KR100847287B1 - -1 Human Short Helical Peptide-1 which has antimicrobial antitumor and immune stimulating activity and uses thereof - Google Patents

Abstract

The present invention is antimicrobial, anticancer, immunostimulatory activity pharmaceutical containing human short helical peptide-1 (hSHP-1), hSHP-1, which is an antibacterial, anticancer, and immunoactive protein expressed in human The present invention relates to a method for the treatment of antimicrobial, anticancer, and immunostimulatory activity comprising administering a host composition, hSHP-1 peptide.

hSHP, short helical peptide, antibacterial anticancer protein

Description

Human Short Helical Peptide-1 which has antimicrobial, antitumor, and immune stimulating activity and uses}

1 shows sequence similarity between hSHP-1 amino acid sequence and other antimicrobial proteins recorded in the Antimicrobial Protein Database (APD; University of Nebraska Medical Center, USA, http://aps.unmc.edu/AP/main.html). It shows the result of searching.

2 shows the immunohistochemical expression of hSHP-1 in human organs.

Figure 3 shows the results of the minimum inhibitory concentration (MIC) of hSHP-1 tested in a variety of bacteria.

Figure 4 shows the results of minimum bactericidal concentration (MBC) along the peptide length of hSHP-1.

Figure 5 shows the hSHP-1 gene nucleotide sequence and amino acid sequence.

FIG. 6 shows that hSHP-1 is rapidly crosslinked by TGase in an experiment to confirm that hSHP and monodansyl cadaverine form crosslinks by transglutaminase (TGase). Figure showing that.

7 is shown as a colony destruction assay (colony killing assay) of hSHP-1 results for Staphylococcus aureus and E. coli, Staphylococcus aureus is shown a hSHP-1 is almost complete sterilization from 2 μM, notably in E. coli The low bactericidal power of hSHP-1 can kill 50% of E. coli at 30 μM.

FIG. 8 shows that the oral exfoliated epithelium was bathed with 6M urea, 1 mM EDTA solution for 1 week to remove all nonspecifically attached components, followed by immunochemical staining with hSHP-1 antibody. This is a local positive test (which means that hSHP-1 in saliva was cross-linked to the fallen epithelium).

Fig. 9 shows that abundant amounts of hSHP-1 were expressed in mucosal membranes and exocrine glands as a result of immunohistochemical staining of hSHP seen in tissues of human organs. HSHP-1 was found mainly concentrated in the oral mucosa and gastrointestinal mucosa.

FIG. 10 is a diagram showing that hSHP-1 was found in the same manner as a result of Western blot using proteins obtained from saliva, tears and semen directly received from the parotid gland.

11 is a diagram showing the results of immunoprecipitation experiments in mixed needles using a chromatographic column made using an antibody that specifically reacts with hSHP-1, where hSHP-1 positive bands of various sizes were observed. This means that hSHP-1 causes protein modification very quickly.

12 is a diagram showing the results of performing mRNA hybridization to identify hSHP-1 mRNA expressed in each organ of the human body, in which expression of hSHP-1 was observed in most glands, in particular gastrointestinal tissue and prostate Very strong expression in the back.

Fig. 13 shows the results of observing the antimicrobial activity of hSHP-1 against Staphyococcus aureus and Candida albican by scanning electron microscopy (hSHP-1 observed that cell membranes were completely destroyed within 30 minutes). .

14 is a diagram illustrating the expression of hSHP-1 during oral cancer in malignant disease in oral precancerous lesions. (In normal oral mucosal epithelium, hSHP-1 expression is weak but precancerous lesions (leukoplakia). ), Increased expression in carcinoma in situ, strong expression in well-differentiated early cancer cells, and increased malignancy, indicating no hSHP-1 expression in poorly differentiated cancer cells).

FIG. 15 shows the expression of hSHP-1 in liver cancer cells and malignant chondroma cells, in which a portion of hSHP-1 moves into the nucleus and attaches to the nucleus.

Fig. 16 is an electron micrograph showing the appearance of hSHP-1 infiltrating into the cytoplasm and the nucleus in hepatic cancer cells (in the cytoplasm, the Golgi apparatus and the endoplasmic reticulum are densely seen and attached to the nucleus indiscriminately).

FIG. 17 shows the results of observing the effect of hSHP-1 on the cell proliferation of human gingival and periodontal ligament fibroblasts. The phenomenon of hSHP-1 significantly inhibiting the proliferation of these cells was observed. .

Figure 18 shows the various genes involved in oncogenes, apoptosis-related genes, and tissue degeneration in order to know the effects of hSHP-1 on gene expression using human gastric cancer cell lines (SNU-1 cells). As a result showing the results of searching for the genes, hSHP-1 was observed to suppress the overall activity of SNU-1 cells.

The present invention relates to human short helical peptide-1 (hereinafter referred to as 'hSHP-1'), which is an antibacterial, anticancer, and immunoactive protein expressed in humans.

The study of antimicrobial and anticancer proteins is a very important area in the life sciences and medicine. All living organisms are known to produce antimicrobial substances for survival. Penicillin, which is extracted from blue mold, is also an antimicrobial substance produced by the fungus to survive. Therapies are said to have their own antibacterial substances in the planar and dandelion extracts. To date, more than 800 antimicrobial substances have been reported from various plants and animals, but there are very few reports on human antimicrobial and anticancer proteins. Defensin, cathelicidin (LL-37), dermicidin, hisstatin and lysozyme have been reported as human antibacterial proteins. Compared to magainin found in toads, antibacterial activity is significantly lower. To date, antibacterial proteins have been found in various animals such as frogs, pigs, snakes, crocodiles, scorpions, and flies, but they are not immediately available because they show an immune response when used in humans. There is a need for a technique for producing an antimicrobial protein having antimicrobial activity.

The present inventors earnestly researched to find a novel antimicrobial protein that is specifically expressed in humans, and as a result, by separating hSHP-1 and confirming that it has antimicrobial, anticancer and immunostimulatory activity, the present invention was completed.

Accordingly, an object of the present invention is to provide a novel hSHP-1 peptide having antibacterial, anticancer and immunostimulatory activity.

It is another object of the present invention to provide an antimicrobial, anticancer, immunostimulatory pharmaceutical composition comprising hSHP-1 peptide as an active ingredient, or an antimicrobial household product comprising the peptide.

Another object of the present invention to provide a method for the treatment of antimicrobial, anticancer, immunostimulatory activity comprising the step of administering hSHP-1 peptide.

The objects and advantages of the invention will become apparent from the following detailed description, claims and drawings.

The present invention provides peptides having antimicrobial, anticancer or immunostimulating activity selected from the following groups:

(a) the N-terminus of the peptide is selected from any one of the 12th to 31st amino acid residues of SEQ ID NO: 2, and the C-terminal of the peptide is 45 or less than SEQ ID NO: 2 A peptide selected from the 46th amino acid residue and is at least 16-mer,

(b) tandem repeat of the peptide (a).

Preferably, the peptide is a peptide of any one of SEQ ID NOs: 3-8, or tandem repeat thereof, and the peptide is glycosylated.

The present invention provides a polynucleotide encoding the peptide.

The present invention comprises the peptide as an active ingredient, oral cavity, eyeball, hair, skin, nasal cavity, gastrointestinal tract, peritoneum, pleura, vagina, prostate, bladder (peebo), urinary tract (pee), articular capsule, pituitary membrane, outer ear, Provided is a pharmaceutical composition for the treatment of an infectious disease occurring in the inner ear, bone marrow, or major internal organs. Preferably, the pathogen of the infectious disease is Gram-negative or Gram-positive bacteria, fungi, encapsulating virus, and more preferably the pathogen of the infectious disease is methicillin resistant bacteria.

The present invention provides a pharmaceutical composition for preventing or treating precancerous lesions or cancer, comprising the peptide as an active ingredient. Preferably, the cancer is selected from skin cancer, osteosarcoma, oral cancer, gastric cancer, intestinal cancer, brain tumor, or liver cancer.

The present invention provides a composition for enhancing immune function comprising the peptide as an active ingredient.

The present invention provides an antibacterial article comprising the peptide as an active ingredient. Preferably, the household product is selected from cosmetics, toothpaste, skin cleansers, feminine cleansers, artificial tears used for dry eyes, artificial saliva used for dry mouth, artificial runny nose used for nasal drying, food additives or feed additives. .

In order to survive, organisms need antibacterial function to kill pathogens first. Antibacterial protein is used to prevent germ invasion and to sterilize primarily in vitro. Every organism produces its own unique antimicrobial proteins, with a few known in humans. Human antibacterial proteins include hisstatin, lysozyme, LL-37, defensin, and dermicidin, but they are still insufficient to explain the effective bactericidal action of the human body. . LL-37 is a 37 amino acid cleaved from a protein called cathelicidin and has a very strong antibacterial effect. hSHP-1 is easily adhered to bacterial biofilms or viral capsule through unique glycosylation, so it can be used to treat serious infectious or viral diseases. In the present invention, the discovery of hSHP-1, a new secretory antibacterial protein produced in humans, researched its characteristics, function, structure, components and methods of use, and treatment of infectious diseases or caused by lack or deficiency of hSHP-1. In order to prevent the disease is to provide a method of using hSHP-1.

Meanwhile, as shown in some antimicrobial proteins, hSHP-1, which is a human antimicrobial protein, has a function of inhibiting target hyperproliferation of mucosal-dermal epithelium by inflammatory or physico-chemical stimulation, so that hSHP-1 forms keloids, It can suppress the development of polyps and precancerous lesions. Therefore, it is intended to provide a method of using hSHP-1 for the treatment and prevention of various hyperproliferative diseases or precancerous lesions.

1. hSHP-1 Gene and Naming

The hSHP-1 gene is recorded in GenBank under the name AY177672 under the name Salvic. The open reading frame (ORF) of hSHP-1 is 141 bp, of which 138 bp designates 46 amino acid sequences (SEQ ID NO: 2) (FIG. 5).

Eleven amino acids at the amino terminus among 46 amino acid sequences are assumed to act as signal peptides as a hydrophobic array. Thus, 35 amino acids at the carboxyl terminus are believed to have antimicrobial and anticancer functions inherent in hSHP-1.

hSHP-1 was called Salvic at the time of discovery as a victorious substance in the saliva, and this substance mainly acts as a bactericidal to bacteria in mucosa. It was renamed mucocidin, but later it was observed that the genetic material had antimicrobial activity as well as anti-cancer and immune-promoting effects on proliferating cells. Recently, cationic short helical peptides (SHPs) SHP is important because it is known that it is a multifuctional peptide showing both antibacterial and anticancer effects. Therefore, this genetic material was first recorded as a cationic short helical peptide of a single gene appearing in human and was renamed hSHP-1 by the inventors.

The SHP found in humans is LL-37, which is derived from a large protein called cathelicidin, which differs from the single hereditary hSHP-1. In reality, however, there are innate immunities of various forms, each of which is presumed to have a large amount of genetic material in the form of SHP. Therefore, it is expected that several hSHPs will be revealed in humans in the future.

2. Modification of hSHP-1 Protein

hSHP-1 is a short protein with an alpha-type helix, which contains eight serine amino acids, two of which have a serine-serine structure, which is believed to be severely glycosylated as it is rapidly cylated. On the other hand, having a cysteine amino acid in the central portion of the protein code (two) glycosylated hSHP-1 disulfide bond (disulfide bond) is present as a relatively large macromolecule (macromolecule). These macromolecules of glycosylated hSHP-1 reduce denaturation by strong proteolytic enzymes present in large quantities in mucus such as saliva and tears, easily adhere to bacterial bacterial walls, and maintain activity in acidic and alkaline solutions. Enables hSHP-1 function. In fact, the pI value of the amino acid sequence of hSHP-1 is 9.45 and the molecular weight of the initial hSHP-1 protein is estimated to be 5.25 kDa, but the isotrophic electrophoresis (iso) of hSHP-1 obtained from actual saliva or tear and prostate Is about 6-6.3 and the molecular weight is dispersed at about 6, 35, 45, 52, 77 kDa, etc. in Western blot by immuno-precipitation, with the strongest presence of 45 kDa hSHP-1 ( 11).

As a result, hSHP-1 is produced in mucin-secreting cells, and citrate and glycosylation continue around the hydrophilic amino acid serine-serine part by the glycosylation process, resulting in high molecular weight, and disulfide bonds centered on cysteine. When it is discharged out of the cell, it forms a helical complex structure, and it is rapidly transduced by transgurutaminase-3, which is present in the mucus, to form a gulutamine-lysine (QK) crosslink with other proteins in the saliva or proteins on the mucosal epithelium. To form a complex.

3. Cross linking ability of hSHP-1 protein

hSHP-1 has one glutamine (Q) and two lysine (K) at the carboxy terminus, which amino acids can crosslink hSHP-1 to mucosal epithelium by transglutaminase. Experimentally, after mixing and incubating hSHP-1, transglutaminase-2, and monodansylcadaverine, electrophoresis using SDS-gel resulted in the combination of hSHP-1 and monodansylcadaverine in one band. As the incubation time was longer, the cross-linked products increased proportionally (FIG. 6). On the other hand, in immunohistochemical staining, hSHP-1 is observed to form a thin film while attached to the mucosal surface (FIG. 8). This pattern was observed not only in the oral mucosa but also in the gastrointestinal, small intestine, large intestine, and ocular mucosa.

4. Production method of hSHP-1 antibody

hSHP-1 is a short protein consisting of 46 amino acids. Since 11 amino acids at the amino terminus are assumed to be cleaved into a signal peptide, antibodies can be produced using 35 amino acids. Although the 23 amino acids in the carboxyl terminus have an actual antimicrobial effect, these 23 amino acids have very low antigenicity, making antibody production difficult. Therefore, an antibody can be produced using the peptide containing the amino acid corresponding to amino terminal 13-23 with high antigenicity of hSHP-1. hSHP-1 antibodies can produce both polyclonal and monoclonal antibodies. In the case of polyclonal antibodies, most animals such as rabbits (New Zealand white rabbit), goats, cows, mice, and white papers can be used. A monospecific antibody is produced when the carboxy terminal 35 amino acids are used. In the case of monoclonal antibodies, highly specific antibodies can be produced. For example, sustained monoclonal antibodies are produced using myeloma hybrids in mice.

5. Expression level and expression pattern of hSHP-1 in human body

The expression pattern of hSHP-1 can be easily observed by immunohistochemical observation and RNA in situ hybridization. The degree of expression and the pattern of expression in each mucosal tissue and organ of human are as follows (FIGS. 2, 9, 12).

1) Oral mucosa and salivary glands

hSHP-1 is mainly produced in the catheter cells of salivary glands and expression is observed in some serous gland cells. Among large salivary glands, the expression is greatest in the parotid gland, and a considerable amount is also observed in the submandibular gland. Small amounts of expression are observed in the sublingual glands or small salivary glands that produce mucus. hSHP-1 is observed in the form of small granules in the catheter cells, usually gathered near the nucleus and gradually released out of the cell. As a result, when saliva is secreted, a large amount of hSHP-1 is included in the saliva. In normal oral mucosal cells, hSHP-1 expression is hardly detected, but a thin membrane-like hSHP-1 reaction is observed on the mucosal surface to which saliva touches, indicating that hSHP-1 in saliva is firmly attached to the mucosal surface. it means.

Collect the dropped oral mucosal keratinocytes into 6M urea, 1 mM EDTA solution, and continue to boil for 1 week in the bath. The immunohistochemical staining of the dropped oral mucosa with hSHP-1 was performed. It was confirmed that the positive side of hSHP-1 was observed more intensively. As a result, since hSHP-1 was firmly attached to the dropped oral keratinized epithelium by cross-linking, hSHP-1 was not isolated even when boiled in 6M urea, 1 mM EDTA solution for 1 week. In addition, it was estimated that the hSHP-1 in saliva was attached to the fallen oral mucosal keratin epithelium, because it was more concentrated on the outer surface of the fallen oral mucosal keratinocytes.

Oral mucosal epithelium is dysplastic deformation gradually leads to vitiligo, but keratinary epithelium is not easily eliminated from the oral mucosa, the mucosal epithelium thickens, and the keratinary epithelium continues to enlarge. At this time, as the production of hSHP-1 increases, the phenomenon of accumulation in cells is observed. In early well-differentiated cancer cells in which oral mucosal keratinizing epithelium has metastasized to cancer, the production of hSHP-1 is greatly increased and is abundantly expressed in the cytoplasm. Some hSHP-1 migrates near the nucleus and accumulates in the nucleus . Thus, cancer cells accumulating hSHP-1 in the nucleus are observed to gradually shrink or die due to phobia in the nucleus. However, when the progression of cancer is increased to become malignant cancer cells that do not differentiate, hSHP-1 expression is almost disappeared and the amount of nuclear material is increased so that the abnormal shape (Fig. 14).

2) eye mucous membranes and tear glands

The most frequent expression of hSHP-1 in the eye is the M'bomian gland, which is the tear gland on the back of the upper eyelid. These tear glands produce very important functional tears, which cleanse the surface of the eye every time the eye blinks, killing attached bacteria and protecting the surface of the eye. hSHP-1 is also secreted in the lacrimal gland, the main secretory gland of the eye, and the expression level of hSHP-1 is mainly increased in the catheter cells and some glandular cells are also observed. In particular, since the expression of hSHP-1 in the lacrimal gland is greatly increased in the presence of inflammatory diseases in the eye, it is speculated that hSHP-1 expression may be closely related to inflammatory diseases in the eye.

3) nasal mucosa and mucous glands

hSHP-1 is diffusely expressed in the mucous secretory cells of the nasal mucosa, especially in the mucus secreting cells distributed in the mucosa of the maxillary sinus. The nasal mucosa consists mainly of elongated columnar cells, the membranes of which are positive on hSHP-1 on their outer surface. This suggests that hSHP-1 is actively secreted from the nasal mucosa and strongly adheres to the surface of the nasal mucosa, thereby continuously protecting the nasal mucosa.

4) gastrointestinal mucosa

hSHP-1 shows strong expression in gastrointestinal mucosa, and very strong expression level is observed mainly in parietal cells of gastric mucosa. In addition, the expression of hSHP-1 is observed in some mucin-secreting cells, which are strongly attached to the surface of the gastrointestinal mucosa and show a positive reaction on the membrane. Especially in the papillary structure of the mucosa, the expression level of the immature cells in the bottom (crypt) is small, but concentrated in the surface of the terminal mature enterocyte (terminal mature enterocyte) on the top of the papillae. In particular, hSHP-1 is continuously expressed in gastric acid-releasing wall cells when the pH of gastric juice is about 2.5-3 in acidic condition. HSHP-1 does not decrease its activity even in acidic conditions. Is supposed to do.

In mucosal epithelium showing chronic gastritis due to inflammatory changes in the gastrointestinal wall, mucin-secreting epithelial cells have a positive response to hSHP-1, and mucosal epithelium shows a reduced hSHP-1 attached to the mucosal surface, but most hSHP- 1 is found in the cytoplasm of phagocytes infiltrated in connective tissue below the mucosal epithelium. It is believed that hSHP-1 adheres firmly to the inducing antigen and acts as an antimicrobial agent or is easily eaten by phagocytes by opsonization while surrounding the antigen. As a result, hSHP-1 is thought to act as a catalyst for promoting immune action against unspecified antigens in the inflammatory process.

When gastrointestinal mucosa metastasizes to cancer, the expression of hSHP-1 is increased when early dysplastic hyperplasia occurs, and some hSHP-1 accumulated in cells in early cancer cells is infiltrated into the nucleus. Is observed. In this way, cancer cells that are indiscriminately attached to hSHP-1 by infiltrating the nucleus are gradually destroyed, and the cancer cells disappear as the nucleus is also destroyed.

5) small and large intestine mucosa

In the papillary mucosal epithelium of the small intestine and the large intestine, the expression of the basal intestinal cells is poor, but the strong concentration of hSHP-1 is observed on the surface of the intestinal cells of the papillary region. This type of expression is very prominent in the large intestine, which is strongly expressed on the surface of the intestinal cells, confined to the apical part of the mucosal papilla where feces collect and reach.

In the case of inflammatory diseases of the small and large intestine, the concentrated hSHP-1 positive at the apex of the papillary epithelium is reduced, and diffuse hSHP-1 positive overall is observed in the mucosal epithelium. As a result, the hSHP-1 membrane, which acts as a protective barrier for feces at the apex of the papillary epithelium of the intestinal membrane, disappears and hSHP-1 disperses as a promoter of chronic inflammatory response of the intestinal membrane.

6) vagina and uterine mucosa

hSHP-1 is observed in the Bartholin's gland of vaginal mucosa and a membrane-like positive reaction is found on the surface of keratinized epithelium of vaginal mucosa. Vaginal fluid is a weak acid of pH 4-5, so hSHP-1 is well activated, antibacterial, anticancer and immune promoting function.

A positive hSHP-1 response is observed in well-developed endometrium mucosal epithelium of the uterine mucosa. In menstrual mucosal epithelium, hSHP-1 expression is drastically reduced, but in the regenerator of uterine mucosa, hSHP-1 expression is increased again.

7) Skin and sweat glands and sebaceous glands

hSHP-1 is abundantly expressed in skin glands and sebaceous glands. The expression of hSHP-1 is mainly found in the catheter cells of the glands. This was separated in large quantities.

hSHP-1 is found concentrated in the sebaceous gland with hair, and is observed in the sebaceous glands in which hSHP-1 is concentrated in the cytoplasm of fat cells. The released hSHP-1 is mixed with oil and spreads to the skin surface. At this time, hSHP-1 binds tightly to the outer surface of keratin epithelium on the surface of the skin, and the hSHP-1 positive reaction on the membrane is observed. Perhaps the hSHP-1 membrane acts as a protective barrier on the skin and continues to be antimicrobial.

8) prostate

The catheter of prostate gland shows the strongest expression of hSHP-1 among human glands, and it is observed that the granules of the granules that show positive hSHP-1 reaction in the catheter cells are slowly discharged. In patients with prostatic hyperplasia, the expression of hSHP-1 is increased, and molecularly biological analysis of the excreted semen resulted in abundance of glycated hSHP-1. This suggests that hSHP-1 secreted from the prostate has inherent antibacterial activity in semen.

9) Tonsils and mucosal immune organs

hSHP-1 shows a positive membrane-like reaction on the surface of normal skin or mucosal epithelium, whereas it is concentrated in the cytoplasm of immune mucosal epithelial cells in the tonsils or intestinal mucosa. In particular, the cryptic epithelium cracked and depressed in the tonsils showed strong positive reactions throughout the cytoplasm, and immune cells such as lymphocytes were infiltrated and proliferated just below the epithelial layer. It is interpreted that hSHP-1 acts rapidly on antigens penetrating from the outside of the mucosa, preferentially attaches to the mucosal epithelium, and transfers it to connective tissue, thereby providing an antigen-transfer function that causes immune cells to produce immune action. As a result, the cationic short helical peptide of hSHP-1 reacts indiscriminately to externally infiltrated antigens, and superficial antigen delivery functions that mucosal epithelial cells directly deliver the antigen to immune cells. This is another example of hSHP-1, which is illustrated as an example of an immune promoting function.

10) liver

Hepatocytes are not exocrine cells, but are cells that produce abundant hSHP-1. In normal hepatocytes, a small amount of hSHP-1 is expressed in the cytoplasm, but the production of hSHP-1 is increased when hepatocytes are stimulated by cholestasis, chronic inflammatory reactions, or cirrhosis of the liver. do. Most of the produced hSHP-1 is passed through the vessel wall of the liver and discharged into the sinusoid-type hepatic vascular system and dispersed throughout the body. Therefore, hSHP-1 is abundantly detected in normal human blood.

When hepatocytes are severely stimulated by cholestasis, chronic inflammatory reactions, or cirrhosis of the liver, hSHP-1 is observed to accumulate excessively in the cytoplasm of hepatocytes. In the case of continuous cholesterosis, chronic inflammatory reactions, or cirrhosis of the liver, some hSHP-1 gathers around the nucleus. However, although no hSHP has yet been infiltrated into the nucleus, when hepatocytes cause tumorous changes and suddenly increase proliferation, a large amount of hSHP-1 is infiltrated into the nucleus and indiscriminately binds to the nucleus. The hSHP-1 is indiscriminately bound to hepatic cancer cells, and the nucleus gradually disappears, causing vacuole degeneration and decreasing cellular granules (FIG. 15, 16).

11) articular capsule

Positive response of hSHP-1 is found in the knee joint fluid of rheumatoid patients. Since much higher amounts of hSHP-1 are detected in the knee joint fluid of rheumatoid patients than in the knee joint fluid of patients with degenerative arthritis, it is described that hSHP-1 is involved in the joint inflammatory response, especially the development of autoimmunity. Immunohistochemical examination of articular capsule tissue to determine the origin of hSHP-1 in joint fluid revealed that hSHP-1 was expressed in articular capsular epithelial cells, and hSHP-1 was membrane-coated on articular capsule epithelial cells and articular or cartilage surfaces. Aspect was observed. It is speculated that hSHP-1 acts as a protective film in the articular capsule, and has antibacterial, antiproliferative and immune promoting functions.

12) cerebrospinal fluid and meninges

Since hSHP-1 is abundantly detected in cerebrospinal fluid, traces of hSHP-1-producing cells are found in abundant expression levels in the spinal fluid-ependymal cells, and the meningeal membranes that cover the brain Expression was observed. Therefore, hSHP-1 is widely present in cerebrospinal fluid and is thought to have various antibacterial, antiproliferative and immune promoting functions.

13) blood vessel

It is observed that hSHP-1 present in the blood eventually adheres to the inner surface of vascular endothelial cells dispersed throughout the body. In older vessels, the attachment of hSHP-1 appears faint, whereas in neovascular vessels, a distinct positive response is observed in the form of a membrane. Therefore, it is assumed that hSHP-1 attaches to the inner surface of vascular endothelial cells on the inner surface of blood vessels, thereby inducing unique antibacterial, anti-proliferative and immune promoting effects. In particular, hSHP-1 may be involved in angiogenesis, since it is strongly expressed on the inner surface of vascular endothelial cells.

In order to know that hSHP-1 is involved in neovascularization, h-HP-1 peptide of C-23 (SEQ ID NO: 6) was added to human umblical vascular endothelial cells (HUVECs) cell culture and VEGF (vascular endothelial). growth factor (5 ng / mL) promotes the migration of HUVECs, and the C-23 peptide has a C-23 peptide concentration of 1 in proportion to the increase in the concentration of C-23 peptide. Cell migration inhibition was observed at 35% for μM and 55% for C-23 peptide concentration of 2.5 μM. This indirectly indicates that C-23 peptides can effectively inhibit cell migration of HUVECs and thus can inhibit the production of neovascularization.

6. Antimicrobial Activity of hSHP-1

1) antibacterial action against bacteria and fungi

hSHP-1 is estimated to have 16 helixes in total, of which 11 helices have unique antimicrobial activity. The hydrophobic helix at the amino terminus is strongly attached to bacteria, and the carboxyl terminus has a glutamine-lysine cross-linking motif by transglutamine, which is firmly attached to the mucosal membrane or the surface of major cells, and thus is expected to have an antimicrobial effect. It is estimated that the major antibacterial action of hSHP-1 is that the helical structure of the 12th-45th amino acid has strong adhesion to the bacterial cell membrane and is inserted into the cell membrane to make holes in the cell wall and destroy the cells. The smallest peptide (16 peptides; SRVLNRSLQVKVVKIT) with the same antimicrobial activity as hSHP-1 has a positive charge value of +4. Using the carboxy terminal end of hSHP-1 23 gae peptide (SEQ ID No. 6, SEQ ID), the observed results of the antibacterial activity, Staphylococcus aureus, Candida albicans, Aspergillus niger, Pseudomonas aeruginosa , etc. pathogens strong sterilizing power (hSHP-1, C with respect to the -23 mer, MIC: about 10 μM / 10 ⁷ cells) and moderate bactericidal power (MIC: about 100 μM / 10 ⁷ cells) for Escherichia coli , a human resident bacteria (FIG. 3, 4).

On the other hand, hSHP-1 showed the same antimicrobial activity as Staphylococcus aureus which is not resistant to Staphylococcus aureus resistant to methicillin (methicillin) (FIG. 7). Helicobacter pylori , a causative agent of human gastritis, showed moderate bactericidal activity (MIC: about 150 μM / 10 ⁷ cells).

2) antibacterial action against viruses

hSHP-1 has two serine-serine (SS) motifs to which citric acid is attached, and when two hSHP-1s are linked by disulfide bonds to form dimers, a number of citric oxidation Glycoprotein is produced by. This glycosylated hSHP-1 has the function of an antivirus (glycosylated basic proline rich protein). Thus, it has strong adhesion to capsular viruses and cationic SHP has an effective antiviral function by damaging the capsular membrane.

3) suppression and removal of biofilm production

Since glycosylated hSHP-1 has adhesion to the biofilm formed by bacteria or fungi, it not only acts as an antimicrobial agent against bacteria in the biofilm, but also inhibits the formation of the biofilm and relaxes the structure of the biofilm, thereby inhibiting the production of new biofilm. It has the function of removing the existing biofilm.

4) Supplementation for hSHP-1 Deficiency

Therefore, antimicrobial activity against bacteria, fungi and viruses can be expected very effectively if the glycosylated hSHP-1 function is continuously maintained in human mucous membranes or skin.

In particular, in the case of senile or degenerative diseases or autoimmune diseases, the secretion of the secretion from the various mucous membranes and skin is remarkable. In this case, multiple infections caused by various pathogens may occur due to the decreased secretion of hSHP-1. In this case, the supply of hSHP-1, one of the components missing in the mucus, is required. In order to determine the hSHP-1 deficiency in the above patients, ELISA, dot blot, Western blot, etc. can be used as an assay for measuring the concentration of hSHP-1 in mucus secretions. As a test that can test the antimicrobial activity against bacteria by using a bacterial killing assay (killing assay) using a medium can be determined by the Minimum Inhibitory Concentration (MIC) value.

7. Anticancer Activity of hSHP-1

In normal cells, the hSHP-1 protein is produced in ribosomes and then gathered in the Golgi apparatus to be glycosylated hSHP-1 protein. At this time, when citric acid is attached to hSHP-1, the protein whose original pI is 9.45 is acidified by citric oxidation, resulting in a weakly acidic protein of about 6.2. By doing so, hSHP-1 is easily discharged out of the cell to have a unique antibacterial effect. However, when cell overproliferation or cancer progresses, metabolic changes in intracellular glucose are caused by anoxia and N-glucosyl sialic acid (NeuGc) -G (M2) by sialin (sialic acid transporter). The overproduction of ganglioside, hSHP-1, is not fully glycosylated by citric acid, so hSHP-1 accumulates in the cell, and the accumulated pH value of hSHP-1 is about 8-9. 1 moves to the acidic nucleus. At this time, the helical structure and repeated hydrophobicity and hydrophilic substrate of hSHP-1 have the property of non-specifically binding to the DNA helix structure. As a result, the accumulation of hSHP-1 was observed in cells of proliferative precancerous or early cancer cells, and some hSHP-1 migrated into the nucleus and indiscriminately attached to the nucleus at the periphery of the nucleus. Found in observation. As a result, when hSHP-1 penetrates into the nucleus of the cell and attaches to the nucleus, it is observed that the proliferation of the cell is inhibited and the cell becomes apoptosis later. Thus, this phenomenon in which hSHP-1 is seen in precancerous tumor cells or cancer cells is explained by the anticancer action on the cells. For example, cytoplasmic accumulation of hSHP-1 is observed in the cell layer where last cell differentiation is not possible in leukoplakia, a precancerous lesion of the oral cavity. In addition to the accumulation, some hSHP-1 migrates into the nucleus and attaches to the nucleus. This phenomenon is observed not only in oral cancer, but also in malignant cells such as gastric cancer and liver cancer. This anticancer activity may be explained as an early stage of anticancer activity, but hSHP-1 production is rapidly stopped in the late-stage cancer cells because hSHP-1 production is rapidly reduced in malignant cells that have not been differentiated. 1 It is difficult to expect continuous anticancer or anticancer action.

The anticancer effect of hSHP-1 is a result of the antiproliferative action of hSHP-1, which inhibits early cell dysplasia with increased proliferation. On the other hand, in abnormally proliferating precancerous lesions or early cancer cells, citric oxidation and glycosylation of hSHP-1 are not easily achieved due to increased production of lactic acid due to hypoxia and excessive consumption of ATP. hSHP-1 is not excreted outside the cell and accumulates in the cytoplasm. And as the accumulated hSHP-1 gradually migrates into the nucleus and attaches indiscriminately to the DNA of the nucleus, the activity of the cell is rapidly decreased and the cell disappears. At this time, the cells severely damaged by the nuclear nucleus by hSHP-1 can be removed by premature dropout from the mucosal epithelium and early cancer cells showing abnormal proliferation by early dropout from the mucosal epithelium before the cell death process.

Investigation of the effect of hSHP-1 on cultured cells in in vitro cell culture showed that the carboxy terminal 23 amino acids with the smallest peptide with antimicrobial activity were slightly weaker in anticancer activity and corresponded to the 12-46th hSHP-1 protein sequence. The 35 amino acid peptide showed stronger anticancer activity.

Cell culture experiments using 35 amino acids of hSHP-1 (C-35: SEQ ID NO : 8) peptides revealed that gingival fibroblasts, periodontal ligament fibroblasts, and human skin Immortalized cell lines obtained from keratinocytes of the epithelium were inhibited by more than 30% of cell proliferation within 1 day, oral cancer cell line, human osteogenic sarcoma (HOS), gastric cancer cell line (SNU-1), etc. In cancer cells of hSHP-1, the C-35 peptide inhibited cell proliferation was observed. In the same cell experiments using the C-23 peptide of hSHP-1, it was observed that the C-23 peptide of hSHP-1 rapidly migrated into the nucleus of the cell, but the inhibition of cell proliferation was somewhat low (FIG. 17).

On the other hand, using SNU-1, HOS, and RHEK, we identified genetic material expressing these cells during anti-cancer activity of C-35 peptide of hSHP-1. As a result, PCNA, CDK4, c were identified by C-35 peptide. -Expression of cell proliferation factors such as erbB2 and EGFR is drastically reduced, and apoptosis such as caspase-9, caspase-8, caspase-3, FAS, p53, Bax, BAD, FADD, etc. Expression of related genetic proteins was greatly increased, and genetic proteins involved in cell degeneration such as MMP-3, MMP-9, and MMP-10 were also rapidly increased. As a result, it is observed that the C-35 peptide of hSHP-1 can effectively inhibit the proliferation of proliferative immortalized cells or cancer cells and kill some cancer cells.

Therefore, hSHP-1 increases cytoplasmic accumulation in proliferative cells or cancer cells based on the above experimental results, and hSHP-1 binds indiscriminately and strongly to the nucleus of these cells, thereby inhibiting cell proliferation and causing cell death. (Fig. 18).

8. Immunostimulating Action of hSHP-1

HSHP-1, a cationic short helical peptide, adheres firmly to antigenic substances, and hSHP-1 has the property of cross-linking to the mucosal epithelial surface. Can act as an attachment. Some mucosal epithelium is responsible for transferring the attached antigenic material by endocytosis and then delivering it to nearby immune cells, such as cells of the cracked mucosal tissue of the tonsil or cracked mucosal tissue of the intestinal membrane. Commonly observed.

9. Classification of hSHP-1 Peptides

Peptides of the useful portion of hSHP-1 depend on the inclusion of unit amino acids involved in antimicrobial or anticancer activity. First, the amino terminus N-11 peptide is very hydrophobic and strongly adheres to the hydrophobic lipid component in the hydrophobic environment. Since the amino acid sequence of N-11 has a structure of a signal peptide, hSHP-1 is outside the cell. It is believed to be used to maintain the safety of hSHP-1 until it is released and delivered to the site of use. The main amino acid sequence with antimicrobial minimum size of ³⁰ SRVL1NRSLQVKVVKIT ⁴⁵ contains two arginine (R) and two lysine (K). Usually C-23 peptide (SEQ ID NO: 6) Since SSVSS has the amino acid sequence, it can be glycosylated by citric acid and is suitable as an antimicrobial peptide because it creates a sustained α-helical structure. In addition, C-23 peptides were tested for immunogenicity, and thus the antigenicity of the C-23 peptides was very low. Therefore, even if the human peptide C-23 is continuously used in a large amount in humans, it is very unlikely that immunological side effects occur for the C-23 peptide.

On the other hand, the C-35 peptide (SEQ ID NO: 8) has a sustained α-helix structure containing the 19th amino acid, cysteine (C), and is very important because it disulfides two hSHP-1 peptides. It can be used as a functional peptide. In particular, since the C-35 peptide shows anticancer function against proliferative cells or cancer cells, the C-35 peptide can be used as a multifunctional peptide.

Since the helical structure of hSHP-1 requires a sequence of at least eight amino acids to form a bi-rotating peptide, theoretically, the smallest peptide that exhibits minimal antimicrobial activity can use eight of the hSHP-1 amino acid sequences. The method allows the design of stronger antimicrobial proteins.

10. Production method of hSHP-1 recombinant protein

1) Recombinant Protein Production in Bacteria Using Plasmid Vectors

Since hSHP-1 is a short peptide with a small sequence, it is possible to construct a vector using a tandem repeated code. In this case, the optimization of DNA code is necessary to make human protein codes well in bacteria. Since hSHP-1 itself can inhibit the proliferation of bacteria that are host cells, fusion proteins such as KSI, His-tag, GFP, and GST can be attached to the hSHP-1 code to inhibit hSHP-1 function and hSHP- The pure separation of 1 should be used as a method to aid in chromatographic adhesion. The vector used in this method can be used most commercially available vectors such as pET32b, pET series. When bacteria produce recombinant proteins, peptides are produced that do not glycosylate or disulfide the protein. Thus, purely isolated hSHP-1 peptides are inevitably glycosylated and sulfided.

2) Pichia pastoris Protein production

Pichia pastoris belonging to yeast has better intracellular glycosylation and relatively low amount of endotoxin compared to bacteria, thus producing pure functional hSHP-1 peptide. The vector used in this method can be used for most yeast vectors that are commercially available, in which case the growth of the yeast is relatively fast, it is possible to produce a relatively large amount of recombinant protein.

3) Recombinant protein production in insect cells using Baculovirus

The bacterial vector baculovirus can be produced by infecting insect cells with a recombinant protein to produce hSHP-1 peptide. In this case, since the glycosylation and sulfidation of the produced peptides are relatively well performed, it is possible to produce a highly functional hSHP-1 recombinant protein. Insect cells have relatively high proliferation, low endotoxin production, and convenient handling in culture, while host cells recognize hSHP as a different functional protein, which promotes antimicrobial, anti-cancer and immunity of hSHP through modified glycation or sulfidation. A tendency to decrease was found. Therefore, attention is required to the conditions of cell culture, harvesting time, and pure purification method.

4) Production of recombinant protein in animal cells using CMV vector

It is a method of producing hSHP-1 recombination protein by inserting a vector using a CMV promoter directly into immortalized cells such as humans, mice, and cows or cancer cells which are commonly used in cell culture. Even in this case, saccharification and sulfidation of hSHP-1 is performed smoothly, but hSHP-1 is not glycosylated in high proliferative host cells and accumulates in the cells, and thus may have an anticancer effect of inhibiting and killing the proliferation of host cells. Production of sufficient amounts of hSHP-1 can be difficult. In addition, animal cells are difficult to produce large amounts of hSHP-1 due to low cell division rate and protein production rate compared to bacteria or yeast.

11. How to separate hSHP-1 in human secretion

It is observed that human secreted hSHP-1 is made into a multifunctional protein through glycosylation and disulfide bond in secreted cells and then discharged out of the cells. Therefore, recombinant proteins made from prokaryote bacteria lack glycosylation and disulfide bonds during the production of general recombinant proteins, and recombinant proteins made from yeast or animal cells, which are eukaryotes, have incomplete glycosylation and disulfide bonds. Because of this, hSHP-1 produced in the human glands has a well-formed molecular structure, so that the antibacterial and anticancer functions necessary in the human body can function very uniquely. Therefore, hSHP-1 can be separated from the body fluids directly secreted directly from humans and used for appropriate purposes.

Among human secretions, hSHP-1 produces a large amount of organs such as salivary glands, lacrimal glands, vaginal fluid, prostate, mammary gland, sebaceous glands, and gastrointestinal fluids. Among these, salivary glands, tears, etc. can be obtained relatively easily. In the case of salivary glands, for example, the amount of saliva produced by adults is about 1.5 L per day, and a large amount of saliva is produced. In the salivary glands, a large amount of hSHP-1 is produced in the parotid gland, and the submandibular, sublingual, And production of hSHP-1 in the order of butadioscrum and the like. Western blot analysis of the components of hSHP-1 contained in the saliva showed that the amount of hSHP-1 compared to the initial mixed saliva collected by collecting parotid saliva collected directly from the parotid catheter at once. Was increased, but there was not much difference in composition. However, compared to parotid and early mixed needles, SDS-gel electrophoresis abnormalities were observed in the mixed needles that had been secreted into the mouth for more than one minute. As a result, a sufficient amount of hSHP-1 can be separated and purified by obtaining a large amount of the initial mixing needle from several people.

There are various methods to purify hSHP-1, in order to prevent the degrading or denaturation of collected saliva, first mix 10 mL of saliva with 1 mL of 0.5 M citric acid (pH 4.0) and 20 μL of 0.5 M EDTA. Deactivates proteolytic enzymes. hSHP-1 is highly acid resistant and can withstand acidity below pH 4.

In order to remove excessively glycated mucin or PRP protein, two-fold 100% ethanol was added, mixed well, and left to stand at -20 ° C. freezer for 1 hour, followed by centrifugation to obtain a precipitate. Shake the precipitate with distilled water to dissolve. At this time, the solid component which is not dissolved is discarded and the supernatant is used for pure separation of hSHP-1. Since a considerable amount of hSHP-1 also exists in this solid component, the pure separation of hSHP-1 can be carried out using the distilled water dissolution solution performed in the 2nd and 3rd case depending on the case.

Ion exchange chromatography, gel filtration chromatography, HPLC and the like can be used to obtain the water-soluble hSHP-1 obtained above. For ion exchange chromatography, glycosylated hSHP-1 can be isolated using Tris-acetate buffer, pH 6.0, mainly using SP columns (Pharmacia, USA). In the case of separating hSHP-1 by gel filtration or HPLC method, the separation and purification were repeated immediately after confirming the peak of hSHP-1 through experiments such as ELISA, dot blot, and western blot. Pure glycosylated hSHP-1 can be obtained.

The glycosylated hSHP-1 of the human secretion thus obtained includes a number of early glycated proteins (6 kDa), heavily glycosylated hSHP-1 (30-50 kDa), and hSHP-1 (53-70 kDa) cross-linked with other proteins. Types are isolated, all of which are positive for hSHP-1 antibodies.

On the other hand, it is possible to isolate and purify hSHP-1 in secretion using the antibody of hSHP-1, in which case the amount of hSHP-1 to be separated is limited. For this method, the glycosylation component in the heavy chain of the antibody is substituted with the amide of the beads to crosslink the hSHP-1 antibody to the beads. Using the prepared hSHP-1 antibody column, hSHP-1 contained in the secretion was attached to the antibody, and then separated by citric acid (citric acid) at pH 2.5 to purify hSHP-1.

Since hSHP-1 is not denatured even in acidic solution of pH 2.5, it can be stored as it is, but it is stored after dialysis and lyophilization to remove acid and base depending on the purpose of use.

12. Application of hSHP-1 Protein

1) antibacterial action

C-16 peptide, the smallest antimicrobial peptide of hSHP-1, may be biochemically synthesized or produced as a recombinant protein and administered directly to the site of infection. Longer hSHP-1 peptides can be used to increase antimicrobial activity and increase antimicrobial duration, approximately C-19 (SEQ ID NO: 4), C-21 (SEQ ID NO: 5), C-23 (SEQ ID NO: 6 sequences), C-30 (SEQ ID NO: 7), C-35 (SEQ ID NO: 8) Use of peptides is effective.

hSHP-1 has strong antimicrobial activity against both Gram-positive and Gram-negative pathogenic bacteria. E. coli , a human resident bacterium, has low antibacterial activity compared to other pathogenic bacteria and can be used as a safe drug against resident bacteria.

hSHP-1 has the same antimicrobial activity against methicillin-resistant bacteria, antimicrobial activity in neutral solutions like saliva in the mouth and tears in the eyes, and hSHP-1 activity is not reduced in acidic solutions such as stomach. Without strong antibacterial action. And since it has the same antimicrobial activity in a hydrophobic environment like sebaceous glands, it is a human antibacterial protein that can be widely used in the body.

Use of glycosylated hSHP-1 with citric acid is necessary to inhibit degradation of hSHP-1 and to enhance antimicrobial activity. In addition, it is speculated that two hSHP-1 form a dimer and have a complex SHP structure for the antibacterial action. Therefore, dimer formation and citrate glycosylation of hSHP-1 are necessary for the continuous maintenance of hSHP-1 in solution. This hSHP-1 is observed in the native form of human secretion (native form) (hSHP-1) is an inherent antimicrobial activity against the virus as well as bacteria, inhibits the formation of bacterial biofilms and remove the biofilms It can be used as a highly functional material.

In general, the synthetic peptide or recombinant protein of hSHP-1 can be used as a wide range of alternative solutions or therapeutics, such as tears, saliva, runny nose, gastrointestinal fluid, vaginal fluid, prostate fluid, sweat. hSHP-1 requires complex formulations to maintain stability and functionality according to the peptide properties mentioned above.

2) anticancer action

The anticancer effect of hSHP-1 is a result of the antiproliferative action of hSHP-1, which inhibits early cell dysplasia with increased proliferation. In abnormally proliferating precancerous and early cancer cells, hSHP-1 is not easily produced by citricization and glycosylation of hSHP-1 due to increased production of lactic acid by hypoxia and excessive consumption of ATP. It is not excreted outside the cell and accumulates in the cytoplasm. As the accumulated hSHP-1 gradually migrates into the nucleus and attaches indiscriminately to the DNA of the nucleus, the activity of the cell is rapidly decreased and the cell is extinguished. At this time, the cells severely damaged by nuclei by hSHP-1 can be removed from the mucous membrane of precancerous lesion cells or early cancer cells showing abnormal proliferation through the early dropout from the mucosal epithelium before the cell death process.

In the case of chemical synthesis peptide or recombinant peptide of hSHP-1, cell culture using hSHP-1 which is artificially glycosylated, hSHP-1 easily enters the cell and gathers into the nucleus, thereby making hSHP-1 a precancerous lesion or anticancer It can be used as a therapeutic.

The anti-cancer effect can be expected by using ointment containing hSHP-1 on skin or easily accessible mucous membranes in the eyes or mouth.If it is difficult to access such as the gastrointestinal tract, hSHP can be taken by taking oral medication periodically. Anticancer effect of -1 can be expected.

The hSHP-1 chemotherapy is effective in precancerous lesions or early cancer cells where hSHP-1 can easily penetrate into the cell, but when the cell wall is thickened by unclear glycoproteins such as malignant cancer cells and anoxia increases in the cytoplasm. HSHP-1 is not easily penetrated into cells, so the effect is difficult to expect. In this case, the anti-cancer activity of hSHP-1 can be induced by transferring hSHP-1 to malignant cancer cells using a mutant virus that selectively infects proliferative cancer cells but not secondary infection using viral vectors.

3) immune promoting action

HSHP-1, a cationic short helical peptide, adheres firmly to antigenic substances, and hSHP-1 has a property of cross-linking to mucosal epithelial surfaces. It can hold a sieve and attach it to mucosal cells. Some mucosal epithelium is responsible for transferring the attached antigenic material by endocytosis and then delivering it to nearby immune cells, such as cells of the cracked mucosal tissue of the tonsil or cracked mucosal tissue of the intestinal membrane. Commonly observed.

When a specific antigen is attached to hSHP-1, taken as a drug, or attached to thin skin or mucous membranes for a long time with an ointment, waxy effects that restore immune function can be obtained.

Depending on the nature of the specific antigen, conditions such as peptide size, glycosylation and disulfide dimer formation of hSHP-1 can be used. For small antigens such as, for example, influenza virus encapsulation, partially glycosylated short hSHP-1 can be used to promote immune delivery through mucosal cells.

Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation of carbohydrate compounds, such as lactose, amylose, dextrose, sucrose, sorbitol, mannitol, starch, cellulose, and the like. ), Acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, salt solution, alcohol, gum arabic, vegetable oil (e.g. corn oil, cotton Seed oil, soy milk, olive oil, coconut oil), polyethylene glycol, methyl cellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil, and the like. The pharmaceutical composition of the present invention further includes, but is not limited to, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, and the like, in addition to the above components.

The pharmaceutical composition of the present invention may be administered transdermally, orally or parenterally, and in the case of parenteral administration, it may be administered by intravenous infusion, subcutaneous infusion, intramuscular infusion, or the like.

Suitable dosages of the pharmaceutical compositions of the invention vary depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, condition of food, time of administration, route of administration, rate of excretion and response to reaction, Usually a skilled practitioner can easily determine and prescribe a dosage effective for the desired treatment or prophylaxis. According to a preferred embodiment of the invention, a suitable dosage is 50 mg-10 g once daily on an adult basis to be.

The pharmaceutical compositions of the present invention may be prepared in unit dosage form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporation into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or an aqueous medium, or may be in the form of extracts, powders, granules, tablets or capsules, and may further include a dispersant or stabilizer.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, and the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention to those skilled in the art to which the present invention pertains. Will be self-evident.

Example 1 Crosslinking Ability and Expression Characteristics of hSHP-1

Since hSHP-1 has a cross-linking ability by transgurutamine at the carboxy terminus, hSHP-1 has a structure capable of firmly binding to cell membranes of mucosal cells in other protein components or mucous membranes in saliva. To test this, oral dropping epithelium was obtained in saliva, and they were boiled at 100 ° C for 2 hours in 8 M urea, 1% SDS, 2 mM EDTA and 20 mM mercaptoethanol solution to observe the dissolution of protein. After boiling repeatedly until no, paraffin tissue samples were prepared by conventional method, and immunohistochemical staining was performed using hSHP-1 antibody. As a result, hSHP-1 was strongly expressed on the surface of the outer epithelium of the oral dropping epithelium. It was. Meanwhile, in order to confirm that hSHP-1 is used as a substrate of transgurutaminase, transgurutamine was added to a solution obtained by mixing hSHP-1 with monodansylcadaverine, a well-known substrate of transgurutaminease. An experiment was conducted to react the naze. As a result, it was observed that hSHP-1 cross-linked with monosingyl cardaberate depending on calcium ion by transgurutaminase (FIG. 6).

On the other hand, hSHP-1 is expressed linearly on the surface of the outer epithelial cells of various human epithelial tissue epithelium, especially in the large intestine mucosa. 9).

Example 2: Antibody Production Against hSHP-1

hSHP-1 is a short protein consisting of 46 amino acids. Since 11 amino acids at the amino terminus are assumed to be cleaved as signal peptides, antibodies can be produced using 35 amino acids. Although the 23 amino acids in the carboxyl terminus have an actual antimicrobial effect, these 23 amino acids have very low antigenicity, making antibody production difficult. Therefore, an antibody can be produced using the peptide containing the amino acid corresponding to amino terminal 13-23 with high antigenicity of hSHP-1. Antibodies to hSHP-1 can produce both dark and monoclonal antibodies. In the case of polyclonal antibodies, most animals such as rabbits, goats, cows, mice, and white papers can be used, and when the carboxy terminal 35 amino acids are used, a monospecific antibody is produced. In the case of monoclonal antibodies, highly specific antibodies can be produced. For example, sustained monoclonal antibodies can be produced using myeloma hybrids in mice.

Example 3: Recombinant hSHP-1 Preparation

1) Recombinant Protein Production in Bacteria Using Plasmid Vectors

Since hSHP-1 is a short peptide with a small sequence, it was possible to construct a vector using a tandem repeated code. In this case, the optimization of DNA code is necessary to make human protein codes well in bacteria. Since hSHP-1 itself can inhibit the proliferation of bacteria that are host cells, fusion proteins such as KSI, His-tag, GFP, and GST are attached to the hSHP-1 code to inhibit hSHP-1 function and It was used as a method to help chromatographic adhesion during separation. The vector used in this method can be used most commercially available vectors such as pET32b, pET series. When bacteria produce recombinant proteins, peptides are produced that do not glycosylate or disulfide the protein. Thus, purely isolated hSHP-1 peptides are inevitably glycosylated and sulfided.

2) Recombinant Protein Production from Pichia pastoris

Yeast Pichia pastoris has better intracellular glycosylation and relatively low endotoxin amount compared to bacteria, which is advantageous for the production of pure functional hSHP-1 peptide. The vector used in this method can be used for most yeast vectors that are commercially available, in which case the growth of the yeast is relatively fast, it is possible to produce a relatively large amount of recombinant protein.

3) Recombinant protein production in insect cells using Baculovirus

HSHP-1 peptides can be produced by infecting insect cells with baculoviruses to produce recombinant proteins. In this case, since the glycosylation and sulfidation of the produced peptides are relatively good, it is possible to produce a highly functional hSHP-1 recombinant protein. Insect cells have relatively high proliferation, low endotoxin production, and convenient handling in culture, while host cells recognize hSHP as a different functional protein, which promotes antimicrobial, anti-cancer and immunity of hSHP through modified glycation or sulfidation. A tendency to decrease was found. Therefore, attention has to be paid to the conditions of cell culture, harvesting time and pure purification method.

4) Production of recombinant protein in animal cells using CMV vector

It is a method of producing hSHP-1 recombinant protein by inserting a vector using a CMV promoter directly into immortalized cells such as humans, mice, and cows or cancer cells commonly used in cell culture. Even in this case, saccharification and sulfidation of hSHP-1 is performed smoothly, but hSHP-1 is not glycosylated in high proliferative host cells and accumulates in the cells, and thus may have an anticancer effect of inhibiting and killing the proliferation of host cells. Production of sufficient amounts of hSHP-1 can be difficult. In addition, animal cells had a difficulty in producing large amounts of hSHP-1 due to the low rate of cell division and protein production compared to bacteria or yeast.

Example 4: Isolation of hSHP-1 Complex from Human Mucus

hSHP-1 has been shown to function as a complex by combining with other proteins in human mucus. It is difficult to separate and purify only hSHP-1 protein from human saliva, so hSHP-1 conjugated saliva protein complex Was extracted. Extraction method has a method of extracting directly from saliva using hSHP-1 antibody column, but there was a limit in mass production. Therefore, the characteristics of hSHP-1 protein were used to make a large amount in mucus.

First, since hSHP-1 has a spiral structure and hydrophobicity and hydrophilicity are repeated, a property of very high adhesion is used. For example, to separate hSHP-1 from parotid or mixed saliva, first use ethanol precipitation to convert 70% ethanol, mucin, and proline rich proteins in saliva into 70% ethanol. The precipitate was removed and the supernatant was precipitated with increasing ethanol in order of 80 and 90%, respectively, to collect hydrophobic proteins and the remaining supernatant was dried to collect strong hydrophobic proteins. Among these proteins, the hSHP-1 content was the highest in the protein dissolved in 90% or more, and the hSHP-1 content was decreased in the order of 90 and 80%. The saliva protein thus obtained was separated using a cationic ion exchange column (SP-column) to show a cationic protein. Among them, histatin and basic proline rich proteins were still included, so HPLC purification was performed using a C-8 column using hSHP-1's non-specific binding properties of the helical structure. Hydrophilic fluid was used as distilled water, hydrophobic fluid was used as methanol and 0.01% of trifluoroacetate was added to each solution. In this case, most of the hSHP-1 binding needle complexes are attached to the C-8 column so that some of them dissolve during the methanol flow to separate and show complex peaks. This means that the hSHP-1 binding needle complex forms a variety of structures with a wide variety of heterologous proteins. A large amount of hSHP-1 deficient saliva complexes were sufficiently attached to the C-8 column, dissolved in acetic acid solution at pH 2.5, collected together, lyophilized, and 0.1 M DTT solution was added, followed by incubation at 70 ° C. for 20 minutes to disulfide bonds. Disconnected. The hSHP-1 binding needle complex was separated and purified using a silicate gel filtration column having low hydrophobic adsorption. The reduced hSHP-1 binding saliva complex has a high activity of multifunctional protein function, so it can be lyophilized or stored at 4 ° C. and immediately tested for antibacterial and anticancer activity.

Example 5: Antimicrobial Activity of hSHP-1

1) antibacterial action against bacteria and fungi

hSHP-1 is estimated to have 16 helixes in total, of which 11 helices have unique antimicrobial activity. The hydrophobic helix of the amino terminus is strongly attached to bacteria and the carboxyl terminus has a glutamine-lysine cross-linking motif by transglutamine, which is firmly attached to the mucosal membrane or the surface of major cells. It is estimated that the major antibacterial action of hSHP-1 is that the helical structure of the 12th-45th amino acid has strong adhesion to the bacterial cell membrane and is inserted into the cell membrane to make holes in the cell wall and destroy the cells. The smallest peptide (16 peptides; SRVLNRSLQVKVVKIT) showing the same antimicrobial activity as hSHP-1 had a positive charge value of +4. After using the carboxy-terminal 23 peptide of hSHP-1 observed antibacterial action, Staphylococcus aureus, Candida albicans, Aspergillus niger, a strong sterilizing power (hSHP-1, C-23 mer, MIC against pathogens Pseudomonas aeruginosa including: about 10 μM / 10 ⁷ cells) and a moderate bactericidal power (MIC: about 100 μM / 10 ⁷ cells) with respect to Escherichia coli , a human resident bacterium (FIG. 7).

On the other hand, hSHP-1 showed the same antibacterial activity and no Staphylococcus aureus is resistant even in Staphylococcus aureus that are resistant to methicillin. Helicobacter pylori , a causative agent of human gastritis, showed moderate bactericidal activity (MIC: about 150 μM / 10 ⁷ cells).

2) antibacterial action against viruses

hSHP-1 has two serine-serine (SS) motifs to which citric acid is attached, and when two hSHP-1s are linked by disulfide bonds to form dimers, glycosylation by multiple citric oxidation (sialidation) Made a glycoprotein. This glycosylated hSHP-1 has the function of an antivirus (glycosylated basic proline rich protein). Thus, it has strong adhesion to capsular viruses and cationic SHP shows effective antiviral function by damaging the capsular membrane.

3) suppression and removal of biofilm production

Since glycosylated hSHP-1 has adhesion to the biofilm formed by bacteria or fungi, it not only acts as an antimicrobial agent against bacteria in the biofilm, but also inhibits the formation of the biofilm and relaxes the structure of the biofilm, thereby inhibiting the production of new biofilm. It has the function of removing the existing biofilm.

4) Treatment for hSHP-1 Deficiency

Therefore, antimicrobial activity against bacteria, fungi and viruses could be expected very effectively if hSHP-1 function was continuously glycated on human mucous membranes or skin.

In particular, in the case of senile or degenerative diseases or autoimmune diseases, the secretion of the secretion from the various mucous membranes and skin is remarkable. In this case, multiple infections caused by various pathogens may occur due to the decreased secretion of hSHP-1. In this case, supply of hSHP-1, one of the components deficient in mucus, is required. In order to determine the hSHP-1 deficiency in the above patients, ELISA, dot blot, Western blot, etc. can be used as an assay for measuring the concentration of hSHP-1 in mucus secretions. As a test that can test the antimicrobial activity against bacteria by using a bacterial killing assay (killing assay) using a medium can be determined by the Minimum Inhibitory Concentration (MIC) value.

C-16 peptide, the smallest antimicrobial peptide of hSHP-1, may be biochemically synthesized or produced as a recombinant protein and administered directly to the site of infection. Longer hSHP-1 peptides can be used to increase antimicrobial activity and increase antimicrobial duration, approximately C-19 (SEQ ID NO: 4), C-21 (SEQ ID NO: 5), C-23 (SEQ ID NO: 6 sequences), C-30 (SEQ ID NO: 7), C-35 (SEQ ID NO: 8) Use of peptides is effective.

hSHP-1 has strong antimicrobial activity against both Gram-positive and Gram-negative pathogenic bacteria. E. coli, a human resident bacterium, has low antibacterial activity compared to other pathogenic bacteria and can be used as a safe drug against resident bacteria.

hSHP-1 showed the same antimicrobial action against methicillin-resistant bacteria, antimicrobial action in neutral solution like saliva in mouth or tear in eye, as well as strong antibacterial activity in hSHP-1 in acidic solution such as stomach. It showed action. And since it has the same antimicrobial activity in a hydrophobic environment like sebaceous glands, it is a human antibacterial protein that can be widely used in the body.

Use of glycosylated hSHP-1 with citric acid is necessary to inhibit degradation of hSHP-1 and to enhance antimicrobial activity. In addition, it is speculated that two hSHP-1s form a dimer and have a complex SHP structure. Therefore, dimer formation and citrate glycosylation of hSHP-1 are necessary for the continuous maintenance of hSHP-1 in solution. This hSHP-1 is observed in the native form of human secretion (native form) (hSHP-1) is an inherent antimicrobial activity against the virus as well as bacteria, inhibits the formation of bacterial biofilms and remove the biofilms It can be used as a highly functional material.

In general, the synthetic peptide or recombinant protein of hSHP-1 can be used as a wide range of alternative solutions or therapeutics, such as tears, saliva, runny nose, gastrointestinal fluid, vaginal fluid, prostate fluid, sweat. hSHP-1 requires complex formulations to maintain stability and functionality according to the peptide properties mentioned above.

   Example 6: Anticancer Activity of hSHP-1

The anticancer effect of hSHP-1 is a result of the antiproliferative action of hSHP-1, which inhibits early cell dysplasia with increased proliferation. In abnormally proliferating precancerous or early cancer cells, the increased production of lactic acid by hypoxia and excessive consumption of ATP prevent the easy cSylation and glycosylation of hSHP-1. It was not excreted and accumulated in the cytoplasm. And as the accumulated hSHP-1 gradually moved into the nucleus and attached to the DNA of the nucleus indiscriminately, the activity of the cell was rapidly decreased and the cell was killed (FIG. 15). At this time, the cells severely damaged by the nucleus by hSHP-1 can be removed from the mucous membrane of precancerous or early cancer cells showing abnormal proliferation through the early dropout of the mucosal epithelium before the cell death process (FIG. 14). .

In the case of chemical synthesis peptide or recombinant peptide of hSHP-1, cell culture using hSHP-1 which is artificially glycosylated, hSHP-1 easily enters the cell and gathers into the nucleus, thereby making hSHP-1 a precancerous lesion or anticancer It can be used as a therapeutic agent (FIGS. 14, 15, 16).

The anti-cancer effect can be expected by using ointment containing hSHP-1 on skin or easily accessible mucous membranes in the eyes or mouth.If it is difficult to access such as the gastrointestinal tract, hSHP can be taken by taking oral medication periodically. Anticancer effect of -1 can be expected.

The hSHP-1 chemotherapy is effective in precancerous lesions or early cancer cells where hSHP-1 can easily penetrate into the cell, but when the cell wall is thickened by unclear glycoproteins such as malignant cancer cells and anoxia increases in the cytoplasm. HSHP-1 is not easily penetrated into cells, so the effect is difficult to expect. In this case, the anti-cancer activity of hSHP-1 can be induced by transferring hSHP-1 to malignant cancer cells using a mutant virus that selectively infects proliferative cancer cells but not secondary infection using viral vectors.

   Example 7: Immunostimulatory Activity of hSHP-1

HSHP-1, a cationic short helical peptide, adheres firmly to antigenic substances. Since hSHP-1 has the property of crosslinking to the mucosal epithelium, it catches antigens flowing around mucosal cells and attaches them to mucosal cells. Can play a role. Some mucosal epithelium plays a role in delivering the attached antigenic material by endocytosis to nearby immune cells, which is commonly found in cells of tonsil fissured mucosa or cells of intestinal mucosa.

When certain antigens are attached to hSHP-1, taken as drugs, or attached to thin skin or mucous membranes for an extended period of time with ointments, waxy effects that restore immune function can be obtained. Depending on the nature of the specific antigen, conditions such as peptide size, glycosylation and disulfide dimer formation of hSHP-1 can be used. For small antigens, such as, for example, influenza virus cysts, partially glycosylated short hSHP-1 can be used to promote immune delivery through mucosal cells.

Since hSHP-1 of the present invention is an antibacterial and anticancer protein secreted by humans, there is no possibility of an allergic reaction immunologically and showed a very strong antimicrobial activity compared to other antibacterial proteins of humans. It has the strongest antimicrobial activity among human antimicrobial proteins found to date and is a broad spectrum of antimicrobial proteins observed in most glands and mucosal tissues of humans as well as saliva, and can cause infectious mucosal diseases when deficient. Thus hSHP-1 could be used critically for the prevention and treatment of infectious mucosal diseases.

In addition, the hSHP-1 of the present invention acts on proliferative precancerous tumor cells or cancer cells by structural specificity, inhibits proliferation, induces early dropout or early apoptosis of cancer cells, thereby inhibiting early cancer occurrence and invading cancer cells. The anticancer effect of hSHP-1 is very high because it is observed that antiproliferative effect is observed by the effect of reducing proliferation.

In addition, the hSHP-1 of the present invention is a structurally helical peptide, strong hydrophobic and positive cationic charge characteristics, and is firmly attached to other proteins while forming disulfide bonds and glutamine-lysine crosslinks. It has been observed that hSHP-1 not only adheres to mucosal epithelium but also cross-links with intercalating proteins, forming a salivary complex. In particular, hSHP-1 reacts rapidly to complex foreign antigens that are difficult to recognize and forms complexes and attaches to the mucosal epithelium. By attaching the antigen complexes to the mucosal epithelium, they can promote mucosa-associated immunity mechanisms.

Attach sequence list electronic file

Claims (12)

Peptides having anticancer activity or immunostimulating activity selected from the group: (a) the N-terminus of the peptide is selected from any of the 12 th to 31 th amino acid residues of SEQ ID NO: 2, and the C-terminus of the peptide is 45 th or A peptide selected from the 46th amino acid residue and is 16-mer to 23-mer, (b) tandem repeat of the peptide (a). Peptide having an antimicrobial activity, anticancer activity or immune promoting activity, characterized in that any one of the peptides of SEQ ID NO: 3 to 7 or tandem repeat thereof. The peptide according to claim 1 or 2, wherein the peptide is glycosylated. A polynucleotide encoding the peptide of claim 1. delete delete delete A pharmaceutical composition for preventing or treating precancerous lesions or cancer, comprising the peptide of claim 1 or 2 as an active ingredient. The pharmaceutical composition of claim 8, wherein the cancer is selected from skin cancer, osteosarcoma, oral cancer, gastric cancer, intestinal cancer, brain tumor or liver cancer. Claim 1 or claim 2, wherein the peptide and the antigen as an active ingredient, mucosal immune function enhancing pharmaceutical composition. The antimicrobial household article comprising the peptide of claim 2 as an active ingredient, which is a cosmetic, toothpaste, skin cleaner, feminine cleanser, artificial tears used for dry eyes, artificial saliva used for dry mouth, nasal dry An antibacterial household article, which is selected from artificial runny nose, food additives or feed additives used. delete

Images