CN111413383B - Method for constructing double-recognition-site nanopore - Google Patents
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Abstract
The invention discloses a method for constructing a nanopore with double recognition sites, which comprises the following steps: performing structural domain function analysis on the fusion expressed two single recognition site nanopores to obtain a structural domain analysis result; selecting a structural domain gene, and designing a fusion protein to obtain a fusion protein with two recognition sites; selecting an expression system which ensures the stable structure and the polymerization of the fusion protein, and sequentially finishing the design of an expression scheme, the optimization of expression conditions, the polymerization of the pore proteins, the purification and separation, the pore embedding and sequencing and the mutation treatment of the fusion protein according to the host type of the expression system; the method uses the naturally-existing single-recognition-site nanopore protein, obtains the double-recognition-site nanopore protein through an engineered fusion protein expression technology, greatly expands the selectable range of the nanopore protein, and solves the problem that the naturally-existing double-sensitive-site protein nanopore is extremely small. And the performance of the pore channel is optimized through mutation, and the detection performance of the pore channel is improved.
Description
Technical Field
The invention relates to the technical field of nanopore detection, in particular to a method for constructing a nanopore with double recognition sites.
Background
A class of transmembrane protein channels exists in natural biological membranes, which have diameters on the nanometer scale and allow ions and molecules of a particular size to pass through, called nanopores.
The nano-porous protein is embedded on an insulating artificial thin film, the thin film is placed in conductive ionic liquid, and voltage is applied to two sides of the thin film, so that charged ions pass through the nano-porous channel to form ionic current. When molecules of a specific size pass through the channel or are bonded to the channel, the channel is blocked, so that ion current is reduced, and the size of the blocking current is related to characteristic information such as molecular size and charge distribution, so that the molecules can be identified by measuring the size of the blocking current.
By applying the principle, when nucleic acid or other analytes instantaneously pass through the protein nanopore under the action of voltage, the nucleic acid sequencing can be realized by measuring the change of the blocking current of the base sequence passing through the pore. The existing high-throughput sequencing technology needs enzyme amplification and fluorescence labeling processes, so that the method has the defects of low speed and high price. Compared with the existing sequencing method, nanopore sequencing can be measured by a single molecule without amplification, and has the characteristics of high speed, low cost, long read length, high sensitivity and the like.
Some protein nanopores have been used for detecting polymers such as nucleic acids, for example, mutant α -hemolysin nanopores for nucleic acid detection (see the following documents:
CN107109479A, Kasiaanowicz et al, Proc Natl Acad Sci USA,1996.93(24):13770, Stoddart et al, Proc Natl Acad Sci USA, 2009.106(19):7702), MspA nanopores (CN107207571A, CN102216783A, CN106459159A, Derrington et al, Proc Natl Acad Sci USA, 2010.107 (37): 16060, Manrao et al, Nat Biotechnol, 2012.30 (4): 349) CsgG nanopore (CN107207570A), FraC nanopore (WO2018/012963a1, Wloka et al, angelw Chem Int Ed Engl, 2016.55 (40): 12494) ClyA nanopores for protein detection (CN105358567A, Biesemans, et al, Nano Lett, 2015.15(9): 6076). These nanopores are all proteins with a single recognition site or three or more recognition sites.
The recognition sites refer to some specific amino acid sites in the protein nanopore and are main contributing sites of current signals when the nanopore detects molecules. The number, shape, charge state, hydrophilicity and hydrophobicity, and the like of the recognition sites are key factors for determining the detection resolution of the nanopore. In terms of the number of recognition sites, mathematical simulation studies on hemolysin-based targets show that nanopores with double recognition sites have better base recognition effects than single recognition sites (Stoddart, D., et al., Angew Chem Int Ed, 2010.49 (3): p.556-9). Three or more recognition sites are limited by signal-to-noise ratio and cannot be effectively base-distinguished.
However, the number of naturally occurring protein nanopores with double recognition sites is very small, and finding a suitable nanopore and performing corresponding modification are the key points for further improving the sequencing accuracy.
The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.
For the above reasons, the present applicant has proposed a method for constructing a nanopore with dual recognition sites, aiming to solve the above problems.
Disclosure of Invention
In order to meet the requirements, the invention aims to provide a method for constructing a nanopore with double recognition sites, relates to a method for changing the number of recognition sites of a protein nanopore by protein fusion expression, and applies the constructed protein with double recognition sites to the detection of analytes such as nucleic acid. The method specifically comprises the following steps: and fusing and expressing the two protein nanopores with the single recognition sites to form a fusion protein nanopore with the two recognition sites, and keeping the hole embedding capacity and the polymerization property of the fusion nanopore. For example, a part or all of functional domains of two single-recognition-site nanopore proteins MspA and FraC are taken, and fusion expression of the proteins is performed in a mode of direct connection or connection of a connecting sequence (linker), and then the fusion is performed to form an MspA-FraC tunnel with two recognition sites. The application also describes that the fusion expressed protein with double recognition sites is mutated to obtain a protein channel with better performance. In another aspect, the invention also discloses the use of protein nanopores with altered numbers of recognition sites and nanopores mutated based thereon for the detection of analytes such as nucleic acids, polypeptides and the like.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method of constructing a nanopore with dual recognition sites, comprising the steps of:
based on the gene sequence of the pre-prepared or fusion expression protein, performing structural domain function analysis on the fusion expressed two single recognition site nanopores by using a structural domain analysis tool to obtain a structural domain analysis result;
according to the result of the structural domain analysis, selecting a structural domain gene, designing a fusion protein to obtain a fusion protein with two recognition sites, and enabling the fusion protein with the two recognition sites to comprise a phospholipid membrane binding structural domain;
selecting an expression system which ensures the stable structure and the polymerization of the fusion protein, carrying out codon optimization on a gene sequence of the fusion protein according to the host type of the expression system, optimizing different expression plasmids and expression strain combinations of the gene sequence of the fusion protein to obtain the successful expression of the fusion protein, and designing a purification tag at the tail end of the fusion protein to realize the purification after protein expression;
obtaining a completely folded fusion protein by optimizing expression conditions;
obtaining high-purity fusion protein monomer or polymer by optimizing a purification method;
inducing the protein expressed as a monomer after the expression of the fusion protein by using a reagent to obtain the protein assembled into a polymer form;
purifying and separating the protein monomer after fusion expression and the protein assembled into a polymer form;
detecting and characterizing an analyte using a means for current characterization using a test system comprising a fusion protein that has been subjected to a purification separation process, a membrane layer, and a current measuring device;
optimizing the structure, charge state and hydrophilicity and hydrophobicity inside the pore channel of the fusion expressed double-recognition-site nanopore protein through mutation, and expressing the mutated protein for analyte detection and characterization.
In one possible embodiment, the step of obtaining a fusion protein having two recognition sites further comprises:
and (3) selecting protein three-dimensional structure simulation software for simulation to obtain the three-dimensional structure of the fusion protein, and predicting the folding effect of the fusion protein according to the three-dimensional structure.
In one possible embodiment, the step of obtaining a fusion protein having two recognition sites further comprises:
the domains derived from different nanopore proteins are linked by a linker sequence or directly linked.
In one possible embodiment, the step of selecting an expression system that ensures structural stability and polymerization of the fusion protein comprises selecting prokaryotic expression systems, eukaryotic expression systems, insect expression systems, animal cell expression systems, cell-free in vitro expression systems, and other systems useful for protein expression.
In one possible embodiment, the step of obtaining the folded intact fusion protein by optimizing the expression conditions comprises optimizing the type of culture medium, optimizing the culture conditions, optimizing the concentration of the inducer, the type of the inducer, the induction temperature and the induction time.
In one possible embodiment, the step of purifying and separating the fusion expressed protein monomer and the protein assembled into a polymer form comprises purifying and separating by using gel filtration chromatography, ion exchange chromatography, hydrophobic chromatography and affinity chromatography.
In a possible embodiment, the step of inducing the protein expressed as a monomer after the expression of the fusion protein by using the reagent further comprises an induction treatment by using rabbit erythrocyte membranes or liposomes or amphiphilic chemical reagents.
In one possible embodiment, the step of purifying and separating the protein monomers after fusion expression and the protein assembled into the polymer form comprises purifying and separating the protein monomers after fusion expression and the protein assembled into the polymer form by using exclusion chromatography or density gradient centrifugation or ultrafiltration membrane separation or gel cutting purification.
In one possible embodiment, the step of detecting and characterizing the analyte using a means of amperometric characterization comprises placing the fusion protein in a membrane layer between a first conductive liquid medium and a second conductive liquid medium, such that at least one of the first conductive liquid medium and the second conductive liquid medium contains the analyte.
In one possible embodiment, the membrane layer is a polymer film, a lipid layer, a solid film or other insulating thin film;
the analyte is one or more of nucleotide, nucleic acid, amino acid, oligopeptide, polypeptide, protein, polymer, medicine, inorganic molecule, ion, pollutant and nano-scale substance.
In one possible embodiment, the step of detecting and characterizing the analyte using a means for current characterization comprises:
a. constructing a test system containing fusion expression protein and a membrane layer;
b. applying a voltage to cause the analyte to interact with or pass through the fused nanopore;
c. upon interaction of an analyte with or passage through a fusion nanopore, at least one current value is obtained indicative of at least one characteristic of the analyte, characterizing the analyte.
In one possible embodiment, the step of constructing a test system comprising the fusion expressed protein and the membrane layer comprises adding the purified protein to a conductive liquid medium, and obtaining a unique ion channel between the two conductive liquid media according to the principle that the protein is spontaneously embedded or induced to be embedded in the membrane layer.
In one possible embodiment, the step of constructing a test system comprising the fusion expressed protein and the membrane layer comprises preparing vesicles from the protein and phospholipid, inserting the protein into the vesicle membrane during vesicle formation, adding the vesicles to a conductive liquid medium, fusing the vesicles with the membrane layer, and inserting the protein into the membrane layer simultaneously to obtain an ion channel between the two conductive liquid media.
In one possible embodiment, the step of optimizing the structure, charge state and hydrophilicity and hydrophobicity inside the pore channel of the fusion expressed double-recognition-site nanopore protein through mutation comprises the step of performing insertion, deletion and replacement operations on a base or a peptide segment of a specific site of the protein.
Compared with the prior art, the invention has the beneficial effects that: by adopting the double-recognition-site nanopore construction method, the nanopore protein of the double recognition sites is obtained by using the naturally-existing single-recognition-site nanopore protein and an engineered fusion protein expression technology, so that the selectable range of the nanopore protein is greatly expanded, and the problem that the protein nanopore of the naturally-existing double-sensitive sites is extremely small is solved. Through mutation of the fusion protein, the performance of the pore channel is further optimized, and the detection performance of the pore channel is improved. Because the nanopore with the double recognition sites has better homopolymer resolution than the pore canal with the single recognition site, the effective distinction of the current signal difference is easier to realize compared with three or more recognition sites. The fusion protein obtained by the method is used for detecting polymers such as nucleic acid and the like, so that the detection accuracy can be greatly improved, and the detection accuracy which cannot be obtained by the detection method in the prior art can be obtained.
The invention is further described below with reference to the accompanying drawings and specific embodiments.
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FIG. 1 is a schematic flow chart of a specific embodiment of a method for constructing a nanopore with dual recognition sites according to the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In the description of the present invention, it is to be understood that the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", "clockwise", "counterclockwise", and the like, indicate orientations and positional relationships based on those shown in the drawings, and are used only for convenience of description and simplicity of description, and do not indicate or imply that the device or element being referred to must have a particular orientation, be constructed and operated in a particular orientation, and thus, should not be considered as limiting the present invention.
Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of the present invention, "a plurality" means two or more unless specifically defined otherwise.
In the present invention, unless otherwise expressly stated or limited, the terms "mounted," "connected," "secured," and the like are to be construed broadly and can, for example, be connected or detachably connected or integrated; can be mechanically or electrically connected; either directly or indirectly through intervening media, either internally or in any other relationship. The specific meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations.
In the present invention, unless otherwise expressly stated or limited, "above" or "below" a first feature means that the first and second features are in direct contact, or that the first and second features are not in direct contact but are in contact with each other via another feature therebetween. Also, the first feature being "on," "above" and "over" the second feature includes the first feature being directly on and obliquely above the second feature, or merely indicating that the first feature is at a higher level than the second feature. A first feature being "under," "below," and "beneath" a second feature includes the first feature being directly under and obliquely below the second feature, or simply meaning that the first feature is at a lesser elevation than the second feature.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above should not be understood to necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples described in this specification can be combined and combined by those skilled in the art.
As shown in the method flowchart of fig. 1, a flowchart of a specific embodiment of a method for constructing a nanopore with dual recognition sites according to the present invention includes the following steps:
step S1, based on the gene sequence of the pre-prepared or fusion expression protein, using a structural domain analysis tool to perform structural domain function analysis on the fusion expressed two single recognition site nanopores to obtain a structural domain analysis result;
step S2, according to the result of the domain analysis, selecting the domain gene, designing the fusion protein, obtaining the fusion protein with two recognition sites, making the fusion protein with two recognition sites include the phospholipid membrane binding domain;
s3, selecting an expression system which ensures the stable structure and the polymerization of the fusion protein, optimizing the codon of the gene sequence of the fusion protein according to the host type of the expression system, optimizing different expression plasmids and expression strain combinations of the gene sequence of the fusion protein to obtain the successful expression of the fusion protein, and designing a purification label at the tail end of the fusion protein to realize the purification after the protein expression;
step S4, obtaining a completely folded fusion protein by optimizing expression conditions;
step S5, obtaining high-purity fusion protein monomer or polymer by optimizing the purification method;
step S6, inducing the protein expressed as monomer after the fusion protein is expressed by using reagent to obtain the protein assembled into polymer form;
step S7, purifying and separating the protein monomer after fusion expression and the protein assembled into polymer form;
step S8, using a test system comprising the fusion protein which is purified and separated, a membrane layer and a current measuring device, and detecting and characterizing the analyte by means of current characterization;
and step S9, optimizing the structure, the charge state and the hydrophilicity and hydrophobicity inside the pore channel of the fusion expressed double-recognition-site nanopore protein through mutation, and expressing the mutated protein for analyte detection and characterization.
Specifically, step S1, step S2, step S3, step S4, step S5, step S6, step S7, step S8, and step S9, aiming at achieving domain analysis, fusion protein design, expression scheme design, expression condition optimization, purification condition optimization, porin polymerization, purification separation, pore-building and sequencing, and fusion protein mutation; because the number of the recognition sites is one of the key factors for determining the sequencing accuracy, the nanopore with the double recognition sites has better homopolymer resolution than the pore with the single recognition site under the same condition, and the effective differentiation of the current signal difference is easier to realize compared with three or more recognition sites.
However, naturally occurring protein nanopores with double sensitive sites are rare. The application uses the steps of S1-S9, uses the naturally-existing single recognition site nanopore protein, and obtains the double recognition site nanopore protein through an engineered fusion protein expression technology, thereby greatly expanding the selectable range of the nanopore protein. Through mutation of the fusion protein, the performance of the pore channel is further optimized, and the detection performance of the pore channel is improved. The fusion protein is used for detecting polymers such as nucleic acid and the like, and the detection accuracy can be effectively improved.
In one example, the domain analysis result obtained in step S1 is a region having a specific structure and an independent function in the protein, and is also a basic functional unit of the protein. Thus, in a nanopore protein, different domains are responsible for different functions such as interaction with a biological membrane or maintenance of a specific polymer structure, respectively, e.g., in MspA nanopores, different domains such as Rim, Stem, Periplasm, etc. (reference data can be found in mahfud, m., et al, J Biol Chem, 2006.281(9): p.5908-15.).
Therefore, in step S1, the gene sequence based on the pre-preparation can include, but is not limited to, the gene sequence reported in the existing literature.
In one embodiment, the step of obtaining a fusion protein having two recognition sites in step S2 further comprises:
and (3) selecting protein three-dimensional structure simulation software for simulation to obtain the three-dimensional structure of the fusion protein, and predicting the folding effect of the fusion protein according to the three-dimensional structure.
In one embodiment, the step of obtaining a fusion protein having two recognition sites further comprises:
the domains derived from different nanopore proteins are linked by a linker sequence or directly linked.
Specifically, in the expression of fusion proteins, the domains derived from different nanopore proteins may be linked by a linker or directly linked. The length of the linker sequence is important for protein folding and stability, and suitable linker sequences can be designed by reference to linker sequence design tools reported in the literature.
In one embodiment, the step of selecting an expression system capable of ensuring the structural stability and polymerization of the fusion protein in step S3 includes selecting a prokaryotic expression system, a eukaryotic expression system, an insect expression system, an animal cell expression system, a cell-free in vitro expression system, and other systems capable of being used for protein expression, wherein the prokaryotic expression system is preferably selected on the premise of ensuring the structural stability and polymerization of the fusion protein, because the fusion protein can be engineered to be expressed in the prokaryotic system or the eukaryotic system, and the prokaryotic expression system is characterized by short time and low cost, because the nanopore protein is used for molecular detection and does not need to maintain the activity of the protein. According to the host type of the expression system, the gene sequence of the fusion protein needs to be optimized correspondingly.
In addition, in order to solve the problem that successful expression and folding of the fusion protein by different expression plasmids and expression strains can affect successful expression of the fusion protein, in this embodiment, a means of optimizing combinations of different expression plasmids and expression strains is adopted to obtain successful expression of the fusion protein.
In one embodiment, to solve the purification problem after protein expression, a purification tag, such as a histidine tag, is designed at the end of the fusion protein for purification after protein expression.
In one embodiment, the step of obtaining the folded and intact fusion protein by optimizing the expression conditions in step S4 includes optimizing the type of culture medium, optimizing the culture conditions, optimizing the concentration of the inducer, the type of the inducer, the induction temperature, and the induction time.
Specifically, for step S4, since the present embodiment adopts an engineered expression-inducing manner, and the protein engineered expression-inducing manner may be expressed as a soluble protein or an inclusion body protein, the expression amount is also uncertain. In order to overcome the problem, expression conditions such as culture medium type, culture conditions, inducer concentration, inducer type induction temperature, induction time and the like are optimized, so that the completely folded fusion protein is obtained.
In one embodiment, since the extracted total protein contains a considerable amount of hetero-proteins possessed by the expression system itself, which has an effect on monomer-induced assembly into polymer form and the nanopore-embedded test, step S5 is performed to purify the protein by a specific purification method, such as separation of fusion protein containing histidine tag by affinity chromatography, according to the characteristics of the fusion protein. The step S5 includes gel filtration chromatography, ion exchange chromatography, hydrophobic chromatography, and affinity chromatography.
In one embodiment, since some of the nanoporous proteins can self-assemble into mature polymeric tunnels, such as MspA, upon expression. Some of the nanopore proteins are expressed as protein monomers, which require specific processing conditions to assemble into a polymeric form, such as Phi 29. Therefore, the step of inducing the protein expressed as a monomer after the expression of the fusion protein using the reagent in step S6 further includes an induction treatment using rabbit erythrocyte membranes, liposomes, other amphiphilic chemical reagents, or the like.
In one embodiment, the step of purifying and separating the protein monomers after fusion expression and the protein assembled into the polymer form in step S7 includes purifying and separating the protein monomers after fusion expression and the protein assembled into the polymer form by using exclusion chromatography, density gradient centrifugation, ultrafiltration membrane separation, gel cutting purification, or other processes.
Specifically, after the nanopore protein is expressed and polymerized, common monomers and polymers can coexist in a solution, part of protein monomers do not have a pore-plugging function, and part of protein monomers can interfere with a pore-plugging test, so that the protein monomers and the polymers after fusion expression need to be purified and separated. The method can effectively solve the problem and achieve the effects of purification and separation.
In one embodiment, the step S8 of detecting and characterizing the analyte by using the current characterization method includes placing the fusion protein in a film layer between a first conductive liquid medium and a second conductive liquid medium, such that at least one of the first conductive liquid medium and the second conductive liquid medium contains the analyte.
In one embodiment, the film is a polymer film, a lipid layer, a solid film or other insulating film.
In one embodiment, the analyte is one or more of a nucleotide, a nucleic acid, an amino acid, an oligopeptide, a polypeptide, a protein, a polymer, a drug, an inorganic molecule, an ion, a contaminant, a nanoscale substance.
In one embodiment, the process of detecting and characterizing the analyte by using the current characterization method in step S8 includes:
a. constructing a test system containing fusion expression protein and a membrane layer;
b. applying a voltage to cause the analyte to interact with or pass through the fused nanopore;
c. upon interaction of an analyte with or passage through a fusion nanopore, at least one current value is obtained indicative of at least one characteristic of the analyte, characterizing the analyte.
In an alternative embodiment, the step of constructing a test system comprising fusion expressed protein and membrane layer comprises adding purified protein to a conductive liquid medium, and obtaining a unique ion channel between the two conductive liquid media according to the principle that protein is spontaneously embedded or induced to be embedded in the membrane layer.
In an alternative embodiment, the process of constructing the test system comprising fusion expressed protein and membrane layer may specifically be that the protein and phospholipid are prepared into vesicles, during the formation of the vesicles, the protein is embedded into the vesicle membrane, the vesicles are added into the conductive liquid medium and fused with the membrane layer, and the protein is embedded into the membrane layer at the same time, so as to obtain an ion channel between the two conductive liquid media.
In one embodiment, the step of optimizing the structure, charge state and hydrophilicity and hydrophobicity inside the pore channel of the fusion expressed nanopore protein by mutation in step S9 includes performing insertion, deletion and substitution operations on a base or a peptide segment of a specific site of the protein.
Due to the fusion expression of the nanopore protein with the double recognition sites, the structure, the charge state and the hydrophilicity and hydrophobicity inside the pore can be further optimized through mutation, so that the detection accuracy is improved. In the embodiment, mutation means is adopted for processing, so that the mutated protein is used for testing after being expressed, and the accuracy is improved conveniently.
The steps in the method of the embodiment of the invention can be sequentially adjusted, combined and deleted according to actual needs.
Various other modifications and changes may be made by those skilled in the art based on the above-described technical solutions and concepts, and all such modifications and changes should fall within the scope of the claims of the present invention.
Claims (11)
1. A method for constructing a nanopore with double recognition sites is characterized by comprising the following steps:
based on the gene sequence of the pre-prepared or fusion expression protein, performing structural domain function analysis on the fusion expressed two single recognition site nanopores by using a structural domain analysis tool to obtain a structural domain analysis result;
according to the result of the structural domain analysis, selecting a structural domain gene, designing a fusion protein to obtain a fusion protein with two recognition sites, and enabling the fusion protein with the two recognition sites to comprise a phospholipid membrane binding structural domain;
selecting an expression system which ensures the stable structure and the polymerization of the fusion protein, carrying out codon optimization on a gene sequence of the fusion protein according to the host type of the expression system, optimizing different expression plasmids and expression strain combinations of the gene sequence of the fusion protein to obtain the successful expression of the fusion protein, and designing a purification tag at the tail end of the fusion protein to realize the purification after protein expression;
obtaining a completely folded fusion protein by optimizing expression conditions;
obtaining high-purity fusion protein monomer or polymer by optimizing a purification method;
inducing the protein expressed as a monomer after the expression of the fusion protein by using a reagent to obtain the protein assembled into a polymer form;
purifying and separating the protein monomer after fusion expression and the protein assembled into a polymer form;
detecting and characterizing an analyte using a means for current characterization using a test system comprising a fusion protein that has been subjected to a purification separation process, a membrane layer, and a current measuring device;
optimizing the structure, charge state and hydrophilicity and hydrophobicity inside the pore channel of the fusion expressed double-recognition-site nanopore protein through mutation, and expressing the mutated protein for analyte detection and characterization.
2. The method of constructing a dual recognition site nanopore according to claim 1, wherein said step of obtaining a fusion protein having two recognition sites is further followed by:
and (3) selecting protein three-dimensional structure simulation software for simulation to obtain the three-dimensional structure of the fusion protein, and predicting the folding effect of the fusion protein according to the three-dimensional structure.
3. The method of constructing a dual recognition site nanopore according to claim 1, wherein said step of obtaining a fusion protein having two recognition sites is further followed by:
the domains derived from different nanopore proteins are linked by a linker sequence or directly linked.
4. The method of claim 1, wherein the step of inducing the protein expressed as a monomer after the expression of the fusion protein using the reagent further comprises inducing the protein using rabbit erythrocyte membrane or liposome or an amphiphilic chemical reagent.
5. The method for constructing a nanopore according to claim 1, wherein the step of purifying and separating the protein monomer after fusion expression and the protein assembled into the polymer form comprises purifying and separating the protein monomer after fusion expression and the protein assembled into the polymer form by using exclusion chromatography, density gradient centrifugation, ultrafiltration membrane separation or gel cutting purification.
6. The method of claim 1, wherein the step of detecting and characterizing the analyte using amperometric features comprises placing the fusion protein in a membrane layer between a first conductive liquid medium and a second conductive liquid medium, such that at least one of the first conductive liquid medium and the second conductive liquid medium contains the analyte.
7. The method for constructing a nanopore according to claim 1, wherein the membrane layer is a polymer membrane, a lipid layer or a solid state membrane;
the analyte is one or more of nucleotide, nucleic acid, amino acid, oligopeptide, polypeptide, protein, polymer, medicine, inorganic molecule, ion and pollutant.
8. The method of constructing a dual recognition site nanopore according to claim 1, wherein said analyte is a nanoscale substance.
9. The method of constructing a dual recognition site nanopore according to claim 1, said step of performing detection and characterization of an analyte using a means of amperometric characterization comprising:
a. constructing a test system containing fusion expression protein and a membrane layer;
b. applying a voltage to cause the analyte to interact with or pass through the fused nanopore;
c. upon interaction of an analyte with or passage through a fusion nanopore, at least one current value is obtained indicative of at least one characteristic of the analyte, characterizing the analyte.
10. The method of claim 9, wherein the step of constructing a test system comprising fusion expressed protein and membrane layer comprises adding purified protein to a conductive liquid medium to obtain a unique ion channel between the two conductive liquid media based on the principle that protein is spontaneously embedded or induced to be embedded in the membrane layer.
11. The method of claim 9, wherein the step of constructing a test system comprising fusion expressed protein and membrane layer comprises preparing vesicles from the protein and phospholipid, inserting the protein into the vesicle membrane during vesicle formation, adding the vesicles to a conductive liquid medium, fusing with the membrane layer, and inserting the protein into the membrane layer simultaneously to obtain an ion channel between the two conductive liquid media.
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