HfaB novel nanopore protein mutant and application thereof in sequencing

Abstract

The invention relates to a novel HfaB nanopore protein mutant and application thereof in sequencing, and belongs to the technical field of nanopores. The polynucleotide for coding the nanopore protein mutant and the recombinant expression vector can express the nanopore protein mutant, and can be used for constructing recombinant cells or recombinant strains for expressing the nanopore protein mutant. The wild-type nanopore protein provided by the invention has relatively high pore stability and is suitable for nanopore sequencing, the sequencing current property of the nanopore protein mutant obtained by modification on the basis of the wild-type nanopore protein is further improved, the sequencing precision is obviously improved, the sequencing signal amplitude and step number are obviously increased, the pore current stability is enhanced, and the nanopore protein mutant can be applied to nanopore sequencing. The spontaneous blocking condition is reduced, and the abnormal noise on the via hole signal is reduced.

Description

Technical Field

[0001] This disclosure relates to novel HfaB nanoporous protein mutants and their applications in sequencing, belonging to the field of nanoporous technology. Background Technology

[0002] Nucleic acids are the carriers of biological genetic information, and nucleic acid sequencing is fundamental to understanding gene function and deciphering the mysteries of life. It is also a core technology in modern life sciences and related industries. Nanopore sequencing, as a fourth-generation sequencing technology, exhibits significant advantages, including real-time processing, portability, potential low cost, ultra-long read lengths, and the ability to detect epigenetic modifications on nucleic acids and perform direct RNA sequencing. It represents the future direction of nucleic acid sequencing technology. Currently, nanopore sequencing is widely used in rapid pathogen detection, genetic disease diagnosis, food safety monitoring, and human genome sequencing. Based on the type of nanopore, nanopore sequencing is divided into solid-state nanopore sequencing and biological nanopore sequencing. Biological nanopore sequencing uses natural pore membrane proteins as nanopores. These proteins possess specific pore structures, biological activities, and the ability to insert into lipid bilayer membranes, offering the advantage of flexible modification. In recent years, biological nanopore sequencing has made significant progress. Biological nanopores are tiny pores, essentially channels formed on a membrane. Under an applied electric field, ions pass through the nanopore, generating a constant current. Charged nucleic acids, under the influence of the electric field, pass through the biological nanopore, causing a blockage of the current. Different bases have different resistance to the generation of electric current. By analyzing the current signal, the base sequence information on the nucleic acid chain can be obtained.

[0003] The properties of nanoporins are key factors determining the accuracy of nanopore sequencing, and the screening and optimization of nanoporins is a major challenge that needs to be overcome in the development of nanopore sequencers. Nanopore sequencing has extremely high requirements for the pore stability, key amino acid composition of the contraction region, pore diameter range, and amino acid properties of the pore wall of nanoporins. Currently, nanoporins suitable for nanopore sequencing are scarce, with only a few types available, such as Mycobacterium smegmatis porin A (MspA) (patent EP3029467A1) and curli-specific transport channel protein (CsgG) (patent US20230295715A1). Therefore, it is necessary to discover and optimize novel nanoporins to improve the accuracy of nanopore sequencing. Summary of the Invention

[0004] The problem the invention aims to solve

[0005] Nanopore sequencing has high requirements for the pore stability of nanopore proteins, the key amino acid composition of the contraction region, the pore diameter range, and the amino acid properties of the pore inner wall. Currently, nanopore proteins suitable for nanopore sequencing are scarce, and the sequencing signal and accuracy are poor.

[0006] Solution for solving the problem

[0007] To address the aforementioned issues, this disclosure presents a novel nanoporin. Through expression and purification of wild-type nanoporin, its pore stability was investigated. Based on this, its atomic-level three-dimensional structure was further elucidated, determining properties such as the pore diameter range. Referring to the wild-type nanoporin, the amino acids were modified, and novel nanoporins suitable for nanopore sequencing were screened using DNA sample sequencing. This enriches the variety of nanoporins, providing more options for nanopore sequencing. Further in-depth analysis and modification of the novel nanoporin provided in this disclosure may also improve the accuracy of fourth-generation nanopore sequencing.

[0008] This disclosure provides nanoporin mutants, wherein the mutants are selected from the group consisting of (I)-(IV): (I) the nanoporin mutant contains a mutation at at least one position corresponding to the 79th and 80th positions of the sequence shown in SEQ ID NO:1 compared to the sequence shown in SEQ ID NO:1; (II) has at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, most preferably at least 99% sequence identity with the amino acid sequence shown in (I), and does not include mutants of the sequence shown in SEQ ID NO:1; (III) a mutant encoded by a polynucleotide hybridized to the polynucleotide shown in (a) or (b) under very stringent conditions: (a) a polynucleotide encoding a mutant of the amino acid sequence shown in (I); (b) a full-length complementary polynucleotide of (a); (IV) a fragment of the mutant shown in any one of (I), (II) or (III), and the fragment still possesses nanoporin activity.

[0009] In some embodiments, the nanoporin mutant corresponds to the sequence shown in SEQ ID NO:1 and has a mutated amino acid at at least one of the following positions: S79N, S79W, S79I, S79A, S79V, S79L, S79Q, S79Y, S79E, S79K, E80Q, E80N, E80S, E80W, E80A, E80I.

[0010] Optionally, the nanoporin mutant is selected from any one of the following groups (V)-(VIII): (V) The nanoporin mutant, compared with the sequence shown in SEQ ID NO:1, also contains a mutation at at least one of the positions corresponding to the sequence shown in SEQ ID NO:1, namely positions 77, 182, 184, 186, 218, 220, 239, and 235; (VI) The amino acid sequence shown in (V) has at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity, and does not include the mutant of the sequence shown in SEQ ID NO:1; (VII) A mutant encoded by a polynucleotide that hybridizes with the polynucleotide shown in (c) or (d) under very stringent conditions: (c) A polynucleotide encoding a mutant of the amino acid sequence shown in (V); (d) Full-length complementary polynucleotides of (c); (VIII) A fragment of the mutant shown in any one of (V), (VI), or (VII), and the fragment still has nanoporin activity.

[0011] In some embodiments, the amino acids having mutations at one or more of the following positions are: Y77I, Y77T, Y77Q, Y77M, Y77D, Y77W, Y77V, Y77F, G182D, R184Q, F186Q, E218Q, F220Q, K239Q, and K235E.

[0012] In some embodiments, the nanoporin mutant includes the deletion or addition of at least one amino acid residue at the N-terminus or C-terminus of the mutant of the sequence shown in (I) or (V), and / or the deletion of amino acid residues 133 to 138 of the sequence shown.

[0013] Preferably, the nanoporin mutant includes a mutant with 5 amino acid residues deleted at the C-terminus of the sequence shown in (I) or (V), and a mutant with the sequence shown having amino acid residues deleted from positions 133 to 138; more preferably, the nanoporin mutant includes a mutant with positions 133 to 138 and positions 288 to 292 deleted from the sequence shown in (I) or (V).

[0014] In some embodiments, the nanoporin mutant is selected from any one of the following (IX)-(VIII): (IX) The nanoporin mutant, compared with the sequence shown in SEQ ID NO:1, further includes a spike at at least one of positions 68, 71, 73, 88, 112, 115, 118, 119, 120, 123, 125, 225 corresponding to the sequence shown in SEQ ID NO:1; and, optionally, the mutant further includes a modification of the β-barrel of the nanoporin as shown in SEQ ID NO:1; (X) has at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, most preferably at least 99% sequence identity with the amino acid sequence shown in (IX), and does not include SEQ ID NO:1. (1) A mutant of the sequence shown in NO:1; (1) A mutant encoded by a polynucleotide that can hybridize with the polynucleotide shown in (e) or (f): (e) A polynucleotide encoding a mutant of the amino acid sequence shown in (IX); (f) A full-length complementary polynucleotide to (e); (VIII) A fragment of a mutant shown in any one of (IX), (X) or (XI), wherein the fragment still has nanoporin activity.

[0015] In some embodiments, the nanoporin mutant also has mutated amino acid residues at one or more of the following positions corresponding to the sequence shown in SEQ ID NO:1: R225D, A119Q, A112Q, E115Q, I118Q, H68Y, T71Q, Q73E, R88Q, R120I, E123A, I125E; and, optionally, the modification of the β-barrel includes modification of amino acid residues at positions 169–186 and / or positions 216–241 of the sequence shown in SEQ ID NO:1; preferably, the modification of the β-barrel includes replacing positions 169–186 of the sequence shown in SEQ ID NO:1 with the sequence shown in SEQ ID NO:198; preferably, the modification of the β-barrel includes replacing positions 216–241 of the sequence shown in SEQ ID NO:1 with the sequence shown in SEQ ID NO:199.

[0016] In some embodiments, the nanoporin mutant corresponds to the sequence shown in SEQ ID NO:1 and has the following (m1) to (m2) sequences. 48 Mutations shown in any of the following: (m1)S79N; (m2)S79W; (m3)S79I; (m4)S79A; (m5)S79V; (m6)S79L; (m7)S79Q; (m8)S79Y; (m9)S79E; (m 10 S79K; (m 11 E80Q;(m 12E80N; (m 13 E80S; (m 14 E80W; (m 15 E80A; (m 16 E80I; (m 17 Y77I, S79I; (m 18 Y77T, S79I; (m 19 Y77Q, S79I; (m 20 Y77M, S79I; (m 21 Y77D, S79I; (m 22 Y77W, S79I; (m 23 Y77V, S79I; (m 24 Y77F, S79I; (m 25 S79I, deletion of amino acid residues at positions 133–138 and 288–292; (m 26 S79I, R184Q; (m 27 S79I, E218Q; (m 28 S79I, K239Q; (m 29 )S79I, R184Q, E218Q, K239Q; (m 30 S79I, R184Q, K235E; (m 31 S79I, G182D, R184Q; (m 32 S79I, R184Q, F186Q; (m 33 S79I, R184Q, F220Q; (m 34 )S79I, R184Q, F186Q, R225D; (m 35 )S79I, R184Q, F186Q, A119Q; (m 36 )S79I, R184Q, F186Q, E115Q; (m 37 )S79I, R184Q, F186Q, A112Q; (m 38 )S79I, R184Q, F186Q, I118Q; (m 39 )S79I, R184Q, F186Q, H68Y; (m 40 )S79I, R184Q, F186Q, T71Q; (m 41 )S79I, R184Q, F186Q, Q73E; (m 42 )S79I, R184Q, F186Q, R88Q; (m 43 )S79I, R184Q, F186Q, R120I; (m 44)S79I, R184Q, F186Q, E123A; (m 45 )S79I, R184Q, F186Q, I125E; (m 46 The amino acid sequences of S79I, R184Q, and F186Q, from position 169 to 186, are shown in SEQ ID NO: 198, and the amino acid sequences from position 216 to 241 are shown in SEQ ID NO: 199; (m 47 )S79I, R184Q, F186Q, T71Q, Q73E, R88Q; (m 48 The amino acid sequences of S79I, R184Q, F186Q, T71Q, Q73E, and R88Q, from position 169 to 186, are shown in SEQ ID NO:198, and the amino acid sequences from position 216 to 241 are shown in SEQ ID NO:199.

[0017] Preferably, the nanoporin mutant corresponds to the amino acid sequence shown in SEQ ID NO:1 and has any of the following mutations: (m3)S79I; (m5)S79V; (m6)S79V; (m79I ... 11 E80Q;(m 12 E80N; (m 25 S79I, deletion of amino acid residues at positions 133–138 and 288–292; (m 26 S79I, R184Q; (m 27 S79I, E218Q; (m 28 S79I, K239Q; (m 29 )S79I, R184Q, E218Q, K239Q; (m 32 S79I, R184Q, F186Q; (m 34 )S79I, R184Q, F186Q, R225D; (m 35 )S79I, R184Q, F186Q, A119Q; (m 36 )S79I, R184Q, F186Q, E115Q; (m 37 )S79I, R184Q, F186Q, A112Q; (m 38 )S79I, R184Q, F186Q, I118Q; (m 39 )S79I, R184Q, F186Q, H68Y; (m 40 )S79I, R184Q, F186Q, T71Q; (m 41 )S79I, R184Q, F186Q, Q73E; (m 42)S79I, R184Q, F186Q, R88Q; (m 43 )S79I, R184Q, F186Q, R120I; (m 44 )S79I, R184Q, F186Q, E123A; (m 45 )S79I, R184Q, F186Q, I125E; (m 46 The amino acid sequences of S79I, R184Q, and F186Q, from position 169 to 186, are shown in SEQ ID NO: 198, and the amino acid sequences from position 216 to 241 are shown in SEQ ID NO: 199; (m 47 )S79I, R184Q, F186Q, T71Q, Q73E, R88Q; (m 48 The amino acid sequences of S79I, R184Q, F186Q, T71Q, Q73E, and R88Q, from position 169 to 186, are shown in SEQ ID NO:198, and the amino acid sequences from position 216 to 241 are shown in SEQ ID NO:199.

[0018] This disclosure provides an isolated polynucleotide, wherein the polynucleotide encodes a nanoporin mutant as described in any of the preceding embodiments. In some embodiments, the polynucleotide may be in DNA or RNA form. The DNA form includes cDNA, genomic DNA, or synthetically produced DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand. The polynucleotide encoding the mutant of this disclosure includes: a coding sequence that encodes only the mutant; a coding sequence of the mutant and various additional coding sequences; a coding sequence of the mutant (and optional additional coding sequences) and a non-coding sequence.

[0019] This disclosure provides a recombinant expression vector comprising the polynucleotides described above.

[0020] This disclosure provides a recombinant host cell comprising a nanoporin mutant as described in any of the preceding claims, an isolated polynucleotide as described above, or a recombinant expression vector as described above.

[0021] In some embodiments, the recombinant host cell is derived from microorganisms of the genera *Escherichia*, *Erwinia*, *Serratia*, *Providencia*, *Enterobacteria*, *Salmonella*, *Streptomyces*, *Pseudomonas*, *Brevibacterium*, *Bacillus*, or *Corynebacterium*. In some optional embodiments, the recombinant host cell or genetically engineered bacteria is derived from *Escherichia coli*, *Corynebacterium glutamicum*, or *Bacillus subtilis*. In some more preferred embodiments, the recombinant host cell or genetically engineered bacteria is derived from *Escherichia coli*, including *Escherichia coli* DH5α, *Escherichia coli* Top10, and *Escherichia coli Trans*. T1, etc.; further preferred is Escherichia coli OMP8, which is disclosed in the non-patent literature Alexanderj Prilipov, Prashant S Phale, Patrick Van Gelder, Jurg P Rosenbusch, Ralf Koebnik, Coupling site-directed mutagenesis with high-level expression: large scale production of mutant porins from E. coli, FEMS Microbiology Letters, Volume 163, Issue 1, June 1998, Pages 65-72, specifically BL21(DE3)omp8(BL21(DE3),ΔlamB ompF::Tn5ΔompAΔompC), which is incorporated herein by reference.

[0022] This disclosure provides a cell culture comprising the recombinant host cell or recombinant genetically engineered bacteria as described above.

[0023] This disclosure provides a kit comprising a nanoporin mutant as described in any of the preceding claims and / or a nanoporin with an amino acid sequence as shown in SEQ ID NO:1.

[0024] In some embodiments, the kit is used to detect the presence or absence of one or more features of a target analyte; optionally, the target analyte is one or more of nucleic acids, peptides, or proteins; optionally, the nucleic acid is at least one of single-stranded DNA, double-stranded DNA, RNA, and miRNA.

[0025] In some alternative implementations, the target analyte features include at least one of the following (A) to (E): (A) the length of the target analyte; (B) the properties of the target analyte; (C) the sequence of the target analyte; (D) the secondary structure of the target analyte; (E) whether the target analyte is modified.

[0026] This disclosure provides the use of nanoporin mutants as described in any of the preceding claims, nanoporins with amino acid sequences as shown in SEQ ID NO:1, isolated polynucleotides as described above, recombinant expression vectors as described above, recombinant host cells as described above, cell cultures as described above, or kits as described in any of the preceding claims in detecting the presence or absence of one or more features of a target analyte.

[0027] In some embodiments, the target analyte is one or more of nucleic acids, peptides, or proteins; optionally, the nucleic acid is at least one of single-stranded DNA, double-stranded DNA, RNA, and miRNA. In some optional embodiments, the target analyte is characterized by at least one of the following (A) to (E): (A) the length of the target analyte; (B) the properties of the target analyte; (C) the sequence of the target analyte; (D) the secondary structure of the target analyte; and (E) whether the target analyte is modified.

[0028] This disclosure provides the use of nanoporin mutants as described above, isolated polynucleotides as described above, recombinant expression vectors as described above, recombinant host cells as described above, cell cultures as described above, kits as described above, or nanoporins with amino acid sequences as shown in SEQ ID NO:1 in detecting the presence, absence, or one or more characteristics of a target analyte.

[0029] In some alternative embodiments, the target analyte is one or more of nucleic acids, peptides, or proteins. In some specific embodiments, the nucleic acid is at least one of single-stranded DNA, double-stranded DNA, RNA, and miRNA.

[0030] In some alternative implementations, the target analyte features include at least one of the following (A) to (E): (A) the length of the target analyte; (B) the properties of the target analyte; (C) the sequence of the target analyte; (D) the secondary structure of the target analyte; (E) whether the target analyte is modified.

[0031] The effects of the invention

[0032] This disclosure provides a novel nanoporin, HfaB, and resolves its atomic-level three-dimensional structure. The HfaB nanoporin exhibits high pore stability, with the narrowest point of the contraction region having a diameter of [missing information]. This invention is applicable to nanopore sequencing. It optimizes the nanopore structure of wild-type HfaB nanopore protein to further improve the sequencing current properties of nanopore proteins and enhance the accuracy of fourth-generation nanopore sequencing. The invention modifies the wild-type HfaB nanopore protein, providing novel HfaB nanopore protein mutants, enriching the variety of nanopore proteins and offering more options for nanopore sequencing. Through modification and optimization, these mutants, while maintaining consistency with the wild type in protein expression levels, thermal stability, and oligomeric state, exhibit significantly improved sequencing current properties, making them suitable for nanopore sequencing. Specifically, mutants H4E and H5C show significantly improved sequencing accuracy, with a marked increase in sequencing signal amplitude and step number. Furthermore, mutant H4E resolves the current instability issue. Building upon this, by accumulating mutation sites, mutants H7M, H7N, H7O, H7R, and H7W are obtained, showing significantly improved sequencing properties, reduced spontaneous blockage, reduced abnormal noise in the pore signal, and more stable pore current. Furthermore, by modifying the mutant H7W, the resulting mutant, while maintaining or even improving upon its performance in terms of protein expression levels, thermal stability, and oligomerization, exhibited further improvements in sequencing properties and more stable pore current. Attached Figure Description

[0033] Figure 1 This is an SDS-PAGE gel image of wild-type HfaB protein.

[0034] Figure 2 The image shows the molecular sieve results for wild-type HfaB protein.

[0035] Figure 3 This is a cryo-electron microscopy density map of wild-type HfaB protein; among which, Figure 3 A is a top view. Figure 3 B is the front view.

[0036] Figure 4 This is a schematic diagram of the wild-type HfaB protein structure.

[0037] Figure 5 This is a diagram of the overall conformation of the contractile region of the wild-type HfaB protein.

[0038] Figure 6 The diagram shows the expression and purification of H4G, H4H, H4I, H4J, H4K, and H4L mutant proteins.

[0039] Figure 7The diagram shows the expression and purification of H4C, H4D, H4E, H4F, H5C, H5D, H5E, H5F, H5G, and H5H mutant proteins.

[0040] Figure 8 This is a diagram of the overall conformation of the contractile region of the H4E mutant protein.

[0041] Figure 9 The diagram shows the expression and purification of H5S, H5T, H5U, H5V, H5W, H5X, H5Y, and H5Z mutant proteins.

[0042] Figure 10 The graph shows the expression and purification of H5J, H7M, H7N, H7O, H7R, H7U, H7V, H7W, and H7X mutant proteins.

[0043] Figure 11 This is a graph showing the current signal of wild-type HfaB nanopores.

[0044] Figure 12 The image shows the nanopore current signals of the H4G, H4H, H4I, H4J, H4K, and H4L mutants.

[0045] Figure 13 The image shows the nanopore current signals of the H4C, H4E, H5C, H5D, H5E, H5F, H5G, and H5H mutants.

[0046] Figure 14 The image shows the nanopore current signals of the H5S, H5T, H5U, H5V, H5W, H5X, H5Y, and H5Z mutants.

[0047] Figure 15A The image shows the current signal of the nanopores in the H4E and H5J mutants, which demonstrates the improved spontaneous blockage of the nanopores in the H5J mutant.

[0048] Figure 15B The graph shows the number of spontaneous blockages of H4E and H5J within a sequencing time of 3600s, indicating that the spontaneous blockage of nanopores is improved in the H5J mutant.

[0049] Figure 16A The images show the nanopore current signals of mutants H7M, H7W, H7R, H7O, H7N, H7U, H7V, and H7X, revealing a significant improvement in spontaneous nanopore blockage in mutants H7M, H7W, and H7R.

[0050] Figure 16B The graph shows the number of spontaneous blockages of mutants H7M, H7W, H7R, H7O, H7N, H7U, H7V, and H7X within a sequencing time of 3600s, indicating that the spontaneous blockage of nanopores is significantly improved in mutants H7M, H7W, and H7R.

[0051] Figure 17A The diagram shows the expression and purification of H8F, H8H, H8I, H8J, and H8K mutant proteins.

[0052] Figure 17B The diagram shows the expression and purification of H10M, H10N, H9O, H9P, and H10L mutant proteins.

[0053] Figure 17C The diagram shows the expression and purification of H9R, H9S, H9T, H9U, and H9W mutant proteins.

[0054] Figure 18A The image shows the nanopore current signals of the H9R, H9P, H9O, and H8K mutants.

[0055] Figure 18B The image shows the nanopore current signals of the H9W, H9U, H9T, and H9S mutants.

[0056] Figure 18C The image shows the nanopore current signals of the H9R, H9P, H9O, and H8K mutants.

[0057] Figure 18D The image shows the nanopore current signals of the H8J, H8I, H8H, and H8F mutants. Detailed Implementation

[0058] Various exemplary embodiments, features, and aspects of this disclosure will be described in detail below. The term "exemplary" as used herein means "serving as an example, embodiment, or illustration." Any embodiment illustrated herein as "exemplary" is not necessarily to be construed as superior or better than other embodiments. Furthermore, numerous specific details are set forth in the following detailed description to better illustrate this disclosure. Those skilled in the art will understand that this disclosure can be practiced without certain specific details. In other instances, methods, means, apparatus, and steps well known to those skilled in the art have not been described in detail in order to highlight the spirit of this disclosure.

[0059] Unless otherwise stated, all units used in this specification are International Standard Units (SI), and all numerical values ​​and ranges appearing in this disclosure should be understood to include systematic errors unavoidable in industrial production. In this specification, the word "may" has both the meaning of performing a certain treatment and the meaning of not performing a certain treatment.

[0060] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to that embodiment, which are included in at least one of the embodiments described herein and may or may not be present in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0061] In this specification, the range of values ​​referred to as "value A to value B" refers to the range including the endpoint values ​​A and B.

[0062] In this specification, the terms "peptide" and "protein" are used interchangeably herein and refer to an amino acid polymer of at least two amino acid residues linked together by covalent bonds (e.g., peptide bonds). The polymer may be linear, branched, or cyclic, may contain modified amino acids, and may be separated by non-amino acid segments. The term also includes amino acid polymers that have been modified (e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, ubiquitination, glycosylation, amidation of C-terminal amino acids, or any other manipulation, such as conjugation with labeled components). The term "amino acid" may include native amino acids, non-native amino acids, amino acid analogs, and all their D and L stereoisomers. The amino acids and their abbreviations and English abbreviations in this disclosure are as follows: Histidine (His, H); Serine (Ser, S); Glutamic acid (Glu, E); Glutamine (Gln, Q); Glycine (Gly, G); Threonine (Thr, T); Phenylalanine (Phe, F); Aspartic acid (Asp, D); Tyrosine (Tyr, Y); Leucine (Leu, L); Isoleucine (Ile, I); Arginine (Arg, R); Alanine (Ala, A); Valine (Val, V); Tryptophan (Trp, W); Methionine (Met, M); Asparagine (Asn, N); Cysteine ​​(Cys, C); Lysine (Lys, K); Proline (Pro, P).

[0063] The term "nanoporin" refers to the HfaB nanoporin derived from Rhizobium sp. RU33AB, Uniprot ACCESSION: A0A1N6RVG5_9HYPH. In some specific embodiments of this disclosure, the HfaB nanoporin comprises the amino acid sequence shown in SEQ ID NO:1, encoded by the nucleotide sequence shown in SEQ ID NO:1. The term "fragment" refers to a polypeptide or a catalytic or carbohydrate-binding module that has one or more (e.g., several) amino acids deleted from the amino and / or carboxyl ends of a mature polypeptide or domain. In the technical solutions of this disclosure, the fragment possesses nanoporin activity.

[0064] The term "wild-type" refers to an object found in nature. For example, a polypeptide or polynucleotide sequence that exists in an organism, can be isolated from a natural source, and has not been intentionally modified by humans in a laboratory is naturally occurring. As used in this disclosure, "naturally occurring" and "wild-type" are synonyms. The term "mutant" refers to a polynucleotide or polypeptide that contains alterations (i.e., substitutions, insertions, and / or deletions) at one or more (e.g., several) positions relative to the "wild-type" or "comparative" polynucleotide or polypeptide, wherein substitution refers to replacing a nucleotide or amino acid occupying a position with a different nucleotide or amino acid. Deletion refers to removing a nucleotide or amino acid occupying a position. Insertion refers to adding a nucleotide or amino acid adjacent to and immediately following the nucleotide or amino acid occupying the position. Exemplarily, a "mutant" in this disclosure is a polypeptide having enhanced nanoporin activity. The term "amino acid mutation" or "nucleotide mutation" includes "substitution, duplication, deletion, or addition of one or more amino acids or nucleotides." In this disclosure, the term "mutation" refers to a change in a nucleotide sequence or amino acid sequence. In one specific embodiment, the term "mutation" refers to "substitution." A "mutation" can also include the addition, deletion, or substitution of amino acids at one or more positions corresponding to the sequence shown in SEQ ID NO:1 that do not affect the activity of the nanoporin. It is well known that changing a few amino acid residues in certain regions of a polypeptide, such as non-critical regions, does not substantially alter its biological activity; for example, appropriately replacing, adding, or deleting certain amino acids results in sequences that do not affect their activity.

[0065] As used herein, the terms “corresponding” and “corresponding” have the meaning commonly understood by one of ordinary skill in the art. Specifically, “corresponding” and “corresponding” mean a position in one sequence that corresponds to a specified position in another sequence after homology or sequence identity alignment. The term “conservative substitution” involves replacing an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art and include those with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), nonpolar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), β-branched chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). A “conservative substitution” typically involves exchanging an amino acid at one or more sites in a protein. This substitution can be conserved. As examples of substitutions considered conservative, exemplified examples include substitutions from Ala to Ser or Thr, Arg to Gln, His, or Lys, Asn to Glu, Gln, Lys, His, or Asp, Asp to Asn, Glu, or Gln, Cys to Ser or Ala, Gln to Asn, Glu, Lys, His, Asp, or Arg, Glu to Gly, Asn, Gln, Lys, or Asp, Gly to Pro, and His to Asn, Lys, Gln, Arg, or Tyr. Substitutions include substitutions such as Ile to Leu, Met, Val, or Phe; Leu to Ile, Met, Val, or Phe; Lys to Asn, Glu, Gln, His, or Arg; Met to Ile, Leu, Val, or Phe; Phe to Trp, Tyr, Met, Ile, or Leu; Ser to Thr or Ala; Thr to Ser or Ala; Trp to Phe or Tyr; Tyr to His, Phe, or Trp; and Val to Met, Ile, or Leu. Furthermore, conserved mutations also include naturally occurring mutations arising from individual differences, strain differences, or species differences in gene origin. In some embodiments, the term "mutation" in this disclosure may be selected from "conserved mutation." In this disclosure, the term "conserved mutation" refers to a mutation that maintains the normal function of a protein. A representative example of a conserved mutation is a conserved substitution.

[0066] In this specification, the terms "sequence identity" or "percentage of identity" in comparisons of two nucleic acids or peptides refer to the percentage of identical sequences or identical sequences when compared and aligned using nucleotide or amino acid residue sequence comparison algorithms or by visual inspection to achieve the highest possible correspondence. In other words, the identity of a nucleotide or amino acid sequence can be defined using a ratio that represents the proportion of identical nucleotides or amino acids in the total number of nucleotides or amino acids in the aligned portion, assuming the maximum number of identical nucleotides or amino acids and omitting gaps as needed. The methods disclosed herein for determining “sequence identity” or “percentage of identity” include, but are not limited to: Computational Molecular Biology, edited by Lesk, AM, Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, edited by Smith, DW, Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, edited by Griffin, AM and Griffin, HG, Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, edited by Gribskov, M. and Devereux, J., M. Stockton Press, New York, 1991; and Carillo, H. and Lipman, D., SIAM J. Applied. Math., 48:1073 (1988). Preferred methods for determining identity aim to achieve the largest possible match between the tested sequences. Methods for determining identity are compiled into publicly available computer programs. Preferred computer program methods for determining identity between two sequences include, but are not limited to: the GCG package (Devereux, J. et al., 1984), BLASTP, BLASTN, and FASTA (Altschul, S., F. et al., 1990). The BLASTX program is publicly available from NCBI and other sources (BLAST manual, Altschul, S. et al., NCBI NLM NIHBethesda, Md. 20894; Altschul, S. et al., 1990). The well-known Smith-Waterman algorithm can also be used for identity determination.In some embodiments, the nanoporin mutant of this disclosure has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or percentage of identity among amino acid residues compared to the nanoporin containing the sequence shown in SEQ ID NO:1. In other embodiments, the polynucleotide encoding the nanoporin mutant of this disclosure has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or percentage of identity among nucleotides compared to the polynucleotide encoding the sequence shown in SEQ ID NO:1 (the sequence of said polynucleotide is the nucleotide sequence shown in SEQ ID NO:2). The determination / calculation of "sequence identity" or "percentage of identity" can be based on any suitable region of the sequence. For example, a region of at least about 50 residues, a region of at least about 100 residues, a region of at least about 200 residues, a region of at least about 400 residues, or a region of at least about 500 residues. In some embodiments, the sequence is substantially identical along the entire length of any one or two compared biopolymers (i.e., nucleic acids or polypeptides).

[0067] In this specification, the term "polynucleotide" refers to a polymer composed of nucleotides. Polynucleotides can be in the form of individual fragments or as a component of a larger nucleotide sequence structure, derived from a nucleotide sequence isolated at least once in number or concentration, and capable of being recognized, manipulated, and recovered using standard molecular biology methods (e.g., using cloning vectors). This also includes an RNA sequence (i.e., A, T, G, C) when a nucleotide sequence is represented by a DNA sequence (i.e., A, U, G, C), where "U" replaces "T". In other words, "polynucleotide" refers to a polymer of nucleotides removed from other nucleotides (individual fragments or entire fragments), or it can be a component or part of a larger nucleotide structure, such as an expression vector or a polycistronic sequence. Polynucleotides include DNA, RNA, and cDNA sequences.

[0068] The term “codon optimization” refers to the configuration of a nucleotide sequence encoding a polypeptide to contain codons preferred by the host cell or organism to improve gene expression and translation efficiency in the host cell or organism. The term “isolated” means a substance in a form or environment not naturally occurring. Non-limiting examples of isolated substances include (1) any substance not naturally occurring, (2) any substance including, but not limited to, any enzyme, mutant, nucleic acid, protein, peptide, or cofactor, which is at least partially removed from one or more naturally occurring components associated with it; (3) any substance artificially modified relative to a naturally found substance; or (4) any substance modified by increasing the amount of the substance relative to other components naturally associated with it (e.g., recombinant generation in a host cell; multiple copies of the gene encoding the substance; and the use of a promoter stronger than that naturally associated with the gene encoding the substance). Isolated substances may be present in fermentation broth samples. For example, host cells may be genetically modified to express the polypeptides disclosed herein. Fermentation broth from host cells will contain isolated polypeptides. “Recombinant polynucleotide” is a type of “polynucleotide”. The term "recombinant polynucleotide" refers to a polynucleotide having a sequence that is not linked together in nature. Recombinant polynucleotides can be contained in a suitable vector, which can then be used to transform into a suitable host cell. A host cell containing the recombinant polynucleotide is called a "recombinant host cell." The polynucleotide is then expressed in the recombinant host cell to produce, for example, a "recombinant polypeptide."

[0069] The term "expression" includes any step involved in polypeptide production, including but not limited to: transcription, post-transcriptional modification, translation, post-translational modification, and secretion. The term "expression vector" refers to a DNA construct containing a DNA sequence operatively linked to a suitable control sequence to express a target gene in a suitable host. "Recombinant expression vector" refers to a DNA structure used to express, for example, a polynucleotide encoding a desired exogenous polypeptide. Recombinant expression vectors may include, for example, a collection of genetic elements that regulate gene expression, such as promoters and enhancers; ii) a structural or coding sequence transcribed into mRNA and translated into protein; and iii) a transcriptional subunit containing appropriate transcription and translation initiation and termination sequences. Recombinant expression vectors are constructed in any suitable manner. The nature of the vector is not important, and any vector, including plasmids, viruses, bacteriophages, and transposons, can be used. Possible vectors used in this disclosure include, but are not limited to, chromosomal, non-chromosomal, and synthetic DNA sequences, such as bacterial plasmids, bacteriophage DNA, yeast plasmids, and vectors derived from combinations of plasmids and bacteriophage DNA, from viruses such as vaccinia, adenovirus, fowlpox, baculovirus, SV40, and pseudorabies. Exemplarily, the expression vectors include, but are not limited to, vectors that can be replicated and expressed in prokaryotic cells, such as the pET series, Duet series, pGEX series, pHY300, pHY300PLK, or pQlink series.

[0070] The term "recombinant gene" is a gene that is not naturally occurring. Recombinant genes are artificial. A recombinant gene comprises a protein-coding sequence operatively linked to an expression control sequence. Embodiments include, but are not limited to, introducing a foreign gene from a microorganism, an endogenous protein-coding sequence operatively linked to a heterologous promoter, and a gene having a modified protein-coding sequence. Recombinant genes are stored in the genome of a microorganism, a plasmid in a microorganism, or a bacteriophage in a microorganism. The term "operatively linked" refers to a configuration in which a regulatory sequence is positioned relative to the coding sequence of a polynucleotide, such that the regulatory sequence directs the expression of the coding sequence. Exemplarily, the regulatory sequence may be selected from sequences encoded by promoters and / or enhancers. The term "host cell" means any cell type readily transformable, transfected, transduced, etc., using a mutant polypeptide, a polynucleotide encoding a mutant polypeptide, or a recombinant expression vector containing the mutant polypeptide of this disclosure. The term "recombinant host cell" encompasses a host cell that differs from the parent cell after the introduction of a polynucleotide or recombinant expression vector encoding a mutant polypeptide, specifically achieved through transformation. The host cell disclosed herein can be a prokaryotic cell or a eukaryotic cell, as long as it is a cell capable of introducing the polypeptide or recombinant polypeptide of the present disclosure that encodes nanoporin activity.

[0071] The term "cell culture" refers to a combination of cells and a cell culture medium, wherein the cells are cultured in a cell culture medium outside of an organism. The terms "transformation, transfection, transduction" have the meaning commonly understood by those skilled in the art, referring to the process of introducing exogenous DNA into a host. The methods of transformation, transfection, and transduction include any method of introducing nucleic acids into cells, including but not limited to electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and lithium acetate-DMSO method.

[0072] The host cell culture disclosed herein can be performed according to conventional methods in the art, including but not limited to plate culture, shake flask culture, batch culture, continuous culture, and fed-batch culture, and various culture conditions such as temperature, time, and pH of the culture medium can be appropriately adjusted according to actual conditions.

[0073] In this specification, the term "highly stringent conditions" refers to pre-hybridization and hybridization at 42°C for 12 to 24 hours in 5X SSPE (saline sodium phosphate EDTA), 0.3% SDS, 200 μg / ml cleaved and denatured salmon sperm DNA, and 50% formamide, following a standard DNA blotting procedure. Finally, the vector material is washed three times at 65°C with 2X SSC and 0.2% SDS for 15 minutes each time. The term "very highly stringent conditions" refers to pre-hybridization and hybridization at 42°C for 12 to 24 hours in 5X SSPE (saline sodium phosphate EDTA), 0.3% SDS, 200 μg / ml cleaved and denatured salmon sperm DNA, and 50% formamide, following a standard DNA blotting procedure. Finally, the vector material is washed three times at 70°C with 2X SSC and 0.2% SDS for 15 minutes each time. An abbreviation for "Angstrom," a commonly used unit of length in crystallography, atomic physics, and ultrastructure analysis. Unless otherwise defined or clearly indicated by the context, all technical and scientific terms in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. The technical solutions of this disclosure are described in detail below:

[0074] This disclosure focuses on the HfaB nanoporous protein derived from Rhizobium sp. RU33AB, and describes the protein expression and purification processes through the construction of an expression vector. Figure 1 and Figure 2Using cryo-electron microscopy and other techniques, the atomic-level three-dimensional structure of HfaB nanoporous protein was resolved. HfaB protein belongs to the same family as CsgG protein. The amino acid sequence similarity between HfaB protein and the CsgG protein of *E. coli* currently used for nanopore sequencing is only 25.1%, making it a novel nanoporous protein. Nine HfaB monomers constitute a stable HfaB nanoporous protein. Figure 3 A, Figure 3 B and Figure 4 The diameter of the narrowest part (contraction zone) of the HfaB nanoporous protein pores is... ( Figure 5 ).

[0075] In a first aspect of this disclosure, a nanoporin mutant is provided. In some preferred embodiments, the nanoporin mutant corresponds to the sequence shown in SEQ ID NO:1 and has a mutated amino acid at position 79, such as S79I or S79V. In other preferred embodiments, the nanoporin mutant corresponds to the sequence shown in SEQ ID NO:1 and has a mutated amino acid at position 80, such as E80Q or E80N.

[0076] Furthermore, based on the above-mentioned nanoporin mutant, the nanoporin mutant further includes the deletion or addition of at least one amino acid residue at the N-terminus or C-terminus of the mutant of the sequence shown in (I) or (V), and / or the deletion of amino acid residues 133 to 138 in the sequence shown. In some preferred embodiments, the nanoporin mutant includes the deletion of 5 amino acid residues at the C-terminus of the mutant of the sequence shown in (I) or (V), and the deletion of amino acid residues 133 to 138 in the mutant of the sequence shown; in some more preferred embodiments, the nanoporin mutant includes the deletion of amino acid residues 133 to 138 and 288 to 292 in the mutant of the sequence shown in (I) or (V).

[0077] In some preferred embodiments, the nanoporin mutant corresponds to the amino acid sequence shown in SEQ ID NO:1, and has the following (m1) to (m2) amino acid sequences. 48 The mutations shown in any of the following are: (m1) S79N (i.e., H4C); (m2) S79W (i.e., H4D); (m3) S79I (i.e., H4D); (m4) S79A (i.e., H4F); (m5) S79V (i.e., H5C); (m6) S79L (i.e., H5D); (m7) S79Q (i.e., H5E); (m8) S79Y (i.e., H5F); (m9) S79E (i.e., H5G); (m 10 S79K (i.e., H5H); (m 11 E80Q (i.e., H4G); (m 12E80N (i.e., H4H); (m 13 E80S (i.e., H4I); (m 14 E80W (i.e., H4J); (m 15 E80A (i.e., H4K); (m 16 E80I (i.e., H4L); (m 17 Y77I, S79I (i.e., H5S); (m 18 Y77T, S79I (i.e., H5T); (m 19 Y77Q, S79I (i.e., H5U); (m 20 Y77M, S79I (i.e., H5V); (m 21 Y77D, S79I (i.e., H5W); (m 22 Y77W, S79I (i.e., H5X); (m 23 Y77V, S79I (i.e., H5Y); (m 24 Y77F, S79I (i.e., H5Z); (m 25 S79I, deletion of amino acid residues at positions 133–138 and 288–292 (i.e., H5J); (m 26 S79I, R184Q (i.e., H7M); (m 27 S79I, E218Q (i.e., H7N); (m 28 S79I, K239Q (i.e., H7O); (m 29 S79I, R184Q, E218Q, K239Q (i.e., H7R); (m 30 S79I, R184Q, K235E (i.e., H7U); (m 31 S79I, G182D, R184Q (i.e., H7V); (m 32 S79I, R184Q, F186Q (i.e., H7W); (m 33 S79I, R184Q, F220Q (i.e., H7X); (m 34 S79I, R184Q, F186Q, R225D (i.e., H8F); (m 35 S79I, R184Q, F186Q, A119Q (i.e., H8H); (m 36 S79I, R184Q, F186Q, E115Q (i.e., H8I); (m 37 S79I, R184Q, F186Q, A112Q (i.e., H8J); (m 38 S79I, R184Q, F186Q, I118Q (i.e., H8K); (m 39S79I, R184Q, F186Q, H68Y (i.e., H10M); (m 40 S79I, R184Q, F186Q, T71Q (i.e., H10N); (m 41 S79I, R184Q, F186Q, Q73E (i.e., H9O); (m 42 S79I, R184Q, F186Q, R88Q (i.e., H9P); (m 43 S79I, R184Q, F186Q, R120I (i.e., H9R); (m 44 S79I, R184Q, F186Q, E123A (i.e., H9S); (m 45 S79I, R184Q, F186Q, I125E (i.e., H9T); (m 46 The amino acid sequences S79I, R184Q, and F186Q, from position 169 to 186, are shown in SEQ ID NO: 198, and the amino acid sequences from position 216 to 241 are shown in SEQ ID NO: 199 (i.e., H10L); (m 47 S79I, R184Q, F186Q, T71Q, Q73E, R88Q (i.e., H9U); (m 48 The amino acid sequences of S79I, R184Q, F186Q, T71Q, Q73E, and R88Q, from position 169 to 186, are shown in SEQ ID NO:198, and the amino acid sequences from position 216 to 241 are shown in SEQ ID NO:199 (i.e., H9W).

[0078] Example

[0079] The embodiments of this disclosure will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of this disclosure. Where specific conditions are not specified in the examples, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0080] Example 1

[0081] 1. Construction of wild-type HfaB nanoporous protein carrier

[0082] Wild-type HfaB nanoporous protein was derived from Rhizobium sp. RU33AB (Uniprot ACCESSION: A0A1N6RVG5_9HYPH). The expression gene for this protein was synthesized artificially, and codon optimization for expression in *E. coli* was performed. Using the synthesized gene (as shown in SEQ ID NO:2) as a template, a Strep-TagII tag was added to the C-terminus of the protein. Forward and reverse primers were designed, and the target gene was amplified by PCR. The amplification product was ligated into the pQlink vector via a seamless cloning reaction to construct an expression vector containing wild-type HfaB nanoporous protein. The primer sequences are as follows:

[0083] HfaB-F:5'-GAGGAGAAATTAACTATGACCTTTGGCAAATTTCGCAGC-3' (SEQ ID NO:196),

[0084] HfaB-R:

[0085] 5'-CAGCTAATTAAGCTTTTACTTTTCGAACTGCGGGTGGCTCCAGTTGGCCGCCGCAACCAC-3' (SEQ ID NO: 197).

[0086] 2. Expression and purification of wild-type HfaB nanoporous protein

[0087] Following Strep column affinity chromatography, high-purity wild-type HfaB protein oligomers were obtained. Figure 1 Molecular sieve chromatography was used to separate HfaB protein oligomers and monomers. Figure 2 ). Take the oligomeric state (pore-forming state) HfaB protein for cryo-electron microscopy sample preparation. The specific steps are as follows: (1) Bacterial expansion culture and induced expression. Transform the expression vector containing wild-type HfaB nanoporous protein obtained above into OMP8 competent cells (OMP8 is disclosed in Coupling site-directed mutationnesis with high-level expression: large scale production of mutantporins from E. coli (2018), specifically BL21(DE3)omp8, which is incorporated herein by reference). Incubate the seed culture overnight at 37℃ and 200rpm, and take 1mL to inoculate into 1L LB medium for expansion culture. 600When the temperature is 1, cool to 26℃ and induce overnight with 0.2mM IPTG (isopropyl thiogalactoside); (2) Collect cell membranes. Collect bacterial cells at 4000rpm, resuspend each 1L of bacteria in 20ml of lysis buffer, and sonicate for 2min. Centrifuge at 18000rpm at 4℃ for 1 hour and collect cell membranes; (3) Solubilize membranes. Resuspend membrane components in a glass homogenizer with a membrane lysis buffer (resuspend each 1L of bacterial cell membrane in 15mL of membrane lysis buffer), and magnetically stir at 4℃ for 1h to fully lyse the membrane. Use detergent to fully extract membrane proteins from the cell membrane. Centrifuge at 18000rpm at 4℃ for 1 hour and collect the supernatant membrane protein components; (4) Strep column affinity chromatography. The supernatant was incubated with Strep beads (Streptactin Beads 4FF, brand: Tiandi Renhe, catalog number: SA053250) at 4℃ for 45 min. The mixture of supernatant and beads was then introduced into the column and flow-through was performed twice by gravity. 10 column volumes of wash buffer were used to remove non-specifically bound contaminating proteins. Finally, 5 column volumes of elution buffer were used to elute the target protein. (5) SDS-PAGE gel analysis was performed to detect protein expression and purification. Two 15 μL wild-type HfaB protein samples were each added to 5 μL of protein loading buffer. One sample was placed at room temperature (25℃) for 10 min and then loaded directly. The other sample was heated in a metal bath at 100℃ for 10 min, centrifuged, and then loaded. Figure 1 As shown, after Strep column affinity chromatography, relatively uniform and stable wild-type HfaB protein oligomers (porous state) were obtained, which dissociated into monomers upon heating. (6) Molecular sieve chromatography. A GE Superose6 Increase™10 / 300GL (model: 29-0915-96) gel filtration chromatography column was equilibrated with molecular sieve buffer. Protein samples were aspirated into the loading loop using a syringe, and the corresponding oligomeric proteins were collected by referring to the molecular sieve result diagram.

[0088] Lysis buffer: 20mM Tris-HCl pH 8.0, 150mM NaCl

[0089] Film-dissolving buffer: 20mM Tris-HCl pH 8.0, 150mM NaCl, 1% LDAO.

[0090] Wash buffer: 20mM Tris-HCl pH 8.0, 150mM NaCl, 0.3% LDAO,

[0091] Elution buffer: 20mM Tris-HCl pH 8.0, 150mM NaCl, 0.1% LDAO, 2.5mM desulfurized biotin.

[0092] Molecular sieve buffer: 20mM Tris-HCl pH 8.0, 150mM NaCl, 0.06% LDAO.

[0093] 3. Atomic-level structural analysis of wild-type HfaB nanoporous protein

[0094] After obtaining wild-type HfaB nanoporous protein with high purity and homogeneity, the inventors conducted sample observation and screening using cryo-electron microscopy, collected and processed data, and constructed atomic models to resolve the atomic-level structure of the wild-type HfaB nanoporous protein. The specific steps are as follows: (1) Cryo-electron microscopy sample preparation. Prepare liquid ethane, set the temperature and humidity parameters of the EMGP instrument, treat the grid with hydrophilicity, add 3 μL of protein sample to the grid, and prepare the cryo-electron microscopy sample using the EMGP instrument. (2) Cryo-electron microscopy sample observation and screening. Load the prepared cryo-electron microscopy sample onto a Talos F200C to observe the cryo-electron microscopy sample, and screen samples with good contrast, good dispersion, and little contamination for data collection. (3) Cryo-electron microscopy sample data collection and processing. After screening and obtaining suitable cryo-electron samples for data collection, use a Titan 1 electron microscope to complete the cryo-electron microscopy data collection. After selecting a suitable sample, save the position to be photographed, and adjust the electron microscope state, mainly including the electron microscope alignment, background subtraction, and basic data collection parameters (underfocus -1.2 μm to -2.0 μm, electronic metrology). 32 frames per second and pixel size After setting up the data collection system, data collection began. Data processing was performed using cryoSPARC, involving particle sorting, 2D classification, and 3D classification, ultimately yielding the desired results. Electron density map ( Figure 3 (4) Construction of the atomic model. Using the predicted structure, the model was adjusted and optimized using Coot, and then refined using Real-space refinement in Phenix software to finally obtain the atomic model of the wild-type HfaB nanoporous protein. Figure 4 ).

[0095] 4. Detection of sequencing current of wild-type HfaB nanoporous proteins

[0096] After obtaining wild-type HfaB nanoporous protein, the inventors first tested its DNA sequencing properties.

[0097] To detect the sequencing properties of wild-type HfaB nanoporin, an artificial membrane and a single nanoporin system were constructed to test the current flowing through the nanoporin. Utilizing the self-assembly of block copolymers / phospholipids into a bilayer, the block copolymers / phospholipids were assembled into a lipid bilayer by passing the oil and liquid phases through the surface of a microwell support array twice. The bilayer membrane was then stably stored in a buffer solution (200 mM KCl, 100 mM K3[Fe(CN)6], 150 mM K4[Fe(CN)6], 25 mM PBS, pH 8.0). After the single nanoporin assembled into the bilayer membrane, 2 mL of the above buffer solution was used to remove excess nanopores. The pore current signal of the wild-type HfaB nanoporin was recorded at 150 mV. Figure 11 Wild-type HfaB nanoporin exhibits relatively stable pore currents, making it suitable for characterizing DNA sequencing properties. Before detecting sequencing properties, T4-Dda (T4-Dda is a helicase, specifically T4 Dda-E94C / C109A / C136A / A360C as described in patent US20170283470A1) was added to the 5' end of the dsDNA construct (final concentration 0.1 nM) using the method described in patent US20170283470A1. Then, TMAD (100 μM) was added to the premix, and the mixture was incubated for 5 minutes. Finally, MgCl2 (final concentration 30 mM), ATP (final concentration 30 mM), KCl (final concentration 500 mM), and potassium phosphate buffer (final concentration 25 mM) were added to the premix to obtain the sequencing buffer. During the detection process, 200 μL of anchoring buffer (10 nM DNA tether, 500 mM KCl, 30 mM MgCl2, 30 mM ATP, 25 mM PBS, pH 8.0) was added to the system and incubated for 5 minutes. Then, 100 μL of sequencing buffer (0.1 nM DNA sample, 500 mM KCl, 30 mM MgCl2, 30 mM ATP, 25 mM PBS, pH 8.0) was added, and the system was run at 150 mV for 2 hours to obtain resolvable DNA perforation signals. Figure 11 ).

[0098] like Figure 11 As shown, wild-type HfaB nanoporin exhibits uniform electrophysiological properties, but suffers from high noise and unstable current states in the pore current. Furthermore, the sequencing signal amplitude is small when single-stranded DNA translocates through the pore protein's contraction region, resulting in a low signal-to-noise ratio. Frequent spontaneous blockage during sequencing further reduces capture efficiency. In conclusion, while wild-type HfaB nanoporin possesses base recognition capabilities and can be used for DNA sequencing, its overall sequencing properties require further improvement and optimization.

[0099] Example 2

[0100] First, we tried to mutate the negatively charged glutamic acid at position 80, and mutated it into six uncharged amino acids Q, N, S, W, A and I, which cover side chains with different properties. These were named H4G, H4H, H4I, H4J, H4K and H4L mutants, respectively.

[0101] The expression, purification (note: molecular sieve chromatography step omitted), and sequencing property detection methods for H4G, H4H, H4I, H4J, H4K, and H4L mutants are the same as those for wild-type HfaB nanoporous protein in Example 1. The construction method of the mutant protein vector is as follows:

[0102] A mutant vector was constructed using single-point mutagenesis PCR. Using an expression vector containing wild-type HfaB nanoporous protein as a template, primers were designed for PCR amplification to obtain PCR products. The PCR products were digested using DpnI enzyme (NEB, catalog number: R0176L), and then transformed into DH5α competent cells for positive clone selection. Single colonies were picked for sequencing, and plasmids extracted using the kit were stored at -20℃ for later use. The primer sequences used for PCR are shown in Table 1 below.

[0103] Table 1

[0104] Primer name Sequence (5'to3') SEQ ID NOs: H4G-F CAGGAAGCGGGCAACTATCTGCC SEQ ID NO:101 H4G-R GTTGCCCGCTTCCTGGCTGCTATAGCGGCCGGTC SEQ ID NO:102 H4H-F AACGAAGCGGGCAACTATCTGCCG SEQ ID NO:103 H4H-R GTTGCCCGCTTCGTTGCTGCTATAGCGGCCGGTC SEQ ID NO:104 H4I-F AGCGAAGCGGGCAACTATCTGCC SEQ ID NO:105 H4I-R GTTGCCCGCTTCGCTGCTGCTATAGCGGCCGGTC SEQ ID NO:106 H4J-F TGGGAAGCGGGCAACTATCTGCCG SEQ ID NO:107 H4J-R GTTGCCCGCTTCCCAGCTGCTATAGCGGCCGGTC SEQ ID NO:108 H4K-F GCGGAAGCGGGCAACTATCTGCCG SEQ ID NO:109 H4K-R GTTGCCCGCTTCCGCGCTGCTATAGCGGCCGGTC SEQ ID NO:110 H4L-F ATTGAAGCGGGCAACTATCTGCC SEQ ID NO:111 H4L-R GTTGCCCGCTTCAATGCTGCTATAGCGGCCGGTC SEQ ID NO:112

[0105] Expression and purification revealed that the expression levels, stability, and oligomeric state of the six mutant proteins were essentially consistent with those of the wild type, and the mutation of Glu80 did not cause significant changes in the biochemical properties of the protein. Figure 6 The sequencing properties of mutant DNA showed that the DNA pore signals of H4G, H4H, H4I, H4J, H4K, and H4L mutants were similar to those of wild-type mutants in terms of sequencing accuracy (signal amplitude, number of steps, etc.). However, the pore current still exhibited some instability and spontaneous blockage issues. Nevertheless, some mutants showed significantly reduced pore current noise and enhanced sequencing stability. Figure 12 Among them, the H4G and H4H mutants showed better results.

[0106] Example 3

[0107] The inventors made various mutations to serine at position 79, covering a variety of amino acids with different properties, including N, W, I, A, V, L, Q, Y, E, and K, which were named H4C, H4D, H4E, H4F, H5C, H5D, H5E, H5F, H5G, and H5H mutants, respectively.

[0108] The expression, purification (note: molecular sieve chromatography step omitted), and sequencing property detection methods for H4C, H4D, H4E, H4F, H5C, H5D, H5E, H5F, H5G, and H5H mutants are the same as those for wild-type HfaB nanoporous protein in Example 1. The construction method of the mutant protein vector is as follows:

[0109] A mutant vector was constructed using single-point mutagenesis PCR. Using an expression vector containing wild-type HfaB nanoporous protein as a template, primers were designed for PCR amplification to obtain PCR products. The PCR products were digested with DpnI enzyme, and then transformed into DH5α competent cells for positive clone selection. Single colonies were picked for sequencing, and plasmids extracted from the kit were stored at -20℃ for later use. The primer sequences used for PCR are shown in Table 2 below.

[0110] Table 2

[0111]

[0112]

[0113] Expression and purification revealed that the expression levels, stability, and oligomeric state of the ten mutant proteins were essentially consistent with those of the wild type, and the Ser79 mutation did not cause significant changes in the biochemical properties of the proteins. Figure 7 DNA sequencing results showed that the sequencing properties of H4E and H5C mutant DNA were significantly improved, with the H4E mutant showing particularly significant improvement. It resolved the issue of unstable current, stabilizing the pore current at 0.25-0.3 nA. Simultaneously, the sequencing accuracy of both H4E and H5C was significantly improved, with a marked increase in both sequencing signal amplitude and step number. However, the problem of spontaneous blockage still exists. Figure 13 Structural analysis revealed () Figure 8 The diameter of the shrinkage region in the H4E mutant is larger than that of the wild-type HfaB. Shrink to It is speculated that reducing the diameter of the shrinkage region within a certain range is the main factor improving the sequencing properties of the H4E mutant, especially its sequencing accuracy.

[0114] Example 4

[0115] Based on the H4E mutant (where the serine residue at position 79 of the wild-type HfaB nanoporin is mutated to isoleucine, S79I), a further accumulating mutation at position 77 was attempted to improve the properties of DNA sequencing current. The accumulating mutations covered a variety of amino acids with different properties, resulting in the following amino acids: I, T, Q, M, D, W, V, and F, named H5S, H5T, H5U, H5V, H5W, H5X, H5Y, and H5Z mutants, respectively.

[0116] The expression, purification (note: molecular sieve chromatography step omitted), and sequencing property detection methods for H5S, H5T, H5U, H5V, H5W, H5X, H5Y, and H5Z mutants are the same as those for wild-type HfaB nanoporous protein in Example 1. The construction method of the mutant protein vector is as follows:

[0117] A mutant vector was constructed using single-point mutation PCR. Using the H4E mutant vector as a template, primers were designed for PCR amplification to obtain PCR products. The PCR products were digested with DpnI enzyme, and the digested products were transformed into DH5α competent cells for positive clone selection. Single colonies were picked for sequencing, and plasmids extracted using the kit were stored at -20℃ for later use. The primer sequences used for PCR are shown in Table 3 below.

[0118] Table 3

[0119] Primer name Sequence (5'to3') SEQ ID NOs: H5S-F ATTAGCATTGAAGAAGCGGGCAAC SEQ ID NO:133 H5S-R TTCTTCAATGCTAATGCGGCCGGTCTGATCGGTAATC SEQ ID NO:134 H5T-F ACCAGCATTGAAGAAGCGGGCAAC SEQ ID NO:135 H5T-R TTCTTCAATGCTGGTGCGGCCGGTCTGATCGGTAATC SEQ ID NO:136 H5U-F CAGAGCATTGAAGAAGCGGGCAAC SEQ ID NO:137 H5U-R TTCTTCAATGCTCTGGCGGCCGGTCTGATCGGTAATC SEQ ID NO:138 H5V-F ATGAGCATTGAAGAAGCGGGCAAC SEQ ID NO:139 H5V-R TTCTTCAATGCTCATGCGGCCGGTCTGATCGGTAATC SEQ ID NO:140 H5W-F GATAGCATTGAAGAAGCGGGC SEQ ID NO:141 H5W-R TTCTTCAATGCTATCGCGGCCGGTCTGATCGGTAATC SEQ ID NO:142 H5X-F GGAGCATTGAAGAAGCGGGCAAC SEQ ID NO:143 H5X-R CTTCTTCAATGCTCCAGCGGCCGGTCTGATCGGTAATC SEQ ID NO:144 H5Y-F GTTAGCATTGAAGAAGCGGGCAAC SEQ ID NO:145 H5Y-R TTCTTCAATGCTAACGCGGCCGGTCTGATCGGTAATC SEQ ID NO:146 H5Z-F TTAGCATTGAAGAAGCGGGCAAC SEQ ID NO:147 H5Z-R CTTCTTCAATGCTAAAGCGGCCGGTCTGATCGGTAATC SEQ ID NO:148

[0120] Expression and purification revealed that the H5W and H5Z mutants exhibited unstable oligomeric states, with monomers predominating on SDS-PAGE gel images. The expression levels, stability, and oligomeric states of the other six mutant proteins were largely consistent with the wild-type. Figure 9 DNA sequencing results showed that adding the mutation at position 77 did not further improve the DNA sequencing properties of the S79I mutant; instead, it made the pore current extremely unstable and noisy, making it difficult to distinguish the DNA pore signal. Figure 14 ).

[0121] Example 5

[0122] The H4E mutant was modified to obtain the H5J mutant. The H5J mutant includes the S79I mutation, V133-S138 truncation, and V288-N292 truncation. The expression, purification (note: molecular sieve chromatography step omitted), and sequencing property detection methods for the H5J mutant were performed according to the expression, purification, and sequencing property detection methods for wild-type HfaB nanoporous protein in Example 1. The construction method of the mutant protein vector is as follows:

[0123] First, using the H4E mutant vector as a template, primers were designed for PCR amplification to construct a truncated H5I mutant vector with accumulated V133-S138. Then, using the H5I mutant vector as a template, primers were designed for PCR amplification to construct a truncated H5J mutant vector with accumulated V288-N292. The primer sequences used for PCR are shown in Table 4 below.

[0124] Table 4

[0125] Primer name Sequence (5'to3') SEQ ID NOs: H4P-F TGGAGCCACCCGCAGTTC SEQ ID NO:149 H4P-R CTGCGGGTGGCTCCACACGGTGGTTTTCCGCGCGATC SEQ ID NO:150 H4Q-F TGGAGCCACCCGCAGTTC SEQ ID NO:149 H4Q-R CTGCGGGTGGCTCCAGCGATCATCTTGGCTCGC SEQ ID NO:151

[0126] Expression and purification revealed that the H5J mutant exhibited relatively few oligomers on SDS-PAGE gel images. Figure 10 DNA sequencing results showed that ( Figure 15A and Figure 15B The H5J mutant has similar overall properties to the H4E mutant, but its sequencing properties are more stable, mainly reflected in the reduced frequency of spontaneous blocking and the reduction of abnormal noise in the through-hole signal.

[0127] Example 6

[0128] This embodiment adds mutations to R184, E218, K235, and K239 on top of the S79I mutation. The H7M mutant includes mutations at two sites: S79I and R184Q; the H7N mutant includes mutations at two sites: S79I and E218Q; the H7O mutant includes mutations at two sites: S79I and K239Q; the H7U mutant includes mutations at three sites: S79I, R184Q, and K235E; and the H7R mutant includes mutations at four sites: S79I, R184Q, E218Q, and K239Q. Furthermore, this embodiment also attempts to cumulatively mutate G182, F186, F220, and V188. The H7V mutant includes mutations at three sites: S79I, R184Q, and G182D; the H7W mutant includes mutations at three sites: S79I, R184Q, and F186Q; and the H7X mutant includes mutations at three sites: S79I, R184Q, and F220Q.

[0129] The expression, purification (note: molecular sieve chromatography step omitted), and sequencing property detection methods for H7M, H7N, H7O, H7R, H7U, H7V, H7W, and H7X mutants are the same as those for wild-type HfaB nanoporous protein in Example 1. The construction method of the mutant protein vector is as follows:

[0130] The H7M, H7N, and H7O mutants were constructed using single-point mutagenesis PCR with primers designed based on the H4E mutant vector. The H7U, H7V, H7W, and H7X mutants were constructed using single-point mutagenesis PCR with primers designed based on the H7M mutant vector. The H7R mutant was constructed in two rounds. First, using the H7M mutant vector as a template, primers were designed to construct the H7R1 mutant vector using single-point mutagenesis PCR (with H7N-F and H7N-R primers). Then, using the H7R1 mutant vector as a template, primers were designed to construct the H7R mutant vector using single-point mutagenesis PCR (with H7O-F and H7O-R primers). The primer sequences used for PCR are shown in Table 5 below.

[0131] Table 5

[0132] Primer name Sequence (5'to3') SEQ ID NOs: H7M-F AACGCTTTGCGGTGACCGC SEQ ID NO:152 H7M-R TCACCGCAAAGCGTTGCGCGCCGCCACCGCCAC SEQ ID NO:153 H7N-F CAAGTGTTTGCGAGCGTGTTTCG SEQ ID NO:154 H7N-R GCTCGCAAACACTTGGCGGCCCACCGCTTGTTTG SEQ ID NO:155 H7O-F CAAAGCCAAGAAGGCCTGCAAG SEQ ID NO:156 H7O-R GCCTTCTTGGCTTTGGGTGCCAATTTTAATATCAAACAGTTCATCGC SEQ ID NO:157 H7U-F GAAATTGGCACCAAAAGCCAAG SEQ ID NO:158 H7U-R TTTGGTGCCAATTTCAATATCAAACAGTTCATCGCTAAAAAAGCG SEQ ID NO:159 H7V-F GATGCGCAACGCTTTGCGGTG SEQ ID NO:160 H7V-R AAAGCGTTGCGCATCGCCACCGCCACCGCCCAC SEQ ID NO:161 H7W-F CAAGCGGTGACCGCGGCGGTGGATATTC SEQ ID NO:162 H7W-R CGCGGTCACCGCTTGGCGTTGCGCGCCGCCAC SEQ ID NO:163 H7X-F CAAGCGAGCGTGTTTCGCTTTTTTAG SEQ ID NO:164 H7X-R AAACACGCTCGCTTGCACTTCGCGGCCCACCGCTTG [[ID=

[0133] Expression and purification revealed that the expression levels, stability, and oligomeric state of the eight mutant proteins were basically consistent with those of the wild type, and the mutations in the barrel wall amino acids did not cause significant changes in the biochemical properties of the proteins. ​ DNA sequencing results showed that the sequencing properties of H7M, H7N, H7O, H7R, and H7W were significantly improved, mainly reflected in a reduced frequency of spontaneous blockage, a reduction in abnormal noise in the pore signal, and more stable pore current. ​ and ​ ).

[0134] ​

[0135] This embodiment adds the following mutations to the H7W mutant: R225D, A119Q, E115Q, A112Q, I118Q, H68Y, T71Q, Q73E, R88Q, R120I, E123A, and I125E, and attempts to replace the entire β bucket of the H7W mutant. The H8F mutant includes mutations at four sites: S79I, R184Q, F186Q, and R225D. The H8H mutant includes mutations at four sites: S79I, R184Q, F186Q, and A119Q. The H8I mutant includes mutations at four sites: S79I, R184Q, F186Q, and E115Q. The H8J mutant includes mutations at four sites: S79I, R184Q, F186Q, and A112Q. The H8K mutant includes mutations at four sites: S79I, R184Q, F186Q, and I118Q. The H10M mutant includes mutations at four sites: S79I, R184Q, F186Q, and H68Y. The H10N mutant includes mutations at four sites: S79I, R184Q, F186Q, and T71Q; the H9O mutant includes mutations at four sites: S79I, R184Q, F186Q, and Q73E; the H9P mutant includes mutations at four sites: S79I, R184Q, F186Q, and R88Q; the H9R mutant includes mutations at four sites: S79I, R184Q, F186Q, and R120I; the H9S mutant includes mutations at four sites: S79I, R184Q, F186Q, and E123A; and the H9T mutant includes mutations at four sites: S79I, R184Q, F186Q, and I125E. H10L involves replacing the entire β-barrel of the H7W mutant with amino acids, specifically replacing 18 amino acids from S169 to F186 with SGGVGARYFGIGADTQYQ (SEQ ID NO:198) and 26 amino acids from G216 to Q241 with SYEVQAGVFRFIDYQRLLEGEVGYTSN (SEQ ID NO:199). The H9U mutant includes mutations at six sites: S79I, R184Q, F186Q, T71Q, Q73E, and R88Q. The H9W mutant includes mutations at six sites: S79I, R184Q, F186Q, T71Q, Q73E, and R88Q, as well as the aforementioned replacement of the entire β-barrel.

[0136] The construction methods for mutant protein vectors are as follows: H8F, H8H, H8I, H8J, H8K, H10M, H10N, H9O, H9P, H9R, H9S, and H9T mutants were constructed using the H7W mutant vector as a template, with primers designed for each, and single-point mutagenesis PCR was used. The H10L mutant was constructed in two rounds. First, using the H7W mutant vector as a template, primers (H10K-F and H10K-R) were designed, and the H10K mutant vector was constructed by PCR. Then, using the H10K mutant vector as a template, primers (H10L-F and H10L-R) were designed, and the H10L mutant vector was constructed by PCR. H9U and H9W mutant vectors were constructed using the same pair of primers (H9U-F and H9U-R) as templates, respectively. The primer sequences used for PCR are shown in Table 6 below.

[0137] Table 6

[0138]

[0139]

[0140] The expression and purification of H8F, H8H, H8I, H8J, H8K, H10M, H10N, H9O, H9P, H9R, H9S, H9T, H10L, H9U, and H9W mutants (note: molecular sieve chromatography step omitted) and sequencing property detection methods are the same as those for wild-type HfaB nanoporous protein in Example 1. Expression and purification revealed that the expression levels, stability, and oligomeric state of H8H, H8I, H8J, H8K, H10M, H10N, H9O, H9P, H9R, H9S, H9T, and H9U mutant proteins were basically consistent with the wild type. The expression levels of H8F, H10L, and H9W mutant proteins were significantly increased, while their stability and oligomeric state were basically consistent with the wild type. ​ DNA sequencing results showed that H10N sequencing properties were significantly improved, mainly reflected in a significant reduction in spontaneous blockage and more stable pore current. ​ ).

[0141] Wild-type HfaB

[0142] >Amino acid sequence (SEQ ID NO:1) Rhizobium sp.RU33AB

[0143] MTFGKFRSLLLAPVLLLAACQSQTAEQAPKEPFLGPVPIATRTPVDAALECLSKTPEVQRYPRMFAVHVITDQTGRYSSEEAGNYLPRDSAGMMVSLLQKAGVKQVNRSNTAVSEWEIARAREQILGDGGNVVVGDESLPFRPLVKGALRGSDYVIDGVITQLDFNTYSGGIEATVGGGGGGARRFAVTAAVDIRVTDTKSTRIVRAKSYSKQAVGREVFASVFRFFSDELFDIKIGTKSQEGLQAAVRWTLADATYDLVKELTGHKGACDVYLPKASQDDRAETTVVAAAN

[0144] >DNA sequence (SEQ ID NO:2)

[0145] ATGACCTTTGGCAAATTTCGCAGCCTGTTACTGGCGCCGGTGCTGTTACTGGCCGCGTGTCAGAGTCAGACCGCGGAACAAGCGCCGAAAGAACCGTTTCTGGGCCCGGTGCCGATTGCGACCCGCACCCCGGTGGATGCGGCGCTGGAATGCCTGAGCAAAACCCCGGAAGTGCAGCGCTATCCGCGCATGTTTGCGGTGCATGTGATTACCGATCAGACCGGCCGCTATAGCAGCGAAGAAGCGGGCAACTATCTGCCGCGCGATAGCGCGGGCATGATGGTGAGCCTGCTGCAGAAAGCGGGCGTGAAACAAGTGAACCGCAGCAACACCGCGGTGAGCGAATGGGAAATTGCGCGCGCGCGCGAACAGATTCTGGGCGATGGCGGCAACGTGGTTGTGGGCGATGAAAGCCTGCCGTTTCGCCCGCTGGTGAAAGGCGCGCTGCGCGGCAGCGATTATGTGATTGATGGCGTGATTACGCAGCTGGATTTTAACACGTACAGCGGCGGTATCGAAGCGACGGTGGGCGGTGGCGGTGGCGGCGCGCGCCGCTTTGCGGTGACCGCGGCGGTGGATATTCGCGTGACCGATACCAAAAGCACCCGCATTGTGCGCGCGAAAAGCTATAGCAAACAAGCGGTGGGCCGCGAAGTGTTTGCGAGCGTGTTTCGCTTTTTTAGCGATGAACTGTTTGATATTAAAATTGGCACCAAAAGCCAAGAAGGCCTGCAAGCGGCGGTGCGCTGGACCCTGGCGGATGCGACCTATGATCTGGTGAAAGAACTGACCGGCCATAAAGGCGCGTGCGATGTGTATCTGCCGAAAGCGAGCCAAGATGATCGCGCGGAAACCACCGTGGTTGCGGCGGCCAACH4G

[0146] >Amino acid sequence (SEQ ID NO:3)

[0147] MTFGKFRSLLLAPVLLLAACQSQTAEQAPKEPFLGPVPIATRTPVDAALECLSKTPEVQRYPRMFAVHVITDQTGRYSSQEAGNYLPRDSAGMMVSLLQKAGVKQVNRSNTAVSEWEIARAREQILGDGGNVVVGDESLPFRPLVKGALRGSDYVIDGVITQLDFNTYSGGIEATVGGGGGGARRFAVTAAVDIRVTDTKSTRIVRAKSYSKQAVGREVFASVFRFFSDELFDIKIGTKSQEGLQAAVRWTLADATYDLVKELTGHKGACDVYLPKASQDDRAETTVVAAAN

[0148] >DNA sequence (SEQ ID NO:4)

[0149] ATGACCTTTGGCAAATTTCGCAGCCTGTTACTGGCGCCGGTGCTGTTACTGGCCGCGTGTCAGAGTCAGACCGCGGAACAAGCGCCGAAAGAACCGTTTCTGGGCCCGGTGCCGATTGCGACCCGCACCCCGGTGGATGCGGCGCTGGAATGCCTGAGCAAAACCCCGGAAGTGCAGCGCTATCCGCGCATGTTTGCGGTGCATGTGATTACCGATCAGACCGGCCGCTATAGCAGCCAGGAAGCGGGCAACTATCTGCCGCGCGATAGCGCGGGCATGATGGTGAGCCTGCTGCAGAAAGCGGGCGTGAAACAAGTGAACCGCAGCAACACCGCGGTGAGCGAATGGGAAATTGCGCGCGCGCGCGAACAGATTCTGGGCGATGGCGGCAACGTGGTTGTGGGCGATGAAAGCCTGCCGTTTCGCCCGCTGGTGAAAGGCGCGCTGCGCGGCAGCGATTATGTGATTGATGGCGTGATTACGCAGCTGGATTTTAACACGTACAGCGGCGGTATCGAAGCGACGGTGGGCGGTGGCGGTGGCGGCGCGCGCCGCTTTGCGGTGACCGCGGCGGTGGATATTCGCGTGACCGATACCAAAAGCACCCGCATTGTGCGCGCGAAAAGCTATAGCAAACAAGCGGTGGGCCGCGAAGTGTTTGCGAGCGTGTTTCGCTTTTTTAGCGATGAACTGTTTGATATTAAAATTGGCACCAAAAGCCAAGAAGGCCTGCAAGCGGCGGTGCGCTGGACCCTGGCGGATGCGACCTATGATCTGGTGAAAGAACTGACCGGCCATAAAGGCGCGTGCGATGTGTATCTGCCGAAAGCGAGCCAAGATGATCGCGCGGAAACCACCGTGGTTGCGGCGGCCAACH4H

[0150] >Amino acid sequence (SEQ ID NO:5)

[0151] MTFGKFRSLLLAPVLLLAACQSQTAEQAPKEPFLGPVPIATRTPVDAALECLSKTPEVQRYPRMFAVHVITDQTGRYSSNEAGNYLPRDSAGMMVSLLQKAGVKQVNRSNTAVSEWEIARAREQILGDGGNVVVGDESLPFRPLVKGALRGSDYVIDGVITQLDFNTYSGGIEATVGGGGGGARRFAVTAAVDIRVTDTKSTRIVRAKSYSKQAVGREVFASVFRFFSDELFDIKIGTKSQEGLQAAVRWTLADATYDLVKELTGHKGACDVYLPKASQDDRAETTVVAAAN

[0152] >DNA sequence (SEQ ID NO:6)

[0153] ATGACCTTTGGCAAATTTCGCAGCCTGTTACTGGCGCCGGTGCTGTTACTGGCCGCGTGTCAGAGTCAGACCGCGGAACAAGCGCCGAAAGAACCGTTTCTGGGCCCGGTGCCGATTGCGACCCGCACCCCGGTGGATGCGGCGCTGGAATGCCTGAGCAAAACCCCGGAAGTGCAGCGCTATCCGCGCATGTTTGCGGTGCATGTGATTACCGATCAGACCGGCCGCTATAGCAGCAACGAAGCGGGCAACTATCTGCCGCGCGATAGCGCGGGCATGATGGTGAGCCTGCTGCAGAAAGCGGGCGTGAAACAAGTGAACCGCAGCAACACCGCGGTGAGCGAATGGGAAATTGCGCGCGCGCGCGAACAGATTCTGGGCGATGGCGGCAACGTGGTTGTGGGCGATGAAAGCCTGCCGTTTCGCCCGCTGGTGAAAGGCGCGCTGCGCGGCAGCGATTATGTGATTGATGGCGTGATTACGCAGCTGGATTTTAACACGTACAGCGGCGGTATCGAAGCGACGGTGGGCGGTGGCGGTGGCGGCGCGCGCCGCTTTGCGGTGACCGCGGCGGTGGATATTCGCGTGACCGATACCAAAAGCACCCGCATTGTGCGCGCGAAAAGCTATAGCAAACAAGCGGTGGGCCGCGAAGTGTTTGCGAGCGTGTTTCGCTTTTTTAGCGATGAACTGTTTGATATTAAAATTGGCACCAAAAGCCAAGAAGGCCTGCAAGCGGCGGTGCGCTGGACCCTGGCGGATGCGACCTATGATCTGGTGAAAGAACTGACCGGCCATAAAGGCGCGTGCGATGTGTATCTGCCGAAAGCGAGCCAAGATGATCGCGCGGAAACCACCGTGGTTGCGGCGGCCAACH4I

[0154] >Amino acid sequence (SEQ ID NO:7)

[0155] MTFGKFRSLLLAPVLLLAACQSQTAEQAPKEPFLGPVPIATRTPVDAALECLSKTPEVQRYPRMFAVHVITDQTGRYSSSEAGNYLPRDSAGMMVSLLQKAGVKQVNRSNTAVSEWEIARAREQILGDGGNVVVGDESLPFRPLVKGALRGSDYVIDGVITQLDFNTYSGGIEATVGGGGGGARRFAVTAAVDIRVTDTKSTRIVRAKSYSKQAVGREVFASVFRFFSDELFDIKIGTKSQEGLQAAVRWTLADATYDLVKELTGHKGACDVYLPKASQDDRAETTVVAAAN

[0156] >DNA sequence (SEQ ID NO:8)

[0157] ATGACCTTTGGCAAATTTCGCAGCCTGTTACTGGCGCCGGTGCTGTTACTGGCCGCGTGTCAGAGTCAGACCGCGGAACAAGCGCCGAAAGAACCGTTTCTGGGCCCGGTGCCGATTGCGACCCGCACCCCGGTGGATGCGGCGCTGGAATGCCTGAGCAAAACCCCGGAAGTGCAGCGCTATCCGCGCATGTTTGCGGTGCATGTGATTACCGATCAGACCGGCCGCTATAGCAGCAGCGAAGCGGGCAACTATCTGCCGCGCGATAGCGCGGGCATGATGGTGAGCCTGCTGCAGAAAGCGGGCGTGAAACAAGTGAACCGCAGCAACACCGCGGTGAGCGAATGGGAAATTGCGCGCGCGCGCGAACAGATTCTGGGCGATGGCGGCAACGTGGTTGTGGGCGATGAAAGCCTGCCGTTTCGCCCGCTGGTGAAAGGCGCGCTGCGCGGCAGCGATTATGTGATTGATGGCGTGATTACGCAGCTGGATTTTAACACGTACAGCGGCGGTATCGAAGCGACGGTGGGCGGTGGCGGTGGCGGCGCGCGCCGCTTTGCGGTGACCGCGGCGGTGGATATTCGCGTGACCGATACCAAAAGCACCCGCATTGTGCGCGCGAAAAGCTATAGCAAACAAGCGGTGGGCCGCGAAGTGTTTGCGAGCGTGTTTCGCTTTTTTAGCGATGAACTGTTTGATATTAAAATTGGCACCAAA

[0158] AGCCAAGAAGGCCTGCAAGCGGCGGTGCGCTGGACCCTGGCGGATGCGACCTATGATCTGGTGAAAGAACTGACCGGCCATAAAGGCGC

[0159] GTGCGATGTGTATCTGCCGAAAGCGAGCCAAGATGATCGCGCGGAAACCACCGTGGTTGCGGCGGCCAAC

[0160] H4J

[0161] >Amino acid sequence (SEQ ID NO:9)

[0162] MTFGKFRSLLLAPVLLLAACQSQTAEQAPKEPFLGPVPIATRTPVDAALECLSKTPEVQRYPRMFAVHVITDQTGRYSSWEAGNYLPRDSAGM

[0163] MVSLLQKAGVKQVNRSNTAVSEWEIARAREQILGDGGNVVVGDESLPFRPLVKGALRGSDYVIDGVITQLDFNTYSGGIEATVGGGGGGARRF

[0164] AVTAAVDIRVTDTKSTRIVRAKSYSKQAVGREVFASVFRFFSDELFDIKIGTKSQEGLQAAVRWTLADATYDLVKELTGHKGACDVYLPKASQD

[0165] DRAETTVVAAAN

[0166] >DNA sequence (SEQ ID NO:10)

[0167] ATGACCTTTGGCAAATTTCGCAGCCTGTTACTGGCGCCGGTGCTGTTACTGGCCGCGTGTCAGAGTCAGACCGCGGAACAAGCGCCGAAA

[0168] GAACCGTTTCTGGGCCCGGTGCCGATTGCGACCCGCACCCCGGTGGATGCGGCGCTGGAATGCCTGAGCAAAACCCCGGAAGTGCAGCG

[0169] CTATCCGCGCATGTTTGCGGTGCATGTGATTACCGATCAGACCGGCCGCTATAGCAGCTGGGAAGCGGGCAACTATCTGCCGCGCGATAGC

[0170] GCGGGCATGATGGTGAGCCTGCTGCAGAAAGCGGGCGTGAAACAAGTGAACCGCAGCAACACCGCGGTGAGCGAATGGGAAATTGCGC

[0171] GCGCGCGCGAACAGATTCTGGGCGATGGCGGCAACGTGGTTGTGGGCGATGAAAGCCTGCCGTTTCGCCCGCTGGTGAAAGGCGCGCTG

[0172] CGCGGCAGCGATTATGTGATTGATGGCGTGATTACGCAGCTGGATTTTAACACGTACAGCGGCGGTATCGAAGCGACGGTGGGCGGTGGC

[0173] GGTGGCGGCGCGCGCCGCTTTGCGGTGACCGCGGCGGTGGATATTCGCGTGACCGATACCAAAAGCACCCGCATTGTGCGCGCGAAAAG

[0174] CTATAGCAAACAAGCGGTGGGCCGCGAAGTGTTTGCGAGCGTGTTTCGCTTTTTTAGCGATGAACTGTTTGATATTAAAATTGGCACCAAA

[0175] AGCCAAGAAGGCCTGCAAGCGGCGGTGCGCTGGACCCTGGCGGATGCGACCTATGATCTGGTGAAAGAACTGACCGGCCATAAAGGCGC

[0176] GTGCGATGTGTATCTGCCGAAAGCGAGCCAAGATGATCGCGCGGAAACCACCGTGGTTGCGGCGGCCAAC

[0177] H4K

[0178] > Amino acid sequence (SEQ ID NO:11)

[0179] MTFGKFRSLLLAPVLLLAACQSQTAEQAPKEPFLGPVPIATRTPVDAALECLSKTPEVQRYPRMFAVHVITDQTGRYSSAEAGNYLPRDSAGMM

[0180] VSLLQKAGVKQVNRSNTAVSEWEIARAREQILGDGGNVVVGDESLPFRPLVKGALRGSDYVIDGVITQLDFNTYSGGIEATVGGGGGGARRFA

[0181] VTAAVDIRVTDTKSTRIVRAKSYSKQAVGREVFASVFRFFSDELFDIKIGTKSQEGLQAAVRWTLADATYDLVKELTGHKGACDVYLPKASQDD

[0182] RAETTVVAAAN

[0183] >DNA sequence (SEQ ID NO:12)

[0184] ATGACCTTTGGCAAATTTCGCAGCCTGTTACTGGCGCCGGTGCTGTTACTGGCCGCGTGTCAGAGTCAGACCGCGGAACAAGCGCCGAAA

[0185] GAACCGTTTCTGGGCCCGGTGCCGATTGCGACCCGCACCCCGGTGGATGCGGCGCTGGAATGCCTGAGCAAAACCCCGGAAGTGCAGCG

[0186] CTATCCGCGCATGTTTGCGGTGCATGTGATTACCGATCAGACCGGCCGCTATAGCAGCGCGGAAGCGGGCAACTATCTGCCGCGCGATAGC

[0187] GCGGGCATGATGGTGAGCCTGCTGCAGAAAGCGGGCGTGAAACAAGTGAACCGCAGCAACACCGCGGTGAGCGAATGGGAAATTGCGC

[0188] GCGCGCGCGAACAGATTCTGGGCGATGGCGGCAACGTGGTTGTGGGCGATGAAAGCCTGCCGTTTCGCCCGCTGGTGAAAGGCGCGCTG

[0189] CGCGGCAGCGATTATGTGATTGATGGCGTGATTACGCAGCTGGATTTTAACACGTACAGCGGCGGTATCGAAGCGACGGTGGGCGGTGGC

[0190] GGTGGCGGCGCGCGCCGCTTTGCGGTGACCGCGGCGGTGGATATTCGCGTGACCGATACCAAAAGCACCCGCATTGTGCGCGCGAAAAG

[0191] CTATAGCAAACAAGCGGTGGGCCGCGAAGTGTTTGCGAGCGTGTTTCGCTTTTTTAGCGATGAACTGTTTGATATTAAAATTGGCACCAAA

[0192] AGCCAAGAAGGCCTGCAAGCGGCGGTGCGCTGGACCCTGGCGGATGCGACCTATGATCTGGTGAAAGAACTGACCGGCCATAAAGGCGC

[0193] GTGCGATGTGTATCTGCCGAAAGCGAGCCAAGATGATCGCGCGGAAACCACCGTGGTTGCGGCGGCCAAC

[0194] H4L

[0195] >Amino acid sequence (SEQ ID NO:13)

[0196] MTFGKFRSLLLAPVLLLAACQSQTAEQAPKEPFLGPVPIATRTPVDAALECLSKTPEVQRYPRMFAVHVITDQTGRYSSIEAGNYLPRDSAGMM

[0197] VSLLQKAGVKQVNRSNTAVSEWEIARAREQILGDGGNVVVGDESLPFRPLVKGALRGSDYVIDGVITQLDFNTYSGGIEATVGGGGGGARRFA

[0198] VTAAVDIRVTDTKSTRIVRAKSYSKQAVGREVFASVFRFFSDELFDIKIGTKSQEGLQAAVRWTLADATYDLVKELTGHKGACDVYLPKASQDD

[0199] RAETTVVAAAN

[0200] >DNA sequence (SEQ ID NO:14)

[0201] ATGACCTTTGGCAAATTTCGCAGCCTGTTACTGGCGCCGGTGCTGTTACTGGCCGCGTGTCAGAGTCAGACCGCGGAACAAGCGCCGAAA

[0202] GAACCGTTTCTGGGCCCGGTGCCGATTGCGACCCGCACCCCGGTGGATGCGGCGCTGGAATGCCTGAGCAAAACCCCGGAAGTGCAGCG

[0203] CTATCCGCGCATGTTTGCGGTGCATGTGATTACCGATCAGACCGGCCGCTATAGCAGCATTGAAGCGGGCAACTATCTGCCGCGCGATAGC

[0204] GCGGGCATGATGGTGAGCCTGCTGCAGAAAGCGGGCGTGAAACAAGTGAACCGCAGCAACACCGCGGTGAGCGAATGGGAAATTGCGC

[0205] GCGCGCGCGAACAGATTCTGGGCGATGGCGGCAACGTGGTTGTGGGCGATGAAAGCCTGCCGTTTCGCCCGCTGGTGAAAGGCGCGCTG

[0206] CGCGGCAGCGATTATGTGATTGATGGCGTGATTACGCAGCTGGATTTTAACACGTACAGCGGCGGTATCGAAGCGACGGTGGGCGGTGGC

[0207] GGTGGCGGCGCGCGCCGCTTTGCGGTGACCGCGGCGGTGGATATTCGCGTGACCGATACCAAAAGCACCCGCATTGTGCGCGCGAAAAG

[0208] CTATAGCAAACAAGCGGTGGGCCGCGAAGTGTTTGCGAGCGTGTTTCGCTTTTTTAGCGATGAACTGTTTGATATTAAAATTGGCACCAAA

[0209] AGCCAAGAAGGCCTGCAAGCGGCGGTGCGCTGGACCCTGGCGGATGCGACCTATGATCTGGTGAAAGAACTGACCGGCCATAAAGGCGC

[0210] GTGCGATGTGTATCTGCCGAAAGCGAGCCAAGATGATCGCGCGGAAACCACCGTGGTTGCGGCGGCCAAC

[0211] H4C

[0212] >Amino acid sequence (SEQ ID NO:15)

[0213] MTFGKFRSLLLAPVLLLAACQSQTAEQAPKEPFLGPVPIATRTPVDAALECLSKTPEVQRYPRMFAVHVITDQTGRYSNEEAGNYLPRDSAGMM

[0214] VSLLQKAGVKQVNRSNTAVSEWEIARAREQILGDGGNVVVGDESLPFRPLVKGALRGSDYVIDGVITQLDFNTYSGGIEATVGGGGGGARRFA

[0215] VTAAVDIRVTDTKSTRIVRAKSYSKQAVGREVFASVFRFFSDELFDIKIGTKSQEGLQAAVRWTLADATYDLVKELTGHKGACDVYLPKASQDD

[0216] RAETTVVAAAN

[0217] >DNA sequence (SEQ ID NO:16)

[0218] ATGACCTTTGGCAAATTTCGCAGCCTGTTACTGGCGCCGGTGCTGTTACTGGCCGCGTGTCAGAGTCAGACCGCGGAACAAGCGCCGAAA

[0219] GAACCGTTTCTGGGCCCGGTGCCGATTGCGACCCGCACCCCGGTGGATGCGGCGCTGGAATGCCTGAGCAAAACCCCGGAAGTGCAGCG

[0220] CTATCCGCGCATGTTTGCGGTGCATGTGATTACCGATCAGACCGGCCGCTATAGCAACGAAGAAGCGGGCAACTATCTGCCGCGCGATAGC

[0221] GCGGGCATGATGGTGAGCCTGCTGCAGAAAGCGGGCGTGAAACAAGTGAACCGCAGCAACACCGCGGTGAGCGAATGGGAAATTGCGC

[0222] GCGCGCGCGAACAGATTCTGGGCGATGGCGGCAACGTGGTTGTGGGCGATGAAAGCCTGCCGTTTCGCCCGCTGGTGAAAGGCGCGCTG

[0223] CGCGGCAGCGATTATGTGATTGATGGCGTGATTACGCAGCTGGATTTTAACACGTACAGCGGCGGTATCGAAGCGACGGTGGGCGGTGGC

[0224] GGTGGCGGCGCGCGCCGCTTTGCGGTGACCGCGGCGGTGGATATTCGCGTGACCGATACCAAAAGCACCCGCATTGTGCGCGCGAAAAG

[0225] CTATAGCAAACAAGCGGTGGGCCGCGAAGTGTTTGCGAGCGTGTTTCGCTTTTTTAGCGATGAACTGTTTGATATTAAAATTGGCACCAAA

[0226] AGCCAAGAAGGCCTGCAAGCGGCGGTGCGCTGGACCCTGGCGGATGCGACCTATGATCTGGTGAAAGAACTGACCGGCCATAAAGGCGC

[0227] GTGCGATGTGTATCTGCCGAAAGCGAGCCAAGATGATCGCGCGGAAACCACCGTGGTTGCGGCGGCCAAC

[0228] H4D

[0229] > Amino acid sequence (SEQ ID NO:17)

[0230] MTFGKFRSLLLAPVLLLAACQSQTAEQAPKEPFLGPVPIATRTPVDAALECLSKTPEVQRYPRMFAVHVITDQTGRYSWEEAGNYLPRDSAGM

[0231] MVSLLQKAGVKQVNRSNTAVSEWEIARAREQILGDGGNVVVGDESLPFRPLVKGALRGSDYVIDGVITQLDFNTYSGGIEATVGGGGGGARRF

[0232] AVTAAVDIRVTDTKSTRIVRAKSYSKQAVGREVFASVFRFFSDELFDIKIGTKSQEGLQAAVRWTLADATYDLVKELTGHKGACDVYLPKASQD

[0233] DRAETTVVAAAN

[0234] >DNA sequence (SEQ ID NO:18)

[0235] ATGACCTTTGGCAAATTTCGCAGCCTGTTACTGGCGCCGGTGCTGTTACTGGCCGCGTGTCAGAGTCAGACCGCGGAACAAGCGCCGAAA

[0236] GAACCGTTTCTGGGCCCGGTGCCGATTGCGACCCGCACCCCGGTGGATGCGGCGCTGGAATGCCTGAGCAAAACCCCGGAAGTGCAGCG

[0237] CTATCCGCGCATGTTTGCGGTGCATGTGATTACCGATCAGACCGGCCGCTATAGCTGGGAAGAAGCGGGCAACTATCTGCCGCGCGATAGC

[0238] GCGGGCATGATGGTGAGCCTGCTGCAGAAAGCGGGCGTGAAACAAGTGAACCGCAGCAACACCGCGGTGAGCGAATGGGAAATTGCGC

[0239] GCGCGCGCGAACAGATTCTGGGCGATGGCGGCAACGTGGTTGTGGGCGATGAAAGCCTGCCGTTTCGCCCGCTGGTGAAAGGCGCGCTG

[0240] CGCGGCAGCGATTATGTGATTGATGGCGTGATTACGCAGCTGGATTTTAACACGTACAGCGGCGGTATCGAAGCGACGGTGGGCGGTGGC

[0241] GGTGGCGGCGCGCGCCGCTTTGCGGTGACCGCGGCGGTGGATATTCGCGTGACCGATACCAAAAGCACCCGCATTGTGCGCGCGAAAAG

[0242] CTATAGCAAACAAGCGGTGGGCCGCGAAGTGTTTGCGAGCGTGTTTCGCTTTTTTAGCGATGAACTGTTTGATATTAAAATTGGCACCAAA

[0243] AGCCAAGAAGGCCTGCAAGCGGCGGTGCGCTGGACCCTGGCGGATGCGACCTATGATCTGGTGAAAGAACTGACCGGCCATAAAGGCGC

[0244] GTGCGATGTGTATCTGCCGAAAGCGAGCCAAGATGATCGCGCGGAAACCACCGTGGTTGCGGCGGCCAAC

[0245] H4E

[0246] >Amino acid sequence (SEQ ID NO:19)

[0247] MTFGKFRSLLLAPVLLLAACQSQTAEQAPKEPFLGPVPIATRTPVDAALECLSKTPEVQRYPRMFAVHVITDQTGRYSIEEAGNYLPRDSAGMM

[0248] VSLLQKAGVKQVNRSNTAVSEWEIARAREQILGDGGNVVVGDESLPFRPLVKGALRGSDYVIDGVITQLDFNTYSGGIEATVGGGGGGARRFA

[0249] VTAAVDIRVTDTKSTRIVRAKSYSKQAVGREVFASVFRFFSDELFDIKIGTKSQEGLQAAVRWTLADATYDLVKELTGHKGACDVYLPKASQDD

[0250] RAETTVVAAAN

[0251] >DNA sequence (SEQ ID NO:20)

[0252] ATGACCTTTGGCAAATTTCGCAGCCTGTTACTGGCGCCGGTGCTGTTACTGGCCGCGTGTCAGAGTCAGACCGCGGAACAAGCGCCGAAA

[0253] GAACCGTTTCTGGGCCCGGTGCCGATTGCGACCCGCACCCCGGTGGATGCGGCGCTGGAATGCCTGAGCAAAACCCCGGAAGTGCAGCG

[0254] CTATCCGCGCATGTTTGCGGTGCATGTGATTACCGATCAGACCGGCCGCTATAGCATTGAAGAAGCGGGCAACTATCTGCCGCGCGATAGC

[0255] GCGGGCATGATGGTGAGCCTGCTGCAGAAAGCGGGCGTGAAACAAGTGAACCGCAGCAACACCGCGGTGAGCGAATGGGAAATTGCGC

[0256] GCGCGCGCGAACAGATTCTGGGCGATGGCGGCAACGTGGTTGTGGGCGATGAAAGCCTGCCGTTTCGCCCGCTGGTGAAAGGCGCGCTG

[0257] CGCGGCAGCGATTATGTGATTGATGGCGTGATTACGCAGCTGGATTTTAACACGTACAGCGGCGGTATCGAAGCGACGGTGGGCGGTGGC

[0258] GGTGGCGGCGCGCGCCGCTTTGCGGTGACCGCGGCGGTGGATATTCGCGTGACCGATACCAAAAGCACCCGCATTGTGCGCGCGAAAAG

[0259] CTATAGCAAACAAGCGGTGGGCCGCGAAGTGTTTGCGAGCGTGTTTCGCTTTTTTAGCGATGAACTGTTTGATATTAAAATTGGCACCAAA

[0260] AGCCAAGAAGGCCTGCAAGCGGCGGTGCGCTGGACCCTGGCGGATGCGACCTATGATCTGGTGAAAGAACTGACCGGCCATAAAGGCGC

[0261] GTGCGATGTGTATCTGCCGAAAGCGAGCCAAGATGATCGCGCGGAAACCACCGTGGTTGCGGCGGCCAAC

[0262] H4F

[0263] >Amino acid sequence (SEQ ID NO:21)

[0264] MTFGKFRSLLLAPVLLLAACQSQTAEQAPKEPFLGPVPIATRTPVDAALECLSKTPEVQRYPRMFAVHVITDQTGRYSAEEAGNYLPRDSAGMM

[0265] VSLLQKAGVKQVNRSNTAVSEWEIARAREQILGDGGNVVVGDESLPFRPLVKGALRGSDYVIDGVITQLDFNTYSGGIEATVGGGGGGARRFA

[0266] VTAAVDIRVTDTKSTRIVRAKSYSKQAVGREVFASVFRFFSDELFDIKIGTKSQEGLQAAVRWTLADATYDLVKELTGHKGACDVYLPKASQDD

[0267] RAETTVVAAAN

[0268] >DNA sequence (SEQ ID NO:22)

[0269] ATGACCTTTGGCAAATTTCGCAGCCTGTTACTGGCGCCGGTGCTGTTACTGGCCGCGTGTCAGAGTCAGACCGCGGAACAAGCGCCGAAA

[0270] GAACCGTTTCTGGGCCCGGTGCCGATTGCGACCCGCACCCCGGTGGATGCGGCGCTGGAATGCCTGAGCAAAACCCCGGAAGTGCAGCG

[0271] CTATCCGCGCATGTTTGCGGTGCATGTGATTACCGATCAGACCGGCCGCTATAGCGCGGAAGAAGCGGGCAACTATCTGCCGCGCGATAGC

[0272] GCGGGCATGATGGTGAGCCTGCTGCAGAAAGCGGGCGTGAAACAAGTGAACCGCAGCAACACCGCGGTGAGCGAATGGGAAATTGCGC

[0273] GCGCGCGCGAACAGATTCTGGGCGATGGCGGCAACGTGGTTGTGGGCGATGAAAGCCTGCCGTTTCGCCCGCTGGTGAAAGGCGCGCTG

[0274] CGCGGCAGCGATTATGTGATTGATGGCGTGATTACGCAGCTGGATTTTAACACGTACAGCGGCGGTATCGAAGCGACGGTGGGCGGTGGC

[0275] GGTGGCGGCGCGCGCCGCTTTGCGGTGACCGCGGCGGTGGATATTCGCGTGACCGATACCAAAAGCACCCGCATTGTGCGCGCGAAAAG

[0276] CTATAGCAAACAAGCGGTGGGCCGCGAAGTGTTTGCGAGCGTGTTTCGCTTTTTTAGCGATGAACTGTTTGATATTAAAATTGGCACCAAA

[0277] AGCCAAGAAGGCCTGCAAGCGGCGGTGCGCTGGACCCTGGCGGATGCGACCTATGATCTGGTGAAAGAACTGACCGGCCATAAAGGCGC

[0278] GTGCGATGTGTATCTGCCGAAAGCGAGCCAAGATGATCGCGCGGAAACCACCGTGGTTGCGGCGGCCAAC

[0279] H5C

[0280] >Amino acid sequence (SEQ ID NO:23)

[0281] MTFGKFRSLLLAPVLLLAACQSQTAEQAPKEPFLGPVPIATRTPVDAALECLSKTPEVQRYPRMFAVHVITDQTGRYSVEEAGNYLPRDSAGMM

[0282] VSLLQKAGVKQVNRSNTAVSEWEIARAREQILGDGGNVVVGDESLPFRPLVKGALRGSDYVIDGVITQLDFNTYSGGIEATVGGGGGGARRFA

[0283] VTAAVDIRVTDTKSTRIVRAKSYSKQAVGREVFASVFRFFSDELFDIKIGTKSQEGLQAAVRWTLADATYDLVKELTGHKGACDVYLPKASQDD

[0284] RAETTVVAAAN

[0285] >DNA sequence (SEQ ID NO:24)

[0286] ATGACCTTTGGCAAATTTCGCAGCCTGTTACTGGCGCCGGTGCTGTTACTGGCCGCGTGTCAGAGTCAGACCGCGGAACAAGCGCCGAAA

[0287] GAACCGTTTCTGGGCCCGGTGCCGATTGCGACCCGCACCCCGGTGGATGCGGCGCTGGAATGCCTGAGCAAAACCCCGGAAGTGCAGCG

[0288] CTATCCGCGCATGTTTGCGGTGCATGTGATTACCGATCAGACCGGCCGCTATAGCGTGGAAGAAGCGGGCAACTATCTGCCGCGCGATAGC

[0289] GCGGGCATGATGGTGAGCCTGCTGCAGAAAGCGGGCGTGAAACAAGTGAACCGCAGCAACACCGCGGTGAGCGAATGGGAAATTGCGC

[0290] GCGCGCGCGAACAGATTCTGGGCGATGGCGGCAACGTGGTTGTGGGCGATGAAAGCCTGCCGTTTCGCCCGCTGGTGAAAGGCGCGCTG

[0291] CGCGGCAGCGATTATGTGATTGATGGCGTGATTACGCAGCTGGATTTTAACACGTACAGCGGCGGTATCGAAGCGACGGTGGGCGGTGGC

[0292] GGTGGCGGCGCGCGCCGCTTTGCGGTGACCGCGGCGGTGGATATTCGCGTGACCGATACCAAAAGCACCCGCATTGTGCGCGCGAAAAG

[0293] CTATAGCAAACAAGCGGTGGGCCGCGAAGTGTTTGCGAGCGTGTTTCGCTTTTTTAGCGATGAACTGTTTGATATTAAAATTGGCACCAAA

[0294] AGCCAAGAAGGCCTGCAAGCGGCGGTGCGCTGGACCCTGGCGGATGCGACCTATGATCTGGTGAAAGAACTGACCGGCCATAAAGGCGC

[0295] GTGCGATGTGTATCTGCCGAAAGCGAGCCAAGATGATCGCGCGGAAACCACCGTGGTTGCGGCGGCCAAC

[0296] H5D

[0297] >Amino acid sequence (SEQ ID NO:25)

[0298] MTFGKFRSLLLAPVLLLAACQSQTAEQAPKEPFLGPVPIATRTPVDAALECLSKTPEVQRYPRMFAVHVITDQTGRYSLEEAGNYLPRDSAGMM

[0299] VSLLQKAGVKQVNRSNTAVSEWEIARAREQILGDGGNVVVGDESLPFRPLVKGALRGSDYVIDGVITQLDFNTYSGGIEATVGGGGGGARRFA

[0300] VTAAVDIRVTDTKSTRIVRAKSYSKQAVGREVFASVFRFFSDELFDIKIGTKSQEGLQAAVRWTLADATYDLVKELTGHKGACDVYLPKASQDD

[0301] RAETTVVAAAN

[0302] >DNA sequence (SEQ ID NO:26)

[0303] ATGACCTTTGGCAAATTTCGCAGCCTGTTACTGGCGCCGGTGCTGTTACTGGCCGCGTGTCAGAGTCAGACCGCGGAACAAGCGCCGAAA

[0304] GAACCGTTTCTGGGCCCGGTGCCGATTGCGACCCGCACCCCGGTGGATGCGGCGCTGGAATGCCTGAGCAAAACCCCGGAAGTGCAGCG

[0305] CTATCCGCGCATGTTTGCGGTGCATGTGATTACCGATCAGACCGGCCGCTATAGCCTGGAAGAAGCGGGCAACTATCTGCCGCGCGATAGC

[0306] GCGGGCATGATGGTGAGCCTGCTGCAGAAAGCGGGCGTGAAACAAGTGAACCGCAGCAACACCGCGGTGAGCGAATGGGAAATTGCGC

[0307] GCGCGCGCGAACAGATTCTGGGCGATGGCGGCAACGTGGTTGTGGGCGATGAAAGCCTGCCGTTTCGCCCGCTGGTGAAAGGCGCGCTG

[0308] CGCGGCAGCGATTATGTGATTGATGGCGTGATTACGCAGCTGGATTTTAACACGTACAGCGGCGGTATCGAAGCGACGGTGGGCGGTGGC

[0309] GGTGGCGGCGCGCGCCGCTTTGCGGTGACCGCGGCGGTGGATATTCGCGTGACCGATACCAAAAGCACCCGCATTGTGCGCGCGAAAAG

[0310] CTATAGCAAACAAGCGGTGGGCCGCGAAGTGTTTGCGAGCGTGTTTCGCTTTTTTAGCGATGAACTGTTTGATATTAAAATTGGCACCAAA

[0311] AGCCAAGAAGGCCTGCAAGCGGCGGTGCGCTGGACCCTGGCGGATGCGACCTATGATCTGGTGAAAGAACTGACCGGCCATAAAGGCGC

[0312] GTGCGATGTGTATCTGCCGAAAGCGAGCCAAGATGATCGCGCGGAAACCACCGTGGTTGCGGCGGCCAAC

[0313] H5E

[0314] >Amino acid sequence (SEQ ID NO:27)

[0315] MTFGKFRSLLLAPVLLLAACQSQTAEQAPKEPFLGPVPIATRTPVDAALECLSKTPEVQRYPRMFAVHVITDQTGRYSQEEAGNYLPRDSAGMM

[0316] VSLLQKAGVKQVNRSNTAVSEWEIARAREQILGDGGNVVVGDESLPFRPLVKGALRGSDYVIDGVITQLDFNTYSGGIEATVGGGGGGARRFA

[0317] VTAAVDIRVTDTKSTRIVRAKSYSKQAVGREVFASVFRFFSDELFDIKIGTKSQEGLQAAVRWTLADATYDLVKELTGHKGACDVYLPKASQDD

[0318] RAETTVVAAAN

[0319] >DNA sequence (SEQ ID NO:28)

[0320] ATGACCTTTGGCAAATTTCGCAGCCTGTTACTGGCGCCGGTGCTGTTACTGGCCGCGTGTCAGAGTCAGACCGCGGAACAAGCGCCGAAA

[0321] GAACCGTTTCTGGGCCCGGTGCCGATTGCGACCCGCACCCCGGTGGATGCGGCGCTGGAATGCCTGAGCAAAACCCCGGAAGTGCAGCG

[0322] CTATCCGCGCATGTTTGCGGTGCATGTGATTACCGATCAGACCGGCCGCTATAGCCAGGAAGAAGCGGGCAACTATCTGCCGCGCGATAGC

[0323] GCGGGCATGATGGTGAGCCTGCTGCAGAAAGCGGGCGTGAAACAAGTGAACCGCAGCAACACCGCGGTGAGCGAATGGGAAATTGCGC

[0324] GCGCGCGCGAACAGATTCTGGGCGATGGCGGCAACGTGGTTGTGGGCGATGAAAGCCTGCCGTTTCGCCCGCTGGTGAAAGGCGCGCTG

[0325] CGCGGCAGCGATTATGTGATTGATGGCGTGATTACGCAGCTGGATTTTAACACGTACAGCGGCGGTATCGAAGCGACGGTGGGCGGTGGC

[0326] GGTGGCGGCGCGCGCCGCTTTGCGGTGACCGCGGCGGTGGATATTCGCGTGACCGATACCAAAAGCACCCGCATTGTGCGCGCGAAAAG

[0327] CTATAGCAAACAAGCGGTGGGCCGCGAAGTGTTTGCGAGCGTGTTTCGCTTTTTTAGCGATGAACTGTTTGATATTAAAATTGGCACCAAA

[0328] AGCCAAGAAGGCCTGCAAGCGGCGGTGCGCTGGACCCTGGCGGATGCGACCTATGATCTGGTGAAAGAACTGACCGGCCATAAAGGCGC

[0329] GTGCGATGTGTATCTGCCGAAAGCGAGCCAAGATGATCGCGCGGAAACCACCGTGGTTGCGGCGGCCAAC

[0330] H5F

[0331] >Amino acid sequence (SEQ ID NO:29)

[0332] MTFGKFRSLLLAPVLLLAACQSQTAEQAPKEPFLGPVPIATRTPVDAALECLSKTPEVQRYPRMFAVHVITDQTGRYSYEEAGNYLPRDSAGMM

[0333] VSLLQKAGVKQVNRSNTAVSEWEIARAREQILGDGGNVVVGDESLPFRPLVKGALRGSDYVIDGVITQLDFNTYSGGIEATVGGGGGGARRFA

[0334] VTAAVDIRVTDTKSTRIVRAKSYSKQAVGREVFASVFRFFSDELFDIKIGTKSQEGLQAAVRWTLADATYDLVKELTGHKGACDVYLPKASQDD

[0335] RAETTVVAAAN

[0336] >DNA sequence (SEQ ID NO:30)

[0337] ATGACCTTTGGCAAATTTCGCAGCCTGTTACTGGCGCCGGTGCTGTTACTGGCCGCGTGTCAGAGTCAGACCGCGGAACAAGCGCCGAAA

[0338] GAACCGTTTCTGGGCCCGGTGCCGATTGCGACCCGCACCCCGGTGGATGCGGCGCTGGAATGCCTGAGCAAAACCCCGGAAGTGCAGCG

[0339] CTATCCGCGCATGTTTGCGGTGCATGTGATTACCGATCAGACCGGCCGCTATAGCTATGAAGAAGCGGGCAACTATCTGCCGCGCGATAGC

[0340] GCGGGCATGATGGTGAGCCTGCTGCAGAAAGCGGGCGTGAAACAAGTGAACCGCAGCAACACCGCGGTGAGCGAATGGGAAATTGCGC

[0341] GCGCGCGCGAACAGATTCTGGGCGATGGCGGCAACGTGGTTGTGGGCGATGAAAGCCTGCCGTTTCGCCCGCTGGTGAAAGGCGCGCTG

[0342] CGCGGCAGCGATTATGTGATTGATGGCGTGATTACGCAGCTGGATTTTAACACGTACAGCGGCGGTATCGAAGCGACGGTGGGCGGTGGC

[0343] GGTGGCGGCGCGCGCCGCTTTGCGGTGACCGCGGCGGTGGATATTCGCGTGACCGATACCAAAAGCACCCGCATTGTGCGCGCGAAAAG

[0344] CTATAGCAAACAAGCGGTGGGCCGCGAAGTGTTTGCGAGCGTGTTTCGCTTTTTTAGCGATGAACTGTTTGATATTAAAATTGGCACCAAA

[0345] AGCCAAGAAGGCCTGCAAGCGGCGGTGCGCTGGACCCTGGCGGATGCGACCTATGATCTGGTGAAAGAACTGACCGGCCATAAAGGCGC

[0346] GTGCGATGTGTATCTGCCGAAAGCGAGCCAAGATGATCGCGCGGAAACCACCGTGGTTGCGGCGGCCAAC

[0347] H5G<000​​​​​​​​​​​​​​​​​​​​​​GAACCGTTTCTGGGCCCGGTGCCGATTGCGACCCGCACCCCGGTGGATGCGGCGCTGGAATGCCTGAGCAAAACCCCGGAAGTGCAGCG

[0356] CTATCCGCGCATGTTTGCGGTGCATGTGATTACCGATCAGACCGGCCGCTATAGCGAAGAAGAAGCGGGCAACTATCTGCCGCGCGATAGC

[0357] GCGGGCATGATGGTGAGCCTGCTGCAGAAAGCGGGCGTGAAACAAGTGAACCGCAGCAACACCGCGGTGAGCGAATGGGAAATTGCGC

[0358] GCGCGCGCGAACAGATTCTGGGCGATGGCGGCAACGTGGTTGTGGGCGATGAAAGCCTGCCGTTTCGCCCGCTGGTGAAAGGCGCGCTG

[0359] CGCGGCAGCGATTATGTGATTGATGGCGTGATTACGCAGCTGGATTTTAACACGTACAGCGGCGGTATCGAAGCGACGGTGGGCGGTGGC

[0360] GGTGGCGGCGCGCGCCGCTTTGCGGTGACCGCGGCGGTGGATATTCGCGTGACCGATACCAAAAGCACCCGCATTGTGCGCGCGAAAAG

[0361] CTATAGCAAACAAGCGGTGGGCCGCGAAGTGTTTGCGAGCGTGTTTCGCTTTTTTAGCGATGAACTGTTTGATATTAAAATTGGCACCAAA

[0362] AGCCAAGAAGGCCTGCAAGCGGCGGTGCGCTGGACCCTGGCGGATGCGACCTATGATCTGGTGAAAGAACTGACCGGCCATAAAGGCGC

[0363] GTGCGATGTGTATCTGCCGAAAGCGAGCCAAGATGATCGCGCGGAAACCACCGTGGTTGCGGCGGCCAAC

[0364] H5H

[0365] >Amino acid sequence (SEQ ID NO:33)

[0366] MTFGKFRSLLLAPVLLLAACQSQTAEQAPKEPFLGPVPIATRTPVDAALECLSKTPEVQRYPRMFAVHVITDQTGRYSKEEAGNYLPRDSAGMM

[0367] VSLLQKAGVKQVNRSNTAVSEWEIARAREQILGDGGNVVVGDESLPFRPLVKGALRGSDYVIDGVITQLDFNTYSGGIEATVGGGGGGARRFA

[0368] VTAAVDIRVTDTKSTRIVRAKSYSKQAVGREVFASVFRFFSDELFDIKIGTKSQEGLQAAVRWTLADATYDLVKELTGHKGACDVYLPKASQDD

[0369] RAETTVVAAAN

[0370] >DNA sequence (SEQ ID NO:34)

[0371] ATGACCTTTGGCAAATTTCGCAGCCTGTTACTGGCGCCGGTGCTGTTACTGGCCGCGTGTCAGAGTCAGACCGCGGAACAAGCGCCGAAA

[0372] GAACCGTTTCTGGGCCCGGTGCCGATTGCGACCCGCACCCCGGTGGATGCGGCGCTGGAATGCCTGAGCAAAACCCCGGAAGTGCAGCG

[0373] CTATCCGCGCATGTTTGCGGTGCATGTGATTACCGATCAGACCGGCCGCTATAGCAAAGAAGAAGCGGGCAACTATCTGCCGCGCGATAGC

[0374] GCGGGCATGATGGTGAGCCTGCTGCAGAAAGCGGGCGTGAAACAAGTGAACCGCAGCAACACCGCGGTGAGCGAATGGGAAATTGCGC

[0375] GCGCGCGCGAACAGATTCTGGGCGATGGCGGCAACGTGGTTGTGGGCGATGAAAGCCTGCCGTTTCGCCCGCTGGTGAAAGGCGCGCTG

[0376] CGCGGCAGCGATTATGTGATTGATGGCGTGATTACGCAGCTGGATTTTAACACGTACAGCGGCGGTATCGAAGCGACGGTGGGCGGTGGC

[0377] GGTGGCGGCGCGCGCCGCTTTGCGGTGACCGCGGCGGTGGATATTCGCGTGACCGATACCAAAAGCACCCGCATTGTGCGCGCGAAAAG

[0378] CTATAGCAAACAAGCGGTGGGCCGCGAAGTGTTTGCGAGCGTGTTTCGCTTTTTTAGCGATGAACTGTTTGATATTAAAATTGGCACCAAA

[0379] AGCCAAGAAGGCCTGCAAGCGGCGGTGCGCTGGACCCTGGCGGATGCGACCTATGATCTGGTGAAAGAACTGACCGGCCATAAAGGCGC

[0380] GTGCGATGTGTATCTGCCGAAAGCGAGCCAAGATGATCGCGCGGAAACCACCGTGGTTGCGGCGGCCAAC

[0381] H5S

[0382] >Amino acid sequence (SEQ ID NO:35)

[0383] MTFGKFRSLLLAPVLLLAACQSQTAEQAPKEPFLGPVPIATRTPVDAALECLSKTPEVQRYPRMFAVHVITDQTGRISIEEAGNYLPRDSAGMMV

[0384] SLLQKAGVKQVNRSNTAVSEWEIARAREQILGDGGNVVVGDESLPFRPLVKGALRGSDYVIDGVITQLDFNTYSGGIEATVGGGGGGARRFAV

[0385] TAAVDIRVTDTKSTRIVRAKSYSKQAVGREVFASVFRFFSDELFDIKIGTKSQEGLQAAVRWTLADATYDLVKELTGHKGACDVYLPKASQDDR

[0386] AETTVVAAAN

[0387] >DNA sequence (SEQ ID NO:36)

[0388] ATGACCTTTGGCAAATTTCGCAGCCTGTTACTGGCGCCGGTGCTGTTACTGGCCGCGTGTCAGAGTCAGACCGCGGAACAAGCGCCGAAA

[0389] GAACCGTTTCTGGGCCCGGTGCCGATTGCGACCCGCACCCCGGTGGATGCGGCGCTGGAATGCCTGAGCAAAACCCCGGAAGTGCAGCG

[0390] CTATCCGCGCATGTTTGCGGTGCATGTGATTACCGATCAGACCGGCCGCATTAGCATTGAAGAAGCGGGCAACTATCTGCCGCGCGATAGC

[0391] GCGGGCATGATGGTGAGCCTGCTGCAGAAAGCGGGCGTGAAACAAGTGAACCGCAGCAACACCGCGGTGAGCGAATGGGAAATTGCGC

[0392] GCGCGCGCGAACAGATTCTGGGCGATGGCGGCAACGTGGTTGTGGGCGATGAAAGCCTGCCGTTTCGCCCGCTGGTGAAAGGCGCGCTG

[0393] CGCGGCAGCGATTATGTGATTGATGGCGTGATTACGCAGCTGGATTTTAACACGTACAGCGGCGGTATCGAAGCGACGGTGGGCGGTGGC

[0394] GGTGGCGGCGCGCGCCGCTTTGCGGTGACCGCGGCGGTGGATATTCGCGTGACCGATACCAAAAGCACCCGCATTGTGCGCGCGAAAAG

[0395] CTATAGCAAACAAGCGGTGGGCCGCGAAGTGTTTGCGAGCGTGTTTCGCTTTTTTAGCGATGAACTGTTTGATATTAAAATTGGCACCAAA

[0396] AGCCAAGAAGGCCTGCAAGCGGCGGTGCGCTGGACCCTGGCGGATGCGACCTATGATCTGGTGAAAGAACTGACCGGCCATAAAGGCGC

[0397] GTGCGATGTGTATCTGCCGAAAGCGAGCCAAGATGATCGCGCGGAAACCACCGTGGTTGCGGCGGCCAAC

[0398] H5T

[0399] >Amino acid sequence (SEQ ID NO:37)

[0400] MTFGKFRSLLLAPVLLLAACQSQTAEQAPKEPFLGPVPIATRTPVDAALECLSKTPEVQRYPRMFAVHVITDQTGRTSIEEAGNYLPRDSAGMM

[0401] VSLLQKAGVKQVNRSNTAVSEWEIARAREQILGDGGNVVVGDESLPFRPLVKGALRGSDYVIDGVITQLDFNTYSGGIEATVGGGGGGARRFA

[0402] VTAAVDIRVTDTKSTRIVRAKSYSKQAVGREVFASVFRFFSDELFDIKIGTKSQEGLQAAVRWTLADATYDLVKELTGHKGACDVYLPKASQDD

[0403] RAETTVVAAAN

[0404] >DNA sequence (SEQ ID NO:38)

[0405] ATGACCTTTGGCAAATTTCGCAGCCTGTTACTGGCGCCGGTGCTGTTACTGGCCGCGTGTCAGAGTCAGACCGCGGAACAAGCGCCGAAA

[0406] GAACCGTTTCTGGGCCCGGTGCCGATTGCGACCCGCACCCCGGTGGATGCGGCGCTGGAATGCCTGAGCAAAACCCCGGAAGTGCAGCG

[0407] CTATCCGCGCATGTTTGCGGTGCATGTGATTACCGATCAGACCGGCCGCACCAGCATTGAAGAAGCGGGCAACTATCTGCCGCGCGATAGC

[0408] GCGGGCATGATGGTGAGCCTGCTGCAGAAAGCGGGCGTGAAACAAGTGAACCGCAGCAACACCGCGGTGAGCGAATGGGAAATTGCGC

[0409] GCGCGCGCGAACAGATTCTGGGCGATGGCGGCAACGTGGTTGTGGGCGATGAAAGCCTGCCGTTTCGCCCGCTGGTGAAAGGCGCGCTG

[0410] CGCGGCAGCGATTATGTGATTGATGGCGTGATTACGCAGCTGGATTTTAACACGTACAGCGGCGGTATCGAAGCGACGGTGGGCGGTGGC

[0411] GGTGGCGGCGCGCGCCGCTTTGCGGTGACCGCGGCGGTGGATATTCGCGTGACCGATACCAAAAGCACCCGCATTGTGCGCGCGAAAAG

[0412] CTATAGCAAACAAGCGGTGGGCCGCGAAGTGTTTGCGAGCGTGTTTCGCTTTTTTAGCGATGAACTGTTTGATATTAAAATTGGCACCAAA

[0413] AGCCAAGAAGGCCTGCAAGCGGCGGTGCGCTGGACCCTGGCGGATGCGACCTATGATCTGGTGAAAGAACTGACCGGCCATAAAGGCGC

[0414] GTGCGATGTGTATCTGCCGAAAGCGAGCCAAGATGATCGCGCGGAAACCACCGTGGTTGCGGCGGCCAAC

[0415] H5U

[0416] >Amino acid sequence (SEQ ID NO: 39)

[0417] MTFGKFRSLLLAPVLLLAACQSQTAEQAPKEPFLGPVPIATRTPVDAALECLSKTPEVQRYPRMFAVHVITDQTGRQSIEEAGNYLPRDSAGMM

[0418] VSLLQKAGVKQVNRSNTAVSEWEIARAREQILGDGGNVVVGDESLPFRPLVKGALRGSDYVIDGVITQLDFNTYSGGIEATVGGGGGGARRFA

[0419] VTAAVDIRVTDTKSTRIVRAKSYSKQAVGREVFASVFRFFSDELFDIKIGTKSQEGLQAAVRWTLADATYDLVKELTGHKGACDVYLPKASQDD

[0420] RAETTVVAAAN

[0421] >DNA sequence (SEQ ID NO: 40)

[0422] ATGACCTTTGGCAAATTTCGCAGCCTGTTACTGGCGCCGGTGCTGTTACTGGCCGCGTGTCAGAGTCAGACCGCGGAACAAGCGCCGAAA

[0423] GAACCGTTTCTGGGCCCGGTGCCGATTGCGACCCGCACCCCGGTGGATGCGGCGCTGGAATGCCTGAGCAAAACCCCGGAAGTGCAGCG

[0424] CTATCCGCGCATGTTTGCGGTGCATGTGATTACCGATCAGACCGGCCGCCAGAGCATTGAAGAAGCGGGCAACTATCTGCCGCGCGATAGC

[0425] GCGGGCATGATGGTGAGCCTGCTGCAGAAAGCGGGCGTGAAACAAGTGAACCGCAGCAACACCGCGGTGAGCGAATGGGAAATTGCGC

[0426] GCGCGCGCGAACAGATTCTGGGCGATGGCGGCAACGTGGTTGTGGGCGATGAAAGCCTGCCGTTTCGCCCGCTGGTGAAAGGCGCGCTG

[0427] CGCGGCAGCGATTATGTGATTGATGGCGTGATTACGCAGCTGGATTTTAACACGTACAGCGGCGGTATCGAAGCGACGGTGGGCGGTGGC

[0428] GGTGGCGGCGCGCGCCGCTTTGCGGTGACCGCGGCGGTGGATATTCGCGTGACCGATACCAAAAGCACCCGCATTGTGCGCGCGAAAAG

[0429] CTATAGCAAACAAGCGGTGGGCCGCGAAGTGTTTGCGAGCGTGTTTCGCTTTTTTAGCGATGAACTGTTTGATATTAAAATTGGCACCAAA

[0430] AGCCAAGAAGGCCTGCAAGCGGCGGTGCGCTGGACCCTGGCGGATGCGACCTATGATCTGGTGAAAGAACTGACCGGCCATAAAGGCGC

[0431] GTGCGATGTGTATCTGCCGAAAGCGAGCCAAGATGATCGCGCGGAAACCACCGTGGTTGCGGCGGCCAAC

[0432] H5V

[0433] >Amino acid sequence (SEQ ID NO:41)

[0434] MTFGKFRSLLLAPVLLLAACQSQTAEQAPKEPFLGPVPIATRTPVDAALECLSKTPEVQRYPRMFAVHVITDQTGRMSIEEAGNYLPRDSAGMM

[0435] VSLLQKAGVKQVNRSNTAVSEWEIARAREQILGDGGNVVVGDESLPFRPLVKGALRGSDYVIDGVITQLDFNTYSGGIEATVGGGGGGARRFA

[0436] VTAAVDIRVTDTKSTRIVRAKSYSKQAVGREVFASVFRFFSDELFDIKIGTKSQEGLQAAVRWTLADATYDLVKELTGHKGACDVYLPKASQDD

[0437] RAETTVVAAAN

[0438] >DNA sequence (SEQ ID NO:42)

[0439] ATGACCTTTGGCAAATTTCGCAGCCTGTTACTGGCGCCGGTGCTGTTACTGGCCGCGTGTCAGAGTCAGACCGCGGAACAAGCGCCGAAA

[0440] GAACCGTTTCTGGGCCCGGTGCCGATTGCGACCCGCACCCCGGTGGATGCGGCGCTGGAATGCCTGAGCAAAACCCCGGAAGTGCAGCG

[0441] CTATCCGCGCATGTTTGCGGTGCATGTGATTACCGATCAGACCGGCCGCATGAGCATTGAAGAAGCGGGCAACTATCTGCCGCGCGATAGC

[0442] GCGGGCATGATGGTGAGCCTGCTGCAGAAAGCGGGCGTGAAACAAGTGAACCGCAGCAACACCGCGGTGAGCGAATGGGAAATTGCGC

[0443] GCGCGCGCGAACAGATTCTGGGCGATGGCGGCAACGTGGTTGTGGGCGATGAAAGCCTGCCGTTTCGCCCGCTGGTGAAAGGCGCGCTG

[0444] CGCGGCAGCGATTATGTGATTGATGGCGTGATTACGCAGCTGGATTTTAACACGTACAGCGGCGGTATCGAAGCGACGGTGGGCGGTGGC

[0445] GGTGGCGGCGCGCGCCGCTTTGCGGTGACCGCGGCGGTGGATATTCGCGTGACCGATACCAAAAGCACCCGCATTGTGCGCGCGAAAAG

[0446] CTATAGCAAACAAGCGGTGGGCCGCGAAGTGTTTGCGAGCGTGTTTCGCTTTTTTAGCGATGAACTGTTTGATATTAAAATTGGCACCAAA

[0447] AGCCAAGAAGGCCTGCAAGCGGCGGTGCGCTGGACCCTGGCGGATGCGACCTATGATCTGGTGAAAGAACTGACCGGCCATAAAGGCGC

[0448] GTGCGATGTGTATCTGCCGAAAGCGAGCCAAGATGATCGCGCGGAAACCACCGTGGTTGCGGCGGCCAAC

[0449] H5W

[0450] >Amino acid sequence (SEQ ID NO:43)

[0451] MTFGKFRSLLLAPVLLLAACQSQTAEQAPKEPFLGPVPIATRTPVDAALECLSKTPEVQRYPRMFAVHVITDQTGRDSIEEAGNYLPRDSAGMM

[0452] VSLLQKAGVKQVNRSNTAVSEWEIARAREQILGDGGNVVVGDESLPFRPLVKGALRGSDYVIDGVITQLDFNTYSGGIEATVGGGGGGARRFA

[0453] VTAAVDIRVTDTKSTRIVRAKSYSKQAVGREVFASVFRFFSDELFDIKIGTKSQEGLQAAVRWTLADATYDLVKELTGHKGACDVYLPKASQDD

[0454] RAETTVVAAAN

[0455] >DNA sequence (SEQ ID NO:44)

[0456] ATGACCTTTGGCAAATTTCGCAGCCTGTTACTGGCGCCGGTGCTGTTACTGGCCGCGTGTCAGAGTCAGACCGCGGAACAAGCGCCGAAA

[0457] GAACCGTTTCTGGGCCCGGTGCCGATTGCGACCCGCACCCCGGTGGATGCGGCGCTGGAATGCCTGAGCAAAACCCCGGAAGTGCAGCG

[0458] CTATCCGCGCATGTTTGCGGTGCATGTGATTACCGATCAGACCGGCCGCGATAGCATTGAAGAAGCGGGCAACTATCTGCCGCGCGATAGC

[0459] GCGGGCATGATGGTGAGCCTGCTGCAGAAAGCGGGCGTGAAACAAGTGAACCGCAGCAACACCGCGGTGAGCGAATGGGAAATTGCGC

[0460] GCGCGCGCGAACAGATTCTGGGCGATGGCGGCAACGTGGTTGTGGGCGATGAAAGCCTGCCGTTTCGCCCGCTGGTGAAAGGCGCGCTG

[0461] CGCGGCAGCGATTATGTGATTGATGGCGTGATTACGCAGCTGGATTTTAACACGTACAGCGGCGGTATCGAAGCGACGGTGGGCGGTGGC

[0462] GGTGGCGGCGCGCGCCGCTTTGCGGTGACCGCGGCGGTGGATATTCGCGTGACCGATACCAAAAGCACCCGCATTGTGCGCGCGAAAAG

[0463] CTATAGCAAACAAGCGGTGGGCCGCGAAGTGTTTGCGAGCGTGTTTCGCTTTTTTAGCGATGAACTGTTTGATATTAAAATTGGCACCAAA

[0464] AGCCAAGAAGGCCTGCAAGCGGCGGTGCGCTGGACCCTGGCGGATGCGACCTATGATCTGGTGAAAGAACTGACCGGCCATAAAGGCGC

[0465] GTGCGATGTGTATCTGCCGAAAGCGAGCCAAGATGATCGCGCGGAAACCACCGTGGTTGCGGCGGCCAAC

[0466] H5X

[0467] >Amino acid sequence (SEQ ID NO:45)

[0468] MTFGKFRSLLLAPVLLLAACQSQTAEQAPKEPFLGPVPIATRTPVDAALECLSKTPEVQRYPRMFAVHVITDQTGRWSIEEAGNYLPRDSAGMM

[0469] VSLLQKAGVKQVNRSNTAVSEWEIARAREQILGDGGNVVVGDESLPFRPLVKGALRGSDYVIDGVITQLDFNTYSGGIEATVGGGGGGARRFA

[0470] VTAAVDIRVTDTKSTRIVRAKSYSKQAVGREVFASVFRFFSDELFDIKIGTKSQEGLQAAVRWTLADATYDLVKELTGHKGACDVYLPKASQDD

[0471] RAETTVVAAAN

[0472] >DNA sequence (SEQ ID NO:46)

[0473] ATGACCTTTGGCAAATTTCGCAGCCTGTTACTGGCGCCGGTGCTGTTACTGGCCGCGTGTCAGAGTCAGACCGCGGAACAAGCGCCGAAA

[0474] GAACCGTTTCTGGGCCCGGTGCCGATTGCGACCCGCACCCCGGTGGATGCGGCGCTGGAATGCCTGAGCAAAACCCCGGAAGTGCAGCG

[0475] CTATCCGCGCATGTTTGCGGTGCATGTGATTACCGATCAGACCGGCCGCTGGAGCATTGAAGAAGCGGGCAACTATCTGCCGCGCGATAGC

[0476] GCGGGCATGATGGTGAGCCTGCTGCAGAAAGCGGGCGTGAAACAAGTGAACCGCAGCAACACCGCGGTGAGCGAATGGGAAATTGCGC

[0477] GCGCGCGCGAACAGATTCTGGGCGATGGCGGCAACGTGGTTGTGGGCGATGAAAGCCTGCCGTTTCGCCCGCTGGTGAAAGGCGCGCTG

[0478] CGCGGCAGCGATTATGTGATTGATGGCGTGATTACGCAGCTGGATTTTAACACGTACAGCGGCGGTATCGAAGCGACGGTGGGCGGTGGC

[0479] GGTGGCGGCGCGCGCCGCTTTGCGGTGACCGCGGCGGTGGATATTCGCGTGACCGATACCAAAAGCACCCGCATTGTGCGCGCGAAAAG

[0480] CTATAGCAAACAAGCGGTGGGCCGCGAAGTGTTTGCGAGCGTGTTTCGCTTTTTTAGCGATGAACTGTTTGATATTAAAATTGGCACCAAA

[0481] AGCCAAGAAGGCCTGCAAGCGGCGGTGCGCTGGACCCTGGCGGATGCGACCTATGATCTGGTGAAAGAACTGACCGGCCATAAAGGCGC

[0482] GTGCGATGTGTATCTGCCGAAAGCGAGCCAAGATGATCGCGCGGAAACCACCGTGGTTGCGGCGGCCAAC

[0483] H5Y

[0484] >Amino acid sequence (SEQ ID NO:47)

[0485] MTFGKFRSLLLAPVLLLAACQSQTAEQAPKEPFLGPVPIATRTPVDAALECLSKTPEVQRYPRMFAVHVITDQTGRVSIEEAGNYLPRDSAGMM

[0486] VSLLQKAGVKQVNRSNTAVSEWEIARAREQILGDGGNVVVGDESLPFRPLVKGALRGSDYVIDGVITQLDFNTYSGGIEATVGGGGGGARRFA

[0487] VTAAVDIRVTDTKSTRIVRAKSYSKQAVGREVFASVFRFFSDELFDIKIGTKSQEGLQAAVRWTLADATYDLVKELTGHKGACDVYLPKASQDD

[0488] RAETTVVAAAN

[0489] >DNA sequence (SEQ ID NO:48)

[0490] ATGACCTTTGGCAAATTTCGCAGCCTGTTACTGGCGCCGGTGCTGTTACTGGCCGCGTGTCAGAGTCAGACCGCGGAACAAGCGCCGAAA

[0491] GAACCGTTTCTGGGCCCGGTGCCGATTGCGACCCGCACCCCGGTGGATGCGGCGCTGGAATGCCTGAGCAAAACCCCGGAAGTGCAGCG

[0492] CTATCCGCGCATGTTTGCGGTGCATGTGATTACCGATCAGACCGGCCGCGTTAGCATTGAAGAAGCGGGCAACTATCTGCCGCGCGATAGC

[0493] GCGGGCATGATGGTGAGCCTGCTGCAGAAAGCGGGCGTGAAACAAGTGAACCGCAGCAACACCGCGGTGAGCGAATGGGAAATTGCGC

[0494] GCGCGCGCGAACAGATTCTGGGCGATGGCGGCAACGTGGTTGTGGGCGATGAAAGCCTGCCGTTTCGCCCGCTGGTGAAAGGCGCGCTG

[0495] CGCGGCAGCGATTATGTGATTGATGGCGTGATTACGCAGCTGGATTTTAACACGTACAGCGGCGGTATCGAAGCGACGGTGGGCGGTGGC

[0496] GGTGGCGGCGCGCGCCGCTTTGCGGTGACCGCGGCGGTGGATATTCGCGTGACCGATACCAAAAGCACCCGCATTGTGCGCGCGAAAAG

[0497] CTATAGCAAACAAGCGGTGGGCCGCGAAGTGTTTGCGAGCGTGTTTCGCTTTTTTAGCGATGAACTGTTTGATATTAAAATTGGCACCAAA

[0498] AGCCAAGAAGGCCTGCAAGCGGCGGTGCGCTGGACCCTGGCGGATGCGACCTATGATCTGGTGAAAGAACTGACCGGCCATAAAGGCGC

[0499] GTGCGATGTGTATCTGCCGAAAGCGAGCCAAGATGATCGCGCGGAAACCACCGTGGTTGCGGCGGCCAAC

[0500] H5Z

[0501] >Amino acid sequence (SEQ ID NO:49)

[0502] MTFGKFRSLLLAPVLLLAACQSQTAEQAPKEPFLGPVPIATRTPVDAALECLSKTPEVQRYPRMFAVHVITDQTGRFSIEEAGNYLPRDSAGMM

[0503] VSLLQKAGVKQVNRSNTAVSEWEIARAREQILGDGGNVVVGDESLPFRPLVKGALRGSDYVIDGVITQLDFNTYSGGIEATVGGGGGGARRFA

[0504] VTAAVDIRVTDTKSTRIVRAKSYSKQAVGREVFASVFRFFSDELFDIKIGTKSQEGLQAAVRWTLADATYDLVKELTGHKGACDVYLPKASQDD

[0505] RAETTVVAAAN

[0506] >DNA sequence (SEQ ID NO:50)

[0507] ATGACCTTTGGCAAATTTCGCAGCCTGTTACTGGCGCCGGTGCTGTTACTGGCCGCGTGTCAGAGTCAGACCGCGGAACAAGCGCCGAAA

[0508] GAACCGTTTCTGGGCCCGGTGCCGATTGCGACCCGCACCCCGGTGGATGCGGCGCTGGAATGCCTGAGCAAAACCCCGGAAGTGCAGCG

[0509] CTATCCGCGCATGTTTGCGGTGCATGTGATTACCGATCAGACCGGCCGCTTTAGCATTGAAGAAGCGGGCAACTATCTGCCGCGCGATAGC

[0510] GCGGGCATGATGGTGAGCCTGCTGCAGAAAGCGGGCGTGAAACAAGTGAACCGCAGCAACACCGCGGTGAGCGAATGGGAAATTGCGC

[0511] GCGCGCGCGAACAGATTCTGGGCGATGGCGGCAACGTGGTTGTGGGCGATGAAAGCCTGCCGTTTCGCCCGCTGGTGAAAGGCGCGCTG

[0512] CGCGGCAGCGATTATGTGATTGATGGCGTGATTACGCAGCTGGATTTTAACACGTACAGCGGCGGTATCGAAGCGACGGTGGGCGGTGGC

[0513] GGTGGCGGCGCGCGCCGCTTTGCGGTGACCGCGGCGGTGGATATTCGCGTGACCGATACCAAAAGCACCCGCATTGTGCGCGCGAAAAG

[0514] CTATAGCAAACAAGCGGTGGGCCGCGAAGTGTTTGCGAGCGTGTTTCGCTTTTTTAGCGATGAACTGTTTGATATTAAAATTGGCACCAAA

[0515] AGCCAAGAAGGCCTGCAAGCGGCGGTGCGCTGGACCCTGGCGGATGCGACCTATGATCTGGTGAAAGAACTGACCGGCCATAAAGGCGC

[0516] GTGCGATGTGTATCTGCCGAAAGCGAGCCAAGATGATCGCGCGGAAACCACCGTGGTTGCGGCGGCCAAC

[0517] H5J

[0518] >Amino acid sequence (SEQ ID NO:51)

[0519] MTFGKFRSLLLAPVLLLAACQSQTAEQAPKEPFLGPVPIATRTPVDAALECLSKTPEVQRYPRMFAVHVITDQTGRYSIEEAGNYLPRDSAGMM

[0520] VSLLQKAGVKQVNRSNTAVSEWEIARAREQILGDGGNVLPFRPLVKGALRGSDYVIDGVITQLDFNTYSGGIEATVGGGGGGARRFAVTAAVDI

[0521] RVTDTKSTRIVRAKSYSKQAVGREVFASVFRFFSDELFDIKIGTKSQEGLQAAVRWTLADATYDLVKELTGHKGACDVYLPKASQDDRAETTV>DNA sequence (SEQ ID NO:52)

[0522] ATGACCTTTGGCAAATTTCGCAGCCTGTTACTGGCGCCGGTGCTGTTACTGGCCGCGTGTCAGAGTCAGACCGCGGAACAAGCGCCGAAA

[0523] GAACCGTTTCTGGGCCCGGTGCCGATTGCGACCCGCACCCCGGTGGATGCGGCGCTGGAATGCCTGAGCAAAACCCCGGAAGTGCAGCG

[0524] CTATCCGCGCATGTTTGCGGTGCATGTGATTACCGATCAGACCGGCCGCTATAGCATTGAAGAAGCGGGCAACTATCTGCCGCGCGATAGC

[0525] GCGGGCATGATGGTGAGCCTGCTGCAGAAAGCGGGCGTGAAACAAGTGAACCGCAGCAACACCGCGGTGAGCGAATGGGAAATTGCGC

[0526] GCGCGCGCGAACAGATTCTGGGCGATGGCGGCAACGTGCTGCCGTTTCGCCCGCTGGTGAAAGGCGCGCTGCGCGGCAGCGATTATGTGA

[0527] TTGATGGCGTGATTACGCAGCTGGATTTTAACACGTACAGCGGCGGTATCGAAGCGACGGTGGGCGGTGGCGGTGGCGGCGCGCGCCGCT

[0528] TTGCGGTGACCGCGGCGGTGGATATTCGCGTGACCGATACCAAAAGCACCCGCATTGTGCGCGCGAAAAGCTATAGCAAACAAGCGGTGG

[0529] GCCGCGAAGTGTTTGCGAGCGTGTTTCGCTTTTTTAGCGATGAACTGTTTGATATTAAAATTGGCACCAAAAGCCAAGAAGGCCTGCAAG

[0530] CGGCGGTGCGCTGGACCCTGGCGGATGCGACCTATGATCTGGTGAAAGAACTGACCGGCCATAAAGGCGCGTGCGATGTGTATCTGCCGA

[0531] AAGCGAGCCAAGATGATCGCGCGGAAACCACCGTG

[0532] H7M

[0533] >Amino acid sequence (SEQ ID NO:53)

[0534] MTFGKFRSLLLAPVLLLAACQSQTAEQAPKEPFLGPVPIATRTPVDAALECLSKTPEVQRYPRMFAVHVITDQTGRYSIEEAGNYLPRDSAGMM

[0535] VSLLQKAGVKQVNRSNTAVSEWEIARAREQILGDGGNVVVGDESLPFRPLVKGALRGSDYVIDGVITQLDFNTYSGGIEATVGGGGGGAQRFA

[0536] VTAAVDIRVTDTKSTRIVRAKSYSKQAVGREVFASVFRFFSDELFDIKIGTKSQEGLQAAVRWTLADATYDLVKELTGHKGACDVYLPKASQDD

[0537] RAETTVVAAAN

[0538] >DNA sequence (SEQ ID NO:54)

[0539] ATGACCTTTGGCAAATTTCGCAGCCTGTTACTGGCGCCGGTGCTGTTACTGGCCGCGTGTCAGAGTCAGACCGCGGAACAAGCGCCGAAA

[0540] GAACCGTTTCTGGGCCCGGTGCCGATTGCGACCCGCACCCCGGTGGATGCGGCGCTGGAATGCCTGAGCAAAACCCCGGAAGTGCAGCG

[0541] CTATCCGCGCATGTTTGCGGTGCATGTGATTACCGATCAGACCGGCCGCTATAGCATTGAAGAAGCGGGCAACTATCTGCCGCGCGATAGC

[0542] GCGGGCATGATGGTGAGCCTGCTGCAGAAAGCGGGCGTGAAACAAGTGAACCGCAGCAACACCGCGGTGAGCGAATGGGAAATTGCGC

[0543] GCGCGCGCGAACAGATTCTGGGCGATGGCGGCAACGTGGTTGTGGGCGATGAAAGCCTGCCGTTTCGCCCGCTGGTGAAAGGCGCGCTG

[0544] CGCGGCAGCGATTATGTGATTGATGGCGTGATTACGCAGCTGGATTTTAACACGTACAGCGGCGGTATCGAAGCGACGGTGGGCGGTGGC

[0545] GGTGGCGGCGCGCAACGCTTTGCGGTGACCGCGGCGGTGGATATTCGCGTGACCGATACCAAAAGCACCCGCATTGTGCGCGCGAAAAG

[0546] CTATAGCAAACAAGCGGTGGGCCGCGAAGTGTTTGCGAGCGTGTTTCGCTTTTTTAGCGATGAACTGTTTGATATTAAAATTGGCACCAAA

[0547] AGCCAAGAAGGCCTGCAAGCGGCGGTGCGCTGGACCCTGGCGGATGCGACCTATGATCTGGTGAAAGAACTGACCGGCCATAAAGGCGC

[0548] GTGCGATGTGTATCTGCCGAAAGCGAGCCAAGATGATCGCGCGGAAACCACCGTGGTTGCGGCGGCCAAC

[0549] H7N

[0550] >Amino acid sequence (SEQ ID NO:55)

[0551] MTFGKFRSLLLAPVLLLAACQSQTAEQAPKEPFLGPVPIATRTPVDAALECLSKTPEVQRYPRMFAVHVITDQTGRYSIEEAGNYLPRDSAGMM

[0552] VSLLQKAGVKQVNRSNTAVSEWEIARAREQILGDGGNVVVGDESLPFRPLVKGALRGSDYVIDGVITQLDFNTYSGGIEATVGGGGGGARRFA

[0553] VTAAVDIRVTDTKSTRIVRAKSYSKQAVGRQVFASVFRFFSDELFDIKIGTKSQEGLQAAVRWTLADATYDLVKELTGHKGACDVYLPKASQDD

[0554] RAETTVVAAAN

[0555] >DNA sequence (SEQ ID NO:56)

[0556] ATGACCTTTGGCAAATTTCGCAGCCTGTTACTGGCGCCGGTGCTGTTACTGGCCGCGTGTCAGAGTCAGACCGCGGAACAAGCGCCGAAA

[0557] GAACCGTTTCTGGGCCCGGTGCCGATTGCGACCCGCACCCCGGTGGATGCGGCGCTGGAATGCCTGAGCAAAACCCCGGAAGTGCAGCG

[0558] CTATCCGCGCATGTTTGCGGTGCATGTGATTACCGATCAGACCGGCCGCTATAGCATTGAAGAAGCGGGCAACTATCTGCCGCGCGATAGC

[0559] GCGGGCATGATGGTGAGCCTGCTGCAGAAAGCGGGCGTGAAACAAGTGAACCGCAGCAACACCGCGGTGAGCGAATGGGAAATTGCGC

[0560] GCGCGCGCGAACAGATTCTGGGCGATGGCGGCAACGTGGTTGTGGGCGATGAAAGCCTGCCGTTTCGCCCGCTGGTGAAAGGCGCGCTG

[0561] CGCGGCAGCGATTATGTGATTGATGGCGTGATTACGCAGCTGGATTTTAACACGTACAGCGGCGGTATCGAAGCGACGGTGGGCGGTGGC

[0562] GGTGGCGGCGCGCGCCGCTTTGCGGTGACCGCGGCGGTGGATATTCGCGTGACCGATACCAAAAGCACCCGCATTGTGCGCGCGAAAAG

[0563] CTATAGCAAACAAGCGGTGGGCCGCCAAGTGTTTGCGAGCGTGTTTCGCTTTTTTAGCGATGAACTGTTTGATATTAAAATTGGCACCAAA

[0564] AGCCAAGAAGGCCTGCAAGCGGCGGTGCGCTGGACCCTGGCGGATGCGACCTATGATCTGGTGAAAGAACTGACCGGCCATAAAGGCGC

[0565] GTGCGATGTGTATCTGCCGAAAGCGAGCCAAGATGATCGCGCGGAAACCACCGTGGTTGCGGCGGCCAAC

[0566] H7O

[0567] >Amino acid sequence (SEQ ID NO:57)

[0568] MTFGKFRSLLLAPVLLLAACQSQTAEQAPKEPFLGPVPIATRTPVDAALECLSKTPEVQRYPRMFAVHVITDQTGRYSIEEAGNYLPRDSAGMM

[0569] VSLLQKAGVKQVNRSNTAVSEWEIARAREQILGDGGNVVVGDESLPFRPLVKGALRGSDYVIDGVITQLDFNTYSGGIEATVGGGGGGARRFA

[0570] VTAAVDIRVTDTKSTRIVRAKSYSKQAVGREVFASVFRFFSDELFDIKIGTQSQEGLQAAVRWTLADATYDLVKELTGHKGACDVYLPKASQDD

[0571] RAETTVVAAAN

[0572] >DNA sequence (SEQ ID NO:58)

[0573] ATGACCTTTGGCAAATTTCGCAGCCTGTTACTGGCGCCGGTGCTGTTACTGGCCGCGTGTCAGAGTCAGACCGCGGAACAAGCGCCGAAA

[0574] GAACCGTTTCTGGGCCCGGTGCCGATTGCGACCCGCACCCCGGTGGATGCGGCGCTGGAATGCCTGAGCAAAACCCCGGAAGTGCAGCG

[0575] CTATCCGCGCATGTTTGCGGTGCATGTGATTACCGATCAGACCGGCCGCTATAGCATTGAAGAAGCGGGCAACTATCTGCCGCGCGATAGC <000149​​​GCGCGCGCGAACAGATTCTGGGCGATGGCGGCAACGTGGTTGTGGGCGATGAAAGCCTGCCGTTTCGCCCGCTGGTGAAAGGCGCGCTG

[0578] CGCGGCAGCGATTATGTGATTGATGGCGTGATTACGCAGCTGGATTTTAACACGTACAGCGGCGGTATCGAAGCGACGGTGGGCGGTGGC

[0579] GGTGGCGGCGCGCGCCGCTTTGCGGTGACCGCGGCGGTGGATATTCGCGTGACCGATACCAAAAGCACCCGCATTGTGCGCGCGAAAAG

[0580] CTATAGCAAACAAGCGGTGGGCCGCGAAGTGTTTGCGAGCGTGTTTCGCTTTTTTAGCGATGAACTGTTTGATATTAAAATTGGCACCCAA

[0581] AGCCAAGAAGGCCTGCAAGCGGCGGTGCGCTGGACCCTGGCGGATGCGACCTATGATCTGGTGAAAGAACTGACCGGCCATAAAGGCGC

[0582] GTGCGATGTGTATCTGCCGAAAGCGAGCCAAGATGATCGCGCGGAAACCACCGTGGTTGCGGCGGCCAAC

[0583] H7R

[0584] > Amino acid sequence (SEQ ID NO:59)

[0585] MTFGKFRSLLLAPVLLLAACQSQTAEQAPKEPFLGPVPIATRTPVDAALECLSKTPEVQRYPRMFAVHVITDQTGRYSIEEAGNYLPRDSAGMM

[0586] VSLLQKAGVKQVNRSNTAVSEWEIARAREQILGDGGNVVVGDESLPFRPLVKGALRGSDYVIDGVITQLDFNTYSGGIEATVGGGGGGAQRFA​​VTAAVDIRVTDTKSTRIVRAKSYSKQAVGRQVFASVFRFFSDELFDIKIGTQSQEGLQAAVRWTLADATYDLVKELTGHKGACDVYLPKASQDD

[0588] RAETTVVAAAN

[0589] >DNA sequence (SEQ ID NO:60)

[0590] ATGACCTTTGGCAAATTTCGCAGCCTGTTACTGGCGCCGGTGCTGTTACTGGCCGCGTGTCAGAGTCAGACCGCGGAACAAGCGCCGAAA

[0591] GAACCGTTTCTGGGCCCGGTGCCGATTGCGACCCGCACCCCGGTGGATGCGGCGCTGGAATGCCTGAGCAAAACCCCGGAAGTGCAGCG

[0592] CTATCCGCGCATGTTTGCGGTGCATGTGATTACCGATCAGACCGGCCGCTATAGCATTGAAGAAGCGGGCAACTATCTGCCGCGCGATAGC

[0593] GCGGGCATGATGGTGAGCCTGCTGCAGAAAGCGGGCGTGAAACAAGTGAACCGCAGCAACACCGCGGTGAGCGAATGGGAAATTGCGC

[0594] GCGCGCGCGAACAGATTCTGGGCGATGGCGGCAACGTGGTTGTGGGCGATGAAAGCCTGCCGTTTCGCCCGCTGGTGAAAGGCGCGCTG <​​​​​

[0597] CTATAGCAAACAAGCGGTGGGCCGCCAAGTGTTTGCGAGCGTGTTTCGCTTTTTTAGCGATGAACTGTTTGATATTAAAATTGGCACCCAA

[0598] AGCCAAGAAGGCCTGCAAGCGGCGGTGCGCTGGACCCTGGCGGATGCGACCTATGATCTGGTGAAAGAACTGACCGGCCATAAAGGCGC

[0599] GTGCGATGTGTATCTGCCGAAAGCGAGCCAAGATGATCGCGCGGAAACCACCGTGGTTGCGGCGGCCAAC

[0600] H7U

[0601] >Amino acid sequence (SEQ ID NO:61)

[0602] MTFGKFRSLLLAPVLLLAACQSQTAEQAPKEPFLGPVPIATRTPVDAALECLSKTPEVQRYPRMFAVHVITDQTGRYSIEEAGNYLPRDSAGMM

[0603] VSLLQKAGVKQVNRSNTAVSEWEIARAREQILGDGGNVVVGDESLPFRPLVKGALRGSDYVIDGVITQLDFNTYSGGIEATVGGGGGGAQRFA

[0604] VTAAVDIRVTDTKSTRIVRAKSYSKQAVGREVFASVFRFFSDELFDIEIGTKSQEGLQAAVRWTLADATYDLVKELTGHKGACDVYLPKASQDD

[0605] RAETTVVAAAN

[0606] >DNA sequence (SEQ ID NO:62)

[0607] ATGACCTTTGGCAAATTTCGCAGCCTGTTACTGGCGCCGGTGCTGTTACTGGCCGCGTGTCAGAGTCAGACCGCGGAACAAGCGCCGAAA

[0608] GAACCGTTTCTGGGCCCGGTGCCGATTGCGACCCGCACCCCGGTGGATGCGGCGCTGGAATGCCTGAGCAAAACCCCGGAAGTGCAGCG

[0609] CTATCCGCGCATGTTTGCGGTGCATGTGATTACCGATCAGACCGGCCGCTATAGCATTGAAGAAGCGGGCAACTATCTGCCGCGCGATAGC

[0610] GCGGGCATGATGGTGAGCCTGCTGCAGAAAGCGGGCGTGAAACAAGTGAACCGCAGCAACACCGCGGTGAGCGAATGGGAAATTGCGC

[0611] GCGCGCGCGAACAGATTCTGGGCGATGGCGGCAACGTGGTTGTGGGCGATGAAAGCCTGCCGTTTCGCCCGCTGGTGAAAGGCGCGCTG

[0612] CGCGGCAGCGATTATGTGATTGATGGCGTGATTACGCAGCTGGATTTTAACACGTACAGCGGCGGTATCGAAGCGACGGTGGGCGGTGGC

[0613] GGTGGCGGCGCGCAACGCTTTGCGGTGACCGCGGCGGTGGATATTCGCGTGACCGATACCAAAAGCACCCGCATTGTGCGCGCGAAAAG

[0614] CTATAGCAAACAAGCGGTGGGCCGCGAAGTGTTTGCGAGCGTGTTTCGCTTTTTTAGCGATGAACTGTTTGATATTGAAATTGGCACCAAA

[0615] AGCCAAGAAGGCCTGCAAGCGGCGGTGCGCTGGACCCTGGCGGATGCGACCTATGATCTGGTGAAAGAACTGACCGGCCATAAAGGCGC

[0616] GTGCGATGTGTATCTGCCGAAAGCGAGCCAAGATGATCGCGCGGAAACCACCGTGGTTGCGGCGGCCAAC

[0617] H7V

[0618] >Amino acid sequence (SEQ ID NO:63)

[0619] MTFGKFRSLLLAPVLLLAACQSQTAEQAPKEPFLGPVPIATRTPVDAALECLSKTPEVQRYPRMFAVHVITDQTGRYSIEEAGNYLPRDSAGMM

[0620] VSLLQKAGVKQVNRSNTAVSEWEIARAREQILGDGGNVVVGDESLPFRPLVKGALRGSDYVIDGVITQLDFNTYSGGIEATVGGGGGDAQRFA

[0621] VTAAVDIRVTDTKSTRIVRAKSYSKQAVGREVFASVFRFFSDELFDIKIGTKSQEGLQAAVRWTLADATYDLVKELTGHKGACDVYLPKASQDD

[0622] RAETTVVAAAN

[0623] >DNA sequence (SEQ ID NO:64)

[0624] ATGACCTTTGGCAAATTTCGCAGCCTGTTACTGGCGCCGGTGCTGTTACTGGCCGCGTGTCAGAGTCAGACCGCGGAACAAGCGCCGAAA

[0625] GAACCGTTTCTGGGCCCGGTGCCGATTGCGACCCGCACCCCGGTGGATGCGGCGCTGGAATGCCTGAGCAAAACCCCGGAAGTGCAGCG

[0626] CTATCCGCGCATGTTTGCGGTGCATGTGATTACCGATCAGACCGGCCGCTATAGCATTGAAGAAGCGGGCAACTATCTGCCGCGCGATAGC

[0627] GCGGGCATGATGGTGAGCCTGCTGCAGAAAGCGGGCGTGAAACAAGTGAACCGCAGCAACACCGCGGTGAGCGAATGGGAAATTGCGC

[0628] GCGCGCGCGAACAGATTCTGGGCGATGGCGGCAACGTGGTTGTGGGCGATGAAAGCCTGCCGTTTCGCCCGCTGGTGAAAGGCGCGCTG

[0629] CGCGGCAGCGATTATGTGATTGATGGCGTGATTACGCAGCTGGATTTTAACACGTACAGCGGCGGTATCGAAGCGACGGTGGGCGGTGGC

[0630] GGTGGCGATGCGCAACGCTTTGCGGTGACCGCGGCGGTGGATATTCGCGTGACCGATACCAAAAGCACCCGCATTGTGCGCGCGAAAAG

[0631] CTATAGCAAACAAGCGGTGGGCCGCGAAGTGTTTGCGAGCGTGTTTCGCTTTTTTAGCGATGAACTGTTTGATATTAAAATTGGCACCAAA

[0632] AGCCAAGAAGGCCTGCAAGCGGCGGTGCGCTGGACCCTGGCGGATGCGACCTATGATCTGGTGAAAGAACTGACCGGCCATAAAGGCGC

[0633] GTGCGATGTGTATCTGCCGAAAGCGAGCCAAGATGATCGCGCGGAAACCACCGTGGTTGCGGCGGCCAAC

[0634] H7W

[0635] >Amino acid sequence (SEQ ID NO:65)

[0636] MTFGKFRSLLLAPVLLLAACQSQTAEQAPKEPFLGPVPIATRTPVDAALECLSKTPEVQRYPRMFAVHVITDQTGRYSIEEAGNYLPRDSAGMM

[0637] VSLLQKAGVKQVNRSNTAVSEWEIARAREQILGDGGNVVVGDESLPFRPLVKGALRGSDYVIDGVITQLDFNTYSGGIEATVGGGGGGAQRQA

[0638] VTAAVDIRVTDTKSTRIVRAKSYSKQAVGREVFASVFRFFSDELFDIKIGTKSQEGLQAAVRWTLADATYDLVKELTGHKGACDVYLPKASQDD

[0639] RAETTVVAAAN

[0640] >DNA sequence (SEQ ID NO:66)

[0641] ATGACCTTTGGCAAATTTCGCAGCCTGTTACTGGCGCCGGTGCTGTTACTGGCCGCGTGTCAGAGTCAGACCGCGGAACAAGCGCCGAAA

[0642] GAACCGTTTCTGGGCCCGGTGCCGATTGCGACCCGCACCCCGGTGGATGCGGCGCTGGAATGCCTGAGCAAAACCCCGGAAGTGCAGCG

[0643] CTATCCGCGCATGTTTGCGGTGCATGTGATTACCGATCAGACCGGCCGCTATAGCATTGAAGAAGCGGGCAACTATCTGCCGCGCGATAGC

[0644] GCGGGCATGATGGTGAGCCTGCTGCAGAAAGCGGGCGTGAAACAAGTGAACCGCAGCAACACCGCGGTGAGCGAATGGGAAATTGCGC

[0645] GCGCGCGCGAACAGATTCTGGGCGATGGCGGCAACGTGGTTGTGGGCGATGAAAGCCTGCCGTTTCGCCCGCTGGTGAAAGGCGCGCTG

[0646] CGCGGCAGCGATTATGTGATTGATGGCGTGATTACGCAGCTGGATTTTAACACGTACAGCGGCGGTATCGAAGCGACGGTGGGCGGTGGC

[0647] GGTGGCGGCGCGCAACGCCAAGCGGTGACCGCGGCGGTGGATATTCGCGTGACCGATACCAAAAGCACCCGCATTGTGCGCGCGAAAAG

[0648] CTATAGCAAACAAGCGGTGGGCCGCGAAGTGTTTGCGAGCGTGTTTCGCTTTTTTAGCGATGAACTGTTTGATATTAAAATTGGCACCAAA

[0649] AGCCAAGAAGGCCTGCAAGCGGCGGTGCGCTGGACCCTGGCGGATGCGACCTATGATCTGGTGAAAGAACTGACCGGCCATAAAGGCGC

[0650] GTGCGATGTGTATCTGCCGAAAGCGAGCCAAGATGATCGCGCGGAAACCACCGTGGTTGCGGCGGCCAAC

[0651] H7X

[0652] >Amino acid sequence (SEQ ID NO:67)

[0653] MTFGKFRSLLLAPVLLLAACQSQTAEQAPKEPFLGPVPIATRTPVDAALECLSKTPEVQRYPRMFAVHVITDQTGRYSIEEAGNYLPRDSAGMM

[0654] VSLLQKAGVKQVNRSNTAVSEWEIARAREQILGDGGNVVVGDESLPFRPLVKGALRGSDYVIDGVITQLDFNTYSGGIEATVGGGGGGAQRFA

[0655] VTAAVDIRVTDTKSTRIVRAKSYSKQAVGREVQASVFRFFSDELFDIKIGTKSQEGLQAAVRWTLADATYDLVKELTGHKGACDVYLPKASQD

[0656] DRAETTVVAAAN

[0657] >DNA sequence (SEQ ID NO:68)

[0658] ATGACCTTTGGCAAATTTCGCAGCCTGTTACTGGCGCCGGTGCTGTTACTGGCCGCGTGTCAGAGTCAGACCGCGGAACAAGCGCCGAAA

[0659] GAACCGTTTCTGGGCCCGGTGCCGATTGCGACCCGCACCCCGGTGGATGCGGCGCTGGAATGCCTGAGCAAAACCCCGGAAGTGCAGCG

[0660] CTATCCGCGCATGTTTGCGGTGCATGTGATTACCGATCAGACCGGCCGCTATAGCATTGAAGAAGCGGGCAACTATCTGCCGCGCGATAGC

[0661] GCGGGCATGATGGTGAGCCTGCTGCAGAAAGCGGGCGTGAAACAAGTGAACCGCAGCAACACCGCGGTGAGCGAATGGGAAATTGCGC

[0662] GCGCGCGCGAACAGATTCTGGGCGATGGCGGCAACGTGGTTGTGGGCGATGAAAGCCTGCCGTTTCGCCCGCTGGTGAAAGGCGCGCTG

[0663] CGCGGCAGCGATTATGTGATTGATGGCGTGATTACGCAGCTGGATTTTAACACGTACAGCGGCGGTATCGAAGCGACGGTGGGCGGTGGC

[0664] GGTGGCGGCGCGCAACGCTTTGCGGTGACCGCGGCGGTGGATATTCGCGTGACCGATACCAAAAGCACCCGCATTGTGCGCGCGAAAAG

[0665] CTATAGCAAACAAGCGGTGGGCCGCGAAGTGCAAGCGAGCGTGTTTCGCTTTTTTAGCGATGAACTGTTTGATATTAAAATTGGCACCAAA

[0666] AGCCAAGAAGGCCTGCAAGCGGCGGTGCGCTGGACCCTGGCGGATGCGACCTATGATCTGGTGAAAGAACTGACCGGCCATAAAGGCGC

[0667] GTGCGATGTGTATCTGCCGAAAGCGAGCCAAGATGATCGCGCGGAAACCACCGTGGTTGCGGCGGCCAAC

[0668] H8F

[0669] >Amino acid sequence (SEQ ID NO: 69)

[0670] MTFGKFRSLLLAPVLLLAACQSQTAEQAPKEPFLGPVPIATRTPVDAALECLSKTPEVQRYPRMFAVHVITDQTGRYSIEEAGNYLPRDSAGMM

[0671] VSLLQKAGVKQVNRSNTAVSEWEIARAREQILGDGGNVVVGDESLPFRPLVKGALRGSDYVIDGVITQLDFNTYSGGIEATVGGGGGGAQRFA

[0672] VTAAVDIRVTDTKSTRIVRAKSYSKQAVGRQVFASVFDFFSDELFDIKIGTQSQEGLQAAVRWTLADATYDLVKELTGHKGACDVYLPKASQDD

[0673] RAETTVVAAAN

[0674] >DNA sequence (SEQ ID NO: 70)

[0675] ATGACCTTTGGCAAATTTCGCAGCCTGTTACTGGCGCCGGTGCTGTTACTGGCCGCGTGTCAGAGTCAGACCGCGGAACAAGCGCCGAAA

[0676] GAACCGTTTCTGGGCCCGGTGCCGATTGCGACCCGCACCCCGGTGGATGCGGCGCTGGAATGCCTGAGCAAAACCCCGGAAGTGCAGCG

[0677] CTATCCGCGCATGTTTGCGGTGCATGTGATTACCGATCAGACCGGCCGCTATAGCATTGAAGAAGCGGGCAACTATCTGCCGCGCGATAGC

[0678] GCGGGCATGATGGTGAGCCTGCTGCAGAAAGCGGGCGTGAAACAAGTGAACCGCAGCAACACCGCGGTGAGCGAATGGGAAATTGCGC

[0679] GCGCGCGCGAACAGATTCTGGGCGATGGCGGCAACGTGGTTGTGGGCGATGAAAGCCTGCCGTTTCGCCCGCTGGTGAAAGGCGCGCTG

[0680] CGCGGCAGCGATTATGTGATTGATGGCGTGATTACGCAGCTGGATTTTAACACGTACAGCGGCGGTATCGAAGCGACGGTGGGCGGTGGC

[0681] GGTGGCGGCGCGCAACGCtttGCGGTGACCGCGGCGGTGGATATTCGCGTGACCGATACCAAAAGCACCCGCATTGTGCGCGCGAAAAGCT

[0682] ATAGCAAACAAGCGGTGGGCCGCcAAGTGTTTGCGAGCGTGTTTGATTTTTTTAGCGATGAACTGTTTGATATTAAAATTGGCACCcAAAGC

[0683] CAAGAAGGCCTGCAAGCGGCGGTGCGCTGGACCCTGGCGGATGCGACCTATGATCTGGTGAAAGAACTGACCGGCCATAAAGGCGCGTG

[0684] CGATGTGTATCTGCCGAAAGCGAGCCAAGATGATCGCGCGGAAACCACCGTGGTTGCGGCGGCCAAC

[0685] H8H

[0686] >Amino acid sequence (SEQ ID NO:71)

[0687] MTFGKFRSLLLAPVLLLAACQSQTAEQAPKEPFLGPVPIATRTPVDAALECLSKTPEVQRYPRMFAVHVITDQTGRYSIEEAGNYLPRDSAGMM

[0688] VSLLQKAGVKQVNRSNTAVSEWEIQRAREQILGDGGNVVVGDESLPFRPLVKGALRGSDYVIDGVITQLDFNTYSGGIEATVGGGGGGAQRFA

[0689] VTAAVDIRVTDTKSTRIVRAKSYSKQAVGRQVFASVFRFFSDELFDIKIGTQSQEGLQAAVRWTLADATYDLVKELTGHKGACDVYLPKASQDD

[0690] RAETTVVAAAN

[0691] >DNA sequence (SEQ ID NO:72)

[0692] ATGACCTTTGGCAAATTTCGCAGCCTGTTACTGGCGCCGGTGCTGTTACTGGCCGCGTGTCAGAGTCAGACCGCGGAACAAGCGCCGAAA

[0693] GAACCGTTTCTGGGCCCGGTGCCGATTGCGACCCGCACCCCGGTGGATGCGGCGCTGGAATGCCTGAGCAAAACCCCGGAAGTGCAGCG

[0694] CTATCCGCGCATGTTTGCGGTGCATGTGATTACCGATCAGACCGGCCGCTATAGCATTGAAGAAGCGGGCAACTATCTGCCGCGCGATAGC

[0695] GCGGGCATGATGGTGAGCCTGCTGCAGAAAGCGGGCGTGAAACAAGTGAACCGCAGCAACACCGCGGTGAGCGAATGGGAAATTCAGC

[0696] GCGCGCGCGAACAGATTCTGGGCGATGGCGGCAACGTGGTTGTGGGCGATGAAAGCCTGCCGTTTCGCCCGCTGGTGAAAGGCGCGCTG

[0697] CGCGGCAGCGATTATGTGATTGATGGCGTGATTACGCAGCTGGATTTTAACACGTACAGCGGCGGTATCGAAGCGACGGTGGGCGGTGGC

[0698] GGTGGCGGCGCGCAACGCtttGCGGTGACCGCGGCGGTGGATATTCGCGTGACCGATACCAAAAGCACCCGCATTGTGCGCGCGAAAAGCT

[0699] ATAGCAAACAAGCGGTGGGCCGCcAAGTGTTTGCGAGCGTGTTTCGCTTTTTTAGCGATGAACTGTTTGATATTAAAATTGGCACCcAAAG

[0700] CCAAGAAGGCCTGCAAGCGGCGGTGCGCTGGACCCTGGCGGATGCGACCTATGATCTGGTGAAAGAACTGACCGGCCATAAAGGCGCGT

[0701] GCGATGTGTATCTGCCGAAAGCGAGCCAAGATGATCGCGCGGAAACCACCGTGGTTGCGGCGGCCAAC

[0702] H8I

[0703] >Amino acid sequence (SEQ ID NO:73)

[0704] MTFGKFRSLLLAPVLLLAACQSQTAEQAPKEPFLGPVPIATRTPVDAALECLSKTPEVQRYPRMFAVHVITDQTGRYSIEEAGNYLPRDSAGMM

[0705] VSLLQKAGVKQVNRSNTAVSQWEIARAREQILGDGGNVVVGDESLPFRPLVKGALRGSDYVIDGVITQLDFNTYSGGIEATVGGGGGGAQRFA

[0706] VTAAVDIRVTDTKSTRIVRAKSYSKQAVGRQVFASVFRFFSDELFDIKIGTQSQEGLQAAVRWTLADATYDLVKELTGHKGACDVYLPKASQDD

[0707] RAETTVVAAAN

[0708] >DNA sequence (SEQ ID NO:74)

[0709] ATGACCTTTGGCAAATTTCGCAGCCTGTTACTGGCGCCGGTGCTGTTACTGGCCGCGTGTCAGAGTCAGACCGCGGAACAAGCGCCGAAA

[0710] GAACCGTTTCTGGGCCCGGTGCCGATTGCGACCCGCACCCCGGTGGATGCGGCGCTGGAATGCCTGAGCAAAACCCCGGAAGTGCAGCG

[0711] CTATCCGCGCATGTTTGCGGTGCATGTGATTACCGATCAGACCGGCCGCTATAGCATTGAAGAAGCGGGCAACTATCTGCCGCGCGATAGC

[0712] GCGGGCATGATGGTGAGCCTGCTGCAGAAAGCGGGCGTGAAACAAGTGAACCGCAGCAACACCGCGGTGAGCCAGTGGGAAATTGCGC

[0713] GCGCGCGCGAACAGATTCTGGGCGATGGCGGCAACGTGGTTGTGGGCGATGAAAGCCTGCCGTTTCGCCCGCTGGTGAAAGGCGCGCTG

[0714] CGCGGCAGCGATTATGTGATTGATGGCGTGATTACGCAGCTGGATTTTAACACGTACAGCGGCGGTATCGAAGCGACGGTGGGCGGTGGC

[0715] GGTGGCGGCGCGCAACGCtttGCGGTGACCGCGGCGGTGGATATTCGCGTGACCGATACCAAAAGCACCCGCATTGTGCGCGCGAAAAGCT

[0716] ATAGCAAACAAGCGGTGGGCCGCcAAGTGTTTGCGAGCGTGTTTCGCTTTTTTAGCGATGAACTGTTTGATATTAAAATTGGCACCcAAAG

[0717] CCAAGAAGGCCTGCAAGCGGCGGTGCGCTGGACCCTGGCGGATGCGACCTATGATCTGGTGAAAGAACTGACCGGCCATAAAGGCGCGT

[0718] GCGATGTGTATCTGCCGAAAGCGAGCCAAGATGATCGCGCGGAAACCACCGTGGTTGCGGCGGCCAAC

[0719] H8J

[0720] >Amino acid sequence (SEQ ID NO:75)

[0721] MTFGKFRSLLLAPVLLLAACQSQTAEQAPKEPFLGPVPIATRTPVDAALECLSKTPEVQRYPRMFAVHVITDQTGRYSIEEAGNYLPRDSAGMMVSLLQKAGVKQVNRSNTQVSEWEIARAREQILGDGGNVVVGDESLPFRPLVKGALRGSDYVIDGVITQLDFNTYSGGIEATVGGGGGGAQRFAVTAAVDIRVTDTKSTRIVRAKSYSKQAVGRQVFASVFRFFSDELFDIKIGTQSQEGLQAAVRWTLADATYDLVKELTGHKGACDVYLPKASQDDRAETTVVAAAN

[0722] >DNA sequence (SEQ ID NO:76)

[0723] ATGACCTTTGGCAAATTTCGCAGCCTGTTACTGGCGCCGGTGCTGTTACTGGCCGCGTGTCAGAGTCAGACCGCGGAACAAGCGCCGAAAGAACCGTTTCTGGGCCCGGTGCCGATTGCGACCCGCACCCCGGTGGATGCGGCGCTGGAATGCCTGAGCAAAACCCCGGAAGTGCAGCGCTATCCGCGCATGTTTGCGGTGCATGTGATTACCGATCAGACCGGCCGCTATAGCATTGAAGAAGCGGGCAACTATCTGCCGCGCGATAGCGCGGGCATGATGGTGAGCCTGCTGCAGAAAGCGGGCGTGAAACAAGTGAACCGCAGCAACACCCAGGTGAGCGAATGGGAAATTGCGCGCGCGCGCGAACAGATTCTGGGCGATGGCGGCAACGTGGTTGTGGGCGATGAAAGCCTGCCGTTTCGCCCGCTGGTGAAAGGCGCGCTGCGCGGCAGCGATTATGTGATTGATGGCGTGATTACGCAGCTGGATTTTAACACGTACAGCGGCGGTATCGAAGCGACGGTGGGCGGTGGCGGTGGCGGCGCGCAACGCtttGCGGTGACCGCGGCGGTGGATATTCGCGTGACCGATACCAAAAGCACCCGCATTGTGCGCGCGAAAAGCTATAGCAAACAAGCGGTGGGCCGCcAAGTGTTTGCGAGCGTGTTTCGCTTTTTTAGCGATGAACTGTTTGATATTAAAATTGGCACCcAAAGCCAAGAAGGCCTGCAAGCGGCGGTGCGCTGGACCCTGGCGGATGCGACCTATGATCTGGTGAAAGAACTGACCGGCCATAAAGGCGCGTGCGATGTGTATCTGCCGAAAGCGAGCCAAGATGATCGCGCGGAAACCACCGTGGTTGCGGCGGCCAACH8K

[0724] >Amino acid sequence (SEQ ID NO:77)

[0725] MTFGKFRSLLLAPVLLLAACQSQTAEQAPKEPFLGPVPIATRTPVDAALECLSKTPEVQRYPRMFAVHVITDQTGRYSIEEAGNYLPRDSAGMMVSLLQKAGVKQVNRSNTAVSEWEQARAREQILGDGGNVVVGDESLPFRPLVKGALRGSDYVIDGVITQLDFNTYSGGIEATVGGGGGGAQRFAVTAAVDIRVTDTKSTRIVRAKSYSKQAVGRQVFASVFRFFSDELFDIKIGTQSQEGLQAAVRWTLADATYDLVKELTGHKGACDVYLPKASQDDRAETTVVAAAN

[0726] >DNA sequence (SEQ ID NO:78)

[0727] ATGACCTTTGGCAAATTTCGCAGCCTGTTACTGGCGCCGGTGCTGTTACTGGCCGCGTGTCAGAGTCAGACCGCGGAACAAGCGCCGAAAGAACCGTTTCTGGGCCCGGTGCCGATTGCGACCCGCACCCCGGTGGATGCGGCGCTGGAATGCCTGAGCAAAACCCCGGAAGTGCAGCGCTATCCGCGCATGTTTGCGGTGCATGTGATTACCGATCAGACCGGCCGCTATAGCATTGAAGAAGCGGGCAACTATCTGCCGCGCGATAGCGCGGGCATGATGGTGAGCCTGCTGCAGAAAGCGGGCGTGAAACAAGTGAACCGCAGCAACACCGCGGTGAGCGAATGGGAACAGGCGCGCGCGCGCGAACAGATTCTGGGCGATGGCGGCAACGTGGTTGTGGGCGATGAAAGCCTGCCGTTTCGCCCGCTGGTGAAAGGCGCGCTGCGCGGCAGCGATTATGTGATTGATGGCGTGATTACGCAGCTGGATTTTAACACGTACAGCGGCGGTATCGAAGCGACGGTGGGCGGTGGCGGTGGCGGCGCGCAACGCtttGCGGTGACCGCGGCGGTGGATATTCGCGTGACCGATACCAAAAGCACCCGCATTGTGCGCGCGAAAAGCTATAGCAAACAAGCGGTGGGCCGCcAAGTGTTTGCGAGCGTGTTTCGCTTTTTTAGCGATGAACTGTTTGATATTAAAATTGGCACCcAAAGCCAAGAAGGCCTGCAAGCGGCGGTGCGCTGGACCCTGGCGGATGCGACCTATGATCTGGTGAAAGAACTGACCGGCCATAAAGGCGCGTGCGATGTGTATCTGCCGAAAGCGAGCCAAGATGATCGCGCGGAAACCACCGTGGTTGCGGCGGCCAACH10M

[0728] >Amino acid sequence (SEQ ID NO:79)

[0729] MTFGKFRSLLLAPVLLLAACQSQTAEQAPKEPFLGPVPIATRTPVDAALECLSKTPEVQRYPRMFAVYVITDQTGRYSIEEAGNYLPRDSAGMMVSLLQKAGVKQVNRSNTAVSEWEIARAREQILGDGGNVVVGDESLPFRPLVKGALRGSDYVIDGVITQLDFNTYSGGIEATVGGGGGGAQRQAVTAAVDIRVTDTKSTRIVRAKSYSKQAVGREVFASVFRFFSDELFDIKIGTKSQEGLQAAVRWTLADATYDLVKELTGHKGACDVYLPKASQDDRAETTVVAAAN

[0730] >DNA sequence (SEQ ID NO:80)

[0731] ATGACCTTTGGCAAATTTCGCAGCCTGTTACTGGCGCCGGTGCTGTTACTGGCCGCGTGTCAGAGTCAGACCGCGGAACAAGCGCCGAAAGAACCGTTTCTGGGCCCGGTGCCGATTGCGACCCGCACCCCGGTGGATGCGGCGCTGGAATGCCTGAGCAAAACCCCGGAAGTGCAGCGCTATCCGCGCATGTTTGCGGTGTATGTGATTACCGATCAGACCGGCCGCTATAGCATTGAAGAAGCGGGCAACTATCTGCCGCGCGATAGCGCGGGCATGATGGTGAGCCTGCTGCAGAAAGCGGGCGTGAAACAAGTGAACCGCAGCAACACCGCGGTGAGCGAATGGGAAATTGCGCGCGCGCGCGAACAGATTCTGGGCGATGGCGGCAACGTGGTTGTGGGCGATGAAAGCCTGCCGTTTCGCCCGCTGGTGAAAGGCGCGCTGCGCGGCAGCGATTATGTGATTGATGGCGTGATTACGCAGCTGGATTTTAACACGTACAGCGGCGGTATCGAAGCGACGGTGGGCGGTGGCGGTGGCGGCGCGCAACGCCAAGCGGTGACCGCGGCGGTGGATATTCGCGTGACCGATACCAAAAGCACCCGCATTGTGCGCGCGAAAAGCTATAGCAAACAAGCGGTGGGCCGCGAAGTGTTTGCGAGCGTGTTTCGCTTTTTTAGCGATGAACTGTTTGATATTAAAATTGGCACCAAAAGCCAAGAAGGCCTGCAAGCGGCGGTGCGCTGGACCCTGGCGGATGCGACCTATGATCTGGTGAAAGAACTGACCGGCCATAAAGGCGCGTGCGATGTGTATCTGCCGAAAGCGAGCCAAGATGATCGCGCGGAAACCACCGTGGTTGCGGCGGCCAACH10N

[0732] >Amino acid sequence (SEQ ID NO: 81)

[0733] MTFGKFRSLLLAPVLLLAACQSQTAEQAPKEPFLGPVPIATRTPVDAALECLSKTPEVQRYPRMFAVHVIQDQTGRYSIEEAGNYLPRDSAGMMVSLLQKAGVKQVNRSNTAVSEWEIARAREQILGDGGNVVVGDESLPFRPLVKGALRGSDYVIDGVITQLDFNTYSGGIEATVGGGGGGAQRQAVTAAVDIRVTDTKSTRIVRAKSYSKQAVGREVFASVFRFFSDELFDIKIGTKSQEGLQAAVRWTLADATYDLVKELTGHKGACDVYLPKASQDDRAETTVVAAAN

[0734] >DNA sequence (SEQ ID NO:82)

[0735] ATGACCTTTGGCAAATTTCGCAGCCTGTTACTGGCGCCGGTGCTGTTACTGGCCGCGTGTCAGAGTCAGACCGCGGAACAAGCGCCGAAAGAACCGTTTCTGGGCCCGGTGCCGATTGCGACCCGCACCCCGGTGGATGCGGCGCTGGAATGCCTGAGCAAAACCCCGGAAGTGCAGCGCTATCCGCGCATGTTTGCGGTGCATGTGATTCAGGATCAGACCGGCCGCTATAGCATTGAAGAAGCGGGCAACTATCTGCCGCGCGATAGCGCGGGCATGATGGTGAGCCTGCTGCAGAAAGCGGGCGTGAAACAAGTGAACCGCAGCAACACCGCGGTGAGCGAATGGGAAATTGCGCGCGCGCGCGAACAGATTCTGGGCGATGGCGGCAACGTGGTTGTGGGCGATGAAAGCCTGCCGTTTCGCCCGCTGGTGAAAGGCGCGCTGCGCGGCAGCGATTATGTGATTGATGGCGTGATTACGCAGCTGGATTTTAACACGTACAGCGGCGGTATCGAAGCGACGGTGGGCGGTGGCGGTGGCGGCGCGCAACGCCAAGCGGTGACCGCGGCGGTGGATATTCGCGTGACCGATACCAAAAGCACCCGCATTGTGCGCGCGAAAAGCTATAGCAAACAAGCGGTGGGCCGCGAAGTGTTTGCGAGCGTGTTTCGCTTTTTTAGCGATGAACTGTTTGATATTAAAATTGGCACCAAAAGCCAAGAAGGCCTGCAAGCGGCGGTGCGCTGGACCCTGGCGGATGCGACCTATGATCTGGTGAAAGAACTGACCGGCCATAAAGGCGCGTGCGATGTGTATCTGCCGAAAGCGAGCCAAGATGATCGCGCGGAAACCACCGTGGTTGCGGCGGCCAACH9O

[0736] >Amino acid sequence (SEQ ID NO:83)

[0737] MTFGKFRSLLLAPVLLLAACQSQTAEQAPKEPFLGPVPIATRTPVDAALECLSKTPEVQRYPRMFAVHVITDETGRYSIEEAGNYLPRDSAGMMVSLLQKAGVKQVNRSNTAVSEWEIARAREQILGDGGNVVVGDESLPFRPLVKGALRGSDYVIDGVITQLDFNTYSGGIEATVGGGGGGAQRQAVTAAVDIRVTDTKSTRIVRAKSYSKQAVGREVFASVFRFFSDELFDIKIGTKSQEGLQAAVRWTLADATYDLVKELTGHKGACDVYLPKASQDDRAETTVVAAAN

[0738] >DNA sequence (SEQ ID NO:84)

[0739] ATGACCTTTGGCAAATTTCGCAGCCTGTTACTGGCGCCGGTGCTGTTACTGGCCGCGTGTCAGAGTCAGACCGCGGAACAAGCGCCGAAAGAACCGTTTCTGGGCCCGGTGCCGATTGCGACCCGCACCCCGGTGGATGCGGCGCTGGAATGCCTGAGCAAAACCCCGGAAGTGCAGCGCTATCCGCGCATGTTTGCGGTGCATGTGATTACCGATGAAACCGGCCGCTATAGCATTGAAGAAGCGGGCAACTATCTGCCGCGCGATAGCGCGGGCATGATGGTGAGCCTGCTGCAGAAAGCGGGCGTGAAACAAGTGAACCGCAGCAACACCGCGGTGAGCGAATGGGAAATTGCGCGCGCGCGCGAACAGATTCTGGGCGATGGCGGCAACGTGGTTGTGGGCGATGAAAGCCTGCCGTTTCGCCCGCTGGTGAAAGGCGCGCTGCGCGGCAGCGATTATGTGATTGATGGCGTGATTACGCAGCTGGATTTTAACACGTACAGCGGCGGTATCGAAGCGACGGTGGGCGGTGGCGGTGGCGGCGCGCAACGCCAAGCGGTGACCGCGGCGGTGGATATTCGCGTGACCGATACCAAAAGCACCCGCATTGTGCGCGCGAAAAGCTATAGCAAACAAGCGGTGGGCCGCGAAGTGTTTGCGAGCGTGTTTCGCTTTTTTAGCGATGAACTGTTTGATATTAAAATTGGCACCAAAAGCCAAGAAGGCCTGCAAGCGGCGGTGCGCTGGACCCTGGCGGATGCGACCTATGATCTGGTGAAAGAACTGACCGGCCATAAAGGCGCGTGCGATGTGTATCTGCCGAAAGCGAGCCAAGATGATCGCGCGGAAACCACCGTGGTTGCGGCGGCCAACH9P

[0740] >Amino acid sequence (SEQ ID NO:85)

[0741] MTFGKFRSLLLAPVLLLAACQSQTAEQAPKEPFLGPVPIATRTPVDAALECLSKTPEVQRYPRMFAVHVITDQTGRYSIEEAGNYLPQDSAGMMVSLLQKAGVKQVNRSNTAVSEWEIARAREQILGDGGNVVVGDESLPFRPLVKGALRGSDYVIDGVITQLDFNTYSGGIEATVGGGGGGAQRQAVTAAVDIRVTDTKSTRIVRAKSYSKQAVGREVFASVFRFFSDELFDIKIGTKSQEGLQAAVRWTLADATYDLVKELTGHKGACDVYLPKASQDDRAETTVVAAAN

[0742] >DNA sequence (SEQ ID NO:86)

[0743] ATGACCTTTGGCAAATTTCGCAGCCTGTTACTGGCGCCGGTGCTGTTACTGGCCGCGTGTCAGAGTCAGACCGCGGAACAAGCGCCGAAAGAACCGTTTCTGGGCCCGGTGCCGATTGCGACCCGCACCCCGGTGGATGCGGCGCTGGAATGCCTGAGCAAAACCCCGGAAGTGCAGCGCTATCCGCGCATGTTTGCGGTGCATGTGATTACCGATCAGACCGGCCGCTATAGCATTGAAGAAGCGGGCAACTATCTGCCGCAGGATAGCGCGGGCATGATGGTGAGCCTGCTGCAGAAAGCGGGCGTGAAACAAGTGAACCGCAGCAACACCGCGGTGAGCGAATGGGAAATTGCGCGCGCGCGCGAACAGATTCTGGGCGATGGCGGCAACGTGGTTGTGGGCGATGAAAGCCTGCCGTTTCGCCCGCTGGTGAAAGGCGCGCTG

[0744] CGCGGCAGCGATTATGTGATTGATGGCGTGATTACGCAGCTGGATTTTAACACGTACAGCGGCGGTATCGAAGCGACGGTGGGCGGTGGC

[0745] GGTGGCGGCGCGCAACGCCAAGCGGTGACCGCGGCGGTGGATATTCGCGTGACCGATACCAAAAGCACCCGCATTGTGCGCGCGAAAAG

[0746] CTATAGCAAACAAGCGGTGGGCCGCGAAGTGTTTGCGAGCGTGTTTCGCTTTTTTAGCGATGAACTGTTTGATATTAAAATTGGCACCAAA

[0747] AGCCAAGAAGGCCTGCAAGCGGCGGTGCGCTGGACCCTGGCGGATGCGACCTATGATCTGGTGAAAGAACTGACCGGCCATAAAGGCGC

[0748] GTGCGATGTGTATCTGCCGAAAGCGAGCCAAGATGATCGCGCGGAAACCACCGTGGTTGCGGCGGCCAAC

[0749] H9R

[0750] >Amino acid sequence (SEQ ID NO:87)

[0751] MTFGKFRSLLLAPVLLLAACQSQTAEQAPKEPFLGPVPIATRTPVDAALECLSKTPEVQRYPRMFAVHVITDQTGRYSIEEAGNYLPRDSAGMM

[0752] VSLLQKAGVKQVNRSNTAVSEWEIAIAREQILGDGGNVVVGDESLPFRPLVKGALRGSDYVIDGVITQLDFNTYSGGIEATVGGGGGGAQRQA

[0753] VTAAVDIRVTDTKSTRIVRAKSYSKQAVGREVFASVFRFFSDELFDIKIGTKSQEGLQAAVRWTLADATYDLVKELTGHKGACDVYLPKASQDD

[0754] RAETTVVAAAN

[0755] >DNA sequence (SEQ ID NO:88)

[0756] ATGACCTTTGGCAAATTTCGCAGCCTGTTACTGGCGCCGGTGCTGTTACTGGCCGCGTGTCAGAGTCAGACCGCGGAACAAGCGCCGAAA

[0757] GAACCGTTTCTGGGCCCGGTGCCGATTGCGACCCGCACCCCGGTGGATGCGGCGCTGGAATGCCTGAGCAAAACCCCGGAAGTGCAGCG

[0758] CTATCCGCGCATGTTTGCGGTGCATGTGATTACCGATCAGACCGGCCGCTATAGCATTGAAGAAGCGGGCAACTATCTGCCGCGCGATAGC

[0759] GCGGGCATGATGGTGAGCCTGCTGCAGAAAGCGGGCGTGAAACAAGTGAACCGCAGCAACACCGCGGTGAGCGAATGGGAAATTGCGA

[0760] TTGCGCGCGAACAGATTCTGGGCGATGGCGGCAACGTGGTTGTGGGCGATGAAAGCCTGCCGTTTCGCCCGCTGGTGAAAGGCGCGCTG

[0761] CGCGGCAGCGATTATGTGATTGATGGCGTGATTACGCAGCTGGATTTTAACACGTACAGCGGCGGTATCGAAGCGACGGTGGGCGGTGGC

[0762] GGTGGCGGCGCGCAACGCCAAGCGGTGACCGCGGCGGTGGATATTCGCGTGACCGATACCAAAAGCACCCGCATTGTGCGCGCGAAAAG

[0763] CTATAGCAAACAAGCGGTGGGCCGCGAAGTGTTTGCGAGCGTGTTTCGCTTTTTTAGCGATGAACTGTTTGATATTAAAATTGGCACCAAA

[0764] AGCCAAGAAGGCCTGCAAGCGGCGGTGCGCTGGACCCTGGCGGATGCGACCTATGATCTGGTGAAAGAACTGACCGGCCATAAAGGCGC

[0765] GTGCGATGTGTATCTGCCGAAAGCGAGCCAAGATGATCGCGCGGAAACCACCGTGGTTGCGGCGGCCAAC

[0766] H9S

[0767] >Amino acid sequence (SEQ ID NO:89)

[0768] MTFGKFRSLLLAPVLLLAACQSQTAEQAPKEPFLGPVPIATRTPVDAALECLSKTPEVQRYPRMFAVHVITDQTGRYSIEEAGNYLPRDSAGMM

[0769] VSLLQKAGVKQVNRSNTAVSEWEIARARAQILGDGGNVVVGDESLPFRPLVKGALRGSDYVIDGVITQLDFNTYSGGIEATVGGGGGGAQRQ

[0770] AVTAAVDIRVTDTKSTRIVRAKSYSKQAVGREVFASVFRFFSDELFDIKIGTKSQEGLQAAVRWTLADATYDLVKELTGHKGACDVYLPKASQD

[0771] DRAETTVVAAAN

[0772] >DNA sequence (SEQ ID NO:90)

[0773] ATGACCTTTGGCAAATTTCGCAGCCTGTTACTGGCGCCGGTGCTGTTACTGGCCGCGTGTCAGAGTCAGACCGCGGAACAAGCGCCGAAA

[0774] GAACCGTTTCTGGGCCCGGTGCCGATTGCGACCCGCACCCCGGTGGATGCGGCGCTGGAATGCCTGAGCAAAACCCCGGAAGTGCAGCG

[0775] CTATCCGCGCATGTTTGCGGTGCATGTGATTACCGATCAGACCGGCCGCTATAGCATTGAAGAAGCGGGCAACTATCTGCCGCGCGATAGC

[0776] GCGGGCATGATGGTGAGCCTGCTGCAGAAAGCGGGCGTGAAACAAGTGAACCGCAGCAACACCGCGGTGAGCGAATGGGAAATTGCGC

[0777] GCGCGCGCGCGCAGATTCTGGGCGATGGCGGCAACGTGGTTGTGGGCGATGAAAGCCTGCCGTTTCGCCCGCTGGTGAAAGGCGCGCTG

[0778] CGCGGCAGCGATTATGTGATTGATGGCGTGATTACGCAGCTGGATTTTAACACGTACAGCGGCGGTATCGAAGCGACGGTGGGCGGTGGC

[0779] GGTGGCGGCGCGCAACGCCAAGCGGTGACCGCGGCGGTGGATATTCGCGTGACCGATACCAAAAGCACCCGCATTGTGCGCGCGAAAAG

[0780] CTATAGCAAACAAGCGGTGGGCCGCGAAGTGTTTGCGAGCGTGTTTCGCTTTTTTAGCGATGAACTGTTTGATATTAAAATTGGCACCAAA

[0781] AGCCAAGAAGGCCTGCAAGCGGCGGTGCGCTGGACCCTGGCGGATGCGACCTATGATCTGGTGAAAGAACTGACCGGCCATAAAGGCGC

[0782] [[ID=2l]]GTGCGATGTGTATCTGCCGAAAGCGAGCCAAGATGATCGCGCGGAAACCACCGTGGTTGCGGCGGCCAAC

[0783] H9T

[0784] >Amino acid sequence (SEQ ID NO:91)

[0785] MTFGKFRSLLLAPVLLLAACQSQTAEQAPKEPFLGPVPIATRTPVDAALECLSKTPEVQRYPRMFAVHVITDQTGRYSIEEAGNYLPRDSAGMM

[0786] VSLLQKAGVKQVNRSNTAVSEWEIARAREQELGDGGNVVVGDESLPFRPLVKGALRGSDYVIDGVITQLDFNTYSGGIEATVGGGGGGAQRQ

[0787] AVTAAVDIRVTDTKSTRIVRAKSYSKQAVGREVFASVFRFFSDELFDIKIGTKSQEGLQAAVRWTLADATYDLVKELTGHKGACDVYLPKASQD

[0788] DRAETTVVAAAN

[0789] >DNA sequence (SEQ ID NO:92)

[0790] ATGACCTTTGGCAAATTTCGCAGCCTGTTACTGGCGCCGGTGCTGTTACTGGCCGCGTGTCAGAGTCAGACCGCGGAACAAGCGCCGAAA

[0791] GAACCGTTTCTGGGCCCGGTGCCGATTGCGACCCGCACCCCGGTGGATGCGGCGCTGGAATGCCTGAGCAAAACCCCGGAAGTGCAGCG

[0792] CTATCCGCGCATGTTTGCGGTGCATGTGATTACCGATCAGACCGGCCGCTATAGCATTGAAGAAGCGGGCAACTATCTGCCGCGCGATAGC

[0793] GCGGGCATGATGGTGAGCCTGCTGCAGAAAGCGGGCGTGAAACAAGTGAACCGCAGCAACACCGCGGTGAGCGAATGGGAAATTGCGC

[0794] GCGCGCGCGAACAGGAACTGGGCGATGGCGGCAACGTGGTTGTGGGCGATGAAAGCCTGCCGTTTCGCCCGCTGGTGAAAGGCGCGCTG

[0795] CGCGGCAGCGATTATGTGATTGATGGCGTGATTACGCAGCTGGATTTTAACACGTACAGCGGCGGTATCGAAGCGACGGTGGGCGGTGGC

[0796] GGTGGCGGCGCGCAACGCCAAGCGGTGACCGCGGCGGTGGATATTCGCGTGACCGATACCAAAAGCACCCGCATTGTGCGCGCGAAAAG

[0797] CTATAGCAAACAAGCGGTGGGCCGCGAAGTGTTTGCGAGCGTGTTTCGCTTTTTTAGCGATGAACTGTTTGATATTAAAATTGGCACCAAA

[0798] AGCCAAGAAGGCCTGCAAGCGGCGGTGCGCTGGACCCTGGCGGATGCGACCTATGATCTGGTGAAAGAACTGACCGGCCATAAAGGCGC

[0799] GTGCGATGTGTATCTGCCGAAAGCGAGCCAAGATGATCGCGCGGAAACCACCGTGGTTGCGGCGGCCAAC

[0800] H10K

[0801] >Amino acid sequence (SEQ ID NO:93)

[0802] MTFGKFRSLLLAPVLLLAACQSQTAEQAPKEPFLGPVPIATRTPVDAALECLSKTPEVQRYPRMFAVHVITDQTGRYSIEEAGNYLPRDSAGMM

[0803] VSLLQKAGVKQVNRSNTAVSEWEIARAREQILGDGGNVVVGDESLPFRPLVKGALRGSDYVIDGVITQLDFNTYSGGVGARYFGIGADTQYQA

[0804] VTAAVDIRVTDTKSTRIVRAKSYSKQAVGREVFASVFRFFSDELFDIKIGTKSQEGLQAAVRWTLADATYDLVKELTGHKGACDVYLPKASQDD

[0805] RAETTVVAAAN

[0806] >DNA sequence (SEQ ID NO:94)

[0807] ATGACCTTTGGCAAATTTCGCAGCCTGTTACTGGCGCCGGTGCTGTTACTGGCCGCGTGTCAGAGTCAGACCGCGGAACAAGCGCCGAAA

[0808] GAACCGTTTCTGGGCCCGGTGCCGATTGCGACCCGCACCCCGGTGGATGCGGCGCTGGAATGCCTGAGCAAAACCCCGGAAGTGCAGCG

[0809] CTATCCGCGCATGTTTGCGGTGCATGTGATTACCGATCAGACCGGCCGCTATAGCATTGAAGAAGCGGGCAACTATCTGCCGCGCGATAGC

[0810] GCGGGCATGATGGTGAGCCTGCTGCAGAAAGCGGGCGTGAAACAAGTGAACCGCAGCAACACCGCGGTGAGCGAATGGGAAATTGCGC

[0811] GCGCGCGCGAACAGATTCTGGGCGATGGCGGCAACGTGGTTGTGGGCGATGAAAGCCTGCCGTTTCGCCCGCTGGTGAAAGGCGCGCTG

[0812] CGCGGCAGCGATTATGTGATTGATGGCGTGATTACGCAGCTGGATTTTAACACGTACAGCggcggTgttggggcaagatattttggcatcggtgccgacacgcaatac

[0813] CAAGCGGTGACCGCGGCGGTGGATATTCGCGTGACCGATACCAAAAGCACCCGCATTGTGCGCGCGAAAAGCTATAGCAAACAAGCGGT

[0814] GGGCCGCGAAGTGTTTGCGAGCGTGTTTCGCTTTTTTAGCGATGAACTGTTTGATATTAAAATTGGCACCAAAAGCCAAGAAGGCCTGCA

[0815] AGCGGCGGTGCGCTGGACCCTGGCGGATGCGACCTATGATCTGGTGAAAGAACTGACCGGCCATAAAGGCGCGTGCGATGTGTATCTGCC

[0816] GAAAGCGAGCCAAGATGATCGCGCGGAAACCACCGTGGTTGCGGCGGCCAAC

[0817] H10L

[0818] >Amino acid sequence (SEQ ID NO:95)

[0819] MTFGKFRSLLLAPVLLLAACQSQTAEQAPKEPFLGPVPIATRTPVDAALECLSKTPEVQRYPRMFAVHVITDQTGRYSIEEAGNYLPRDSAGMM

[0820] VSLLQKAGVKQVNRSNTAVSEWEIARAREQILGDGGNVVVGDESLPFRPLVKGALRGSDYVIDGVITQLDFNTYSGGVGARYFGIGADTQYQA

[0821] VTAAVDIRVTDTKSTRIVRAKSYSKQAVSYEVQAGVFRFIDYQRLLEGEVGYTSNEGLQAAVRWTLADATYDLVKELTGHKGACDVYLPKASQ

[0822] DDRAETTVVAAAN [[ID=2,6]]

[0823] >DNA sequence (SEQ ID NO:96)

[0824] ATGACCTTTGGCAAATTTCGCAGCCTGTTACTGGCGCCGGTGCTGTTACTGGCCGCGTGTCAGAGTCAGACCGCGGAACAAGCGCCGAAA

[0825] GAACCGTTTCTGGGCCCGGTGCCGATTGCGACCCGCACCCCGGTGGATGCGGCGCTGGAATGCCTGAGCAAAACCCCGGAAGTGCAGCG

[0826] CTATCCGCGCATGTTTGCGGTGCATGTGATTACCGATCAGACCGGCCGCTATAGCATTGAAGAAGCGGGCAACTATCTGCCGCGCGATAGC

[0827] GCGGGCATGATGGTGAGCCTGCTGCAGAAAGCGGGCGTGAAACAAGTGAACCGCAGCAACACCGCGGTGAGCGAATGGGAAATTGCGC

[0828] GCGCGCGCGAACAGATTCTGGGCGATGGCGGCAACGTGGTTGTGGGCGATGAAAGCCTGCCGTTTCGCCCGCTGGTGAAAGGCGCGCTG

[0829] CGCGGCAGCGATTATGTGATTGATGGCGTGATTACGCAGCTGGATTTTAACACGTACAGCggcggTgttggggcaagatattttggcatcggtgccgacacgcaatac

[0830] CAAGCGGTGACCGCGGCGGTGGATATTCGCGTGACCGATACCAAAAGCACCCGCATTGTGCGCGCGAAAAGCTATAGCAAACAAGCGGT

[0831] GtcctatgaagttcaggccggggttttccgctttattgactaccagcgcttgcttgaaggggaagtgggttacacctcgaacGAAGGCCTGCAAGCGGCGGTGCGCTGGACCCTGGCGGAT

[0832] GCGACCTATGATCTGGTGAAAGAACTGACCGGCCATAAAGGCGCGTGCGATGTGTATCTGCCGAAAGCGAGCCAAGATGATCGCGCGGAA

[0833] ACCACCGTGGTTGCGGCGGCCAAC

[0834] H9U

[0835] >Amino acid sequence (SEQ ID NO:97)

[0836] MTFGKFRSLLLAPVLLLAACQSQTAEQAPKEPFLGPVPIATRTPVDAALECLSKTPEVQRYPRMFAVHVIQDETGRYSIEEAGNYLPQDSAGMM

[0837] VSLLQKAGVKQVNRSNTAVSEWEIARAREQILGDGGNVVVGDESLPFRPLVKGALRGSDYVIDGVITQLDFNTYSGGIEATVGGGGGGAQRQA

[0838] VTAAVDIRVTDTKSTRIVRAKSYSKQAVGREVFASVFRFFSDELFDIKIGTKSQEGLQAAVRWTLADATYDLVKELTGHKGACDVYLPKASQDD

[0839] RAETTVVAAAN

[0840] >DNA sequence (SEQ ID NO:98)

[0841] ATGACCTTTGGCAAATTTCGCAGCCTGTTACTGGCGCCGGTGCTGTTACTGGCCGCGTGTCAGAGTCAGACCGCGGAACAAGCGCCGAAA

[0842] ]>GAACCGTTTCTGGGCCCGGTGCCGATTGCGACCCGCACCCCGGTGGATGCGGCGCTGGAATGCCTGAGCAAAACCCCGGAAGTGCAGCG

[0843] CTATCCGCGCATGTTTGCGGTGCATGTGATTCAAGATGAAACCGGCCGCTATAGCATTGAAGAAGCGGGCAACTATCTGCCGCAAGATAGC

[0844] GCGGGCATGATGGTGAGCCTGCTGCAGAAAGCGGGCGTGAAACAAGTGAACCGCAGCAACACCGCGGTGAGCGAATGGGAAATTGCGC

[0845] GCGCGCGCGAACAGATTCTGGGCGATGGCGGCAACGTGGTTGTGGGCGATGAAAGCCTGCCGTTTCGCCCGCTGGTGAAAGGCGCGCTG

[0846] CGCGGCAGCGATTATGTGATTGATGGCGTGATTACGCAGCTGGATTTTAACACGTACAGCGGCGGTATCGAAGCGACGGTGGGCGGTGGC

[0847] GGTGGCGGCGCGCAACGCCAAGCGGTGACCGCGGCGGTGGATATTCGCGTGACCGATACCAAAAGCACCCGCATTGTGCGCGCGAAAAG

[0848] CTATAGCAAACAAGCGGTGGGCCGCGAAGTGTTTGCGAGCGTGTTTCGCTTTTTTAGCGATGAACTGTTTGATATTAAAATTGGCACCAAA

[0849] AGCCAAGAAGGCCTGCAAGCGGCGGTGCGCTGGACCCTGGCGGATGCGACCTATGATCTGGTGAAAGAACTGACCGGCCATAAAGGCGC

[0850] GTGCGATGTGTATCTGCCGAAAGCGAGCCAAGATGATCGCGCGGAAACCACCGTGGTTGCGGCGGCCAAC

[0851] H9W

[0852] >Amino acid sequence (SEQ ID NO:99)

[0853] MTFGKFRSLLLAPVLLLAACQSQTAEQAPKEPFLGPVPIATRTPVDAALECLSKTPEVQRYPRMFAVHVIQDETGRYSIEEAGNYLPQDSAGMM

[0854] VSLLQKAGVKQVNRSNTAVSEWEIARAREQILGDGGNVVVGDESLPFRPLVKGALRGSDYVIDGVITQLDFNTYSGGVGARYFGIGADTQYQA

[0855] VTAAVDIRVTDTKSTRIVRAKSYSKQAVSYEVQAGVFRFIDYQRLLEGEVGYTSNEGLQAAVRWTLADATYDLVKELTGHKGACDVYLPKASQ

[0856] DDRAETTVVAAAN

[0857] >DNA sequence (SEQ ID NO:100)

[0858] ATGACCTTTGGCAAATTTCGCAGCCTGTTACTGGCGCCGGTGCTGTTACTGGCCGCGTGTCAGAGTCAGACCGCGGAACAAGCGCCGAAA

[0859] GAACCGTTTCTGGGCCCGGTGCCGATTGCGACCCGCACCCCGGTGGATGCGGCGCTGGAATGCCTGAGCAAAACCCCGGAAGTGCAGCG

[0860] CTATCCGCGCATGTTTGCGGTGCATGTGATTCAAGATGAAACCGGCCGCTATAGCATTGAAGAAGCGGGCAACTATCTGCCGCAAGATAGC

[0861] GCGGGCATGATGGTGAGCCTGCTGCAGAAAGCGGGCGTGAAACAAGTGAACCGCAGCAACACCGCGGTGAGCGAATGGGAAATTGCGC

[0862] GCGCGCGCGAACAGATTCTGGGCGATGGCGGCAACGTGGTTGTGGGCGATGAAAGCCTGCCGTTTCGCCCGCTGGTGAAAGGCGCGCTG

[0863] CGCGGCAGCGATTATGTGATTGATGGCGTGATTACGCAGCTGGATTTTAACACGTACAGCGGCGGTgttggggcaagatattttggcatcggtgccgacacgcaat

[0864] acCAAGCGGTGACCGCGGCGGTGGATATTCGCGTGACCGATACCAAAAGCACCCGCATTGTGCGCGCGAAAAGCTATAGCAAACAAGCGG

[0865] TGtcctatgaagttcaggccggggttttccgctttattgactaccagcgcttgcttgaaggggaagtgggttacacctcgaacGAAGGCCTGCAAGCGGCGGTGCGCTGGACCCTGGCGGA

[0866] TGCGACCTATGATCTGGTGAAAGAACTGACCGGCCATAAAGGCGCGTGCGATGTGTATCTGCCGAAAGCGAGCCAAGATGATCGCGCGGA

[0867] AACCACCGTGGTTGCGGCGGCCAAC

[0868] It should be noted that although specific examples have been used to illustrate the technical solutions of this disclosure, those skilled in the art will understand that this disclosure is not limited thereto. Various embodiments of this disclosure have been described above; the above descriptions are exemplary and not exhaustive, and are not limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical applications, or technical improvements to the embodiments in the market, or to enable other those skilled in the art to understand the embodiments disclosed herein.

Claims

1. Nanoporin mutants, among which, The mutant is selected from any one of the following groups (I)-(IV): (I) The nanoporin mutant contains a mutation at at least one of the positions corresponding to the 79th and 80th positions of the sequence shown in SEQ ID NO:1, compared to the sequence shown in SEQ ID NO:1; (II) has at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the amino acid sequence shown in (I), and does not include mutants of the sequence shown in SEQ ID NO:1; (III) A mutant encoded by a polynucleotide that can hybridize with the polynucleotide shown in (a) or (b): (a) A polynucleotide encoding a mutant with the amino acid sequence shown in (I); (b) and (a) full-length complementary polynucleotides; (IV) A fragment of a mutant shown in any one of (I), (II) or (III), wherein the fragment still possesses nanoporin activity.

2. The nanoporin mutant according to claim 1, wherein, The nanoporin mutant corresponds to the sequence shown in SEQ ID NO:1 and has a mutated amino acid at at least one of the following positions: S79N, S79W, S79I, S79A, S79V, S79L, S79Q, S79Y, S79E, S79K, E80Q, E80N, E80S, E80W, E80A, E80I.

3. The nanoporin mutant according to claim 1 or 2, wherein, The nanoporin mutant is selected from any one of the following groups (V)-(VIII): (V) The nanoporin mutant, compared with the sequence shown in SEQ ID NO:1, also contains a mutation at at least one of the positions 77, 182, 184, 186, 218, 220, 239, and 235 corresponding to the sequence shown in SEQ ID NO:1; The amino acid sequences shown in (VI) and (V) have at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity, and do not include mutants of the sequence shown in SEQ ID NO:1; (VII) A mutant encoded by a polynucleotide that can hybridize with the polynucleotide shown in (c) or (d): (c) Polynucleotides encoding mutants with the amino acid sequence shown in (V); Full-length complementary polynucleotides of (d) and (c); (VIII) A fragment of a mutant shown in any one of (V), (VI) or (VII), wherein the fragment still possesses nanoporin activity; Optionally, the nanoporin mutant corresponds to the sequence shown in SEQ ID NO:1 and has mutated amino acids at one or more of the following positions: Y77I, Y77T, Y77Q, Y77M, Y77D, Y77W, Y77V, Y77F, G182D, R184Q, F186Q, E218Q, F220Q, K239Q, K235E.

4. The nanoporin mutant according to any one of claims 1-3, wherein, The nanoporin mutants include the deletion or addition of at least one amino acid residue at the N-terminus or C-terminus of the mutants shown in (I) or (V), and / or the deletion of amino acid residues 133 to 138 in the sequences shown. Preferably, the nanoprotein pore mutant includes a mutant with 5 amino acid residues deleted at the C-terminus of the sequence shown in (I) or (V), and a mutant with the sequence shown has amino acid residues deleted from positions 133 to 138. More preferably, the nanoprotein pore mutant includes a mutant of the sequence shown in (I) or (V) that omits amino acid residues at positions 133-138 and 288-292; Optionally, the nanoporin mutant is selected from any one of the following groups (IX)-(VIII): (IX) The nanoporin mutant described herein, compared to the sequence shown in SEQ ID NO:1, further includes a protrusion at at least one of the following positions corresponding to positions 68, 71, 73, 88, 112, 115, 118, 119, 120, 123, 125, and 225 of the sequence shown in SEQ ID NO:1; and, Optionally, the mutant further comprises a modification of the β-barrel of the nanoporin as shown in SEQ ID NO:1; The amino acid sequences shown in (X) and (IX) have at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity, and do not include mutants of the sequence shown in SEQ ID NO:1; (XI) A mutant encoded by a polynucleotide that can hybridize with the polynucleotide shown in (e) or (f): (e) Polynucleotides encoding mutants with the amino acid sequence shown in (IX); (f) and (e) are full-length complementary polynucleotides; (VIII) A fragment of a mutant shown in any one of (IX), (X) or (XI), wherein the fragment still possesses nanoporin activity.

5. The nanoporin mutant according to claim 4, wherein, The nanoporin mutant also has mutated amino acid residues at one or more of the following positions corresponding to the sequence shown in SEQ ID NO:1: R225D, A119Q, E115Q, A112Q, I118Q, H68Y, T71Q, Q73E, R88Q, R120I, E123A, I125E; Optionally, the modification of the β-barrel includes modification of amino acid residues at positions 169–186 and / or positions 216–241 of the sequence shown in SEQ ID NO:1; Preferably, the modification of the β-bucket includes replacing positions 169 to 186 of the sequence shown in SEQ ID NO:1 with the sequence shown in SEQ ID NO:198; Preferably, the modification of the β-barrel includes replacing positions 216 to 241 of the sequence shown in SEQ ID NO:1 with the sequence shown in SEQ ID NO:

199.

6. The nanoporin mutant according to any one of claims 1-5, wherein, The nanoporin mutant corresponds to the amino acid sequence shown in SEQ ID NO:1, and has the following (m1)~( ... 48 Any mutation shown in any of the following: (m1)S79N; (m2)S79W; (m3)S79I; (m4)S79A; (m5)S79V; (m6)S79L; (m7)S79Q; (m8)S79Y; (m9)S79E; (m 10 )S79K; (m 11 )E80Q; (m 12 )E80N; (m 13 )E80S; (m 14 )E80W; (m 15 )E80A; (m 16 )E80I; (m 17 )Y77I、S79I; (m 18 )Y77T、S79I; (m 19 )Y77Q、S79I; (m 20 )Y77M、S79I; (m 21 )Y77D、S79I; (m 22 )Y77W、S79I; (m 23 )Y77V、S79I; (m 24 )Y77F、S79I; (m 25 S79I, deletion of amino acid residues at positions 133-138 and 288-292; (m 26 )S79I、R184Q; (m 27 )S79I、E218Q; (m 28 )S79I、K239Q; (m 29 )S79I、R184Q、E218Q、K239Q; (m 30 )S79I、R184Q、K235E; (m 31 )S79I、G182D、R184Q; (m 32 )S79I、R184Q、F186Q; (m 33 )S79I、R184Q、F220Q; (m 34 )S79I、R184Q、F186Q、R225D; (m 35 )S79I、R184Q、F186Q、A119Q; (m 36 )S79I、R184Q、F186Q、E115Q; (m 37 )S79I、R184Q、F186Q、A112Q; (m 38 )S79I、R184Q、F186Q、I118Q; (m 39 )S79I、R184Q、F186Q、H68Y; (m 40 )S79I、R184Q、F186Q、T71Q; (m 41 )S79I、R184Q、F186Q、Q73E; (m 42 )S79I、R184Q、F186Q、R88Q; (m 43 )S79I、R184Q、F186Q、R120I; (m 44 )S79I、R184Q、F186Q、E123A; (m 45 )S79I、R184Q、F186Q、I125E; (m 46 The amino acid sequences of S79I, R184Q, and F186Q, from position 169 to 186, are shown in SEQ ID NO:198, and the amino acid sequences from position 216 to 241 are shown in SEQ ID NO:

199. (m 47 )S79I、R184Q、F186Q、T71Q、Q73E、R88Q; (m 48 S79I, R184Q, F186Q, T71Q, Q73E, R88Q, the amino acid sequences from position 169 to 186 are shown in SEQ ID NO:198, and the amino acid sequences from position 216 to 241 are shown in SEQ ID NO:

199. Preferably, the nanoporin mutant corresponds to the amino acid sequence shown in SEQ ID NO:1 and has any of the following mutations: (m3)S79I; (m5)S79V; (m 11 )E80Q; (m 12 )E80N; (m 25 S79I, deletion of amino acid residues at positions 133-138 and 288-292; (m 26 )S79I、R184Q; (m 27 )S79I、E218Q; (m 28 )S79I、K239Q; (m 29 )S79I、R184Q、E218Q、K239Q; (m 32 )S79I、R184Q、F186Q; (m 34 )S79I、R184Q、F186Q、R225D; (m 35 )S79I、R184Q、F186Q、A119Q; (m 36 )S79I、R184Q、F186Q、E115Q; (m 37 )S79I、R184Q、F186Q、A112Q; (m 38 )S79I、R184Q、F186Q、I118Q; (m 39 )S79I、R184Q、F186Q、H68Y; (m 40 )S79I、R184Q、F186Q、T71Q; (m 41 )S79I、R184Q、F186Q、Q73E; (m 42 )S79I、R184Q、F186Q、R88Q; (m 43 )S79I、R184Q、F186Q、R120I; (m 44 )S79I、R184Q、F186Q、E123A; (m 45 )S79I、R184Q、F186Q、I125E; (m 46 The amino acid sequences of S79I, R184Q, and F186Q, from position 169 to 186, are shown in SEQ ID NO:198, and the amino acid sequences from position 216 to 241 are shown in SEQ ID NO:

199. (m 47 )S79I、R184Q、F186Q、T71Q、Q73E、R88Q; (m 48 The amino acid sequences of S79I, R184Q, F186Q, T71Q, Q73E, and R88Q, from position 169 to 186, are shown in SEQ ID NO:198, and the amino acid sequences from position 216 to 241 are shown in SEQ ID NO:

199.

7. Which of the following is correct (A) or (B): (A) Encoding isolated polynucleotides of the nanoporous protein mutant as described in any one of claims 1 to 6; (B) contains a recombinant expression vector of (A).

8. A recombinant host cell, wherein, The recombinant host cell comprises the nanoporin mutant as described in any one of claims 1 to 6, the isolated polynucleotide as described in claim 7, or the recombinant expression vector described therein. Optionally, the recombinant host cell is derived from microorganisms of the genera Escherichia, Erwinia, Serratia, Providencia, Enterobacteria, Salmonella, Streptomyces, Pseudomonas, Brevibacterium, Bacillus, or Corynebacterium. Preferably, the recombinant host cell is derived from Escherichia coli.

9. A cell culture comprising the recombinant host cell as described in claim 8.

10. A reagent kit, wherein, The kit is used to detect the presence or absence of one or more features of a target analyte, and it comprises the nanoporin mutant of any one of claims 1 to 6 and / or the nanoporin with the amino acid sequence shown in SEQ ID NO:1; Optionally, the target analyte is one or more of nucleic acids, peptides, or proteins; Optionally, the nucleic acid is at least one of single-stranded DNA, double-stranded DNA, RNA, and miRNA; Optionally, the target analyte is characterized by at least one of the following (A) to (E): (A) Length of the target analyte; (B) Characteristics of the target analyte; (C) Sequence of the target analyte; (D) Secondary structure of the target analyte; (E) Whether the target analyte is modified.

11. The use of the nanoporin mutant as described in any one of claims 1 to 6, the nanoporin with an amino acid sequence as shown in SEQ ID NO:1, the isolated polynucleotide or recombinant expression vector as described in claim 7, the recombinant host cell as described in claim 8, the cell culture as described in claim 9, or the kit as described in claim 10 in detecting the presence or absence of one or more features of the target analyte; Optionally, the target analyte is one or more of nucleic acids, peptides, or proteins; Optionally, the nucleic acid is at least one of single-stranded DNA, double-stranded DNA, RNA, and miRNA; Optionally, the target analyte is characterized by at least one of the following (A) to (E): (A) Length of the target analyte; (B) Characteristics of the target analyte; (C) Sequence of the target analyte; (D) Secondary structure of the target analyte; (E) Whether the target analyte is modified.

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