the protein band is near the spotting hole when the target protein is not boiled and has a larger molecular weight. After boiling, the size is about 30KD, which is the same as the expected target protein. This shows that the target protein is in a polymer state before boiling and in a monomer state after boiling.

After testing, the concentration of the target protein solution was 1.0 mg/mL.

Example 3: Application of BCP22 protein and its mutants as porins in nanopore sequencing

1. Library Construction

Two partially complementary DNA chains (top strand (SEQ ID No. 2, where Y = iSP18) and bottom strand (SEQ ID No. 3) were annealed to form a linker (as shown in FIG. 9 ), and then connected to the double-stranded target fragment to be tested (SEQ ID No. 6) using T4 DNA ligase at room temperature and purified to prepare a sequencing library. The sequencing library was then incubated with helicase BCH105 (SEQ ID No. 5) at 25°C for 1 h (the molar concentration ratio of the sequencing library to the helicase BCH105 was 1:8) to form a sequencing library containing the BCH105 motor protein (as shown in FIG. 9A ). During sequencing, the sequencing library can further complementarily pair with single-stranded DNA with cholesterol (SEQ ID NO: 4, cholesterol connected to the 5' end of the DNA) to form a structure as shown in FIG. 9B .

2. Construction of nanopore biosensors using porin BCP22 and its mutants

The current signal is collected using a patch clamp amplifier. Ag/AgCl electrodes are immersed in sequencing buffer and are located in the cis and trans regions of the electrolytic cell. Sequencing libraries and nanopore reagents are added to the cis region.

After the porin BCP22 obtained in Example 2 was diluted a certain multiple using 1xPBS buffer, a single nanopore protein was inserted into a phospholipid bilayer composed of diacylphosphatidylcholine (DPhPC, 1,2-diphytanoyl-sn-glycero-3-phosphocholine) under the action of an external electric field to form a nanopore biosensor.

An external voltage was applied and the current amplitude value of a single porin was obtained.

Figure 10 shows the nanopore biosensor current of wild-type BCP22 when voltages of 0.02V, 0.04V, 0.10V, 0.14V and 0.18V are applied (the figures from left to back are voltages of 0.02V, 0.04V, 0.10V, 0.14V and 0.18V, respectively), indicating that the BCP22 protein can be inserted into the membrane, and then the pore in the middle can allow ions to pass through, generating an open current, and the noise of the open current is relatively small.

3. Using Porin BCP22 for DNA Sequencing

The sequencing library containing the BCH105 motor protein prepared in the first step and the single-stranded DNA with cholesterol (SEQ ID No. 4) were mixed with the sequencing buffer and added to the nanopore biosensor prepared in the second step; after applying an external 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 produce different blocking current amplitude values. The single-stranded DNA with cholesterol can bind to the phospholipid bilayer, which helps the nanopore capture the sequencing library and reduce the amount of sequencing library loading.

Sequencing buffer: 0.47M KCl, 25mM HEPES, 1mM EDTA, 5mM ATP, 25mM MgCl2, balance water, pH 7.6.

FIG. 11 shows the current change when the library DNA passes through the nanopore protein BCP22 under the action of an applied voltage of 0.14 V, indicating that the wild-type protein BCP22 can be used as a nanopore for nanopore sequencing.

Industrial Applications

The present invention obtains a nanopore protein BCP22 from the deep-sea metagenome through gene mining with computer-aided structure prediction. Protein preparation and nanopore sequencing verification show that it has the ability to be applied to nanopore sequencing and can detect small molecules, DNA, RNA and polypeptides.

Claims

A porin is (a), (b) or (c):

a) a protein having the amino acid sequence shown in SEQ ID NO: 1;

b) a protein that has more than 70% identity to SEQ ID NO:1 and has the function of SEQ ID NO:1;

c) A fusion protein obtained by adding a tag to the end of the protein sequence shown in a) or b).
The porin according to claim 1, characterized in that:

The protein shown in b) is a mutant of the protein shown in a), and compared with the amino acid sequence shown in SEQ ID NO.1, the mutant has mutations in the amino acid residues at one or more of the following sites: 66, 67, 71 and 72.
The porin according to claim 2, characterized in that:

The mutation modes of each site are as follows:

The N at position 66 mutated to A, G or S;

The D at position 67 mutated to A, G, S, T, N, or Q;

E at position 71 mutated to A, G, S, T, N, or Q;

The Y at position 72 is mutated to A, G, S, T, N or Q.
The porin according to claim 1, characterized in that:

The protein shown in b) is a mutant of the protein shown in a), and compared with the amino acid sequence shown in SEQ ID NO.1, the mutant has mutations in amino acid residues at one or more of the following sites: 102, 111, 112, 113, 116, 119, 120, 123, 129, 162, 170, 173, 175, 204, 212, 217, 218, 221 and 224.
The porin according to claim 2 or 3, characterized in that:

The porin protein is a porin protein according to claim 2 or 3, wherein the amino acid residues at one or more of the following positions are mutated: 102, 111, 112, 113, 116, 119, 120, 123, 129, 162, 170, 173, 175, 204, 212, 217, 218, 221 and 224.
The porin according to claim 5, characterized in that:

The mutation modes of each site are as follows:

The R at position 102 mutated to A, G, S, T, N, or Q;

E at position 111 mutated to R, K, A, G, S, T, N, or Q;

The R at position 112 mutated to A, G, S, T, N, or Q;

The K at position 113 mutated to A, G, S, T, N, or Q;

The R at position 116 mutated to A, G, S, T, N, or Q;

The R at position 119 mutated to A, G, S, T, N, or Q;

The mutation of D at position 120 is A, G, S, T, N or Q;

The K at position 123 mutated to A, G, S, T, N or Q;

The K at position 129 mutated to A, G, S, T, N, or Q;

The R at position 162 mutated to A, G, S, T, N, or Q;

The S at position 170 mutated to A, G, V, L, I, Y, F, or W;

The R at position 173 mutated to A, G, S, T, N, or Q;

The D at position 175 mutated to A, G, S, T, N, or Q;

The S at position 204 mutated to A, G, V, L, I, Y, F, or W;

The K at position 212 mutated to D, E, A, G, S, T, N, or Q;

The D at position 217 mutated to A, G, S, T, N, or Q;

The K at position 218 mutated to D, E, A, G, S, T, N, or Q;

E at position 221 mutated to A, G, S, T, N, or Q;

The T at position 224 is mutated to A, G, V, L, I, Y, F or W.
According to any one of claims 1 to 6, the porin is characterized in that:

The porin exists in the form of a polymer;

Further, the porin exists in the form of 9-mers;

Or, the pore size formed by the porin is 0.5-3 nm;

Furthermore, the pore size formed by the porin is 1-2 nm.
A nucleic acid molecule encoding the porin of any one of claims 1-7.
An expression cassette, a recombinant vector or a transgenic cell line containing the nucleic acid molecule of claim 8.
A recombinant bacterium comprising the nucleic acid molecule according to claim 8.
Use of the nucleic acid molecule according to claim 8, the expression cassette, the recombinant vector or the transgenic cell line according to claim 9, or the recombinant bacteria according to claim 10 in the preparation of porins.
Use of the porin according to any one of claims 1 to 7 in the preparation of a nanobiosensor.
A nano biosensor comprising the porin according to any one of claims 1 to 7 and a lipid bilayer.
Use of the porin according to any one of claims 1 to 7, the nucleic acid molecule according to claim 8, the expression cassette, recombinant vector or transgenic cell line according to claim 9, the recombinant bacteria according to claim 10 or the nanobiosensor according to claim 13 in nucleic acid nanopore sequencing.
A method for preparing and producing the porin described in any one of claims 1-7, comprising the following steps: culturing the recombinant bacteria described in claim 10, and then performing an induction treatment to obtain the porin described in any one of claims 1-7.
A kit comprising the porin according to any one of claims 1 to 7.
The kit according to claim 16 is characterized in that: the kit further comprises at least one of the following: a linker containing a helicase recognition site, a helicase, and a single-stranded DNA containing cholesterol.
A method for nanopore sequencing of nucleic acids, comprising the following steps:

1) Preparation of sequencing library and nanopore biosensor containing helicase;

The nanopore biosensor is prepared according to the following method: inserting the porin described in any one of claims 1 to 7 into a lipid bilayer to form a nanopore channel, thereby constructing a nanopore biosensor;

2) The sequencing library containing the helicase, the single-stranded DNA containing cholesterol and the sequencing buffer are mixed and added to the nanopore biosensor, and a voltage is applied to capture the sequencing library.
The method according to claim 18, characterized in that:

The sequencing library containing the helicase is prepared according to the following method: the nucleic acid to be tested is connected to a connector to prepare a sequencing library, and then the sequencing library is combined with a helicase to obtain a sequencing library containing the helicase; wherein the connector is obtained by annealing two single-stranded DNA molecules, and the single-stranded DNA molecules contain a helicase binding site.
The method according to claim 18 or 19, characterized in that:

The pore size of the nanopore channel is 0.5-3 nm.