JP2671911B2 - Antibacterial protein, production method and use thereof - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an antibacterial protein that is induced in the body fluid of larvae of an insect belonging to the order Coleoptera, its production method and its use.

[0002]

BACKGROUND ART Inoculation of insects with bacteria and different types of blood cells,
It is known that various antibacterial proteins are induced in body fluids when a stimulus such as simply damaging the body surface is given. These substances are considered to have an important role in host defense of animals without antibody-producing ability. Among these, for example, a member of the family Tenebrionidae (Zophobas at
ratus) as an active protein induced in larval body fluid, a protein having antibacterial activity (J. Boil. Chem., 266, 245
20-24525 (1991)) and the like are fixed. The protein with its antibacterial activity is named Coleoptericin, a protein whose physicochemical properties have been clarified, and also obtained from the larval fluid of a member of the family Tenebrionidae (Zophobas atratus). And proteins whose physicochemical properties are also known. Since they have a broad antibacterial spectrum, they are considered to be bioprotective substances, and further, these antibacterial proteins have strong antibacterial activity against Escherichia coli, which is a clam-negative bacterium, and Micrococcus, which is a gram-positive bacterium.

On the other hand, in recent years, the existence of antibacterial proteins having a high antibacterial activity and a broad spectrum has been revealed in semen and serum even in higher mammals, and antimicrobial proteins in general animal body fluids have been regarded as important. ing.

However, the antibacterial proteins of other insects belonging to the order Coleoptera have not been purified or separated, and physicochemical properties such as properties of substances have not been clarified.

[0005]

In view of the above circumstances, the present invention has established a method for purifying and separating an antibacterial protein of the beetle Beetle belonging to the order Coleoptera, and has established the physical and chemical properties of the substance, N-terminal amino acid sequence and the like. The purpose is to clarify. Therefore, if this proteinaceous substance can be purified / isolated into a single substance, its amino acid sequence is determined, and these insect-derived proteins act on the biological defense mechanism of humans and other vertebrates, for example, medical / pharmaceutical There is a great contribution from the point of view.

[0006]

[Means for Solving the Problems] As a result of intensive studies, the present inventors have surprisingly found that an antibacterial protein is induced in the body fluid of insects belonging to the order Coleoptera, especially the larva of the swan, Beetle. It was found that they were carried out, the purification and separation of them were successful, and the present invention was completed.

That is, the present invention is as follows: (1) The N-terminal amino acid sequence H ₂ is induced in the body fluid of an insect larva belonging to the order Coleoptera upon body surface injury.
N-Ala-Leu-Ser-Pro-Gly-Ala-Pro-Asn-
Phe-Pro-Asn-Pro-Gly-, which is an antibacterial protein having the following properties, or an antibacterial protein having an immunological common antigenicity therewith (i) molecular weight of about 7,300 to 7,800 ( ii) Thermal stability (5-10 minutes, 100 ° C) (2) Insects belonging to the order Coleoptera are the beetle Beetle (Or
yctes rhinoceros), the antibacterial protein according to (1), (3) the larva is a third-instar larva, (1) or the antibacterial protein according to (2), (4) the body fluid of the larva is 0 after body surface injury. The antibacterial protein according to any one of (1) to (3), which is after 10 days, and

(5) The body surface of the larva of an insect belonging to the order Coleoptera is damaged, and the body fluid obtained after removing body fluid cells and fat pads has an N-terminal amino acid sequence of H ₂
N-Ala-Leu-Ser-Pro-Gly-Ala-Pro-Asn
-Phe-Pro-Asn-Pro-Gly-, which is an antibacterial protein having the following properties, or an antibacterial protein having an immunological common antigenicity therewith (I) Molecular weight of about 7,300 to 7,800 (ii) Thermal stability (5 to 10 minutes, 100 ° C) (6) Insects belonging to the order Coleoptera are the beetle Beetle (Or
yctes rhinoceros) (5) The production method according to (5) The larva is a third-instar larva, the production method according to (5) or (6) (8) The body fluid of the larva is 0 to 10 after body surface injury. The production method according to any one of (5) to (7), which is after a lapse of days, and

(9) The N-terminal amino acid sequence of H ₂ N-Ala-Leu-Ser-Pro-Gly-Ala, which is induced in the body fluid of larvae of insect larvae belonging to the order Coleoptera, is induced.
-Pro-Asn-Phe-Pro-Asn-Pro-Gly-, which is an edible antibacterial protein containing an antibacterial protein having the following properties, or an antibacterial protein having an immunological common antigenicity therewith Agent (i) Molecular weight: approx. 7,300 to 7,800 (ii) Thermal stability (5 to 10 minutes, 100 ° C) (10) Insects belonging to the order Coleoptera are beetle beetles
(Oryctes rhinoceros), the edible antibacterial agent according to (9). (11) The larva is a third-instar larva, (9) or (1).
(12) The edible antibacterial agent according to any one of (9) to (11), wherein the body fluid of the larva is 0 to 10 days after the body surface injury. To do.

Hereinafter, the present invention will be described in detail. In the present invention, "antibacterial protein having immunological common antigenicity"
The term “antibacterial protein” in which one or more amino acids are substituted or deleted and which has a common antigenicity.

The insects targeted by the present invention are insects belonging to the order Coleoptera. Specifically, there are scarab beetles, beetles, etc. Among these, the scarab beetles of the family Scarabaeidae, particularly the Chinese beetle, are preferred.

[0012] The target insect must be a larva. Here, the term “larva” refers to those after hatching and before pupation in the process of complete metamorphosis of insects. Suitable in the present invention are larvae that are synchronized with the third instar, in particular larvae of the beetle that are synchronized with the third instar.

The procedure of damaging insect body fluids to induce antimicrobial peptides can be any method that results in an increased risk of infection. It is needless to say that the term "body surface" does not mean only the body surface, but also includes an operation such as penetration of an injection needle that causes damage to the body. The body surface to be injured may be anywhere in the body of the insect, but it is usually the part excluding the head.

0 to 10 days after the injury is given, preferably about 1 day after, the body fluid is squeezed out and the body fluid cells are removed by centrifugation to obtain a starting material.

For the isolation and purification of the antibacterial protein of the present invention, various methods commonly used for fractionation and purification of general proteins can be applied. In the present invention, in particular, when using the larvae of the Taiwanese beetle, the centrifugal supernatant obtained above is fractionated by reverse phase chromatography, and a fraction showing significant activity according to the method for measuring antibacterial activity described below for each fraction is obtained. It can be separated and further purified by reverse phase chromatography to obtain a single peak.

In the case of purifying from the centrifugal fraction to the single substance of the present invention, various methods other than those described above are conceivable. For example, ion exchange chromatography, hydrophobic chromatography, electrophoresis, etc. are appropriately used to achieve the purpose. It is possible.

Since the antibacterial substance thus obtained is inactivated by the trypsin treatment, it is clear that it is a protein substance. This antibacterial protein is obtained as a white powder when freeze-dried.

Further, since the antibacterial protein of the present invention has good heat resistance, it can be purified by heating a crude sample to enhance its specific activity. That is, 80 to
It is convenient to perform heat treatment at 120 ° C., preferably around 100 ° C. for about 10 to 60 minutes.

The method for measuring the antibacterial activity of the antibacterial protein according to the present invention is, for example, in the case of Gram-positive bacteria, Staphylococcus aureus
(Staphylococcus aureus) or hemolytic streptococcus (Str
eptococcus pyogenes), in the case of Gram-negative bacteria, Escherichia coli K12 can be used, and its growth inhibition rate can be measured as an index.

The antibacterial protein according to the present invention has a broad antibacterial spectrum as described above and extends to both Gram positive and negative. Therefore, it is useful as an antibacterial agent by taking advantage of its antibacterial property. That is, alone or in combination with a suitable liquid, solid or gaseous carrier or diluent,
Alternatively, it can be used as an external or internal antibacterial agent in the form of being combined with other drugs.

Therefore, the antibacterial peptide of the present invention is
For example, it can be used as a drug against bacterial diseases. In this case, the dosage form and the dosage of the administration can be arbitrarily selected in consideration of the type and symptoms of the patient and the disease, as long as the antibacterial effect according to the present invention is recognized.

Since the antibacterial protein of the present invention is a protein itself, it is considered that its toxicity is almost negligible at least when it is orally inoculated. Therefore, this antibacterial peptide can be used as a drug for humans and animals and a food or feed additive. For example, the antibacterial protein according to the present invention is useful as an antibacterial agent as a food additive, in other words, an edible antibacterial agent.

[0023]

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

1) Material and measuring method (1) Starting material As the larva of the Taiwan beetle, a third instar larva was used. For the operation of injuring the body surface, a method was used in which the larva was placed on ice for several minutes to slow down the movement, and then a hypodermic needle was penetrated.
The injured larvae were bred at 25 ° C. for 24 to 48 hours in a container containing mulch. After that, cut off the larval leg,
Body droplets were collected in a tube on ice by pressing down on the abdomen and squeezing out. Usually about 1.5 ml from one larva
Body fluid was obtained. The obtained body fluid is 39,000 × g / 5
The blood cells were removed by centrifugation for 0 minutes, and the clear supernatant was stored at -20 ° C.

(2) Fractionation of body fluid Body fluid (about 15 ml) collected from about 10 larvae was used as solvent A.
(20% acetonitrile / 0.05% trifluoroacetic acid)
Sep-Pak C ₁₈ cartridge (Wa
ters Associates). After washing the column with solvent A, the antibacterial activity was eluted with 60% acetonitrile / 0.05% trifluoroacetic acid. The active fraction was freeze-dried to remove acetonitrile. This dry powder was dissolved in 0.05% trifluoroacetic acid and applied to a ProRPC HR5 / 10 C ₈ column (Pharmacia, 5 × 100 mm) equilibrated with 0.05% trifluoroacetic acid. After washing the column with 0.05% trifluoroacetic acid, gradient elution was carried out under the condition of 0% → 50% acetonitrile / 0.05% trifluoroacetic acid (75 minutes). At this stage, antibacterial activity was eluted in the 26-31% fraction of acetonitrile. The active fraction was freeze-dried to remove acetonitrile. Furthermore, this dry powder was dissolved in solvent A and equilibrated with solvent A to produce ProRPC HR5 / 1.
It was applied to a 0 C ₈ column (Pharmacia, 5 × 100 mm). After washing the column with solvent A, 20% → 40% acetonitrile / 0.05% trifluoroacetic acid (120 minutes)
The elution was performed under mild gradient conditions. At this stage, the antibacterial activity is 22-24% of acetonitrile fraction
It was separated into two fractions (fraction B) and 24-27% acetonitrile (fraction C). For these active fractions,
Lyophilization was performed to remove the acetonitrile. The present invention relates to an antibacterial protein (lineasalacin) from the C fraction.
It was made by purifying.

(3) Measurement of antibacterial activity Staphylococcus aureus, Streptococcus pyogenes or Escheric
Hia coli K12) was cultured, exponentially growing cells were selected, and 30 mM phosphate buffer (Na ₂ containing 60 mM NaCl) was selected.
HPO ₄ / NaH ₂ PO ₄ , pH 7.0) (Bec
kman DU-650 spectrometer A ₅₅₀ , 0.1 (1.5
× 10 ⁷ cells / ml)). Sample (50 μl), Mueller Hinton medium (40 μl, Difco) and bacterial suspension (1
0 μl) into a microtube and shake to 3
Incubated at 7 ° C for 20 hours. Thereafter, the mixture was quenched and _A550 was measured.

(4) Tricine-SDS-polyacrylamide gel electrophoresis Slab-type tricine-SDS-polyacrylamide gel electrophoresis was performed by the method of Schagger and Jagow (Anal. Bioc.
hem. 166, 368-379 (1987)). Protein samples were dissolved in SDS gel-loading buffer (50 mM Tris-HCl (pH 6.8), 10% β-mercaptoethanol, 2% SDS (sodium dodecyl sulfate), 0.1% promophenol blue, 10% glycerol). After heating at 100 ° C. for 5 minutes, it was subjected to tricine-SDS-polyacrylamide gel electrophoresis. The acrylamide content of the gel was 16.2%.
After electrophoresis, the gel was loaded with the method of Fairbanks et al. (Biochemi
stry, Vol. 10, 2606-2617 (1971)) and stained with Coomassie Brilliant Blue R-250.

2) Purification of linasalacin About 140 μg of the C fraction obtained from about 10 larvae was
10 mM phosphate buffer containing 130 mM NaCl (Na ₂ H
PO ₄ / NaH ₂ PO ₄ , pH 6.0) and heat treatment at 100 ° C. for 5 minutes in order to completely remove the heat-precipitating protein. Then, the preparation was subjected to centrifugation for 10 minutes / 39,000 × g, and the resulting supernatant was finally prepared so as to contain 30% acetonitrile / 0.05% trifluoroacetic acid, and the solvent was added. B (30% acetonitrile / 0.05%
Reversed phase column (2.
1 x 100 mm, Pharmacia μRPC C2 / C1
8) was applied. This reversed-phase column was connected to a Pharmacia SMART system and used. After washing the column with solvent B, 30% → 40% acetonitrile / 0.0
Elution was performed under mild gradient conditions of 5% trifluoroacetic acid (60 minutes). Fraction 200 μl each,
Lyophilize to remove acetonitrile,
Each fraction was measured. The antibacterial activity was eluted as a single peak corresponding to the protein peak as shown in FIG. The active fraction was subjected to tricine-SDS-polyacrylamide gel electrophoresis to give a single protein band (molecular weight: about 7,30).
0-7800) was given and it was named linasalacin.
The electropherogram of purified liner salacin is shown in FIG. In the figure, the arrow indicates the indicator protein (myoglobin I & II (1
4.1kDa), myoglobin I (8.2kDa), myoglobin II (6.2kDa) and myoglobin III (2.
The position of 5 kDa)) is shown. The amount of protein can be determined according to the method of Raleigh (J.
Biol. Chem. , 193, pp. 265-275 (195
1)), using synthetic silkworm cecropin B as a standard. The purified physiologically active protein linasalacin of the present invention was subjected to Edman degradation and analyzed for PTH-amino acid, whereby the 13 amino acid sequence at the N-terminal (SEQ ID NO: 1) was determined.

3) Measurement of linasalacin concentration and antibacterial activity It was measured using the antibacterial protein linasalacin purified in 2). In the measurement of the antibacterial activity of 1)-(3), the amount of protein in 50 μl of the test liquid was changed to 0 to 10 μg / ml. FIG. 3 shows the results. From this result, the antibacterial activity of the physiologically active protein linasalacin of the present invention was strongly recognized against both Gram-positive bacteria and negative bacteria.

Further, the physiologically active protein linasalacin of the present invention retained its original antibacterial activity even after being heat-treated at 100 ° C. for 5 to 10 minutes.

[0031]

INDUSTRIAL APPLICABILITY The antibacterial peptide according to the present invention has antibacterial properties by itself, and has almost no toxicity. Therefore, there are various possible applications that can utilize this property,
In particular, it can be expected to be applied as a pharmaceutical preparation containing these substances as active ingredients or used as a food additive.
In particular, the antibacterial peptide obtained from the larva of the Taiwanese beetle is thermostable and can be particularly expected to be used for applications including a processing step by heat.

[0032]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 13 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence: Ala Leu Ser Pro Gly Ala Pro Asn Phe Pro Asn Pro Gly 1 5 10

[Brief description of the drawings]

FIG. 1 is an explanatory diagram showing the results of reverse phase SMART analysis of purified liner salacin.

FIG. 2 is a copy of an electropherogram of liner salacin.

FIG. 3 is a view showing an antibacterial action of the liner salacin of the present invention.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication C07K 7/08 ZNA A61K 37/02 ADZ

Claims (12)

(57) [Claims] 1. An N-terminal amino acid sequence which is induced in the body fluid of a larva of an insect belonging to the order Coleoptera, H ₂ N-Ala-Leu-Ser-Pro-Gly-Ala-Pro.
An antibacterial protein which is -Asn-Phe-Pro-Asn-Pro-Gly- and has the following properties. (i) Molecular weight about 7,300 to 7,800 (ii) Thermal stability (5 to 10 minutes, 100 ° C) 2. The antibacterial protein according to claim 1, wherein the insect belonging to the order Coleoptera is the beetle Oryctes rhinoceros. 3. The antibacterial protein according to claim 1 or 2, wherein the larva is a third-instar larva. 4. The antibacterial protein according to any one of claims 1 to 3, wherein the body fluid of the larva is from 0 to 10 days after the body surface injury. 5. The body surface of an insect larva belonging to the order Coleoptera is injured, and body fluids and fat pads are removed from the body fluid obtained thereafter, and the N-terminal amino acid sequence is H ₂ N-Al.
a-Leu-Ser-Pro-Gly-Ala-Pro-Asn-Phe
A method for producing an antibacterial protein, which comprises -Pro-Asn-Pro-Gly- and obtains an antibacterial protein having the following properties. (i) Molecular weight about 7,300 to 7,800 (ii) Thermal stability (5 to 10 minutes, 100 ° C) 6. The method according to claim 5, wherein the insect belonging to the order Coleoptera is the beetle Oryctes rhinoceros. 7. The production method according to claim 5, wherein the larva is a third-instar larva. 8. The method according to claim 5, wherein the body fluid of the larva is from 0 to 10 days after the body surface injury. 9. induced body fluid of the larvae when insect larvae body injuries belonging to Coleoptera, N-terminal amino acid sequence _{H 2 N-Ala-Leu-} Ser-Pro-Gly-Ala-Pro
-Asn-Phe-Pro-Asn-Pro-Gly-, which is an edible antibacterial agent containing an antibacterial protein having the following properties as an active ingredient. (i) Molecular weight about 7,300 to 7,800 (ii) Thermal stability (5 to 10 minutes, 100 ° C) 10. The edible antibacterial agent according to claim 9, wherein the insect belonging to the order Coleoptera is the beetle Oryctes rhinoceros. 11. The edible antibacterial agent according to claim 9, wherein the larva is a third-instar larva. 12. The edible antibacterial agent according to claim 9, wherein the body fluid of the larva is 0 to 10 days after the body surface injury.