A method for constructing dual recognition site nanopores

Abstract

The invention discloses a method for constructing a double-recognition site nanopore, which includes: performing structural domain function analysis on two kinds of single-recognition site nanopores fused and expressed to obtain a structural domain analysis result; selecting a structural domain gene and performing a fusion protein Design and obtain a fusion protein with two recognition sites; select an expression system that ensures the stability and polymerizability of the fusion protein structure, and complete the expression scheme design, expression condition optimization, porin polymerization, purification and separation in sequence according to the host type of the expression system , pore embedding and sequencing, and fusion protein mutation processing; this application uses the naturally occurring nanopore protein with a single recognition site, and obtains a nanopore protein with dual recognition sites through engineered fusion protein expression technology, which greatly expands the scope of nanopores. The selectable range of porins solves the problem of few naturally occurring protein nanopores with dual sensitive sites. By mutating the performance of the optimized channel, the detection performance of the channel 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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