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Protein, transmembrane nucleic acid unwinding nanopore, construction method and application thereof

A protein and DNA helicase technology, applied in the fields of application, nanotechnology, nanotechnology, etc.

Active Publication Date: 2019-01-15
GUANGZHOU KONGQUE GENE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The technical problem to be solved by the present invention is to overcome the deficiency that the existing small-aperture protein nanopore needs external unwinding active components when transporting double-stranded nucleic acid

Method used

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  • Protein, transmembrane nucleic acid unwinding nanopore, construction method and application thereof
  • Protein, transmembrane nucleic acid unwinding nanopore, construction method and application thereof
  • Protein, transmembrane nucleic acid unwinding nanopore, construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0174] Example 1 E1 protein and its mutant recombinant vector construction and expression purification:

[0175] Amino acid sequence (SEQ ID No.3) of truncation E1-1 (306-577):

[0176]LQTEKFDFGTMVQWAYDHKYAEESKIAYEYALAAGSDSNARAFLATNSQAKHVKDCATMVRHYLRAETQALSMPAYIKARCKLATGEGSWKSILTFFNYQNIELITFINALKLWLKGIPKKNCLAFIGPPNTGKSMLCNSLIHFLGGSVLSFANHKSHFWLASLADTRAALVDDATHACWRYFDTYLRNALDGYPVSIDRKHKAAVQIKAPPLLVTSNIDVQAEDRYLYLHSRVQTFRFEQPCTDESGEQPFNITDADWKSFFVRLWGRLDL。

[0177] The coding gene sequence (SEQ ID No.4) of the truncated body E1-1 (306-577):

[0178] Ttgcagaccgagaaattcgacttcggaactatggtgcaatgggcctatgatcacaaatatgctgaggagtctaaaatagcctatgaatatgctttggctgcaggatctgatagcaatgcacgggcttttttagcaactaacagccaagctaagcatgtgaaggactgtgcaactatggtaagacactatctaagagctgaaacacaagcattaagcatgcctgcatatattaaagctaggtgcaagctggcaactggggaaggaagctggaagtctatcctaactttttttaactatcagaatattgaattaattacctttattaatgctttaaagctctggctaaaaggaattccaaaaaaaaactgtttagcatttattggccctcc aaacacaggcaagtctatgctctgcaactcattaattcattttttgg...

Embodiment 2

[0215] Embodiment 2. Fluorescent labeling and membrane fusion experiments of protein truncated bodies and mutants thereof:

[0216] Fluorescence-labeled truncated E1-2 (306-605) protein: the material used was Fluorine-labeled Fluorescein Isothiocyanate (FITC) Conjugate Kit (Sigma, St. Louis, Missouri). According to the description of the kit, excess FITC was removed by column chromatography, and FITC-linked E1 was eluted with protein buffer (20 mM tris-HCl (Ph7.3), 200 mM NaCl). The FITC-labeled E1 (306-605) was verified by SDS-PAGE (figure omitted, the position of FITC-labeled E1-2 on SDS-PAGE is slightly higher than that of unlabeled E1-2).

[0217]Preparation of small vesicles (SUVs) containing truncated E1-2 (306-605): To prepare small vesicles containing E1 protein with a size of about 0.1 μm, 1 mg / ml DOPC was added to a vial, and nitrogen gas was used to After drying the chloroform, add an aqueous solution containing E1 protein (concentration is about 0.5 mg / ml), rehydr...

Embodiment 3 E1

[0222] Embodiment 3, E1 protein and its mutant electrophysiological experiment:

[0223] Electrophysiological measurement: the instrument is HEKA EPC-10USB, which integrates the amplifier and digital-to-analog conversion part. The two electrodes are the pressure (voltage clamp mode) electrode and the reference electrode, which are a pair of silver / silver chloride electrodes. . It is used to measure the transmembrane current across both sides of the phospholipid bilayer membrane. All sampling frequencies are at 2kHz, and the low-pass filtering frequency is 1kHz. Data collection was done by Patch-master software, and data analysis was done by Clamfit.

[0224] Construction of lipid bilayer: use horizontal standard lipid bilayer builder and vertical standard lipid bilayer box (BCH-13A, purchased from Warner.No.64-0451. Customized 50μm CUPs) to construct plane Lipid bilayer. The builder and the bilayer box were divided into cis-type by using a thin polytetrafluoroethylene mate...

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Abstract

The invention belongs to the field of biotechnology, in particular to a protein, a transmembrane nucleic acid unwinding nanopore, a construction method and an application thereof. The technical problem to be solved by the invention is to overcome the shortcoming that the existing small pore protein nanopore needs an external dissociative active component when transporting the double-stranded nucleic acid. The scheme of the invention for solving the defect is to provide a truncated body E1-1 (306-577) protein and E1-2 (306-605) protein and variants thereof of a double-stranded DNA helicase protein of bovine papillomavirus, as well as variant of homologous proteins thereof, which can be used for preparing conductive channel-containing film, which provides a new and effective choice for thatfield.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a transmembrane nucleic acid untwisting nanopore and its construction method and application. Background technique [0002] The high-precision detection of biological and chemical active substances has long been one of the common goals of industry and scientific research, and this requires robust and stable sensing devices, from the first application of α-hemolysin α-HL protein to single molecules Since the beginning of the detection field, nanopores have been playing an increasingly important role as a technical platform for single-molecule detection. [0003] Binding or "capturing" detection of analytes of interest through intermolecular affinity (e.g., covalent binding or non-covalent attraction) such as histidine insertion into the β-barrel region of α-HL for specific discrimination quantification Divalent metal ions such as nickel, copper, and zinc; use genetic engi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/14C12N15/55G01N33/487C12Q1/6869A61K47/42A61K9/70
CPCA61K47/42G01N33/48721A61K9/7007C12N9/14C12Q1/6869C12Y306/04012G01N2333/914C12Q2565/631C07K14/00C12Q1/68C07K14/005C12N9/90C12N2710/20022B82Y5/00
Inventor 耿佳孙柯逯光文魏霞蔚应斌武
Owner GUANGZHOU KONGQUE GENE TECH CO LTD
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