Promoter, promoter library, gene expression cassette, recombinant expression vector, recombinant cell and preparation method

Abstract

The invention discloses a promoter, a promoter library, a gene expression cassette, a recombinant expression vector, a recombinant cell and a preparation method. The provided promoter comprises a feature fragment 1 and / or a feature fragment 2; the sequence of the feature fragment 1 comprises VBCGHCDW, and the sequence of the feature fragment 2 comprises SNNSWWBBWB. The promoter provided by the invention shows the promoter activity in rhodococcus in a stable phase specific mode, can express a target gene in a stable phase without additionally adding an inducer, and does not influence the growth of cells.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a promoter, a promoter library, a gene expression cassette, a recombinant expression vector, a recombinant cell and a preparation method. Background Art

[0002] Rhodococcus erythropolis is a Gram-positive actinomycete with characteristics such as strong adaptability, high tolerance to organic solvents, and rich metabolic activity, and is widely used in the fields of biocatalysis, environmental remediation, and lignin utilization. However, in the production process of Rhodococcus whole-cell biocatalysts, the improvement of cell catalytic performance includes the improvement of cell permeability, cell confidentiality, cell morphology, etc., and there is a certain conflict between the expression of related genes and the realization of functions and cell growth. Summary of the Invention

[0003] Based on this, it is necessary to provide a promoter, a promoter library, a gene expression cassette, a recombinant expression vector, a recombinant cell and a preparation method.

[0004] In some embodiments, a promoter is provided, and the promoter includes characteristic fragment 1 and / or characteristic fragment 2;

[0005] The sequence of the characteristic fragment 1 includes VBCGHCDW, and the sequence of the characteristic fragment 2 includes SNNSWWBBWB;

[0006] Wherein, each S independently represents a G base or a C base, each W independently represents an A base or a T base, each H independently represents an A base, a T base or a C base, each B independently represents a G base, a T base or a C base, each V independently represents a G base, an A base or a C base, each D independently represents a G base, an A base or a T base, and each N independently represents an A base, a T base, a G base or a C base.

[0007] In some embodiments, the nucleotide sequence of the promoter includes the nucleotide sequence shown in Formula I or Formula II, wherein,

[0008] Formula I is (N) p VBCGHCDW(N) q SNNSWWBBWB(N) r ,

[0009] Formula II is (N) p SNNSWWBBWB(N) q VBCGHCDW(N) r ,

[0010] In each formula, p, q, and r represent the number of random bases, and p, q, and r are each independently selected from integers between 1 and 350:

[0011] Optionally, in each formula, p, q, and r represent the number of random bases, and p, q, and r are each independently selected from integers between 3 and 310.

[0012] In some embodiments, the nucleotide sequence of the promoter is shown as one of SEQ ID NO: 1 to SEQ ID NO: 54.

[0013] In some embodiments, a promoter library is provided, and the promoter library includes the promoter described above;

[0014] Optionally, the promoter library includes multiple promoters with nucleotide sequences shown as SEQ ID NO: 1 to SEQ ID NO: 54;

[0015] Optionally, the promoter library includes promoters with nucleotide sequences shown as SEQ ID NO: 1 to SEQ ID NO: 54.

[0016] In some embodiments, a gene expression cassette is provided, and the gene expression cassette includes the promoter and a target gene;

[0017] Optionally, the target gene includes one or more genes that inhibit cell growth;

[0018] Optionally, the target gene includes one or more of a gene encoding cavin, a gene encoding a nucleic acid degrading enzyme, and a gene encoding a cell elongation protein;

[0019] Optionally, in the gene expression cassette, the target gene is located downstream of the promoter.

[0020] In some embodiments, a recombinant expression vector is provided, and the recombinant expression vector includes the promoter or the gene expression cassette described above;

[0021] Optionally, the recombinant expression vector includes one or more of a plasmid vector, a phage vector, and a viral vector;

[0022] Optionally, the plasmid vector includes one or more of pNV18.1, pBNV, pNV18.1-derived plasmids, and pBNV-derived plasmids.

[0023] In some embodiments, a recombinant cell is provided, and the provided recombinant cell satisfies one or more of the following conditions:

[0024] The recombinant cell carries the promoter;

[0025] The recombinant cell carries the gene expression cassette; and,

[0026] The recombinant cell carries the recombinant expression vector.

[0027] In some embodiments, the host cell of the recombinant cell includes Rhodococcus;

[0028] Optionally, the Rhodococcus includes Rhodococcus ruber;

[0029] Optionally, the Rhodococcus ruber includes Rhodococcus ruber TH.

[0030] In some embodiments, a method for constructing a recombinant cell is provided, including the following steps: introducing the promoter or the gene expression cassette or the recombinant expression vector into the host cell to construct the recombinant cell.

[0031] In some embodiments, a method for preparing a target protein is provided, including the following steps: culturing at least one of the recombinant cell and the recombinant cell constructed by the construction method.

[0032] The promoter provided above shows promoter activity in a stationary-phase specific manner in Rhodococcus, and can express the target gene in the stationary phase without the need to additionally add an inducer, without affecting the growth of the cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the technical solutions in the embodiments and examples of the present application, and to more fully understand the present application and its beneficial effects, the following will briefly introduce the drawings required for description in the embodiments or examples. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative efforts.

[0034] Figure 1 It is a graph showing the changes in OD460 and glucose concentration during the fermentation of Rhodococcus ruber TH in Example 2;

[0035] Figure 2 It is an expression curve of the promoter P1-P9 expressing Mcherry in Example 2;

[0036] Figure 3 It is an expression curve of the promoter P10-P18 expressing Mcherry in Example 2;

[0037] Figure 4 It is an expression curve of the promoter P19-P27 expressing Mcherry in Example 2;

[0038] Figure 5 Expression curve of Mcherry expressed by promoter P28 - P36 in Example 2;

[0039] Figure 6 Expression curve of Mcherry expressed by promoter P37 - P45 in Example 2;

[0040] Figure 7 Expression curve of Mcherry expressed by promoter P46 - P54 in Example 2;

[0041] Figure 8 Expression curve of Mcherry expressed by promoter Pa2 in Example 2;

[0042] Figure 9 Effect of holin expressed by promoter Pa2 and late promoter P1 - P5 on cell growth in Example 3;

[0043] Figure 10 Effect of holin expressed by promoter Pa2 and late promoter P1 - P5 on cell permeability in Example 3;

[0044] Figure 11 Effect of Ddel expressed by promoter Pa2 and promoter P10, P15, P20, P25, P30 on cell growth in Example 4;

[0045] Figure 12 Effect of Ddel expressed by promoter Pa2 and promoter P10, P15, P20, P25, P30 on cell viability in Example 4;

[0046] Figure 13 Effect of DivIVA expressed by promoter Pa2 and promoter P6, P12, P18, P24 on cell growth in Example 5;

[0047] Figure 14 Effect of DivIVA expressed by promoter Pa2 and promoter P6, P12, P18, P24 on cell morphology in Example 5;

[0048] Figure 15 Effect of DivIVA expressed by promoter Pa2 and promoter P6, P12, P18, P24 on cell length in Example 5;

[0049] Figure 16 Expression curve of Mcherry expressed by promoter T1 to T4 in Comparative Example 1. Detailed implementation manners

[0050] To facilitate the understanding of the present invention, the present invention will be described more comprehensively below with reference to the relevant accompanying drawings. Preferred embodiments of the present invention are shown in the accompanying drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided so that the understanding of the disclosure of the present invention is more thorough and comprehensive.

[0051] It should be noted that when an element is referred to as being "fixed to" another element, it can be directly on the other element or there can also be an intermediate element. When an element is considered to be "connected" to another element, it can be directly connected to the other element or there may be an intermediate element at the same time.

[0052] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs. The terms used in the description of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0053] The term

[0054] Unless otherwise stated or there is a contradiction, the terms or phrases used herein have the following meanings:

[0055] The selection scope of the terms "and / or", "or / and", and "and / or" used in this application includes any one of two or more related listed items, and also includes any and all combinations of the related listed items. The said any and all combinations include combinations of any two related listed items, any more related listed items, or all related listed items. It should be noted that when at least two conjunctions selected from "and / or", "or / and", and "and / or" are used to connect at least three items, it should be understood that in this application, this technical solution undoubtedly includes the technical solution connected by "logical AND", and undoubtedly also includes the technical solution connected by "logical OR". For example, "A and / or B" includes three parallel solutions: A, B, and "the combination of A and B".

[0056] In this application, when it comes to "multiple", "diverse", "multiple times", "pluralistic", etc., unless otherwise specifically defined, it means greater than 2 or equal to 2 in quantity. For example, "one or more" means one or greater than or equal to two.

[0057] The "combinations thereof", "any combinations thereof", "any combination manners thereof", etc. used in this application include all suitable combination manners of any two or any two or more of the listed items.

[0058] In this application, the "suitable combination method", "suitable method", "any suitable method", etc., the "suitable" mentioned therein shall be based on being able to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0059] In this application, "preferred", "better", "more preferable", "preferably" are only used to describe the implementation manners or embodiments with better effects. It should be understood that they do not constitute a limitation on the protection scope of this application.

[0060] In this application, "further", "even further", "especially", etc. are used for descriptive purposes, indicating differences in content, but should not be construed as a limitation on the protection scope of this application.

[0061] In this application, "optionally", "optional", "option", mean that it can be either present or absent, that is, it refers to any one of the two parallel options of "present" or "absent". If "optional" appears multiple times in a technical solution, without special instructions and without contradictions or mutual restrictions, each "optional" is independent of each other.

[0062] In this invention, regarding "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc., the terms "first", "second", "third", "fourth", etc. are only used for descriptive purposes, and cannot be understood as indicating or implying relative importance or quantity, nor can it be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumerative description, and it should be understood that they do not constitute a closed limitation on quantity.

[0063] In this application, for the technical features described in an open-ended manner, it includes the closed technical solutions composed of the listed features, as well as the open technical solutions containing the listed features.

[0064] In this application, regarding the numerical interval (that is, the numerical range), without special instructions, the optional numerical values are considered continuous within the above numerical interval, and include the two numerical endpoints of this numerical range (that is, the minimum value and the maximum value), as well as each numerical value between these two numerical endpoints. Without special instructions, when the numerical interval only refers to the integers within this numerical interval, it includes the two endpoint integers of this numerical range, as well as each integer between the two endpoints. In this article, it is equivalent to directly listing each integer. For example, t is an integer selected from 1 to 10, indicating that t is any integer selected from the integer group composed of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In addition, when providing multiple range descriptions of features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed in this article should be understood to include any and all sub-ranges subsumed therein.

[0065] The temperature parameters in this application, unless otherwise specified, allow for either constant temperature treatment or variation within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within ranges such as ±5°C, ±4°C, ±3°C, ±2°C, ±1°C are allowed.

[0066] In this application, %(w / w) and wt% both represent weight percentage, %(v / v) refers to volume percentage, and %(w / v) refers to mass-volume percentage.

[0067] "Room temperature" in this application generally refers to 5°C to 30°C, preferably 25 ± 5°C.

[0068] In this application, the "target gene" refers to any gene that is linked to a promoter herein for regulating its transcription level.

[0069] In this application, the term "vector" refers to a nucleic acid molecule that can transport or transfer foreign nucleic acid molecules. This term encompasses both expression vectors and transcription vectors. The term "expression vector" refers to a vector that can express an insert in a target cell and usually contains control sequences (such as enhancer, promoter, and terminator sequences) that drive the expression of the insert. The term "transcription vector" refers to a vector that can be transcribed but not translated. Transcription vectors are used to amplify their inserts. Foreign nucleic acid molecules are referred to as "inserts" or "transgenes". Vectors usually consist of an insert and a larger sequence that serves as the backbone of the vector. Based on the structure or source of the vector, the main types of vectors include plasmid vectors, cosmid vectors, phage vectors (such as λ phage), viral vectors (such as adenovirus vectors), and artificial chromosomes.

[0070] In this application, the term "plasmid" refers to an extrachromosomal element that often carries genes not part of the cell's core metabolic machinery and is usually in the form of a circular double-stranded DNA molecule. These elements can be autonomously replicating sequences, genomic integration sequences, phages, or nucleotide sequences of any origin, linear, circular, or supercoiled single-stranded or double-stranded DNA or RNA. Usually, a plasmid contains an origin of replication that functions in a host cell (such as Escherichia coli), as well as a selection marker for detecting host cells containing the plasmid. In some embodiments, the plasmid is a closed-loop DNA molecule. A "co-expression plasmid" refers to different types of plasmids that can be expressed in the same host bacterium.

[0071] In this application, the term "nucleic acid" refers to any linear or sequentially arranged nucleotides and nucleosides, for example, cDNA, genomic DNA, mRNA, tRNA, oligonucleotides, oligonucleosides, and their derivatives. Nucleic acids can include bacterial plasmid vectors, which include expression, cloning, cosmid, and transformation vectors, and nucleic acids can include modified or derivatized nucleotides and nucleosides.

[0072] In the present application, the term "nucleotide sequence" refers to oligonucleotides, nucleotides or polynucleotides and their fragments or portions, and refers to DNA or RNA of genomic or synthetic origin, which may be single-stranded or double-stranded and represents the sense or antisense strand. The terms "polynucleotide", "oligonucleotide", "nucleotide sequence" and "nucleic acid" may be used interchangeably herein and include, but are not limited to, coding sequences. That is, one or more polynucleotides or one or more nucleic acid sequences that are transcribed and translated into polypeptides in vitro or in vivo when placed under the control of appropriate regulatory or control sequences; control sequences such as translation initiation and termination codons, promoter sequences, ribosome binding sites, polyadenylation signals, transcription factor binding sites, transcription termination sequences, upstream and downstream regulatory domains, enhancers, silencers, DNA sequences to which one or more transcription factors bind and positively (induce) or negatively (repress) alter the promoter activity of a gene, etc.

[0073] In the present application, the term "expression" refers to the conversion of sequence information into the corresponding expression product, including direct transcription products (such as mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA or any other type of RNA) or proteins produced by translation of mRNA.

[0074] In the present application, the term "encoding" means that a DNA polynucleotide sequence can be transcribed into RNA (mRNA) that translates into a protein; or can be transcribed into RNA that does not translate into a protein (non-coding RNA such as tRNA, rRNA, etc.); or an RNA polynucleotide sequence that can be translated into a protein.

[0075] In the present application, the term "DNA", "RNA" or "nucleic acid" or "nucleic acid fragment" or "polynucleotide" refers to any one or more nucleic acid segments present in a polynucleotide or construct. The nucleic acid or its fragment can be provided in linear (such as mRNA) or circular (such as plasmid) form, and in double-stranded or single-stranded form. An "isolated" nucleic acid or polynucleotide refers to a nucleic acid molecule, DNA or RNA that has been isolated from its natural environment. For example, in the context of the present invention, the recombinant polynucleotide contained in the vector is isolated. Other examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or purified polynucleotides in solution, etc.

[0076] In the present application, "gene" refers to a polynucleotide containing nucleotides encoding a functional molecule, where the functional molecule includes a functional molecule produced only by transcription (bioactive RNA) or a functional molecule produced by transcription and translation (e.g., polypeptide). The term "gene" includes cDNA and genomic DNA nucleic acids, and also refers to a nucleic acid fragment that expresses a specific RNA, protein, or polypeptide, which contains regulatory sequences before (5' non-coding sequence) and after (3' non-coding sequence) the coding sequence.

[0077] As used herein, "host cell" refers to any cell type that is readily transformable, transfectable, transducible, etc. with a transcription initiation element containing a promoter or an expression vector. "Recombinant cell" encompasses a host cell that is different from the parental cell after introduction of a transcription initiation element or a recombinant expression vector.

[0078] A promoter with temporal variation may be affected by changes in the concentration of culture medium components (carbon source, nitrogen source, phosphorus source) in terms of the principle of gene expression regulation, may be induced by stress such as starvation and dissolved oxygen, may be regulated by a quorum sensing system, may also be induced by metabolites during fermentation, and others may change following the fermentation cycle under the regulation of the σ factor network.

[0079] The promoter induced by nutrient source starvation introduced exogenously, as well as the homologous elements of the dynamic regulation system in other strains, did not show obvious changes in temporal characteristics in Rhodococcus. When the exogenous promoter was introduced, the relevant regulatory elements were missing, or the regulatory elements and regulatory modes of these promoters in Rhodococcus were different from those of the source strains.

[0080] In some embodiments, a promoter is provided, and the provided promoter includes feature fragment 1 and / or feature fragment 2;

[0081] The sequence of feature fragment 1 includes VBCGHCDW, and the sequence of feature fragment 2 includes SNNSWWBBWB;

[0082] Wherein, each S independently represents a G base or a C base, each W independently represents an A base or a T base, each H independently represents an A base, a T base, or a C base, each B independently represents a G base, a T base, or a C base, each V independently represents a G base, an A base, or a C base, each D independently represents a G base, an A base, or a T base, and each N independently represents an A base, a T base, a G base, or a C base.

[0083] In some embodiments, the nucleotide sequence of the promoter includes the nucleotide sequence shown in Formula I or Formula II, wherein,

[0084] Formula I is (N) p VBCGHCDW(N)q SNNSWWBBWB(N) r ,

[0085] Formula II is (N) p SNNSWWBBWB(N) q VBCGHCDW(N) r ,

[0086] In each formula, p, q, and r represent the number of random bases, and p, q, and r are each independently selected from integers between 1 and 350.

[0087] In some embodiments, in each formula, p, q, and r represent the number of random bases, and p, q, and r are each independently selected from integers between 3 and 310.

[0088] In some embodiments, the nucleotide sequence of the promoter is as shown in one of SEQ ID NO: 1 to SEQ ID NO: 54.

[0089] In some embodiments, a promoter library is provided, and the promoter library includes the aforementioned promoters.

[0090] In some embodiments, the promoter library includes multiple promoters with nucleotide sequences as shown in SEQ ID NO: 1 to SEQ ID NO: 54.

[0091] In some embodiments, the promoter library includes promoters with nucleotide sequences as shown in SEQ ID NO: 1 to SEQ ID NO: 54.

[0092] In some embodiments, a gene expression cassette is provided, and the gene expression cassette includes a promoter and a target gene.

[0093] In some embodiments, the target gene includes one or more genes that inhibit cell growth.

[0094] In some embodiments, the target gene includes one or more of a gene encoding cavin, a gene encoding a nucleic acid degrading enzyme, and a gene encoding a cell elongation protein.

[0095] In some embodiments, in the gene expression cassette, the target gene is located downstream of the promoter.

[0096] In some embodiments, a recombinant expression vector is provided, and the recombinant expression vector includes a promoter or a gene expression cassette.

[0097] In some embodiments, the recombinant expression vector includes one or more of a plasmid vector, a phage vector, and a viral vector.

[0098] In some embodiments, the plasmid vector includes one or more of pNV18.1, pBNV, pNV18.1-derived plasmids, and pBNV-derived plasmids.

[0099] In some embodiments, a recombinant cell is provided, and the provided recombinant cell carries the aforementioned promoter.

[0100] In some embodiments, a recombinant cell is provided, and the provided recombinant cell carries the aforementioned gene expression cassette.

[0101] In some embodiments, a recombinant cell is provided, and the provided recombinant cell carries the aforementioned recombinant expression vector.

[0102] In some embodiments, the host cell of the provided recombinant cell includes Rhodococcus.

[0103] In some embodiments, Rhodococcus includes Rhodococcus ruber.

[0104] In some embodiments, Rhodococcus ruber includes Rhodococcus ruber TH.

[0105] In some embodiments, a method for constructing a recombinant cell is provided, including the following steps: introducing the aforementioned promoter, or the aforementioned gene expression cassette, or the aforementioned recombinant expression vector into a host cell to construct a recombinant cell.

[0106] In some embodiments, a method for preparing a target protein is provided, including the following steps: culturing at least one of the recombinant cell and the recombinant cell constructed by the construction method.

[0107] Using the provided promoter, a method for Rhodococcus ruber to express a gene with an inhibitory effect on growth was developed and tested and evaluated for the Rhodococcus ruber chassis. When Rhodococcus ruber carries functional genes such as caveolin, nucleic acid degrading enzyme, and cell elongation protein, etc., the expression intensity of the corresponding protein in the late fermentation stage (when Rhodococcus ruber grows to the stationary phase) is more than 10 times that in the early and middle fermentation stages (when Rhodococcus ruber grows to the logarithmic phase), reducing the impact of expressing the gene with an inhibitory effect on growth on the cell growth of Rhodococcus ruber; and further achieving a maximum increase in cell permeability of up to 345% with little impact on growth, reducing the cell survival rate to 10 -4 , and extending the cell length up to 2.43 times. The provided promoter enables Rhodococcus to realize the gene function while not affecting cell growth during the process of expressing the gene with an inhibitory effect on growth.

[0108] The following examples facilitate a better understanding of the present invention, but are not limited thereto.

[0109] The following experimental methods are all conventional methods unless otherwise specified; experimental reagents can all be obtained from commercial sources unless otherwise specified.

[0110] The Rhodococcus ruber TH used in the examples was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on February 27, 2008, with the deposit accession number CGMCC No. 2380. This strain has been disclosed in the prior art CN101663389A.

[0111] The plasmid vector pNV18.1 used in the examples has been disclosed in Chiba, et al., Jpn J Infect Dis 2007, 60(1), 45 - 47.

[0112] The plasmid vector pNV18.1 - Pa2 used in the examples has been disclosed in the prior art CN110499274A. The plasmid vector pNV18.1 - Pa2 in this application is pNV18 - Pa2 in this prior art.

[0113] The formula of the kanamycin seed medium used in the examples is: glucose 10 - 50 g / L, yeast extract 1 - 4 g / L, peptone 1 - 10 g / L, KH 2 PO 4 0.2 - 3 g / L, K 2 HPO 4 0.2 - 3 g / L, MgSO 4 ·7H 2 O 0.2 - 3 g / L, monosodium glutamate 1 g / L, pH value 7.5.

[0114] The formula of the fermentation medium used in the examples is: glucose 10 - 50 g / L, urea 5 - 20 g / L, yeast extract 1 - 4 g / L, peptone 1 - 10 g / L, KH 2 PO 4 0.2 - 3 g / L, K 2 HPO 4 0.2 - 3 g / L, MgSO 4 ·7H 2 O 0.2 - 3 g / L, monosodium glutamate 1 g / L, CoCl 2 0.03 - 0.5 mM, pH value 7.5.

[0115] The nucleotide sequences of promoter sequences P1 to P54, as well as the mcherry gene, the holin gene, the nucleic acid degrading enzyme Ddel gene, and the cell elongation protein divIVA gene are as follows. The underlined and bold parts in the promoter sequences P1 to P54 are characteristic sequence 1 and characteristic sequence 2, where the italic part is characteristic sequence 1 and the non-italic part is characteristic sequence 2.

[0116] SEQ ID NO:1 (P1)

[0117] GACCAAACAGGAGAACCCTTCATGTACGGCGTCGCCGCGACCGCCGCCGACGCCGACC

[0118] GGATCCGCCTTGAGGTGGTATCCGGTGGAGTGGCCGCATTGCCCACCTG

[0119] TTCGATCCGACGTGCTGCGAGGTGGCTCTGGCCCCCAGCTCCGGGGAGCGCTGCACC

[0120] GTAGGCCCTGCCGAGCGTGACCCCGGTCCGATGTGAAAAGCCCATATACCACATAACA

[0121] TTTCGGTTTCACGGTATGTCATTCGCGCTGTAGTCCCCCTCCGCCTGAATAGCATTCCA

[0122] GTCGACCGGTTTTCATGGACGTCCGCC GTCGATTTCGGAAGGGAGGGAACC。

[0123] SEQ ID NO:2 (P2)

[0124] GGCCAGGACGAGGGCGGATTCCACTCGCTCGGATCGTCGCATCAGGGGATTGCCGCTC

[0125] CACGGAGCGCGGCGCCACAACCGGAGGGCCAGGGGCTGCTCTGCGTTCATCTCGATAC

[0126] TCCTGCAGTTG CAGTGTTCCGCCGCCGACGGCCGGTGGTGTAGAGCACAA

[0127] GGTCACCGCTCGATTCGGGTGGTCGCGAGAAGGAGCCGGGGCCTCCGCCCGGCGGTGA

[0128] GATCGAG CCCTGGGGAGAGGCCGGTCGCGGTGTCATTCTCGATGTGAG

[0129] TCGCCCGGGTCGTCGGGAGCTCCGCCCCACCCGGGGTCCGCACACAGAGAGGTGCACATC。

[0130] SEQ ID NO:3(P3)

[0131] GACTTTTCGAGCCGGGCCGCGAAATCGTGCATGAACTGCATGTGGGCCGAGATCTCCG

[0132] CCGGCGTCCACTGGTCCATGGGCACGTCGTTGACGGCAGCCGGGGCGCCGCGGTAGTG

[0133] CTTGAGCAGCAGGTACTTGGCCATCGTGTTCTCCTCGGTGCTGGTGCGACCCATTGTGG

[0134] TCGCATTCGATTCGGGGACGGAGCCGGTCGCGGGTT CGCCGTCCGAATTCT

[0135] CTCGAATTCGGCCCGAATCCGGGATGCGCCGCCCGGAGCCGAAAGGTCGCGTTTCTCC

[0136] GATATGCG GTGGCGCAGGGACTACCATCGAATACGGCGAGGGAACCCG。

[0137] SEQ ID NO:4(P4)

[0138] CGGTGACGAGATAGACGCCGCGCTGGAAGCCGGTCAG ACCGGCTCTGGAA

[0139] CGGCAGGGTGAGCAGGAAGCCGGTGAGGAGCTGGACGCCGGTCTGCACGACGCGCAG

[0140] CTCCTGCAGCAGACTCGACCAGTTCCGGTCCAGTCGCTGCACGGGCGTCTCGTGCCGG

[0141] ACCCGCTTGTTCCAGTCCTCGTTCGGAACCGCTGTGGGCTGCACTCTCTCGGCATGCCC

[0142] CGTACGGCGCTC CCCGGTAGCACCACCGTCCTCTTGTGACCTGTGTCA

[0143] CTGTGGGGGTACTGTCGGCTCCACCAGGGACAACGTCGCTCCGAGGAAAGGAGGGCGGTC。

[0144] SEQ ID NO:5(P5)

[0145] CTGGTACAGCGGGTAGAGCGGGGAGGGAGCGGACGACGACGCGGTGAGCAGGAAGCT

[0146] GGCCGTGCCGACCGCCAGGAAACCGTGACGGCGTGGCAGGGAGCGACCC

[0147] CTCCGGCATCGGACCTCCTCGGAACTCGTGGGAACGGTCGACAGTTCGTTGACCAAGG

[0148] CAACGAACTAGTGGAACATGCCGCCCTCGGCG GCCCGGTCACTTCCCG

[0149] GTAAAAACCGCCTCTCACCTGCGCTCGAGTGTTATCGCAGTGCAACGTCCCGGCCGTTG

[0150] ACCGACCGGTCGATCAGGTCTATACCGAAGGCAGGACCCGATACGCACGAGGAGGCTCGAC。

[0151] SEQ ID NO:6(P6)

[0152] GGTGTGATCGCGGAACAGGTCGACCAAGATCTGGAACAGCACGATCAGGTAGGCCAC

[0153] GAAGGCGAA GTGTACCACACGTAATCCCAGAACGAATCCATGATGTG

[0154] CACCTCTCTGTGTGCGGACCCCGGGTGGGGCGGAGCT CCCGGGCGACTCA

[0155] CATCGAGAATGACACCGCGACCGGCCTCTCCCCAGGGGACAAAGACCCTCGATCTCAC

[0156] CGCCGGGCGGAGGCCCCGGCTCCTTCTCGCGACCACCCGAATCGAGCGGTGACCTTGT

[0157] GCTCTACACCACCGGCCGTCGGCGGCGGAACACTGTAGTCGAGCAACTGCAGGAGTATCGAG。

[0158] SEQ ID NO:7(P7)

[0159] GAGGCCTGGCAGCGGGTGGCCGAACTCGAGGCCGATCTCGCGATGGTGCGCGCCGAAC

[0160] TG GCGGGCCACCCAGCTGCGCGAGGCGCGGATCCACATCGCCGAGCTGCG

[0161] GATGCAGGTGGCGGAACTGCGGCGCGAACGCGGCCGCCCCGGACCGGATCCCGACT

[0162] CGCCGAACGCGTAGGTCGAATCCGAACGATCGTTTGGAGGGCTACCGG

[0163] GGGTGCGAACGTTCGGATCTCGGGGGTCGGGACTTCCGGCCCTGTCGGGCGCGTCCCG

[0164] CACGGAGTCGAATGGACACGGACCGACCTTCCGCCGATCCGTGTCCCAGGAGAGGAGCAGAC。

[0165] SEQ ID NO:8(P8)

[0166] CGATGCCGCG GATCGCGCAGCAGGGGCAGCGCCGACCCGTCGGGCGGAC

[0167] GGTCGGCGGGGAACTCGATGACGAGATAGTCGACCGGACCCATCTCGTCGATGTCGGA

[0168] ATCGGTCACCGGGTGCCTCGTTTCAGCGACGGGATCGGTCTTCGGCAGCAGTTCGGGC

[0169] GGTTTCACGGCGCCACACCCGCATGGTGGCGACGCCGACCGGGGAACGGAGAACCGG

[0170] TCCACTG GATAGCTCGCACATGTCTGCGCGCATCCGGTTTTCGGGCAG

[0171] GCTGTCTTCGGGCAGGCAACCGGATTGCTCCCGCCTCGTCGCCGAAAGAAGGAGCGCGAGGG。

[0172] SEQ ID NO:9(P9)

[0173] GACGTCAGGTCGGCCAGCGCGGCGGCCCACCGACCCTCCCGCAGGGCCTGGTCCCCAC

[0174] GTAGGCGCACTGCATCCACAG CGCCGCCGTGACCTGTGCCGATACCTC

[0175] AATTTCGTCTCCGGCGCGCACGTTTGCTACAGTTTGCGGGCGGTTCCGAACGGAACCCGCGGGGCTGTGGCGCAGTTGGTAGCGCGCTTCGTTCGCATCGAAGAGGTCAGGGGTTCGATTCCCCTCAGCTCCACCACCGAAGGGCGTCTCCCGGGCCGGGAGGCGCCTTTC TGTCGTGCCGCCGATTGCGGGCCGAACAGCCGCGCGGTCGGGGAGACTGTGTGAC。

[0176] SEQ ID NO:10(P10)

[0177] GGCAGGT CGCGGCCAGGAAGCGTTCGCACGGTGGCGTGGCACCGAGA

[0178] TCGAGCACGCTGAGGAGCCGCTCCGATTCGCACAGTCGGCACTTCATTGATCACACCC

[0179] CTCGGGCAGTCGGCCGGGGACCGCGCTTGCGAGATTTTGCAACATCGGGTCCGACTCA

[0180] ACTAGATAACCATACTGAGAGATCGAACGATAGCCTTAAACAAACCTCAATATTGTTT

[0181] TGGCGGCTGTTT TCGGTAGACACACCTAGACTCGGGACTATTCACTCCCCC

[0182] GCTCGTGGGTCGGGCACGCCGGGGGCGAGGCGTACTGTGAGGTCCTGGGGAGGTCTCCCG。

[0183] SEQ ID NO:11(P11)

[0184] AAGCCCATATACCACATAACATTTCGGTTTCACGGTATGTCATTCGCGCTGTAGTCCCC

[0185] CTCCGCCTGAATAGCATTCCAGTCGACCGGTTTTCATGGACGTCCGCC GTC

[0186] GATTTCGGAAGGGAGGGAACCATGAA TACACAGCTCCCACGCTGCTC

[0187] AGCCGCGGGGCCTTTTCCCAGATCACCTCTGGGGGAGGGATCTGGTGGGTCGAGTGGG

[0188] TAGGTAAGTTCAACTGACTCACGCGAGGTGCTCATGGACAGCGACGTCGATTCGTTCG

[0189] TGCTCATCCGAGGCGACCATCAGCCGGCCGTGGTCCGTTAGCACCTCGCTCAGGCACGA。

[0190] SEQ ID NO:12(P12)

[0191] CGGTTCCGACCGACGGACTGCGGGCCGCACGCAGCGGCCCGTTCCGGCAGGTCCGCCA

[0192] GTTCTGCCGGATTTGTCAGAAGCCGGGAGTACCCTGAAGTGGCGCCGGCCGGTTCCGG

[0193] GTCGGGGCGCCTTGGGGAACTGCT TGCGCACCCCGCCGTGCCGGGGC

[0194] ACCGTCGTCCACGGCCGGACCACGGTCCGCGCCGCGGGGGCGCTCGCCGCCCGGATCC

[0195] ACCGCCGGATCGCGCCGATAGACCACATCTGACGGACGCCGCCCCCGCCACCACCGGC

[0196] GGCACGGATGTCCCCCACCGGATTCACTCCAT CGTTCCCGAAAGGACGTTGTC。

[0197] SEQ ID NO:13(P13)

[0198] GATCTCCTCGGTGCTCATGGCCGAGCCGGCAGTGGCCGAGGCGCGGCGGGAGGCGGCG

[0199] GAGGCGAACAAGGTGGTCTGCATGACGTTTCTCTGTCCTAACTCTCTTCAGCGCGTGCT

[0200] GCTTTCACAGTGGCATCCAAGTCTTCCCGGAGCACAGGTTTTCGAAACCTGTAGCAGA

[0201] GATTTCACATTTCCTGGTCCGTTCTGTCCGGATCCGGCTCGGCATCCCGTTCGGTCACG

[0202] GCCCGTG TACCCGGATTCGCCTGCGAGGCCG GTACTGTGA

[0203] TAGCTGTCACACAGAGTCGGCAGGTGTCGACGTGCCGACCGACCTCTCGGAGGTACGA。

[0204] SEQ ID NO:14(P14)

[0205] CACGGGCAGGGTGGAGGTGCCCTTCGGTGCGAAACCCGGCGA ATCTG

[0206] GATCGACTCGAACGGTGATCCCGTCGCCGCGCCCAGAACAGGCTCGGAC A

[0207] GCCGGTGTCGTTGCCGCGCTCACGGTGTGGGGGATGGGCTCCGGTGTCGCGCTGCTGCT

[0208] CCTCTACGGAGTGCACCGGGCCGGTGCGAAGCGGAGGATCTCGGCCCTGGATCGGGAG

[0209] TGGAACGACGTCGGAAGGAGCCCGGGATGGTCGATGGGTTGACCGTCGGGATGCCGGT

[0210] CGGCGCGAAGCGGCATTCTCGATCTTCTTGCCGGGTAAGATCTTGCGTCGGGAGGCCCGG。

[0211] SEQ ID NO:15(P15)

[0212] CGGCCGAGCCGTCGGCCTGCGGGGAA CGAGACGGTCGACGGAACCCAGGATGTCGGCATCGCTGATGGGATTCCTCTTTCCACTGTGCCGGGAACGGGTGGTCGGGCCGCGTCCGGCTGTGTCGACGGGGTGACTGGTGCGCACGACCGGCAGTGCCCGTTGTTCGCCGCTCGGCCGCGGACGACTCGCGCTTCGGTCGATGTGGTGACCGAAGACCTTTCGGATCCCTCCCGCGCAGCCGTGCGGTCGGGATCTCAC AACTCGATGCGGGCCGGTCGGTGCCGCGCGCACAATACCGTACGTCGGGCCGCATCGAAGGGAAGGCGATTGGC。

[0213] SEQ ID NO:16(P16)

[0214] CACCACACTGGCAGTGTGGGGGTCAGGGGTTCGAGTCCCCTTAGCTCCACTCCCGCAC

[0215] CCTCTCCACCCCCTCCGGGACGC GTGCGGCCGGACCGGTATCCCGGGC

[0216] AAT TTTCCCCCTCGGAACCTGCTTCATGCTCTCTCGTAGCGGCGCACACTG

[0217] TGATGGATCGCTTCGAAAACGGGAGAGGTGAGCTGTCGTGTCCGAAGGTTCGAACATT

[0218] ACCCCGGGTTCTTCTGCCGGTGCCGGTGCCGGTGCCGCACCGGGTGCGGGTGCTACTGCCGGTTCGGGTGCTACAGCCGGTTCGGGTGCTACTGCCGGTTCGGGTGCTACTGCCGGT. SEQ ID NO:17(P17)

[0219] ATGTACGGTCGTCCCGCAAAAGGGGCCACATGGATGCGAGATGAGCATGCCGGGTGCT

[0220] GGTGCCGACGCTGTCACG TTACCACCGATTTCGGTGGCGATGTACCAC

[0221] CCGCGGTCCTCGAGGCCGCGGTGGTGGTGGGCGTCGGTAATTCGAGCGGGGTTCCGGA

[0222] GTCGGATCTGCCGGAACCCGGGGGACGGTTCCTCCGTCGGGGTCCGCTGAACTCGGTC

[0223] GAGTCGCGGACGACGGCGGTACCGTAGCGCGCCGGG AGCAGGATCCGAG

[0224] AATTTCATTCCGGCCGGCTTCGGGAAATCACCTTGCCGGTCTGGTCGAGGAGGCTGACCTG。

[0225] SEQ ID NO:18(P18)

[0226] CTAGGAGGACCCCATGTGTTACCCCGTCACCTGCCCGAACTGTGGGAAGACCGGCTGG

[0227] GGCGGCTGCGGCCAGCATGTCGAT GCGTTCCGTGCCCGCACCCGAGCGCT

[0228] GCACCTGCGGCCAGGACACCACCCCCGGTGCGCAGGCACACACCCTCGGCGGACTCTT

[0229] TCGCCGCTGACCGCACCTCGACGAGACTGCGCCTCCACCGCATTCTCCGCCGCGGCAA

[0230] CACCCCGCCCTGCGCGAGCTGGGCGGGGTGTTCGCGTCGCGGTCCGAG

[0231] CCTTCCGCGGTCCGCGTGCCGGTGAAAAACTCGTAGACGCCCACTCGAGAAGGGACATCC。

[0232] SEQ ID NO:19(P19)

[0233] GACTTCGACGTCGCCCCCGTGCTGCGCGCCGTGGCCGACCGGAATCCCCACGCCCTGA

[0234] TGTTCGGCACCGATCTACCGTCCACCCGGGCCCCGCGGCCGTTCCTCGACGCGGACGT

[0235] ACCGACACCCTGGGCCCGGAACTCGCCGCGCGGGCACTCCACGACAAT

[0236] GCCGCGCAGTTCTACCGCGTGCGTTGACATCCCCGGCAACCAAGATCTGCGAAACGTC

[0237] GTCTGCTCCGCCGTCCGCGATCTACACTCGGG TCGGTGCGCGGGGCGCGCC

[0238] GCCGGCGGGGCGCGGCGGTCGGCACCCGGACGTGACATTCCGATCCCAGGAGCATCAGC。

[0239] SEQ ID NO:20(P20)

[0240] GATGCCCCTGCTGATGAGCTCGATCAACCGGGACCTCGGCCTGACCGGCACCGTGTGG

[0241] GAAAACCTCCCGCAGACCACGGGTCAGGGTCTGATCGTCTTCATGTCCGACGGC

[0242] GCGCGCCACCGTCGACCGCGCGCCCGTCGTCGAGGTCGACGGGTACGGCACCCC

[0243] CGAGGCCCTGGTCGAGGCGAACCCGGGCACCGACCGGCT GCCGGCCA

[0244] CTGATCCGCCCCGGTGGGACCTCCACCCGCCCGATTCGAGGCCCAATGGCCATTTTCCG

[0245] ACGGCGCTGCGGGTGAGACGCTGGACCCGGCACCCACCGATCGGCGAAAGGGCCGAACCA。SEQ IDNO:21(P21)

[0246] TCGAGCAGTTGTCGTTCGGTGACGCGTAGTT GATCCGGCC

[0247] CGTAGATCAGGACTGTCAGCAGTAGTACGACAGGTACGGACGCAAGGAATTCCATGTC

[0248] GGCCTCCAGCGGTGGTCCGCGTAGGCGGGAAGGTGGAGGTGCTCGGGTGCGTCGTCGG

[0249] TGACCACTCCAGCGTACGGACCCGCACGGGGTGCCTGTCGGGACGGCCACCGCGCCGC

[0250] TCCGTGATCTGTCGCACACCCTCGGGTTACTCGCCGGTACCGATCCGGCGAATATCGTG

[0251] ATGCACGTCACAAAACGGGTGGATACTCGCCGGTAGCAGTCCTATCGGGGAGGCATCAC。

[0252] SEQ ID NO:22(P22)

[0253] GAGTGTGCGCGCCAGCCCGAAGAACGTGATGGCGCACACCAAGCAGACCGTGATGGA

[0254] GAAGGTGAACCA AAGTACGCGCTCGTCGCGGTGGGC CGG

[0255] TGCGCTGGTGCTGCGCAAGCTGTTGCGCTGAATTCTGTCGCGCCGAGTTCTGTCGCGTT

[0256] GAGTTCCGGCCCCGAAGGGCACCGTCGTCCGGCGTACCGGACCGCGGTGCCCTTCGTC

[0257] GTCTCCGGGCGGCGTCGGCGCGCGGGCCGATCGGTGACCTTGGACCGTTCCGAGTTCG

[0258] ATCATGGCGTGATTGACTGAGAGTGAGCCACACCGCGCCATCTCGGAGGGGGCTGACGGCC。

[0259] SEQ ID NO:23(P23)

[0260] GCGGCATT TTGCCGGGTAAGATCTTGCGTCGGGAGGCCCGGATGAGCG

[0261] ACTGGGTGTGCCGGCTGATGATGCCGAAGGATTTCGGCGACAGTCCGGCTCAACTGCA

[0262] TCTGGTGCTCCGTGACGATCTCACGGACGCGGAGGTGCTCGCCCGCGTCCTCGGCCTGG

[0263] TTCACGAGTTCTACGGTCCCCGTGTGCAG TCTGAGGCCCGAGTCCTGGGCG

[0264] CAGACCCGAGTCGGGTCCCGGCTCCGAAGCTCACCCGCCGGCTGAGGAACGGAGGCG

[0265] ATACGACGGGTCGTTTCACCGCAGATCACCGAGCCGGATCTCAGGAAAGGGGACGGGTC。

[0266] SEQ ID NO:24(P24)

[0267] CGAATCCGGCGATTCCGATCGCCACCCCTGCCGCGAGTGCGGCCACAGTCCTGCGCCG

[0268] TGTGTTCATTGCCGGCACCACCTTTCTCTCCCCACCGAGTCAACGCACCGTCGGTGAAG

[0269] ACTTCCCGTTCCGGAATGTGAAGCTTGTGTGAGAGCA CCCTCCTCCCGGCG

[0270] GCCTCGGATCCGAGCCCGGCGACACGAGCGGGACCGCCTACGCTGCGGCGTCACCGAA

[0271] CGCCGATCCTCAGG GGGCAGGGGCCGAACTGTGGGCTTTCGGCCCTT

[0272] GAGGGCCGGTGCAGGGATTCGTAGCGTCGGAGTCGGGCATCGTGACGAGGAGGCCGGCG。

[0273] SEQ ID NO:25(P25)

[0274] TGCCCTCGATGCCCACGGCGGTGAACGCGTCGCGCACCCATTCCGCGGCCGCGACACA

[0275] CTGTTCGGGCGGGAACAGACGCGGATCCGCCACCGACCGGAAGGCCACGAGTTCCGCG

[0276] AGGTCCGCGCGCGCTGCCGGCAGGAGCCGGCGCACCCGCTCGCACATCTC

[0277] CCACGGCACCT GTGTCTCGTTTTCGGTACGGGTACAGCCCGTCCCGAT

[0278] CGAAGCATGCCCGGCGCGCCGATATCCGGCGTACATTTCGCGATGTCGGGGTCGCCAA

[0279] CGGGCGGAACGGTCCACGGGAGGCGGGGGGCCGGCCGACCCTCGAACGGAAGGCACTGCG。

[0280] SEQ ID NO:26(P26)

[0281] GAGGCCGCGGTGCCGCGGCCACTCGACCCGTTCCGC GACGGTCGTCGCGC

[0282] TGCTGCGCGGCCGCAGCACCACCTGACGGCGCCGCGGGACGCCCGCCCCGGCGGGCCG

[0283] CGCGAGTGGGCCGGTCCGCGAACGGTCGGGCGAAACGGAGAAATCGCGCCGCGTGGT

[0284] ATCCGCCTCGGGGAGACACCCCGCCGGTAGGCTGAGCCGTACATCACAATGATGCGGT

[0285] TGAAGTGACCCACGGCACCGACACCGCCT CGGCCCCAGCGGCCGAGAGGTCGGCAAGGGCAACGGAGTCGGCGCGGGTGGCCGGCTCGCACGGAGAGGAGCCAGGCGCC。

[0286] SEQ ID NO:27(P27)

[0287] CACTGCCCGGATCCGCGCGGCGGCGGCCCGGTCCTGCCAGATGTCGGT CC

[0288] TCGATGCCGCGCCTGTGCAGGCCCCGGCGCAGGCGCGAGCAGAACGGACACCCGGGC

[0289] CGCCACAACACCTCGACTCCGTCGACGAAGTC ATCCGGTTTCTCCTTC

[0290] CGCCTACGTCTCGTCCATATACCCAGAGGGGTATACGACCTCACTGTACCGGCCCGCCC

[0291] GGCGCTGCGCCGCGCGGCCGGCGCCGTTCCCCGGCACGGTGACCTTCGACCCTTTCCG

[0292] GCGACGCCCGAGGGGTGGACCGTGGAACCACAGCCGCGAGGCGATGCGGAAGGAGAAGCC。

[0293] SEQ ID NO:28(P28)

[0294] ACATGCTGCTCGAACGCCG GAG GCTGCTCAAGGAGCGCCT

[0295] CGAACTGCTCAGGACCAAGCGGCGCGGCGCCTGACCTCAGGTCGACCGGTACGCACGA

[0296] GGCCGCCCCCGGGATCCGTCCCGGGGGCGGCCTCGTGCGTACCGGGGGCGGTGCGTGT

[0297] CCTGCCGGGTCGGCCGTGTTGCGCGCACGGCACAAATGGATCAAACGCCGACAGCGGC

[0298] TCCGCGCGCGTGTCATCATGATCGAACACCGGCCGGATTCTCGGACCGGCGACCTCGG

[0299] ACGACGCGAAACGGAATGACCCACTGGGGTTTCGGGCCGCGGTACAGGGGGCGAGAAGCG。

[0300] SEQ ID NO:29(P29)

[0301] TGCATCGGGCGCTCCCGCGACTCGGCGAGCAGGAACATCGAGTCCGTCACCGGCATGT

[0302] ACCCCACAGGGACTGGCCCCTTTCGTCCGCCGTCGCGCGCTGGGATGG

[0303] GGTCCGGCACATGCCGTACCGAATAGGTAACAGAAGGGCCCGCGACCTGCGGAAATCC

[0304] CGATGTCCGGTCTCCATCGAGTCACGGTGCTTCGCCCCGGCGGATC ACTGT

[0305] CGATCAACCGACCCGGACGAACGTCACGAAAGCCACGGGCAGAAGGACCGACGAAGG

[0306] CGAACCGGAGCCGACGACGAGAGCAGCTCCGTCCGAGCGGACGAGGACGAGGAGGTCCCG。

[0307] SEQ ID NO:30(P30)

[0308] CGGTGCTTGCCCACGGGGTCACTGCCGCTCCCCTCGCGGCCCGCTACGCGCACTGGTTC

[0309] GAACAGCATCCCCGTCGGCTGTCCCCGCCCCTGAAGGGCGGAGGGACAGCCGGGCAG

[0310] GCAGATGCGGCGCATCCTCACAGATCCGGCGGCCTGGGCGATTCGACCGCCGACTCTT

[0311] C TAGGCTCGGGACACGGAGGGCACACGCT GCGATCACCA

[0312] GCTCGCCCTCCCCACCCGAGAGGCCGTGGCGATGTCTCCGAACCGGGCGTTCCCCCGC

[0313] GGGATCGTCGACCGGTACCCCGGGACGCCCAGTCGATCGATCAGCAAGGGTGAGCAGCGAA。

[0314] SEQ ID NO:31(P31)

[0315] GCACGGCCTCGAGCAG TCTGATCGCGCGCGACGATCACCTGGTCGATGCG

[0316] GTCCTGGACCTCCGCGAGCAGCTCCCGCAGCCGCAGTTGGGACAGCGTGCCCGAGAGC

[0317] AACGGTTCCTCGTCGGCAGCCATCGCTCACCCCTTCAGTGGATCCCGGGCCCCGGGCCC

[0318] CGCCCCGGCCGCTTCGGAAGGCCCGGTTTCTCCCATTGTGCCGAGGGTAATCCGCGGA

[0319] CCGCCGCCGCCGCGGTGACTTGCGCCCCCACGGTGACGACTTTCGACCCTGCTGCTTGC

[0320] GGACACGACGGACCACTGTGGAGTC TCCACAGGTGAAGAAGGTCCAGG。

[0321] SEQ ID NO:32(P32)

[0322] GCTGGGTCGGGCTCGGA GTACGTGGCGTCCTCGGCGCAACTCGCCCGCTG

[0323] GCTCGAGGAGCTGGGTGTGCCGCCGGCGCCCGTCCGGTCTCTGCCGATCCGGGAGGTC

[0324] GCGCCCGGGGTGACGGTCGAGGGTCCGGTGCCGGAACTCGTCGACGAGGTGCGTGCGCTGCACCGGCGATGCCACGGCGTGA ACCCACTGCGTCACGGTGAGATGACGGACGACCGCAGTCGATCACGGGTTGTGATCGACTGCGGCGCTTTGCAGTTCCTGTACCCGTTCCTGAGCTGACCATTGAACCGGTCGGGGCTGGTCGACGGTCGAAAGGAACGCCC。

[0325] SEQ ID NO:33(P33)

[0326] CGGCCCG CGGCGCGCGATCGAGCGTGGCGAACTGCCGTCGGGTGTCC

[0327] CGGTGAGCCTGCTGCTCGAGATGGTCACGGGCGCCGCGCTCAGCCACGTGCTGTTCTCC

[0328] CTCGGCGGCCCGGCGCACGGCGCCGAGGACACCGCCTCTGCCGGGGACACCGCCTATG

[0329] CCCGCACG GGCCGCAGTCCGGGCGTTGGTCGCGGATCGAGAACAGAGTCC

[0330] AGGCGCCGTGACCTAACTGCAATCGAATTGCCCGGCGAGCCGCCGGAATCGATACCGC

[0331] TGCCGCGTCGCCGCGACTAGCCTGGCTGACAGGTCTTACCCGTGGTGGGAGGCGCGAACG。

[0332] SEQ ID NO:34(P34)

[0333] GACGACGTTCATGTCGTCGGGCTTGTCGATCGCCACGATGTGCAACTCCACGATCGCCT

[0334] CCTCGGATGCCCTGCGGACCGACAGACGTCGCCAGTCAGTCTGCCCGGCCGCGCCCCT

[0335] CG CCCGCAGTC ACGGAAGCTCTACCGCCGAACCTTCGGC

[0336] GGGCGTCCCGGCACGGAGCATCCCGATCCGACCGTCCGGAGGACTTTTTCCCTCGGAC

[0337] CCGGGCGGGTCGACCGCCGGCACCGCACGCCCACGATGCGGGGCGTGAGGACCAATG

[0338] CCCCTATGCCGGTCGTCCGGGGCGGCGCATGATCGGCTCGACGCGAACGGAGGGAGCGGCC。

[0339] SEQ ID NO:35(P35)

[0340] AGCCAAGCAGGCC GTACAT GCGGCCGGCAAGTCCCCGGC

[0341] CGAGCAGATCGCCGACGCCAAGGCGCTACTCGACTCGGGTGCGATCACGGCCGCCGAG

[0342] TTCGAGCACCTCAAAGCTCAGGCGCTGGGACGGGGTACCGCTGGTCGTGACGGCGTCC

[0343] CCGTCGGCTGAGTCGCGAGCGGGACCGGAAGTCCGGTGCGATCGTCCTTTCATCATGT

[0344] CGGTGAGGGCAGGTGGCCGGCAGTCGAATTCGTGATCGGGTCCGTTGCGGCAGAAGTA

[0345] GATTCCAACTCTCGGGGACGGATCCGGCGGAGTCGGCCGGTGCACGCGGAAGGAGTGCGAC。

[0346] SEQ ID NO:36(P36)

[0347] CATGCACGTTGTGTGACACGGTCGTCTCCCTCTCCGAACGGATCTGACGGGTCGAGTGT

[0348] GCGCCGGGTTGCACAGGTCCAGCAGGGACTTTCGTCCCATTCTCGGTATCGGCGCGGTC

[0349] ACGCACCCCTCGCG C GGCGGGAGCAATCCGGTTGCCTGCC

[0350] CGAAGACAGCCTGCCCGAAAACCGGATGCGCGCAGACATGTGCGAGCTATCGTCGAAC

[0351] ATGCAGTGGACCGGTTCTCCGTTCCCCGGTCGGCGTCGCCACCATGCGGGTGTGGCGCCGTGAAACCGCCCGAACTGCTGCCGAAGACCGATCCCGTCGCTGAAACGAGGCACCCG。SEQ ID NO:37(P37)

[0352] CCACCACCGACGGCGAATCCCTGTGGGTCTTCCGGTACTCGAGCGTGGGCCGCAGCCG

[0353] CTC TCCACGGCG GGTCCGGGCGCTGCATCCGGAGGTGGAG

[0354] GTGCTGCATCGCCTCGGCCCCGAGACCCGCTTCGTGGTCTCCGAACCACTGCGCGACCT

[0355] GCCGGGTGCCTGGAACGAGGTGCCCGAGGCGTCCGTCGGAATCGTCCGGCCCGGGGCG

[0356] GACGAGATCCTCCCGTTCCGGCCGCTCGAGCCGGTCTGACATTCACCGGAAGGAATGT

[0357] CGCGAGTCGTGCTGGGCACCCGGCCCGGCGGACACATCCGGCAATCAGGAGGAGGCGGC。

[0358] SEQ ID NO:38(P38)

[0359] GATCCGGACGGCTCCGTCGC ATC CGGGCTCGAAGGCGAG

[0360] GTCGATGTCACGCTGTTCGCGGAGGCCGCCTCCGGTCTGCTCGACGACACCGACTTCGCCGAAGCCGGCGCCGCACTCGAGCCGGGCTGTTCGGCGGCTGTGCTCGTGTACGAGAACACGTGGGCAGCACCCTTTGCTCGCGCTCTGCGCCGCAACGGCGCCCAGCTCGTCGCGTCGGGCCGGATTCCCGTGCAGAGCATCCTGTCGTCCCTGGATCGGATCGAGTCCGAAAGCTGAGAACAGACAGAGGGATTCGGCTACGTCGATCCTTCGAGTATGCAGAGGAGTGCGTC。

[0361] SEQ ID NO:39(P39)

[0362] TCGGCACCAGCGCGACCCGCCGCTC TGGTTT GCCGACGGT

[0363] GGGTCGCGCTGTCACCGCACGTCCCGTACCCATATCCTCGGTTTCCAAAGTTTTCGACT

[0364] CCTGCGTTGAATCGGCACCTCACCGCGATGAGCGGCGGGTCATGTGCGACGAGCGGCC

[0365] GACTCGGGCCGTGCACTATGGGCGATGAGCCCAGTCAGACGTACCGCGCCCAGGTCGG

[0366] GGGCGCAACCGTTCGGTTTCGTCTGAATCGAGTCCGAACAGTGAGTCGGTGCCAGAAT

[0367] GTGAGGTAGTTCACTTCACAATTCTGCCGTATCGGCCGTGAGGGAGGAGGCACGAGTCC。

[0368] SEQ ID NO:40(P40)

[0369] GGGCCGAACGCCGTGTCGTCGCTGTCGAGCGTGACGGACGAGACCTGCTTGTCCACGA

[0370] GCGAGAGCAGCACCGGTTTCCACCCGAGATCCAC TCGTGCCGAGTTCGCT

[0371] TGCGCGAGGCAGTTGGTGGCACGGTTGCGGACGTACACCTCGGCGCC

[0372] GACGAGCACCGCGACCAGCGCGACGGCGAGGACGATGCCGATGGTGGCGAAGGGGAC

[0373] ACGCTTCCGGTTCGTCATGGTTCCGGATTCTCCCGAAGAACTCTGAGGAAGGTCTGTGA

[0374] AGATGCGCCGCCGGGACCGGAAAGTGTGAGGTGTACCACAGTTGTTCGGTACTGTGGCGGC。

[0375] SEQ ID NO:41(P41)

[0376] GGGCGCCAGTCAACCGC TCGGGGCCGCGGCTGACTGGGAACGCCGCGGAT

[0377] ACTGGACCCCGGCCGGAGGCCGGCAGTGTCACCGCGGCCGGAGGCCGGCAGTGTCAC

[0378] GGTGCCCCCGGCAGGA CGACCTAGAGATTAGAAGGCTCTTGCTCTATC

[0379] CAGCTGAGCTACGGAGGCGCGGCCAGAATCTTAACTGTTTGCCGGTGCGTGCCCGACC

[0380] GAGGCCCCGCCGCTGCCGGCGCGGTGGGCGCGTGCGTCCCGATCGGGCGGCGAATCCG

[0381] CCCGTCGCGCCGCGAGCGCGGGCATACACTCCGTAGGTAGCTGAAATGTGAGGTATGCCAC。

[0382] SEQ ID NO:42(P42)

[0383] CTGTACCCGCTGGGGGCGAAGTCCAGAGCGTGCACGATGTGCAGCGGCAACCCGCGGT

[0384] CGGCGGCGGTCGCGGCGGCCCACCGGAC CGGAGGCCGGAGAGCCGTCGA

[0385] TCCCGACCACCAC CGCACCGCCTCGTCGTGCGCGGGTTCGTGGTGCGT

[0386] CTTGCTGCCGGGGAACTTCACGTCGGTCACCGCTCTCGTCGCAGGGATCGTCCTCGGCC

[0387] TCGAGGTTAACCCGCTCCCCCTGGGCCAGGAATGGTCTTTGGTCCTTCTCCGGGAAGTC

[0388] CGGAGCCACTGGTCCGGCGGGGAGGATGCGGCCACAATCGTGGGGAGAGGAGGCGCGTC。

[0389] SEQ ID NO:43(P43)

[0390] TGCTGATCGCGCTGACCGGACTCGC CGGCCCACGATGGCAGCG

[0391] CGCGATGGGTGTGCTCGGCGTGCTGTTGTTCCTGTCCCCCTGGGTCATGGGCTACA

[0392] CCGAGTACAGCGGTGCATCCTGGACGGCCTGGGTGGTGGGCGTGCTCACCGTGGTCGT

[0393] CGCGGTAGCAGCGCTGCCCGCGATGAACGCCCGGATGCACGGACACGGCGGACTGGC

[0394] CACGCACCACTGATCACACACCCCGCGTCACACACCCCGCGGACGGCCGGCATGCCGG

[0395] CCGCCCGCCCACGCCCCTGGCGCCGTCACATTCCCGCCCGTGCCCCCGGAGGTGAACGCC。SEQ IDNO:44(P44)

[0396] AAACCTGTGGGCCGCACCGTTCGCATCGGCGGTACGCCGCGCCGGCGGACAACTGGTG

[0397] GCCAGCGGCCGCATCCCCGTGCAGGCCCTGCTGGCCGCAATCGAA ATCGG

[0398] AAGGAGACTGACATGCCCCTCGCAGCCAGGCGAATGCGCCGCGCCGCGGTGATCGGAC

[0399] CCGCACCCGTGGCTCGTACAGCCACGACTGTCGCAACCGCAGCGGTGGTCGCTCACGG

[0400] CGTCCGCCGGCGTTCCGACCGCCGGGAAGATCGGCGAGAAGACCGCAGAGACCGGCG

[0401] CTGACCCGACGGCCGGAACACGG CGTAAGCCCGACAGTGGAAGGGACTGCC。

[0402] SEQ ID NO:45(P45)

[0403] CCCATGCTG CGTCGCCGCCGACTTCCCCGACCT ATGGCCC

[0404] ATCCCGCGGTACCGTGGGTCGACGCGCAGATCTCCATCGCCACCCACAAGGCCAACGT

[0405] CTACATCGACCTCTCCGGCTGGTCCCCCAAGTACTTCCCGCCGCAACTGGTCAAGGCCG

[0406] CGAACACGATGCTGCGCCACAAGGTGCTGTTCGGCTCCGACTTCCCCGTCATCCAAGTG

[0407] GATCGATGGCGCAACGATTTCGCCACTCTCGACATCAAACCCGACATCGCACCGATGA

[0408] TCTTCAAGCAGAACGCACTGCGCGTTCTCGGCATCAAGCGCTGAGCACAGGAGGCCCC。

[0409] SEQ ID NO:46(P46)

[0410] TACCGATTCTCAGTTCCGCTGTGCTGCCGTGTGCAGTCCTCGGGACCGCGCTGCTGGGA

[0411] TGGCTCGAGCCGAGGACCGCCCAGATCGTGGCCGAAGTCGCGGTGCTGGTCCGCATCG

[0412] GAGGAATCGTGTGGGTCATCGGCCGGTTGAAG CCACCCGTGCGTCGG

[0413] TGGTTGCTGCTGCGCTCTTGACCGCGATCGCGACC GATCGTGAAGATCGTG

[0414] CTCACTCACTGACCCTTGTCACGAGGGGGTACCGGACCGCTCGGTTCCGGCGGCAACC

[0415] TCGGCATTCTCGAGCCGGGGATGGCGCCGGTGTGATGCAATGAGGTGGTGTGCAGATGG。

[0416] SEQ ID NO:47(P47)

[0417] ACTTCGCCGGGAAGTCGCCGGCCGAAGCCGAGTTCCGCAC TACCGGG

[0418] TGGTGCGCCATCCGCTGTATCTGGGTTTCCTGATCGCGTTCTGGGTGGC TG

[0419] AGCGTGGGGCACCTGCTGTTCGCCGCCGTCACCACCGGATACATCCTCGTCGCACTGCG

[0420] GTTCGAGGAACACGATCTGACCGAGACGTTCGGCGACCGGTACCGCGACTACAGCGCC

[0421] CGCGTGCCCCGGCTGGTACCGCGCCCGCGCGCGGTGAGCCGAACCTGACCATGTTCCG

[0422] GGCCGGCTCCGGTGCGGGCGCCCCGCACCGGAGCCGTGCAACGCCGTAGGGTGCTTCGC。

[0423] SEQ ID NO:48(P48)

[0424] GAGTACTGGCGGGTGATG CGACGGCGGGAGGCTGCTGCGCTTGGCGAGAC

[0425] CGACCCACTACGAGGACGGCAGCTTCTCGTTCGC GTCGACGACGTACC

[0426] TGTCGAATTCGTCTCGCCCCACGGCTCGCGTCGTCCCGACGAGAAGTAGACCGCGACG

[0427] ACCACGGCGGCGAGCGAATCGAACCGGCCGAACTGCCGGTGGCGTGCCGGCGCCCCGT

[0428] CGCGTGTTCTCGTCGATCCGGCAGACCGTGGGGCAGTAACGGTCCACACTGAGTGGGT

[0429] GGCGCAAGCGGTCGCGTGGGCCGCGGATCCCGGCAGGGGCTGTCGACACAGGAGGACTCC。

[0430] SEQ ID NO:49(P49)

[0431] TTCGGCGCCGCCAACACGCCCGTCACCGGGGTCGGCGGTGAGCACCC

[0432] GGCAGCGACGACGGGGCCCGCGATCCGCTGGTCGCCGAGTTGCGCGCACGAGGTCATC

[0433] AGGTGGCGGTGCTCCCCCAGTCCAGCGGGCTCAGCGCGCTGCGGCGGGACGAGACCGG

[0434] GTGGATCGGCGGCGCGGACC GAGGGCGCCGTGATGGGCGACACCACCGG

[0435] ATAACGATCCGGGGCGAAGGTCCGGGCGCCGAGTCACGACTGAATAACCACACGGGGTTTCGCCGATCTGCGGGAATAAGATCGACAACCATCGATCGCCGTCACGGCGGGTGGGAACGG。

[0436] SEQ ID NO:50(P50)

[0437] AACTGTGCCAGAGCGAGCGGCACGAGCATCCGCCGCAGGGTCGAGGACGGGCGCGGA

[0438] GAGTCGAGACTGTCCGGCATGGCGAGCCCTTCCG AGAGATGCACTGACTC

[0439] GTGCACAACGTGGCCAGTGACATTCTGGGGCATTCGAAGAACCCAAGGTGGCAATTTC

[0440] GAGCGTCCACCGCCGCCGTTGCGTCGCCCCGATCGAGCCGGCGAAATGGTCGAAATCC

[0441] GGCGGATCTCTCGGCACTGATGGAAAATCGCTTCCATGCACCTTCCGGCGATCGCTCCG

[0442] GAAGTGCCG GAGGCCCCTCGCCTCCAGTCCACACATCGGGAGATTCGGTC。

[0443] SEQ ID NO:51(P51)

[0444] GCAGCACCGG CGCCGCCCCAACGGCACCCCGTGGCCAGCCAGCCACC

[0445] TGTCCCGCATCGCCTCCGAGCACTTCAAGAGCCTCGGAATGAGCTGGACCCTCCACAC

[0446] CCTTCGCCACCGCTTCGCCACACGACTGTGCGACGCCGGC CCGCGACGTCC

[0447] AGGCGCTACTCGGACATTCCAGCCTCGCGACCACAACCGTGTACCTGTCCCAGGCCAC

[0448] CCGACACGCGGCAGCTTCGGTCGACAAGCTCGGCGAAGGCGTCAGCCTTCTCACTCGA

[0449] CGCCAGCACGCCACACGCAAGAAGAGATGACCACGATCCACCATAACGAAAGGCACTGCC。

[0450] SEQ ID NO:52(P52)

[0451] CCCCACACCGACCCCGACCGCGCCTACGAGATCATGCGGACCGTCGCCGGTGTGCCCG

[0452] ATGAACACTGACCCGCTTCC TTACCACCACAGAACAGGACACCATGCAGC

[0453] ACCACGAGTA CTTCAGCCCGGCACCCCCGCCGACGCGGACACTCCCAC

[0454] CGAGGCCGAGACGGATCCCCGCCGGCACGGACCGCACGACTACTCCGGGCTCTTCTCG

[0455] GCTCCGAACTGACCGGCATATCCCCGCTGCGGGGGGGAACATGATCGAATCCGGCGCA

[0456] TGACCCCGGCGCAGACGTCACACTGAAATCCGGAGAAATCCCCTGAACTCTGGAGACGCC。

[0457] SEQ ID NO:53(P53)

[0458] GGTACCGCCGACCAGGCACAACAGCGCCACCCCGGCCCCGACCATCCCGGCGCCGAGC

[0459] CGCCGCACCACCGCACCGGACACCCTCGCCACACCCACACCCATCACGCACTCCTT

[0460] GGCACACCCCTGCAC ACCGCCGCCGCGGGAGCGGGCAGCCC

[0461] CTCGTGTGTAGCACCGAAATGCCGGGATTGCCCTGGTTTTCACAAAAGTACCGGACGA

[0462] ACGGTTTCTGTGATGTAAGCGTTATCCGATACGGGTGTGGTTGGTGGGAAACCGGTTA

[0463] GGTTTCCAGGCAGCGATGGCGCCACCGCTGACCTTGTTCCTGCGAAAGACGGTAGGTCTCA。

[0464] SEQ ID NO:54(P54)

[0465] CGGGCACGGTCTGGATCAACTGCCACAA ACCGCACTGCCCTTCGGGG

[0466] GCTACAAGCAGTCGGGCTGGGGGCGGGAACTCGGTGAGGGAGCCCTGGCGGAGTACA

[0467] CCCAGACCAAGTCGGTCAACATCGCACTCTGACCGGCCGGGGCGGTGCCCGGCGCCGG

[0468] ATGGGCACCGCCGCCCGTACTGCCGGCCGAACGAGAACCCGGCACCGAGGCGTTCTCG

[0469] TTT CGAACCGTGACGCCGCGGTGACTGCACCGACAACGGCCGGGCGCACG

[0470] CTTCTTCCGGCGCTCGGCACCGAACCGCCGGCACACCCCCTCTCCCGAAAGTCGAAGCACCG。

[0471] SEQ ID NO:55(mcherry)

[0472] ATGGTGAGCAAGGGCGAGGAGGATAACATGGCCATCATCAAGGAGTTCATGCGCTTCA

[0473] AGGTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCG

[0474] AGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGTGGCC

[0475] CCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCTAC

[0476] GTGAAGCACCCCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAA

[0477] GTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCC

[0478] TCCCTGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCT

[0479] CCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCTCCGAGCGGAT

[0480] GTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGA

[0481] CGGCGGCCACTACGACGCTGAGGTCAAGACCACCTACAAGGCCAAGAAGCCCGTGCA

[0482] GCTGCCCGGCGCCTACAACGTCAACATCAAGTTGGACATCACCTCCCACAACGAGGAC

[0483] TACACCATCGTGGAACAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAGTGA。

[0484] SEQ ID NO:56(holin)

[0485] ATGTCCGTCACCAACGGCAACCGCAAGCAGGGCGACGGCGTCCCCACCTCCATCTCCT

[0486] CCATCCCCCTCACCGAGGTCCGCGCCCGCACCCCCCAGGAGGCCTCCATCGGCGACCT

[0487] CGTCCGCGACGCCACCGCCCAGGTCTCCACCCTCTTCCGCGCCGAGGTCGAGCTCGCCA

[0488] AGGCCGAGGTCACCGGCGAGGTCAAGAAGGGCCTCCAGGGCTCCCTCTTCTTCATCCT

[0489] CGCCCTCGCCGTCCTCGTCTTCTCCTCCTTCTTCTTCTTCTTCTTCCTCGCCGAGCTCATC

[0490] GACATCTGGACCCCCCGCTGGCTCGCCTTCCTCATCGTCTTCCTCATCATGGTCGTCGTC

[0491] ACCGCCATCTTCGCCCTCATCGGCTACCTCCGCGTCCGCAAGCTCCGCGCCCCCGAGAA

[0492] GACCATCGACTCCCTCCGCCAGGCCCGCACCGTCCTCCCCCAGCAGCACGACGCCGAGCCCGCCGCCGGCCCCCGCCACGCCCGCTGA。

[0493] SEQ ID NO:57(ddel)

[0494] ATGAAGGCCGCCACCGACCAGGAACTGAGGAAACTGATTGTGCTGTACAACAACGTGA

[0495] TGGAGGTCATGGAGCACGACGCCGCGAAGTCGATGCGCGACGACAACCGGGCATACG

[0496] GCGGCTTCGTCCGCGCCGCCAAGGGTAAGATCCAAGAACTCATCACCGAGCGGCTCGT

[0497] GCGAACCGTCTGGGACGTCGAGATGGGCGAGAACCCGGAACGGCTGAGCATCAACTC

[0498] GAAGAAGATCAAGATCCCGATCTTGCGTTCCTACGTCGATTCCATCAATGACGAGAAC

[0499] CTCAAGAAGTACATCTCCAGCAACATCCTCAAGTACTCGTATGGTCTGTCCGTCGACAA

[0500] GCACGTGTTCATCGACAATAAATTCGTGCTCGGCATCGAGTGCAAGGCGTACACGGAG

[0501] AACGCGATGCTGAAGCGGATCCTCGTGGACTTCTACCTCCTGAAGACCAAGTTCCCCA

[0502] AGCTGAACTGCTTCCTGTTCCAGTTGGAAAGCCAGCTCGGGGGCGACTACTCGGAATG

[0503] CAACAAGTTCCCGATCGGGTCCTACCCGACGCGCACGATCATGAGTTACTTCAAGAAC

[0504] GTCGACCTCAACATCGTCACCCTGCTCGAAGGAGAGCGCAAGGTGGATCGCCCCATTA

[0505] ACAAGCCGCAGTTCTTCAAACCCCTGAAGGTCGAGCACCTCGAGGTTGCGATCGGCTACCTGCAGGAGTCGCTGTCGGAGATCTAA。

[0506] SEQ ID NO:58(divIVA)

[0507] ATGCTGACGGTTCTGCTCTACCTGCTCGTCGTGGTGGTCGTCGGCGCGGTGCTGTTCCT

[0508] CGTGGCGAGCGCGGTGTTCGGTCGGGGGGAGACCCTGGCGCCGATCCCGCCGGGCGCC

[0509] ACGGCGACGGCGCTTCCGGTCACCGACGTGACCGGCGCGGACGTCCTCGAGCTGCGTT

[0510] TCCAGCAGGTGCTGCGGGGCTACAAGACCGCCGAGGTGGACTGGGCGCTCGACCGGCT

[0511] CGCGCACGAGATCGACGATCTGCGGGCCCGGCTCGCCGAGTACGAGAACACCGGATAC

[0512] GACAACACCGAGTTCGAGAACACCGAGTACGAGAACACCGAATACGACAACACCGGT

[0513] GGGGTCACTCCGGGCGGCGACGAGGCGGACAGCGGCCGGACCGGGGCATCCGTCGCGGCGGAGGCCGCGCCGGACCGTCGGACAGGGGGGAGTGACGATGGGTGA。

[0514] SEQ ID NO:59(T1)

[0515] ACCCGCGAACCTAGTGGGCGCGGCCCGGCGTCGTGCAGATTGTGGTTCGTGCGGCGCG

[0516] TGCCGGGGCGCCTCGCCGCTGTTGTCGGGGCGCTCGCCGCCGTTGCAGGGGCGGCTCG

[0517] CCGCCGTTGCCCGGGCGGCTCGCCGCCGTGCGGAAGCAGCGCCGCGCCGCGCCACGTGGCGTAGGTTCGGCGGAGAGGACACGCTACGACGAAAGGGACCGATCG。

[0518] SEQ ID NO:60(T2)

[0519] TCCGAAGCCTTTCTCTCGGACTTGAGTCTTTCTCACTCAACTAACGTGGCGGGCCGCCT

[0520] GTCAATTCCACTCGGAGCGTGAGGTGCGTCACGTCGGAGCCCCGCCGATTTCAGTTGTGCACAACATAGCGGTGCGCTACTTTATTTCTC。

[0521] SEQ ID NO:61(T3)

[0522] GGGTGCGGTTACCCGGTCCGGTGGGTGTCAATCCCCGGCCGCGGGTTTCCCCGGGCTC

[0523] GGTTCGGGCACCCGGTTGATCGCATCGGTGCCGCGGCGAGCGGCGCCGGCGCTCAGAGCGAAGTGCGGACCGGAGGAACACCTG。

[0524] SEQ ID NO:62(T4)

[0525] GCCGGCCGACCTGCGCTTCGCCCTCCTGCTCCGGGTGGCGCGGGCGCGACCGGAACGG

[0526] ATTCGCAGTGCGCGTCGCTCGGATAGAGTTCGAAAACATCCCTGATCCGGTCCGCTCGCGTCGAATGTGCTGTTCCCAAGGGGTTTGAG。

[0527] Example 1: Summary and Re - mining of the Characteristics of the Specific Promoter in the Stationary Phase of Rhodococcus erythropolis

[0528] In Rhodococcus erythropolis, after testing, the promoter sequences P1 - P27 (SEQ ID NO: 1 - SEQ ID NO: 27) are promoters in the late fermentation stage (when Rhodococcus erythropolis grows to the stationary phase), and their expression levels in the late fermentation stage are more than 5 times that in the early and middle stages. According to the sequence alignment tool, the promoter sequences P1 - P27 (with a length of 350 bp) were analyzed, and several specific promoter characteristic sequences in the stationary phase that can cover 27 promoters were obtained, including characteristic sequence 1: VBCGHCDW; characteristic sequence 2: SNNSWWBBWB. Degenerate base codes are R (A / G), Y (C / T), M (A / C), K (G / T), S (G / C), W (A / T), H (A / T / C), B (G / T / C), V (G / A / C), D (G / A / T), N (A / T / G / C).

[0529] Secondly, the promoter sequences P1 - P27 contain (N) p VBCGHCDW(N) q SNNSWWBBWB(N) r or (N) p SNNSWWBBWB(N) q VBCGHCDW(N) r characteristic sequences, where p, q, and r represent the number of random bases, and p, q, and r are each independently selected from integers between 1 and 350. According to the characteristic sequences of the specific promoter in the stationary phase, screening and searching were carried out in the Rhodococcus genome, and further promoters that meet the characteristic requirements were obtained. Their sequences are P28 - P54 (SEQ ID NO: 28 - SEQ ID NO: 54). According to the further detection and characterization in Example 2, it was proved that the promoters P28 - P54 also conform to the expression rule of the specific promoter in the stationary phase of Rhodococcus, where the expression level in the stationary phase is more than 5 times that in the lag phase and logarithmic phase, and they are specific promoters in the stationary phase that meet the standards. Therefore, the characteristic sequence of the obtained specific promoter in the stationary phase is (N) p VBCGHCDW(N)q SNNSWWBBWB(N) r or (N) p SNNSWWBBWB(N) q VBCGHCDW(N) r 。

[0530] Example 2: Characterization of the stationary-phase specific promoter of Rhodococcus ruber

[0531] The purpose of this example is to construct an expression system based on the stationary-phase specific promoter of Rhodococcus ruber.

[0532] Rhodococcus ruber TH was cultured in a fermentation medium. Cells were sampled every three hours to collect cell mass and measure OD460 and glucose concentration until the fermentation ended at 60 hours.

[0533] The changes in OD460 and glucose concentration during the fermentation process are as Figure 1 shown. Based on OD460, the fermentation period from 0 - 12 h was the growth lag phase, the fermentation period from 15 - 33 h was the logarithmic growth phase, and after 36 h, it basically entered the stationary phase (late fermentation phase). Similarly, according to the glucose consumption curve, the glucose concentration decreased rapidly from about 15 h, at the beginning of the logarithmic growth phase of the cells, and was exhausted at about 33 h. After that, the cell growth entered the late fermentation phase. Thus, according to the cell growth stage, it was divided into three time periods: early fermentation phase, mid-fermentation phase, and late fermentation phase.

[0534] First, construct the plasmid vector pNV18.1-mcherry carrying the mcherry gene of red fluorescent protein, the method is as follows: The plasmid vectors pNV18.1 and pNV18.1-Pa2 were digested by KpnI and EcoRI to obtain the plasmid backbone; the mcherry gene (SEQ ID NO:55) was amplified and synthesized using mcherry-up and mcherry-down. The plasmid backbone and the mcherry gene were ligated by Gibson and transformed into Top10 competent cells, and then spread on a plate containing 25 μg / mL kanamycin to construct the pNV18.1-mcherry and pNV18.1-Pa2-mcherry expression plasmids respectively.

[0535] Next, construct the characterization plasmid pNV18.1-Px-mcherry of the stationary-phase specific promoter of Rhodococcus ruber. The plasmid vector pNV18.1-mcherry was digested with HindIII and KpnI to obtain the plasmid backbone; it was ligated with the synthesized P1-P54 promoter sequences by Gibson assembly, and then transformed into Top10 competent cells. The cells were spread on plates containing 25 μg / mL kanamycin to construct the expression plasmids pNV18.1-P1-mcherry to pNV18.1-P54-mcherry. The expression plasmids pNV18.1-P1-mcherry to pNV18.1-P54-mcherry, the pNV18.1 plasmid that does not express the mcherry gene, and the pNV18.1-Pa2-mcherry plasmid expressing mcherry under the Pa2 promoter were respectively introduced into Rhodococcus ruber TH to obtain the recombinant strains TH-P1-mcherry to TH-P54-mcherry, and the control strains TH-Pa2 and TH-control.

[0536] The primers used in the above process are shown in Table 1.

[0537] Table 1 Primers and sequences used in Example 1

[0538]

[0539] The recombinant strains TH-P1-mcherry to TH-P54-mcherry and the control strains TH-Pa2 and TH-control were respectively inoculated into seed media supplemented with 25 μg / mL kanamycin and cultured at 25 - 37 °C and 100 - 200 rpm for 48 hours. Then, they were transferred to fermentation media at a ratio of 10% and fermented at 28 °C and 200 rpm for 48 hours. Samples were taken at 12 h, 24 h, 36 h, and 48 h respectively, and the relative fluorescence intensity of the fluorescent protein Mcherry was measured. The specific method for detecting the relative fluorescence intensity of the fluorescent protein Mcherry is as follows: Take 1 mL of the fermentation broth and centrifuge it at 13000 rpm for 2 min; remove the supernatant as much as possible, and resuspend the cell pellet with an equal volume of 10 mM PBS solution; measure the OD460 of the resuspended solution and dilute the resuspended solution to an OD460 of 1.0; Take 200 μL of the diluted bacterial solution and add it to a black 96-well microplate, and measure the fluorescence intensity of the bacteria in the black well plate with a microplate reader. The measured fluorescence intensity minus the fluorescence intensity of the control strain TH-control is the relative fluorescence intensity of the cells. The changes in the relative fluorescence intensity of the recombinant strains TH-P1-mcherry to TH-P54-mcherry and the control strain TH-Pa2 are respectively as Figures 2 to 8 shown.

[0540] Combined with Figure 1 andFigures 2 to 8 It can be seen that the provided promoter shows promoter activity in Rhodococcus ruber in a stationary-phase specific manner.

[0541] The results show that for the common promoter Pa2 of Rhodococcus ruber, the expression level of the fluorescent protein at 24 h in the logarithmic phase can reach 1 / 3 of that at 48 h in the stationary phase. Therefore, for some proteins that have a negative effect on cell growth, when expressed using the Pa2 promoter, their growth will be inhibited. For the promoters P1 - P54 obtained by mining, it can be observed that their expression intensity at 24 h in the logarithmic phase is weak, all below 1 / 10 of the expression level at 48 h in the stationary phase, and the expression intensity of the fluorescent protein ranges from the highest 13574 to the lowest 248, with a 50-fold difference. Therefore, the newly obtained stationary-phase specific promoter of Rhodococcus ruber has obvious characteristics of high expression in the stationary phase and a large span of expression intensity.

[0542] Example 3: Application of the stationary-phase specific promoter of Rhodococcus ruber in the expression of holin

[0543] The purpose of this example is to construct a holin expression system based on the stationary-phase specific promoter of Rhodococcus ruber.

[0544] First, construct a plasmid vector carrying the holin gene. The method is as follows: The plasmid vectors pNV18.1 - Pa2 - mcherry, pNV18.1 - P1 - mcherry, pNV18.1 - P2 - mcherry, pNV18.1 - P3 - mcherry, pNV18.1 - P4 - mcherry, pNV18.1 - P5 - mcherry in Example 2 were digested with KpnI and EcoRI to obtain plasmid backbones; the synthesized holin gene (SEQ ID NO:56) was used, and the plasmid backbone and the holin gene were ligated by Gibson and transformed into Top10 competent cells, which were then spread on plates containing 25 μg / mL kanamycin to construct the pNV18.1 - Pa2 - holin, pNV18.1 - P1 - holin, pNV18.1 - P2 - holin, pNV18.1 - P3 - holin, pNV18.1 - P4 - holin, pNV18.1 - P5 - holin expression plasmids respectively.

[0545] The expression plasmids pNV18.1-Pa2-holin, pNV18.1-P1-holin, pNV18.1-P2-holin, pNV18.1-P3-holin, pNV18.1-P4-holin, pNV18.1-P5-holin and the pNV18.1 plasmid that does not express the holin gene were respectively introduced into Rhodococcus ruber TH to obtain the recombinant strains TH-Pa2-holin, TH-P1-holin, TH-P2-holin, TH-P3-holin, TH-P4-holin, TH-P5-holin, and the control strain TH-control.

[0546] The recombinant strains TH-Pa2-holin, TH-P1-holin, TH-P2-holin, TH-P3-holin, TH-P4-holin, TH-P5-holin and the control strain TH-control were respectively inoculated into a seed medium supplemented with 25 μg / mL kanamycin and cultured at 25-37 °C and 100-200 rpm for 48 h. Then, they were transferred to the fermentation medium at a ratio of 10% and fermented at 28 °C and 200 rpm for 48 h. The OD460 of the fermentation broth was measured at 24 h and 48 h, and the cell permeability was measured by sampling at 48 h. The specific method for detecting cell permeability is as follows: The Rhodococcus cells were diluted with water at pH 7.2-7.5 to an OD460 of 1.0; the FAM-labeled cyclic peptide (FAM-CG6C) was dissolved in water and diluted to 0.1 mg / mL; after centrifuging 1 mL of the diluted Rhodococcus cells at 8000 rpm for 2 min, 1 mL of the FAM-CG6C solution was added, and the mixture was incubated and stained at 28 °C in the dark for 1 h; after staining, it was centrifuged at 8000 rpm for 2 min, washed 3 times with water, resuspended with PBS, and detected with an excitation wavelength of 488 nm and an emission wavelength of 520 nm.

[0547] The effects of the promoters Pa2 and P1-P5 expressing the pore-forming protein Holin on cell growth and cell permeability are respectively as Figure 9 、 Figure 10 shown.

[0548] The results showed that expressing holin using the strong promoter Pa2 had a significant inhibitory effect on cell growth, and the OD460 at 24 h and 48 h was only 33.0% and 24.6% of the normal level, respectively. While expressing holin using promoters P1 - P5 had no obvious effect on cell growth at 24 h, and had a partial effect on cell growth at 48 h, with the OD460 reaching over 83.6% of the normal level. In terms of improving cell permeability, the permeabilities of TH-P1-holin, TH-P2-holin, TH-P3-holin, TH-P4-holin, and TH-P5-holin were increased by 345%, 321%, 302%, 276%, and 266% respectively compared to the control. Although lower than 351% of TH-Pa2-holin, there was also a significant improvement. Overall, it can be seen that using the provided promoters can successfully achieve the expression of holin without obvious impact on growth, and can also realize the ability of holin to improve cell permeability.

[0549] Example 4: Application of the specific promoter during the stationary phase of Rhodococcus erythropolis in the expression of nucleic acid degrading enzymes

[0550] The purpose of this example is to construct a nucleic acid degrading enzyme Ddel expression system based on the specific promoter during the stationary phase of Rhodococcus erythropolis.

[0551] First, construct a plasmid vector carrying the nucleic acid degrading enzyme Ddel gene. The method is as follows: The plasmid vectors pNV18.1-Pa2-mcherry, pNV18.1-P10-mcherry, pNV18.1-P15-mcherry, pNV18.1-P20-mcherry, pNV18.1-P25-mcherry, and pNV18.1-P30-mcherry in Example 2 were digested with KpnI and EcoRI to obtain the plasmid backbone; the synthesized nucleic acid degrading enzyme ddel gene (SEQ ID NO:57) was used, and the two were ligated by Gibson and transformed into Top10 competent cells, which were then spread on plates containing 25 μg / mL kanamycin to respectively construct the expression plasmids pNV18.1-Pa2-Ddel, pNV18.1-P10-Ddel, pNV18.1-P15-Ddel, pNV18.1-P20-Ddel, pNV18.1-P25-Ddel, and pNV18.1-P30-Ddel.

[0552] The expression plasmids pNV18.1-Pa2-Ddel, pNV18.1-P10-Ddel, pNV18.1-P15-Ddel, pNV18.1-P20-Ddel, pNV18.1-P25-Ddel, pNV18.1-P30-Ddel, and the pNV18.1 plasmid that does not express the ddel gene were respectively introduced into Rhodococcus erythropolis to obtain the recombinant strains TH-Pa2-Ddel, TH-P10-Ddel, TH-P15-Ddel, TH-P20-Ddel, TH-P25-Ddel, TH-P30-Ddel, and the control strain TH-control.

[0553] The recombinant strains TH-Pa2-Ddel, TH-P10-Ddel, TH-P15-Ddel, TH-P20-Ddel, TH-P25-Ddel, TH-P30-Ddel, and the control strain TH-control were respectively inoculated into a seed medium supplemented with 25 μg / mL kanamycin and cultured at 25 - 37°C and 100 - 200 rpm for 48 hours. Then, they were transferred to a fermentation medium at a ratio of 10% and fermented at 28°C and 200 rpm for 48 hours. The OD460 of the fermentation broth was measured at 24 h and 48 h, and samples were taken at 48 h to measure the cell survival rate. The specific method for detecting the cell survival rate is as follows: The Rhodococcus erythropolis cells were diluted with water at pH 7.2 - 7.5 to an OD460 of 1.0; the diluted recombinant bacterial solution was further diluted 1000 times, and the control bacterial solution was further diluted 100000 times. 100 μL of each bacterial solution was spread on an LB plate containing 25 μg / mL kanamycin; after culturing for 48 h, the colonies were counted.

[0554] The effects of Pa2 and promoters P10, P15, P20, P25, P30 on expressing the nucleic acid degrading enzyme Ddel on cell growth and cell survival rate are respectively as Figure 11 , Figure 12 shown.

[0555] The results showed that expressing the nucleic acid degrading enzyme Ddel using the strong promoter Pa2 had an obvious inhibitory effect on cell growth, and the OD460 at 24 h and 48 h was only 19.2% and 12.9% of the normal value. However, expressing the nucleic acid degrading enzyme Ddel using promoters P10, P15, P20, P25, P30 had no obvious effect on cell growth at 24 h and had a partial effect on cell growth at 48 h, and the OD460 could reach more than 87.8% of the normal value. In terms of cell survival rate, the cell survival rates of TH-Pa2-Ddel, TH-P10-Ddel, TH-P15-Ddel, TH-P20-Ddel, TH-P25-Ddel, TH-P30-Ddel could be reduced to 10 -4In summary, the use of the provided promoter can successfully achieve the expression of nucleolytic enzymes without significant impact on growth, and can also realize the ability of nucleolytic enzymes to reduce the cell viability at the end of fermentation.

[0556] Example 5: Application of the stationary-phase specific promoter of Rhodococcus ruber in the expression of cell elongation protein

[0557] The purpose of this example is to construct a cell elongation protein DivIVA expression system based on the stationary-phase specific promoter of Rhodococcus ruber.

[0558] First, construct a plasmid vector carrying the cell elongation protein DivIVA gene as follows: The plasmid backbones of pNV18.1-Pa2-mcherry, pNV18.1-P6-mcherry, pNV18.1-P12-mcherry, pNV18.1-P18-mcherry, and pNV18.1-P24-mcherry in Example 2 were obtained by digestion with KpnI and EcoRI; the synthesized cell elongation protein divIVA gene (SEQ ID NO:58) was used, and the two were ligated by Gibson and transformed into Top10 competent cells, which were then spread on plates containing 25 μg / mL kanamycin to construct the pNV18.1-Pa2-Div, pNV18.1-P6-Div, pNV18.1-P12-Div, pNV18.1-P18-Div, and pNV18.1-P24-Div expression plasmids respectively.

[0559] The pNV18.1-Pa2-Div, pNV18.1-P6-Div, pNV18.1-P12-Div, pNV18.1-P18-Div, and pNV18.1-P24-Div expression plasmids, and the pNV18.1 plasmid that does not express the holin gene were respectively introduced into Rhodococcus ruber to obtain the recombinant strains TH-Pa2-Div, TH-P6-Div, TH-P12-Div, TH-P18-Div, TH-P24-Div, and the control strain TH-control.

[0560] Recombinant bacteria TH-Pa2-Div, TH-P6-Div, TH-P12-Div, TH-P18-Div, TH-P24-Div, and the control strain TH-control were respectively inoculated into the seed medium supplemented with 25 μg / mL kanamycin, cultured at 25 - 37°C and 100 - 200 rpm for 48 hours, transferred to the fermentation medium at a ratio of 10%, and fermented at 28°C and 200 rpm for 48 hours. The OD460 of the fermentation broth was measured at 24 h and 48 h respectively, and samples were taken at 48 h to measure cell morphology and cell length. The specific methods for detecting cell morphology and cell length are as follows: Take 2 μL of the bacterial solution and evenly coat it on a glass slide, and air-dry it at room temperature; Add 0.1 mL of the dye to the air-dried bacteria, after staining for 3 min, bake it with an alcohol lamp until the liquid completely evaporates; Gently wash off the dye with deionized water, and dry the glass slide in an oven at 45°C; Drop 20 μL of cedar oil on the fixed bacteria, directly observe the cell morphology with a 100× objective lens, and take pictures for preservation; Measure the cell lengths of 10 cells in each of 2 fields of view, and calculate the average cell length.

[0561] The effects of promoter Pa2 and the provided promoters P6, P12, P18, P24 expressing the cell elongation protein DivIVA on cell growth, cell morphology, and cell length are respectively as Figure 13 , Figure 14 and Figure 15 shown.

[0562] The results showed that expressing the cell elongation protein DivIVA using the strong promoter Pa2 had an obvious inhibitory effect on cell growth, and the OD460 at 24 h and 48 h was only 53.7% and 55.9% of the normal value. While expressing the cell elongation protein DivIVA using the provided promoters P6, P12, P18, P24 had no obvious effect on cell growth at 24 h, and had a partial effect on cell growth at 48 h, but the OD460 could also reach more than 90.5% of the normal value. In terms of cell morphology, the cell size of the recombinant bacteria had obvious changes compared with the control bacteria, and the cell length increased significantly. According to the measurement of the average cell length, the cell lengths of TH-Pa2-Div, TH-P6-Div, TH-P12-Div, TH-P18-Div, TH-P24-Div increased from 1.20 μm in the control group to 2.92 μm, 2.67 μm, 2.50 μm, 2.12 μm, and 1.58 μm respectively. Generally speaking, using the provided promoters can successfully achieve the expression of the cell elongation protein and have no obvious effect on growth, and can also achieve the ability of the cell elongation protein to increase cell length.

[0563] Comparative Example 1

[0564] Recombinant bacteria expressing the red fluorescent protein mcherry were constructed using stationary-phase specific promoters from other strains. The construction method was similar to that in Example 2, except that the promoter sequences T1 - T3 (SEQ ID NO:59 - SEQ ID NO:61) from Bacillus subtilis and the promoter sequence T4 (SEQ ID NO:62) from Mycobacterium smegmatis were used for construction, resulting in recombinant bacteria TH-T1 to TH-T4.

[0565] The recombinant bacteria TH-T1 to TH-T4 were fermented and cultured in the same method as in Example 2, and the fluorescence intensity of the bacterial cells was detected in the same method as in Example 2. The results are as Figure 16 shown.

[0566] As Figure 16 shown by the results, the late-stage promoters T1 - T4 from other strains did not show the characteristics of late-stage expression in Rhodococcus.

[0567] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity in description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as within the scope described in this specification.

[0568] The above-described embodiments only represent several implementation manners of the present invention. Their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent should be subject to the appended claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. A promoter, characterized in that The promoter includes characteristic fragment 1 and / or characteristic fragment 2; The sequence of the characteristic fragment 1 includes VBCGHCDW, and the sequence of the characteristic fragment 2 includes SNNSWWBBWB; Among them, each S independently represents a G base or a C base, each W independently represents an A base or a T base, each H independently represents an A base, a T base or a C base, each B independently represents a G base, a T base or a C base, each V independently represents a G base, an A base or a C base, each D independently represents a G base, an A base or a T base, and each N independently represents an A base, a T base, a G base or a C base.

2. The promoter according to claim 1, characterized in that The nucleotide sequence of the promoter includes the nucleotide sequence shown in Formula I or Formula II, wherein: (N) p VBCGHCDW(N) q SNNSWWBBWB(N) r , (N) p SNNSWWBBWB(N) q VBCGHCDW(N) r , In each formula, p, q, and r represent the number of random bases, and p, q, and r are each independently selected from integers between 1 and 350: Optionally, in each formula, p, q, and r represent the number of random bases, and p, q, and r are each independently selected from integers between 3 and 310.

3. The promoter according to any one of claims 1 to 2, characterized in that The nucleotide sequence of the promoter is shown in one of SEQ ID NO: 1 to SEQ ID NO:

54.

4. A promoter library, characterized in that The promoter library comprises the promoter according to any one of claims 1 to 3; Optionally, the promoter library includes multiple promoters whose nucleotide sequences are shown in SEQ ID NO: 1 to SEQ ID NO: 54; Optionally, the promoter library includes promoters with nucleotide sequences as shown in SEQ ID NO: 1 to SEQ ID NO:

54.

5. A gene expression cassette, characterized in that The gene expression cassette comprises the promoter according to any one of claims 1 to 3 and a target gene; Optionally, the target gene includes one or more genes that have an inhibitory effect on cell growth; Optionally, the target gene includes one or more of a gene encoding a holin, a gene encoding a nucleic acid degrading enzyme, and a gene encoding a cell extension protein; Optionally, in the gene expression cassette, the target gene is located downstream of the promoter.

6. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the promoter according to any one of claims 1 to 3 or the gene expression cassette according to claim 5; Optionally, the recombinant expression vector includes one or more of a plasmid vector, a phage vector and a viral vector; Optionally, the plasmid vector comprises one or more of pNV18.1, pBNV, a pNV18.1-derived plasmid and a pBNV-derived plasmid.

7. A recombinant cell, characterized in that One or more of the following conditions are met: The recombinant cell carries the promoter according to any one of claims 1 to 3; The recombinant cell carries the gene expression cassette of claim 5; and The recombinant cell carries the recombinant expression vector according to claim 6.

8. The recombinant cell according to claim 7, characterized in that The host cell of the recombinant cell includes Rhodococcus; Optionally, the genus Rhodococcus includes Rhodococcus rubrum; Optionally, the Rhodococcus rubrum includes Rhodococcus rubrum TH.

9. A method for constructing a recombinant cell, characterized in that: The method comprises the following steps: introducing the promoter according to any one of claims 1 to 3, the gene expression cassette according to claim 5, or the recombinant expression vector according to claim 6 into the host cell to construct the recombinant cell.

10. A method for preparing a target protein, characterized in that: The method comprises the following steps: culturing at least one of the recombinant cells described in claim 7 or 8 and the recombinant cells constructed by the construction method described in claim 9.

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