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.