KR100859586B1 - The novel M cell target peptide - Google Patents

Abstract

The present invention comprises the steps of forming a phage display peptide library; in establishing an in vitro M cell model; Searching for M cell target peptides; in in vitro Phage transcytosis analysis step; Identifying the peptide sequence; In the step of re-validating the M cell target candidate peptide and selecting the target peptide in In vitro phage display method and the above in CKSTHPLSC peptide selected using in vitro phage display method. Since the CKSTHPLSC peptide selected in the present invention can induce the uptake of macromolecular substances through M cells in the mucosal layer of the small intestine, there is an excellent effect of providing an oral vaccine delivery system.

Oral Vaccine Delivery System, Transcytosis, Phase Patch, Phage Display, Absorption Inducing Peptide

Description

The novel M cell target peptide

1 is a schematic diagram illustrating a process for selecting a transcytosis-inducing peptide functional group using a trans-well cell culture system.

Figure 2 shows a schematic diagram of the in vitro phage display biopanning process of the present invention.

3 is a graph measuring the electrical resistance value of the Caco-2 cells of the present invention by culturing in a trans-well.

Figure 4a is a mixed culture of Caco-2 cells and Raji cells of the present invention in in vitro FITC-bound latex beads that passed through M cells were measured using a flow cytometer. FIG. 4B is a photograph showing the expression of beta-1 integrin in Caco-2 cells, and FIG. 4C is a diagram of beta-1 integrin in M cells. 4D is a photograph showing the expression of microvilli in Caco-2 cells, and FIG. 4E is a photograph showing the movement of Raji cells to Caco-2 cells.

Figure 5 shows a schematic diagram of the M cell target peptide selection process of the present invention.

Figure 6 is a graph showing the binding assay of M cell target peptide to Raji cells of the present invention.

Figure 7 of the present invention in The exploration of M cell target peptide functional groups using in vitro M cell model and phage transcytosis technique is shown schematically.

Figure 8a is a graph showing the biopanning of Caco-2 cells of the present invention, Figure 8b is a graph showing the biopanning of M cells.

9 is a schematic diagram showing the process of identifying the phage population recovered in the biopanning process of the present invention.

Figure 10a is a graph showing the transcytosis analysis in Caco-2 cells of the M cell target candidate peptide functional group of the present invention, Figure 10b is a graph showing the transcytosis analysis in M cells.

11 is a schematic diagram showing the immunohistochemical analysis of the CKSTHPLSC phage of the present invention.

12A and 12B are fluorescence micrographs showing the influx of peptides when the CKSTHPLSC peptide is injected into the villi of the small intestine of the present invention, and FIGS. 13C and 13D are M cell sites when the CSKSSDYQC peptide is injected into the villi of the small intestine of the rat This is a fluorescence microscope picture of peptides introduced.

FIG. 13A is a schematic diagram of an oral protein vaccine incorporating the transcytosis inducing peptide of the present invention, and FIG. 13B is a schematic diagram of absorption of M protein of a protein vaccine using transsitesis.

The present invention relates to a highly efficient absorption inducing peptide that passes through M cells without passing through the epithelial cell layer of the small intestine. More specifically, the present invention in The present invention relates to a highly efficient absorption inducing peptide that passes through M cells without passing through the epithelial cell layer of the small intestine selected using in vitro phage display method.

In general, the path of infectious agents from the outside into the body is divided into the oral cavity, respiratory and genital tract and skin. Receptor mediated through the M cells in the follicle associated epithelium (FAE) of the Peyer's patch. It can be divided into pathways that enter between tight junctions between endicytosis and enterocytes of the small intestine that constitute villus. The mucosal immune response, unlike the systemic immune response, absorbs macromolecules and microorganisms that are transported into the intestinal tract, resulting in antigen-specific immune responses that finally pass through the small intestine's epithelial cells. Induce secretion of type IgA (secretory IgA).

The most effective route for the administration of vaccines for the prevention of diseases that enter the mucous membranes of the small intestine through M cells, such as waterborne infectious diseases such as cholera, typhoid fever, bacterial dysentery, etc., is the oral drug delivery that targets M cells. Is known to induce a mucosal immune response. Therefore, if it is possible to secure a functional (ligand) that specifically induces transcytosis in M cells, it can be applied to an efficient oral vaccine delivery system that can activate mucosal immune response. Development of an efficient drug and vaccine delivery system should be preceded in order to improve the low absorption rate, which is the biggest problem faced in the development of oral administration of protein-based drugs and vaccines, and to improve the compliance of patients or livestock.

Water-borne infectious diseases are transmitted through the digestive system through the food medium, such as typhoid fever, paratyphoid, bacterial dysentery, cholera, vibrio sepsis, etc., and are classified as group 1 statutory infectious diseases according to Article 2 of the Prevention Act. Until the industrialization, the damage caused by these infectious diseases spread around the world, and since then, it has been somewhat reduced, but the damage caused by these diseases is reported every summer. In recent years, the incidence of water-borne infectious diseases in Korea has increased significantly each year.

In the case of water-borne infectious diseases, high-efficiency oral vaccines are needed because they can only act to alleviate diarrhea or dehydration, which are symptoms of various therapeutic drugs.

Recently, several alternatives have been proposed to improve and solve these problems. Among these methods, the oral dosage method is the dosage form that best compensates for the disadvantages of the injection method because patients have less objection and do not need a separate device such as a skilled worker or a syringe. However, the oral dosage method has a number of advantages, but there are problems in practical use. The first is the restriction on the size of the substance, and protein-based drugs have macromolecules (macromolecule) to limit the membrane permeation. Next, the intestinal epithelium that passes through the digestive system of the living body loses its activity by the action of various digestive enzymes secreted from the digestive system, including the stomach. It is very difficult to be absorbed into the body by blocking the cell layer and to function properly. Oral administration should improve the problems such as low absorption rate of less than 1%, instability through the digestive tract, low membranous membrane permeability, even though it is the most ideal route of administration in terms of ease of administration and high drug compliance.

Recently, due to the development of biotechnology, various protein drugs are being developed as a next-generation drug to replace chemotherapy. Developing a highly efficient oral dosage system is an urgent task.

Specialized cells called M cells exist to absorb various antigens in the intestine through specific and nonspecific pathways, and play a pivotal role in secreting secreted IgA, the final product of mucosal immune response, through epithelial cells of the small intestine. .

Selecting a peptide targeting M cells and applying it to an oral vaccine may be a more efficient way to improve the low transport efficiency of existing protein-based drugs and vaccines. To date, the most desirable model of oral vaccines has been ideally thought to target M cells, but progress has been slow due to several technical limitations. In order to establish an efficient oral vaccine delivery system, an efficient cell culture model should be established in addition to the above-described technique for inducing transcytosis-inducing ligands. According to the previous study, human adenocarcinoma Cell line) Ca and M cells present in the small intestine phase patch when mixed culture of lymphocytes (primary culture) in the Peyer`s patch of BALB / c mice Several mechanisms that occur in cells reduce alkaline phosphatase, a brush-border enzyme secreted at the apical surfce of epithelial cells, and cytoskeletal secreted at the tip of microvilli. The rearrangement of the protein villin was reproduced and subsequent studies validated the M cell model (Kerneis, 1997). . Subsequently, studies to find peptide functional groups targeting M cells have been conducted using rat and phage display techniques (Higgins, 2004), but are limited to the process of selecting peptide functional groups targeting M cells and transporting candidate peptides. The efficiency was not considered. It is very difficult to mask the superiority by comparing the absorption efficiency at the cellular level individually for each known substance, so in order to overcome this, it is necessary to target the receptor that efficiently induces transcytosis. There is a need to establish new research techniques for exploring materials quickly and effectively.

Therefore, in addition to being used to select binding specific peptides, phage display technology may be usefully applied when selecting specific peptide sequences that induce transcytosis. In the prior art, the transcytosis of MDCK (Madin-Darby canine kidney) epithelial cells through a three-dimensional cell culture method using a M13 phage library is used to identify specific peptide sequences and integrin. It has been shown to have a motif that binds to (integrin) (Vasily, 2000). In the process, MDCK epithelial cells were cultured on a trans-well membrane to confirm that these cells formed an epithelial cell lining by tight bonding, and then a phage library was added to the top or the bottom of the opposite well ( The phage molecules passed through the wells are identified. Phage detected through the epithelial cell layer in the opposite well is likely to have a transcytotic peptide that induces transcytosis. It is selecting (refer FIG. 1). Therefore, this method was applied to an in vitro M cell model to devise a new method for selecting peptide sequences that induce M cell specific transcytosis.

Accordingly, the present invention is devised in view of the points described above, an object of the present invention in In vitro phage display method is used to provide specific peptides that induce the uptake of macromolecular substances through M cells.

Another object of the present invention is to select a specific peptide to induce the uptake of macromolecular substances through the M cells in in vitro A phage display method is provided.

Still another object of the present invention is to provide an oral vaccine delivery system in which a specific peptide is introduced into a protein vaccine to induce uptake of macromolecular substances through the M cells.

The above object of the invention is to form a phage display peptide library andin in vitro Establish an M cell modelin in vitro Phage Transcytosis Mechanismin in vitro This was accomplished by searching for a peptide ligand that crosses the M cell layer and selecting a high absorption uptake inducing peptide that passes through the M cells without passing through the epithelial cell layer of the small intestine.

Hereinafter, the specific configuration of the present invention will be described in detail with reference to the drawings and embodiments.

The present invention comprises the steps of forming a phage display peptide library; establishing an in vitro M cell model; Searching for M cell target peptides; in vitro phage transcytosis assay step; Identifying the peptide sequence; Revalidation of the M cell target candidate peptide and selection of the target peptide.
EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail according to preferable embodiment. However, this does not limit the scope of the present invention.

< Example >

Phage display Peptide  library( Phage display peptide library )

The phage display peptide library used in the present invention is 'ph.D.-C7CTM (New England BioLab.)', Which contains seven random amino acid sequences at the end of the gene producing pIII, a kind of coat protein, in the genome of M13 bacteriophage. It consists of recombinant bacteriophage expressing hundreds of millions of different peptides obtained by infecting E. coli by artificially inserting a gene sequence to express a peptide of random amino acid sequence.

Meanwhile, in the present invention, the seven random amino acid sequences introduced into the M13 phage are designed to have cysteine residues on both sides, thereby naturally forming disulfide bonds during peptide expression, thereby forming a ring shape to induce stronger binding with the target protein. (See FIG. 2).

In in vitro  Establishment of an M Cell Model

In order to produce a high-efficiency oral vaccine, the target peptide functional group should be selected after the M cell model was established.

Human colon cancer-derived Caco-2 cells were cultured in transwells to determine whether the cells formed a tight bond to prevent permeation of foreign substances (transepithelial electrical resistance (TER)). Was measured. As shown in Figure 3, as a result of measuring the TER value at intervals of 2 days during the culture period of 24 days it was confirmed that the increase to 350 Ω.cm ² .

In general, epithelial cells of the small intestine are judged to form a close bond when the TER value is maintained at 200 Ω.cm ² or more. Based on this result, the cells form a close bond after 16 days of culture. Was observed. In addition, TER values were measured at 48 hours intervals for 4 days after the mixed culture of Caco-2 cells and Raji cells after 20 days of cultivation to transform them into M cells, and the cells maintained the electrical resistance even after the mixed culture. Could.

As one of the important functional characteristics of M cells is to induce mucosal immune response through antigen presentation-treatment process after absorbing macromolecules present in the intestinal tract, the following experiment is used as the final evaluation item to evaluate the effectiveness of the model system. Built in in vitro Functional characteristics of the M cell model were confirmed.

C2Bbe1 cells and cells cultured 2 and 4 days after the mixed culture were prepared as test materials, and 1.6 × 10 ¹⁰ FITC-conjugated latex beads (diameter 200 μm) were put in the front end surface of each cell. The present invention is divided into two experimental zones, one experimental zone is 37 ℃ (90 minutes)-> 4 ℃ (90 minutes)-> 37 ℃ (60 minutes), the other experimental zone is 4 ℃ (90 minutes)-> 37 After treatment with a temperature change of &lt; RTI ID = 0.0 &gt; C &lt; / RTI &gt; (90 minutes)-> 4 [deg.] C. (60 minutes), the number of beads transferred to the lower trans-well chamber was detected by flowcytometry. For 4 days after the mixed culture, it was confirmed that the bead movement occurred when the temperature was maintained at 37 ° C., and when the cell was maintained at 4 ° C., the movement of the beads did not occur. On the contrary, it was confirmed that the movement of the beads was significantly lower in the cells 2 days after the mixed culture with C2Bbe1 cells. Based on these results, the optimal incubation period for inducing functional characteristics of M cells was determined to be 4 days (see FIG. 4A). Next, the expression of integrin, which is a transmembrane glycoprotein (transmembrane glycoprotein) that is specifically expressed on the end surface of M cells, was confirmed. As mentioned above, it was confirmed that the expression of integrin was distributed on the side of the cell (basolateral side) in Caco-2 cells (see FIG. 4B), and in the case of M cells, β-1 integrin was specifically expressed at the front end of the cell. It was confirmed (see FIG. 4C).

Next in terms of morphology, Transmission electron microscopy (TEM) was used to determine whether M cells induced differentiation in vitro showed expression patterns of irregular microvilli, which are inherent in vivo . As shown in FIG. 4D, in the case of Caco-2 cells, the expression of microvilli was uniform and strong binding was observed at the intercellular binding site. Therefore, it was confirmed that the differentiation induction of cells occurred under normal conditions. in In vitro M cells (Fig. 4e) was confirmed that the Raji cells were moved to the Caco-2 cell site, and the irregular expression of microvilli occurred at the front end surface of the site where the Raji cells are present. Based on the above results in In vitro differentiation of M cells was determined to be normally induced. As a result of evaluating the effectiveness of the M cell model based on the functional, morphological and cellular biological evaluation, it was confirmed that the M cell model was transformed into a normal M cell model.

M cell target Peptide  quest

In established in Example 2 above To explore peptide functional groups targeting M cells using in vitro M cell models and phage display techniques in vitro jeonbyeon on to the M cells is because the induced by mixed culture of differentiated cells and Raji cells C2Bbe1 C2Bbe1 cells and in In vitro M cell model was carried out using a phage peptide library to perform a transcytosis assay to select peptides with significantly higher transcytosis ability and to select Raji cell binding assays. After analysis of the binding specific peptides, these peptide analysis tables were compared to select M cell specific peptide sequences (see FIG. 5).

Raji  Cell binding assay

The first step in selecting M cell target peptides was a Raji cell binding assay. Biopanning was performed after injection into 1.0 × 10 ⁷ cell / ml Raji cell suspension prepared with a 1.0 × 10 ¹¹ pfu phage library. Insertless phage was used. As a result of performing biopanning four times in total, it was confirmed that the optimal concentration of phage was reduced in the second biopanning compared to the first. This is consistent with the results of a typical phage display, predicting that a phage population that would specifically bind to Raji cells would be selected. Subsequent 3 to 4 biopannings showed that the titer concentration of the 4th was increased by 872 times compared to that of the 1st, and the insertless phage used as a control was significantly lower than the phage encoding the peptide. It confirmed that it was seen (refer FIG. 6).

 In general, when biopanning through phage display technique is performed, the phage population having strong affinity with the target target increases as the number of biopanning cycles increases. It can be said to be the most basic parameter to determine whether it is going in the intended direction. As shown in FIG. 6, biopanning was performed using the phage display method. As the biopanning was repeated, the titer concentration of phage bound to Raji cells rapidly increased to the fourth order. As the number of biopanning cycles increases, the number of phages that bind to cells increases because the percentage of phages that bind to the cell's receptors increases gradually as the number of biopanning cycles increases.

In in vitro  scrap paper Transcytosis  analysis: Caco -2 cells and in in vitro  M cell Transcytosis  analysis

As described above in The transmutation into M cells in vitro was induced by Raji cells by the functional-receptor interactions of chemokine 20 secreted from Caco-2 cells and mixed chemokine receptor 6 expressed on the surface of Raji cells in mixed culture. By moving to the cell layer of Caco-2 cells, the characteristics of the cells were changed. But in Induction of differentiation in vitro does not induce differentiation to have all the characteristics of M cells, but some populations have disadvantages inherent in Caco-2 cells.

Thus to the present invention relates to starting a Caco-2 cell transport system analysis and Saito M cell targeting peptide to secure control information for searching for a specific functional group in the peptide in cell M In vitro M cell transcytosis assay was performed (see FIG. 7).

The results of four biopanning tests showed that the titer concentrations of the four cells increased significantly in both cell types compared to the first one.In contrast, the insertless phage used as a control was significantly higher than that of the peptide-encoded phage. As a result, it was confirmed to show low binding (see FIGS. 8A and 8B).

Peptide  Identification of sequences

Raji Cell Binding Assay with Caco-2 Cells in In vitro M cell transcytosis analysis was carried out in three independent experiments, in which the phage populations recovered in the fourth biopanning process were plated on solid medium, and each individual phage plaque was extracted to extract genomic DNA. Sequence analysis was used to identify which peptide sequences each possessed (see FIG. 9).

A total of 520 effective peptide sequences (except insertless phage) recovered from each of the three cells were identified and the results are shown in Table 1 and 20 peptide sequences were expected to be M cell specific. The optimal concentration of phage obtained during the 4th biopanning process was from 10 ⁵ to Considering 10 ⁷ (pfu / 10ul), the frequency of appearance in Table 2 from 520 effective samplings in this large population can be interpreted as a very high percentage of phages with these peptide sequences in the population. These peptide sequences were found to have a very high potential to effectively induce binding and uptake in Raji, Caco-2, and in vitro M cells.

Table 1 Peptide Sequence Sorting Results

analysis Concentration ratio Insertion Ratio Raji Cell Binding Assay Peptide R1: 10/45 17/62 (27.4%) Peptide R2: 4/45 Peptide R3 ~ R6: 3/45 Caco-2 Cell Transcytosis Assay Peptide C1: 7/230 114/344 (33.2%) Peptide C2: 6/230 Peptide C3: 5/230 Peptide C4 ~ C7: 4/230 Peptide C8 ~ C15: 3/230 M cell transcytosis assay Peptide M1: 5/245 83/328 (25.4%) Peptide M2: 4/245 Peptide M, M4: 3/245 Peptide M445: 2/245

M cell target candidate Peptide  Revalidation and Target Peptide  Selection

After identification of the peptide sequence, the caco-2 was selected using 20 peptides selected for re-validation of the target M cells of the 20 selected M cell target peptides and for selecting peptides showing M cell-specific high transcytosis efficiency. Transcytosis analysis was performed using cells and in vitro M cells.

As a result, clone M445 (amino acid sequence: CKSTHPLSC) is the Caco-2 cells do not induce cytochrome system in trans It was confirmed to have the highest transcytotic capacity in M cells in vitro (see FIGS. 10A and 10B).

Based on these results, it was determined that the CKSTHPLSC peptide sequence of the present invention exhibits a high transcytosis efficiency specifically for M cells, and the peptide sequence of the present invention has a very high potential for effectively inducing uptake in M cells. there was.

CKSTHPLSC  M cell specificity assay for sequence

In the present invention, in The CKSTHPLSC peptide sequence of the present invention identified using the in vitro phage display technique was performed as follows to test whether the effective absorption in the mucosal layer of the small intestine and the presence of a specific absorption pathway when absorbed.

(One) In - vivo  Phage binding analysis Trafficking  analysis

In Peptide sequences selected through in vitro transcytosis analysis and re-validation experiments using 20 M cell target candidate peptides were obtained in It is an overly logical leap to conclude that M cells in the small intestine will be specific in vivo . Therefore, in the present invention, after fasting the rat (Sprajud-Daurei male (350g) fasted in the intestinal lumen of the small intestine (intestinal tract) using a closed-ileal loop technique After sealing to include the earth patch portion, the phage and insertless phage of the CSKSSDYQC peptide sequence showing the phage and small intestinal epithelial cell specific passage efficiency of the CKSTHPLSC peptide sequence of the present invention were injected at 1.0x10 ¹¹ pfu / 500 μl, respectively, and then 30 After minutes, the phage patch site where M cells are known to be present and the epithelial cell site of the small intestine (EC: site not including the phase patch) were excised to quantify phage binding to each site.

As a result, the phage having the end of the CKSTHPLSC peptide sequence of the present invention showed a significantly higher level of binding ability in the phase patch site than the phage and insertless phage of the CSKSSDYQC peptide sequence. In the CSKSSDYQC peptide sequence was confirmed to show a low binding capacity compared to the phage. These results indicate that the selected M cell target CKSTHPLSC peptide sequence of the present invention in These results suggest the possibility of becoming M cell specific peptide sequences in vivo .

For the next to verify whether or not applicable to peptide vaccine delivery of a high efficiency based on the results as described above in In vivo phage trafficking assay was performed. As described above, the antigen introduced through the M cells is taken up by the antigen presenting cells and migrated to mescentric lymph nodes (MLNs). Thus, phage with M cell specific peptide sequences will be higher in migration to MLNs than phages with nonspecific peptides. Therefore, a lung-ileal loop was made at the phase patch site and the epithelial cell site of the small intestine (the site not including the phase patch), so that the phage of the CKSTHPLSC peptide sequence of the present invention, the phage of the CSKSSDYQS peptide sequence, and the insertless phage were injected for 30 minutes. Phage transferred to MLNs was then quantified.

As a result, it was confirmed that the phage having the CKSTHPLSC peptide sequence of the present invention was moved to MLNs at a higher level in the phase patch site than the phage and insertless phage of the CSKSSDYQC peptide sequence, and the epithelium of the general small intestine without M cells was present. It was confirmed that the cell site showed a lower binding capacity than the phage of the CSKSSDYQC peptide sequence. On the contrary, the phage of the CSKSSDYQC peptide sequence showing the specific absorption efficiency of the small intestine was higher than that of the phase patch where M cells exist. This result is consistent with the phage lung-ileal loop binding assay, and thus the M cell target CKSTHPLSC peptide sequence selected in Another result suggests the possibility of being M cell specific peptide sequences in vivo .

Immunohistologic Analysis

Recombinant phage expressing the CKSTHPLSC peptide sequence of the present invention at the end of the insertless phage that does not express a specific peptide sequence at the terminal was confirmed that the absorption efficiency in the phase patch site where the M cells in the small intestine is greatly enhanced . In the present invention, one step further, it was examined whether the phage of the CKSTHPLSC peptide sequence of the present invention passes through a specific pathway called M cell through the immunohistochemical assay.

Briefly describing the process, recombinant synthesis in which biotin is mixed with fasted rats is injected into the lung-bowel loop of the present invention CKSTHPLSC and CSKSSDYQC peptide sequence, and after 30 minutes, the patch phase and the general small intestine After removal of a portion of the ileum area, it was fixed with 4% paraformaldehyde and the small intestine chorionic tissue was prepared using a freezing flake, followed by confocal injection with streptavidin introduced with Alexa488 with green fluorescence. The position of the peptide was confirmed through a microscope (see FIG. 11).

As a result, in the phase patch region (red) where small cells of the small intestine of the rat injected with the CKSTHPLSC peptide of the present invention were found, a high green fluorescence signal was observed, but in the small intestine chorionic tissue, the specific signal was observed at the same position. Could not (see FIGS. 12C and 12D). On the contrary, in the chorionic tissues of the small intestine of rats injected with the CSKSSDYQC peptide, specific green fluorescence signals flowing into the body along the epithelial cell layer were observed, but at the phase patch site, the green fluorescent signal at a relatively low level compared to the CKSTHPLSC peptide of the present invention. It could be confirmed (see FIGS. 12A and 12B). Compared to CSKSSDYQC, which is an epithelial cell specific absorption peptide of the small intestine, at the phase patch site injected with the CKSTHPLSC peptide of the present invention, a high level of green fluorescence signal is observed, and the signal of the CKSTHPLSC peptide of the present invention is low in the small intestinal chorionic tissue. It could be assumed that there was a pathway through which the cells could be absorbed. Based on the above results, it was confirmed that the present invention CKSTHPLSC peptide injected into the lung-intestinal loop effectively enters the body through M cells present in the face patch after reaching the small intestine.

oral- White food  Delivery system

Above in Peptide sequences that effectively induce transcytosis in M cells selected using the in vitro phage transcytosheet method are conjugated into a single gene by protein-based vaccines and genetic recombination techniques, and a single-chain recombinant protein Can be produced with a drug (see FIGS. 14A and 14B). The length of the peptide functional group introduced in this way is a very short sequence of about 7 to 9 amino acids, whereas in the previous studies in which a protein of a polymer having a known transcytosis capacity was introduced into a vaccine, a drug deterioration of the drug was pointed out as a problem. The interference with inherent conformation or function can be minimized. On the other hand, when using a known transcytosis inducer it is necessary to further verify whether it was the optimal choice in terms of absorption efficiency in M cells, the present invention in In vitro phage display method is expected to solve this problem because it selects the peptide sequence showing the optimal absorption efficiency through the M cells at the highest efficiency.

Existing protein-based functional substances such as insulin, growth hormones such as growth hormones, cytokines such as interferon and interleukin, and various enzymes that regulate physiological activity, which depend on the administration method by injection to the peptide selected through the present invention Introduced in the present invention, an efficient oral dosage system that dramatically improves the absorption efficiency in the biofilm can predict the proliferation effect in the fields of medicine, pharmacy, animal husbandry and veterinary along with the expansion of the availability of protein drugs. Practically, oral administration in medicine and pharmacy can reduce unnecessary labor and incidental costs of injectable drugs, and anticipate pain relief from patient administration. When the vaccine is administered by injection, it is possible to avoid unnecessary labor costs and unnecessary increase in production cost due to the need for skilled labor.

In addition, if the research on the membrane receptors via these peptides is supported in the future, it is expected to contribute to deepening the academic knowledge by identifying the mechanism of mass transport in small intestine M cells.

Since the CKSTHPLSC peptide of the present invention can induce the uptake of macromolecular substances through the M cells in the mucosal layer of the small intestine, by establishing an oral vaccine delivery system that enhances the absorption efficiency in the biological membrane, the use of vaccines, medical, It is a very useful invention in the fields of pharmacy, animal husbandry and veterinary medicine.

Claims (3)

delete An absorption promoting inducing peptide that passes through M cells in the mucosal layer of the small intestine having the amino acid sequence of CKSTHPLSC. delete

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