Colletotrichum protein nanopore system and application thereof in protein sequencing

Abstract

The invention relates to an anthrax protein nanopore system and application thereof in protein sequencing, and belongs to the technical field of nanopore detection. The invention aims to solve the problems of low flux, insufficient accuracy and limited nanopore resolution in the existing protein sequencing technology. According to the invention, an anthrax protein PA63 heptamer nanopore electrochemical detection device is constructed, an asymmetric pH buffer system is adopted, Trypsin, Calpain-1, Pancreative Elatase, CarboxyceptidaseA enzyme and the like are added step by step for polypeptide enzyme digestion, voltage is applied to drive amino acid to pass through nanopores, current signals are collected, amplitude, retardation time and frequency characteristics are analyzed, and polypeptide amino acid sequence detection is realized. The nanopore is small in opening current (fA level) and high in resolution ratio, detection accuracy and sensitivity are enhanced by combining multi-enzyme step-by-step cutting and carving, and a direct and efficient sequencing means is provided for protein function research.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of nanopore detection, and relates to a nanometer pore system of anthrax protein and application thereof in protein sequencing. BACKGROUND

[0002] The research history of anthrax virus protein (PA63) can be traced back to the 1980s. Anthrax toxin protective antigen (PA) is a key component and "gateway" for anthrax toxin to exert toxicity. Its active form PA63 is a heptameric nanopore assembled by 7 monomers. It is non-toxic by itself, but its core function is to form a pore on the cell membrane to mediate the entry of two other toxic factors (lethal factor and edema factor) into the cell, thereby causing cell death and the typical symptoms of anthrax. Its unique heptameric structure, clear mechanism of action and engineering characteristics make it continue to play an important role in the field of nanobiotechnology.

[0003] Proteins are the main executors of life functions: enzyme catalysis, signal transduction, structural support, immune response, and almost all cellular processes cannot do without proteins. Specific domains in the sequence determine how proteins interact with other molecules. The central dogma describes the flow of genetic information from DNA to RNA and then to protein. However, the DNA sequence cannot completely predict the final shape and function of the protein. Processes such as alternative splicing of mRNA and post-translational modification (PTM) allow a gene to produce multiple protein variants (Proteoforms) with different shapes and functions. Therefore, directly sequencing proteins is the only way to obtain their most authentic and functional information.

[0004] Current protein sequencing technology has gone through three generations of development. The first generation is the traditional chemical degradation method, but its throughput is low, slow, time-consuming and labor-intensive, and it is not effective for long-chain proteins (>50 aa) (because the efficiency decreases with the increase of the number of cycles). The second generation is mass spectrometry (MS), which has the advantages of high throughput and high sensitivity, making it the mainstream method of protein sequencing. However, it has not been able to achieve a very high level of sequencing accuracy, and it is an indirect sequencing method.

[0005] The third generation of sequencing technology is currently at the forefront, aiming to directly and long-read sequence single protein molecules like nanopore DNA sequencing. Nanopore technology can directly read protein sequences and simultaneously detect various PTMs. Currently, there are many nanopores that have achieved protein sequencing function, but the resolution needs to be improved. SUMMARY

[0006] In view of the above, one of the purposes of the present application is to provide a anthrax protein nanopore system, the second purpose is to provide a anthrax protein nanopore system detection method, the third purpose is to provide a anthrax protein nanopore system application.

[0007] In order to achieve the above-mentioned purpose, the present application provides the following technical solutions: The present application provides a anthrax protein nanopore system, the anthrax protein nanopore system is composed of anthrax protein nanopore device and buffer system, the anthrax protein nanopore device is composed of electrode, membrane, chamber, the nanopore formed by embedding anthrax protein in the membrane, the nanopore is connected with chamber A and chamber B; the buffer system includes buffer A applied to chamber A and buffer B applied to chamber B, the pH value of buffer A and buffer B is different, forming an asymmetric pH system, and further comprising a protease system; Preferably, the protease system includes one or more of recombinant human trypsin, calpain-1, elastase and carboxypeptidase A; Preferably, the anthrax protein is PA83 which is cut by the membrane endoenzyme of Furin family, releasing 63kDa C-terminal fragment PA63 oligomer to form a heptamer, the membrane is made of Teflon membrane or silicon nitride solid material, and the electrode material is silver electrode coated with silver chloride on the surface; Preferably, the buffer A is a mixed aqueous solution of 1-butyl-3-methylimidazolium chloride with a concentration of 0.5M-1M and tris-hydroxymethyl aminomethane with a concentration of 10mM, and the pH is 5.6; the buffer B is a mixed aqueous solution of 1-butyl-3-methylimidazolium chloride with a concentration of 0.5M-1M and tris-hydroxymethyl aminomethane with a concentration of 10mM, and the pH is 7.6; Further, the detection method of the anthrax protein nanopore system comprises the following steps: S1: adding the polypeptide or protein solution to be detected into chamber A of the anthrax protein nanopore device, adding buffer solution A into chamber A, and adding buffer solution B into chamber B; S2: adding recombinant human trypsin, calpain-1 and elastase into chamber A in sequence for stepwise enzymatic reaction, and each enzyme has a reaction time of 45 minutes; during the reaction, an electric field force is formed by applying voltage to the device, and the current signal is collected; S3: amplifying the current signal collected in step S2 through a low-noise current amplifier, selecting characteristic current signals by using ClampFit software, and importing the data into Origin software for statistical analysis to obtain the polypeptide or protein amino acid sequence; Further, the detection method of the anthrax protein nanopore system comprises the following steps: S1: Add the solution of the polypeptide or protein to be tested into chamber A of the anthrax protein nanopore device, add buffer solution A to chamber A, and add buffer solution B to chamber B; S2: First, add the ZnCl2 solution to the carboxypeptidase A and react for 15 minutes, then add it to chamber A for enzyme cutting reaction, the reaction time is 45 minutes, and an electric field force is applied to the device during the reaction, and the current signal is collected; S3: The current signal collected in step S2 is amplified by a low-noise current amplifier, the characteristic current signal is selected by ClampFit software, and the data is imported into Origin software for statistical analysis, and the amino acid sequence of the polypeptide or protein is obtained; Preferably, the molar ratio of the recombinant human trypsin, calpain-1, elastase and carboxypeptidase A to the polypeptide or protein to be tested is 1:20; Further, the anthrax protein nanopore system is applied to polypeptide or protein sequencing.

[0008] The beneficial effects of the present application are: 1. Precise protein sequencing: The present application uses a stable anthrax virus antigen protein (PA63) nanopore channel combined with polypeptide protein combined enzyme cutting method, which can sequence polypeptides or proteins of different sequences. The current signal generated by the amino acid after enzyme cutting is collected by an electrochemical detection device, and the amplitude, block time and signal frequency of the signal are analyzed, so that the small current change and signal appearance order of different amino acids are accurately recorded, and the detection of polypeptide or protein sequence is realized.

[0009] 2. High throughput and direct detection: Compared with traditional protein sequencing methods (such as low throughput of chemical degradation method and indirect detection of mass spectrometry), the present application directly sequences single protein molecules with long read length using nanopore technology, with resolution up to fA level, improving the accuracy and sensitivity of detection.

[0010] 3. Stability and reliability: The nanopore electrochemical detection device constructed in the present application uses a specific buffer system (such as BMIMCl and Tris buffer, pH gradient difference), which ensures the stability of the nanopore channel, thereby improving the repeatability and reliability of the detection.

[0011] 4. Significance in life science and medical research: Protein sequencing is the basis for understanding protein function. Proteins have multiple key roles in the human body, including structure and support, catalyzing biochemical reactions, transportation and storage, providing the basis for movement, immune defense, and regulation and communication. The present application directly sequences polypeptide sequences, which helps to reveal the structure and function relationship of proteins, and promotes the development of life science and medical research.

[0012] Additional advantages, objects, and features of the application will be apparent to those skilled in the art upon examination of the following specification. It is intended that the application not be limited by any of the details of the specification. Instead, the true scope of the application is to be determined by the full width of the claims, along with their equivalents. BRIEF DESCRIPTION OF DRAWINGS

[0013] In order to make the objectives, technical solutions and advantages of the present application clearer, the preferred embodiments of the present application will be described in detail below with reference to the drawings, in which: Figure 1 Schematic diagram of the process of polypeptide being hydrolyzed by three enzymes and then driven through anthrax nanopore by voltage; Figure 2 Scatter plot of the blocking current ratio (ΔI / Io) of the product of polypeptide being hydrolyzed by three enzymes; Figure 3 Schematic diagram of the product signal and Gaussian plot of the blocking current ratio of the product of RMSDDDDDDD polypeptide being hydrolyzed by three enzymes separately and in combination; Figure 4 Schematic diagram of the product signal and Gaussian plot of the blocking current ratio of the product of KMSDDDDDDD polypeptide being hydrolyzed by three enzymes separately and in combination; Figure 5 Schematic diagram of the product signal and Gaussian plot of the blocking current ratio of the product of RYADDDDDDD polypeptide being hydrolyzed by three enzymes separately and in combination; Figure 6 Schematic diagram of the product signal and Gaussian plot of the blocking current ratio of the product of KYADDDDDDD polypeptide being hydrolyzed by three enzymes separately and in combination; Figure 7 Schematic diagram of the product signal and Gaussian plot of the blocking current ratio of the product of KMSDDDDDDD,KYADDDDDDD polypeptide being hydrolyzed by three enzymes simultaneously, and two-dimensional scatter plot of the blocking current ratio and the residence time in the pore; Figure 8 Statistical diagram of the difference analysis of the amino acids produced by KMSDDDDDDD,KYADDDDDDD being hydrolyzed by three enzymes and the amino acid control group; Figure 9 Schematic diagram of the product signal and Gaussian plot of the blocking current ratio of the product of KMSDDDDDDD,KYADDDDDDD polypeptide being hydrolyzed by three enzymes simultaneously, and two-dimensional scatter plot of the blocking current ratio and the residence time in the pore; Figure 10 Schematic diagram of the product signal of HAEGTF (glucagon-like peptide-1 abnormal fragment) polypeptide being hydrolyzed by carboxypeptidase A. DETAILED DESCRIPTION

[0014] Following embodiments of the present application will be described in detail by specific examples. Other advantages and effects of the present application can be easily understood by those skilled in the art from this disclosure. The present application can also be implemented or applied by other different embodiments, and various modifications or changes can be made to the details based on different views and applications without departing from the spirit of the present application. It should be noted that the drawings provided in the following examples only illustrate the basic concept of the present application in a schematic manner, and the following examples and features in the examples can be combined with each other without conflict.

[0015] The drawings are only used for exemplary illustration, and the representation is only a schematic diagram, not a physical diagram, and cannot be understood as a limitation of the present application. In order to better illustrate the embodiments of the present application, some components in the drawings may be omitted, enlarged or reduced, and do not represent the size of the actual product. It is understandable for those skilled in the art that some well-known structures and their descriptions in the drawings may be omitted.

[0016] The same or similar reference numerals in the drawings of the embodiments of the present application correspond to the same or similar components. In the description of the present application, it should be understood that if the terms "upper", "lower", "left", "right", "front", "back" and the like indicate the orientation or positional relationship shown in the drawings, they are only for the convenience of describing the present application and simplifying the description, and do not indicate or imply that the devices or elements referred to must have a specific orientation, be constructed and operated in a specific orientation. Therefore, the terms describing the positional relationship in the drawings are only used for exemplary illustration, and cannot be understood as a limitation of the present application. For those skilled in the art, the specific meaning of the above terms can be understood according to the specific circumstances.

[0017] Example 1 A stable anthrax virus antigen protein nanopore channel is constructed, specifically as follows: 1. Preparation of solution: Preparation of basic buffer solution: 1-butyl-3-methylimidazolium chloride (BMIMCL) and tris were dissolved in deionized water, and the pH was adjusted to form different basic buffer solutions containing 1-butyl-3-methylimidazolium chloride (BMIMCL) and tris (buffer A: 1-butyl-3-methylimidazolium chloride concentration of 0.5M, tris concentration of 10mM, pH=5.6; buffer B: 1-butyl-3-methylimidazolium chloride concentration of 0.5M, tris concentration of 10mM, pH=7.6; buffer C: 1-butyl-3-methylimidazolium chloride concentration of 1M, tris concentration of 10mM, pH=7.6; buffer D: 1-butyl-3-methylimidazolium chloride concentration of 1M, tris concentration of 10mM, pH=5.6). Finally, filtration was performed using a 0.22μM filter before use; 2. Construction of a nanopore electrochemical detection and analysis device: The anthrax virus antigen protein biological nanopore was embedded in a Teflon membrane, and the membrane was placed in a resin chamber containing a basic buffer solution. The membrane divided the entire resin chamber into two small chambers, and two electrodes (silver electrodes coated with silver chloride) were placed in the two small chambers. The electrodes were connected in order to a power supply and an ammeter to form a current loop, thereby forming a nanopore electrochemical detection and analysis device (as shown in Figure 2 ).

[0018] 3. Single enzyme digestion and multiple enzyme combination digestion of three polypeptides and detection: (1) The three polypeptides were divided into three parts each, and one enzyme, two enzymes, and three enzymes were added to each part, respectively. The samples were incubated at 38°C for 30 minutes to form nine test samples. Then, the test samples were added to the chamber containing the buffer (cis end), and a -100mv voltage was applied to make the test samples pass through the nanopore and generate an electrical signal.

[0019] (2) The current signal collected in step (1) is amplified by a low-noise current amplifier (Axon Axopatch 200B), and the amplitude, current block time and signal generation frequency of the collected current signal are analyzed (the collected current signal file is imported into the clampfit software to select the characteristic current signal, and the amplitude, current block time and signal generation frequency of the collected current signal are analyzed (the collected current signal file is imported into the clampfit software to select the characteristic current signal, and the selected current signal data is imported into the origin software for statistical analysis and drawing of the mathematical statistics graph of the signal), and finally the corresponding product signal statistics graph is obtained.

[0020] The product signal graphs produced by hydrolysis of different polypeptides are shown in FIG. 1, and three samples treated by different enzymes produce different amino acid signals. The current signal amplitude is processed to obtain the blocking current ratio ΔI / Io, which is consistent with the amino acid blocking current ratio detected alone. This proves that the enzyme digestion produces corresponding amino acids, and the amino acid pore current signal produced by enzyme digestion is consistent with the amino acid signal detected alone. According to the types of enzymes added and the different amino acid signals produced, the sequence of the polypeptide can be determined. Figures 3-6 Figure 2 The product signal graphs produced by hydrolysis of different polypeptides are shown in FIG. 1, and three samples treated by different enzymes produce different amino acid signals. The current signal amplitude is processed to obtain the blocking current ratio ΔI / Io, which is consistent with the amino acid blocking current ratio detected alone. This proves that the enzyme digestion produces corresponding amino acids, and the amino acid pore current signal produced by enzyme digestion is consistent with the amino acid signal detected alone. According to the types of enzymes added and the different amino acid signals produced, the sequence of the polypeptide can be determined.

[0021] Example 2 Three enzymes are used to simultaneously digest polypeptides, and different amino acid signals are produced after the treated polypeptide samples are detected by nanopore electrochemistry. The specific process is as follows: 1. Solution preparation: (1) Prepare the basic buffer solution: dissolve 1-butyl-3-methylimidazolium chloride (BMIMCL) and tris-hydroxymethyl aminomethane (Tris) in deionized water to form a basic buffer solution containing 1-butyl-3-methylimidazolium chloride (BMIMCL) and tris-hydroxymethyl aminomethane (Tris) (wherein the concentration of 1-butyl-3-methylimidazolium chloride in buffer A is 0.5M, the concentration of tris-hydroxymethyl aminomethane is 10mM, pH=5.6; the concentration of 1-butyl-3-methylimidazolium chloride in buffer B is 0.5M, the concentration of tris-hydroxymethyl aminomethane is 10mM, pH=7.6;). Finally, filter with a 0.22uM filter and then use; (2) Prepare the polypeptide solution: dissolve three polypeptide dry powders in deionized water to form a polypeptide solution with a concentration of 10mM.

[0022] ​(3) enzyme digestion: trypsin, calpain-1 and pancreatic elastase were added into the polypeptide solution, the ratio of enzyme to polypeptide was controlled at 1:20, and CaCl2 was added to activate calpain-1, the concentration of CaCl2 was controlled at 5 mM. The reagents were prepared and placed in a 38°C environment for 40 minutes.

[0023] 2. Constructing a nanopore electrochemical detection and analysis device: The anthrax virus antigen protein biological nanopore was embedded in a Teflon membrane, and the membrane was placed in a resin chamber containing a basic buffer solution. The membrane divided the entire resin chamber into two small chambers A and B. Buffer A and B were added to the two small chambers, respectively, and two electrodes (silver electrodes coated with silver chloride) were placed in the two small chambers. A power supply, ammeter, and other devices were connected in sequence between the two electrodes to form a current loop, thereby forming the nanopore electrochemical detection and analysis device (as shown in Figure 1 ).

[0024] 3. Detecting the three enzyme-digested polypeptides: (1) The enzyme-digested polypeptides were added to chamber A of the nanopore electrochemical detection device constructed above, and the basic buffer solutions A and B were used as electrolytes. An electric field was formed in the solution by applying a voltage to the device, which pushed the amino acids produced by enzyme digestion to interact with the nanopore, and the current signal was collected. (2) The current signal collected in step (1) was amplified using a low-noise current amplifier (Axon Axopatch 200B), and the amplitude, current block time, and signal generation frequency of the collected current signal were analyzed (the collected current signal file was imported into clampfit software to select the characteristic current signal, and the selected current signal data was imported into origin software for statistical analysis and drawing of the mathematical statistics graph), and finally the amino acid signal statistical graph produced by enzyme digestion was obtained.

[0025] The amino acids contained in the polypeptides were detected individually as a control group, and the blocking current ratios of various amino acids were analyzed and summarized into a scatter plot. As shown in Figure 2 , the amino acids were distinguished mainly from the specific peak value and blocking current ratio, and the parameters of different amino acids were recorded. As shown in Figure 7 , the blocking current ratio parameters of the enzyme-digested amino acids were compared. As shown in Figure 8 , there was no significant difference between the enzyme-digested amino acids and the amino acids detected in the control group after differential analysis.

[0026] Example 3 The endonuclease reaction is divided into two types, endonuclease reaction and exonuclease reaction. The buffer solution in the chamber is set as the reaction environment for the enzyme digestion. The polypeptide is first added to the chamber. The endonuclease reaction involves three enzymes, which are added in different time steps. The single reaction time is 45 minutes. The sequence of the polypeptide is determined according to the order of the amino acid appearance. The exonuclease reaction involves Carboxypeptidase A enzyme. After adding the enzyme, the reaction and detection can be performed. The specific process is as follows: 1. Preparation of solution (1) Preparation of basic buffer solution: 1-butyl-3-methylimidazolium chloride (BMIMCL) and tris are dissolved in deionized water to form a basic buffer solution containing 1-butyl-3-methylimidazolium chloride (BMIMCL) and tris (wherein the concentration of 1-butyl-3-methylimidazolium chloride in buffer solution A is 0.5M, the concentration of tris is 10mM, pH=5.6; the concentration of 1-butyl-3-methylimidazolium chloride in buffer solution B is 0.5M, the concentration of tris is 10mM, pH=7.6;). Finally, filter with a 0.22uM filter before use; (2) Preparation of polypeptide solution: dissolve the polypeptide in deionized water to form a polypeptide solution with a concentration of 10mM.

[0027] 2. Construction of nanopore electrochemical detection and analysis device The anthrax virus antigen protein biological nanopore is embedded in the membrane (the membrane used here is a Teflon membrane). The membrane is placed in a resin chamber containing a basic buffer solution. The membrane divides the entire resin chamber into two small chambers A and B. Buffer solutions A and B are added to the two small chambers, respectively. Two electrodes (the two electrodes are silver electrode materials coated with silver chloride) are placed in the two small chambers. The power supply, ammeter and other devices are connected in sequence between the two electrodes to form a current loop, i.e. the nanopore electrochemical detection and analysis device (as shown in Figure 1 ).

[0028] 3. Real-time endonuclease reaction and detection of polypeptide (1) The prepared polypeptide solution is added to chamber A of the nanopore electrochemical detection device constructed above. The basic buffer solutions A and B are used as electrolytes in the device. Trypsin enzyme is added for reaction and detection for the first 45 minutes. Then Calpain-1 enzyme and Pancreatic Elastase enzyme are added in sequence. Each reaction and detection time is 45 minutes. An electric field is formed in the solution by applying voltage to the device. Under the action of the electric field, the digested amino acids interact with the nanopore. The current signal is collected. (2) the current signal collected in step (1) is amplified by a low-noise current amplifier (Axon Axopatch 200B), and the amplitude, current block time and signal generation frequency of the collected current signal are analyzed (the collected current signal file is imported into clampfit software to select the characteristic current signal, and the selected current signal data is imported into origin software for statistical analysis and drawing of the mathematical statistics graph of the signal), and finally the corresponding amino acid sample statistics graph is obtained.

[0029] As shown in Figure 9 , different amino acid signals appear in the signal graph within 0-45 minutes, 45-90 minutes and 90-145 minutes according to the time of adding the enzyme, and the sequence of the polypeptide can be determined according to the order of the appearance of the amino acids in each time period.

[0030] 4, polypeptide is subjected to real-time exonuclease reaction and detection: (1) the prepared HAEGTF polypeptide solution is added to chamber A in the above-mentioned nano-pore electrochemical detection device, the basic buffer solution A and B are used as electrolyte in the device, ZnCl2 solution is added to the CarboxypeptidaseA enzyme to increase its activity for 15 minutes, then the reacted solution is added to chamber A for exonuclease reaction, at the same time, voltage is applied to the device to form an electric field in the solution, under the action of the electric field, the enzyme-cleaved amino acids interact with the nano-pore, and the current signal is collected; (2) the current signal collected in step (1) is amplified by a low-noise current amplifier (Axon Axopatch 200B), and the amplitude, current block time and signal generation frequency of the collected current signal are analyzed (the collected current signal file is imported into clampfit software to select the characteristic current signal, and the selected current signal data is imported into origin software for statistical analysis and drawing of the mathematical statistics graph of the signal), and finally the corresponding amino acid sample statistics graph is obtained.

[0031] As shown in Figure 10 , different amino acid signals appear in a certain period of time after the polypeptide is hydrolyzed by exonuclease, which is obviously consistent with the sequence of the polypeptide.

[0032] In summary: the present application designs a new protein sequencing method based on anthrax nano-pore, which detects and verifies the sequence of the protein by combining various enzymes, the anthrax virus antigen protein channel has the characteristics of small opening current and high resolution, has high sensitivity for amino acid detection, and has important practical significance for amino acid sequence detection in the protein.

[0033] Finally, it is to be explained that the above embodiments are only used to illustrate the technical solutions of the present application but not to limit the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or equivalently replaced without departing from the purpose and scope of the present application, and all of them should be covered in the scope of the claims of the present application.

Claims

1. Anthrax protein nanopore system characterized in that: The anthrax protein nanopore system is composed of an anthrax protein nanopore device and a buffer system, wherein the anthrax protein nanopore device comprises electrodes, a membrane, and chambers, the membrane is embedded with nanopores formed by anthrax proteins, and the nanopores are connected to chamber A and chamber B; the buffer system comprises buffer A applied to chamber A and buffer B applied to chamber B, the pH values of the buffer A and the buffer B are different, forming an asymmetric pH system, and further comprising a protease system.

2. Anthrax protein nanopore system according to claim 1, characterized in that: The protease system comprises one or more of recombinant human trypsin, calpain-1, elastase, and carboxypeptidase A.

3. The anthrax protein nanopore system of claim 1, wherein: The anthrax protein is PA83 which is cleaved by a membrane endopeptidase of the Furin family to release a 63kDa C-terminal fragment PA63 which oligomerizes to form a heptamer, the membrane is made of Teflon or silicon nitride solid material, and the electrode material is a silver electrode coated with silver chloride.

4. The anthrax protein nanopore system of claim 1, wherein: The buffer A is a mixed aqueous solution of 1-butyl-3-methylimidazolium chloride with a concentration of 0.5M-1M and tris(hydroxymethyl)aminomethane with a concentration of 10mM, and has a pH of 5.6; the buffer B is a mixed aqueous solution of 1-butyl-3-methylimidazolium chloride with a concentration of 0.5M-1M and tris(hydroxymethyl)aminomethane with a concentration of 10mM, and has a pH of 7.

6.

5. The detection method of the anthrax protein nanopore system according to any one of claims 1-4, characterized in that: S1: adding a polypeptide or protein solution to be detected into chamber A of the anthrax protein nanopore device, adding buffer solution A into chamber A, and adding buffer solution B into chamber B; S2: sequentially adding recombinant human trypsin, calpain-1, and elastase into chamber A for stepwise enzymatic reaction, each enzyme has a reaction time of 45 minutes, an electric field force is formed by applying a voltage to the device during the reaction, and current signals are collected; S3: amplifying the current signals collected in step S2 through a low-noise current amplifier, selecting characteristic current signals by using ClampFit software, and importing the data into Origin software for statistical analysis to obtain the amino acid sequence of the polypeptide or protein.

6. The detection method of the anthrax protein nanopore system according to any one of claims 1-4, characterized in that: S1: adding a polypeptide or protein solution to be detected into chamber A of the anthrax protein nanopore device, adding buffer solution A into chamber A, and adding buffer solution B into chamber B; S2: adding ZnCl2 solution into carboxypeptidase A first, reacting for 15 minutes, and then adding into chamber A for enzymatic reaction, the reaction time is 45 minutes, an electric field force is formed by applying a voltage to the device during the reaction, and current signals are collected; S3: amplifying the current signals collected in step S2 through a low-noise current amplifier, selecting characteristic current signals by using ClampFit software, and importing the data into Origin software for statistical analysis to obtain the amino acid sequence of the polypeptide or protein.

7. A detection method according to claim 5 or 6, characterised in that: The molar ratio of any one of the recombinant human trypsin, calpain-1, elastase, and carboxypeptidase to the polypeptide or protein to be detected is 1:

20.

8. Use of the anthrax protein nanopore system of any of claims 1-4 in polypeptide or protein sequencing.

Images