Use of a cel-iii protein and mutants thereof in nanopore detection

Abstract

The application belongs to the technical field of biomolecules, and provides application of a CEL-III protein mutant in nanopore detection. The CEL-III protein is applied to nanopore detection, has the advantages of good nanopore size uniformity, high stability, strong controllability and low cost, and provides a new core material for single molecule detection technology, and has a wide application prospect in the fields of environmental monitoring, clinical diagnosis, drug screening and the like. The CEL-III protein mutant introduces charge residues or hydrophobic groups through amino acid mutation, and the capture efficiency of anion molecules and hydrophobic molecules is specifically improved; the CEL-III protein mutant can improve the current signal quality in the application of nanopore detection, compared with the wild-type CEL-III protein, the generated current property is stable, the current signal width is narrower, the burr is less, and the recognition efficiency and detection performance are improved.

Description

Technical Field

[0001] This invention belongs to the field of biomolecular technology, and in particular relates to the application of a CEL-III protein and its mutants in nanopore detection. Background Technology

[0002] Nanopore technology is a single-molecule-level detection technique that utilizes nanoscale pores (biological nanopores or solid-state nanopores) to achieve real-time detection of individual molecules. Biological nanopores have shown great potential in fields such as nucleic acid sequencing, protein analysis, and small molecule detection due to their advantages of good size uniformity, high biocompatibility, and strong controllability. Currently, commonly used biological nanopores mainly consist of bacterial pore-forming proteins such as α-hemolysin (α-HL) and MspA protein. However, these proteins suffer from difficulties in controlling pore size and poor compatibility with complex biological samples, limiting their application scope.

[0003] CEL-III protein, derived from the marine invertebrate Cucumaria echinata, possesses a unique three-domain architecture: two N-terminal carbohydrate-binding domains (domains 1 and 2, both β-cloverleaf structures) and a C-terminal oligomerization pore-forming domain (domain 3, containing two α-helices and a β-sandwich structure). Under specific conditions (such as lactose binding, high pH, ​​and high ionic strength), CEL-III protein undergoes conformational changes, with seven monomers assembling to form a transmembrane β-barrel structure (approximately 25 Å in diameter and 75 Å in length) containing 14 β-chains. This β-barrel structure exhibits ion permeability and controllable size. However, there are currently no reports on the application of CEL-III protein in nanopore experiments, and its unique pore-forming properties have yet to be fully explored for its potential applications in the field of nanopores. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides an application of CEL-III protein and its mutants in nanopore detection, aiming to solve the problems mentioned in the background art.

[0005] In a first aspect, the present invention provides an application of a CEL-III protein mutant in nanopore detection, wherein the CEL-III protein mutant is obtained by mutation of wild-type CEL-III protein, and the amino acid sequence of wild-type CEL-III protein is shown in SEQ ID NO:1;

[0006] The CEL-III protein mutant is selected from A, B, or C below:

[0007] A: The wild-type CEL-III protein has an amino acid mutation at V322 to V322F;

[0008] B: The wild-type CEL-III protein has an amino acid mutation at E325 to E325R;

[0009] C: The amino acid I335 of the wild-type CEL-III protein is mutated to I335W.

[0010] Furthermore, the nucleotide sequence encoding the wild-type CEL-III protein is shown in SEQ ID NO:2.

[0011] Furthermore, the nanopore detection is a single-molecule detection.

[0012] Furthermore, the single-molecule detection includes nucleic acid single-base resolution.

[0013] Furthermore, the target analytes for single-molecule detection include polysaccharides, polypeptides, or nucleotides.

[0014] Furthermore, the CEL-III protein mutant nanopore was constructed, including the following steps:

[0015] Step S1: Construct a recombinant vector for the encoding gene of the CEL-III protein mutant;

[0016] Step S2: Express and purify the CEL-III protein mutant;

[0017] Step S3: Prepare the CEL-III protein mutant nanopore.

[0018] Secondly, the present invention provides a nanopore detection device for CEL-III protein mutants, comprising an electrolyte cell, an electrode system, and the CEL-III protein mutant nanopore; the electrolyte cell is divided into left and right chambers, each injected with a 10mM Tris-HCl buffer solution containing 1M KCl, and the CEL-III protein mutant nanopore is embedded in a support membrane; the electrode system consists of Ag / AgCl electrodes, which are placed in the left and right electrolyte cells respectively and connected to a current amplifier to record ion current signals.

[0019] Thirdly, the present invention provides a product comprising the CEL-III protein mutant, said product being a composition, complex, or kit.

[0020] The present invention has the following beneficial effects:

[0021] (1) CEL-III protein, when applied to nanopore detection, offers advantages such as good nanopore size uniformity, high stability, strong tunability, and low cost. Its unique structural stability, tunability, and biocompatibility are significantly superior to existing nanopore materials, providing a new core material for single-molecule detection technology and showing broad application prospects in environmental monitoring, clinical diagnosis, and drug screening. The heptamer structure of CEL-III protein is doubly stable through the calcium ion binding site in domain 1 / 2 and the hydrophobic interaction in domain 3, maintaining activity in the range of 4-50℃ and stability in the pH range of 5.0-9.0. The ultra-high structural stability allows CEL-III protein to be directly used in complex samples (such as soil leachate and undiluted saliva) without strict pH / temperature control, eliminating the need for cumbersome sample pretreatment and reducing detection costs. In addition, CEL-III protein has a natural membrane insertion capability, spontaneously embedding into lipid bilayer membranes under physiological conditions, improving efficiency.

[0022] (2) CEL-III protein mutants introduce charged residues (such as E325R introducing positive charge) or hydrophobic groups (such as V322F enhancing the aromatic ring effect) through amino acid mutations, which specifically improves the capture efficiency of anionic molecules and hydrophobic molecules. CEL-III protein mutants can improve the quality of current signals in nanopore detection applications. Compared with wild-type CEL-III protein, the current generated is stable, the current signal width is narrower, and there are fewer spikes, which improves recognition efficiency and detection performance. Attached Figure Description

[0023] Exemplary embodiments of the present invention can be more fully understood by referring to the following figures:

[0024] Figure 1 This is a top view of the CEL-III protein channel ribbon structure model simulated by ALPHAFOLD in Example 1 of the present invention, with monomers marked in red.

[0025] Figure 2 This is a side view of the CEL-III protein channel ribbon structure model simulated by ALPHAFOLD in Example 1 of the present invention. Monomers are marked in red.

[0026] Figure 3 This is a top view of the CEL-III protein channel ribbon structure model simulated by ALPHAFOLD in Example 1 of the present invention, with monomers marked in red.

[0027] Figure 4 This is a top view of the surface structure model of the CEL-III protein channel simulated by ALPHAFOLD in Example 1 of the present invention, with monomers marked in red.

[0028] Figure 5This is a side view of the surface structure model of the CEL-III protein channel simulated by ALPHAFOLD in Example 1 of the present invention. Monomers are marked in red.

[0029] Figure 6 This is a top view of the surface structure model of the CEL-III protein channel simulated by ALPHAFOLD in Example 1 of the present invention, with monomers marked in red.

[0030] Figure 7 This is a purification electrophoresis diagram of the CEL-III protein (wild type) from Example 1 and the CEL-III V322F, CEL-III E325R, and CEL-III I335W protein mutants from Example 3 of the present invention; in the diagram, lane 1: protein marker; lane 2: eluted CEL-III protein (wild type); lane 3: eluted CEL-III V322F protein mutant; lane 4: eluted CEL-III E325R protein mutant; lane 5: eluted CEL-III I335W protein mutant.

[0031] Figure 8 This is a graph showing the detection results of the nanopore opening current of CEL-III protein (wild type) in Example 2 of the present invention.

[0032] Figure 9 This is a graph showing the current detection results during single-stranded DNA piercing in Example 4 of the present invention.

[0033] Figure 10 This is a graph showing the current detection results during single-stranded RNA pore piercing in Example 5 of the present invention.

[0034] Figure 11 This is a graph showing the current detection results during polypeptide perforation in Example 6 of the present invention.

[0035] Figure 12 This is a graph showing the current detection results during disaccharide perforation in Embodiment 7 of the present invention. Detailed Implementation

[0036] To make the technical problems to be solved, the technical solutions, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0038] This invention provides an application of a CEL-III protein mutant in nanopore detection. The CEL-III protein mutant is obtained by mutating wild-type CEL-III protein, and the amino acid sequence of wild-type CEL-III protein is shown in SEQ ID NO:1.

[0039] CEL-III protein mutants are selected from A, B, or C below:

[0040] A: The wild-type CEL-III protein has an amino acid mutation at V322 to V322F;

[0041] B: The wild-type CEL-III protein has an amino acid mutation at E325 to E325R;

[0042] C: The amino acid I335 of the wild-type CEL-III protein is mutated to I335W.

[0043] In some embodiments, the nucleotide sequence encoding the wild-type CEL-III protein is shown in SEQ ID NO:2.

[0044] In some embodiments, nanopore detection is single-molecule detection.

[0045] In some embodiments, single-molecule detection includes nucleic acid single-base resolution.

[0046] In some embodiments, the target analytes for single-molecule detection include polysaccharides, peptides, or nucleotides.

[0047] In some embodiments, constructing a CEL-III protein mutant nanopore includes the following steps:

[0048] Step S1: Construct a recombinant vector for the gene encoding the CEL-III protein mutant;

[0049] Step S2: Express and purify the CEL-III protein mutant;

[0050] Step S3: Prepare and obtain CEL-III protein mutant nanopores.

[0051] In some embodiments, the present invention provides a nanopore detection device for CEL-III protein mutants, including an electrolyte cell, an electrode system, and a CEL-III protein mutant nanopore; the electrolyte cell is divided into left and right chambers, each injected with a 10mM Tris-HCl buffer solution containing 1M KCl, and the CEL-III protein mutant nanopore is embedded in a support membrane; the electrode system consists of Ag / AgCl electrodes, which are placed in the left and right electrolyte cells respectively and connected to a current amplifier to record ion current signals.

[0052] In some embodiments, the present invention provides a product comprising a CEL-III protein mutant, the product being a composition, a complex, or a kit.

[0053] Experimental materials:

[0054] Strains and vectors: Escherichia coli BL21 CodonPlus (DE3) RIL (Agilent), pET-32a vector (Novagen), CEL-III-LS1 cDNA.

[0055] Reagents: IPTG (Sigma), lactose-cellulose chromatography medium (Seikagaku Kogyo), 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC, Avanti Polar Lipids), QuickChange site-directed mutagenesis kit (Agilent Technologies, for mutant construction), patch-clamp amplifier (Axopatch 200B, Molecular Devices); all chemical reagents were of analytical grade, and buffer solutions were prepared with ultrapure water.

[0056] Buffer formulation:

[0057] Lysis buffer: 10 mM Tris-HCl (pH=7.5), 150 mM NaCl, 1 mM PMSF;

[0058] Inclusion body washing buffer: 10 mM Tris-HCl (pH=7.5), 150 mM NaCl, 2 M urea;

[0059] Inclusion body lysis buffer: 10 mM Tris-HCl (pH=7.5), 6 M guanidine hydrochloride, 10 mM DTT;

[0060] Refolding buffer: 10 mM Tris-HCl (pH=7.5), 0.5 M L-arginine hydrochloride, 2 mM reduced glutathione, 0.2 mM oxidized glutathione;

[0061] Affinity chromatography equilibration buffer: 10 mM Tris-HCl (pH=7.5), 150 mM NaCl, 10 mM CaCl2;

[0062] Affinity chromatography elution buffer: equilibration buffer + 100 mM lactose;

[0063] Nanopore preparation buffer: 10 mM Tris-HCl (pH=7.8), 150 mM NaCl, 10 mM CaCl2, 100 mM lactose;

[0064] Lipid bilayer membrane (BLM) detection buffer: 10 mM Tris-HCl (pH=7.8), 150 mM NaCl, 10 mM CaCl2;

[0065] Nucleic acid detection buffer: 10mM Tris-HCl (pH=7.5), 1M KCl, 1mM MgCl2.

[0066] Example 1: Expression and purification of CEL-III protein (wild type)

[0067] 1. Construction of recombinant plasmids

[0068] (1) PCR amplification: Using CEL-III-LS1 cDNA as a template, the mature region (Gln1-Ile432) gene was amplified. PCR reaction system (50μL): 2×PrimeSTAR Max Premix 25μL, template cDNA 1μL, forward and reverse primers (10μM) 2μL each, ultrapure water 20μL. Reaction conditions: 98℃ pre-denaturation for 3min; 98℃ denaturation for 10s, 58℃ annealing for 30s, 72℃ extension for 1min (35 cycles); 72℃ final extension for 5min.

[0069] (2) Vector ligation and transformation: The PCR product was verified by 1% agarose gel electrophoresis (target fragment approximately 1300 bp), purified using a gel extraction kit, and then digested with the pET-32a vector using Nco I and Xho I (reacted at 37℃ for 3 h). After purification, the digested product was ligated overnight at 16℃ using T4 DNA ligase (vector to insert molar ratio 1:3). The ligation product was transformed into E. coli DH5α competent cells, plated on LB agar plates containing 50 μg / mL ampicillin, and incubated at 37℃ for 12 h.

[0070] (3) Sequencing verification: Pick a single colony, inoculate it into LB liquid medium (containing ampicillin), shake culture at 37℃ for 8h, extract plasmid (plasmid mini-prep kit, QIAGEN), send it to the sequencing company for verification (sequencing primer is T7 promoter primer), ensure that the CEL-III gene sequence is correct and without mutation, and obtain recombinant plasmid pET-32a-CEL-III.

[0071] 2. Induced expression

[0072] (1) Competent transformation: The verified recombinant plasmid was transformed into Escherichia coli BL21 CodonPlus (DE3) RIL competent cells, incubated on ice for 30 min, heat-shocked at 42℃ for 90 s, incubated on ice for 2 min, and 800 μL of antibiotic-free LB medium was added. The cells were then cultured at 37℃ with shaking for 1 h. 200 μL of the bacterial culture was spread onto LB agar plates containing 50 μg / mL ampicillin and cultured at 37℃ for 12 h.

[0073] (2) Scale-up and induction: Pick a single colony and inoculate it into 5 mL of LB liquid medium (containing ampicillin), and culture at 37°C with shaking for 12 h (seed culture). Inoculate the seed culture into 1 L of LB liquid medium (containing ampicillin) at a ratio of 1:100, and culture at 37°C with shaking until... (Approximately 3-4 hours). Add IPTG to a final concentration of 1 mM, and continue incubation at 37°C with shaking for 6 hours.

[0074] (3) Collection of bacterial cells: Transfer the cultured bacterial solution to a centrifuge bottle, centrifuge at 4℃ and 6000g for 10min, discard the supernatant, resuspend the bacterial cell pellet in lysis buffer (1g bacterial cells / 10mL buffer), and store at -80℃ for later use.

[0075] 3. Inclusion body extraction and processing

[0076] (1) Ultrasonic disruption: Thaw the frozen bacterial suspension, add PMSF to a final concentration of 1mM, and disrupt it with an ultrasonic disruptor (Ningbo Xinzhi) (power 300W, working time 3s, interval 5s, total time 30min). During the process, use an ice bath to cool down to avoid protein denaturation.

[0077] (3) Inclusion body centrifugation collection: Centrifuge the lysate at 4℃ and 12000g for 20min, discard the supernatant (soluble protein portion), resuspend the precipitate with washing buffer, centrifuge at 4℃ and 10000g for 15min, repeat the washing 3 times until the precipitate is white (to remove membrane proteins and other impurities), and finally obtain the inclusion body precipitate.

[0078] (4) Inclusion body dissolution: The inclusion body precipitate was resuspended in dissolution buffer (100 mg inclusion body / 1 mL buffer) and stirred magnetically overnight at 4°C to ensure complete dissolution of the inclusion body (the protein dissolution was confirmed by measuring the absorbance at 280 nm using a UV spectrophotometer).

[0079] 4. Protein refolding and purification

[0080] (1) Gradient dilution refolding: The dissolved protein solution was slowly injected into the refolding buffer (final protein concentration 0.1 mg / mL) using a peristaltic pump, and magnetically stirred at 4°C for 30 min. Then it was transferred to a dialysis bag (molecular weight cutoff 10 kDa) and dialyzed at 4°C for 48 h. The refolding buffer was changed every 12 h during this period.

[0081] (2) Lactose-cellulose affinity chromatography: ① Column equilibration: Load 5 mL of lactose-cellulose chromatography medium into the chromatography column and equilibrate the column with affinity chromatography equilibration buffer at a flow rate of 1 mL / min until the pH and conductivity of the eluent are consistent with the equilibration buffer. ② Sample loading: Filter the refolded protein solution through a 0.22 μm filter membrane and load it onto the chromatography column at a flow rate of 0.5 mL / min. Collect the breakthrough solution (for SDS-PAGE to detect the sample loading efficiency). ③ Elution: Elute impurities with equilibration buffer containing 50 mM lactose (10 column volumes), and then elute the target protein with equilibration buffer containing 100 mM lactose. Collect 1 mL of each sample in one tube and detect the absorbance at 280 nm using a UV spectrophotometer. Collect the peak fraction with protein absorption.

[0082] 5. Protein purity and activity verification: SDS-PAGE detection, take 10 μL of each collected peak fraction, add 5×SDS loading buffer (without β-mercaptoethanol to avoid Trx tag dissociation from CEL-III), 10% SDS-PAGE electrophoresis, Coomassie brilliant blue staining, and observe the target band (CEL-III molecular weight is about 64.8 kDa).

[0083] A top view of the CEL-III protein channel ribbon structure model simulated by ALPHAFOLD is shown below. Figure 1 As shown; a side view of the CEL-III protein channel ribbon structure model simulated by ALPHAFOLD. Figure 2 As shown; A top view of the CEL-III protein channel ribbon structure model simulated by ALPHAFOLD is shown below. Figure 3 As shown; A top view of the CEL-III protein channel surface structure model simulated by ALPHAFOLD is shown below. Figure 4 As shown; a side view of the surface structure model of the CEL-III protein channel simulated by ALPHAFOLD is shown below. Figure 5 As shown; A top view of the CEL-III protein channel surface structure model simulated by ALPHAFOLD is shown below. Figure 6 As shown. The results indicate that the CEL-III protein consists of seven identical subunits that assemble symmetrically around a central axis that forms a transmembrane channel, forming a uniform and stable 14-chain transmembrane β-barrel structure (approximately 25 Å in diameter and 75 Å in length).

[0084] The amino acid sequence of the wild-type CEL-III protein is shown in SEQ ID NO:1, and the nucleotide sequence encoding the wild-type CEL-III protein is shown in SEQ ID NO:2.

[0085] amino acid sequence:

[0086] SEQ ID NO:1:

[0087] QVLCTNPLDIGELRSFKSKQCVDIVGNQGSGNIATYDCDGLSDQQIIICGDGTIRNEARNYCFTPDGSGNANVMSSPCTLYPEIPSSQRWRQGRRKTFTDNGGIEQVATEIINLASGKCLDIEGSDGTGDIGVYDCQNLDDQYFYVRSRGPELFYGRLRNEKSDLCLDVEGSDGKGNVLMYSCEDNLDQWFRYYENGEIVNAKSGMCLDVEGSDGSGNVGIYRCDDLRDQMWSRPNAYCNGDYCSFLNKESNKCLDVSGDQGTGDVGTWQCDGLPDQRFKWVFDDWEVPTATWNMVGCDQNGKVSQQISNTISFSSTVTAGVAVEVSSTIEKGVIFAKATVSVKVTASLSKAWTNSQSGTTAITYTCDNYDSDEEFTRGCMWQLAIETTEVKSGDLLVWNPQIVKCTRSNTAPGCAPFTKCANEDCTFCTDI;

[0088] Nucleotide sequence:

[0089] SEQ ID NO:2:

[0090]

[0091] Example 2: Preparation and characterization of CEL-III protein (wild type) nanopores

[0092] 1. Nanopore preparation (heptamer assembly)

[0093] Oligopolymerization reaction: Take the purified CEL-III protein (0.2 mg / mL) and mix it with nanopore preparation buffer, and incubate it in a water bath at 25°C for 1.5 h, gently mixing once every 30 min during the process.

[0094] 2. Characterization of ion current in lipid bilayer membrane (BLM)

[0095] (1) Preparation of BLM: Dissolve DPPC in n-decane (concentration 10mg / mL), take 1μL and coat it on the micropores (diameter 100μm) of the polytetrafluoroethylene chip, place at room temperature for 10min to form a lipid bilayer membrane.

[0096] (2) Nanopore embedding and current recording: The BLM chip was installed in a dual-channel electrolyte cell. 2 mL of BLM detection buffer was added to each of the left and right chambers. An Ag / AgCl electrode was inserted, and a patch-clamp amplifier (Axopatch 200B) and a data acquisition system were connected. A transmembrane voltage of 50 mV was applied. After the baseline current stabilized (<5 pA), 10 μL of CEL-III oligomer solution (final concentration 0.01 mg / mL) was added to the upper chamber, and the change in current signal was recorded (sampling frequency 1 kHz, bandwidth 100 Hz).

[0097] (3) Current data analysis: The current trajectory is analyzed using Clampfit software.

[0098] The results of CEL-III protein (wild type) nanopore opening current detection are as follows: Figure 8 As shown, the results indicate that the nanopore opening current signal of CEL-III protein (wild type) has large noise and many current spikes.

[0099] Example 3: Construction and Characterization of CEL-III Protein Mutant Nanopores

[0100] 1. Construction of protein mutant plasmids

[0101] Using pET-32a-CEL-III as a template, a key site mutant was constructed using the QuickChange site-directed mutagenesis kit. A 25 μL mutagenesis reaction mixture was prepared according to the kit instructions. The reaction conditions were: 95℃ denaturation for 30 s; 95℃ denaturation for 30 s, 55℃ annealing for 1 min, and 68℃ extension for 10 min (18 cycles). After the reaction, 1 μL of Dpn I enzyme was added, and the mixture was digested at 37℃ for 1 h (to remove the template plasmid). The mixture was then transformed into DH5α competent cells, and sequencing was used to verify the correctness of the mutation site.

[0102] The nucleotide sequences of the forward and reverse primers for the CEL-III protein mutant gene are shown in Table 1. The CEL-III protein mutant is as follows:

[0103] (1) The amino acid V322 of the wild-type CEL-III protein is mutated to V322F;

[0104] (2) The amino acid E325 of the wild-type CEL-III protein is mutated to E325R;

[0105] (3) The amino acid I335 of the wild-type CEL-III protein is mutated to I335W.

[0106] Table 1. Nucleotide sequences of forward and reverse primers

[0107]

[0108] 2. Expression, purification, and nanopore characteristic detection of protein mutants

[0109] (1) Expression and purification: Express and purify CEL-III V322F, CEL-III E325R and CEL-III I335W protein mutants to ensure purity ≥90%.

[0110] (2) Nanopore preparation and current detection: CEL-III protein mutant nanopores were prepared and ion current was detected by BLM system.

[0111] The purification electrophoresis results of the CEL-III protein (wild type) from Example 1 and the CEL-III V322F, CEL-III E325R, and CEL-III I335W protein mutants from Example 3 are as follows: Figure 7 As shown.

[0112] Example 4: Application of CEL-III protein (wild-type) nanopores in DNA resolution

[0113] 1. Nanopore preparation: The purified CEL-III protein (concentration 0.1-0.5 mg / mL) was mixed with a buffer containing 100 mM lactose (10 mM Tris-HCl, pH=7.8, 150 mM NaCl, 10 mM CaCl2) and incubated at 25 °C for 1.5 h to obtain a CEL-III oligomer solution.

[0114] 2. Assembly of the detection system: A lipid bilayer membrane system (Black Lipid Membrane, BLM) was used. 1,2-Dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC) was dissolved in n-decane (concentration 10 mg / mL) and coated onto the micropores (diameter 100 μm) of the polytetrafluoroethylene chip to form a lipid bilayer membrane. 2 mL of 10 mM Tris-HCl buffer (pH=7.5) containing 1 M KCl was added to each of the left and right chambers. An Ag / AgCl electrode was inserted, and the current amplifier and data acquisition system were connected. Buffer containing CEL-III oligomers (0.1 mg / mL) was added.

[0115] 3. Sample preparation: Dilute 1-10 nM single-stranded DNA (5'-3' ATATA) with detection buffer and add 1 mM MgCl2.

[0116] 4. Detection and Data Analysis:

[0117] (1) Sample injection and signal recording: 10 μL of single-stranded DNA was injected into the electrolyte cell and the current signal was recorded;

[0118] (2) Current signal analysis: Extract current blocking events using Clampfit software and analyze the blocking amplitude and duration.

[0119] Current detection results during single-stranded DNA pore drilling are as follows Figure 9 As shown, the results indicate that when CEL-III protein (wild type) nanopores detect single-stranded DNA, the current signal noise is relatively large, there are many current spikes, and the current baseline fluctuation is large.

[0120] Example 5: Application of CEL-III I335W protein mutant nanopores in RNA resolution

[0121] 1. Nanopore preparation: The purified CEL-III I335W protein mutant (concentration 0.5 mg / mL) was mixed with a buffer containing 100 mM lactose (10 mM Tris-HCl, pH=7.8, 150 mM NaCl, 10 mM CaCl2) and incubated at 25 °C for 1.5 h to obtain a CEL-III I335W oligomer solution.

[0122] 2. Detection System Assembly: A lipid bilayer membrane system was used. 1,2-Dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC) was dissolved in n-decane (concentration 10 mg / mL) and coated onto the micropores (diameter 100 μm) of the polytetrafluoroethylene chip to form a lipid bilayer membrane. 2 mL of 10 mM Tris-HCl buffer (pH=7.5) containing 1 M KCl was added to each of the left and right chambers. An Ag / AgCl electrode was inserted, and the current amplifier and data acquisition system were connected. Buffer containing CEL-III I335W oligomer (0.1 mg / mL) was added.

[0123] 3. Sample preparation: Dilute 1-10 nM single-stranded RNA (5'-3' AAUAA) with detection buffer and add 1 mM MgCl2;

[0124] 4. Detection and Data Analysis:

[0125] (1) Sample injection and signal recording: 10 μL of single-stranded RNA was injected into the electrolyte cell and the current signal was recorded;

[0126] (2) Current signal analysis: Extract current blocking events using Clampfit software and analyze the blocking amplitude and duration.

[0127] Current detection results during single-stranded RNA pore drilling are as follows Figure 10 As shown, the results indicate that when the CEL-III I335W protein mutant nanopore detects single-stranded RNA, the generated current properties are stable, the current signal width is narrower, and there are fewer spikes. The principle behind the improved current signal quality is that I335 is located inside the β-barrel (8 Å from the pore center). After the isoleucine (small side chain) is mutated to tryptophan (large side chain containing an indole ring), it extends 3 Å towards the pore center, reducing the pore size from 25 Å to 19-20 Å. The space occupancy effect inside the β-barrel reduces the pore size, making it more suitable for small molecule analysis.

[0128] Example 6: Application of CEL-III V322F protein mutant nanopores in peptide resolution

[0129] (1) Nanopore preparation: The purified CEL-III V322F protein mutant (concentration 0.1 mg / mL) was mixed with a buffer containing 100 mM lactose (10 mM Tris-HCl, pH=7.8, 150 mM NaCl, 10 mM CaCl2) and incubated at 25 °C for 1.5 h to obtain CEL-III V322F oligomer solution;

[0130] (2) Assembly of the detection system: A lipid bilayer membrane system was used. 1,2-Dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC) was dissolved in n-decane (concentration 10 mg / mL) and coated onto the micropores (diameter 100 μm) of the polytetrafluoroethylene chip to form a lipid bilayer membrane. 2 mL of 10 mM Tris-HCl buffer (pH=7.5) containing 1 M KCl was added to the left and right chambers respectively. An Ag / AgCl electrode was inserted, and the current amplifier and data acquisition system were connected. Buffer containing CEL-III V322F oligomer (0.1 mg / mL) was added.

[0131] 3. Sample preparation: Dilute 1-10 nM of the peptide Lys-Arg-Ala with detection buffer and add 1 mM MgCl2;

[0132] 4. Detection and Data Analysis:

[0133] (1) Sample injection and signal recording: 10 μL of the peptide Lys-Arg-Ala was injected into the electrolyte cell and the current signal was recorded;

[0134] (2) Current signal analysis: Extract current blocking events using Clampfit software and analyze the blocking amplitude and duration.

[0135] Current detection results during peptide pore perforation are as follows Figure 11 As shown, the results indicate that the CEL-III V322F protein mutant nanopores exhibit stable current characteristics, a narrower current signal width, and fewer spikes when detecting peptides. The principle behind this improved current signal quality is that V322 is a small side-chain residue with a hydrophobic core in domain 3. The mutation of valine to a more hydrophobic phenylalanine enhances the hydrophobic interaction with adjacent L349 residues through π-π stacking, thereby increasing the stability of the heptamer.

[0136] Example 7: Application of CEL-III E325R protein mutant nanopores in disaccharide resolution

[0137] 1. Nanopore preparation: The purified CEL-III E325R protein (concentration 0.4 mg / mL) was mixed with a buffer containing 100 mM lactose (10 mM Tris-HCl, pH=7.8, 150 mM NaCl, 10 mM CaCl2) and incubated at 25 °C for 1.5 h to obtain a CEL-III E325R oligomer solution.

[0138] 2. Detection System Assembly: A lipid bilayer membrane system was used. 1,2-Dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC) was dissolved in n-decane (concentration 10 mg / mL) and coated onto the micropores (diameter 100 μm) of the polytetrafluoroethylene chip to form a lipid bilayer membrane. 2 mL of 10 mM Tris-HCl buffer (pH=7.5) containing 1 M KCl was added to each of the left and right chambers. An Ag / AgCl electrode was inserted, and the current amplifier and data acquisition system were connected. Buffer containing CEL-III E325R oligomer (0.1 mg / mL) was added.

[0139] 3. Sample preparation: Dilute 1-10 nM of disaccharide Gal-β1,4-Glc (β-1,4-galactosyl glucose, concentration) with detection buffer and add 1 mM MgCl2;

[0140] 4. Detection and Data Analysis:

[0141] (1) Sample injection and signal recording: 10 μL of disaccharide Gal-β1,4-Glc was injected into the electrolyte cell and the current signal was recorded;

[0142] (2) Current signal analysis: Extract current blocking events using Clampfit software and analyze the blocking amplitude and duration.

[0143] The current detection results during disaccharide perforation are as follows: Figure 12 As shown, the results indicate that the CEL-III E325R protein mutant nanopore provides stable current characteristics, a narrower current signal width, and fewer spikes when detecting disaccharides. The principle behind the improved current signal quality is as follows: E325 is located inside the β-barrel. Glutamic acid, an acidic amino acid with a negatively charged side chain, forms an electrostatic attraction with the adjacent L344. Replacing glutamic acid with arginine (a basic amino acid with a positively charged side chain) increases the positive charge density within the pore, reversing ion selectivity (from K⁺ preference to Cl⁻ preference) and expanding the scope of anion analysis.

[0144] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An application of a CEL-III protein mutant in nanopore detection, characterized in that: The CEL-III protein mutant was obtained by mutating the wild-type CEL-III protein, and the amino acid sequence of the wild-type CEL-III protein is shown in SEQ ID NO:1; The CEL-III protein mutant is selected from A, B, or C below: A: The wild-type CEL-III protein has an amino acid mutation at V322 to V322F; B: The wild-type CEL-III protein has an amino acid mutation at E325 to E325R; C: The amino acid I335 of the wild-type CEL-III protein is mutated to I335W.

2. The application as described in claim 1, characterized in that: The nucleotide sequence encoding the wild-type CEL-III protein is shown in SEQ ID NO:

2.

3. The application as described in claim 1, characterized in that: The nanopore detection is a single-molecule detection.

4. The application as described in claim 3, characterized in that: The single-molecule detection includes nucleic acid single-base resolution.

5. The application as described in claim 3, characterized in that: The target analytes for single-molecule detection include polysaccharides, peptides, or nucleotides.

6. The application as described in claim 1, characterized in that: It also includes the preparation of CEL-III protein mutant nanopores, the preparation of which includes the following steps: Step S1: Construct a recombinant vector for the encoding gene of the CEL-III protein mutant; Step S2: Express and purify the CEL-III protein mutant; Step S3: Mix the purified CEL-III protein mutant with nanopore preparation buffer to obtain the CEL-III protein mutant nanopore.

7. A nanopore detection device for CEL-III protein mutants, comprising an electrolyte cell, an electrode system, and a CEL-III protein mutant nanopore as described in claim 6, characterized in that: The electrolyte pool is divided into left and right chambers, each filled with a 10mM Tris-HCl buffer solution containing 1M KCl. The CEL-III protein mutant nanopore is embedded in the support membrane. The electrode system consists of Ag / AgCl electrodes, which are placed in the left and right electrolyte pools respectively and connected to a current amplifier to record the ion current signal.

8. A CEL-III protein mutant as described in claim 1.

Images