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
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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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