WO2024138382A1 - Porin monomer, porin, mutant thereof and use thereof - Google Patents

Abstract

Provided in the present invention are a porin monomer, a porin, a mutant thereof and the use thereof. The porin monomer comprises: (a) a protein composed of an amino acid sequence as shown in SEQ ID NO: 1; or (b) a protein mutant, wherein the amino acid sequence of the protein mutant is subjected to substitution, deletion and/or addition of one or several amino acids at at least one of the following positions in the amino acid sequence as shown in SEQ ID NO: 1: position 63, position 64, etc., and the protein mutant has the function of forming a pore channel structure via polymerization; or (c) a porin monomer having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to the protein of (a) or (b), and having the function of forming a pore channel structure via polymerization. By means of the present invention, the problem in the prior art of the poor stability of a pore channel of the porin can be solved; and the present invention is applicable to the field of single-molecule sequencing.

Description

Porin monomer, porin and its mutant and its application Technical Field

The present invention relates to the field of single molecule sequencing, and in particular to a porin monomer, a porin and a mutant thereof, and applications thereof.

Background technique

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 sequencing accuracy, throughput, chip stability and applicable scenarios, 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-based single-molecule sequencer is a highly integrated detection system 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 to build a high-precision single-molecule nanopore 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. So far, only a few natural pore proteins such as Mycobacterium smegmatis porin A (MspA) and curli-specific transporter (CsgG) can meet the requirements of becoming single-molecule detectors in the industry. How to find more excellent pore proteins that can be used for single-molecule sequencing through gene mining methods is still an unresolved problem.

Summary of the invention

The main purpose of the present invention is to provide a porin monomer, a porin and a mutant thereof and applications thereof, so as to solve the problem of poor pore stability of porin in the prior art.

To achieve the above object, according to the first aspect of the present invention, there is provided a porin monomer, which comprises: (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1; or (b) a protein mutant, wherein the amino acid sequence of the protein mutant is substituted, deleted and/or one or more amino acids are added at at least one of the following positions of the amino acid sequence shown in SEQ ID NO: 1: 63, 64, 65, 66, 67, 68, 69, 103, 107, 108, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135 8, 109, 113, 116, 117, 123, 126, 153, 156, 167, 169, 201, 206, 209, 213, 216, 218, and the protein mutant has the function of forming a pore structure by polymerization; or (c) a pore protein monomer that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with the protein in (a) or (b), and has the function of forming a pore structure by polymerization.

Further, in (b), the type of amino acid substituted at each position is independently selected from the following: G63 is mutated into G63A, G63S or G63T; E64 is mutated into E64G, E64A, E64S, E64T, E64N or E64Q; K65 is mutated into K65G, K65A, K65S, K65T, K65N or K65Q; F66 is mutated into F66G, F66A, F66S, F66T, F66N or F66Q; A67 is mutated into A67G, A67S, A67T, A67N or A67Q; N68 is mutated into N68G, N68A, N68S, N68T or N68Q; I69 mutated to I69G, I69A, I69S, I69T, I69N or I69Q; D103 mutated to D103N, D103A, D103G, D103S, D103T, D103Q, D103R or D103K; K107 mutated to K107N, K107A, K107G, K107S, K107T or K107Q; R109 mutated to R109W, R109F, R109Y, R109N, R109Q, R109S, R109T, R109A or R109G; R113 mutated to R113W, R113F, R113Q 3Y, R113N, R113Q, R113S, R113T, R113A or R113G; R116 is mutated to R116N, R116A, R116G, R116S, R116T or R116Q; E117 is mutated to E117N, E117A, E117G, E117S, E117T, E117Q, E117R or E117K; E123 is mutated to E123N, E123A, E123G, E123S, E123T, E123Q, E123R or E123K; K126 is mutated to K126N, K126Q, K126S, K126T, K126A or K126G; D15 3 mutated to D153A, D153G, D153V, D153L, D153I, D153Y, D153F or D153W; R156 mutated to R156A, R156G, R156N, R156Q, R156S, R156T, R156D or R156E; R167 mutated to R167N, R167Q, R167S, R167T, R167A or R167G; D169 mutated to D169N, D169Q, D169S, D169T, D169A or D169G; D201 mutated to D201N, D201Q, D201S, D201T, D201A or D201G; R 206 mutated to R206N, R206Q, R206S, R206T, R206A, R206G, R206D or R206E; D209 mutated to D209N, D209Q, D209S, D209T, D209A, D209G, D209R or D209K; E213 mutated to E213N, E213Q, E213S, E213T, E213A or E213G; E216 mutated to E216N, E216Q, E216S, E216T, E216A or E216G; E218 mutated to E218N, E218Q, E218S, E218T, E218A or E218G.

In order to achieve the above object, according to the second aspect of the present invention, a protein construct is provided. The protein construct is composed of two or more porin monomers mentioned above, connected by covalent or non-covalent bonding.

In order to achieve the above object, according to the third aspect of the present invention, a porin is provided, wherein the porin is composed of 7 to 11 porin monomers mentioned above linked covalently or non-covalently.

Furthermore, the porin is composed of 9 porin monomers linked non-covalently.

Furthermore, the pore diameter of the porin is 0.5 to 3 nm.

In order to achieve the above object, according to the fourth aspect of the present invention, a kit is provided, the kit comprising the above porin monomer, or the above protein construct, or the above porin.

Furthermore, the kit also includes a membrane layer, and the membrane layer includes a lipid layer or an artificial polymer membrane.

Furthermore, the kit also includes a sequencing buffer and/or single-stranded DNA linked to cholesterol.

To achieve the above object, according to the fourth aspect of the present invention, an isolated DNA molecule is provided, which has a nucleotide sequence encoding the above porin monomer, or encoding the above protein construct, or encoding the above porin.

Further, the DNA molecule has the nucleotide sequence shown in SEQ ID NO: 8.

Furthermore, a DNA molecule that has more than 70%, preferably more than 80%, more preferably more than 90%, and further preferably more than 95% identity with the nucleotide sequence shown in SEQ ID NO: 8 and encodes a protein with the same function.

In order to achieve the above object, according to the fifth aspect of the present invention, a recombinant vector is provided, which comprises the above DNA molecule.

In order to achieve the above object, according to the sixth aspect of the present invention, a host cell is provided, wherein the host cell is transformed with the above recombinant vector.

In order to achieve the above-mentioned purpose, according to the seventh aspect of the present invention, a nanopore sensor is provided, which comprises: a membrane layer; and a pore protein inserted in the middle of the membrane layer to form a pore, and when a voltage is applied across the membrane layer, the pore generates current; wherein the pore protein comprises the above-mentioned pore protein.

Further, the membrane layer includes a lipid layer or an artificial polymer membrane; preferably, the lipid layer includes amphiphilic lipids; preferably, the amphiphilic lipids contain a phospholipid bilayer; preferably, the lipid layer includes a planar membrane layer or a liposome; preferably, the liposome includes a multilayer liposome or a unilamellar liposome; preferably, the lipid layer includes a phospholipid bilayer composed of diphytylphosphatidylcholine.

In order to achieve the above objective, according to an eighth aspect of the present invention, a nanopore sequencing device is provided, which comprises the above nanopore sensor.

Furthermore, the nanopore sequencing device includes: an electrolytic cell, the electrolytic cell contains a sequencing buffer; a nanopore sensor, the nanopore sensor is located in the center of the electrolytic cell and divides the electrolytic cell and the sequencing buffer into a positive electrolyte area and a negative electrolyte area; a first electrode and a second electrode, the first electrode and the second electrode are respectively arranged in the positive electrolyte area and the negative electrolyte area, and the first electrode and the second electrode are connected to the signal processing chip; preferably, the first electrode and the second electrode include metal or composite electrode materials; preferably, the first electrode and the second electrode are different, silver and silver chloride, respectively; or the first electrode and the second electrode are the same, and each is independently selected from gold, platinum, graphene or titanium nitride.

In order to achieve the above-mentioned purpose, according to the ninth aspect of the present invention, a sequencing method is provided, which utilizes the above-mentioned pore protein, or the above-mentioned nanopore sensor, or the above-mentioned nanopore sequencing device to detect and analyze the electrical signal generated when the biological molecule to be tested passes through the pore of the pore protein, so as to determine the sequence of the biological molecule to be tested.

Furthermore, the biomolecule to be detected includes any one of the following modified or unmodified biomolecules: DNA, RNA or polypeptide.

Further, the biological molecule to be detected is a target nucleic acid sequence, and the sequencing method includes: (a) contacting the target nucleic acid sequence with a nucleic acid binding protein, which controls the movement speed of the target nucleic acid sequence through the pore of the pore protein; (b) when a voltage is applied across the pore, the target nucleic acid sequence moves through the pore, and the electrical signal passing through the pore is measured, wherein different types of nucleotides generate different electrical signals when passing through the pore, thereby determining the sequence information of the target nucleic acid based on the electrical signal; preferably, the nucleic acid binding protein is selected from nucleases, polymerases, topoisomerases, ligases, helicases and single-stranded binding proteins; preferably, the electrical signal includes an electric current.

In order to achieve the above-mentioned purpose, according to the tenth aspect of the present invention, there is provided the above-mentioned porin monomer, the above-mentioned porin, the above-mentioned kit, the above-mentioned DNA molecule, the above-mentioned recombinant vector, the above-mentioned host cell, the above-mentioned nanopore sensor, or the above-mentioned nanopore sequencing device, for use in small molecule detection, DNA sequencing, RNA sequencing or polypeptide sequencing.

The present invention provides a new porin monomer, which can be polymerized to form porin BCP20. The protein and its mutants have good stability and can meet the requirements of single-molecule nanopore porin sequencing, and can realize the detection of small molecules such as nucleotides, amino acids, sugars, vitamins, DNA, RNA and polypeptides.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

FIG. 1 shows a side view of the three-dimensional structure of BCP20 predicted according to Example 1 of the present invention.

FIG. 2 shows a top view of the three-dimensional structure of BCP20 predicted according to Example 1 of the present invention.

Figure 3 shows a schematic diagram of the predicted side chain structure of the key amino acids in the gating region (sensor region) of BCP20 according to Example 1 of the present invention, wherein A shows the distance between the key amino acids in the sensor region, and B shows an enlarged view of the key amino acids in the sensor region.

FIG. 4 shows an SDS-PAGE image obtained by purification of BCP20 protein according to Example 4 of the present invention.

Figure 5 shows a schematic diagram of the sequencing library structure according to Example 5 of the present invention, wherein a: positive strand (top strand); b: antisense strand (bottom strand); c: double-stranded target fragment to be tested; d: helicase BCH105.

FIG. 6 shows a graph of the pore opening current of BCP20 according to Example 6 of the present invention at different voltages in a phospholipid bilayer.

FIG. 7 shows a graph showing the current variation when the DNA to be tested passes through the nanopore BCP20 according to Example 7 of the present invention.

Figure 8 shows a schematic diagram of the structure of a sequencing library combined with a single-stranded DNA containing cholesterol according to Example 7 of the present invention, wherein a: positive strand (top strand); b: antisense strand (bottom strand); c: double-stranded target fragment to be tested; d: helicase BCH105; e: single-stranded DNA containing cholesterol.

FIG. 9 shows the specific structure of iSp18 according to Embodiment 5 of the present invention.

Detailed ways

It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

As mentioned in the background technology, there are very few existing porins that can be used for single-molecule nanosequencing, and the protein stability is poor. Therefore, in this application, the inventors try to develop a new porin, and through the gene mining method of computer-assisted structure prediction, a new porin monomer is excavated from the deep-sea metagenome (derived from samples of the Mariana Trench at a depth of 11,000 meters). Multiple monomers can be covalently linked or non-covalently polymerized into porins with pores. The porin can be used as a detection protein and applied to the detection of small molecules such as nucleotides, amino acids, sugars, vitamins, or in nanopore-based DNA, RNA or polypeptide sequencing. Therefore, a series of protection schemes of this application are proposed.

In a first typical embodiment of the present application, a porin monomer is provided, the porin monomer comprising: (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1; or (b) a protein mutant, the amino acid sequence of the protein mutant undergoes substitution, deletion and/or addition of one or more amino acids at at least one of the following positions of the amino acid sequence shown in SEQ ID NO: 1: 63, 64, 65, 66, 67, 68, 69, 103, 107, 108, 109, 113, 116, 117, 123, 126, 153, 156, 167, 169, 201, 206, 209, 213, 216, 218, and the protein mutant has or (c) a porin monomer that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the porin monomer in (a) or (b) and has the function of forming a pore structure by polymerization.

SEQ ID NO: 1:

The porin monomers defined in (a) above can polymerize to form porin BCP20 with a pore structure, and when applied to nanopore sequencing, can allow the biomolecules to be tested to pass through the pore one by one, generating a current signal. Based on the sequence of (a), the protein is mutated, for example, at other positions such as the mutation sites disclosed in (b) or (c), after substitution and/or deletion and/or addition of one or more amino acids, the pore structure and function of the porin can still be obtained. Mutation of the porin monomers may affect the stability of the protein and aggregates, the inner diameter of the pore, and the amino acid residues on the inner wall of the pore, thereby affecting its physicochemical properties and the performance of the biomolecules to be tested. However, the conventional operation mode of mutation, and the method of screening and obtaining proteins with nanopore structure and functional activity are well known to those skilled in the art.

Identity in this specification refers to the "identity" between amino acid sequences, that is, the total ratio of the same type of amino acid residues in the amino acid sequence. The identity of amino acid sequences can be determined using alignment programs such as BLAST (Basic Local Alignment Search Tool) and FASTA.

Proteins with 70%, 75%, 80%, 85%, 90%, 95%, 99% or more (e.g. 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or more, or even 99.9% or more) identity and the same function, whose active sites, active pockets, active mechanisms, protein structures, etc. are most likely the same as the proteins provided by the sequence in a), are homologous proteins obtained by amino acid mutations.

As used herein, amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y) and valine (Val; V).

Substitution and replacement rules. Generally speaking, the effects of replacing amino acids with similar properties are similar. For example, conservative amino acid replacements may occur in the above homologous proteins. "Conservative amino acid replacements" include but are not limited to:

Hydrophobic amino acids (Ala, Cys, Gly, Pro, Met, Val, Ile, Leu) are replaced by other hydrophobic amino acids;

The hydrophobic amino acids with bulky side chains (Phe, Tyr, Trp) are replaced by other hydrophobic amino acids with bulky side chains;

Amino acids with positively charged side chains (Arg, His, Lys) are replaced by other amino acids with positively charged side chains;

Amino acids with polar, uncharged side chains (Ser, Thr, Asn, Gln) are replaced by other amino acids with polar, uncharged side chains.

A person skilled in the art may also perform conservative substitutions on amino acids according to amino acid substitution rules well known to those skilled in the art, such as the "blosum62 scoring matrix" in the prior art.

The "AlphaFold2-Multimer" used in this application is a public artificial intelligence model that can predict the conformation of protein complexes. The prediction of protein three-dimensional structure can be very close to the level observed by cryo-electron microscopes and other equipment in real experiments. It can obtain a more realistic protein structure, thereby guiding the exploration of protein structure and protein activity.

In a preferred embodiment, in (b), the types of amino acids substituted at each position are independently selected from the following: G63 mutates to G63A, G63S, G63T; E64 mutates to E64G, E64A, E64S, E64T, E64N, E64Q; K65 mutates to K65G, K65A, K65S, K65T, K65N, K65Q; F66 mutates to F66G, F66A, F66T, F66N, F66Q; 66S, F66T, F66N, F66Q; A67 mutated to A67G, A67S, A67T, A67N, A67Q; N68 mutated to N68G, N68A, N68S, N68T, N68Q; I69 mutated to I69G, I69A, I69S, I69T, I69N, I69Q; D103 mutated to D103N, D103A, D103G, D103S, D 103T, D103Q, D103R, D103K; K107 mutated to K107N, K107A, K107G, K107S, K107T, K107Q; R109 mutated to R109W, R109F, R109Y, R109N, R109Q, R109S, R109T, R109A, R109G; R113 mutated to R113W, R113F, R113S 13Y, R113N, R113Q, R113S, R113T, R113A, R113G; R116 mutated to R116N, R116A, R116G, R116S, R116T, R116Q; E117 mutated to E117N, E117A, E117G, E117S, E117T, E117Q, E117R, E117K; E123 mutated to E123 N, E123A, E123G, E123S, E123T, E123Q, E123R, E123K; K126 mutated to K126N, K126Q, K126S, K126T, K126A, K126G; D153 mutated to D153A, D153G, D153V, D153L, D153I, D153Y, D153F, D153W; R156 mutated R156A, R156G, R156N, R156Q, R156S, R156T, R156D, R156E; R167 mutated to R167N, R167Q, R167S, R167T, R167A, R167G; D169 mutated to D169N, D169Q, D169S, D169T, D169A, D169G; D201 mutated to D201N, D 201Q, D201S, D201T, D201A, D201G; R206 mutated to R206N, R206Q, R206S, R206T, R206A, R206G, R206D, R206E; D209 mutated to D209N, D209Q, D209S, D209T, D209A, D209G, D209R, D209K; E213 mutated to E2 13N, E213Q, E213S, E213T, E213A, E213G; E216 mutated to E216N, E216Q, E216S, E216T, E216A, E216G; E218 mutated to E218N, E218Q, E218S, E218T, E218A, E218G; among them, the letters before the numbers represent the original amino acids, and the letters after the numbers represent the mutated amino acids.

Among the above-mentioned mutation sites, three types of sites are mainly included: mutation sites in the gate region (sensor region), mutation sites at the entrance of the porin, and mutation sites in the transmembrane region of the porin. Among them, the mutation sites in the sensor region mainly determine the opening of the porin, thereby directly determining the opening current. Because the nucleic acid to be tested is negatively charged, by adjusting the amino acids at the mutation sites at the entrance of the porin, including but not limited to mutating uncharged or negatively charged amino acids to positively charged amino acids, or mutating positively charged amino acids to other types of amino acids, this mutation can adjust the capture rate of the library. Mutations in the transmembrane region of the porin can enhance the insertion stability of the porin on lipid or polymer membranes. In addition, charged amino acids located at the inner wall or exit of the pore structure of the porin can also affect the perforation of the sample to be tested.

Among the above mutation sites, G63, N68, I69, E64, K65, F66 and A67 are located in the sensor region. D103, K107, R109, R113, R116, E117, E123 and K126 are located at the entrance of the porin protein. Mutations in their charged properties can play an important role in regulating the capture rate of the library and sequencing noise. D153 is located on the outer wall of the porin transmembrane region. Mutations to hydrophobic amino acids can enhance the stability of the porin pore. R156, R167, D169, D201, E216 and E218 are located on the inner wall of the pore structure. They are charged amino acids on the inner wall of the porin barrel. Mutations in their charges can promote the smooth passage of the DNA molecules to be tested through the porin. R206, D209 and E213 are located in the exit loop region of the pore structure. Mutations to them can cause the sequenced nucleic acid chain to leave the porin.

The above mutation sites and the mutated amino acids can affect the pore structure, passing performance, library capture ability and other properties of the pore protein monomer and the pore protein formed by polymerization, especially G63, N68 and I69 located in the sensor area, which are located in the center of the pore protein channel and have a greater impact on the diameter, stability, affinity with the passing molecules, and ability to generate electrical signals of the protein pore structure. Among them, N68 and I69 are the most central amino acids in the sensor area. Mutations to them can directly determine the current size and the resolution of the perforated analyte. Mutations to the above two sites include but are not limited to mutations to polar uncharged or small side chain amino acids such as N, Q, S, T, G or A. G63 is located below N68 and I69. In order to reduce the impact on sequencing current and nucleic acid perforation, it can be mutated to a small side chain amino acid, including but not limited to mutations to A, S or T.

In a second typical embodiment of the present application, a protein construct is provided. The protein construct is composed of two or more of the above-mentioned porin monomers linked covalently or non-covalently.

In a third typical embodiment of the present application, a porin is provided, wherein the porin is composed of 7 to 11 porin monomers mentioned above, connected covalently or non-covalently.

In a preferred embodiment, the porin is composed of 9 porin monomers linked non-covalently.

Porin monomers can spontaneously aggregate together through hydrogen bonds, ionic bonds, hydrophobic interactions, etc. to form porins. Therefore, the porin monomers obtained by expression and purification exist in the form of polymers, especially nonamers, under non-denaturing conditions, while porin monomers exist after protein denaturation.

In a preferred embodiment, the pore diameter of the porin is 0.5-3 nm.

Within a certain range, the smaller the pore diameter of the porin, the higher its accuracy when used for sequencing. If the pore diameter of the porin is too large (more than one molecule may pass through the pore at a time), it is difficult to meet the needs of single-molecule sequencing. When the biological molecule to be tested passes through the overly large pore, the current signal generated may be missed or erroneous, resulting in low sequencing accuracy. In single-molecule sequencing, accurate sequencing results are obtained by sequencing the same molecule multiple times. Therefore, the higher the sequencing accuracy, the shorter the number and time of sequencing required. Using porin with high sequencing accuracy for sequencing can greatly reduce sequencing time and reduce costs. This advantage is particularly evident in high-throughput sequencing.

In a fourth typical embodiment of the present application, a kit is provided, which includes the above-mentioned porin monomer, or protein construct, or porin.

To further improve the convenience of operation, in a preferred embodiment, the kit further comprises a membrane layer, and the membrane layer comprises a lipid layer or an artificial polymer membrane.

Preferably, the lipid layer comprises amphiphilic lipids; preferably, the amphiphilic lipids comprise a phospholipid bilayer; preferably, the lipid layer comprises a planar membrane layer or a liposome; preferably, the liposome comprises a multilayer liposome or a unilamellar liposome; preferably, the lipid layer comprises a phospholipid bilayer composed of diphytylphosphatidylcholine.

Artificial polymer membranes include, but are not limited to, polysiloxanes, polyolefins, perfluoropolyethers, perfluoroalkyl polyethers, polystyrenes, polyoxypropylenes, polyvinyl acetates, polyoxybutylenes, polyisoprene, polybutadiene, polyvinyl chlorides, polyalkyl acrylates, polyalkyl methacrylates, polyacrylonitrile, polypropylene, PTHF, polymethacrylates, polyacrylates, polysulfones, polyethylene ethers, poly(propylene oxide) and copolymers thereof, substituted C1-C6 alkyl acrylates and methacrylates, acrylamides, methacrylamides, (C1-C6 alkyl) acrylamides and methacrylamides, N,N-dialkyl-acrylamides, ethoxy acrylates and methacrylates, polyethylene glycol monomethacrylates and polyethylene glycol monomethyl ether methacrylates, hydroxy-substituted (C1-C6 alkyl) acrylamides and methacrylamides, hydroxy-substituted C1-C6 alkyl vinyl ethers, sodium vinyl sulfonates, sodium styrene sulfonates, 2-acrylamide-2-methyl The invention can be one or more of 2-hydroxypropyl 2-nitropropanesulfonic acid, N-vinylpyrrole, N-vinyl-2-pyrrolidone, 2-vinyloxazoline, 2-vinyl-4,4′-bisalkyloxazolinyl-5-one, 2,4-vinylpyridine, ethylenically unsaturated carboxylic acids having a total of 3 to 5 carbon atoms, amino(C1-C6 alkyl)-, mono(C1-C6 alkylamino)(C1-C6 alkyl)- and bis(C1-C6 alkylamino)(C1-C6 alkyl)-acrylates and methacrylates, allyl alcohol, 3-trimethylammonium methacrylate 2-hydroxypropyl ester chloride, dimethylaminoethyl methacrylate (DMAEMA), dimethylaminoethyl methacrylamide, glycerol methacrylate, N-(1,1-dimethyl-3-oxobutyl)acrylamide, cyclic imino ethers, vinyl ethers, cyclic ethers containing epoxy derivatives, cyclic unsaturated ethers, N-substituted ethylenimines, β-lactones and β-lactams, vinyl ketone acetals, vinyl acetals or phosphoranes.

In a preferred embodiment, the kit further comprises a sequencing buffer and/or single-stranded DNA linked to cholesterol.

By using the porins, membrane layer and sequencing buffer in the above kit, one or more porins can be inserted into the membrane layer in the sequencing buffer to form a nanopore sensor. The nanopore experiment buffer can provide a neutral environment to maintain the stability of the porins and the membrane layer, and the metal ions contained in the nanopore experiment buffer have good conductivity. There are many options for the selection of the membrane layer. Porin can be inserted into a planar membrane layer or a spherical liposome, or on a lipid layer formed by different components to form a nanopore sensor.

The cholesterol on the single-stranded DNA, RNA and other test molecules can bind to the above-mentioned lipid layer or artificial polymer membrane, which helps the nanopore capture the sequencing library and reduces the amount of sequencing library loading. When actually used, the cholesterol in the above-mentioned kit can first bind to the test molecule and then be added to the space where the nanopore sensor is located for sequencing.

In a fifth typical embodiment of the present application, an isolated DNA molecule is provided, wherein the DNA molecule has a nucleotide sequence encoding the above-mentioned porin monomer, or encoding the above-mentioned protein construct, or encoding the above-mentioned porin.

In a preferred embodiment, the DNA molecule has a nucleotide sequence shown in SEQ ID NO: 8.

In a preferred embodiment, the DNA molecule has more than 70%, preferably more than 80%, more preferably more than 90%, and further preferably more than 95% identity with the nucleotide sequence shown in SEQ ID NO: 8 (for example, it can be 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or more, or even 99.9% or more) and encodes a protein with the same function.

SEQ ID NO: 8:

The above-mentioned DNA molecule can encode a porin monomer having the above-mentioned structure and function of the present application. The nucleotides are mutated on the basis of the sequence shown in SEQ ID NO: 8, and hybridized with the DNA molecule defined in (a) under strict conditions, and no frameshift mutation occurs. If the mutation occurs in the nucleotides encoding the porin pore, it may result in the encoding of a porin with an altered pore, affecting the pore size of the porin and the properties of the amino acid residues on the inner wall of the pore; if the mutation occurs in the nucleotides encoding the non-pore part of the protein, it may affect the folding mode, three-dimensional structure and other properties of the encoded protein, thereby affecting the physicochemical properties and stability of the protein.

"Isolated" in this application means changed "by the hand of man" from its natural state, i.e., if it occurs in nature, it is changed and/or separated from its original environment. For example, a polynucleotide or polypeptide naturally present in a living organism is not "isolated", however, the same polynucleotide or polypeptide separated from its coexisting state in its natural state is "isolated" (as the term is used in this article).

In a sixth typical embodiment of the present application, a recombinant vector is provided, which comprises the above-mentioned DNA molecule.

The above DNA molecule, i.e., the porin expression gene, is inserted into a recombinant vector, and the porin expression gene is replicated in large quantities by utilizing the ability of the recombinant vector to replicate itself in large quantities. "Recombinant" here refers to genetically engineered DNA prepared by transplanting or splicing a gene from one species into the cells of a host organism of a different species. This DNA becomes part of the host's gene structure and is replicated.

In a seventh typical embodiment of the present application, a host cell is provided, wherein the host cell is transformed with the above-mentioned recombinant vector.

The above recombinant vector is transformed into a host cell, and the host cell is used to replicate, transcribe, and translate the porin expression gene on the recombinant vector, so that a large amount of porin can be produced. The host cell includes common host cells such as Escherichia coli, yeast, mammalian cells, and insect cells. The host cell is used to fold the porin to form a correct three-dimensional structure, and a porin with normal structure and function is obtained.

In an eighth typical embodiment of the present application, a nanopore sensor is provided, which includes: a membrane layer; and a pore protein inserted in the middle of the membrane layer to form a pore, and when a voltage is applied across the membrane layer, the pore generates current; wherein the pore protein includes the above-mentioned pore protein.

The nanopore sensor in this application specifically refers to a membrane layer with porin inserted. This type of nanopore sensor inserts a porin with a pore in the membrane layer, which can fix the pore direction of the porin. When a voltage is applied across the membrane layer, the pore diameter is perpendicular to the direction of the electric field force, and the ions on both sides of the membrane layer pass through the pore of the porin under the action of the electric field force, generating current.

In a preferred embodiment, the membrane layer comprises a lipid layer or an artificial polymer membrane; preferably, the lipid layer comprises amphiphilic lipids; preferably, the amphiphilic lipids comprise a phospholipid bilayer; preferably, the lipid layer comprises a planar membrane layer or a liposome; preferably, the liposome comprises a multilayer liposome or a unilamellar liposome; preferably, the lipid layer comprises a phospholipid bilayer composed of diphytanoylphosphatidylcholine (DPhPC, 1,2-diphytanoyl-sn-glycero-3-phosphocholine).

There are many options for the selection of lipid layers. Porin can be inserted into planar membrane layers or spherical liposomes, or into lipid layers formed of different components to form nanopore sensors.

When an electric field force is applied across the membrane layer, the biomolecule to be tested passes through the pore protein through the pore under the action of the voltage. The biomolecule to be tested includes biomacromolecules carrying biological genetic information such as DNA, RNA, polypeptides or proteins. The biomolecule to be tested can carry a group molecule for modification. The group molecule includes but is not limited to cholesterol, polyethylene glycol with different polymerization degrees, biotin or fluorescent group molecules.

In a ninth typical embodiment of the present application, a nanopore sequencing device is provided, wherein the nanopore sequencing device comprises the above-mentioned nanopore sensor.

In a preferred embodiment, the nanopore sequencing device includes: an electrolytic cell, the electrolytic cell contains a sequencing buffer; a nanopore sensor, the nanopore sensor is located in the center of the electrolytic cell and divides the electrolytic cell and the sequencing buffer into a positive electrolyte region and a negative electrolyte region; a first electrode and a second electrode, the first electrode and the second electrode are respectively arranged in the positive electrolyte region and the negative electrolyte region, and the first electrode and the second electrode are connected to a signal processing chip; preferably, the first electrode and the second electrode include metal or composite electrode materials; preferably, the first electrode and the second electrode are different, and are silver and silver chloride, respectively; or the first electrode and the second electrode are the same, and are each independently selected from gold, platinum, graphene or titanium nitride.

In the nanopore sequencing device, there are an electrolytic cell containing an electrolyte, a nanopore sensor, a first electrode, and a second electrode. The nanopore sensor is placed in the center of the electrolytic cell containing an electrolyte, and the electrolytic cell is decomposed into a positive electrolyte area and a negative electrolyte area. Two electrodes are respectively provided in the two areas, and the two electrodes are used to form an electric field applied to the nanopore sensor. The biological molecules to be tested pass through the pore protein on the membrane, generating a current amplitude. By receiving this current amplitude and transmitting the current amplitude to a signal processing chip connected to the electrode. According to the difference in current amplitude, the signal processing chip, that is, the nanopore sequencing device including the signal processing chip, can perform data analysis and determination on the sequence of the biological molecules to be tested.

In the tenth typical embodiment of the present application, a sequencing method is provided, which utilizes the above-mentioned pore protein, or nanopore sensor, or nanopore sequencing device to detect and analyze the electrical signal generated when the biological molecule to be tested passes through the pore of the pore protein, so as to determine the sequence of the biological molecule to be tested.

In a preferred embodiment, the biomolecule to be detected includes any one of the following modified or unmodified biomolecules: DNA, RNA or polypeptide.

In a preferred embodiment, the biological molecule to be detected is a target nucleic acid sequence, and the sequencing method comprises: (a) contacting the target nucleic acid sequence with a nucleic acid binding protein, which controls the movement speed of the target nucleic acid sequence through the pore of the pore protein; (b) when a voltage is applied across the pore, the target nucleic acid sequence moves through the pore, and the electrical signal passing through the pore is measured, wherein different types of nucleotides generate different electrical signals when passing through the pore, thereby determining the sequence information of the target nucleic acid based on the electrical signal; preferably, the nucleic acid binding protein is selected from nucleases, polymerases, topoisomerases, ligases, helicases and single-stranded binding proteins; preferably, the electrical signal comprises an electric current.

When voltage is applied across the pore, ions in the solution pass through the central gating region of the pore protein, generating an electric current. When the target nucleic acid sequence (including DNA or RNA) moves through the pore, the electrical signal passing through the pore is measured. Since different types of nucleotides have different sizes, the degree of blockage to the current is different, so that when the analyte composed of different nucleotides passes through the pore, different current signals are generated. By analyzing this current signal, the sequence information of the target nucleic acid can be determined.

In the eleventh typical embodiment of the present application, there is provided an application of the above-mentioned porin monomer, porin, kit, DNA molecule, recombinant vector, host cell, nanopore sensor, or nanopore sequencing device in small molecule detection, DNA sequencing, RNA sequencing or polypeptide sequencing.

The above-mentioned small molecules include but are not limited to small molecule compounds such as nucleotides, amino acids, polysaccharides or vitamins.

One major advantage of nanopore sequencing over traditional sequencing is that it does not affect accuracy due to accumulated errors, so it can achieve extremely long read lengths. This can make up for the gaps that are unavoidable when assembling short sequencing fragments in traditional sequencing, determine whether long fragments are missing, repeated, inverted, or translocated in chromosomes, and cover the full length of the transcriptome, which is typically several kb in length, thus providing a new solution for scientific research such as genome assembly, structural variation, and variable splicing.

Since nanopore sequencing does not require PCR amplification, the original base modification information on the nucleic acid molecule to be tested can be retained, and the type, site and abundance of the modified base can be directly sequenced at one time. Therefore, the pore protein of the present application can also detect several nucleic acid molecules with DNA/RNA modified bases: including 5-methylcytosine (5mC), 6-methyladenine (m6A), 7-methylguanine (m7G), pseudouracil (pseudouridine, Ψ), etc. Through specific model training and algorithm development for various modified bases, nanopore sequencing can complete the identification and positioning of more modified bases, thereby constructing a more complete genome/transcriptome modification map.

In addition, from the perspective of clinical application, nanopore sequencing has the characteristics of long read length, high portability, fast sequencing speed and real-time readout, so it is suitable for major epidemic monitoring and rapid detection of pathogens (for example, large-scale epidemic operations such as Zika virus, Ebola virus, Dengue virus and Coronavirus), which is very timely. In addition to viruses, nanopore sequencing can also be used for rapid detection of other pathogens such as bacteria and fungi.

Based on the common composition of proteins and nucleic acid molecules, the nanopore sequencing platform also has great application potential in the field of protein sequencing. For example, according to the exploration that has been carried out so far, by using protein unfolding enzymes as rate control tools, the characteristic signals of proteins have been successfully observed, and the initial identification of protein types and modification states has been achieved, verifying the possibility of pore protein sequencing. In future development, by further optimizing the rate control system and developing adapted pore proteins and signal analysis algorithms, it will eventually be possible to achieve fingerprint recognition and even sequence identification of proteins at the single-molecule level.

In addition to being used in the field of sequencing, the nanopore platform can also be used as a basic detection platform, combined with sensing methods, to complete the metabolomics detection of various small and large molecules. In combination with genomics, proteomics, and metabolomics, the nanopore platform can eventually develop into a universal measurement platform that meets the needs of full-omics analysis, providing a powerful research tool for a deeper understanding of the laws of life and the mechanisms of disease occurrence.

The beneficial effects of the present application will be further explained in detail below in conjunction with specific embodiments.

Example 1 Predicted structure of AlphaFold2-Multimer of wild-type BCP20

N68之间的距离为

G63之间的距离为

图3中B示出了侧链结构的放大图。 Nine porin monomers (SEQ ID NO: 1) are non-covalently polymerized into nonamers to obtain porin BCP20, and the structure of BCP20 is predicted using AlphaFold2-Multimer. The prediction results are shown in Figures 1, 2, and 3. Figure 1 is a side view of the predicted structure of BCP20, Figure 2 is a top view of the predicted structure of BCP20, and Figure 3 is the side chain structure of important amino acids in the sensor region of the predicted structure of BCP20, showing that the three amino acids in the amino acid side chain are G63, N68, and I69, and Figure 3 A shows that the distance between I69 is

The distance between N68 is

The distance between G63 is

FIG3B shows an enlarged view of the side chain structure.

SEQ ID NO: 1:

Example 2 Construction of expression vectors for porin monomers and their mutants

By the In-fusion method, the DNA sequence encoding the porin monomer (SEQ ID NO: 8) was inserted into the multiple cloning region of the vector pET24a after digestion with NdeI and XhoI. StrepII amino acid was added to the C-terminus of the amino acid sequence of the porin monomer (SEQ ID NO: 1) as a purification tag, wherein the screening tag was kanamycin, and the constructed vector was named pET24a-BCP20. By the site-directed mutagenesis method, the Agilent site-directed mutagenesis kit was used to construct the corresponding mutants with the expression vector of the porin monomer as a template. In this application, porin monomer mutants of G63A, N68Q and I69N were constructed, and the construction method of the mutants was consistent with that of the wild type.

Example 3 Cultivation and induction of porin monomer strains

The constructed porin monomer or its mutant expression plasmids were independently transformed into the E. coli expression strain E. coli BL21 (DE3), and the bacterial solution was 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 inoculated into 5 mL LB liquid culture medium containing 50 μg/mL kanamycin, and cultured at 37°C, 200 rpm, overnight. The above-obtained bacterial solution was inoculated into 50 mL LB liquid culture medium containing 50 μg/mL kanamycin at a volume ratio of 1:100, and cultured at 37°C, 200 rpm for 4 hours. The expanded cultured bacterial solution was inoculated into 2 L LB liquid culture medium containing 50 μg/mL kanamycin at a volume ratio of 1:100 and cultured at 37°C, 200 rpm. When the _OD600 value reaches about 0.6-0.8, add IPTG with a final concentration of 0.5mM, culture at 16℃, 200rpm for about 16-18h. Collect the bacterial solution by centrifugation at 8000rpm and freeze the bacteria at -20℃ for later use.

Example 4 Extraction and purification of recombinant porin monomers

(1) Preparation of purification buffer

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

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

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

(2) Purification step

Resuspend the cells thoroughly at a ratio of 10 mL Buffer A per 1 g of cells, and ultrasonically disrupt the cells until the cell solution is clear. Then, place the cell on a rotator and rotate it at 4°C overnight. Centrifuge at 18,000 rpm at 4°C for 1 hour the next day, take the supernatant, filter it 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 obtained protein was concentrated to 1 mL, passed through a Superdex 6increase 10/300GL (Cytiva) column equilibrated with buffer B, and the target protein was collected and then stored at -80°C. The target protein obtained after purification was subjected to SDS-PAGE electrophoresis, and the results are shown in Figure 4. It includes electrophoresis bands before cooking (porin nonamer BCP20) and after cooking at 95°C (porin monomer after denaturation). The results show that the target protein is in a polymer state before cooking and in a monomer state after cooking. The SDS-PAGE results of the mutant are consistent with those of the wild-type protein, which will not be specifically displayed here.

Example 5 Library Construction

The sense strand and antisense strand of two partially complementary DNA strands (SEQ ID NO: 4) were annealed to form a linker, and then connected to the double-stranded target fragment pUC57 (SEQ ID NO: 5) to be tested 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: 6) at 25°C for 1 hour (molar concentration ratio 1:8) to form a sequencing library containing BCH105 motor protein (as shown in Figure 5). During sequencing, the sequencing library can further complementarily pair with single-stranded DNA with cholesterol (SEQ ID NO: 7, cholesterol is connected to the 5′ end of DNA) to form a structure as shown in Figure 8.

Linker sequence sense strand: S1-(iSp18) ₄ -S2.

The sequence of S1 is shown in SEQ ID NO: 2, the sequence of S2 is shown in SEQ ID NO: 3, and the structure of iSp18 is shown in Figure 9.

SEQ ID NO:2:tttttttttttttttttttttttttttttttttttttttttttt.

SEQ ID NO:3:ggttgtttctgttggtgctgatattgct.

SEQ ID NO: 4 (linker sequence antisense strand):

gcaatatcagcaccaacagaaacaacctttgaggcgagcggtcaa.

SEQ ID NO: 5:

SEQ ID NO: 6:

Example 6 Construction of nanopore biosensor using porin BCP20 and its mutants

Use a patch clamp amplifier or other electrical signal amplifier to collect current signals. According to the method disclosed in the literature (Ji Z, Guo P. Channel from bacterial virus T7 DNA packaging motor for the differentiation of peptides composed of a mixture of acidic and basic amino acids. Biomaterials. 2019 May 21; 214: 119-222), a single-channel nanopore detection system based on patch clamp and signal amplifier was constructed. Ag/AgCl electrodes were immersed in sequencing buffer and the electrodes were located in the cis and trans regions of the electrolytic cell, respectively. After diluting the porin (i.e., the porin BCP20 purified in Example 4) by a certain multiple using 1xPBS buffer, a single nanoporin BCP20 is inserted into a phospholipid bilayer composed of diacylphosphatidylcholine (DPhPC, 1,2-diphytanoyl-sn-glycero-3-phosphocholine) under the action of an external electric field force to form a nanopore biosensor. The dilution multiple is based on whether the porin is embedded in the membrane (i.e., embedded in the pore). Generally, a protein concentration of 0.1 mg/ml is used and diluted 100 times, 50 times, or other times with PBS for an attempt. If a certain dilution concentration fails to embed the pore, it is necessary to reduce the dilution multiple and continue to try until the nanoporin is successfully embedded in the membrane layer. An external voltage is applied to obtain the current amplitude value of a single porin. Figure 6 shows the biosensor current generated by the pore of the nanoporin BCP20 when voltages of 0.02V, 0.04V, 0.10V, 0.14V, and 0.18V are applied. Similarly, porin BCP20 mutants (G63A, N68Q, and I69N) also generated biosensing currents.

Example 7 Use of Porin BCP20 and Its Mutants for DNA Sequencing

The sequencing library containing the pUC57 sequence prepared in Example 5 (SEQ ID NO: 5) and single-stranded DNA with cholesterol (SEQ ID NO: 7, cholesterol is connected to the 5′ end of DNA) were mixed with sequencing buffer and added to the nanopore biosensor; 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. 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. Figure 7 shows the current changes generated when the library DNA passes through the porin BCP20 under the action of an applied voltage of 0.14V. Similarly, current changes were also generated when the library DNA passed through the porin BCP20 mutants (G63A, N68Q and I69N).

Single-stranded DNA sequence with cholesterol (SEQ ID NO: 7):

ttgaccgctcgcctc.

The above-mentioned single-stranded DNA with cholesterol can bind to the membrane, and then complement the bottom chain of the library, thereby pulling the library to the vicinity of the nanopore, as shown in Figure 8, thereby increasing the capture efficiency during nanopore sequencing.

From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: the present invention has discovered a new porin monomer, which is polymerized into porin BCP20, and the porin BCP20 and its mutants have good stability and can meet the needs of single-molecule nanopore sequencing. By using the above porin and its mutants, a nanopore sensor and a further nanopore sequencing device can be formed to realize the detection of samples such as DNA, RNA, polypeptides, and proteins.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims (22)

A porin monomer, characterized in that the porin monomer comprises: (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1; or (b) a protein mutant, wherein the amino acid sequence of the protein mutant undergoes substitution, deletion and/or addition of one or more amino acids at at least one of the following positions of the amino acid sequence shown in SEQ ID NO: 1: 68, 69, 63, 64, 65, 66, 67, 103, 107, 108, 109, 113, 116, 117, 123, 126, 153, 156, 167, 169, 201, 206, 209, 213, 216, 218, and the protein mutant has the function of forming a pore structure through polymerization; or (c) a porin monomer having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to the protein described in (a) or (b) and having the function of forming a pore structure through polymerization. The porin monomer according to claim 1, characterized in that, in (b), the type of amino acid substituted at each site is independently selected from the following: G63 mutation to G63A, G63S, or G63T; E64 mutation is E64G, E64A, E64S, E64T, E64N, or E64Q; K65 mutation to K65G, K65A, K65S, K65T, K65N, or K65Q; F66 mutation to F66G, F66A, F66S, F66T, F66N, or F66Q; A67 mutation to A67G, A67S, A67T, A67N, or A67Q; N68 mutation is N68G, N68A, N68S, N68T, or N68Q; I69 mutation is I69G, I69A, I69S, I69T, I69N or I69Q; D103 mutation is D103N, D103A, D103G, D103S, D103T, D103Q, D103R or D103K; K107 mutation is K107N, K107A, K107G, K107S, K107T, or K107Q; R109 is mutated to R109W, R109F, R109Y, R109N, R109Q, R109S, R109T, R109A or R109G; R113 mutation is R113W, R113F, R113Y, R113N, R113Q, R113S, R113T, R113A or R113G; R116 mutation to R116N, R116A, R116G, R116S, R116T, or R116Q; E117 mutation is E117N, E117A, E117G, E117S, E117T, E117Q, E117R, or E117K; E123 mutation is E123N, E123A, E123G, E123S, E123T, E123Q, E123R or E123K; K126 mutations are K126N, K126Q, K126S, K126T, K126A, or K126G; D153 mutation is D153A, D153G, D153V, D153L, D153I, D153Y, D153F or D153W; R156 is mutated to R156A, R156G, R156N, R156Q, R156S, R156T, R156D or R156E; R167 mutation to R167N, R167Q, R167S, R167T, R167A, or R167G; D169 mutation is D169N, D169Q, D169S, D169T, D169A or D169G; D201 mutated to D201N, D201Q, D201S, D201T, D201A, or D201G; R206 is mutated to R206N, R206Q, R206S, R206T, R206A, R206G, R206D or R206E; D209 is mutated to D209N, D209Q, D209S, D209T, D209A, D209G, D209R or D209K; E213 mutation is E213N, E213Q, E213S, E213T, E213A or E213G; E216 mutation is E216N, E216Q, E216S, E216T, E216A or E216G; E218 mutation is E218N, E218Q, E218S, E218T, E218A or E218G. A protein construct, characterized in that the protein construct is formed by covalently or non-covalently linking two or more porin monomers according to claim 1 or 2. A porin, characterized in that the porin is composed of 7 to 11 porin monomers according to claim 1 or 2, connected covalently or non-covalently. The porin according to claim 4, characterized in that the porin is composed of 9 porin monomers connected non-covalently. The porin according to claim 4 or 5, characterized in that the pore diameter of the porin is 0.5 to 3 nm. A kit, characterized in that the kit comprises the porin monomer according to claim 1 or 2, or the protein construct according to claim 3, or the porin according to any one of claims 4 to 6. The kit according to claim 7 is characterized in that the kit further comprises a membrane layer, and the membrane layer comprises a lipid layer or an artificial polymer membrane. The kit according to claim 7 or 8, characterized in that the kit further comprises a sequencing buffer and/or single-stranded DNA linked to cholesterol. An isolated DNA molecule, characterized in that the DNA molecule has: A nucleotide sequence encoding the porin monomer according to claim 1 or 2, or encoding the protein construct according to claim 3, or encoding the porin according to any one of claims 4 to 6. The DNA molecule according to claim 10 is characterized in that the DNA molecule has a nucleotide sequence shown in SEQ ID NO: 8. The DNA molecule according to claim 11 is characterized in that it has more than 70%, preferably more than 80%, more preferably more than 90%, and further preferably more than 95% identity with the nucleotide sequence shown in SEQ ID NO: 8 and encodes a DNA molecule with the same functional protein. A recombinant vector, characterized in that the recombinant vector comprises the DNA molecule according to any one of claims 10 to 12. A host cell, characterized in that the host cell is transformed with the recombinant vector according to claim 13. A nanopore sensor, characterized in that the nanopore sensor comprises: film layer; and a porin protein inserted in the middle of the membrane layer to form a pore, and when a voltage is applied across the membrane layer, the pore generates an electric current; Wherein, the porin comprises the porin according to any one of claims 4 to 6. The nanopore sensor according to claim 15, characterized in that the membrane layer comprises a lipid layer or an artificial polymer membrane; Preferably, the lipid layer comprises amphiphilic lipids; Preferably, the amphiphilic lipid comprises a phospholipid bilayer; Preferably, the lipid layer comprises a planar membrane layer or a liposome; Preferably, the liposomes comprise multilamellar liposomes or unilamellar liposomes; Preferably, the lipid layer comprises a phospholipid bilayer composed of diphytylphosphatidylcholine. A nanopore sequencing device, characterized in that the nanopore sequencing device comprises the nanopore sensor according to claim 15 or 16. The nanopore sequencing device according to claim 17, characterized in that the nanopore sequencing device comprises: an electrolytic cell containing a sequencing buffer; A nanopore sensor, wherein the nanopore sensor is located in the center of the electrolytic cell and divides the electrolytic cell and the sequencing buffer into a positive electrode electrolyte region and a negative electrode electrolyte region; A first electrode and a second electrode, wherein the first electrode and the second electrode are respectively arranged in the positive electrode electrolyte region and the negative electrode electrolyte region, and the first electrode and the second electrode are connected to a signal processing chip; Preferably, the first electrode and the second electrode comprise metal or composite electrode materials; Preferably, the first electrode and the second electrode are different, being silver and silver chloride respectively; or the first electrode and the second electrode are the same, and are each independently selected from gold, platinum, graphene or titanium nitride. A sequencing method, characterized in that the sequencing method uses the porin according to any one of claims 4 to 6, or the nanopore sensor according to claim 15 or 16, or the nanopore sequencing device according to claim 17 or 18 to detect and analyze the electrical signal generated when the biological molecule to be tested passes through the pore of the porin, so as to determine the sequence of the biological molecule to be tested. The sequencing method according to claim 19 is characterized in that the biological molecule to be detected includes any one of the following modified or unmodified biological molecules: DNA, RNA or polypeptide. The sequencing method according to claim 19, wherein the biomolecule to be detected is a target nucleic acid sequence, and the sequencing method comprises: (a) contacting the target nucleic acid sequence with a nucleic acid binding protein, wherein the nucleic acid binding protein controls the speed at which the target nucleic acid sequence moves through the pore of the porin; (b) when a voltage is applied across the pore, the target nucleic acid sequence moves through the pore, and an electrical signal passing through the pore is measured, wherein different types of nucleotides generate different electrical signals when passing through the pore, thereby determining the sequence information of the target nucleic acid based on the electrical signal; Preferably, the nucleic acid binding protein is selected from the group consisting of a nuclease, a polymerase, a topoisomerase, a ligase, a helicase and a single-stranded binding protein; Preferably, the electrical signal comprises an electric current. Use of the porin monomer of claim 1 or 2, the porin of any one of claims 4 to 6, the kit of any one of claims 7 to 9, the DNA molecule of any one of claims 10 to 12, the recombinant vector of claim 13, the host cell of claim 14, the nanopore sensor of claim 15 or 16, or the nanopore sequencing device of claim 17 or 18 in small molecule detection, DNA sequencing, RNA sequencing or polypeptide sequencing.

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