CN115974984A - Bimengated channel protein, channel protein mutant, nucleotide sequence and application thereof - Google Patents

Abstract

The application provides a double-gate channel protein, a channel protein mutant, a nucleotide sequence and application thereof, belonging to the technical field of nanopore single molecule detection. The amino acid sequence of the double-gate channel protein is shown as SEQ ID NO. 1. The amino acid sequence of the porin mutant is as follows: the sequence shown in SEQ ID No.2, the first mutant sequence or the second mutant sequence. The first mutant sequence and the second mutant sequence are respectively sequences with homology of more than or equal to 75 percent with SEQ ID NO.1 and SEQ ID NO. 2. The double-gate channel protein and the channel protein mutant are favorable for improving the detection accuracy of substances to be detected and the integrated assembly effect of the channel protein, and also favorable for effectively detecting continuous same base sequences, have better structural stability and electrophysiological stability, and are also favorable for promoting the effective capture and/or translocation of substances to be detected of cross-channel proteins to be detected.

Description

Bimengated channel protein, channel protein mutant, nucleotide sequence and application thereof

Technical Field

The application relates to the technical field of nanopore single molecule detection, in particular to a double-gate pore protein, a pore protein mutant, a nucleotide sequence and application thereof.

Background

The nano-pore single molecule detection technology is a novel detection method developed on the basis of electrophysiology. The basic principle of the method is as follows: the species to be tested (nucleotides, proteins, polypeptides, etc.) are transported through a thin, small nanopore to produce a restricted flow of ions. Due to the difference of physicochemical properties of the substances to be detected, the substances to be detected have differentiation on the blocking effect of the nano Kong Dianliu when staying in the nano holes; therefore, through distinguishing the blocking current, the related physicochemical information of the substance to be detected can be obtained. The method has the advantages of no mark, high flux, low cost, small sample demand and the like, so that the nanopore single molecule detection technology has wide prospect in different means of gene sequencing and substance structure analysis at present.

Biological nanopores (namely, pore proteins) have become the most important focus in nanopore monomolecular detection technology due to the characteristics of high sensitivity, high repeatability and the like; the biological nanopore is embedded in a phospholipid bilayer to be assembled into a biochip, and the detection of an electric signal and/or an optical signal of a substance to be detected is realized by means of a computer processor and sensing equipment.

The research shows that different protein nanopores such as alpha-hemolysin (alpha-HL), mycobacterium smegmatis toxin protein A (MspA), aerolysin (aerolysin), bacteriophage phi29connector motor protein (phi 29 connector), outer membrane protein (OmpG), secretin nanopores (GspD, invG) and bacterial amyloid fiber (beta-amyloids) secretion channel structures (CsgG, csgG-CsgF and CsgG-CsgE) can detect nucleic acid, polypeptide, protein, metal ions, antibiotics and organic pollutants and analyze the valence state and matter configuration direction of the metal ions. However, the existing bio-nanopore has low detection accuracy for a continuous same base sequence, low differentiation for electrically different substances, poor structural stability of the porin (for example, depolymerization of the porin occurs in base sequence sequencing), electrophysiological instability (switching of the porin between open pore and closed pore) or poor integration and assembly effect of the porin (stability of a biochip formed by assembling the bio-nanopore and a phospholipid bilayer).

Disclosure of Invention

The application aims to provide a double-gate channel protein, a channel protein mutant, a nucleotide sequence and application thereof, and aims to solve the technical problems that the detection accuracy of continuous same base sequences in nucleic acid sequencing of the existing biological nanopore is low, the detection effect on substances with electrical property differences is poor, the structure and single-channel electrophysiology of the channel protein are unstable, and the integrated assembly effect of the channel protein is poor.

In a first aspect, the present application provides a bi-gated pore protein, the amino acid sequence of which is shown in SEQ ID No. 1.

In the technical scheme, the amino acid sequence shown in SEQ ID NO.1 has a central gate characteristic sequence and a cap gate characteristic sequence, so that the porthole protein is a double-gated porthole protein with a central gate structure and a cap gate structure; wherein, the length of the cap characteristic sequence is up to 70 amino acids, so that the double-gate channel protein has a longer cap structure; the length from the cap gate contraction area to the central gate contraction area is up to 90 angstroms, so that the interference of the cap gate and the central gate on signal reading of the substance to be detected can be reduced, and the detection accuracy of the double-gate pore channel protein on the substance to be detected can be improved; the constriction region of the central gate is 4 angstroms, which can realize the recognition of nucleotide from single base, and is favorable to the effective sequencing detection of continuous same base sequence. In addition, the double-gate pore channel protein central gate structure provided by the application is clear, has better structural stability, has linear relation of single-channel current and voltage, has electrophysiological stability, can promote effective capture and/or translocation of substances to be detected of cross-pore channel protein, and has better integrated assembly effect (namely, the stability of the biochip assembled by the double-gate pore channel protein and phospholipid bilayer is better), so that the double-gate pore channel protein central gate structure has good application prospect in the technical field of nanopore single molecule detection.

Optionally, the bi-gated channel protein comprises a multimer of subunit polypeptides, the amino acid sequence of which is shown in SEQ ID No. 1.

Alternatively, the multimer comprises a 12-16 mer.

The polymer comprises a 12-16 polymer, namely the polymer can contain 12-16 subunit polypeptides, and the pore size of the double-gate channel protein can be regulated.

In a second aspect, the present application provides a porin mutant, the porin mutant having an amino acid sequence of: the sequence shown in SEQ ID NO.2, the first mutant sequence or the second mutant sequence.

Wherein, the first mutant sequence has a sequence with homology of more than or equal to 30 percent with SEQ ID NO.1, and the second mutant sequence has a sequence with homology of more than or equal to 30 percent with SEQ ID NO. 2.

In the above technical solution, since the sequence shown in SEQ ID No.2 is a truncation of the sequence shown in SEQ ID No.1 (which also has a central gate characteristic sequence and a cap characteristic sequence identical to the sequence shown in SEQ ID No. 1), when the amino acid sequence of the porin mutant is the sequence shown in SEQ ID No.2, it can also achieve the improvement of the detection accuracy of the substance to be detected and the integrated assembly effect of porin, and can achieve the effective detection of the continuous same base sequence, and the porin mutant thereof also has better structural stability and better electrophysiological stability, and can also promote the effective capture and/or translocation of the substance to be detected across porin; and the function of the channel protein can be further optimized after mutation.

Because the first mutant sequence is a sequence with homology of more than or equal to 30 percent with the SEQ ID NO.1, and the second mutant sequence is a sequence with homology of more than or equal to 30 percent with the SEQ ID NO.2, the first mutant sequence and the second mutant sequence are favorable for improving the detection accuracy of the substances to be detected and the integrated assembly effect of the channel proteins, can be favorable for effectively detecting continuous same base sequences, have better structural stability and better electrophysiological stability, and are also favorable for promoting the effective capture and/or translocation of the substances to be detected of the cross-channel proteins, so that the method is suitable for detection under different conditions.

Therefore, the porin mutant provided by the application can also be used for analyte detection and analysis, and has good application prospect in the technical field of nanopore single molecule detection.

In an alternative embodiment of the present application in combination with the second aspect, the mutant porin comprises a multimer of subunit polypeptides, the multimer being a homomultimer or a heteromultimer. The amino acid sequence of at least one subunit polypeptide in the multimer is: the sequence shown in SEQ ID NO.1, the sequence shown in SEQ ID NO.2, the first mutant sequence or the second mutant sequence.

Alternatively, the multimer comprises a 9-20 mer.

The polymer comprises 9-20 polymers, namely the polymer can contain 9-20 subunit polypeptides, and the pore size of the mutant of the porin can be regulated.

In an alternative embodiment of the present application in combination with the second aspect, the first mutant sequence satisfies at least one of the following conditions a-D:

a: at least one amino acid in the sequence shown in SEQ ID NO.1 is substituted.

B: at least one amino acid is inserted into the sequence shown in SEQ ID NO. 1.

C: at least one amino acid is deleted in the sequence shown in SEQ ID NO. 1.

D: at least one amino acid in the sequence shown in SEQ ID NO.1 is modified.

Or, the second mutant sequence satisfies at least one of the following E-H conditions:

e: at least one amino acid in the sequence shown in SEQ ID NO.2 is substituted.

F: at least one amino acid is inserted into the sequence shown in SEQ ID NO. 2.

G: at least one amino acid is deleted in the sequence shown in SEQ ID NO. 2.

H: the side chain group of at least one amino acid in the sequence shown in SEQ ID NO.2 is modified.

Wherein the substitution comprises changing the species of amino acid or changing the configuration of amino acid; changing the type of amino acid includes: altering the electrical properties of the amino acid, altering the hydrophobicity of the amino acid, altering the structural rigidity of the amino acid, or altering the size of the side chain group of the amino acid.

Optionally, the modification comprises at least one of a glycosylation modification, a phosphorylation modification, a ubiquitination modification, an S-nitrosylation modification, an N-methylation modification, an O-methylation modification, an N-acetylation modification, and a lipidation modification; and/or, the modification comprises: at least one of porphyrin, cholesterol and dodecyl glucoside molecules is added to the side chain group.

The modification is carried out on the side chain group of at least one amino acid in the sequence shown in SEQ ID NO.1 or the sequence shown in SEQ ID NO.2, which is favorable for improving the structural stability of the channel protein mutant.

In an alternative embodiment of the present application, in combination with the second aspect, the first mutant sequence satisfies the following condition: the amino acid at a specific site in the sequence shown in SEQ ID NO.1 is replaced by an electrically neutral or positively charged amino acid.

Wherein the specific site includes at least one of 85 th site, 102 th site, 158 th site, 169 th site and 253 th site.

In the technical scheme, the method can enhance the hole-entering rate of the negatively charged substance to be detected (such as nucleic acid and the like).

In an alternative embodiment of the present application, in combination with the second aspect, the first mutant sequence satisfies the following condition: the amino acids in the first region and the amino acids in the second region in the sequence shown in SEQ ID NO.1 are respectively replaced by amino acids with different electric properties.

Wherein the amino acids in the first region are from position 526 to position 547 and the amino acids in the second region are from position 400 to position 490.

In the technical scheme, the cap gate and the central gate can realize selective detection on substances to be detected with different charges respectively.

In an alternative embodiment of the present application in combination with the second aspect, the first mutant sequence satisfies at least one of the following I-K conditions:

i: the amino acids of the first region of the sequence shown in SEQ ID NO.1 are deleted in whole or in part.

J: changing the electrical property of the amino acid in the first region in the sequence shown in SEQ ID NO. 1.

K: the size of the side chain group of the amino acid of the first region in the sequence shown in SEQ ID NO.1 is changed.

Or, the first mutant sequence satisfies at least one of the following L-N conditions:

l: the amino acids of the second region of the sequence shown in SEQ ID NO.1 are deleted in whole or in part.

M: changing the electrical property of the amino acid in the second region in the sequence shown in SEQ ID NO. 1.

N: the size of the side chain group of the amino acid of the second region in the sequence shown in SEQ ID NO.1 is changed.

Wherein the amino acids in the first region are from position 526 to position 547, and the amino acids in the second region are from position 400 to position 490.

In the technical scheme, the pore diameters of the double pores of the double-gate pore channel protein can be respectively regulated to obtain the pore channel protein mutant so as to meet different requirements.

In an alternative embodiment of the present application, in combination with the second aspect, the first mutant sequence satisfies at least one of the following O — S conditions:

o: deletion of amino acids from position 1 to position 50-152 in the sequence shown in SEQ ID NO.1, deletion of amino acids from position 1 to position 221 in the sequence shown in SEQ ID NO.1, or deletion of amino acids from position 1 to position 294 in the sequence shown in SEQ ID NO. 1.

P: deletion of the amino acids from position 320 to position 339 in the sequence shown in SEQ ID NO. 1.

Q: the amino acid at least one position from 661 st position to 705 th position in the sequence shown in SEQ ID NO.1 is mutated to cysteine.

R: the amino acid at least one position from 661 th position to 705 th position in the sequence shown in SEQ ID NO.1 is mutated into cysteine, and the cysteine is connected with maleimide porphyrin and/or cysteine-added cholesterol.

S: any one of the 627 th site, 628 th site and 629 th site in the sequence shown in SEQ ID NO.1 is mutated into phenylalanine.

In the technical scheme, when the first mutant sequence meets the condition of O (namely, the nitrogen end of the sequence shown in SEQ ID NO.1 is deleted), the length of the double-gate channel protein can be regulated and controlled. When the first mutant sequence satisfies P (namely, the free chain at the N3 end of the sequence shown in SEQ ID NO.1 is deleted), the situation that the double-gate channel protein blocks a substance to be detected can be reduced. When the first mutant sequence satisfies Q, conditions are provided for connecting other molecules (including hydrophobic protein AspS and the like) to the channel protein. When the first mutant sequence meets the R condition, on one hand, the channel protein can be quickly anchored on the surface of the phospholipid bilayer, the interaction between the double-gate channel protein and a membrane (namely the phospholipid bilayer in a biochip for subsequent detection) is promoted, and the assembly of the protein in the phospholipid bilayer is accelerated; on the other hand, the newly added compound (namely cysteine connected maleimide porphyrin and/or cysteine-acidified cholesterol) forms a bridge between the channel protein and the phospholipid bilayer, stabilizes the structure of the channel protein and phospholipid layer complex, and improves the stability of the biochip formed by assembling the double-gate channel protein and the phospholipid bilayer. When the first mutant sequence meets the S condition, the interaction between the double-portal protein and a membrane (namely a phospholipid bilayer in a biochip for subsequent formation detection) can be promoted, and the efficiency of assembling the double-portal protein in the phospholipid bilayer to form a portal is improved.

In a third aspect, the present application provides a nucleotide sequence, the nucleotide sequence being: a sequence shown in SEQ ID NO.3, a sequence shown in SEQ ID NO.4, a third mutant sequence or a fourth mutant sequence.

Wherein, the third mutant sequence has homology of more than or equal to 30 percent with SEQ ID NO.3, and the fourth mutant sequence has homology of more than or equal to 30 percent with SEQ ID NO. 4; the sequence shown by SEQ ID NO.3 is the sequence shown by coding SEQ ID NO.1, and the sequence shown by SEQ ID NO.4 is the sequence shown by coding SEQ ID NO. 2.

In the technical scheme, the channel protein formed by the amino acid sequence coded by the nucleotide sequence is beneficial to improving the detection accuracy of the substance to be detected and the integrated assembly effect of the channel protein, can be beneficial to effectively detecting the continuous same base sequence and is also beneficial to promoting the effective capture and/or translocation of the substance to be detected of the cross-channel protein; has good application prospect in the technical field of nano-pore single molecule detection.

In a fourth aspect, the present application provides the use of a dual gated porin as provided in the first aspect above, a porin mutant as provided in the second aspect above, or a nucleotide sequence as provided in the third aspect above, for the preparation of a biochip; the biochip can detect electrical and/or optical signals of the substance to be detected.

Optionally, the test agent includes at least one of a nucleic acid, a protein, a polysaccharide, a neurotransmitter, a chiral compound, a heavy metal, and a toxin.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are required to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present application and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained from the drawings without inventive effort.

FIG. 1 is an electrophoretogram and a molecular sieve image of Bimengated protein.

FIG. 2 is a diagram of structural analysis of a bi-gated pore protein.

FIG. 3 is a graph of size analysis of a bi-gated channel protein.

Figure 4 is a graph of channel resistivity for the first porin mutant.

FIG. 5 is a graph of translocation of a first porin mutant to double-stranded DNA.

Figure 6 is a graph of translocation of the first porin mutant to single-stranded DNA.

FIG. 7 is a 3D structural diagram of a cryoelectron microscope of a second porin mutant.

FIG. 8 is a graph of channel resistivity for the third porin mutant.

Fig. 9 is a channel resistivity graph of the fourth porin mutant.

Detailed Description

The application provides a double-portal protein, and the amino acid sequence of the double-portal protein is shown in SEQ ID NO. 1.

The amino acid sequence shown in SEQ ID NO.1 has a central gate signature sequence (i.e., from position 526 to position 547 of SEQ ID NO. 1) and a cap gate signature sequence (i.e., from position 400 to position 490 of SEQ ID NO. 1), so that the porin is a double-gate porin with central gate and cap gate structures; the bi-gated protein has a lumen between the cis-opening and the trans-opening to interact with the phospholipid bilayer.

Wherein, the length of the cap gate characteristic sequence is up to 70 amino acids, so that the double-gate channel protein has a longer cap gate structure; the length from the cap gate contraction area to the central gate contraction area is up to 90 angstroms, so that the interference of the cap gate and the central gate on signal reading of the substance to be detected can be reduced, and the detection accuracy of the double-gate pore channel protein on the substance to be detected can be improved; the constriction region of the central gate is 4 angstroms, which can realize the recognition of nucleotide from single base, and is favorable to the effective sequencing detection of continuous same base sequence. In addition, the double-gated-pore protein central gate structure provided by the application is clear, has better structural stability, has linear relation of single-channel current and voltage, has electrophysiological stability, can promote effective capture and/or translocation of substances to be detected of cross-pore protein, and has better integrated assembly effect (namely, the stability of the biochip assembled by the double-gated-pore protein and phospholipid bilayers is better), so that the double-gated-pore protein central gate structure has good application prospect in the technical field of nanopore single molecule detection.

The double-gate channel protein comprises a polymer formed by subunit polypeptides, and the amino acid sequence of the subunit polypeptides is shown in SEQ ID NO. 1. Further, in the present application, a bi-gated protein includes a 12-16 mer; namely, the polymer can contain 12-16 subunit polypeptides, and the pore size of the double-gate channel protein can be regulated.

It is noted that in some possible embodiments, a bi-gated protein can include any number of subunit polypeptides, such that the bi-gated protein can form a sufficiently large cavity structure to permit passage of a target analyte, such as a polynucleotide.

Aiming at the double-gate channel protein, the application also provides a channel protein mutant, and the amino acid sequence of the channel protein mutant is as follows: the sequence shown in SEQ ID NO.2, the first mutant sequence or the second mutant sequence. Wherein, the first mutant sequence has a sequence with homology of more than or equal to 30 percent with SEQ ID NO.1, and the second mutant sequence has a sequence with homology of more than or equal to 30 percent with SEQ ID NO. 2.

Because the sequence shown in SEQ ID NO.2 is a truncated body of the sequence shown in SEQ ID NO.1 (the truncated body also has a central gate characteristic sequence and a cap characteristic sequence which are the same as the sequence shown in SEQ ID NO.1, and compared with the sequence shown in SEQ ID NO.1, the truncated body does not have an N0-N1 structural domain), when the amino acid sequence of the porin mutant is the sequence shown in SEQ ID NO.2, the detection accuracy of a substance to be detected and the integrated assembly effect of porin can be improved, the continuous same base sequence can be effectively detected, the porin mutant also has better structural stability and better electrophysiological stability, and the effective capture and/or translocation of the substance to be detected of the cross-porin can be promoted.

The first mutant sequence has a sequence with homology of more than or equal to 30 percent with SEQ ID NO.1, and the second mutant sequence has a sequence with homology of more than or equal to 30 percent with SEQ ID NO. 2; the first mutant sequence and the second mutant sequence are also beneficial to improving the detection accuracy of the substance to be detected and the integrated assembly effect of the channel protein, can be beneficial to effectively detecting the continuous same base sequence, have better structural stability and better electrophysiological stability, and are also beneficial to promoting the effective capture and/or translocation of the substance to be detected of the cross-channel protein; and can be further endowed with new functions after mutation so as to be suitable for detection of different conditions.

Therefore, the porin mutant provided by the application can also be used for analyte detection and analysis, and has good application prospect in the technical field of nanopore single molecule detection.

Illustratively, the first mutant sequence may have greater than or equal to 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% homology to SEQ ID No.1, and so forth; the second mutant sequence has homology of more than or equal to 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% with SEQ ID NO.2, etc.

Furthermore, the sequence of the first mutant is a sequence with homology of more than or equal to 75 percent with SEQ ID NO.1, and the sequence of the second mutant is a sequence with homology of more than or equal to 75 percent with SEQ ID NO. 2.

In some alternative embodiments, the first mutant sequence is a sequence having greater than or equal to 95% homology to SEQ ID No. 1.

Under the above conditions, the first mutant sequence has higher homology with SEQ ID No.1, which not only can be beneficial to ensure that the tunnel protein mutant can fully or partially retain the advantages of the double-gate tunnel protein (the amino acid sequence of which is shown in SEQ ID No. 1), but also can optimize the function of the tunnel protein mutant through mutation so as to be suitable for detection under different conditions.

In some alternative embodiments, the second mutant sequence is a sequence having greater than or equal to 95% homology to SEQ ID No. 2. Under the above conditions, the second mutant has higher homology with SEQ ID No.2, which not only can be beneficial to ensure that the tunnel protein mutant can fully or partially retain the advantages of the double-gate tunnel protein (the amino acid sequence of which is shown in SEQ ID No. 2), but also can optimize the function of the tunnel protein mutant through mutation so as to be suitable for detection under different conditions.

In the present application, the porin mutant includes a multimer of subunit polypeptides, either a homomultimer or a heteromultimer. The amino acid sequence of at least one subunit polypeptide in the multimer is: the sequence shown in SEQ ID NO.1, the sequence shown in SEQ ID NO.2, the first mutant sequence or the second mutant sequence.

It should be noted that homomultimers refer to: the amino acid sequences of all subunit polypeptides in the porin mutant are the same; heteromultimer refers to: the amino acid sequence of at least one subunit polypeptide in the porin mutant is different from the amino acid sequence of other subunit polypeptides.

Further, in the present application, the porin mutant comprises a 9-20 mer; namely, the porin mutant can contain 9-20 subunit polypeptides, and the pore diameter of the porin mutant can be regulated.

It is noted that, in some possible embodiments, the porin mutant may comprise any number of subunit polypeptides, such that the porin mutant may form a sufficiently large cavity to permit passage of a target analyte, such as a polynucleotide.

In the present application, the first mutant sequence satisfies at least one of the following A-D conditions, or the second mutant sequence satisfies at least one of the following E-H conditions.

A: at least one amino acid in the sequence shown in SEQ ID NO.1 is substituted.

B: at least one amino acid is inserted into the sequence shown in SEQ ID NO. 1.

C: at least one amino acid is deleted in the sequence shown in SEQ ID NO. 1.

D: at least one amino acid in the sequence shown in SEQ ID NO.1 is modified.

E: at least one amino acid in the sequence shown in SEQ ID NO.2 is substituted.

F: at least one amino acid is inserted into the sequence shown in SEQ ID NO. 2.

G: at least one amino acid is deleted in the sequence shown in SEQ ID NO. 2.

H: the side chain group of at least one amino acid in the sequence shown in SEQ ID NO.2 is modified.

Wherein the substitution comprises changing the species of amino acid or changing the configuration of amino acid; changing the type of amino acid includes: altering the electrical properties of the amino acid, altering the hydrophobicity of the amino acid, altering the structural rigidity of the amino acid, or altering the size of the side chain groups of the amino acid.

Illustratively, altering the configuration of an amino acid may include: replacing the D-form amino acid with the L-form amino acid; or, the amino acid in the L form is replaced with the amino acid in the D form.

Altering the electrical property of the amino acid can include: replacing the negatively charged amino acid with a positively or neutrally charged amino acid; alternatively, a negatively or electrically neutral amino acid is substituted for a positively charged amino acid.

Altering the hydrophobicity of the amino acid can include: replacing hydrophobic amino acid with hydrophilic amino acid; alternatively, a hydrophilic amino acid is substituted for a hydrophobic amino acid.

Altering the structural rigidity of an amino acid includes: replacing an amino acid having a flexible group as a side chain group with an amino acid having a rigid group as a side chain group; or, an amino acid having a rigid group as a side chain group is replaced with an amino acid having a flexible group as a side chain group.

Varying the size of the side chain groups of amino acids includes: replacing the amino acid with a larger side chain group with an amino acid with a smaller side chain group; alternatively, an amino acid with a smaller side chain group is substituted for an amino acid with a larger side chain group.

Further, in the application, glycosylation modification, phosphorylation modification, ubiquitination modification, S-nitrosylation modification, N-methylation modification, O-methylation modification, N-acetylation modification or lipidation modification are performed on a side chain group of at least one amino acid in a sequence shown in SEQ ID No.1 or a sequence shown in SEQ ID No.2, so that the structural stability of the tunnel protein mutant is improved.

Or, in some feasible embodiments, porphyrin, cholesterol or dodecyl glucoside molecules can be added to the side chain group of at least one amino acid in the sequence shown in SEQ ID No.1 or the sequence shown in SEQ ID No.2, which is favorable for improving the structural stability of the tunnel protein mutant.

In the present application, the porin mutant may be a mutation in one or more amino acids at the surface of the lumen of a double gated porin (the amino acid sequence of which is shown in SEQ ID No. 1).

In the prior art, when the amino acid sequence on the inner surface of the cavity of the channel protein and the substance to be detected have the same charge, translocation of the substance to be detected through the inner surface of the cavity of the channel protein is ineffective; the application can promote substances to be detected with different electric properties (such as positively charged protein and negatively charged nucleic acid) to enter the pore canal (i.e. inner cavity) of the porthole protein by changing the electric property of the amino acid sequence of the central gate and/or cap gate structure of the double-gate porthole protein (the amino acid sequence of which is shown as SEQ ID NO. 1).

The application can also regulate and control the size of the pore of the central gate and/or the cap gate of the double-gate pore channel protein (the amino acid sequence of the double-gate pore channel protein is shown as SEQ ID NO. 1) so as to be suitable for substances to be detected with different sizes. For example, the amino acids of the central gate and/or cap gate structure of a bi-gated pore channel protein (the amino acid sequence of which is shown in SEQ ID NO. 1) are replaced with larger or smaller amino acids; or, the amino acid sequence of the central gate and/or cap gate structure is deleted in whole or in part.

In some embodiments, the cis-opening of the porin mutant may have a diameter of 60 angstroms to 150 angstroms; alternatively, the porin mutant trans-opening may have a diameter of 30 angstroms to 150 angstroms.

In some embodiments, the porin mutant (having a central gate and a cap gate structure) constriction can have a diameter of about 2.5 angstroms to about 25 angstroms.

The application can also inhibit or prevent the opening of the central gate and/or the cap gate of a bigated channel protein (the amino acid sequence of which is shown in SEQ ID NO. 1). For example, the amino acids of the central gate and/or the cap gate structure of a bi-gated channel protein (the amino acid sequence of which is shown in SEQ ID NO. 1) are replaced with amino acids with more rigid side chain groups.

By way of illustration, in the present application, the first mutant sequence may satisfy the following condition: the amino acid at a specific position in the sequence shown in SEQ ID NO.1 is replaced by a neutral or positively charged amino acid. Wherein the specific site includes at least one of 85 th site, 102 th site, 158 th site, 169 th site and 253 th site.

In the above technical solution, the contraction of the bi-portal protein (the amino acid sequence of which is shown in SEQ id No. 1) can be limited to enhance the rate of the hole-entry of negatively charged substances to be tested (e.g. nucleic acids).

Illustratively, the first mutant sequence may satisfy the following condition:

substitution of aspartic acid (D) at position 85 in the sequence shown in SEQ ID NO.1 with asparagine (N), glutamine (Q), threonine (T), serine (S), glycine (G), arginine (R), or lysine (K), or the like.

Or, the glutamic acid (E) at position 102 in the sequence shown in SEQ ID NO.1 is replaced with asparagine (N), glutamine (Q), threonine (T), alanine (A), serine (S), glycine (G), proline (P), histidine (H), phenylalanine (F), tyrosine (Y), arginine (R), or lysine (K), or the like.

Or, in the sequence shown by SEQ ID NO.1, arginine (R) at position 158 is replaced by asparagine (N), glutamine (Q), threonine (T), alanine (A), serine (S), glycine (G), proline (P), histidine (H), phenylalanine (F), tyrosine (Y), lysine (K), or valine (V), or the like.

Or, the glutamic acid (E) at position 169 in the sequence shown in SEQ ID NO.1 is replaced with asparagine (N), glutamine (Q), threonine (T), alanine (A), serine (S), glycine (G), proline (P), histidine (H), arginine (R), or lysine (K), or the like.

Or, the substitution of aspartic acid (E) at position 253 in the sequence shown in SEQ ID NO.1 with asparagine (N), glutamine (Q), threonine (T), serine (S), glycine (G), arginine (R), or lysine (K), or the like.

In the present application, the first mutant sequence may also satisfy the following condition: the amino acid in the first region and the amino acid in the second region in the sequence shown in SEQ ID NO.1 are respectively replaced by amino acids with different electric properties. Wherein the amino acids in the first region are from position 526 to position 547, and the amino acids in the second region are from position 400 to position 490.

In the technical scheme, the cap gate and the central gate can realize selective detection on substances to be detected with different charges respectively.

Illustratively, the first mutant sequence may also satisfy the following condition: in the sequence shown in SEQ ID NO.1, the amino acids from the 526 th site to the 547 th site are replaced by negatively charged amino acids, and the amino acids from the 400 th site to the 490 th site are replaced by positively charged amino acids; or, the amino acids from position 526 to position 547 and the amino acids from position 400 to position 490 in the sequence shown in SEQ ID NO.1 are replaced with positively charged amino acids.

In the present application, the first mutant sequence satisfies at least one of the following I-K conditions; or, the first mutant sequence satisfies at least one of the following L-N conditions.

I: the amino acids of the first region of the sequence shown in SEQ ID NO.1 are deleted in whole or in part.

J: changing the electrical property of the amino acid in the first region in the sequence shown in SEQ ID NO. 1.

K: the size of the side chain group of the amino acid of the first region in the sequence shown in SEQ ID NO.1 is changed.

L: the amino acids of the second region of the sequence shown in SEQ ID NO.1 are deleted in whole or in part.

M: changing the electrical property of the amino acid in the second region in the sequence shown in SEQ ID NO. 1.

N: the size of the side chain group of the amino acid of the second region in the sequence shown in SEQ ID NO.1 is changed.

Wherein the amino acids in the first region are from position 526 to position 547 and the amino acids in the second region are from position 400 to position 490.

In the technical scheme, the pore diameters of the double pores of the double-gate pore channel protein can be respectively regulated to obtain the pore channel protein mutant so as to meet different requirements.

Further, in some alternative embodiments, the respective control of the pore sizes of the two pores of the bi-gated channel protein may also be performed by the following scheme: the charged amino acid between the 436 st site and the 451 th site in the sequence shown in SEQ ID NO.1 is replaced.

Illustratively, the first mutant sequence satisfies the following condition: increasing the size of the central gate channel can be achieved by replacing phenylalanine (F) at position 542 in the sequence shown in SEQ ID No.1 with alanine (a) (i.e. by mutating the central gate of the sequence shown in SEQ ID No. 1).

Illustratively, the first mutant sequence satisfies the following condition: promotion of insertion can be achieved by replacing proline (P) at position 444 in the sequence shown in SEQ ID NO.1 with phenylalanine (F) (i.e., by mutating the cap gate of the sequence shown in SEQ ID NO. 1).

In the present application, the first mutant sequence also satisfies at least one of the following O-S conditions:

o: deletion of amino acids from position 1 to position 152 in the sequence shown in SEQ ID NO.1, deletion of amino acids from position 1 to position 221 in the sequence shown in SEQ ID NO.1, or deletion of amino acids from position 1 to position 294 in the sequence shown in SEQ ID NO. 1.

P: deletion of the amino acids from position 320 to position 339 in the sequence shown in SEQ ID NO. 1.

Q: the amino acid from 661 st position to 705 th position in the sequence shown in SEQ ID NO.1 is mutated into cysteine.

R: the amino acid at least one position from 661 th position to 705 th position in the sequence shown in SEQ ID NO.1 is mutated into cysteine, and the cysteine is connected with maleimide porphyrin and/or cysteine-added cholesterol.

S: any one of the 627 th site, 628 th site and 629 th site in the sequence shown in SEQ ID NO.1 is mutated into phenylalanine.

In the technical scheme, when the first mutant sequence meets the condition of O (namely, the nitrogen end of the sequence shown in SEQ ID NO.1 is deleted), the length of the double-gate channel protein can be regulated and controlled. When the first mutant sequence satisfies P (namely, the free chain at the N3 end of the sequence shown in SEQ ID NO.1 is deleted), the situation that the double-gate channel protein blocks a substance to be detected can be reduced. When the first mutant sequence meets Q, conditions are provided for connecting other molecules (including hydrophobic protein AspS and the like) to the pore channel protein. When the first mutant sequence meets the R condition, on one hand, the channel protein can be quickly anchored on the surface of the phospholipid bilayer, the interaction between the double-gated channel protein and a membrane (namely the phospholipid bilayer in a biochip for subsequent detection) is promoted, and the assembly of the protein in the phospholipid bilayer is accelerated; on the other hand, the newly added compound (namely cysteine connected maleimide porphyrin and/or cysteine-acidified cholesterol) forms a bridge between the channel protein and the phospholipid bilayer, stabilizes the structure of the channel protein and phospholipid layer complex, and improves the stability of the biochip formed by assembling the double-gate channel protein and the phospholipid bilayer. When the first mutant sequence meets the S condition, the interaction between the double-gate pore channel protein and a membrane (namely a phospholipid bilayer in a biochip for subsequent formation detection) can be promoted, and the efficiency of assembling the pore channel protein in the phospholipid bilayer to form a pore channel is improved.

Illustratively, the first mutant sequence satisfies the following condition: the amino acids from the 1 st site to the 294 st site in the sequence shown in SEQ ID NO.1 are completely deleted (namely, the N0-N2 end of the sequence shown in SEQ ID NO.1 is deleted), so that the length of the pore channel can be shortened, and the pore-entering rate of the substance to be detected is improved.

Illustratively, the first mutant sequence satisfies the following condition: the amino acids from the 1 st site to the 294 st site in the sequence shown in SEQ ID NO.1 are completely deleted (namely, the N0-N2 end of the sequence shown in SEQ ID NO.1 is deleted), and the aspartic acid (D) from the 447 th site to the 449 th site in the sequence shown in SEQ ID NO.1 is replaced by asparagine (N), so that the effects of promoting the hole entrance of an object to be detected, reducing the gate control of the hole and enhancing the action signal of the object to be detected and the hole can be realized.

Illustratively, the first mutant sequence satisfies the following condition: the amino acids from the 320 th site to the 322 th site in the sequence shown in SEQ ID NO.1 are completely deleted (namely, the N3 end of the sequence shown in SEQ ID NO.1 is deleted), so that the effects of reducing orifice obstruction and reducing gating can be realized.

The application also provides a nucleotide sequence which is: a sequence shown in SEQ ID NO.3, a sequence shown in SEQ ID NO.4, a third mutant sequence or a fourth mutant sequence. Wherein, the third mutant sequence has homology of more than or equal to 30 percent with SEQ ID NO.3, and the fourth mutant sequence has homology of more than or equal to 30 percent with SEQ ID NO. 4; the sequence shown by SEQ ID NO.3 is the sequence shown by coding SEQ ID NO.1, and the sequence shown by SEQ ID NO.4 is the sequence shown by coding SEQ ID NO. 2.

The channel protein formed by the amino acid sequence coded by the nucleotide sequence is beneficial to improving the detection accuracy of a substance to be detected and the integrated assembly effect of the channel protein, can be beneficial to effectively detecting the same continuous base sequence, and is also beneficial to promoting the effective capture and/or translocation of the substance to be detected of the cross-channel protein; has good application prospect in the technical field of nanopore single molecule detection.

Illustratively, the third mutant sequence may have greater than or equal to 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% homology to SEQ ID No.3, and so forth; the fourth mutant sequence has homology of more than or equal to 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% with SEQ ID NO.4, etc.

Further, the third mutant sequence has a homology of more than or equal to 75 percent with SEQ ID NO.3, and/or the fourth mutant sequence has a homology of more than or equal to 75 percent with SEQ ID NO. 4.

Still further, the third mutant sequence has a homology of more than or equal to 95% with SEQ ID NO.3, and/or the fourth mutant sequence has a homology of more than or equal to 95% with SEQ ID NO. 4.

The application also provides a recombinant vector, an expression cassette or a recombinant bacterium, wherein the genome of the recombinant vector, the expression cassette or the recombinant bacterium contains the nucleotide sequence (namely the sequence shown by SEQ ID NO.3, the sequence shown by SEQ ID NO.4, a third mutant sequence or a fourth mutant sequence) provided by the above.

The application also provides an application of the double-gated pore channel protein, the pore channel protein mutant or the nucleotide sequence in preparing a biochip; the biochip can detect electrical and/or optical signals of the substance to be detected.

The amino acid sequence coded by the nucleotide sequence forms the double-gate channel protein or the channel protein mutant provided by the application and is embedded in a phospholipid bilayer to be assembled into a biochip, and the detection of an electric signal and/or an optical signal of a substance to be detected is realized by means of a computer processor and sensing equipment.

Further, the substance to be tested may include at least one of nucleic acid, protein, polysaccharide, neurotransmitter, chiral compound, heavy metal, and toxin.

In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions of the embodiments of the present application will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

Example 1

And (3) expressing and purifying the protein.

Expression of the protein: synthesizing a gene sequence (shown as SEQ ID NO. 1) for coding the bi-portal pore channel protein, transforming an N-terminal strep II-tag sequence (the sequence is WSHPQFEK) of the gene sequence into an E.coli C43 expression strain, and screening on an agar plate containing 100 mu g/mL of antibiotics to obtain a single colony.

Single colonies were picked and cultured at 37 ℃ to OD at 200rpm ₆₀₀ 1.6, the culture was expanded according to 1 ₆₀₀ After 0.6, IPTG was added and the temperature was lowered to 20 ℃ to continue the culture for 14 hours, and the cells were collected by centrifugation (4000 g in the centrifugation condition) and washed once with a phosphate buffer solution having a pH of 7.4.

And (3) protein purification: adding the cleaned thallus into a buffer solution A, wherein the mass concentration of the thallus in the buffer solution A is 0.1g/mL; the cells were then lysed by sonication, the cell debris removed by centrifugation (4000 g centrifugation conditions) and centrifuged for 1.5h at 200000g (Beckman, 70Ti rotor). The membrane was sufficiently dissolved in buffer B in the centrifuged system, mixed at 4 ℃ for 1 hour, and filtered through a 0.22 μm filter to obtain a supernatant. Wherein the buffer A contains NaCl and Tris-HCl, the concentration of the NaCl in the buffer A is 300mM, the concentration of the Tris-HCl in the buffer A is 50mM, and the pH value of the buffer A is 8.0. Buffer B contained NaCl, tris-HCl, LDAO (lauryl dimethyl amine oxide) and DDM (dodecyl-. Beta. -D-maltoside), and the concentration of NaCl in buffer B was 300mM, the concentration of Tris-HCl in buffer B was 50mM, the concentration of LDAO in buffer B was 0.01g/mL, the concentration of DDM in buffer B was 0.01g/mL, and the pH of buffer A was 8.0.

Then, the supernatant is injected into a Strep-Tactin XT Resin chromatographic column, washed by the solution C, and added with eluent to collect the protein. The collected protein is further subjected to polymer and monomer separation by gel chromatography molecular sieve. Wherein the solution C comprises NaCl, tris-HCl and LDAO, the concentration of the NaCl and the Tris-HCl in the solution C is 300mM and 50mM respectively, and the concentration of the LDAO in the solution C is 0.001g/mL; the eluent comprises NaCl, tris-HCl, LDAO and desthiobiotin, the concentration of the NaCl, the Tris-HCl and the desthiobiotin in the eluent is 300mM, 50mM and 5mM respectively, and the concentration of the LDAO in the eluent is 0.001g/mL.

FIG. 1 is an electrophoretogram and a molecular sieve image of the multimeric protein (designated as a bi-gated channel protein) obtained after the above purification. Part A in figure 1 is an electrophoresis diagram of purified double-gate channel protein, wherein M is marker, L is lysate, P is purified polymer protein, and S is molecular sieve-treated polymer protein. In FIG. 1, part B is a molecular sieve diagram of the bi-gated channel protein obtained after purification, wherein the abscissa represents the peak volume and the ordinate represents the peak intensity.

As can be seen from FIG. 1, the expression and purification method of the protein of example 1 can obtain multimeric protein.

Example 2

The double-gated channel protein obtained after purification in example 1 was analyzed for the structure and size of the multimer, and the results are shown in FIGS. 2 and 3.

The left part in FIG. 2 is the structural diagram of Cryo-EM obtained by the single particle cryoelectron microscopy technology for the bi-porthole protein obtained after purification in example 1; part A in FIG. 2 is a cryoelectron micrograph; part B in FIG. 2 is a structural diagram of protein 3D, and part C in FIG. 2 is a sectional diagram of protein 3D. In FIG. 3, part A is a region of the 3D structure size distribution of the protein of hole2.0 statistics; FIG. 3, section B, is a graph comparing the calculated pore size of hole2.0 with that of Vibrio cholerae VcGspD (PDB ID:5WQ 8), wherein the abscissa represents the transverse diameter of the protein 3D structure, the ordinate represents the longitudinal height of the 3D structure, and the cat represents the bi-gated pore protein obtained after purification in example 1.

As can be seen from FIG. 2, the structure of the bi-gated protein of the present application has a cap gate sequence as long as 70 amino acids, which is longer than 10 amino acids of VcGspD (PDB ID:5WQ 8), so that the cap gate of the bi-gated protein provided by the present application has a longer structure.

Furthermore, the Cryo-EM structure is analyzed by taking the obtained structure as a template through HOLE2 v2.2.005, the distance between the cap gate contraction area and the central gate contraction area is about 90 angstroms, and compared with VcGspD, the length is 15 angstroms, the interference of the cap gate and the central gate on signal reading of a substance to be detected can be reduced, and the detection accuracy of the bi-gate channel protein on the substance to be detected can be improved.

Still further, the central gate has a constriction size of 4 angstroms, which facilitates recognition of nucleotides from a single base, thereby facilitating efficient sequencing of consecutive identical base sequences.

Example 3

Electrophysiological analysis was performed on the biportal protein mutants.

Obtaining a purified polymer protein mutant (marked as a first channel protein mutant) by referring to the method for expressing and purifying the protein in the example 1; the difference from example 1 is that: the gene sequence for synthesizing and coding the first channel protein mutant is shown as SEQ ID NO. 2.

Electrophysiological analysis was performed on the purified first porin mutant, and the analysis results are shown in fig. 4 to 6. Fig. 4 is a channel resistivity diagram of the purified first porin mutant, tested by the method of: the wells were performed in a buffer containing 1M KCl and 50Mm Tri-HCl, and the channel current-voltage relationship shown in FIG. 4 was measured; the dashed line indicated by reference character a in fig. 4 is in the third quadrant, with a slope of 1.8332; the dashed line indicated by reference number B in FIG. 4 is in the first quadrant and has a slope of 1.6895; the dashed line indicated by reference character C in fig. 4 is in the first quadrant and has a slope of 0.25261. FIG. 5 is an easy-to-map of the purified first porin mutant on double-stranded DNA, wherein the data portion of the curve in FIG. 5 corresponds to the data on the left ordinate, and the data portion of the straight line in FIG. 5 corresponds to the data on the right ordinate. FIG. 6 is a graph showing the translocation of the purified first porin mutant to single-stranded DNA.

As can be seen from FIG. 4, the purified first mutant porin has a pore in the closed state at a forward voltage greater than 50Mv, a channel electrophysiology of 0.25nS, and a pore size of about 0.25nm; and under the negative voltage, the current voltage of the channel is 1.8nS.

As can be seen from FIG. 5, under the condition of-100 Mv, the well failed to translocate the double-stranded DNA, and the well was in a clogged state. As can be seen from FIG. 6, the purified multimeric protein mutant was able to achieve single-stranded DNA translocation.

The results of fig. 4-6 all show that the purified first-channel protein mutant can be used as a double-gated nanopore for single-stranded DNA sequencing.

Example 4

Different mutants of the bi-gated channel protein were analyzed and the results are shown in FIGS. 7 to 9.

The amino acid sequence of the double gated channel protein of example 1 was mutated (denoted as second channel protein mutant): all amino acids from position 1 to position 294 in the sequence shown in SEQ ID NO.1 are deleted; FIG. 7 is a 3D structural diagram of a cryo-electron microscope of a second porin mutant.

The amino acid sequence of the double gated channel protein of example 1 was mutated (denoted as third channel mutant): all of the amino acids from position 1 to 294 in the sequence shown in SEQ ID NO.1 were deleted, and the aspartic acid (D) from position 447 to position 449 in the sequence shown in SEQ ID NO.1 was replaced with asparagine (N); fig. 8 is a channel resistivity chart of the third porin mutant, tested as follows: performing a jack in a buffer solution containing 1M KCl and 50Mm Tri-HCl, and measuring the channel current-voltage relationship as shown in FIG. 8, wherein the dotted line marked with the reference character A in FIG. 8 is in the third quadrant, and the slope is 2.9107; the dashed line in FIG. 8, labeled B, is in the first quadrant and has a slope of 2.8837.

The amino acid sequence of the double gated channel protein of example 1 was mutated (designated as fourth channel protein mutant): substitution of phenylalanine (F) at position 542 in the sequence represented by SEQ ID NO.1 with alanine (A); fig. 9 is a channel resistivity diagram of a fourth porin mutant, tested as follows: performing the plugging in a buffer solution containing 1M KCl and 50Mm Tri-HCl, and measuring the channel current voltage relationship as shown in FIG. 9, wherein the dotted line denoted by reference character A in FIG. 9 is in the third quadrant, and the slope is 4.6018; in the first quadrant, designated by reference numeral B in FIG. 9, the slope is 4.7067.

As can be seen from FIG. 7, the second porin mutant, in which all of the amino acids from position 1 to position 294 in the sequence shown in SEQ ID NO.1 are deleted, can achieve an increase in the size of the central portal.

As can be seen from FIG. 8, the amino acids from position 1 to position 294 in the sequence shown in SEQ ID No.1 were all deleted, and asparagine (N) was substituted for aspartic acid (D) from position 447 to position 449 in the sequence shown in SEQ ID No.1, and the resulting third porin mutant no longer exhibited gating.

As can be seen from FIG. 9, the size of the central pore becomes 2-fold after mutation (i.e., the fourth pore mutant) as compared to before mutation (i.e., the double-gated pore) in the fourth pore mutant formed by replacing phenylalanine (F) at position 542 with alanine (A) in the sequence shown in SEQ ID NO. 1.

In conclusion, the double-gate tunnel protein and the tunnel protein mutant provided by the application are beneficial to improving the detection accuracy of substances to be detected and the integrated assembly effect of the tunnel protein, are also beneficial to effectively detecting continuous same base sequences, have better structural stability and electrophysiological stability, and are also beneficial to promoting the effective capture and/or translocation of substances to be detected of the cross-tunnel protein.

The embodiments described above are some, but not all embodiments of the present application. The detailed description of the embodiments of the present application is not intended to limit the scope of the claimed application, but is merely representative of selected embodiments of the application. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.

Claims (10)

1. A bi-gated pore protein, characterized in that the amino acid sequence of the bi-gated pore protein is shown in SEQ ID NO. 1;

optionally, the bi-gated channel protein comprises a polymer composed of subunit polypeptides, wherein the amino acid sequence of the subunit polypeptides is shown as SEQ ID NO. 1;

optionally, the multimer comprises a 12-16 mer.

2. A porin mutant, wherein the porin mutant has the amino acid sequence: the sequence shown in SEQ ID NO.2, the first mutant sequence or the second mutant sequence;

wherein, the first mutant sequence is a sequence with homology of more than or equal to 30 percent with SEQ ID NO.1, and the second mutant sequence is a sequence with homology of more than or equal to 30 percent with SEQ ID NO. 2.

3. The porin mutant of claim 2, wherein said porin mutant comprises a multimer of subunit polypeptides, said multimer being a homomultimer or a heteromultimer;

the amino acid sequence of at least one subunit polypeptide in the multimer is: the sequence shown in SEQ ID NO.1, the sequence shown in SEQ ID NO.2, the first mutant sequence or the second mutant sequence;

optionally, the multimer comprises a 9-20 mer.

4. The porin mutant of claim 2, wherein said first mutant sequence satisfies at least one of the following conditions a-D:

a: at least one amino acid in the sequence shown in SEQ ID NO.1 is replaced;

b: at least one amino acid is inserted into the sequence shown in SEQ ID NO. 1;

c: at least one amino acid is deleted in the sequence shown in SEQ ID NO. 1;

d: at least one amino acid in the sequence shown in SEQ ID NO.1 is modified;

or, the second mutant sequence satisfies at least one of the following E-H conditions:

e: at least one amino acid in the sequence shown in SEQ ID NO.2 is replaced;

f: at least one amino acid is inserted into the sequence shown in SEQ ID NO. 2;

g: at least one amino acid is deleted in the sequence shown in SEQ ID NO. 2;

h: the side chain group of at least one amino acid in the sequence shown in SEQ ID NO.2 is modified;

wherein the substitution comprises altering the species of amino acid or altering the configuration of the amino acid; the variety of the changed amino acid includes: changing the electrical property of the amino acid, changing the hydrophobicity of the amino acid, changing the structural rigidity of the amino acid or changing the size of a side chain group of the amino acid;

optionally, the modification comprises at least one of a glycosylation modification, a phosphorylation modification, a ubiquitination modification, an S-nitrosylation modification, an N-methylation modification, an O-methylation modification, an N-acetylation modification, and a lipidation modification; and/or, the modification comprises: at least one of porphyrin, cholesterol and dodecyl glucoside molecules is added to the side chain group.

5. The porin mutant of claim 4, wherein said first mutant sequence satisfies the following condition: the amino acid at a specific position in the sequence shown in SEQ ID NO.1 is replaced by a neutral or positively charged amino acid;

wherein the specific site includes at least one of 85 th site, 102 th site, 158 th site, 169 th site and 253 th site.

6. The porin mutant of claim 4, wherein said first mutant sequence satisfies the following conditions: the amino acid in the first region and the amino acid in the second region in the sequence shown in SEQ ID NO.1 are respectively replaced by amino acids with different electric properties;

wherein the amino acids in the first region are from position 526 to position 547, and the amino acids in the second region are from position 400 to position 490.

7. The porin mutant of claim 4, wherein said first mutant sequence satisfies at least one of the following I-K conditions:

i: the amino acids of the first region in the sequence shown in SEQ ID NO.1 are totally or partially deleted;

j: changing the electrical property of amino acid in a first region in the sequence shown in SEQ ID NO. 1;

k: changing the size of a side chain group of amino acids in a first region in the sequence shown in SEQ ID NO. 1;

or, the first mutant sequence satisfies at least one of the following L-N conditions:

l: the amino acids of the second region in the sequence shown in SEQ ID NO.1 are totally or partially deleted;

m: changing the electrical property of amino acids in a second region in the sequence shown in SEQ ID NO. 1;

n: changing the size of the side chain group of the amino acid of the second region in the sequence shown in SEQ ID NO. 1;

wherein the amino acids in the first region are from position 526 to position 547, and the amino acids in the second region are from position 400 to position 490.

8. The porin mutant of claim 4, wherein said first mutant sequence satisfies at least one of the following O-S conditions:

o: deletion of amino acids from position 1 to position 50-152 in the sequence shown in SEQ ID NO.1, deletion of amino acids from position 1 to position 221 in the sequence shown in SEQ ID NO.1, or deletion of amino acids from position 1 to position 294 in the sequence shown in SEQ ID NO. 1;

p: deletion of amino acids from position 320 to position 339 in the sequence shown in SEQ ID NO. 1;

q: mutating amino acid at least one position from 661 th position to 705 th position in the sequence shown in SEQ ID NO.1 into cysteine;

r: mutating amino acids at least one position from 661 th position to 705 th position in a sequence shown in SEQ ID NO.1 into cysteine, wherein the cysteine is connected with maleimide porphyrin and/or cysteine-added cholesterol;

s: any one of the 627 th site, 628 th site and 629 th site in the sequence shown in SEQ ID NO.1 is mutated into phenylalanine.

9. A nucleotide sequence, wherein the nucleotide sequence is: a sequence shown as SEQ ID NO.3, a sequence shown as SEQ ID NO.4, a third mutant sequence or a fourth mutant sequence;

wherein, the third mutant sequence has a homology of more than or equal to 30 percent with SEQ ID NO.3, and the fourth mutant sequence has a homology of more than or equal to 30 percent with SEQ ID NO. 4; the sequence shown by SEQ ID NO.3 is the sequence shown by coding SEQ ID NO.1, and the sequence shown by SEQ ID NO.4 is the sequence shown by coding SEQ ID NO. 2.

10. Use of a bi-gated channel protein according to claim 1, a channel protein mutant according to claims 2-8 or a nucleotide sequence according to claim 9 for the preparation of a biochip; the biochip can detect the electrical and/or optical signals of the substance to be detected;

optionally, the test agent includes at least one of a nucleic acid, a protein, a polysaccharide, a neurotransmitter, a chiral compound, a heavy metal, and a toxin.

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