KR0161146B1 - Novel antibiotic hyphancin-3 derived from hyphntria cunea - Google Patents

Abstract

The present invention relates to a novel antibiotic hyphancin 3 isolated from the body fluid of Hyphantria cunea, wherein the hypancin 3 family antibiotics of the present invention have a molecular weight of about 4 kd and a mature hypansin 3 family. 37 for antibiotics, or 35 amino acids for amidation. In addition, the thermal stability is high, even if treated at 100 ℃ for 8 hours or more, the antimicrobial properties are maintained and shows a stable activity at a wide range of pH, for example, pH 2.0 to pH 12.0.

Description

Novel Antibiotic Hyphansin 3 from White Bull Moth

FIG. 1 shows the results of genomic Southern blot experiments on Hypansin III from white fire moths.

Figure 2 shows the nucleotide sequence and amino acid sequence of hypansin III-5,

3 shows the nucleotide sequence and amino acid sequence of hypansin III-10,

Figure 4 shows the nucleotide sequence and amino acid sequence of hypansin III-12,

Figure 5 shows the nucleotide sequence and amino acid sequence of hypansin III-14.

The present invention relates to a novel antibiotic hyphancin 3 isolated from the body fluids of Hyphantria cunea.

Insects use methods such as fleeing, stealth, and denial to protect themselves from the enemy, or they can control their enemies by means such as bee venom, ant acid, or stink of stink bugs. In addition, invaders that enter the body through food or wounds protect themselves by phagocytic cells such as blood cells forming nodules or by ingesting invaders or by producing humoral immune-related substances. These humoral immune-related substances include lectins or phenol oxidases present as bodily fluids, and lysozyme, cecropin, defensin, and atacin, which are newly produced or amplified upon invasion of the enemy. And peptides having antibiotic ability, such as peptides rich in proline or glycine.

Secroffin was found from the moth, the North American silk moth (Hyalophora cecropia) (Hultmark et al., Eur. J. Biochem., 106, 7 (1980); Steiner, Nature, 292, 246 (1981)) Studies on sexual immune related substances have been actively conducted. In particular, Callaway et al. Prepared secropin and its derivatives by genetic recombination (Callaway et al., Antimicro. Agent Chemother., 37, 1614 (1993). ).

Hyphantria cunea, also called the American White Bull Moth, is a carnivorous insect belonging to the Lepidoptera Arctidae family, and is mainly distributed in Western regions such as the United States, Canada, Hungary, and Italy. In Korea, it occurs two to three times a year. After wintering as a pupa, the first adult emerges in mid-May, and hatches in July-September, and it passes through the larvae for 25-30 days and the pupa for 13 days. Allegory survives for 3 to 4 days, life cycle is 48 to 56 days (Dolomite et al., Agriculture and Pestology, Hyangmunsa, (1990)).

The present inventors have applied for a patent application by separating high-pansine peptides having antibiotic ability from the body fluid of white fire moth (Korean Patent Application No. 93-32007), and continued research to separate another antibiotic from white fire moth. As a result, the antibiotics of the high-pansin 3-series were isolated and cloned to determine the DNA sequence to complete the present invention.

It is an object of the present invention to provide novel antibiotics and genes encoding them.

Another object of the present invention is to provide an antibiotic composition comprising the novel antibiotic.

In accordance with the above object, the present invention provides a polypeptide antibiotic hypansin 3 having a molecular weight of about 4 kd isolated from the body fluid of a white worm moth and a gene encoding the same.

According to another object of the present invention, the present invention provides an antibiotic composition comprising an antibiotic of the third series of hypansin.

The present invention will be described in detail as follows.

The novel antibiotic hypansin 3 of the present invention is a strong inducer produced in the body fluid (haemolymph) after a certain time after injecting microorganisms, for example, E. coli, Enterobacter, etc. It can be obtained by separating and purifying antibiotics, or from natural white moth larvae or larvae with physical stimuli.

More specifically, 10 ⁵ to 10 ⁶ CFU (colony forming unit) microorganisms, preferably 3 × 10 ⁵ to 3 × 10 ⁶ CFU microorganisms are injected per larvae, a predetermined time, preferably 18 to 24 After a period of time, the body fluid is recovered from the larvae's larvae and separated and purified only by fractions having anti-bioactivity using known methods such as centrifugation and chromatography to obtain 20 to 40 μg of antibiotics per 1 ml of white fire larva larvae. Can be.

The high-pansin 3-series antibiotics of the present invention isolated by the above method have a molecular weight of about 4 kd, 37 amino acids for the mature high-pansin 3-series antibiotics, or 35 amino acids when the C-terminal part is amidated. Is done. In addition, the thermal stability is high, even if treated at 100 ℃ for 8 hours or more, the antimicrobial properties are maintained and shows a stable activity at a wide range of pH, for example, pH 2.0 to pH 12.0.

On the other hand, the hypancin 3-series antibiotics of the present invention are amino acids 2, tryptophan, and 3, 6, 7 amino acids are lysine, amino acid 8 isoleucine, amino acid 9 is glutamic acid It is similar to antibiotic secroffins, but other amino acids are new antibiotics.

The DNA encoding the high-pansin 3-series antibiotics of the present invention can be cloned from the cDNA library of white moths. For example, by a plaque hybridization method, a fragment of DNA encoding a high-pansin 3-series antibiotic isolated from a body fluid of a white fire moth, preferably a fragment consisting of 32 bases encoding an N-terminus is probed. Cloning from cDNA libraries of white moths is possible, and the nucleotide sequences of the four DNAs obtained by this method are shown in FIGS. The isolated peptide is not necessarily identical to the DNA sequence, and in the present invention, high pansin 3 is used as a peptide and high pansin III is used as a gene.

The antibacterial activity of hypansin in the present invention can be measured by a method such as paper disk method (Lambert et al., Proc, Natl. Acad. Sci., 86, 262 (1989)), various Gram-negative bacteria and Gram-positive It can be assured that it exhibits strong antibacterial activity against bacteria.

Accordingly, the present invention provides an antibiotic composition comprising an effective amount of a high-pansin 3-series antibiotic as an active ingredient.

Such antibiotic compositions may include pharmaceutically acceptable excipients, carriers, diluents, and the like, along with the hypansin 3-series antibiotics, and may be formulated according to known methods using readily available ingredients. The formulations may be mixed with the carrier, diluted with the carrier, or contained in a carrier in the form of a capsule, sachet, paper or other container. If the carrier serves as a diluent, it may be a solid, semisolid or liquid substance which acts as a vehicle, excipient or medium for the active ingredient. The formulations may be in the form of tablets, pills, powders, sachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols, soft and hard gelatin capsules, sterile injectable solutions, sterile packaged powders and the like.

Examples of suitable carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, Cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydrocybenzoate, talc, magnesium stearate, mineral oil, and the like. The composition may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives and the like in addition to the above components. Compositions of the present invention can be formulated using techniques known in the art to release the active ingredient rapidly, continuously or delayed after administration to a patient.

Antibiotic compositions of the invention can be administered orally or parenterally, and the active compounds are effective for a wide range of dosages. For example, the daily dosage of the active compound typically ranges from about 1 to 100 mg, preferably 5 to 50 mg per kg of body weight. However, since the amount of the compound actually administered will be determined by the physician in light of the relevant circumstances, including the condition of the disease to be treated, the route of administration chosen, the age, weight and response of the individual patient, and the severity of the disease, the range of administration may be It is not intended to limit the scope in any way.

The present invention will be described more specifically with reference to Examples and Examples. However, the scope of the present invention is not limited by the following reference examples and examples.

Example 1

Isolation and Purification of Antibiotics from the Body Fluid of White Bull Moth

(Step 1)

Insect resources from the Korea Institute of Science and Technology, Biotechnology Research Institute, were fed silkworm artificial feed (oriental flowr) with a photoperiod of 25 to 27 ° C, 55 to 75% relative humidity, 14 to 16 hours of cancer, and 8 to 10 hours of cancer. 30,000 6-year-old white-fly moth larvae were bred in the chamber, and 100,000 E. coli (KCTC 2597) cultured in LB medium were injected into the body cavity of each larva and maintained for 18 to 24 hours. The larvae's leg was punctured with a fine needle, wounded, and then a few drops of body fluids were collected in a plastic tube cooled with ice and heat-treated at 100 ° C for 3 minutes. The heat-treated body fluid was centrifuged at 3,000 g to obtain a supernatant, which was named immune hemolym.

200 μl of immune hemolymph was dissolved in 50 mM phosphate buffer (pH 7.5), and proteinase K (from Tritirachium album, Sigma) and XIV (from Streptomyces griseus, Sigma) were each 10 IU / mg was added and reacted for 1 hour. Hemolymph was measured by measuring the antimicrobial activity of E. coli of hemolymph and protease-treated hemolymph by paper disk method (Lambert et al., Proc. Ntal. Acad. Sci., 86, 262 (1989)). Showed antimicrobial activity, but hemolymph treated with protease did not show antimicrobial activity. As a result, it was confirmed that the substance showing antimicrobial activity in hemolymph was peptide.

(Step 2)

20 ml of hemolymph obtained in (Step 1) was loaded into a column (length 240 mm, inner diameter 34 mm) filled with CM-Sepharose CCF-100 (manufactured by Sigma) pretreated with 50 mM citric acid buffer (pH 5.2). Then, the mixture was eluted with an eluent having a NaCl concentration gradient of 0 to 0.5 M (50 mM citric acid buffer (pH 5.2)) at a flow rate of 1 ml per minute, and 200 µl of each fraction was obtained and vacuum dried. After dissolving the vacuum dried in 20 μl of distilled water, the antimicrobial activity was measured. Antibiotic fractions were collected and concentrated by ultrafiltration (molecular weight cut-off value 1000, spectrum).

The concentrate was analyzed by reverse phase HPLC (Hewlett Packard, HP 1050), and the analysis conditions were as follows. That is, after loading the concentrate into LiChrosorb RP-18 (length 200mm, inner diameter 4.6mm), acidified water having acetonitrile concentration gradient increased by 1% per minute (trifluoroacetic acid 0.05% Was absorbed at 220 nm while flowing at a rate of 4 ml per minute. As a result, the novel high-pansin 3-antibiotics were obtained between the acetonitrile concentration gradient 40 to 50%, and the high-pansin 3-antibiotics separated at retention time of 44.5 minutes, 45.5 minutes, 46.5 minutes and 48 minutes, respectively. Named 3a, 3b, 3c and 3d.

In the reverse phase HPLC purification process, high pansin 1 (Korean Patent Application No. 93-32007) at acetonitrile concentration of 35%, high pansin 2 (Korean Patent Application No. 93-32007) at 55%, and lysozyme at 50%. Respectively obtained.

Example 2

Determination of Antimicrobial Activity of Hypansin 3 Series Antibiotic

The antibacterial activity of the highpansin 3a, 3b, 3c and 3d separated above was measured by the paper disk method as follows.

Gram-negative bacteria Escherichia coli KCTC 2597, Escherichia coli KCTC 1682, Enterobacter cloacae KCTC 2361, Klebsi cultured in nutrient medium (tryptone 0.5%, yeast extract 0.3%, beef extract 0.15%, glucose 0.1%) Klebsiella pneumoniae KCTC 2208 and Pseudomonas aeruginosa KCTC 2004; And Gram-positive bacteria Staphylococcus aurreus KCTC 1621, Staphylococcus epidermis KCTC 1917, Bacillus thuringiensis KCTC 1033, Streptococcus lactis 5 ml each of KCTC 1913 and Micrococcus luteus KCTC 3065 were aliquoted into plate medium and allowed to coagulate. A paper disc (Watmansa) having a diameter of 6.1 mm was placed on the medium, and 10 μl of 1 mg / ml of purified high-pansin 3a, 3b, 3c, or 3d was added to each of the discs, and incubated at 37 ° C. for about 18 hours. The diameter of the ring was measured.

On the other hand, Saccharomyces cerevisiae KCTC 522, Candida albicans KCTC 1940, Penicillium citrium (fungi) grown in potato-glucose medium (potato 3.3%, glucose 0.33%) Penicillium citrinum) KCTC 1255, Aspergillus niger (Aspergillus niger) KCTC 2119 was confirmed by the same method for the antibacterial activity of hypansin 3a, 3b, 3c or 3d.

The measurement results of the antimicrobial activity are shown in Table 1 below.

In the above results, hypansin 3a, 3b, 3c and 3d was shown to have an antimicrobial effect only against Gram-negative bacteria at the concentration of 6μM, but also showed antimicrobial activity against Gram-positive bacteria at a concentration of 20μM or more. However, the bacterium did not show anti-bioactivity.

Example 3

Molecular Weight Measurement of Hypansin 3-series Antibiotic

The molecular weights of the high-pansin 3-series antibiotics were determined by Tricine SDS-PAGE (Schagger Jagow, Anal. Biochem. 166, 368 (1987)) as follows.

The lyophilizate (3 μg) of the high pansin 3-series antibiotics purified by reversed phase HPLC in Example 1 (step 2) was dissolved in distilled water (3 μl) and the same amount of reaction solution (10% SDS, 20% glycerol, 10%) 2-mercaptoethanol, 0.12M tris-hydrochloric acid, 0.02% Coomassie Brilliant Blue G-250, pH 6.8), and then reacted at 65 ° C for 5 minutes. The resulting solution was loaded onto a discontinuous SDS gel (resolving gel: length 4 cm, 16.5% T, 3% C; stacking gel: length 1 cm, 4% T, 3% C), and then 0.2M tris-hydrochloric acid buffer as negative buffer. (pH 8.9) was electrophoresed using 0.1M Tris-tricine buffer (pH 8.25) containing 0.1% SDS as the positive buffer. After electrophoresis, it was fixed with 10% trichloroacetic acid and stained with a staining solution (colloidal blue, ISS, USA). As the molecular weight standard (ISS), insulin (3.5kd), aprotinin (6.1kd), lysozyme (14kd), and trisine inhibitor (20.4kd) were used. It became.

Example 4

(Change of antimicrobial activity according to pH change)

Dissolve 3 μg of highpansin 3b obtained in Example 1 in 1 ml of distilled water, and 5 μl of this solution was dissolved in 0.1 M HC1-KC1 buffer (pH 2.0), 50 mM citric acid-sodium citrate buffer (pH 4.0 and 6.0), 0.1 M phosphoric acid buffer (pH 8.0), mixed with 15 μl of sodium carbonate-sodium hydroxide buffer (pH 10.0) or disodium phosphate-sodium hydroxide buffer (pH 12.0) and maintained at 25 ° C. for 30 minutes, followed by antibacterial activity against E. coli KCTC 2597 by paper disk method. Was measured.

As a result, since the diameter of the transparent ring for E. coli was 11, 12, 12, 12, 11 and 10 mm, respectively, it was found that there was no difference in the antimicrobial activity due to the pH change.

Example 5

(Measurement of Antimicrobial Activity According to Temperature Change)

3 μg of the highpansin 3b obtained in Example 1 was dissolved in 1 ml of distilled water, and 5 μl of this solution was mixed with 15 μl of citric acid-sodium citrate buffer (pH 6.0) for 8 hours at 80 ° C., followed by the paper disc method. As a result of measuring the antimicrobial activity, the diameter of the transparent ring was 13 mm.

On the other hand, after treating the solution for 8 hours at 100 ℃, the antimicrobial activity was measured by the same method, the diameter of the transparent ring was 10mm. In other words, it was found that there is no change in the antimicrobial activity by temperature, it can be seen that high-pansin 3b is a heat stable material.

Example 6

Amino acid sequence of Hypansin 3

The N-terminal amino acid sequences of Hypansin 3a, 3b, 3c and 3d obtained in Example 1 were determined using a peptide sequencer (ABI, 476A) to obtain the following sequences:

As can be seen from the above, hypansin 3a, 3b, 3c and 3d are tryptophan amino acids 2 and 3, 6 and 7 lysine, amino acid isoleucine 8 and amino acid glutamic acid 9 It was confirmed that it is a kind of antibiotic peptide secroffin, which is preserved unchanged. However, since other amino acids differ from the secropins, it was determined to be a novel high-pansin antibiotic, and the DNA sequence encoding the high-pansin 3-antibiotic of the present invention was published on March 31, 1995. It was registered as u23831 (III-5), u23832 (III-10), u23833 (III-12) and u23834 (III-14) in the Gene Bank of the Institute for Biotechnology Research.

From the above results, the DNA sequence 5'-TGG AA (A / G), which encodes the amino acid sequence Trp Lys Val Phe Lys Lys Ile Glu Lys Val Gly, was used as a probe for gene cloning of the high pansin antibiotics. / T) AA (A / G) AA (A / G) AT (A / C / T) GA (A / G) AA (A / G) GT (N) GG-3 '(32-mer) It was. In this sequence, N is any one of A, T, G, C and the base in parenthesis means that any one of them is selected, so that the 32-mer codes for the same amino acid due to the degeneracy of the codon. It means a plurality of base sequences.

Example 7

(Preparation of cDNA Library of White Bull Moth)

100,000 E. coli (KCTC 2597) cultured in LB medium per 100-year-old white worm moth larvae were injected into the cavities of the larvae, finely pulverized under liquid nitrogen after 3 hours, and then pulverized (4M guanidium isocyanate, 25 mM EDTA). 50 mM Tris-Cl, pH 7.5, 23 ml of β-mercaptoethanol added to 2 ml) was dispersed, and disrupted for 1 minute using a homogenizer (Tekmar, Tissuemizer). N-lauroyl sarcosine was added to the crushing liquid so as to have a concentration of 0.5%, and the mixed solution was centrifuged at 4,000 g for 10 minutes at 4 ° C, and then a supernatant was obtained. This was again ultracentrifuged for 24 hours at 100,000 g in 5.7 M CsCl solution to obtain 500 μg total RNA. MRNA was isolated using a rapid mRNA separation kit (Stratagene).

Using the ZAP-cDNA synthesis kit (Stratagene), cDNA was synthesized from 5 μg of the mRNA isolated above, and purified through a Sephacryl S-400 column to obtain 100ng of cDNA. This was inserted into the restriction enzyme XhoI-EcoRI position of the lambda ZAP II vector (Stratagene), and then, using the Gigapack II Gold packaging extract (Stratagene), E. coli XL1-Blue MRF '(Stratagene) was transformed. The transformed Escherichia coli was incubated overnight at 37 ° C. in NZY solid medium containing 0.5% NaCl, 002% magnesium sulfate, 0.5% yeast extract, 1% NZ amine, pH 7.5 containing IPTG and X-gal. CDNA libraries were completed by obtaining plaques.

Example 8

(Check the base sequence of the gene encoding the high-pansin 3-series antibiotics)

The degenerate oligomers of Example 6 were labeled with gamma- ³² P according to the method of Sambrook et al. (Sambrook et al., Molecular Cloning, CSH, (1989)).

E. coli XL1-Blue MRF '(10 ⁵ ) was inoculated with 300,000 plaques obtained in Example 7, transferred to a Hybond N ⁺ membrane (Amasham), and then each 10 pM of labeled oligomer was added to the reaction solution ( Hybridization reaction was performed overnight at 37 ° C. in 6 × SSC, 20 mM NaH ₂ PO ₄ , 0.4% SDS, 5X Denhardt's, 500 μg / ml denatured salmon sperm DNA). Fifteen positive plaques were obtained in the first round.

Ten positive clones obtained in the second screening carried out in the same manner as above using a nitrocellulose membrane (Schleicher Schuell) were transferred to plasmid pBluescript (manufactured by Stratagene, ExAssist / SOLR), and the base sequence was determined.

Four non-overlapping genes out of the 10 base sequences were named Hipansin III-5, III-10, III-12 and III-14, respectively, and their base sequences are shown in FIGS. 2 to 5.

From the base sequence of FIGS. 2 to 5, the antipeptide composed of 37 amino acids was removed by removing the signal peptide consisting of 26 amino acids from preprohyphansin 3 precursor composed of 63 amino acids. It can be seen that it is mature hypansin 3 with activity.

Example 9

Southern blotting analysis using white light moth genomic DNA

10 μg of genomic DNA extracted from the worm larvae by phenol extraction (Sambrook et al., Molecular Cloning, CSH, (1989)) was digested with restriction enzymes SalI, XbaI, XhoI or HindIII at 37 ° C. overnight. The digested fragments were electrophoresed on a 0.8% agarose gel, and the DNA fragments separated on the gel were treated with denaturing solution (0.5N NaOH, 1.5M NaCl) and transferred to a nylon membrane (Amarsham).

Probes prepared using the DIG-Random Primed DNA Labeling Kit (Boehringer Mannheim) from 300ng each of the cDNAs of the highpansine III-5, III-10, III-12 and III-14 genes obtained in Example 8 were prepared using the nylon It was added to the membrane and hybridized at 68 ° C. overnight. After the reaction was completed, the membranes were washed with wash solution (0.1X SSC, 0.1% SDS) and anti-DIG alkaline phosphatase (Boehringer Mannheim) was treated according to the manufacturer's instructions. The result of the Southern blotting is shown in FIG.

In FIG. 1, A is an electrophoretic gel, B is a bloated nylon membrane, in column A and B the first row is a control treated with lambd DNA with HindIII, and columns 2-5 are genomic DNA. Samples treated with Sali, Xbai, XhoI and Hind III are shown.

From the above results, it was confirmed that the highpansin III-5, III-10, III-12, III-14 genes isolated in the present invention are the genotypes of the whiteworm moth larva itself, and the whiteworm moths at least the highpansin III family genes. It is estimated to have about 20.

(Toxicity test)

20-week-old females weighing 15-18 g and 20 males weighing 17-20 g as 5-week-old specific pathogens-free ICR mice with temperature 22 ± 1 ° C, humidity 55 ± 5%, and lighting 12L / 12D Breeding indoors. Feed for experimental animals (Korean experimental animals) and drinking water were supplied after sterilization and free ingestion.

Hypansin 3a, 3b, 3c or 3d of Example 1 was prepared at a concentration of 100 mg / ml using sterile distilled water as a solvent, and five female and male mice were orally administered 0.2 ml per 20 g of body weight. Samples were administered orally once and observed for side effects or lethality for 10 days after administration. That is, on the day of administration, changes in general symptoms and the presence or absence of dead animals were observed at least once in the morning, at least once every afternoon from 1 hour, 4 hours, 8 hours, 12 hours, and from 2 to 10 days after administration. In addition, the animals were killed and dissected at 10 days of administration, and the internal organs were visually examined. Changes in body weight were measured at daily intervals from the day of administration to observe the weight loss phenomenon of animals by high-pansin 3a, 3b, 3c or 3d.

As a result, in the case of acute oral toxicity, no lethal animals were observed for 10 days at a dose of 1,000 mg / kg of highpansin 3a, 3b, 3c or 3d. An autopsy of the surviving animals was performed 10 days later, and there were no gross lesions, and normal body weight gain was observed without any weight loss for 10 days from the day after the administration.

In conclusion, hypancin 3a, 3b, 3c or 3d was not toxic upon oral administration at the above concentrations.

Formulation Example: Preparation of antibiotics containing high-pansin 3-series antibiotics

[1] preparation of capsules

Hard gelatin capsules were prepared using an antibiotic composition comprising the following ingredients:

The ingredients were mixed thoroughly and filled into hard gelatin capsules in a total amount of 500 mg.

[2] tablets manufacturing

Tablets each containing 500 mg of active ingredient were prepared as follows:

Active ingredient, starch and cellulose are passed through No. 45 mesh sieve and mixed thoroughly. An aqueous solution containing polyvinylpyrrolidone was mixed with the powder obtained, and then the mixture was passed through No. 14 mesh sieve. The granules obtained were dried at 50 ° C. and passed through a No. 18 mesh sieve. Sodium carboxymethyl cellulose, magnesium stearate and talc, which were previously passed through a 60 mesh sieve, were added to the granules, mixed, and then compressed into tablets to obtain tablets with a weight of 500 mg each.

Claims (4)

An antibiotic polypeptide having any one amino acid sequence selected from the group consisting of the following amino acid sequences: DNA encoding the polypeptide of claim 1. According to claim 2, having one base sequence selected from the group consisting of the following base sequences An antibiotic composition comprising the antibiotic polypeptide of claim 1 as an active ingredient.