KR20100050141A - An anti-fungal peptide gene isolated from beetle, protaetia brevitarsis, larvae ,said anti-fungal peptide and its analogue - Google Patents

Abstract

PURPOSE: A novel antifungal peptide gene isolated from Protaetia brevitarsis larva is provided to use as an agent for preventing and treating fungal diseases. CONSTITUTION: A novel antifungal peptide isolated from Protaetia brevitarsis larva comprises a base of sequence number 1. The novel antipeptide isolated from Protaetia brevitarsis larva has an amino acid sequence of sequence number 2. A novel antifungal peptide derivative isolated from Protaetia brevitarsis larva has an amino acid of sequence number 4(9Pbm1; ALWLAIGRG), 5(9Pbm2; ALWLAIGKG), 6(9Pbm3; RLWLAIGKG), 7(9Pbm4; RLLLAIGRG), 8(9Pbm5; RLWLRIGRG), or 9(9Pbm6; RLWLAIGRG).

Description

Novel antifungal peptide gene isolated from white spotted flower larva and its antifungal peptide and its derivatives {An anti-fungal peptide gene isolated from beetle, Protaetia brevitarsis, larvae, said anti-fungal peptide and its analogue}

The present invention is a new antifungal peptide gene ( protatiamycin ), the antifungal peptide and isolated from the larva of the coleoptera ( Protaetia brevitarsis ) belonging to the coleopteran which can be usefully used as a prophylactic and treatment of fungal diseases of humans and plants To derivatives thereof. The derivative of the antifungal peptide gene according to the present invention can be usefully used as an agent for preventing and treating fungal diseases of humans and plants.

In a broad sense, antigen-antibody responses, called immune responses, have played a major role in biological defense in invertebrates, including humans. But insects protect themselves by strengthening innate immune mechanisms, which are different from acquired immune mechanisms such as antigen-antibody responses. As such, innate immunity plays an important role as an important defense against early infection in vertebrates including humans as well as insects. A very important means of the innate immune mechanism of insects is to obtain substances with bactericidal and inactivating effects on various microorganisms. Among them, peptides and proteins with antimicrobial activity centered on antimicrobial peptides are highly available for disease control such as domestic animals. Such insect-derived anti-pathogenic protein may be very useful for the prevention and treatment of damage caused by the infection of various pathogens as well as the value of diseases of humans and livestock.

These antimicrobial proteins are divided into five types according to their structure and size. First, Cecropin, Second, Defensin, Third, Large glycine-rich protein, Fourth, Small proline-rich protein, Finally Lysozyme to be.

Insect-derived antibacterial proteins to date have been identified as sacootoxin III found in Sarcophaga peregrina (Baba et al, 1987) and diptericin found in Drosophila melanogaster . al 1990), holotricin 3 found in Holotrichia diomphalia (Lee et al., Biol. Pharm. Bull., 18: 1049-1052, 1995), Tenebrio molitor Tenecin 3 (Lee et al., Biochem. Biophys. Res. Commun. 218: 6-11, 1996), defensin, correop, found in Allomirina dichotoma Coleoptericin A and B (Miyanoshita et al., 1996; Sakisaca et al., 2001) Heliomycin found in Heliothis virescens (Lamberty et al. , J. Biol. Chem. 274: 9320-9326, 1999), rhinocerosine (Scarabaec), found in Oryctes rhinoceros in) (Yang et al., 1998; Tomie et al., 2003), cicadin (Wang and Ng, Peptides, 23: 7-11, 2002), found in Cicada flammata , Cicada (Cecropin (saito et al., 2005) found from Acalolepta luxuriosa ), and morphine analogue (Orizumi et al., 2005) found from Spodoptera litura . Studies on white spotted blotch have been reported for proteatin 1, 2 and 3 from immune-induced larvae (Yoon et al., 2003). Since there is no report of activity, the present inventors made cDNA gene bank after immunization induction from larvae of immunized white spotted flower larvae to select antifungal peptide genes having high homology with previously reported antimicrobial-related genes. And amino acid sequences, while some amino acid sequences and their derivatives Synthetic construct by Candida Albi confirmed that high antifungal activity against a fungal disease, including crops (Candida albicans) hameuroseo and completed the present invention.

The present invention synthesizes deduced amino acids and derivatives thereof of a partial region of cDNA from which a new antifungal peptide gene (protatiamycin) has been isolated from coleopteran insects, white spotted larvae, and includes Candida albicans . The purpose of this study is to identify antifungal activity against four species, including Aspergillus niger .

In order to achieve the above object, in the present invention, a new antifungal peptide gene having antibacterial activity is effectively isolated from the larva of white spotted radish and synthesizes deduced amino acids and derivatives thereof for some regions of the isolated gene to exhibit antifungal activity. To reveal.

Therefore, the present invention provides a novel antifungal peptide gene isolated from the white spotted flower larva of SEQ ID NO: 1.

The present invention also provides a novel antifungal peptide isolated from white spotted flower larva of SEQ ID NO: 2.

Preferably, it provides a novel antifungal peptide isolated from the white spotted flower larva of the alpha helix region having the amino acid sequence of SEQ ID NO: 3 of the amino acid sequence of SEQ ID NO: 2.

The present invention also provides a derivative of the antifungal peptide having the amino acid sequence of SEQ ID NO: 4 to 9.

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

White spotted flower plain gram-negative bacteria as immunity yudowon the first white-spotted flowers plain larvae to separate the antimicrobial peptide gene from larval (Escherichia coli JM109, hereinafter "E.coli" referred to) the scanning and after 24 hours the white spotted flower plain larvae Was rapidly frozen with liquid nitrogen to prepare a cDNA gene bank. Randomly, 1000 EST analyzes selected defencin-related genes with high homology with previously reported antimicrobial genes.

Separating total RNA from the white spotted flower plain larvae immunity induced by E.coli and was by the cDNA of the selected anti-fungal peptide gene in the above as a probe, Northern blot analysis after 4 hours E.coli scanning maximize the amount of expression, and Since then, the amount of expression was gradually reduced.

As a result of determining the total base sequence of this gene, the cDNA was composed of 79 amino acids (SEQ ID NO: 2) having a total size of 633 bp (SEQ ID NO: 1) and a coding region starting at the 149 th base and ending at the 389 th position. In addition, it was confirmed that the preliminary transcription termination signal 'AATAA' exists at the 591th position, and the structure was analyzed by the PSIPRED protein structure prediction program, and the structure resulted in the α-helix region. Could confirm.

Anti-fungal activity was synthesized by synthesizing deduced amino acids of a partial region of the antifungal peptide gene and derivatives thereof according to the present invention against four kinds of crop fungi, Aspergillus niger , including Candida albicans . Assay. As a result, the growth of the five fungi disclosed when the derivative of the antifungal peptide according to the present invention was inhibited, the MIC analysis method was confirmed that the activity of the fungus is suppressed even at a concentration of 2㎍ / ㎖.

On the other hand, the red blood cell destructive ability was examined to confirm whether the derivatives having antifungal activity showed cytotoxicity, whereas the synthetic derivatives did not destroy red blood cells, whereas melittin, a bee venom used as a positive control, was highly cytotoxic. I could confirm it.

As described above, the present invention provides a cDNA of a new antifungal peptide gene isolated from white spotted larvae and an amino acid encoding the same. The antifungal peptide gene (protatiamycin) derivative of the present invention was inhibited in growth against four kinds of crops including Aspergillus niger , including crops including Candida albicans . It was confirmed that fungal activity was inhibited even at a concentration of μg / ml or less. It had 10 times more antifungal activity than melittin treated with the positive control.

On the other hand, the red blood cell destructive ability was examined to confirm whether the derivatives having antifungal activity showed cytotoxicity, whereas the synthetic derivatives did not destroy red blood cells, whereas melittin, a bee venom used as a positive control, was highly cytotoxic. I could confirm it.

Therefore, the derivative of the antifungal peptide gene isolated from the white spotted larva larvae presented in the present invention is Aspergillus niger that causes onion black mold disease of crops, including Candida albicans , which causes candidiasis. Prevention of Botrytis alli causing onion ash fungus, Fusarium oxysporum causing vegetable wilting disease, Penicillium expasum causing apple green fungus It may be useful as a control agent.

The present invention will be described in more detail with reference to the following Examples. These examples are only to specifically describe the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples. In addition, although not shown in specific examples, it will be apparent to those skilled in the art that a pharmaceutical composition for an antifungal agent containing a derivative of the antifungal peptide gene according to the present invention having an antifungal action as an active ingredient is possible.

Example 1 Antifungal Peptide Gene Selection

(1) Induction of immunity of white spotted larva

To select new antifungal peptide genes from white spotted larvae, white spotted larvae were immunized. That is, 5 μl injection of each individual at a concentration of 1 mg / ml laminin was induced for immunization, and then reared at 25 ° C. for 24 hours.

(2) Gene Bank Construction and Antifungal Peptide Related Gene Selection

After 24 hours of immunization, the larvae were crushed under liquid nitrogen to prepare cDNA gene bank, and one of the antimicrobial genes was selected as a result of 1,000 EST assays.

Example 2 cDNA Cloning and Deduction Amino Acid Sequence of Antifungal Peptide Gene

Sequencing of the selected clones was performed by an automatic base sequencer (Perkin Elmer Co., ABI370), which cDNA (SEQ ID NO: 1) has a total size of 633 bp, is initiated at base 149 and is located at base 389. It had an open reading frame terminated at and the nucleotide sequence of the 3 'end was confirmed that the presence of the preliminary transcription termination signal' AAATAA 'including the poly A base (see Fig. 1).

79 amino acids (SEQ ID NO: 2) were deduced from the ORF of cDNA. The 33rd to 36th amino acids (Arg-Gln-Lys-Arg) are cleavage sites and 43 amino acids from the 37th amino acid valine to the 79th amino acid arginine (mature protein) The molecular weight of the protein is estimated to be 4.317 KDa. A comparison of the homology of amino acid sequences of different insects to the mature protein consisting of 43 amino acids was carried out using the CLUSTAL W program. As a result, it was 65% compared to Copris tripartitus defensin and Anomala cuprea. ) And southern horn beetle ( Oryctes rhinoceros ) showed 76% and 79% homology, respectively. The newly discovered gene from the white spotted larva was named proteatiamycin.

Example 3 Expression Pattern of Antibacterial Peptide Gene

In order to see the expression patterns of the selected genes, the vesicle total RNA was evaluated by 4 hours, 8 hours, 16 hours, and 24 hours after immunization with gram-negative bacteria (Esherichia coli JM109) as an immunogenic source. Total RNA was extracted using a SV Total RNA Isolation System (Promega, USA). The total RNA isolated was electrophoresed in 1.2% formaldehyde gel, the gel was transferred to nylon membrane with 20 X SSC solution (3M NaCl, 0.3M Sodium citrate), and the RNA was fixed by UV crosslink. The treated nylon membrane was identified after reacting the radioactive isotope [α-32P] dCTP-labeled antifungal peptide gene cDNA with a probe.

As shown in FIG. 2, the expression level of the gene was increased after 4 hours of immunization.

Example 4 Antifungal Peptide Gene Synthesis

Antimicrobial proteins reported so far have α-helix structures, contain disulfide linkages in their structures, and contain a large number of proline and glycine amino acids. Can be divided into: Structural analysis of proteatiamycin revealed that the 19th to 30th amino acids of the proteathiacin mature protien contained α-helix moieties, and thus, α-helix moiety peptides and their Derivatives were synthesized and assayed for antifungal activity against Candida albicans and crop fungal diseases.

Peptides were synthesized in peptron (Daejeon, Korea) using an automated solidphase peptide synthesizer (Pioneer Apploed Biosystems, Foster, Caclif.).

Table 1 shows a peptide synthesized with α-helix 12 amino acid sequence (12Pbn: Ala19-Gly30-NH2) and its derivative amino acid sequence of protitamycin.

TABLE 1

Peptide Amino acid sequence 12 Pbn ACAAHCLAIGRG-NH2 (SEQ ID NO: 3) 9Pbm1 ALWLAIGRG-NH2 (SEQ ID NO: 4) 9Pbm2 ALWLAIGKG-NH2 (SEQ ID NO: 5) 9Pbm3 RLWLAIGKG-NH2 (SEQ ID NO: 6) 9Pbm4 RLLLAIGRG-NH2 (SEQ ID NO: 7) 9Pbm5 RLWLRIGRG-NH2 (SEQ ID NO: 8) 9Pbm6 RLWLAIGRG-NH2 (SEQ ID NO: 9)

Example 5 Antifungal Activity Analysis of Antifungal Peptide Gene (Protatiamycin)

Antifungal activity analysis was performed using two methods: radial diffusion assay (RDA) and minimum inhibitory concentration (MIC).

(1) Culture conditions of various strains

Yeast bacteria Candida albicans was grown overnight at 30 ° C. and 200 rpm in YPD medium (1% yeast extract, 2% peptone, 2% dextrose), then inoculated in new medium and grown for 2 hours. It is logarithmic.

Mold fungus Aspergillus niger , Botrytis alli , Fusarium oxysporum , Penicillium expasum were found in PDA medium (0.4% potatoes-). infusion from 200g, 2% dextrose, 1.5% agar) was incubated for 3-7 days at 25 ℃.

(2) Antifungal activity of proteatiamycin against Candida albicans by RDA analysis

In order to verify the antifungal activity of the novel peptide protaetiamycin of the present invention, 4X106 CFU (colony forming units) / ml Candida albicans was mixed with a sterile underlay gel for RDA and 100 mm square. Hardened to the plate. At this time, a hole having a diameter of 3 mm was inserted into the proteatiacin peptide (5 µg / 5 µl), and the buffer used to dissolve the peptide was placed in a negative control group, and a well-known secreted antibiotic peptide along with melittin. Cecropin B was used as a positive control. Peptides were incubated at 37 ° C. for 3 hours to diffuse onto the gel, onto which an overlay gel for RDA was poured, hardened and incubated overnight at 37 ° C. The antifungal activity of each peptide can be confirmed by the size of the clear zone appearing on the gel.

The results are shown in Fig. As shown in FIG. 3, the negative control group did not draw a ring, and all of the protitamycin peptides except 12Pbn of the present invention were clearer than melittin and cecropin B used as positive control groups. Clear zones are shown. Therefore, the antifungal activity of the peptide of the present invention was verified.

(3) Antifungal activity of various crop fungal diseases of proteatiamycin through MIC analysis

Aspergillus, which causes pathogenic fungi Candida albicans and onion black fungal disease, to identify the minimum inhibitory concentration (MIC) values of various fungi of the protetiamycin, a novel peptide of the present invention Aspergillus niger , Botrytis alli causing onion mildew disease, Fusarium oxysporum causing vegetable wilting disease, Penicillium expasum causing apple green fungal disease ) And cultured by adding the protitamycin peptide of the present invention.

Each strain was inoculated at 90 μl into 96-well plates at a concentration of 2 × 103 cells, and 10 μl of protatimycin diluted to various concentrations was added to each well plate and incubated at 28 ° C. for 24 hours. In addition, melittin (Melittin) was treated under the same conditions as the peptide and used as a positive control.

After the incubation, each well plate was observed under a microscope, and the MIC value was determined based on the peptide concentrations of the wells and the wells in which the bacteria were grown.

The results are shown in Table 2 below. As shown in Table 2, 12Pbn was inactivated compared to melittin, which was used as a positive control, but 9Pbm1 ~ 6 were on average 4 to 10 times higher antifungal than melittin. Showed activity. Therefore, it was confirmed that the peptide of the present invention is a peptide having antibiotic activity with excellent antifungal activity.

[Table 2] MIC for various fungi of proteathiamycin

Peptide MIC (μg / ml) C. albicans A. niger B. alli F. oxysporum P. expasum Melittin 20-40 20-40 8-20 8-15 8-15 12 Pbn > 100 > 40 > 40 > 20 > 20 9Pbm1 5-10 4-8 4-8 8-15 2-4 9Pbm2 5-10 4-8 4-8 > 20 4-8 9Pbm3 <5 <2 <2 2-4 2-4 9Pbm4 <5 2-4 <2 4-8 2-4 9Pbm5 <5 2-4 2-4 2-4 2-4 9Pbm6 <5 <2 <2 2-4 2-4

In addition, Figure 4 is a microscopic observation of the minimum growth inhibitory concentration for Aspergillus niger bacteria causing onion black mold disease using the derivative 9Pbm3.

Figure 4a is used for the melittin (melittin) as a positive control, Figure 4b is using the derivative 9Pbm3. As shown in Figures 4a and 4b, in the case of the derivative 9Pbm3 it was not possible to confirm the growth of bacteria at a concentration of less than 2㎍ / ㎖, but in the case of melittin used as a positive control, some bacteria at a concentration of 20㎍ / ㎖ You can see it grows.

(4) Cytotoxicity Assay of Synthetic Peptides

In order to assay the cytotoxicity of the novel peptide protaetiamycin of the present invention, blood was collected from mice to investigate the degree of destruction of erythrocytes. The red blood cells were separated from the blood, diluted to a maximum of 4% with PBS buffer, and then cultured with the addition of peptides. The blood samples were incubated at 90 μl in 96-well plates and 10 μl of protetiamycin diluted to different concentrations in each well and incubated at 37 ° C. for 4 hours. In addition, melittin (Melittin) was treated under the same conditions as the peptide and used as a positive control. After incubation, 405 nm was measured with an ELISA plate reader to observe the degree of erythrocyte destruction.

5 is an illustration of the cytotoxicity of the peptide showing the antifungal activity antifungal activity. Figure 5a is a photograph of the experimental results of the derivative 9Pbm3 and melittin. The right melittin destroys red blood cells in culture, exposing hemoglobin to red. B (Buffer) was used as a negative control that did not destroy red blood cells, and T (Triton X-100) was used as a positive control that destroyed 100% red blood cells. FIG. 5B is a graph numerically based on FIG. 5A. In the case of melittin, a positive control, red blood cells were destroyed at a concentration of 5 µg / ml, and 100% at 20 µg / ml. You can see that you do not.

In other words, all proteamycin was found to be non-toxic, in contrast to the positive control melittin, which showed severe toxicity. In conclusion, the peptides of the present invention were confirmed to be peptides having excellent antifungal activity without toxicity.

Figure 1 shows the cDNA base sequence and deduced amino acid sequence of the antifungal peptide gene isolated from white spotted larva larvae.

Figure 2 shows the results of the isolation and Northern blot analysis of the total RNA after the induction of immunity to determine the induced expression pattern by the immunization of the antifungal peptide gene isolated from white spotted larva larvae.

Figure 3 synthesizes the deduced amino acid sequence (12Pbn) and its derivatives (9Pbm1 ?? 9Pbm6) of a partial region of the antifungal peptide gene isolated from white spotted larva larvae and to Candida albicans bacteria using RDA method This is a picture of the antifungal activity assay. At the far right of the upper line, 0.01% acetic acid (acetic acid) as a control, Cecropin B (Ce. B) and melittin (Melittin) were used as a positive control.

In addition, Figure 4 is a microscopic observation of the minimum growth inhibition concentration for Aspergillus niger causing onion black mold disease using the derivative 9Pbm3, Figure 4a is a melittin (melittin as a positive control) ), And FIG. 4B uses the derivative 9Pbm3.

FIG. 5 is a diagram illustrating the cytotoxicity of peptides showing antifungal activity against antifungal activity. FIG. 5A is a photograph showing the cytotoxicity test results of the derivative 9Pbm3 and melittin, and FIG. 5B is a numerical value based on FIG. 5A.

<110> Republic of Korea <120> An anti-fungal protein isolated from beetle, Protaetia          brevitarsis, larvae and its analogue <160> 9 <170> KopatentIn 1.71 <210> 1 <211> 633 <212> DNA <213> Protaetia brevitarsis <400> 1 tttacttaaa ttaataatac ttaagatatt tacatataca gtagtacctt tttatttaaa 60 agcagataac tttaccgcaa caatctggcc tcgtgccgaa ttcggcacga ggataatttg 120 gaaggagacc ttgctagtaa tacaagacaa tgtccaaatt caccgtatta gctttcatag 180 tcgccatgtg tgtgatgcac actttcgcca taccagtacc tgaagaaatt gaagggagca 240 tcataaggca gaaaagggtt acatgtgacc ttttaagttt tgagattctg ggcgttgctt 300 taaaccacag cgcctgtgca gctcattgtt tagccattgg tcgtgggggt ggtgcatgcc 360 aaggtggcat atgtgtatgt agaaggtaac tgtttatttg tatacgaact aaaaaaatac 420 tctttcactt attgaacata gttcaataaa ttatgtttta ttgactctac atttcattca 480 aaatagttta cattttatat aacacaagct attgatatcg cattaagagt tgaatacata 540 tgtatattat ataacccctt aacaggttct gaaaattgtg tatgtaaatg taaataatgt 600 aatgtaaata aataaaatat tttttatata aaa 633 <210> 2 <211> 79 <212> PRT <213> Protaetia brevitarsis <400> 2 Met Ser Lys Phe Thr Val Leu Ala Phe Ile Val Ala Met Cys Val Met   1 5 10 15 His Thr Phe Ala Ile Pro Val Pro Glu Glu Ile Glu Gly Ser Ile Ile              20 25 30 Arg Gln Lys Arg Val Thr Cys Asp Leu Leu Ser Phe Glu Ile Leu Gly          35 40 45 Val Ala Leu Asn His Ser Ala Cys Ala Ala His Cys Leu Ala Ile Gly      50 55 60 Arg Gly Gly Gly Ala Cys Gln Gly Gly Ile Cys Val Cys Arg Arg  65 70 75 <210> 3 <211> 12 <212> PRT <213> Protaetia brevitarsis <400> 3 Ala Cys Ala Ala His Cys Leu Ala Ile Gly Arg Gly   1 5 10 <210> 4 <211> 9 <212> PRT <213> Protaetia brevitarsis <400> 4 Ala Leu Trp Leu Ala Ile Gly Arg Gly   1 5 <210> 5 <211> 9 <212> PRT <213> Protaetia brevitarsis <400> 5 Ala Leu Trp Leu Ala Ile Gly Lys Gly   1 5 <210> 6 <211> 9 <212> PRT <213> Protaetia brevitarsis <400> 6 Arg Leu Trp Leu Ala Ile Gly Lys Gly   1 5 <210> 7 <211> 9 <212> PRT <213> Protaetia brevitarsis <400> 7 Arg Leu Leu Leu Ala Ile Gly Arg Gly   1 5 <210> 8 <211> 9 <212> PRT <213> Protaetia brevitarsis <400> 8 Arg Leu Trp Leu Arg Ile Gly Arg Gly   1 5 <210> 9 <211> 9 <212> PRT <213> Protaetia brevitarsis <400> 9 Arg Leu Trp Leu Ala Ile Gly Arg Gly   1 5  

Claims (9)

A novel antifungal peptide gene isolated from white spotted flower larvae having the nucleotide sequence of SEQ ID NO: 1 below.

A novel antifungal peptide isolated from white spotted flower larvae having the amino acid sequence of SEQ ID NO: 2 below.

The method of claim 2, A novel antifungal peptide isolated from white spotted flower larvae of the alpha helix region having the amino acid sequence of SEQ ID NO: 3 among the amino acid sequence of SEQ ID NO: 2. 12 Pbn (SEQ ID NO: 3): ACAAHCLAIGRG A derivative of the novel antifungal peptide isolated from the white spotted radish larva having the amino acid sequence of SEQ ID NO: 4 below. 9Pbm1 (SEQ ID NO: 4): ALWLAIGRG Derivative of a novel antifungal peptide isolated from white spotted flower larvae having the amino acid sequence of SEQ ID NO: 5 below. 9Pbm2 (SEQ ID NO: 5): ALWLAIGKG A derivative of the novel antifungal peptide isolated from the white spotted flower larva having the amino acid sequence of SEQ ID NO: 6 below. 9Pbm3 (SEQ ID NO: 6): RLWLAIGKG Derivative of a novel antifungal peptide isolated from white spotted flower larvae having the amino acid sequence of SEQ ID NO: 7 below. 9Pbm4 (SEQ ID NO: 7): RLLLAIGRG A derivative of the novel antifungal peptide isolated from the white spotted flower larvae having the amino acid sequence of SEQ ID NO: 8. 9Pbm5 (SEQ ID NO: 8): RLWLRIGRG Derivative of a novel antifungal peptide isolated from white spotted flower larvae having the amino acid sequence of SEQ ID NO: 9 below. 9Pbm6 (SEQ ID NO: 9): RLWLAIGRG

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