CN117384260A

CN117384260A - Novel nanoporous protein mutant and application thereof

Info

Publication number: CN117384260A
Application number: CN202310040893.1A
Authority: CN
Inventors: 张宁
Original assignee: Beijing Puyi Biotechnology Co ltd
Current assignee: Beijing Puyi Biotechnology Co ltd
Priority date: 2023-01-12
Filing date: 2023-01-12
Publication date: 2024-01-12
Anticipated expiration: 2043-01-12
Also published as: CN117384260B

Abstract

The invention discloses a novel nanopore protein mutant and application thereof, mainly comprising expression purification, structural analysis and mutant construction of shortening of a contraction zone of the protein, current property of a phospholipid membrane of the mutant protein and determination of nucleic acid substrate detection capability. The PslD mutant nanoporous protein truncated in the contraction area provided by the invention is a novel nanoporous protein with stability and good current property.

Description

Novel nanoporous protein mutant and application thereof

Technical Field

The invention relates to a method for representing target polynucleotide, in particular to PslD mutant membrane protein and application thereof, belonging to the fields of genetic engineering and genetic engineering.

Background

Nanopore sequencing is an emerging sequencing technology, has the characteristics of real-time, portability, potential low cost, ultra-long reading length, capability of detecting epigenetic modification information on nucleic acid and the like, and as a third generation sequencing technology, shows great superiority and becomes the future development direction of the nucleic acid sequencing technology. Nanopore sequencing is widely used in the fields of rapid pathogen detection, food safety monitoring, human genome sequencing, antibiotic resistance detection and the like. According to the difference of the nanopores, nanopore sequencing is classified into solid state nanopore sequencing and biological nanopore sequencing. The biological nanopore sequencing adopts natural pore canal membrane protein as a nanopore, and the natural pore canal membrane protein has a specific pore diameter structure, biological activity and capability of being inserted into a lipid bilayer membrane, has the advantage of being capable of being flexibly modified, and has been greatly progressed in recent years. Biological nanopores are miniature small pores that essentially form channels in a membrane through which ions pass under the application of an applied electric field, producing a constant current. The charged nucleic acid passes through the biological nano-hole under the action of electric field force, so that current is blocked. The inhibition effect of the current generation of different base pairs is different, and the base sequence information on the nucleic acid strand can be obtained by analyzing the current signal.

Compared with second generation sequencing, the current nanopore sequencing accuracy is low, and the property of the nanopore protein is a key factor for determining the sequencing accuracy, so that the screening and optimization of the nanopore protein are difficulties that need to be overcome in the process of developing a nanopore sequencer. The size of the bases constituting the nucleic acid strand is in the order of 1nm, and only the nanopores having comparable pore diameters can realize effective signal detection when the nucleic acid strand passes through the pores. In addition, the thickness of the narrowest part (constriction region) of the nanopore protein channel is also a key factor affecting the sequencing accuracy, and the distance between adjacent bases on a nucleic acid strand is aboutIf the constriction is too thick, the current is hindered from multiple bases, and the difficulty of signal decoding increases significantly, thereby affecting sequencing accuracy. At present, the porin suitable for nanopore sequencing is rare, and the conventional CsgG porin has the defect of lower sequencing accuracy than that of second generation sequencing, so that a novel nanopore protein needs to be discovered and optimized, and the nanopore sequencing accuracy is improved.

Disclosure of Invention

Aiming at the problem that porin suitable for nanopore sequencing is rare at present, the invention discovers a novel nanopore protein PslD, analyzes the atomic level three-dimensional structure of the novel nanopore protein PslD, enriches the types of the nanopore proteins, and provides more choices for nanopore sequencing. The PslD protein is a unique nanopore protein different from MspA and CsgG, and has a bottle-shaped structure as a whole. The bottleneck is a transmembrane region formed by a creeping alpha spiral and a positive parallel beta barrel, and the shrinkage region is positioned at the bottom of the bottle. The wild type PslD protein cannot be directly used for sequencing, and the mutant protein after shortening modification of the contraction region has sequencing capability. The PslD protein is optimized through further transformation, and the third generation nanopore sequencing accuracy is improved.

In a first aspect, the invention provides a PslD mutant nanoporous protein, which is obtained by truncating or truncating the contraction region of the wild type PslD shown in SEQ ID NO. 1.

In some embodiments, the truncation is a truncation of 1-10 amino acids from M65 to the C-terminus, preferably 2 (M65-P66), 4 (M65-L68), 6 (M65-a 70) or 8 (M65-N72) amino acids, or a substitution from M65 to the C-terminus with a short amino acid sequence, e.g. a substitution of 8, 9, 10 or 11 amino acids with 3 amino acids, preferably a substitution of 11 amino acids with GSG (M65-T75).

In some embodiments, the PslD mutant nanoporous protein has an amino acid sequence as shown in SEQ ID NOS.2-6.

Preferably, the amino acid sequence of the PslD mutant nano-pore protein is shown as SEQ ID NO. 5.

In a second aspect, the present invention provides a nucleic acid sequence encoding the above PslD mutant nanopore protein.

In some embodiments, the nucleic acid sequence is selected from the group consisting of:

1) SEQ ID NO. 8-12;

2) A nucleic acid sequence which has at least 85% homology with the nucleic acid sequence shown in SEQ ID NO. 8-12 and which encodes a protein of the amino acid sequence shown in SEQ ID NO. 2-6;

3) A nucleic acid sequence complementary to 1) or 2).

In some embodiments, the homology is between 85% and 99%.

In some embodiments, the homology is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%.

In a third aspect, the present invention provides a recombinant vector comprising the nucleic acid sequence described above.

In a fourth aspect, the present invention provides a genetically engineered cell or microorganism comprising the recombinant vector described above, or having integrated in its genome the nucleic acid sequence described above; preferably, the microorganism is E.coli.

In a fifth aspect, the invention provides a product comprising a mutant of the nanoporous protein PslD described above.

In some embodiments, the product is a composition, complex, kit.

In a sixth aspect, the present invention provides a method for preparing the above-mentioned nanoporous protein PslD mutant, comprising the steps of:

1) Constructing a nano-pore protein PslD mutant vector;

2) And expressing and purifying the nano-pore protein PslD mutant.

In a seventh aspect, the present invention provides a membrane layer in which the above-described nanoporous protein PslD mutant is embedded.

In some embodiments, the membrane layer is a phospholipid membrane layer.

In some embodiments, the membrane layer is a phospholipid monolayer membrane layer.

In an eighth aspect, the invention provides a detection system comprising a nanoporous protein PslD mutant as described above or a membrane layer as described above.

In a ninth aspect, the present invention provides a device comprising a nanoporous protein PslD mutant as described above, a membrane layer as described above or a detection system as described above.

In a tenth aspect, the present invention provides the use of a nanoporous protein PslD mutant as described above, a membrane layer as described above or a detection system as described above for preparing a device for sequencing and/or detecting a sample.

In some embodiments, the sample is one or more of a nucleotide, a nucleic acid, an amino acid, an oligopeptide, a polypeptide, a protein.

In an eleventh aspect, the present invention provides the use of a nanoporous protein PslD mutant as described above, a membrane layer as described above, a detection system as described above or a device as described above for sequencing and/or detecting a sample.

Advantageous effects

The inventor discovers a novel nano-porous protein PslD and analyzes the atomic level three-dimensional structure(FIG. 3). The PslD nano-pore assembled by the octamers is in a bottle-shaped structure and consists of three structural domains R1, R2 and R3 (shown in figure 4). R1 and R2 are located in the periplasm space, R3 is a unique transmembrane region domain and consists of eight creeping alpha helices and a forward parallel beta barrel. The total length of the nano holes is->Bottom R1 domain width->Beta barrel inner diameter is about>(FIG. 4), the diameter of the pore canal surrounded by alanine (Ala) at position 74 in the existing amino acid residue in the bottom contraction region is +.>(FIG. 5) is the narrowest point of the entire nanochannel that can be seen at present.

The inventors found that the wild-type PslD protein constriction was too small in diameter to block the pore canal and thus could not be embedded (FIG. 7). Mutation experiments show that mutant proteins with properly truncated contractile regions can be expressed normally and have stable properties (figure 6). The PslD mutant deleted by M65-N72 can be well embedded into a membrane, the pore current is about 0.25nA, the spontaneous plugging condition is few, the current property is relatively stable, the capability of sequencing biological macromolecules such as nucleic acid substrates is provided, and the subsequent optimization can be continued to further reduce the current noise (figure 8 e).

Drawings

FIG. 1 is a diagram of a SDS-PAGE gel of a wild-type PslD protein;

FIG. 2 is a graph showing the results of a wild-type PslD protein molecular sieve;

FIG. 3 is a graph of the density of wild type PslD protein by electron microscopy;

FIG. 4 is a schematic diagram of the structure of a wild-type PslD protein;

FIG. 5 is an overall conformational diagram of the wild-type PslD protein contraction region;

FIG. 6 is a diagram of mutant PslD protein expression purification;

FIG. 7 is a plot of the current signal of the wild-type PslD protein;

FIG. 8 is a plot of the current signal of the mutant PslD protein.

Detailed Description

The technical scheme of the invention will be further described in detail below with reference to specific embodiments. It is to be understood that the following examples are illustrative only and are not to be construed as limiting the scope of the invention. All techniques implemented based on the above description of the invention are intended to be included within the scope of the invention.

Unless otherwise indicated, the starting materials and reagents used in the following examples were either commercially available or may be prepared by known methods.

Example 1: construction of wild PslD nanopore protein expression vector and expression and purification of protein

1. Construction of wild type PslD nanoporous protein vector

The target gene is amplified by PCR using Pseudomonas aeruginosa (PAO 1, SEQ ID 1) genome as a template, designing a primer, and adding Strep-TagII tag at the protein C end. The amplified product was ligated into the pQlink vector by a seamless cloning reaction.

The carrier construction steps are as follows:

the target gene fragment is amplified by PCR using a forward and reverse primer by taking a Pseudomonas aeruginosa (PAO 1, SEQ ID 1) genome as a template, and the gel recovery product is connected to a linearized pQlink vector by a seamless cloning method and is transferred into DH5 alpha competent cells for positive cloning screening. 2 clones were picked for sequencing, plasmids were extracted after correct sequencing, and plasmids were stored at-20℃for use.

primer F:

TTAACTATGGGATCCATGAAACGCACCCTCCTCATG

primer R:

AGCTCAGCTAATTAAGCTTCACTTTTCGAACTGCGGGTGGCTCCAGCGATCATTGTTGACGGTGTA

The PCR system was as follows (50. Mu.L):

the PCR procedure was as follows:

the seamless cloning system (10. Mu.L) was as follows:

2 Xseamless clone buffer 5. Mu.L

3. Mu.L of fragment of interest (50 ng/. Mu.L)

Linearization vector (10 ng/. Mu.L) 2. Mu.L

After 30min of reaction at 50 ℃, 3 mu L of the mixture is transferred into DH5 alpha cells, and monoclonal sequencing is performed.

2. Expression and purification of wild type PslD nanoporous protein

After Strep column affinity chromatography, high purity wild type PslD protein oligomers and monomers were obtained (FIG. 1). Molecular sieve chromatography separated PslD protein oligomers and monomers (FIG. 2). The oligomer state (pore-forming state) PslD protein is taken and used for preparing a frozen electron microscope sample.

1) And (5) culturing bacteria in an enlarged mode and inducing expression. Wild type PslD nanopore proteins were transferred into BL21 (DE 3) competent cells. Seed was obtained at 37℃and 200rpm1mL of the seed solution is inoculated into 1L of LB culture medium for expansion culture, and OD ₆₀₀ 1, cooling to 20 ℃, and inducing with 0.2mM IPTG (isopropyl thiogalactoside) overnight;

2) Cell membranes were collected. The cells were collected at 4000rpm, resuspended in 20ml lysis buffer per L of bacteria and sonicated by a sonicator for 2min. Centrifuging at 18000rpm and 4 ℃ for 1 hour, and collecting cell membranes;

3) And (5) dissolving the film. The membrane component is resuspended by a membrane dissolving buffer (15 mL/L bacteria) by means of a glass homogenizer, the membrane is fully dissolved by magnetic stirring at a low temperature of 4 ℃ for 1h, and the membrane protein is fully extracted from the cell membrane by using a detergent. Centrifuging at 18000rpm and 4 ℃ for 1 hour, and collecting a supernatant protein component;

4) Strep column affinity chromatography. The supernatant was incubated with Strep beads at 4℃for 30min, the supernatant and beads were introduced into the column, gravity flow was passed through twice, and Wash buffer at 10 column volumes removed non-specifically bound heteroproteins. Finally, eluting the target protein by using an absorption buffer with the volume of 5 times of the column volume;

5) Molecular sieve chromatography. The Superose6 TM10/300GL gel filtration chromatographic column is equilibrated with molecular sieve buffer, the protein is sucked by a syringe and is loaded on a loading ring, the oligomer protein is collected and concentrated, and the proportion and purity of each component are detected by SDS-PAGE gel.

Splitting buffer:20mM Tris-HCl pH 8.0, 150mM NaCl

Dissolving membrane buffer:20mM Tris-HCl pH 8.0, 150mM NaCl,1% DDM

Wash buffer：20mM Tris-HCl pH 8.0，150mM NaCl，0.2％DDM

An execution buffer: desulphated biotin with 20mM Tris-HCl pH 8.0, 150mM NaCl,0.05%DDM,2.5mM

Molecular sieve buffer:20mM Tris-HCl pH 8.0, 150mM NaCl,0.03%DDM

Example 2: atomic level structural analysis of wild type PslD nanoporous proteins

After obtaining wild PslD nanoporous protein with higher purity and uniformity (example 1), the inventors obtained the wild PslD nanoporous protein atomic level structure after constructing atomic model by observation screening, data collection and processing of a frozen electron microscope sample

1) And (5) preparing a frozen electron microscope sample.

Preparing liquid ethane, setting parameters such as temperature and humidity of an EMGP instrument, carrying out hydrophilic treatment on a net, sucking 3ul protein samples, adding the protein samples to the net, and preparing a frozen electron microscope sample by using the EMGP instrument.

2) And (5) observing and screening the frozen electron microscope samples.

And loading the prepared frozen electron microscope sample to Talos F200C for observing the frozen electron microscope sample, and screening the sample with good contrast, good dispersion and less pollution for data collection.

3) And (5) collecting and processing the data of the frozen electron microscope sample.

After screening to obtain frozen samples suitable for data collection, the frozen data collection is completed by utilizing an Arctic electron microscope, the position to be photographed is stored after the proper sample is selected, and the state of the electron microscope is regulated, wherein the method mainly comprises the steps of closing an axis of the electron microscope, deducting the back and the data collection basic parameters (the under-focus quantity is-1.5 mu m to-2.5 mu m, and the electron meteringPicture frame number 32 frames and pixl size->) Is set, and data collection is started. Performing data processing by RELION software, and selecting granule, two-dimensional classifying and three-dimensional classifying to obtain +.> Electron density map.

4) And (6) constructing an atomic model.

Firstly, an initial model is obtained through Phenix software, then the model is optimized through Coot, and finally, real-space definition in the Phenix software is utilized for refinement.

Example 3: detection of current properties of wild-type and mutant PslD nanopore protein pore channels

In this example, the inventors tried to examine the channel current properties of the wild type PslD nanopore protein on an artificial membrane, and found that it could not be embedded in the artificial membrane due to the constriction blocking the nanopore channel. Thus, truncations of different lengths are made for their constriction. Extending from M65 to the C-terminus, truncating 2 amino acids (M65-P66), 4 amino acids (M65-L68), 6 amino acids (M65-A70), 8 amino acids (M65-N72) and replacing 11 amino acids (M65-T75) with three amino acids (GSG), respectively. Expression purification shows that the protein expression quantity, stability and oligomerization state of the five mutants are basically consistent with those of the wild type. When not heated, the protein has two states of monomer and oligomer, and after heating, the oligomer is dissociated, and only the monomer is left. The truncation mutations did not result in a significant change in the biochemical properties of the protein.

Construction of PslD mutant vector and purification of protein expression

The wild PslD protein vector is used as a template, a primer is designed, a mutation vector is constructed in a PCR mode, a PCR product is digested by DPN1, and the PCR product is transferred into DH5 alpha competent cells for positive clone screening. Single colony sequencing was picked up and the plasmid was extracted from the kit and stored at-20℃for further use. Five truncated mutant proteins (. DELTA.M 65-P66,. DELTA.M 65-L68,. DELTA.M 65-A70,. DELTA.M 65-N72, and M65-T75 were replaced with GSG) were purified in the same manner as the expression of the wild-type PslD protein (described in example 1).

The primers were designed as follows:

ΔM65-P66

primer F：cgcgatgccggggagaccctctcggcgttcaacg

primer R：ctccccggcatcgcggac

ΔM65-L68

primer F：cgcgatgccggggagtcggcgttcaacgtcgcc

primer R：ctccccggcatcgcggac

ΔM65-A70

primer F：cgcgatgccggggagttcaacgtcgccaccatctatg

primer R：ctccccggcatcgcggac

ΔM65-N72

primer F：cgcgatgccggggaggtcgccaccatctatgaactgac

primer R：ctccccggcatcgcggac

M65-T75 is replaced by GSG

primer F：ggggagggcagcggaatctatgaactgacgctgtacacc

primer R：tccgctgccctccccggcatcgcggac

2. Cell current property detection

After obtaining wild-type and mutant PslD nanopore proteins, the inventors examined the current properties of these proteins. A phospholipid monolayer (DPhPC) was artificially formed and after a single nanopore protein was embedded in the phospholipid monolayer, the pore current change of the nanopore protein was recorded at a voltage of 150 mV.

In buffer (600 mM KCl,75mM K) ₃ [Fe(CN) ₆ ，25mM K ₄ [Fe(CN) ₆ ]·3H ₂ O,100mM Hepes,pH 8.0), electrical signal measurements are obtained from nanopores embedded in the DPhPC phospholipid bilayer. After single well insertion of the phospholipid bilayer was achieved, the phospholipid bilayer was incubated with 2mL buffer (600 mM KCl,75mM K ₃ [Fe(CN) ₆ ，25mM K ₄ [Fe(CN) ₆ ]·3H ₂ O,100mM Hepes,pH 8.0) is flowed through the system to remove residual excess nanopores. The current signals of the wild type and mutant PslD nanopore proteins on the phospholipid membrane were recorded separately, and the results were recorded as shown (fig. 7-8).

In the experiments, no embedding of wild type PslD nanopores into phospholipid membranes was found (FIG. 7), and deletion of two, four or six amino acid ΔM65-P66, ΔM65-L68 or ΔM65-A70 mutants could only cause membrane fluctuations, but not (FIGS. 8 a-c). The PslD mutant with M65-T75 replaced by GSG can be embedded into a membrane, the pore current is about 0.35nA, the current noise is relatively small, but the spontaneous blocking phenomenon exists, and the subsequent optimization is needed to be eliminated (figure 8 d). The DeltaM 65-N72 mutant can be well embedded, the pore current is about 0.25nA at 150mV, the current noise is small, the spontaneous blocking phenomenon is less, the current property is stable, the capability of sequencing biological macromolecules such as nucleic acid substrates is provided, and the subsequent optimization can be continued to further reduce the current noise (figure 8 e). Experimental results show that the PslD nanopore sequencing performance can be improved by modifying and optimizing the PslD nanopore protein contraction region.

P01

SEQ ID1 protein

MKRTLLMLAMLALAACNTPARIPAPDSDTVDSGKRALEELARLPPAMERVRVGDTLRIVR

DAGEMPTLSAFNVATIYELTLYTVLNDGSIYYPFIGRIQAAHRTPQEIANELTTKLAPIYREP

RVTVNINQAPGNTVFVGGAVRNPSAVPIPAANNMEQAILGAGGILPVGDARRVALMREDS

EGRYHAYFLDFSQLMKIGPEGRKPLAMQRGDIVFVPKSMVGDRIEGVDVYLNQLLPFAKSI

GVGVSYTVNNDR

>SEQ ID7 DNA

atgaaacgcaccctcctcatgctcgccatgctcgccctggccgcatgcaacacccccgcacggattcccgcaccggacagcgacaccgtgga

cagcggcaagcgtgccctggaagaactcgccaggctaccgccggcgatggagcgggtgcgcgtcggggacaccctgcggatcgtccgcg

atgccggggagatgccgaccctctcggcgttcaacgtcgccaccatctatgaactgacgctgtacaccgtgctcaacgacggcagcatctacta

tccgttcatcggtcgcatccaggccgcgcaccgcacgccgcaggaaatcgccaacgagctgaccaccaagctcgcgccgatctaccgcgag

ccgcgggtcacggtgaacatcaaccaggcgccgggcaatacggtgttcgtcggcggcgcggtgcgcaacccgtcggccgtgccgatcccc

gccgccaacaacatggagcaggcgatcctcggcgccggcggcatcctgccggtgggcgacgcccgccgcgtggcgctcatgcgcgagga

ctcggaaggccgctatcacgcctacttcctcgacttcagccagttgatgaagatcgggccggaaggccgcaagcccctggccatgcagcgtg

gcgacatcgtcttcgtgcccaagtcgatggtcggcgaccgtatcgagggcgtcgacgtctacctgaaccagttgctgcccttcgccaagtccatc

ggcgtcggcgtcagctacaccgtcaacaatgatcgcΔM65-P66

SEQ ID2 protein

MKRTLLMLAMLALAACNTPARIPAPDSDTVDSGKRALEELARLPPAMERVRVGDTLRIVR

DAGETLSAFNVATIYELTLYTVLNDGSIYYPFIGRIQAAHRTPQEIANELTTKLAPIYREPRV

TVNINQAPGNTVFVGGAVRNPSAVPIPAANNMEQAILGAGGILPVGDARRVALMREDSEG

RYHAYFLDFSQLMKIGPEGRKPLAMQRGDIVFVPKSMVGDRIEGVDVYLNQLLPFAKSIGV

GVSYTVNNDR

>SEQ ID8 DNA

atgccggggagaccctctcggcgttcaacgtcgccaccatctatgaactgacgctgtacaccgtgctcaacgacggcagcatctactatccgttc

atcggtcgcatccaggccgcgcaccgcacgccgcaggaaatcgccaacgagctgaccaccaagctcgcgccgatctaccgcgagccgcgg

gtcacggtgaacatcaaccaggcgccgggcaatacggtgttcgtcggcggcgcggtgcgcaacccgtcggccgtgccgatccccgccgcca

acaacatggagcaggcgatcctcggcgccggcggcatcctgccggtgggcgacgcccgccgcgtggcgctcatgcgcgaggactcggaa

ggccgctatcacgcctacttcctcgacttcagccagttgatgaagatcgggccggaaggccgcaagcccctggccatgcagcgtggcgacatc

gtcttcgtgcccaagtcgatggtcggcgaccgtatcgagggcgtcgacgtctacctgaaccagttgctgcccttcgccaagtccatcggcgtcg

gcgtcagctacaccgtcaacaatgatcgcΔM65-L68

SEQ ID3 protein

MKRTLLMLAMLALAACNTPARIPAPDSDTVDSGKRALEELARLPPAMERVRVGDTLRIVR

DAGESAFNVATIYELTLYTVLNDGSIYYPFIGRIQAAHRTPQEIANELTTKLAPIYREPRVTV

NINQAPGNTVFVGGAVRNPSAVPIPAANNMEQAILGAGGILPVGDARRVALMREDSEGRY

HAYFLDFSQLMKIGPEGRKPLAMQRGDIVFVPKSMVGDRIEGVDVYLNQLLPFAKSIGVGV

SYTVNNDR

>SEQ ID9 DNA

atgccggggagtcggcgttcaacgtcgccaccatctatgaactgacgctgtacaccgtgctcaacgacggcagcatctactatccgttcatcggt

cgcatccaggccgcgcaccgcacgccgcaggaaatcgccaacgagctgaccaccaagctcgcgccgatctaccgcgagccgcgggtcac

ggtgaacatcaaccaggcgccgggcaatacggtgttcgtcggcggcgcggtgcgcaacccgtcggccgtgccgatccccgccgccaacaa

catggagcaggcgatcctcggcgccggcggcatcctgccggtgggcgacgcccgccgcgtggcgctcatgcgcgaggactcggaaggcc

gctatcacgcctacttcctcgacttcagccagttgatgaagatcgggccggaaggccgcaagcccctggccatgcagcgtggcgacatcgtctt

cgtgcccaagtcgatggtcggcgaccgtatcgagggcgtcgacgtctacctgaaccagttgctgcccttcgccaagtccatcggcgtcggcgt

cagctacaccgtcaacaatgatcgcΔM65-A70

SEQ ID4 protein

MKRTLLMLAMLALAACNTPARIPAPDSDTVDSGKRALEELARLPPAMERVRVGDTLRIVR

DAGEFNVATIYELTLYTVLNDGSIYYPFIGRIQAAHRTPQEIANELTTKLAPIYREPRVTVNI

NQAPGNTVFVGGAVRNPSAVPIPAANNMEQAILGAGGILPVGDARRVALMREDSEGRYHA

YFLDFSQLMKIGPEGRKPLAMQRGDIVFVPKSMVGDRIEGVDVYLNQLLPFAKSIGVGVSY

TVNNDR

>SEQ ID10 DNA

atgccggggagttcaacgtcgccaccatctatgaactgacgctgtacaccgtgctcaacgacggcagcatctactatccgttcatcggtcgcatc

caggccgcgcaccgcacgccgcaggaaatcgccaacgagctgaccaccaagctcgcgccgatctaccgcgagccgcgggtcacggtgaa

catcaaccaggcgccgggcaatacggtgttcgtcggcggcgcggtgcgcaacccgtcggccgtgccgatccccgccgccaacaacatgga

gcaggcgatcctcggcgccggcggcatcctgccggtgggcgacgcccgccgcgtggcgctcatgcgcgaggactcggaaggccgctatca

cgcctacttcctcgacttcagccagttgatgaagatcgggccggaaggccgcaagcccctggccatgcagcgtggcgacatcgtcttcgtgcc

caagtcgatggtcggcgaccgtatcgagggcgtcgacgtctacctgaaccagttgctgcccttcgccaagtccatcggcgtcggcgtcagctac

accgtcaacaatgatcgcΔM65-N72

SEQ ID5 protein

MKRTLLMLAMLALAACNTPARIPAPDSDTVDSGKRALEELARLPPAMERVRVGDTLRIVR

DAGEVATIYELTLYTVLNDGSIYYPFIGRIQAAHRTPQEIANELTTKLAPIYREPRVTVNINQ

APGNTVFVGGAVRNPSAVPIPAANNMEQAILGAGGILPVGDARRVALMREDSEGRYHAYF

LDFSQLMKIGPEGRKPLAMQRGDIVFVPKSMVGDRIEGVDVYLNQLLPFAKSIGVGVSYTV

NNDR

>SEQ ID11 DNA

atgccggggaggtcgccaccatctatgaactgacgctgtacaccgtgctcaacgacggcagcatctactatccgttcatcggtcgcatccaggc

cgcgcaccgcacgccgcaggaaatcgccaacgagctgaccaccaagctcgcgccgatctaccgcgagccgcgggtcacggtgaacatcaa

ccaggcgccgggcaatacggtgttcgtcggcggcgcggtgcgcaacccgtcggccgtgccgatccccgccgccaacaacatggagcaggc

gatcctcggcgccggcggcatcctgccggtgggcgacgcccgccgcgtggcgctcatgcgcgaggactcggaaggccgctatcacgccta

cttcctcgacttcagccagttgatgaagatcgggccggaaggccgcaagcccctggccatgcagcgtggcgacatcgtcttcgtgcccaagtc

gatggtcggcgaccgtatcgagggcgtcgacgtctacctgaaccagttgctgcccttcgccaagtccatcggcgtcggcgtcagctacaccgtc

aacaatgatcgcGSG substitute for M65-T75

SEQ ID6 protein

MKRTLLMLAMLALAACNTPARIPAPDSDTVDSGKRALEELARLPPAMERVRVGDTLRIVR

DAGEGSGIYELTLYTVLNDGSIYYPFIGRIQAAHRTPQEIANELTTKLAPIYREPRVTVNINQ

APGNTVFVGGAVRNPSAVPIPAANNMEQAILGAGGILPVGDARRVALMREDSEGRYHAYF

LDFSQLMKIGPEGRKPLAMQRGDIVFVPKSMVGDRIEGVDVYLNQLLPFAKSIGVGVSYTV

NNDR

>SEQ ID12 DNA

atgccggggagggcagcggaatctatgaactgacgctgtacaccgtgctcaacgacggcagcatctactatccgttcatcggtcgcatccagg

ccgcgcaccgcacgccgcaggaaatcgccaacgagctgaccaccaagctcgcgccgatctaccgcgagccgcgggtcacggtgaacatca

accaggcgccgggcaatacggtgttcgtcggcggcgcggtgcgcaacccgtcggccgtgccgatccccgccgccaacaacatggagcagg

cgatcctcggcgccggcggcatcctgccggtgggcgacgcccgccgcgtggcgctcatgcgcgaggactcggaaggccgctatcacgcct

acttcctcgacttcagccagttgatgaagatcgggccggaaggccgcaagcccctggccatgcagcgtggcgacatcgtcttcgtgcccaagt

cgatggtcggcgaccgtatcgagggcgtcgacgtctacctgaaccagttgctgcccttcgccaagtccatcggcgtcggcgtcagctacaccgt

caacaatgatcgc

The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A PslD mutant nano-pore protein, which is obtained by truncating or truncating the contraction region of the wild PslD protein shown in SEQ ID NO. 1;

preferably, the truncation is a truncation of 1-10 amino acids from M65 towards the C-terminus, preferably 2 (M65-P66), 4 (M65-L68), 6 (M65-a 70) or 8 (M65-N72) amino acids, or a substitution from M65 towards the C-terminus with a short amino acid sequence, e.g. 8, 9, 10 or 11 amino acids with 3 amino acids, preferably 11 amino acids with GSG (M65-T75);

preferably, the amino acid sequence of the PslD mutant nano-pore protein is shown as SEQ ID NO. 2-6;

2. A nucleic acid sequence encoding the PslD mutant nanopore protein of claim 1;

1) As shown in SEQ ID NO. 8-12;

3) A nucleic acid sequence complementary to 1) or 2);

preferably, the homology is between 85% and 99%;

preferably, the homology is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%.

3. A recombinant vector comprising the nucleic acid sequence of claim 2.

4. A genetically engineered cell or microorganism containing the recombinant vector of claim 3, or having integrated into its genome the nucleic acid sequence of claim 2; preferably, the microorganism is E.coli.

5. A composition, complex, kit comprising the PslD mutant nanopore protein of claim 1.

6. The method for preparing the PslD mutant nano-pore protein as set forth in claim 1, comprising the steps of:

1) Constructing a PslD mutant nano-pore protein carrier;

2) And expressing and purifying the PslD mutant nano-pore protein.

7. A membrane layer having embedded therein the PslD mutant nanopore protein of claim 1;

preferably, the membrane layer is a phospholipid membrane layer;

preferably, the membrane layer is a phospholipid monolayer membrane layer.

8. A detection system comprising the PslD mutant nanopore protein of claim 1 or the membrane layer of claim 7.

9. A device comprising the PslD mutant nanopore protein of claim 1, the membrane layer of claim 7, or the detection system of claim 8.

10. Use of the PslD mutant nanopore protein of claim 1, the membrane layer of claim 7, or the detection system of claim 8 in the preparation of a device for sample sequencing and/or detection;

preferably, the sample is one or more of a nucleotide, a nucleic acid, an amino acid, an oligopeptide, a polypeptide, a protein.

11. Use of the PslD mutant nanopore protein of claim 1, the membrane layer of claim 7, or the detection system of claim 8, or the device of claim 9 in sample sequencing and/or detection;