KR20040061963A - Sialic acid-specific lectin protein prepared from paecilomyces japonica, process for preparing the same and the use thereof - Google Patents

Abstract

PURPOSE: A sialic acid-specific lectin protein prepared from Paecilomyces japonica, a process for preparing the same and the use thereof are provided, which sialic acid-specific lectin protein is used as a reagent for carbohydrate-related studies based on its N-acetylneuramic acid recognizing principle, and for diagnosis of diseases associated with distribution change of N-acetylneuramic acid, and is used as a carrier protein for delivery of drug into a local area and an inhibitor for growth of cancer cells. CONSTITUTION: The sialic acid-specific lectin protein, PJA(Paecilomyces japonica agglutinin) protein, is prepared from Paecilomyces japonica, and consists of single subunit with molecular weight of 16.0 kDa, wherein PJA protein specifically binds to N-acetylneuramic acid or N-acetylneuramic acid containing glycoprotein. The process for preparing the PJA protein comprises the steps of: finely chopping Paecilomyces japonica; adding NaCl solution into chopped Paecilomyces japonica and filtering the mixture; and subjecting the filtered solution to fetuin-affinity chromatography.

Description

Sialic acid-specific lectin protein prepared from Paecilomyces japonica, process for preparing the same and the use

The present invention relates to a lectin protein isolated and purified from Paecilomyces japonica mushrooms, a method for producing and use thereof, and more specifically, to N-acetylneuraminic acid isolated and purified from snow cauliflower fungus. Lectin PJA, a binding protein component, a method for preparing the same, and various uses based on the biological activity of an isolated PJA.

In recent years, as the technology for analyzing and controlling the structure and function of biological materials has been rapidly developed in genetic engineering and protein engineering, sugars such as oligosaccharides have been recognized as an important research subject for the clarification of bioregulatory mechanisms. In order to proceed with the related research, studies on the effect of oligosaccharides on the structure and function of living organisms and the role of mutual information communication between cells should be comprehensively studied.

Oligosaccharides, one of the important components of the living body, form complex molecules such as glycoproteins or glycolipids that bind to proteins or lipids, which are indispensable for maintaining the biological activity of an individual such as cell adhesion, communication between cells, and morphology of tissue individuals. It is in charge of function.

The functions of oligosaccharides known to date are largely three. First, oligosaccharides bind to other proteins, and their interactions play an important role in cell-specific recognition. Second, when the oligosaccharides bound to the protein itself significantly change its specific function. For example, N-CAM, which is a cell adhesion molecule of oligosaccharides specifically expressed in the brain, such as polysialic acid. Third, most proteins are glycoproteins, and oligosaccharides are bound through Asn or Ser / Thr of the protein itself to activate the function of the protein. In other words, oligosaccharides can be thought of as important substances that can cause changes in their function not only by themselves but also by binding to other biological substances.

Intercellular recognition occurs mainly through sugars on the cell surface, and in order to mediate this smoothly, molecules that recognize sugar must exist on the cell surface. One example is the presence of carbohydrate-binding proteins such as lectins on the cell surface.

Paecilomyces japonica, which means insects in winter and grasses in summer, refers to mushrooms parasitic from insects.The mushroom spores float in the air and enter parasitic insects through the respiratory organs. It comes from wild cordyceps. Snow fungus is an incomplete fungus that forms conidia, and is caused by almost all insects without affecting the habitat environment such as temperature and humidity. To date, research has been conducted on snow cordyceps to isolate small molecules such as ergosterol peroxide, acetoxyscirpenediol, and others, which have anticancer activity. Presence of a polymer of the protein in fungi lectin is already known, to have a sugar binding characteristics of these known is Agaricus bisporus lectin derived βGal1-3GalNAC bond, Agrocybe aergerita derived galactose binding lectins, fucose-derived Alueuria aurantia lectin binding, Fomes fomentarius N-acetyl-D-galactosamine-binding lectins derived from, N-acetyl-D-glucosamine-binding lectins derived from Psathyrella velutina , etc. are known (Kawagishi H. Food Reviews International 11, 63-68, 1995). However, the presence of lectins that recognize sialic acid, such as N-acetylneuraminic acid, is very rare in mushrooms.In particular, a single substance is obtained by isolating and purifying lectins that have confirmed the sugar-binding properties from Snow Cordyceps. There is little information on sugar, anticancer effects, etc.

The present inventors have isolated and purified the protein lectin PJA, which has not been known for a while, from the Snow Clover, and characterized the substance and tested its biological activity, and it specifically binds to N-acetylneuraminic acid. By having various beneficial biochemical properties, it has been confirmed that the present invention can be used for research reagents related to various sugar-binding proteins and inhibitors of cancer cell proliferation.

Accordingly, an object of the present invention in one aspect is to provide a PJA which is a lectin component which is a kind of protein that recognizes N-acetylneuraminic acid, extracted from Paecilomyces japonica.

In another aspect, an object of the present invention is to provide a method for preparing the lectin PJA.

In a further aspect, an object of the present invention is to provide a diagnostic reagent capable of detecting when the structure of the sugar in the cell containing the lectin PJA contains N-acetylneuraminic acid and its distribution changes during the onset .

In another aspect, an object of the present invention is to provide a use of the lectin PJA as an anticancer agent that exhibits a proliferation inhibitory effect on cancer cells.

In still another aspect, an object of the present invention is to provide a use of the lectin PJA in immunobiochemistry research materials or in aggregation of cancer cells.

1 is a graph showing UV (280 nm) spectroscopy chromatogram and erythrocyte agglutination reaction of a pettuin-affinity column of lectin PJA prepared according to the present invention. Absorbance at 280 nm; Hemagglutination.

Figure 2 is a photograph showing the results of electrophoresis of lectin PJA prepared according to the method of the present invention. Lane 1: molecular marker (66, 45, 36, 29, 24, 20.1, 14.2 kDa); Lane 2: cordyceps protein stock solution; Lane 3: fetuin-affinity column unbound; Lane 4: lectin PJA, a fetuin-affinity column fraction; Lane 5: lectin PJA (+ 2-mercaptoethanol) as a fetuin-affinity column fraction; Lane 6: molecular marker (66, 45, 36, 29, 24, 20.1, 14.2 kDa).

Figure 3 is a graph showing the molecular weight of the lectin PJA prepared according to the method of the present invention by MALDI-TOF mass spectrometer.

Hereinafter, the present invention will be described in more detail.

Lectin is a protein that selectively binds to carbohydrates. It is isolated and purified from plants, microorganisms, viruses, invertebrates, vertebrates, etc., and is widely used as a material for research on various diseases. To date, about 300 species have been reported. Lectin has a variety of functions, such as agglutination of erythrocytes and sedimentation is well known, and blood types are classified, and recently, a specific aggregation of cancer cells is revealed. In other words, lectins bind to sugars with specific structures and allow selective exchange of information between cells with lectin substances and cells with sugars that bind to them.

Lectin research can be considered as an important field for elucidating the mechanism of information transfer between cells through molecular recognition.Neutral system, immune system, inflammatory response, etc. In terms of research and the relationship between infections, diseases (cancer, rheumatism) and sugar, research is being actively conducted.

In particular, sugar chains on the surface of cells have been found to be an important factor in determining the metastasis of cancer to experimental models in connection with cancer. This fact indicates that lectins have different binding properties to surface sugar chains of different cancer cell variants. Metastasis is different in cancer cells selected in vitro, and the possibility of changing metastasis by modifying the sugar chains of cancer cells has been confirmed from studies such as the report. These properties are known to be common to all types of cancer cells and to organs that target metastasis. Metastatic tumors appear frequently over time even after the primary tumor is completely removed by surgical treatment such as surgery, which is one of the biggest causes of difficulty in treating malignant tumors. In view of this, recently, the use of asialoglycoprotein as a carrier, the method using sugar chain-modified polymers, the targeting of protein-modified sugars by sugar modification, and the introduction of sugar chains by particulate carriers have been associated with cancer. It has been studied. In particular, glucose recognition mechanisms frequently used for systemic drug targeting include galactose receptors on the surface of hepatic parenchymal cells, mannose receptors on various macrophages, including cooper cells, and the like. Since these receptors have very high binding and expressing each organ and cell, it is expected to target cell-specific drugs using this apparatus.

Recently, structural changes in oligosaccharides of proteins in the body during rheumatoid, cancer, and AIDS have been reported, and attempts have been made to interpret the results of these diseases from structural changes in oligosaccharides. In particular, the significant change of sialyl epitope in the sialoconjugate on the cell surface of pathogens or tumors is related to the type of sialic acid in the oligosaccharides and the sugar binding method to adjacent sugars. It is believed to be attributable. Therefore, lectins that specifically recognize only various sialic acids and their sugar binding methods can be used as useful tools in identifying various sialyl epitopes in pathogens and malignant tumor biopsies.

Sialic acid refers to about 30 derivatives such as N-acetylneuraminic acid or N-glycolylneuraminic acid, an acyl substituent of the C-5 amino group. Compound. Most other sialic acids have one or more O-acetyl substituents on one or more C-4, C-7, C-8, C-9 hydroxyl groups. Such substituted sialic acid may cause transformation or other changes in the cellular environment, and by inhibiting or weakening sialic acid hydrolase of bacteria or virus, altering their immune capacity, affecting the enzymatic ability of the complex sugars. It is known to give.

Most lectins have been reported to specifically recognize mannose (D-mannose) and galactose (D-galactose) and specific N-acetylneuraminic acid, which plays a structural and functional role in the non-reducing end of sugar. Lectin is recognized as a species. Lectins that bind to N-acetylneuraminic acid are Wheat germ agglutinin (WGA), Maackia amurensis agglutinin (MAA, from elm), Limulus polyphemus lectin (limulin, from Sambucus), Sambucus niger agglutinin (SNA, Elderberry) Derived) and the like. There are many reports on the anti-tumor effect of plant-derived lectins that inhibit cancer growth and the inhibition of cancer by carcinogens. WGA and MAA are known to have cytotoxic effects on liver cancer, melanoma, chorionic cancer, and osteosarcoma. In vivo, murine ascitic lymphoma and murine ascitic lymphoma tumors are known. In vitro, the effects of inhibiting human nasal septum tumor, human colon adenocarcinoma, and mouse embryonal carcinoma are known. MAA and SNA are reported to be selective for receptors of influenza A virus. WGA has also been used to analyze sugar structure in HIV and SNA has been used to identify N-acetylneuraminic acid in liver cancer cells (Abdullaev F. et al. Natural Toxin 5, 157-163, 1997).

In addition, many cancer-related experimental models have been reported to have a significant relationship with the increase or dramatic change in sugar chains containing N-acetylneuraminic acid and the corresponding increase in lectin binding. This increase in N-acetylneuraminic acid is simultaneously observed in the N- or O-linked oligosaccharides of glycoproteins. Inhibition of the formation of sugar chains by glycosynthesis inhibitors such as tunicamycin shows a decrease in metastatic properties. Increasing the number of sugars, sugar chains containing a large number of neuramic acid is shown. Recently, IGR39 from primary tumors and IGR37 from metastatic lymph nodes in human melanoma cell lines from the same cancer patients have been reported to bind strongly with lectins. HM7 melanoma Affinity columns that combine lectins with sepharose have also been used to separate and purify mucin-typeglycoproteins from the plasma membrane of cells (melanoma cells). In addition, a substance combining fluorochrome tetramethyltamine isothiocyanate with lectin is used for effective detection in B16F10 melanoma and Lewis lung carcinoma cells. On the other hand, there has been an example of the possibility of a carrier protein that binds doxorubicin to lectin and then becomes an acid-labile chemotherapy prodrug for colon carcinoma.

The present inventors conducted a variety of experiments by selecting the Snow Cordyceps as a material in order to search for useful substances from natural organisms.

In one aspect, the present invention provides a PJA, which is a lectin component which is a kind of protein that recognizes N-acetylneuraminic acid, extracted from Snow Cordyceps sinensis.

The lectin component is a protein consisting of a subunit having a molecular weight of about 16.0 kDa by electrophoresis and a mass spectrometer and a molecular weight of 16235.0 Da by a MALDI-TOF mass spectrometer, and N by erythrocyte agglutination and hemagglutination inhibition. It has been shown to bind to glycoproteins comprising acetylneuraminic acid or N-acetylneuraminic acid.

In another aspect, the present invention provides a method for preparing lectin PJA from snow cauliflower.

Specifically, the lectin PJA was finely cut in the Cordyceps sinensis, 0.15M NaCl aqueous solution was added and stirred at 4 ° C. for 24 hours, and the precipitate was filtered through a filter to separate the protein component through a petuin-affinity column. It is manufactured by.

In a further aspect, the present invention provides a use as a diagnostic reagent for diagnosing a disease in which the structure of a sugar in a cell, including the lectin PJA, contains N-acetylneuraminic acid and its amount changes. Examples of such a disease include, but are not limited to, pancreatic cancer, gastric cancer, liver cancer, melanoma, chorionic cancer, bone cancer, colon cancer and the like.

As a result of testing the biological activity of the PJA, it specifically binds to N-acetylneuraminic acid as a monosaccharide and to fetuin containing N-acetylneuraminic acid as a glycoprotein. .

In another aspect, the present invention provides a use of the lectin PJA as an anticancer agent that exhibits a proliferation inhibitory effect on cancer cells.

The lectin PJA material of the present invention selectively recognizes and binds to a glycoprotein comprising N-acetylneuraminic acid or N-acetylneuraminic acid as confirmed by hemagglutination and hemagglutination inhibition. Not only can be used, but also has an anticancer effect as evidenced in the examples described below. Therefore, the lectin PJA of the present invention can be effectively used as an active ingredient of cancer cell proliferation inhibitors (or anticancer agents) in diseases in which N-acetylneuraminic acid is present. Examples of the target disease include, but are not limited to, pancreatic cancer and gastric cancer.

The pharmaceutical composition of the cancer cell proliferation inhibitor according to the present invention can be formulated in combination with a known pharmaceutical carrier. Generally, the active ingredient is blended with a pharmaceutically acceptable liquid or solid carrier, and a solvent, a dispersant, an emulsifier, a buffer, a stabilizer, an excipient, a binder, a disintegrant, a lubricant and the like are added as necessary and purified. And solid solutions such as granules, powders, powders, and capsules, liquids such as ordinary liquids, suspensions, and emulsions. Moreover, it can also be made into the dry goods which can be made into a liquid state by addition of a suitable carrier before use.

The pharmaceutical composition of the present invention can be administered by any of parenteral agents such as oral preparations, injections, and drops.

A medical carrier can be selected according to the said dosage form and formulation, and, in the case of an oral preparation, starch, lactose, white sugar, mannitol, carboxymethylcellulose, corn starch, an inorganic salt, etc. are used, for example. Moreover, in preparation of an oral preparation, a binder, a disintegrating agent, surfactant, a lubricating agent, a fluidity promoter, a mating agent, a coloring agent, a fragrance | flavor, etc. can also be mix | blended further.

On the other hand, in the case of parenteral preparations, the composition comprising lectin PJA, which is an active ingredient of the present invention, is used as a diluent for distilled water for injection, physiological saline solution, glucose aqueous solution, vegetable oil for injection, sesame oil, peanut oil, soybean oil, and corn oil. It is prepared by dissolving or suspending in propylene glycol, polyethylene glycol or the like and adding a fungicide, stabilizer, isotonicity agent, analgesic agent and the like as necessary.

The pharmaceutical composition of the present invention is administered by a suitable route of administration according to the formulation form. The administration method need not be particularly limited and can be administered by internal content, external application, and injection. Injections can be administered, for example, intravenously, intramuscularly, subcutaneously, intradermally or the like.

The dosage of the pharmaceutical composition of the present invention is appropriately set depending on the form of the preparation, the method of administration, the purpose of use, and the age, weight and symptoms of the patient to be applied thereto. Amounts are, for example, 10 μg-200 mg / kg body weight per adult. Of course, since the dose varies depending on various conditions, an amount smaller than the dose may be sufficient, or it may be necessary beyond the range.

In a still further aspect, the present invention provides a drug conjugate in which the lectin PJA is combined as a carrier protein for a topical delivery agent, and the agonist is a selective drug as an anticancer agent. Examples of diseases targeted by such anticancer agents include, but are not limited to, pancreatic cancer, gastric cancer, liver cancer, melanoma, chorionic cancer, bone cancer, and colorectal cancer.

Hereinafter, the present invention is described in more detail by the following representative examples, but the present invention is not limited in any way by these examples.

<Example>

Example 1 Isolation of Protein Solution Containing Lectin PJA from Cordyceps Sinensis

After removing the pupa part, 15g of the snowflake Cordyceps sinensis was collected and washed with tertiary distilled water, and then rinsed with 0.15M NaCl solution using a grinder and mixed well at 4 ° C for 24 hours. The mixed solution was subjected to a primary filter with a 6 μm membrane filter and secondary filtration with a 0.45 μm membrane filter.

Example 2: Isolation of Lectin PJA from Cordyceps Sinensis Solution

The protein solution obtained in Example 1 was applied to a petuin-affinity column, and chromatograms were prepared by UV / VIS spectroscopy at 70 fractions of 280 nm. The results are shown in FIG. 1. The fractions were placed on a petuine affinity column and allowed to stand at room temperature for 1 hour, and then aliquoted with 1 ml while slowly flowing 10 mM Tris-HCl and 150 mM NaCl (pH 7.5). Subsequently, 10 mM Tris-HCl and 150 mM NaCl (pH 7.5) were flowed at 280 nm until the absorbance value became 0.0. Received fractions 2-11 with absorbance values greater than 0.1 and these were identified by erythrocyte agglutination reactions, which are not lectin components, and from fraction 30, where the absorbance values were completely 0.0, 200 mM NaOH-0.2M KCl buffer (pH 12.5). 1 mL aliquots were flowed quickly and immediately neutralized with 1M Tris-HCl (pH 8.5). As a result of measuring absorbance at 280 nm for these fractions, fractions 31-37 showed high absorbance. In addition, the results of the hemagglutination reaction experiments described below with respect to these fractions, it was confirmed that the lectin having a high activity.

The results of the total protein amount, activity, and recovery rate of the lectin PJA obtained in Example 1 and Example 2 are shown in Table 1 below.

Total protein amount (mg) Active (unit / 1mg / ml) PJA recovery (%) Protein extracts 504.0 83 100.0 Fetuin-affinity column 31-37 fraction (PJA) 127.0 257 25.2

In Table 1, activity represents a unit of lectin contained in 1 mg / ml protein, and unit represents a dilution factor causing erythrocyte aggregation. The recovery of final PJA in the protein extract was 25.2% from the protein extract.

Example 3: Confirmation of Purity and Molecular Weight of Lectin PJA

In order to confirm the molecular weight and purity of the lectin obtained in Example 2, an electrophoresis, MALDI-TOF mass spectrometer was applied.

First, electrophoresis was performed on a 12% polyacrylamide gel containing 0.1% SDS to compare with a molecular weight standard. After completion of development, the protein bands were stained with Coomassie blue staining reagent. This experimental result is shown in FIG. In Figure 2, lane 1 is molecular marker (66, 45, 36, 29, 24, 20.1, 14.2 kDa); Lane 2 is Cordyceps sinensis stock; Lane 3 is a fetuin-affinity column unbound material; Lane 4 is lectin PJA, a fetuin-affinity column fraction; Lane 5 is lectin PJA (+ 2-mercaptoethanol) which is a fetuin-affinity column fraction; Lane 6 is molecular markers 66, 45, 36, 29, 24, 20.1, 14.2 kDa.

As can be seen from the electrophoresis result of FIG. 3, one band having a molecular weight of about 16.0 kDa in the non-reduced state without adding 2-mercaptoethanol was identified. In addition, a band of about 16.0 kDa was observed in the reduced state where 2-mercaptoethanol was added. From this result, it was confirmed that PJA is a protein having one subunit and its molecular weight is 16.0 kDa.

On the other hand, in order to confirm the molecular weight more accurately, MALDI-TOF mass spectrometer (manufacturer: Perceptive; product name: Voyager ^TM RP) was carried out according to the manufacturer's instructions to analyze the molecular weight to obtain a value of 16235.0 m / z The results are shown in FIG.

From the results of the electrophoresis and mass spectrometry, it was confirmed that the lectin PJA was a single component of about 16.0 kDa in electrophoresis, and became a subunit having a molecular weight of exactly 16235.0 Da by mass spectrometry.

Example 4 Hemagglutination of Lectin PJA

Using the lectin PJA obtained in Example 2, serum of humans, mice, rats, and rabbits was diluted with 10 mM phosphate buffer, 0.15 M NaCl (pH 7.2) to 6.25%, and then hemagglutination was performed. Fractions positive in this reaction were collected and subjected to electrophoresis as shown in Example 3 to confirm that it was a single substance of a single band, which was named PJA (Paecilomyces japonica agglutinin). Erythrocytes of human A, B, O, mouse, rat, and rabbit were diluted in the same manner as above, and 50 μl was left at room temperature with 50 μl of lectin PPA to check for hemagglutination. As a result, erythrocytes of humans having only N-acetylneuraminic acid were aggregated. However, in the red blood cells, N-glycolylneuraminic acid-containing mice, rat erythrocytes were 32-fold, rabbit erythrocytes were up to 256-fold dilutions, the results are shown in Table 2 below.

Red blood cells Present sialic acid class Minimal hemagglutination inhibitory activity Person A type Neuac 64 Person B type Neuac 64 Person O type Neuac 64 mouse Neu, Gc, NeuAc, O-acetylsialic acid 32 Rat Neu, Gc, NeuAc, O-acetylsialic acid 32 rabbit Neu, Gc, NeuAc, O-acetylsialic acid 256 Note: The minimum hemagglutination inhibitory activity is the minimum erythrocyte agglutination inhibition dilution factor obtained by diluting lectin PJA by 2 times for various red blood cells diluted to 6.25%, NeuGc is N-glycolylneuraminic acid, and NeuAc is N-acetyl Represents neuramic acid.

These results indicate that PJA selectively recognizes N-acetylneuraminic acid. In human erythrocytes, aggregation of lectin PJA was inhibited up to a 64-fold dilution, resulting in agglutination when the lectin solution was about 1 μg / ml. This is the minimum concentration that occurs, indicating that it is present at a concentration of about 64 μg / ml.

Example 5 Hemagglutination Inhibition of Lectin PJA

In order to confirm the fact that the lectin PJA of the present invention recognizes N-acetylneuraminic acid, hemagglutination inhibition reaction using monosaccharide, disaccharide, and glycoprotein was performed. 50 μl of PJA lectin solution dissolved in 0.15 M NaCl in each well of a 96 well microplate and various monosaccharides (β-D-glucose, D-galactose, L-fucose, D-mannose, N-acetylneuraminic acid, Methyl-α-D-glucopyranoside), disaccharides (α-lactose, β-lactose, D-lactose, β-gentioose, maltose, D-rapinose, D-cellobiose, N-acetyllactozamine ) And 50 μl of a solution of glycoproteins (pettuin, asialofetuin) in 0.15 M NaCl, respectively, reacted at room temperature for 1 hour or more, and then 50 μl of red blood cells suspended in 0.15 M NaCl was added to each well. The reaction was carried out at room temperature for 1 hour. As a result, Petuin containing N-acetylneuraminic acid as a monosaccharide and N-acetylneuraminic acid as a glycoprotein inhibited the aggregation, and other disaccharides and glycoproteins did not inhibit the aggregation. As shown in Table 3.

Classification carbohydrate Sialic acid type Minimal hemagglutination inhibition concentration Monosaccharides β-D-glucose - － D-galactose - － L-fucose - － D-Mannose - － N -acetylneuraminic acid Neuac 0.0125mM Methyl-α-D-glucopyranoside - － saccharose α-lactose - － β-lactose - － D-lactose - － β-gentiviose - － maltose - － D-rapinose - － D-cellobiose - － N-acetyllactozamin - － Glycoprotein Petuin Neuac 0.05mM Asialopetuin - －

In Table 3, the minimum concentration of erythrocyte aggregation inhibition was diluted by 32 times PJA showing the minimum aggregation in 6.25% erythrocytes of rats showing minimal hemagglutination inhibition, and the concentration of monosaccharides, disaccharides, and glycoproteins was twice as high as each concentration. Dilution represents the minimum concentration that indicates aggregation inhibition,-indicates no aggregation inhibition. Monosaccharides and disaccharides (except N-acetyllactozamine) did not show coagulation inhibition at 100 mM and asialopettuin at 100 μM, and N-acetyllactozamine did not show aggregation inhibition at 10 mM. NeuAc stands for N-acetylneuraminic acid.

Example 6: Amino Acid Composition Analysis of Lectin PJA

To analyze the amino acid composition of Cordyceps lectin PJA, an amino acid composition analyzer (manufacturer: Pharmacia; product name: Biochrom 20 Auto-amino acid analyzer) was performed according to the manufacturer's instructions and analyzed as shown in Table 4 below.

amino acid Furtherance(%) Asx 10.07 Glx 6.01 Ser 5.71 Thr 6.24 Pro 9.17 Gly 9.99 Ala 6.46 Cys 0.53 Val 5.48 Met ND Ile 2.55 Leu 7.14 Tyr 5.26 Phe 6.91 His 2.55 Lys 9.77 Arg 6.24

Asx represents aspartic acid, asparagine. Glx stands for glutamic acid and glutamine. ND indicates not detected.

Example 7 Effect of Temperature of Lectin PJA

In order to confirm the activity of cordyceps lectin PJA by temperature, the activity was confirmed after 1 hour at 4 ° C, 15 ° C, 25 ° C, 37 ° C, 45 ° C, 50 ° C, 55 ° C, 60 ° C and 65 ° C. As shown in below, the activity was 100% below 55 ° C.

Temperature activation 4 ℃ 15 ℃ 25 ℃ 37 ℃ 45 ℃ 50 ℃ 55 ℃ 60 ℃ 65 ℃ 100% 100% 100% 100% 100% 100% 100% 50% 0%

Example 8: Effect of Lectin PJA on pH Change

To determine the effects of lectin PJA on pH changes, 25 mM KCl-HCl (pH 1.96, 3.16), 20 mM glycine-HCl (pH 4.04, 5.63, 6.00), 20 mM Tris-HCl (pH 7.34, 8.00, 9.06), 25 mM As a result of checking the activity in sodium carbonate-HCl (pH 10.08, 10.89), as shown in Table 6, the activity was 100% at pH 4.04 to pH 8.00.

Buffer (pH) activation 25 mM KCl-HCl (pH 1.96) 25 mM KCl-HCl (pH 3.16) 20 mM glycine-HCl (pH 4.04) 20 mM glycine-HCl (pH 5.63) 20 mM glycine-HCl (pH 6.00) 20 mM Tris-HCl (pH 7.34) -HCl (pH 8.00) 20 mM Tris-HCl (pH 9.06) 25 mM sodium carbonate-HCl (pH 10.08) 25 mM sodium carbonate-HCl (pH 10.89) 0% 0% 100% 100% 100% 100% 100% 50% 25% 0%

Example 9 Inhibition of Cancer Cell Proliferation by Lectin PJA

Proliferation inhibitory effect on cancer cells was performed using MTT (3- [4,5-dimethyldiazol-2-yl] -2,5-diphenyl tetrazolium bromide) assay (Mosmann T et al. J. Immunol. Methods 65, pp 55-63 (1983). After culturing each cancer cell in RPMI 1640 90%, FBS (fetal bovine serum) 10% medium and using a hematocytometer to confirm that the number is about 1 x 10 ^{4 in} 100μl put into a 96 well plate 8, 0.8, 0.08 100 μl of μM lectin PJA was added and left for 72 hours in a 37 ° C. incubator maintaining 5% CO ₂ . 50 μl of MTT reagent was added to each well, and the mixture was left in a CO ₂ incubator for 4 hours. The supernatant was discarded, 150 μl of dimethyl sulfoxide was added, mixed for 10 minutes, and the absorbance was measured at 540 nm using a microplate. When the experiment was conducted in the same manner without adding the lectin PJA under the experimental conditions, the reduction ratio was confirmed by comparing with the case where the lectin PJA was added at 100%, and the same experiment was repeated three times. The reduction ratio was expressed as the mean value ± standard deviation value. As a result, as shown in Table 7, lectin PJA showed a cell proliferation inhibitory effect on human human pancreatic cancer AcPC-1 and human gastric cancer SNU-1.

Cancer cell origin Cancer cell common name Badge composition Lactin PJA Concentration Cancer cell growth inhibition% Human pancreatic cancer AsPC-1 RPMI 1640 90%, FBS 10% 1 μM0.1 μM0.01 μM 46.1 ± 3.9% 37.0 ± 7.1% 24.2 ± 3.6% Human stomach cancer SNU-1 RPMI 1640 90%, FBS 10% 1 μM0.1 μM0.01 μM 56.5 ± 8.7% 34.1 ± 7.6% 31.7 ± 6.3%

Example 9: Pharmaceutical Formulations

Form of the formulation (eg tablet)

Active ingredient (Lectin PJA) 10 mg

Lactose 80 mg

Starch 17 mg

Magnesium Stearate 3 mg

10 mg of crystalline cellulose

Tablets were prepared by conventional methods and contain the above ingredients as raw material in one tablet. Tablets may be added as usual, enteric coat (eg hydroxypropylmethylcellulose phthalate), sugar coating or film (ethyl cellulose).

According to the present invention, using a lectin PJA that specifically recognizes N-acetylneuraminic acid, it can be used as a reagent for carbohydrate-related research based on the principle of recognizing N-acetylneuraminic acid and the distribution of N-acetylneuraminic acid. It can be used as a cancer cell proliferation inhibitor for pancreatic cancer and gastric cancer as well as carrier proteins for the diagnosis of changing diseases and delivery of selective drugs that minimize side effects by combining anticancer agents with lectins.

Claims (8)

PJA (Paecilomyces japonica agglutinin), a lectin component extracted from Cordyceps sinensis, consisting of a single subunit of about 16.0 kDa molecular weight by electrophoresis and mass spectrometry. The PJA of claim 1, wherein the PJA specifically binds to a glycoprotein comprising N-acetylneuraminic acid or N-acetylneuraminic acid by erythrocyte agglutination or erythrocyte agglutination. A method of preparing PJA, which is a lectin component from Cordyceps sinensis, comprising the steps of finely cutting Cordyceps sinensis, adding NaCl aqueous solution to the mixture, filtering the mixture with a filter, and applying filtrate to the filtrate. A diagnostic reagent comprising a lectin PJA according to claim 1, wherein the structure of the sugar in the cell comprises N-acetylneuraminic acid and its distribution can detect a change in the onset. A drug conjugate in which a lectin PJA according to claim 1 as a carrier protein for topical delivery agent is combined with a selective drug as an agonist. Use of lectin PJA according to claim 1 as a research material by immunobiochemistry or in aggregation of cancer cells and the like. A cancer cell proliferation inhibitor comprising lectin PJA according to claim 1 as an active ingredient. The cancer cell proliferation inhibitor according to claim 7, wherein the cancer is pancreatic cancer or gastric cancer.