WO2017116005A1 - Small rna gene for regulating extracellular vesicle generation and host immune response, and use thereof - Google Patents

Abstract

It was ascertained that a vector expressing an E. coli-derived small RNA (sRNA), of the present invention, overexpresses an sRNA, an sRNA overexpression strain increases extracellular vesicle generation, and an sRNA overexpression strain-derived extracellular vesicle induces Th1 and Th17 immune responses similar to those of live bacterial infection, and thus it was ascertained that the vector expressing an E. coli-derived sRNA can be useful as an immunoregulatory composition. In addition, it was ascertained that treating an E. coli-derived extracellular vesicle with polymyxin B allows toxicity caused by the endotoxin within the vesicle to be removed and simultaneously allows the immunoregulatory effect of the vesicle to be maintained, and thus it was ascertained that an extracellular vesicle containing the vector expressing an E. coli-derived sRNA can be useful as an immunoregulatory composition.

Description

SMALL RNA gene and its use to modulate extracellular vesicle formation and host immune response

The present invention relates to an immunomodulatory pharmaceutical composition, health functional food, cosmetic composition and the like containing a vector expressing E. coli-derived small RNA (sRNA) as an active ingredient.

Immunity is largely divided into innate immunity that has been born since birth and acquired immunity that is acquired by adapting to life. Innate immunity, also known as 'natural immunity', responds nonspecifically to the antigen and does not have a special memory function. Innate immune systems include skin, mucous tissue, acidic acid, and complement that are present in the blood to block antigen invasion. Cells include macrophage and polymorphonuclear leukocytes, which are responsible for phagocytosis, and natural killer (NK) cells that can kill infected cells. In fact, most infections are protected by innate immunity. In addition, acquired immunity is also known as 'acquired immunity' and can remember the first invading antigen, and the characteristic that can effectively remove the antigen by reacting specifically when invading again serves to reinforce innate immunity. The commonly used definition of immunity says this. This acquired immunity is understood to be divided into humoral immunity and cell-mediated immunity for convenience. Humoral immunity differentiates after B lymphocytes recognize an antigen to secrete an antibody, which shows its function mainly to remove infected bacteria. Antibodies are present in body fluids and consist of glycoproteins called immunoglobulins (Ig). These include IgG, IgM, IgA, IgD, and IgE, each of which performs unique functions and sometimes duplicates some functions. IgA antibodies are characterized by their delivery to the fetus via the placenta. This immunity is called maternal immunity, which is why it does not infect well for many months after birth. Cellular immunity plays a role in the thymus-derived T lymphocytes recognizing antigens to secrete lymphokine or to directly kill infected cells. Secreted lymphokines may also activate phagocytes to aid phagocytosis. Such cellular immunity primarily functions to remove cells infected with viruses or bacteria that can grow within cells. Acquired immunity can be obtained by immunization of a pathogen or its toxin with an immunogen, and such immunity is called artificial immunity.

Allergies are characterized by the memory of foreign antigens, which can cause disease if hypersensitivity reactions occur when useful antigens enter the body. Hypersensitivity reactions can be divided into four types based on expression time and reaction type. Type I (immediate) is typical of most allergic reactions, with symptoms such as asthma, seasonal or perennial rhinitis, allergic sinusitis, conjunctivitis, food and drug allergies, atopic dermatitis, urticaria, and bee stings. Reaction and the like. Type II (antibody involvement) causes cytolytic or inflammatory reactions of humoral immunity. Symptoms include hemolysis of red blood cells due to inadequate blood transfusion, and neonatal hemolytic disease. Type III is caused by reactions by antigen-antibody binding or by antigens of small molecular weight. Causes acute glomerulonephritis, irritable pneumonia. Finally, type IV (delayed) is a type of cellular immune response induced by T lymphocytes and macrophages that causes important reactions such as tuberculin response, tuberculosis, and microorganisms such as viruses. .

An immune response occurs to enter the body and effectively remove the pathogenic antigen, which is an example of a useful allergic reaction. Immunotherapy against pathogenic antigens is largely active immunotherapy that injects a substance such as a vaccine into the body to induce an antigen-specific immune response, and a manual method of modulating the immune response by administering a substance such as a monoclonal antibody to the antigen. It may be divided into passive immunotherapy. Vaccines, which are active immunotherapy, induce immunity to specific diseases by administering antigenic substances, together with an adjuvant for inducing not only information about the antigen but also a desired immune response.

There are a total of 101 known E. coli sRNAs (small RNA), and their mechanisms are currently being studied. To date, a large number of sRNAs, although differing in sequence, form specific secondary structures that Hfq proteins recognize and bind to. The function of Hfq is to increase the half-life of sRNA so that it functions effectively in a small amount, to help it bind quickly with the target mRNA, and to function quickly, and to bind the RNase to promote the degradation of the target mRNA to further enhance gene expression. Effectively reducing it. Random mutagenesis and screening for producing sRNAs of E. coli have been used to produce effective sRNAs (V. Sharma et al., Engineering artificial small RNAs for conditional gene silencing in Escherichia coli.ACS Synthetic Biology, 2012; 1 (1) ): 6-13). In addition, the basic function of E. coli sRNA is continuously studied, but at this stage the exact function is little known.

In the 1960s, electron microscopy revealed that Gram-negative bacteria secreted extracellular vesicles (EVs or outer membrane vesicles (OMV)). Extracellular vesicles are spherical, phospholipid bilayer, 20-200 nm in size. Gram-negative bacterial-derived extracellular vesicles contain LPS as well as several outer membrane proteins. There have been reports of meningococcal-derived endoplasmic reticulum in the blood of patients who died of severe sepsis, and it has been reported that extracellular vesicles derived from meningococcal bacteria secrete inflammatory mediators in vitro.

In recent years, attempts have been made to use extracellular vesicles secreted by bacteria as vaccine vehicles. In addition, it has recently been reported that Gram-negative bacteria-derived extracellular vesicles cause sepsis. The Gram-negative bacterial extracellular membrane contains endotoxin (lipopolysaccharide; LPS), which causes bacteria-derived vesicles to be absorbed into the blood and acts on vascular endothelial cells to induce systemic inflammatory reactions to cause sepsis. From these facts, in order to use Gram-negative bacterial-derived extracellular vesicles as a vaccine delivery agent, there remains a problem of overcoming the toxic effects of endotoxins present in the vesicles. There is a problem of obtaining extracellular vesicles in large quantities.

Thus, the inventors of the present invention, while studying to develop a substance that modulates the immune response, when the recombinant E. coli-derived sRNA that regulates various cellular functions introduced into the strain, the sRNA overexpressing strain increases the production of extracellular vesicles, sRNA Extracellular vesicles derived from overexpressing strains were found to increase the induction of immune responses and induce Th1 and Th17 immune responses similar to live bacterial infections. In addition, it was confirmed that toxicity of Gram-negative bacteria-derived extracellular vesicles could be eliminated by co-administering a substance that inhibited endotoxin activity in bacterial-derived extracellular vesicles. Through this, the present invention was completed by revealing that the vector expressing the E. coli-derived sRNA can be usefully used as an active ingredient of the immunomodulatory composition.

An object of the present invention is to provide an immunomodulatory pharmaceutical composition, health functional food, and cosmetic composition containing a vector expressing E. coli-derived small RNA (sRNA) as an active ingredient.

The technical problem to be achieved by the present invention is not limited to the above-mentioned problem, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the above object, the present invention provides a pharmaceutical composition for immunomodulation containing a vector expressing E. coli-derived small RNA (sRNA) as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for immunomodulation comprising a strain transformed with a vector expressing E. coli-derived sRNA, a culture medium thereof, or an extracellular vesicle isolated from the strain or culture medium thereof as an active ingredient. do.

The present invention also provides a dietary supplement for immunomodulation containing a vector expressing E. coli-derived sRNA as an active ingredient.

In another aspect, the present invention provides a health functional food for immunomodulation comprising a strain transformed with a vector expressing E. coli-derived sRNA, a culture thereof, or an extracellular vesicle isolated from the strain or culture medium thereof as an active ingredient.

The present invention also provides a cosmetic composition for immunomodulation containing a vector expressing E. coli-derived sRNA as an active ingredient.

In another aspect, the present invention provides a cosmetic composition for immunomodulation comprising a strain transformed with a vector expressing E. coli-derived sRNA, a culture medium thereof, or an extracellular vesicle isolated from the strain or culture medium thereof as an active ingredient.

In addition, the present invention provides an immunomodulatory method comprising administering to a subject a pharmaceutical composition containing a vector expressing E. coli-derived small RNA (sRNA) as an active ingredient.

The present invention also provides an immunomodulatory use of a vector expressing E. coli-derived small RNA (sRNA).

In addition, the present invention is administered to a subject a pharmaceutical composition containing a strain transformed with a vector expressing E. coli-derived sRNA, a culture thereof, or an extracellular vesicle isolated from the strain or culture thereof as an active ingredient. It provides an immunomodulation method comprising the step of.

The present invention also provides an immunomodulatory use of a strain transformed with a vector expressing E. coli-derived sRNA, a culture thereof, or an extracellular vesicle isolated from the strain or the culture thereof.

Vector expressing E. coli-derived sRNA (small RNA) overexpress sRNA, sRNA overexpressing strain increases the extracellular vesicle production, sRNA overexpressing strain-derived extracellular vesicles Th1, similar to live infection By confirming the induction of the Th17 immune response, it was confirmed that the vector expressing the E. coli-derived sRNA can be usefully used as an immunomodulatory composition.

1 is a diagram showing the expression of sRNA (small RNA) through the Northern blot (northern blot) analysis.

2 is a diagram confirming the immune response by sRNA overexpressing strain culture through the amount of IL-6 inducing Th17 immune response:

NT: Badge that did not process anything.

Figure 3 is the result of measuring the amount of extracellular vesicle (extracellular vesicle) in the sRNA overexpressing strain:

EV: extracellular vesicles.

Figure 4 is a diagram observing the morphological characteristics of sRNA overexpressing strain-derived extracellular vesicles by dynamic light scattering method and electron microscopy.

Figure 5 is a measure of the immune response caused by extracellular vesicles derived from sRNA overexpression strain by the secretion amount of TNF-alpha and IL-6 in inflammatory cells (macrophage lines):

Media: Badge that did not process anything;

LPS: control group;

EV: extracellular vesicles;

sup: culture containing extracellular vesicles; And

sup-EV: culture medium in which extracellular vesicles were removed.

6 is a diagram confirming the immune response and apoptosis caused by sRNA overexpressing strain-derived extracellular vesicles through colon epithelial cell line:

Media: Badge that did not process anything;

LPS: control group;

EV: extracellular vesicles;

sup: culture containing extracellular vesicles; And

sup-EV: culture medium in which extracellular vesicles were removed.

Figure 7 is a diagram confirming the immune response in the body by the extracellular vesicles derived from the sRNA overexpressing strain through a test animal.

8 is a diagram illustrating the characteristics of E. coli strain-derived extracellular vesicles after heat treatment or polymyxin B (PMB), followed by electron microscopy and dynamic light scattering.

9 is a diagram illustrating the measurement of vesicle-specific antibodies in serum by treatment of E. coli-derived extracellular vesicles with heat treatment or polymyxin B (PMB), followed by administration of extracellular vesicles (5 ug) to the mouse abdominal cavity.

FIG. 10 is a diagram illustrating the survival rate of mice by injecting E. coli intraperitoneally three times after injecting E. coli-derived extracellular vesicles (5 ug) treated with heat treatment or polymyxin B (PMB).

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for immunomodulation containing a vector expressing E. coli-derived small RNA (sRNA) as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for immunomodulation comprising a strain transformed with a vector expressing E. coli-derived sRNA, a culture medium thereof, or an extracellular vesicle isolated from the strain or culture medium thereof as an active ingredient. do.

In addition, the present invention provides a dietary supplement for immunomodulation containing a vector expressing E. coli-derived small RNA (sRNA) as an active ingredient.

In another aspect, the present invention provides a health functional food for immunomodulation comprising a strain transformed with a vector expressing E. coli-derived sRNA, a culture medium thereof, or an extracellular vesicle isolated from the strain or culture medium thereof as an active ingredient. do.

In addition, the present invention provides a cosmetic composition for immunomodulation containing a vector expressing E. coli-derived small RNA (sRNA) as an active ingredient.

In another aspect, the present invention provides a cosmetic composition for immunomodulation comprising a strain transformed with a vector expressing E. coli-derived sRNA, a culture medium thereof, or an extracellular vesicle isolated from the strain or culture medium thereof as an active ingredient. .

E. coli-derived sRNA is SgrS, RybD, RybB, FnrS, MicC, MicF, GlmY, RprA, CyaR, MicA, OmrA, MgrR, RyhB, GadY, GlmZ, OxyS, DicF, DsrA, Spot42, RseX, IS118, ArcZ, Omr , RyeB, ChiX and GcvB are preferred, DsrA, MicF, GlmY, RprA, CyaR, MicA, OmrA, MgrR, GadY, GlmZ, OxyS, DicF, RseX, ArcZ, OmrB and RyeB, more preferably RseX and Most preferred is MicA.

The SgrS the nucleotide sequence (ATAAATGTGAGCGGATAACATTGACATTGTGAGCGGATAACAAGATACTGAGCACGATGAAGCAAGGGGGTGCCCCATGCGTCAGTTTTATCAGCACTATTTTACCGCGACAGCGAAGTTGTGCTGGTTGCGTTGGTTAAGCGTCCCACAACGATTAACCATGCTTGAAGGACTGATGCAGTGGGATGACCGCAATTCTGAAAGTTGACTTGCCTGCATCATGTGTGACTGAGTATTGGTGTAAAATCACCCGCCAGCAGATTATACCTGCTGGTTTTTTTTATTCTCGCCGCGCTAAA) described in SEQ ID NO: 1, RybB the nucleotide sequence (GCCACTGCTTTTCTTTGATGTCCCCATTTTGTGGAGCCCATCAACCCCGCCATTTCGGTTCAAGGTTGATGGGTTTTTTGTTATCTAAAACTTATCTACTACC), RybD described in SEQ ID NO: 2 is the nucleotide sequence (ATATCTGTAATAAGAAATAGCCCTCGCCGCTTCCCTCTACAGGAATGGCGAAGGGCTGTCGGTTTCGACATGGTTGGCCATCGTATGATGGCCTTTTTTGTGCTTATCGCGATGATTT) described in SEQ ID NO: 3, FnrS is described in SEQ ID NO: 4 Nucleotide sequence (GCAGGTGAATGCAACGTCAAGCGATGGGCGTTGCGCTCCATATTGTCTTACTTCCTTTTTTGAATTACTGCATAGCACAATTGATTCGTACGACGCCGACTTTGATGAGTCGGCTTTTTTTTGCCTGTTATTTATCAGCGTCTTCTCCCTCTCTCCTCATCTACTCATTCTCAT) C is a nucleotide sequence (GTTATATGCCTTTATTGTCACAGATTTTATTTTCTGTTGGGCCATTGCATTGCCACTGATTTTCCAACATATAAAAAGACAAGCCCGAACAGTCGTCCGGGCTTTTTTTTTAGAATTGGATAATCCTTATCC) described in SEQ ID NO: 5, MicF the nucleotide sequence (GCTATCATCATTAACTTTATTTATTACCGTCATTCATTTCTGAATGTCTGTTTACCCCTATTTCAACCGGATGCCTCGCATTCGGTTTTTTTTACCCTTCTTTACACACT), GlmY described in SEQ ID NO: 6 is the nucleotide sequence (AGTGGCTCATTCACCGACTTATGTCAGCCCCTTCGGGACGTGCTACATAAAATACGAATGACGCACAACAAGGTGCCTGCCGTCCAACTTCTGATATCAGCGTAGCTATATCAACCATCGGGCGAAACGTCGAGTTAGGCACCGCCTTATTCCATAACAAAGCCGGGTAATTCCCGGCTTTGTTGTATCTGAACTTCCCCTCGGTTAGCATCAGGCTATTCGCGTCTGACGAGAGTAACAC), RprA described in SEQ ID NO: 7 is represented by SEQ. ID. NO: 8 Nucleotide sequence (ACGGTTATAAATCAACATATTGATTTATAAGCATGGAAATCCCCTGAGTGAAACAACGAATTGCTGTGTGTAGTCTTTGCCCATCTCCCACGATGGGCTTTTTTTTAACATTTTTCCGCTTCGCTACCTCGCCCCTCTTTTTCCTACCTCCCCCCCCCCCCCCCCCCCCCCCCTCTCCCCCCCCCTCTCTCTCCCCCCCTC TACAATGAAATAAAAGG), MicA the nucleotide sequence (GAAAGACGCGCATTTGTTATCATCATCCCTGAATTCAGAGATGAAATTTTGGCCACTCACGAGTGGCCTTTTTCTTTTCTGTCAGGCGTG) described in SEQ ID NO: 10, OmrA the nucleotide sequence (CCCAGAGGTATTGATTGGTGAGATTATTCGGTACGCTCTTCGTACCCTGTCTCTTGCACCAACCTGCGCGGATGCGCAGGTTTTTTTTCGCACCTAATTTACTGTCGC), MgrR the nucleotide sequence (GATTCGTTATCAGTGCAGGAAAATGCCTGTTAGCGTAAAAGCAAAACACAAATCTATCCATGCAAGCATTCACCGCCGGTTTACTGGCGGTTTTTTTTCGCCGTCATAAAAATCAGGCCCCTTGTACACAACTGTAACAA), RyhB described in SEQ ID NO: 12 which is described in SEQ ID NO: 11, SEQ ID NO: 13 the base to be described in SEQ ID NO: (GCGATCAGGAAGACCCTCGCGGAGAACCTGAAAGCACGACATTGCTCACATTGCTTCCAGTATTACTTAGCCAGCCGGGTGCTGGCTTTTTTTTT), GadY the base sequence described in SEQ ID NO: 14 (ACTGAGAGCACAAAGTTTCCCGTGCCAACAGGGAGTGTTATAACGGTTTATTAGTCTGGAGACGGCAGACTATCCTCTTCCCGGTCCCCTATGCCGGGTTTTTTT), GlmZ the nucleotide sequence (GTAGATGCTCATTCCATCTCTTATGTTCGCCTTAGTGCCTCATAAACTCCGGAATGACGCAGAGCCGTTTACGGTGCTTATCGTCCACTGACAGATG represented by SEQ. ID. NO: 15 TCGCTTATGCCTCATCAGACACCATGGACACAACGTTGAGTGAAGCACCCACTTGTTGTCATACAGACCTGTTTTAACGCCTGCTCCGTAATAAGAGCAGGCGTTTTTTTATGTATCAGGAAGGCCCCGGAGG), OxyS the nucleotide sequence (GAAACGGAGCGGCACCTCTTTTAACCCTTGAAGTCACTGCCCGTTTCGAGAGTTTCTCAACTCGAATAACTAAAGCCAACGTGAACTTTTGCGGATCTCCAGGATCCGCTTTTTTTTGCCATAAAAAAGCCCGGCG) described in SEQ ID NO: 16, DicF the nucleotide sequence (TTTCTGGTGACGTTTGGCGGTATCAGTTTTACTCCGTGACTGCTCTGCCGCCCTTTTTAAAGTGAATTTTGTGATGTGGTGAATGCGGCTGAGCGC), DsrA the nucleotide sequence (AACACATCAGATTTCCTGGTGTAACGAATTTTTTAAGTGCTTCTTGCTTAAGCAAGTTTCATCCCGACCCCCTCAGGGTCGGGATTTTTTT), Spot42 described in SEQ ID NO: 18 which is described in SEQ ID NO: 17, SEQ ID NO: 19 nucleotide represented by SEQ. ID. (GTAGGGTACAGAGGTAAGATGTTCTATCTTTCAGACCTTTTACTTCACGTAATCGGATTTGGCTGAATATTTTAGCCGCCCCAGTCAGTAATGACTGGGGCGTTTTTTATTGGGCGAAAGAAAAGATCCGTAATGCCTGATGCGC), RseX the nucleotide sequence (TTTTTATTATTCTGTGTCATGATGCTTCCGTTATTAGCCTTTTATCGTCTTGTTTATATTTTTTGGGCCGGCATGATGCCGGCTTTTTTTTATGCCTTCATTAATG described in SEQ ID NO: 20 TGCGCCTGATCACACCAGCCGTTTGGCGCAACAATCATTGATACCCCCTATGT), IS118 is the nucleotide sequence (GGTTCTGGAGGGGGTTTGTTGTGGGCAATGATGCATTTAAGTTATCGTCTGCAGATAGAGGAGATATTACAATAAACAACGAATCAGGGCATTTGATAGTCAATACCGCAATTCTATCAGGAGATATAGTCACTCTAAGAGGAGGAGAAATTAGGTTGGTATTATAGCTTGTGCGCGCCATGATTGGCGCGCAATTTAAACTTAGTGCTTTACATCGCTATTGTCTTGATTTCTTTGAATTATTTTATAAATTAA), ArcZ the nucleotide sequence (GTGCGGCCTGAAAAACAGTGCTGTGCCCTTGTAACTCATCATAATAATTTACGGCGCAGCCAAGATTTCC CTGGTGTTGGCGCAGTATTCGCGCACCCCGGTCTAGCCGGGGTCATTTTTTAGTGGCTTTTGCCACCCACG CTTTCAGCACTTCTAC), OmrB the nucleotide sequence (CCCAGAGGTATTGATAGGTGAAGTCAACTTCGGGTTGAGCACATGAATTACACCAGCCTGCGCAGATGCGCAGGTTTTTTTTGCCGGTCATCAATCTGTAAC), RyeB described in SEQ ID NO: 23 which is described in SEQ ID NO: 22 which is described in SEQ ID NO: 21 SEQ ID NO: SEQ ID NO: 24 (GGCAAGGCAACTAAGCCTGCATTAATGCCAACTTTTAGCGCACGGCTCTCTCCCAAGAGCCATTTCCCTGGACCGAATACAGGAATCGTGTTCGGTCTCTTTTTATCTGTTAAAAGCCAGAAGCATTTCCTTCGC), ChiX is the base sequence of SEQ ID NO: 25 (GTGC CTGAAAAACAGTGCTGTGCCCTTGTAACTCATCATAATAATTTACGGCGCAGCCAAGATTTCCCTGGTGTTGGCGCAGTATTCGCGCACCCCGGTCTAGCCGGGGTCATTTTTTAGTGGCTTTTGCCACCCACGCTTTCAGCACTTCTAC) and GcvB is preferably a nucleotide sequence (ACTTCCTGAGCCGGAACGAAAAGTTTTATCGGAATGCGTGTTCTGGTGAACTTTTGGCTTACGGTTGTGATGTTGTGTTGTTGTGTTTGCAATTGGTCTGCGATTCAGACCATGGTAGCAAAGCTACCTTTTTTCACTTCCTGTACATTTACCCTGTCTGTCCATAGTGATTAATGTAGCACCGCCTAATTGCGGTGCTTTTTTTTACCTTGCGATCGCGAATTAC) described in SEQ ID NO: 26.

The vector is preferably a vector that overexpresses sRNA.

The vector is preferably any one selected from the group consisting of plasmid DNA, linear DNA and recombinant viral vectors.

The extracellular vesicles can be separated using methods such as centrifugation, ultracentrifugation, filtration by filter, gel filtration chromatography, pre-flow electrophoresis, capillary electrophoresis, and combinations thereof. In addition, it may further include a process for washing to remove impurities, concentration of the obtained extracellular vesicles and the like.

The extracellular vesicles separated by the method is preferably 15 nm to 100 nm in size.

This strain is Salmonella (Salmonella), E. coli (Escherichia coli), keurep when Ella bacteria (Klebsiella pneumoniae) and Pseudomonas species that the (Pseudomonas) strain are preferred, and most preferably Salmonella and E. coli strains.

In a specific embodiment of the present invention, the present inventors prepared a vector expressing E. coli-derived sRNA, and confirmed that it is overexpressed by introducing it into a Salmonella strain (see FIG. 1), as a result of confirming the induction of the immune response of the sRNA overexpressing strain, It was confirmed that the sRNA overexpressing strain induces an immune response more than the control (see FIG. 2).

In addition, the present inventors confirmed the extracellular vesicle generation amount of the sRNA overexpressing strain, the sRNA overexpressing strain was confirmed to produce more extracellular vesicles than the control group (see Figure 3), the physical properties of the resulting extracellular vesicles As a result, it was confirmed that the size and shape are similar to the extracellular vesicles generated in the control group (see Fig. 4).

In addition, the present inventors confirmed the immune response of the sRNA overexpressing strain-derived extracellular vesicles, and confirmed that the immune response is efficiently induced by the sRNA overexpressing strain-derived extracellular vesicles (see FIG. 5). The control group and the sRNA overexpressing strain was confirmed that there is no significant difference (see Figure 6).

In addition, the present inventors confirmed the ability of the sRNA overexpressing strain-derived extracellular vesicles to induce an immune response, inducing Th1 and Th17 immune responses similar to probiotic infection (see FIG. 7).

In addition, the present inventors heat-treated on the E. coli-derived extracellular vesicles, or treated with polymyxin B, confirmed that the shape and size of the vesicles similar to the original vesicles (see Fig. 8), as a result of administering them to mice, It was confirmed that the vesicle-specific IgG antibody was well prepared by extracellular vesicles pretreated with heat treatment or polymyxin B (see FIG. 9).

In addition, the present inventors heat treated the E. coli-derived extracellular vesicles, or treated with polymyxin B to induce an immune response, and then observed whether to suppress the mortality caused by E. coli infection, when treated with polymyxin B , Similar to the case without pretreatment, it was confirmed that the vaccine efficacy (see Figure 10).

 Therefore, the strain expressing E. coli-derived sRNA (small RNA) and the vector expressing E. coli-derived sRNA transformed strain, its culture or extracellular vesicles isolated from the strain or its culture Overexpressing sRNA, sRNA overexpressing strains increase immune response and extracellular vesicle production, and sRNA overexpressing strain-derived extracellular vesicles express the E. coli-derived sRNA by confirming that they induce a Th1, Th17 immune response similar to live infection. The vector was confirmed that it can be usefully used as an immunomodulatory pharmaceutical composition, health functional food, and cosmetic composition.

In addition, when treated with Gram-negative bacteria-derived extracellular vesicles, polymyxin B, which inhibits LPS activity, generated vesicular-specific antibodies and induced vaccine efficacy similarly to untreated vesicles. It was confirmed that the extracellular vesicles derived from Gram-negative bacteria containing the vector expressing can be useful as an immunomodulatory pharmaceutical composition, health functional food, and cosmetic composition.

The pharmaceutical composition of the present invention may be administered orally or parenterally and may be administered in various formulations. When formulated, a diluent or excipient such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactants that are commonly used may be used. Are prepared using.

The pharmaceutical compositions of the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, oral formulations, external preparations, suppositories, and sterile injectable solutions, respectively, according to a conventional method. Can be used. Carriers, excipients and diluents that may be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, Methylcellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used.

Solid preparations for oral administration include tablets, pills, powders, granules and capsules, and the like, which may be used in the pharmaceutical composition of the present invention at least one excipient such as starch, calcium carbonate, sucrose, lactose And gelatin etc. are mixed and prepared. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions and syrups, and may include various excipients such as wetting agents, sweeteners, fragrances and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. have.

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerol and gelatin may be used.

The preferred dosage of the pharmaceutical composition of the present invention depends on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. The administration may be administered once a day, or may be divided several times. The dosage does not limit the scope of the invention in any aspect.

On the other hand, in order to use the composition of the present invention as a dietary supplement for immunomodulation, it can be prepared by a variety of methods known in the food science or pharmaceutical field and mixed with itself or a food-acceptable carrier, excipient, diluent, etc. It can be prepared in any food form that can be taken orally. Preferably in the form of beverages, pills, granules, tablets or capsules.

The health functional food of the present invention may further include ingredients that are commonly added during food production and are food acceptable. For example, when prepared in a beverage, one or more components from citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, etc. may be further included in addition to the composition of the present invention.

The amount that can be included as an active ingredient of the health functional food according to the present invention may be appropriately selected according to the age, sex, weight, condition, symptoms of the disease of a person who wants an immune enhancing health functional food, preferably adult standard 1 It is good to include about 0.01 g to 10.0 g per day, and by ingesting a health functional food having such a content, an immune enhancing effect can be obtained.

In addition, the cosmetic composition of the present invention may be prepared in any formulation commonly prepared in the art, for example, solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactants- Containing cleansing, oils, powder foundations, emulsion foundations, wax foundations, sprays and the like, but is not limited thereto. More specifically, it may be prepared in the form of a flexible lotion, nutrition lotion, nutrition cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray or powder.

As the cosmetically effective carrier contained in the cosmetic composition of the present invention, a carrier commonly used in the art may be used depending on the dosage form. When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, trakant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide, etc. may be used as carrier components. Can be.

 When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used, in particular, in the case of spray, additionally chloro fluorohydrocarbon, propane Propellant such as butane or dimethyl ether.

 When the formulation of the present invention is a solution or emulsion, a solvent, solubilizer or emulsifier is used as the carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 Fatty acid esters of, 3-butylglycol oil, glycerol aliphatic ester, polyethyleneglycol or sorbitan.

 When the formulation of the present invention is a suspension, liquid carrier diluents such as water, ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystals Soluble cellulose, aluminum metahydroxy, bentonite, agar or tracant and the like can be used.

 When the formulation of the present invention is a surfactant-containing cleansing, the carrier component is an aliphatic alcohol sulfate, an aliphatic alcohol ether sulfate, a sulfosuccinic acid monoester, an isethionate, an imidazolinium derivative, a methyltaurate, a sarcosinate, a fatty acid amide. Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters and the like can be used.

 Ingredients included in the cosmetic composition of the present invention, in addition to the active ingredient and the carrier ingredient, include ingredients commonly used in cosmetic compositions, for example, conventional adjuvants such as antioxidants, stabilizers, solubilizers, vitamins, pigments and flavorings. It may include

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited by the following examples.

Example 1 Construction of Hfq-binding small RNA (Hfq-dependent sRNA) Library

About 30% of E. coli sRNA binds to Hfq, a global regulatory protein. One of the drawbacks of sRNA is that it does not show phenotypes without overexpression. With this in mind, we attempted to build a library made by inserting plasmids containing sRNAs with Hfq-related into pLac. The method was fabricated and used in the same way based on the backbone vector built by Susan Gottesman.

<1-1> Preparation of pLac

pLac was designed to insert P _L lacO-1 (a slightly modified form of the lac transcriptional promoter) into pBR322 to obtain RNA from +1, which is regulated by pLac and the transcriptional start of this promoter. P _L lacO-1 is Lutz and Bujard (R. Lutz, H. Bujard, Independent and tight regulation of transcriptional units in Escherichia coli via the LacR / O, the TetR / O and AraC / I1-I2 regulatory elements. Nucleic Acids Research , 1997; 25 (6): 1203-1210) was used as the artificial promoter sequence (ATAAATGTGAGCGGATAACATTGACATTGTGAGCGGATAACAAGATACTGACGT (SEQ ID NO: 27)). In particular, 35 regions and 10 regions (underlined) of pLac were transformed from TTTACA to TTGACA and TATGAT to GATACT, respectively, to promote more efficient expression.

Specifically, two oligos were designed to be cleaved by Ssp I and Aat II by slightly modifying the sequence of P _L lacO-1 at the positions of Ssp I and Aat II of pBR322 (Oligo 1: ATT ATA AAT GTG AGC). GGA TAA CAT TGA CAT TGT GAG CGG ATA ACA AGA TAC TGA CGT (SEQ ID NO: 28), Oligo 2: TAA TAT TTA CAC TCG CCT ATT GTA ACT GTA ACA CTC GCC TAT TGT TCT ATG AC (SEQ ID NO: 29) and ligated to pBR322 ( Ssp I / Aat II) to obtain pLac.

<1-2> Construction of Hfq-binding sRNA Library

Hfq-binding sRNA library was constructed using pLac obtained in Example <1-1>.

Specifically, the obtained pLac was cleaved into Aat II and Eco RI, Aat II and Hin dIII (for MicA) or Bam HI and Hin dIII (for SgrS), and the primer containing sRNA in pLac (Table 1; Restriction enzyme cleavage sites were prepared by underlining), and MG1655 chromosomal DNA was used as a template for PCR amplification using Pfu polymerase (PCR reaction condition: 5 minutes at 94 ° C, 30 sec at 94 ° C., 30 sec at 59 ° C., 30 sec at 72 ° C., 30 min extension at 72 ° C., 5 min extension at 72 ° C.), cleavage using the above-mentioned restriction enzymes, and 10 × ligation buffer in 5 ul of PCR product. The total 10 ul volume is added to room temperature by adding 1 ul of ligation buffer, 1 ul of pLac cleavage product (20 ng / ul), 2 ul of sterile distilled water, and 1 ul of Ligase (Roche, 1 unit / ul). Ligation at 1 hour. 10 ul of the ligation mixture (mixture) was mixed well with XL1-blue 100 ul for 10 minutes on ice (ice) and then gave a heat-shock (heat-shock) for 1 minute 30 seconds at 42 ℃ and placed on ice again for 5 minutes. 900 ul of LB media was added to re-generation at 37 ° C. for 1 hour, and then centrifuged at 12,000 rpm for 30 seconds to spread the whole cells onto an LB plate. The next day colonies were identified and then Mini-prep was performed.

SRNA primer designation order SEQ ID NO: Omra OmrA_F (AatII) GCGT GACGTC CCCAGAGGTATTGATTGGTGAGATTATTCGGTACG SEQ ID NO: 30 OmrA_R (EcoRI) CCGA GAATTC GCGACAGTAAATTAGGTGCGAAAAAAAACC SEQ ID NO: 31 Ommrb OmrB_F (AatII) GCGT GACGTC CCCAGAGGTATTGATAGGTGAAGTCAACTTCGGG SEQ ID NO: 32 OmrB_R (EcoRI) CCGA GAATTC GTTACAGATTGATGACCGGCAAAAAAAACC SEQ ID NO: 33 RprA RprA_F (AatII) GCGT GACGTC ACGGTTATAAATCAACATATTGATTA SEQ ID NO: 34 RprA_R (EcoRI) GCGA GAATTC GAAAGAGTGAGGGGCGAGGTAGC SEQ ID NO: 35 Arcz ArcZ_F (AatII) GCGT GACGTC GTGCGGCCTGAAAAACAGTGC SEQ ID NO: 36 ArcZ_R (EcoRI) GCGA GAATTC GTAGAAGTGCTGAAAGCGTGG SEQ ID NO: 37 Glmz GlmZ_F (AatII) GCGT GACGTC GTAGATGCTCATTCCATCTC SEQ ID NO: 38 GlmZ_R (EcoRI) GCGA GAATTC CCTCCGGGGCCTTCCTGATAC SEQ ID NO: 39 Glmy GlmY_F (AatII) GCGT GACGTC AGTGGCTCATTCACCGACTTATG SEQ ID NO: 40 GlmY_R (EcoRI) GCGA GAATTC GTGTTACTCTCGTCAGACGCG SEQ ID NO: 41 Oxys OxyS_F (AatII) GCGT GACGTC GAAACGGAGCGGCACCTCTTTTAACCC SEQ ID NO: 42 OxyS_R (EcoRI) GCGA GAATTC CGCCGGGCTTTTTTATGGCA SEQ ID NO: 43 DicF DicF_F (AatII) GCGT GACGTC TTTCTGGTGACGTTTGGCGG SEQ ID NO: 44 DicF_R (EcoRI) GCGA GAATTC GCGCTCAGCCGCATTCACCACA SEQ ID NO: 45 Spot42 Spot42_F (AatII) GCGT GACGTC GTAGGGTACAGAGGTAAGATGTTCTATC SEQ ID NO: 46 Spot42_R (EcoRI) GCGA GAATTC GCGCATCAGGCATTACGGATC SEQ ID NO: 47 RseX RseX_F (AatII) GCGT GACGTC tttTTATTATTCTGTGTCATGATGC SEQ ID NO: 48 RseX_R (EcoRI) GCGA GAATTC ACATAGGGGGTATCAATGATTGTTG SEQ ID NO: 49 FnrS FnrS_F (AatII) GCGT GACGTC GCAGGTGATGCAACGTCAAGCGATG SEQ ID NO: 50 FnrS_R (EcoRI) GCGA GAATTC CTGGAACAGGATCGCCAGGAATC SEQ ID NO: 51 DsrA DsrA_F (AatII) GGCCAA GACGTC AACACATCAGATTTCCTGGTGTAACG SEQ ID NO: 52 DsrA_R (EcoRI) GCAGCA GAATTC AAAAAAATCCCGACCCTGAGGG SEQ ID NO: 53 RyhB RyhB_F (AatII) GGCCAA GACGTC GCGATCAGGAAGACCCTCGCGGAG SEQ ID NO: 54 RyhB_R (EcoRI) GCAGCA GAATTC AAAAAAAAAGCCAGCACCCGGCTGGC SEQ ID NO: 55 MicC MicC_F (AatII) CGAT GACGTC GTTATATGCCTTTATTGTCACAG SEQ ID NO: 56 MicC_R (EcoRI) CGTAC GAATTC GGATAAGGATTATCCAATTCTAAA SEQ ID NO: 57 MicF MicF_F (AatII) CGAT GACGTC GCTATCATCATTAACTTTATTTAT SEQ ID NO: 58 MicF_R (EcoRI) CGTAC GAATTC AGTGTGTAAAGAAGGGTAAAAAA SEQ ID NO: 59 GcvB GcvB_F (AatII) CGAT GACGTC ACTTCCTGAGCCGGAAACGAAAAG SEQ ID NO: 60 GcvB_R (EcoRI) CGTAC GAATTC GTAATTCGCGATCGCAAGGTAA SEQ ID NO: 61 ChiX ChiX_F (AatII) TAA GACGTC ACACCGTCGCTTAAAGTGAC SEQ ID NO: 62 ChiX_R (EcoRI) TGG GAATTC AAAAAAATGGCCAATATCGCTATTG SEQ ID NO: 63 GadY GadY_F (AatII) TAA GACGTC ACTGAGAGCACAAAGTTTCCCG SEQ ID NO: 64 GadY_R (EcoRI) TGG GAATTC AAAAAAACCCGGCATAGGGG SEQ ID NO: 65 MicA MicA_F (AatII) CGAT GACGTC GAAAGACGCGCATTTGTTATCATC SEQ ID NO: 66 MicA_R (HindIII) CGTAC AAGCTT CACGCCTGACAGAAAAGAAAAAGGC SEQ ID NO: 67 RybB RybB_F (AatII) CGAT GACGTC GCCACTGCTTTTCTTTGATGTCC SEQ ID NO: 68 RybB_R (EcoRI) CGTAC GAATTC GGTAGTAGATAAGTTTTAGATAAC SEQ ID NO: 69 MgrR MgrR_F (AatII) AATC GACGTC ACAGTAGCATCAGTTTTCTCAATGAATGTTAAAC SEQ ID NO: 70 MgrR_R (EcoRI) GTAACAA GAATTC GGTTAGGTGAGGGATTATCTCCGTT SEQ ID NO: 71 CyaR CyaR_F (AatII) GCGT GACGTC GCTGAAAAACATAACCCATAAAATGCTAG SEQ ID NO: 72 CyaR_R (EcoRI) GA GAATTC CCTTTTATTTCATTGTATTACGCGTAAAAAATAAGC SEQ ID NO: 73 Ryeb RyeB_F (AatII) GCGT GACGTC GGCAAGGCAACTAAGCCTGCATTAATGCCAACT SEQ ID NO: 74 RyeB_R (EcoRI) GA GAATTC GCGAAGGAAATGCTTCAGGCTTTTAACA SEQ ID NO: 75 IS118 IS118_F (AatII) GCGT GACGTC GGTTCTGGAGGGGGTTTGTTGT SEQ ID NO: 76 IS118_R (EcoRI) GA GAATTC TTAATTTATAAAATAATTCAAAGAAATC SEQ ID NO: 77 FnrS FnrS_F (AatII) GCGT GACGTC GCAGGTGAATGCAAGCAACGT SEQ ID NO: 78 FnrS_R (EcoRI) GA GAATTC CTGGAACAGGATCGCCAGGAATCGGT SEQ ID NO: 79 SgrS SgrS_F (BamHI) CG GGATCC ATAAATGTGAGCGGATAACATTGA SEQ ID NO: 80 SgrS_R (HindIII) CGTAC AAGCTTT TTAGCGGCGAGAATAAAAAAAACCA SEQ ID NO: 81

The mini-preps were grown in 3 ml LB medium and transferred to 1.5 ml microtubes and centrifuged at 12,000 rpm for 30 seconds to bring down the cells. The down cells were obtained by a manufacturer's method using a Mini-prep kit (Thermo Scientific). Thus obtained DNA was confirmed whether the desired sRNA entered through sequencing, 26 pBRplac-sRNA was obtained. This is listed in Table 2 along with the size of the sRNA.

sRNA type Size (nt) sRNA type Size (nt) SgrS To 220 RyhB 90 ChiX 88 GadY 105, 90, 59 RybB 80 Glmz 210 FnrS 122 Oxys 109 MicC 109 DicF 53 MicF 93 DsrA 85 Glmy 150, 180 Spot42 109 RprA 105 RseX 91 CyaR 86 IS118 194 MicA To 70 Arcz ~ 55, 88, 120 Omra 88 Ommrb 82 MgrR 98 Ryeb 104, 74 RybD 130 GcvB 205

Example 2 Confirmation of Overexpression of sRNA

<2-1> Salmonella introduction of E. coli sRNA overexpression plasmid

Hfq-binding sRNAs are excellent in species conservation. Since the nucleotide sequence alone could be a method of expressing the same morphology in Salmonella and exploring its function, the same species as the E. coli sRNA sequence was utilized by utilizing a species-preserved sRNA among Hfq-linked sRNAs. PBRplac-sRNA plasmid containing eggplant sRNA was introduced into Salmonella.

Specifically, pBRplac-sRNA was introduced into Salmonella Typhimurium 14028S species by electroporation.

<2-2> Confirmation of Overexpression

It was confirmed whether each sRNA is properly expressed in the obtained strain.

Specifically, Salmonella strain containing each sRNA was incubated with Ampicillin (50 ug / ml) and IPTG (0.1 mM) in LB liquid medium, and when OD ₆₀₀ = 0.5, hot-phenol ( phenol) was used to obtain total RNA, and some of them were subjected to 8% polyacrylamide gel electrophoresis. Electrophoresis-completed samples were transferred to a semi-dry blotting method using a Hybond-N + membrane, followed by biotinylated oligos at 5 'that specifically bind to each sRNA. Northern blot analysis was performed using Table 3). Detection of overexpressed RNA was performed according to the manufacturer's method using the Bright Star system (Ambion), oligos were synthesized from Bioneer and then used.

sRNA Oligo (5 '→ 3') SEQ ID NO: MicF TCAACCGGATGCCTCGCATTCGGTTTTTTTT SEQ ID NO: 82 RydC CTAAAACCGACCCGTGGTACAGGCGAAGAATACGGGTCT SEQ ID NO: 83 Arcz CGCCGTAAATTATTATGATGAGTTACAAGGGCACAGCAC SEQ ID NO: 84 Glmz GTGGACGATAAGCACCGTAAACGGCTCTGCGTCATTCCGG SEQ ID NO: 85 Glmy CATTCGTATTTTATGTAGCACGTCCCGAAGGGGCTG SEQ ID NO: 86 RseX GATAAAAGGCTAATAACGGAAGCATCATGACACAG SEQ ID NO: 87 Omra GAGACAGGGTACGAAGAGCGTACCGAATAATCTCACC SEQ ID NO: 88 GadY GGGGACCGGGAGAGGATAGTCTGCCGTCTCCAGAC SEQ ID NO: 89 DsrA TGAGGGGGTCGGGATGAAACTTGCTTAAGCAAGAAGCACT SEQ ID NO: 90 MicA CATCTCTGAATTCAGGGATGATGATAACAAATGCGCGTCTTT SEQ ID NO: 91 Ryeb CGGTCCAGGGAAATGGCTCTTGGGAGAGAGCCGTGCGC SEQ ID NO: 92 MgrR CAGTAAACCGGCGGTGAATGCTTGCATGGATAGAT SEQ ID NO: 93 Oxys AAACTCTCGAAACGGGCAGTGACTTCAAGGGTTAAA SEQ ID NO: 94 RprA CAAAATGGGGACATCAAAGAAAAGCAGTGGC SEQ ID NO: 95 CyaR TGGTTCCTGGTACAGCTAGCATTTTATGGGTTATG SEQ ID NO: 96 Ommrb CATGTGCTCAACCCGAAGTTGACTTCACCTATCAATACC SEQ ID NO: 97 DicF GGCAGAGCAGTCACGGAGTAAAACTGATACCGCC SEQ ID NO: 98

As a result, as shown in Figure 1, it was confirmed that all kinds of sRNA is overexpressed (Figure 1).

Example 3 Confirmation of Immune Response of sRNA Overexpressing Salmonella Strains

<3-1> Confirmation of Increased Immune Response by sRNA Overexpressing Strain Culture

Salmonella cell cultures overexpressing sRNA were obtained, and the same amount was treated in mouse peritoneal macrophage line (RAW 264.7), and the amount of IL-6 as an indicator of immune response was confirmed.

Specifically, sRNA-containing Salmonella strains were cultured with Ampicillin (50 ug / ml) and IPTG (0.1 mM) in TSB (Tryptic soy broth) medium, and grown to OD ₆₀₀ = 1.5, followed by 2 ul of each culture medium. Cell lines (RAW 264.7) were treated with 1 × 10 ⁵ cell numbers and cultured for 12 hours. Then, IL-6, an inflammatory cytokine, was measured by an Enzyme-linked immunosorbent assay (ELISA) technique. The ELISA adds 100 ul of primary antibody (BD Bioscience, USA) that binds to the cytokine to be searched in a 96-well microplate for 12 hours at room temperature. After washing three times with wash buffer (PBS / Tween-20 0.05%), the assay solution (Reagent diluent; BD Bioscience, USA) was dispensed at 300 ul / well and then blocked for 1 hour at room temperature. It was. Then, after washing again using the washing buffer, the sample and the quantitative material to be analyzed are put together (BD Bioscience, USA) and reacted for 2 hours. Again washed three times with wash buffer and 100 ul of avidin-HRP bound detection antibody was added to each well. After 1 hour of reaction at room temperature, the solution was washed three times with washing buffer, and then a set of Substrate Solution (TMB Substrate Reagent; Pharmingen, BD Bioscience, USA) was added to each well of 100 ul. After reacting for 30 minutes in a dark place at room temperature, 50 ul of 2NH ₂ SO ₄ was added, and then measured at a wavelength of 450 nm / 570 nm with a Microplate reader (Molecular Devices, Sunnyvale, USA) within 30 minutes. It was.

As a result, as shown in Figure 2, it was confirmed that RyeB, ArcZ, MicA, MgrR, OmrA, MicF, GadY, RseX and OmrB overexpressing strains induce IL-6 more than the control pBRplac (Fig. 2).

<3-2> Confirmation of Extracellular Vesicle Production of sRNA Overexpressing Strains

In Example <3-1>, the amount of extracellular vesicles was confirmed using RseX and MicA strains that repeatedly increased the amount of IL-6 among sRNA overexpressing strains affecting the immune response.

Specifically, the amount of extracellular vesicles was confirmed using BCA protein assay (Pierce, USA), and directly through nanosite tracking analysis (LM-10HS) to determine whether the number of extracellular vesicles increased. The extracellular vesicle sample is adjusted to 500 ng / ml and 2 mL is prepared. 0.3-0.4 ml of the sample is placed in the chamber of the LM-10HS instrument and the camera is focused to ensure that the particles are clearly visible. When the camera is in focus, the camera is raised to the maximum level, and the sample is captured by decreasing the level by one step to confirm that there is no drift of the sample. After confirming that the sample was free of concentration, focus and drift, the capture interval was set to 30 seconds and data was confirmed.

As a result, as shown in Figure 3, when the overexpression of RseX and MicA it was confirmed that the extracellular vesicle production more than six times higher than the control pBRplac, the number of extracellular vesicles increased 1.5 ~ 4 times per 1 ug It was confirmed (FIG. 3).

<3-3> Physical Characterization of Extracellular Vesicles Derived from sRNA Overexpressing Strains

The physical properties of the extracellular vesicles produced in Example <3-2> were confirmed using dynamic light scattering and transmission electron microscopy.

As a result, as shown in Table 4 and FIG. 4, it was confirmed that the extracellular vesicles derived from the sRNA overexpressing strain were about 20 nm in size and similar in shape to those of the control pBRplac (FIG. 4).

Strain (sRNA) Average size (nm) Salmonella typhimurium (pBRplac) 26.07 ± 5.98 Salmonella typhimurium (RseX) 19.93 ± 2.503 Salmonella typhimurium (MicA) 21.39 ± 1.92

<3-4> Identification of Immune Responses Using Cell Lines of Extracellular Vesicles Derived from sRNA Overexpressing Strains

In order to confirm that extracellular vesicles play an important role in inducing immune responses, each strain-derived extracellular vesicles (EV), extracellular vesicles (supernatant, sup) and extracellular vesicles removed (sup-EV) 1 Inflammatory cytokines IL-6 and TNF-α 12 hours after treatment with ug / ml of mouse peritoneal macrophage (RAW 264.7) 1 × 10 ⁵ cell number in the same manner as in Example <3-1> It was confirmed by measurement.

As a result, as shown in Figure 5, it was confirmed that the immune response by the extracellular vesicle derived from the sRNA overexpressing strain is the largest (Fig. 5).

In addition, since Salmonella is a bacterium causing enteritis, the mouse colon epithelial cell line (CT-26) was used to confirm the immune response inducing ability and cell death. Immune responses were confirmed in the same manner as above, and cell death experiments were confirmed using MTT assay.

As a result, as shown in Figure 6, it was confirmed that the induction of the immune response by the extracellular vesicles derived from the sRNA overexpressing strain, the apoptosis of the sRNA overexpressing strain was confirmed that there is no significant difference when compared with the control (Fig. 6 ).

<3-5> Confirmation of Immune Response Using Experimental Animals of Extracellular Vesicles Derived from sRNA Overexpressing Strain

Salmonella strains are known to induce enteritis and induce Th1 immune responses. Thus, the ability to induce Th1 immune response of extracellular vesicles obtained through sRNA overexpression was confirmed.

Specifically, 100 ug / hd of extracellular vesicles were administered to the five mice per group (C67BL / 6) at two-day intervals through the intraperitoneal route. After dissecting on day 5, cells were isolated from intraperitoneal lymph nodes and subjected to T cell restimulation for 12 hours using anti-CD3 and CD28, and confirmed in the same manner as in Example <3-1>.

As a result, as shown in Figure 7, the cytokines involved in the Th1, Th17 immune response, IFN-γ, IL-17 was increased compared to the control group when administered extracellular vesicles derived from sRNA overexpressing strains, cytokines involved in Th2 immune response Caine IL-4 was confirmed that no difference from the control group when administered extracellular vesicles derived from sRNA overexpression strain (Fig. 7).

Through this, it was confirmed that the sRNA overexpressing strain Salmonella-derived extracellular vesicles can induce Th1 and Th17 immune responses similar to probiotic infection.

Example 4 Evaluation of Immune Response and Vaccine Efficacy by Polymyxin B Treatment in E. coli Strain-derived Extracellular Vesicles

<4-1> Confirmation of physical properties of heat treatment or polymyxin B treatment on E. coli strain-derived extracellular vesicles

E. coli was incubated in a test tube containing 3 ml LB solution for 4 hours at 37 ℃, 10 μl of each was transferred to 8 2L Erlenmeyer flask containing 500 ml LB solution and incubated for 37 hours, 4 hours. The culture solution was divided into 12 350 ml high-speed centrifuge tubes, and then performed twice in succession at 4 ° C. and 5,000 × g for 15 minutes. Four liters of supernatant are passed through a membrane filter with a pore size of 0.45 μm once and then up to 300 ml using a Quixstand system that can only pass molecules up to 100 kDa. Concentrated. The concentrate was passed once through a membrane filter with a pore size of 0.22 μm, then divided into 50 ml ultracentrifuge tubes, followed by ultratrace separation for 3 hours at 150,000 xg at 4 ° C. It was. The supernatant was discarded and the intestinal coliform-derived extracellular vesicles were extracted by dissolving the precipitate under the tube with PBS.

After treatment with extracellular vesicles separated by the above method (10 minutes at 100 ° C) or polymyxin B for 6 hours, changes in the physical properties of the extracellular vesicles were determined by dynamic light scattering and electron microscopy. It was confirmed using (Transmission electron microscopy). As a result, as shown in Fig. 8, the size of the extracellular vesicles treated with heat treatment or polymyxin B was about 20 nm in size and similar in appearance to the vesicles without any treatment as a control (Fig. 8). .

<4-2> Antibody Production by Heat Treatment or Polymyxin B Treated Escherichia Coli-derived Extracellular Vesicles

E. coli-derived extracellular vesicles isolated by 4-1 were treated with heat treatment or polymyxin B, and then extracellular vesicles (5 μg) were injected three times a week into the abdominal cavity of mice. 72 hours after injection, mouse serum was isolated to determine IgG antibodies specific for vesicles.

As a result, it was confirmed that the IgG antibody was generated similarly to the case where the vesicle-specific antibody was not pretreated regardless of the heat treatment or polymyxin B administration from the first vesicle administration (see FIG. 9).

<4-3> Vaccine efficacy of heat treated or polymyxin B-treated E. coli-derived extracellular vesicles to prevent E. coli infection

After treatment with E. coli-derived extracellular vesicles isolated by 4-1, heat treatment or polymyxin B, immunotherapy was performed by injecting extracellular vesicles (5 μg) three times a week into the abdominal cavity of mice. One week after the last vaccination, E. coli was administered intraperitoneally to evaluate the effect of preventing E. coli death.

As a result, it was not effective when heat treated bacteria-derived vesicles, but when treated with polymyxin B in the vesicles, death by E. coli infection effectively prevented similarly to the vesicles without pretreatment (see Figure 10).

The description of the present invention set forth above is for illustrative purposes, and one of ordinary skill in the art may understand that the present invention may be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. There will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

The vector expressing sRNA (small RNA) according to the present invention overexpresses sRNA and increases the production of extracellular vesicles of the strain, thereby inducing a Th1, Th17 immune response by the extracellular vesicles, which expresses the sRNA. Vectors may be usefully used in the fields of medicines, nutraceuticals, and cosmetics for the prevention, improvement, or treatment of various diseases related to lowered immune function through immunomodulatory effects.

Claims (21)

A pharmaceutical composition for immunomodulation comprising a vector expressing E. coli-derived small RNA (sRNA) as an active ingredient. According to claim 1, wherein the E. coli-derived sRNA is SgrS, RybD, RybB, FnrS, MicC, MicF, GlmY, RprA, CyaR, MicA, OmrA, MgrR, RyhB, GadY, GlmZ, OxyS, DicF, DsrA, Spot42, RseX , IS118, ArcZ, OmrB, RyeB, ChiX and GcvB, characterized in that any one selected from the group consisting of, pharmaceutical composition for immunomodulation. According to claim 2, wherein the SgrS is a base sequence described in SEQ ID NO: 1; RybB is the nucleotide sequence set forth in SEQ ID NO: 2; RybD is the nucleotide sequence set forth in SEQ ID NO: 3; FnrS is the nucleotide sequence set forth in SEQ ID NO: 4; MicC is the nucleotide sequence set forth in SEQ ID NO: 5; MicF is the nucleotide sequence set forth in SEQ ID NO: 6; GlmY is the nucleotide sequence set forth in SEQ ID NO: 7; RprA is the nucleotide sequence set forth in SEQ ID NO: 8; CyaR is the nucleotide sequence set forth in SEQ ID NO: 9; MicA has the nucleotide sequence set forth in SEQ ID NO: 10; OmrA is the nucleotide sequence set forth in SEQ ID NO: 11; MgrR is the nucleotide sequence set forth in SEQ ID NO: 12; RyhB is the nucleotide sequence set forth in SEQ ID NO: 13; GadY is the nucleotide sequence set forth in SEQ ID NO: 14; GlmZ is the nucleotide sequence set forth in SEQ ID NO: 15; OxyS is the nucleotide sequence set forth in SEQ ID NO: 16; DicF is a nucleotide sequence set forth in SEQ ID NO: 17; DsrA is the nucleotide sequence set forth in SEQ ID NO: 18; Spot42 has the nucleotide sequence set forth in SEQ ID NO: 19; RseX is the nucleotide sequence set forth in SEQ ID NO: 20; IS118 is a nucleotide sequence set forth in SEQ ID NO: 21; ArcZ is the nucleotide sequence set forth in SEQ ID NO: 22; OmrB is the nucleotide sequence set forth in SEQ ID NO: 23; RyeB is the nucleotide sequence set forth in SEQ ID NO: 24; ChiX is the nucleotide sequence set forth in SEQ ID NO: 25; And GcvB is a nucleotide sequence of SEQ ID NO: 26, characterized in that the immunomodulatory pharmaceutical composition. The pharmaceutical composition for immunomodulation of claim 1, wherein the vector is a vector that overexpresses sRNA. The pharmaceutical composition for immunomodulation of claim 1, wherein the vector is any one selected from the group consisting of plasmid DNA, linear DNA, and recombinant viral vectors. A pharmaceutical composition for immunomodulation comprising a strain transformed with a vector expressing E. coli-derived sRNA, a culture medium thereof, or an extracellular vesicle isolated from the strain or culture medium thereof as an active ingredient. The method of claim 6, wherein the E. coli-derived sRNA is SgrS, RybD, RybB, FnrS, MicC, MicF, GlmY, RprA, CyaR, MicA, OmrA, MgrR, RyhB, GadY, GlmZ, OxyS, DicF, DsrA, Spot42, RseX , IS118, ArcZ, OmrB, RyeB, ChiX and GcvB, characterized in that any one selected from the group consisting of, pharmaceutical composition for immunomodulation. 7. The method of claim 6 wherein the strain of Salmonella (Salmonella), E. coli (Escherichia coli), keurep when Ella bacteria (Klebsiella pneumoniae) and Pseudomonas species (Pseudomonas), characterized in that any one of selected from the group consisting of strain, the immune Controlled pharmaceutical composition. 7. The immunomodulatory pharmaceutical composition of claim 6, wherein the extracellular vesicles are treated with heat or a compound to eliminate toxicity. The pharmaceutical composition for immunomodulation of claim 9, wherein the compound is polymyxin B. The pharmaceutical composition for immunomodulation of claim 1 or 6, wherein the immunomodulation increases an immune response to a bacterial or viral infection. The pharmaceutical composition for immunomodulation according to claim 1 or 6, wherein the immunomodulation increases an immune response to cancer cell specific antigens. The pharmaceutical composition for immunomodulation according to claim 1 or 6, wherein the immunomodulation inhibits hypersensitivity to allergens. Health functional food for immunomodulation containing a vector expressing E. coli-derived sRNA as an active ingredient. A health functional food for immunomodulation comprising a strain transformed with a vector expressing E. coli-derived sRNA, a culture medium thereof, or an extracellular vesicle isolated from the strain or culture medium thereof as an active ingredient. An immunomodulating cosmetic composition containing a vector expressing E. coli-derived sRNA as an active ingredient. An immunomodulating cosmetic composition comprising a strain transformed with a vector expressing E. coli-derived sRNA, a culture medium thereof, or an extracellular vesicle isolated from the strain or culture medium thereof as an active ingredient. An immunomodulatory method comprising administering to a subject a pharmaceutical composition containing a vector expressing E. coli-derived small RNA (sRNA) as an active ingredient. An immunomodulatory use of a vector expressing E. coli-derived small RNA (sRNA). Administering to the individual a strain transformed with a vector expressing E. coli-derived sRNA, a culture thereof, or a pharmaceutical composition containing an extracellular vesicle isolated from the strain or culture thereof as an active ingredient. , Immunomodulation method. An immunomodulatory use of a strain transformed with a vector expressing E. coli-derived sRNA, a culture thereof, or an extracellular vesicle isolated from the strain or the culture thereof.

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