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WO2020029101A1 - Composant de nanopore à base de canal de protéine de queue, procédé de préparation de celui-ci et applications associées - Google Patents

Composant de nanopore à base de canal de protéine de queue, procédé de préparation de celui-ci et applications associées Download PDF

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Publication number
WO2020029101A1
WO2020029101A1 PCT/CN2018/099294 CN2018099294W WO2020029101A1 WO 2020029101 A1 WO2020029101 A1 WO 2020029101A1 CN 2018099294 W CN2018099294 W CN 2018099294W WO 2020029101 A1 WO2020029101 A1 WO 2020029101A1
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Prior art keywords
phage
tail protein
protein channel
tail
channel
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Ceased
Application number
PCT/CN2018/099294
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English (en)
Chinese (zh)
Inventor
王少英
法金·哈克
杨玲娜
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Shenzhen P&z Bio Medicine Co Ltd
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Shenzhen P&z Bio Medicine Co Ltd
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Priority to PCT/CN2018/099294 priority Critical patent/WO2020029101A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing

Definitions

  • Nanopore device based on tail protein channel and preparation process and application thereof
  • the present application belongs to the technical field of nanopore detection, and particularly relates to a nanopore based on a tail protein channel and a preparation process and application thereof.
  • Nanopore technology is a new type of single-molecule detection technology. It is an emerging detection technology that is a multi-disciplinary fusion of nanometer, biology, chemistry, and electronic information. Its principle is in a cavity filled with electrolyte solution. The cavity is divided into two small chambers by an insulating layer. There is a nano-sized hole on the insulating layer for ions or other small molecules to pass. A voltage is used to drive a single molecule through a nano-scale channel to form a detectable ion Current changes. Analyzing the current changes can identify the nature of the translocation molecule and reveal information about the translocation of the molecule. Nanopore detection technology has the advantages of low cost, simple and fast operation, real-time detection, high-throughput, and non-labeling, so that it has developed rapidly in various fields.
  • nanopores generally include solid nanopores and biological nanopores.
  • the solid nanopores are nano-scale channels obtained by physical or chemical drilling or etching of solid-based systems, and biological nanopores are formed in lipid membranes.
  • Protein channels in nanoscale pipes There are a large number of membrane channels in organisms, which are used to regulate the transport of biological macromolecules such as proteins and nucleic acids, and as a metabolic pathway. Many processes in organisms involve the passage of biological polymer chains through the channels on biofilms. These channels and biological macromolecules The spatial structure coincides with nanotechnology, which provides more possibilities for the research on the recognition of biological macromolecules such as proteins and DNA.
  • Nanopores Some protein channels in organisms have been explored as nanopores, such as ot-hemolysin, MspA, aerolysin, FluA, Omp F / G, CsgG, ClyA, PA63, and non-membrane proteins such as viruses. Phage phi29, SPP1, T3 and T4. These nanochannels have been successfully used to detect small molecules, polymers, peptides, and DNA / RNA.
  • protein engineering such as site-directed mutagenesis, amino acid insertions and deletions
  • functional modules have been widely used to adjust the properties of nanopores. For different analytes, nanopores are required to have different shapes.
  • this application is to solve the above technical problems, so as to propose a tail protein channel-based nanopore device with higher stability, sensitivity, accuracy, and better embedding, and a preparation process and application.
  • the present application provides a nanoporous device based on a tail protein channel, which is characterized in that it includes a first conductive liquid region and a second conductive liquid region, and the first conductive liquid region and the second conductive liquid region pass through.
  • the two-layer insulating film is separated, and the two-layer insulating film is provided with a tail protein channel.
  • the tail protein channel is one of a combination of a wild type tail protein channel, a mutant tail protein channel, a wild type tail protein channel homolog, and a mutant tail protein channel homolog.
  • the wild-type tail protein channel is selected from the group consisting of phage 29, phage T4, phage T3, phage T5, phage T7, phage SPP1, phage P22, phage P2, phage P3, X-phage caudin phage Mu, Mu, At least one of phage G, phage HK97, and phage P2.
  • the mutant tail protein channel is composed of a bacterial cell 29, a bacterial cell T4, a bacterial cell T3, a bacterial cell T5, a phage T7, a phage SPP1, a phage P22, a phage P2, a phage P3, and X phage Tail protein phage Mu, phage G, phage HK97 and at least one of phage P2 mutations.
  • the mutated tail protein channel of phage 29 is gp-9 del 417-491, Mut-a,
  • the Mut-a is K134I, D138, 139L
  • the Mut-b is K134I, D138, 139L, D158L, E163V
  • the Mut-c is K134I, D138, 139L, D158L, E163V , E309 V, D311V.
  • the double-layer insulating film is a lipid or a chemical film.
  • the present application also provides a process for preparing the tail protein channel-based nanopore device, which includes the following steps:
  • S2. Apply a voltage to a tail protein channel to detect a change in current or optical signal when an analyte passes through the tail protein channel.
  • the step S1 is specifically: separating a lipid or a chemical membrane of 0.1-0.5 mg / L dissolved in the organic phase
  • the seed is applied or allowed to flow through the pores with a pore diameter of 20-40 (Vm) to form a bilayer membrane, which separates the liquid on its two sides, and then adds a tail protein channel to embed it in the bilayer membrane.
  • the application also provides an application of the tail protein channel-based nanopore device for detecting nucleic acids, amino acids, peptides, proteins, polymers, and chemical molecules.
  • the tail protein channel-based nanopore device includes a first conductive liquid region and a second conductive liquid region, and the first conductive liquid region and the second conductive liquid region are insulated by a double layer.
  • the membrane is separated, and a tail protein channel is provided on the double-layer insulating membrane.
  • the tail protein channel is derived from a virus cell phage, which can be inserted into a lipid-based or polymer-based film layer as a nanopore.
  • tail protein channels can have stronger hydrophobicity and detection ability through mutation, which can greatly improve device stability, sensitivity, detection accuracy, and embedding, and have a wider application range.
  • FIG. 1 is a schematic diagram of a nanopore based on a tail protein channel according to an embodiment of the present application
  • FIG. 2 Wild type gp9 colony per identification results of phage 29;
  • FIG. 3 phage 29 wild-type gp9 protein expression, purification SDS-PAGE;
  • 5 is a gp-9 tail protein channel wild-type and mutant expression and purification results of phage 29 described in Example 1 of the present application;
  • FIG. 6 shows the assembly results of wild type and mutant gp-9 tail protein channels of phage 29 according to Example 1 of the present application;
  • FIG. 7 is a result of electrical signals detected after insertion of a pore-containing gp-9 tail protein channel mutant nanopore device into a membrane.
  • This embodiment provides a nanoporous device based on a tail protein channel. As shown in FIG. 1, it includes a first conductive liquid region and a second conductive liquid region. The first conductive liquid region and the second conductive liquid region The conductive liquid region is separated by a double-layer insulating film, and the double-layer insulating film is provided with a tail protein channel.
  • the tail protein channel is a wild-type tail protein channel and is a gp-9 protein of phage 29.
  • a process for preparing the tail protein channel-based nanopore device is also provided:
  • a double-layer insulating film is prepared, and a tail protein channel is inserted into the double-layer insulating film. Specifically, 0.1 mg / L of conventional lipid or chemical film molecules dissolved in an organic phase are applied to a pore diameter of 20 1 A double-layer membrane is formed on both sides of the pore, and the double-layer membrane separates the liquid on both sides of the pore, and then adds a tail protein channel to embed it in the double-layer membrane.
  • S2. Apply a voltage to the inserted tail protein channel to detect a change in current or optical signal when the analyte passes through the tail protein channel.
  • the tail protein channel is prepared by the following process:
  • a BamH I restriction site (G / GATCC) is added upstream of the wild-type Phi29-gp9 gene fragment, and a stop codon (TGA) and Xho I restriction site (C / TCGAG) are added downstream.
  • the constructed DNA sequence such as the sequence list NO:
  • GGAAATTACAGCGTCGAGAATGAATTGAGGTGACTCGAG uses the recombinant plasmid containing the gp-9 gene as a template to amplify this sequence by PCR. The results are shown in Figure 4.
  • the Phi29-gp9 gene containing the BamH I and Xho I digestion sites and the modified pET28a plasmid containing the TEV digestion site were subjected to double digestion reactions with BamH I and Xho I enzymes, respectively, followed by T4 DNA ligase
  • the recovered Phi29-gp9 gene was ligated with a pET28a plasmid containing the corresponding cohesive end (reacted at 25 ° C for 1 h).
  • the constructed recombinant plasmid containing the Phi29-gp9 gene was transformed into E.
  • the recombinant pET28a plasmid transformed BL21 (DE3) strain (the plasmid contains the DNA sequence described in SEQ ID NO: 1). Incubate on ice for 30 min, heat shock at 42 ° C for 90 s, leave on ice for 2 min, centrifuge at 5000 rpm for 1 min at room temperature, then coat the plate (50 pg / mL kanamycin sulfate), and place in a 37 ° C incubator Incubate overnight. Take 2.5 g of LB Borth powder and 1.5 g of Agar and dissolve in 100 mL of dd H2O. Sterilize by autoclaving at high temperature and pressure.
  • a single colony expressing strain BL21 (DE3) was picked and cultured in a 5 mL LB liquid medium (50 / mL kanamycin sulfate) at 37 ° C, 220 rpm in a shaker overnight, and 25 g of LB Borth was taken.
  • the powder was dissolved with 1 L of dd H20, divided into 5 conical flasks, and sterilized by autoclaving at high temperature and pressure.
  • the OD600 value was measured. When the OD600 value was about 0.8, IPTG induced protein expression was added at a final concentration of 0.5 mM, and induced at 16 ° C, 200 rpm for 24 h. Samples of the bacterial solution without IPTG inducer were used as a negative control. Collect the bacterial solution in a 50 mL centrifuge tube and centrifuge at 6000 rpm and 4 ° C for 10 min. Discard the supernatant and collect the bacterial cells.
  • the collected bacterial cells were resuspended in 20 mL of crushed buffer (25 mM Tris, pH 8.0, 500 mM NaCl), placed in an ice-water bath to sonicate the bacterial cells, and treated for 15 min (ultrasonic for 2s, suspended for 2s for a cycle ).
  • crushed buffer 25 mM Tris, pH 8.0, 500 mM NaCl
  • This embodiment provides a nanopore device based on a tail protein channel and a preparation process thereof.
  • the difference from Embodiment 1 is that the tail protein channel is a mutant tail protein channel: gp-9 del 417-491.
  • gp-9 protein The expression and purification method of gp-9 protein are the same as those in Example 1, except that: a gene including the nucleotide sequence shown in the sequence list NO: 2 is used: GGATCCATGGCATATGTACCATTATCAGGA
  • This embodiment provides a nanopore device based on a tail protein channel and a preparation process thereof.
  • the difference from Embodiment 1 is that the tail protein channel is a mutant tail protein channel: Mut-a: K134I, D138, 139L
  • This embodiment provides a nanopore device based on a tail protein channel and a preparation process thereof.
  • the difference from Embodiment 1 is that the tail protein channel is a mutant tail protein channel: Mut-b: K134I, D138,139L, D158L, E163V.
  • the collected bacterial cells were resuspended in 20 mL of crushed buffer (25 mM Tris, pH 8.0, 500 mM NaCl), placed in an ice-water bath to sonicate the bacterial cells, treated for 15 min (ultrasonic for 2s, suspended for 2s for one cycle) ). After crushing and ending, centrifuge at 1000 rpm and 4 ° C for 40 min, discard the supernatant and collect the pellet.
  • crushed buffer 25 mM Tris, pH 8.0, 500 mM NaCl
  • the collected supernatant was slowly added dropwise to 100 mL of protein renaturing buffer (50 mM Tris, pH 8.0, 500 mM NaCl, 200 mM L-arginine, 15% v / v glycerol, 1 mM GSSG, 10 mM GSH). Put on a magnetic stirrer and stir slowly, refold at 4 ° C overnight.
  • protein renaturing buffer 50 mM Tris, pH 8.0, 500 mM NaCl, 200 mM L-arginine, 15% v / v glycerol, 1 mM GSSG, 10 mM GSH.
  • the renaturation reaction solution is transferred to a dialysis bag and placed in 2 L of dialysis buffer (50 mM
  • Tris pH 8.0, 500 mM NaCl), dialyzed at 4 ° C for more than 12 h.
  • Ni-NTA affinity purification of the protein of interest was the same as the Ni-NTA affinity purification method in Example 1.
  • This embodiment provides a nanopore device based on a tail protein channel and a preparation process thereof, which is different from Example 1 in that the tail protein channel is a mutant tail protein channel: Mut-c: K134I,

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Abstract

La présente invention concerne un composant de nanopore à base de canal de protéine de queue, qui comprend une première zone liquide électroconductrice et une seconde zone liquide électroconductrice. La première zone liquide électroconductrice est séparée de la seconde zone liquide électroconductrice par une membrane isolante à double couche. Un canal de protéine de queue est disposé sur la membrane isolante à double couche. Le canal de protéine de queue est issu d'un bactériophage viral et cellulaire et sert de nanopore inséré dans la membrane à base de lipide ou de polymère. Lorsqu'une tension est appliquée à la membrane isolante à double couche, un courant est généré avec des ions passant à travers le nanopore, les caractéristiques d'un analyte peuvent être déterminées par détection d'un signal de courant ou d'un signal de lumière; ceci est applicable dans la détection de maladies et dans le séquençage d'acides nucléiques et de protéines. De plus, le canal de protéine de queue peut fournir une capacité d'hydrophobicité et de détection plus forte au moyen d'une mutation, ce qui permet d'augmenter la stabilité, la sensibilité, la précision de détection et l'incorporation du composant, et d'étendre la gamme d'applications.
PCT/CN2018/099294 2018-08-08 2018-08-08 Composant de nanopore à base de canal de protéine de queue, procédé de préparation de celui-ci et applications associées Ceased WO2020029101A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040157304A1 (en) * 2002-09-18 2004-08-12 Purdue Research Foundation Molecular rotary nanomotor and methods of use
CN103392008A (zh) * 2010-09-07 2013-11-13 加利福尼亚大学董事会 通过持续性酶以一个核苷酸的精度控制dna在纳米孔中的移动

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040157304A1 (en) * 2002-09-18 2004-08-12 Purdue Research Foundation Molecular rotary nanomotor and methods of use
CN103392008A (zh) * 2010-09-07 2013-11-13 加利福尼亚大学董事会 通过持续性酶以一个核苷酸的精度控制dna在纳米孔中的移动

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
GENG, J.: "Three reversible and controllable discrete steps of channel ga- ting of a viral DNA packaging motor", BIOMATERIALS, 31 July 2011 (2011-07-31), pages 8234 - 8242, XP028277133, DOI: 10.1016/j.biomaterials.2011.07.034 *
HAQUE, F.: "Real-time sensing and discrimination of single chemicals using the channel of phi29 DNA packaging nanomotor", ACS NANO, vol. 6, no. 4, 9 April 2012 (2012-04-09) - 24 April 2012 (2012-04-24), pages 3251 - 3261, XP055318253 *
PENG, J.: "One-Way Traffic of a Viral Motor Channel for Double-Stranded DNA Translocation", NANO LETT., vol. 10, no. 9, 19 August 2010 (2010-08-19), pages 3620 - 3627, XP055684598 *
SCHWARTZ, C.: "Sequential action of ATPase, ATP, ADP, Pi and dsDNA in procapsid-free system to enlighten mechanism in viral dsDNA packaging", NUCLEIC ACIDS RESEARCH, vol. 40, no. 6, 22 November 2011 (2011-11-22) - March 2012 (2012-03-01), pages 2577 - 2586, XP055684595 *
WENDELL, D.: "Translocation of double-stranded DNA through membrane-ada- pted phi29 motor protein nanopores", NATURE NANOTECHNOLOGY, vol. 4, no. 11, 27 November 2009 (2009-11-27), pages 765 - 772, XP055062618 *
XU, J. W.: "The bacteriophage PHI 29 tail possesses a pore-forming loop for cell membrane penetration", NATURE, vol. 534, no. 7608, 20 June 2016 (2016-06-20) - 23 June 2016 (2016-06-23), pages 544 - 547, XP055684605 *

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