Method for constructing double-recognition-site nanopores

Abstract

The invention discloses a method for constructing double-recognition-site nanopores, which comprises the following steps of carrying out structural domain function analysis on two single-recognition-site nanopores subjected to fusion expression to obtain a structural domain analysis result, selecting a structural domain gene, and carrying out fusion protein design to obtain a fusion protein with two recognition sites, selecting an expression system capable of ensuring the stable and polymerizable structure of the fusion protein, and sequentially finishing expression scheme design, expression condition optimization, pore protein polymerization, purification and separation, pore embedding and sequencing and fusion protein mutation treatment according to the host type of the expression system. The naturally existing nanopore protein with the single recognition site is used, and the nanopore protein with the double recognition sites is obtained through an engineered fusion protein expression technology, so that the selectable range of the nanopore protein is greatly expanded, and the problem that the naturally existing protein nanopores with the double sensitive sites are extremely fewis solved. By mutating and optimizing the performance of the pore channels, the detection performance of the pore channels is improved.

Description

technical field [0001] The invention relates to the technical field of nanopore detection, in particular to a method for constructing a double recognition site nanopore. Background technique [0002] There is a type of transmembrane protein channel in natural biological membranes. The protein channel has a diameter of nanometer scale and allows ions and molecules of specific size to pass through, which is called nanopore. [0003] Embed the nanoporous protein on an insulating artificial film, place it in a conductive ionic liquid, and apply a voltage on both sides of the film, then the charged ions will pass through the nanopore to form an ionic current. When a molecule of a specific size passes through the channel or binds to the channel, it will block the channel and reduce the ion flow. The size of the blocked current is related to the characteristic information such as molecular size and charge distribution. Therefore, the molecule can be identified by measuring the size...

AI Technical Summary

Problems solved by technology

However, three or more recognition sites are limited by the signal-to-noise ratio and cannot perform effective base discrimination

[0008] However, there are very few naturally occurring protein nanopores with dual recognition sites. Finding suitable nanopores and making corresponding modifications is the key to further improving the accuracy of sequencing.

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