KR0154493B1 - Novel phellinus linteus strain which produces anti-cancer immunoactive material - Google Patents

Abstract

The present invention relates to a novel Pelinus linteus strain that produces an anticancer immunoactive substance. The pelinus linteus KCTC 0173BP strain of the present invention produces a novel substance that exhibits excellent anticancer immune activity, and is a restriction enzyme of mitochondrial DNA. Judging from the fact that it is segmented, it is a novel strain that is clearly distinguished from other strains of the genus Mud mushroom and other strains of the same species of Felinus linteus.

Description

New Felinus linteus strain produces anticancer immunoactive substances

1 to 6 show the results of electrophoresis after cleaving the mitochondrial DNA of the novel Felinus linteus strain of the present invention with restriction enzymes BglII, Clal, EcoRI, PvuII, SpeI and NsiI, respectively, and FIG. The evolutionary system of the Felinus linteus KCTC 0173BP strain is shown.

The present invention relates to a novel Pelinus linteus strain that produces an anticancer immunoactive substance.

It has been known for a long time that many kinds of rhizome belonging to Aphyllophorales, which is a kind of Basidiomycetes, have a therapeutic effect on various diseases, especially tumors, and they are mainly transmitted as herbal medicine or folk medicine. Among them, Pelinus linteus, which belongs to the Phellunus of the Polyporaceae family, has been commonly used as a valuable medicine because it is called a situation in Chinese medicine. Felinus linteus, however, is a rare species in the natural world, and it is very difficult to obtain fruiting bodies, and it is very difficult to artificially culture mycelium. . In addition, no studies have been conducted to determine the microbiological or genetic characteristics of the Fellinus linteus strain.

In the case of microorganisms, not only the same species differ greatly in the type and amount of the active substance produced by each strain, but also in the culture conditions, the stability of the strain, and the frequency of genetic variation. Therefore, it is important to select safe microorganisms capable of producing large amounts of powerful anti-cancer immunoactive substances, cultivating artificial liquids, and having low genetic variation.

Mud mushroom genus belonging to the species Pellinus linteus can be distinguished from other genus mushrooms by physiological biochemical and morphological characteristics.However, in muds, the species have different morphological similarities between different species or species. It is known to be difficult to identify, and there is no unified money classification system.

Accordingly, the present inventors completed the present invention by establishing a classification identification system of mud mushrooms using molecular biological genetic information for the first time, and separately identifying and identifying a novel Felinus linteus fungus that produces a large amount of anticancer immunoactive substances.

It is an object of the present invention to provide novel Felinus linteus strains.

In order to achieve the above object, the present invention provides a novel Felinus linteus strain KCTC 0173BP for producing an anticancer immunoactive polysaccharide.

The present invention will be described in more detail as follows.

The novel Felinus linteus strain of the present invention is obtained by selecting a strain having high anticancer activity after collecting mushrooms determined to be natural in nature, and their microbiological characteristics, in particular, the characteristics of fruiting bodies are shown in Table 1 below.

Whether or not the selected strain is a new strain is determined by restriction fragment length polymorphism (RFLP) for each species or strain when mitochondrial DNA isolated from each strain is isolated from each strain and then digested with restriction enzymes. ) Is identified by its distinctive specificity. In other words, the total nucleic acid is isolated from the culture mycelium of the P. linus strain, the mitochondrial DNA is separated therefrom, cleaved with a restriction enzyme, and the DNA fragmentation is confirmed by electrophoresis, and the DNA segment of the strain to be compared. Genetic variation characteristics of each strain can be identified by calculating restriction enzyme segment similarity using the number of fragments with the same size and the number of fragments with different sizes.

Felinus linteus strain of the present invention screened by the above method is based on the morphological characteristics of the fruiting body, and the existing situation based on simple Japanese color book (Issue Hoiisha, 189, 1989) in which Imasege and Honggo etc. Although the morphological characteristics are very similar to the strains, they are clearly systematically different from the existing strains as judged by the restriction enzyme fragmentation pattern of mitochondrial DNA and the size of total mitochondrial DNA. In addition, unlike beta-glucan, which is an anticancer immunoactive substance isolated from basidiomycetes, both glycoside bonds of alpha type and beta type exist, (1 → 3), (1 → 4) and (1 → 6) produce acidic complex polysaccharides with a novel structure that exhibits strong anticancer immunity with a specific structure with various types of monosaccharide bonds.

Therefore, the strain was deposited as the accession number KCTC 0173BP dated June 12, 1995 to the Center for Genetic Resources, Institute of Biotechnology, attached to the Korea Institute of Science and Technology.

The anticancer immunoactive material produced from Felinus linteus KCTC 0173BP was extracted by hydrothermal extraction of the cultured mycelium of the strain, and then sequentially extracted with 80% ethanol and 60% ethanol and separated by ion exchange chromatography, etc. It can be separated and purified by confirming. At this time, the anti-cancer immune activity was treated by splicocytes extracted from B ₆ C ₃ F mice, which were collected in the laboratory of the Genetic Resource Center, Laboratory of Biotechnology, and treated with LPS (lipopoly saccharide) as a positive control group. The number of cells to form can be confirmed by measuring by a plaque forming assay (Barington, T. and Heimann, C., J. Immuno.Methods, 146, 129-137 (1992)).

Hereinafter, the present invention will be described in detail by the following examples, but the following examples are merely to illustrate the present invention, but are not intended to limit the scope of the invention.

Example 1

Isolation of Mitochondrial DNA from Felinus Linteus Strains

[Step 1]

Culture of Strains and Isolation of Total Nucleic Acids

Pelinus linteus KCTC 0173BP strains were transformed into malt extract agar medium containing 2% malt extract, 0.5% peptone, 0.5% glucose, and 2% agar, incubated for 2 weeks at 24 ° C, and then the mycelium was scraped and the malt extract liquid medium was After re-inoculation, the terminated mycelium was collected. Total nucleic acid was isolated from mycelium according to the modified method of leather and Brada DNA separation method (Raeder and Brada, Lett, Microbiol, 1, 17-20 (1985)).

The culture fungus was filtered with gauze, washed with 20 mM EDTA solution and freeze dried. 2 g of dry mycelium was placed in a mortar and pestle, and ground with a pestle to break up the mycelium, and then suspended in 20 ml of extraction buffer solution. 14 ml of phenol and 6 ml of chloroform were added to the suspension, followed by stirring and centrifugation (10,000 × g, 1 hour). The supernatant was recovered.

500 μl of RNase A (10 mg / mL) was added to the supernatant to remove RNA, and extracted with 20 mL of chloroform, followed by 0.54 times of isopropanol to precipitate DNA at room temperature, followed by washing with 70% ethanol. Was separated.

[Step 2]

Isolation of Mitochondrial DNA

The total nucleic acid isolated in [Step 1] was dissolved in 10 ml of TE buffer (10 mM Tris-HC1 (pH 7.5), 1 mM EDTA), and cesium chloride bisbenzimide was added to the mixture for 10 hours at 100,000 × g. Centrifuged. The mitochondrial DNA band formed on the upper layer was recovered by syringe, separated, precipitated with ethanol, and placed in 50 µl of TE buffer for use in restriction enzyme reaction and analysis.

Example 2

Restriction Cleavage Band Pattern of Mitochondrial DNA of Felinus Linteus KCTC 0173BP

The mitochondrial DNA of Felinus linteus KCTC 0173BP was digested with restriction enzymes BglII, Clal, EcoRI, PvuII, SpeI and NsiI (purchased by Cosco and Amersham), respectively, and then TAE buffer solution (40 mM Tris-acetate, 1 mM EDTA). Electrophoresis was performed on 0.6% agarose gel using.

After electrophoresis, the gel was stained and observed in 0.5 µl / ml of Ethidium bromide (EtBr) solution. As a result, mitochondrial DNA fragments were shown in Table 2 and FIGS. 1 to 6, respectively. The KCTC 0173BP strain was visually identified as a unique strain of DNA cutting bands that were very different from other strains of the mud mushroom as well as the strains of Felinus linteus.

In Figures 1-6, M is a standard DNA size marker, LIK is Felinus linteus KCTC 0173BP, LA5 is Felinus laevigatus FP-564998-T, and LA1 is Felinus lavigatus FP -125082-T CH1 is Felinus chrisoloma HHB-12766-T PU is Pelinus punctatus SNU-930719-6 LIU is Felinus Linteus L13202 LIJ is LIJ The Linteus WD1222 is replaced by the Pellinus ribis SNU-930919-5 for the RI and the Pellinus igniarius FP-125027-T for the RI, and the CH3 for the Felinus crissoloma HHB-3585-SP. Indicates.

* Very large and very small cuts in the stomach may make slight variations in the size of the mitochondrial DNA because it is difficult to measure the band size.

Example 3

Evolutionary Schematic of the Felinus Linteus KCTC 0173BP Strain

The evolutionary tree was created by calculating the restriction enzyme fragmentation similarity (F) and base substitution degree per base position (P) by substituting the results of restriction enzyme cleavage patterns of mitochondrial DNA of each Felinus linteus strain obtained in Example 2 into the following equation. It was. That is, by comparing the restriction enzyme segmentation pattern formed by one type of restriction enzyme in pairs with the segmentation pattern of the comparison target strain, the number of fragments having the same size and the number of fragments having different sizes are calculated, and The restriction enzyme fragment similarity of was calculated.

Restriction fragment similarity (F) = 2nxy / (n _x + n _y )

(Where n _x and n _y represent the number of DNA fragments of strains x and y respectively and n _xy represents the number of common DNA fragments of both strains.)

In addition, using the calculated F value, the base sequence per degree (necleotide sequence divergence value) was calculated as follows.

Base substitution degree per base position (P) =-(In F) / r

(Wherein r represents the number of recognized base pairs of restriction enzymes.)

As a result, the strain Felinus Linteus KCTC 0173BP was located very far from the strain Felinus linteus WD1222 as well as other strains as shown in FIG. 7 and clearly different from the strain Felinus linteus L13202. The evolutionary strain was identified as a completely different strain.

In addition, all species of the genus Phellinus investigated in the present invention was measured to be 80 to 100kb in size of mitochondrial DNA similar to the size of mitochondrial DNA of common fungi, but the size of about 42.5kb in the case of Felinus linteus strain It is estimated that this strain has been differentiated early from other species.

Example 4

Isolation and Purification of Anticancer Immunoactive Substances from the Felinus Linteus KCTC 0173BP Strain

20 l of a culture medium containing 50 g of glucose, 10 g of peptone, 10 g of yeast extract, and 0.5 g of dipotassium phosphate was added to a 30 L fermenter, and sterilized at 121 ° C. for 20 minutes. Inoculate 0.5 liter of seedlings of Pelinus linteus KCTC 0173BP prepared in advance in the same culture medium aseptically into the culture medium, and incubate for 3 days under conditions of temperature 28 ° C, aeration rate 1vvm, and agitation speed 100-200ppm. 80 g of mycelium was obtained.

1 L of water was added to the obtained mycelium, and hydrothermal extraction was repeated three times at 95 ° C. for 3 hours, followed by removing the mycelium cake and recovering the hydrothermal extract. Ethanol was added to the hot water extract at a final concentration of 80%, and treated at 4 ° C. for 24 hours. This process was repeated three times. The ethanol treated mole was then centrifuged at 12,000 rpm for 30 minutes. The obtained precipitate was dissolved in water and dialyzed at 4 ° C for 72 hours using a dialysis membrane (cellulose tubing, manufactured by Nippon Samgwang Pure Chemical Co., Ltd.), and ethanol was added to the dialyzed crude extract at a final concentration of 60%. Treatment during the start followed by centrifugation. The supernatant was lyophilized to obtain crude polysaccharide insoluble in 80% ethanol solution and soluble in 60% ethanol, and its anticancer immune activity was measured as follows.

In other words, the anticancer activity of mushroom-derived polysaccharides is not due to direct cytotoxicity, but due to the formation of antibodies by macrophage T cells, natural killer cells and B cells of macrophages, immune activity is increased and consequently anticancer activity. Therefore, anti-cancer immune activity can be indirectly demonstrated by counting antibody-forming lymphocytes. Therefore, as a result of investigating the number of antibody-forming cells by the crude polysaccharide obtained above, it was confirmed that it exhibits strong anticancer immune activity as shown in Table 3 below.

LPS used as a positive standard control is a polysaccharide showing strong anticancer immune activity

As can be seen in the above examples, the Felinus linteus KCTC 0173BP strain of the present invention produces a novel substance that exhibits excellent anticancer immune activity, and is determined by the restriction enzyme segmentation of mitochondrial DNA in the evolutionary system. It is a novel strain that is clearly distinguished from the strains and other strains of its kind, Pelinus linteus.

Claims (1)

Pelinus linteus KCTC 0173 BP strain producing anti-cancer immunoactive substances.