CN112505126B - Method for detecting influence of metal ions on induction of HIV-1 Tat proteolysis - Google Patents

Abstract

The invention relates to the application and detection method of metal ions in affecting the hydrolysis of Tat proteolysis induced by HIV‑1, and belongs to the fields of biological detection and biomedicine. The invention discloses the application of metal ions in affecting the hydrolysis of Tat proteolysis induced by HIV‑1, by recording the addition of metal ions (copper ions or calcium ions) on the Tat protein induced by HIV‑1 before and after trypsin is added to the nanopore electrochemistry Detect the change of the current signal in the analysis device, and observe the influence of metal ions on the Tat proteolysis induced by HIV-1 in real time; and disclose a nanopore electrochemical detection and analysis device and method for detecting the influence, which fully utilizes the nanopore The advantages of real-time detection of electrochemical detection devices, accurate recording of small current signal changes, simple operation, high reproducibility of implementation, and reliable detection results.

Description

Method for Detecting Effects of Metal Ions on Inducing HIV-1 Tat Proteolysis

technical field

The invention belongs to the field of biological detection and biomedicine, and specifically relates to the application of metal ions in the hydrolysis of Tat proteolysis induced by HIV-1 and its detection method.

Background technique

Acquired immunodeficiency syndrome, or AIDS (AIDS), is a disease caused by human immunodeficiency virus (HIV) infection. This disease attacks human CD4T lymphocytes, and the CD4T cells of AIDS patients show progressive or irregular decrease, and the patients lose part or all of their immune function, which leads to other diseases, and the clinical manifestations of patients are various. The disease has attracted widespread attention from all walks of life due to its rapid spread, high fatality rate, and incurability. HIV virus reverse transcription activator protein Tat (hereinafter collectively referred to as the Tat protein that induces HIV-1), transactivates viral transcription and promotes viral replication. It can cross the cell membrane structure, carry foreign virus molecules into normal cells, and can promote the high expression of foreign proteins, playing a key role in HIV replication. HIV-1-inducing Tat protein is one of the earliest proteins expressed by HIV. The detection of HIV-1-inducing Tat protein can advance the window period of HIV infection and provide assistance for early diagnosis of the disease. At the same time, the study of the hydrolysis of its reverse transcription activating protein Tat protein will help to understand the regulatory role of the Tat protein that induces HIV-1 in the transcription process of HIV-1, and destroys the transmembrane transport of the Tat protein that induces HIV-1. Provide theoretical and experimental support for the HIV-1-inducing Tat protein as a new target for anti-AIDS drugs.

Currently, routine HIV detection methods are cumbersome to operate, and the most common method is enzyme-linked immunosorbent assay (ELISA), including western blotting (WB), strip immunoassay (LIATKE HIVⅢ), radioimmunoprecipitation assay (RIPA) and immunoassay. Fluorescence assay (IFA), etc. These detection methods can provide a basis for screening and confirmation to a certain extent, but in different stages of HIV infection, the large-scale fluctuations and false positives brought about by conventional detection methods will further affect the diagnosis and treatment of the disease.

Therefore, it is necessary to further study the factors affecting the hydrolysis process of HIV retroactivator protein (Tat protein that induces HIV-1) and the corresponding detection methods.

Contents of the invention

In view of this, one of the purposes of the present invention is to provide the application of metal ions in the Tat proteolysis affecting the induction of HIV-1; the second purpose of the present invention is to provide a method for detecting the Tat proteolysis of HIV-1 induced by metal ions method.

To achieve the above object, the present invention provides the following technical solutions:

1. The application of metal ions in affecting the Tat proteolysis of HIV-1 induction, the application is specifically: copper ions have the effect of hindering the Tat proteolysis of HIV-1 induction, and calcium ions have the effect of promoting the Tat protein of HIV-1 induction The role of hydrolysis.

Preferably, the application is specifically: adding a basic buffer solution containing metal ions to the HIV-1-inducing Tat protein sample and the buffer solution containing protease, so that the metal ions can affect the HIV-1-inducing Tat protein sample. Protein hydrolysis.

Preferably, the HIV-1 induced Tat protein sample is prepared by dissolving HIV-1 Tat protein in enzyme-free water.

Further preferably, the metal ion is copper ion or calcium ion.

Preferably, the basic buffer solution is a solution containing alkali metal chloride and 4-hydroxyethylpiperazineethanesulfonic acid (HEPES).

Preferably, the protease has the effect of causing the proteolysis of Tat induced by HIV-1.

Further preferably, the protease is any one or more of trypsin, chymotrypsin or apoptosis protease.

Preferably, the basic buffer solution containing metal ions is a solution formed by dissolving the metal chloride in the basic buffer solution;

When the metal ion is copper ion, the concentration of copper ion is 1.0-160 μM;

When the metal ion is calcium ion, the concentration of calcium ion is 1.0-9.0 mM.

Preferably, the concentration of alkali metal chloride in the basic buffer solution is 0.1-1M, the concentration of 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) is 1-10mM, and the solvent in the buffer solution is deionized water .

Preferably, the alkali metal chloride is any one of potassium chloride, lithium chloride or sodium chloride.

2. A method for detecting metal ions affecting the hydrolysis of Tat proteolysis inducing HIV-1, said method being specifically:

(1) The Tat protein sample that induces HIV-1 is placed in the nanopore electrochemical detection and analysis device, and the basic buffer solution is used as the electrolyte. Under the effect of an external electric field, the current signal is collected. Analyze the characteristics of the current block time and obtain the detection result I of Tat protein;

(2) After the signal frequency of the current signal in step (1) is stable, continue to add buffer solution containing protease in the nanopore detection and analysis device, collect the current signal, and analyze the amplitude and current block time characteristics of the current signal, Get Tat proteolysis test result II;

(3) Place the Tat protein sample that induces HIV-1 in the same nanopore electrochemical detection and analysis device as in step (1), adopt a basic buffer solution containing metal ions as the electrolyte, and under the action of an external electric field, Collect the current signal, analyze the amplitude and current block time characteristics of the current signal, and obtain the Tat protein detection result III;

(4) After the signal frequency of the current signal in step (1) is stable, continue to add the buffer solution containing protease in the nanopore detection and analysis device, collect the current signal, analyze the amplitude and current block time characteristics of the current signal, Get the Tat protein detection result IV;

(5) Compare the Tat protein detection result II with the Tat protein detection result I, Tat protein detection result II, Tat protein detection result I and Tat protein detection result IV, and the two comparison results can reflect the effect of metal ions on the induction of HIV- 1 Effect of Tat proteolysis.

Preferably, the alkali metal chloride concentration in the basic buffer solution in step (1) is 0.1-1M, and the concentration of 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) is 1-10mM;

The solvent in the buffer solution is deionized water, and the pH of the basic buffer solution is 8.0;

The alkali metal chloride is any one of potassium chloride, lithium chloride or sodium chloride; the basic buffer solution containing metal ions described in step (3) is that the metal chloride is dissolved in the basic A solution formed in a buffer solution, the metal ion is calcium ion or copper ion, when the metal ion is copper ion, the concentration of copper ion is 1.0-160 μm; when the metal ion is calcium ion, the concentration of calcium ion is 1.0 ~9.0mM;

The protease has the effect of causing the hydrolysis of the HIV-1-inducing Tat protein, and the protease is any one or more of trypsin, chymotrypsin or apoptosis protease.

Preferably, the nanopore electrochemical detection and analysis device is specifically: placing the support film 6 containing the nanopore 7 in the solution chamber 5 containing the electrolyte, and placing the electrode I1, the ammeter 2, the negative pole, the positive pole, and the electrode of the power supply 3 II4 are connected in sequence, wherein the electrode I1 and the electrode II4 are placed in the solution chamber 5 and respectively located on both sides of the support film 6, forming a nanopore electrochemical detection and analysis device;

The material of the support film is any one of graphene, graphene oxide, silicon nitride, molybdenum disulfide or biological nanopores and glass nanopores.

Preferably, a low-noise current amplifier (Axon Axopatch 200B) is used to amplify the current signal before analyzing the amplitude and current block time characteristics of the current signal.

Preferably, the molar ratio of the HIV-1-inducing Tat protein in the HIV-1-inducing Tat protein sample to the protease in the buffer containing protease is 5000:1-1000:1;

The molar ratio of the HIV-1-inducing Tat protein in the HIV-1-inducing Tat protein sample to the metal ion in the basic buffer solution containing metal ions is 1:0.1-16.

The beneficial effects of the present invention are:

1. The present invention discloses the application of metal ions in affecting the hydrolysis of Tat proteolysis induced by HIV-1. By recording the addition of metal ions (copper ions or calcium ions), the Tat protein induced by HIV-1 is nanopored before and after trypsin is added. Electrochemical detection and analysis of current signal changes in the device, real-time observation of the impact of metal ions on the induction of HIV-1 Tat proteolysis, and thus concluded that copper ions have the effect of hindering the induction of HIV-1 Tat proteolysis, and calcium ions can promote the induction of HIV-1 -1 Tat proteolysis.

2. The present invention uses a nanopore electrochemical detection and analysis device to detect the effect of metal ions (copper ions or calcium ions) on the Tat proteolysis of HIV-1, fully utilizing the advantages of the nanopore electrochemical detection device for real-time detection, accurate Record small current signal changes. The detection method provided by the invention is simple to operate, highly reproducible, and reliable in detection results. On this basis, the HIV-1 can be inhibited by destroying the translocation of the Tat protein that induces HIV-1 on the basis of an enzyme kinetics method. Infection has broad application prospects.

Other advantages, objects and features of the present invention will be set forth in the following description to some extent, and to some extent, will be obvious to those skilled in the art based on the investigation and research below, or can be obtained from It is taught in the practice of the present invention. The objects and other advantages of the invention may be realized and attained by the following specification.

Description of drawings

In order to make the purpose of the present invention, technical solutions and advantages clearer, the present invention will be described in detail below in conjunction with the accompanying drawings, wherein:

Fig. 1 is the nanopore electrochemical detection analysis device that the present invention adopts, wherein 1 is electrode I, 2 is ammeter, 3 is power supply, 4 is electrode II, 5 is solution chamber, 6 is supporting film, 7 is nanopore;

Fig. 2 is the amplitude of current signal under different stages in embodiment 1 and the analysis result of current block time characteristic;

Fig. 3 is the influence of copper ion on the Tat proteolysis rate of inducing HIV-1;

Fig. 4 is the amplitude of current signal under different stages in embodiment 2 and the analysis result of current block time characteristic;

Figure 5 is the effect of calcium ions on the Tat proteolysis rate inducing HIV-1;

Fig. 6 is the detection process of the present invention's research metal ion (copper ion or calcium ion) to the Tat proteolysis influence that induces HIV-1;

Fig. 7 shows the mechanism by which metal ions (copper ions or calcium ions) affect the hydrolysis of Tat proteolysis induced by HIV-1.

Detailed ways

Embodiments of the present invention are described below through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific implementation modes, and various modifications or changes can be made to the details in this specification based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, in the case of no conflict, the following embodiments and features in the embodiments can be combined with each other.

Example 1

To study the effect of copper ions on the Tat proteolysis induced by HIV-1, the specific method is as follows:

1. Prepare the solution:

(1) Prepare basic buffer solution: dissolve potassium chloride and 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) in deionized water, and adjust the pH=8.0 of the solution to form potassium chloride at a concentration of 1M, 4 The concentration of -hydroxyethylpiperazineethanesulfonic acid (HEPES) is a basic buffer solution of 5 mM;

(2) Preparation of a basic buffer solution containing copper ions: dissolving copper chloride in the basic buffer solution prepared above to form a solution with a copper ion concentration of 1.0uM;

(3) Preparation of HIV-1-inducing Tat protein samples: dissolving HIV-1 Tat protein in enzyme-free water to form a solution with a concentration of 10 mM inducing HIV-1 Tat protein, and placing it in a refrigerator at -20°C for later use;

(4) Preparation of buffer solution containing trypsin: Dissolve trypsin in enzyme-free water to obtain a stock solution with a concentration of 4.2uM, and store it in a -20°C refrigerator for later use.

2. Prepare the nanopore electrochemical detection and analysis device: place the supporting film 6 (using silicon nitride as material) containing the nanopore 7 (the aperture of the nanopore is 2 to 10 nm) in the solution chamber 5, place the electrodes I1, The ammeter 2, the negative pole of the power supply 3, the positive pole of the power supply 3, and the electrode II4 are connected in sequence, wherein the electrode I and the electrode II are placed in the solution chamber and are respectively located on both sides of the supporting film to form a nanopore electrochemical detection and analysis device. As shown in Figure 1.

3. Detect the hydrolysis of HIV-1-inducing Tat egg in trypsin:

(1) Add the prepared HIV-1-inducing Tat protein sample to the above-mentioned nanopore electrochemical detection and analysis device, adopt the basic buffer solution prepared above as the electrolyte, and collect the current signal through the ammeter under the action of an external electric field (induces the Tat protein of HIV-1);

(2) After the signal frequency of the above-mentioned current signal is stable, continue to add buffer solution containing trypsin to the nanopore detection and analysis device in step (1) (wherein the Tat protein sample that induces HIV-1 induces HIV-1 Tat protein and trypsin in the buffer solution containing trypsin are 5000:1), collect current signal by ammeter (add trypsin);

(3) Add the Tat protein sample prepared to induce HIV-1 to the above-mentioned nanopore electrochemical detection and analysis device (as shown in Figure 1, wherein 1 is electrode I, 2 is ammeter, 3 is power supply, 4 is electrode II, 5 is a solution chamber, 6 is a supporting film, and 7 is a nanopore), the above-mentioned basic buffer solution containing copper ions is used as the electrolyte (the Tat protein that induces HIV-1 in the Tat protein sample that induces HIV-1 and the mol ratio of copper ions in the basic buffer solution containing copper ions is 10:1), and acts under an applied electric field;

(4) After the signal frequency of the current signal in the above step (3) is stable, use the same nanopore detection and analysis device as in the step (1), and continue to add a buffer solution containing trypsin (wherein the Tat protein of HIV-1 is induced The molar ratio of the Tat protein that induces HIV-1 in the sample to the trypsin in the buffer containing trypsin is 5000:1), and the current signal is collected by an ammeter (adding copper ions+trypsin);

(5) Use a low-noise current amplifier (Axon Axopatch 200B) to amplify the current signals collected in step (1), step (2) and step (4), and then perform the amplitude and current block time characteristics of the current signal Analysis, obtain corresponding Tat protein detection result respectively, as shown in Figure 2;

(6) Comparing the detection results of Tat protein with the detection results of Tat protein added with trypsin, it can be seen that the Tat protein that induces HIV-1 is under the action of trypsin, and the current signal produced by the Tat protein that induces HIV-1 decreases, indicating that adding trypsin Protease can hydrolyze the Tat protein that induces HIV-1; comparing the detection results of Tat protein, the detection result of Tat protein with trypsin and the detection result of Tat protein with copper ion+trypsin, it can be seen that after adding copper ion, The Tat protein that induces HIV-1 still has more Tat protein signals under the action of trypsin, indicating that the addition of copper ions reduces the hydrolysis of the Tat protein that induces HIV-1, as shown in Figure 2. It can be seen that copper ions The addition of can hinder the hydrolysis of Tat protein induced by HIV-1.

Fig. 3 shows the hydrolysis rate of HIV-1-inducing Tat protein under the action of trypsin over time and the change of hydrolysis rate of HIV-1-inducing Tat protein under the action of trypsin in the presence of copper ions over time. It can be seen from Figure 3 that the Tat proteolysis rate of HIV-1 was significantly decreased after adding copper ions under the same conditions, further indicating that copper ions have the effect of hindering the Tat proteolysis of HIV-1.

Example 2

To study the effect of calcium ions on the hydrolysis of Tat proteolysis induced by HIV-1, the specific method is as follows:

1. Prepare the solution:

(1) Prepare basic buffer solution: dissolve potassium chloride and 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) in deionized water, and adjust the pH=8.0 of the solution to form potassium chloride at a concentration of 1M, 4 The concentration of -hydroxyethylpiperazineethanesulfonic acid (HEPES) is a basic buffer solution of 5 mM;

(2) Preparation of a basic buffer solution containing calcium ions: magnesium chloride is dissolved in the basic buffer solution prepared above, and the calcium ion concentration formed is a solution of 1.0 mM;

(3) Preparation of HIV-1-inducing Tat protein sample: dissolving HIV-1-inducing Tat protein in enzyme-free water to form a solution with a concentration of HIV-1-inducing Tat protein of 10 mM, and placing it in a refrigerator at -20°C for later use ;

(4) Preparation of buffer solution containing trypsin: Dissolve trypsin in enzyme-free water to obtain a stock solution with a concentration of 4.2uM, and store it in a -20°C refrigerator for later use.

2. Prepare the nanopore electrochemical detection and analysis device: place the supporting film 6 (using silicon nitride as a material) containing the nanopore 7 (the aperture of the nanopore is 2 to 10nM) in the solution chamber 5, place the electrode I1, the ammeter 2. The negative pole of the power supply 3, the positive pole of the power supply 3, and the electrode II4 are connected in sequence, wherein the electrode I and the electrode II are placed in the solution chamber and are respectively located on both sides of the supporting film to form a nanopore electrochemical detection and analysis device, such as Figure 1 shows.

3. Detect the hydrolysis of HIV-1-inducing Tat egg in trypsin:

(1) Add the prepared HIV-1-inducing Tat protein sample to the above-mentioned nanopore electrochemical detection and analysis device, adopt the basic buffer solution prepared above as the electrolyte, and collect the current signal through the ammeter under the action of an external electric field (induces the Tat protein of HIV-1);

(2) After the signal frequency of the above-mentioned current signal is stable, continue to add buffer solution containing trypsin to the nanopore detection and analysis device in step (1) (wherein the Tat protein sample that induces HIV-1 induces HIV-1 Tat protein and trypsin in the buffer solution containing trypsin are 5000:1), collect current signal by ammeter (add trypsin);

(3) Add the prepared Tat protein sample for inducing HIV-1 to the above-mentioned nanopore electrochemical detection and analysis device, and use the above-mentioned basic buffer solution containing calcium ions as the electrolyte (wherein the Tat protein sample for inducing HIV-1 The molar ratio of calcium ions in the Tat protein that induces HIV-1 in the basic buffer solution containing calcium ions is 1:100), and acts under the applied electric field;

(4) After the signal frequency of the above-mentioned current signal is stable, use the same nanopore detection and analysis device as in step (1), continue to add buffer solution containing trypsin (wherein the Tat protein sample that induces HIV-1 induces HIV-1 The molar ratio of the Tat protein of 1 and the trypsin in the buffer containing trypsin is 5000:1), and the current signal is collected by the ammeter (adding calcium ion+trypsin);

(5) Use a low-noise current amplifier (Axon Axopatch 200B) to amplify the current signals collected in step (1), step (2) and step (4), and then perform the amplitude and current block time characteristics of the current signal analysis, respectively to obtain the corresponding Tat protein detection results, as shown in Figure 4;

(6) Comparing the detection results of Tat protein with the detection results of Tat protein added with trypsin, it can be seen that the Tat protein that induces HIV-1 is under the action of trypsin, and the current signal produced by the Tat protein that induces HIV-1 decreases, indicating that adding trypsin Protease can hydrolyze the Tat protein that induces HIV-1; comparing the detection results of Tat protein, the detection result of Tat protein with trypsin, and the detection result of Tat protein with calcium ion+trypsin, it can be seen that after adding calcium ion, The Tat protein signal that induces HIV-1 under the action of trypsin almost disappears, indicating that after adding calcium ions, the degree of hydrolysis of the Tat protein that induces HIV-1 is further enhanced under the action of trypsin, as shown in Figure 4 . It can be seen that the addition of calcium ions can promote the hydrolysis of Tat protein induced by HIV-1.

Fig. 5 shows the hydrolysis rate of HIV-1-inducing Tat protein under the action of trypsin over time and the hydrolysis rate of HIV-1-inducing Tat protein under the action of trypsin in the presence of calcium ions. It can be seen from Figure 5 that the addition of calcium ions under the same conditions induces a significant increase in the Tat proteolysis rate of HIV-1, further illustrating that calcium ions can promote the Tat proteolysis of HIV-1.

Potassium chloride in the basic buffer solution in the above-mentioned embodiment 1 and embodiment 2 can also be replaced by any one in lithium chloride or sodium chloride, and the material of the supporting film can also be replaced by graphene, graphene oxide, bismuth Any one of molybdenum sulfide or glass nanopores, the concentration of metal ions in the base buffer solution containing metal ions is replaced within the range of 1.0-9.0mM, and the Tat protein that induces HIV-1 in the Tat protein sample that induces HIV-1 and contains The molar ratio of trypsin in the buffer solution of trypsin is replaced within the scope of 5000～1000:1, and the trypsin adopted in the embodiment can be replaced with chymotrypsin or apoptosis protease etc. and can trigger and induce HIV-1 Tat protein The hydrolyzed protease and the above substitutions can all obtain the same effect of metal ions on the hydrolysis of Tat protein induced by HIV-1.

In addition, the concentration of copper ions in the basic buffer solution containing copper ions in Example 1 can be in the range of 1.0-160 μm. During the detection process, the Tat protein that induces HIV-1 and the Tat protein containing copper The molar ratio of copper ions in the basic buffer solution of ions can hinder the hydrolysis of the Tat protein sample induced by HIV-1 in the range of 1:0.1～16; the copper ions in the basic buffer solution containing calcium ions in Example 2 The concentration can be in the range of 1.0 ~ 9.0mM. During the detection process, the molar ratio of the Tat protein that induces HIV-1 in the Tat protein sample that induces HIV-1 to the calcium ion in the basic buffer solution containing calcium ion is 1:100. In the range of ~900, it can induce the hydrolysis of HIV-1 Tat protein samples.

The present invention studies the influence of metal ions (copper ions or calcium ions) on the Tat proteolysis induced by HIV-1, and the detection process is shown in FIG. 6 . HIV virus reverse transcription activator protein Tat (induces HIV-1 Tat protein), transactivates viral transcription and promotes viral replication. It can pass through the cell membrane structure, carry foreign virus molecules into normal cells, and can promote the high expression of foreign proteins, which plays a key role in HIV replication, so in the process of inducing the hydrolysis of HIV-1 Tat protein through Adding metal ions (copper ions or calcium ions) can improve the high-efficiency expression of foreign proteins that can affect the Tat protein that induces HIV-1 through the mechanism of action shown in Figure 7 .

In summary, the present invention discloses the application of metal ions in the hydrolysis of Tat protein affecting the induction of HIV-1 and its detection method. The current signals in the nanopore electrochemical detection and analysis device before and after hydrolysis are respectively recorded to induce HIV-1. 1's Tat proteolysis; and by replacing the basic buffer solution with metal ions (copper ions or calcium ions), the effects of different metal ions on the Tat proteolysis process induced by HIV-1 were studied.

The present invention uses a nanopore electrochemical detection and analysis device to detect the effect of metal ions (copper ions or calcium ions) on the hydrolysis of HIV-1 Tat proteolysis, fully utilizes the advantages of real-time detection by the nanopore electrochemical detection device, and accurately records tiny The current signal changes. The detection method provided by the invention is simple to operate, highly reproducible, and reliable in detection results. On this basis, the HIV-1 can be inhibited by destroying the translocation of the Tat protein that induces HIV-1 on the basis of an enzyme kinetics method. Infection has broad application prospects.

Finally, it is noted that the above embodiments are only used to illustrate the technical solutions of the present invention without limitation. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be carried out Modifications or equivalent replacements, without departing from the spirit and scope of the technical solution, should be included in the scope of the claims of the present invention.

Claims (5)

1. A method for detecting influence of metal ions on induction of Tat proteolysis of HIV-1 is characterized by comprising the following steps:

(1) Placing a Tat protein sample for inducing HIV-1 in a nanopore electrochemical detection analysis device, collecting a current signal by using a basic buffer solution as an electrolyte under the action of an external electric field, and analyzing the amplitude and the current blocking time characteristics of the current signal to obtain a Tat protein detection result I;

(2) After the signal frequency of the current signal in the step (1) is stable, continuing adding a buffer solution containing protease into the nanopore detection analysis device, collecting the current signal, and performing amplitude and current blocking time characteristic analysis on the current signal to obtain a Tat proteolysis detection result II;

(3) Placing a Tat protein sample for inducing HIV-1 in a nanopore electrochemical detection analysis device which is the same as that in the step (1), collecting a current signal by using a basic buffer solution containing metal ions as an electrolyte under the action of an external electric field, and analyzing the amplitude and the current blocking time characteristic of the current signal to obtain a Tat protein detection result III;

(4) After the signal frequency of the current signal in the step (1) is stable, continuing adding a buffer solution containing protease into the nanopore detection analysis device, collecting the current signal, and analyzing the amplitude and the current blocking time characteristic of the current signal to obtain a Tat protein detection result IV;

(5) And respectively comparing the Tat protein detection result II with the Tat protein detection result I, the Tat protein detection result II and the Tat protein detection result I with the Tat protein detection result IV, wherein the two comparison results can reflect the influence of metal ions on the induction of the hydrolysis of the Tat protein of the HIV-1.

2. The method according to claim 1, wherein the concentration of alkali metal chloride in the basic buffer solution in step (1) is 0.1 to 1M, and the concentration of 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) is 1 to 10mM;

the solvent in the buffer solution is deionized water, and the pH of the basic buffer solution is =8.0;

the alkali metal chloride is any one of potassium chloride, lithium chloride or sodium chloride; the basic buffer solution containing metal ions in the step (3) is a solution formed by dissolving the metal chloride in the basic buffer solution, the metal ions are calcium ions or copper ions, and when the metal ions are copper ions, the concentration of the copper ions is 1.0-160 μm: when the metal ion is calcium ion, the concentration of the calcium ion is 1.0-9.0 mM;

the protease has the function of inducing the hydrolysis of the Tat protein of the HIV-1, and the protease is any one or more of trypsin, chymotrypsin or apoptosis protease.

3. The method according to claim 1, wherein the nanopore electrochemical detection analysis device is specifically: placing a supporting film (6) containing a nanopore (7) in a solution chamber (5) containing electrolyte, and sequentially connecting an electrode I (1), an ammeter (2), a negative electrode, a positive electrode and an electrode II (4) of a power supply (3), wherein the electrode I (1) and the electrode II (4) are placed in the solution chamber (5) and are respectively positioned on two sides of the supporting film (6) to form a nanopore electrochemical detection analysis device;

the material of the support film is any one of graphene, graphene oxide, silicon nitride, molybdenum disulfide or biological nano-pores and glass nano-pores.

4. The method of claim 1, wherein the current signal is first amplified using a low noise current amplifier before the analysis of the amplitude and current block time characteristics of the current signal is performed.

5. The method according to claim 1, wherein the molar ratio of HIV-1 inducing Tat protein in the HIV-1 inducing Tat protein sample to protease in the protease containing buffer is 5000 to 1000;

the molar ratio of the HIV-1-inducing Tat protein in the HIV-1-inducing Tat protein sample to the metal ions in the basic buffer solution containing the metal ions is 1.

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