KR0139091B1 - Novel Antimicrobial Protein Isolated from the Larvae of Chinese Cabbage (Pieris rapae) - Google Patents

Abstract

The present invention relates to a novel antimicrobial protein isolated from the cabbage butterfly (Pieris rapae) larvae.

The antimicrobial protein of the present invention showed antimicrobial activity against both Gram-negative bacteria and Gram-positive bacteria by lysing bacteria, and particularly, showed stronger antimicrobial activity against Gram-negative bacteria Escherichia coli. In addition, the antimicrobial protein of the present invention has a molecular weight of about 4,000 Daltons, and it was confirmed that it belongs to the secropin system by amino acid analysis. Since the antimicrobial protein of the present invention exhibits antimicrobial activity against bacteria, it may be used as an antimicrobial agent.

Description

Novel Antimicrobial Protein Isolated from the Larvae of Chinese Cabbage (Pieris rapae)

FIG. 1 is a graph showing that the immune hemolymph of cabbage butterfly larvae has antimicrobial activity against E. coli.

Figure 2 is a graph showing that the immune hemolymph of cabbage butterfly larvae have antibacterial activity against Bacillus subtilis.

3 is a graph showing that the immune hemolymph of cabbage butterfly larva lyses bacteria.

Figure 4 is a photograph showing the results of measuring the antimicrobial activity of the treatment of immune hemolymph with trypsin.

5 is a graph showing the results of reverse phase FPLC of the eluate obtained from the CM-Sepharose column using a PepRPC HR 5/5 column.

Figure 6 is a photograph showing the results of acidic PAGE of the active fraction of the antimicrobial protein obtained by each purification step.

Figure 7 (a) is a photograph showing the result of SDS-PAGE of the purified antimicrobial protein.

FIG. 7 (b) is a graph showing molecular weight determination by SDS-PAGE.

8 shows the amino acid sequence of the purified antimicrobial protein.

The present invention relates to a novel antimicrobial protein isolated from the cabbage larvae (Pieris rapae) larvae. More specifically, the present invention relates to an antimicrobial protein that exhibits antimicrobial activity against various bacteria isolated from larvae of Chinese cabbage butterfly.

Every animal on the planet has a biodefense mechanism that can protect itself or protect itself from infection by microorganisms such as viruses and bacteria. This phenomenon also applies to invertebrates including insects. Unlike higher animals, insects do not have an immune response due to antibodies, so they must protect themselves with other effective defenses. In other words, insects are exposed to a variety of environments and are more likely to become infected, including infections from contaminated food, infections through the respiratory tract, infections through wounds, and infections from intestinal bacteria during metamorphosis. It can be infected through the bar, it is necessary to have a defense mechanism against this external infection.

Insects respond with cellular and humoral immunity to effectively defend themselves from these infections.

Cellular immune responses have already been studied by morphologists since the 1900's and have been described as hemocyte responses such as phagocytosis, nodule formation, and encapsulation (see NA). Ratcliffe et al., Int. Rev. Cytol., 97: 183-350 (1985)).

In the humoral immune response, Boman et al., Sweden, identified antibacterial protein (ABP) in Drosophila in the early 1970s (HG Boman et al., Nature (London), 237: 232-). 235 (1972)), an inducible bacteriolytic protein in the immune haemolymph of the cecropia moth, which was tested by the antimicrobial proteins, cecropin and lysozyme. Was isolated for the first time (D. Hultmark et al., Eur. J. Biochem., 106: 7-16 (1980)), followed by identification of atacin (D. Hultmark et al. , EMBO J., 2: 571-576 (1983)). Subsequently, the structures of the genes for atasin, lysozyme and cecropin were identified (K. Kockum et al., EMBO. J., 3: 2071-2075 (1984); A. Engstrom et al., EMBO). J., 4: 2119-2122 (1985); P. von Hofsten et al, Proc. Natl. Acad. Sci., USA. 82: 2240-2243 (1985)), recently hemolin and andro Pins were identified (SC Sun et al., Science, 250: 225-230 (1990)); C. Samakovlis et al., EMBO. J., 10: 163-169 (1991).

Other antimicrobial proteins include sarcotoxin (M. Okada et al., J. Biol. Chem., 260: 7174-7177 (1985)), diftericin, J. Dimarcq et al. , Eur. J. Biochem., 171: 17-21 (1988)), apidaecin (see, P. Casteels et al., EMBO. J., 8: 2387-2391 (1989)), absinine ( abaecin, P. Casteels et al., Eur. J. Biochem., 187: 381-386 (1990)), and defensin (P. Bulet et al., Eur. J. Biochem., 209: 977-984 (1992).

On the other hand, the inventors of the present invention intensively searched for new antibiotics from a variety of insects, as a result of inducing and separating a highly active antimicrobial protein from the larvae of Chinese cabbage butterfly (Pieris rapae), characteristics, structure and function As a result of the discovery, it was found that the novel antimicrobial protein was completed the present invention.

After all, it is an object of the present invention to provide a novel antimicrobial protein isolated from the cabbage butterfly (Pieris rapae) larvae.

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

In order to prepare an immune haemolymph from cabbage butterfly larvae, E. coli was injected into the larvae of the larvae, centrifuged with hemolymph, and the supernatant was obtained.

The antimicrobial activity of the immune hemolymph obtained in the above was determined by the inhibition zone measurement using a thin agarose plate inoculated with Gram-negative Escherichia coli and Gram-positive Bacillus subtilis. As a result, the immune hemolymph of cabbage butterfly larvae showed stronger antibacterial activity against Gram-negative Escherichia coli. On the other hand, in order to find out how the antimicrobial factors in the hemolymph lymphocytes actually act on the bacteria, the hemolymph was treated with a transmission electron microscope (TEM) and a scanning electron microscope (SEM). Observation of E. coli showed that the antimicrobial factors in the immune hemolymph lysed the bacteria.

In this regard, as a result of measuring the degree of lysis of bacteria by immune hemolymph, the immune hemolymph of cabbage white butterfly larva significantly reduced the cell number of gram-negative and gram-positive bacteria, especially in E. coli, a gram-negative bacterium. It showed stronger lysis.

In addition, in order to identify what kind of antimicrobial factors in the immune blood lymphocytes, trypsin treatment and antimicrobial activity was measured in the immune blood lymphocytes, trypsin-treated immune blood lymphocytes showed no antibacterial activity, but trypsin treatment Antimicrobial activity in the immune hemolymph was not seen, it was found that the antimicrobial factor in the immune hemolymph is a protein.

In order to isolate the former antimicrobial protein, the hemolymph obtained from Chinese cabbage larvae was heat-treated and centrifuged, and then the supernatant was injected into a CM-Sepharose column, washed and eluted with an active fraction showing antimicrobial activity. These fractions were then injected into a PepRPC HR5 / 5 column to run reverse phase FPLC. An antimicrobial protein eluting at a concentration of 30.1 to 31.8% acetonitrile was obtained.

It was confirmed by acidic-PAGE that the previously isolated antimicrobial protein was purified. The molecular weight of the antimicrobial protein was about 4,000 Da, similar to that of Secropin A. In addition, amino acid sequence determination of the antimicrobial protein confirmed that 23 amino acids among the 40 amino acids of the antimicrobial protein of the present invention were identical to those of known secropin A.

Therefore, it was found that the antimicrobial protein of the present invention belongs to the secropin system.

As described above, since the antimicrobial protein of the present invention exhibits antimicrobial activity against both Gram-negative bacteria and Gram-positive bacteria, it may be used as an antimicrobial agent against various bacteria.

Amino acid sequences used in the present invention are abbreviated as follows according to the IUPAC-IUB nomenclature:

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example 1

Production of Immune Hemolymph from Chinese Cabbage Butterfly

The Chinese cabbage butterfly (Pieris rapae) was collected outdoors in Cheonan, Chungcheongnam-do, South Korea, and reared in a greenhouse.The fourth larvae were transferred to the laboratory and fed with kale to feed at 27 ℃, relative humidity of 70-75%, and photoperiod 16L: It was raised in 8D and used as a sample. About 2 × 104 Escherichia coli in log phase was injected into the larvae's chest, hemolymph was taken and centrifuged, and the supernatant was used as a sample for antimicrobial activity measurement.

Example 2

Measurement of antimicrobial activity

Example 2-1

Suppression Band Measurement

Antimicrobial activity of immune hemolymphs was measured using a thin agarose plate inoculated with Gram-negative E. coli and Gram-positive Bacillus subtilis. Measurements were made using standard curves (see H. Steiner et al., Nature, 292: 246-248 (1981)). That is, at 37 ° C., E. coli of 50 log of the logarithm grown in LB liquid medium and 25 times of Bacillus subtilis were mixed with 0.05 ml of the dilution solution and 6 ml of the 1% agarose solution dissolved in the LB liquid medium. It was poured into a Petri dish having a diameter of 9 cm and hardened. After making a hole with a diameter of 3 mm in a solid plate medium, 3 μl of superimmune blood supernatant was added thereto, and incubated at 30 ° C. for 15 to 20 hours, and the inhibition zone of bacteria was measured (see FIGS. 1 and 2). Degree). Hemolymphs taken from larvae injected with sterile distilled water, including saline, and hemolymphs taken after wounding the larvae with a needle. As shown in FIGS. 1 and 2, the immune hemolymphs of cabbage larva larvae exhibit antimicrobial activity against both Escherichia coli and Bacillus subtilis and, in particular, have a stronger antimicrobial activity against Gram-negative Escherichia coli. .

On the other hand, in order to see how the antimicrobial factor in the immune hemolymph actually acts on bacteria, E. coli treated with immune hemolymph was observed using TEM and SEM. SEM observation showed that abnormal projections appeared in the cell membrane of Escherichia coli.

Example 2-2

Lysis of bacteria

The degree of dissolution of bacteria by immune hemolymph was diluted with 0.1 M phosphate buffer solution (0 ° C, pH 6.4) to adjust absorbance at 0.3 to 0.5 at 570 nm, followed by 1 ml of bacterial solution and immune hemolymph. 20 µl was mixed and reacted with each time from 2 to 15 minutes, and the absorbance was measured at 570 nm (see FIG. 3). As shown in FIG. 3, the immune hemolymph of cabbage white butterfly reduced the number of cells of three bacteria, Escherichia coli K12, Escherichia coli K12 D21 and Bacillus megaterium Bmll within 50 minutes, in particular 50%. , E. coli, a Gram-negative bacterium, showed stronger lytic activity.

Example 3

Identification of Antimicrobial Proteins

In order to identify what the antimicrobial factors in the immune hemolymphs prepared in Example 1 were treated with trypsin and hemolymphs, the antimicrobial activity was measured as in Example 2-1 (see reference Example 2-1). 4). In Figure 4, NH is a normal blood lymph; T. NH was normal blood lymphocytes treated with trypsin; IH is immune hemolymph; And T.IH represent immunohemolymphs treated with trypsin, respectively. As shown in Figure 4, the normal blood lymphocytes, trypsin-treated normal blood lymphocytes and trypsin-treated immune blood lymphocytes did not show any antimicrobial activity at all, but immune blood lymphocytes, antimicrobial activity appears to appear, It can be seen that the antibacterial factor is a protein.

Example 4

Purification of Antimicrobial Proteins

In order to isolate the antimicrobial protein, the hemolymph obtained from the cabbage white butterfly larvae at the end of 5 years was centrifuged at 10,000 g for 10 minutes to remove blood cells, followed by centrifugation at 100 ° C for 10 minutes, and then the supernatant was 0.1M. A sample was prepared by diluting with ammonium acetate buffer solution (pH 6.0, hereinafter, 'buffer solution A') and 1: 1. The prepared sample was injected into a CM-Sepharose column (10 x 1.5 cm) previously equilibrated with Buffer A, and the antimicrobial protein was eluted at an elution rate of 0.75 ml / min in an ammonium acetate concentration gradient of 0.1 to 1 M. Then, the solution containing the antimicrobial protein was re-injected into the CM-Sepharose column to elute the antimicrobial protein as before, and fractions showing antimicrobial activity against both E. coli and Bacillus megaterium were collected from these eluted fractions. These fractions were injected into a PepRPC HR5 / 5 column equilibrated with 0.1% trifluoroacetate (TFA) (Pharmacia, Sweden), and twice reversed FPLC with acetonitrile containing 0.1% TFA and 0.1% TFA. (See Fig. 5). In FIG. 5, (−) is the absorbance at 214 nm; (---) is the concentration gradient of acetonitrile; (-●-) is an antimicrobial activity against Escherichia coli; And (-○-) show antibacterial activity against Bacillus megaterium, respectively. As shown in Figure 5, a total of four peaks eluted were identified at 30-34% acetonitrile concentrations. Among these peaks, the antimicrobial protein eluting at the concentration of 30.1 to 31.8% acetonitrile, namely the first peak that exhibits stronger antimicrobial activity to gram negative bacteria, was named 'Hinnavin I'.

In order to confirm that the Hinnavin I isolated from the former was purified, West et al. (MHP West et al., Electrophoresis., 5: 133-138 (1984) were partially modified to obtain 15% at pH 4). Electrophoresis was performed using polyacrylamide gel for about 150 minutes at 200 V. Then, 5 ml of 0.6% agar solution containing logarithmic bacteria was poured on the gel (8 × 8 cm) to confirm the antibacterial activity. The cells were incubated for about 37 ° C. Coomassie blue was stained as in the case of normal protein (see Fig. 6) In Fig. 6, lane A was normal blood lymph; Immune hemolymph; Lane C is heat treated immune blood lymph; Lane D is an active fraction eluted from the CM-Sepharose column; Lane E is the fraction corresponding to the first peak obtained from the first reversed phase FPLC; Each represents Hinnavin I obtained from the second reversed phase FPLC. As shown in Figure 6, Hinnavin I appears in a single band, indicating that the antimicrobial protein has been purified.

The purification process of Hinnavin I is shown in Table 1 below. The amount of protein was measured as absorbance at 595 nm by Bradford's method (MM Bradford, Anal. Biochem., 72: 248-254 (1976)). Hultmark et al. (Hultmark, D. et al., Eur. J. Biochem., 106: 7-16 (1980)).

Table 1: Hinnavin I Purification Process

As shown in Table 1, the total activity of Hinnavin I was 3.87 kU, the yield was 0.53%, and the specific activity was 11.06 kU.

Example 5

Characterization of Hinnavin I

Example 5-1

Determination of Molecular Weight of Hinnavin I

The molecular weight of Hinnavin I was measured using standard molecular weight markers according to the method of Schagger and Jagow (see H. Schagger et al., Anal. Biochem., 166: 368-379 (1987)). (See also Figures 7 (a) and 7 (b)). In FIG. 7 (a), the first lane is Hinnavin I; The second lane comprises commercially available cecropin A; And lane 3 represents a standard molecular weight marker. In Figures 7 (a) and 7 (b), A is myoglobin residues 1-153 (16,950 Da), B is myoglobin residues 1-131 (14,440 Da), C is myoglobin residues 56-153 (10,600 Da), D represents myoglobin residues 56-131 (8,160 Da), E represents myoglobin residues 1-55 (6,210 Da), F represents standard molecular weight markers of glucagon (3,480 Da), and HI represents Hinnavin I and C _A represents Means pin A. As shown in Figures 7 (a) and 7 (b), the molecular weight of Hinnavin I was about 4,000 Da, similar to that of Secropin A.

In addition, the molecular weight of Hinnavin I was measured using an electrospray ionization mass spectrometer (Fisons, UK) to determine the molecular weight of Hinnavin I. The molecular weight of Hinnavin I was determined to be 4,139.94 ± 10.91.

Example 5-2

Amino Acid Sequence Determination of Hinnavin I

The amino acid sequence of Hinnavin I as determined by Ed Sequence degradation using Pro Sequencer 6600B (Milligen, USA) is shown in FIG. 8 along with the amino acid sequences of several known cecropin As. Hinnavin I of the present invention was found to have the following sequence:

GWKIGKKLEHMGQNIRDGLISAGPAVFAVGQAATIYAAAK

In Fig. 8, the amino acids shown in bold correspond to the amino acids of known secropin A. As shown in FIG. 8, since 23 amino acids among the 40 amino acids of Hinnavin I of the present invention are the same as those of known secropin A, it can be seen that Hinnavin I belongs to the secropin family. In addition, the first half of the amino acid up to 20 is mainly a lot of hydrophilic amino acids, the second half after 20 it can be seen that the distribution of a large number of amino acids mainly hydrophobic.

As described and demonstrated in detail above, the present invention provides a novel antimicrobial protein isolated from the larvae of Chinese cabbage butterfly. The antimicrobial protein of the present invention showed antimicrobial activity against both Gram-negative and Gram-positive bacteria by lysing bacteria, and particularly, showed stronger antimicrobial activity against Gram-negative bacteria Escherichia coli. In addition, the antimicrobial protein of the present invention has a molecular weight of about 4,000 Daltons, and it was confirmed that it belongs to the secropin system by amino acid analysis. Since the antimicrobial protein of the present invention exhibits antimicrobial activity against bacteria, it may be used as an antimicrobial agent.

Claims (1)

Antibacterial protein Hinnavin I isolated from the larvae of the cabbage butterfly (Pieris rapae) having the following amino acid sequence: GWKIGKKLEHMGQNIRDGLISAGPAVFAVGQAATIYAAAK (In the above, the amino acid sequence is described according to the nomenclature of IUPAC-IUB.)