WO2023123370A1 - Nanopore protein and related use thereof in sequencing - Google Patents

Nanopore protein and related use thereof in sequencing Download PDF

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WO2023123370A1
WO2023123370A1 PCT/CN2021/143722 CN2021143722W WO2023123370A1 WO 2023123370 A1 WO2023123370 A1 WO 2023123370A1 CN 2021143722 W CN2021143722 W CN 2021143722W WO 2023123370 A1 WO2023123370 A1 WO 2023123370A1
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nanoporin
nanopore
protein
sequencing
electrode
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PCT/CN2021/143722
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French (fr)
Chinese (zh)
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董宇亮
胡兆龙
王子
郭斐
刘欢欢
吴蔚
季州翔
曾涛
章文蔚
徐讯
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深圳华大生命科学研究院
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Priority to PCT/CN2021/143722 priority Critical patent/WO2023123370A1/en
Publication of WO2023123370A1 publication Critical patent/WO2023123370A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/35—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the invention relates to the field of single-molecule sequencing, in particular to a nanopore protein and related applications in sequencing.
  • Biomacromolecules such as DNA, RNA, and protein are the basic substances that constitute life, and their primary sequence structure and group post-modification determine their biological functions.
  • the abundance of biological macromolecules in organisms is also closely related to biological phenotype.
  • the technology of sequencing and quantifying biological macromolecules is a core tool for understanding the laws of life.
  • the existing technologies cannot completely restore the sequence, modification, abundance and other information of these biomacromolecules.
  • nucleic acids neither the early Sanger sequencing method nor the current mainstream sequencing-by-synthesis sequencing method can achieve continuous and accurate detection of long-segment DNA sequences and direct measurement of original base modifications and RNA sequences.
  • the necessary PCR Amplification will introduce errors and biases; for proteins, the detection of their primary amino acid sequence and modification can only be satisfied in an indirect way, and no matter the Edman (Edman) degradation sequencing method or mass spectrometry sequencing method, the additional processing steps are not enough. Avoid reducing the resolution and introducing errors.
  • the traditional biomacromolecular sequencing measurement technology is ensemble measurement, which obtains averaged information and cannot reflect the individual state differences of individual molecules, which often determine the fate of cells. The direct and complete determination of the sequence, modification and abundance of a single biological macromolecule can truly restore the details of the state of the biological system, which is an important challenge for future biotechnology. On the basis of this demand, single-molecule sequencing technology emerged as the times require.
  • Single-molecule sequencing technologies are mainly divided into two categories: one is optical-based zero-mode waveguide sequencing, represented by Pacific Biosciences (Pacbio) in the United States; the other is electrical-based nanopore sequencing, represented by Oxford Nanotechnology Co. Represented by Oxford Nanopore Technologies (ONT).
  • the former is applied to DNA sequencing, and the sequence and modification information of the template strand can be inferred based on the pulsed fluorescent signal generated when the polymerase extends the DNA primer chain to synthesize new bases; the latter can be applied to both DNA and RNA sequencing.
  • the base unit on the RNA molecule is analyzed and sequenced by analyzing the continuous current signal generated when the base unit passes through the nanoporin one by one.
  • Nanopore sequencing has more advantages in sequencing speed, throughput, portability, and direct RNA sequencing, and has gained widespread attention in recent years.
  • Natural nanoporins generally have the ability to form pores, but in the in vitro expression and purification system of recombinant proteins, the stability of the pores of recombinant nanoporins may not meet the needs of single-molecule detector-related instrument products.
  • natural nanoporins have a wide range of pore size distributions, which may not necessarily meet the needs of single-molecule detection.
  • the properties of the amino acid residues in the pore walls of natural nanoporins, especially the charge properties do not necessarily meet the properties of a specific analyte.
  • the main purpose of the present invention is to provide a nanoporin and its related application in sequencing, so as to solve the problem of poor channel stability of the nanoporin in the prior art.
  • a kind of nanopore protein comprising (a) MP964, MP964 is the protein with the amino acid sequence shown in SEQ ID NO: 1; Or (b) MP964Mut, MP964Mut is a protein having the amino acid sequence shown in SEQ ID NO: 2; or (c) at least one of the following positions of the amino acid sequence in (a) or (b): 97th, 98th, and 127th , No. 143, No.
  • 148 a protein with a pore structure after substitution and/or deletion and/or addition of one or several amino acids; or (d) and any of (a), (b) and (c)
  • the defined amino acid sequence has more than 80% homology and has the same function protein.
  • the types of amino acids substituted at each site are independently selected from the following: No. 97: Q/Y/A/S/H; No. 98: Q/Y/S/H; No. 127 Bit: K/H/Y; No. 143: K/H/Y; No. 148: R/H/Y; Among them, "/" represents "or”;
  • any defined amino acid sequence has more than 85%, preferably more than 90%, more preferably more than 95%, more preferably more than 99% homology and have the same function protein; preferably, Nanoporins are derived from mycobacteria.
  • the pore diameter of the nanoporin is 1.2-1.6nm; preferably, the current amplitude value of the nanoporin at a voltage of 150mV is 145-155pA; preferably, the current amplitude value of the nanoporin at a voltage of 180mV is 170pA ⁇ 190pA; Preferably, the conductance of the nanoporin is 0.8 ⁇ 1.2nS.
  • a kit which includes the above-mentioned nanoporin.
  • the kit also includes a lipid layer or an artificial polymer membrane; preferably, the lipid layer includes amphiphilic lipids; preferably, the amphiphilic lipids include a phospholipid bilayer; preferably, the lipid layer includes a planar membrane layer or liposome; preferably, the liposome includes multilamellar liposomes or unilamellar liposomes; preferably, the lipid layer includes a phospholipid bilayer composed of diphytylphosphatidylcholine.
  • the kit also includes a nanoporin experimental buffer; preferably, the nanoporin experimental buffer is HEPES buffer; preferably, the nanoporin experimental buffer contains 0.1-1.0M KCl; preferably, the nanoporin
  • the assay buffer is 0.5M KCl, 10mM HEPES, 1mM EDTA, pH 7.8.
  • an isolated DNA molecule has (a) a nucleotide sequence encoding the above-mentioned nanoporin; or (b) under stringent conditions with (a) the nucleotide sequence of hybridization of the defined DNA molecule; or (c) has the nucleotide sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4; or (d) with (a) to (c) Any one of the nucleotide sequences defined in has more than 70% homology and encodes a DNA molecule with the same functional protein.
  • it has more than 75%, preferably more than 85%, more preferably more than 95%, and more preferably more than 99% homology with any of the nucleotide sequences defined in (a) to (c), and the encoding has the same function Protein DNA molecule.
  • a recombinant vector which comprises the above DNA molecule.
  • a host cell is provided, the host cell is transformed with the above recombinant vector.
  • a nanoporous biofilm which includes: a membrane layer; and a nanoporin inserted into the middle of the membrane layer to form a channel, when crossing the membrane When an electric field force is applied, conduction occurs in the channel; wherein, the nanoporin includes the above-mentioned nanoporin.
  • the membrane layer includes a lipid layer; preferably, the lipid layer includes amphiphilic lipids; preferably, the amphiphilic lipids include a phospholipid bilayer; preferably, the lipid layer includes a planar membrane layer or a liposome; Preferably, the liposome includes multilamellar liposomes or unilamellar liposomes; preferably, the lipid layer includes a phospholipid bilayer composed of diphytylphosphatidylcholine.
  • the nanoporin is mobile in the membrane layer; preferably, when an electric field force is applied across the membrane layer, the nanopore biomembrane can displace the biomolecules to be tested through the pores; preferably, the biomolecules to be tested include DNA, RNA, polypeptide or protein; Preferably, the biomolecule to be tested has a modified group molecule, more preferably the group molecule is selected from cholesterol, polyethylene glycol, biotin or fluorescent group molecule; Preferably, DNA and /or RNA includes any one or more of the following modified bases: 5-methylcytosine, 6-methyladenine, 7-methylguanine or pseudouracil.
  • a nanopore sequencing device includes the above-mentioned nanopore biofilm.
  • the nanopore sequencing device includes: an electrolytic cell containing an electrolyte; a nanopore biofilm located in the center of the electrolytic cell, and dividing the electrolytic cell and the electrolyte into a positive electrolyte area and a negative electrolyte area; the first electrode and The second electrode, the first electrode and the second electrode are respectively arranged in the positive electrode electrolyte area and the negative electrode electrolyte area; the receiving electrode includes two receiving electrodes respectively located in the positive electrode liquid area and the negative electrode electrolyte area, the receiving electrode and signal processing
  • the chip is connected; preferably, the electrolyte is a nanoporin experimental buffer; preferably, the nanoporin experimental buffer is a HEPES buffer; preferably, the nanoporin experimental buffer contains 0.1-1.0M KCl; more preferably,
  • the nanoporin assay buffer includes 0.5M KCl, 10mM HEPES, 1mM EDTA, pH 7.8; preferably, the first electrode and the second electrode include metal or composite
  • a sequencing method utilizes the above-mentioned nanopore protein, or the above-mentioned nanopore biofilm, or the above-mentioned nanopore sequencing device by analyzing the biomolecules to be tested through The electrical signal generated by the pores of nanoporins is used to sequence the biomolecules to be tested.
  • the biomolecules to be tested include modified or unmodified DNA, RNA, polypeptide or protein; preferably, under the action of an electric field force, a single molecule of the biomolecules to be tested passes through the pores of the nanoporin to generate electrical signals; preferably ground, the electrical signal includes a blocking current amplitude.
  • a single nanoporin is inserted into a lipid layer, preferably a phospholipid bilayer, to form a nanoporous biofilm, and the nanoporin is sequenced using the structure of the nanoporous biofilm.
  • the pore stability of the mutated nanoporin is improved, which facilitates the improvement of sequencing accuracy and data throughput in subsequent sequencing.
  • Figure 1 shows a schematic diagram of the SDS-PAGE purification results of nanoporin MP964 according to Example 5 of the present invention
  • Figure 2 shows a schematic diagram of the SDS-PAGE purification results of nanoporin MP964Mut according to Example 6 of the present invention
  • Fig. 3 shows a schematic diagram of the homology modeling three-dimensional structure of nanoporin MP964 according to Example 7 of the present invention
  • Fig. 4 shows a schematic diagram of the homology modeling three-dimensional structure of the nanoporin MP964Mut according to Example 7 of the present invention
  • Fig. 5 shows the current retardation percentage distribution graph of the oligonucleotide sample of nanoporin MP964 according to Example 8 of the present invention
  • Fig. 6 shows the current retardation percentage distribution diagram of the oligonucleotide sample of nanoporin MP964Mut according to Example 8 of the present invention
  • Figure 7 shows a statistical schematic diagram of the DNA capture rate of nanoporins MP964 and MP964Mut according to Example 8 of the present invention
  • Figure 8 shows a schematic diagram of the conductance distribution of the nanoporin MP964Mut in different experiments according to Example 9 of the present invention
  • Figure 9 shows a schematic diagram of the current characteristics of the oligonucleotide sample A according to Example 9 of the present invention passing through the nanoporin mutant MP964Mut under an applied voltage of 180mV;
  • Fig. 10 shows a schematic diagram of the percentage distribution diagram of current blockage of oligonucleotide sample A according to Example 9 of the present invention
  • Figure 11 shows a schematic diagram of the current characteristics of the oligonucleotide sample B according to Example 9 of the present invention passing through the nanoporin mutant MP964Mut under an applied voltage of 180mV;
  • Figure 12 shows a schematic diagram of the current block percentage distribution diagram of oligonucleotide sample B according to Example 9 of the present invention.
  • Fig. 13 shows a schematic diagram of controlling DNA to pass through the nanoporin mutant MP964Mut under an applied voltage of 180mV, and the current amplitudes of different amplitudes are generated as the DNA moves, wherein, graph (A) is a current characteristic graph. Figure (B) is an enlarged current diagram;
  • Fig. 14 shows that when the DNA is controlled to pass through the nanoporin mutant MP964Mut under an applied voltage of 150mV, current amplitude changes of different magnitudes are produced as the DNA moves, wherein, graph (A) is a current characteristic graph.
  • Figure (B) is an enlarged current diagram.
  • Natural nanoporins generally have the ability to form pores, but in the in vitro expression and purification system of recombinant proteins, the stability of the pores of recombinant nanoporins may not meet the needs of single-molecule detector-related instrument products. At the same time, natural nanoporins have a wide range of pore size distributions, which may not necessarily meet the needs of single-molecule detection.
  • a nanopore protein including (a) MP964, MP964 is a protein having the amino acid sequence shown in SEQ ID NO: 1; or (b) MP964Mut, MP964Mut is A protein having the amino acid sequence shown in SEQ ID NO: 2; or (c) at least one of the following positions of the amino acid sequence in (a) or (b): 97th, 98th, 127th, 143rd, 148th, a protein having a pore structure through substitution and/or deletion and/or addition of one or several amino acids; or any one of (d) and (a), (b) and (c) defined
  • the amino acid sequence has more than 80% homology and has the same function protein.
  • the nanoporin defined in (a) or (b) above has a pore structure, and when applied to nanopore sequencing, it can allow the biomolecules to be tested to pass through the pore one by one to generate a current signal.
  • MP964Mut is a nanopore protein obtained by mutation based on MP964. On the basis of (a) or (b) sequence, the protein is mutated, for example, on the basis of retaining the mutation site of MP964Mut, after substitution and/or deletion and/or addition of one or several amino acids at other positions , can still obtain and maintain the pore structure and function of the above-mentioned nanoporin.
  • Nanopore proteins may affect the stability of proteins and aggregates, the inner diameter of pores, and the amino acid residues on the inner wall of pores, thereby affecting their physical and chemical properties and the passage performance of biomolecules to be tested.
  • the methods for proteins with nanopore structure and functional activity are known to those skilled in the art, or can be realized by further combining with the similar screening steps of MP964Mut in this application.
  • amino acid sequence of MP964 is as follows: SEQ ID NO: 1:
  • the amino acid sequence of MP964Mut is as follows: SEQ ID NO: 2:
  • the types of amino acids substituted at each site are independently selected from the following: No. 97: Q/Y/A/S/H; No. 98: Q/Y/ S/H; No. 127: /K/H/Y; No. 143: K/H/Y; No.
  • homologous proteins with the same function, their properties such as protein and aggregate stability, pore inner diameter, pore inner wall amino acid residues, and the passing performance of biomolecules to be tested are all It has the same probability as the protein provided by the sequence (a) or (b), and is a homologous protein obtained by amino acid mutation.
  • the nanoporin is derived from mycobacteria.
  • the pore diameter of the nanoporin is 1.2-1.6 nm.
  • the pore diameter range is smaller than that of existing nanoporins, so the pore stability is better and the sequencing accuracy is also higher.
  • the current amplitude value of the nanoporin at a voltage of 150mV is 145-155pA; preferably, the current amplitude value of the nanoporin at a voltage of 180mV is 170-190pA; preferably, the conductance of the nanoporin is 0.8-1.2 nS.
  • the smaller the pore diameter of the nanoporin the higher the accuracy when it is used for sequencing. If the pore diameter of the nanoporin 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 biomolecule to be tested passes through the too large pore, the current signal generated may be missed or generated Errors lead to low sequencing accuracy. In single-molecule sequencing, the same molecule is sequenced multiple times to obtain accurate sequencing results. Therefore, the higher the accuracy of sequencing, the shorter the number and time required for sequencing. Using nanopore proteins with high sequencing accuracy for sequencing can greatly reduce the time and cost of sequencing, and this advantage is especially obvious in high-throughput sequencing.
  • a kit which includes the above-mentioned nanoporin.
  • the kit containing the above-mentioned nanoporin for sequencing, because the pore diameter of the above-mentioned nanoporin is relatively smaller, the pore stability is better, and the sequencing accuracy is relatively higher, so the sequencing time and cost can be reduced.
  • the kit also includes a lipid layer or an artificial polymer membrane; preferably, the lipid layer includes amphiphilic lipids; preferably, the amphiphilic lipids include phospholipids bilayer; preferably, the lipid layer comprises a planar membrane layer or a liposome; preferably, the liposome comprises a multilamellar liposome or a unilamellar liposome; preferably, the lipid layer comprises a diphytylphosphatidyl Phospholipid bilayers composed of choline.
  • the lipid layer includes amphiphilic lipids; preferably, the amphiphilic lipids include phospholipids bilayer; preferably, the lipid layer comprises a planar membrane layer or a liposome; preferably, the liposome comprises a multilamellar liposome or a unilamellar liposome; preferably, the lipid layer comprises a diphytylphosphatidyl Phospholipid bilayers composed of choline.
  • Artificial polymer films include but are not limited to polysiloxane, polyolefin, perfluoropolyether, perfluoroalkyl polyether, polystyrene, polyoxypropylene, polyvinyl acetate, polyoxybutylene, polyisoprene , polybutadiene, polyvinyl chloride, polyalkylacrylate, polyalkylmethacrylate, polyacrylonitrile, polypropylene, PTHF, polymethacrylate, polyacrylate, polysulfone, polyvinyl ether, Poly(propylene oxide) and its copolymers, radical-substituted C1-C6 alkyl acrylates and methacrylates, acrylamides, methacrylamides, (C1-C6 alkyl)acrylamides and methacrylamides, N,N-dialkyl-acrylamides, ethoxylated acrylates and methacrylates, polyethylene glycol monomethacrylate and polyethylene glycol mono
  • the kit also includes a nanoporin experimental buffer; preferably, the nanoporin experimental buffer is a HEPES buffer; preferably, the nanoporin experimental buffer
  • the solution contains 0.1-1.0M KCl; preferably, the nanopore assay buffer includes 0.5M KCl, 10mM 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), 1mM ethylenediaminetetraacetic acid (EDTA), pH 7.8.
  • nanoporin lipid layer and nanoporin experimental buffer in the above kit
  • one or more nanoporins can be inserted into the lipid layer in the nanoporin experimental buffer to form a nanoporous organism membrane.
  • the nanopore experimental buffer can provide a neutral environment to maintain the stability of the nanopore protein and lipid layer, and the metal ions contained in it make the nanopore experimental buffer have good conductivity.
  • lipid layer There are many options for the choice of lipid layer.
  • nanoporin can be inserted to form a nanopore biomembrane.
  • an isolated DNA molecule has (a) a nucleotide sequence encoding the aforementioned nanoporin; or (b) under stringent conditions with (a ) the nucleotide sequence of the hybridized DNA molecule as defined; or (c) has the nucleotide sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4; or (d) is defined in (a) to (c) Any nucleotide sequence has more than 70% (preferably more than 80%, more preferably more than 85%, more preferably more than 90%, most preferably more than 95%, such as 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, Even more than 99.9%) homologous DNA molecules that encode proteins with the same function.
  • the above-mentioned DNA molecule can encode the nanopore protein having the above-mentioned structure and function of the present application.
  • the gene shares more than 75%, preferably more than 85%, more preferably more than 95%, and more preferably more than 99% of any nucleotide sequence defined in (a) to (c). DNA molecules that are homologous and encode proteins with the same function.
  • DNA molecules with 75%, 85%, 90%, 95%, 99% or more homology and encoding nanopore proteins with the same function, the active sites, active pockets, and active mechanisms of the encoded proteins are equal to (a)
  • the genes provided by the sequences have the same high probability and are homologous genes obtained through nucleotide mutation.
  • isolated in this application means altered “by the hand of man” from its natural state, ie, if it occurs in nature, it is altered and/or separated from its original environment.
  • a polynucleotide or polypeptide naturally occurring in a living organism is not “isolated”, whereas the same polynucleotide or polypeptide separated from its natural state coexistence is “isolated” (as used herein the term).
  • the DNA molecule in the present invention hybridizes with the gene encoding the nanoporin of the present invention under "stringent conditions", which refers to the conditions under which the presence of the gene encoding the nanopore protein of the present invention can be identified by means of nucleic acid hybridization.
  • stringent conditions refers to the conditions under which the presence of the gene encoding the nanopore protein of the present invention can be identified by means of nucleic acid hybridization.
  • two DNA molecules can form an antiparallel double-stranded nucleic acid structure, it can be said that the two DNA molecules can specifically hybridize to each other.
  • One DNA molecule is said to be the "complement” of the other if two DNA molecules exhibit perfect complementarity.
  • two DNA molecules are said to be "complementary” if they are capable of hybridizing to each other with sufficient stability so that they anneal and bind to each other under conventional "high stringency" conditions. Deviations from perfect complementarity are permissible as long as the deviation does not completely prevent the two molecules from forming a double-stranded structure.
  • a DNA molecule In order for a DNA molecule to serve as a primer or probe, it only needs to be sufficiently complementary in sequence to form a stable double-stranded structure under the particular solvent and salt concentration employed.
  • a substantially homologous sequence is a DNA molecule that can specifically hybridize with a matching complementary strand of another DNA molecule under highly stringent conditions.
  • Suitable stringent conditions to promote DNA hybridization for example, treatment with 6.0 ⁇ sodium chloride/sodium citrate (SSC) at about 45° C., followed by washing with 2.0 ⁇ SSC at 50° C., are known to those skilled in the art. is well known.
  • the salt concentration in the washing step can be selected from about 2.0 ⁇ SSC, 50°C for low stringency conditions to about 0.2 ⁇ SSC, 50°C for high stringency conditions.
  • the temperature conditions in the washing step can be increased from about 22°C at room temperature for low stringency conditions to about 65°C for high stringency conditions.
  • Both the temperature condition and the salt concentration can be changed, or one can be kept constant while the other variable is changed.
  • the stringent conditions in the present invention can specifically hybridize with the nucleotide sequence encoding the nanoporin of the present application in 6 ⁇ SSC, 0.5% SDS solution at 65° C., and then use 2 ⁇ SSC, Wash the membrane once with 0.1% SDS, 1 ⁇ SSC, and 0.1% SDS.
  • a recombinant vector comprises the above-mentioned DNA molecule, that is, a nanoporin expression gene.
  • the nanoporin expression gene is inserted into the recombinant vector, and the nanoporin expression gene is copied in large quantities by utilizing the self-replicating function of the recombinant vector.
  • "Recombinant” herein refers to genetically engineered DNA prepared by transplanting or splicing genes from one species into cells of a host organism of a different species. This DNA becomes part of the host's genetic makeup and is replicated.
  • a host cell transformed with the above-mentioned recombinant vector is provided.
  • the above recombinant vector is transformed into a host cell, and the host cell is used to replicate, transcribe, and translate the nanoporin expression gene on the recombinant vector, so that a large amount of nanoporin can be produced.
  • Host cells include common host cells such as Escherichia coli, yeast, mammalian cells, and insect cells. The host cells are used to fold the nanoporin to form a correct three-dimensional structure, and obtain a nanoporin with normal structure and function.
  • a nanoporous biofilm which includes: a membrane layer; and a nanoporin inserted into the middle of the membrane layer to form a channel, when an electric field is applied across the membrane layer When the force is applied, the conduction occurs in the channel; wherein, the nanoporin includes the above-mentioned nanoporin.
  • the nanoporous biomembrane in this application specifically refers to the membrane layer with nanoporin inserted.
  • This kind of nanoporous biomembrane inserts nanoporin with pores in the membrane layer, which can fix the direction of the pores of the nanoporin.
  • the diameter of the pores is perpendicular to the direction of the electric field, and the pores Conductance occurs to generate an electrical signal.
  • the membrane layer comprises a lipid layer; 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 liposome; preferably, the liposome includes multilamellar liposomes or unilamellar liposomes; preferably, the lipid layer includes a phospholipid bilayer composed of diphytylphosphatidylcholine.
  • lipid layer There are many options for the choice of lipid layer.
  • nanoporin can be inserted to form a nanopore biomembrane.
  • the nanoporin is mobile within the membrane layer; preferably, the nanoporous biomembrane is capable of translocating the biomolecules to be tested through the pores when an electric field force is applied across the membrane layer; preferably , the biomolecules to be tested include DNA, RNA, polypeptide or protein; preferably, the biomolecules to be tested have modified group molecules, more preferably the group molecules are selected from cholesterol, polyethylene glycol with different degrees of polymerization, biotin or Fluorophore molecules; preferably, DNA and/or RNA include any one or more of the following modified bases: 5-methylcytosine (5mC), 6-methyladenine (m6A), 7-methylguanine Purine (m7G), pseudouracil (pseudouridine, ⁇ ).
  • 5mC 5-methylcytosine
  • m6A 6-methyladenine
  • m7G 7-methylguanine Purine
  • pseudouracil pseudouracil
  • the nanoporin is movable in the membrane layer, and can automatically adjust its position and orientation under the action of an electric field.
  • the biomolecules to be tested pass through the nanoporin through the pores under the action of the electric field force.
  • the biomolecules to be tested include biomacromolecules such as DNA, RNA, polypeptide or protein that carry biological genetic information.
  • the biomolecules to be tested can have moieties for modification. Molecules include, but are not limited to, cholesterol, polyethylene glycols of different degrees of polymerization, biotin, or fluorophore molecules.
  • a nanopore sequencing device is provided, and the nanopore sequencing device includes the above-mentioned nanopore biofilm.
  • the nanopore sequencing device can be used to perform single-molecule sequencing of the biomolecules to be tested.
  • the nanopore sequencing device includes: an electrolytic cell containing an electrolyte; a nanopore biofilm located in the center of the electrolytic cell and dividing the electrolytic cell and the electrolyte into a positive electrolyte area and a negative electrolyte area; the first electrode and the second electrode, the first electrode and the second electrode are respectively arranged in the positive electrode electrolyte area and the negative electrode electrolyte area; the receiving electrode includes two and are respectively located in the positive electrode liquid area and the negative electrode electrolyte area, the The receiving electrode is connected to the signal processing chip; preferably, the electrolyte is a nanopore experimental buffer; preferably, the nanoporin experimental buffer is a HEPES buffer; preferably, the nanoporin experimental buffer contains 0.1-1.0M KCl; More preferably, nanopore experiment buffer comprises 0.5M KCl, 10mM HEPES, 1mM EDTA, pH 7.8; Preferably, first electrode and second electrode comprise metal or composite electrode material; Preferably,
  • the nanopore sequencing device includes an electrolytic cell containing electrolyte, a nanopore biomembrane, a first electrode and a second electrode. Put the nanoporous biofilm into the center of the electrolytic cell containing the electrolyte, and decompose the electrolytic cell to form the positive electrolyte area and the negative electrolyte area. The two areas are respectively equipped with two electrodes, and the two electrodes are used to form and apply to the nanoporous biofilm. The electric field on the membrane.
  • the biomolecules to be tested pass through the nanopore proteins on the membrane, generating a current amplitude. The current amplitude can be received by the receiving electrode, and the current amplitude is transmitted to a signal processing chip connected to the receiving 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 biomolecules to be tested.
  • a sequencing method uses the above-mentioned nanopore protein, or nanopore biomembrane, or nanopore sequencing device to analyze the biomolecules passing through the nanopore protein.
  • the electrical signal generated during the pore channel is used to sequence the biomolecules to be tested.
  • the biomolecules to be tested include modified or unmodified DNA, RNA, polypeptide or protein; preferably, under the action of an electric field force, a single molecule of the biomolecules to be tested passes through the pores of the nanoporin , generating an electrical signal; preferably, the electrical signal includes a blocking current amplitude.
  • a single nanoporin is inserted into a lipid layer, preferably a phospholipid bilayer, to form a nanoporous biomembrane, and the nanoporin is sequenced using the structure of the nanoporous biomembrane.
  • the accuracy will not be affected by the accumulation of errors, so extremely long read lengths can be achieved.
  • it can make up for the unavoidable gap (Gap) problem in the assembly of short sequencing fragments in traditional sequencing, determine whether there are deletions, duplications, inversions, and translocations of long fragments in the chromosome, and cover the entire length of the transcriptome with a typical length of several kb, so as to provide Scientific research such as genome assembly, structural variation, and alternative splicing provides new solutions.
  • Gap unavoidable gap
  • nanopore sequencing does not require PCR amplification, the original base modification information on the nucleic acid molecule to be tested can be retained, and then the type, site and abundance of the modified base can be directly obtained through one-time sequencing. Therefore, the nanoporin 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, ⁇ ), etc.
  • 5mC 5-methylcytosine
  • m6A 6-methyladenine
  • m7G 7- Methylguanine
  • pseudouracil pseudouracil
  • 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, Zika virus ) virus, Ebola virus (Ebola virus), Dengue virus (Dengue virus) and novel coronavirus (Coronavirus) and other large-scale epidemic operations), very time-sensitive.
  • pathogens for example, Zika virus
  • Ebola virus Ebola virus
  • Dengue virus Dengue virus
  • coronavirus novel coronavirus
  • nanopore sequencing can also be used for the rapid detection of other pathogens such as bacteria and fungi.
  • the nanopore sequencing platform 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 explorations that have been carried out so far, it can be known that by using protein unfolding enzyme as a rate-controlling tool, the characteristic signal of protein was successfully observed, and the preliminary identification of protein type and modification state was realized, which verified the possibility of nanopore protein sequencing. In future development, by further optimizing the rate control system and developing adapted nanopore proteins and signal analysis algorithms, the fingerprinting and even sequence identification of proteins at the single-molecule level can be finally realized.
  • the nanopore platform can also be used as a basic detection platform, combined with sensing means, to complete the metabolomic detection of various small molecules and macromolecules. Combining genomics, proteomics, and metabolomics, the nanopore platform can eventually develop into a general-purpose measurement platform that meets the needs of full-omics analysis, providing a powerful research tool for a deeper understanding of the law of life and the mechanism of disease occurrence .
  • the gene sequence (SEQ ID NO: 3) encoded by nanoporin MP964 was inserted into the cloning region of the vector pET24a after digestion with NdeI and XhoI.
  • the six His at the N-terminal of the amino acid sequence of MP964 were used as purification tags, the screening tag in the expression vector was kanamycin, and the constructed vector was named pET24a-MP964.
  • the Agilent site-directed mutagenesis kit was used, and the expression vector of nanoporin MP964 was used as a template to construct the expression gene of the corresponding mutant protein MP964Mut.
  • the N-terminal 6 His of the amino acid sequence of the nanoporin MP964 mutant was used as a purification tag, the screening tag in the expression vector was kanamycin, and the constructed vector was named pET24a-MP964Mut.
  • Embodiment 3 expresses the cultivation and induction of MP964 bacterial strain
  • LB liquid medium tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L.
  • the recombinant expression vector pET24a-MP964 was transformed into Escherichia coli expression strain E.coli BL21(DE3), and the bacterial solution was evenly spread on a plate with 50 ⁇ g/mL kanamycin, and cultured overnight at 37°C. Pick a single colony and culture it in 5 mL LB liquid medium (containing 50 ⁇ g/mL kanamycin) at 37°C, 200 rpm, and culture overnight. Inoculate the bacterial solution obtained above into 50 mL LB (containing 50 ⁇ g/mL kanamycin) at a ratio of 1:100 and culture at 37°C, 200 rpm, for 4 hours.
  • OD600 value reaches about 0.6-0.8
  • IPTG isopropyl- ⁇ -D-thiogalactoside
  • Embodiment 4 expresses the cultivation and induction of MP964Mut bacterial strain
  • LB liquid medium tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L.
  • the recombinant expression vector pET24a-MP964Mut was transformed into E. coli expression strain E.coli BL21(DE3), and the bacterial solution was evenly spread on a plate with 50 ⁇ g/mL kanamycin, and cultivated overnight at 37°C. Pick a single colony and culture it in 5 mL LB liquid medium (containing 50 ⁇ g/mL kanamycin) at 37°C, 200 rpm, and culture overnight. Inoculate the bacterium solution obtained above into 50 mL LB liquid medium (containing 50 ⁇ g/mL kanamycin) at a ratio of 1:100 and culture at 37°C, 200 rpm, for 4 hours.
  • Buffer A equilibration buffer 20mM Tris-HCl+250mM NaCl+0.5% Tween-20+5% glycerol, pH 7.9.
  • Buffer B elution buffer 20mM Tris-HCl+250mM NaCl+0.5% Tween-20+5% glycerol+500mM imidazole, pH 7.9.
  • Buffer C equilibration buffer 20mM Tris-HCl+50mM NaCl+0.5% Tween-20+5% glycerol, pH 6.5.
  • Buffer D elution buffer 20mM Tris-HCl+1000mM NaCl+0.5% Tween-20+5% glycerol, pH 6.5.
  • Buffer E dilution 20mM Tris-HCl+0.5% Tween-20+5% glycerol, pH 6.5.
  • the single-channel current detection in this experiment is based on Axon Digidata 1550B low-noise data acquisition system and Axopatch 200B patch clamp amplifier.
  • a phospholipid bilayer composed of diphytylphosphatidylcholine (DPhPC, 1,2-diphytanoyl-sn-glycero-3-phosphocholine) was formed in the center of the electrolytic cell containing 150 ⁇ m small pores of Teflon material; the Ag and AgCl electrodes were respectively Placed in the positive electrolyte area and the negative electrolyte area of the electrolytic cell separated by the phospholipid bilayer membrane, and the two areas are filled with nanopore experimental buffer (0.5M KCl, 10mM HEPES, 1mM EDTA, pH 7.8).
  • nanopore experimental buffer 0.5M KCl, 10mM HEPES, 1mM EDTA, pH 7.8.
  • DNA passes through the nanoporin under the action of an electric field force, resulting in a blocking current amplitude.
  • FIG. 5 for the percentage distribution of the current blockade of the nanoporin MP964 to the oligonucleotide sample
  • FIG. 6 for the percentage distribution of the current blockade of the nanopore protein MP964Mut to the oligonucleotide sample.
  • Figure 7 for the statistical comparison of the DNA capture rates of nanoporins MP964 and MP964Mut. It shows that compared with MP964, the mutated nanopore protein MP964Mut has better DNA capture ability.
  • G-quadruplex is a high-level structure folded by DNA containing tandem repeated guanine. It exists widely in prokaryotic and eukaryotic cells and participates in multiple functions such as gene replication, recombination, and regulation. Plays an important role in the life activities of cells. Therefore, the basic research on the structure and biological function of G-quadruplex is of great significance.
  • the single channel current detection of this embodiment is based on Axon Digidata 1550B low noise data acquisition system and Axopatch200B patch clamp amplifier.
  • a phospholipid bilayer composed of diphytylphosphatidylcholine (DPhPC, 1,2-diphytanoyl-sn-glycero-3-phosphocholine) is formed in the center of the electrolytic cell containing 150 ⁇ m small pores of Teflon material; Ag/AgCl electrodes are placed In the positive and negative electrolyte areas of the electrolyzer separated by a phospholipid bilayer membrane, the two areas are filled with nanopore experimental buffer (0.5M KCl, 10mM HEPES, 1mM EDTA, pH 7.8).
  • nanopore experimental buffer 0.5M KCl, 10mM HEPES, 1mM EDTA, pH 7.8.
  • Figure 8 shows the conductance distribution of the nanopore protein MP964Mut in different batches of experiments, which is used to investigate the uniformity of the nanopore.
  • the distribution of conductivity in Figure 8 has an obvious main peak, which reflects the uniformity of the opening current in the single-molecule experiment. The more concentrated the distribution of the opening current, the more uniform the formed pores, and the more stable the pores. good.
  • Oligonucleotide sample A (SEQ ID NO: 5: ggttggtgtggttgg) or sample B (SEQ ID NO: 6: ggttggtgtggttggttttttttttttt) was dissolved in nanopore buffer and annealed, cooled to 4°C before use. After a single nanoporin is inserted into the phospholipid bilayer, add an appropriate amount of oligonucleotide sample A or oligonucleotide sample B to the positive electrolyte area of the electrolytic cell. DNA passes through the nanopore under the action of an electric field force, generating a blocking current amplitude.
  • Quadruplex DNA polymers with different structures produce different amplitudes of blocking currents, so the current amplitudes can be used to distinguish G-quadruplexes composed of different sequences.
  • the percent current block is the ratio of the blocked current (Ib) to the open pore current (Io) associated with the porosity event. The results are shown in Figure 9, Figure 10, Figure 11 and Figure 12.
  • Example 10 The nanopore biofilm was constructed using nanopore protein MP964Mut and used for DNA sequencing.
  • Plasmid pUC57 was digested with EcoRI and HindIII restriction enzymes at 37°C for 2 hours; then purified using 0.4-0.6X AMPure XP magnetic beads (Beckman) to obtain double-stranded DNA fragments with high purity.
  • a sequencing library was constructed for the target DNA fragments using the ligation sequencing 109 kit (SQK-LSK109, Oxford Nanopore Technologies Ltd). Sequencing buffer: 0.5M KCl, 10 mM HEPES, 1 mM EDTA, 10 mM MgCl 2 , 2 mM ATP, pH 7.8.
  • the sequencing library containing the pUC57 sequence and the single-stranded DNA with cholesterol (FLT reagent) were mixed with the sequencing buffer and added to the nanopore sequencing device; after applying an applied voltage of 150mV or 180mV, it was observed that the DNA was captured by the nanopore, resulting in characteristic The amplitude value of the blocking current. And as the helicase moves the DNA through the nanopore, the amplitude of the current changes stepwise. Different DNA sequences produce different blocking current amplitude values.
  • the single-stranded DNA with cholesterol can be combined with the phospholipid bilayer, which helps the nanopore capture the sequencing library and reduces the loading amount of the sequencing library.
  • FIG. 13 shows that the helicase controls DNA passing through the nanoporin mutant MP964Mut under an applied voltage of 180mV, and current amplitudes of different magnitudes are generated as the DNA moves.
  • FIG. 13(A) is a current characteristic diagram
  • FIG. 13(B) is an enlarged current diagram.
  • FIG. 14 shows that the helicase controls DNA to pass through the nanoporin mutant MP964Mut under an applied voltage of 150 mV, and the current amplitude changes with different amplitudes are produced as the DNA moves.
  • FIG. 14(A) is a current characteristic diagram
  • FIG. 14(B) is an enlarged current diagram.
  • nanopore protein MP964Mut can construct a successful nanopore biofilm, and can realize the sequencing of DNA fragments.

Abstract

Provided in the present invention are a nanopore protein and the related use thereof in sequencing. The nanopore protein comprises: (a) a MP964 protein; or (b) a MP964 MUT protein; or (c) a protein subjected to substitution and/or deletion and/or addition of one or more amino acids at at least one of the following positions of the amino acid sequence of the protein in (a) or (b) and having a pore channel structure: position 97, position 98, position 127, position 143, and position 148; or (d) a protein having 80% or more homology with the amino acid sequence of the protein defined in any one of (a), (b) and (c) and having same function as the protein.

Description

纳米孔蛋白及其在测序中的相关应用Nanopore proteins and their related applications in sequencing 技术领域technical field
本发明涉及单分子测序领域，具体而言，涉及一种纳米孔蛋白及其在测序中的相关应用。The invention relates to the field of single-molecule sequencing, in particular to a nanopore protein and related applications in sequencing.
背景技术Background technique
DNA、RNA、蛋白质等生物大分子是构成生命体的基础物质，其一级序列结构及基团后修饰(post-modification)决定了其生物学功能。生物大分子在生物体内的丰度也与生物表型紧密相关。对生物大分子进行测序和定量的技术，是理解生命运行规律的核心工具。然而，当前已有的技术并不能完整还原这些生物大分子的序列、修饰、丰度等信息。例如，对于核酸，早期的桑格(Sanger)测序法与现在主流的边合成边测序测序法均无法实现长片段DNA序列的连续准确检测与原始碱基修饰及RNA序列的直接测量，必需的PCR扩增会引入错误与偏好性；对于蛋白质，其一级氨基酸序列及修饰的检测只能通过间接的方式满足，无论埃德曼(Edman)降解测序法或质谱测序法，增加的处理步骤都不可避免得会降低分辨率，引入误差。此外，传统生物大分子测序测量技术为系宗(ensemble)测量，获得的是平均化信息，无法反应单个分子的个体状态差异，而这种差异往往决定着细胞的命运。对单个生物大分子的序列、修饰与丰度的直接完整测定，可真实还原生物***状态细节，是未来生物技术的重要挑战。在此需求基础上，单分子测序技术应运而生。Biomacromolecules such as DNA, RNA, and protein are the basic substances that constitute life, and their primary sequence structure and group post-modification determine their biological functions. The abundance of biological macromolecules in organisms is also closely related to biological phenotype. The technology of sequencing and quantifying biological macromolecules is a core tool for understanding the laws of life. However, the existing technologies cannot completely restore the sequence, modification, abundance and other information of these biomacromolecules. For example, for nucleic acids, neither the early Sanger sequencing method nor the current mainstream sequencing-by-synthesis sequencing method can achieve continuous and accurate detection of long-segment DNA sequences and direct measurement of original base modifications and RNA sequences. The necessary PCR Amplification will introduce errors and biases; for proteins, the detection of their primary amino acid sequence and modification can only be satisfied in an indirect way, and no matter the Edman (Edman) degradation sequencing method or mass spectrometry sequencing method, the additional processing steps are not enough. Avoid reducing the resolution and introducing errors. In addition, the traditional biomacromolecular sequencing measurement technology is ensemble measurement, which obtains averaged information and cannot reflect the individual state differences of individual molecules, which often determine the fate of cells. The direct and complete determination of the sequence, modification and abundance of a single biological macromolecule can truly restore the details of the state of the biological system, which is an important challenge for future biotechnology. On the basis of this demand, single-molecule sequencing technology emerged as the times require.
单分子测序技术主要分为两类：一类是基于光学的零模波导测序，以美国太平洋生物科学公司(Pacific Biosciences，Pacbio)为代表；一类是基于电学的纳米孔测序，以英国牛津纳米孔技术公司(Oxford Nanopore Technologies，ONT)为代表。其中，前者应用于DNA测序，依据聚合酶延伸DNA引物链合成新碱基时产生的脉冲荧光信号来推测模板链的序列与修饰信息；后者则可同时应用于DNA与RNA测序，通过对DNA/RNA分子上碱基单元逐一通过纳米孔蛋白时产生的连续电流信号进行解析而测序。纳米孔测序在测序速度、通量、便携性、直接RNA测序上更有优势，近年来获得了广泛关注。Single-molecule sequencing technologies are mainly divided into two categories: one is optical-based zero-mode waveguide sequencing, represented by Pacific Biosciences (Pacbio) in the United States; the other is electrical-based nanopore sequencing, represented by Oxford Nanotechnology Co. Represented by Oxford Nanopore Technologies (ONT). Among them, the former is applied to DNA sequencing, and the sequence and modification information of the template strand can be inferred based on the pulsed fluorescent signal generated when the polymerase extends the DNA primer chain to synthesize new bases; the latter can be applied to both DNA and RNA sequencing. The base unit on the RNA molecule is analyzed and sequenced by analyzing the continuous current signal generated when the base unit passes through the nanoporin one by one. Nanopore sequencing has more advantages in sequencing speed, throughput, portability, and direct RNA sequencing, and has gained widespread attention in recent years.
目前，仅有耻垢分枝杆菌孔蛋白A(MspA)、curli-特异性转运通道(CsgG)等少数几种天然蛋白符合需求。天然的纳米孔蛋白一般具有成孔道的能力，但是在重组蛋白的体外表达纯化***中，重组纳米孔蛋白的孔道稳定性不一定可以满足单分子检测器相关仪器产品的需求。同时，天然纳米孔蛋白的孔径分布范围较广，不一定满足单分子检测的需求。天然纳米孔蛋白孔道内壁氨基酸残基的性质，尤其是带电性质，不一定满足特定待测物的性质。Currently, only a few natural proteins such as Mycobacterium smegmatis porin A (MspA) and curli-specific transport channel (CsgG) meet the demand. Natural nanoporins generally have the ability to form pores, but in the in vitro expression and purification system of recombinant proteins, the stability of the pores of recombinant nanoporins may not meet the needs of single-molecule detector-related instrument products. At the same time, natural nanoporins have a wide range of pore size distributions, which may not necessarily meet the needs of single-molecule detection. The properties of the amino acid residues in the pore walls of natural nanoporins, especially the charge properties, do not necessarily meet the properties of a specific analyte.
发明内容Contents of the invention
本发明的主要目的在于提供一种纳米孔蛋白及其在测序中的相关应用，以解决现有技术中纳米孔蛋白的孔道稳定性差的问题。The main purpose of the present invention is to provide a nanoporin and its related application in sequencing, so as to solve the problem of poor channel stability of the nanoporin in the prior art.
为了实现上述目的，根据本发明的第一个方面，提供了一种纳米孔蛋白，包括(a)MP964，MP964为具有SEQ ID NO：1所示的氨基酸序列的蛋白质；或(b)MP964Mut，MP964Mut为具有SEQ ID NO：2所示的氨基酸序列的蛋白质；或(c)在(a)或(b)中的氨基酸序列的如下至少一个位点：第97位、第98位、第127位、第143位、第148位，经过取代和/或缺失和/或添加一个或几个氨基酸且具有孔道结构的蛋白质；或(d)与(a)、(b)和(c)中任一所限定的氨基酸序列具有80％以上同源性且具有相同功能的蛋白质。In order to achieve the above object, according to the first aspect of the present invention, a kind of nanopore protein is provided, comprising (a) MP964, MP964 is the protein with the amino acid sequence shown in SEQ ID NO: 1; Or (b) MP964Mut, MP964Mut is a protein having the amino acid sequence shown in SEQ ID NO: 2; or (c) at least one of the following positions of the amino acid sequence in (a) or (b): 97th, 98th, and 127th , No. 143, No. 148, a protein with a pore structure after substitution and/or deletion and/or addition of one or several amino acids; or (d) and any of (a), (b) and (c) The defined amino acid sequence has more than 80% homology and has the same function protein.
进一步地，(c)中，各位点取代的氨基酸的类型各自独立地选自如下：第97位：Q/Y/A/S/H；第98位：Q/Y/S/H；第127位：K/H/Y；第143位：K/H/Y；第148位：R/H/Y；其中，“/”代表“或”；优选地，(d)中，与(a)、(b)和(c)中任一所限定的氨基酸序列具有85％以上，优选90％以上，更优选95％以上，进一步优选99％以上同源性且具有相同功能的蛋白质；优选地，纳米孔蛋白来源于分枝杆菌。Further, in (c), the types of amino acids substituted at each site are independently selected from the following: No. 97: Q/Y/A/S/H; No. 98: Q/Y/S/H; No. 127 Bit: K/H/Y; No. 143: K/H/Y; No. 148: R/H/Y; Among them, "/" represents "or"; Preferably, in (d), and (a) , (b) and (c) any defined amino acid sequence has more than 85%, preferably more than 90%, more preferably more than 95%, more preferably more than 99% homology and have the same function protein; preferably, Nanoporins are derived from mycobacteria.
进一步地，纳米孔蛋白的孔道直径为1.2～1.6nm；优选地，纳米孔蛋白在150mV电压下的电流振幅值为145～155pA；优选地，纳米孔蛋白在180mV电压下的电流振幅值为170～190pA；优选地，纳米孔蛋白的电导为0.8～1.2nS。Further, the pore diameter of the nanoporin is 1.2-1.6nm; preferably, the current amplitude value of the nanoporin at a voltage of 150mV is 145-155pA; preferably, the current amplitude value of the nanoporin at a voltage of 180mV is 170pA ~190pA; Preferably, the conductance of the nanoporin is 0.8~1.2nS.
为了实现上述目的，根据本发明的第二个方面，提供了一种试剂盒，该试剂盒包括上述纳米孔蛋白。In order to achieve the above purpose, according to the second aspect of the present invention, a kit is provided, which includes the above-mentioned nanoporin.
进一步地，试剂盒还包括脂质层或人造高分子膜；优选地，脂质层包括两亲脂类；优选地，两亲脂类包含磷脂双分子层；优选地，脂质层包括平面膜层或脂质体；优选地，脂质体包括多层脂质体或单层脂质体；优选地，脂质层包括二植酰磷脂酰胆碱组成的磷脂双分子层。Further, the kit also includes a lipid layer or an artificial polymer membrane; preferably, the lipid layer includes amphiphilic lipids; preferably, the amphiphilic lipids include a phospholipid bilayer; preferably, the lipid layer includes a planar membrane layer or liposome; preferably, the liposome includes multilamellar liposomes or unilamellar liposomes; preferably, the lipid layer includes a phospholipid bilayer composed of diphytylphosphatidylcholine.
进一步地，试剂盒还包括纳米孔蛋白实验缓冲液；优选地，纳米孔蛋白实验缓冲液为HEPES缓冲液；优选地，纳米孔蛋白实验缓冲液含有0.1-1.0M KCl；优选地，纳米孔蛋白实验缓冲液为0.5M KCl，10mM HEPES，1mM EDTA，pH 7.8。Further, the kit also includes a nanoporin experimental buffer; preferably, the nanoporin experimental buffer is HEPES buffer; preferably, the nanoporin experimental buffer contains 0.1-1.0M KCl; preferably, the nanoporin The assay buffer is 0.5M KCl, 10mM HEPES, 1mM EDTA, pH 7.8.
为了实现上述目的，根据本发明的第三个方面，提供了一种分离的DNA分子，该DNA分子具有(a)编码上述纳米孔蛋白的核苷酸序列；或(b)在严格条件下与(a)限定的DNA分子杂交的核苷酸序列；或(c)具有SEQ ID NO：3或SEQ ID NO：4所示的核苷酸序列；或(d)与(a)至(c)中限定的任一种核苷酸序列具有70％以上同源性且编码具有相同功能蛋白质的DNA分子。In order to achieve the above object, according to a third aspect of the present invention, an isolated DNA molecule is provided, the DNA molecule has (a) a nucleotide sequence encoding the above-mentioned nanoporin; or (b) under stringent conditions with (a) the nucleotide sequence of hybridization of the defined DNA molecule; or (c) has the nucleotide sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4; or (d) with (a) to (c) Any one of the nucleotide sequences defined in has more than 70% homology and encodes a DNA molecule with the same functional protein.
进一步地，与(a)至(c)中限定的任一种核苷酸序列具有75％以上，优选85％以上，更优选95％以上，进一步优选99％以上同源性且编码具有相同功能蛋白质的DNA分子。Further, it has more than 75%, preferably more than 85%, more preferably more than 95%, and more preferably more than 99% homology with any of the nucleotide sequences defined in (a) to (c), and the encoding has the same function Protein DNA molecule.
为了实现上述目的，根据本发明的第四个方面，提供了一种重组载体，该重组载体包含上述DNA分子。In order to achieve the above object, according to the fourth aspect of the present invention, a recombinant vector is provided, which comprises the above DNA molecule.
为了实现上述目的，根据本发明的第五个方面，提供了一种宿主细胞，该宿主细胞转化有上述重组载体。In order to achieve the above object, according to the fifth aspect of the present invention, a host cell is provided, the host cell is transformed with the above recombinant vector.
为了实现上述目的，根据本发明的第六个方面，提供了一种纳米孔生物膜，该纳米孔生物膜包括：膜层；以及***膜层中间以形成孔道的纳米孔蛋白，当跨越膜层施加电场力时，孔道发生电导；其中，纳米孔蛋白包括上述纳米孔蛋白。In order to achieve the above object, according to the sixth aspect of the present invention, a nanoporous biofilm is provided, which includes: a membrane layer; and a nanoporin inserted into the middle of the membrane layer to form a channel, when crossing the membrane When an electric field force is applied, conduction occurs in the channel; wherein, the nanoporin includes the above-mentioned nanoporin.
进一步地，膜层包括脂质层；优选地，脂质层包括两亲脂类；优选地，两亲脂类包含磷脂双分子层；优选地，脂质层包括平面膜层或脂质体；优选地，脂质体包括多层脂质体或单层脂质体；优选地，脂质层包括二植酰磷脂酰胆碱组成的磷脂双分子层。Further, the membrane layer includes a lipid layer; preferably, the lipid layer includes amphiphilic lipids; preferably, the amphiphilic lipids include a phospholipid bilayer; preferably, the lipid layer includes a planar membrane layer or a liposome; Preferably, the liposome includes multilamellar liposomes or unilamellar liposomes; preferably, the lipid layer includes a phospholipid bilayer composed of diphytylphosphatidylcholine.
进一步地，纳米孔蛋白在膜层中是可移动的；优选地，当跨越膜层施加电场力时，纳米孔生物膜能够通过孔道将待测生物分子移位；优选地，待测生物分子包括DNA、RNA、多肽或蛋白质；优选地，待测生物分子带有修饰的基团分子，更优选基团分子选自胆固醇、聚乙二醇、生物素或荧光基团分子；优选地，DNA和/或RNA包括如下任意一种或多种修饰碱基：5-甲基胞嘧啶、6-甲基腺嘌呤、7-甲基鸟嘌呤或假尿嘧啶。Further, the nanoporin is mobile in the membrane layer; preferably, when an electric field force is applied across the membrane layer, the nanopore biomembrane can displace the biomolecules to be tested through the pores; preferably, the biomolecules to be tested include DNA, RNA, polypeptide or protein; Preferably, the biomolecule to be tested has a modified group molecule, more preferably the group molecule is selected from cholesterol, polyethylene glycol, biotin or fluorescent group molecule; Preferably, DNA and /or RNA includes any one or more of the following modified bases: 5-methylcytosine, 6-methyladenine, 7-methylguanine or pseudouracil.
为了实现上述目的，根据本发明的第七个方面，提供了一种纳米孔测序装置，该纳米孔测序装置包括上述纳米孔生物膜。In order to achieve the above object, according to the seventh aspect of the present invention, a nanopore sequencing device is provided, the nanopore sequencing device includes the above-mentioned nanopore biofilm.
进一步地，纳米孔测序装置包括：电解槽，含有电解液；纳米孔生物膜，位于电解槽的中央，并将电解槽及电解液分割为正极电解液区和负极电解液区；第一电极和第二电极，第一电极和第二电极分别设置在正极电解液区和负极电解液区；接收电极，接收电极包括两个且分别位于正极电极液区和负极电解液区，接收电极与信号处理芯片相连；优选地，电解液为纳米孔蛋白实验缓冲液；优选地，纳米孔蛋白实验缓冲液为HEPES缓冲液；优选地，纳米孔蛋白实验缓冲液含有0.1-1.0M KCl；更优选地，纳米孔蛋白实验缓冲液包括0.5M KCl，10mM HEPES，1mM EDTA，pH 7.8；优选地，第一电极和第二电极包括金属或复合电极材料；优选地，第一电极和第二电极不同，分别为银和氯化银；优选地，第一电极和第二电极相同，包括金、铂、石墨烯或氮化钛。Further, the nanopore sequencing device includes: an electrolytic cell containing an electrolyte; a nanopore biofilm located in the center of the electrolytic cell, and dividing the electrolytic cell and the electrolyte into a positive electrolyte area and a negative electrolyte area; the first electrode and The second electrode, the first electrode and the second electrode are respectively arranged in the positive electrode electrolyte area and the negative electrode electrolyte area; the receiving electrode includes two receiving electrodes respectively located in the positive electrode liquid area and the negative electrode electrolyte area, the receiving electrode and signal processing The chip is connected; preferably, the electrolyte is a nanoporin experimental buffer; preferably, the nanoporin experimental buffer is a HEPES buffer; preferably, the nanoporin experimental buffer contains 0.1-1.0M KCl; more preferably, The nanoporin assay buffer includes 0.5M KCl, 10mM HEPES, 1mM EDTA, pH 7.8; preferably, the first electrode and the second electrode include metal or composite electrode materials; preferably, the first electrode and the second electrode are different, respectively are silver and silver chloride; preferably, the first electrode and the second electrode are the same, comprising gold, platinum, graphene or titanium nitride.
为了实现上述目的，根据本发明的第八个方面，提供了一种测序方法，该测序方法利用上述纳米孔蛋白，或者上述纳米孔生物膜，或者上述纳米孔测序装置通过解析待测生物分子通过纳米孔蛋白的孔道时产生的电信号，对待测生物分子进行测序。In order to achieve the above object, according to the eighth aspect of the present invention, a sequencing method is provided. The sequencing method utilizes the above-mentioned nanopore protein, or the above-mentioned nanopore biofilm, or the above-mentioned nanopore sequencing device by analyzing the biomolecules to be tested through The electrical signal generated by the pores of nanoporins is used to sequence the biomolecules to be tested.
进一步地，待测生物分子包括修饰或未修饰的DNA、RNA、多肽或蛋白质；优选地，在电场力的作用下，待测生物分子的单个分子通过纳米孔蛋白的孔道，产生电信号；优选地，电信号包括阻滞电流振幅。Further, the biomolecules to be tested include modified or unmodified DNA, RNA, polypeptide or protein; preferably, under the action of an electric field force, a single molecule of the biomolecules to be tested passes through the pores of the nanoporin to generate electrical signals; preferably ground, the electrical signal includes a blocking current amplitude.
进一步地，单个纳米孔蛋白***脂质层中，优选为磷脂双分子层中，形成纳米孔生物膜，纳米孔蛋白利用纳米孔生物膜的结构进行测序。Furthermore, a single nanoporin is inserted into a lipid layer, preferably a phospholipid bilayer, to form a nanoporous biofilm, and the nanoporin is sequenced using the structure of the nanoporous biofilm.
为了实现上述目的，根据本发明的第九个方面，提供了上述纳米孔蛋白，或者上述试剂盒，或者上述DNA分子，或者上述重组载体，或者上述宿主细胞，或者上述纳米孔生物膜，或者上述纳米孔测序装置，或者上述测序方法在小分子检测、单分子DNA测序、单分子RNA测序、多肽测序或蛋白质测序中的应用。In order to achieve the above object, according to the ninth aspect of the present invention, the above-mentioned nanopore protein, or the above-mentioned kit, or the above-mentioned DNA molecule, or the above-mentioned recombinant vector, or the above-mentioned host cell, or the above-mentioned nanopore biofilm, or the above-mentioned A nanopore sequencing device, or the application of the above-mentioned sequencing method in small molecule detection, single-molecule DNA sequencing, single-molecule RNA sequencing, polypeptide sequencing or protein sequencing.
应用本发明的技术方案，通过对纳米孔蛋白MP964进行突变，使得突变后的纳米孔蛋白的孔道稳定性提高，便于在后续的测序中提高测序准确度和测序的数据通量。By applying the technical solution of the present invention, by mutating the nanoporin MP964, the pore stability of the mutated nanoporin is improved, which facilitates the improvement of sequencing accuracy and data throughput in subsequent sequencing.
附图说明Description of drawings
构成本申请的一部分的说明书附图用来提供对本发明的进一步理解，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。在附图中：The accompanying drawings constituting a part of the present application are used to provide a further understanding of the present invention, and the schematic embodiments and descriptions of the present invention are used to explain the present invention, and do not constitute an improper limitation of the present invention. In the attached picture:
图1示出了根据本发明实施例5的纳米孔蛋白MP964的SDS-PAGE纯化结果示意图；Figure 1 shows a schematic diagram of the SDS-PAGE purification results of nanoporin MP964 according to Example 5 of the present invention;
图2示出了根据本发明实施例6的纳米孔蛋白MP964Mut的SDS-PAGE纯化结果示意图；Figure 2 shows a schematic diagram of the SDS-PAGE purification results of nanoporin MP964Mut according to Example 6 of the present invention;
图3示出了根据本发明实施例7的纳米孔蛋白MP964的同源建模三维结构示意图；Fig. 3 shows a schematic diagram of the homology modeling three-dimensional structure of nanoporin MP964 according to Example 7 of the present invention;
图4示出了根据本发明实施例7的纳米孔蛋白MP964Mut的同源建模三维结构示意图；Fig. 4 shows a schematic diagram of the homology modeling three-dimensional structure of the nanoporin MP964Mut according to Example 7 of the present invention;
图5示出了根据本发明实施例8的纳米孔蛋白MP964的寡核苷酸样品的电流阻滞百分比分布图；Fig. 5 shows the current retardation percentage distribution graph of the oligonucleotide sample of nanoporin MP964 according to Example 8 of the present invention;
图6示出了根据本发明实施例8的纳米孔蛋白MP964Mut的寡核苷酸样品的电流阻滞百分比分布图；Fig. 6 shows the current retardation percentage distribution diagram of the oligonucleotide sample of nanoporin MP964Mut according to Example 8 of the present invention;
图7示出了根据本发明实施例8的纳米孔蛋白MP964及MP964Mut的DNA捕获率的统计示意图；Figure 7 shows a statistical schematic diagram of the DNA capture rate of nanoporins MP964 and MP964Mut according to Example 8 of the present invention;
图8示出了根据本发明实施例9的纳米孔蛋白MP964Mut在不同次实验的电导分布示意图；Figure 8 shows a schematic diagram of the conductance distribution of the nanoporin MP964Mut in different experiments according to Example 9 of the present invention;
图9示出了根据本发明实施例9的寡核苷酸样品A在180mV外加电压下穿过纳米孔蛋白突变体MP964Mut，随时间变化的电流特征示意图；Figure 9 shows a schematic diagram of the current characteristics of the oligonucleotide sample A according to Example 9 of the present invention passing through the nanoporin mutant MP964Mut under an applied voltage of 180mV;
图10示出了根据本发明实施例9的寡核苷酸样品A的电流阻滞百分比分布图示意图；Fig. 10 shows a schematic diagram of the percentage distribution diagram of current blockage of oligonucleotide sample A according to Example 9 of the present invention;
图11示出了根据本发明实施例9的寡核苷酸样品B在180mV外加电压下穿过纳米孔蛋白突变体MP964Mut，随时间变化的电流特征示意图；Figure 11 shows a schematic diagram of the current characteristics of the oligonucleotide sample B according to Example 9 of the present invention passing through the nanoporin mutant MP964Mut under an applied voltage of 180mV;
图12示出了根据本发明实施例9的寡核苷酸样品B的电流阻滞百分比分布图示意图；Figure 12 shows a schematic diagram of the current block percentage distribution diagram of oligonucleotide sample B according to Example 9 of the present invention;
图13示出了在外加电压180mV下控制DNA穿过纳米孔蛋白突变体MP964Mut，随着DNA移动产生不同幅度大小的电流振幅的示意图，其中，图(A)为电流特征图。图(B)为放大电流图；Fig. 13 shows a schematic diagram of controlling DNA to pass through the nanoporin mutant MP964Mut under an applied voltage of 180mV, and the current amplitudes of different amplitudes are generated as the DNA moves, wherein, graph (A) is a current characteristic graph. Figure (B) is an enlarged current diagram;
图14示出了在外加电压150mV下控制DNA穿过纳米孔蛋白突变体MP964Mut，随着DNA移动产生不同幅度大小的电流振幅变化，其中，图(A)为电流特征图。图(B)为放大电流图。Fig. 14 shows that when the DNA is controlled to pass through the nanoporin mutant MP964Mut under an applied voltage of 150mV, current amplitude changes of different magnitudes are produced as the DNA moves, wherein, graph (A) is a current characteristic graph. Figure (B) is an enlarged current diagram.
具体实施方式Detailed ways
需要说明的是，在不冲突的情况下，本申请中的实施例及实施例中的特征可以相互组合。下面将结合实施例来详细说明本发明。It should be noted that, in the case of no conflict, the embodiments in the present application and the features in the embodiments can be combined with each other. The present invention will be described in detail below in conjunction with examples.
如背景技术所提到的，目前，仅有耻垢分枝杆菌孔蛋白A(MspA)、curli-特异性转运通道(CsgG)等少数几种天然蛋白符合纳米孔测序的应用需求。天然的纳米孔蛋白一般具有形成孔道的能力，但是在重组蛋白的体外表达纯化***中，重组纳米孔蛋白的孔道稳定性不一定可以满足单分子检测器相关仪器产品的需求。同时，天然纳米孔蛋白的孔径分布范围较广，不一定满足单分子检测的需求。天然纳米孔蛋白孔道内壁氨基酸残基的性质，尤其是带电性质，不一定满足特定待测物的性质。因而，在本申请中发明人通过对蛋白MP964进行突变，对其聚体稳定性，孔道内径及孔道内壁性质进行了优化，使其可以作为检测的纳米孔蛋白，因而提出了本申请的一系列保护方案。As mentioned in the background art, at present, only a few natural proteins such as Mycobacterium smegmatis porin A (MspA) and curli-specific transport channel (CsgG) meet the application requirements of nanopore sequencing. Natural nanoporins generally have the ability to form pores, but in the in vitro expression and purification system of recombinant proteins, the stability of the pores of recombinant nanoporins may not meet the needs of single-molecule detector-related instrument products. At the same time, natural nanoporins have a wide range of pore size distributions, which may not necessarily meet the needs of single-molecule detection. The properties of the amino acid residues in the pore walls of natural nanoporins, especially the charge properties, do not necessarily meet the properties of a specific analyte. Therefore, in the present application, the inventors mutated the protein MP964, optimized its polymer stability, pore inner diameter and pore inner wall properties, so that it can be used as a nanoporin for detection, and thus proposed a series of protection scheme.
在本申请第一种典型的实施方式中，提供了一种纳米孔蛋白，包括(a)MP964，MP964为具有SEQ ID NO：1所示的氨基酸序列的蛋白质；或(b)MP964Mut，MP964Mut为具有SEQ ID NO：2所示的氨基酸序列的蛋白质；或(c)在(a)或(b)中的氨基酸序列的如下至少一个位点：第97位、第98位、第127位、第143位、第148位，经过取代和/或缺失和/或添加一个或几个氨基酸且具有孔道结构的蛋白质；或(d)与(a)、(b)和(c)中任一所限定的氨基酸序列具有80％以上同源性且具有相同功能的蛋白质。In the first typical embodiment of the present application, a nanopore protein is provided, including (a) MP964, MP964 is a protein having the amino acid sequence shown in SEQ ID NO: 1; or (b) MP964Mut, MP964Mut is A protein having the amino acid sequence shown in SEQ ID NO: 2; or (c) at least one of the following positions of the amino acid sequence in (a) or (b): 97th, 98th, 127th, 143rd, 148th, a protein having a pore structure through substitution and/or deletion and/or addition of one or several amino acids; or any one of (d) and (a), (b) and (c) defined The amino acid sequence has more than 80% homology and has the same function protein.
上述(a)或(b)限定的纳米孔蛋白，具有孔道结构，应用于纳米孔测序时，能够使待测生物分子逐一从孔道中穿过，产生电流信号。MP964Mut是以MP964为基础进行突变而获得的纳米孔蛋白。在(a)或(b)序列的基础上，对蛋白进行突变，比如，在保留MP964Mut的突变位点的基础上，在其他位置上取代和/或缺失和/或添加一个或几个氨基酸后，仍能够获得保持上述纳米孔蛋白的孔道结构和功能。对纳米孔蛋白进行突变，可能对蛋白及聚集体稳定性、孔道内径、孔道内壁氨基酸残基产生影响，从而影响其理化性质和待测生物分子通过性能，但突变的常规操作方式，以及筛选获得具有纳米孔道结构和功能活性的蛋白的方法，是本领域技术人员所公知，或者进一步结合本申请中MP964Mut类似的筛选步骤进行操作即可实现。The nanoporin defined in (a) or (b) above has a pore structure, and when applied to nanopore sequencing, it can allow the biomolecules to be tested to pass through the pore one by one to generate a current signal. MP964Mut is a nanopore protein obtained by mutation based on MP964. On the basis of (a) or (b) sequence, the protein is mutated, for example, on the basis of retaining the mutation site of MP964Mut, after substitution and/or deletion and/or addition of one or several amino acids at other positions , can still obtain and maintain the pore structure and function of the above-mentioned nanoporin. Mutations on nanopore proteins may affect the stability of proteins and aggregates, the inner diameter of pores, and the amino acid residues on the inner wall of pores, thereby affecting their physical and chemical properties and the passage performance of biomolecules to be tested. The methods for proteins with nanopore structure and functional activity are known to those skilled in the art, or can be realized by further combining with the similar screening steps of MP964Mut in this application.
其中，MP964的氨基酸序列如下：SEQ ID NO：1：Wherein, the amino acid sequence of MP964 is as follows: SEQ ID NO: 1:
MHHHHHHELDSQLGVLDEQGRTLTIQQWDTEFQGVYPMDRNRLTREWFYSGQGAYIVSGPNADAFTGELMLGYQIGFPWSLGVGINFSYTSPNILLDATNFEPGPDFAPLAYVITPNFFPGLSISADLGNGPGVQEVVTFTAEVTGAQGNVVVSDAHGTVTGAAGGVLLRPFVRLVSSQGATVTTYGEPWNMK。MHHHHHHELDSQLGVLDEQGRTLTIQQWDTEFQGVYPMDRNRLTREWFYSGQGAYIVSGPNADAFTGELMLGYQIGFPWSLGVGINFSYTSPNILLDATNFEPGPDFAPLAYVITPNFFPGLSISADLGNGPGVQEVVTFTAEVTGAQGNVVVSDAHGTVTGAAGGVLLRPFVRLVSSQGATVTTYG EPWNMK.
MP964Mut的氨基酸序列如下：SEQ ID NO：2：The amino acid sequence of MP964Mut is as follows: SEQ ID NO: 2:
MHHHHHHELDSQLGVLDEQGRTLTIQQWDTEFQGVYPMDRNRLTREWFYSGQGAYIVSGPNADAFTGELMLGYQIGFPWSLGVGINFSYTSPNILLNNTNFEPGPDFAPLAYVITPNFFPG LSISARLGNGPGVQEVVTFTARVTGAKGNVVVSDAHGTVTGAAGGVLLRPFVRLVSSQGATVTTYGEPWNMK。MHHHHHHELDSQLGVLDEQGRTLTIQQWDTEFQGVYPMDRNRLTREWFYSGQGAYIVSGPNADAFTGELMLGYQIGFPWSLGVGINFSYTSPNILLNNTNFEPGPDFAPLAYVITPNFFPG LSISARLGNGPGVQEVVTFTARVTGAKGNVVVSDAHGTVTGAAGGVLLRPFVRLVSSQGATVTT YGEPWNMK.
在一种优选的实施例中，(c)中，各位点取代的氨基酸的类型各自独立地选自如下：第97位：Q/Y/A/S/H；第98位：Q/Y/S/H；第127位：/K/H/Y；第143位：K/H/Y；第148位：/R/H/Y；其中，“/”代表“或”；优选地，(d)中，与(a)、(b)和(c)中任一所限定的氨基酸序列具有85％以上(比如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％以上，甚至99.9％以上)，优选90％以上，更优选95％以上，进一步优选99％以上同源性且具有相同功能的蛋白质。在保留MP964Mut突变位点的基础上，对MP964Mut的氨基酸序列进行突变，能够获得活性不变或提高的具有不同程度同源性的变体。In a preferred embodiment, in (c), the types of amino acids substituted at each site are independently selected from the following: No. 97: Q/Y/A/S/H; No. 98: Q/Y/ S/H; No. 127: /K/H/Y; No. 143: K/H/Y; No. 148: /R/H/Y; Among them, "/" represents "or"; preferably, ( In d), having more than 85% (such as 85%, 86%, 87%, 88%, 89%, 90%, 91%) of the amino acid sequence defined in any one of (a), (b) and (c) , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, even 99.9%), preferably 90% Proteins with the above, more preferably 95% or more, even more preferably 99% or more homology and the same function. On the basis of retaining the MP964Mut mutation site, the amino acid sequence of MP964Mut is mutated to obtain variants with different degrees of homology with unchanged or improved activity.
上述85％、90％、95％、99％以上同源性且具有相同功能的蛋白质，其蛋白及聚集体稳定性、孔道内径、孔道内壁氨基酸残基、待测生物分子通过性能等性质，均和(a)或(b)序列提供的蛋白质大概率相同，为通过氨基酸突变获得的同源蛋白，优选地，纳米孔蛋白来源于分枝杆菌。For the above 85%, 90%, 95%, 99% or more homologous proteins with the same function, their properties such as protein and aggregate stability, pore inner diameter, pore inner wall amino acid residues, and the passing performance of biomolecules to be tested are all It has the same probability as the protein provided by the sequence (a) or (b), and is a homologous protein obtained by amino acid mutation. Preferably, the nanoporin is derived from mycobacteria.
在一种优选的实施例中，纳米孔蛋白的孔道直径为1.2～1.6nm。该孔径范围较现有纳米孔蛋白的孔径小，因而孔道稳定性更好，测序准确性也更高。优选地，纳米孔蛋白在150mV电压下的电流振幅值为145～155pA；优选地，纳米孔蛋白在180mV电压下的电流振幅值为170～190pA；优选地，纳米孔蛋白的电导为0.8～1.2nS。In a preferred embodiment, the pore diameter of the nanoporin is 1.2-1.6 nm. The pore diameter range is smaller than that of existing nanoporins, so the pore stability is better and the sequencing accuracy is also higher. Preferably, the current amplitude value of the nanoporin at a voltage of 150mV is 145-155pA; preferably, the current amplitude value of the nanoporin at a voltage of 180mV is 170-190pA; preferably, the conductance of the nanoporin is 0.8-1.2 nS.
在一定范围上，纳米孔蛋白的孔道直径越小，其在用于测序时的准确度越高。若纳米孔蛋白的孔道直径过大(一次可能不止一个分子通过孔道)，难以满足单分子测序的需求，待测生物分子在过大的孔道中穿过时，产生的电流信号有可能会遗漏或产生错误，导致测序的准确度低。在单分子测序中，利用对同一分子进行多次测序的方式，获得准确的测序结果。因此测序的准确度越高，所需的测序次数和时间越短。利用测序准确度高的纳米孔蛋白进行测序，能够大大减少测序的时间，降低成本，这种优势在高通量的测序中尤为明显。In a certain range, the smaller the pore diameter of the nanoporin, the higher the accuracy when it is used for sequencing. If the pore diameter of the nanoporin 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 biomolecule to be tested passes through the too large pore, the current signal generated may be missed or generated Errors lead to low sequencing accuracy. In single-molecule sequencing, the same molecule is sequenced multiple times to obtain accurate sequencing results. Therefore, the higher the accuracy of sequencing, the shorter the number and time required for sequencing. Using nanopore proteins with high sequencing accuracy for sequencing can greatly reduce the time and cost of sequencing, and this advantage is especially obvious in high-throughput sequencing.
在本申请第二种典型的实施方式中，提供了一种试剂盒，该试剂盒包括上述纳米孔蛋白。利用含有上述纳米孔蛋白的试剂盒进行测序时，由于上述纳米孔蛋白的孔道直径相对更小，孔道稳定性更好，进而测序准确性也相对更高，因而能够减少测序时间和成本。In the second typical embodiment of the present application, a kit is provided, which includes the above-mentioned nanoporin. When using the kit containing the above-mentioned nanoporin for sequencing, because the pore diameter of the above-mentioned nanoporin is relatively smaller, the pore stability is better, and the sequencing accuracy is relatively higher, so the sequencing time and cost can be reduced.
为进一步提高操作便利性，在一种优选的实施例中，试剂盒还包括脂质层或人造高分子膜；优选地，脂质层包括两亲脂类；优选地，两亲脂类包含磷脂双分子层；优选地，脂质层包括平面膜层或脂质体；优选地，脂质体包括多层脂质体或单层脂质体；优选地，脂质层包括二植酰磷脂酰胆碱组成的磷脂双分子层。In order to further improve the convenience of operation, in a preferred embodiment, the kit also includes a lipid layer or an artificial polymer membrane; preferably, the lipid layer includes amphiphilic lipids; preferably, the amphiphilic lipids include phospholipids bilayer; preferably, the lipid layer comprises a planar membrane layer or a liposome; preferably, the liposome comprises a multilamellar liposome or a unilamellar liposome; preferably, the lipid layer comprises a diphytylphosphatidyl Phospholipid bilayers composed of choline.
人造高分子膜包括但不限于聚硅氧烷、聚烯烃、全氟聚醚、全氟烃基聚醚、聚苯乙烯、聚氧丙烯、聚乙酸乙烯酯、聚氧丁烯、聚异戊二烯、聚丁二烯、聚氯乙烯、聚烷基丙烯酸酯、聚烷基甲基丙烯酸酯、聚丙烯腈、聚丙烯、PTHF、聚甲基丙烯酸酯、聚丙烯酸酯、聚砜、聚乙烯醚、聚(环氧丙烷)及其共聚物、基取代的C1-C6烷基丙烯酸酯和甲基丙烯酸酯、丙烯 酰胺、甲基丙烯酰胺、(C1-C6烷基)丙烯酰胺和甲基丙烯酰胺、N,N-二烷基-丙烯酰胺、乙氧基丙烯酸酯和甲基丙烯酸酯、聚乙二醇单甲基丙烯酸酯和聚乙二醇单甲基醚甲基丙烯酸酯、羟基取代的(C1-C6烷基)丙烯酰胺和甲基丙烯酰胺、羟基取代的C1-C6烷基乙烯基醚、乙烯基磺酸钠、苯乙烯基磺酸钠、2-丙烯酰胺-2-甲基丙磺酸、N-乙烯基吡咯、N-乙烯基-2-吡咯烷酮、2-乙烯基恶唑啉、2-乙烯基-4,4'–双烷基恶唑啉基-5-酮、2,4-乙烯基吡啶、总共具有3-5个碳原子的乙烯化不饱和羧酸，氨基(C1-C6烷基)-、单(C1-C6烷氨基)(C1-C6烷基)-和双(C1-C6烷氨基)(C1-C6烷基)-丙烯酸酯和甲基丙烯酸酯、烯丙醇、3-三甲基铵甲基丙烯酸2-羟丙基酯氯化物、二甲基氨乙基甲基丙烯酸酯(DMAEMA)、二甲基氨乙基甲基丙烯酰胺、甘油甲基丙烯酸酯、N-(1,1-二甲基-3-氧代丁基)丙烯酰胺、环亚氨基醚、乙烯基醚、包含环氧衍生物的环醚、环不饱和醚、N-取代环乙亚胺、β-内酯和β-内酰胺、乙烯酮缩醛、乙烯基缩醛或正膦中的一种或多种。Artificial polymer films include but are not limited to polysiloxane, polyolefin, perfluoropolyether, perfluoroalkyl polyether, polystyrene, polyoxypropylene, polyvinyl acetate, polyoxybutylene, polyisoprene , polybutadiene, polyvinyl chloride, polyalkylacrylate, polyalkylmethacrylate, polyacrylonitrile, polypropylene, PTHF, polymethacrylate, polyacrylate, polysulfone, polyvinyl ether, Poly(propylene oxide) and its copolymers, radical-substituted C1-C6 alkyl acrylates and methacrylates, acrylamides, methacrylamides, (C1-C6 alkyl)acrylamides and methacrylamides, N,N-dialkyl-acrylamides, ethoxylated acrylates and methacrylates, polyethylene glycol monomethacrylate and polyethylene glycol monomethyl ether methacrylate, hydroxyl-substituted (C1 -C6 alkyl)acrylamide and methacrylamide, hydroxyl substituted C1-C6 alkyl vinyl ether, sodium vinyl sulfonate, sodium styryl sulfonate, 2-acrylamide-2-methylpropanesulfonic acid , N-vinylpyrrole, N-vinyl-2-pyrrolidone, 2-vinyloxazoline, 2-vinyl-4,4'-dialkyloxazolinyl-5-one, 2,4- Vinylpyridine, ethylenically unsaturated carboxylic acids having a total of 3-5 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, 2-hydroxypropyl 3-trimethylammonium methacrylate chloride, dimethylaminoethyl methyl Acrylic acid ester (DMAEMA), dimethylaminoethyl methacrylamide, glycerol methacrylate, N-(1,1-dimethyl-3-oxobutyl) acrylamide, cyclic imino ether, Vinyl ethers, cyclic ethers containing epoxy derivatives, cyclic unsaturated ethers, N-substituted ethyleneimines, β-lactones and β-lactams, ketene acetals, vinyl acetals or phosphoranes one or more.
为方便构建纳米孔生物膜，在一种优选的实施例中，试剂盒还包括纳米孔蛋白实验缓冲液；优选地，纳米孔蛋白实验缓冲液为HEPES缓冲液；优选地，纳米孔蛋白实验缓冲液含有0.1-1.0M KCl；优选地，纳米孔实验缓冲液包括0.5M KCl，10mM 4-羟乙基哌嗪乙磺酸(HEPES)，1mM乙二胺四乙酸(EDTA)，pH 7.8。In order to facilitate the construction of nanoporous biofilms, in a preferred embodiment, the kit also includes a nanoporin experimental buffer; preferably, the nanoporin experimental buffer is a HEPES buffer; preferably, the nanoporin experimental buffer The solution contains 0.1-1.0M KCl; preferably, the nanopore assay buffer includes 0.5M KCl, 10mM 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), 1mM ethylenediaminetetraacetic acid (EDTA), pH 7.8.
利用上述试剂盒中的纳米孔蛋白、脂质层和纳米孔蛋白实验缓冲液，能够在纳米孔蛋白实验缓冲液中，将一个或多个纳米孔蛋白***到脂质层中，形成纳米孔生物膜。纳米孔实验缓冲液能够提供维持纳米孔蛋白和脂质层稳定的中性环境，其中包含的金属离子使纳米孔实验缓冲液具有良好的导电性。对于脂质层的选择可以有多种选择，在平面膜层或球形的脂质体上，在不同成分形成的脂质层上，纳米孔蛋白均能够***，形成纳米孔生物膜。Using the nanoporin, lipid layer and nanoporin experimental buffer in the above kit, one or more nanoporins can be inserted into the lipid layer in the nanoporin experimental buffer to form a nanoporous organism membrane. The nanopore experimental buffer can provide a neutral environment to maintain the stability of the nanopore protein and lipid layer, and the metal ions contained in it make the nanopore experimental buffer have good conductivity. There are many options for the choice of lipid layer. On the flat membrane layer or spherical liposome, on the lipid layer formed by different components, nanoporin can be inserted to form a nanopore biomembrane.
在本申请第三种典型的实施方式中，提供了一种分离的DNA分子，该DNA分子具有(a)编码上述纳米孔蛋白的核苷酸序列；或(b)在严格条件下与(a)限定的DNA分子杂交的核苷酸序列；或(c)具有SEQ ID NO：3或SEQ ID NO：4所示的核苷酸序列；或(d)与(a)至(c)中限定的任一种核苷酸序列具有70％以上(优选80％以上，更优选85％以上，进一步优选90％以上，最优选95％以上，比如可以是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％以上，甚至99.9％以上)同源性且编码具有相同功能蛋白质的DNA分子。In the third typical embodiment of the present application, an isolated DNA molecule is provided, the DNA molecule has (a) a nucleotide sequence encoding the aforementioned nanoporin; or (b) under stringent conditions with (a ) the nucleotide sequence of the hybridized DNA molecule as defined; or (c) has the nucleotide sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4; or (d) is defined in (a) to (c) Any nucleotide sequence has more than 70% (preferably more than 80%, more preferably more than 85%, more preferably more than 90%, most preferably more than 95%, such as 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, Even more than 99.9%) homologous DNA molecules that encode proteins with the same function.
上述DNA分子，能够编码具有本申请上述结构和功能的纳米孔蛋白。在(a)序列的基础上对核苷酸进行突变，在严格条件下与(a)限定的DNA分子杂交，且不发生移码突变，若突变发生在编码纳米孔蛋白孔道上的核苷酸，可能会导致编码出孔道发生改变的纳米孔蛋白，影响该纳米孔蛋白的孔径和孔道内壁的氨基酸残基的性质；若突变发生在编码蛋白质非孔道部分的核苷酸上，可能会影响编码蛋白质的折叠方式、三维结构等性质，从而影响蛋白质的理化性质和稳定性。(b)限定的在严格条件下的DNA分子杂交的核苷酸序列，包括与(a)限定的DNA分子具有80％、85％、90％、95％、98％、99％、99.9％或100％互补的核苷酸序列。The above-mentioned DNA molecule can encode the nanopore protein having the above-mentioned structure and function of the present application. Mutate the nucleotides based on the sequence of (a), hybridize with the DNA molecule defined in (a) under stringent conditions, and no frameshift mutation occurs, if the mutation occurs in the nucleotide encoding the nanopore protein channel , may lead to the encoding of nanoporins with changed channels, affecting the pore size of the nanopore proteins and the properties of amino acid residues on the inner wall of the channels; if the mutation occurs in the nucleotides encoding the non-channel part of the protein, it may affect the coding The folding mode, three-dimensional structure and other properties of proteins affect the physicochemical properties and stability of proteins. (b) defined nucleotide sequences that hybridize to DNA molecules under stringent conditions, including 80%, 85%, 90%, 95%, 98%, 99%, 99.9%, or 100% complementary nucleotide sequences.
SEQ ID NO：3：SEQ ID NO: 3:
atgcaccaccaccaccaccacgaactggatagccagctgggcgtgctggatgaacagggccgtaccttaaccattcagcagtgggataccgaatttcagggcgtgtatccgatggatcgcaaccgcctgacccgcgaatggttttatagcggccagggcgcgtatattgtgagcggccctaatgcggatgcgtttaccggcgaactgatgctgggctatcagattggctttccgtggagcctgggcgtgggcattaattttagctataccagcccgaacattctgctggatgcgaccaactttgaaccgggcccggattttgcgccgctggcgtatgttattaccccgaactttttcccgggcctgagcattagcgcggatctgggcaatggcccgggcgttcaggaagttgttacctttaccgcggaagtgaccggtgcgcagggtaatgtggttgttagcgatgcgcatggcaccgtgaccggcgcggcgggtggtgttttattacgtccttttgtgcgcctggtgagcagccaaggcgcgaccgtgaccacttatggcgaaccgtggaatatgaaataa。atgcaccaccaccaccaccacgaactggatagccagctgggcgtgctggatgaacagggccgtaccttaaccattcagcagtgggataccgaatttcagggcgtgtatccgatggatcgcaaccgcctgacccgcgaatggttttatagcggccagggcgcgtatattgtgagcggccctaatgcggatgcgttta ccggcgaactgatgctgggctatcagattggctttccgtggagcctgggcgtgggcattaattttagctataccagcccgaacattctgctggatgcgaccaactttgaaccgggcccggattttgcgccgctggcgtatgttattaccccgaacttttcccgggcctgagcattagcgcggatctgggcaatggcccggg cgttcaggaagttgttacctttaccgcggaagtgaccggtgcgcagggtaatgtggttgttagcgatgcgcatggcaccgtgaccggcgcggcgggtggtgttttattacgtccttttgtgcgcctggtgagcagccaaggcgcgaccgtgaccacttatggcgaaccgtggaat atgaaataa.
SEQ ID NO：4：SEQ ID NO: 4:
atgcatcatcatcatcatcacgaactggatagccagctgggcgtgctggatgaacagggccgtaccttaaccattcagcagtgggataccgaatttcagggcgtgtatccgatggatcgcaaccgcttaacccgcgaatggttttatagcggccagggcgcgtatattgtgagcggccctaatgcggatgcgtttaccggtgaactgatgctgggctatcagattggctttccgtggagcctgggcgtgggtattaattttagctataccagcccgaacattctgctgaacaacaccaactttgaaccgggcccggattttgcgccgctggcgtatgttattaccccgaacttttttccgggcctgagcattagcgcgcgcctgggcaatggtcctggtgtgcaagaagttgttacctttaccgcgcgcgttaccggcgcgaaaggtaatgttgtggttagcgatgcgcatggcaccgttaccggcgcggcaggcggtgtgttattacgtccttttgttcgcttagtgagcagccagggcgcgaccgttaccacctatggtgaaccttggaacatgaaataa。atgcatcatcatcatcatcacgaactggatagccagctgggcgtgctggatgaacagggccgtaccttaaccattcagcagtgggataccgaatttcagggcgtgtatccgatggatcgcaaccgcttaacccgcgaatggttttatagcggccagggcgcgtatattgtgagcggccctaatgcggatgcgtttaccggt gaactgatgctgggctatcagattggctttccgtggagcctgggcgtgggtattaattttagctataccagcccgaacattctgctgaacaacaccaactttgaaccgggcccggattttgcgccgctggcgtatgtatttaccccgaactttttccgggcctgagcattagcgcgcgcctgggcaatggtcctggt gtgcaagaagttgttacctttaccgcgcgcgttaccggcgcgaaaggtaatgttgtggttagcgatgcgcatggcaccgttaccggcgcggcaggcggtgtgttattacgtccttttgttcgcttagtgagcagccagggcgcgaccgttaccacctatggtgaaccttggaacatga aataa.
在一种优选的实施例中，该基因与(a)至(c)中限定的任一种核苷酸序列具有75％以上，优选85％以上，更优选95％以上，进一步优选99％以上同源性且编码具有相同功能蛋白质的DNA分子。In a preferred embodiment, the gene shares more than 75%, preferably more than 85%, more preferably more than 95%, and more preferably more than 99% of any nucleotide sequence defined in (a) to (c). DNA molecules that are homologous and encode proteins with the same function.
75％、85％、90％、95％、99％以上同源性且编码具有相同功能纳米孔蛋白的DNA分子，其编码的蛋白质的活性位点、活性口袋、活性机制等均和(a)序列提供的基因大概率相同，为通过核苷酸突变获得的同源基因。DNA molecules with 75%, 85%, 90%, 95%, 99% or more homology and encoding nanopore proteins with the same function, the active sites, active pockets, and active mechanisms of the encoded proteins are equal to (a) The genes provided by the sequences have the same high probability and are homologous genes obtained through nucleotide mutation.
需要说明的是，本申请中的“同源性”是指任意两个核苷酸序列或氨基酸序列之间进行比较，从相应基因编码的第一个氨基酸到最后一个氨基酸之间的同一性。It should be noted that "homology" in this application refers to the comparison between any two nucleotide sequences or amino acid sequences, from the first amino acid to the last amino acid encoded by the corresponding genes.
本申请中“分离的”是指“通过人工”从其天然状态改变，即，如果它在自然界中发生，则将其改变和/或从其原始环境中分离出来。例如，天然存在于生命有机体中的多核苷酸或多肽不是“分离的”，然而从其天然状态的共存物中分离的相同的多核苷酸或多肽是“分离的”(如在本文中使用的术语)。"Isolated" in this application means altered "by the hand of man" from its natural state, ie, if it occurs in nature, it is altered and/or separated from its original environment. For example, a polynucleotide or polypeptide naturally occurring in a living organism is not "isolated", whereas the same polynucleotide or polypeptide separated from its natural state coexistence is "isolated" (as used herein the term).
本发明中的DNA分子在“严格条件下”与本发明的纳米孔蛋白编码基因杂交，是指能够通过的核酸杂交的方式来鉴定本发明纳米孔蛋白编码基因的存在的条件。本发明中，如果两个DNA分子能形成反平行的双链核酸结构，就可以说这两个DNA分子彼此间能够进行特异性杂交。如果两个DNA分子显示出完全的互补性，则称其中一个DNA分子是另一个DNA分子的“互补物”。本发明中，如果两个DNA分子能够以足够的稳定性相互杂交从而使它们在常规的“高度严格”条件下退火且彼此结合，则称这两个DNA分子具有“互补性”。从完全互补性中偏离是可以允许的，只要这种偏离不完全阻止两个分子形成双链结构。为了使一个DNA分子能够作为引物或探针，仅需保证其在序列上具有充分的互补性，以使得在所采用的特定溶剂和盐浓度下能形成稳定的双链结构。The DNA molecule in the present invention hybridizes with the gene encoding the nanoporin of the present invention under "stringent conditions", which refers to the conditions under which the presence of the gene encoding the nanopore protein of the present invention can be identified by means of nucleic acid hybridization. In the present invention, if two DNA molecules can form an antiparallel double-stranded nucleic acid structure, it can be said that the two DNA molecules can specifically hybridize to each other. One DNA molecule is said to be the "complement" of the other if two DNA molecules exhibit perfect complementarity. In the present invention, two DNA molecules are said to be "complementary" if they are capable of hybridizing to each other with sufficient stability so that they anneal and bind to each other under conventional "high stringency" conditions. Deviations from perfect complementarity are permissible as long as the deviation does not completely prevent the two molecules from forming a double-stranded structure. In order for a DNA molecule to serve as a primer or probe, it only needs to be sufficiently complementary in sequence to form a stable double-stranded structure under the particular solvent and salt concentration employed.
本发明中，基本同源的序列是一段DNA分子，该DNA分子在高度严格条件下能够和相匹配的另一段DNA分子的互补链发生特异性杂交。促进DNA杂交的适合的严格条件，例如，大约在45℃条件下用6.0×氯化钠/柠檬酸钠(SSC)处理，然后在50℃条件下用2.0×SSC洗涤，这些条件对本领域技术人员是公知的。例如，在洗涤步骤中的盐浓度可以选自低度严格条件的约2.0×SSC、50℃到高度严格条件的约0.2×SSC、50℃。此外，洗涤步骤中的温度条件可以从低度严格条件的室温约22℃，升高到高度严格条件的约65℃。温度条件和盐浓度可以都发生改变，也可以其中一个保持不变而另一个变量发生改变。优选地，本发明中的严格条件可为在6×SSC、0.5％SDS溶液中，在65℃下与编码本申请的纳米孔蛋白的核苷酸序列发生特异性杂交，然后用2×SSC、0.1％SDS和1×SSC、0.1％SDS各洗膜1次。In the present invention, a substantially homologous sequence is a DNA molecule that can specifically hybridize with a matching complementary strand of another DNA molecule under highly stringent conditions. Suitable stringent conditions to promote DNA hybridization, for example, treatment with 6.0× sodium chloride/sodium citrate (SSC) at about 45° C., followed by washing with 2.0× SSC at 50° C., are known to those skilled in the art. is well known. For example, the salt concentration in the washing step can be selected from about 2.0×SSC, 50°C for low stringency conditions to about 0.2×SSC, 50°C for high stringency conditions. In addition, the temperature conditions in the washing step can be increased from about 22°C at room temperature for low stringency conditions to about 65°C for high stringency conditions. Both the temperature condition and the salt concentration can be changed, or one can be kept constant while the other variable is changed. Preferably, the stringent conditions in the present invention can specifically hybridize with the nucleotide sequence encoding the nanoporin of the present application in 6×SSC, 0.5% SDS solution at 65° C., and then use 2×SSC, Wash the membrane once with 0.1% SDS, 1×SSC, and 0.1% SDS.
在本申请第四种典型的实施方式中，提供了一种重组载体，该重组载体包含上述DNA分子，即纳米孔蛋白表达基因。In the fourth typical embodiment of the present application, a recombinant vector is provided, and the recombinant vector comprises the above-mentioned DNA molecule, that is, a nanoporin expression gene.
在重组载体上***纳米孔蛋白表达基因，利用重组载体能够大量自我复制的功能，大量复制纳米孔蛋白表达基因。此处的“重组”是指通过将来自一个物种的基因移植或剪接到不同物种的宿主有机体的细胞中而制备的基因工程化的DNA。这种DNA成为宿主基因结构的一部分并被复制。The nanoporin expression gene is inserted into the recombinant vector, and the nanoporin expression gene is copied in large quantities by utilizing the self-replicating function of the recombinant vector. "Recombinant" herein refers to genetically engineered DNA prepared by transplanting or splicing genes from one species into cells of a host organism of a different species. This DNA becomes part of the host's genetic makeup and is replicated.
在本申请第五种典型的实施方式中，提供了一种宿主细胞，该宿主细胞转化有上述重组载体。In the fifth typical embodiment of the present application, a host cell transformed with the above-mentioned recombinant vector is provided.
将上述重组载体转化入宿主细胞中，利用宿主细胞对重组载体上的纳米孔蛋白表达基因进行复制、转录、翻译，能够大量产生纳米孔蛋白。宿主细胞包括大肠杆菌、酵母菌、哺乳动物细胞、昆虫细胞等常用宿主细胞，利用宿主细胞对纳米孔蛋白进行折叠使之形成正确的三维结构，获得结构和功能正常的纳米孔蛋白。The above recombinant vector is transformed into a host cell, and the host cell is used to replicate, transcribe, and translate the nanoporin expression gene on the recombinant vector, so that a large amount of nanoporin can be produced. Host cells include common host cells such as Escherichia coli, yeast, mammalian cells, and insect cells. The host cells are used to fold the nanoporin to form a correct three-dimensional structure, and obtain a nanoporin with normal structure and function.
在本申请第六种典型的实施方式中，提供了一种纳米孔生物膜，该纳米孔生物膜包括：膜层；以及***膜层中间以形成孔道的纳米孔蛋白，当跨越膜层施加电场力时，孔道发生电导；其中，纳米孔蛋白包括上述纳米孔蛋白。本申请中的纳米孔生物膜特指有纳米孔蛋白***的膜层。In the sixth typical embodiment of the present application, a nanoporous biofilm is provided, which includes: a membrane layer; and a nanoporin inserted into the middle of the membrane layer to form a channel, when an electric field is applied across the membrane layer When the force is applied, the conduction occurs in the channel; wherein, the nanoporin includes the above-mentioned nanoporin. The nanoporous biomembrane in this application specifically refers to the membrane layer with nanoporin inserted.
此种纳米孔生物膜，在膜层中***中***带有孔道的纳米孔蛋白，能够将纳米孔蛋白的孔道朝向进行固定，在跨越膜层施加电场力时，孔道直径垂直于电场方向，孔道发生电导产生电信号。This kind of nanoporous biomembrane inserts nanoporin with pores in the membrane layer, which can fix the direction of the pores of the nanoporin. When the electric field force is applied across the membrane layer, the diameter of the pores is perpendicular to the direction of the electric field, and the pores Conductance occurs to generate an electrical signal.
在一种优选的实施例中，膜层包括脂质层；优选地，脂质层包括两亲脂类；优选地，两亲脂类包含磷脂双分子层；优选地，脂质层包括平面膜层或脂质体；优选地，脂质体包括多层脂质体或单层脂质体；优选地，脂质层包括二植酰磷脂酰胆碱组成的磷脂双分子层。In a preferred embodiment, the membrane layer comprises a lipid layer; 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 liposome; preferably, the liposome includes multilamellar liposomes or unilamellar liposomes; preferably, the lipid layer includes a phospholipid bilayer composed of diphytylphosphatidylcholine.
对于脂质层的选择可以有多种选择，在平面膜层或球形的脂质体上，在不同成分形成的脂质层上，纳米孔蛋白均能够***，形成纳米孔生物膜。There are many options for the choice of lipid layer. On the flat membrane layer or spherical liposome, on the lipid layer formed by different components, nanoporin can be inserted to form a nanopore biomembrane.
在一种优选的实施例中，纳米孔蛋白在膜层中是可移动的；优选地，当跨越膜层施加电 场力时，纳米孔生物膜能够通过孔道将待测生物分子移位；优选地，待测生物分子包括DNA、RNA、多肽或蛋白质；优选地，待测生物分子带有修饰的基团分子，更优选基团分子选自胆固醇、不同聚合度的聚乙二醇、生物素或荧光基团分子；优选地，DNA和/或RNA包括如下任意一种或多种修饰碱基：5-甲基胞嘧啶(5mC)、6-甲基腺嘌呤(m6A)、7-甲基鸟嘌呤(m7G)、假尿嘧啶(pseudouridine，Ψ)。In a preferred embodiment, the nanoporin is mobile within the membrane layer; preferably, the nanoporous biomembrane is capable of translocating the biomolecules to be tested through the pores when an electric field force is applied across the membrane layer; preferably , the biomolecules to be tested include DNA, RNA, polypeptide or protein; preferably, the biomolecules to be tested have modified group molecules, more preferably the group molecules are selected from cholesterol, polyethylene glycol with different degrees of polymerization, biotin or Fluorophore molecules; preferably, DNA and/or RNA include any one or more of the following modified bases: 5-methylcytosine (5mC), 6-methyladenine (m6A), 7-methylguanine Purine (m7G), pseudouracil (pseudouridine, Ψ).
因为膜层具有一定的流动性，所以纳米孔蛋白在膜层是可移动的，能够在电场的作用下自动调整位置和朝向。当跨越膜层施加电场力时，待测生物分子在电场力的作用下经由孔道穿过纳米孔蛋白。待测生物分子包括DNA、RNA、多肽或蛋白质等携带生物遗传信息的生物大分子。待测生物分子可以带有用于修饰的基团分子。基团分子包括但不限于胆固醇、不同聚合度的聚乙二醇、生物素或荧光基团分子。Because the membrane layer has a certain fluidity, the nanoporin is movable in the membrane layer, and can automatically adjust its position and orientation under the action of an electric field. When an electric field force is applied across the membrane layer, the biomolecules to be tested pass through the nanoporin through the pores under the action of the electric field force. The biomolecules to be tested include biomacromolecules such as DNA, RNA, polypeptide or protein that carry biological genetic information. The biomolecules to be tested can have moieties for modification. Molecules include, but are not limited to, cholesterol, polyethylene glycols of different degrees of polymerization, biotin, or fluorophore molecules.
在本申请第七种典型的实施方式中，提供了一种纳米孔测序装置，该纳米孔测序装置包括上述纳米孔生物膜。利用上述纳米孔测序装置能够对待测生物分子进行单分子测序。In the seventh typical embodiment of the present application, a nanopore sequencing device is provided, and the nanopore sequencing device includes the above-mentioned nanopore biofilm. The nanopore sequencing device can be used to perform single-molecule sequencing of the biomolecules to be tested.
在一种优选的实施例中，纳米孔测序装置包括：电解槽，含有电解液；纳米孔生物膜，位于电解槽的中央，并将电解槽及电解液分割为正极电解液区和负极电解液区；第一电极和第二电极，第一电极和第二电极分别设置在正极电解液区和负极电解液区；接收电极，包括两个且分别位于正极电极液区和负极电解液区，该接收电极与信号处理芯片相连；优选地，电解液为纳米孔实验缓冲液；优选地，纳米孔蛋白实验缓冲液为HEPES缓冲液；优选地，纳米孔蛋白实验缓冲液含有0.1-1.0M KCl；更优选地，纳米孔实验缓冲液包括0.5M KCl，10mM HEPES，1mM EDTA，pH 7.8；优选地，第一电极和第二电极包括金属或复合电极材料；优选地，第一电极和第二电极不同，分别为银和氯化银；优选地，第一电极和第二电极相同，包括金、铂、石墨烯或氮化钛。In a preferred embodiment, the nanopore sequencing device includes: an electrolytic cell containing an electrolyte; a nanopore biofilm located in the center of the electrolytic cell and dividing the electrolytic cell and the electrolyte into a positive electrolyte area and a negative electrolyte area; the first electrode and the second electrode, the first electrode and the second electrode are respectively arranged in the positive electrode electrolyte area and the negative electrode electrolyte area; the receiving electrode includes two and are respectively located in the positive electrode liquid area and the negative electrode electrolyte area, the The receiving electrode is connected to the signal processing chip; preferably, the electrolyte is a nanopore experimental buffer; preferably, the nanoporin experimental buffer is a HEPES buffer; preferably, the nanoporin experimental buffer contains 0.1-1.0M KCl; More preferably, nanopore experiment buffer comprises 0.5M KCl, 10mM HEPES, 1mM EDTA, pH 7.8; Preferably, first electrode and second electrode comprise metal or composite electrode material; Preferably, first electrode and second electrode different, respectively silver and silver chloride; preferably, the first electrode and the second electrode are the same, including gold, platinum, graphene or titanium nitride.
在纳米孔测序装置中，包括含有电解液的电解槽、纳米孔生物膜、第一电极和第二电极。将纳米孔生物膜放入含有电解液的电解槽中央，将电解槽分解形成正极电解液区和负极电解液区，2个区域分别设置有2个电极，利用2个电极形成施加在纳米孔生物膜上的电场。待测生物分子通过膜上的纳米孔蛋白，产生电流振幅。通过接收电极能够接收此种电流振幅，并将电流振幅传送至与接收电极相连的信号处理芯片。根据电流振幅的不同，该信号处理芯片，即包含信号处理芯片的纳米孔测序装置，能对待测生物分子的序列进行数据分析和测定。The nanopore sequencing device includes an electrolytic cell containing electrolyte, a nanopore biomembrane, a first electrode and a second electrode. Put the nanoporous biofilm into the center of the electrolytic cell containing the electrolyte, and decompose the electrolytic cell to form the positive electrolyte area and the negative electrolyte area. The two areas are respectively equipped with two electrodes, and the two electrodes are used to form and apply to the nanoporous biofilm. The electric field on the membrane. The biomolecules to be tested pass through the nanopore proteins on the membrane, generating a current amplitude. The current amplitude can be received by the receiving electrode, and the current amplitude is transmitted to a signal processing chip connected to the receiving 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 biomolecules to be tested.
在本申请第八种典型的实施方式中，提供了一种测序方法，该测序方法利用上述纳米孔蛋白，或者纳米孔生物膜，或者纳米孔测序装置通过解析待测生物分子通过纳米孔蛋白的孔道时产生的电信号，对待测生物分子进行测序。In the eighth typical embodiment of the present application, a sequencing method is provided. The sequencing method uses the above-mentioned nanopore protein, or nanopore biomembrane, or nanopore sequencing device to analyze the biomolecules passing through the nanopore protein. The electrical signal generated during the pore channel is used to sequence the biomolecules to be tested.
在一种优选的实施例中，待测生物分子包括修饰或未修饰的DNA、RNA、多肽或蛋白质；优选地，在电场力的作用下，待测生物分子的单个分子通过纳米孔蛋白的孔道，产生电信号；优选地，电信号包括阻滞电流振幅。In a preferred embodiment, the biomolecules to be tested include modified or unmodified DNA, RNA, polypeptide or protein; preferably, under the action of an electric field force, a single molecule of the biomolecules to be tested passes through the pores of the nanoporin , generating an electrical signal; preferably, the electrical signal includes a blocking current amplitude.
在一种优选的实施例中，单个纳米孔蛋白***脂质层中，优选为磷脂双分子层中，形成纳米孔生物膜，纳米孔蛋白利用纳米孔生物膜的结构进行测序。In a preferred embodiment, a single nanoporin is inserted into a lipid layer, preferably a phospholipid bilayer, to form a nanoporous biomembrane, and the nanoporin is sequenced using the structure of the nanoporous biomembrane.
上述纳米孔蛋白，或者试剂盒，或者DNA分子，或者重组载体，或者宿主细胞，或者纳米孔生物膜，或者纳米孔测序装置，或者测序方法在单分子生物传感器、小分子检测、单分子DNA测序、单分子RNA测序、多肽测序或蛋白质测序中的应用。The aforementioned nanopore proteins, or kits, or DNA molecules, or recombinant vectors, or host cells, or nanopore biofilms, or nanopore sequencing devices, or sequencing methods in single-molecule biosensors, small molecule detection, and single-molecule DNA sequencing , single-molecule RNA sequencing, peptide sequencing, or protein sequencing.
基于目前纳米孔测序相较于传统测序的一大优势：不会因为错误累积而影响准确率，因此可达到极长的读长。进而可以弥补传统测序短测序片段组装时无法避免的空缺(Gap)问题，判断染色体中是否发生长片段的缺失、重复、倒位、易位，覆盖典型长度为数kb的转录组全长，从而为基因组组装、结构变异、可变剪切等科学研究提供全新的解决方案。Based on one of the advantages of nanopore sequencing compared with traditional sequencing: the accuracy will not be affected by the accumulation of errors, so extremely long read lengths can be achieved. In turn, it can make up for the unavoidable gap (Gap) problem in the assembly of short sequencing fragments in traditional sequencing, determine whether there are deletions, duplications, inversions, and translocations of long fragments in the chromosome, and cover the entire length of the transcriptome with a typical length of several kb, so as to provide Scientific research such as genome assembly, structural variation, and alternative splicing provides new solutions.
由于纳米孔测序无需PCR扩增，因此可保留待测核酸分子上的原始碱基修饰信息，进而直接一次性测序获知修饰碱基的种类、位点及丰度。因而，本申请的纳米孔蛋白同样能够对数种带有DNA/RNA修饰碱基的核酸分子进行检测：包括5-甲基胞嘧啶(5mC)、6-甲基腺嘌呤(m6A)、7-甲基鸟嘌呤(m7G)、假尿嘧啶(pseudouridine，Ψ)等。通过对各种修饰碱基进行特定模型训练与算法开发，纳米孔测序可完成更多修饰碱基的识别定位，从而构建出更为完备的基因组/转录组修饰图谱。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 then the type, site and abundance of the modified base can be directly obtained through one-time sequencing. Therefore, the nanoporin 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.
此外，从临床应用的角度考虑，纳米孔测序长读长、高便携性、快测序速度与实时读出的特点，因而适合应用于重大疫情监测及病原快速检测中(比如，寨卡(Zika virus)病毒、埃博拉病毒(Ebola virus)、登革热病毒(Dengue virus)及新型冠状病毒(Coronavirus)等大规模流行病的行动中)，极具时效性。除病毒外，纳米孔测序还可用于细菌、真菌等其它病原体的快速检测。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, Zika virus ) virus, Ebola virus (Ebola virus), Dengue virus (Dengue virus) and novel coronavirus (Coronavirus) and other large-scale epidemic operations), very time-sensitive. In addition to viruses, nanopore sequencing can also be used for the rapid detection of other pathogens such as bacteria and fungi.
基于蛋白质与核酸分子的共性组成，纳米孔测序平台在蛋白质测序领域也具有巨大的应用潜力。比如，根据目前已经进行的探索可知：通过使用蛋白解折叠酶作为控速工具，成功观察到蛋白质特征性信号，并实现蛋白质种类与修饰状态的初步识别，验证了纳米孔蛋白质测序的可能性。在未来的发展中，通过进一步优化控速体系、开发适配的纳米孔蛋白与信号解析算法，可最终实现在单分子水平对蛋白质进行指纹图谱识别甚至序列鉴定。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 explorations that have been carried out so far, it can be known that by using protein unfolding enzyme as a rate-controlling tool, the characteristic signal of protein was successfully observed, and the preliminary identification of protein type and modification state was realized, which verified the possibility of nanopore protein sequencing. In future development, by further optimizing the rate control system and developing adapted nanopore proteins and signal analysis algorithms, the fingerprinting and even sequence identification of proteins at the single-molecule level can be finally realized.
除应用于测序领域外，纳米孔平台还可作为基础检测平台，结合传感手段，完成各种小分子与大分子的代谢组学检测。结合基因组学、蛋白组学、代谢组学，纳米孔平台最终可发展为一种满足全组学分析需求的通用型测量平台，为更深刻得理解生命规律与疾病发生机制提供强有力的研究工具。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 means, to complete the metabolomic detection of various small molecules and macromolecules. Combining genomics, proteomics, and metabolomics, the nanopore platform can eventually develop into a general-purpose measurement platform that meets the needs of full-omics analysis, providing a powerful research tool for a deeper understanding of the law of life and the mechanism of disease occurrence .
下面将结合具体的实施例来进一步详细解释本申请的有益效果。The beneficial effects of the present application will be further explained in detail below in conjunction with specific embodiments.
实施例1纳米孔蛋白MP964表达载体的构建Example 1 Construction of Nanoporin MP964 Expression Vector
通过In-fusion的方法，采用NdeI和XhoI酶切后，将纳米孔蛋白MP964编码的基因序列(SEQ ID NO：3)***到载体pET24a的克隆区。将MP964氨基酸序列的N端6个His作为纯化标签，表达载体中筛选标签为卡那霉素，将构建好的载体命名为pET24a-MP964。Through the In-fusion method, the gene sequence (SEQ ID NO: 3) encoded by nanoporin MP964 was inserted into the cloning region of the vector pET24a after digestion with NdeI and XhoI. The six His at the N-terminal of the amino acid sequence of MP964 were used as purification tags, the screening tag in the expression vector was kanamycin, and the constructed vector was named pET24a-MP964.
实施例2孔蛋白MP964突变体(MP964Mut)的表达载体的构建Construction of the expression vector of embodiment 2 porin MP964 mutant (MP964Mut)
通过定点突变的方法，采用Agilent定点突变试剂盒，以纳米孔蛋白MP964的表达载体为模板，构建相应的突变蛋白MP964Mut的表达基因。纳米孔蛋白MP964突变体氨基酸序列的N端6个His作为纯化标签，表达载体中筛选标签为卡那霉素，将构建好的载体命名为pET24a-MP964Mut。Through the method of site-directed mutagenesis, the Agilent site-directed mutagenesis kit was used, and the expression vector of nanoporin MP964 was used as a template to construct the expression gene of the corresponding mutant protein MP964Mut. The N-terminal 6 His of the amino acid sequence of the nanoporin MP964 mutant was used as a purification tag, the screening tag in the expression vector was kanamycin, and the constructed vector was named pET24a-MP964Mut.
实施例3表达MP964菌株的培养和诱导Embodiment 3 expresses the cultivation and induction of MP964 bacterial strain
LB液体培养基：胰蛋白胨10g/L，酵母提取物5g/L，NaCl 10g/L。LB liquid medium: tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L.
将重组表达载体pET24a-MP964转化到大肠杆菌表达菌株E.coli BL21(DE3)中，将菌液均匀涂抹在50μg/mL卡那霉素的平板上，37℃过夜培养。挑取单菌落，于5mL LB液体培养基(含有50μg/mL卡那霉素)培养，37℃，200rpm，过夜培养。将上述所得菌液，按1:100接种于50mL LB(含有50μg/mL卡那霉素)中培养，37℃，200rpm，4h。将扩大培养的菌液，按1:100接种于2L LB液体培养基(含有50μg/mL卡那霉素)中培养，37℃，200rpm，待OD600值达0.6-0.8左右，加入异丙基-β-D-硫代半乳糖苷(IPTG)至终浓度为0.5mM，18℃，200rpm，培养过夜，约16-18h。将长好的菌体于8000rpm离心收集，菌体冻存于-20℃待用。The recombinant expression vector pET24a-MP964 was transformed into Escherichia coli expression strain E.coli BL21(DE3), and the bacterial solution was evenly spread on a plate with 50 μg/mL kanamycin, and cultured overnight at 37°C. Pick a single colony and culture it in 5 mL LB liquid medium (containing 50 μg/mL kanamycin) at 37°C, 200 rpm, and culture overnight. Inoculate the bacterial solution obtained above into 50 mL LB (containing 50 μg/mL kanamycin) at a ratio of 1:100 and culture at 37°C, 200 rpm, for 4 hours. Inoculate the expanded cultured bacterial solution in 2L LB liquid medium (containing 50μg/mL kanamycin) at a ratio of 1:100, and cultivate at 37°C, 200rpm. When the OD600 value reaches about 0.6-0.8, add isopropyl- β-D-thiogalactoside (IPTG) to a final concentration of 0.5mM, 18°C, 200rpm, cultivate overnight, about 16-18h. The grown bacteria were collected by centrifugation at 8000 rpm, and the bacteria were frozen and stored at -20°C until use.
实施例4表达MP964Mut菌株的培养和诱导Embodiment 4 expresses the cultivation and induction of MP964Mut bacterial strain
LB液体培养基：胰蛋白胨10g/L，酵母提取物5g/L，NaCl 10g/L。LB liquid medium: tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L.
将重组表达载体pET24a-MP964Mut转化到大肠杆菌表达菌株E.coli BL21(DE3)中，将菌液均匀涂抹在50μg/mL卡那霉素的平板上，37℃过夜培养。挑取单菌落，于5mL LB液体培养基(含有50μg/mL卡那霉素)培养，37℃，200rpm，过夜培养。将上述所得菌液，按1:100接种于50mL LB液体培养基(含有50μg/mL卡那霉素)中培养，37℃，200rpm，4h。将扩大培养的菌液，按1:100接种于2L LB液体培养基(含有50μg/mL卡那霉素)中培养，37℃，200rpm，待OD600值达0.6-0.8左右，加入IPTG至终浓度为0.5mM，18℃，200rpm，培养过夜，约16-18h。将长好的菌体于8000rpm离心收集，菌体冻存于-20℃待用。The recombinant expression vector pET24a-MP964Mut was transformed into E. coli expression strain E.coli BL21(DE3), and the bacterial solution was evenly spread on a plate with 50 μg/mL kanamycin, and cultivated overnight at 37°C. Pick a single colony and culture it in 5 mL LB liquid medium (containing 50 μg/mL kanamycin) at 37°C, 200 rpm, and culture overnight. Inoculate the bacterium solution obtained above into 50 mL LB liquid medium (containing 50 μg/mL kanamycin) at a ratio of 1:100 and culture at 37°C, 200 rpm, for 4 hours. Inoculate the expanded cultured bacterial solution in 2L LB liquid medium (containing 50μg/mL kanamycin) at a ratio of 1:100 and cultivate at 37°C, 200rpm. When the OD600 value reaches about 0.6-0.8, add IPTG to the final concentration 0.5mM, 18°C, 200rpm, cultivate overnight, about 16-18h. The grown bacteria were collected by centrifugation at 8000 rpm, and the bacteria were frozen and stored at -20°C until use.
实施例5 MP964蛋白提取及纯化Example 5 MP964 protein extraction and purification
纯化Buffer配制：Purification Buffer preparation:
Ni柱亲和层析Ni column affinity chromatography
Buffer A平衡缓冲液：20mM Tris-HCl+250mM NaCl+0.5％Tween-20+5％甘油，pH 7.9。Buffer A equilibration buffer: 20mM Tris-HCl+250mM NaCl+0.5% Tween-20+5% glycerol, pH 7.9.
Buffer B洗脱缓冲液：20mM Tris-HCl+250mM NaCl+0.5％Tween-20+5％甘油+500mM咪唑，pH 7.9。Buffer B elution buffer: 20mM Tris-HCl+250mM NaCl+0.5% Tween-20+5% glycerol+500mM imidazole, pH 7.9.
离子交换层析ion exchange chromatography
Buffer Dilute稀释缓冲液：Buffer Dilute dilution buffer:
Buffer C平衡缓冲液：20mM Tris-HCl+50mM NaCl+0.5％Tween-20+5％甘油，pH 6.5。Buffer C equilibration buffer: 20mM Tris-HCl+50mM NaCl+0.5% Tween-20+5% glycerol, pH 6.5.
Buffer D洗脱缓冲液：20mM Tris-HCl+1000mM NaCl+0.5％Tween-20+5％甘油，pH 6.5。Buffer D elution buffer: 20mM Tris-HCl+1000mM NaCl+0.5% Tween-20+5% glycerol, pH 6.5.
蛋白样品稀释液protein sample diluent
Buffer E稀释液：20mM Tris-HCl+0.5％Tween-20+5％甘油，pH 6.5。Buffer E dilution: 20mM Tris-HCl+0.5% Tween-20+5% glycerol, pH 6.5.
按1g菌体加10mL亲和A液的比例重悬菌体，超声破碎细胞，直至菌体溶液至澄清。将破碎后的菌体12000rpm，4℃离心30min，取上清，0.22μm滤膜过滤后于4℃储存。Resuspend the cells according to the ratio of 1 g of cells to 10 mL of Affinity A solution, and disrupt the cells by ultrasonic until the cell solution is clear. The crushed bacteria were centrifuged at 12000 rpm at 4°C for 30 min, and the supernatant was taken, filtered through a 0.22 μm filter membrane and stored at 4°C.
将Ni柱亲合层析柱水洗5CV，Buffer B清洗5CV，Buffer A进行平衡10CV后，进行上样。上样完成后，平衡15CV，使用Buffer B进行线性洗脱(0-8％Buffer B，30CV)去除杂质，使用Buffer B进行线性洗脱(8-100％Buffer B，5CV)收集目的蛋白。Wash the Ni column affinity chromatography column with water for 5CV, wash Buffer B for 5CV, and equilibrate Buffer A for 10CV before loading the sample. After loading the sample, balance 15CV, use Buffer B for linear elution (0-8% Buffer B, 30CV) to remove impurities, and use Buffer B for linear elution (8-100% Buffer B, 5CV) to collect the target protein.
将Ni柱收集到的蛋白用Buffer E稀释2倍，将阴离子交换Q柱水洗5CV，Buffer C平衡5CV，蛋白样品上样。用Buffer C平衡5CV后，用洗脱缓冲液Buffer D线性洗脱(0-9％Buffer D，30CV)，用洗脱缓冲液Buffer D线性洗脱(9-100％Buffer D，10CV)并收集蛋白。收集蛋白进行4℃过夜透析，透析液为Buffer A，随后储存于-80℃。如图1所示，SDS-PAGE结果显示目标蛋白主要以单体形式存在，表明纳米孔蛋白MP964的聚体稳定性较差。Dilute the protein collected by the Ni column 2 times with Buffer E, wash the anion exchange Q column with water for 5CV, equilibrate with Buffer C for 5CV, and load the protein sample. After equilibrating 5CV with Buffer C, linearly eluted with elution buffer Buffer D (0-9% Buffer D, 30CV), linearly eluted with elution buffer Buffer D (9-100% Buffer D, 10CV) and collected protein. Proteins were collected and dialyzed overnight at 4°C with Buffer A as the dialysate, and then stored at -80°C. As shown in Figure 1, the results of SDS-PAGE showed that the target protein mainly existed in the form of monomers, indicating that the polymer stability of nanoporin MP964 was poor.
实施例6纳米孔蛋白MP964Mut蛋白提取及纯化Example 6 Extraction and Purification of Nanoporin MP964Mut Protein
按1g菌体加10mL亲和A液的比例重悬菌体，超声破碎细胞，直至菌体溶液至澄清。将破碎后的菌体12000rpm，4℃离心30min，取上清，0.22μm滤膜过滤后于4℃储存。Resuspend the cells according to the ratio of 1 g of cells to 10 mL of Affinity A solution, and disrupt the cells by ultrasonic until the cell solution is clear. The crushed bacteria were centrifuged at 12000 rpm at 4°C for 30 min, and the supernatant was taken, filtered through a 0.22 μm filter membrane and stored at 4°C.
将Ni柱亲合层析柱水洗5CV，Buffer B清洗5CV，Buffer A进行平衡10CV后，进行上样。上样完成后，平衡15CV，使用Buffer B进行线性洗脱(0-8％Buffer B，30CV)去除杂质，使用Buffer B进行线性洗脱(8-100％Buffer B，5CV)收集目的蛋白。Wash the Ni column affinity chromatography column with water for 5CV, wash Buffer B for 5CV, and equilibrate Buffer A for 10CV before loading the sample. After loading the sample, balance 15CV, use Buffer B for linear elution (0-8% Buffer B, 30CV) to remove impurities, and use Buffer B for linear elution (8-100% Buffer B, 5CV) to collect the target protein.
将Ni柱收集到的蛋白用Buffer E稀释2倍，将阴离子交换Q柱水洗5CV，Buffer C平衡5CV，蛋白样品上样。用Buffer C平衡5CV后，用洗脱缓冲液Buffer D线性洗脱(0-9％Buffer D，30CV)，用洗脱缓冲液Buffer D线性洗脱(9-100％Buffer D，10CV)并收集蛋白。收集蛋白进行4℃过夜透析，透析液为Buffer A，随后储存于-80℃。重组蛋白体外表达纯化后得到的蛋白是以单体形式还是多聚体的形式存在，多聚体含量越多，说明其以多聚体形式形成的孔道的稳定性越好。如图2所示，SDS-PAGE结果显示蛋白的纯化效果很好，目标蛋白主要以聚体形式存在，表明纳米孔蛋白MP964Mut的聚体稳定性有大幅提升，满足应用需求。Dilute the protein collected by the Ni column 2 times with Buffer E, wash the anion exchange Q column with water for 5CV, equilibrate with Buffer C for 5CV, and load the protein sample. After equilibrating 5CV with Buffer C, linearly eluted with elution buffer Buffer D (0-9% Buffer D, 30CV), linearly eluted with elution buffer Buffer D (9-100% Buffer D, 10CV) and collected protein. Proteins were collected and dialyzed overnight at 4°C with Buffer A as the dialysate, and then stored at -80°C. Whether the protein obtained after in vitro expression and purification of the recombinant protein exists in the form of a monomer or a multimer, and the higher the content of the multimer, the better the stability of the pores formed by it in the form of a multimer. As shown in Figure 2, the results of SDS-PAGE show that the purification effect of the protein is very good, and the target protein mainly exists in the form of aggregates, indicating that the stability of the aggregates of the nanoporous protein MP964Mut has been greatly improved, which meets the application requirements.
实施例7对比MP964和MP964Mut的孔径差异Example 7 Contrasting the pore size difference between MP964 and MP964Mut
用Rosetta软件建模方法构建MP964(图3)与MP964Mut(图4)的分子结构，得到两 个孔蛋白的孔径分别为
Figure PCTCN2021143722-appb-000001
和
Figure PCTCN2021143722-appb-000002
说明突变的引入成功地缩小了纳米孔蛋白的孔径，导致突变体MP964Mut具有更好的测序准确度。 The molecular structures of MP964 (Fig. 3) and MP964Mut (Fig. 4) were constructed by Rosetta software modeling method, and the pore diameters of the two porins were obtained as
Figure PCTCN2021143722-appb-000001
and
Figure PCTCN2021143722-appb-000002
It shows that the introduction of the mutation successfully reduced the pore size of the nanoporin, resulting in better sequencing accuracy of the mutant MP964Mut.
实施例8对比MP964和MP964Mut的捕获率差异Example 8 Contrasting the capture rate difference between MP964 and MP964Mut
本实验单通道电流检测基于Axon Digidata 1550B低噪音数据采集***和Axopatch 200B膜片钳放大器。在含有150μm小孔的Teflon材料的电解槽中央形成由二植酰磷脂酰胆碱(DPhPC，1，2-diphytanoyl-sn-glycero-3-phosphocholine)组成的磷脂双分子层；Ag和AgCl电极分别放置在由磷脂双分子层膜隔开的电解槽的正极电解液区和负极电解液区，并且2个区域充满纳米孔实验缓冲液(0.5M KCl，10mM HEPES，1mM EDTA，pH 7.8)。将用PBS缓冲液稀释的纳米孔蛋白加入电解槽正极电解液区后；施加电压180mV，待单个纳米孔蛋白***到磷脂双分子层中，形成纳米孔生物膜。The single-channel current detection in this experiment is based on Axon Digidata 1550B low-noise data acquisition system and Axopatch 200B patch clamp amplifier. A phospholipid bilayer composed of diphytylphosphatidylcholine (DPhPC, 1,2-diphytanoyl-sn-glycero-3-phosphocholine) was formed in the center of the electrolytic cell containing 150 μm small pores of Teflon material; the Ag and AgCl electrodes were respectively Placed in the positive electrolyte area and the negative electrolyte area of the electrolytic cell separated by the phospholipid bilayer membrane, and the two areas are filled with nanopore experimental buffer (0.5M KCl, 10mM HEPES, 1mM EDTA, pH 7.8). Add the nanoporin diluted with PBS buffer into the positive electrolyte area of the electrolytic cell; apply a voltage of 180mV, and wait for a single nanoporin to insert into the phospholipid bilayer to form a nanoporous biofilm.
DNA在电场力作用下穿过纳米孔蛋白，产生阻滞电流振幅。纳米孔蛋白MP964对寡核苷酸样品的电流阻滞百分比分布图参见图5，纳米孔蛋白MP964Mut对寡核苷酸样品的电流阻滞百分比分布图参见图6。纳米孔蛋白MP964及MP964Mut的DNA捕获率的统计比较参见图7。说明相较于MP964，突变后的纳米孔蛋白MP964Mut具有更好的DNA捕获能力。DNA passes through the nanoporin under the action of an electric field force, resulting in a blocking current amplitude. See FIG. 5 for the percentage distribution of the current blockade of the nanoporin MP964 to the oligonucleotide sample, and FIG. 6 for the percentage distribution of the current blockade of the nanopore protein MP964Mut to the oligonucleotide sample. See Figure 7 for the statistical comparison of the DNA capture rates of nanoporins MP964 and MP964Mut. It shows that compared with MP964, the mutated nanopore protein MP964Mut has better DNA capture ability.
实施例9利用纳米孔蛋白MP964Mut构建纳米孔生物膜并应用于G-四链体结构区分Example 9 Constructing Nanopore Biomembrane Using Nanoporin MP964Mut and Applying to G-quadruplex Structural Discrimination
G-四链体(G-quadruplex)是由含有串联重复的鸟嘌呤的DNA折叠而成的高级结构，在原核和真核细胞中广泛存在，参与基因复制、重组、调控等多种功能，在细胞的生命活动中扮演者重要的作用。因此对G-四链体的结构、生物学功能的基础研究具有重要意义。G-quadruplex (G-quadruplex) is a high-level structure folded by DNA containing tandem repeated guanine. It exists widely in prokaryotic and eukaryotic cells and participates in multiple functions such as gene replication, recombination, and regulation. Plays an important role in the life activities of cells. Therefore, the basic research on the structure and biological function of G-quadruplex is of great significance.
本实施例的单通道电流检测基于Axon Digidata 1550B低噪音数据采集***和Axopatch200B膜片钳放大器。在含有150μm小孔的Teflon材料的电解槽中央形成由二植酰磷脂酰胆碱(DPhPC，1，2-diphytanoyl-sn-glycero-3-phosphocholine)组成的磷脂双分子层；Ag/AgCl电极放置在由磷脂双分子层膜隔开的电解槽的正极和负极电解液区，并且2个区域充满纳米孔实验缓冲液(0.5M KCl，10mM HEPES，1mM EDTA，pH 7.8)。将用PBS缓冲液稀释的孔蛋白加入正极电解液区后；施加电压180mV，待单个纳米孔蛋白***到磷脂双分子层中，形成纳米孔生物膜。通过电流的变化能够判断是否是单个蛋白***磷脂双分子层中。The single channel current detection of this embodiment is based on Axon Digidata 1550B low noise data acquisition system and Axopatch200B patch clamp amplifier. A phospholipid bilayer composed of diphytylphosphatidylcholine (DPhPC, 1,2-diphytanoyl-sn-glycero-3-phosphocholine) is formed in the center of the electrolytic cell containing 150 μm small pores of Teflon material; Ag/AgCl electrodes are placed In the positive and negative electrolyte areas of the electrolyzer separated by a phospholipid bilayer membrane, the two areas are filled with nanopore experimental buffer (0.5M KCl, 10mM HEPES, 1mM EDTA, pH 7.8). After the porin diluted with PBS buffer solution is added to the positive electrode electrolyte area; a voltage of 180mV is applied, and a single nanoporin is inserted into the phospholipid bilayer to form a nanoporous biofilm. Whether a single protein is inserted into the phospholipid bilayer can be judged by the change of the current.
图8展示了纳米孔蛋白MP964Mut在不同批次实验中的电导分布情况，用于考察该纳米孔的均一性。图8中电导率的分布有一个明显的主峰，反应了在单分子实验中的开孔电流的均一性，开孔电流的分布越集中，说明形成的孔道越均一，也可以说明孔道稳定性越好。Figure 8 shows the conductance distribution of the nanopore protein MP964Mut in different batches of experiments, which is used to investigate the uniformity of the nanopore. The distribution of conductivity in Figure 8 has an obvious main peak, which reflects the uniformity of the opening current in the single-molecule experiment. The more concentrated the distribution of the opening current, the more uniform the formed pores, and the more stable the pores. good.
将寡核苷酸样品A(SEQ ID NO：5：ggttggtgtggttgg)或样品B(SEQ ID NO：6：ggttggtgtggttggtttttttttt)溶解在纳米孔缓冲液中并对其退火，冷却到4℃后待用。待单个纳米孔蛋白***磷脂双分子层中，加入适量的寡核苷酸样品A或寡核苷酸样品B到电解槽正极电解液区。DNA在电场力作用下穿过纳米孔，产生阻滞电流振幅。不同结构的四链体DNA聚合物产生的阻滞电流振幅不同，因此电流振幅可以用来区分不同序列组成的G-四链体。电流阻 滞百分比是与穿孔事件相关的阻滞电流(Ib)与开孔电流(Io)的比值。结果见图9、图10、图11和图12。Oligonucleotide sample A (SEQ ID NO: 5: ggttggtgtggttgg) or sample B (SEQ ID NO: 6: ggttggtgtggttggtttttttttt) was dissolved in nanopore buffer and annealed, cooled to 4°C before use. After a single nanoporin is inserted into the phospholipid bilayer, add an appropriate amount of oligonucleotide sample A or oligonucleotide sample B to the positive electrolyte area of the electrolytic cell. DNA passes through the nanopore under the action of an electric field force, generating a blocking current amplitude. Quadruplex DNA polymers with different structures produce different amplitudes of blocking currents, so the current amplitudes can be used to distinguish G-quadruplexes composed of different sequences. The percent current block is the ratio of the blocked current (Ib) to the open pore current (Io) associated with the porosity event. The results are shown in Figure 9, Figure 10, Figure 11 and Figure 12.
图9和图11中的Io与Ib是两个不同的电流值台阶。Io and Ib in Figure 9 and Figure 11 are two different current value steps.
本实施例说明该蛋白MP964Mut能够构建纳米孔生物膜，对不同的特殊结构DNA的检测中，可以实现不同的特征阻滞电流的区分。This example shows that the protein MP964Mut can construct a nanopore biomembrane, and in the detection of DNA with different special structures, it can realize the distinction of different characteristic blocking currents.
实施例10利用纳米孔蛋白MP964Mut构建纳米孔生物膜并用于DNA测序。Example 10 The nanopore biofilm was constructed using nanopore protein MP964Mut and used for DNA sequencing.
文库构建：利用EcoRI和HindIII限制性内切酶对质粒pUC57在37℃进行酶切2小时；然后使用0.4-0.6X AMPure XP磁珠(Beckman)进行纯化获得纯度较高的双链DNA片段。利用连接测序109试剂盒(SQK-LSK109，Oxford Nanopore Technologies Ltd)对目的DNA片段构建测序文库。测序缓冲液：0.5M KCl，10mM HEPES，1mM EDTA，10mM MgCl 2，2mM ATP，pH 7.8。 Library construction: Plasmid pUC57 was digested with EcoRI and HindIII restriction enzymes at 37°C for 2 hours; then purified using 0.4-0.6X AMPure XP magnetic beads (Beckman) to obtain double-stranded DNA fragments with high purity. A sequencing library was constructed for the target DNA fragments using the ligation sequencing 109 kit (SQK-LSK109, Oxford Nanopore Technologies Ltd). Sequencing buffer: 0.5M KCl, 10 mM HEPES, 1 mM EDTA, 10 mM MgCl 2 , 2 mM ATP, pH 7.8.
单通道纳米孔电流测量基于数字化装置的放大器。Ag/AgCl电极浸润在测序缓冲液中并且电极分别位于正极电解液区和负极电解液区。测序文库和纳米孔等试剂加入到正极电解液区中。使用1xPBS缓冲液将纳米孔蛋白稀释一定的倍数后(10 5-10 7稀释，本实施例中的稀释倍数为10 6)在外加电场力作用下将单个纳米孔***由二植酰磷脂酰胆碱(DPhPC，1，2-diphytanoyl-sn-glycero-3-phosphocholine)组成的磷脂双分子层中，形成纳米孔生物膜。施加外加电压，获得单个孔蛋白的电流振幅值。本实验中施加电压范围为50-200mV，图13和图14分别展示了在180mV和150mV的电压条件下，DNA移动产生的电流振幅。 Single-channel nanopore amperometric measurements based on digitizer amplifiers. The Ag/AgCl electrode is soaked in the sequencing buffer and the electrodes are respectively located in the positive electrolyte area and the negative electrode electrolyte area. Reagents such as sequencing libraries and nanopores are added to the catholyte zone. After diluting the nanopore protein with 1xPBS buffer for a certain number of times (10 5 -10 7 dilution, the dilution factor in this example is 10 6 ), under the action of an external electric field, a single nanopore was inserted into the diphytylphosphatidylcholine In the phospholipid bilayer composed of alkali (DPhPC, 1,2-diphytanoyl-sn-glycero-3-phosphocholine), a nanoporous biofilm is formed. Applied voltage is applied and current amplitude values are obtained for individual porins. In this experiment, the applied voltage range was 50-200mV. Figure 13 and Figure 14 show the current amplitudes generated by DNA movement under the voltage conditions of 180mV and 150mV, respectively.
将含有pUC57序列的测序文库和带有胆固醇的单链DNA(FLT试剂)与测序缓冲液混合并加入纳米孔测序装置中；施加外加电压150mV或180mV后，观察到DNA被纳米孔捕获，产生特征的阻滞电流振幅值。并且随着解旋酶拉扯DNA通过纳米孔进行移动，电流振幅值逐步改变。不同的DNA序列产生不同的阻滞电流振幅值。带有胆固醇的单链DNA可以与磷脂双分子层进行结合，有助于纳米孔捕获测序文库，降低测序文库上样量。The sequencing library containing the pUC57 sequence and the single-stranded DNA with cholesterol (FLT reagent) were mixed with the sequencing buffer and added to the nanopore sequencing device; after applying an applied voltage of 150mV or 180mV, it was observed that the DNA was captured by the nanopore, resulting in characteristic The amplitude value of the blocking current. And as the helicase moves the DNA through the nanopore, the amplitude of the current changes stepwise. Different DNA sequences produce different blocking current amplitude values. The single-stranded DNA with cholesterol can be combined with the phospholipid bilayer, which helps the nanopore capture the sequencing library and reduces the loading amount of the sequencing library.
其中，图13示出了解旋酶在外加电压180mV下控制DNA穿过纳米孔蛋白突变体MP964Mut，随着DNA移动产生不同幅度大小的电流振幅。图13(A)为电流特征图，图13(B)为放大电流图。图14示出了解旋酶在外加电压150mV下控制DNA穿过纳米孔蛋白突变体MP964Mut，随着DNA移动产生不同幅度大小的电流振幅变化。图14(A)为电流特征图，图14(B)为放大电流图。Wherein, Fig. 13 shows that the helicase controls DNA passing through the nanoporin mutant MP964Mut under an applied voltage of 180mV, and current amplitudes of different magnitudes are generated as the DNA moves. FIG. 13(A) is a current characteristic diagram, and FIG. 13(B) is an enlarged current diagram. FIG. 14 shows that the helicase controls DNA to pass through the nanoporin mutant MP964Mut under an applied voltage of 150 mV, and the current amplitude changes with different amplitudes are produced as the DNA moves. FIG. 14(A) is a current characteristic diagram, and FIG. 14(B) is an enlarged current diagram.
本实施例说明纳米孔蛋白MP964Mut可以构建出一个成功的纳米孔生物膜，并且可以实现DNA片段的测序。This example shows that the nanopore protein MP964Mut can construct a successful nanopore biofilm, and can realize the sequencing of DNA fragments.
从以上的描述中，可以看出，本发明上述的实施例实现了如下技术效果：通过对纳米孔蛋白MP964进行突变，使得突变后的纳米孔蛋白的稳定性提高、孔径减小、对待测生物分子捕获能力提高，便于在后续的测序中提高测序准确度和测序的数据通量。From the above description, it can be seen that the above-mentioned embodiments of the present invention have achieved the following technical effects: by mutating the nanoporin MP964, the stability of the mutated nanoporin is improved, the pore size is reduced, and the tested biological The improved molecular capture ability facilitates the improvement of sequencing accuracy and sequencing data throughput in subsequent sequencing.
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims (19)

  1. 一种纳米孔蛋白，其特征在于，包括：A nanopore protein, characterized in that, comprising:
    (a)MP964，所述MP964为具有SEQ ID NO：1所示的氨基酸序列的蛋白质；或(a) MP964, said MP964 is a protein having the amino acid sequence shown in SEQ ID NO: 1; or
    (b)MP964Mut，所述MP964Mut为具有SEQ ID NO：2所示的氨基酸序列的蛋白质；或(b) MP964Mut, said MP964Mut is a protein having the amino acid sequence shown in SEQ ID NO: 2; or
    (c)在(a)或(b)中的所述氨基酸序列的如下至少一个位点：第97位、第98位、第127位、第143位、第148位，经过取代和/或缺失和/或添加一个或几个氨基酸且具有孔道结构的蛋白质；或(c) At least one of the following positions in the amino acid sequence in (a) or (b): 97th, 98th, 127th, 143rd, 148th, substituted and/or deleted And/or add one or several amino acids and have a protein with a pore structure; or
    (d)与(a)、(b)和(c)中任一所限定的所述氨基酸序列具有80％以上同源性且具有相同功能的蛋白质。(d) A protein having more than 80% homology with the amino acid sequence defined in any one of (a), (b) and (c) and having the same function.
  2. 根据权利要求1所述的纳米孔蛋白，其特征在于，所述(c)中，各位点取代的氨基酸的类型各自独立地选自如下：Nanoporin according to claim 1, characterized in that, in (c), the types of amino acids substituted at each site are independently selected from the following:
    第97位：Q/Y/A/S/H；No. 97: Q/Y/A/S/H;
    第98位：Q/Y/S/H；No. 98: Q/Y/S/H;
    第127位：K/H/Y；The 127th place: K/H/Y;
    第143位：K/H/Y；No. 143: K/H/Y;
    第148位：R/H/Y；Bit 148: R/H/Y;
    其中，“/”代表“或”；Among them, "/" stands for "or";
    优选地，所述(d)中，与(a)、(b)和(c)中任一所限定的所述氨基酸序列具有85％以上，优选90％以上，更优选95％以上，进一步优选99％以上同源性且具有相同功能的蛋白质；Preferably, in (d), the amino acid sequence as defined in any one of (a), (b) and (c) has more than 85%, preferably more than 90%, more preferably more than 95%, even more preferably Proteins with more than 99% homology and the same function;
    优选地，所述纳米孔蛋白来源于分枝杆菌。Preferably, the nanoporin is derived from mycobacteria.
  3. 根据权利要求1所述的纳米孔蛋白，其特征在于，所述纳米孔蛋白的孔道直径为1.2～1.6nm；The nanoporin according to claim 1, wherein the pore diameter of the nanoporin is 1.2 to 1.6 nm;
    优选地，所述纳米孔蛋白在150mV电压下的电流振幅值为145～155pA；Preferably, the current amplitude value of the nanoporin at a voltage of 150mV is 145-155pA;
    优选地，所述纳米孔蛋白在180mV电压下的电流振幅值为170～190pA；Preferably, the current amplitude value of the nanoporin at a voltage of 180mV is 170-190pA;
    优选地，所述纳米孔蛋白的电导为0.8～1.2nS。Preferably, the conductance of the nanoporin is 0.8-1.2 nS.
  4. 一种试剂盒，其特征在于，所述试剂盒包括权利要求1至3中任一项所述的纳米孔蛋白。A kit, characterized in that the kit comprises the nanoporin according to any one of claims 1-3.
  5. 根据权利要求4所述的试剂盒，其特征在于，所述试剂盒还包括脂质层或人造高分子膜；The kit according to claim 4, wherein the kit also includes a lipid layer or an artificial polymer membrane;
    优选地，所述脂质层包括两亲脂类；Preferably, said lipid layer comprises amphiphilic lipids;
    优选地，所述两亲脂类包含磷脂双分子层；Preferably, said amphiphilic lipid comprises a phospholipid bilayer;
    优选地，所述脂质层包括平面膜层或脂质体；Preferably, the lipid layer comprises a planar membrane layer or a liposome;
    优选地，所述脂质体包括多层脂质体或单层脂质体；Preferably, the liposomes comprise multilamellar liposomes or unilamellar liposomes;
    优选地，所述脂质层包括二植酰磷脂酰胆碱组成的磷脂双分子层。Preferably, the lipid layer includes a phospholipid bilayer composed of diphytylphosphatidylcholine.
  6. 根据权利要求4或5所述的试剂盒，其特征在于，所述试剂盒还包括纳米孔蛋白实验缓冲液；The kit according to claim 4 or 5, wherein the kit also includes a nanoporin experiment buffer;
    优选地，所述纳米孔蛋白实验缓冲液为HEPES缓冲液；Preferably, the nanoporin experiment buffer is HEPES buffer;
    优选地，所述纳米孔蛋白实验缓冲液含有0.1-1.0M KCl；Preferably, the nanoporin assay buffer contains 0.1-1.0M KCl;
    优选地，所述纳米孔蛋白实验缓冲液为0.5M KCl，10mM HEPES，1mM EDTA，pH 7.8。Preferably, the nanoporin experiment buffer is 0.5M KCl, 10mM HEPES, 1mM EDTA, pH 7.8.
  7. 一种分离的DNA分子，其特征在于，所述DNA分子具有An isolated DNA molecule, characterized in that the DNA molecule has
    (a)编码权利要求1或2所述的纳米孔蛋白的核苷酸序列；或(a) a nucleotide sequence encoding the nanoporin of claim 1 or 2; or
    (b)在严格条件下与(a)限定的DNA分子杂交的核苷酸序列；或(b) a nucleotide sequence that hybridizes under stringent conditions to the DNA molecule defined in (a); or
    (c)具有SEQ ID NO：3或SEQ ID NO：4所示的核苷酸序列；或(c) has the nucleotide sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4; or
    (d)与(a)至(c)中限定的任一种所述核苷酸序列具有70％以上同源性且编码具有相同功能蛋白质的DNA分子。(d) A DNA molecule having more than 70% homology with any one of the nucleotide sequences defined in (a) to (c) and encoding a protein with the same function.
  8. 根据权利要求7所述的DNA分子，其特征在于，与(a)至(c)中限定的任一种所述核苷酸序列具有75％以上，优选85％以上，更优选95％以上，进一步优选99％以上同源性且编码具有相同功能蛋白质的DNA分子。The DNA molecule according to claim 7, characterized in that it has more than 75%, preferably more than 85%, more preferably more than 95% of any of the nucleotide sequences defined in (a) to (c), Further preferred are DNA molecules that are more than 99% homologous and encode proteins with the same function.
  9. 一种重组载体，其特征在于，所述重组载体包含权利要求7或8所述的DNA分子。A recombinant vector, characterized in that the recombinant vector comprises the DNA molecule according to claim 7 or 8.
  10. 一种宿主细胞，其特征在于，所述宿主细胞转化有权利要求9所述的重组载体。A host cell, characterized in that the host cell is transformed with the recombinant vector according to claim 9.
  11. 一种纳米孔生物膜，其特征在于，所述纳米孔生物膜包括：A nanoporous biofilm, characterized in that the nanoporous biofilm comprises:
    膜层；以及film layer; and
    ***所述膜层中间以形成孔道的纳米孔蛋白，当跨越所述膜层施加电场力时，所述孔道发生电导；a nanoporin inserted into the middle of the membrane layer to form a channel that conducts electricity when an electric field force is applied across the membrane layer;
    其中，所述纳米孔蛋白包括权利要求1至3中任一项所述的纳米孔蛋白。Wherein, the nanoporin comprises the nanoporin according to any one of claims 1-3.
  12. 根据权利要求11所述的纳米孔生物膜，其特征在于，所述膜层包括脂质层；The nanopore biofilm according to claim 11, wherein the membrane layer comprises a lipid layer;
    优选地，所述脂质层包括两亲脂类；Preferably, said lipid layer comprises amphiphilic lipids;
    优选地，所述两亲脂类包含磷脂双分子层；Preferably, said amphiphilic lipid comprises a phospholipid bilayer;
    优选地，所述脂质层包括平面膜层或脂质体；Preferably, the lipid layer comprises a planar membrane layer or a liposome;
    优选地，所述脂质体包括多层脂质体或单层脂质体；Preferably, the liposomes comprise multilamellar liposomes or unilamellar liposomes;
    优选地，所述脂质层包括二植酰磷脂酰胆碱组成的磷脂双分子层。Preferably, the lipid layer includes a phospholipid bilayer composed of diphytylphosphatidylcholine.
  13. 根据权利要求12所述的纳米孔生物膜，其特征在于，所述纳米孔蛋白在所述膜层中是可移动的；The nanoporous biofilm of claim 12, wherein the nanoporin is mobile in the membrane layer;
    优选地，当跨越所述膜层施加电场力时，所述纳米孔生物膜能够通过所述孔道将待测生物分子移位；Preferably, said nanoporous biofilm is capable of translocating a biomolecule to be tested through said pores when an electric field force is applied across said membrane layer;
    优选地，所述待测生物分子包括DNA、RNA、多肽或蛋白质；Preferably, the biomolecules to be tested include DNA, RNA, polypeptide or protein;
    优选地，所述待测生物分子带有修饰的基团分子，更优选所述基团分子选自胆固醇、聚乙二醇、生物素或荧光基团分子；Preferably, the biomolecule to be tested has a modified group molecule, more preferably the group molecule is selected from cholesterol, polyethylene glycol, biotin or fluorescent group molecule;
    优选地，所述DNA和/或RNA包括如下任意一种或多种修饰碱基：5-甲基胞嘧啶、6-甲基腺嘌呤、7-甲基鸟嘌呤或假尿嘧啶。Preferably, the DNA and/or RNA includes any one or more of the following modified bases: 5-methylcytosine, 6-methyladenine, 7-methylguanine or pseudouracil.
  14. 一种纳米孔测序装置，其特征在于，所述纳米孔测序装置包括权利要求11至13中任一项所述的纳米孔生物膜。A nanopore sequencing device, characterized in that the nanopore sequencing device comprises the nanopore biofilm according to any one of claims 11-13.
  15. 根据权利要求14所述的纳米孔测序装置，其特征在于，所述纳米孔测序装置包括：The nanopore sequencing device according to claim 14, wherein the nanopore sequencing device comprises:
    电解槽，含有电解液；an electrolytic cell containing an electrolytic solution;
    所述纳米孔生物膜，位于所述电解槽的中央，并将所述电解槽及所述电解液分割为正极电解液区和负极电解液区；The nanoporous biofilm is located in the center of the electrolytic cell, and divides the electrolytic cell and the electrolyte into a positive electrolyte area and a negative electrolyte area;
    第一电极和第二电极，所述第一电极和所述第二电极分别设置在所述正极电解液区和所述负极电解液区；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;
    接收电极，所述接收电极包括两个且分别位于所述正极电极液区和所述负极电解液区，所述接收电极与信号处理芯片相连；Receiving electrodes, the receiving electrodes include two and are respectively located in the positive electrode liquid area and the negative electrode electrolyte area, and the receiving electrodes are connected to the signal processing chip;
    优选地，所述电解液为纳米孔蛋白实验缓冲液；Preferably, the electrolyte is a nanoporin experiment buffer;
    优选地，所述纳米孔蛋白实验缓冲液为HEPES缓冲液；Preferably, the nanoporin experiment buffer is HEPES buffer;
    优选地，所述纳米孔蛋白实验缓冲液含有0.1-1.0M KCl；Preferably, the nanoporin assay buffer contains 0.1-1.0M KCl;
    更优选地，所述纳米孔蛋白实验缓冲液包括0.5M KCl，10mM HEPES，1mM EDTA，pH 7.8；More preferably, the nanoporin experiment buffer comprises 0.5M KCl, 10mM HEPES, 1mM EDTA, pH 7.8;
    优选地，所述第一电极和所述第二电极包括金属或复合电极材料；Preferably, said first electrode and said second electrode comprise a metal or a composite electrode material;
    优选地，所述第一电极和所述第二电极不同，分别为银和氯化银；Preferably, the first electrode and the second electrode are different, being silver and silver chloride respectively;
    优选地，所述第一电极和所述第二电极相同，包括金、铂、石墨烯或氮化钛。Preferably, the first electrode and the second electrode are identical, comprising gold, platinum, graphene or titanium nitride.
  16. 一种测序方法，其特征在于，所述测序方法利用权利要求1至3中任一项所述的纳米孔蛋白，或者权利要求11至13中任一项所述的纳米孔生物膜，或者权利要求14或15所述的纳米孔测序装置通过解析待测生物分子通过所述纳米孔蛋白的孔道时产生的电信号，对所述待测生物分子进行测序。A sequencing method, characterized in that, the sequencing method utilizes the nanopore protein according to any one of claims 1 to 3, or the nanopore biofilm according to any one of claims 11 to 13, or the nanopore biofilm according to any one of claims 11 to 13, or the The nanopore sequencing device described in claim 14 or 15 performs sequencing on the biomolecules to be tested by analyzing the electrical signal generated when the biomolecules to be tested pass through the pores of the nanopore protein.
  17. 根据权利要求16所述的测序方法，其特征在于，所述待测生物分子包括修饰或未修饰的DNA、RNA、多肽或蛋白质；The sequencing method according to claim 16, wherein the biomolecule to be tested comprises modified or unmodified DNA, RNA, polypeptide or protein;
    优选地，在电场力的作用下，所述待测生物分子的单个分子通过所述纳米孔蛋白的所述孔道，产生所述电信号；Preferably, under the action of an electric field force, a single molecule of the biomolecule to be tested passes through the channel of the nanoporin to generate the electrical signal;
    优选地，所述电信号包括阻滞电流振幅。Preferably, said electrical signal comprises a blocking current amplitude.
  18. 根据权利要求17所述的测序方法，其特征在于，单个所述纳米孔蛋白***脂质层中，优选为磷脂双分子层中，形成纳米孔生物膜，所述纳米孔蛋白利用所述纳米孔生物膜的结构进行测序。The sequencing method according to claim 17, wherein a single nanopore protein is inserted into a lipid layer, preferably a phospholipid bilayer, to form a nanopore biofilm, and the nanopore protein utilizes the nanopore Biofilm structures are sequenced.
  19. 权利要求1至3中任一项所述的纳米孔蛋白，或者权利要求4至6中任一项所述的试剂盒，或者权利要求7或8所述的DNA分子，或者权利要求9所述的重组载体，或者权利要求10所述的宿主细胞，或者权利要求11至13中任一项所述的纳米孔生物膜，或者权利要求14或15所述的纳米孔测序装置，或者权利要求16至18中任一项所述的测序方法在小分子检测、单分子DNA测序、单分子RNA测序、多肽测序或蛋白质测序中的应用。The nanoporin described in any one of claims 1 to 3, or the kit described in any one of claims 4 to 6, or the DNA molecule described in claim 7 or 8, or the described DNA molecule in claim 9 or the host cell described in claim 10, or the nanopore biofilm described in any one of claims 11 to 13, or the nanopore sequencing device described in claim 14 or 15, or claim 16 Application of the sequencing method described in any one of to 18 in small molecule detection, single molecule DNA sequencing, single molecule RNA sequencing, polypeptide sequencing or protein sequencing.
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105372303A (en) * 2015-09-15 2016-03-02 西北大学 Single-molecule analysis method for detecting methylated DNA
CN106459159A (en) * 2014-05-02 2017-02-22 牛津纳米孔技术公司 Mutant pores
CN109207454A (en) * 2017-01-24 2019-01-15 四川大学 Albumen, cross-film nucleic acid untwist nano-pore and its construction method and application
CN109295187A (en) * 2018-10-31 2019-02-01 南京大学 It is a kind of based on nano-pore to the dislocation sequencing approach of unnatural nucleic acids direct Sequencing
CN111499705A (en) * 2019-01-30 2020-08-07 深圳市梅丽纳米孔科技有限公司 Mutant NfpAB nanopore, test system, manufacturing method and application
CN113480620A (en) * 2021-08-18 2021-10-08 成都齐碳科技有限公司 Mutant of porin monomer, protein pore and application thereof

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106459159A (en) * 2014-05-02 2017-02-22 牛津纳米孔技术公司 Mutant pores
CN105372303A (en) * 2015-09-15 2016-03-02 西北大学 Single-molecule analysis method for detecting methylated DNA
CN109207454A (en) * 2017-01-24 2019-01-15 四川大学 Albumen, cross-film nucleic acid untwist nano-pore and its construction method and application
CN109295187A (en) * 2018-10-31 2019-02-01 南京大学 It is a kind of based on nano-pore to the dislocation sequencing approach of unnatural nucleic acids direct Sequencing
CN111499705A (en) * 2019-01-30 2020-08-07 深圳市梅丽纳米孔科技有限公司 Mutant NfpAB nanopore, test system, manufacturing method and application
CN113480620A (en) * 2021-08-18 2021-10-08 成都齐碳科技有限公司 Mutant of porin monomer, protein pore and application thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DATABASE Protein 6 October 2020 (2020-10-06), ANONYMOUS : "MspA family porin [Mycolicibacterium sp. P9-64]", XP093075951, retrieved from NCBI Database accession no. WP_149371538.1 *

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