CN118561971B - Protein mutant, nanopore detection system and use thereof - Google Patents

Abstract

The invention relates to the field of valproic acid detection, in particular to a protein mutant, a nanopore detection system and application thereof. The invention provides a mechanical force sensitive channel protein mutant of corynebacterium glutamicum, which at least comprises the following mutations: the amino acid sequences of the protein mutants A52R, T, R, A, R, Q, 45, R, L, 67R and A63R are SEQ ID NO. 2 or SEQ ID NO. 5. The invention also provides a nanopore detection system comprising the protein mutant, and application of the protein mutant or the nanopore detection system in preparation of a kit for directly detecting a sample comprising a valproic acid medicament. Compared with a wild type, the sensitivity of the protein mutant for detecting the valproic acid medicaments can be improved by 400 times, and further, the direct detection of a plasma sample and a saliva sample is realized.

Description

Protein mutant, nanopore detection system and use thereof

Technical Field

The present invention relates to the field of valproic acid detection, and in particular to a protein mutant, a nanopore detection system and uses thereof.

Background Art

Valproic acid (VPA) is a first-line broad-spectrum antiepileptic drug commonly used in clinical practice. It can effectively treat systemic, partial or other epilepsy. The blood concentration of valproic acid is the result of the combined effects of multiple factors such as the patient's pathological and physiological conditions, environment, and genetics. In addition to patient factors (for example, individual absorption and metabolism vary greatly), the blood concentration of valproic acid is susceptible to drug interactions, such as other drugs for mental illnesses such as neuroleptics and antidepressants, and other antiepileptic drugs such as phenobarbital and carbamazepine. In clinical practice, the blood concentration of valproic acid is usually controlled within the therapeutic window (50-100 μg/mL) to balance clinical efficacy and tolerability. The risk of adverse reactions increases significantly when the blood concentration is greater than 100 μg/mL, and <50 μg/mL may lead to clinical treatment failure. It can be seen that the clinical dosage of valproic acid is actually difficult to grasp. In order to maximize the balance between its clinical efficacy and adverse reactions, therapeutic drug monitoring (TDM) is essential for patients taking valproic acid. The implementation of TDM can maximize the safety and effectiveness of clinical medication and reduce possible adverse reactions.

Patients who take valproic acid for a long period of time, patients with large individual differences in pharmacodynamics and pharmacokinetic, patients with a narrow therapeutic window, and special patients (such as the elderly, pregnant women, and children) are all clear indications for TDM of valproic acid. However, for the above-mentioned patients who need close monitoring of therapeutic drugs, it is often necessary to collect plasma samples from them multiple times, which will further reduce the acceptance and cooperation of the above-mentioned patients, and is not conducive to close monitoring of therapeutic drugs for the above-mentioned patients. Chinese patent application CN115728353A discloses the use of MscCG in the preparation of a kit for detecting valproic acid drugs, and its detection limit for valproic acid drugs is 4 mM. In other words, wild-type MscCG cannot directly detect clinical samples (especially saliva samples with extremely low concentrations of valproic acid), thus limiting its practical application in clinical practice.

Summary of the invention

In the first aspect, the present invention provides a mutant of a mechanosensitive channel protein of Corynebacterium glutamicum, characterized in that the protein mutant comprises at least the following mutations compared with the mechanosensitive channel protein of the wild-type Corynebacterium glutamicum: A52R, T59R, A55R, Q45R, L67R and A63R (that is, the original amino acids at the following sites of the mechanosensitive channel protein of the wild-type Corynebacterium glutamicum are replaced with arginine: 52, 59, 55, 45, 67 and 63), and the amino acid sequence of the mechanosensitive channel protein of the wild-type Corynebacterium glutamicum is SEQ ID NO: 1. Compared with the mechanosensitive channel protein of the wild-type Corynebacterium glutamicum, the protein mutant has improved sensitivity in detecting valproic acid drugs.

In some embodiments, the amino acid sequence of the protein mutant is SEQ ID NO:2 or SEQ ID NO:5.

In some embodiments, the protein mutant has a sensitivity of 10 μM for detecting valproate drugs.

In a second aspect, the present invention provides a nanopore detection system, characterized in that the system comprises the above-mentioned protein mutant, an insulating membrane, a first medium and a second medium; wherein the protein mutant is embedded in the insulating membrane, the insulating membrane separates the first medium from the second medium, the protein mutant provides a channel connecting the first medium and the second medium, and the first medium and the second medium include an electrolyte solution.

In some embodiments, the first medium and the second medium include a lithium chloride solution, and a concentration of the lithium chloride solution includes 300 mM - 600 mM.

In some embodiments, the system further comprises a valproate drug, and the valproate drug is added to the first medium.

In some embodiments, the valproate drugs include valproic acid and pharmaceutically acceptable salts thereof.

In some embodiments, the concentration of the valproate drug is at least 10 μM.

In some embodiments, the pH of the first medium and the second medium is 8.0 - 8.5.

In a third aspect, the present invention provides use of the above-mentioned protein mutant or system in preparing a kit for directly detecting a sample containing valproic acid drugs.

In some embodiments, the sample is a plasma sample or a saliva sample.

In some embodiments, the valproate drug is sodium valproate.

In some embodiments, the sample is derived from a subject who has been treated with the valproate drug.

In some embodiments, the kit is used to determine the presence of the valproate drug in the sample.

In some embodiments, the kit is used to determine the concentration of the valproic acid drug in the sample. In some embodiments, the kit determines the concentration of the valproic acid drug based on the following linear equation: y=1.39x-1.33, where x represents the concentration of the valproic acid drug and y represents the signal frequency.

Compared with the prior art, the beneficial effects of the present invention include at least the following aspects:

The present invention provides a mechanosensitive channel protein mutant of Corynebacterium glutamicum. The mutation direction of the mechanosensitive channel protein of Corynebacterium glutamicum in the prior art is to enhance its ability to secrete glutamate, so as to increase the yield of glutamate. The prior art has not yet reported on improving the sensitivity of the mechanosensitive channel protein of Corynebacterium glutamicum as a nanopore.

The present invention breaks through the conventional mutation idea of mutating the original amino acids in the narrower area of the nanopore, and replaces arginine at specific sites (i.e., sites 52, 59, 55, 45, 67, and 63) in the middle of the transmembrane end of the mechanosensitive channel protein of wild-type Corynebacterium glutamicum. Compared with the mechanosensitive channel protein of wild-type Corynebacterium glutamicum, the sensitivity of the protein mutant provided by the present invention to detect valproic acid drugs can be increased by 400 times, thereby realizing direct detection of plasma samples and saliva samples. Therefore, in addition to being able to accurately reflect the level of free valproic acid in the subject and its fluctuation, the present invention also improves the acceptance and cooperation of the subject to a certain extent, which helps to more conveniently and long-term monitor the therapeutic drugs of subjects taking valproic acid.

In addition, the present invention alleviates the problem that valproic acid drugs easily cause blockage of the mechanical force-sensitive channel protein of Corynebacterium glutamicum in actual detection by optimizing the electrolyte solution and pH conditions of the first medium and the second medium, and further improves the detection effect of the protein mutant provided by the present invention on valproic acid drugs.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, each element or part is not necessarily drawn according to the actual scale. Obviously, the drawings described below are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without paying creative labor.

FIG1 is a graph showing the results of electrophysiological testing of three mutants designed in Example 1 and detection of 40 μM valproic acid;

FIG2 is a result diagram of potential key sites for the interaction between wild-type MscCG protein and valproic acid at the transmembrane end;

FIG3 is a diagram showing the results of screening the optimal mutant amino acid combination using kinetic simulation;

FIG4 is a result graph showing that the signal frequency obtained by transmembrane end mutant 2 detecting low concentration valproic acid is linearly related to the corresponding concentration of valproic acid;

FIG5 is a result graph showing that the transmembrane end mutant 2 cannot detect oxcarbazepine, levetiracetam and lamotrigine;

FIG6 is a graph showing the results of direct detection of clinical samples by transmembrane end mutant 2;

FIG7 is a result diagram showing the background signal of wild-type MscCG protein;

FIG8 is a graph showing the results that the wild-type MscCG protein cannot directly detect valproic acid at concentrations of 40 μM and below.

DETAILED DESCRIPTION

In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

Herein, "and/or" includes any and all combinations of one or more of the associated listed items.

Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

As used in this specification, the term "about" typically means +/- 5% of the stated value, more typically +/- 4% of the stated value, more typically +/- 3% of the stated value, more typically +/- 2% of the stated value, even more typically +/- 1% of the stated value, even more typically +/- 0.5% of the stated value.

In this specification, some embodiments may be disclosed in a format of being in a certain range. It should be understood that this description of "being in a certain range" is only for convenience and brevity, and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and independent numerical values within this range. For example, the description of the range 1-6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, such as 1, 2, 3, 4, 5 and 6. Regardless of the breadth of the range, the above rules apply.

Nanopore

The nanopore used in the present invention is a mechanosensitive channel protein of Corynebacterium glutamicum (MscCG) mutant. In some embodiments, the mutant is a non-naturally occurring variant produced by recombinant technology. In some embodiments, compared with the mechanosensitive channel protein of wild-type Corynebacterium glutamicum, the mutant includes at least the following mutations: A52R, T59R, A55R, Q45R, L67R and A63R. In some embodiments, the amino acid sequence of the mutant is SEQ ID NO: 2 or SEQ ID NO: 5. The following abbreviations are used for the 20 naturally occurring amino acids: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

Analytes

The analyte is a charged substance. If the analyte has a net charge, it is charged. The analyte can be negatively charged or positively charged. If the analyte has a net negative charge, it is negatively charged. If the analyte has a net positive charge, it is positively charged. As used herein, "pharmaceutically acceptable salt" refers to a salt of a specified compound that is safe, effective, and has the desired biological activity when applied topically to an individual. Pharmaceutically acceptable salts include salts of acidic or basic groups present in a specified compound. In some embodiments, the analyte is a valproate drug, which includes valproic acid and pharmaceutically acceptable salts thereof, such as sodium valproate and magnesium valproate.

Nanopore Detection System

The "nanopore detection system (referred to as "nanopore system")" includes a pore with nanoscale size (referred to as "nanopore"), an insulating membrane, a first medium and a second medium. In some embodiments, the pore with nanoscale size is a mutant of the mechanosensitive channel protein (MscCG) of Corynebacterium glutamicum. The pore with nanoscale size allows the analyte to translocate from one side of the insulating membrane to the other side.

In some embodiments, the pores with nanoscale dimensions are embedded in the insulating film, and the insulating film (which can also be understood as a complex of the pores with nanoscale dimensions and the insulating film) separates the first medium from the second medium, and the pores with nanoscale dimensions provide a channel connecting the first medium and the second medium; after applying a driving force between the first medium and the second medium, the analyte located in the first medium interacts with the nanopore to form a current (i.e., an electrical signal). In the present invention, "first medium" refers to the medium where the analyte is located when it is added to the nanopore system; "second medium" refers to the other side of the "first medium" in the two parts of the medium separated by the insulating film. As used herein, "driving force" refers to the force that drives the interaction between the analyte and the nanopore by means of voltage, electroosmotic flow, concentration gradient, etc. In some embodiments, the driving force is preferably voltage.

The first medium and the second medium may be the same or different, and the first medium and the second medium may include an electrolyte solution. In some embodiments, the electrolyte solution is an aqueous solution of an alkali metal halide, such as sodium chloride, lithium chloride, cesium chloride, potassium chloride, sodium bromide. In some embodiments, the electrolyte solution is a lithium chloride solution. In some embodiments, the concentration range of lithium chloride in the first medium and/or the second medium may be 300 mM- 600 mM. In some embodiments, the concentration of the electrolyte solution contained in the first medium and the second medium is different, in other words, there is a difference in the concentration of the electrolyte solution in the first medium and the second medium, for example, the concentration of lithium chloride in the first medium may be 300 mM, and the concentration of lithium chloride in the second medium may be 600 mM. In some embodiments, the first medium and/or the second medium may also include a buffer salt, such as MOPS. In some embodiments, the first medium and/or the second medium is alkaline, and its pH range may be 8.0 – 8.5.

As used herein, "insulating membrane" refers to a membrane having the ability to carry nanopores and block ionic currents passing through non-nanopores. The insulating membrane may include a lipid membrane and/or a polymer membrane. Exemplary lipid membranes include DPHPC, DOPC, and E. coli polar lipids (E. coli lipid), and exemplary polymer membranes include triblock copolymer polymer membranes.

In some specific embodiments, the nanopore system includes two electrolyte chambers, which are separated by an insulating membrane to form a trans compartment and a cis compartment, the hole of the nanopore is embedded in the insulating membrane, and only the nanopore on the insulating membrane connects the two electrolyte chambers. When an electric potential is applied to the two electrolyte chambers, the electrolyte ions in the solution in the electrolyte chamber move through electrophoresis and pass through the nanopore.

The interaction between the nanopore and the analyte

The analyte may contact the nanopore on either side of the insulating membrane. The analyte may contact either side of the insulating membrane so that the analyte passes through the channel of the nanopore to reach the other side of the insulating membrane. In this case, the analyte interacts with the nanopore as it passes through the insulating membrane via the channel of the hole. Alternatively, the analyte may contact the side of the insulating membrane, which may allow the analyte to interact with the nanopore, separate from the nanopore and remain on the same side of the insulating membrane. The analyte may interact with the nanopore in any manner and at any site. The analyte may also impact the nanopore, interact with the nanopore, separate from the nanopore and remain on the same side of the insulating membrane.

During the interaction between the analyte and the nanopore, the analyte affects the current flowing through the nanopore in a manner specific to the analyte, i.e., the current flowing through the nanopore is characteristic for the specific analyte. A control experiment can be performed to determine the effect of a specific analyte on the current flowing through the nanopore, and then to identify the specific analyte in the sample or determine whether the specific analyte is present in the sample. More specifically, the current pattern obtained by detecting the analyte can be compared with a known current pattern obtained using a known analyte under the same conditions to identify the presence or absence of the analyte, the concentration, or the degree of deviation from the known current pattern.

The nanopore system of the present invention may also include one or more measuring devices for measuring the current flowing through the nanopore, such as a patch clamp amplifier or a data acquisition device.

sample

The analyte may be present in any suitable sample. The present invention is typically performed on a sample known to contain or suspected of containing the analyte. The present invention may be performed on a sample containing one or more analytes of unknown species. Alternatively, the present invention may confirm the species of the one or more analytes known to be present or expected to be present in the sample.

The sample may include a biological sample. The present invention may be performed in vitro on a sample obtained from or extracted from any organism or microorganism. Preferably, the sample is a fluid sample. The sample generally includes a body fluid. The sample may be a body fluid sample, such as urine, blood, serum, plasma, lymph, cyst fluid, pleural fluid, ascites fluid, peritoneal fluid, amniotic fluid, epididymal fluid, cerebrospinal fluid, bronchoalveolar lavage fluid, breast milk, tears, saliva, sputum or a combination thereof. The sample may be derived from humans or other mammals. The sample may include a non-biological sample. The non-biological sample is preferably a fluid sample, such as drinking water, sea water, river water and a reagent for laboratory testing.

In some embodiments, the sample may not be processed before analysis, i.e., the sample containing the valproic acid drug is directly detected. As used herein, "direct detection" means that the sample is detected without additional processing of the analyte (e.g., derivatization, formation of polypeptides) and the sample (e.g., by centrifugation, filtration, dilution, precipitation, enrichment or other physical or chemical means known in the art). In some embodiments, the sample is a plasma sample or a saliva sample.

A kit for direct detection of samples containing valproate

A kit refers to a group of packaged related components, usually one or more compounds or compositions. In some embodiments, the kit provided by the present invention includes a MscCG protein mutant, one or more electrolyte solutions, an insulating film or a substance capable of generating an insulating film. The electrolyte solution is an aqueous solution of an alkali metal halide, such as sodium chloride, lithium chloride, cesium chloride, potassium chloride, and sodium bromide. In some embodiments, the electrolyte solution is a lithium chloride solution. In some embodiments, the concentration range of the lithium chloride solution can be 300 mM - 600 mM. In some embodiments, the kit may also include a buffer salt, such as MOPS. The substance capable of generating an insulating film may be a lipid or a triblock copolymer.

In some specific embodiments, the MscCG protein mutant is located in an insulating film that separates a first medium from a second medium and provides a channel connecting the first medium and the second medium. After a sample is added to the first medium and a driving force is applied to the first medium and the second medium, the valproic acid drug in the sample interacts with the MscCG protein mutant, and then the MscCG protein mutant detects the valproic acid drug. In some embodiments, the valproic acid drug includes valproic acid and a pharmaceutically acceptable salt thereof, such as sodium valproate. The kit can be used to determine the presence of the valproic acid drug in the sample to be tested.

In some specific embodiments, the sample is derived from a subject who has received the valproic acid drug treatment, and the type of the sample includes one or more of urine, blood, serum, plasma, lymph, cyst fluid, pleural fluid, ascites fluid, peritoneal fluid, amniotic fluid, epididymal fluid, cerebrospinal fluid, bronchoalveolar lavage fluid, breast milk, tears, saliva, and sputum.

In some specific embodiments, the kit further includes a standard curve for determining the concentration or concentration range of the valproic acid drug. The standard curve is determined by the signal frequency generated by the MscCG protein mutant detecting different concentrations of the valproic acid drug standard solution containing the valproic acid drug. That is to say, the concentration of the valproic acid drug contained in the valproic acid drug standard solution is known, such as 10, 20, 30, 40 and 50 μM valproic acid drugs. The MscCG protein mutant detects valproic acid drugs with different concentration gradients, and different signal frequencies are generated. The concentration of valproic acid drugs is linearly related to the corresponding signal frequency, and can be fitted into a standard curve of valproic acid drugs (see Figure 4), and the corresponding linear equation is y=1.39 x-1.33, where x represents the concentration of valproic acid drugs and y represents the signal frequency.

The kit provided by the present invention requires a small amount of sample (e.g., about 2-20 μL) and a short detection time (e.g., about 5 min) in practical applications, and can directly achieve specific detection (e.g., qualitative and quantitative) of low concentrations (e.g., 10 μM) of valproic acid drugs in samples.

Embodiment 1

Design of MscCG protein mutants

MscCG (mechanosensitive channel of Corynebacterium glutamicum) is a mechanosensitive channel protein of Corynebacterium glutamicum. It is a heptamer protein consisting of a transmembrane region and a cytoplasmic region. The longitudinal center of the protein is the channel, and the channel openings are located at the upper end of the transmembrane region and the lower end of the cytoplasmic region respectively. There are seven smaller openings on the side of the cytoplasmic region. The amino acid sequence of the wild-type MscCG protein is shown in SEQ ID NO: 1.

MRIIKRRVESAADADTTKNQLAFAGVGVYIAQIVAFFMLAVSAMQAFGFSLAGAAIPATIASAAIGLGAQSIVADFLAGFFILTEKQFGVGDWVRFEGNGIVVEGTVIEITMRATKIRTIAQETVIIPNSTAKVCINNSNNWSRAVVVIPIPMLGSENITDVIARSEAATRRALGQEKIAPEILGELDVHPATEVTPPTVVGMPWMVTMRFLV QVTAGNQWLVERAIRTEIISEFWEEYGSATTTSG TLIDSLHVEHEPKTSLIDASPQALKEPKPEAAATVASLAASSNDDADNADASVINAGNPEKELDSDVLEQELSSEEPEETAKPDHSLRGFFRTDYYPNRWQKILSFGGRVRMSTSLLLGALLLLSLFKVMTVEPSENWQNSSGWLSPSTATSTAVTTSETSAPVSTPSMTVPTTVEETPTMESNVETQQETSTPATATPQRADTIEPTEEATSQEETTASQTQSP AVEAPTAVQETVAPTSTP (SEQ ID NO:1)

In this example, potential key sites in the wild-type MscCG protein channel were optimized for mutation, and the following mutants were designed:

(1) Transmembrane end mutant 1: S50R; L51R; I56R; A52R; T59R; A55R; Q45R; L67R; A63R (amino acid sequence as shown in SEQ ID NO: 2);

(2) Cytoplasmic end bottom mutants: L253R; 257R; V255R; D251R; E258R (amino acid sequence as shown in SEQ ID NO: 3);

(3) Cytoplasmic side pore mutants: I120R; W93R; W221R; T106R (amino acid sequence is shown in SEQ ID NO: 4).

MRIIKRRVESAADADTTKNQLAFAGVGVYIAQIVAFFMLAVSAMRAFGFRRRGARRPARIASRAIGRGAQSIVADFLAGFFILTEKQFGVGDWVRFEGNGIVVEGTVIEITMRATKIRTIAQETVIIPNSTAKVCINNSNNWSRAVVVIPIPMLGSENITDVIARSEAATRRALGQEKIAPEILGELDVHPATEVTPPTVVGMPWMVTMRFLV QVTAGNQWLVERAIRTEIISEFWEEYGSATTTSG TLIDSLHVEHEPKTSLIDASPQALKEPKPEAAATVASLAASSNDDADNADASVINAGNPEKELDSDVLEQELSSEEPEETAKPDHSLRGFFRTDYYPNRWQKILSFGGRVRMSTSLLLGALLLLSLFKVMTVEPSENWQNSSGWLSPSTATSTAVTTSETSAPVSTPSMTVPTTVEETPTMESNVETQQETSTPATATPQRADTIEPTEEATSQEETTASQTQSP AVEAPTAVQETVAPTSTP (SEQ ID NO:2)

MRIIKRRVESAADADTTKNQLAFAGVGVYIAQIVAFFMLAVSAMQAFGFSLAGAAIPATIASAAIGLGAQSIVADFLAGFFILTEKQFGVGDWVRFEGNGIVVEGTVIEITMRATKIRTIAQETVIIPNSTAKVCINNSNNWSRAVVVIPIPMLGSENITDVIARSEAATRRALGQEKIAPEILGELDVHPATEVTPPTVVGMPWMVTMRFLV QVTAGNQWLVERAIRTEIISEFWEEYGSATTTSG TLIRSRHRERREPKTSLIDASPQALKEPKPEAAATVASLAASSNDDADNADASVINAGNPEKELDSDVLEQELSSEEPEETAKPDHSLRGFFRTDYYPNRWQKILSFGGRVRMSTSLLLGALLLLSLFKVMTVEPSENWQNSSGWLSPSTATSTAVTTSETSAPVSTPSMTVPTTVEETPTMESNVETQQETSTPATATPQRADTIEPTEEATSQEETTASQTQSPAVE APTAVQETVAPTSTP (SEQ ID NO:3)

MRIIKRRVESAADADTTKNQLAFAGVGVYIAQIVAFFMLAVSAMQAFGFSLAGAAIPATIASAAIGLGAQSIVADFLAGFFILTEKQFGVGDRVRFEGNGIVVEGRVIEITMRATKIRTRAQETVIIPNSTAKVCINNSNNWSRAVVVIPIPMLGSENITDVIARSEAATRRALGQEKIAPEILGELDVHPATEVTPPTVVGMPWMVTMRFLVQV TAGNQRLVERAIRTEIISEFWEEYGSATTTSG TLIDSLHVEHEPKTSLIDASPQALKEPKPEAAATVASLAASSNDDADNADASVINAGNPEKELDSDVLEQELSSEEPEETAKPDHSLRGFFRTDYYPNRWQKILSFGGRVRMSTSLLLGALLLLSLFKVMTVEPSENWQNSSGWLSPSTATSTAVTTSETSAPVSTPSMTVPTTVEETPTMESNVETQQETSTPATATPQRADTIEPTEEATSQEETTASQTQSP AVEAPTAVQETVAPTSTP (SEQ ID NO:4)

Embodiment 2

Electrophysiological testing of MscCG mutants

Experimental conditions:

Conductivity buffer: -Cis (cis end, the same below): 300 mM LiCl, 10 mM MOPS, pH 8.0; -Trans (trans end, the same below): 600 mM LiCl, 10 mM MOPS, pH 8.0

Embedded hole voltage: +200～+300 mV

Detection voltage: +50 mV (for cytoplasmic end bottom mutants and cytoplasmic end side hole mutants) or -50 mV (for transmembrane end mutants)

Lipid membrane: Escherichia coli polar lipids

Detection device: Warner vertical sample trough

In this example, the three mutants were characterized by single-channel electrophysiological testing. The results are shown in FIG1 . These three mutants can be stably inserted into the insulating membrane and have the ability of single-molecule sensing.

Embodiment 3

Direct detection of low concentrations of valproic acid drugs by MscCG mutants

This example tests the ability of the three mutants described above to detect 40 μM valproic acid.

Experimental conditions:

Conductivity buffer: -Cis: 300 mM LiCl, 10 mM MOPS, pH 8.0; -Trans: 600 mM LiCl, 10 mM MOPS, pH 8.0

Embedded hole voltage: +200～+300 mV

Detection voltage: +50 mV (for cytoplasmic end bottom mutants and cytoplasmic end side hole mutants) or -50 mV (for transmembrane end mutants)

Lipid membrane: Escherichia coli polar lipids

Detection device: Warner vertical sample trough

The test results are shown in Figure 1. The cytoplasmic end bottom mutant (Figure 1C) and the cytoplasmic end side hole variant (Figure 1A) cannot detect 40 μM valproic acid. The signals generated by the cytoplasmic end bottom mutant and the cytoplasmic end side hole variant may belong to the fluctuation range of their own solution environment. As shown in Figure 1B, the transmembrane end mutant 1 produced a characteristic electrical signal and was able to detect 40 μM valproic acid.

Embodiment 4

This embodiment combines the experimental results of the above embodiment, and the three-dimensional configuration of the wild-type MscCG protein and valproic acid, and uses molecular simulation technology to locate the potential key sites of the interaction between the wild-type MscCG protein and valproic acid at the transmembrane end, as shown in Figure 2. The results of Figure 2 show that there are many potential key sites for the interaction between the wild-type MscCG protein and valproic acid at the transmembrane end. Based on this, this embodiment further designed some transmembrane end mutants, and the test results showed that among these transmembrane end mutants, only mutants that mutated at sites 52, 59, 55, 45, 67 and 63 at the same time should have a better ability to detect low-concentration valproic acid drugs.

Furthermore, this embodiment uses kinetic simulation to screen the optimal mutant amino acid combination, as shown in Figure 3. Compared with histidine (mut_HIS) and lysine (mut_LYS), the mutant in which positions 52, 59, 55, 45, 67 and 63 are simultaneously mutated to arginine (mut_ARG) should have a better ability to detect low-concentration valproic acid drugs. The optimized transmembrane end mutant (transmembrane end mutant 2) obtained: A52R; T59R; A55R; Q45R; L67R; A63R (amino acid sequence is shown in SEQ ID NO: 5).

MRIIKRRVESAADADTTKNQLAFAGVGVYIAQIVAFFMLAVSAMRAFGFSLRGARIPARIASRAIGRGAQSIVADFLAGFFILTEKQFGVGDWVRFEGNGIVVEGTVIEITMRATKIRTIAQETVIIPNSTAKVCINNSNNWSRAVVVIPIPMLGSENITDVIARSEAATRRALGQEKIAPEILGELDVHPATEVTPPTVVGMPWMVTMRFLV QVTAGNQWLVERAIRTEIISEFWEEYGSATTTSG TLIDSLHVEHEPKTSLIDASPQALKEPKPEAAATVASLAASSNDDADNADASVINAGNPEKELDSDVLEQELSSEEPEETAKPDHSLRGFFRTDYYPNRWQKILSFGGRVRMSTSLLLGALLLLSLFKVMTVEPSENWQNSSGWLSPSTATSTAVTTSETSAPVSTPSMTVPTTVEETPTMESNVETQQETSTPATATPQRADTIEPTEEATSQEETTASQTQSP AVEAPTAVQETVAPTSTP (SEQ ID NO:5)

Embodiment 5

Transmembrane end mutant 2 directly detects low concentrations of valproic acid drugs

This example further tests whether the optimized transmembrane end mutant can detect low concentrations of valproic acid drugs (including 10, 20, 30, 40 and 50 μM valproic acid).

Experimental conditions:

Conductivity buffer: -Cis: 300 mM LiCl, 10 mM MOPS, pH 8.0; -Trans: 600 mM LiCl, 10 mM MOPS, pH 8.0

Embedded hole voltage: +200～+300 mV

Detection voltage: -50 mV

Lipid membrane: Escherichia coli polar lipids

Detection device: Warner vertical sample trough

The results showed that the optimized transmembrane end mutant was able to detect 10, 20, 30, 40 and 50 μM valproic acid, and the concentration of valproic acid drugs was linearly related to the corresponding signal frequency (the corresponding linear equation was y=1.39x-1.33, where x represented the concentration of valproic acid drugs and y represented the signal frequency), as shown in Figure 4.

The above experimental results show that the present invention significantly improves the detection sensitivity of MscCG protein to valproic acid drugs by introducing appropriate mutations (amino acid substitutions) at the transmembrane end, increasing the sensitivity from 4 mM to 10 μM.

Embodiment 6

Transmembrane end mutant 2 specific detection of valproic acid drugs

This example tests whether the optimized transmembrane end mutants can detect other anti-epileptic drugs, wherein the concentration of valproic acid, oxcarbazepine, levetiracetam and lamotrigine is 40 μM.

Experimental conditions:

Conductivity buffer: -Cis: 300 mM LiCl, 10 mM MOPS, pH 8.0; -Trans: 600 mM LiCl, 10 mM MOPS, pH 8.0

Embedded hole voltage: +200～+300 mV

Detection voltage: -50 mV

Lipid membrane: Escherichia coli polar lipids

Detection device: Warner vertical sample trough

The results are shown in Figure 5 , and the optimized transmembrane end mutant can only specifically detect valproic acid, but cannot detect other anti-epileptic drugs, such as oxcarbazepine, levetiracetam, and lamotrigine.

Embodiment 7

Transmembrane end mutant 2 directly detects clinical samples containing low concentrations of valproic acid drugs

This example tests the ability of the optimized transmembrane end mutant to directly detect clinical samples, wherein the clinical samples include healthy human plasma samples, plasma samples of patients taking valproic acid, and saliva samples of patients taking valproic acid. The plasma samples of patients taking valproic acid and the saliva samples of patients taking valproic acid were prepared by adding 30 μM valproic acid to 1 mL healthy human plasma samples and healthy human saliva samples, respectively.

Experimental conditions:

Conductivity buffer: -Cis: 300 mM LiCl, 10 mM MOPS, pH 8.0; -Trans: 600 mM LiCl, 10 mM MOPS, pH 8.0

Embedded hole voltage: +200～+300 mV

Detection voltage: -50 mV

Lipid membrane: Escherichia coli polar lipids

Detection device: Warner vertical sample trough

The results are shown in Figure 6. The optimized transmembrane end mutant has the ability to directly detect low concentrations of valproic acid drugs in clinical samples, and will not be affected by other substances in the clinical samples, and has strong anti-interference ability.

Comparative Example

Wild-type MscCG protein cannot directly detect low concentrations of valproic acid drugs

Experimental conditions:

Buffer: -Cis: 300 mM LiCl, 10 mM MOPS, pH 8.0; -Trans: 600 mM LiCl, 10 mM MOPS, pH 8.0

Embedded hole voltage: +200～+300 mV

Detection voltage: +50 mV

Lipid membrane: Escherichia coli polar lipids

Detection device: Warner vertical sample trough

The results are shown in Figures 7 and 8. 40 μM valproic acid cannot cause the wild-type MscCG protein to produce a characteristic electrical signal, that is, the wild-type MscCG protein does not have the ability to directly detect samples containing valproic acid drugs at a concentration of 40 μM or below.

The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the enlightenment of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the purpose of the present invention and the claims, which all fall within the protection of the present invention.

Claims (9)

1. A mechano-sensitive channel protein mutant of corynebacterium glutamicum, comprising the following mutations compared to the mechano-sensitive channel protein of a wild-type corynebacterium glutamicum: A52R, T, R, A, R, Q, 45, R, L R and A63R, and the amino acid sequence of the protein mutant is SEQ ID NO. 5.

2. A nanopore detection system comprising the protein mutant of claim 1, an insulating membrane, a first medium, and a second medium; wherein the protein mutant is embedded in the insulating film, the insulating film separating the first medium from the second medium, the protein mutant providing a channel communicating the first medium with the second medium, the first medium and the second medium comprising an electrolyte solution.

3. The system of claim 2, wherein the electrolyte solution is a lithium chloride solution, and wherein the concentration of the lithium chloride solution comprises 300 mM to 600 mM.

4. The system of claim 3, further comprising a valproic acid drug added to the first medium, the valproic acid drug being valproic acid and pharmaceutically acceptable salts thereof.

5. The system of claim 4, wherein the concentration of the valproic acid is at least 10 μm.

6. The system of claim 2, wherein the pH of the first medium and the second medium is between 8.0 and 8.5.

7. Use of the protein mutant of claim 1 or the system of any one of claims 3-6 in the preparation of a kit for directly detecting a sample comprising a valproic acid drug, said valproic acid drug being valproic acid and pharmaceutically acceptable salts thereof.

8. The use according to claim 7, wherein the sample is a plasma sample or a saliva sample.

9. The use according to claim 7, wherein the kit is for determining the concentration of the valproic acid drug in the sample.