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Mutated NfpAB nanopore, test system, manufacturing method and application

A test system and nanopore technology, applied in the field of nanopores, can solve problems such as unknowns, achieve the effect of improving accuracy and reducing error rate

Active Publication Date: 2020-08-07
SHENZHEN MERRIME NANOPORE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] NfpAB is a new type of nanoporin derived from Nocardia farcinica. The diameter of the channel is about 1.4-1.6nm. It is an octameric channel composed of two monomer proteins, NfpA and NfpB. More negative charge distribution, wild-type NfpAB nanopore has been reported to test positively charged polypeptides, but whether it can be used for nucleic acid sequencing, peptide, protein detection and sequencing is still unknown

Method used

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  • Mutated NfpAB nanopore, test system, manufacturing method and application
  • Mutated NfpAB nanopore, test system, manufacturing method and application
  • Mutated NfpAB nanopore, test system, manufacturing method and application

Examples

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Embodiment 1

[0058] This embodiment provides a mutant NfpAB nanopore, such as figure 1 As shown, the nanopore is a protein complex composed of at least one NfpA protein mutant and at least one NfpB protein mutant, or a protein complex composed of mutant monomers expressed by at least two NfpA and NfpB fusion mutant genes . The charge distribution of the nanopore is as follows figure 2 shown.

[0059] (1) First construct NfpA and NfpB wild-type vectors:

[0060] Obtain the wild-type NfpA sequence (number: nfa15890) and NfpB sequence (number: nfa15900) from the NCBI website, the NfpA mutant monomer is composed of a polypeptide with at least 70% identical amino acid sequence to the sequence SEQ ID NO: 1, and the NfpB mutation The monomer consists of a polypeptide having an amino acid sequence at least 70% identical to the sequence SEQ ID NO:2. Wherein, the monomeric protein sequence (SEQ ID NO:1) of wild-type NfpA is:

[0061] DTFVPLPDGQKVGPGVTITRTGEHAVISPSMAANGAGRVAWVSGNATADVTVTPEGEVGP...

Embodiment 2

[0114] This embodiment provides a mutant NfpAB nanopore and a test system containing the nanopore, which are basically the same as those in Comparative Document 1, except that the known single-stranded nucleic acid ssDNA1 (SEQ ID NO :3) detection, detection results such as Figure 11 shown. The gene sequence (SEQ ID NO: 3) of the single-stranded nucleic acid ssDNA1 is:

[0115]

Embodiment 3

[0117] This embodiment provides a mutant NfpAB nanopore and a test system containing the nanopore, which are basically the same as in Example 1, except that the NfpA-M2 (SEQ ID NO: 8) and NfpB-M2 (SEQ ID NO: 8) are used. ID NO: 9) nucleotide sequence, and finally NfpAB-M2 comprising the amino acid sequences shown in NfpA-M2 (SEQ ID NO: 10) and NfpB-M2 (SEQ ID NO: 11) was prepared. The obtained NfpAB-M2 is used to detect known single-stranded nucleic acid ssDNA1 (SEQ ID NO: 3), which increases the number of positive charges inside the pore, reduces the rate of single-stranded DNA passing through the hole to a certain extent, and increases the detection accuracy. Test results such as Figure 12 shown.

[0118] Wherein the NfpA mutant monomer DNA sequence (SEQ ID NO: 8) of the mutant NfpAB-M2 is:

[0119] GAATTCATGGATACCTTTGTTCCGCTGCCGGACGGTCAAAAAGTTGGTCCGGGCGTTACCATTACCCGTACCGGTGAACACGCAGTTATTTCTCCGAGTATGGCAGCAAACGGCGCAGGTCGCGTTGCTTGGGTTTCTGGTAACGCAACCGCAGACGTAACCGTTACCCCGAAAG...

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Abstract

The invention discloses a mutant NfpAB nanopore which is a protein complex composed of at least one NfpA protein mutant monomer and at least one NfpB protein mutant monomer, or a protein complex composed of at least two mutant monomers expressed by NfpA and NfpB fusion mutant genes. The nanopore probe has a structure and recognition sites different from those of known nanopores, can be used for recognition, concentration detection and sequencing of polymers such as nucleic acid, polypeptide and protein, allows capture and transfer of single-stranded DNA and distinguishes adenine (A), thymine (T), cytosine (C) and guanine (G). The NfpAB mutant is formed by combining two different monomers, the modification space in a pore channel is enriched, the pore channel has a conical pore channel structure similar to MspA, and the NfpAB mutant is an ideal pore channel for nucleic acid sequencing. The invention also discloses a test system containing the nanopore, a manufacturing method and an application, through engineering site-specific mutagenesis in the nanopore, charge distribution in a pore channel is adjusted, the speed of nucleic acid passing through the pore channel is reduced, and accurate recognition of a basic group is realized.

Description

technical field [0001] The invention belongs to the field of nanopore technology, and relates to a novel protein nanopore and its application, in particular to a mutant NfpAB nanopore, a test system containing the nanopore and a manufacturing method thereof, and the detection system of the test system in the detection of polymers. and applications in sequencing. Background technique [0002] Protein nanopore is a transmembrane molecular channel composed of polypeptides and protein complexes that naturally exists on biological membranes, which allows ions or macromolecules to pass through, thereby playing the role of molecular transport and signal transduction. Nano (10 -9 meter) scale diameter, so called nanopores. [0003] Nanopore has become a powerful tool for protein detection and nucleic acid sequencing. The nanopore is placed on a biomimetic membrane, and a certain potential is applied to both ends of the membrane. The ion flow formed by ions passing through the nano...

Claims

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Application Information

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IPC IPC(8): C07K14/355C12Q1/6869G01N33/68
CPCC07K14/355G01N33/68C12Q1/6869G01N27/447
Inventor 李伟周智文豪
Owner SHENZHEN MERRIME NANOPORE TECH CO LTD
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