Double mutant heat labile enterotoxin gene comprising spacer

Abstract

The present invention relates to a double mutant heat labile enterotoxin gene comprising a spacer and, more specifically, to a use of a heat labile enterotoxin protein, encoded by the gene, for enhancing immunity. It was found that when the double mutant heat labile enterotoxin gene comprising a spacer according to the present invention is introduced to host cells, the translations of LTA and LTB constituting the labile enterotoxin gene do not affect each other, and thus LTB expression increases more than fivefold. In addition, it was experimentally confirmed that the expressed double mutant heat labile enterotoxin protein exhibits weak enterotoxic activity and simultaneously activates humoral and cellular immunity. Thus, the double mutant heat labile enterotoxin gene according to the present invention can be variously utilized in the fields of vaccines and vaccine adjuvants.

Description

Double mutant heat-resistant toxin gene containing a spacer

[0001] The present invention relates to a double mutant heat-resistant toxin gene comprising a spacer, and more particularly, to the use of a heat-resistant toxin protein encoded by the gene for enhancing immunity.

[0002] Heat-labile enterotoxin (LT) produced by enterotoxigenic E. coli and cholera toxin (CT) produced by Vibrio cholerae promote secretory diarrhea (intestinal secretion) during infections with these microorganisms by similar mechanisms.

[0003] LT is used as an Escherichia coli vaccine antigen and has been studied as an immunostimulant in various other vaccine compositions. However, clinical studies to utilize LT's immune-activating function as an immunostimulant in vaccine compositions have been challenging due to the neurotoxicity induced by LT when administered intranasally with vaccine antigens, making clinical application difficult. Because LT is an enterotoxin, its application as a vaccine antigen requires an inactivation process to remove the toxicity. However, chemical treatment or heating to inactivate LT can denature it, potentially impairing its function as a vaccine antigen and its immune-activating function.

[0004] Recently, the development of dmLT (double mutant LT), a detoxified mutant of LT, for use as an LT vaccine and vaccine adjuvant, has been reported. LT consists of LTA, an enzymatic molecule that promotes cAMP production, and the LTB pentamer (pentamer), which participates in cell binding and membrane penetration, formed by noncovalent association.

[0005] dmLT is a variant of LTA in which arginine at position 192 is substituted with glycine (R192G) and leucine at position 211 is substituted with alanine (L211A). This variant eliminates the intestinal toxin toxicity but maintains the immune-activating function. In other words, dmLT has eliminated the intestinal toxin function that promotes cAMP production by A1 due to the mutations in LTA (R192G, L211A), but it has been reported that the overall structure of LT is maintained and the immune-activating function induced by it is maintained.

[0006] Examples of cases in which dmLT is being developed as an immunogenic adjuvant in vaccine compositions include the development of a pathogenic E. coli vaccine (NCT02531802) that activates mucosal immunity by including dmLT as a mucosal immunogenic adjuvant in an oral E. coli vaccine composition, and the development of a polio virus vaccine (NCT04232943) that includes dmLT as an immunogenic adjuvant in an inactivated polio virus vaccine and is being developed via the intramuscular route.

[0007] Accordingly, the present inventors designed a recombinant dmLT containing a sequence encoding a spacer inserted between LTA and LTB. When the recombinant dmLT of the present invention was transfected into a host cell, LTB expression increased by more than fivefold, and the produced protein maintained its physiological function, thereby completing the present invention.

[0008] Accordingly, the purpose of the present invention is to provide a nucleic acid comprising LTA (heat labile enterotoxin A) represented by the base sequence of SEQ ID NO: 6; LTB represented by the base sequence of SEQ ID NO: 7; and a spacer; and a protein encoding the nucleic acid.

[0009] Another object of the present invention is to provide a recombinant vector comprising the nucleic acid; and a host cell into which the recombinant vector is transduced.

[0010] Another object of the present invention is to provide a composition comprising a protein encoded by the nucleic acid.

[0011] Another object of the present invention is to provide a method for producing a recombinant double mutant LT, comprising the steps of: (a) culturing the host cell in a medium; and (b) inducing recombinant protein expression in the cultured host cell of step (a).

[0012] To achieve the above purpose, the present invention provides a nucleic acid comprising LTA represented by the base sequence of SEQ ID NO: 6; LTB represented by the base sequence of SEQ ID NO: 7; and a spacer.

[0013] The present invention also provides a protein encoded by the nucleic acid.

[0014] The present invention also provides a recombinant vector comprising the nucleic acid.

[0015] The present invention also provides a host cell into which the recombinant vector is transduced.

[0016] The present invention also provides an immunogenic composition comprising the protein.

[0017] The present invention also provides an immunostimulant composition comprising the above protein.

[0018] The present invention also provides a vaccine composition comprising the above protein.

[0019] The present invention also provides a method for producing a recombinant double mutant LT, comprising the steps of: (a) culturing the host cell in a medium; and (b) inducing recombinant protein expression in the cultured host cell of step (a).

[0020] When the double mutant heat-labile toxin gene comprising the spacer according to the present invention was introduced into a host cell, the translation of LTA and LTB, which constitute the heat-labile toxin, did not affect each other, and the expression of LTB was confirmed to increase by more than 5 times. In addition, it was experimentally confirmed that the expressed double mutant heat-labile toxin protein had a weak enterotoxin function and simultaneously activated humoral and cellular immunity. Therefore, the double mutant heat-labile toxin gene of the present invention can be utilized in various fields such as vaccines and immune adjuvants.

[0021] Figure 1 shows a cleavage map of the expression vector NS_dmLT_pET-30a(+) containing the recombinant dmLT gene of the invention.

[0022] Figure 2 is a diagram showing the results of confirming the LTA and LTB expression levels of E. coli expressing the recombinant dmLT of the present invention through SDS-PAGE and Western blotting.

[0023] Figure 3 is a diagram showing the results of confirming the amount of LTA and LTB expression according to the culture temperature in E. coli expressing the recombinant dmLT of the present invention.

[0024] Figure 4 is a diagram showing the results of confirming the amount of LTA and LTB expression according to culture time in E. coli expressing the recombinant dmLT of the present invention.

[0025] Figure 5 is a diagram showing the results of confirming the amount of LTA and LTB expression according to the culture medium in E. coli expressing the recombinant dmLT of the present invention.

[0026] Figure 6 is a diagram showing the results of measuring blood IgG, IgG1, IgG2a, and IgA antibody levels induced by the recombinant dmLT of the present invention (***, P<0.001).

[0027] Figure 7 is a diagram showing the results of analyzing the expression of cellular immune factors IFN-γ, TNF-α, IL-4, and IL-17 induced by the recombinant dmLT of the present invention (*, P<0.05; **, P<0.01; ***, P<0.001).

[0028] Figure 8 is a diagram showing the results of measuring blood IgG, IgG1, IgG2a, and IgA antibody titers following vaccination with a vaccine composition containing the recombinant dmLT of the present invention (*, P<0.05; **, P<0.01; ***, P<0.001).

[0029] Figure 9 is a diagram showing the results of analyzing the expression of cellular immune factors IFN-γ, TNF-α, IL-4, and IL-17 following vaccination with a vaccine composition containing the recombinant dmLT of the present invention (*, P<0.05; **, P<0.01; ***, P<0.001).

[0030] Figure 10 is a diagram showing the results of confirming whether LTB among the recombinant dmLT of the present invention binds to GM1.

[0031] Figure 11 is a diagram showing the results of confirming the cAMP induction ability of the recombinant dmLT of the present invention.

[0032] Hereinafter, the present invention will be described in detail.

[0033] According to an aspect of the present invention, the present invention provides a nucleic acid comprising LTA (heat labile enterotoxin A) represented by the base sequence of SEQ ID NO: 6; LTB represented by the base sequence of SEQ ID NO: 7; and a spacer; and a protein encoding the nucleic acid.

[0034] In a specific embodiment of the present invention, the nucleic acid is arranged in the form of structural formula 1, and it is preferable that each component is operably linked.

[0035]

[0036] [Structural formula 1]

[0037] LTA-Spacer-LTB

[0038]

[0039] The LTA and LTB that make up wild-type LT partially overlap at the LTA end and LTB start sites on the mRNA. This suggests that LTA and LTB compete with each other at the translational level during the expression of wild-type LT. In contrast, the nucleic acid of the present invention (i.e., recombinant dmLT) contains a spacer between LTA (R192G, L211A) and LTB, so that their translations do not interfere with each other.

[0040] In a specific embodiment of the present invention, it is preferable that the nucleic acid further comprises a signal sequence, and the signal sequence may be represented by one or more base sequences selected from the group consisting of SEQ ID NOs: 1 to 3. More preferably, the signal sequence represented by the base sequence of SEQ ID NO: 1 may be operably linked to the 5' end of LTA, and the signal sequence represented by the base sequence of SEQ ID NO: 2 may be operably linked to the 5' end of LTB. In addition, the signal sequence represented by the base sequence of SEQ ID NO: 3 may be applied to both the 5' ends of LTA and LTB. Accordingly, the signal sequence represented by SEQ ID NO: 1 and the signal sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3; the signal sequence represented by SEQ ID NO: 2 and the signal sequence represented by signal sequence 1 or SEQ ID NO: 3; A combination of may be used, and in one embodiment of the present invention, a combination of a signal sequence represented by sequence number 1 and a signal sequence represented by sequence number 2, and a combination of simultaneously introducing sequence number 3 to the 5' end of LTA and the 5' end of LTB were used.

[0041] In the present invention, the signal sequence is a short peptide of about 16 to 30 letters, and is mainly located at the N-terminus of a newly synthesized protein. The main function of the signal sequence is to transport the protein to the cell membrane.

[0042] In a specific embodiment of the present invention, the spacer is a base sequence of a certain length inserted between LTA and LTB, which further includes a ribosome binding site (RBS) of LTB so that LTA and LTB are independently translated and their respective translation levels are independently controlled. The spacer described above includes an RBS and can be represented by the base sequence of SEQ ID NO: 4.

[0043] In the present invention, the ribosome binding site refers to a sequence region of mRNA that helps the ribosome find the initiation codon and initiate translation. Specifically, in E. coli, the ribosomal binding site (RBS) refers to a consensus sequence (5'-AGGAGG-3') that binds complementarily to the 16S RNA of the ribosome 30S subunit as a base sequence on the mRNA, and a change in the base sequence of this region significantly affects the translation level of the adjacent downstream protein. In addition, factors affecting protein translation include a) the length and composition of the base sequence between the RBS and the first codon of the adjacent downstream expressed protein; b) the composition of the upstream base sequence adjacent to the RBS, particularly the adnine ratio; c) the formation of secondary structures in the sequence adjacent to the RBS, etc. In the present invention, a spacer sequence including an RBS was designed in consideration of the above a) to c) and inserted between LTA and LTB.

[0044] Originally, LTA is structurally unstable as a protein and cannot exist alone. It can exist as a complete LT only when associated with LTB. Therefore, in order to increase the expression level of complete LT, it is essential to maintain the expression of LTB higher than that of LTA. However, if LTB expression is increased beyond the limit, complete LT is produced, and the remaining surplus LTB forms a stable LTB pentamer, which must be removed to produce pure LT. Therefore, in the present invention, a spacer sequence including the RBS of LTB was designed in consideration of the factors that the base sequence around the RBS mentioned above affects the expression to maintain the expression of LTB at an appropriate level.

[0045] In a preferred embodiment of the present invention, the nucleic acid may further include a ribosome binding site at the 5' end of LTA. The ribosome binding site introduced into the spacer and the ribosome binding site introduced at the 5' end of LTA may use the same sequence or different sequences.

[0046] The nucleic acid of the present invention may be represented by the base sequence of SEQ ID NO: 5, but the scope of the present invention is not limited thereto. In addition, variants of the base sequence are included within the scope of the present invention. Specifically, the nucleic acid of the present invention has a sequence homology of 70% or more, more preferably 80% or more, even more preferably 90% or more, and most preferably 95% or more with SEQ ID NO: 5, and means a sequence that exhibits substantially the same physiological activity as the base sequence represented by SEQ ID NO: 5. The "% of sequence homology" for a polynucleotide is determined by comparing a comparison region with two optimally aligned sequences, and a portion of the polynucleotide sequence in the comparison region may include additions or deletions (i.e., gaps) compared to a reference sequence for the optimal alignment of the two sequences (which does not include additions or deletions).

[0047] As mentioned above, "substantially identical physiological activity" refers to immune-enhancing activity. The scope of "functional equivalents" of the present invention includes derivatives that maintain the basic structure and immune-enhancing activity of the nucleic acid according to the present invention, but have modified chemical structures. For example, this includes structural modifications to alter the stability, storability, volatility, or solubility of the nucleic acid.

[0048] In the present invention, "operably linked" means linked in a manner that enables gene expression when an appropriate molecule binds to an expression regulatory sequence.

[0049] The scope of the present invention also includes functional equivalents of proteins encoded by the nucleic acids. The term "functional equivalent" refers to a peptide that has at least 80%, preferably 90%, and more preferably 95% sequence homology (i.e., identity) with the protein encoded by the nucleic acids as a result of addition, substitution, or deletion of amino acids, for example, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% sequence homology, and exhibits substantially the same physiological activity as the protein encoded by the nucleic acids. Sequence homology and identity herein are defined as the percentage of amino acid residues in a candidate sequence relative to a protein encoded by said nucleic acid, after aligning the candidate sequence with the protein encoded by said nucleic acid and introducing gaps. Where necessary, conservative substitutions are not considered part of the sequence homology to obtain the maximum percent sequence homology. N-terminal, C-terminal, or internal extensions, deletions, or insertions in the protein encoded by said nucleic acid are not construed as sequences that affect sequence homology or homology.

[0050] Additionally, the sequence identity can be determined by commonly used standard methods for comparing similar portions of the amino acid sequences of two polypeptides. Computer programs such as BLAST or FASTA align two polypeptides so that each amino acid matches optimally (along the full length of one or both sequences or along predicted portions of one or both sequences). The programs provide default opening penalties and default gap penalties, and provide scoring matrices such as PAM250 (a standard scoring matrix) that can be used in conjunction with the computer program. For example, the percent identity can be calculated as follows: the total number of identical matches is multiplied by 100, and then divided by the sum of the length of the longer sequence within the matched span and the number of gaps introduced into the longer sequence to align the two sequences.

[0051] In an embodiment of the present invention, when the nucleic acid according to the present invention (i.e., recombinant dmLT) was introduced into a host cell, translation did not affect each other, and the expression of LTB increased by more than 5 times. In addition, it was confirmed that the recombinant dmLT increased blood IgG, IgG1, IgG2a, and IgA antibodies; and the secretion of cellular immune factors IFN-γ, TNF-α, IL-4, and IL-17; and did not induce cAMP. This means that the recombinant dmLT of the present invention has a weak enterotoxin function and simultaneously activates humoral and cellular immunity, and can be utilized in various fields such as vaccines and immune boosters.

[0052]

[0053] According to another aspect of the present invention, the present invention provides a recombinant vector comprising the nucleic acid; and a host cell into which the recombinant vector is transduced.

[0054] In the present invention, a vector refers to a means for expressing a target gene in a host cell. For example, it includes a plasmid vector, a cosmid vector, a bacteriophage vector, an adenovirus vector, a retrovirus vector, and a viral vector such as an adeno-associated virus vector. A vector that can be used as the recombinant vector can be produced by manipulating a plasmid (e.g., pGLS, pSC101, pGV1106, pACYC177, ColE1, pKT230, ME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, and pUC19, etc.), a phage (e.g., λgt4λB, λCharon, λΔz1, and M13, etc.) or a virus (e.g., CMV, SV40, etc.) that is frequently used in the art.

[0055] In the recombinant vector, the nucleic acid may be operably linked to a promoter. The term "operably linked" refers to a functional linkage between a nucleotide expression regulatory sequence (e.g., a promoter sequence) and another nucleotide sequence. Thus, the regulatory sequence can regulate the transcription and / or translation of the other nucleotide sequence.

[0056] The recombinant vector may be constructed typically as a cloning vector or an expression vector. The expression vector may be any vector commonly used in the art to express foreign proteins in plants, animals, or microorganisms. The recombinant vector may be constructed using various methods known in the art.

[0057] The above recombinant vector can be constructed using a prokaryotic cell or a eukaryotic cell as a host. For example, when a eukaryotic cell is used as a host, the replication origin that operates in the eukaryotic cell included in the vector includes, but is not limited to, the f1 replication origin, the SV40 replication origin, the pMB1 replication origin, the adeno replication origin, the AAV replication origin, the CMV replication origin, and the BBV replication origin. In addition, a promoter derived from the genome of a mammalian cell (e.g., a metallothionine promoter) or a promoter derived from a mammalian virus (e.g., an adenovirus late promoter, a vaccinia virus 7.5K promoter, an SV40 promoter, a cytomegalovirus (CMV) promoter, and a tk promoter of HSV) can be used, and generally has a polyadenylation sequence as a transcription termination sequence.

[0058] Any host cell known in the art can be used as the host cell, and as prokaryotic cells, for example, Bacillus strains such as Escherichia coli (BL21(DE), BL21(DE3)plysS, BL21(DE3)star, BL21(DE3)soluble), Bacillus subtilis, Bacillus thuringiensis, and enterobacteria and strains such as Salmonella typhimurium, Serratia marcescens, and various Pseudomonas species, and in the case of transformation into eukaryotic cells, yeast (Saccharomyce cerevisiae), insect cells, plant cells, and animal cells, for example, SP2 / 0, CHO (Chinese hamster ovary) K1, CHO DG44, PER.C6, W138, BHK, COS-7, 293, HepG2, Huh7, 3T3, RIN, and MDCK cell lines, can be used as the host cell. there is.

[0059] Insertion of the nucleic acid or a recombinant vector containing the same into a host cell can be accomplished using an insertion method widely known in the art. For example, if the host cell is a prokaryotic cell, the CaCl2 method or electroporation can be used, and if the host cell is a eukaryotic cell, the microinjection method, calcium phosphate precipitation method, electroporation, liposome-mediated transfection method, and gene bombardment can be used, but are not limited thereto.

[0060] The method for selecting the transformed host cells can be easily performed using methods widely known in the art, utilizing the phenotype expressed by the selection marker. For example, if the selection marker is a specific antibiotic resistance gene, the transformants can be easily selected by culturing them in a medium containing the antibiotic.

[0061]

[0062] According to another aspect of the present invention, the present invention provides a composition comprising a protein encoded by the nucleic acid. The composition may be an immunogenic composition; an immunoadjuvant composition; or a vaccine composition.

[0063] In an embodiment of the present invention, it was confirmed that the protein of the present invention described above increases blood IgG, IgG1, IgG2a, and IgA antibodies; and secretion of cellular immune factors IFN-γ, TNF-α, IL-4, and IL-17; and does not induce cAMP. This means that the protein of the present invention has a weak enterotoxin function and simultaneously activates humoral and cellular immunity, and thus can be utilized in various fields such as vaccines and immune enhancers.

[0064] In the present invention, immunostimulation is one of the important therapeutic strategies that reinforces the body's defense mechanisms against various diseases such as infectious diseases, cancer, and inflammatory diseases, and can achieve an immune-enhancing effect by increasing the activity of immune cells to stimulate the immune response. For example, phagocytes play a key role in the immune response. The main role of macrophages, phagocytosis, is to absorb microorganisms and other pyrogenic particles, and also secrete numerous cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-12 (IL-12), and cytotoxic and inflammatory substances such as nitric oxide (NO), thereby stimulating the immune response. Therefore, increasing macrophage activity can be one means of immune-enhancement. Since the occurrence of infections and diseases mainly occurs when the immune function is weakened, various studies are being conducted to enhance these immune responses through immune-enhancing substances when the function of the body's immune system is weakened.

[0065] The composition for enhancing immunity according to the present invention may contain a pharmaceutically effective amount of a protein encoded by the nucleic acid of the present invention and an excipient or diluent.

[0066] The pharmaceutically effective amount described above refers to an amount sufficient to exert an immune-enhancing effect. The term "pharmaceutically acceptable" refers to a composition that is physiologically acceptable and, when administered to humans, does not typically cause allergic reactions, such as gastrointestinal upset or dizziness, or similar reactions.

[0067] In the present invention, the vaccine is a composition containing an antigenic substance, and is administered for the purpose of inducing specific active or passive immunity against the antigen. The vaccine composition according to the present invention may be administered in an immunologically effective amount. The "immunologically effective amount" refers to an amount sufficient to exhibit a preventive effect against a related disease and an amount that does not cause side effects or serious or excessive immune responses. The exact administration concentration varies depending on the specific immunogen to be administered, and can be easily determined by those skilled in the art based on factors well known in the medical field, such as the age, weight, health, sex, drug sensitivity of the subject of vaccination, administration route, and administration method, and may be administered once or multiple times.

[0068] The vaccine composition according to the present invention may comprise one or more suitable immunostimulants, excipients or carriers.

[0069] An adjuvant that may be included in the vaccine composition of the present invention refers to a substance that enhances the immune response of an injected animal, and many different adjuvants are known to those skilled in the art. The adjuvants include, but are not limited to, Freund's complete and incomplete adjuvants, vitamin E, non-ionic blocking polymers, muramyl dipeptide, Quil A, mineral oil and mineral-free oil, Carbopol, water-in-oil emulsion adjuvants, and the like.

[0070] Carriers that may be included in the vaccine composition of the present invention are well known to those skilled in the art and include, but are not limited to, proteins, sugars, and the like. The carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous carriers include propylene glycol, polyethylene glycol, edible oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcohol / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral carriers include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous carriers include electrolyte replenishers, liquids, and nutritional replenishers, such as those based on Ringer's dextrose.

[0071] The vaccine composition of the present invention may further comprise preservatives and other additives, such as antimicrobial agents, antioxidants, chelating agents, inert gases, etc. Preservatives include formalin, thimerosal, neomycin, polymyxin B, and amphotericin B. The vaccine composition of the present invention may comprise one or more suitable emulsifiers, such as Span or Tween. In addition, the vaccine composition of the present invention may comprise a protective agent, and any protective agent known in the art may be used without limitation, including, but not limited to, lactose (LPGG) or trehalose (TPGG).

[0072]

[0073] According to another aspect of the present invention, the present invention provides a method for producing a recombinant double mutant LT, comprising the steps of: (a) culturing the host cell in a medium; and (b) inducing recombinant protein expression in the cultured host cell of step (a).

[0074] In the present invention, "cultivation" refers to growing cells under appropriately artificially controlled environmental conditions. The cells can be grown in a conventional medium. A medium contains nutrients required by the target cells, i.e., the cells that will become the cultured organism, for culturing specific cells. It may be a mixture in which substances for special purposes are additionally added. The medium is also called a culture medium or culture solution, and encompasses natural media, synthetic media, and selective media.

[0075] The medium and other culture conditions used for culturing the microorganism of the present invention may be any medium commonly used for culturing Escherichia microorganisms, but must adequately satisfy the requirements of the microorganism of the present invention. Preferably, the microorganism of the present invention is cultured under aerobic conditions in a conventional medium containing appropriate carbon sources, nitrogen sources, amino acids, vitamins, etc., while controlling temperature, pH, etc.

[0076] In a preferred embodiment of the present invention, the medium may include glucose, pyruvate, etc. as a carbon source. Inorganic compounds may include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, and calcium carbonate, and other compounds may also include amino acids, vitamins, and appropriate precursors. These media or precursors may be added to the culture in batch or continuous mode.

[0077] During cultivation, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid can be added to the culture in an appropriate manner to adjust the pH of the culture. Furthermore, foaming can be suppressed during cultivation using antifoaming agents such as fatty acid polyglycol esters. Furthermore, to maintain aerobic conditions in the culture, oxygen or oxygen-containing gas can be injected into the culture. To maintain both anaerobic and aerobic conditions, nitrogen, hydrogen, or carbon dioxide can be injected without gas injection.

[0078] In a specific embodiment of the present invention, the medium of step (a) is preferably LB (Luria Bertani) or 2xYT (2x Yeast Extract Tryptone medium), and more preferably LB.

[0079] In a specific embodiment of the present invention, the step (b) is preferably performed at a temperature of 10 to 25°C, more preferably 15 to 20°C, and most preferably 17°C.

[0080] In a specific embodiment of the present invention, the step (b) may be performed for 1 to 150 hours, preferably 10 to 100 hours, more preferably 72 hours, but the culture period may continue until the desired amount of useful material is obtained.

[0081] The recombinant double mutant LT produced in the above culturing step of the present invention may further include a step of purifying or recovering, and a method of recovering the recombinant double mutant LT from a microorganism or culture may be a method known in the art, such as centrifugation, filtration, anion exchange chromatography, crystallization, and HPLC, but is not limited to these examples.

[0082] The above recovery step may include a purification process, and a person skilled in the art may select and utilize one of several known purification processes as needed.

[0083]

[0084] Duplicate contents are omitted in consideration of the complexity of this specification, and terms not otherwise defined in this specification have meanings commonly used in the technical field to which the present invention belongs.

[0085] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

[0086]

[0087] Example 1. Design of recombinant dmLT and production of expression vector containing it.

[0088] The present inventors designed a recombinant dmLT having a sequence encoding a spacer containing a ribosome binding site inserted between LTA and LTB. The spacer containing the ribosome binding site is represented by the nucleotide sequence of SEQ ID NO: 4. Specifically, a DNA sequence (SEQ ID NO: 5) was obtained from the protein sequence of dmLT. The 5' and 3' ends of the obtained dmLT DNA sequence were ligated to the NdeI and HindIII cleavage sites of PET-30a(+) via NdeI and HindIII linkers, respectively, to complete the expression vector NS_dmLT_PET-30a(+). In the above expression vector NS_dmLT_PET-30a(+), LTA (R192G, L211A) (SEQ ID NO: 6) and LTB (SEQ ID NO: 7) are transcribed into a single mRNA through a single T7 promoter and have their own ribosome binding site (RBS) and stop codon. The gene inserted into the expression vector and the cleavage map of the expression vector NS_dmLT_pET-30a(+) are shown in Fig. 1. Specifically, the 5' end of LTA (R192G, L211A) of the dmLT includes a Native signal sequence (SEQ ID NO: 1) represented by SEQ ID NO: 1, and the 5' end of LTB includes a Native signal sequence represented by SEQ ID NO: 2. In addition, a 'spacer (SEQ ID NO: 4) including a ribosome binding site' is inserted between the 5' end of LTA (R192G, L211A) and the 5' end of the native signal sequence of LTB. In addition, the 5' end of the native signal sequence of LTA (R192G, L211A) includes a ribosome binding site represented by SEQ ID NO: 8.

[0089] LTA and LTB, which comprise wild-type LT, partially overlap the LTA end and LTB start regions on the mRNA. This suggests that LTA and LTB compete with each other at the translational level during wild-type LT expression. In contrast, the recombinant dmLT designed in this example contains a spacer between LTA (R192G, L211A) and LTB, preventing their translation from interfering with each other.

[0090] In addition to dmLT (NS_dmLT_PET-30a(+)) that introduced a native signal sequence into LTA (R192G, L211A) and LTB, PS_dmLT_PET-30a(+) that introduced the same pelB signal sequence (SEQ ID NO: 3) into the 5'-end native signal sequence position of LTA (R192G, L211A) and LTB was produced.

[0091]

[0092] Example 2. Production of a recombinant dmLT expression strain

[0093] Escherichia coli strains (BL21(DE), BL21(DE3)plysS, BL21(DE3)star, BL21(DE3)soluble) were transformed with the expression vector NS_dmLT_PET-30a(+) constructed in Example 1. Specifically, 0.5-1μg of the expression vector was mixed with the E. coli strain, left on ice for 30 minutes, and then heat-shocked at 42°C for 1 minute and 30 seconds. After that, the strain was left on ice again for 1 minute, and 500μL of LB medium was seeded. After that, the strain was cultured in a 37°C shaking incubator for 1 hour, and the transformants were isolated by culturing on LB solid medium containing kanamycin. The isolated transformants (i.e., recombinant E. coli expressing dmLT) were cultured in LB liquid medium containing kanamycin at 37°C and 200 RPM for 18 hours, and then 10% (w / v) glycerol stock was prepared and stored at -70°C.

[0094]

[0095] Example 3. Culturing and purification of recombinant dmLT

[0096] The recombinant E. coli expressing the recombinant dmLT produced in Example 2 was seed-cultured in 10 mL LB liquid culture medium containing kanamycin (50 ug / mL) at 37°C for 12 to 18 hours. Thereafter, the culture was transferred to 1 L LB main culture liquid medium with the same medium composition and cultured with shaking at 37°C for 12 to 18 hours. When the OD reached 0.6 during culture, 1 mM IPTG was added to induce dmLT expression. After the main culture was completed, the cells were recovered from the liquid culture medium by centrifugation and suspended in TEAN buffer (0.2 M NaCl, 50 mM Tris, 1 mM EDTA, 3 mM NaN3, pH 7.5). The E. coli suspended in the TEAN buffer was disrupted by ultrasonication. The homogenate was eluted with TEAN buffer to obtain dmLT present inside the E. coli cell and the periplasmic space. The eluted dmLT was bound to a galactose affinity agarose resin equilibrated with TEAN buffer, and impurities were removed by washing with TEAN buffer. dmLT was eluted with TEAN buffer containing 300 mM galactose to purify it. The purified dmLT was analyzed by Superdex 200 size exclusion chromatography HPLC to quantify free LTB pentamers that were not associated with dmLT, and the ratio of intact dmLT excluding free LTB pentamers in the purified dmLT sample was determined. Cell growth during cultivation was measured by the OD (600 nm) of the final culture broth.

[0097]

[0098] Example 4. Expression of recombinant dmLT

[0099] Recombinant dmLT was expressed in recombinant E. coli strains introduced with NS_dmLT_PET-30a(+) (including the native signal sequence) or PS_dmLT_PET-30a(+) (including the pelB signal sequence). The E. coli strains used in this experiment are listed in Table 1 below.

[0100] HostGenotypeBL21(DE3)F - ompThsdS B (r B - ,m B - )gal dcm(DE3)SoluBL21 + F-ompThsdS B (r B -m B -)galdcm(DE3)BL21(DE3) pLysSF - ompThsdS B (r B - , m B - )gal dcm(DE3) pLysS(Cam R )BL21 Star (DE3)F - ompThsdS B (r B - , m B - )gal dcm rne131 (DE3)+The SoluBL21 strain contains uncharacterized mutations obtained through special selection criteria

[0101] Specifically, recombinant dmLT-expressing Escherichia coli was cultured in LB liquid medium at 37°C. When the OD (600 nm) reached 0.6 during culture, 1 mM IPTG was added to induce dmLT expression. The cells were harvested from the E. coli culture and resuspended in TEAN buffer. The resuspended cells were disrupted by ultrasonication and centrifuged to collect the supernatant. The supernatant contained dmLT expressed in the periplasmic space and within the cell. The expression of dmLT was analyzed using SDS-PAGE and Western blotting of the cell lysate supernatant, and the results are shown in Figure 2.

[0102] As shown in Fig. 2, it was confirmed that dmLT was expressed in the produced recombinant E. coli. In particular, it was confirmed that the protein expression of LTA (R192G, L211A) and LTB was high in recombinant E. coli strains 5837, 5840, 5841, and 5843.

[0103]

[0104] Example 5. Expression conditions of recombinant dmLT

[0105] The expression vector NSdmLT_PET-30a(+) designed in Example 1 was transformed into E. coli BL21(DE3). In this example, the recombinant dmLT expression conditions of the transformed recombinant E. coli were confirmed.

[0106]

[0107] 5-1. Temperature

[0108] Recombinant E. coli was cultured under the same conditions as Example 2, inoculated into 1 L LB medium of the same composition, and cultured with shaking at 37°C. When the OD (600 mm) reached 0.6, 1 mM IPTG was added, and the culture was performed under various temperature conditions (17°C or 37°C). The expression amount of recombinant dmLT was analyzed as in Example 3. In addition, the expressed recombinant dmLT was purified using galactose affinity agarose resin, and then quantified by SDS-PAGE and BCA. In addition, the association of LTA (R192G L211A) and LTB was confirmed through Superdex 200 size exclusion chromatography. The results confirming the expression and association of recombinant dmLT according to temperature conditions are shown in Table 2 and Fig. 3.

[0109] Expression induction at 37°C Expression induction at 17°C Cell growth (final 600 nm OD) 2.11.5 Recombinant dmLT expression amount 2.5 mg / L 3.5 mg / LLTA (R192G L211A) and LTB association ratio 15% 55%

[0110] As shown in Table 2 and Figure 3, recombinant E. coli cultured under 17°C conditions showed slower bacterial growth than those cultured under 37°C conditions, but the recombinant dmLT expression level and association ratio were high.

[0111]

[0112] 5-2. Incubation time

[0113] Recombinant E. coli was cultured under the same conditions as Example 2, inoculated into 1 L LB medium of the same composition, and cultured with shaking at 37°C. When the OD (600 mm) reached 0.6, 1 mM IPTG was added, and cultured at 17°C for 18 or 72 hours. The expression amount of recombinant dmLT was analyzed as in Example 3. In addition, the expressed recombinant dmLT was purified using galactose affinity agarose resin, and then quantified by SDS-PAGE and BCA. In addition, the association of LTA (R192G L211A) and LTB was confirmed through Superdex 200 size exclusion chromatography. The results of confirming the expression and association of recombinant dmLT according to the culture time conditions are shown in Table 3 and Fig. 4.

[0114] 18-hour expression 72-hour expression Cell growth 1.6 1.8 Recombinant dmLT expression amount 2.7 mg / L 10.0 mg / LLTA (R192G, L211A) and LTB association ratio 65% 73%

[0115] As shown in Table 3 and Figure 4, the recombinant E. coli cultured for 72 hours had higher cell growth, expression level of recombinant dmLT, and association ratio than the recombinant E. coli cultured for 18 hours.

[0116]

[0117] 5-3. Badge

[0118] Recombinant E. coli was cultured under the same conditions as Example 2, and then inoculated into 1 L of LB or 2xYT medium of the same composition, followed by shaking culture at 37°C. When the OD (600 mm) reached 0.6, 1 mM IPTG was added, and the medium was cultured at 17°C for 72 hours. The expression amount of recombinant dmLT was analyzed as in Example 3. In addition, the expressed recombinant dmLT was purified using galactose affinity agarose resin, and then quantified by SDS-PAGE and BCA. In addition, the association of LTA (R192G L211A) and LTB (whether LTB pentamer was formed) was confirmed through Superdex 200 size exclusion chromatography. The results of confirming the expression and association of recombinant dmLT according to the medium conditions are shown in Table 4 and Fig. 5.

[0119] LB medium (1L) 2XYT medium (1L) Composition Yeast extract 5g Yeast extract 10g Trypton 10g Trypton 16g NaCl 5g NaCl 5g Cell growth 1.732.23 Recombinant dmLT expression amount 11mg 5mg Association ratio of LTA (R192G, L211A) and LTB 78% 78%

[0120] As shown in Table 4 and Fig. 5, the growth of the cells was high under 2xYT medium conditions, but the recombinant dmLT expression level was higher under LB medium conditions.

[0121]

[0122] Example 6. Immunogenicity test of recombinant dmLT

[0123] To confirm the immunogenicity of recombinant dmLT, 50 μg of purified recombinant dmLT was administered intramuscularly to Balb / c mice on days 0 and 21.

[0124]

[0125] To measure humoral immunity induced by recombinant dmLT, blood samples were collected at weeks 0, 3, and 5 of recombinant dmLT administration. dmLT-specific IgG, IgG1, IgG2a, and IgA were measured in the blood, and the results are shown in Figure 6.

[0126] As shown in Figure 6, the recombinant dmLT administration group was confirmed to have formed antibodies IgG, IgG1, IgG2a, and IgA from the third week of administration. In contrast, the negative control group (untreated group) was confirmed to have no formation of antibodies IgG, IgG1, IgG2a, and IgA.

[0127]

[0128] To measure cellular immune activation, spleen cells were isolated from mice 5 weeks after recombinant dmLT administration. The isolated spleen cells were cultured in RPMI medium, and the cellular immune factors secreted into the culture medium when sensitized with dmLT, such as IFN-γ, TNF-α, IL-4, and IL-17, were measured by ELISA. The results of measuring cellular immune activation by ELISA are shown in Figure 7.

[0129] As shown in Figure 7, it was confirmed that immune cells of mice sensitized with recombinant dmLT secreted high levels of dmLT-specific cellular immune factors IFN-γ, TNF-α, IL-4, and IL-17.

[0130]

[0131] The above results indicate that recombinant dmLT simultaneously activates humoral and cellular immunity.

[0132]

[0133] Example 7. Evaluation of vaccine efficacy of recombinant dmLT

[0134] To evaluate the vaccine efficacy of recombinant dmLT, the experimental groups were designed as shown in Table 5.

[0135] Test group dmLT Dosage (dose / mouse) MONTANAIDETM (ISA206) ​​Dose Number of individuals G1 Negative control group -5G2 10 μg -5G3 5 μg -5G4 2 μg -5G5 1 μg -5G6 0.5 μg -5G7 2 μg ISA206 50% 5G8 1 μg ISA206 50% 5G9 0.5 μg ISA206 50% 5

[0136] Specifically, purified recombinant dmLT was intramuscularly inoculated into 6-week-old female BALB / c mice at doses of 0.5, 1, 2, 5, and 10 μg per mouse on days 0 and 3. Blood samples were collected from the mice on weeks 0, 3, and 5. dmLT-specific IgG, IgG1, IgG2a, and IgA antibody titers were measured from the serum. In addition, when mouse spleen cells were isolated and cultured on week 5 and then sensitized with recombinant dmLT, the cellular immune factors secreted into the culture medium, such as IFN-γ, TNF-α, IL-4, and IL-17, were measured.

[0137] Additionally, in the low-dose dmLT (0.5, 1, 2 μg / mouse) administration group, MONTANAIDETM ISA206 was co-administered to confirm the effect of the immunostimulant on the immunogenicity of dmLT.

[0138]

[0139] - Induction of humoral immunity by recombinant dmLT vaccine vaccination

[0140] To assess whether the target level of antibodies was induced by dmLT vaccine administration, IgG, IgG1, IgG2a, and IgA antibody titers were measured. The antibody titer measurement results are shown in Figure 8.

[0141] As shown in Figure 8, IgG antibody titers significantly increased compared to the negative control group from 3 weeks after the first vaccination with the recombinant dmLT vaccine, and further increases were observed during the second boost. In particular, the 2 μg recombinant dmLT vaccination groups (G4 and G7) showed significantly higher antibody titers than the other groups. Furthermore, a comparison of the ratios of dmLT-specific IgG2a and IgG1 induced in the blood revealed that the ratios were close to 1 in all administration groups.

[0142] In addition, IgA antibody titers significantly increased in all administration groups compared to the negative control group, and in particular, the groups vaccinated with 2 μg of recombinant dmLT (G4 and G7) and the group vaccinated with 1 μg of recombinant dmLT plus ISA206 (G8) showed significantly higher IgA antibody titers. In addition, the IgA antibody titers of all administration groups further increased during the second boost, and in particular, the experimental group including the ISA206 adjuvant showed significantly higher IgA antibody titers.

[0143] The above results confirmed that dmLT-specific IgG and IgA antibody titers significantly increased when the recombinant dmLT vaccine was administered, suggesting that sufficient humoral immunity and mucosal immunity were induced when the recombinant dmLT vaccine was administered.

[0144]

[0145] - Induction of cellular immunity by recombinant dmLT vaccination

[0146] To determine whether dmLT vaccine administration induces cellular immunity, cellular immune factors such as IFN-γ, TNF-α, IL-4, and IL-17 were measured. The results of measuring the concentrations of cellular immune factors are shown in Figure 9.

[0147] As shown in Figure 9, the secretion amounts of representative Th1 cytokines, IFN-γ and TNF-α, significantly increased in the dmLT alone administration group (G2-G5) compared to the control group. In addition, the secretion amount of IL-4, a representative Th2 cytokine, significantly increased in all test groups compared to the control group, and the secretion amount increased further in the dmLT alone administration group (G2-G5). IL-17A, which affects mucosal immunity, also significantly increased in the dmLT alone administration group (G2-G5) compared to the control group.

[0148] Meanwhile, the dmLT administration group containing the immune-enhancing agent ISA206 barely secreted dmLT cytokines. This appears to be because the protein structure of dmLT was denatured by the oil component ISA206, which impaired the immune-activating function of dmLT. Although humoral immunity to dmLT was induced even when the structure was denatured by ISA206, it was determined that the unique protein structure of dmLT and the immune-activating function of dmLT derived from it must be maintained in order to activate cellular immunity to dmLT.

[0149]

[0150] Example 8. Confirmation of GM1 binding of recombinant dmLT

[0151] LTB binds to GM1 (monosialotetrahexosylganglioside) on the cell surface and mediates the delivery of toxic LTA into cells. The binding of LTB to GM1 in the produced recombinant dmLT was confirmed through GM1-ELISA (ganglioside enzyme-linked immunosorbent assays). Specifically, an indirect ELISA developed in-house was performed using a GM1-coated ELISA plate (Human anti-ganglioside (GM1) antibody (IgG) ELISA Kit (CSB-E09694h, Cusabio, China)) and a detection antibody (Anti-LT subunit (A+B) antibody (ab188541, Abcam, UK)). The test group was treated with recombinant dmLT at various concentrations, and as a positive control, CT (cholera toxin; List Labs, USA), which has an amino acid sequence similar to LT and binds to GM1, was treated. The results of the GM1 binding test are shown in Fig. 10.

[0152] As shown in Figure 10, purified recombinant dmLT showed increased binding to GM1 in a concentration-dependent manner, similar to CT (positive control). This result suggests that recombinant dmLT retains biological activity.

[0153]

[0154] Example 9. cAMP induction test of recombinant dmLT

[0155] CHO cells cultured in EMEM (Eagle's minimum essential medium) containing 10% FBS were seeded in 6-well plates. Before treatment with recombinant dmLT, CHO cells were cultured in EMEM (+1% FBS, 1 mM 3-isobutyl-1-methylznathine (IBMX)) for 30 min at 37°C in 5% CO2. To induce cAMP, 10 μg of recombinant dmLT and 10 μg of CT (cholerae toxin) were each treated with trypsin and activated at 37°C for 45 min. Activated recombinant dmLT or CT was added to the medium at a concentration of 0.001 to 10 μg / ml and cultured for 3 h. The induced cAMP was extracted from the cells. cAMP induced by recombinant dmLT or CT was quantified using a cAMP ELISA kit, and the results are shown in Fig. 11.

[0156] As shown in Figure 11, the positive control, CT, sufficiently induced cAMP, but recombinant dmLT did not induce cAMP even at a 10,000-fold higher concentration. This result suggests that recombinant dmLT has a weakened enterotoxin function (i.e., is attenuated).

[0157]

[0158] In summary, the present inventors designed a recombinant dmLT having a sequence encoding a spacer inserted between LTA and LTB. When introduced into a host cell, the recombinant dmLT did not interfere with each other's translation, resulting in a more than fivefold increase in LTB expression. Furthermore, the recombinant dmLT was confirmed to increase serum IgG, IgG1, IgG2a, and IgA antibodies, and the secretion of cellular immune factors IFN-γ, TNF-α, IL-4, and IL-17, while not inducing cAMP. This suggests that the recombinant dmLT of the present invention has a weak enterotoxin function and simultaneously activates humoral and cellular immunity, and thus can be utilized in various fields such as vaccines and immune boosters.

[0159]

[0160] While specific aspects of the present invention have been described in detail, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. LTA (heat labile enterotoxin A) represented by the base sequence of sequence number 6; LTB represented by the base sequence of sequence number 7; and A nucleic acid comprising a spacer.

2. In the first paragraph, the nucleic acid is arranged in the form of structural formula 1. [Structural formula 1] LTA-SPACER-LTB 3. A nucleic acid according to claim 1, wherein the nucleic acid further comprises a signal sequence.

4. In the third paragraph, the nucleic acid comprises a signal sequence at the 5' end of LTA or the 5' end of LTB.

5. In the third paragraph, the signal sequence is a nucleic acid represented by one or more base sequences selected from the group consisting of sequence numbers 1 to 3.

6. A nucleic acid in the first paragraph, wherein the spacer comprises a ribosome binding site.

7. A nucleic acid in claim 1, wherein the nucleic acid further comprises a ribosome binding site at the 5' end of LTA.

8. A protein encoded by a nucleic acid of any one of claims 1 to 7.

9. A recombinant vector comprising a nucleic acid of any one of claims 1 to 7.

10. A host cell transfected with the recombinant vector of clause 9.

11. An immunogenic composition comprising the protein of clause 8.

12. An adjuvant composition comprising the protein of clause 8.

13. A vaccine composition comprising the protein of clause 8. 14.(a) A step of culturing the host cell of clause 10 in a medium; and (b) a step of inducing recombinant protein expression in the cultured host cell of step (a); A method for producing a recombinant double mutant LT.

15. A method in claim 14, wherein the medium in step (a) is LB (Luria Bertani) or 2xYT (2x Yeast Extract Tryptone medium).

16. A method according to claim 14, wherein step (b) is performed at a temperature of 10 to 25°C.

17. A method according to claim 14, wherein step (b) is performed for 1 to 150 hours.

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