WO2021056598A1 - 一种Mmup单体变体及其应用 - Google Patents

一种Mmup单体变体及其应用 Download PDF

Info

Publication number
WO2021056598A1
WO2021056598A1 PCT/CN2019/109332 CN2019109332W WO2021056598A1 WO 2021056598 A1 WO2021056598 A1 WO 2021056598A1 CN 2019109332 W CN2019109332 W CN 2019109332W WO 2021056598 A1 WO2021056598 A1 WO 2021056598A1
Authority
WO
WIPO (PCT)
Prior art keywords
mmup
monomer
monomer variant
porin
variant
Prior art date
Application number
PCT/CN2019/109332
Other languages
English (en)
French (fr)
Inventor
刘少伟
周雅
陈呈尧
Original Assignee
北京齐碳科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 北京齐碳科技有限公司 filed Critical 北京齐碳科技有限公司
Priority to IL291787A priority Critical patent/IL291787A/en
Priority to CN201980002430.8A priority patent/CN111164096B/zh
Priority to EP19947045.1A priority patent/EP4036107A4/en
Publication of WO2021056598A1 publication Critical patent/WO2021056598A1/zh
Priority to ZA2022/03618A priority patent/ZA202203618B/en

Links

Images

Classifications

    • 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
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • 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
    • 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 present invention relates to the technical field of characterization of nucleic acid characteristics, in particular to a Mmup monomer variant, a porin and a construct containing the Mmup monomer variant, and the use of the Mmup monomer variant or porin to characterize target polynucleosides Sour method.
  • Nanopore sequencing technology is a kind of single-stranded nucleic acid molecule as the sequencing unit, using a nanopore that can provide an ion current channel, so that the single-stranded nucleic acid molecule is driven by the electric field to pass through the nanopore.
  • the polynucleotide passes through the nanopore, it is easy to pass through the nanopore.
  • the corresponding blocking current will be generated due to the physical occupancy effect, and the different signals generated are read in real time and then analyzed to obtain the gene sequencing technology of the polynucleotide sequence information.
  • Nanopore sequencing technology has the following advantages: it can easily build a library without amplification; the reading speed is fast, and the reading speed of single-stranded molecules can reach tens of thousands of bases per hour; the reading length is longer, usually Up to thousands of bases; can directly measure methylated DNA or RNA.
  • Patent US20150065354A1 discloses a method for characterizing target polynucleotides using XPD helicase, which uses pores and XPD helicase.
  • the XPD helicase of the invention can control the movement of the target polynucleotide through the pore.
  • Patent US20170268055A1 discloses a composition and method for polynucleotide sequencing.
  • the method uses a step-by-step translocation step of translocation through a pore with the target polynucleotide to characterize the target polynucleotide, including characterizing the sequence of the polynucleotide Method and composition.
  • Patent CN106103741A discloses a method for linking one or more polynucleotide binding proteins to a target polynucleotide, and the invention also relates to a new method for characterizing the target polynucleotide.
  • Patent CN102216783B discloses a Mycobacterium smegmatis porin (Msp) nanopore and the use of the nanopore for sequencing, wherein the 90 or 91 position of wild-type Msp is mutated to improve the conductance of the analyte during sequencing, and Reduce the translocation speed of analytes in sequencing.
  • Msp Mycobacterium smegmatis porin
  • the present invention further provides a new nanoporin.
  • Mmup monomer variants were prepared by mutating wild-type Mmup proteins that could not be used for sequencing, and confirmed that the Mmup monomer variants are effective in sequencing. Features.
  • the present invention proves that the preparation of Mmup monomer variants with specific site mutations of Mmup mutant proteins can be used for nanopore sequencing, but the wild-type Mmup monomer does not have this function.
  • the porins of the present invention to nanopore sequencing, the difference in current signals of various nucleotides can be clearly seen, and the sequencing accuracy is relatively high.
  • the "Mmup” in the present invention is derived from Mycobacterium mucinae.
  • the “Mmup” is derived from Mycolicibacterium mucogenicum.
  • the first aspect of the present invention provides a Mmup monomer variant, the Mmup monomer variant comprising an amino acid sequence with any one or more amino acid mutations at positions 91-99 of SEQ ID NO:1.
  • the Mmup monomer variant comprises a mutation of aspartic acid (D) at position 91 and/or alanine (A) at position 99.
  • the monomer variant of Mmup comprises a mutation of aspartic acid (D) at position 91.
  • the Mmup monomer variant comprises a mutation of alanine (A) at position 99.
  • the Mmup monomer variant comprises the mutations of aspartic acid (D) at position 91 and alanine (A) at position 99.
  • the Mmup monomer variant contains at least one of the following mutations:
  • D91 mutations are: proline (P), tryptophan (W), arginine (R), glutamine (Q), lysine (K), phenylalanine (F), serine (S ), asparagine (N), cysteine (C), isoleucine (I), leucine (L) or valine (V), or unnatural amino acids; or,
  • A99 mutations are: proline (P), phenylalanine (F), isoleucine (I), leucine (L), valine (V), lysine (K) or arginine Acid (R), or unnatural amino acid.
  • the Mmup monomer variant contains D91K and/or A99K mutations.
  • the Mmup monomer variant comprises a D91K mutation.
  • the monomer variant of Mmup comprises a mutation of A99K.
  • the Mmup monomer variant contains mutations of D91K and A99K.
  • the Mmup monomer variant further comprises an amino acid sequence of any one or more amino acids at positions 80-90 and/or 100-120 of SEQ ID NO:1.
  • the Mmup monomer variant further comprises an amino acid sequence with any one or more amino acid mutations from positions 1-79 and/or 121-186 of SEQ ID NO:1.
  • the Mmup monomer variant further comprises a mutation of leucine (L) at position 89, a mutation of asparagine (N) at position 110, a mutation of aspartic acid (D) at position 120, One or a combination of two or more of the mutation of asparagine (N) at position 136 or the mutation of serine (S) at position 141.
  • L leucine
  • N asparagine
  • D aspartic acid
  • S serine
  • the Mmup monomer variant contains at least one of the following mutations:
  • L89 mutations are: asparagine (N), alanine (A), glutamine (Q), glycine (G), serine (S), threonine (T), lysine (K) or proline Amino acid (P), or, unnatural amino acid; or,
  • N110 mutation is: proline (P), alanine (A), isoleucine (I) or leucine (L), or unnatural amino acid; or,
  • D120 mutations are: arginine (R), lysine (K), asparagine (N), glutamine (Q), alanine (A), serine (S), glycine (G) or threon Amino acid (T), or unnatural amino acid; or,
  • N136 mutations are: arginine (R), lysine (K), glutamine (Q), alanine (A), serine (S) or threonine (T), or unnatural amino acids; or,
  • S141 mutations are: arginine (R), lysine (K), glutamine (Q), alanine (A), serine (S) or threonine (T), or unnatural amino acids.
  • the Mmup monomer variant may also include other mutation types in addition to the above mutation types, as long as the mutation does not affect the differentiation of different polynucleotides when the polynucleotide passes through the porin.
  • the Mmup monomer variant may also include a mutation that introduces cysteine to connect a molecule for sequencing, such as a nucleic acid binding protein.
  • the Mmup monomer variant may only contain the constriction region and loop region fragment sequences of the porin forming domain, and retain the pore forming activity.
  • the excess residues can be removed or other amino acid residues can be added while retaining the pore forming activity.
  • the fragment length can be at least 12, 20, 40, 50, 100 or 150 amino acids.
  • the Mmup monomer variant may be modified to facilitate identification or purification. For example: by adding aspartic acid residues (asp tag), streptavidin tag, flag tag or histidine residue (His tag).
  • asp tag aspartic acid residues
  • streptavidin tag streptavidin tag
  • flag tag flag tag
  • histidine residue His tag
  • the Mmup monomer variant may carry a display marker.
  • a display marker for example: fluorescent molecules, radioactive isotope 125 I, radioactive isotope 35 S, polynucleotide, biotin, antigen or antibody.
  • the Mmup monomer variant further includes a molecular motor.
  • the molecular engine is an enzyme.
  • the enzyme is polymerase, exonuclease or Klenow fragment.
  • the second aspect of the present invention provides a construct comprising at least one Mmup monomer variant described in the present invention. Wherein, the construct retains the ability to form pores.
  • the construct contains 1-50 Mmup monomer variants, wherein the Mmup monomer variants are the same or different.
  • the constructs include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 , 47, 48, 49 or 50 of the Mmup monomer variants, wherein the Mmup monomer variants are the same or different.
  • the construct contains 1-20 Mmup monomer variants, wherein the Mmup monomer variants are the same or different.
  • the construct comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 Mmup Monomer variants, wherein the Mmup monomer variants are the same or different.
  • the construct further contains a wild-type Mmup monomer.
  • the construct contains 1-50 wild-type Mmup monomers.
  • the constructs include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 , 47, 48, 49 or 50 wild-type Mmup monomers.
  • the construct contains 1-20 wild-type Mmup monomers. Specifically, the construct contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 wild Type Mmup monomer.
  • the construct contains 4-10 identical or different Mmup monomer variants.
  • the construct contains 4, 6, 8, 10 identical or different Mmup monomer variants.
  • the Mmup monomer variant and the Mmup monomer variant, the wild-type Mmup monomer and the wild-type Mmup monomer, and the Mmup monomer variant and the wild-type Mmup monomer are covalently connected.
  • the Mmup monomer variant and the Mmup monomer variant, the wild-type Mmup monomer and the wild-type Mmup monomer, and the Mmup monomer variant and the wild-type Mmup monomer are genetically fused.
  • the third aspect of the present invention provides a porin comprising at least one Mmup monomer variant, said Mmup monomer variant comprising SEQ ID NO: 1 with any one or more amino acid mutations in positions 91-99
  • the amino acid sequence, the mutation results in that when the polynucleotide single strand passes through the porin comprising at least one Mmup monomer variant, the result is caused by the difference in the physical or chemical properties of different kinds of nucleotides The difference in resistance within the hole.
  • the mutation results in a change in charge properties or hydrophobic properties of amino acids.
  • the difference in resistance refers to the characteristics that can be used to characterize the polynucleotide, and the characteristics include the source, length, size, molecular weight, identity, sequence, secondary structure, concentration, or target polynucleotide of the polynucleotide. Whether the amino acid is modified. Further preferably, the difference in resistance refers to the sequence feature that can be used to characterize the polynucleotide, that is, the porin can be used for sequencing to accurately distinguish different bases of the polynucleotide.
  • the polynucleotide may be naturally occurring or artificially synthesized. Further preferably, the polynucleotide may be natural DNA, RNA, or modified DNA or RNA.
  • one or more nucleotides in the target polynucleotide may be modified, such as methylated, oxidized, damaged, abasic, protein-labeled, tagged or polynucleoside A spacer is connected in the middle of the acid sequence.
  • the artificially synthesized nucleic acid is selected from peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), locked nucleic acid (LNA) or other synthetic polymers with nucleoside side chains.
  • PNA peptide nucleic acid
  • GNA glycerol nucleic acid
  • TAA threose nucleic acid
  • LNA locked nucleic acid
  • the target polynucleotide is single-stranded, double-stranded, or at least part of it is double-stranded.
  • the Mmup monomer variant comprises a mutation of aspartic acid (D) at position 91 and/or alanine (A) at position 99.
  • the monomer variant of Mmup comprises a mutation of aspartic acid (D) at position 91.
  • the Mmup monomer variant comprises a mutation of alanine (A) at position 99.
  • the Mmup monomer variant comprises the mutations of aspartic acid (D) at position 91 and alanine (A) at position 99.
  • the Mmup monomer variant contains at least one of the following mutations:
  • D91 mutations are: proline (P), tryptophan (W), arginine (R), glutamine (Q), lysine (K), phenylalanine (F), serine (S ), asparagine (N), cysteine (C), isoleucine (I), leucine (L) or valine (V), or unnatural amino acids; or,
  • A99 mutations are: proline (P), phenylalanine (F), isoleucine (I), leucine (L), valine (V), lysine (K) or arginine Acid (R), or unnatural amino acid.
  • the Mmup monomer variants comprise mutations of D91K and/or A99K.
  • the monomer variant of Mmup comprises mutations of D91K and A99K.
  • the Mmup monomer variant further comprises an amino acid sequence of any one or more amino acids at positions 80-90 and/or 100-120 of SEQ ID NO:1.
  • the Mmup monomer variant further comprises an amino acid sequence in which any one or more amino acids are mutated at positions 1-79 and/or 121-186 of SEQ ID NO:1.
  • the Mmup monomer variant further comprises a mutation of leucine (L) at position 89, a mutation of asparagine (N) at position 110, a mutation of aspartic acid (D) at position 120, One or a combination of two or more of the mutation of asparagine (N) at position 136 or the mutation of serine (S) at position 141.
  • L leucine
  • N asparagine
  • D aspartic acid
  • S serine
  • the Mmup monomer variant further comprises at least one of the following mutations:
  • L89 mutations are: asparagine (N), alanine (A), glutamine (Q), glycine (G), serine (S), threonine (T), lysine (K) or proline Amino acid (P), or, unnatural amino acid; or,
  • N110 mutation is: proline (P), alanine (A), isoleucine (I) or leucine (L), or unnatural amino acid; or,
  • D120 mutations are: arginine (R), lysine (K), asparagine (N), glutamine (Q), alanine (A), serine (S), glycine (G) or threon Amino acid (T), or unnatural amino acid; or,
  • N136 mutations are: arginine (R), lysine (K), glutamine (Q), alanine (A), serine (S) or threonine (T), or unnatural amino acids; or,
  • S141 mutations are: arginine (R), lysine (K), glutamine (Q), alanine (A), serine (S) or threonine (T), or unnatural amino acids.
  • the porin comprising at least one Mmup monomer variant comprises at least 1-50 Mmup monomer variants, wherein the Mmup monomer variants are the same or different.
  • the porin comprising at least one Mmup monomer variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42 , 43, 44, 45, 46, 47, 48, 49 or 50 of the Mmup monomer variants, and the Mmup monomer variants are the same or different.
  • the porin comprising at least one Mmup monomer variant comprises at least 1-20 Mmup monomer variants, wherein the Mmup monomer variants are the same or different.
  • the porin comprising at least one Mmup monomer variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19 or 20 of the Mmup monomer variants, and the Mmup monomer variants are the same or different.
  • the porin comprising at least one Mmup monomer variant further comprises a wild-type Mmup monomer.
  • the porin comprising at least one Mmup monomer variant comprises 1-50 wild-type Mmup monomers.
  • the porin comprising at least one Mmup monomer variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42 , 43, 44, 45, 46, 47, 48, 49 or 50 wild-type Mmup monomers.
  • the porin comprising at least one Mmup monomer variant comprises 1-20 wild-type Mmup monomers.
  • the porin comprising at least one Mmup monomer variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19 or 20 wild-type Mmup monomers.
  • the porin comprising at least one Mmup monomer variant comprises 4-10 identical or different Mmup monomer variants.
  • the porin comprising at least one Mmup monomer variant comprises 4, 6, 8, 10 identical or different Mmup monomer variants.
  • the Mmup monomer variant and the Mmup monomer variant, the wild-type Mmup monomer and the wild-type Mmup monomer, and the Mmup monomer variant and the wild-type Mmup monomer are covalently connected.
  • the Mmup monomer variant and the Mmup monomer variant, the wild-type Mmup monomer and the wild-type Mmup monomer, and the Mmup monomer variant and the wild-type Mmup monomer are genetically fused.
  • the Mmup monomer variants contained in the porin comprising at least one Mmup monomer variant are the same or different.
  • the porin comprising at least one Mmup monomer variant may comprise eight identical or different Mmup monomer variants.
  • the porin comprising at least one Mmup monomer variant comprises one Mmup monomer variant and seven identical monomers, wherein the Mmup monomer variant is different from the same monomer.
  • the porin comprising at least one Mmup monomer variant comprises two identical or different Mmup monomer variants and six identical monomers, wherein the Mmup monomer variant is different from the same monomer.
  • the porin comprising at least one Mmup monomer variant comprises three identical or different Mmup monomer variants and five identical monomers, wherein the Mmup monomer variant is different from the same monomer .
  • the porin comprising at least one Mmup monomer variant comprises four identical or different Mmup monomer variants and four identical monomers, wherein the Mmup monomer variant is different from the same monomer .
  • the porin comprising at least one Mmup monomer variant comprises five identical or different Mmup monomer variants and three identical monomers, wherein the Mmup monomer variant is different from the same monomer .
  • the porin comprising at least one Mmup monomer variant comprises six identical or different Mmup monomer variants and two identical monomers, wherein the Mmup monomer variant is different from the same monomer .
  • the porin comprising at least one Mmup monomer variant comprises seven identical or different Mmup monomer variants and one monomer, wherein the Mmup monomer variant is different from one monomer.
  • the porin comprising at least one Mmup monomer variant comprises eight identical or different Mmup monomer variants.
  • the porin comprising at least one Mmup monomer variant may be homologous or heterologous.
  • the porin comprising at least one Mmup monomer variant comprises a contraction zone and a loop zone.
  • the pore diameter of the constriction zone of the porin comprising at least one Mmup monomer variant is smaller than the constriction zone pore diameter of the porin composed of wild-type Mmup monomer. Further preferably, the pore diameter of the constriction zone of the porin comprising at least one Mmup monomer variant is less than or More preferably, the pore diameter of the constriction zone of the porin comprising at least one Mmup monomer variant is less than or
  • the pore diameter of the constriction zone of the porin comprising at least one Mmup monomer variant is approximately equal to or
  • the Mmup monomer variant may also include other mutation types in addition to the above mutation types, as long as the mutation does not affect the differentiation of different polynucleotides when the polynucleotide passes through the porin.
  • the monomer variant may also include a mutation to introduce a cysteine to connect a molecule for sequencing, such as a nucleic acid binding protein.
  • the Mmup monomer variant may only contain the constriction region and loop region fragment sequences of the porin forming domain, and retain the pore forming activity.
  • the excess residues can be removed or other amino acid residues can be added while retaining the pore forming activity.
  • the fragment length can be at least 12, 20, 40, 50, 100 or 150 amino acids.
  • the Mmup monomer variant may be modified to facilitate identification or purification. For example: by adding aspartic acid residues (asp tag), streptavidin tag, flag tag or histidine residue (His tag).
  • asp tag aspartic acid residues
  • streptavidin tag streptavidin tag
  • flag tag flag tag
  • histidine residue His tag
  • the Mmup monomer variant may carry a display marker.
  • a display marker for example: fluorescent molecules, radioactive isotope 125 I, radioactive isotope 35 S, polynucleotide, biotin, antigen or antibody.
  • the Mmup monomer variant further includes a molecular motor.
  • the molecular engine is an enzyme.
  • the enzyme is polymerase, exonuclease or Klenow fragment.
  • the porin comprising at least one Mmup monomer variant further includes a cap-shaped body forming region and/or a barrel-shaped body forming region.
  • the porin containing at least one Mmup monomer variant allows hydrated ions to flow from one side of the membrane to the other layer of the membrane under the driving of an applied electric potential.
  • the membrane is a double-layer membrane, more preferably a lipid double-layer membrane.
  • the fourth aspect of the present invention provides a porin encoding any one of the Mmup monomer variants of the present invention, any one of the Mmup monomer variants of the present invention, or any one of the constructs of the present invention The nucleotide sequence of the body.
  • the nucleotide sequence encoding the Mmup monomer variant and the sequence shown in SEQ ID NO: 2 or SEQ ID NO: 11 have 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% homology, and the nucleotide sequence encoding the Mmup monomer variant sequence.
  • nucleotide sequence encoding the Mmup monomer variant is shown in SEQ ID NO: 11.
  • the fifth aspect of the present invention provides a vector containing a nucleotide sequence encoding the Mmup monomer variant of the present invention, the porin of the present invention, or the construct of the present invention.
  • the vector may be provided with a plasmid, virus or phage vector that is provided with an origin of replication, a promoter optionally used to express the nucleotide sequence, and a regulatory signal gene of the promoter.
  • the vector may contain one or more selectable marker genes, such as tetracycline resistance genes.
  • the promoter and other expression regulatory signals can be selected to be compatible with the host cell for which the expression vector is designed.
  • the promoter is selected from T7, trc, lac, ara or ⁇ L promoter.
  • the Mmup monomer variants of the present invention can be prepared by chemical synthesis or recombinant methods, and are preferably prepared by recombinant methods.
  • the vector contains and encodes any of the porins of the present invention comprising at least one Mmup monomer variant, any of the Mmup monomer variants of the present invention, or any of the constructs of the present invention.
  • a promoter to which the nucleotide sequence is operably linked is operably linked.
  • the promoter is an inducible promoter or a constitutive promoter, wherein the inducible promoter is an acetamide inducible promoter.
  • the nucleotide sequence encoding the porin comprising at least one monomer variant of Mmup comprises at least one nucleotide sequence encoding the monomer variant of Mmup.
  • the nucleotide sequence encoding the porin comprising at least one Mmup monomer variant further comprises at least one nucleotide sequence encoding a wild-type Mmup monomer.
  • nucleotide sequence encoding the Mmup monomer variant and the nucleotide sequence encoding the Mmup monomer variant the nucleotide sequence encoding the Mmup monomer variant and the nucleotide sequence encoding the wild-type Mmup monomer
  • the nucleotide sequence, or, the nucleotide sequence encoding the wild-type Mmup monomer and the nucleotide sequence encoding the wild-type Mmup monomer are connected by a sequence encoding an amino acid linker.
  • Protein mutant bacteria said bacteria include:
  • the bacterium comprises a vector with a promoter operably linked to a nucleotide sequence encoding a Mmup monomer variant, a construct comprising a Mmup monomer variant, or a porin comprising a Mmup monomer variant.
  • the Mmup monomer variants include paralogues or homologs of the Mmup monomer variants.
  • constructs containing Mmup monomer variants include paralogues or homologous constructs or monomers of Mmup monomer variants.
  • the porins containing Mmup monomer variants include paralogues or homologs porins or monomers of the Mmup monomer variants.
  • the bacterium may also comprise a promoter operably linked to a nucleotide sequence encoding a wild-type Mmup monomer, a construct containing a wild-type Mmup monomer, or a porin containing a wild-type Mmup monomer. Carrier.
  • the wild-type Mmup monomer includes a paralogue or homologous monomer of the wild-type Mmup monomer.
  • the construct containing the wild-type Mmup monomer is a wild-type Mmup monomer paralogue or homologous construct or monomer.
  • the porin containing the wild-type Mmup monomer is a paralogue or homologous porin or monomer of the wild-type Mmup monomer.
  • the bacterium is Mycobacterium mucinae.
  • the seventh aspect of the present invention provides a method for producing Mmup porin, the method comprising transforming any one of the bacteria of the present invention with a vector comprising any one of the present invention, and inducing the bacteria to express Mmup porin.
  • the eighth aspect of the present invention provides a method for preparing Mmup monomer variants.
  • the vector can be introduced into a suitable host cell by inserting the nucleotide sequence encoding the Mmup monomer variant into the vector. , Introducing the vector into a compatible bacterial host cell and culturing the host cell under conditions that allow the expression of the nucleotide to produce the Mmup monomer variant of the present invention.
  • the ninth aspect of the present invention provides a cell comprising the nucleotide sequence of the present invention or the vector.
  • the cell may be Escherichia coli or the like. More preferably, the cell is a dam+ type strain (for example, a DH5 ⁇ strain).
  • the tenth aspect of the present invention provides a method for characterizing a target polynucleotide, including:
  • steps (a) and (b) are repeated one or more times.
  • the target polynucleotide in the step (a) can be combined with a polynucleotide processing enzyme derived from it to control the translocation speed.
  • the polynucleotide processing enzyme is a polypeptide capable of interacting with the polynucleotide and modifying at least one of its properties.
  • the polynucleotide processing enzyme may or may not have enzymatic activity, as long as the enzyme binds to the polynucleotide and controls its translocation speed in the pore.
  • the nucleic acid can be combined with one or more polynucleotide processing enzymes.
  • the polynucleotide processing enzyme is a nucleolytic enzyme.
  • the polynucleotide processing enzymes include, but are not limited to, nucleic acid binding protein, helicase, polymerase, exonuclease, telomerase, reverse transcriptase, translocator or topoisomerase.
  • the polynucleotide processing enzyme is a gyrase.
  • the step (a) also includes the target polynucleotide and nucleic acid binding protein, helicase, exonuclease, telomerase, topoisomerase, reverse transcriptase, translocator and/or
  • the step of contacting one or a combination of two or more of the polymerases makes the translocation speed of the target polynucleotide sequence through the porin lower than that of the nucleic acid binding protein, helicase, exonuclease, telomerase, The rate of translocation in the absence of topoisomerase, reverse transcriptase, translocator, and/or polymerase.
  • the nucleic acid binding protein includes but is not limited to modified or wild eukaryotic single-stranded binding protein, bacterial single-stranded binding protein, ancient single-stranded binding protein, viral single-stranded binding protein or double-stranded binding protein One or a combination of two or more.
  • the nucleic acid binding protein includes, but is not limited to, SSBEco from Escherichia coli, SSBBhe from Bartonella henselae, SSBCbu from Coxiella burnetii, SSBTma from Thermotoga maritima, SSBHpy from Helicobacter pylori, SSBHpy from Deinococcus radiodurans, SSBDaqua from Deinococcus radiodurans SSBTaq, SSBMsm from Mycobacterium smegmatis, SSBSso from Sulfolobus solfataricus, SSBSso7D from Sulfolobus solfataricus, SSBMHsmt from Homo sapiens, SSBMle from Mycobacterium leprae, SSBMle from BacteriohageioT4, gp32 from BacteriohageioT4, gp32 from BacteriohageioT4, gp32 from Bacteriohageio
  • the helicase may be any Hel308 family helicase and modified Hel308 family helicase, RecD helicase and its variants, TrwC helicase and its variants, Dda helicase And its variants, TraI Eco and its variants, XPD Mbu and its variants.
  • the polymerase includes but is not limited to modified or wild DNA polymerase, including but not limited to Phi29 DNA polymerase, Tts DNA polymerase, M2 DNA polymerase, VENT DNA polymerase, T5 DNA polymerase, PRD1 DNA polymerase , Bst DNA polymerase or REPLI-gscDNA polymerase.
  • the exonuclease includes, but is not limited to, modified or wild exonuclease I from E. coli, exonuclease III from E. coli, exonuclease from bacteriophage lambda or from thermophilic RecJ of Thermus.
  • the step (a) includes the step of contacting the target polynucleotide with a helicase, the helicase is EF8813, and the amino acid sequence of the helicase is SEQ ID NO: 3, the nucleotide sequence of the helicase is shown in SEQ ID NO: 4.
  • the target polynucleotide can be contacted with one or more helicases.
  • the target polynucleotide can be contacted with 2-20 helicases, or even more helicases.
  • the helicases that bind to the target polynucleotide may be the same or different.
  • a plurality of helicases bound to the target polynucleotide are covalently connected to each other.
  • the one or more characteristics are selected from the source, length, size, molecular weight, identity, sequence, secondary structure, concentration, or whether the target polynucleotide is modified.
  • the feature is a sequence.
  • the one or more features in the step (b) are performed by electrical measurement and/or optical measurement.
  • electrical and/or optical signals are generated by electrical measurement and/or optical measurement, and each nucleotide corresponds to a signal level, and then the electrical signals and/or optical signals are converted into sequence characteristics of nucleotides .
  • the electrical measurement in the present invention is selected from current measurement, impedance measurement, field effect transistor (FET) measurement, tunnel measurement or wind tunnel measurement.
  • FET field effect transistor
  • the electrical signal of the present invention is selected from the measured values of current, voltage, tunneling, resistance, potential, conductivity or lateral electrical measurement.
  • the electrical signal is a current passing through the hole. That is, the current passes through the pore in a nucleotide-specific manner, and if a characteristic current related to nucleotides is detected to flow through the pore, the nucleotide is present. Otherwise, it does not exist. However, the distinction between similar nucleotides or modified nucleotides is determined by the magnitude of the current.
  • the conductance generated during the characterization of polynucleotides using the porins of the present invention is higher than that of the pores formed by the wild-type Mmup monomer.
  • the method further includes the step of applying a potential difference across the porin contacted by the target polynucleotide.
  • the potential difference is sufficient to translocate the target polynucleotide from the channel of the porin.
  • the target polynucleotide may be natural DNA, RNA, or modified DNA or RNA.
  • the target polynucleotide of the present invention is a macromolecule containing one or more nucleotides.
  • the target polynucleotide of the present invention may be naturally occurring or artificially synthesized.
  • one or more nucleotides in the target polynucleotide may be modified, such as methylation, oxidation, damage, abasic, protein labeling, tag or polynucleotide sequence
  • a spacer is connected in the middle.
  • the artificially synthesized nucleic acid is selected from peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), locked nucleic acid (LNA), or other synthetic polymers with nucleoside side chains.
  • the porin allows hydrated ions to flow from one side of the membrane to the other layer of the membrane under the driving of an applied electric potential.
  • the membrane can form a barrier to the flow of ions, nucleotides and nucleic acids.
  • the membrane is a double-layer membrane, further preferably a lipid bilayer membrane.
  • the lipid bilayer membrane includes, but is not limited to, one or a mixture of two or more of phospholipids, glycolipids, cholesterol, and mycolic acid.
  • the porin channel is located between the first conductive liquid medium and the second conductive liquid medium, wherein at least one conductive liquid medium contains the target polynucleotide, and the first conductive liquid medium and the second conductive liquid medium
  • the two conductive liquid media may be the same or different, as long as the purpose of analyzing one or more characteristics of the target polynucleotide can be achieved.
  • the target polynucleotide is single-stranded, double-stranded, or at least part of it is double-stranded.
  • the target polynucleotide is double-stranded.
  • the double-stranded part constitutes a Y adapter structure
  • the Y adapter structure contains a leader sequence that is preferentially screwed into the porin, and the 3'end of the leader sequence is connected with thiol, biotin or cholesterol, and To combine with a layer of the lipid bilayer membrane to point the target polynucleotide in the correct direction and have a pulling effect.
  • the 3'end of the leader sequence is connected to cholesterol for binding to a layer of the lipid bilayer membrane.
  • Adjusting the voltage, salt concentration, buffer, additives or temperature in the process of characterizing the target polynucleotide can control the degree of discrimination of different nucleotides of the porin of the present invention in characterizing the target polynucleotide.
  • the additive is selected from DTT, urea or betaine.
  • the voltage range is -250mV to +250mV.
  • the voltage is selected from -250mV, -210mV, -180mV, -140mV, -110mV, -90mV, -70mV, -40mV, 0mV, +40mV, +70mV, +90mV, +110mV, +140mV , +180mV, +210mV, +250mV.
  • the voltage is -180mV to +180mV.
  • the method includes: inserting the porin into the membrane, and then combining the target polynucleotide with the porin, nucleic acid binding protein, polymerase, and exonuclease. , Telomerase, topoisomerase, reverse transcriptase, translocase and/or helicase contact, apply a potential difference across the porin contacting the target polynucleotide, so that the target polynucleotide sequence passes through Porin; and
  • the method for inserting the porin into the membrane can be any method known in the art that can achieve the purpose of characterizing polynucleotides.
  • the porin may be suspended in a solution containing a lipid bilayer in a purified form, so that it diffuses into the lipid bilayer and is inserted into the lipid bilayer by binding to the lipid bilayer and assembling into a functional state.
  • the lipid bilayer is a solution containing a lipid bilayer in a purified form, so that it diffuses into the lipid bilayer and is inserted into the lipid bilayer by binding to the lipid bilayer and assembling into a functional state.
  • the eleventh aspect of the present invention provides a porin comprising at least one Mmup monomer variant according to the present invention, any Mmup monomer variant according to the present invention, and any construct according to the present invention.
  • the twelfth aspect of the present invention provides a kit for characterizing a target polynucleotide.
  • the kit includes any of the porins of the present invention comprising at least one Mmup monomer variant, and any of the porins of the present invention.
  • the Mmup monomer variant, the construct, the nucleotide sequence, the vector, the cell or the porin can be multiple.
  • the kit also includes one or more nucleic acid binding proteins, helicases, exonucleases, telomerases, topoisomerases, reverse transcriptases, transposases and/or polymerases.
  • one or a combination of two or more is particularly advantageous.
  • the kit further includes a lipid bilayer chip, and the pore protein spans the lipid bilayer.
  • the kit contains one or more lipid bilayers, and each lipid bilayer contains one or more porins.
  • the kit also includes reagents or devices for characterizing the target polynucleotide.
  • the reagents include buffers and tools required for PCR amplification.
  • a device for characterizing a target polynucleotide comprising any of the porins of the present invention comprising at least one Mmup monomer variant, and any of the porins of the present invention Mmup monomer variants, any of the constructs of the present invention, the nucleotide sequence of the present invention, any of the vectors of the present invention, or any of the mutant bacteria of the present invention.
  • the device further includes one or more of nucleic acid binding proteins, helicases, exonucleases, telomerases, topoisomerases, reverse transcriptases, transposases and/or polymerases.
  • nucleic acid binding proteins helicases, exonucleases, telomerases, topoisomerases, reverse transcriptases, transposases and/or polymerases.
  • the device further includes a sensor that supports the porin and can transmit the signal that the porin interacts with the polynucleotide, at least one memory for storing the target polynucleotide, and a solution required to implement the characterization process .
  • the device further includes a patch clamp amplifier and/or a data acquisition device.
  • the fourteenth aspect of the present invention provides a sensor for characterizing target polynucleotides.
  • the sensor includes any of the porins of the present invention comprising at least one Mmup monomer variant, and any of the porins of the present invention. Mmup monomer variants, any of the constructs of the present invention, the nucleotide sequence of the present invention, any of the vectors of the present invention, or any of the mutant bacteria of the present invention.
  • the "unnatural amino acid” in the present invention is a compound containing amino and carboxyl groups that is not naturally found in proteins.
  • the unnatural amino acid is any unnatural amino acid known in the art.
  • the unnatural amino acids include, but are not limited to, N-ethyl aspartyl, hydroxylysine, 3-hydroxyproline, 2-aminobutyric acid, ⁇ -alanine, ⁇ -amino Propionic acid, 2-aminoadipate, 3-aminoadipate, 4-aminobutyric acid, 6-aminocaproic acid, 2-aminoheptanoic acid, allo-isoleucine, isochain lysine, 4- Hydroxyproline, allo-hydroxylysine, 2-aminoisobutyric acid, N-methylglycine, N-methylisoleucine, 3-aminoisobutyric acid, 6-N-methyllysine , 2,4-Diaminobutyric acid, N-methylvaline, ornith
  • modified...-based acid in the present invention is an amino acid whose side chain is chemically modified.
  • side chain is chemically modified.
  • novel functional groups such as sulfhydryl, amino, or carboxyl
  • signal-generating moieties such as fluorescent groups or radioactive labels
  • nucleotides in the present invention include but are not limited to: adenosine monophosphate (AMP), guanosine monophosphate (GMP), thymidine monophosphate (TMP), uridine monophosphate (UMP), cytosine Nucleoside monophosphate (CMP), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), deoxyadenosine monophosphate (dAMP), deoxyguanosine monophosphate (dGMP), deoxythymidine monophosphate (dTMP), deoxyuridine monophosphate (dUMP) and deoxycytidine monophosphate (dCMP).
  • the nucleotide is selected from AMP, TMP, GMP, CMP, UMP, dAMP, dTMP, dGMP or dCMP.
  • the "about” in the present invention is used to indicate the standard deviation allowed by the value and the device or method used to determine the value.
  • the "homology" in the present invention means that in terms of using protein sequences or nucleotide sequences, those skilled in the art can adjust the sequences according to actual work needs, so that the used sequences are similar to those obtained in the prior art. Ratio, with (including but not limited to) 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% , 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 70%, 80%, 81%, 8
  • the "Mmup monomer variant" in the present invention refers to having at least or at most 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, and wild-type Mmup monomer. 99, 99.5 or 99.9% or more, or can come from any range therebetween, but less than 100% identity and remain channel-forming when combined with one or more other Mmup monomer variants or wild-type Mmup monomer Ability of Mmup monomer variant.
  • the Mmup monomer variant is further determined to include a mutation in the sequence portion that promotes the formation of the contraction zone and/or loop zone of the fully formed channel forming porin.
  • the Mmup monomer variant may be, for example, a recombinant protein.
  • Mmup monomer variants can contain any of the mutations described herein.
  • paralogue or homologue porin of Mmup monomer variant in the present invention means that the paralogue or homologue porin of the wild-type Mmup monomer has at least or at most 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or 99.9% or more, or can be from any range in between, but less than 100% identity and maintain the channel Forms a paralogue or homolog porin of a monomeric variant of Mmup that is capable of.
  • the paralogue or homologue porin of the Mmup monomer variant is further determined to contain a mutation in this part of the sequence that promotes the fully formed channel to form the constriction and/or loop of the porin The formation of the zone.
  • the paralogue or homologue porin of the Mmup monomer variant can be, for example, a recombinant protein. Any paralogue or homologue porin of any Mmup monomer variant can optionally be used in any of the embodiments herein.
  • the "paralog or homolog construct of the Mmup monomer variant" in the present invention refers to the paralogue or homolog construct of the wild-type Mmup monomer having at least or at most 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or 99.9% or more, or can be from any range in between, but less than 100% identity and maintain the channel Form a paralogue or homologous construct of a capable Mmup monomer variant.
  • the paralogue or homologous construct of the Mmup monomer variant is further determined to contain a mutation in this part of the sequence that promotes the fully formed channel to form the constriction and/or loop of the porin The formation of the zone.
  • the paralogue or homologous construct of the Mmup monomer variant may, for example, be a recombinant protein. Any paralogue or homologous construct of any Mmup monomer variant can optionally be used in any of the embodiments herein.
  • Figure 1 Purification results of Mmup-(D91K/A99K) monomer variant protein molecular exclusion chromatography. Lanes 1-6 show the SDS-PAGE electrophoresis detection results of different components separated by molecular sieves.
  • Figure 2 A stick model of nanoporins (D91K/A99K) containing Mmup monomer variants.
  • the figure mainly shows the amino acid distribution characteristics of the constriction and loop regions of the channel, especially the key amino acid residues in the constriction and loop regions.
  • Group distribution pointing to the lysine at position 91 and asparagine at position 92 of the amino acid residue in the center of the pore, the diameter of the pore is about
  • the lysine at position 99 is involved in the correct assembly of the channel complex.
  • the homology modeling is completed by SWISS MODEL, and the template pdb is located at 1uun.
  • Figure 3 A stick model of a nanopore channel containing wild-type Mmup monomers.
  • the figure mainly shows the amino acid distribution characteristics of the constriction and circular region of the pore, especially the key amino acid residues in the constriction and circular region, mainly at position 91 Aspartic acid, asparagine at position 92, valine at position 93 and serine at position 94, the diameters of the contraction zone formed by D91 and N92 are respectively with Among them, homology modeling is completed by SWISS MODEL, and the template pdb is 1uun.
  • Figure 4 A cartoon schematic diagram of a nanopore containing a wild-type Mmup monomer based on homology modeling.
  • the area 1 corresponds to the cap-shaped body forming area
  • the area 2 corresponds to the barrel-shaped body forming area
  • the area 3 corresponds to the contraction area and the annular area.
  • Figure 5 The structure diagram of the tested DNA construct X2&cX2-80-15, where segment a corresponds to SEQ ID NO: 7, and b corresponds to the helicase EF8813-1 (containing the N-terminal histidine tag and its fusion A variant protein of the TOPV-HI domain, SEQ ID NO: 3-4), the helicase can bind to the segment labeled a, and the segment c corresponds to SEQ ID NO: 6, and the segment d corresponds to SEQ ID NO: 5, segment e corresponds to SEQ ID NO: 8, its 5'end 45 bases are complementary to the test strand c segment region, and the 3'end contains 40 thymines and 3'cholesterol corresponding to g TEG mark, segment f corresponds to SEQ ID NO: 9.
  • Figure 6 The structure diagram of the DNA construct S1T&S1MC to be tested, where segment a corresponds to SEQ ID NO: 10, and b corresponds to helicase EF8813-1 (containing N-terminal histidine tag and fused with TOPV-HI structure) Domain variant protein, SEQ ID NO: 3-4), the helicase can bind to the segment marked a, segment h refers to the dspacer that only retains the phosphate backbone, marked as x, segment c Corresponds to SEQ ID NO: 12, segment d corresponds to SEQ ID NO: 13, and segment e corresponds to SEQ ID NO: 14, and its 5'end 45 bases are complementary to the test strand c segment region, and the 3'end Containing 20 thymines and a 3'cholesterol TEG marker corresponding to g, segment f corresponds to SEQ ID NO: 15.
  • the protein sequence of Mmup monomer variants is optimized by the corresponding amino acid codons, and appropriate restriction enzyme cleavage sites are added at both ends of the gene.
  • the specific 5'end adds the NcoI site ccatgg, and the 3'end adds the xhoI site.
  • Click ctcgag then proceed to gene synthesis, and clone the synthesized gene into the expression vector pET24b.
  • Induced mutation gene takes the plasmid to be mutated as a template, and uses the designed primers and KOD plus high-fidelity enzyme to carry out PCR amplification reaction to induce the target gene mutation.
  • the PCR amplification reaction is completed, and the nucleotide sequence of the Mmup monomer variant (SEQ ID NO: 11) is obtained, and then placed in an ice bath for 5 minutes, and then placed at room temperature (avoid repeated freezing and thawing).
  • the methylated plasmid is digested with DpnI enzyme to select mutant plasmid DNA (plasmid containing SEQ ID NO: 11).
  • DpnI enzyme to select mutant plasmid DNA (plasmid containing SEQ ID NO: 11).
  • DH5 ⁇ is selected when transferring this plasmid DNA into E. coli.
  • the specific steps are: add 4 ⁇ L mutant plasmid DNA sample to 50 ⁇ L DH5 ⁇ competent cells, then place on ice for 30min, heat shock at 42°C for 90s, then immediately ice bath for 2min, add 500 ⁇ L SOC medium and incubate at 37°C for 1 hour, and finally Take 100 ⁇ L of bacterial solution to coat the resistance screening plate.
  • the Mmup monomer variant plasmid verified by sequencing was transferred to BL21 (DE3) for culture. Then the protein is purified, and the reagent formula for protein purification is shown in Table 1.
  • the cells were collected by centrifugation at 6000 rpm and 4°C for 15 minutes.
  • the collected penetrant samples were added with ammonium sulfate with a final concentration of 40%, and precipitated in an ice bath for 2 hours, and then centrifuged at 14000 rpm and 4°C for 30 minutes to collect floating precipitates.
  • the floating precipitate was re-dissolved by adding a certain volume of molecular sieve buffer containing 0.5% C8E4 (tetraethylene glycol monooctyl ether) detergent, and incubated overnight at 4°C.
  • the cells were collected by centrifugation at 6000 rpm and 4°C for 15 minutes.
  • the collected penetrant samples were added with ammonium sulfate with a final concentration of 40%, and precipitated in an ice bath for 2 hours, and then centrifuged at 14000 rpm and 4°C for 30 minutes to collect floating precipitates.
  • the floating precipitate was re-dissolved by adding a certain volume of molecular sieve buffer containing 0.5% C8E4 detergent, and incubated overnight at 4°C.
  • the sample incubated overnight was centrifuged at 47000g for 30min at 4°C the next day, and the supernatant was collected.
  • the supernatant is subjected to the last step of molecular exclusion chromatography purification, and the collected target component is the Mmup monomer variant.
  • a single nanoporin was inserted into the phospholipid bilayer, and electrical measurements were obtained from the single nanoporin.
  • the buffer 400mM KCl, 10 mM HEPES pH 8.0, 50 mM MgCl 2 ) flow through the system to remove any excess Mmup monomer variant porin.
  • DNA construct X2&cX2-80-15 or S1T&S1MC (1 ⁇ 2nM final concentration) to the Mmup monomer variant porin experimental system, mix well, make the buffer (400mM KCl, 10mM HEPES pH 8.0, 50mM MgCl 2 ) Flow through the system to remove any excess DNA construct X2&cX2-80-15 or S1T&S1MC. Then the premix of helicase (EF8813-1, 15nM final concentration) and fuel (ATP 3mM final concentration) was added to the porin experimental system of the single Mmup monomer variant, and the Mmup monomer was monitored at a voltage of +180mV. Sequencing of porin variants.
  • the control group is the same as the above steps, except that the porin of the Mmup monomer variant is replaced with the nanopore of the wild-type Mmup monomer (the stick model and three-dimensional structure are shown in Figures 3 and 4).
  • the rod model of the nanopore of the wild-type Mmup monomer shows the amino acid distribution characteristics of the pore contraction zone and the ring zone, especially the key amino acid residues in the contraction zone and the ring zone, mainly aspartic acid at position 91, 92 Asparagine at position, valine at position 93 and serine at position 94, the diameters of the contraction zone formed by D91 and N92 are respectively with
  • the nanoporins containing Mmup monomer variants (the stick model is shown in Figure 2)
  • the stick model shows the amino acid distribution characteristics of the constriction zone and annular zone of the channel after mutation, especially The distribution of key amino acid residues in the constriction zone and circular zone, pointing to the
  • Figure 7 shows the single channel behavior characteristics of the nanopore channel containing wild-type Mmup monomer at a voltage of ⁇ 180mV.
  • the fully open current of the nanopore channel containing the wild-type Mmup monomer in the test system is about 380pA under the +180mV condition, with obvious gate control and strong residual nucleic acid through-hole signal; under the -180mV condition, the fully open current is close to -350pA, the gate The control is stronger, and the disturbance to the membrane is very severe.
  • the nanopore channel containing the wild-type Mmup monomer cannot meet the requirements for sequencing nanoporins, and cannot complete the sequencing purpose.
  • Figure 8 shows the nanoporin containing the Mmup monomer variant (D91K/A99K) at voltages of +180mV, 0mV and -180mV and its gating characteristics. The positive gating disappears and the porin can Maintain a stable open state under applied voltage. At 180mv, 400mm KCl salt concentration, it can generate an opening current of about 160pA.
  • Fig. 9 shows the signal of nucleic acid passing through the nanopore at voltages of +180 mV and 0 mV for nanoporins containing Mmup monomer variants (D91K/A99K).

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Physics & Mathematics (AREA)
  • General Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Medicinal Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Plant Pathology (AREA)
  • Cell Biology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Food Science & Technology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)

Abstract

本发明提供了一种包含SEQ ID NO：1第91-99位任意一个或多个氨基酸突变的氨基酸序列的Mmup单体变体、以及包含至少一个Mmup单体变体的孔蛋白或构建体及其用途。本发明还提供了一种表征目标多核苷酸的方法。

Description

一种Mmup单体变体及其应用 技术领域
本发明涉及核酸特性的表征技术领域，具体涉及一种Mmup单体变体、包含Mmup单体变体的孔蛋白及构建体，及应用所述Mmup单体变体或孔蛋白进行表征目标多核苷酸的方法。
背景技术
纳米孔测序技术是一种以单链核酸分子作为测序单元，利用一个能够提供离子电流通道的纳米孔，使得单链核酸分子在电场力驱动下通过该纳米孔，当多核苷酸通过纳米孔易位时，会由于物理占位效应而产生相对应的阻塞电流，对产生的不同信号实时读取进而分析得到多核苷酸序列信息的基因测序技术。纳米孔测序技术具有以下优势：在无需扩增的情况下，即可简便的建库；阅读速度快，单链分子的阅读速度能够达到每小时数万个碱基；阅读长度更长，通常可以达到数千个碱基；可以直接进行甲基化的DNA或RNA的测量。
但是，每一个或一系列核苷酸在电场力的作用下通过纳米孔蛋白时，会产生一种特定的阻塞电流，此时记录的电流信号与多核苷酸的序列虽是对应关系，却通常是3-4个核苷酸控制某些级别的电流水平，所以仍然需要提高精确度。目前，可以通过改变多核苷酸结构、在纳米孔处的持续时间以及开发新的纳米孔从而控制多核苷酸的易位来提高精确度。
例如：专利US20150065354A1公开了一种使用XPD解旋酶表征目标多核苷酸的方法，所述方法利用孔和XPD解旋酶。该发明所述的XPD解旋酶可以控制目标多核苷酸穿过所述孔的运动。
专利US20170268055A1公开了一种用于多核苷酸测序的组合物和方法，该方法利用用目标多核苷酸通过孔的易位分步易位步骤来表征目标多核苷酸，包括表征多核苷酸的序列的方法和组合物。
专利CN106103741A公开了将一个或多个多核苷酸结合蛋白连接到靶多核苷酸的方法，该发明还涉及表征靶多核苷酸的新方法。
专利CN102216783B公开了一种耻垢分枝杆菌孔蛋白(Msp)纳米孔以及使用该纳米 孔进行测序，其中，将野生型Msp的90或91位点突变以提高分析物在测序时的电导，并降低分析物在测序中的转位速度。
然而，现有技术中并未提到Mmup单体变体及在测序中的应用，且现有技术中可以用于测序的孔蛋白种类很少。
因此，本发明进一步提供了一种新的纳米孔蛋白，通过将不能被用于测序的野生型Mmup蛋白进行突变制备了Mmup单体变体，并证实了该Mmup单体变体在测序中的功能。
发明内容
本发明证明了制备Mmup突变型蛋白特定位点突变的Mmup单体变体，可以用于纳米孔测序，然而野生型Mmup单体并没有该功能。并且，应用本发明所述的孔蛋白进行纳米孔测序，可以明显的看出各种不同核苷酸电流信号的差别，具备较高的测序精确度。
本发明所述的“Mmup”来源于产粘液分枝杆菌。优选的，所述的“Mmup”来源于Mycolicibacterium mucogenicum。
具体的，本发明的第一方面，提供了一种Mmup单体变体，所述的Mmup单体变体包含SEQ ID NO：1第91-99位任意一个或多个氨基酸突变的氨基酸序列。
优选的，所述的Mmup单体变体包含第91位天冬氨酸(D)和/或第99位丙氨酸(A)的突变。
在本发明的一个具体实施方式中，所述的Mmup单体变体包含第91位天冬氨酸(D)的突变。
在本发明的另一个具体实施方式中，所述的Mmup单体变体包含第99位丙氨酸(A)的突变。
在本发明的还一个具体实施方式中，所述的Mmup单体变体包含第91位天冬氨酸(D)和第99位丙氨酸(A)的突变。
优选的，所述的Mmup单体变体包含至少一种如下突变：
D91突变为：脯氨酸(P)、色氨酸(W)、精氨酸(R)、谷氨酰胺(Q)、赖氨酸(K)、苯丙氨酸(F)、丝氨酸(S)、天冬酰胺(N)、半胱氨酸(C)、异亮氨酸(I)、 亮氨酸(L)或缬氨酸(V)，或者，非天然氨基酸；或者，
A99突变为：脯氨酸(P)、苯丙氨酸(F)、异亮氨酸(I)、亮氨酸(L)、缬氨酸(V)、赖氨酸(K)或精氨酸(R)，或者，非天然氨基酸。
进一步优选的，所述的Mmup单体变体包含D91K和/或A99K的突变。
在本发明的一个具体实施方式中，所述的Mmup单体变体包含D91K的突变。
在本发明的另一个具体实施方式中，所述的Mmup单体变体包含A99K的突变。
在本发明的还一个实施方式中，所述的Mmup单体变体包含D91K和A99K的突变。
优选的，所述的Mmup单体变体还包含SEQ ID NO：1第80-90位和/或100-120位任意一个或多个氨基酸突变的氨基酸序列。
进一步优选的，所述的Mmup单体变体还包含SEQ ID NO：1第1-79位和/或121-186位任意一个或多个氨基酸突变的氨基酸序列。
优选的，所述的Mmup单体变体还包含第89位亮氨酸(L)的突变、第110位天冬酰胺(N)的突变、第120位天冬氨酸(D)的突变、第136位天冬酰胺(N)的突变或第141位丝氨酸(S)的突变中的一种或两种以上的组合。
进一步优选的，所述的Mmup单体变体包含至少一种如下突变：
L89突变为：天冬酰胺(N)、丙氨酸(A)、谷氨酰胺(Q)、甘氨酸(G)、丝氨酸(S)、苏氨酸(T)、赖氨酸(K)或脯氨酸(P)，或者，非天然氨基酸；或者，
N110突变为：脯氨酸(P)、丙氨酸(A)、异亮氨酸(I)或亮氨酸(L)，或者，非天然氨基酸；或者，
D120突变为：精氨酸(R)、赖氨酸(K)、天冬酰胺(N)、谷氨酰胺(Q)、丙氨酸(A)、丝氨酸(S)、甘氨酸(G)或苏氨酸(T)，或者，非天然氨基酸；或者，
N136突变为：精氨酸(R)、赖氨酸(K)、谷氨酰胺(Q)、丙氨酸(A)、丝氨酸(S)或苏氨酸(T)，或者，非天然氨基酸；或者，
S141突变为：精氨酸(R)、赖氨酸(K)、谷氨酰胺(Q)、丙氨酸(A)、丝氨酸(S)或苏氨酸(T)，或者，非天然氨基酸。
优选的，所述的Mmup单体变体还可以包含除上述突变类型以外的其他突变型，只要所述的突变不影响多核苷酸通过孔蛋白时对不同多核苷酸的区分即可。
优选的，所述的Mmup单体变体还可以包括引入半胱氨酸的突变，以连接用于测序的分子，例如核酸结合蛋白等。
优选的，所述的Mmup单体变体可以只包含孔蛋白形成结构域的收缩区和环形区片段序列，且保留孔形成活性。可以去除多余的残基或者增加其他氨基酸残基，且保留孔形成活性。所述的片段长度可以为至少12、20、40、50、100或150个氨基酸。
优选的，所述的Mmup单体变体可以是经过修饰的，以便于鉴定或纯化。例如：通过添加天冬氨酸残基(asp标签)、链霉亲和素标签、flag标签或组氨酸残基(His标签)。
优选的，所述Mmup单体变体可带有显示标记物。例如：荧光分子、放射性同位素 125I、放射性同位素 35S、多核苷酸、生物素、抗原或抗体。
优选的，所述的Mmup单体变体还包括分子发动机。优选的，所述的分子发动机是酶。进一步优选的，所述的酶是聚合酶、外切核酸酶或Klenow片段。
本发明的第二方面，提供了一种包含至少一个本发明任一所述Mmup单体变体的构建体。其中，所述的构建体保留了形成孔的能力。
优选的，所述的构建体包含1-50个Mmup单体变体，其中，所述的Mmup单体变体相同或不同。具体的，所述的构建体包含1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49或50个所述Mmup单体变体，其中，所述的Mmup单体变体相同或不同。
进一步优选的，所述的构建体包含1-20个Mmup单体变体，其中，所述的Mmup单体变体相同或不同。具体的，所述的构建体包含1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20个Mmup单体变体，其中，所述的Mmup单体变体相同或不同。
优选的，所述的构建体还包含野生型Mmup单体。
进一步优选的，所述的构建体包含1-50个野生型Mmup单体。具体的，所述的构建体包含1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49或50个野生型Mmup单体。
在本发明的一个具体实施方式中，所述的构建体包含1-20个野生型Mmup单体。具体的，所述的构建体包含1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20个野生型Mmup单体。
最为优选的，所述的构建体包含4-10个相同或不同的Mmup单体变体。
在本发明的一个具体实施方式中，所述的构建体包含4、6、8、10个相同或不同的Mmup单体变体。
优选的，所述的Mmup单体变体与Mmup单体变体、野生型Mmup单体与野生型Mmup单体、Mmup单体变体与野生型Mmup单体之间通过共价连接。
优选的，所述的Mmup单体变体与Mmup单体变体、野生型Mmup单体与野生型Mmup单体、Mmup单体变体与野生型Mmup单体之间是遗传上融合的。
本发明的第三方面，提供了一种包含至少一个Mmup单体变体的孔蛋白，所述的Mmup单体变体包含SEQ ID NO：1第91-99位任意一个或多个氨基酸突变的氨基酸序列，所述的突变导致当多聚核苷酸单链通过所述包含至少一个Mmup单体变体的孔蛋白时，产生由于不同种类的核苷酸的物理或化学性质的不同而造成的孔内电阻的差异。
优选的，所述的突变导致电荷性质或者氨基酸疏水性质变化。
优选的，所述的电阻的差异是指可用于表征多核苷酸的特征，所述的特征包括多核苷酸的来源、长度、大小、分子量、同一性、序列、二级结构、浓度或目标多核苷酸是否被修饰。进一步优选的，所述的电阻的差异是指可用于表征多核苷酸的序列特征，即所述的孔蛋白可以用于测序，精确的区分多核苷酸的不同碱基。
优选的，所述的多核苷酸可以是是天然存在的或人工合成的。进一步优选的，所述的多核苷酸可以是天然的DNA、RNA或者经过修饰的DNA或RNA。
更进一步优选的，所述的目标多核苷酸中的一个或多个核苷酸可以是经过修饰的，例如甲基化、氧化、损伤、脱碱基的、蛋白标记、带有标签或多核苷酸序列中间连接一段间隔物。
更进一步优选的，所述人工合成的核酸选自肽核酸(PNA)、甘油核酸(GNA)、苏糖核酸(TNA)、锁定核酸(LNA)或其他具有核苷侧链的合成聚合物。
优选的，所述的目标多核苷酸为单链、双链或至少一部分是双链的。
优选的，所述的Mmup单体变体包含第91位天冬氨酸(D)和/或第99位丙氨酸(A)的突变。
在本发明的一个具体实施方式中，所述的Mmup单体变体包含第91位天冬氨酸(D)的突变。
在本发明的另一个具体实施方式中，所述的Mmup单体变体包含第99位丙氨酸(A)的突变。
在本发明的还一个具体实施方式中，所述的Mmup单体变体包含第91位天冬氨酸(D)和第99位丙氨酸(A)的突变。
进一步优选的，所述的Mmup单体变体包含至少一种如下突变：
D91突变为：脯氨酸(P)、色氨酸(W)、精氨酸(R)、谷氨酰胺(Q)、赖氨酸(K)、苯丙氨酸(F)、丝氨酸(S)、天冬酰胺(N)、半胱氨酸(C)、异亮氨酸(I)、亮氨酸(L)或缬氨酸(V)，或者，非天然氨基酸；或者，
A99突变为：脯氨酸(P)、苯丙氨酸(F)、异亮氨酸(I)、亮氨酸(L)、缬氨酸(V)、赖氨酸(K)或精氨酸(R)，或者，非天然氨基酸。
更进一步优选的，所述的Mmup单体变体包含D91K和/或A99K的突变。
在本发明的一个具体实施方式中，所述的Mmup单体变体包含D91K和A99K的突变。
优选的，所述的Mmup单体变体还包含SEQ ID NO：1第80-90位和/或100-120位任意一个或多个氨基酸突变的氨基酸序列。
进一步优选的，所述的Mmup单体变体还包含SEQ ID NO：1第1-79位和/或121-186 位任意一个或多个氨基酸突变的氨基酸序列。
优选的，所述的Mmup单体变体还包含第89位亮氨酸(L)的突变、第110位天冬酰胺(N)的突变、第120位天冬氨酸(D)的突变、第136位天冬酰胺(N)的突变或第141位丝氨酸(S)的突变中的一种或两种以上的组合。
进一步优选的，所述的Mmup单体变体还包含至少一种如下突变：
L89突变为：天冬酰胺(N)、丙氨酸(A)、谷氨酰胺(Q)、甘氨酸(G)、丝氨酸(S)、苏氨酸(T)、赖氨酸(K)或脯氨酸(P)，或者，非天然氨基酸；或者，
N110突变为：脯氨酸(P)、丙氨酸(A)、异亮氨酸(I)或亮氨酸(L)，或者，非天然氨基酸；或者，
D120突变为：精氨酸(R)、赖氨酸(K)、天冬酰胺(N)、谷氨酰胺(Q)、丙氨酸(A)、丝氨酸(S)、甘氨酸(G)或苏氨酸(T)，或者，非天然氨基酸；或者，
N136突变为：精氨酸(R)、赖氨酸(K)、谷氨酰胺(Q)、丙氨酸(A)、丝氨酸(S)或苏氨酸(T)，或者，非天然氨基酸；或者，
S141突变为：精氨酸(R)、赖氨酸(K)、谷氨酰胺(Q)、丙氨酸(A)、丝氨酸(S)或苏氨酸(T)，或者，非天然氨基酸。
优选的，所述包含至少一个Mmup单体变体的孔蛋白包含至少1-50个Mmup单体变体，其中，所述的Mmup单体变体相同或不同。
具体的，所述包含至少一个Mmup单体变体的孔蛋白包含1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49或50个所述Mmup单体变体，所述的Mmup单体变体相同或不同。
进一步优选的，所述包含至少一个Mmup单体变体的孔蛋白包含至少1-20个Mmup单体变体，其中，所述的Mmup单体变体相同或不同。
具体的，所述包含至少一个Mmup单体变体的孔蛋白包含1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20个所述Mmup单体变体，所述的Mmup 单体变体相同或不同。
优选的，所述包含至少一个Mmup单体变体的孔蛋白还包含野生型Mmup单体。
优选的，所述包含至少一个Mmup单体变体的孔蛋白包含1-50个野生型Mmup单体。
具体的，所述包含至少一个Mmup单体变体的孔蛋白包含1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49或50个野生型Mmup单体。
进一步优选的，所述包含至少一个Mmup单体变体的孔蛋白包含1-20个野生型Mmup单体。
具体的，所述包含至少一个Mmup单体变体的孔蛋白包含1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20个野生型Mmup单体。
在本发明的一个具体实施方式中，所述包含至少一个Mmup单体变体的孔蛋白包含4-10个相同或不同的Mmup单体变体。
在本发明的一个具体实施方式中，所述包含至少一个Mmup单体变体的孔蛋白包含4、6、8、10个相同或不同的Mmup单体变体。
优选的，所述的Mmup单体变体与Mmup单体变体、野生型Mmup单体与野生型Mmup单体、Mmup单体变体与野生型Mmup单体之间通过共价连接。
优选的，所述的Mmup单体变体与Mmup单体变体、野生型Mmup单体与野生型Mmup单体、Mmup单体变体与野生型Mmup单体之间是遗传上融合的。
优选的，所述包含在包含至少一个Mmup单体变体的孔蛋白中的Mmup单体变体相同或不同。例如：所述包含至少一个Mmup单体变体的孔蛋白可以包含八个相同或者不同的Mmup单体变体。优选的，所述包含至少一个Mmup单体变体的孔蛋白包含一个Mmup单体变体和七个相同单体，其中，所述Mmup单体变体与相同单体不同。或者，所述包含至少一个Mmup单体变体的孔蛋白包含两个相同或不同的Mmup单体变体和六个相同单体，其中，所述的Mmup单体变体与相同单体不同。或者，所述包含至少一个Mmup单体变体 的孔蛋白包含三个相同或不同的的Mmup单体变体和五个相同单体，其中，所述的Mmup单体变体与相同单体不同。或者，所述包含至少一个Mmup单体变体的孔蛋白包含四个相同或不同的的Mmup单体变体和四个相同单体，其中，所述的Mmup单体变体与相同单体不同。或者，所述包含至少一个Mmup单体变体的孔蛋白包含五个相同或不同的的Mmup单体变体和三个相同单体，其中，所述的Mmup单体变体与相同单体不同。或者，所述包含至少一个Mmup单体变体的孔蛋白包含六个相同或不同的的Mmup单体变体和两个相同单体，其中，所述的Mmup单体变体与相同单体不同。或者，所述包含至少一个Mmup单体变体的孔蛋白包含七个相同或不同的的Mmup单体变体和一个单体，其中，所述的Mmup单体变体与一个单体不同。
优选的，所述包含至少一个Mmup单体变体的孔蛋白包含八个相同或不同的所述的Mmup单体变体。
优选的，所述包含至少一个Mmup单体变体的孔蛋白可以为同源的或异源的。
优选的，所述包含至少一个Mmup单体变体的孔蛋白包含收缩区和环形区。
优选的，所述包含至少一个Mmup单体变体的孔蛋白的收缩区孔道直径小于野生型Mmup单体组成的孔蛋白的收缩区孔道直径。进一步优选的，所述包含至少一个Mmup单体变体的孔蛋白的收缩区孔道直径小于
Figure PCTCN2019109332-appb-000001
Figure PCTCN2019109332-appb-000002
Figure PCTCN2019109332-appb-000003
或
Figure PCTCN2019109332-appb-000004
更进一步优选的，所述包含至少一个Mmup单体变体的孔蛋白的收缩区孔道直径小于
Figure PCTCN2019109332-appb-000005
Figure PCTCN2019109332-appb-000006
或
Figure PCTCN2019109332-appb-000007
在本发明的一个具体实施方式中，所述包含至少一个Mmup单体变体的孔蛋白的收缩区孔道直径约等于
Figure PCTCN2019109332-appb-000008
或
Figure PCTCN2019109332-appb-000009
优选的，所述的Mmup单体变体还可以包含除上述突变类型以外的其他突变型，只要所述的突变不影响多核苷酸通过孔蛋白时对不同多核苷酸的区分即可。
优选的，所述的单体变体还可以包括引入半胱氨酸的突变，以连接用于测序的分子， 例如核酸结合蛋白等。
优选的，所述的Mmup单体变体可以只包含孔蛋白形成结构域的收缩区和环形区片段序列，且保留孔形成活性。可以去除多余的残基或者增加其他氨基酸残基，且保留孔形成活性。所述的片段长度可以为至少12、20、40、50、100或150个氨基酸。
优选的，所述的Mmup单体变体可以是经过修饰的，以便于鉴定或纯化。例如：通过添加天冬氨酸残基(asp标签)、链霉亲和素标签、flag标签或组氨酸残基(His标签)。
优选的，所述Mmup单体变体可带有显示标记物。例如：荧光分子、放射性同位素 125I、放射性同位素 35S、多核苷酸、生物素、抗原或抗体。
优选的，所述的Mmup单体变体还包括分子发动机。优选的，所述的分子发动机是酶。进一步优选的，所述的酶是聚合酶、外切核酸酶或Klenow片段。
优选的，所述包含至少一个Mmup单体变体的孔蛋白还包括帽状体形成区和/或桶状体形成区域。
优选的，所述包含至少一个Mmup单体变体的孔蛋白允许水合离子在施加的电势的驱动下从膜的一侧流向膜的另一层。其中，所述的膜为双层膜，进一步优选为脂质双层膜。
本发明的第四方面，提供了一种编码本发明任一所述包含至少一个Mmup单体变体的孔蛋白、本发明任一所述的Mmup单体变体或者本发明任一所述构建体的核苷酸序列。
优选的，所述的编码所述Mmup单体变体的核苷酸序列与SEQ ID NO：2或SEQ ID NO：11所示序列具有70％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、99.5％、99.9％同源性，且编码所述Mmup单体变体的核苷酸的序列。
在本发明的一个具体实施方式中，所述的编码所述Mmup单体变体的核苷酸序列为SEQ ID NO：11所示。
本发明的第五方面，提供了一种包含编码本发明所述的Mmup单体变体、本发明所述的孔蛋白或者本发明所述构建体的核苷酸序列的载体。
优选的，所述的载体可以提供有复制起点、任选用于表达所述核苷酸序列的启动子以及任选所述启动子的调节信号基因的质粒、病毒或噬菌体载体。所述载体可含有一个或多 个选择性标记基因，例如四环素抗性基因。启动子和其他表达调节信号可被选择为与所述表达载体设计用于的宿主细胞相容。所述的启动子选自T7、trc、lac、ara或λL启动子。
本发明所述的Mmup单体变体可以采用化学合成或者重组方式制备，优选为重组方式制备。
优选的，所述的载体包含与编码本发明任一所述包含至少一个Mmup单体变体的孔蛋白、本发明任一所述的Mmup单体变体或者本发明任一所述构建体的核苷酸序列可操作性连接的启动子。
进一步优选的，所述的启动子为诱导型启动子或组成型启动子，其中，所述的诱导型启动子为乙酰胺诱导型启动子。
优选的，所述编码包含至少一个Mmup单体变体的孔蛋白的核苷酸序列包含至少一条编码Mmup单体变体的核苷酸序列。
进一步优选的，所述编码包含至少一个Mmup单体变体的孔蛋白的核苷酸序列还包含至少一条编码野生型Mmup单体的核苷酸序列。
更进一步优选的，所述编码Mmup单体变体的核苷酸序列与编码Mmup单体变体的核苷酸序列，编码Mmup单体变体的核苷酸序列与编码野生型Mmup单体的核苷酸序列，或者，编码野生型Mmup单体的核苷酸序列与编码野生型Mmup单体的核苷酸序列之间通过编码氨基酸连接体序列连接。
本发明的第六方面，提供了一种表达本发明任一所述的Mmup单体变体、本发明任一所述构建体或本发明任一所述包含至少一个Mmup单体变体的孔蛋白的突变细菌，所述的细菌包含：
(a)野生型Mmup单体的缺失；和(b)本发明任一所述的载体。
优选的，所述的细菌包含与编码Mmup单体变体、包含Mmup单体变体的构建体或者包含Mmup单体变体的孔蛋白的核苷酸序列可操作性连接的启动子的载体。
进一步优选的，所述的Mmup单体变体包括Mmup单体变体的旁系同源物或同系物。
进一步优选的，所述的包含Mmup单体变体的构建体包括Mmup单体变体的旁系同源 物或同系物构建体或单体。
进一步优选的，所述的包含Mmup单体变体的孔蛋白包括Mmup单体变体的旁系同源物或同系物孔蛋白或单体。
优选的，所述的细菌还可以包含与编码野生型Mmup单体、包含野生型Mmup单体的构建体或者包含野生型Mmup单体的孔蛋白的核苷酸序列可操作性连接的启动子的载体。
进一步优选的，野生型Mmup单体包括野生型Mmup单体的旁系同源物或同系物单体。
进一步优选的，所述包含野生型Mmup单体的构建体为野生型Mmup单体旁系同源物或同系物构建体或单体。
进一步优选的，所述包含野生型Mmup单体的孔蛋白为野生型Mmup单体的旁系同源物或同系物孔蛋白或单体。
优选的，所述的细菌为产粘液分枝杆菌。
本发明的第七方面，提供了一种产生Mmup孔蛋白的方法，所述的方法包括用包含本发明任一所述的载体转化本发明任一所述的细菌，诱导细菌表达Mmup孔蛋白。
本发明的第八方面，提供了一种Mmup单体变体的制备方法，所述的载体可引入适合的宿主细胞中，通过将所述编码Mmup单体变体的核苷酸序列插入载体中，将所述载体引入相容的细菌宿主细胞并且在允许所述核苷酸表达的条件下培养所述宿主细胞来产生本发明所述的Mmup单体变体。
本发明的第九方面，提供了一种包含本发明所述核苷酸序列或所述载体的细胞。
优选的，所述的细胞可以为大肠杆菌等等。更优选的，所述的细胞为dam+型菌株(例如DH5α菌株)。
本发明的第十方面，提供了一种表征目标多核苷酸的方法，包括：
(a)将目标多核苷酸与本发明任一所述包含至少一个Mmup单体变体的孔蛋白接触，使得所述目标多核苷酸序列穿过包含至少一个Mmup单体变体的孔蛋白；和
(b)获取目标多核苷酸穿过包含至少一个Mmup单体变体的孔蛋白时核苷酸与包含至少一个Mmup单体变体的孔蛋白相互作用的一个或多个特征，以表征所述目标多核苷酸。
优选的，重复步骤(a)、(b)一次或多次。
优选的，所述的步骤(a)中目标多核苷酸可以与衍生自多核苷酸处理酶结合，从而控制转位速度。进一步优选的，所述的多核苷酸处理酶是能够与多核苷酸相互作用并修饰其至少一种性质的多肽。其中，所述的多核苷酸处理酶可以具有酶活性也可以不具有酶活性，只要该酶结合多核苷酸且控制其在孔中的转位速度即可。其中，所述的核酸可以与一个或多个多核苷酸处理酶。
优选的，所述的多核苷酸处理酶为溶核酶。进一步优选的，所述的多核苷酸处理酶包括但不限于核酸结合蛋白、解旋酶、聚合酶、核酸外切酶、端粒酶、反转录酶、转位酶或拓扑异构酶。
在本发明的一个具体实施方式中，所述的多核苷酸处理酶为促旋酶。
优选的，所述步骤(a)中还包括目标多核苷酸与核酸结合蛋白、解旋酶、核酸外切酶、端粒酶、拓扑异构酶、反转录酶、转位酶和/或聚合酶中的一种或两种以上的组合接触的步骤，使得所述目标多核苷酸序列穿过孔蛋白的转位速度小于核酸结合蛋白、解旋酶、核酸外切酶、端粒酶、拓扑异构酶、反转录酶、转位酶和/或聚合酶不存在时的转位速度。
进一步优选的，所述的核酸结合蛋白包括但不限于修饰或者野生的真核单链结合蛋白、细菌单链结合蛋白、古生单链结合蛋白、病毒单链结合蛋白或双链结合蛋白中的一种或两种以上的组合。所述的核酸结合蛋白包括但不限于来自Escherichia coli的SSBEco、来自Bartonella henselae的SSBBhe、来自Coxiella burnetii的SSBCbu、来自Thermathoga maritima的SSBTma、来自Helicobacter pylori的SSBHpy、来自Deinococcus radiodurans的SSBDra、来自Thermus aquaticus的SSBTaq、来自Mycobacterium smegmatis的SSBMsm、来自Sulfolobus solfataricus的SSBSso、来自Sulfolobus solfataricus的SSBSso7D、来自Homo sapiens的SSBMHsmt、来自Mycobacterium leprae的SSBMle、来自Bacteriohage T4的gp32T4、来自Bacteriophage RB69的gp32RB69或来自Bacteriohage T7的gp2.5T7。
进一步优选的，所述的解旋酶可以为任一Hel308家族解旋酶及修饰的Hel308家族解旋酶、RecD解旋酶及其变体、TrwC解旋酶及其变体、Dda解旋酶及其变体、TraI Eco及 其变体、XPD Mbu及其变体。
进一步优选的，所述的聚合酶包括但不限于修饰或者野生的DNA聚合酶，包括但不限于Phi29DNA聚合酶、Tts DNA聚合酶、M2DNA聚合酶、VENT DNA聚合酶、T5DNA聚合酶、PRD1DNA聚合酶、Bst DNA聚合酶或REPLI-gscDNA聚合酶。
进一步优选的，所述的核酸外切酶包括但不限于修饰或者野生的来自大肠杆菌的核酸外切酶I、来自大肠杆菌的核酸外切酶III、来自噬菌体λ核酸外切酶或者来自嗜热栖热菌的RecJ。在本发明的一个具体实施方式中，所述步骤(a)中包括目标多核苷酸与解旋酶接触的步骤，所述的解旋酶为EF8813，所述的解旋酶的氨基酸序列为SEQ ID NO：3所示，所述解旋酶的核苷酸序列为SEQ ID NO：4所示。优选的，所述的目标多核苷酸可以与一个或多个解旋酶接触。进一步优选的，所述的目标多核苷酸可以与2-20个解旋酶、甚至更多个的解旋酶接触。其中，所述与目标多核苷酸结合的解旋酶可以相同也可以不同。且与目标多核苷酸结合的多个解旋酶彼此共价连接。
优选的，所述的一个或多个特征选自目标多核苷酸的来源、长度、大小、分子量、同一性、序列、二级结构、浓度或目标多核苷酸是否被修饰。
在本发明的一个具体实施方式中，所述的特征为序列。
优选的，所述步骤(b)中所述一个或多个特征通过电测量和/或光学测量进行。
进一步优选的，通过电测量和/或光测量产生电信号和/或光信号，而每种核苷酸对应一种信号水平，继而将电信号和/或光信号转化为核苷酸的序列特征。
本发明所述的电测量选自电流测量、阻抗测量、场效应晶体管(FET)测量、隧道测量或风洞测量。
本发明所述的电信号选自电流、电压、隧穿、电阻、电位、电导率或横向电测量的测量值。
在本发明的一个具体实施方式中，所述的电信号为穿过所述孔的电流。即所述电流以核苷酸特异性方式通过所述孔，如果检测到与核苷酸相关特征性电流流经所述孔，则存在所述的核苷酸。反之，则不存在。然而，对于相似核苷酸或者修饰的核苷酸之间的区分， 则根据电流的幅度确定。
优选的，采用本发明所述的孔蛋白进行多核苷酸的表征过程中产生的电导高于野生型Mmup单体形成的孔。
优选的，所述的方法还包括横跨目标多核苷酸接触的孔蛋白施加电势差的步骤。其中，所述的电势差足以将目标多核苷酸从孔蛋白的通道中转位。
优选的，所述的目标多核苷酸可以是天然的DNA、RNA或者经过修饰的DNA或RNA。
本发明所述的目标多核苷酸为含有一个或多个核苷酸的大分子。
本发明所述的目标多核苷酸可以是天然存在的或人工合成的。优选的，所述的目标多核苷酸中的一个或多个核苷酸可以是经过修饰的，例如甲基化、氧化、损伤、脱碱基的、蛋白标记、带有标签或多核苷酸序列中间连接一段间隔物。优选的，所述人工合成的核酸选自肽核酸(PNA)、甘油核酸(GNA)、苏糖核酸(TNA)、锁定核酸(LNA)、或其他具有核苷侧链的合成聚合物。
优选的，所述的孔蛋白允许水合离子在施加的电势的驱动下从膜的一侧流向膜的另一层。其中，所述的膜可形成离子、核苷酸和核酸流的屏障。进一步优选的，所述的膜为双层膜，进一步优选为脂质双层膜。所述的脂质双层膜包括但不限于磷脂、糖脂、胆固醇、分枝菌酸中的一种或两种以上的混合物。
优选的，所述的孔蛋白通道位于第一导电性液体介质与第二导电性液体介质之间，其中，至少一种导电性液体介质包含目标多核苷酸，并且第一导电性液体介质与第二导电性液体介质可以相同，也可以不同，只要可以达到分析目标多核苷酸一个或多个特征的目的即可。
优选的，所述的目标多核苷酸为单链、双链或至少一部分是双链的。
在本发明所述的一个具体实施方式中，所述的目标多核苷酸为至少一部分是双链的。其中所述的双链部分构成Y衔体结构，所述的Y衔体结构包含优先螺入所述孔蛋白的前导序列，所述的前导序列3’端连接硫醇、生物素或胆固醇，用来与脂质双层膜的一层膜结合，以为目标多核苷酸指向正确的方向并具有拉动的作用。
在本发明的一个具体实施方式中，所述的前导序列3’端连接胆固醇，用来与脂质双层膜的一层膜结合。
调节表征目标多核苷酸过程中的电压、盐浓度、缓冲液、添加剂或温度可以控制本发明所述的孔蛋白在表征目标多核苷酸中对不同核苷酸的区分程度。其中，所述的添加剂选自DTT、脲或甜菜碱。
优选的，所述的电压范围为-250mV至+250mV。进一步优选的，所述的电压选自-250mV、-210mV、-180mV、-140mV、-110mV、-90mV、-70mV、-40mV、0mV、+40mV、+70mV、+90mV、+110mV、+140mV、+180mV、+210mV、+250mV。
在本发明的一个具体实施方式中，所述的电压为-180mV至+180mV。
在本发明的一个具体实施方式中，所述的方法包括：将所述的孔蛋白插入膜中，然后将目标多核苷酸与所述的孔蛋白、核酸结合蛋白、聚合酶、核酸外切酶、端粒酶、拓扑异构酶、反转录酶、转位酶和/或解旋酶接触，向横跨目标多核苷酸接触的孔蛋白施加电势差，使得所述目标多核苷酸序列穿过孔蛋白；和
获取目标多核苷酸穿过孔蛋白时核苷酸与孔蛋白相互作用的电流特征，以鉴别多核苷酸为是否存在、为何种核苷酸或者是否经过修饰。
优选的，所述的孔蛋白插入膜中的方法可以为任何本领域已知的可以达到表征多核苷酸目的的方法。进一步优选的，所述孔蛋白可以以纯化形式悬浮于含有脂双层的溶液中，使得其扩散到所述脂双层并且通过结合到所述脂双层并组装为有功能的状态而插入到所述脂双层中。
本发明的第十一方面，提供了一种本发明任一所述包含至少一个Mmup单体变体的孔蛋白、本发明任一所述的Mmup单体变体、本发明任一所述构建体、本发明所述的核苷酸序列、本发明任一所述的载体或者本发明任一所述的突变细菌在表征目标多核苷酸中的应用。
本发明的第十二方面，提供了一种表征目标多核苷酸的试剂盒，所述的试剂盒包括本发明任一所述包含至少一个Mmup单体变体的孔蛋白、本发明任一所述的Mmup单体变体、 本发明任一所述构建体、本发明所述的核苷酸序列、本发明任一所述的载体或者本发明任一所述的突变细菌。
优选的，所述的Mmup单体变体、所述的构建体、所述的核苷酸序列、所述的载体、所述的细胞或所述的孔蛋白均可以为多个。
优选的，所述的试剂盒还包括一个或多个核酸结合蛋白、解旋酶、核酸外切酶、端粒酶、拓扑异构酶、反转录酶、转位酶和/或聚合酶中的一种或两种以上的组合。
优选的，所述的试剂盒还包括脂质双层的芯片，所述的孔蛋白横跨脂质双层。
优选的，所述的试剂盒包含一个或多个脂质双层，每个脂质双层包含一个或多个所述的孔蛋白。
优选的，所述的试剂盒还包括实施表征目标多核苷酸的试剂或装置。进一步优选的，所述的试剂包括缓冲剂、PCR扩增所需的工具。
本发明的第十三方面，提供了一种表征目标多核苷酸的装置，所述的装置包括本发明任一所述包含至少一个Mmup单体变体的孔蛋白、本发明任一所述的Mmup单体变体、本发明任一所述构建体、本发明所述的核苷酸序列、本发明任一所述的载体或者本发明任一所述的突变细菌。
优选的，所述的装置还包括一个或多个核酸结合蛋白、解旋酶、核酸外切酶、端粒酶、拓扑异构酶、反转录酶、转位酶和/或聚合酶中的一种或两种以上的组合。
优选的，所述的装置还包括支撑所述孔蛋白并可传输孔蛋白与多核苷酸相互作用的信号的传感器，至少一个用于存储目标多核苷酸的存储器，和实施表征过程所需的溶液。
优选的，所述的装置还包括膜片钳放大器和/或数据获取装置。
本发明的第十四方面，提供了一种表征目标多核苷酸的传感器，所述的传感器包括本发明任一所述包含至少一个Mmup单体变体的孔蛋白、本发明任一所述的Mmup单体变体、本发明任一所述构建体、本发明所述的核苷酸序列、本发明任一所述的载体或者本发明任一所述的突变细菌。
本发明所述的“非天然氨基酸”为非在蛋白质中天然发现的包含氨基和羧基的化合物。 优选的，所述的非天然氨基酸为本领域已知的任何非天然氨基酸。进一步优选的，所述的非天然氨基酸包括但不限于N-乙基天冬氨酰、羟基赖氨酸、3-羟基脯氨酸、2-氨基丁酸、β-丙氨酸、β-氨基丙酸、2-氨基己二酸、3-氨基己二酸、4-氨基丁酸、6-氨基己酸、2-氨基庚酸、别-异亮氨酸、异锁链赖氨酸、4-羟基脯氨酸、别-羟基赖氨酸、2-氨基异丁酸、N-甲基甘氨酸、N-甲基异亮氨酸、3-氨基异丁酸、6-N-甲基赖氨酸、2,4-二氨基丁酸、N-甲基缬氨酸、鸟氨酸、正亮氨酸、正缬氨酸、锁链素、2,2’-二氨基庚二酸、2,3-二氨基丙酸、N-乙基甘氨酸或2-氨基庚二酸等等。
本发明所述的“修饰的……基酸”为侧链被化学修饰的氨基酸。例如：翻译后修饰的氨基酸，或者，侧链包含新型官能团(如硫氢基、氨基或羧基)，或者，侧链包含产生信号的部分(如荧光基团或放射性标记)。
本发明所述的“核苷酸”包括但不局限于：腺苷单磷酸(AMP)、鸟苷单磷酸(GMP)、胸苷单磷酸(TMP)、尿苷单磷酸(UMP)、胞嘧啶核苷单磷酸(CMP)、环状腺苷单磷酸(cAMP)、环状鸟苷单磷酸(cGMP)脱氧腺苷单磷酸(dAMP)、脱氧鸟苷单磷酸(dGMP)、脱氧胸苷单磷酸(dTMP)、脱氧尿苷单磷酸(dUMP)和脱氧胞苷单磷酸(dCMP)。优选的，所述核苷酸选自AMP、TMP、GMP、CMP、UMP、dAMP、dTMP、dGMP或dCMP。
本发明所述的“和/或”包括择一列出的项目以及任何数量的项目组合。
本发明所述的“包括”是开放式的描述，含有所描述的指定成分或步骤，以及不会实质上影响的其他指定成分或步骤。
本发明所述的“约”用于表示该数值和用于测定该数值的装置或方法所允许的标准差。
本发明所述的“同源性”，是指在使用蛋白序列或核苷酸序列的方面，本领域技术人员可以根据实际工作需要对序列进行调整，使使用序列与现有技术获得的序列相比，具有(包括但不限于)1％，2％，3％，4％，5％，6％，7％，8％，9％，10％，11％，12％，13％，14％，15％，16％，17％，18％，19％，20％，21％，22％，23％，24％，25％，26％，27％，28％，29％，30％，31％，32％，33％，34％，35％，36％，37％，38％，39％，40％，41％，42％，43％，44％，45％，46％，47％，48％，49％，50％，51％，52％，53％，54％，55％， 56％，57％，58％，59％，60％，70％，80％，81％，82％，83％，84％，85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％，99.1％，99.2％，99.3％，99.4％，99.5％，99.6％，99.7％，99.8％，99.9％的序列一致性。
本发明所述的“Mmup单体变体”是指与野生型Mmup单体具有至少或至多70、75、80、85、90、91、92、93、94、95、96、97、98、99、99.5或99.9％或更多、或可来自其间的任何范围、但小于100％的同一性并且当与一个或多个其他Mmup单体变体或野生型Mmup单体结合时保持形成通道的能力的Mmup单体变体。任选地，Mmup单体变体被进一步确定为在促进完全形成的通道形成孔蛋白的收缩区和/或环形区的形成的序列部分中包含突变。Mmup单体变体可以是例如重组蛋白。Mmup单体变体可包含本文中描述的任何突变。
本发明所述的″Mmup单体变体的旁系同源物或同系物孔蛋白″是指与野生型Mmup单体的旁系同源物或同系物孔蛋白具有至少或至多70、75、80、85、90、91、92、93、94、95、96、97、98、99、99.5或99.9％或更多，或可来自其间的任何范围，但小于100％的同一性并且保持通道形成能力的Mmup单体变体的旁系同源物或同系物孔蛋白。任选地，Mmup单体变体的旁系同源物或同系物孔蛋白被进一步确定为在序列的该部分包含突变，所述部分促进完全形成的通道形成孔蛋白的收缩区和/或环形区的形成。Mmup单体变体的旁系同源物或同系物孔蛋白可以例如是重组蛋白质。任何Mmup单体变体的旁系同源物或同系物孔蛋白可以任选地用于本文中的任何实施方案。
本发明所述的“Mmup单体变体的旁系同源物或同系物构建体”是指与野生型Mmup单体的旁系同源物或同系物构建体具有至少或至多70、75、80、85、90、91、92、93、94、95、96、97、98、99、99.5或99.9％或更多，或可来自其间的任何范围，但小于100％的同一性并且保持通道形成能力的Mmup单体变体的旁系同源物或同系物构建体。任选地，Mmup单体变体的旁系同源物或同系物构建体被进一步确定为在序列的该部分包含突变，所述部分促进完全形成的通道形成孔蛋白的收缩区和/或环形区的形成。Mmup单体变体的旁系同源物或同系物构建体可以例如是重组蛋白质。任何Mmup单体变体的旁系同源物或 同系物构建体可以任选地用于本文中的任何实施方案。
附图说明
以下，结合附图来详细说明本发明的实施例，其中：
图1：Mmup-(D91K/A99K)单体变体蛋白分子排阻层析的纯化结果，其中，1-6泳道显示的是分子筛分离的不同组分的SDS-PAGE电泳检测结果。
图2：包含Mmup单体变体的纳米孔蛋白(D91K/A99K)的棍棒模型，图中主要显示了通道孔道收缩区和环形区的氨基酸分布特征，尤其是收缩区和环形区的关键氨基酸残基分布，指向孔道中心的氨基酸残基91位的赖氨酸和92位的天冬酰胺，孔道直径约为
Figure PCTCN2019109332-appb-000010
99位的赖氨酸参与通道复合物的正确组装，其中，同源建模通过SWISS MODEL完成，模板pdb位1uun。
图3：包含野生型Mmup单体的纳米孔通道的棍棒模型，图中主要显示了孔道收缩区和环形区的氨基酸分布特征，尤其是收缩区和环形区的关键氨基酸残基，主要有91位的天冬氨酸，92位的天冬酰胺，93位的缬氨酸和94位的丝氨酸，由D91和N92形成的收缩区直径分别为
Figure PCTCN2019109332-appb-000011
和
Figure PCTCN2019109332-appb-000012
其中，同源建模通过SWISS MODEL完成，模板pdb位1uun。
图4：包含野生型Mmup单体的纳米孔基于同源建模的卡通示意图。其中，区域1对应于帽状体形成区，区域2对应于桶状体形成区域，区域3对应于收缩区和环形区。
图5：待测DNA构建体X2&cX2-80-15的结构图，其中区段a对应于SEQ ID NO：7，b对应于解旋酶EF8813-1(含有N端组氨酸标签及其融合有TOPV-HI结构域的变体蛋白，SEQ ID NO：3-4)，所述解旋酶可以结合到标记为a的区段，区段c对应于SEQ ID NO：6，区段d对应于SEQ ID NO：5，区段e对应于SEQ ID NO：8，其5′端45个碱基与测试链c段区域互补配对，3′端含有40个胸腺嘧啶和对应于g的3′胆固醇TEG标记，区段f对应于SEQ ID NO：9。
图6：待测DNA构建体S1T&S1MC的结构图，其中区段a对应于SEQ ID NO：10，b对应于解旋酶EF8813-1(含有N端组氨酸标签及其融合有TOPV-HI结构域的变体蛋白，SEQ ID NO：3-4)，所述解旋酶可以结合到标记为a的区段，区段h指的是仅保留磷酸骨 架的dspacer，标注为x，区段c对应于SEQ ID NO：12，区段d对应于SEQ ID NO：13，区段e对应于SEQ ID NO：14，其5′端45个碱基与测试链c段区域互补配对，3′端含有20个胸腺嘧啶和对应于g的3′胆固醇TEG标记，区段f对应于SEQ ID NO：15。
图7：包含野生型Mmup单体的纳米孔通道在±180mV电压的单通道行为特征，其中，y轴坐标＝电流(pA)，x轴坐标＝时间(s)。
图8：包含Mmup单体变体(D91K/A99K)的纳米孔蛋白在+180mV，0mV和-180mV电压下开孔电流及其门控特征，其中，y轴坐标＝电流(pA)，x轴坐标＝时间(s)。
图9：包含Mmup单体变体(D91K/A99K)的纳米孔蛋白在+180mV和0mV电压下核酸通过纳米孔的信号特征，其中，y轴坐标＝电流(pA)，x轴坐标＝时间(s)。
图10：当解旋酶(EF8813-1)控制DNA构建体X2穿过包含Mmup单体变体(D91K/A99K)的纳米孔蛋白移位时的部分示例电流轨迹，其中，两条轨迹的y轴坐标(左-20至180，右10至80)＝电流(pA)，x轴坐标(左15:04:25.5至15:04:37.5，右15:04:34.1至15:04:37.1)＝时间(s)，右图为左图虚线部分显示了电流轨迹的放大结果。
图11：当解旋酶(EF8813-1)控制DNA构建体X2穿过包含Mmup单体变体(D91K/A99K)的纳米孔蛋白移位时的电流轨迹的全过程电流变化结果，其中，y轴坐标＝电流(pA)，x轴坐标＝时间(s)。
图12：当解旋酶(EF8813-1)控制DNA构建体S1T穿过包含Mmup单体变体(D91K/A99K)的纳米孔蛋白移位时的部分示例电流轨迹，其中，两条轨迹的y轴坐标＝电流(pA)，x轴坐标＝时间(s)，右图为左图虚线部分显示的电流轨迹的放大结果，箭头指示的电流最大值显示的是dspacer的特征峰。
图13：当解旋酶(EF8813-1)控制DNA构建体S1T穿过包含Mmup单体变体(D91K/A99K)的纳米孔蛋白移位时的电流轨迹的全过程电流变化结果，其中，y轴坐标＝电流(pA)，x轴坐标＝时间(s)。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地 描述，显然，所描述的实施例仅是本发明的部分实施例，而不是全部。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1 Mmup单体变体的制备
一、质粒的构建
Mmup单体变体蛋白质序列通过对应氨基酸的密码子优化，并在基因两端添加合适的限制性内切酶酶切位点，具体的5’端添加NcoI位点ccatgg，3’端添加xhoI位点ctcgag，之后进行基因合成，合成后的基因克隆到表达载体pET24b中。
二、目的基因的定点突变，制备Mmup单体变体核苷酸序列
诱导突变基因(PCR反应)以待突变的质粒为模板，用设计的引物及KOD plus高保真酶进行PCR扩增反应，诱导目的基因突变。
具体步骤如下：
1、设计点突变引物，准备模板质粒DNA(包含SEQ ID NO：2的质粒DNA)，进行50μL PCR反应体系扩增。用DH5α菌株作为宿主菌。在end+型菌株中常有克隆数低的现象，但是对突变效率没有影响。其中，提取模板质粒DNA使用QIGEN质粒提纯试剂盒。点突变引物：
SEQ ID NO：16
Figure PCTCN2019109332-appb-000013
SEQ ID NO：17
Figure PCTCN2019109332-appb-000014
50μL PCR反应体系：
Figure PCTCN2019109332-appb-000015
Figure PCTCN2019109332-appb-000016
PCR扩增反应
循环温度反应时间：
Figure PCTCN2019109332-appb-000017
PCR扩增反应完成，获得Mmup单体变体核苷酸序列(SEQ ID NO：11)，冰浴5min，然后置于室温(避免反复冻融)。
2、模板消化，提取Mmup单体变体基因
PCR反应结束后使用DpnI酶消化甲基化质粒从而选择突变质粒DNA(含有SEQ ID NO：11的质粒)。准备PCR反应产物。具体步骤为：加入1μL(10U/μL)DpnI酶37℃温育2小时。(当质粒DNA用量过多时DpnI酶可能发生与样品反应不完全的现象。如果突变率低，可以适当延长反应时间或增加DpnI酶用量)
3、转化，获得含有Mmup单体变体基因的菌株
反应完毕后在质粒DNA上会产生缺口，因此当把这个质粒DNA转入E.coli中时选择DH5α。具体步骤为：将4μL突变质粒DNA样品加到50μL DH5α感受态细胞里，然后放置在冰上30min，42℃热击90s，之后立即冰浴2min，加入500μL SOC培养基37℃培养1小时，最后取100μL菌液涂布抗性筛选平板。
4、测序验证
挑取4个转化子培养测序，选取突变正确的阳性转化子提取质粒保存备用。
三、制备Mmup单体变体
将测序验证正确的Mmup单体变体质粒转入BL21(DE3)中培养。然后蛋白纯化，其中，蛋白纯化用试剂配方见表1。
吸取20μL包含Mmup单体变体质粒的BL21(DE3)甘油菌接于20mL(1：1000)含有终浓度50mg/mL卡那霉素的新鲜LB培养基，37℃，200rpm摇菌过夜活化；次日按1％接种量扩大培养至含有终浓度50mg/mL卡那霉素的2L的LB培养基中。37℃，220rpm培养至OD600＝0.6-0.8后冰浴迅速降温，之后向培养体系中加入IPTG至终浓度1mM，15℃，220rpm诱导表达过夜。次日6000rpm，4℃离心15min收集菌体，按照菌体：裂解缓冲液＝1：10(m/v)比例重悬菌体，之后加入混合蛋白酶抑制剂和终浓度2％的Triton X-100，高压破碎至菌液变清。
室温，搅拌增溶1～2h，14000rpm，4℃离心30min，收集上清液。上清用0.45μm滤膜过滤后用阴离子交换柱纯化，离子柱预先用Buffer B平衡，上清使用5mL/min流速过柱，收集穿透液。之后用Buffer B洗脱杂蛋白，最后用Buffer C:0-1M盐浓度线性梯度洗脱，收集洗脱组分。收集的穿透液样品加入终浓度40％的硫酸铵，冰浴沉淀2h，之后14000rpm，4℃离心30min，收集漂浮的沉淀物。漂浮的沉淀物加入一定体积含有0.5％C8E4(四甘醇单辛基醚)去污剂的分子筛缓冲液重新溶解，4℃孵育过夜。
次日将过夜孵育的样品47000g，4℃离心30min，收集上清液。上清液进行最后一步的分子排阻层析纯化，收集目的组分即为Mmup单体变体。分子排阻层析结果见图1。
表1蛋白纯化用试剂的配方
Figure PCTCN2019109332-appb-000018
Figure PCTCN2019109332-appb-000019
实施例2孔蛋白的制备
吸取20μL包含Mmup单体变体质粒的BL21(DE3)甘油菌接于20mL(1：1000)含有终浓度50mg/mL卡那霉素的新鲜LB培养基，37℃，200rpm摇菌过夜活化；次日按1％接种量扩大培养至含有终浓度50mg/mL卡那霉素的2L的LB培养基中。37℃，220rpm培养至OD600＝0.6-0.8后冰浴迅速降温，之后向培养体系中加入IPTG至终浓度1mM，15℃，220rpm诱导表达过夜。次日6000rpm，4℃离心15min收集菌体，按照菌体：裂解缓冲液＝1：10(m/v)比例重悬菌体，之后加入混合蛋白酶抑制剂和终浓度2％的Triton X-100，高压破碎至菌液变清。
室温，搅拌增溶1～2h，14000rpm，4℃离心30min，收集上清液。上清用0.45μm滤膜过滤后用阴离子交换柱纯化，离子柱预先用Buffer B平衡，上清使用5mL/min流速过柱，收集穿透液。之后用Buffer B洗脱杂蛋白，最后用Buffer C:0-1M盐浓度线性梯度洗脱，收集洗脱组分。收集的穿透液样品加入终浓度40％的硫酸铵，冰浴沉淀2h，之后14000rpm，4℃离心30min，收集漂浮的沉淀物。漂浮的沉淀物加入一定体积含有0.5％C8E4去污剂的分子筛缓冲液重新溶解，4℃孵育过夜。
次日将过夜孵育的样品47000g，4℃离心30min，收集上清液。上清液进行最后一步的分子排阻层析纯化，收集目的组分即为Mmup单体变体。
实施例3孔蛋白的测序应用
在缓冲液(400mM KCl，10mM HEPES pH 8.0，50mM MgCl 2)中，将单个纳米孔蛋白插入磷脂双分子层中，并从单个纳米孔蛋白获得电测量值。
具体步骤如下：
在将氨基酸序列为D91K/A99K突变的SEQ ID NO：1的单个孔蛋白(Mmup单体变体孔蛋白，棍棒模型如图2所示)插入所述磷脂双分子层之后，使缓冲液(400mM KCl，10mM HEPES pH 8.0，50mM MgCl 2)流经该系统，以除去任何过量的Mmup单体变体孔蛋白。将DNA构建体X2&cX2-80-15或S1T&S1MC(1～2nM终浓度)加入所述Mmup单体变体孔蛋白实验系统中，混匀后，使缓冲液(400mM KCl，10mM HEPES pH 8.0，50mM MgCl 2)流经该系统，以除去任何过量的DNA构建体X2&cX2-80-15或S1T&S1MC。然后将解旋酶(EF8813-1，15nM终浓度)、燃料(ATP 3mM终浓度)预混物加入所述单个Mmup单体变体的孔蛋白实验系统中，并在+180mV电压下监测Mmup单体变体的孔蛋白的测序情况。
对照组与上述步骤相同，仅将Mmup单体变体的孔蛋白替换为野生型Mmup单体的纳米孔(棍棒模型及立体结构如图3、4所示)。其中，野生型Mmup单体的纳米孔的棍棒模型显示了孔道收缩区和环形区的氨基酸分布特征，尤其是收缩区和环形区的关键氨基酸残基，主要有91位的天冬氨酸，92位的天冬酰胺，93位的缬氨酸和94位的丝氨酸，由D91和N92形成的收缩区直径分别为
Figure PCTCN2019109332-appb-000020
和
Figure PCTCN2019109332-appb-000021
与野生型Mmup单体的纳米孔对比，包含Mmup单体变体的纳米孔蛋白(棍棒模型如图2所示)棍棒模型显示了突变后通道孔道收缩区和环形区的氨基酸分布特征，尤其是收缩区和环形区的关键氨基酸残基分布，指向孔道中心的氨基酸残基91位的赖氨酸和92位的天冬酰胺，孔道直径约为
Figure PCTCN2019109332-appb-000022
99位的赖氨酸参与通道复合物的正确组装。
其中，X2&cX2-80-15(具体结构如图5)具体序列如下：
X2：
Figure PCTCN2019109332-appb-000023
Figure PCTCN2019109332-appb-000024
Figure PCTCN2019109332-appb-000025
cX2-80-15：
Figure PCTCN2019109332-appb-000026
Figure PCTCN2019109332-appb-000027
其中，S1T&S1MC(具体结构如图6)具体序列如下：
S1T：
Figure PCTCN2019109332-appb-000028
X(区段h)
Figure PCTCN2019109332-appb-000029
Figure PCTCN2019109332-appb-000030
S1MC：
Figure PCTCN2019109332-appb-000031
Figure PCTCN2019109332-appb-000032
测试结果见图7-13，图7示出了包含野生型Mmup单体的纳米孔通道在±180mV电压的单通道行为特征。测试体系中包含野生型Mmup单体的纳米孔通道在+180mV条件下全开放电流约为380pA，门控明显，有强烈的残留核酸过孔信号；-180mV条件下全开放电流接近-350pA，门控更强，对膜的扰动很剧烈。显然，包含野生型Mmup单体的纳米孔通道无法满足测序纳米孔蛋白的要求，无法完成测序目的。
图8示出了包含Mmup单体变体(D91K/A99K)的纳米孔蛋白在在+180mV，0mV和-180mV电压下开孔电流及其门控特征，其正向门控消失，孔蛋白可以在外加电压下保持稳定的开放状态。在180mv，400mm KCl盐浓度下可以产生160pA左右的开孔电流。图9示出了包含Mmup单体变体(D91K/A99K)的纳米孔蛋白在+180mV和0mV电压下核酸通过纳米孔的信号。
当解旋酶(EF8813-1)控制DNA构建体X2穿过包含Mmup单体变体(D91K/A99K)的纳 米孔蛋白移位时的部分示例电流轨迹，右图为左图(15:04:25.5至15:04:37.5)中虚线部分显示的时间为15:04:34.1至15:04:37.1的电流轨迹的放大结果(参见图10)。当解旋酶(EF8813-1)控制DNA构建体X2穿过包含Mmup单体变体(D91K/A99K)的纳米孔蛋白移位时的电流轨迹的全过程电流变化结果参见图11。
当解旋酶(EF8813-1)控制DNA构建体S1T穿过包含Mmup单体变体(D91K/A99K)的纳米孔蛋白移位时的部分示例电流轨迹，右图为左图虚线部分显示的电流轨迹的放大结果，其中箭头指示的电流最大值显示的是dspacer的特征峰(参见图12)。当解旋酶(EF8813-1)控制DNA构建体S1T穿过包含Mmup单体变体(D91K/A99K)的纳米孔蛋白移位时的电流轨迹的全过程电流变化结果参见图13。
以上详细描述了本发明的优选实施方式，但是，本发明并不限于上述实施方式中的具体细节，在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，这些简单变型均属于本发明的保护范围。
另外需要说明的是，在上述具体实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本发明对各种可能的组合方式不再另行说明。
Figure PCTCN2019109332-appb-000033
Figure PCTCN2019109332-appb-000034
Figure PCTCN2019109332-appb-000035
Figure PCTCN2019109332-appb-000036
Figure PCTCN2019109332-appb-000037
Figure PCTCN2019109332-appb-000038
Figure PCTCN2019109332-appb-000039
Figure PCTCN2019109332-appb-000040
Figure PCTCN2019109332-appb-000041
Figure PCTCN2019109332-appb-000042
Figure PCTCN2019109332-appb-000043
Figure PCTCN2019109332-appb-000044
Figure PCTCN2019109332-appb-000045
Figure PCTCN2019109332-appb-000046
Figure PCTCN2019109332-appb-000047
Figure PCTCN2019109332-appb-000048
Figure PCTCN2019109332-appb-000049
Figure PCTCN2019109332-appb-000050
Figure PCTCN2019109332-appb-000051
Figure PCTCN2019109332-appb-000052
Figure PCTCN2019109332-appb-000053
Figure PCTCN2019109332-appb-000054
Figure PCTCN2019109332-appb-000055

Claims (49)

  1. 一种Mmup单体变体，其特征在于，所述的Mmup单体变体包含SEQ ID NO：1第91-99位任意一个或多个氨基酸突变的氨基酸序列。
  2. 根据权利要求1所述的Mmup单体变体，其特征在于，所述的Mmup单体变体包含第91位天冬氨酸(D)和/或第99位丙氨酸(A)的突变。
  3. 根据权利要求1或2所述的Mmup单体变体，其特征在于，所述的Mmup单体变体包含至少一种如下突变：
    D91突变为：脯氨酸(P)、色氨酸(W)、精氨酸(R)、谷氨酰胺(Q)、赖氨酸(K)、苯丙氨酸(F)、丝氨酸(S)、天冬酰胺(N)、半胱氨酸(C)、异亮氨酸(I)、亮氨酸(L)或缬氨酸(V)，或者，非天然氨基酸；或者，
    A99突变为：脯氨酸(P)、苯丙氨酸(F)、异亮氨酸(I)、亮氨酸(L)、缬氨酸(V)、赖氨酸(K)或精氨酸(R)，或者，非天然氨基酸。
  4. 根据权利要求1-3任一所述的Mmup单体变体，其特征在于，所述的Mmup单体变体包含D91K和/或A99K的突变。
  5. 根据权利要求1-4任一所述的Mmup单体变体，其特征在于，所述的Mmup单体变体包含D91K和A99K的突变。
  6. 根据权利要求1-5任一所述的Mmup单体变体，其特征在于，所述的Mmup单体变体还包含SEQ ID NO：1第80-90位和/或100-120位任意一个或多个氨基酸突变的氨基酸序列。
  7. 根据权利要求1-6任一所述的Mmup单体变体，其特征在于，所述的Mmup单体变体还包含SEQ ID NO：1第1-79位和/或121-186位任意一个或多个氨基酸突变的氨基酸序列。
  8. 根据权利要求1-7任一所述的Mmup单体变体，其特征在于，所述的Mmup单体变体还包含第89位亮氨酸(L)的突变、第110位天冬酰胺(N)的突变、第120位天冬氨酸(D)的突变、第136位天冬酰胺(N)的突变或第141位丝氨酸(S)的突变中的一种或两种以上的组合。
  9. 根据权利要求1-8任一所述的Mmup单体变体，其特征在于，所述的Mmup单体变体包含至少一种如下突变：
    L89突变为：天冬酰胺(N)、丙氨酸(A)、谷氨酰胺(Q)、甘氨酸(G)、丝氨酸(S)、苏氨酸(T)、赖氨酸(K)或脯氨酸(P)，或者，非天然氨基酸；或者，
    N110突变为：脯氨酸(P)、丙氨酸(A)、异亮氨酸(I)或亮氨酸(L)，或者，非天然氨基酸；或者，
    D120突变为：精氨酸(R)、赖氨酸(K)、天冬酰胺(N)、谷氨酰胺(Q)、丙氨酸(A)、丝氨酸(S)、甘氨酸(G)或苏氨酸(T)，或者，非天然氨基酸；或者，
    N136突变为：精氨酸(R)、赖氨酸(K)、谷氨酰胺(Q)、丙氨酸(A)、丝氨酸(S)或苏氨酸(T)，或者，非天然氨基酸；或者，
    S141突变为：精氨酸(R)、赖氨酸(K)、谷氨酰胺(Q)、丙氨酸(A)、丝氨酸(S)或苏氨酸(T)，或者，非天然氨基酸。
  10. 一种包含至少一个权利要求1-9任一所述Mmup单体变体的构建体。
  11. 根据权利要求10所述的构建体，其特征在于，所述的构建体还包含野生型Mmup单体。
  12. 根据权利要求10或11所述的构建体，其特征在于，所述的构建体包含1-20个Mmup单体变体，其中，所述的Mmup单体变体相同或不同。
  13. 根据权利要求10-12任一所述的构建体，其特征在于，所述的构建体包含1-20个野生型Mmup单体。
  14. 根据权利要求10-13任一所述的构建体，其特征在于，所述的Mmup单体变体与Mmup单体变体、野生型Mmup单体与野生型Mmup单体、Mmup单体变体与野生型Mmup单体之间通过共价连接。
  15. 根据权利要求10-14任一所述的构建体，其特征在于，所述的构建体包含4-10个相同或不同的Mmup单体变体。
  16. 一种包含至少一个Mmup单体变体的孔蛋白，其特征在于，所述的Mmup单体变 体包含SEQ ID NO：1第91-99位任意一个或多个氨基酸突变的氨基酸序列，所述的突变导致当多聚核苷酸单链通过所述包含至少一个Mmup单体变体的孔蛋白时，产生由于不同种类的核苷酸的物理或化学性质的不同而造成的孔内电阻的差异。
  17. 根据权利要求16所述包含至少一个Mmup单体变体的孔蛋白，其特征在于，所述的Mmup单体变体包含第91位天冬氨酸(D)和/或第99位丙氨酸(A)的突变。
  18. 根据权利要求16或17所述包含至少一个Mmup单体变体的孔蛋白，其特征在于，所述的Mmup单体变体包含至少一种如下突变：
    D91突变为：脯氨酸(P)、色氨酸(W)、精氨酸(R)、谷氨酰胺(Q)、赖氨酸(K)、苯丙氨酸(F)、丝氨酸(S)、天冬酰胺(N)、半胱氨酸(C)、异亮氨酸(I)、亮氨酸(L)或缬氨酸(V)，或者，非天然氨基酸；或者，
    A99突变为：脯氨酸(P)、苯丙氨酸(F)、异亮氨酸(I)、亮氨酸(L)、缬氨酸(V)、赖氨酸(K)或精氨酸(R)，或者，非天然氨基酸。
  19. 根据权利要求16-18任一所述包含至少一个Mmup单体变体的孔蛋白，其特征在于，所述的Mmup单体变体包含D91K和/或A99K的突变。
  20. 根据权利要求16-19任一所述包含至少一个Mmup单体变体的孔蛋白，其特征在于，所述的Mmup单体变体包含D91K和A99K的突变。
  21. 根据权利要求16-20任一所述包含至少一个Mmup单体变体的孔蛋白，其特征在于，所述的Mmup单体变体还包含SEQ ID NO：1第80-90位和/或100-120位任意一个或多个氨基酸突变的氨基酸序列。
  22. 根据权利要求16-21任一所述包含至少一个Mmup单体变体的孔蛋白，其特征在于，所述的Mmup单体变体还包含SEQ ID NO：1第1-79位和/或121-186位任意一个或多个氨基酸突变的氨基酸序列。
  23. 根据权利要求16-22任一所述包含至少一个Mmup单体变体的孔蛋白，其特征在于，所述的Mmup单体变体还包含第89位亮氨酸(L)的突变、第110位天冬酰胺(N)的突变、第120位天冬氨酸(D)的突变、第136位天冬酰胺(N)的突变或第141位丝氨 酸(S)的突变中的一种或两种以上的组合。
  24. 根据权利要求16-23任一所述包含至少一个Mmup单体变体的孔蛋白，其特征在于，所述的Mmup单体变体包含至少一种如下突变：
    L89突变为：天冬酰胺(N)、丙氨酸(A)、谷氨酰胺(Q)、甘氨酸(G)、丝氨酸(S)、苏氨酸(T)、赖氨酸(K)或脯氨酸(P)，或者，非天然氨基酸；或者，
    N110突变为：脯氨酸(P)、丙氨酸(A)、异亮氨酸(I)或亮氨酸(L)，或者，非天然氨基酸；或者，
    D120突变为：精氨酸(R)、赖氨酸(K)、天冬酰胺(N)、谷氨酰胺(Q)、丙氨酸(A)、丝氨酸(S)、甘氨酸(G)或苏氨酸(T)，或者，非天然氨基酸；或者，
    N136突变为：精氨酸(R)、赖氨酸(K)、谷氨酰胺(Q)、丙氨酸(A)、丝氨酸(S)或苏氨酸(T)，或者，非天然氨基酸；或者，
    S141突变为：精氨酸(R)、赖氨酸(K)、谷氨酰胺(Q)、丙氨酸(A)、丝氨酸(S)或苏氨酸(T)，或者，非天然氨基酸。
  25. 根据权利要求16-24任一所述包含至少一个Mmup单体变体的孔蛋白，其特征在于，所述包含至少一个Mmup单体变体的孔蛋白包含至少1-20个Mmup单体变体，其中，所述的Mmup单体变体相同或不同。
  26. 根据权利要求16-25任一所述包含至少一个Mmup单体变体的孔蛋白，其特征在于，所述包含至少一个Mmup单体变体的孔蛋白包含野生型Mmup单体。
  27. 根据权利要求16-26任一所述包含至少一个Mmup单体变体的孔蛋白，其特征在于，所述包含至少一个Mmup单体变体的孔蛋白包含1-20个野生型Mmup单体。
  28. 根据权利要求16-27任一所述包含至少一个Mmup单体变体的孔蛋白，其特征在于，所述包含至少一个Mmup单体变体的孔蛋白包含4-10个相同或不同的Mmup单体变体。
  29. 根据权利要求16-28任一所述包含至少一个Mmup单体变体的孔蛋白，其特征在于，所述的Mmup单体变体与Mmup单体变体、野生型Mmup单体与野生型Mmup单体、 Mmup单体变体与野生型Mmup单体之间通过共价连接。
  30. 根据权利要求16-29任一所述包含至少一个Mmup单体变体的孔蛋白，其特征在于，所述包含至少一个Mmup单体变体的孔蛋白包含收缩区和环形区。
  31. 根据权利要求30所述包含至少一个Mmup单体变体的孔蛋白，其特征在于，所述包含至少一个Mmup单体变体的孔蛋白的收缩区孔道直径小于野生型Mmup单体组成的孔蛋白的收缩区孔道直径。
  32. 根据权利要求30或31所述包含至少一个Mmup单体变体的孔蛋白，其特征在于，所述包含至少一个Mmup单体变体的孔蛋白的收缩区孔道直径小于
    Figure PCTCN2019109332-appb-100001
    优选的，所述包含至少一个Mmup单体变体的孔蛋白的收缩区孔道直径小于
    Figure PCTCN2019109332-appb-100002
  33. 根据权利要求30-32任一所述包含至少一个Mmup单体变体的孔蛋白，其特征在于，所述包含至少一个Mmup单体变体的孔蛋白还包括帽状体形成区和/或桶状体形成区域。
  34. 一种编码权利要求1-9任一所述的Mmup单体变体、权利要求10-15任一所述构建体或者权利要求16-33任一所述包含至少一个Mmup单体变体的孔蛋白的核苷酸序列。
  35. 一种包含权利要求34所述核苷酸序列的载体。
  36. 根据权利要求35所述的载体，其特征在于，所述的载体包含与编码权利要求1-9任一所述的Mmup单体变体、权利要求10-15任一所述构建体或者权利要求16-33任一所述包含至少一个Mmup单体变体的孔蛋白的核苷酸序列可操作性连接的启动子。
  37. 根据权利要求36所述的载体，其特征在于，所述的启动子为诱导型启动子或组成型启动子，其中，所述的诱导型启动子为乙酰胺诱导型启动子。
  38. 根据权利要求35-37任一所述的载体，其特征在于，所述编码包含至少一个Mmup单体变体的孔蛋白的核苷酸序列包含至少一条编码Mmup单体变体的核苷酸序列。
  39. 根据权利要求38所述的载体，其特征在于，所述编码包含至少一个Mmup单体变体的孔蛋白的核苷酸序列还包含至少一条编码野生型Mmup单体的核苷酸序列。
  40. 根据权利要求38或39所述的载体，其特征在于，所述编码Mmup单体变体的核 苷酸序列与编码Mmup单体变体的核苷酸序列，编码Mmup单体变体的核苷酸序列与编码野生型Mmup单体的核苷酸序列，或者，编码野生型Mmup单体的核苷酸序列与编码野生型Mmup单体的核苷酸序列之间通过编码氨基酸连接体序列连接。
  41. 一种表达权利要求1-9任一所述的Mmup单体变体、权利要求10-15任一所述构建体或权利要求16-33任一所述包含至少一个Mmup单体变体的孔蛋白的突变细菌，其特征在于，所述的细菌包含：
    (a)野生型Mmup单体的缺失；和(b)权利要求35-40任一所述的载体。
  42. 根据权利要求41所述的突变细菌，其特征在于，所述的细菌为产粘液分枝杆菌。
  43. 一种产生Mmup孔蛋白的方法，其特征在于，所述的方法包括用包含权利要求35-40任一所述的载体转化权利要求41-42任一所述的细菌，诱导细菌表达Mmup孔蛋白。
  44. 一种表征目标多核苷酸的方法，其特征在于，包括：
    (a)将目标多核苷酸与权利要求16-33任一所述包含至少一个Mmup单体变体的孔蛋白接触，使得所述目标多核苷酸序列穿过包含至少一个Mmup单体变体的孔蛋白；和
    (b)获取目标多核苷酸穿过包含至少一个Mmup单体变体的孔蛋白时核苷酸与包含至少一个Mmup单体变体的孔蛋白相互作用的一个或多个特征，以表征所述目标多核苷酸。
  45. 根据权利要求44所述的方法，其特征在于，所述步骤(a)中还包括目标多核苷酸与核酸结合蛋白、解旋酶、核酸外切酶、端粒酶、拓扑异构酶、反转录酶、转位酶和/或聚合酶中的一种或两种以上的组合接触的步骤，使得所述目标多核苷酸序列穿过孔蛋白的转位速度小于核酸结合蛋白、解旋酶、核酸外切酶、端粒酶、拓扑异构酶、反转录酶、转位酶和/或聚合酶不存在时的转位速度。
  46. 根据权利要求44或45所述的方法，其特征在于，所述的方法还包括横跨目标多核苷酸接触的孔蛋白施加电势差的步骤。
  47. 一种权利要求16-33任一所述包含至少一个Mmup单体变体的孔蛋白、权利要求1-9任一所述的Mmup单体变体、权利要求10-15任一所述构建体、权利要求34所述的核苷酸序列、权利要求35-40任一所述的载体或者权利要求41-42任一所述的突变细菌在表 征目标多核苷酸中的应用。
  48. 一种表征目标多核苷酸的试剂盒，其特征在于，所述的试剂盒包括权利要求16-33任一所述包含至少一个Mmup单体变体的孔蛋白、权利要求1-9任一所述的Mmup单体变体、权利要求10-15任一所述构建体、权利要求34所述的核苷酸序列、权利要求35-40任一所述的载体或者权利要求41-42任一所述的突变细菌。
  49. 一种表征目标多核苷酸的装置，其特征在于，所述的装置包括权利要求16-33任一所述包含至少一个Mmup单体变体的孔蛋白、权利要求1-9任一所述的Mmup单体变体、权利要求10-15任一所述构建体、权利要求34所述的核苷酸序列、权利要求35-40任一所述的载体或者权利要求41-42任一所述的突变细菌。
PCT/CN2019/109332 2019-09-29 2019-09-30 一种Mmup单体变体及其应用 WO2021056598A1 (zh)

Priority Applications (4)

Application Number Priority Date Filing Date Title
IL291787A IL291787A (en) 2019-09-29 2019-09-30 mmup monomer variant and its application
CN201980002430.8A CN111164096B (zh) 2019-09-29 2019-09-30 一种Mmup单体变体及其应用
EP19947045.1A EP4036107A4 (en) 2019-09-29 2019-09-30 MMUP MONOMER VARIANT AND ITS APPLICATION
ZA2022/03618A ZA202203618B (en) 2019-09-29 2022-03-29 Mmup monomer variant and application thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201910936950 2019-09-29
CN201910936950.8 2019-09-29

Publications (1)

Publication Number Publication Date
WO2021056598A1 true WO2021056598A1 (zh) 2021-04-01

Family

ID=75165389

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/109332 WO2021056598A1 (zh) 2019-09-29 2019-09-30 一种Mmup单体变体及其应用

Country Status (1)

Country Link
WO (1) WO2021056598A1 (zh)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102216783A (zh) 2008-09-22 2011-10-12 华盛顿大学 Msp纳米微孔和相关方法
US20150065354A1 (en) 2011-12-29 2015-03-05 Oxford Nanopore Technologies Limited Method for characterising a polynucelotide by using a xpd helicase
CN106103741A (zh) 2014-01-22 2016-11-09 牛津纳米孔技术公司 将一个或多个多核苷酸结合蛋白连接到靶多核苷酸的方法
US20170268055A1 (en) 2013-11-26 2017-09-21 Illumina, Inc. Compositions and methods for polynucleotide sequencing
CN107207571A (zh) * 2014-04-16 2017-09-26 Uab研究基金会 Msp纳米孔及其用途

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102216783A (zh) 2008-09-22 2011-10-12 华盛顿大学 Msp纳米微孔和相关方法
US20150065354A1 (en) 2011-12-29 2015-03-05 Oxford Nanopore Technologies Limited Method for characterising a polynucelotide by using a xpd helicase
US20170268055A1 (en) 2013-11-26 2017-09-21 Illumina, Inc. Compositions and methods for polynucleotide sequencing
CN106103741A (zh) 2014-01-22 2016-11-09 牛津纳米孔技术公司 将一个或多个多核苷酸结合蛋白连接到靶多核苷酸的方法
CN107207571A (zh) * 2014-04-16 2017-09-26 Uab研究基金会 Msp纳米孔及其用途

Similar Documents

Publication Publication Date Title
CN111164096B (zh) 一种Mmup单体变体及其应用
US12024541B2 (en) Transmembrane pore consisting of two CsgG pores
US10976300B2 (en) Modified nanopores, compositions comprising the same, and uses thereof
CN111164097B (zh) 一种Mnep单体变体及其应用
JP7499761B2 (ja) 細孔
CN106459159B (zh) 突变孔
US7960312B2 (en) Method and agent for immobilizing protein via protein bound to silicon oxide-containing substance
KR20200030070A (ko) 신규한 단백질 포어
CN104136631A (zh) 使用xpd解旋酶表征多核苷酸的方法
CN114957412B (zh) 一种新型孔蛋白单体及其应用
WO2022126304A1 (zh) 一种经修饰的解旋酶及其应用
WO2021056598A1 (zh) 一种Mmup单体变体及其应用
WO2021056599A1 (zh) 一种Mnep单体变体及其应用
US11845779B2 (en) Mutant aerolysin and uses thereof
WO2023060419A1 (zh) 孔蛋白单体的突变体、蛋白孔及其应用
WO2023050031A1 (zh) 孔蛋白单体的突变体、蛋白孔及其应用
WO2023060421A1 (zh) 孔蛋白单体的突变体、蛋白孔及其应用
WO2023060420A1 (zh) 孔蛋白单体的突变体、蛋白孔及其应用
WO2023019471A1 (zh) 孔蛋白单体的突变体、蛋白孔及其应用
WO2023019470A1 (zh) 孔蛋白单体的突变体、蛋白孔及其应用
WO2023060418A1 (zh) 孔蛋白单体的突变体、蛋白孔及其应用
Gupta Toward an understanding of the role of the L1 stalk in translocation by the Escherichia coli ribosome
WO2011039390A2 (es) Mutantes de la adn polimerasa mu

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19947045

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 2019947045

Country of ref document: EP