Shuttle vector, prokaryotic host cell containing shuttle vector, and method for producing protein using host cell

Abstract

The present application provides a shuttle vector, which can be applied to various host cells for operation, thereby providing a novel tool in the field of genetic engineering. The present application further provides a prokaryotic host cell containing the shuttle vector, so as to use the shuttle vector to construct an expression vector containing a gene to be expressed, thereby allowing mass production of proteins in different hosts by using a single vector.

Description

Shuttle vector, prokaryotic host cell containing the same, and method for producing protein using the host cell Technical Field

[0001] The present application relates to a vector for genetic engineering, in particular to a shuttle vector capable of replicating in different hosts, a host cell comprising the shuttle vector, and a method for producing proteins using these host cells. Background Art

[0002] Plasmids are extrachromosomal genetic material found in microorganisms. They are often used as vectors in genetic engineering to carry exogenous genes into host cells for expression. Vectors typically contain: (a) a replicon gene, which enables the vector to replicate within the host; (b) multiple cloning sites, containing various restriction enzyme sites, allowing the target gene to be inserted into the vector; and (c) a drug-resistance gene, which allows the host to acquire a drug-resistant phenotype and allows selection of clones containing the correct target gene. Common vector types include shuttle vectors, cloning vectors, and expression vectors.

[0003] Among them, a shuttle vector refers to a plasmid that can replicate or express proteins in different organisms, such as between different prokaryotic cells, or between prokaryotic and eukaryotic cells. In order to enable it to replicate or express proteins in different hosts, it usually contains replication origins corresponding to different hosts to obtain the ability to replicate in different hosts, thereby effectively improving the convenience of genetic manipulation. For example, gene cloning is often carried out using easy-to-operate Escherichia coli. After the shuttle vector with the target gene fragment is cloned, it can be transformed into another host that can express proteins normally and in large quantities for gene expression. In view of the advantage that shuttle vectors can be used for genetic manipulation in two or more hosts, the field continues to need a variety of new shuttle vectors to meet research and commercial needs.

[0004] Brevibacillus choshinensis is an obligate aerobic Gram-positive bacillus that can produce spores. Currently, the Br. choshinensis HPD31 strain has been developed as a gene expression host for the production of various recombinant proteins. Using it as a gene expression host has several advantages, including: Br. choshinensis is a non-pathogenic, highly safe strain with no safety concerns in operation and application; Br. choshinensis has the characteristic of strong protein secretion and can be used for extracellular production of recombinant proteins. Compared with the Escherichia coli expression system, it is less likely to form inclusion bodies; Br. choshinensis has extremely low extracellular protease activity, and secreted recombinant proteins are not easily degraded.

[0005] Although the Br. choshinensis HPD31 strain has been widely used to express diverse heterologous proteins, the selectivity of shuttle vectors used in Brevibacillus choshinensis is still relatively low.

[0006] Summary of the Invention

[0007] To provide the art with more diverse vector options, this application provides a novel shuttle vector capable of replicating and expressing proteins between different hosts. In one embodiment, the host cells may include at least Escherichia coli, Bacillus subtilis, and Brevibacillus choshinensis.

[0008] In one aspect, the shuttle vector can comprise: (a) a replicon region of the E. coli plasmid pBR322 having the sequence shown in SEQ ID NO: 1; and (b) a replicon region of a plasmid of a Gram-positive bacterium.

[0009] Preferably, the Gram-positive bacteria plasmid replicon region of the shuttle vector may include: (a) the replicon region of the asexual plasmid pBC1 of Bacillus coagulans, which has the sequence shown in SEQ ID NO: 3; (b) the replicon region of the plasmid pC194 of Staphylococcus aureus, which has the sequence shown in SEQ ID NO: 4; or (c) the replicon region of the asexual plasmid pLP31-8 of Lactobacillus plantarum, which has the sequence shown in SEQ ID NO: 5.

[0010] Preferably, the shuttle vector may further comprise a regulatory protein gene of the Escherichia coli plasmid pBR322, which has the sequence shown in SEQ ID NO: 2.

[0011] Preferably, the shuttle vector may further comprise at least one screening gene. In one embodiment, the screening gene may comprise a drug resistance screening gene, a non-drug resistance screening gene, or a combination thereof. In a preferred embodiment, the drug resistance screening gene may comprise a chloramphenicol resistance gene or a neomycin resistance gene.

[0012] Preferably, the shuttle vector may further comprise a multiple cloning site for cloning target genes.

[0013] Preferably, the shuttle vector may further comprise an expression signal, including: (a) an expression signal P01 HPD52 , which has a sequence as shown in SEQ ID NO: 8; (b) expression signal P02 HPD52 , which has a sequence as shown in SEQ ID NO: 9; (c) expression signal P03 HPD52 , which has the sequence shown in SEQ ID NO: 10; (d) expression signal P04 HPD52 , which has a sequence as shown in SEQ ID NO: 11; (e) expression signal P05 HPD52 , which has the sequence shown in SEQ ID NO: 12; (f) expression signal P06 HPD52 , which has the sequence shown in SEQ ID NO: 13; (g) expression signal P07 HPD52 , which has the sequence shown in SEQ ID NO: 14; (h) expression signal P08 HPD52 , which has the sequence shown in SEQ ID NO: 15; (i) expression signal P09 HPD52 , which has the sequence shown in SEQ ID NO: 16; (j) expression signal P10 HPD52 , which has the sequence shown in SEQ ID NO: 17; (k) expression signal P11 HPD52 , which has a sequence as shown in SEQ ID NO: 18; (1) expression signal P13 HPD52 , which has the sequence shown in SEQ ID NO: 20; (m) expression signal P14 HPD52 , which has the sequence shown in SEQ ID NO: 21; (n) expression signal P15 HPD52 , which has the sequence shown in SEQ ID NO: 22; (o) expression signal P16 HPD52 , which has the sequence shown in SEQ ID NO: 23; (p) expression signal P17 HPD52, which has the sequence shown in SEQ ID NO: 24; or (q) expression signal P19 HPD52 , which has a sequence as shown in SEQ ID NO: 26. In a preferred embodiment, the expression signal may further include an inducible expression signal, including: (a) an inducible expression signal P06 HPD52 / lacO-1, which has the sequence shown in SEQ ID NO: 69; (b) inducible expression signal P06 HPD52 / lacO-2, which has the sequence shown in SEQ ID NO: 70; (c) inducible expression signal P06 HPD52 / lacO-3, which has the sequence shown in SEQ ID NO: 71; (d) inducible expression signal P06 HPD52 / lacO-4, which has the sequence shown in SEQ ID NO: 72; (e) inducible expression signal P06 HPD52 / lacO-5, which has the sequence shown in SEQ ID NO: 73; (f) inducible expression signal P06 HPD52 / lacO-6, which has the sequence shown in SEQ ID NO: 74; or (g) an inducible expression signal P06 HPD52 / lacO-7, which has the sequence shown in SEQ ID NO: 75. In a more preferred embodiment, the shuttle vector further comprises a regulatory gene of an inducible expression signal.

[0014] Preferably, the shuttle vector may further comprise a reporter gene. In one embodiment, the reporter gene comprises: (a) the cellulase gene bglC from Bacillus subtilis; (b) the α-amylase gene amyL from Bacillus licheniformis; or (c) the α-amylase gene amyS from Geobacillus stearothermophilus.

[0015] Preferably, the shuttle vector may further comprise a signal peptide comprising: (a) a signal peptide SP02 having a sequence as shown in SEQ ID NO: 92; (b) a signal peptide SP04 having a sequence as shown in SEQ ID NO: 94; (c) a signal peptide SP07 having a sequence as shown in SEQ ID NO: 97; (d) a signal peptide SP08 having a sequence as shown in SEQ ID NO: 98; (e) a signal peptide SP10 having a sequence as shown in SEQ ID NO: 100; (f) a signal peptide SP11 having a sequence as shown in SEQ ID NO: 101; or (g) a signal peptide SP13 having a sequence as shown in SEQ ID NO: 103.

[0016] Preferably, the shuttle vector may further contain a protein gene.

[0017] Another object of the present application is to provide a prokaryotic host cell, which may contain the aforementioned shuttle vector.

[0018] Preferably, the host cell may include Escherichia coli (E. coli), Bacillus subtilis (B. subtilis) and / or Brevibacillus choshinensis (Br. choshinensis).

[0019] Another object of the present application is to provide a method for producing a protein using the aforementioned prokaryotic host cell, which method may comprise the following steps: placing the aforementioned prokaryotic host cell in a culture medium, and allowing the prokaryotic host cell to express the protein under appropriate culture conditions; and recovering the protein from the prokaryotic host cell or the culture medium of the prokaryotic host cell.

[0020] Preferably, the protein in the aforementioned method may comprise a Momordica charantia trypsin inhibitor; more preferably, the Momordica charantia trypsin inhibitor may comprise the sequence shown in SEQ ID NO: 169. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1. General structure of a signal peptide.

[0022] Figure 2. Schematic representation of vectors pEBR-1 to pEBR-4.

[0023] Figure 3. Confirmation of plasmids in Br. choshinensis HPD52 transformants. Agarose gel electrophoresis of plasmid DNA and restriction enzyme-cleaved DNA from (A) Br. choshinensis HPD52, (B) Br. choshinensis HPD52(pNCMO2), (C) Br. choshinensis HPD52(pEBR-1), (D) Br. choshinensis HPD52(pEBR-2), and (E) Br. choshinensis HPD52(pEBR-3).

[0024] Figure 4.P01 HPD52 ~P19 HPD52 A simplified diagram of the expression signal.

[0025] Figure 5. E. coli ECOS with green fluorescent protein expression vectors with different expression signals TM Colony morphology of 9-5 after 24 hours of culture on LB plate. Control group: E. coli ECOS TM 9-5(pEBR-1).

[0026] Figure 6. Analysis of different expression signals of green fluorescent protein gene in E. coli ECOS TM The expression efficiency of 9-5. The figure records the OD of the transformant at different time periods. 600 Signal (open circle) and fluorescence intensity (solid square). Control group: E. coli ECOS TM 9-5(pEBR-1).

[0027] Figure 7. E. coli ECOS with different expression signal green fluorescent protein expression vectors TM 9-5 Fluorescence intensity after 24 hours of culture. Control group: E. coli ECOS TM 9-5(pEBR-1).

[0028] Figure 8. Colony morphology of Br. choshinensis HPD52 cells expressing different green fluorescent protein expression vectors after 48 hours of culture on MT plates. Control group: Br. choshinensis HPD52 (pEBR-1).

[0029] Figure 9. Analysis of expression efficiency of different expression signals in Br. choshinensis HPD52 using green fluorescent protein gene. The figure records the OD of the transformant at different time periods. 600Control group: Br. choshinensis HPD52 (pEBR-1).

[0030] Figure 10. Fluorescence intensity of Br. choshinensis HPD52 cells carrying different expression signal green fluorescent protein expression vectors after 48 hours of culture. Control group: Br. choshinensis HPD52 (pEBR-1).

[0031] Figure 11.P06 HPD52 Engineered to be an inducible expression signal sequence.

[0032] Figure 12. Colony morphology of Br. choshinensis HPD52 cells expressing green fluorescent protein with different inducible expression vectors after culturing on MT plate medium for 48 hours.

[0033] Figure 13. Analysis of different P06 using green fluorescent protein gene HPD52 / lacO inducible expression signal in Br. choshinensis HPD52. The figure records the OD of the transformant at different time points. 600 Control group: Br. choshinensis HPD52 (pEBR-1).

[0034] Figure 14. With different P06 HPD52 Fluorescence intensity of Br. choshinensis HPD52 cells expressing the / lacO inducible expression signal vector after 48 hours of culture.

[0035] Figure 15. Analysis of expression efficiency of different expression signals in B. subtilis DB430 using green fluorescent protein gene. The figure records the OD of transformants at different time periods. 600 The signal (open circles) and fluorescence intensity (filled circles) of the control group: B. subtilis DB430 (pEBR-1).

[0036] FIG16 shows the fluorescence intensity of B. subtilis DB430 cells expressing green fluorescent protein with different expression signal vectors after 48 hours of culture.

[0037] Figure 17. Analysis of different P06 using green fluorescent protein gene HPD52 / lacO inducible expression signal in B.subtilis DB430. The figure records the OD of the transformant at different time points. 600The signal (open circles) and fluorescence intensity (filled circles) of the control group: B. subtilis DB430 (pEBR-1).

[0038] Figure 18. With different P06 HPD52 Fluorescence intensity of B. subtilis DB430 expressing the lacO-inducible expression signal after 48 h of culture.

[0039] Figure 19. The activities of BSBglC, BLAmyL and GSAmyS of Br. choshinensis HPD52 transformants were measured in plate culture medium after 48 hours of culture. (A) BSBglC: Br. choshinensis HPD52 (pEBR-1-P06 HPD52 / lacO-1-BSBglC). (B) BLAmyL: Br.choshinensis HPD52 (pEBR-1-P06 HPD52 / lacO-1-BLAmyL). (C) GSAmyS: Br.choshinensis HPD52 (pEBR-1-P06 HPD52 Control group: Br. choshinensis HPD52 (pEBR-1).

[0040] Figure 20. Protein electrophoresis analysis of BSBglC, BLAmyL, and GSAmyS secreted by Br. choshinensis HPD52 transformants. (A) Control: Br. choshinensis HPD52 (pEBR-1). (B) BSBglC: Br. choshinensis HPD52 (pEBR-1-P06 HPD52 / lacO-1-BSBglC). (C) BLAmyL: Br.choshinensis HPD52 (pEBR-1-P06 HPD52 / lacO-1-BLAmyL). (D) GSAmyS: Br.choshinensis HPD52 (pEBR-1-P06 HPD52 / lacO-1-GSAmyS).

[0041] FIG21 . Effects of different signal peptides on the secretory expression of GSAmyS in Br. choshinensis HPD52.

[0042] Figure 22. Nucleotide and amino acid sequences of momordica charantia trypsin inhibitor TI-4.

[0043] Figure 23. Protein electrophoresis analysis of the secretion of momordica charantia trypsin inhibitor expressed in Br. choshinensis HPD52 transformants. (A) Control group: Br. choshinensis HPD52 (pEBR-1). (B) Br. choshinensis HPD52 (pEBR-1-P06 HPD52 / lacO-3-sec-TI). (C)Br.choshinensis HPD52(pEBR-1- P06 HPD52 / lacO-3-SP02-TI). (D)Br.choshinensis HPD52(pEBR-1-P06 HPD52 / lacO-3-SP04-TI). (E)Br.choshinensis HPD52(pEBR-1-P06 HPD52 / lacO-3-SP13-TI). M:SeeBlue TM Pre-stained Protein Standard.

[0044] Figure 24. Protein electrophoresis analysis of Br. choshinensis HPD52 (pEBR-1-P06 HPD52 / lacO-3-SP13-TI) culture supernatant to obtain a momordica charantia trypsin inhibitor.

[0045] Figure 25. Characterization of purified recombinant TI-4 using linear ion trap mass spectrometry. DETAILED DESCRIPTION

[0046] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by those skilled in the art. In case of conflict, the definitions contained in this specification will control.

[0047] When referring to concentration in this application, "percentage", "%", etc., as used herein, may refer to mass concentration or volume percentage concentration; mass concentration is defined as the mass of the solute divided by the percentage of the volume of the corresponding mixture (including the volume of the solute), and may be expressed herein by "percentage", "%", etc. followed by "(w / v)"; volume percentage concentration is defined as the percentage of the volume of the solute to the total volume of the solution, and may be expressed herein by "percentage", "%", etc. followed by "(v / v)".

[0048] As used in this application, the term "produced from" is synonymous with "comprising." As used herein, the terms "includes," "including," "comprises," "comprising," "has," "having," "contains," "containing," or any other variations thereof, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that contains a plurality of listed elements is not necessarily limited to only those listed elements but may include other elements not expressly listed but inherent to the composition, process, method, article, or apparatus. The term "comprising" is generally used in an inclusive sense, that is, allowing for the presence of one or more additional features or components.

[0049] The indefinite articles "a" and "an" preceding an element or component in the present application are intended to indicate the number of instances (i.e., the number of occurrences) of the element or component without limitation. Therefore, "a" or "an" should be understood to include one or at least one, and the singular form of the element or component also includes the plural unless the number is obviously intended to be singular.

[0050] The present application provides many sequences, including primer sequences, gene DNA sequences and amino acid sequences. When the functions and effects of these sequences in organisms are not changed, they should be allowed to have some mutations. That is to say, even if the sequences are different, but the functions and effects produced are the same as those of the sequences provided in the present application, they should be considered to be the same sequence. For example: in primer sequences, even if the primer sequences are slightly different, primers that can amplify the same product should be considered to be the same; in gene DNA sequences, if a gene undergoes a silent mutation, the amino acid sequence transcribed from it is the same as before the mutation, it should be considered to be the same sequence; for example, in amino acid sequences, amino acid bases with similar physical properties (such as the same electrical properties, the same hydrophilicity or hydrophobicity, benzene rings, etc.) mutate with each other without affecting their original structure and function, and should be considered to be the same sequence. The above situations are only examples and should not be interpreted as limitations.

[0051] The present application aims to provide a shuttle vector capable of replicating in at least one prokaryotic host cell, namely, Escherichia coli, Bacillus subtilis, and Brevibacillus brigittatus. This shuttle vector carries the gene sequence for a desired protein and thereby expresses the protein. By enabling replication in two or more host cell types, this shuttle vector overcomes the disadvantage of being unable to express a protein in a particular host cell, thus offering advantages in genetic engineering applications.

[0052] The terms "shuttle vector" and "expression vector" are used interchangeably herein to refer to vectors containing plasmid replicon regions for both E. coli and various Gram-positive bacteria, enabling the vector to replicate and proliferate in at least E. coli and various Gram-positive bacteria, thus overcoming the species-specific limitations of plasmids. An expression vector specifically refers to a shuttle vector capable of expressing a protein in a host.

[0053] As used herein, "replicon" refers to a region where DNA replication originates, which includes at least one origin of replication.

[0054] In one embodiment, the shuttle vector further comprises a regulatory protein gene from the E. coli plasmid pBR322. Preferably, the regulatory protein gene is a repressor gene, including the rop gene, which can regulate the replication number of the vector and maintain the stability of the vector replication.

[0055] In one embodiment, the shuttle vector comprises a selection gene, such as a neomycin resistance gene marker or a chloramphenicol resistance gene marker, but is not limited thereto.

[0056] In one embodiment, the shuttle vector further comprises a multiple cloning site comprising various restriction enzyme sites for inserting genes to be expressed.

[0057] In one embodiment, the shuttle vector further comprises an expression signal. The term "expression signal" as used herein refers to a DNA sequence that enables transcription of a specific gene, which includes: at least one or more promoters. Because different expression signals have different strengths in attracting RNA polymerase, the transcription strength is also different, which in turn affects the amount of protein expression. Preferably, the expression signal can be made into an inducible expression signal by inserting a regulatory sequence (operator) near it. The inducible expression signal can be negatively or positively regulated, that is, by adding or removing an inducer, the transcription can be promoted or inhibited. Common inducers include sugars and amino acid molecules. Preferably, the shuttle vector further comprises a regulatory gene for an inducible expression signal.

[0058] As used herein, the term "regulatory gene of an inducible expression signal" refers to an essential gene required for the proper functioning of the inducible expression signal. For example, in the lactose operon (lac operon) of the E. coli expression system, the lactose regulatory sequence (lac operator, lacO) is present within the expression signal and is specifically bound by the lactose repressor (lacI). LacI is an essential regulatory gene for regulating the inducible expression signal. In E. coli expression systems, since E. coli can naturally express the lacI gene, vectors used in E. coli only need to contain the lacO sequence to achieve the desired inducible expression signal.

[0059] In one embodiment, the shuttle vector further comprises a regulatory gene for an inducible expression signal. Because the host used in this application is unable to express this regulatory gene on its own, to ensure the proper functioning of the inducible expression signal, the regulatory gene is inserted into the shuttle vector, allowing the host to express the regulatory gene normally. Preferably, the regulatory gene is the repressor lacI gene in the E. coli expression system.

[0060] In one embodiment, the shuttle vector further comprises a reporter gene. The expression levels of these reporter genes can be used to assess changes in protein expression resulting from the insertion of different genes into the shuttle vector in this application. Preferably, these reporter genes include: the cellulase gene bglC from Bacillus subtilis; the α-amylase gene amyL from Bacillus licheniformis; or the α-amylase gene amyS from Bacillus stearothermophilus.

[0061] The term "reporter gene" as used herein refers to a gene that produces a specific protein. This protein is easily detectable, sensitive, and quantifiable. It can be used to investigate the expression efficiency of expression signals, analyze the secretion efficiency of signal peptides, study the localization of target genes in cells, monitor the success of molecular cloning, identify protein-protein interactions, and analyze the efficiency of gene delivery systems. Currently, a variety of reporter genes, such as lacZ, gusA, and gfp, are widely used in various research fields.

[0062] In one embodiment, the shuttle vector comprises a signal peptide. In the experimental results of this application, different signal peptides used have different degrees of influence on the amount of protein production.

[0063] The term "signal peptide" as used herein refers to a polypeptide that participates in the protein secretion process and facilitates protein translocation. A typical signal peptide can be divided into three regions: the N-region, the H-region, and the C-region (Figure 1). The N-region typically contains a positively charged amino acid, such as arginine or lysine, which binds to transport-related proteins or negatively charged phospholipids on the cell membrane. The H-region is often composed of hydrophobic amino acids, such as leucine, which form a stable α-helical structure in the cell membrane. The central amino acid of the H-region is usually glycine or proline, which enables the signal peptide to form a hairpin-like structure and embed into the cell membrane. The C-region forms a β-sheet structure and contains the signal peptidase cleavage site, with the conserved amino acid sequence of alanine-X-alanine. When a protein with a signal peptide is transported outside the cell, a signal peptidase recognizes this site and cleaves the signal peptide, releasing the protein outside the cell. Because signal peptides facilitate post-translational protein transfer and localization, they are crucial for increasing the secretory yield of recombinant proteins. In one embodiment, a prokaryotic host cell is provided, comprising the aforementioned shuttle vector. In one possible embodiment, the prokaryotic host cell may comprise Escherichia coli (E. coli), Bacillus subtilis (B. subtilis), and / or Brevibacillus choshinensis (Br. choshinensis).

[0064] The terms "host" and "host cell" are used interchangeably herein and may refer to prokaryotic cells. One or more vectors described herein may be introduced into such host cells, or such vectors may be genetically engineered to insert a gene to be expressed. As those skilled in the art will appreciate, these terms refer not only to specific cells but also to the progeny or potential progeny of such cells. Subsequent generations may exhibit changes due to genetic mutations or environmental influences, so the progeny of such cells may not be substantially identical to the parent cell; however, they are still encompassed within the scope of the terms as used herein.

[0065] In one embodiment, the shuttle vector contains a protein gene. The gene sequence for the protein can be any gene sequence, provided that the expression vector can successfully insert the protein gene sequence. Those skilled in the art will appreciate that vectors used in various biotechnological applications can all be used to insert genes for different proteins, provided the conditions allow for the vector.

[0066] In one embodiment, a method for producing a protein using the aforementioned prokaryotic host cell is provided. For example, the protein may comprise Momordica charantia trypsin inhibitor; in one aspect, the Momordica charantia trypsin inhibitor may comprise the sequence shown in SEQ ID NO: 169.

[0067] The examples provided herein for producing a momordica charantia trypsin inhibitor using a shuttle vector are intended only to illustrate the ability of the shuttle vectors provided in this application to mass-produce proteins in various hosts. Therefore, any protein can be inserted into the shuttle vector and expressed in the host.

[0068] The following disclosures are examples of the claimed invention, which serve as specific examples of the present application and should not limit the present application by the following examples.

[0069] Example 1: A shuttle vector comprising:

[0070] (a) the replicon region of the E. coli plasmid pBR322, which has the sequence shown in SEQ ID NO: 1; and

[0071] (b) Plasmid replicon region of Gram-positive bacteria.

[0072] Example 2: The shuttle vector as described in the preceding example, wherein the plasmid replicon region of the Gram-positive bacteria comprises:

[0073] (a) The replicon region of the B. coagulans asexual plasmid pBC1, having the sequence shown in SEQ ID NO: 3;

[0074] (b) the replicon region of the S. aureus plasmid pC194 having the sequence shown in SEQ ID NO: 4; or

[0075] (c) The replicon region of the L. plantarum traitless plasmid pLP31-8, having the sequence shown in SEQ ID NO: 5.

[0076] Example 3: The shuttle vector described in the above example further comprises a regulatory protein gene from the E. coli plasmid pBR322, which has the sequence shown in SEQ ID NO: 2.

[0077] Example 4: The shuttle vector as described in the above example further comprises at least one selection gene.

[0078] Example 5: The shuttle vector as described in the above example, wherein the at least one selection gene comprises a drug-resistance selection gene, a non-drug-resistance selection gene, or a combination thereof.

[0079] Example 6: The shuttle vector as described in the above examples, wherein the drug resistance selection gene comprises a chloramphenicol resistance gene or a neomycin resistance gene.

[0080] Example 7: The shuttle vector as described in the above example further comprises a multiple cloning site.

[0081] Example 8: The shuttle vector described in the above example further comprises an expression signal comprising:

[0082] (a) Expression signal P01 HPD52 , which has the sequence shown in SEQ ID NO: 8;

[0083] (b) Expression signal P02 HPD52 , which has the sequence shown in SEQ ID NO: 9;

[0084] (c) Expression signal P03 HPD52 , which has the sequence shown in SEQ ID NO: 10;

[0085] (d) Expression signal P04 HPD52 , which has the sequence shown in SEQ ID NO: 11;

[0086] (e) Expression signal P05 HPD52 , which has the sequence shown in SEQ ID NO: 12;

[0087] (f) Expression signal P06 HPD52 , which has the sequence shown in SEQ ID NO: 13;

[0088] (g) Expression signal P07 HPD52 , which has the sequence shown in SEQ ID NO: 14;

[0089] (h) Expression signal P08 HPD52 , which has the sequence shown in SEQ ID NO: 15;

[0090] (i) Expression signal P09 HPD52 , which has the sequence shown in SEQ ID NO: 16;

[0091] (j) Expression signal P10 HPD52 , which has the sequence shown in SEQ ID NO: 17;

[0092] (k) Expression signal P11 HPD52 , which has the sequence shown in SEQ ID NO: 18;

[0093] (l) Expression signal P13 HPD52, which has the sequence shown in SEQ ID NO: 20;

[0094] (m) Expression signal P14 HPD52 , which has the sequence shown in SEQ ID NO: 21;

[0095] (n) Expression signal P15 HPD52 , which has the sequence shown in SEQ ID NO: 22;

[0096] (o) Expression signal P16 HPD52 , which has the sequence shown in SEQ ID NO: 23;

[0097] (p) expression signal P17 HPD52 , which has the sequence shown in SEQ ID NO: 24; or

[0098] (q) Expression signal P19 HPD52 , which has the sequence shown in SEQ ID NO: 26.

[0099] Example 9: The shuttle vector as described in the previous example, wherein the expression signal is an inducible expression signal, comprising:

[0100] (a) Inducible expression signal P06 HPD52 / lacO-1, which has the sequence shown in SEQ ID NO: 69;

[0101] (b) Inducible expression signal P06 HPD52 / lacO-2, which has the sequence shown in SEQ ID NO: 70;

[0102] (c) Inducible expression signal P06 HPD52 / lacO-3, which has the sequence shown in SEQ ID NO: 71;

[0103] (d) Inducible expression signal P06 HPD52 / lacO-4, which has the sequence shown in SEQ ID NO: 72;

[0104] (e) Inducible expression signal P06 HPD52 / lacO-5, which has the sequence shown in SEQ ID NO: 73;

[0105] (f) Inducible expression signal P06 HPD52 / lacO-6, which has the sequence shown in SEQ ID NO: 74; or

[0106] (g) Inducible expression signal P06 HPD52 / lacO-7, which has the sequence shown in SEQ ID NO:75.

[0107] Example 10: The shuttle vector as described in the above example further comprises a regulatory gene of an inducible expression signal.

[0108] Example 11: The shuttle vector as described in the above example further comprises a reporter gene, comprising:

[0109] (a) Bacillus subtilis (B. subtilis) cellulase gene bglC;

[0110] (b) the α-amylase gene amyL from Bacillus licheniformis; or

[0111] (c) α-amylase gene amyS from G. stearothermophilus.

[0112] Example 12: The shuttle vector as described in the above example further comprises a signal peptide.

[0113] (a) a signal peptide SP02 having the sequence shown in SEQ ID NO: 92;

[0114] (b) a signal peptide SP04 having the sequence shown in SEQ ID NO: 94;

[0115] (c) a signal peptide SP07 having the sequence shown in SEQ ID NO: 97;

[0116] (d) a signal peptide SP08 having the sequence shown in SEQ ID NO: 98;

[0117] (e) a signal peptide SP10 having the sequence shown in SEQ ID NO: 100;

[0118] (f) a signal peptide SP11 having the sequence shown in SEQ ID NO: 101; or

[0119] (g) Signal peptide SP13 having the sequence shown in SEQ ID NO: 103.

[0120] Example 13: The shuttle vector as described in the previous example further comprises a protein gene.

[0121] Example 14: A prokaryotic host cell comprising the shuttle vector of any one of the preceding examples.

[0122] Example 15: The prokaryotic host cell as described in the previous example, wherein the prokaryotic host cell comprises Escherichia coli (E. coli), Bacillus subtilis (B. subtilis) and / or Brevibacillus choshinensis (Br. choshinensis).

[0123] Example 16: A method for producing a protein using the prokaryotic host cell described in the previous example, the method comprising the following steps:

[0124] placing the prokaryotic host cell described in the above example into a culture medium, and allowing the prokaryotic host cell to express the protein under appropriate culture conditions; and

[0125] The protein is recovered from the cells or the culture medium of the cells.

[0126] Example 17: The method of the preceding example, wherein the protein comprises Momordica charantia trypsin inhibitor.

[0127] Example 18: The method of the preceding example, wherein the momordica charantia trypsin inhibitor comprises the sequence shown in SEQ ID NO: 169.

[0128] The materials, methods and examples in this application are illustrative only and, unless otherwise specified, are not intended to limit the present invention. Although methods or materials similar or equivalent to those described herein can be used to implement or test the present invention, those described herein are more appropriate methods and materials.

[0129] Example 1: Related experimental materials

[0130] 1.1. Bacterial strain

[0131] Escherichia coli ECOS TM 9-5: E. coli ECOS TM 9-5 was purchased from Yeastern Company in Taiwan, China; its genotype is recA1 endA1 gyrA96 thi-1 hsdR17 (rK - mK + )e14 - (mcrA - )supE44 relA1 Δ(lac-proAB) / F′[traD36, proAB + , lacI q , lacZΔM15], and used it as a host cell for DNA cloning.

[0132] Brevibacillus brines HPD52: purchased from the Bioresource Collection and Research Center (BCRC) of the Food Industry Development Institute, BCRC number 15920; this is a soil-isolated strain used as a host cell for recombinant protein expression.

[0133] 1.2. Carrier

[0134] pJET1.2: purchased from Thermo Fisher Scientific, USA, is a blunt-end cloning vector. It is 2,974 bp in size and carries the ampicillin resistance gene (Ap R ). It is used as a vector for DNA cloning.

[0135] pNCMO2: purchased from TaKaRa Co., Ltd., Japan, is a shuttle vector for E. coli and Br. choshinensis. It is 5,225 bp in size and carries Ap R Neomycin resistance gene (Nm R ), where Ap R Can be expressed in E. coli, Nm R choshinensis. The vector also contains a multiple cloning site (MCS), sec signal peptide DNA, and the powerful P2 promoter. The MCS contains restriction enzyme sites including PstI, BamHI, SalI, XbaI, XhoI, EcoRI, KpnI, SmaI, BstBI, ClaI, and HindIII. It was used to test electroporation conditions for Br. choshinensis HPD52 and served as a source of P2 promoter and sec signal peptide DNA.

[0136] pNY326: purchased from TaKaRa, Japan, is an expression vector for Br. choshinensis. It is 3,438 bp in size and carries Nm R The vector also contains the MCS, sec signal peptide DNA, and the less efficient P5 promoter. The MCS contains restriction enzyme sites including PstI, BamHI, SalI, XbaI, XhoI, EcoRI, KpnI, SmaI, BstBI, ClaI, and HindIII. This serves as the source of P5 promoter DNA.

[0137] The pEBR-1 described in the pEBR-1 application has a sequence as shown in SEQ ID NO: 170, which is composed of the replication region (replicon-pBR322), the pBR322 regulatory protein gene (rop-pBR322), the chloramphenicol resistance gene (Cm R ), Nm R , an artificially synthesized MCS and the replication region of the asexual plasmid pBC1 of Bacillus coagulans (B. coagulans). Its size is 4,766 bp, of which Cm R Can be expressed in E. coli and B. subtilis, Nm R It can be expressed in B. subtilis and Br. choshinensis. The restriction enzyme sites contained in the MCS include BglII, EcoRI, SpeI, NdeI, BamHI, XmaI, PstI, SalI, HindIII, XbaI, and XhoI. It was used to test electroporation conditions for Br. choshinensis HPD52 and as a vector for DNA cloning.

[0138] The pEBR-2 described in the pEBR-2 application has a sequence as shown in SEQ ID NO: 171, which is a product of replicon-pBR322, rop-pBR322, Cm R 、Nm R A synthetic MCS and the replication region of Staphylococcus aureus plasmid pC194 (replicon-pC194) are constructed. Its size is 4,865 bp; the MCS carries the same restriction enzyme sites as pEBR-1. It was used to test electroporation conditions for Br. choshinensis HPD52.

[0139] The pEBR-3 described in the pEBR-3 application has a sequence as shown in SEQ ID NO: 172, which is a product of replicon-pBR322, rop-pBR322, Cm R 、Nm RA synthetic MCS and the trait-free plasmid pLP31-8 from Lactobacillus plantarum ATIT-031, obtained by site-directed mutagenesis of EcoRI. Its size is 5,236 bp; the MCS carries the same restriction enzyme sites as pEBR-1. It was used to test electroporation conditions for Br. choshinensis HPD52.

[0140] The pEBR-4 described in the pEBR-4 application has a sequence as shown in SEQ ID NO: 173, which is a product of replicon-pBR322, rop-pBR322, Cm R 、Nm R This vector consists of a synthetic MCS and the replication region of Corynebacterium xerosis pTZ12 (replicon-pTZ12). This vector is 5,204 bp in size; the MCS carries the same restriction enzyme sites as pEBR-1. It was used to test electroporation conditions for Br. choshinensis HPD52.

[0141] pMUTIN-GFP+E. coli carrying this vector was purchased from the Bacillus Genetic Stock Center (BGSC) in the United States and is numbered ECE149. It was used as the source of the E. coli lactose repressor gene lacI.

[0142] The pBRLP-8-P23-GFPT vector described in the pBRLP-8-P23-GFPT application has the sequence shown in SEQ ID NO: 174 and is 5,852 bp in size. The vector carries a mutant green fluorescent protein gene, in which NdeI and SalI have been site-directedly mutagenized, serving as the source of the green fluorescent protein gene.

[0143] pJET1.2-BSBglC: The pJET1.2-BSBglC described in the application has the sequence shown in SEQ ID NO: 175 and is 4,495 bp in size. This vector carries the mutant B. subtilis cellulase gene bglC, in which EcoRI and NdeI have been site-directedly mutagenized, serving as the source of bglC.

[0144] pJET1.2-BLAmyL: pJET1.2-BLAmyL described in the application has a sequence as shown in SEQ ID NO: 176 and a size of 4,534 bp. The vector carries the α-amylase gene amyL from Bacillus licheniformis BCRC 10494, which serves as the source of amyL.

[0145] pJET1.2-GSAmyS: pJET1.2-GSAmyS described in the application has a sequence as shown in SEQ ID NO: 177 and a size of 4,645 bp. The vector carries the α-amylase gene amyS from Geobacillus stearothermophilus BCRC 10285, which serves as the source of amyS.

[0146] pBRCM-MCS: The pBRCM-MCS described in the application is 2,353 bp in size and consists of the replication region of the E. coli vector pBR322, the Cm R and an artificially synthesized multiple cloning site, which serves as the source of the multiple cloning site.

[0147] pJET1.2-TI-4: The pJET1.2-TI-4 vector described in the application is 3,199 bp in size and carries the Momordica charantia trypsin inhibitor (TI-4) gene designed according to the codon preference of B. subtilis , serving as the source of the TI-4 gene.

[0148] 1.3. Pharmaceuticals and Reagents

[0149] The drugs used in this application were mainly purchased from Sigma-Aldrich, BD, VWR Life Science AMRESCO, JTBaker, and Merck. Other brands of drugs are marked separately. TM 100bp plus DNA Ladder, GeneRuler TM 1kb DNA Ladder, 6x DNA loading dye, DreamTaq DNA polymerase, PageRuler TM Prestained Protein Ladder,Pierce TM660nm Protein Assay, Albumin standard, 20x MES SDS running buffer, and NuPAGE 4-12% (w / v) Bis-Tris gel were purchased from Thermo Fisher Scientific, USA; Q5 High-Fidelity DNA polymerase was purchased from New England Biolabs, USA; 50x Tris-acetate-EDTA (TAE) buffer and 6x EZ-Vision TM Loading buffer was purchased from VWR Life Science Amresco, USA; GDP-HiFi DNA polymerase was purchased from Genedirex, USA; HiTrap TM SP Sepharose FF was purchased from GE Healthcare, Sweden; Quick Coomassie Stain was purchased from Protein Ark, UK; DNeasy Blood & Tissue Kit was purchased from Qiagen, Germany; T4 DNA ligase was purchased from Yeastern, Taiwan; Plasmid Miniprep Purification Kit II, PCR Clean Up Kit, and Plus Gel Eluted Kit were purchased from GMbiolab, Taiwan; and 10x Tris-glycine-SDS (TGS) buffer was purchased from Biokit, Taiwan.

[0150] Culture medium

[0151] E. coli and B. subtilis were cultured in Luria-Bertani (LB) medium, while Br. choshinensis HPD52 was cultured in MT medium. Appropriate amounts of antibiotics or 1.5% (w / v) agar were added as needed for the experiment. LB medium powder was purchased from BD Bioscience (USA); agar was purchased from Focus Bioscience (Australia). The polypeptone in MT medium was purchased from Bio Basic (Canada); meat extract and yeast extract were purchased from BD Bioscience (USA); and all other ingredients were purchased from Sigma-Aldrich (USA). The formulations of the culture media used in this application are described in Table 1.

[0152] Table 1. Culture medium formula

[0153] 1.5. Preparation of Antibiotic Stock Solutions and Usage Concentrations

[0154] Ampicillin (Ap) stock solution: Dissolve ampicillin powder in deionized water to a concentration of 25 mg / mL. Sterilize by filtration using a 0.22 μm sterile syringe filter made of hydrophilic polyethersulfone (PES) (Merck Millipore, USA). Store the ampicillin stock solution at -20°C until use. Use at a concentration of 100 μg / mL.

[0155] Chloramphenicol (Cm) stock solution: Dissolve chloramphenicol powder in absolute alcohol to a concentration of 10 mg / mL. Sterilize by filtration using a 0.22 μm sterile syringe filter made of hydrophilic polyamide (nylon, NY) (Sartorius, Germany). Store the chloramphenicol stock solution at -20°C until use. Use at a concentration of 25 μg / mL.

[0156] Neomycin (Nm) stock solution: Dissolve neomycin powder in deionized water to a concentration of 50 mg / mL. Sterilize by filtration through a 0.22 μm sterile syringe filter made of hydrophilic polyethersulfone (PES). Store the neomycin stock solution at -20°C until used. Use at a concentration of 10 μg / mL.

[0157] 1.6. Primers used

[0158] Table 2. Primers used to amplify the green fluorescent protein gene, lactose repressor gene, reporter gene, bitter melon trypsin inhibitor gene, and MCS sequence

[0159] a The restriction enzyme sites in bold are: CATATG (NdeI), GGATCC (BamHI), GTCGAC (SalI), TCTAGA (XbaI), CTCGAG (XhoI); the mutated bases used for site-directed mutagenesis are in bold italics.

[0160] Example 2: Method

[0161] DNA extraction

[0162] E. coli plasmid DNA extraction: E. coli plasmid DNA was extracted using the Plasmid Miniprep Purification Kit II. The protocol was modified from the manufacturer's protocol. Solution I, Solution II, Solution III, Washing Solution A, Washing Solution B, and Elution Solution mentioned in the protocol are all reagents included with the kit. Place 1.8 mL of E. coli culture after 14-18 hours of culture in a 2.0 mL microcentrifuge tube and centrifuge at 20,630 × g at 4°C for 5 minutes. Remove the supernatant and collect the cells. Repeat this step once. Add 200 μL of Solution I to resuspend the cells. Next, add 200 μL of Solution II and gently mix several times to lyse the cells. Add 200 μL of Solution III and gently mix several times. Centrifuge at 20,630 × g at room temperature for 10 minutes and collect the supernatant. Place the spin column into a collection tube and transfer the supernatant to the separation tube. Centrifuge at 20,630 × g for 1 minute at room temperature. Discard the solution from the collection tube and add 500 μL of Washing Solution A to the separation tube. Centrifuge at 20,630 × g for 1 minute at room temperature and discard the solution from the collection tube. This step effectively reduces endonuclease contamination. Next, add 600 μL of Washing Solution B to the separation tube and centrifuge at 20,630 × g for 1 minute at room temperature. Discard the solution from the collection tube and repeat this step once. Centrifuge at 20,630 × g for 5 minutes at room temperature to remove any residual alcohol. Place the separation tube into a 1.5 mL microcentrifuge tube and add 35 μL of Elution Solution. After standing at room temperature for 2 minutes, centrifuge at 20,630 × g for 2 minutes at room temperature to extract the plasmid DNA. Store the plasmid DNA at -20°C until use.

[0163] Br. choshinensis HPD52 plasmid DNA extraction: Br. choshinensis HPD52 plasmids were extracted using the Plasmid Miniprep Purification Kit II, following the manufacturer's protocol with modifications. 1.8 mL of Br. choshinensis HPD52 culture, after 14-17 hours of culture, was placed in a 2.0 mL microcentrifuge tube and centrifuged at 7,000 × g and 4°C for 5 minutes. The supernatant was discarded and the cells were collected. Repeat this step once. 200 μL of Solution I containing 10 mg / mL lysozyme was added to suspend the cells and incubated at 37°C for 30 minutes. Subsequent steps were the same as those for E. coli plasmid DNA extraction.

[0164] Extraction of Br. choshinensis HPD52 genomic DNA: Genomic DNA from Br. choshinensis HPD52 was extracted using the DNeasy Blood & Tissue Kit. The protocol was modified from the manufacturer's protocol. Proteinase K solution, Buffer AL, Buffer AW1, Buffer AW2, and Buffer AE mentioned in the protocol are all included with the kit. 1.8 mL of Br. choshinensis HPD52 culture, after 14-17 hours of culture, was placed in a 2.0 mL microcentrifuge tube and centrifuged at 20,630 × g and 4°C for 5 minutes. The supernatant was removed and the cells were collected. This step was repeated once. 1 mL of TSE buffer (10 mM Tris, 10 mM EDTA, 300 mM NaCl, pH 8.0) was added to resuspend the cells. After centrifugation at 20,630 × g and 4°C for 5 minutes, the supernatant was removed and the cells were collected. Next, add 180 μL of lysis buffer (50 mM Tris-HCl, 10 mM EDTA; 25% (w / v) sucrose, 100 μg / mL RNase A, 10 mg / mL lysozyme, pH 8.0) to suspend the cells. After incubation at 37°C for 30 minutes, add 25 μL of Protease K solution and 200 μL of buffer AL. After thorough mixing, incubate at 56°C for 30 minutes, gently shaking the reaction mixture up and down several times every 5 minutes. Then, add 200 μL of absolute ethanol and mix thoroughly. Transfer the above solution to a separation tube and place it in a collection tube. Centrifuge at 20,630 × g and 4°C for 1 minute, and discard the solution from the collection tube. Add 500 μL of buffer AW1 to the separation tube, centrifuge at 20,630 × g and 4°C for 1 minute, and discard the solution from the collection tube. Add 500 μL of Buffer AW2 to the separation tube and centrifuge at 20,630 × g and 4°C for 1 minute. Discard the solution in the collection tube. Centrifuge at 20,630 × g and 4°C for 5 minutes to remove any remaining alcohol. Place the separation tube in a 1.5 mL microcentrifuge tube and add 100 μL of Buffer AE. Allow to stand at room temperature for 2 minutes, then centrifuge at 20,630 × g and room temperature for 2 minutes to extract the genomic DNA. Store the genomic DNA at -20°C until needed.

[0165] Polymerase chain reaction

[0166] The polymerase chain reaction (PCR) is a technique that uses a thermostable DNA polymerase, a specific primer combination, a specific template, deoxyribonucleoside triphosphates (dNTPs), and a reaction buffer in vitro to amplify specific DNA fragments in large quantities through a series of heating and cooling reactions. This application primarily utilizes this technique to amplify or mutate specific DNA fragments.

[0167] Before PCR, primer combinations must be designed for the DNA fragments to be amplified and mutated. Therefore, the primer Tm values ​​must be calculated. Standard primer Tm values ​​are analyzed using Vector NTI (Thermo Fisher Scientific, USA) at a temperature set between 55°C and 65°C. Mutant primer Tm values ​​are calculated using the formula provided by Thermo Fisher Scientific: [Tm = 81.5 + 0.41 (%GC) - 675 / N - %mismatch], which must be above 75°C. Here, %GC is the percentage of GC nucleotides in the mutant primer, N is the length of the mutant primer, and %mismatch is the percentage of the mutant bases in the mutant primer. To meet DNA cloning requirements, restriction enzyme sites are designed into the 5' end of some primers.

[0168] When performing PCR, heat-resistant DNA polymerase, primers, specific template, dNTPs, reaction buffer and an appropriate amount of sterile deionized water are mixed and the reaction is carried out using a polymerase chain reaction instrument Biometra T-Gradient Thermocycler ( Germany) to amplify DNA fragments under specific reaction conditions. The use of Q5 High-Fidelity DNA Polymerase and GDP-HiFi DNA Polymerase and PCR conditions were set according to the manufacturer's instructions. After the PCR reaction, the PCR results were analyzed by agarose gel electrophoresis.

[0169] 2.3. Agarose gel electrophoresis

[0170] Use agarose gel for electrophoresis of DNA molecules, and prepare 0.7% or 2% (w / v) gel according to the experimental requirements. TMThe loading buffer was mixed at a ratio of 5:1. The mixture was then injected into the sample well of the agarose gel. Electrophoresis was performed at 100 V for 30 minutes using 1x TAE buffer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA, pH 7.6) as the running buffer. After electrophoresis, the gel was placed in an E-BOX CX5 film image acquisition and analysis system (Vilber Lourmat, France) to observe and photograph the DNA electrophoresis results.

[0171] 2.4. Recovery of PCR Products

[0172] Recover PCR products using the PCR Clean-Up Kit or Plus Gel Eluted Kit. Follow the manufacturer's instructions for the PCR Clean-Up Kit and Plus Gel Eluted Kit. The binding buffer, wash solution, and elution solution mentioned in the procedures are included with the kits.

[0173] PCR Clean-Up Kit Procedure: Mix 100 μL of PCR product with 300 μL of Binding Buffer in a 1.5 mL microcentrifuge tube and transfer to a separate tube. Place the tube containing the solution in a collection tube and centrifuge at 20,630 × g at room temperature for 1 minute. Discard the solution from the collection tube. Next, add 700 μL of Wash Solution to the tube and centrifuge at 20,630 × g at room temperature for 1 minute. Discard the solution from the collection tube and repeat this step once. Centrifuge at 20,630 × g at room temperature for 5 minutes. Place the tube in a 1.5 mL microcentrifuge tube and add 35 μL of Solution Solution. Let stand at room temperature for 2 minutes, then centrifuge at 20,630 × g at room temperature for 2 minutes to elute the PCR product. Store the PCR product at -20°C until needed.

[0174] Plus Gel Eluted Kit Procedure: After subjecting the DNA sample to agarose gel electrophoresis, the location of the desired DNA fragment within the gel is determined using the E-BOX CX5 film image capture and analysis system. The agarose gel containing the desired DNA fragment is excised with a clean razor blade and placed in a 1.5 mL microcentrifuge tube. 500 μL of Binding Buffer is then added and incubated at 60°C for 10 minutes until the gel is completely dissolved. The melted gel is transferred to a separation tube and placed in a collection tube. Centrifuge at 20,630 × g at room temperature for 1 minute, then discard the solution from the collection tube. Add 500 μL of Binding Buffer to the separation tube, centrifuge at 20,630 × g at room temperature for 1 minute, then discard the solution from the collection tube. Add 700 μL of Wash Solution to the separation tube, centrifuge at 20,630 × g at room temperature for 1 minute, and repeat this step once. Centrifuge at 20,630 × g for 5 minutes at room temperature. Place the separation tube in a 1.5 mL microcentrifuge tube and add 35 μL of the elution solution. Allow to stand at room temperature for 2 minutes, then centrifuge at 20,630 × g for 2 minutes at room temperature to elute the PCR product. Store the PCR product at -20°C until needed.

[0175] 2.5. DNA molecule shearing and recovery

[0176] DNA molecules were sheared using Thermo Fisher Scientific FastDigest restriction enzymes according to the manufacturer's protocol. An appropriate amount of DNA sample, 10x FastDigest buffer, sterile deionized water, and the appropriate amount of restriction enzyme were mixed and incubated at 37°C for 15 minutes. DNA shearing was then monitored by agarose gel electrophoresis. Sheared DNA products were recovered using the Plus Gel Eluted Kit or PCR CleanUp Kit.

[0177] 2.6. DNA molecule adhesion

[0178] DNA molecules were ligated using the YB Rapid Ligation Kit or the CloneJET PCR Cloning Kit according to the manufacturer's protocol. Buffer A, Buffer B, and T4 DNA ligase (3 U / μL) were included with the YB Rapid Ligation Kit; pJET1.2 (50 ng / μL), 2x Reaction Buffer, and T4 DNA ligase (5 U / μL) were included with the CloneJET PCR Cloning Kit.

[0179] YB Rapid Ligation Kit procedure: Mix the recovered vector DNA fragment with the DNA fragment to be inserted into the vector (insert DNA) at an appropriate molar ratio. Add 1 μL of buffer A, 1 μL of buffer B, and 1 μL of T4 DNA ligase (3 U / μL) to a total reaction volume of 10 μL and mix gently. Incubate at 22°C for 30 minutes. This ligation mixture is ready for transformation.

[0180] CloneJET PCR Cloning Kit Procedure: Transfer 3 μL of recovered PCR product to a 1.5 mL microcentrifuge tube. Add 1 μL of pJET1.2 (50 ng / μL), 10 μL of 2x Reaction Buffer, 5 μL of sterile deionized water, and 1 μL of T4 DNA ligase (5 U / μL). Mix gently. Incubate at 22°C for 30 minutes. This ligation mixture is ready for transformation.

[0181] 2.7.E. coli ECOS TM 9-5 thermal shock conversion

[0182] E. coli ECOS TM Heat shock transformation of 9-5 was performed according to the manufacturer's method. E. coli ECOS was taken out from -80℃ freezer. TM After thawing, add 2 μL of carrier or binding mixture and place in an ice bath for 30 minutes. After incubating at 42°C for 45 seconds, shake and place in an ice bath for 5 minutes. Then, add 200 μL of Select APS TM Culture medium (BD, USA). Incubate at 37°C and 180 rpm for 1 hour. Apply an appropriate amount of bacterial solution to LB solid medium containing an appropriate amount of antibiotics and incubate at 37°C for 14-17 hours. Observe the growth of the colonies.

[0183] 2.8. Colony polymerase chain reaction

[0184] Colony polymerase chain reaction (Colony PCR) is a rapid technique for screening transformants for the presence of the target DNA fragment. First, prepare a 100 μL PCR reaction mixture containing 1x Dream Taq buffer, 200 μM dNTPs, 0.5 μM amplification primers, 2.5 U Dream Taq DNA polymerase, and an appropriate amount of sterile deionized water. Aliquot the PCR reaction mixture into 0.2 mL microcentrifuge tubes (10 μL / tube). Under a sterile operating table, pick selected colonies with a sterile toothpick and place them in the microcentrifuge tubes for agitation. Place the 0.2 mL microcentrifuge tubes in a polymerase chain reaction (PCR) instrument and perform the PCR reaction. Reaction conditions are: 95°C for 5 minutes (1 cycle); 95°C for 30 seconds, X°C for 30 seconds, 72°C for Y seconds (25 cycles); and 72°C for 7 minutes (1 cycle). X represents the temperature at which the primers bind, which is set as the Tm value of the primers. Y is the extension time of the thermostable DNA polymerase. If amplifying a 1 kb DNA fragment, set Y to 60 seconds. After the PCR reaction is complete, observe the PCR results using agarose gel electrophoresis.

[0185] 2.9.DNA sequencing

[0186] After screening E. coli colonies carrying target DNA using colony PCR, the colonies were inoculated into 5 mL of Superior broth containing appropriate amounts of antibiotics. TM Culture medium (AthenaES, USA) was added and incubated at 37°C and 180 rpm for 14-17 hours. E. coli plasmids were extracted using the Plasmid Miniprep Purification Kit II. DNA sequencing was performed by Source International Biotech Co., Ltd.

[0187] 2.10. Methods for detecting green fluorescent protein expression intensity in different hosts

[0188] E. coli expression system: Inoculate the E. coli colony carrying the green fluorescent protein expression vector into LB medium containing chloramphenicol and culture at 37°C and 220 rpm for 14 to 17 hours. Inoculate the overnight culture into fresh LB medium containing chloramphenicol and set the starting OD 600After adjusting the pH to 0.1, incubate at 37°C and 220 rpm. At various incubation times, 0.5 mL of the bacterial solution was placed in a 1.5 mL sterile microcentrifuge tube and centrifuged at 7,000 × g at 4°C for 5 minutes to collect the cells. The cells were washed once with 0.5 mL of phosphate buffer (58 mM Na₂HPO₄, 17 mM NaH₂PO₄, 68 mM NaCl) and resuspended in 0.5 mL of phosphate buffer. 100 μL of the bacterial solution was added to a 96-well clear plate and the absorbance was measured at 600 nm using an INFINITE M200 microplate reader (Tecan, USA). Another 100 μL of the bacterial solution was added to a 96-well black plate and the fluorescence was measured at excitation and emission wavelengths of 482 nm and 512 nm, respectively. If necessary, the bacterial solution was diluted appropriately. Fluorescence intensity is expressed as fluorescence value per unit cell (fluorescence / OD 600 )express.

[0189] Br. choshinensis HPD52 expression system: Inoculate Br. choshinensis HPD52 colonies carrying a green fluorescent protein expression vector into MT medium containing neomycin and culture at 37°C and 220 rpm for 14 to 17 hours. Add the overnight culture solution to fresh MT medium containing neomycin and adjust the starting OD 600 After adjusting the pH to 0.1, the cells were cultured at 37°C and 220 rpm. The subsequent operation procedures were the same as those for the E. coli expression system.

[0190] B. subtilis DB430 expression system: Inoculate the B. subtilis DB430 colony carrying the green fluorescent protein expression vector into LB medium containing chloramphenicol and culture at 37°C and 220 rpm for 14 to 17 hours. Add the overnight culture solution to fresh LB medium containing chloramphenicol and adjust the starting OD 600 After adjusting the pH to 0.1, the cells were cultured at 37°C and 220 rpm. The subsequent operation procedures were the same as those for the E. coli expression system.

[0191] Br. choshinensis HPD52 inducible expression system: Inoculate Br. choshinensis HPD52 colonies carrying the green fluorescent protein inducible expression vector into MT medium containing neomycin and culture at 37°C and 220 rpm for 14 to 17 hours. Add the overnight culture solution to fresh MT medium containing neomycin and adjust the starting OD 600After adjusting to 0.1, culture was carried out at 37°C and 220 rpm. 600 At approximately 0.4-0.6, 1 mM isopropyl-β-D-thiogalactoside (IPTG) was added and the culture was continued at 37°C and 220 rpm. The subsequent operation process was the same as that of the E. coli expression system.

[0192] 2.11. Protein-related experimental methods

[0193] Glycine-SDS PAGE protein electrophoresis: First, prepare polyacrylamide gel, which is divided into two layers of gel, namely 4% stacking gel and 12.5% ​​separation gel. The concentration of separation gel can be adjusted according to experimental requirements. Place the electrophoresis film in the electrophoresis tank equipment (ATTO, Japan) and pour in 1x TGS buffer (25mM Tris, 250mM glycine, 0.1% SDS, pH 8.3). Mix the sample with 5x sample buffer (Biokit, Taiwan) at a ratio of 1:4 and heat at 100℃ for 5 minutes. TM After loading the prestained protein ladder and sample into the sample well, electrophoresis was performed at 80V for 60 minutes, followed by 145V for another 60 minutes. After electrophoresis, the gel was removed and protein staining was performed using Quick Coomassie Stain. After staining for 16 hours, the stain was removed with deionized water.

[0194] NuPAGE TM 4-12% (w / v) Bis-Tris protein electrophoresis: After removing the outer packaging and the seal below the precast gel, place the precast gel in an electrophoresis tank (Thermo Fisher Scientific, USA) and add 1x MES SDS Running buffer (Thermo Fisher Scientific, USA) (50mM MES, 50mM Tris, 0.1% SDS, 1mM EDTA, pH 7.3). Mix the sample with 5x sample buffer at a ratio of 1:4 and heat at 100°C for 5 minutes. TMAfter loading the sample and pre-stained protein standard (Invitrogen, USA) into the sample well, electrophoresis was performed at 130V for 60 minutes. After electrophoresis, the gel was removed and protein staining was performed using Quick Coomassie Stain. After staining for 16 hours, the stain was removed with deionized water.

[0195] Protein purification: using 1mL HiTrap TM SP Sepharose FF column with protein liquid chromatography system The target protein was purified using Prime Plus (GE Healthcare, Sweden) using the following purification steps: First, the column was equilibrated with 5 mL of equilibration buffer (20 mM sodium phosphate, pH 7.0). The column was then washed with 5 mL of elution buffer (20 mM sodium phosphate, 1 M sodium chloride, pH 7.0). The elution buffer was then removed from the column with 10 mL of equilibration buffer. This step activates the SP Sepharose FF resin.

[0196] The culture supernatant was mixed with the equilibration buffer at a ratio of 1:4, and the pH of the solution was adjusted to 7.0 using 0.1N HCl solution, and then filtered using a 0.2 μm sterile filter cup (Thermo Fisher Scientific, USA). TM After the sample injection is completed, the non-specifically bound proteins are washed with 20 mL of equilibration buffer. Then, the target protein is eluted using a linear NaCl gradient (0-1 M). TM The protein purification was observed by 4-12% Bis-Tris protein electrophoresis.

[0197] Protein concentration was determined using Pierce TM Determine protein concentration using the 660nm Protein Assay. Place 10μL of protein sample into a 96-well plate, add 150μL of protein assay reagent, and mix thoroughly. Incubate at room temperature for 5 minutes. Then, measure absorbance at 660nm using an INFINITE M200 microplate analyzer. Construct a protein concentration standard curve using bovine serum albumin (BSA) as the standard. Compare the sample absorbance to the standard curve to determine the protein sample concentration.

[0198] Protein Identification by Mass Spectrometry: Purified target proteins were subjected to protein electrophoresis and stained using Quick Coomassie Stain. After 16 hours of staining, the protein sample to be analyzed was removed from the film using a micropipette and placed in a 1.5 mL microcentrifuge tube. The stain was then destained with 20% (v / v) ethanol. The protein sample was then shipped frozen to the Biotechnology Development Center of National Chung Hsing University for protein identification using a Finnigan LTQ XL linear ion trap mass spectrometer (Thermo Electron, USA).

[0199] Example 3: Applicability of different shuttle vectors to Br. choshinensis HPD52 strain

[0200] 3.1. Electrotransformation of Br. choshinensis HPD52

[0201] Electrocompetent cells were prepared according to the method described by Okamoto et al. (Biosci. Biotechnol. Biochem., 61:202-203, 1997) with modifications and stored at -80°C until use. For DNA electroporation experiments, the electrocompetent cells were removed from the -80°C freezer and thawed. 0.5 μg of vector DNA and 100 μL of 15% PEG 6000 (Sigma-Aldrich, USA) solution were added to 100 μL of the electrocompetent cells and gently mixed. The cells were then placed in a pre-chilled electroporation cuvette (Bio-Rad, USA) and allowed to stand in an ice bath for 5 minutes. Electroporation was then performed using a MicroPulser Electroporator (Bio-Rad, USA) at the specified resistance, capacitance, and electric field strength. Add the transformed bacterial suspension to 1 mL of MT medium and shake at 37°C and 180 rpm for 1 hour. Apply an appropriate amount of the bacterial suspension to MT solid medium supplemented with an appropriate amount of neomycin. Incubate at 37°C for 24 hours and observe colony growth.

[0202] 3.2. Transformation of shuttle vectors containing different replication regions into Br. choshinensis HPD52 strain

[0203] This application primarily investigates the suitability of four shuttle vectors, pERB-1 through pERB-4 (Figure 2), for use in Br. choshinensis HPD52. These vectors carry the replication regions of Gram-positive bacterial plasmids, including pBC1, pC194, pLP31-8, and pTZ12, respectively. To prevent misinterpretation of experimental results due to improper competent cell preparation or inappropriate transformation conditions, the conventional Br. choshinensis expression vector pNCMO2, which carries the pUB110 replication region, was used as a positive control during transformation.

[0204] Br. choshinensis HPD52 electrocompetent cells were prepared according to the method described by Okamoto et al. with modifications. After mixing the electrocompetent cells with various vectors, electroporation was performed at 200Ω, 25μF, and 7.5kV / cm. The results showed that the transformation efficiency achieved with pNCMO2, pERB-1, pERB-2, and pERB-3 was 1.9×10 3 , 4.9×10 2 , 8.8×10 1 and 3.5×10 1 transformants / μg DNA; pEBR-4 was unable to transform into Br. choshinensis HPD52. Information on each vector and its transformation efficiency are summarized in Table 3.

[0205] Table 3. Electroporation efficiency of different shuttle vectors in Br. choshinensis HPD52

[0206] a A replicon is a cell that contains the replication origin and all replication-related components.

[0207] b ATRI, Agricultural Science and Technology Research Institute;

[0208] c RCR, rolling circle replication.

[0209] To verify that the transformed Br. choshinensis HPD52 strains harbored the vectors, the vectors were further extracted and digested with the SalI restriction enzyme. The results (Figure 3) showed that pNCMO2, pERB-1, pERB-2, and pERB-3 were indeed present in Br. choshinensis HPD52, and the molecular weights of the vectors were consistent with expectations, indicating no post-transformation deletion or rearrangement.

[0210] Since pERB-1, pERB-2, and pERB-3 can be transformed into Br. choshinensis HPD52, they can be used as genetic manipulation tools for Br. choshinensis HPD52. Since pERB-1 has higher electroporation efficiency than pERB-2 and pERB-3, the pERB-1 shuttle vector was selected for subsequent expression vector construction.

[0211] Example 4: Expression efficiency of different expression signals

[0212] To diversify the selection of expression signals, in this application, novel expression signals were developed using the whole genome sequencing data of Br. choshinensis HPD52.

[0213] Based on the complete genome sequence of Br. choshinensis HPD52 published by NCBI, 19 DNA fragments with potential expression signals were identified; a schematic diagram of each expression signal is shown in Figure 4. Specific primers were designed for these DNA fragments and amplified using PCR. Furthermore, specific primers were used to amplify the P2 promoter using pNCM02 and pNY326, which carry the P2 and P5 promoters, as templates. pNCMO2 With P5 pNY326 Different expression signals were embedded into pEBR-1-GFP to construct 21 green fluorescent protein expression vectors. These expression vectors were transformed into E. coli ECOS TM 9-5, Br. choshinensis HPD52 and B. subtilis DB430. Using the green fluorescent protein gene as a reporter gene, the expression efficiency of each expression signal in different hosts was investigated.

[0214] 4.1. Amplification of different expression signals and construction of green fluorescent protein expression vector

[0215] 4.1.1. Amplification of expression signals

[0216] Using pNY326 and pNCM02 as templates, specific primers (Table 4) were used with GDP-HiFi DNA polymerase to amplify P2 pNCMO2 With P5 pNY326Expression signal. Based on the complete genome sequence of Br. choshinensis HPD52 (GenBank: LJJB01000010.1) published by the National Center for Biotechnology Information (NCBI), we searched for DNA fragments that might be expression signals and designed specific primers. Using Br. choshinensis HPD52 genomic DNA as a template, we used specific primers (Table 4) with GDP-HiFi DNA polymerase to generate expression signal P01. HPD52 ~P19 HPD52 PCR products were recovered using a PCR Clean Up Kit.

[0217] Table 4. Primers used to amplify expression signals

[0218] a The bold areas are restriction enzyme recognition sites: GAATTC (EcoRI), CATATG (NdeI).

[0219] 4.1.2. Construction of green fluorescent protein expression vector

[0220] pBRLP-8-P23-GFPT (Chen et al., Agricultural Chemistry and Food Science, Taiwan, 54: 236-246, 2016) was digested with NdeI and XbaI, and the DNA fragment containing the mutant green fluorescent protein gene, E. coli phage Lambda t0 terminator, and E. coli rrnB T1 terminator was recovered using the Plus Gel Eluted Kit. The recovered DNA fragment was ligated into pEBR-1 digested with the same restriction enzymes using the YB Rapid Ligation Kit and transformed into E. coli ECOS. TM 9-5. Colony polymerase chain reaction was performed using the GFPF (SEQ ID NO: 146) / GFPR (SEQ ID NO: 147) primer combination to select transformants that may carry the inserted DNA. Plasmids from the transformants were extracted and DNA sequenced. pEBR-1, carrying the mutant green fluorescent protein gene, the E. coli phage Lambda t0 terminator, and the E. coli rrnB T1 terminator, was designated pEBR-1-GFP.

[0221] The expression signal DNA fragment described in 4.1.1 was cut with EcoRI and NdeI and recovered using PCRCleanUp Kit. The recovered DNA fragment was inserted into pEBR-1-GFP cut with the same restriction enzymes using YB Rapid Ligation Kit and transformed into E. coli ECOS TM 9-5. Use appropriate specific primer combinations to perform colony polymerase chain reaction to select transformants that may carry external inserted DNA. Extract the plasmids from the transformants and perform DNA sequencing. pNCMO2 With P5 pNY326 The green fluorescent protein expression vectors expressing the signal were named pEBR-1-P2 pNCMO2 -GFP and pEBR-1-P5 pNY326 -GFP. The green fluorescent protein expression vectors with different Br. choshinensis HPD52 expression signals were named pEBR-1-P01 in sequence. HPD52 -GFP~pEBR-1-P19 HPD52 -GFP.

[0222] 4.2. Comparison of different expression signals in E. coli ECOS TM Expression efficiency in 9-5

[0223] After the green fluorescent protein expression vector was transformed into the E. coli host, the expression of green fluorescent protein was initially determined by the colony color of the transformant. The results showed (Figure 5) that E. coli (pEBR-1-P07 HPD52 -GFP) colonies were most noticeably green. The transformants were then cultured in LB liquid medium, and cells were collected at specific time points for fluorescence intensity analysis. Figure 6 shows the growth and fluorescence intensity of each transformant. Figure 7 summarizes the fluorescence intensity analysis results of the E. coli transformants after 24 hours of culture. HPD52 、P05 HPD52 、P06 HPD52 、P07 HPD52 、P08 HPD52 and P12 HPD52 The expression efficiency in E. coli was better, and the fluorescence intensity of the bacteria was higher than 5,000. HPD52 The expression efficiency was the highest, and the bacterial fluorescence intensity was as high as 36,514, which was consistent with the observation results of colony color.

[0224] Comparison of expression efficiency of different expression signals in Br. choshinensis HPD52

[0225] The green fluorescent protein expression vector was introduced into the Br. choshinensis HPD52 host by electroporation. The expression of green fluorescent protein was initially determined by the colony color of the transformant. The results showed (Figure 8) that Br. choshinensis HPD52 (pEBR-1-P2 pNCMO2 -GFP), Br.choshinensis HPD52(pEBR-1-P5 pNY326 -GFP) and Br.choshinensis HPD52 (pEBR-1-P06 HPD52 -GFP) and other transformants showed a more obvious green colony. The transformants were further cultured in MT liquid medium, and the cells were collected at specific culture time points for fluorescence intensity analysis. The growth status of each transformant and the results of the cell fluorescence intensity measurement are shown in Figure 9. The fluorescence intensity analysis results of the Br. choshinensis HPD52 transformant after 48 hours of culture are summarized in Figure 10. The results show that except for P12 HPD52 With P18 HPD52 The signal is relatively low, while other groups all have obvious signals and can be used as options for constructing expression systems.

[0226] Among them, P2 pNCMO2 、P5 pNY326 、P06 HPD52 、P07 HPD52 and P10 HPD52 The expression efficiency in Br. choshinensis HPD52 was high, and the fluorescence intensity of the bacteria was higher than 50,000. HPD52 The expression efficiency is the highest, so the subsequent examples of transforming expression vectors in this application all use P06 HPD52 As an expression signal, it is only one example and shall not limit the present application.

[0227] The expression efficiency of the expression signal in Br. choshinensis HPD52 is P2 pNCMO2 >P5 pNY326 >P06 HPD52 >P10 HPD52 >P07 HPD52 >P13 HPD52 >P16 HPD52 >P09 HPD52 >P05 HPD52 >P11 HPD52 >P01 HPD52 >P08 HPD52 >P03 HPD52 >P14HPD52 >P19 HPD52 >P02 HPD52 >P04 HPD52 >P15 HPD52 >P17 HPD52 >P12 HPD52 >P18 HPD52 .

[0228] The results show that, except for P12 HPD52 With P18 HPD52 Except for the extremely low expression efficiency in Br. choshinensis HPD52, other expression signals can be used as options for constructing Br. choshinensis HPD52 expression systems. The less efficient expression signals can be used to express cell-toxic proteins or for metabolic engineering.

[0229] It is worth noting that P06 HPD52 The expression efficiency is higher than that of the conventional vector P2 pNCMO2 1.5 times lower, but similar to P5 pNY326 It is similar to the Br. choshinensis HPD52 gene and can be used as an option for constructing the Br. choshinensis HPD52 expression system.

[0230] Comparison of expression efficiency of different expression signals in B. subtilis DB430

[0231] The expression vector carrying the expression signal was introduced into another host, B. subtilis DB430, by electroporation. At specific culture time points, the cells were collected for fluorescence intensity analysis. The growth status and fluorescence intensity of each transformant are shown in Figure 15. The fluorescence intensity analysis results of the B. subtilis DB430 transformants after 48 hours of culture are summarized in Figure 16. The results show that P2 pNCMO2 、P5 pNY326 、P01 HPD52 、P02 HPD52 、P05 HPD52 、P06 HPD52 、P07 HPD52 and P17 HPD52 The expression efficiency in B. subtilis DB430 was better, and the bacterial fluorescence intensity was higher than 50,000.

[0232] The results indicate that the shuttle vector used in this application can be expressed not only in E. coli and Br. choshinensis, but also in another host, B. subtilis, as an alternative.

[0233] Example 5: Modification of expression signals and their application in the establishment of an inducible expression system

[0234] The expression signal used in Example 4 eliminates the need for an induction step when producing recombinant proteins, saving inducer costs and simplifying the fermentation process. However, expressing large amounts of certain recombinant proteins may affect bacterial growth, even causing bacterial death, and may also lead to plasmid instability. To avoid these problems, a tightly regulated inducible expression signal can be used for recombinant protein production. This application also attempts to modify P06 HPD52 The expression signal becomes an inducible expression signal to construct an inducible expression system.

[0235] 5.1. Modification of expression signals

[0236] 5.1.1. P06 HPD52 Expression signal transformed into inducible expression signal

[0237] Fourteen primers were designed (Table 5) and the sequences of the E. coli operon lacO or SacI-lacO were inserted into the P06 by overlap extension polymerase chain reaction (OEPCR). HPD52 Different positions in the expression signal, or further deletion of specific sequences, can transform P06 HPD52 Become an inducible expression signal. HPD52 / lacO-1~P06 HPD52 / lacO-7 and other 7 inducible expression signals (Figure 11).

[0238] Table 5, Renovation P06 HPD52 Primers used for inducible expression signals

[0239] a The bold part is the restriction enzyme recognition site: GAGCTC (SacI).

[0240] 5.1.2.P06 HPD52 / lacO-1~P06 HPD52 / lacO-7 amplification

[0241] pEBR-1-P06 HPD52-GFP was used as a template, and two primer combinations were used: P06F (SEQ ID NO: 41) / P06LACO1R (SEQ ID NO: 77) and P06LACO1F (SEQ ID NO: 76) / P06R (SEQ ID NO: 42); P06F (SEQ ID NO: 41) / P06SACILACO2R (SEQ ID NO: 79) and P06SACILACO2F (SEQ ID NO: 78) / P06R (SEQ ID NO: 42); P06F (SEQ ID NO: 41) / P06SACILACO3R (SEQ ID NO: 81) and P06SACILACO3F (SEQ ID NO: 80) / P06R (SEQ ID NO: 42); P06F (SEQ ID NO: 41) / P06SACILACO4R (SEQ ID NO: 83) and P06SACILACO4F (SEQ ID NO: 84). PCR reactions were performed using GDP-HiFi DNA polymerase with P06F (SEQ ID NO: 82) / P06R (SEQ ID NO: 42); P06F (SEQ ID NO: 41) / P06SACILACO5R (SEQ ID NO: 85) and P06SACILACO5F (SEQ ID NO: 84) / P06R (SEQ ID NO: 42); P06F (SEQ ID NO: 41) / P06SACILACO6R (SEQ ID NO: 87) and P06SACILACO6F (SEQ ID NO: 86) / P06R (SEQ ID NO: 42); and P06F (SEQ ID NO: 41) / P06SACILACO7R (SEQ ID NO: 89) and P06SACILACO7F (SEQ ID NO: 88) / P06R (SEQ ID NO: 42). PCR products were recovered using a Plus Gel Eluted Kit. Then, the two recovered PCR products were used as templates and the P06F (SEQ ID NO: 41) / P06R (SEQ ID NO: 42) primer combination was used with GDP-HiFi DNA polymerase for PCR reaction. HPD52 / lacO-1~P06 HPD52 The PCR products were recovered using the Plus Gel Eluted Kit.

[0242] Table 6: P06HPD52 / lacO-1 to P06HPD52 / lacO-7 expression signals

[0243] 5.1.3. Construction of inducible expression vector

[0244] The modified inducible expression signal P06 HPD52 / lacO-1~P06 HPD52 After cleavage of / lacO-7 with EcoRI and NdeI, the DNA fragment was recovered using a PCR Clean Up Kit. The recovered DNA fragment was inserted into pEBR-1-GFP cut with the same restriction enzymes using a YB Rapid Ligation Kit and transformed into E. coli ECOS TM 9-5. Colony polymerase chain reaction was performed using the P06F (SEQ ID NO: 41) / P06R (SEQ ID NO: 42) primer combination to select transformants that may carry the inserted DNA. Plasmids from the transformants were extracted and sequenced. The green fluorescent protein expression vectors carrying the modified expression signal were named pEBR-1-P06. HPD52 / lacO-1-GFP~pEBR-1-P06 HPD52 / lacO-7-GFP.

[0245] 5.2. Adding the lactose repressor gene to the expression vector

[0246] 5.2.1. Mutation of the lactose repressor gene

[0247] Because lacI contains the NdeI cleavage site commonly used in gene cloning, site-directed mutagenesis was performed using OEPCR to avoid affecting subsequent gene cloning. First, four primers were designed: LACIF (SEQ ID NO: 148), LACIM1 (SEQ ID NO: 149), LACIM2 (SEQ ID NO: 150), and LACIR (SEQ ID NO: 151). LACIF and LACIR served as amplification primers, while LACIM1 and LACIM2 served as mutation primers. During the mutagenesis process, PCR reactions were performed using pMUTIN-GFP+ as a template with the LACIF / LACIM2 and LACIM1 / LACIR primer combinations, respectively, and Q5 High-Fidelity DNA Polymerase. PCR products were recovered using the Plus Gel Eluted Kit. Next, PCR reactions were performed using the LACIF / LACIR primer combination and Q5 High-Fidelity DNA Polymerase, using the two recovered PCR products as templates. This step resulted in the generation of the full-length site-directed mutant lacIM. The PCR products were recovered using the Plus Gel Eluted Kit.

[0248] 5.2.2. Construction of expression vectors carrying suppressor genes

[0249] The site-directed mutagenesis lacIM was cut with XbaI and XhoI and recovered using a PCR Clean Up Kit. The recovered DNA fragment was then ligated into pEBR-1-P06 cut with the same restriction enzymes using a YB Rapid Ligation Kit. HPD52 / lacO-3-GFP and transformed into E. coli ECOS TM 9-5. Colony polymerase chain reaction was performed using the LACIF (SEQ ID NO: 148) / LACIR (SEQ ID NO: 151) primer combination to select transformants that may carry the inserted DNA. The plasmids of the transformants were extracted and sequenced. The green fluorescent protein expression vector carrying lacIM was named pEBR-1-P06. HPD52 / lacO-3-lacIM-GFP.

[0250] 5.3. Discussion of P06 HPD52 Inducible expression signal expression efficiency

[0251] pEBR-1-P06 HPD52 / lacO-1-GFP~pEBR-1-P06 HPD52 After electroporation of expression vectors such as lacO-7-GFP into the Br. choshinensis HPD52 host, the color of the transformant colonies was observed. The results (Figure 12) showed apple-green or yellow-green colonies, indicating that Br. choshinensis HPD52 is unable to produce the LacI repressor or similar regulatory proteins to regulate gene expression.

[0252] The transformants were further cultured in MT liquid medium, and the cells were collected at specific culture time points for fluorescence intensity analysis. The growth status of each transformant and the results of the cell fluorescence intensity measurement are shown in Figure 13. The fluorescence intensity analysis results of the Br. choshinensis HPD52 transformant after 48 hours of culture are summarized in Figure 14. The results show that the insertion of lacO or SacI-lacO into P06 HPD52 In the case of PCR, the expression efficiency will be significantly reduced, and the effect will vary depending on the insertion position. HPD52 / lacO-1~P06 HPD52 / lacO-5 has signals in all hosts, and P06 HPD52 / lacO-3 had the highest expression efficiency, and the bacterial fluorescence intensity could reach 47,790.

[0253] Further P06 HPD52 / lacO-2 and P06 HPD52 / lacO-3 specific sequence is deleted to become P06 HPD52 / lacO-6 and P06 HPD52 / lacO-7, was found to have the effect of improving expression efficiency; among them, P06 HPD52 The expression efficiency of / lacO-6 was higher than that of P06 HPD52 / lacO-2 was 2.2 times higher, P06 HPD52 The expression efficiency of / lacO-7 was higher than that of P06 HPD52 / lacO-3 is 2.3 times higher.

[0254] In summary, the expression signal P06 used in this application HPD52 / lacO-1~P06 HPD52 / lacO-7 has signals in the host and P06 HPD52 / lacO-7 has the best expression efficiency, and the bacterial fluorescence intensity can reach 111,286; this expression efficiency is close to P2 pNCMO2 , has the potential to serve as a powerful expression signal.

[0255] Similarly, pEBR-1-P06 HPD52 / lacO-1-GFP~pEBR-1-P06 HPD52 The expression vectors of lacO-7-GFP were introduced into another host, B. subtilis DB430, by electroporation. The growth and fluorescence intensity of each transformant were observed. The results are shown in Figure 17. The fluorescence intensity analysis results of the B. subtilis DB430 transformants after 48 hours of culture are summarized in Figure 18. The results show that P06 HPD52 / lacO-1~P06 HPD52 / lacO-7 has signals in all hosts, and P06 HPD52 / lacO-7 had the highest expression efficiency, which was the same as that in the Br. choshinensis HPD52 host.

[0256] 5.4. Exploring the expression efficiency of expression vectors carrying suppressor genes

[0257] Since Br. choshinensis HPD52 cannot express LacI repressor, in order to achieve the purpose of gene expression regulation, the expression vector pEBR-1-P06 containing the repressor gene was inserted into the HPD52 / lacO-3-lacIM-GFP, the expression vector was introduced into the Br. choshinensis HPD52 host by electroporation, and the transformant was inoculated into MT liquid culture medium. Green fluorescent protein expression was induced using different inducers, including IPTG and lactose. The results (Table 7) showed that IPTG was an effective inducer, while lactose was not. When 1 mM IPTG was added as an inducer, the fluorescence intensity of the bacteria after 48 hours of culture was 48843.6, a 6.7-fold increase compared to the fluorescence intensity of 7312.2 when no inducer was added. These results demonstrate that an inducible expression system for Br. choshinensis HPD52 can be successfully constructed using an expression signal containing lacO in combination with the LacI repressor gene.

[0258] Table 7 Effects of different inducers on P06 HPD52 Effects of lacO-3-lacIM inducible expression signals on expression efficiency in Br. choshinensis HPD52

[0259] Example 6: Different enzyme genes as reporter genes

[0260] 6.1. Construction of different enzyme expression vectors, transformation, and plate culture activity analysis methods

[0261] 6.1.1. Cellulase gene bglC

[0262] After pJET1.2-BSBglC was cut with NdeI and SalI, the cellulase gene bglC was recovered using the Plus Gel Eluted Kit. The recovered DNA fragment was then ligated into pEBR-1-P06 cut with the same restriction enzymes using the YB Rapid Ligation Kit. HPD52 / lacO-1-GFP and transformed into E. coli ECOS TM 9-5. Colony polymerase chain reaction was performed using the BGLCF (SEQ ID NO: 152) / BGLCR (SEQ ID NO: 153) primer combination to select transformants that may have the inserted DNA. The plasmids of the transformants were extracted and sequenced. The vector with the correct sequence was named pEBR-1-P06 HPD52 / lacO-1-BSBglC. This vector was introduced into Br. choshinensis HPD52 by electroporation. After colonies were established, selected colonies were picked with a sterile toothpick and spotted onto MT solid medium containing 0.1% (w / v) AZCL-HE-cellulose (Megazyme, USA) and an appropriate amount of neomycin. The cells were cultured at 37°C for 48 hours. AZCL-HE-cellulose is an insoluble polysaccharide substrate. If the colony secretes cellulase, it will hydrolyze the AZCL-HE-cellulose in the solid medium and release a water-soluble blue dye, forming a blue halo around the colony.

[0263] 6.1.2. α-Amylase Genes amyL and amyS

[0264] pJET1.2-BLAmyL and pJET1.2-GSAmyS were cut with NdeI and XhoI, respectively, and the α-amylase gene was recovered using the Plus Gel Eluted Kit. The recovered DNA fragments were ligated into pEBR-1-P06 cut with NdeI and SalI using the YB Rapid Ligation Kit. HPD52 / lacO-1-GFP and transformed into E. coli ECOS TM 9-5. Colony polymerase chain reaction was performed using the BLAMYF (SEQ ID NO: 154) / BLAMYR (SEQ ID NO: 155) and GSAMYF1 (SEQ ID NO: 156) / GSAMYR1 (SEQ ID NO: 157) primer combinations, respectively, to select transformants that may carry the inserted DNA. Plasmids from the transformants were extracted and sequenced. The expression vector carrying the B. lichenifromis α-amylase gene amyL was named pEBR-1-P06. HPD52 / lacO-1-BLAmyL; the expression vector carrying the G. stearothermophilus α-amylase gene amyS was named pEBR-1-P06 HPD52 / lacO-1- GSAmyS. The aforementioned α-amylase expression vectors were introduced into Br. choshinensis HPD52 using electroporation. After colonies were established, selected colonies were picked up with a sterile toothpick and spotted onto MT solid culture medium containing 1% (w / v) soluble starch and an appropriate amount of neomycin. After incubation at 37°C for 48 hours, the colonies on the solid culture medium were washed with sterile deionized water and 10 mL of iodine solution (BASO, Taiwan) was added. After incubation at 100 rpm for 15 minutes, the iodine solution was removed. Decolorization was then performed with 10 mL of 1 M sodium chloride. If the colonies have the ability to secrete α-amylase, they will hydrolyze the starch in the solid culture medium. Iodine solution can only bind to starch and not to starch hydrolysis products; therefore, a clear zone will appear around the colonies.

[0265] 6.2. Different enzyme genes as reporter genes

[0266] This example mainly explores the feasibility of using the cellulase gene bglC from B. subtilis DB430, the α-amylase gene amyL from B. licheniformis BCRC 10494, and the α-amylase gene amyS from G. stearothermophilus BCRC 10285 as reporter genes in Br. choshinensis HPD52. HPD52 / lacO-1-BSBglC、pEBR-1-P06 HPD52 / lacO-1-BLAmyL and pEBR-1-P06 HPD52 / lacO-1-GSAmyS and other three enzyme expression vectors.

[0267] The enzyme expression vector was introduced into the host strain Br. choshinensis HPD52 by electroporation, and enzyme production by Br. choshinensis HPD52 was observed using plate culture activity assays and protein electrophoresis. The results of the plate culture activity assay (Figure 19) showed that the transformants carrying the enzyme expression vectors were capable of producing B. subtilis BglC (BSBglC), B. licheniformis AmyL (BLAmyL), and G. stearothermophilus AmyS (GSAmyS). Protein electrophoresis results (Figure 20) further verified that the transformants secreted the expressed enzymes.

[0268] The above results indicate that bglC, amyL, and amyS can all be used as reporter genes for Br. choshinensis HPD52.

[0269] Example 7: Applicability of different signal peptides

[0270] 7.1. Construction of α-amylase expression vectors containing different signal peptide DNAs

[0271] 7.1.1.pEBR-1-P06 HPD52 Construction of lacO-3-MCS

[0272] Using pBRCM-MCS as a template, PCR was performed using the MCSF (SEQ ID NO: 164) / MCSR (SEQ ID NO: 165) primer combination with GDP-HiFi DNA polymerase. The PCR product was recovered using the Plus Gel Eluted Kit. HPD52 After cleavage of lacO-3-GFP with NdeI and SalI, the DNA fragments were recovered using the Plus Gel Eluted Kit. The recovered DNA fragments were ligated using the YB Rapid Ligation Kit and transformed into E. coli ECOS. TM 9-5. Colony polymerase chain reaction was performed using the MCSF (SEQ ID NO: 164) / MCSR (SEQ ID NO: 165) primer combination to select transformants that may carry the inserted DNA. The plasmids of the transformants were extracted and sequenced. The vector with the correct sequence was named pEBR-1-P06 HPD52 / lacO-3-MCS.

[0273] 7.1.2. Amplification and modification of α-amylase protein gene and construction of expression vector

[0274] G. stearothermophilus α-amylase carries a 34-amino acid signal peptide at its N-terminus. This application amplifies the DNA sequence of the mature G. stearothermophilus α-amylase amyS protein, i.e., the amino acid portion without the signal peptide, and also performs site-directed mutagenesis on the BamHI and SalI residues within the gene.

[0275] First, six primers were designed: GSAMYSF2 (SEQ ID NO: 158), GSAMYSM1 (SEQ ID NO: 159), GSAMYSM2 (SEQ ID NO: 160), GSAMYSM3 (SEQ ID NO: 161), GSAMYSM4 (SEQ ID NO: 162), and GSAMYSR2 (SEQ ID NO: 163). GSAMYSF2 and GSAMYSR2 served as amplification primers, while GSAMYSM1, GSAMYSM2, GSAMYSM3, and GSAMYSM4 served as mutagenesis primers. PCR reactions were performed using pJET1.2-GSAmyS as a template with the GSAMYSF / GSAMYSM2, GSAMYSM1 / GSAMYSM4, and GSAMYSM3 / GSAMYSR primer combinations and Q5 High-Fidelity DNA Polymerase.

[0276] After the PCR products were recovered using the Plus Gel Eluted Kit, the three recovered PCR products were used as templates to perform PCR reactions using the GSAMYSF2 and GSAMYSR2 primer combinations with Q5 High-Fidelity DNA Polymerase to obtain site-directed mutations of the mature α-amylase gene.

[0277] The PCR products were recovered with the Plus Gel Eluted Kit, and the genes were cloned using the CloneJET PCR Cloning Kit. The ligated products were then transformed into E. coli ECOS TM 9-5. Colony polymerase chain reaction was performed using the GSAMYSF2 / GSAMYSR2 primer combination to identify transformants that may carry the inserted DNA. Plasmids from the transformants were extracted and sequenced. The vector carrying the site-directed mutagenesis of the mature α-amylase gene was named pJET1.2-GSAmySM.

[0278] After pJET1.2-GSAmySM was cut with BamHI and SalI, the site-directed mutagenesis mature α-amylase gene was recovered using the Plus Gel Eluted Kit. The recovered DNA fragment was ligated into pEBR-1-P06 cut with the same restriction enzymes using the YB Rapid Ligation Kit. HPD52 / lacO-3-MCS. The ligation product was transformed into E.coli ECOS TM9-5. Colony polymerase chain reaction was performed using the GSAMYSF2 / GSAMYSR2 primer combination to select transformants that may have inserted DNA. The plasmids of the E. coli transformants were extracted and sequenced. The expression vector with the correct sequence was named pEBR-1-P06 HPD52 / lacO-3-GSAmySM.

[0279] 7.1.3. Amplification of Signal Peptide DNA and Construction of α-Amylase Expression Vectors Carrying Different Signal Peptide DNAs

[0280] Based on information on the Br. choshinensis HPD52 protein from the Signal Peptide website (http: / / www.signalpeptide.de / index.php) and NCBI, we selected 13 secretory protein signal peptide DNAs (Table 9) for specific primer design (Table 8). Specific primers were also designed for the modified signal peptide sec(R2L6) DNA from pNCMO2.

[0281] PCR reactions were performed using Br. choshinensis HPD52 genomic DNA and pNCMO2 as templates, respectively, using specific primers (Table 8) with GDP-HiFi DNA polymerase. The PCR products were recovered using a PCR Clean-Up Kit and then sheared with NdeI and BamHI. The sheared DNA was recovered using a PCR Clean-Up Kit. The recovered DNA fragments were then ligated into pEBR-1-P06, which had been sheared with the same restriction enzymes, using a YB Rapid Ligation Kit. HPD52 / lacO-3-GSAmySM.

[0282] The ligation product was transformed into E. coli ECOS TM 9-5. Perform colony polymerase chain reaction using specific primer combinations to select transformants that may carry the inserted DNA. Extract and sequence the plasmids of the E. coli transformants.

[0283] The α-amylase expression vector carrying the sec signal peptide DNA was named pEBR-1-P06 HPD52 / lacO-3-sec-GSAmySM; the α-amylase expression vector carrying the signal peptide DNA of the secretory protein of Br. choshinensis HPD52 was named pEBR-1-P06 PD52 / lacO-3-SP01-GSAmySM~pEBR-1-P06 HPD52 / lacO-3-SP13-GSAmySM.

[0284] Table 8. Primers used to amplify signal peptide DNA

[0285] a Bold characters indicate restriction enzyme recognition sites: CATATG (NdeI), GGATCC (BamHI); the mutated bases used for site-directed mutagenesis are indicated in bold italics.

[0286] Table 9. Signal peptides used in this application

[0287] a D-score, discrimination score. This score is used to distinguish signal peptides from non-signal peptides.

[0288] b MWP, middle wall protein, middle wall protein.

[0289] 7.2. Effects of Different Signal Peptides on the Expression of α-Amylase Gene in Br. choshinensis HPD52

[0290] The expression vector pEBR-1-P06 HPD52 / lacO-3-sec-GSAmySM and pEBR-1-P06 PD52 / lacO-3-SP01-GSAmySM~pEBR-1-P06 HPD52 The signal peptides / lacO-3-SP13-GSAmySM were introduced into a Br. choshinensis HPD52 host cell via electroporation, and the transformants were inoculated into MT medium containing an appropriate amount of neomycin. After 48 hours of culture, extracellular α-amylase activity was analyzed. The results (Figure 21) showed that SP02, SP04, and SP13 were most effective in expressing α-amylase, with efficiency levels 2.1, 1.6, and 2.6 times that of sec, respectively. These experiments demonstrate that the signal peptides provided in this application can enhance α-amylase expression.

[0291] Example 8: Expression of Momordica charantia trypsin inhibitor using Br. choshinensis HPD52

[0292] 8.1. Construction of Momordica charantia trypsin inhibitor expression vector

[0293] The synthetic bitter melon trypsin inhibitor (TI-4) gene (Figure 22) was inserted into expression vectors with signal peptide DNAs such as sec, SP02, SP04, and SP13, respectively. Four TI-4 secretory expression vectors were constructed, namely, pEBR-1-P06 HPD52 / lacO-3-sec-TI, pEBR-1-P06 HPD52 / lacO-3-SP02-TI, pEBR-1-P06 HPD52 / lacO-3-SP04-TI and pEBR-1-P06 HPD52 / lacO-3-SP13-TI.

[0294] Using pJET1.2-TI-4 as a template, the TI-4F (SEQ ID NO: 166) / TI-4R (SEQ ID NO: 167) primer combination and Q5 High-Fidelity DNA Polymerase were used to amplify the bitter melon trypsin inhibitor (TI-4) gene. After the PCR reaction, the PCR product was recovered using the Plus Gel Eluted Kit. The recovered PCR product was then mixed with different vectors including pEBR-1-P06 HPD52 / lacO-3-sec-GSAmySM, pEBR-1-P06 HPD52 / lacO-3-SP02-GSAmySM, pEBR-1-P06 HPD52 / lacO-3-SP04-GSAmySM and pEBR-1-P06 HPD52 After cleavage of the lacO-3-SP13-GSAmySM with BamHI and SalI, the specific DNA fragment was recovered using a PCR Clean Up Kit. The recovered DNA was ligated using a YB Rapid Ligation Kit, and the ligated product was transformed into E. coli ECOS. TM 9-5. Colony polymerase chain reaction was performed using the TI-4F (SEQ ID NO: 166) / TI-4R (SEQ ID NO: 167) primer combination to select transformants that may carry the inserted DNA. The plasmids of the E. coli transformants were extracted and sequenced. The expression vectors carrying the bitter melon trypsin inhibitor gene were named pEBR-1-P06 and pEBR-2-P07. HPD52 / lacO-3-sec-TI, pEBR-1-P06 HPD52 / lacO-3-SP02-TI, pEBR-1-P06 HPD52 / lacO-3-SP04-TI and pEBR-1-P06 HPD52 / lacO-3-SP13-TI.

[0295] 8.2. Expression of Momordica charantia Trypsin Inhibitor in Host

[0296] pEBR-1-P06 HPD52 / lacO-3-sec-TI, pEBR-1-P06 HPD52 / lacO-3-SP02-TI, pEBR-1-P06 HPD52 / lacO-3-SP04-TI and pEBR-1-P06 HPD52 The Br. choshinensis HPD52 transformant containing lacO-3-SP13-TI was inoculated into MT medium containing neomycin and cultured at 37°C and 180 rpm. After culturing for 14 to 17 hours, an appropriate amount of the bacterial solution was transferred to fresh MT medium containing neomycin and the initial OD was adjusted to 600 After culturing for 24, 48, and 72 hours, the cells were centrifuged at 7,000 × g and 4°C for 30 minutes, and the supernatant fractions were collected and subjected to protein electrophoresis.

[0297] The results (Figure 23) show that all transformants carrying the TI-4 secretion expression vector were able to secrete recombinant TI-4, with distinct bands observed at the expected TI-4 molecular weight. SP13 showed the best results in secretory expression of TI-4.

[0298] 8.3. Purification of the expressed Momordica charantia trypsin inhibitor

[0299] pEBR-1-P06 HPD52 A Br. choshinensis HPD52 transformant expressing TI / lacO-3-SP13-TI was inoculated into MT medium containing neomycin. After 48 hours of culture, the supernatant was collected and purified using ion exchange chromatography for recombinant TI-4. The results (Figure 24) showed that recombinant TI-4 could be purified using a cation exchange resin with a yield of 16.63 mg / L and a purity of 99.93%. Protein identification (Figure 25) confirmed that the purified recombinant protein was indeed TI-4.

[0300] The above results indicate that the shuttle vector provided in this application can be used with the Br. choshinensis HPD52 host to express the target protein, and the expressed protein is also consistent with expectations after identification.

[0301] All of the shuttle vectors, prokaryotic host cells containing the same, and methods for producing proteins using the host cells disclosed and claimed herein can be made and implemented without undue experimentation based on the present disclosure. Although the technical content of this application has been disclosed above with reference to preferred embodiments, it is not intended to limit the claimed invention. Any minor changes and modifications made by anyone skilled in the art without departing from the spirit of the present invention are intended to be within the scope of the present invention.

Claims

1. A shuttle vector comprising: a replicon region of an Escherichia coli plasmid pBR322 having the sequence shown in SEQ ID NO: 1; and A plasmid replicon region from a Gram-positive bacterium.

2. The shuttle vector of claim 1, wherein the plasmid replicon region of the Gram-positive bacteria comprises: a replicon region of the asexual plasmid pBC1 of Bacillus coagulans having the sequence shown in SEQ ID NO: 3; a replicon region of Staphylococcus aureus plasmid pC194 having the sequence shown in SEQ ID NO: 4; or A replicon region of the Lactobacillus plantarum traitless plasmid pLP31-8, which has the sequence shown in SEQ ID NO:

5. 3 . The shuttle vector of claim 1 , further comprising a regulatory protein gene of an Escherichia coli plasmid pBR322 having a sequence as shown in SEQ ID NO:

2. The shuttle vector of claim 1 , further comprising at least one selection gene.

5. The shuttle vector of claim 4, wherein the at least one selection gene comprises a drug resistance selection gene, a non-drug resistance selection gene, or a combination thereof.

6. The shuttle vector of claim 5, wherein the drug resistance selection gene comprises a chloramphenicol resistance gene or a neomycin resistance gene.

7. The shuttle vector of claim 1, further comprising a multiple cloning site.

8. The shuttle vector of claim 1, further comprising an expression signal comprising: (a) Expression signal P01 HPD52 , which has the sequence shown in SEQ ID NO: 8; (b) Expression signal P02 HPD52 , which has the sequence shown in SEQ ID NO: 9; (c) Expression signal P03 HPD52 , which has the sequence shown in SEQ ID NO: 10; (d) Expression signal P04 HPD52 , which has the sequence shown in SEQ ID NO: 11; (e) Expression signal P05 HPD52 , which has the sequence shown in SEQ ID NO: 12; (f) Expression signal P06 HPD52 , which has the sequence shown in SEQ ID NO: 13; (g) Expression signal P07 HPD52 , which has the sequence shown in SEQ ID NO: 14; (h) Expression signal P08 HPD52 , which has the sequence shown in SEQ ID NO: 15; (i) Expression signal P09 HPD52 , which has the sequence shown in SEQ ID NO: 16; (j) Expression signal P10 HPD52 , which has the sequence shown in SEQ ID NO: 17; (k) Expression signal P11 HPD52 , which has the sequence shown in SEQ ID NO: 18; (l) Expression signal P13 HPD52 , which has the sequence shown in SEQ ID NO: 20; (m) Expression signal P14 HPD52 , which has the sequence shown in SEQ ID NO: 21; (n) Expression signal P15 HPD52 , which has the sequence shown in SEQ ID NO: 22; (o) Expression signal P16 HPD52 , which has the sequence shown in SEQ ID NO: 23; (p) Expression signal P17 HPD52 , which has the sequence shown in SEQ ID NO: 24; or (q) Expression signal P19 HPD52 , which has the sequence shown in SEQ ID NO:

26.

9. The shuttle vector of claim 8, wherein the expression signal is an inducible expression signal comprising: (a) Inducible expression signal P06 HPD52 / lacO-1, which has the sequence shown in SEQ ID NO: 69; (b) Inducible expression signal P06 HPD52 / lacO-2, which has the sequence shown in SEQ ID NO:70; (c) Inducible expression signal P06 HPD52 / lacO-3, which has the sequence shown in SEQ ID NO:71; (d) Inducible expression signal P06 HPD52 / lacO-4, which has the sequence shown in SEQ ID NO: 72; (e) Inducible expression signal P06 HPD52 / lacO-5, which has the sequence shown in SEQ ID NO: 73; (f) Inducible expression signal P06 HPD52 / lacO-6, which has the sequence shown in SEQ ID NO: 74; or (g) Inducible expression signal P06 HPD52 / lacO-7, which has the sequence shown in SEQ ID NO:

75.

10. The shuttle vector of claim 9, further comprising a regulatory gene of an inducible expression signal.

11. The shuttle vector of claim 1, further comprising a reporter gene, comprising: (a) cellulase gene bglC of Bacillus subtilis; (b) the α-amylase gene amyL from Bacillus licheniformis; or (c) α-amylase gene amyS from Geobacillus stearothermophilus.

12. The shuttle vector of claim 1, further comprising a signal peptide comprising: (a) a signal peptide SP02 having the sequence shown in SEQ ID NO: 92; (b) a signal peptide SP04 having the sequence shown in SEQ ID NO: 94; (c) a signal peptide SP07 having the sequence shown in SEQ ID NO: 97; (d) a signal peptide SP08 having the sequence shown in SEQ ID NO: 98; (e) a signal peptide SP10 having the sequence shown in SEQ ID NO: 100; (f) a signal peptide SP11 having the sequence shown in SEQ ID NO: 101; or (g) Signal peptide SP13 having the sequence shown in SEQ ID NO:

103.

13. The shuttle vector of claim 1, further comprising a gene for a protein.

14. A prokaryotic host cell comprising the shuttle vector according to any one of claims 1 to 13.

15. The prokaryotic host cell of claim 14, wherein the prokaryotic host cell comprises Escherichia coli, Bacillus subtilis and Brevibacillus choshinensis.

16. A method for producing a protein using the prokaryotic host cell according to claim 15, the method comprising the following steps: Placing the prokaryotic host cell according to claim 15 in a culture medium, and allowing the prokaryotic host cell to express the protein under appropriate culture conditions; and The protein is recovered from the cells or the culture medium of the cells.

17. The method of claim 16, wherein the protein comprises a Momordica charantia trypsin inhibitor.

18. The method of claim 17, wherein the Momordica charantia trypsin inhibitor comprises the sequence shown in SEQ ID NO: 169.

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