WO2021212561A1 - 一种构建双识别位点纳米孔的方法 - Google Patents

一种构建双识别位点纳米孔的方法 Download PDF

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WO2021212561A1
WO2021212561A1 PCT/CN2020/089519 CN2020089519W WO2021212561A1 WO 2021212561 A1 WO2021212561 A1 WO 2021212561A1 CN 2020089519 W CN2020089519 W CN 2020089519W WO 2021212561 A1 WO2021212561 A1 WO 2021212561A1
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protein
fusion
fusion protein
nanopore
expression
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French (fr)
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李伟
周智
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深圳市梅丽纳米孔科技有限公司
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
    • G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/48—Systems using polarography, i.e. measuring changes in current under a slowly-varying voltage

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  • This application relates to the technical field of nanopore detection, and in particular to a method for constructing a dual recognition site nanopore.
  • the protein channel has a nanometer-scale diameter and allows ions and molecules of specific sizes to pass through, called nanopores.
  • nanoporins When nanoporins are embedded on an insulating artificial film, placed in a conductive ionic liquid, and voltage is applied to both sides of the film, the charged ions will pass through the nanopores to form an ionic current.
  • a molecule of a certain size passes through the channel or binds to the pore, it will block the pore and reduce the ion current.
  • the size of the blocking current is related to the molecular size, charge distribution and other characteristic information, so the molecule can be identified by measuring the size of the blocking current .
  • nucleic acid sequencing can be achieved by measuring the change in blocking current of the base sequence passing through the pores.
  • the existing high-throughput sequencing technology needs to go through the process of enzyme amplification and fluorescent labeling, so it has the disadvantages of slow speed and high price.
  • nanopore sequencing does not require amplification, and can be measured with a single molecule, which has the characteristics of fast speed, low cost, long read length, and high sensitivity.
  • Some protein nanopores have been used for the detection of nucleic acids and other polymers, such as mutant ⁇ -hemolysin nanopores for nucleic acid detection (refer to the following documents:
  • CN107109479A Kasianowicz et al., Proc Natl Acad Sci USA, 1996.93(24): 13770, Stoddart et al., Proc Natl Acad Sci USA, 2009.106(19): 7702), MspA nanopore (CN107207571A, CN102216783A, CN102216783A, 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, Wlokaet al., Angew Chem Int Ed Engl, 2016.55(40): 12494), ClyA nanopore for protein detection (CN105358567A, Biesemans, et al., Nano Lett, 2015.15(9): 6076). These nanopores are proteins with a
  • Recognition sites refer to specific amino acid sites in the protein nanopore, which are the main contribution sites of current signals when the nanopore detects molecules.
  • the number, shape, charge state, hydrophilicity and hydrophobicity of recognition sites are the key factors that determine the resolution of nanopore detection.
  • mathematical simulation studies with hemolysin as the object show that nanopores with dual recognition sites have a better base recognition effect than single recognition sites (Stod dart, D., et al., Angew Chem) Int Ed Engl, 2010.49(3): p.556-9).
  • three or more recognition sites are limited by the signal-to-noise ratio, and effective base discrimination cannot be performed.
  • the applicant proposed a method for constructing dual recognition site nanopores, which aims to solve the above problems.
  • the purpose of this application is to provide a method for constructing dual recognition site nanopores, which involves a method for changing the number of protein nanopore recognition sites through protein fusion expression, and the constructed dual recognition site protein Applied to the detection of analytes such as nucleic acids. Specifically, it includes: fusing and expressing two protein nanopores with single recognition sites to form a fusion protein nanopore with two recognition sites, and maintaining the pore size and polymerization properties of the fusion nanopore. For example, take part or all of the functional domains of two single recognition site nanoporins, MspA and FraC, and perform the fusion expression of the protein by direct connection or linker connection, and then polymerize to form two recognition sites.
  • the MspA-FraC channel of the site This application also records the mutation of the fusion-expressed double recognition site protein to obtain a protein channel with better performance.
  • the present application also discloses the use of protein nanopores that change the number of recognition sites and the mutated nanopores to detect nucleic acids, polypeptides and other analytes.
  • a method for constructing a dual recognition site nanopore includes the following steps:
  • the domain analysis tool is used to analyze the domain function of the two single recognition site nanopores expressed by the fusion to obtain the domain analysis results;
  • the structural domain gene select the structural domain gene and design the fusion protein to obtain a fusion protein with two recognition sites, so that the fusion protein with two recognition sites includes a phospholipid membrane binding domain;
  • the fusion protein gene sequence is codon optimized, and different expression plasmids and expression strain combinations of the fusion protein gene sequence are optimized to obtain the fusion protein.
  • test system that includes the fusion protein that has been purified and separated, the membrane layer, and the current measurement device, and use the current characterization method to detect and characterize the analyte;
  • the structure, charge state, and hydrophilicity of the pores of the fusion-expressed dual recognition site nanoporins are optimized by mutation, and the mutant protein is expressed for analyte detection and characterization.
  • the method further includes:
  • the protein three-dimensional structure simulation software is selected for simulation to obtain the three-dimensional structure of the fusion protein, and the folding effect of the fusion protein is predicted based on the three-dimensional structure.
  • the method further includes:
  • the domains derived from different nanoporins are connected by linking sequences or directly connected.
  • the step of selecting an expression system that guarantees the structural stability and polymerizability of the fusion protein 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 that can be used for protein expression.
  • the step of obtaining a fully folded fusion protein by optimizing expression conditions 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.
  • the step of purifying and separating the fusion-expressed protein monomer and the protein assembled into a polymer form includes using gel filtration chromatography, ion exchange chromatography, hydrophobic chromatography, and affinity chromatography. And chromatographic treatment for purification and separation.
  • the step of using a reagent to induce a protein expressed as a monomer after the expression of the fusion protein further includes using rabbit red blood cell membranes or liposomes or an amphipathic chemical reagent to induce treatment.
  • the step of purifying and separating the fusion-expressed protein monomer and the protein assembled into a polymer form includes using size exclusion chromatography or density gradient centrifugation or ultrafiltration membrane separation or cutting.
  • the gel purification process purifies and separates the fusion-expressed protein monomer and the protein assembled into a polymer form.
  • the step of detecting and characterizing an analyte by means of current characterization includes placing the fusion protein in a film layer between the first conductive liquid medium and the second conductive liquid medium , So that at least one of the first conductive liquid medium and the second conductive liquid medium contains an analyte.
  • the membrane layer is a polymer membrane, a lipid layer, a solid membrane or other insulating thin films
  • the analyte is one or more of nucleotides, nucleic acids, amino acids, oligopeptides, polypeptides, proteins, polymers, drugs, inorganic molecules, ions, pollutants, and nano-scale substances.
  • the step of detecting and characterizing an analyte by means of current characterization includes:
  • the current value indicates at least one characteristic of the analyte and characterizes the analyte.
  • the step of constructing a test system including a fusion expressed protein and a membrane layer includes adding the purified protein to a conductive liquid medium, and obtaining the protein according to the principle of spontaneous or induced embedding in the membrane layer. The only ion channel between two conductive liquid media.
  • the step of constructing a test system including a fusion expressed protein and a membrane layer includes preparing the protein and phospholipids into a vesicle, and during the formation of the vesicle, the protein is embedded in the vesicle membrane, The vesicles are added to the conductive liquid medium to fuse with the membrane layer, and the protein is embedded in the membrane layer at the same time to obtain ion channels between the two conductive liquid media.
  • the step of optimizing the structure, charge state, and hydrophilicity of the pores of the fusion-expressed dual recognition site nanoporin through mutation includes: Segment inserts, deletes, and replaces operations.
  • the beneficial effect of the present application lies in: adopting the dual recognition site nanopore construction method of this solution, using naturally occurring single recognition site nanoporins, and obtaining through engineered fusion protein expression technology
  • Nanoporins with dual recognition sites greatly expand the range of options for nanoporins, and solve the problem that there are few naturally occurring protein nanopores with dual sensitive sites.
  • the performance of the channel is further optimized and the detection performance of the channel is improved.
  • the dual recognition site nanopore has a better homopolymer resolution than the single recognition site channel, it is easier to effectively distinguish the current signal difference than three or more recognition sites.
  • the fusion protein obtained by the method of the present application is used for the detection of nucleic acid and other polymers, which can greatly improve the accuracy of detection, and obtain detection accuracy that cannot be obtained by detection methods in the current technology.
  • Fig. 1 is a schematic flow diagram of a specific embodiment of a method for constructing a dual recognition site nanopore according to the present application.
  • first and second are only used for descriptive purposes, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features. Thus, the features defined with “first” and “second” may explicitly or implicitly include one or more of these features. In the description of the present application, “multiple” means two or more than two, unless otherwise specifically defined.
  • the terms “installed”, “connected”, “connected”, “fixed” and other terms should be understood in a broad sense. For example, they may be connected or detachable. Or integrated; it can be a mechanical connection or an electrical connection; it can be directly connected or indirectly connected through an intermediate medium, and it can be the internal communication between two elements or the interaction relationship between two elements.
  • installed may be connected or detachable. Or integrated; it can be a mechanical connection or an electrical connection; it can be directly connected or indirectly connected through an intermediate medium, and it can be the internal communication between two elements or the interaction relationship between two elements.
  • the "on" or “under” of the first feature of the second feature may include direct contact between the first and second features, or may include the first and second features Not in direct contact but through other features between them.
  • the "above”, “above” and “above” of the first feature on the second feature include the first feature directly above and obliquely above the second feature, or it simply means that the first feature is higher in level than the second feature.
  • the “below”, “below” and “below” of the second feature of the first feature include the first feature directly below and obliquely below the second feature, or it simply means that the level of the first feature is smaller than the second feature.
  • the method flow chart shown in FIG. 1 is a schematic flow diagram of a specific embodiment of a method for constructing a dual recognition site nanopore proposed in this application, including the following steps:
  • Step S1 based on pre-preparation or fusion of the gene sequence of the expressed protein, the domain analysis tool is used to analyze the domain function of the two single recognition site nanopores expressed by the fusion to obtain the domain analysis result;
  • Step S2 According to the analysis result of the structural domain, select the structural domain gene and design the fusion protein to obtain a fusion protein with two recognition sites, so that the fusion protein with two recognition sites includes a phospholipid membrane binding domain ;
  • Step S3 Select an expression system that guarantees the stable and polymerizable structure of the fusion protein.
  • codon optimization is performed on the fusion protein gene sequence, and different expression plasmids and expression strain combinations of the fusion protein gene sequence are optimized.
  • Step S4 obtain a fully folded fusion protein by optimizing expression conditions
  • Step S5 obtaining a high-purity fusion protein monomer or polymer by optimizing the purification method
  • Step S6 using a reagent to induce the protein expressed as a monomer after the fusion protein is expressed, to obtain a protein assembled into a polymer form;
  • Step S7 purifying and separating the fused and expressed protein monomer and the protein assembled into a polymer form
  • Step S8 using a test system that includes the fusion protein that has been purified and separated, the membrane layer, and the current measuring device, using current characterization means to detect and characterize the analyte;
  • Step S9 optimizing the structure, charge state, and hydrophilicity and hydrophobicity of the pores of the fusion-expressed dual recognition site nanoporin through mutation, and expressing the mutated protein for analyte detection and characterization.
  • step S1, step S2, step S3, step S4, step S5, step S6, step S7, step S8, and step S9 are aimed at realizing domain analysis, fusion protein design, expression scheme design, expression condition optimization, and purification Condition optimization, pore protein polymerization, purification and separation, pore and sequencing, and mutation of fusion protein; since the number of recognition sites is one of the key factors determining the accuracy of sequencing, under the same conditions, nanopores with dual recognition sites have more The single recognition site channel has better homopolymer resolution, and it is easier to effectively distinguish current signal differences than three or more recognition sites.
  • the domain analysis result obtained in step S1 because the domain is a region with a specific structure and independent function in the protein, it is also the basic functional unit of the protein. Therefore, in nanoporins, different domains are responsible for different functions such as interacting with biomembranes or maintaining specific polymer structures. For example, MspA nanopores are divided into Rim, Stem, Periplasm and other different domains (for reference, see Mahfo ud , M., et al., J Biol Chem, 2006.281(9): p.5908-15.).
  • the pre-prepared gene sequence may include, but is not limited to, the gene sequence reported in the existing literature.
  • the method further includes:
  • the protein three-dimensional structure simulation software is selected for simulation to obtain the three-dimensional structure of the fusion protein, and the folding effect of the fusion protein is predicted based on the three-dimensional structure.
  • the method further includes:
  • the domains derived from different nanoporins are connected by linking sequences or directly connected.
  • the domains derived from different nanoporins can be connected by linkers or directly connected.
  • the length of the linking sequence is very important for protein folding and stability.
  • a suitable linking sequence can be designed.
  • the step of selecting an expression system that guarantees the structural stability and polymerizability 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, and a cell-free in vitro expression system And other systems that can be used for protein expression.
  • the fusion protein can be engineered and expressed in a prokaryotic system or a eukaryotic system, and the prokaryotic expression system is characterized by short time and low cost.
  • the prokaryotic expression system is preferred under the premise of ensuring the stable and polymerizable structure of the fusion protein. According to the host type of the expression system, the corresponding codon optimization of the fusion protein gene sequence is required.
  • the method of optimizing the combination of different expression plasmids and expression strains is used. To obtain the successful expression of the fusion protein.
  • a purification tag such as a histidine tag, is designed at the end of the fusion protein for purification after protein expression.
  • the step of obtaining a fully folded 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.
  • the engineered-induced expression protein may be expressed as a soluble protein or an inclusion body protein, and the expression amount is also very uncertain.
  • expression conditions such as medium type, culture conditions, inducer concentration, inducer type induction temperature, and induction time are optimized to obtain a fully folded fusion protein.
  • step S5 Purify the protein by specific purification methods according to the characteristics of the fusion protein, such as using affinity chromatography to separate the fusion protein containing histidine tag.
  • the step S5 includes gel filtration chromatography, ion exchange chromatography, hydrophobic chromatography and affinity chromatography.
  • some nanoporins can self-assemble into mature polymer pores, such as MspA, when expressed.
  • Some nanoporins are expressed in the form of protein monomers and need to be assembled into a polymer form under specific processing conditions, such as Phi29. Therefore, in step S6, the step of using reagents to induce the protein expressed as a monomer after the expression of the fusion protein also includes using rabbit red blood cell membranes or liposomes or other amphipathic chemical reagents to induce treatment.
  • the step of purifying and separating the fusion-expressed protein monomer and the protein assembled into a polymer form in the step S7 includes using size exclusion chromatography or density gradient centrifugation or ultrafiltration membrane separation or Treatments such as gel-cutting and purification purify and separate the fusion-expressed protein monomer and the protein assembled into a polymer form.
  • nanoporins are expressed and polymerized, generally monomers and polymers will coexist in the solution. Some protein monomers do not have the hole function, and some protein monomers will interfere with the hole test. Therefore, it is necessary to check the fusion expression. Protein monomers and polymers are purified and separated. The above methods can effectively solve this problem and achieve the effect of purification and separation.
  • the process of detecting and characterizing an analyte by means of current characterization described in step S8 includes placing the fusion protein in a film between the first conductive liquid medium and the second conductive liquid medium.
  • the layer at least one of the first conductive liquid medium and the second conductive liquid medium contains an analyte.
  • the film layer is a polymer film, a lipid layer, a solid film or other insulating film.
  • the analyte is one or more of nucleotides, nucleic acids, amino acids, oligopeptides, polypeptides, proteins, polymers, drugs, inorganic molecules, ions, pollutants, and nanoscale substances. kind.
  • the process of detecting and characterizing an analyte by means of current characterization described in step S8 includes:
  • the current value indicates at least one characteristic of the analyte and characterizes the analyte.
  • the step of constructing a test system that includes a fusion expressed protein and a membrane layer includes adding the purified protein to a conductive liquid medium, and according to the principle that the protein is spontaneously embedded or induced to be embedded in the membrane layer, Obtain the only ion channel between the two conductive liquid media.
  • the process of constructing a test system including a fusion expressed protein and a membrane layer can also be specifically: preparing the protein and the phospholipid into a vesicle, and during the formation of the vesicle, the protein is embedded In the vesicle membrane, the vesicles are added to the conductive liquid medium to fuse with the membrane layer, and the protein is embedded in the membrane layer at the same time to obtain ion channels between the two conductive liquid media.
  • the step of optimizing the structure, charge state, and hydrophilicity and hydrophobicity of the fusion-expressed dual recognition site nanoporin through mutation in the step S9 includes: Peptides are inserted, deleted and replaced.
  • the structure, charge state, and hydrophilicity and hydrophobicity inside the pore can be further optimized through mutation, thereby improving the accuracy of detection.
  • a mutation method is adopted to make the mutated protein expressed and used for testing, so as to improve the accuracy.

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Abstract

本申请公开了一种构建双识别位点纳米孔的方法，包括：对融合表达的两种单识别位点纳米孔进行结构域功能分析，获得结构域分析结果；选择结构域基因，进行融合蛋白设计，获得具有两个识别位点的融合蛋白；选择保证融合蛋白结构稳定及可聚合的表达系统，根据表达系统的宿主种类，并依次完成表达方案设计、表达条件优化、孔道蛋白聚合、纯化分离、嵌孔及测序以及融合蛋白突变的处理；本申请使用天然存在的单识别位点的纳米孔蛋白，通过工程化的融合蛋白表达技术，获得双识别位点的纳米孔道蛋白，大大扩展了纳米孔蛋白的可选择范围，解决了天然存在的双敏感位点的蛋白纳米孔极少的问题。通过突变了优化孔道的性能，提高孔道的检测性能。

Description

一种构建双识别位点纳米孔的方法
本申请是以申请号为202010312465.6、申请日为2020年4月20日的中国专利申请为基础，并主张其优先权，该申请的全部内容在此作为整体引入本申请中。
技术领域
本申请涉及纳米孔检测技术领域，尤其涉及一种构建双识别位点纳米孔的方法。
背景技术
天然生物膜上存在一类跨膜蛋白通道，该蛋白通道具有纳米尺度的直径，允许离子及特定大小分子穿过，称为纳米孔。
将纳米孔蛋白嵌在绝缘的人工薄膜上，置于导电离子液体中，并在薄膜两侧施加电压，则带电离子将穿越纳米孔道形成离子电流。当有特定大小的分子穿过通道或结合到孔道上时，会阻塞孔道从而降低离子流，阻塞电流的大小与分子尺寸、电荷分布等特征信息相关，因此可通过测量阻塞电流的大小来识别分子。
运用这一原理，当核酸或者其他分析物在电压的作用下瞬时穿过蛋白纳米孔道时，通过测量碱基序列穿过孔道的阻塞电流变化就能实现核酸测序。现有的高通量测序技术需通过酶扩增和荧光标记的过程，因此具有速度慢、价格贵的缺点。相较于现有的测序方法，纳米孔测序无需扩增，单分子即可测量，具有快速、低成本、长读长、高灵敏度等特点。
已有部分蛋白纳米孔道用于核酸等多聚物的检测，例如用于核酸检测的突变体α-溶血素纳米孔(可参考下列文件：
CN107109479A，Kasianowicz et al.，Proc Natl Acad Sci USA，1996.93(24)：13770，Stoddart et al.，Proc Natl Acad Sci USA，2009.106(19)：7702)、MspA纳米孔(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纳米孔(CN107207570A)， FraC纳米孔(WO2018/012963A1，Wloka et al.，Angew Chem Int Ed Engl，2016.55(40)：12494)，用于蛋白检测的ClyA纳米孔(CN105358567A，Biese mans，et al.，Nano Lett，2015.15(9)：6076)。这些纳米孔均为单识别位点或三个及以上识别位点的蛋白。
识别位点是指蛋白纳米孔中一些特定的氨基酸位点，是纳米孔检测分子时电流信号的主要贡献位点。识别位点的数量、形状、电荷状态、亲疏水性等是决定纳米孔检测分辨率的关键因素。识别位点数量方面，以溶血素为对象的数学模拟研究表明，双识别位点的纳米孔比单识别位点具有更好的碱基识别效果(Stod dart，D.，et al.，Angew Chem Int Ed Engl，2010.49(3)：p.556-9)。而三个及以上的识别位点则受限于信噪比，无法进行有效的碱基区分。
然而，天然存在的双识别位点的蛋白纳米孔极少，寻找合适的纳米孔并进行相应的改造，是进一步提高测序准确度的关键。
公开于该背景技术部分的信息仅仅旨在加深对本申请的总体背景技术的理解，而不应当被视为承认或以任何形式暗示该信息构成已为本领域技术人员所公知的现有技术。
基于上述原因，本申请人提出了一种构建双识别位点纳米孔的方法，旨在解决上述问题。
申请内容
为了满足上述要求，本申请的目的在于提供一种构建双识别位点纳米孔的方法，涉及一种通过蛋白融合表达改变蛋白纳米孔识别位点数量的方法，并将构建的双识别位点蛋白应用于核酸等分析物的检测。具体包括：融合表达两种单识别位点的蛋白纳米孔，形成具有两个识别位点的融合蛋白纳米孔，并保持融合纳米孔的嵌孔能力及聚合性质。举例来说，取MspA和FraC两种单识别位点纳米孔蛋白的部分或全部功能域，以直接连接或连接序列(linker)连接的方式进行蛋白的融合表达，然后进行聚合后形成两个识别位点的MspA-FraC孔道。本申请还记载了到将融合表达的双识别位点的蛋白进行突变，以获得具有更好性能的蛋白孔道。在另一方面，本申请还公开了使用改变识别位点数量的蛋白纳米孔及在此基础上进行突变后的纳米孔进行核酸、多肽等分析物的检测。
为了实现上述目的，本申请采用以下技术方案：
一种构建双识别位点纳米孔的方法，包括以下步骤：
基于预先准备或融合表达蛋白的基因序列，使用结构域分析工具对融合表达的两种单识别位点纳米孔进行结构域功能分析，获得结构域分析结果；
根据所述结构域分析结果，选择结构域基因，进行融合蛋白设计，获得具有两个识别位点的融合蛋白，使所述具有两个识别位点的融合蛋白包括磷脂膜结合结构域；
选择保证融合蛋白结构稳定及可聚合的表达系统，根据表达系统的宿主种类，对融合蛋白基因序列进行密码子优化，对融合蛋白基因序列的不同的表达质粒及表达菌株组合进行优化，获得融合蛋白的成功表达，在融合蛋白末端设计纯化标签以实现蛋白表达后的纯化；
通过优化表达条件，获得折叠完整的融合蛋白；
通过优化纯化方法，获得高纯度的融合蛋白单体或聚合体；
使用试剂对在融合蛋白表达后为单体表达的蛋白进行诱导，获得组装成聚合体形式的蛋白；
对融合表达后的蛋白单体和组装成聚合体形式的蛋白进行纯化分离；
使用包含已经进行纯化分离处理的融合蛋白、膜层和电流测量装置的测试系统，利用电流表征的手段进行分析物的检测和表征；
通过突变对融合表达的双识别位点纳米孔蛋白的孔道内部的结构、电荷状态及亲疏水性进行优化，对突变后的蛋白进行表达后用于分析物检测与表征。
在一个可能的实施方式中，所述获得具有两个识别位点的融合蛋白的步骤之后还包括：
选用蛋白三维结构模拟软件进行模拟，获得融合蛋白的三维结构，根据三维结构预测融合蛋白的折叠效果。
在一个可能的实施方式中，所述获得具有两个识别位点的融合蛋白的步骤之后还包括：
将来源于不同纳米孔蛋白的结构域通过连接序列连接或进行直接连接。
在一个可能的实施方式中，所述选择保证融合蛋白结构稳定及可聚合的表达系 统的步骤包括选择原核表达系统、真核表达系统、昆虫表达系统、动物细胞表达系统、无细胞体外表达系统以及其他可用于蛋白表达的系统。
在一个可能的实施方式中，所述通过优化表达条件，获得折叠完整的融合蛋白的步骤包括，优化培养基种类，优化培养条件，优化诱导剂浓度、诱导剂种类、诱导温度、诱导时间。
在一个可能的实施方式中，所述对融合表达后的蛋白单体和组装成聚合体形式的蛋白进行纯化分离的步骤包括，利用凝胶过滤层析、离子交换层析、疏水层析以及亲和层析的处理进行纯化分离。
在一个可能的实施方式中，所述使用试剂对在融合蛋白表达后为单体表达的蛋白进行诱导的步骤还包括，使用兔红细胞膜或脂质体或两亲性的化学试剂进行诱导处理。
在一个可能的实施方式中，所述对融合表达后的蛋白单体和组装成聚合体形式的蛋白进行纯化分离的步骤包括，使用排阻色谱层析或密度梯度离心或超滤膜分离或切胶纯化的处理对融合表达后的蛋白单体和组装成聚合体形式的蛋白进行纯化分离。
在一个可能的实施方式中，所述利用电流表征的手段进行分析物的检测和表征的步骤包括，将所述融合蛋白置于第一导电液体介质与第二导电液体介质之间的膜层中，使所述第一导电液体介质与第二导电液体介质至少一项中含有分析物。
在一个可能的实施方式中，所述膜层为高分子膜、脂质层、固态膜或其他绝缘薄膜；
所述分析物为核苷酸、核酸、氨基酸、寡聚肽、多肽、蛋白质、多聚物、药物、无机分子、离子、污染物、纳米级物质中的一种或几种。
在一个可能的实施方式中，所述利用电流表征的手段进行分析物的检测和表征的步骤包括：
a.组建包含融合表达蛋白及膜层的测试系统；
b.施加电压，使分析物与融合纳米孔相互作用或穿过纳米孔；
c.分析物与融合纳米孔相互作用或穿过纳米孔时，获取至少一个电流值，所述 电流值指示所述分析物的至少一个特征，表征所述分析物。
在一个可能的实施方式中，所述组建包含融合表达蛋白及膜层的测试系统的步骤包括，将纯化后的蛋白加入导电液体介质，根据蛋白自发嵌入或被诱导嵌入膜层中的原理，获得两种导电液体介质之间的唯一离子通道。
在一个可能的实施方式中，所述组建包含融合表达蛋白及膜层的测试系统的步骤包括，将所述蛋白与磷脂制备成囊泡，在囊泡形成的过程中，蛋白嵌入囊泡膜，将囊泡加入导电液体介质中，与膜层融合，蛋白同时嵌入膜层，获得两种导电液体介质之间的离子通道。
在一个可能的实施方式中，所述通过突变对融合表达的双识别位点纳米孔蛋白的孔道内部的结构、电荷状态及亲疏水性进行优化的步骤包括，对蛋白特定位点的碱基或肽段进行插入、删除及替换操作。
相比于现有技术，本申请的有益效果在于：采用本方案的双识别位点纳米孔构建方法，使用天然存在的单识别位点的纳米孔蛋白，通过工程化的融合蛋白表达技术，获得双识别位点的纳米孔道蛋白，大大扩展了纳米孔蛋白的可选择范围，解决了天然存在的双敏感位点的蛋白纳米孔极少的问题。通过对融合蛋白的突变，进一步优化孔道的性能，提高孔道的检测性能。由于双识别位点的纳米孔具有比单识别位点孔道更好的同聚物分辨率，相较于三个及以上的识别位点，更容易实现电流信号差异的有效区分。利用本申请的方法得到的融合蛋白用于核酸等多聚物的检测，可以极大地提高了检测的准确度，获得当前技术中的检测方法所不能获得的检测准确性。
下面结合附图和具体实施例对本申请作进一步描述。
附图说明
图1是本申请一种构建双识别位点纳米孔的方法的具体实施例流程示意图。
具体实施方式
下面将结合本申请实施例中的附图，对本申请实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本申请一部分实施例，而不是全部的实施例。基于本申请中的实施例，本领域技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本申请保护的范围。
在本申请的描述中，需要理解的是，术语“中心”、“纵向”、“横向”、“长度”、“宽度”、“厚度”、“上”、“下”、“前”、“后”、“左”、“右”、“竖直”、“水平”、“顶”、“底”“内”、“外”、“顺时针”、“逆时针”等指示的方位或位置关系为基于附图所示的方位或位置关系，仅是为了便于描述本申请和简化描述，而不是指示或暗示所指的装置或元件必须具有特定的方位、以特定的方位构造和操作，因此不能理解为对本申请的限制。
此外，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括一个或者更多个该特征。在本申请的描述中，“多个”的含义是两个或两个以上，除非另有明确具体的限定。
在本申请中，除非另有明确的规定和限定，术语“安装”、“相连”、“连接”、“固定”等术语应做广义理解，例如，可以是连接，也可以是可拆卸连接，或成一体；可以是机械连接，也可以是电连接；可以是直接相连，也可以通过中间媒介间接相连，可以是两个元件内部的连通或两个元件的相互作用关系。对于本领域的普通技术人员而言，可以根据具体情况理解上述术语在本申请中的具体含义。
在本申请中，除非另有明确的规定和限定，第一特征在第二特征之“上”或之“下”可以包括第一和第二特征直接接触，也可以包括第一和第二特征不是直接接触而是通过它们之间的另外的特征接触。而且，第一特征在第二特征“之上”、“上方”和“上面”包括第一特征在第二特征正上方和斜上方，或仅仅表示第一特征水平高度高于第二特征。第一特征在第二特征“之下”、“下方”和“下面”包括第一特征在第二特征正下方和斜下方，或仅仅表示第一特征水平高度小于第二特征。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本申请的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不应理解为必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任何的一个或多个实施例或示例 中以合适的方式结合。此外，本领域的技术人员可以将本说明书中描述的不同实施例或示例进行接合和组合。
如图1所示的方法流程图，为本申请提出的一种构建双识别位点纳米孔的方法的具体实施例流程示意图，包括以下步骤：
步骤S1，基于预先准备或融合表达蛋白的基因序列，使用结构域分析工具对融合表达的两种单识别位点纳米孔进行结构域功能分析，获得结构域分析结果；
步骤S2，根据所述结构域分析结果，选择结构域基因，进行融合蛋白设计，获得具有两个识别位点的融合蛋白，使所述具有两个识别位点的融合蛋白包括磷脂膜结合结构域；
步骤S3，选择保证融合蛋白结构稳定及可聚合的表达系统，根据表达系统的宿主种类，对融合蛋白基因序列进行密码子优化，对融合蛋白基因序列的不同的表达质粒及表达菌株组合进行优化，获得融合蛋白的成功表达，在融合蛋白末端设计纯化标签以实现蛋白表达后的纯化；
步骤S4，通过优化表达条件，获得折叠完整的融合蛋白；
步骤S5，通过优化纯化方法，获得高纯度的融合蛋白单体或聚合体；
步骤S6，使用试剂对在融合蛋白表达后为单体表达的蛋白进行诱导，获得组装成聚合体形式的蛋白；
步骤S7，对融合表达后的蛋白单体和组装成聚合体形式的蛋白进行纯化分离；
步骤S8，使用包含已经进行纯化分离处理的融合蛋白、膜层和电流测量装置的测试系统，利用电流表征的手段进行分析物的检测和表征；
步骤S9，通过突变对融合表达的双识别位点纳米孔蛋白的孔道内部的结构、电荷状态及亲疏水性进行优化，对突变后的蛋白进行表达后用于分析物检测与表征。
具体地，步骤S1、步骤S2、步骤S3、步骤S4、步骤S5、步骤S6、步骤S7、步骤S8以及步骤S9，目的在于实现结构域分析、融合蛋白设计、表达方案设计、表达条件优化、纯化条件优化、孔道蛋白聚合、纯化分离、嵌孔及测序以及融合蛋白突变；由于识别位点的数量是决定测序准确度的关键因素之一，在同等条件下，双识别位点的纳米孔具有比单识别位点孔道更好的同聚物分辨率，相 较于三个及以上的识别位点，更容易实现电流信号差异的有效区分。
然而，天然存在的双敏感位点的蛋白纳米孔极少。本申请使用上述步骤S1-S9的步骤，使用天然存在的单识别位点的纳米孔蛋白，通过工程化的融合蛋白表达技术，获得双识别位点的纳米孔道蛋白，大大扩展了纳米孔蛋白的可选择范围。通过对融合蛋白的突变，进一步优化孔道的性能，提高孔道的检测性能。并将融合蛋白用于核酸等多聚物的检测，能够有效提高检测的准确度。
在一实施例中，步骤S1获得的结构域分析结果，由于结构域是蛋白中具有特异结构和独立功能的区域，也是蛋白质的基本功能单元。因此，在纳米孔蛋白中，不同结构域分别负责与生物膜相互作用或维持特定聚合体结构等不同功能，如MspA纳米孔中分为Rim、Stem、Periplasm等不同结构域(参考资料可见Mahfo ud，M.，et al.，J Biol Chem，2006.281(9)：p.5908-15.)。
因此，步骤S1中，所述基于预先准备的基因序列可包括但不限于已有的文献报道的基因序列。
在一实施例中，步骤S2中获得具有两个识别位点的融合蛋白的步骤之后还包括：
选用蛋白三维结构模拟软件进行模拟，获得融合蛋白的三维结构，根据三维结构预测融合蛋白的折叠效果。
在一实施例中，所述获得具有两个识别位点的融合蛋白的步骤之后还包括：
将来源于不同纳米孔蛋白的结构域通过连接序列连接或进行直接连接。
具体地，融合蛋白表达中，来源于不同纳米孔蛋白的结构域可通过连接序列(linker)连接或直接连接。连接序列的长度对蛋白折叠和稳定性非常重要，参照现有文献报道的连接序列设计工具，可设计合适的连接序列。
在一实施例中，所述步骤S3中选择保证融合蛋白结构稳定及可聚合的表达系统的步骤包括选择原核表达系统、真核表达系统、昆虫表达系统、动物细胞表达系统、无细胞体外表达系统以及其他可用于蛋白表达的系统，其中，由于融合蛋白可在原核系统或真核系统中工程化表达，而原核表达系统的特点是时间短、费用低，鉴于纳米孔蛋白用于分子检测并不需要保持蛋白活性，因此在保证融合蛋白结构稳定及可聚合的前提下，优先选择原核表达系统。根据表达系统 的宿主种类，需对融合蛋白基因序列进行相应的密码子优化。
此外，为解决不同的表达质粒及表达菌株对蛋白的成功表达及折叠均会对融合蛋白成功表达产生影响的问题，在本实施例中，采取优化不同的表达质粒及表达菌株组合的手段，用于获得融合蛋白的成功表达。
在一实施例中，为解决蛋白表达后的纯化问题，采取在融合蛋白末端设计纯化标签，如组氨酸标签，用于蛋白表达后的纯化。
在一实施例中，所述步骤S4中通过优化表达条件，获得折叠完整的融合蛋白的步骤包括，优化培养基种类，优化培养条件，优化诱导剂浓度、诱导剂种类、诱导温度、诱导时间。
具体地，对于步骤S4，由于本方案采取了工程化诱导表达的方式，而工程化诱导表达的蛋白可能表达为可溶性蛋白或包涵体蛋白，表达量也有很大不确定性。为克服该问题，本实施例中采取了通过优化培养基种类，培养条件，诱导剂浓度、诱导剂种类诱导温度、诱导时间等表达条件，从而获得折叠完整的融合蛋白。
在一实施例中，由于提取出的总蛋白中含有表达系统本身所具有的相当数量的杂蛋白，对于单体诱导组装成聚合体形式及纳米孔蛋白嵌孔测试均有影响，因此，步骤S5根据融合蛋白特点通过特定的纯化方法进行蛋白的纯化，如用亲和层析分离包含组氨酸标签的融合蛋白。所述步骤S5包括凝胶过滤层析、离子交换层析、疏水层析以及亲和层析。
在一实施例中，由于部分纳米孔蛋白可在表达时自组装成成熟的聚合体孔道，如MspA。部分纳米孔蛋白则以蛋白单体的形式表达，需要特定的处理条件下组装成聚合体形式，如Phi29。因此，步骤S6中使用试剂对在融合蛋白表达后为单体表达的蛋白进行诱导的步骤还包括，使用兔红细胞膜或脂质体或其他两亲性的化学试剂等进行诱导处理。
在一实施例中，所述步骤S7中对融合表达后的蛋白单体和组装成聚合体形式的蛋白进行纯化分离的步骤包括，使用排阻色谱层析或密度梯度离心或超滤膜分离或切胶纯化等的处理对融合表达后的蛋白单体和组装成聚合体形式的蛋白进行纯化分离。
具体地，由于纳米孔蛋白在表达聚合后，一般单体和聚合体会共存于溶液中，部分蛋白单体不具有嵌孔功能，部分蛋白单体会干扰嵌孔测试，因此需要对融合表达后的蛋白单体和聚合体进行纯化分离。采用上述手段可有效解决这一问题，达到纯化分离的作用。
在一实施例中，所述步骤S8记载的利用电流表征的手段进行分析物的检测和表征的过程包括，将所述融合蛋白置于第一导电液体介质与第二导电液体介质之间的膜层中，使所述第一导电液体介质与第二导电液体介质至少一项中含有分析物。
在一实施例中，所述膜层为高分子膜、脂质层、固态膜或其他绝缘薄膜。
在一实施例中，所述分析物为核苷酸、核酸、氨基酸、寡聚肽、多肽、蛋白质、多聚物、药物、无机分子、离子、污染物、纳米级物质中的一种或几种。
在一实施例中，所述步骤S8记载的利用电流表征的手段进行分析物的检测和表征的过程包括：
a.组建包含融合表达蛋白及膜层的测试系统；
b.施加电压，使分析物与融合纳米孔相互作用或穿过纳米孔；
c.分析物与融合纳米孔相互作用或穿过纳米孔时，获取至少一个电流值，所述电流值指示所述分析物的至少一个特征，表征所述分析物。
在一个可选的实施方式中，所述组建包含融合表达蛋白及膜层的测试系统的步骤包括，将纯化后的蛋白加入导电液体介质，根据蛋白自发嵌入或被诱导嵌入膜层中的原理，获得两种导电液体介质之间的唯一离子通道。
在一个可选的实施方式中，所述组建包含融合表达蛋白及膜层的测试系统的过程具体还可以为，将所述蛋白与磷脂制备成囊泡，在囊泡形成的过程中，蛋白嵌入囊泡膜，将囊泡加入导电液体介质中，与膜层融合，蛋白同时嵌入膜层，获得两种导电液体介质之间的离子通道。
在一实施例中，所述步骤S9中通过突变对融合表达的双识别位点纳米孔蛋白的孔道内部的结构、电荷状态及亲疏水性进行优化的步骤包括，对蛋白特定位点的碱基或肽段进行插入、删除及替换操作。
由于融合表达的双识别位点纳米孔蛋白，可进一步通过突变优化孔道内部的结 构、电荷状态及亲疏水性，从而提高检测的准确度。本实施例中采取了突变的手段处理，使突变后的蛋白进行表达后用于测试，以便于提高准确度。
本申请实施例方法中的步骤可以根据实际需要进行顺序调整、合并和删减。
对本领域的技术人员来说，可根据以上描述的技术方案以及构思，做出其他各种相应的改变以及形变，而所有的这些改变以及形变应该属于本申请权利要求的保护范围之内。
发明概述
技术问题
问题的解决方案
发明的有益效果

Claims (10)

  1. 一种构建双识别位点纳米孔的方法，其特征在于，包括以下步骤：
    基于预先准备或融合表达蛋白的基因序列，使用结构域分析工具对融合表达的两种单识别位点纳米孔进行结构域功能分析，获得结构域分析结果；
    根据所述结构域分析结果，选择结构域基因，进行融合蛋白设计，获得具有两个识别位点的融合蛋白，使所述具有两个识别位点的融合蛋白包括磷脂膜结合结构域；
    选择保证融合蛋白结构稳定及可聚合的表达系统，根据表达系统的宿主种类，对融合蛋白基因序列进行密码子优化，对融合蛋白基因序列的不同的表达质粒及表达菌株组合进行优化，获得融合蛋白的成功表达，在融合蛋白末端设计纯化标签以实现蛋白表达后的纯化；
    通过优化表达条件，获得折叠完整的融合蛋白；
    通过优化纯化方法，获得高纯度的融合蛋白单体或聚合体；
    使用试剂对在融合蛋白表达后为单体表达的蛋白进行诱导，获得组装成聚合体形式的蛋白；
    对融合表达后的蛋白单体和组装成聚合体形式的蛋白进行纯化分离；
    使用包含已经进行纯化分离处理的融合蛋白、膜层和电流测量装置的测试系统，利用电流表征的手段进行分析物的检测和表征；
    通过突变对融合表达的双识别位点纳米孔蛋白的孔道内部的结构、电荷状态及亲疏水性进行优化，对突变后的蛋白进行表达后用于分析物检测与表征。
  2. 根据权利要求1所述的构建双识别位点纳米孔的方法，其特征在于，所述获得具有两个识别位点的融合蛋白的步骤之后还包括：
    选用蛋白三维结构模拟软件进行模拟，获得融合蛋白的三维结构 ，根据三维结构预测融合蛋白的折叠效果。
  3. 根据权利要求1所述的构建双识别位点纳米孔的方法，其特征在于，所述获得具有两个识别位点的融合蛋白的步骤之后还包括：
    将来源于不同纳米孔蛋白的结构域通过连接序列连接或进行直接连接。
  4. 根据权利要求1所述的构建双识别位点纳米孔的方法，其特征在于，所述使用试剂对在融合蛋白表达后为单体表达的蛋白进行诱导的步骤还包括，使用兔红细胞膜或脂质体或两亲性的化学试剂进行诱导处理。
  5. 根据权利要求1所述的构建双识别位点纳米孔的方法，其特征在于，所述对融合表达后的蛋白单体和组装成聚合体形式的蛋白进行纯化分离的步骤包括，使用排阻色谱层析或密度梯度离心或超滤膜分离或切胶纯化的处理对融合表达后的蛋白单体和组装成聚合体形式的蛋白进行纯化分离。
  6. 根据权利要求1所述的构建双识别位点纳米孔的方法，其特征在于，所述利用电流表征的手段进行分析物的检测和表征的步骤包括，将所述融合蛋白置于第一导电液体介质与第二导电液体介质之间的膜层中，使所述第一导电液体介质与第二导电液体介质至少一项中含有分析物。
  7. 根据权利要求1所述的构建双识别位点纳米孔的方法，其特征在于，所述膜层为高分子膜、脂质层、固态膜或绝缘薄膜；
    所述分析物为核苷酸、核酸、氨基酸、寡聚肽、多肽、蛋白质、多聚物、药物、无机分子、离子、污染物、纳米级物质中的一种或多种。
  8. 根据权利要求1所述的构建双识别位点纳米孔的方法，所述利用电流表征的手段进行分析物的检测和表征的步骤包括：
    a.组建包含融合表达蛋白及膜层的测试系统；
    b.施加电压，使分析物与融合纳米孔相互作用或穿过纳米孔；
    c.分析物与融合纳米孔相互作用或穿过纳米孔时，获取至少一个电流值，所述电流值指示所述分析物的至少一个特征，表征所述分析物。
  9. 根据权利要求8所述的构建双识别位点纳米孔的方法，其特征在于，所述组建包含融合表达蛋白及膜层的测试系统的步骤包括，将纯化后的蛋白加入导电液体介质，根据蛋白自发嵌入或被诱导嵌入膜层中的原理，获得两种导电液体介质之间的唯一离子通道。
  10. 根据权利要求8所述的构建双识别位点纳米孔的方法，其特征在于，所述组建包含融合表达蛋白及膜层的测试系统的步骤包括，将所述蛋白与磷脂制备成囊泡，在囊泡形成的过程中，蛋白嵌入囊泡膜，将囊泡加入导电液体介质中，与膜层融合，蛋白同时嵌入膜层，获得两种导电液体介质之间的离子通道。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115877018A (zh) * 2022-08-05 2023-03-31 四川大学华西医院 一种孔蛋白在制备检测去氢表雄酮硫酸酯的试剂盒中的应用

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117886907A (zh) * 2022-10-14 2024-04-16 北京普译生物科技有限公司 一种pht纳米孔突变体蛋白及其应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009046094A1 (en) * 2007-10-01 2009-04-09 Nabsys, Inc. Biopolymer sequencing by hybridization of probes to form ternary complexes and variable range alignment
CN106133513A (zh) * 2014-02-19 2016-11-16 华盛顿大学 蛋白质特征的基于纳米孔的分析
US20170306356A1 (en) * 2012-07-24 2017-10-26 Technische Universität Dresden Protein with Recombinase Activity for Site-Specific DNA-Recombination
CN108226249A (zh) * 2018-01-09 2018-06-29 深圳市梅丽纳米孔科技有限公司 一次性纳米孔生物传感器及其制作方法
CN109069662A (zh) * 2016-03-24 2018-12-21 吉尼亚科技公司 可用于纳米孔系统的位点特异性生物缀合方法和组合物

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100291548A1 (en) * 2006-03-12 2010-11-18 Applera Corporation Methods of Detecting Target Nucleic Acids
GB0905140D0 (en) * 2009-03-25 2009-05-06 Isis Innovation Method
CN106471369B (zh) * 2014-05-05 2019-11-15 奥特拉公司 利用纳米细孔的目标检测
JP6942129B2 (ja) * 2015-11-25 2021-09-29 エフ．ホフマン−ラ ロシュ アーゲーＦ． Ｈｏｆｆｍａｎｎ−Ｌａ Ｒｏｃｈｅ Ａｋｔｉｅｎｇｅｓｅｌｌｓｃｈａｆｔ ポリメラーゼ複合体の精製
CA3030704A1 (en) * 2016-07-12 2018-01-18 Rijksuniversiteit Groningen Biological nanopores for biopolymer sensing and sequencing based on frac actinoporin
CN110386986B (zh) * 2018-06-22 2024-01-26 深圳琅技生命科技有限公司 人工改造蛋白质及其构建方法与应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009046094A1 (en) * 2007-10-01 2009-04-09 Nabsys, Inc. Biopolymer sequencing by hybridization of probes to form ternary complexes and variable range alignment
US20170306356A1 (en) * 2012-07-24 2017-10-26 Technische Universität Dresden Protein with Recombinase Activity for Site-Specific DNA-Recombination
CN106133513A (zh) * 2014-02-19 2016-11-16 华盛顿大学 蛋白质特征的基于纳米孔的分析
CN109069662A (zh) * 2016-03-24 2018-12-21 吉尼亚科技公司 可用于纳米孔系统的位点特异性生物缀合方法和组合物
CN108226249A (zh) * 2018-01-09 2018-06-29 深圳市梅丽纳米孔科技有限公司 一次性纳米孔生物传感器及其制作方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DAVID STODDART ET AL.: "Multiple Base-Recognition Sites in a Biological Nanopore: Two Heads are Better than One", 《ANGEWANDTE CHEMIE》, vol. 122, no. 3, 12 January 2010 (2010-01-12), XP055860407, ISSN: 0044-8249 *
SHAOYING WANG, FARZIN HAQUE, PIOTR G. RYCHAHOU, B. MARK EVERS, PEIXUAN GUO: "Engineered Nanopore of Phi29 DNA-Packaging Motor for Real-Time Detection of Single Colon Cancer Specific Antibody in Serum", ACS NANO, vol. 7, no. 11, 26 November 2013 (2013-11-26), US , pages 9814 - 9822, XP055468743, ISSN: 1936-0851, DOI: 10.1021/nn404435v *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115877018A (zh) * 2022-08-05 2023-03-31 四川大学华西医院 一种孔蛋白在制备检测去氢表雄酮硫酸酯的试剂盒中的应用

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