KR101749317B1 - Compositions for prevention, improvement and treatment of Aging Phenotypes Comprising Recombinant Fusion Protein with Vibrio vulnificus Flagellin and Pathogenic Antigen - Google Patents

Abstract

The present invention relates to a composition for preventing, ameliorating or treating aging, which comprises as an active ingredient a recombinant protein obtained by fusing a flagellin, a component of the flagella of the sepsis Vibrio vulnificus ( Vibrio vulnificus ), and a protein antigen of a pathogen. According to the present invention, there is an advantage that the recombinant protein of the present invention can be used to improve external and internal functions caused by aging and to enhance immunity. In addition, the composition of the present invention can easily immunize through mucosal administration.

Description

Compositions for prevention, improvement and treatment of Aging Phenotypes Comprising Recombinant Fusion Protein with Vibrio vulnificus Flagellin and Pathogenic Antigen}

The present invention is an aging comprising a recombinant protein in which the flagellin component of Vibrio vulnificus , which is an agonist of Toll-like receptor-5 (TLR-5), which is effective as a vaccine adjuvant, is fused with an antigen of a pathogen. It relates to a composition for prevention, improvement or treatment.

With the advent of an aging society due to the prolonged life expectancy of mankind, mankind is facing a variety of homework that has not been encountered before. Socioeconomically, the increase in the elderly population per capita is expected due to an increase in the elderly population and a decrease in the working age group, and interest in improving the quality of life of the elderly is also increasing. As the social demand for a healthy and happy old life increases, research on changes in disease patterns and prevention of aging-related diseases according to aging is being actively conducted.

The changes that appear as aging progresses are very diverse. Outwardly, in addition to various external changes such as skin wrinkles, hair bleaching, spine flexion and movement changes, internally reduced functions of each major tissue, food intake and digestive disorders, brain function decline, including memory loss, and cardiovascular There are various changes, such as a decrease in the function of the body. In addition, since these changes induce a disease of each tissue along with a decrease in function, it is very important to understand the cause of the decrease in external and internal function according to aging and develop a technology to control it.

In addition to this, one of the major functional changes with aging is the decrease in immune function. This is called immunosenescence (Immunosenescence). When pathogens invade the host, the host defense functions by two immune systems: innate immunity and adaptive immunity. Among them, the innate immune system is activated immediately after infection, and is an immune system that can quickly regulate against infectious pathogens. It is responsible for the initial stage of infection until the acquired immune system is activated. Receptors that recognize'pathogen associated molecular patterns (PAMPs)' present in pathogens in such innate immunity are called'pattern recognition receptors' (PRRs), and in mammals, these receptors are Toll-like. Receptors (TLRs: Toll-like receptors) are called. Thirteen types of TLRs have been identified so far, and studies on the agonists of each TLRs are actively being conducted (Shizuo Akira et al, Cell, Pathogen Recognition. and Innate Immunity , 124(4):783-801, 2006). PRRs such as TLRs exist on the cell surface or in the cytoplasm, and are known to regulate the innate immune response by stimulation of various PAMPs, and further regulate the adaptive immune response. Therefore, agonists of TLRs can be suitable target substances for the development of various immune modulators and vaccine adjuvants.

Among the research on aging, the field that has been receiving the most attention until recently is the study on the lifespan control of aging or the recovery of aging functions. In studies using Drosophila models or nematodes, there are various methods, such as suppressing or overexpressing the expression of a specific gene, extending the lifespan, prolonging the lifespan through dietary restrictions, and prolonging the lifespan through recent treatments such as rapamycin. Research on lifespan extension is increasing rapidly, and interest in function maintenance or function recovery, etc., rather than simple lifespan extension, is also increasing. However, controlling the expression of a specific gene by referring to the results shown in lower animal models may cause other functional side effects, so its application to humans is limited, and treatment with drugs such as rapamycin has a great effect on immune function. The limit of giving is being pointed out.

Throughout this specification, a number of papers and patent documents are referenced and citations are indicated. The disclosure contents of the cited papers and patent documents are incorporated herein by reference as a whole, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly described.

The present inventors have made diligent research efforts to develop a substance capable of preventing, improving, or treating deterioration in function due to aging. As a result, while the expression of most of the Toll-like receptors in senescent immune cells decreases, it was confirmed that Toll-like receptor-5 was well expressed and maintained its function. In the case of immunization using a recombinant protein fused with an antigenic protein of a pathogen, it was confirmed that not only immunity of aging mice was activated, but also external and internal functions were improved, thereby completing the present invention.

Accordingly, an object of the present invention is to provide a composition for preventing, improving or treating aging, comprising as an active ingredient a recombinant protein obtained by fusion of a flagellin component of Vibrio vulnificus and a protein antigen of a pathogen.

Another object of the present invention is to provide a composition for preventing, improving or treating hair-related diseases comprising the recombinant protein of the present invention as an active ingredient.

Another object of the present invention is to provide a composition for preventing, improving or treating eye-related diseases comprising the recombinant protein of the present invention as an active ingredient.

Another object of the present invention is to provide a composition for preventing, improving or treating intestinal diseases comprising the recombinant protein of the present invention as an active ingredient.

Another object of the present invention is to provide a composition for preventing, improving or treating bone diseases comprising the recombinant protein of the present invention as an active ingredient.

Other objects and advantages of the present invention will become more apparent by the following detailed description, claims and drawings.

According to one aspect of the present invention, the present invention provides a composition for preventing, improving, or treating aging comprising as an active ingredient a recombinant protein obtained by fusion of flagellin, which is a flagellum component of Vibrio vulnificus, and a protein antigen of a pathogen as an active ingredient. to provide.

The present inventors have made diligent research efforts to develop a substance capable of preventing, improving, or treating deterioration in function due to aging. As a result, while the expression of most of the Toll-like receptors in senescent immune cells decreases, it was confirmed that Toll-like receptor-5 was well expressed and maintained its function. In the case of immunization using a recombinant protein fused with an antigenic protein of a pathogen, it was confirmed that not only immunity of aging mice was activated, but also external and internal functions were improved.

The greatest feature of the present invention is to use a recombinant protein in which flagellin, a component of the flagellum of Vibrio vulnificus, is fused with a protein antigen of a pathogen.

In the present specification, the term "flagelin" refers to a unit molecule constituting the flagella, a cilia that determines the motility of bacteria. The flagellin of the present invention includes all of the flagellins acting as a TLR-5 agonist of septic vibrio bacteria, and preferably, the flagellin is FlaB, which is a flagellin of septic vibrio bacteria.

Examples of protein antigens of pathogens that can be used in the present invention are Streptococcus pneumoniae ) surface protein A (PspA) of the alpha helix domain (α-helix domain) and pneumococcal surface adhesion factor A (PsaA: pneumococcal surface protein A); Subunits of influenza virus hemagglutinin (HA) and neuraminidase (NA); And the spike (S) protein of severe acute respiratory syndrome virus (SARS virus), but is not limited thereto. Preferably, the protein antigen of the pathogen of the present invention is pneumococcal surface protein A (PspA).

According to a preferred embodiment of the present invention, the recombinant protein of the present invention is a FlaB-PspA protein having the amino acid sequence of SEQ ID NO: 1.

As used herein, the term "aging" refers to a functional, structural and biochemical process that occurs continuously from birth to death. It occurs in the entire cell and body tissues that make up an individual, and shows a decrease in metabolic rate, an increase in disease, and a decrease in adaptability, which ultimately leads to the death of cells and the entire body. For example, external changes such as increased wrinkles in the skin, decreased gloss of the hair, hair bleaching, hair loss, reduction in the thickness of the dermal layer where hair follicles exist, decrease in the number of hair follicles, decrease in spine curvature and momentum, and decrease in immune function, major tissues It includes, but is not limited to, internal changes, such as a decrease in the function of, and the occurrence of diseases of each tissue.

According to a preferred embodiment of the present invention, the recombinant protein of the present invention enhances immunity. As used herein, the term "immune enhancement" means maintaining the immune response or activity of the immune system in vivo based on an unaged control group or enhancing an aging control group. More preferably, the recombinant protein of the present invention is hematopoietic stem cells, which are important for the increase of secretory IgA (sIgA) antibody production, and the differentiation of T cells based on the aged control group. By increasing the frequency, preventing the degeneration of the thymus gland, or preventing the mesenteric lymphnode (MLN) or spleen from becoming enlarged, the function of the immune-related organs is not reduced by aging or is strengthened compared to the aging control group.

According to a preferred embodiment of the present invention, the recombinant protein of the present invention prevents, improves or treats metabolic function, skin tissue function, intestinal tissue function, muscle tissue function, brain function or cardiovascular function decline. More preferably, metabolic function, function of skin tissue, function of intestinal tissue, function of muscle tissue are prevented, improved or treated, and most preferably function of dermal tissue in which hair follicles exist, function of colon tissue or muscle tissue To prevent, improve, or treat the deterioration of the function of the body.

According to another aspect of the present invention, there is provided a composition for preventing, improving or treating metabolic diseases comprising the recombinant protein of the present invention as an active ingredient.

As used herein, the term "metabolic disease" refers to a syndrome in which risk factors such as obesity, diabetes, hypertriglyceridemia, high blood pressure, cardiovascular disease, and blood coagulation abnormalities appear together. The symptom itself is not fatal, but it is a disease that seriously threatens modern people because it is predisposed to develop into serious diseases such as diabetes or ischemic cardiovascular disease. However, it was officially named as metabolic syndrome or insulin resistance syndrome through the Adult Treatment Program III (ATP III) established by the World Health Organization and the National Institute of Health Research. Preferably, the metabolic disease of the present invention is obesity.

According to another aspect of the present invention, there is provided a composition for preventing, improving or treating hair-related diseases comprising the recombinant protein of the present invention as an active ingredient.

The hair-related diseases that the composition of the present invention can prevent, improve, or treat include, for example, reduction of gloss of hair, decolorization of hair, hair loss, reduction in the thickness of the dermal layer in which hair follicles exist, and reduction in the number of hair follicles, but are limited thereto. It is not.

According to another aspect of the present invention, there is provided a composition for preventing, improving or treating intestinal diseases comprising the recombinant protein of the present invention as an active ingredient.

The intestinal diseases that the composition of the present invention can prevent, ameliorate, or treat include, for example, irritable bowel syndrome (IBS), uncontrolled diarrhea-related irritable bowel syndrome (dIBS), Crohn's disease, traveler's diarrhea, ulcerative colitis, enteritis. , Small intestinal bacteria overgrowth, chronic pancreatitis, pancreatic insufficiency, colitis, diverticulosis, hepatic encephalopathy and hernia, but are not limited thereto. Preferably, the intestinal disease is enteritis or hernia, more preferably colitis or hernia.

According to another aspect of the present invention, there is provided a composition for preventing, improving or treating bone diseases comprising the recombinant protein of the present invention as an active ingredient.

The bone diseases that the composition of the present invention can prevent or treat include, for example, osteoporosis, osteomalacia, rickets, bone metastasis of cancer cells, bone damage due to bone metastasis of cancer cells, osteolysis due to bone metastasis of cancer cells, fibrotic bone dysplasia. , Amorphous bone disease, metabolic bone disease, rheumatoid arthritis, osteoarthritis, degenerative arthritis and discs, but are not limited thereto. Preferably, the bone-related disease is osteoporosis, osteomalacia, metabolic bone disease, rheumatoid arthritis, osteoarthritis or degenerative arthritis, more preferably osteoporosis or osteomalacia.

Since the composition for the prevention, improvement or treatment of the above-described diseases of the present invention contains the above-described recombinant protein as an active ingredient, the description in common with the recombinant protein is omitted in order to avoid excessive complexity of the present specification. do.

The composition of the present invention can be prepared as a pharmaceutical composition.

According to a preferred embodiment of the present invention, the composition of the present invention comprises (a) a pharmaceutically effective amount of the recombinant protein of the present invention described above; And (b) a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically effective amount" means an amount sufficient to achieve the efficacy or activity of the above-described recombinant protein.

When the composition of the present invention is prepared as a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are commonly used in formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc. It does not become. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention can be administered mucosally, orally or parenterally, and is preferably applied by mucosal administration.

A suitable dosage of the pharmaceutical composition of the present invention is formulated in various ways depending on factors such as the formulation method, the mode of administration, the patient's age, weight, sex, pathological condition, food, administration time, route of administration, excretion rate and response sensitivity. Can be. A typical dosage of the pharmaceutical composition of the present invention is in the range of 0.001-100 mg/kg on an adult basis.

The pharmaceutical composition of the present invention is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person having ordinary knowledge in the art. Or it can be prepared by incorporating it into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension, syrup, or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, powder, granule, tablet or capsule, and may additionally include a dispersant or a stabilizer.

The composition of the present invention may be provided as a food composition.

According to a preferred embodiment of the present invention, the composition of the present invention comprises (a) a food-effective amount of the recombinant protein of the present invention described above; And (b) a food physiologically acceptable carrier.

When the composition of the present invention is prepared as a food composition, it contains not only recombinant protein as an active ingredient, but also ingredients commonly added during food production, for example, proteins, carbohydrates, fats, nutrients, flavoring agents, and flavoring agents. Includes. Examples of the aforementioned carbohydrates include monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, oligosaccharides, and the like; And polysaccharides, for example, common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents, natural flavoring agents [taumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.]) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used. For example, when the food composition of the present invention is prepared as a drink, in addition to the water sprout extract of the present invention, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, headworm extract, jujube extract, licorice extract, etc. may be additionally included. I can.

The composition of the present invention may be provided as a cosmetic composition.

When the composition of the present invention is prepared as a cosmetic composition, the composition of the present invention includes not only the above-described recombinant protein, but also components commonly used in cosmetic compositions, such as antioxidants, stabilizers, solubilizers, vitamins, pigments, and It includes conventional adjuvants such as fragrances, and carriers.

As the carrier, purified water, monohydric alcohols (ethanol or propyl alcohol), polyhydric alcohols (glycerol, 1,3-butylene glycol or propylene glycol), higher fatty acids (palmitylic acid or linolenic acid), oils and fats (wheat germ oil, Camellia oil, jojoba oil, olive oil, squalene, sunflower oil, macadamia peanut oil, avocado oil, soybean hydrogenated lecithin or fatty acid glyceride), etc. may be used, but the present invention is not limited thereto. In addition, surfactants, disinfectants, antioxidants, ultraviolet absorbers, anti-inflammatory agents, and refreshing agents may be added as needed.

Surfactants are polyoxyethylene, hydrogenated castor oil, polyoxyethylene, oleyl ether, polyoxyethylene monooleate, polyoxyethylene, glyceryl monostearate, sorbitan monostearate, polyoxyethylene monooleate, sorbitan, sucrose. Fatty acid ester, hexaglycerin monolaurate, polyoxyethylene reduced lanolin, POE, glyceryl pyroglutamic acid, isostearic acid, diester, N-acetyl glutamine, and isostearyl ester.

The fungicide may be optionally included from the group consisting of hinocthiol, tricrosan, rolhexidine gluconate, phenoxyethanol, resorcin, isopropylmethylphenol, azulene, salicylic acid, and zinc pyritaone. .

Antioxidants can be used any of butylhydroxyanisole, molsyx, propyl and erythorbic acid.

Ultraviolet absorbers include benzophenones such as dihydroxybenzophenone, melanin, ethyl paraaminobenzoate, 2-ethylhexyl paradimethylaminobenzoic acid, synoxite, 2-ethylhexyl paramethoxycinnamic acid, and 2-(2-hydroxyl). Any of hydroxy-5-methylphenyl) benzotriazole, urocanic acid and metal oxide fine particles can be used.

Dipotassium glytilithate or allantoin may be used as an anti-inflammatory agent, and red pepper tincture or 1-menthol may be used as a refreshing agent.

The formulation of the composition is an arbitrary formulation capable of blending a recombinant protein as an active ingredient, and in the form of hair loss prevention cosmetics, hair tonic, hair cream, hair lotion, hair shampoo, hair rinse, hair conditioner, hair spray, hair aerosol, It may be prepared in various forms such as solutions, sol gels, emulsions, oils, waxes, aerosols, such as pomades, powders, and gels, but is not limited thereto.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a composition for preventing, improving, or treating aging, comprising as an active ingredient a recombinant protein obtained by fusion of flagellin, a component of the flagellum of Vibrio vulnificus, and a protein antigen of a pathogen as an active ingredient.

(b) The present invention provides a composition for preventing, improving or treating metabolic diseases, hair related diseases, eye diseases, intestinal diseases or bone diseases comprising the recombinant protein of the present invention as an active ingredient.

(c) The present invention has the advantage of improving immunity and improving external and internal functions that occur according to aging by using the recombinant protein of the present invention.

(d) The present invention can be easily immunized through the mucosal administration of the composition of the present invention.

1 shows a schematic diagram of an experimental plan and an experimental method for immunizing general aging mice.
2A and 2B show graphs of measurement results of changes in food intake and weight in aging mice according to immunity of FlaB-PspA recombinant protein.
3 shows a graph of the IgA response in aging mice by sustained immunity.
4 shows the result of morphological changes in aging mice through continuous immunization of antigens.
5 shows histopathological findings on the histologic changes in skin of aged mice by FlaB-PspA recombinant protein immunity.
6 shows changes in hematopoietic stem cells in bone marrow cells of senescent mice by FlaB-PspA recombinant protein.
7 shows organs showing lymphatic system changes in senescent mice by FlaB-PspA recombinant protein immunity.
8 shows the change in bone mineral density of aged mice by FlaB-PspA recombinant protein.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for describing the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

Throughout this specification, "%" used to indicate the concentration of a specific substance is (weight/weight)% for solids/solids, (weight/volume)% for solids/liquids, and Liquid/liquid is (vol/vol)%.

Experiment method and experiment material

With the purified and isolated protein and recombinant protein, aging mice (C57BL/6J, 23 months or longer) were continuously immunized with the protein intranasal (Intranasal) every 2 weeks. The PspA protein and FlaB-PspA recombinant protein were purchased from the laboratory of Professor Jun-haeng Lee of Chonnam National University Clinical Vaccine Development Project Group.

All immunization tests were conducted in SPF (Specific Pathogen Free) facilities. As a control group, young mice (8-10 weeks old) were used, and the immunization test group was a group of non-immunized aging mice, a group immunized intranasally at least 8 times with PBS (16 µl/mouse) at 2 weeks intervals, PspA ( 2.5 µg/16µl/mouse) aging mice intranasally immunized more than 8 times with protein only at 2 weeks intervals, and aging mice with FlaB-PspA (6.5 µg/16µl/mouse) recombinant protein at least 8 times at 2 weeks apart A group immunized intranasally was used.

During continuous immunization, changes in the shape and behavior of aging mice were observed.

After starting immunization, each mouse was individually managed to measure the weight and food intake of mice every week. To accurately measure the change according to immunity, each mouse was separately managed. And after giving 50 g of food per animal, the amount of food remaining after eating at weekly intervals was accurately measured. In addition, the weight of each mouse was accurately measured using an animal scale every week.

In order to compare the morphological changes of aging mice according to immunity, the appearance of the mice, that is, hair condition, hair loss, and discoloration were observed every week, and the anus and eyes were also continuously observed to collect the changed patterns.

In order to compare the behavioral changes of aging mice due to immunity, the behavioral changes of the mice were observed at some time points during immunization. To this end, in order to confirm the behavioral performance of the mice, the aging mice of each group were placed in a limited space and the movement of the mice was observed for a certain period of time. These movements were compared on the basis of movement, which are the standard items of behavioral performance of mice, the number of times to stand up with the hind legs, and the number of supports and nose touches using the forelimbs.

At the intermediate point of continuous immunization, blood and feces were collected from some mice in order to check the change in the immune response according to the number of immunizations, and then the change in antibody production was measured by enzyme-linked immunosorbent assay (ELISA). The measurements were made.

After immunization for an appropriate period, several samples were collected from mice collectively, and then the overall change of aging mice due to immunity was measured through various experimental methods.

Secretion-type immunity to compare mucosal immune responses by collecting serum and various mucosal samples (feces, saliva, vaginal lavage, etc.) from mice in order to check the changes in the immune response in aging mice due to continuous immunity. Changes in the production of globulin A (secretory IgA, sIgA) antibodies were measured by ELISA.

In order to compare the morphological changes of aging mice due to immunity, various tissues including skin tissues were collected, fixed in formalin, and then a paraffin-block was made, and then hematoxylin and eosin (H&E: Hematoxylin). and Eosin) staining to compare histopathological findings. In relation to the effect of immunity through mucosal immunity and food intake, the tissues of the small and large intestine, which are largely occupied by mucosal immunity, were stained with H&E to compare histopathological findings.

To compare the cellular immune response by antigen by isolating lymphocytes from immune-related tissues, lymphocytes are separated from the cervical lymph nodes and the spleen, and the cellular immune response by the antigen is evaluated by ELISA method. Compared with. In addition, in order to separate cells from the bone marrow and observe the changes of various bone marrow cells, bone marrow cells were isolated from the bones of the hind limbs of aging mice and compared using a flow cytometry (FACS: Beckman Coulter). . In particular, the frequency of hematopoietic stem cells, which are importantly related to the differentiation of T cells, was compared and observed through immunostaining using CD34, a hematopoietic stem cell marker.

To compare the changes in bone density of aging mice due to immunity by separating the spine of the mouse, the spine of the mouse was removed and then using a microCT (Micro Computer Tomography) Skyscan 1172 (Micro Photonics Inc., USA). Bone mineral density (BMD) of aging mice according to persistent immunity and kyphosis according to aging were compared and observed.

Next, in the ongoing study, changes in the shape and behavior of aging mice, which have been changed through continuous immunity, were compared and confirmed in relation to metabolism. To this end, blood was collected from Noho mice, serum or plasma was isolated, and changes in hormone-related metabolic genes were compared. In addition, in order to compare and observe changes in mucosal immunity due to immunity, the intestinal-related microenvironment was compared and observed using a normal microbiota. In addition, blood and feces from aging mice that had been continuously immunized with antigens were collected, and then the gene expression patterns were compared and observed through advanced analysis methods such as microarray.

Experiment result

Example One. Flagellin - PspA Changes in food intake and weight in aged mice according to immunity of recombinant protein

During immunization into the nasal cavity of aging mice 8 times at intervals of 2 weeks, the weight and food intake of mice in each group were measured every week, and aging mice that were not immunized were used as controls.

Into the nasal cavity of aged mice, PBS (phosphate buffered saline), the prepared 2.5 μg PspA and 6.5 μg FlaB-PspA recombinant proteins were immunized 8 times at intervals of 2 weeks, and each group of mice was immunized every week, each week. The weight and food intake of the mice in the groups were measured. The results are shown in FIGS. 2A and 2B.

As a result of measuring the body weight, it was confirmed that there was no change in the body weight of the mice due to immunity in all of the mice that were not immunized, the group immunized with PBS, and the group immunized with PspA alone and FlaB-PspA recombinant protein (Fig. 2a). ).

As a result of comparing the food intake of aging mice due to immunity, there was no significant difference in food intake over time in the non-immunized mouse group. There was no significant difference in intake. On the other hand, in the group treated with PspA alone, it was confirmed that food intake gradually increased during continuous immunization.In particular, in the group immunized with FlaB-PspA recombinant protein, food intake increased due to continuous immunity. The result was significantly increased compared to the group treated alone (Fig. 2b).

As can be seen from the results of Example 1, when continuously immunizing aging mice with antigens, there was no significant change in weight change, but it was confirmed that the amount of food intake was remarkably increased. In particular, it was found that when immunization with a recombinant protein in which a vaccine adjuvant and an antigen of a pathogen were fused, the amount of food intake was significantly increased.

Example 2. Observation of mouse morphological changes through continuous antigen immunity

The morphological change of aging mice by immunity was checked every week while immunizing continuously into the nasal cavity of aging mice 8 times at intervals of 2 weeks, and the results are shown in FIG. 3, and aging mice not immunized were used as a control.

The aging mice before the start of immunization had no apparent abnormalities. Hair condition and gloss, bleaching and hair loss, abnormal findings in the anus (colitis or hernia), and abnormal findings in the eyes (cataracts) were not confirmed at all.

However, as a result of observing aging mice continuously after starting immunization, it was confirmed that the hair condition was very poor in the aging mouse group, which was not immunized at all, compared to the group immunized 8 times every two weeks. It was confirmed that the overall appearance was deteriorated, such as severe progression. In addition, abnormal findings such as a hernia were severely observed in the anus, and abnormal findings suspected of cataract findings were found to be severe in both eyes (O-control group). Similarly, in the group immunized with PBS, the condition of the hair was not good, and the appearance was not very good, such as discoloration and hair loss intensifying. In addition, a slight colitis or hernia was observed in the anus, and a suspicious finding of cataract was severely observed in one eye (O-PBS).

On the other hand, as a result of immunization 8 times with PspA, the appearance of aging mice showed slight discoloration, but the hair condition was not very busy, and no abnormal findings were observed in the anus or eyes (O-PspA). In particular, in the group immunized with FlaB-PspA recombinant protein, it was confirmed that the hair condition of aging mice was very similar to that of aging mice before immunization, and no abnormal findings were observed in the anus or eyes at all ( O-FlaB-PspA).

As can be seen from the results of Example 2, it was confirmed that the appearance of aging mice improved a lot through continuous immunity. With aging, the progression of bleaching or hair loss becomes severe, and abnormal findings such as cataracts occur in the eyes. In rodents, abnormal findings such as a hernia were seen in the anus due to a decrease in muscle mass, but it was confirmed that this phenomenon does not occur well when continuous immunization with antigens is performed. In particular, in the group immunized with a recombinant protein fused with a vaccine adjuvant and an antigen of a pathogen, it was found that the appearance of aging mice remained as it was over time or the condition improved.

Example 3. Continuous antigen immunity Uihwan Aging mouse IgA Response change

During continuous immunization, the feces of mice from each group were immunized and collected, and the IgA response of aging mice according to immunity was confirmed by ELISA, and the results are shown in Fig. 4, and the results of aging mice without immunization Was used as a control.

There was no significant difference in IgA response during immunization in aged mice that were not immunized (O-control group). There was no significant difference in the group immunized with PBS alone, but the response slightly increased after 6 immunizations, but there was no significant difference (O-PBS). In the group treated with PspA alone, the IgA response appeared after 4 immunizations, and there was no significant difference thereafter (O-PspA). On the other hand, in the group immunized with FlaB-PspA recombinant protein, it was confirmed that the IgA response was remarkably increased according to the number of immunizations (O-FlaB-PspA).

As can be seen from the results of Example 3, as a result of confirming the IgA response to the mucosal immune response of aging mice through continuous immunity, in other groups, the IgA response did not differ significantly or showed a slight response depending on the immunity, whereas FlaB-PspA In the group immunized with the recombinant protein, it was confirmed that the IgA response was remarkably increased depending on the immunity. In light of this result, it can be thought that the mucosal immunity of aging mice is activated by the recombinant protein.

Example 4. Histological changes of skin in aged mice by recombinant protein immunity

In the result of Example 2, it was observed that the hair condition of aging mice became very good in a group that was continuously immunized with an antigen, especially a recombinant protein.To prove this in more detail, it was confirmed through H&E staining, and the result is shown in FIG. And young mice were used as controls.

As a result of collecting the dorsal skin tissue of an aging mouse and checking it through H&E staining, it is a common phenomenon that hair loss progresses when aging and the dermis layer becomes thinner. In young mice, it can be seen that the dermal layer is thick and hairy and grows, that is, the number of hair follicles exists in the dermal layer (Y-control). On the other hand, in aging mice, it can be seen that the dermal layer becomes thinner and the number of hair follicles is significantly reduced (O-control group). When the aging mouse was continuously immunized with antigen (O-PspA) and recombinant protein (O-FlaB-PspA), it was confirmed that the dermal layer was thickened and the number of hair follicles increased. In particular, it was confirmed that the number of hair follicles was remarkably increased in the group immunized with the FlaB-PspA recombinant protein.

As can be seen from the results of Example 4, it was confirmed through histological analysis that the morphological findings of aging mice were improved when immunization with persistent antigens was performed (Example 2 result). It was confirmed by the histological analysis method that the hair follicles are maintained in the aging mice by continuous immunity that the decolorization and the progression of hair loss in the aging mice shown in the results of Example 2 are improved. In light of this result, it can be considered that immunity with recombinant protein continuously slows or prevents the progression of depigmentation and hair loss that occur with aging.

Experimental example 5. In the bone marrow by recombinant protein immunity Hematopoietic change

Changes in bone marrow cells in the bone marrow of aging mice due to continuous immunity were confirmed, and the results are shown in Fig. 6, and aging mice not immunized were used as a control.

As a result of observing changes in bone marrow cells by separating cells from the bone marrow of senescent mice, there was no difference between the groups in cellularity.

As a result of checking the ratio of bone marrow cells (megakaryocyte, myeloid and erythroid lineages), it was confirmed that bone marrow cells were well maintained and differentiated in aging mice as a whole.

In order to confirm the frequency of hematopoietic stem cells, as a result of confirming through immunostaining with the hematopoietic stem cell marker CD34, it was confirmed that CD34 increased compared to other groups when aging mice were continuously immunized with the FlaB-PspA recombinant protein.

As can be seen from the results of Example 5, it was confirmed that, when continuous immunization with the recombinant protein was performed, not only the shape of the aging mouse was changed, but also the effect was related to the immunity.

Example 6. Changes in lymphatic system organs by recombinant protein immunity

After continuous immunization for an appropriate period, organs were removed from mice in each group, and the shape and weight were measured, and the results are shown in FIG. 7, and young mice were used as controls.

As a result of organ morphology change and weight measurement after the entire tissue was removed from aging mice, differences were observed in lymphatic system-related organs compared to other organs due to continuous immunity. In particular, it was confirmed that the shape and weight of the organs of young mice were very similar in one group immunized with the FlaB-PspA recombinant protein.

It is a common phenomenon that the thymus degenerates with age. It was confirmed that the weight of the thymus gland increased in the group that continuously immunized aging mice with antigens. In particular, immunization with FlaB-PspA recombinant protein was observed. In the group, it was confirmed that the weight was significantly increased. In addition, the mesenteric lymph node (MLN) and spleen, which are widely used in systemic inflammatory reactions, are persistent infections as aging progresses, and most of them become very enlarged. FlaB-PspA in aging mice When continuously immunized with recombinant protein, it was confirmed that the weight of the thymus gland was higher than that of other groups, and the spleen also weighed less than the spleen of aging mice of other groups, and showed a shape similar to the spleen of young mice. Was confirmed.

As can be seen from the results of Example 6, it was confirmed that the morphological characteristics of immune-related organs were improved when immunizing aging mice with persistent antigens. In particular, aging mice immunized with FlaB-PspA recombinant protein had morphological findings that were very similar to the organs of young mice.

Example 7. Changes in Bone Mineral Density in Aged Mice by Recombinant Protein Immunity

The vertebrae were removed from aging mice, and the change in bone density of aging mice through immunity with recombinant protein was measured using a small animal computed tomography (microCT), and the results are shown in FIG. 8, and young mice were used as controls.

After continuously immunizing 8 times, the vertebrae of aging mice were excised, and as a result of measuring the change in bone density due to immunity using microCT, it can be confirmed that the bone density was significantly improved in the group immunized with the recombinant protein. To explain this in relation to Example 1, the body weight did not change according to the number of immunizations, and the result that the food intake increased only in the group immunized with the recombinant protein was thought to be related to the exercise amount of aging mice, and the bone density was also different. Compared to the aging mouse group, it can be explained that it was significantly increased in the group continuously immunized with the recombinant protein.

As described above, specific parts of the present invention have been described in detail, and for those of ordinary skill in the art, it is clear that these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereto. Therefore, it will be said that the practical scope of the present invention is defined by the appended claims and their equivalents.

<110> INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY <120> Compositions for prevention, improvement and treatment of Aging Phenotypes Comprising Recombinant Fusion Protein with Vibrio vulnificus Flagellin and Pathogenic Antigen <130> PN120534D1 <160> 1 <170> KopatentIn 2.0 <210> 1 <211> 612 <212> PRT <213> FlaB-PspA <400> 1 Met Ala Val Asn Val Asn Thr Asn Val Ala Ala Met Thr Ala Gln Arg 1 5 10 15 Tyr Leu Asn Asn Ala Asn Ser Ala Gln Gln Thr Ser Met Glu Arg Leu 20 25 30 Ser Ser Gly Phe Lys Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly Leu 35 40 45 Gln Ile Ser Asn Arg Leu Asn Val Gln Ser Arg Gly Leu Asp Val Ala 50 55 60 Val Arg Asn Ala Asn Asp Gly Ile Ser Ile Ala Gln Thr Ala Glu Gly 65 70 75 80 Ala Met Asn Glu Thr Thr Asn Ile Leu Gln Arg Met Arg Asp Leu Ser 85 90 95 Leu Gln Ser Ala Asn Gly Ser Asn Ser Lys Ser Glu Arg Val Ala Ile 100 105 110 Gln Glu Glu Val Thr Ala Leu Asn Asp Glu Leu Asn Arg Ile Ala Glu 115 120 125 Thr Thr Ser Phe Gly Gly Asn Lys Leu Leu Asn Gly Thr Tyr Gly Thr 130 135 140 Lys Ala Met Gln Ile Gly Ala Asp Asn Gly Glu Ala Val Met Leu Ser 145 150 155 160 Leu Lys Asp Met Arg Ser Asp Asn Val Met Met Gly Gly Val Ser Tyr 165 170 175 Gln Ala Glu Glu Gly Lys Asp Lys Asn Trp Asn Val Ala Ala Gly Asp 180 185 190 Asn Asp Leu Thr Ile Ala Leu Thr Asp Ser Phe Gly Asn Glu Gln Glu 195 200 205 Ile Glu Ile Asn Ala Lys Ala Gly Asp Asp Ile Glu Glu Leu Ala Thr 210 215 220 Tyr Ile Asn Gly Gln Thr Asp Leu Val Lys Ala Ser Val Gly Glu Gly 225 230 235 240 Gly Lys Leu Gln Ile Phe Ala Gly Asn Asn Lys Val Gln Gly Glu Ile 245 250 255 Ala Phe Ser Gly Ser Leu Ala Gly Glu Leu Gly Leu Gly Glu Gly Lys 260 265 270 Asn Val Thr Val Asp Thr Ile Asp Val Thr Thr Val Gln Gly Ala Gln 275 280 285 Glu Ser Val Ala Ile Val Asp Ala Ala Leu Lys Tyr Val Asp Ser His 290 295 300 Arg Ala Glu Leu Gly Ala Phe Gln Asn Arg Phe Asn His Ala Ile Ser 305 310 315 320 Asn Leu Asp Asn Ile Asn Glu Asn Val Asn Ala Ser Lys Ser Arg Ile 325 330 335 Lys Asp Thr Asp Phe Ala Lys Glu Thr Thr Gln Leu Thr Lys Thr Gln 340 345 350 Ile Leu Ser Gln Ala Ser Ser Ser Ile Leu Ala Gln Ala Lys Gln Ala 355 360 365 Pro Asn Ser Ala Leu Ser Leu Leu Gly Val Asp Ser Pro Val Ala Ser 370 375 380 Gln Ser Lys Ala Glu Lys Asp Tyr Asp Ala Ala Lys Lys Asp Ala Lys 385 390 395 400 Asn Ala Lys Lys Ala Val Glu Asp Ala Gln Lys Ala Leu Asp Asp Ala 405 410 415 Lys Ala Ala Gln Lys Lys Tyr Asp Glu Asp Gln Lys Lys Thr Glu Glu 420 425 430 Lys Ala Ala Leu Glu Lys Ala Ala Ser Glu Glu Met Asp Lys Ala Val 435 440 445 Ala Ala Val Gln Gln Ala Tyr Leu Ala Tyr Gln Gln Ala Thr Asp Lys 450 455 460 Ala Ala Lys Asp Ala Ala Asp Lys Met Ile Asp Glu Ala Lys Lys Arg 465 470 475 480 Glu Glu Glu Ala Lys Thr Lys Phe Asn Thr Val Arg Ala Met Val Val 485 490 495 Pro Glu Pro Glu Gln Leu Ala Glu Thr Lys Lys Lys Ser Glu Glu Ala 500 505 510 Lys Gln Lys Ala Pro Glu Leu Thr Lys Lys Leu Glu Glu Ala Lys Ala 515 520 525 Lys Leu Glu Glu Ala Glu Lys Lys Ala Thr Glu Ala Lys Gln Lys Val 530 535 540 Asp Ala Glu Glu Val Ala Pro Gln Ala Lys Ile Ala Glu Leu Glu Asn 545 550 555 560 Gln Val His Arg Leu Glu Gln Glu Leu Lys Glu Ile Asp Glu Ser Glu 565 570 575 Ser Glu Asp Tyr Ala Lys Glu Gly Phe Arg Ala Pro Leu Gln Ser Lys 580 585 590 Leu Asp Ala Lys Lys Ala Lys Leu Ser Lys Leu Glu Glu Leu Ser Asp 595 600 605 Lys Ile Asp Glu 610

Claims (1)

A composition for preventing, improving or treating obesity comprising FlaB-PspA protein having the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

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