CN112505126A - Application of metal ions in influencing HIV-1 induced Tat proteolysis and detection method thereof - Google Patents
Application of metal ions in influencing HIV-1 induced Tat proteolysis and detection method thereof Download PDFInfo
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Abstract
The invention relates to application of metal ions in influencing induction of hydrolysis of Tat protein of HIV-1 and a detection method thereof, belonging to the field of biological detection and biological medicine. The invention discloses an application of metal ions in influence induction of the hydrolysis of Tat protein of HIV-1, wherein the influence of the metal ions on the induction of the hydrolysis of Tat protein of HIV-1 is observed in real time by recording the change of current signals of Tat protein of HIV-1 induced in a nanopore electrochemical detection analysis device before and after trypsin is added due to the addition of the metal ions (copper ions or calcium ions); the nanopore electrochemical detection analysis device and the nanopore electrochemical detection analysis method for detecting the influence effect fully play the advantages of real-time detection of the nanopore electrochemical detection device, accurately record tiny current signal changes, and are simple to operate, high in implementation reproducibility and reliable in detection result.
Description
Technical Field
The invention belongs to the field of biological detection and biological medicine, and particularly relates to application of metal ions in influencing induction of HIV-1 Tat proteolysis and a detection method thereof.
Background
Acquired immunodeficiency syndrome, AIDS, is a disease caused by infection with the Human Immunodeficiency Virus (HIV). The disease attacks human CD4T lymphocytes, the CD4T cells of AIDS patients show progressive or irregular reduction, partial or whole immune function of the patients is lost, other diseases are caused secondarily, and the clinical manifestations of the patients are various. The disease is widely concerned in all social circles due to the reasons of high propagation speed, high fatality rate, incapability of curing and the like. HIV virus reverse transcription activator protein Tat (hereinafter collectively referred to as Tat protein for inducing HIV-1) transactivates viral transcription, and promotes viral replication. It can cross cell membrane structure, carry exogenous virus molecule into normal cell, promote high-efficiency expression of exogenous protein and play a key role in HIV replication. The Tat protein for inducing HIV-1 is taken as one of the earliest expressed proteins of HIV, and the detection of the Tat protein for inducing HIV-1 can advance the window phase of HIV infection and provide assistance for early diagnosis of diseases. Meanwhile, the research on the hydrolysis of the reverse transcription activator protein Tat protein is helpful for understanding the regulation effect of the Tat protein for inducing HIV-1 in the transcription process of HIV-1, the transmembrane transport of the Tat protein for inducing HIV-1 is damaged, and the Tat protein for inducing HIV-1 is used as a new target of anti-AIDS drugs to provide theoretical and experimental support.
At present, the conventional detection method based on HIV is complex in operation, and the most common method is enzyme-linked immunosorbent assay (ELISA), including immunoblotting assay (WB), strip immunoassay (LIATKE HIV III), radioimmunoprecipitation assay (RIPA), immunofluorescence assay (IFA) and the like. The detection methods can provide screening and confirmation bases to a certain extent, but in different HIV infection stages, the wide-range fluctuation and false positive brought by the conventional detection methods further influence the diagnosis and treatment of diseases.
Therefore, further studies on the influencing factors in the hydrolysis of the HIV virus reverse transcriptase activator protein (Tat protein which induces HIV-1) and the corresponding detection methods are needed.
Disclosure of Invention
In view of the above, an object of the present invention is to provide the use of metal ions in inducing Tat proteolysis in HIV-1; the second objective of the invention is to provide a method for detecting the metal ion-induced hydrolysis of Tat protein of HIV-1.
In order to achieve the purpose, the invention provides the following technical scheme:
1. the application of metal ions in influencing induction of HIV-1 Tat proteolysis specifically comprises the following steps: the copper ions have the function of preventing the induction of the hydrolysis of the Tat protein of the HIV-1, and the calcium ions have the function of promoting the induction of the hydrolysis of the Tat protein of the HIV-1.
Preferably, the application specifically comprises: the hydrolysis of HIV-1-inducing Tat protein can be influenced by adding metal ions to the HIV-1-inducing Tat protein sample and the protease-containing buffer solution in the basic buffer solution.
Preferably, the HIV-1-inducing Tat protein sample is prepared by dissolving the Tat protein of HIV-1 in enzyme-free water.
Further preferably, the metal ion is a copper ion or a calcium ion.
Preferably, the base buffer solution is a solution containing alkali metal chloride and 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES).
Preferably, said protease has the effect of causing said induction of Tat proteolysis by HIV-1.
Further preferably, the protease is any one or more of trypsin, chymotrypsin and 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 ions are copper ions, the concentration of the copper ions is 1.0-160 mu M;
when the metal ion is calcium ion, the concentration of calcium ion is 1.0-9.0 mM.
Preferably, the concentration of the alkali metal chloride in the basic buffer solution is 0.1-1M, the concentration of the 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) is 1-10 mM, 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 influence of metal ions on induction of Tat proteolysis of HIV-1 comprises 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 characteristic 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.
Preferably, the concentration of the alkali metal chloride in the basic buffer solution in the step (1) is 0.1-1M, and the concentration of 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) is 1-10 mM;
the solvent in the buffer solution is deionized water, and the pH value 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 ions are calcium ions, the concentration of the calcium ions 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.
Preferably, the nanopore electrochemical detection analysis device specifically comprises: placing a supporting film 6 containing a nanopore 7 in a solution chamber 5 containing electrolyte, and sequentially connecting an electrode I1, an ammeter 2, a negative electrode, a positive electrode and an electrode II 4 of a power supply 3, wherein the electrode I1 and the electrode II 4 are placed in the solution chamber 5 and are respectively positioned on two sides of the supporting film 6, so as to form a nanopore electrochemical detection analysis device;
the material of the supporting film is any one of graphene, graphene oxide, silicon nitride, molybdenum disulfide or biological nano-pores and glass nano-pores.
Preferably, the current signal is first amplified using a low noise current amplifier (Axon Axomatch 200B) before the amplitude and current block time signature analysis of the current signal is performed.
Preferably, the molar ratio of the HIV-1-inducing Tat protein in the HIV-1-inducing Tat protein sample to the protease in the protease-containing buffer solution is 5000: 1-1000: 1;
the molar ratio of the Tat protein for inducing HIV-1 in the Tat protein sample for inducing HIV-1 to the metal ions in the basic buffer solution containing the metal ions is 1: 0.1-16.
The invention has the beneficial effects that:
1. the invention discloses an application of metal ions in influence induction of HIV-1 Tat proteolysis, which is characterized in that the influence of the metal ions on the induction of HIV-1 Tat proteolysis is observed in real time by recording the current signal change of the HIV-1 Tat protein induced by the addition of the metal ions (copper ions or calcium ions) in a nanopore electrochemical detection analysis device before and after the addition of trypsin, so that the copper ions have the effect of preventing the induction of HIV-1 Tat proteolysis, and the calcium ions have the effect of promoting the induction of HIV-1 Tat proteolysis.
2. The invention adopts the nanopore electrochemical detection analysis device to detect the effect of the influence of metal ions (copper ions or calcium ions) on the hydrolysis of the Tat protein of the HIV-1, fully exerts the advantage of real-time detection of the nanopore electrochemical detection device and accurately records the change of a tiny current signal. The detection method provided by the invention is simple to operate, high in implementation reproducibility and reliable in detection result, can inhibit the infection of the HIV by destroying and inducing the transfer of the Tat protein of the HIV-1 on the basis of an enzyme kinetics method, and has wide application prospect.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention. The objectives and other advantages of the invention may be realized and attained by the means of the instrumentalities and combinations particularly pointed out hereinafter.
Drawings
For the purposes of promoting a better understanding of the objects, aspects and advantages of the invention, reference will now be made to the following detailed description taken in conjunction with the accompanying drawings in which:
FIG. 1 is a nanopore electrochemical detection analysis device employed in the present invention, wherein 1 is an electrode I, 2 is an ammeter, 3 is a power supply, 4 is an electrode II, 5 is a solution chamber, 6 is a support membrane, and 7 is a nanopore;
FIG. 2 shows the analysis results of the amplitude and current block time characteristics of the current signals at different stages in example 1;
FIG. 3 is a graph of the effect of copper ions on the induction of the rate of Tat proteolysis in HIV-1;
FIG. 4 shows the analysis results of the amplitude and current block time characteristics of the current signals at different stages in example 2;
FIG. 5 is a graph of the effect of calcium ions on the induction of the rate of Tat proteolysis in HIV-1;
FIG. 6 is a test procedure for investigating the effect of metal ions (copper or calcium) on the induction of Tat proteolysis in HIV-1 according to the present invention;
FIG. 7 is a mechanism by which metal ions (copper or calcium) influence the induction of Tat proteolysis by HIV-1.
Detailed Description
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention. It should be noted that, in the following embodiments, features in the embodiments may be combined with each other without conflict.
Example 1
The influence of copper ions on the induction of Tat proteolysis of HIV-1 was studied, using the following specific method:
1. preparing a solution:
(1) preparing a basic buffer solution: dissolving potassium chloride and 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) in deionized water, and adjusting the pH of the solution to 8.0 to form a basic buffer solution with a potassium chloride concentration of 1M and a 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) concentration of 5 mM;
(2) preparing a basic buffer solution containing copper ions: dissolving copper chloride in the prepared basic buffer solution to form a solution with the copper ion concentration of 1.0 uM;
(3) preparation of HIV-1-inducing Tat protein samples: dissolving the Tat protein of HIV-1 in enzyme-free water to form a solution with the concentration of inducing the Tat protein of HIV-1 being 10mM, and placing the solution in a refrigerator at the temperature of minus 20 ℃ for later use;
(4) preparing a buffer containing trypsin: trypsin was dissolved in enzyme-free water to give a stock solution with a concentration of 4.2uM, which was placed in a freezer at-20 ℃ for use.
2. Preparing a nanopore electrochemical detection analysis device: placing a supporting film 6 (made of silicon nitride) containing a nanopore 7 (the pore diameter of the nanopore is 2-10 nm) in a solution chamber 5, and sequentially connecting an electrode I1, an ammeter 2, a negative electrode of a power supply 3, a positive electrode of the power supply 3 and an electrode II 4, wherein the electrode I and the electrode II are placed in the solution chamber and are respectively positioned on two sides of the supporting film to form the nanopore electrochemical detection analysis device, as shown in FIG. 1.
3. Determination of HIV-1-inducing hydrolysis of Tat eggs in trypsin:
(1) adding the prepared HIV-1-induced Tat protein sample into the nanopore electrochemical detection analysis device, and collecting a current signal (HIV-1-induced Tat protein) through an ammeter under the action of an external electric field by using the prepared basic buffer solution as an electrolyte;
(2) after the signal frequency of the current signal is stabilized, continuously adding a buffer solution containing trypsin (wherein the ratio of trypsin in the Tat protein sample for inducing HIV-1 and the Tat protein sample for inducing HIV-1 in the buffer solution containing trypsin is 5000:1) into the nanopore detection analysis device in the step (1), and collecting the current signal through an ammeter (adding trypsin);
(3) adding the prepared HIV-1-inducing Tat protein sample into the nanopore electrochemical detection analysis device (as shown in figure 1, wherein 1 is an electrode I, 2 is an ammeter, 3 is a power supply, 4 is an electrode II, 5 is a solution chamber, 6 is a supporting film and 7 is a nanopore), adopting the prepared basic buffer solution containing copper ions as an electrolyte (wherein the molar ratio of the HIV-1-inducing Tat protein in the HIV-1-inducing Tat protein sample to the copper ions in the basic buffer solution containing the copper ions is 10:1), and acting under an external electric field;
(4) after the signal frequency of the current signal in the step (3) is stabilized, adopting a nanopore detection analysis device which is the same as that in the step (1), continuously adding a buffer solution containing trypsin (wherein the molar ratio of the Tat protein for inducing HIV-1 in the Tat protein sample for inducing HIV-1 to the trypsin in the buffer solution containing trypsin is 5000:1), and collecting the current signal through an ammeter (adding copper ions and trypsin);
(5) amplifying the current signals collected in the steps (1), (2) and (4) by using a low-noise current amplifier (Axon Azopatch 200B), and then respectively carrying out characteristic analysis on the amplitude and the current blocking time of the current signals to respectively obtain corresponding Tat protein detection results, as shown in FIG. 2;
(6) comparing the detection result of the Tat protein with the detection result of the Tat protein added with the trypsin shows that the current signal generated by the Tat protein of the induced HIV-1 is reduced under the action of the trypsin by the Tat protein of the induced HIV-1, which indicates that the Tat protein of the induced HIV-1 can be hydrolyzed by adding the trypsin; comparing the Tat protein detection result, the Tat protein detection result added with trypsin and the Tat protein detection result added with copper ions and trypsin, it can be known that after the copper ions are added, the Tat protein inducing HIV-1 still has more Tat protein signals under the action of trypsin, which indicates that the hydrolysis of the Tat protein inducing HIV-1 is reduced by the addition of the copper ions, as shown in FIG. 2, and therefore, the addition of the copper ions can block the hydrolysis of the Tat protein inducing HIV-1.
The change in the rate of hydrolysis of the Tat protein inducing HIV-1 by trypsin and the change in the rate of hydrolysis of the Tat protein inducing HIV-1 by trypsin in the presence of copper ions over time are shown in FIG. 3. As can be seen from FIG. 3, the Tat proteolysis rate of HIV-1 induced by the addition of copper ions under the same conditions is significantly reduced, further illustrating the effect of copper ions on inhibiting the Tat proteolysis of HIV-1.
Example 2
The influence of calcium ions on the induction of the hydrolysis of Tat protein of HIV-1 is studied by the following specific method:
1. preparing a solution:
(1) preparing a basic buffer solution: dissolving potassium chloride and 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) in deionized water, and adjusting the pH of the solution to 8.0 to form a basic buffer solution with a potassium chloride concentration of 1M and a 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) concentration of 5 mM;
(2) preparing a basic buffer solution containing calcium ions: dissolving magnesium chloride in the prepared basic buffer solution to form a solution with the calcium ion concentration of 1.0 mM;
(3) preparation of HIV-1-inducing Tat protein samples: dissolving the Tat protein of the induced HIV-1 into enzyme-free water to form a solution with the concentration of 10mM of the Tat protein of the induced HIV-1, and placing the solution in a refrigerator at the temperature of minus 20 ℃ for later use;
(4) preparing a buffer containing trypsin: trypsin was dissolved in enzyme-free water to give a stock solution with a concentration of 4.2uM, which was placed in a freezer at-20 ℃ for use.
2. Preparing a nanopore electrochemical detection analysis device: placing a supporting film 6 (silicon nitride is used as a material) containing a nanopore 7 (the pore diameter of the nanopore is 2-10 nM) in a solution chamber 5, and sequentially connecting an electrode I1, an ammeter 2, a negative electrode of a power supply 3, a positive electrode of the power supply 3 and an electrode II 4, wherein the electrode I and the electrode II are placed in the solution chamber and are respectively positioned on two sides of the supporting film to form the nanopore electrochemical detection analysis device, as shown in FIG. 1.
3. Determination of HIV-1-inducing hydrolysis of Tat eggs in trypsin:
(1) adding the prepared HIV-1-induced Tat protein sample into the nanopore electrochemical detection analysis device, and collecting a current signal (HIV-1-induced Tat protein) through an ammeter under the action of an external electric field by using the prepared basic buffer solution as an electrolyte;
(2) after the signal frequency of the current signal is stabilized, continuously adding a buffer solution containing trypsin (wherein the ratio of trypsin in the Tat protein sample for inducing HIV-1 and the Tat protein sample for inducing HIV-1 in the buffer solution containing trypsin is 5000:1) into the nanopore detection analysis device in the step (1), and collecting the current signal through an ammeter (adding trypsin);
(3) adding the prepared HIV-1-inducing Tat protein sample into the nanopore electrochemical detection analysis device, using the prepared basic buffer solution containing calcium ions as electrolyte (wherein the molar ratio of the Tat protein inducing HIV-1 in the HIV-1-inducing Tat protein sample to the calcium ions in the basic buffer solution containing the calcium ions is 1:100), and acting under an external electric field;
(4) after the signal frequency of the current signal is stable, adopting a nanopore detection analysis device which is the same as that in the step (1), continuously adding a buffer solution containing trypsin (wherein the molar ratio of Tat protein for inducing HIV-1 in a Tat protein sample for inducing HIV-1 to trypsin in the buffer solution containing trypsin is 5000:1), and collecting the current signal through an ammeter (calcium ions and trypsin are added);
(5) amplifying the current signals collected in the steps (1), (2) and (4) by using a low-noise current amplifier (Axon Axomatch 200B), and then respectively carrying out characteristic analysis on the amplitude and the current blocking time of the current signals to respectively obtain corresponding Tat protein detection results, as shown in FIG. 4;
(6) comparing the detection result of the Tat protein with the detection result of the Tat protein added with the trypsin shows that the current signal generated by the Tat protein of the induced HIV-1 is reduced under the action of the trypsin by the Tat protein of the induced HIV-1, which indicates that the Tat protein of the induced HIV-1 can be hydrolyzed by adding the trypsin; comparing the Tat protein detection result, the Tat protein detection result with trypsin and the Tat protein detection result with calcium ion and trypsin, it can be seen that after the calcium ion is added, the Tat protein signal of induced HIV-1 almost disappears under the action of trypsin, which shows that after the calcium ion is added, the hydrolysis degree of the Tat protein of induced HIV-1 under the action of trypsin is further enhanced, as shown in FIG. 4. It can be seen that the addition of calcium ions can promote the induction of hydrolysis of the Tat protein of HIV-1.
The change in the rate of hydrolysis of the Tat protein inducing HIV-1 by trypsin and the change in the rate of hydrolysis of the Tat protein inducing HIV-1 by trypsin in the presence of calcium ions over time are shown in FIG. 5. As can be seen from FIG. 5, the Tat proteolysis rate of HIV-1 induced by the addition of calcium ion under the same conditions was significantly increased, further illustrating the effect of calcium ion in promoting the Tat proteolysis of HIV-1.
In the above embodiments 1 and 2, the potassium chloride in the basic buffer solution can be replaced by any one of lithium chloride or sodium chloride, the material of the supporting film can be replaced by any one of graphene, graphene oxide, molybdenum disulfide or glass nanopores, the concentration of metal ions in a basic buffer solution containing metal ions is replaced within the range of 1.0-9.0 mM, the molar ratio of Tat protein inducing HIV-1 in a Tat protein sample inducing HIV-1 to trypsin in a buffer solution containing trypsin is replaced within the range of 5000-1000: 1, the trypsin adopted in the embodiment can be replaced by protease such as chymotrypsin or apoptosis protease which can initiate the hydrolysis of Tat protein inducing HIV-1, and the effect of the same metal ions on the hydrolysis of Tat protein inducing HIV-1 can be obtained by the replacement.
In addition, the concentration of copper ions in the basic buffer solution containing copper ions in the embodiment 1 can be within the range of 1.0-160 μm, and in the detection process, the molar ratio of the Tat protein inducing HIV-1 in the Tat protein sample of the induced HIV-1 to the copper ions in the basic buffer solution containing copper ions is within the range of 1: 0.1-16, so that the inhibition effect on the hydrolysis of the Tat protein sample inducing HIV-1 can be generated; in example 2, the concentration of copper ions in the basic buffer solution containing calcium ions can be in the range of 1.0-9.0 mM, and in the detection process, the molar ratio of the Tat protein inducing HIV-1 in the Tat protein sample inducing HIV-1 to the calcium ions in the basic buffer solution containing calcium ions is in the range of 1: 100-900, and the hydrolysis of the Tat protein sample inducing HIV-1 can be promoted.
The present inventors studied the effect of metal ions (copper ions or calcium ions) on the induction of Tat proteolysis in HIV-1, and the detection process is shown in FIG. 6. The HIV virus reverse transcription activator protein Tat (inducing Tat protein of HIV-1) transactivates virus transcription and promotes virus replication. The protein can penetrate through a cell membrane structure, carries exogenous virus molecules into normal cells, can promote the high-efficiency expression of exogenous proteins, and plays a key role in HIV replication, so that the high-efficiency expression of the Tat proteins capable of inducing HIV-1 to the exogenous proteins can be improved by adding metal ions (copper ions or calcium ions) in the hydrolysis process of the Tat proteins for inducing HIV-1 and through an action mechanism shown in figure 7.
In conclusion, the invention discloses the application of metal ions in influencing the induction of the hydrolysis of the Tat protein of HIV-1 and a detection method thereof, and the real-time observation of the induction of the hydrolysis of the Tat protein of HIV-1 is carried out by respectively recording current signals in a nanopore electrochemical detection analysis device before and after the hydrolysis; and the influence of different metal ions on the induction of the Tat proteolysis process of HIV-1 was studied by replacing the basic buffer solution with one containing metal ions (copper or calcium ions).
The invention adopts the nanopore electrochemical detection analysis device to detect the effect of the influence of metal ions (copper ions or calcium ions) on the hydrolysis of the Tat protein of the HIV-1, fully exerts the advantage of real-time detection of the nanopore electrochemical detection device and accurately records the change of a tiny current signal. The detection method provided by the invention is simple to operate, high in implementation reproducibility and reliable in detection result, can inhibit the infection of the HIV by destroying and inducing the transfer of the Tat protein of the HIV-1 on the basis of an enzyme kinetics method, and has wide application prospect.
Finally, the above embodiments are only intended to illustrate the technical solutions of the present invention and not to limit the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions, and all of them should be covered by the claims of the present invention.
Claims (10)
1. The application of metal ions in influencing induction of hydrolysis of Tat protein of HIV-1 is characterized in that the application specifically comprises the following steps: the copper ions have the function of preventing the induction of the hydrolysis of the Tat protein of the HIV-1, and the calcium ions have the function of promoting the induction of the hydrolysis of the Tat protein of the HIV-1.
2. The application according to claim 1, characterized in that it is specifically: adding a basic buffer solution containing metal ions into a Tat protein sample for inducing HIV-1 and a buffer solution containing protease to enable the metal ions to influence the hydrolysis of Tat protein for inducing HIV-1;
the HIV-1-induced Tat protein sample is prepared by dissolving the Tat protein of HIV-1 in enzyme-free water;
the metal ions are copper ions or calcium ions;
the basic buffer solution is a solution containing alkali metal chloride and 4-hydroxyethyl piperazine ethanesulfonic acid;
the protease has the effect of causing the induction of Tat proteolysis by HIV-1.
3. The use according to claim 2, wherein the base buffer solution containing metal ions is a solution of metal chloride dissolved in the base buffer solution;
when the metal ions are copper ions, the concentration of the copper ions is 1.0-160 mu M;
when the metal ion is calcium ion, the concentration of calcium ion is 1.0-9.0 mM.
4. The use according to claim 3, wherein the concentration of alkali chloride in the basic buffer solution is 0.1-1M, the concentration of 4-hydroxyethylpiperazine ethanesulfonic acid is 1-10 mM, and the solvent in the buffer solution is deionized water.
5. Use according to claim 4, wherein the alkali metal chloride is any one of potassium chloride, lithium chloride or sodium chloride.
6. 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 characteristic 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.
7. The method according to claim 6, 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-hydroxyethylpiperazine ethanesulfonic acid (HEPES) is 1 to 10 mM;
the solvent in the buffer solution is deionized water, and the pH value 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 ions are calcium ions, the concentration of the calcium ions 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.
8. The method according to claim 6, 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 of a power supply (3), a positive electrode of the power supply and an electrode II (4), wherein the electrode I (1) and the electrode II (4) are placed in the solution chamber (5) and are respectively positioned at two sides of the supporting film (6) to form a nanopore electrochemical detection analysis device;
the material of the supporting film is any one of graphene, graphene oxide, silicon nitride, molybdenum disulfide or biological nano-pores and glass nano-pores.
9. The method of claim 6, 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.
10. The method according to claim 6, 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: 1-1000: 1;
the molar ratio of the Tat protein for inducing HIV-1 in the Tat protein sample for inducing HIV-1 to the metal ions in the basic buffer solution containing the metal ions is 1: 0.1-16.
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Citations (5)
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