JP2008099635A - New antimicrobial protein, method for producing the same and use thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a new antimicrobial protein and to provide a method for producing the same and a use thereof. <P>SOLUTION: The gene comprises a DNA composed of 288 specific nucleotide sequences obtained from Lactobacillus gasseri JCM1131<SP>T</SP>. The protein comprises a specific amino acid sequence. The recombinant plasmid contains the DNA. The host cell is transformed with the recombinant plasmid. The method for producing the protein comprises culturing the host cell and recovering the protein. The composition comprises the protein as an active ingredient. The composition is especially a reagent for research, a food additive, a preservative, an industrial germicide, an agricultural germicide or a medical antibiotic. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

The present invention relates to a novel antibacterial protein, a method for producing the same, and use thereof. More specifically, the present invention relates to a novel antibacterial protein encoded by a gene obtained from Lactobacillus gasseri JCM1131 ^T, which is a kind of lactobacillus having lysogen phage φgaY.

An example in which a structural gene of LysgaY, a novel lytic enzyme, was isolated from Lactobacillus gasseri JCM1131 ^T, which is a kind of lactobacilli, has been reported. (For example, patent document 1).
LysgaY is a protein consisting of 310 amino acids, belongs to the muramidase family, and has been shown to exhibit a broad lysis spectrum against gram-positive bacteria such as Lactobacillus and Staphylococcus.
The amino acid sequence of LysgaY and the nucleotide sequence of 933 of DNA encoding the protein have also been reported.

The results of homology comparison and three-dimensional structure prediction between LysgaY and other lytic enzymes suggest that the N-terminal region, which is the active domain of LysgaY, has a β / α-barrel structure, while the C-terminal region is It was speculated to form a cell wall recognition domain (SH3b).
Several literatures on other lytic enzymes having a cell wall recognition domain (SH3b) similar to the cell wall recognition domain (SH3b), which is the C-terminal region of LysgaY, in the C-terminal region and their ability to bind to the cell wall have been reported ( For example, Non-Patent Document 1, Non-Patent Document 2, Non-Patent Document 3, and Non-Patent Document 4).
However, there has been no report on the antibacterial activity of these similar peptide regions.
JP 2006-121993 A Lu JZ, Fujiwara T, Komatsuzawa H, Sugai M, Sakon J. Cell wall-targeting domain of glycylglycine endopeptidase distinguishes among peptidoglycan cross-bridges.J. Biol. Chem., 2006, Jan 6; 281 (1): p.549 -58. Low LY, Yang C, Perego M, Osterman A, Liddington RC.Structure and lytic activity of a Bacillus anthracis prophage endolysin.J. Biol. Chem. 2005 Oct 21; 280 (42): p.35433-9. Donovan, DM, Foster-Frey, J., Dong, S., Rousseau, GM, Moineau, S., Pritchard, DG, The Cell Lysis Activity of the Streptococcus agalactiae Bacteriophage B30 Endolysin Relies on the Cysteine, Histidine-Dependent Amidohydrolase / Peptidase Domain. Appl. Environ. Microbiol. 2006. 72, p.5108-12. Donovan, DM, Dong, S., Garrett, W., Rousseau, GM, Moineau, S., Pritchard, DG, Peptidoglycan hydrolase fusions maintain their parental specificities.Appl.Environ.Microbiol. 2006. 72, p.2988-96 .

  An object of the present invention is to provide a novel antibacterial protein, a method for producing the same, and a use thereof.

As a result of intensive studies in view of the above-mentioned problems, the present inventors have found that the C-terminal cell wall recognition domain (SH3b) of the structural gene of LysgaY, a lytic enzyme isolated from Lactobacillus gasseri JCM1131 ^T, which is a kind of Lactobacillus. The protein encoded by) (hereinafter referred to as SH3bgaY) was found to have strong and broad spectrum antibacterial activity against phytopathogenic bacteria, thereby completing the present invention.

That is, the present invention relates to the following aspects 1 to 12;
1. A gene consisting of the DNA of any one of the following a) to d);
a) DNA comprising the nucleotide sequence represented by nucleotide numbers 1 to 288 of SEQ ID NO: 1 below,
b) DNA comprising the nucleotide sequence represented by nucleotide numbers 1 to 288 of SEQ ID NO: 1 below.
c) DNA comprising a region encoding a protein having antibacterial activity, which hybridizes with a single-stranded DNA comprising a nucleotide sequence complementary to the nucleotide sequence of the DNA described in a) above under stringent conditions;
d) DNA comprising a region encoding a protein having 95% nucleotide sequence homology with the DNA described in a) and having antibacterial activity.
atg tct gtt gca caa tca gca tca gaa aaa aca tgg act gat gta caa ggt atg act tgg cat gaa gaa cat ggt act ttc atc act ggt gga gcg att aat ctt cgc tgg ggc gct aat acg caa agc aca accg ctca acc gca ggt tca gaa gtt aaa tac aat gct tgg gct aga gat agt gct ggg cgt gta tgg tta cag caa ccg aga gaa aat ggt aag aat ggc tat tta gtt ggt cgt gtc ggc agt gag ccc tgg gga act ttc aaa
2. A gene encoding any one of the following proteins a) to c);
a) a protein comprising an amino acid sequence represented by SEQ ID NO: 2 below,
b) a protein comprising the amino acid sequence represented by SEQ ID NO: 2 below,
c) A protein having an antibacterial activity, comprising an amino acid sequence in which one or several amino acids are deleted, substituted and / or added in the amino acid sequence represented by SEQ ID NO: 2 below.
Met Ser Val Ala Gln Ser Ala Ser Glu Lys Thr Trp Thr Asp Val Gln Gly Met Thr Trp His Glu Glu His Gly Thr Phe Ile Thr Gly Gly Ala Ile Asn Leu Arg Trp Gly Ala Asn Thr Gln Ser Thr Leu Ile Thr Thr Leu Pro Ala Gly Ser Glu Val Lys Tyr Asn Ala Trp Ala Arg Asp Ser Ala Gly Arg Val Trp Leu Gln Gln Pro Arg Glu Asn Gly Lys Asn Gly Tyr Leu Val Gly Arg Val Gly Ser Glu Pro Trp Gly Thr Phe Lys
3. Any one of the following proteins a) to d);
a) a protein comprising an amino acid sequence represented by SEQ ID NO: 2 below,
b) a protein comprising the amino acid sequence represented by SEQ ID NO: 2 below,
c) a protein having an antibacterial activity, comprising an amino acid sequence in which one or several amino acids are deleted, substituted and / or added in the amino acid sequence represented by SEQ ID NO: 2 below,
d) 1 above. A protein encoded by the described DNA and having antibacterial activity.
Met Ser Val Ala Gln Ser Ala Ser Glu Lys Thr Trp Thr Asp Val Gln Gly Met Thr Trp His Glu Glu His Gly Thr Phe Ile Thr Gly Gly Ala Ile Asn Leu Arg Trp Gly Ala Asn Thr Gln Ser Thr Leu Ile Thr Thr Leu Pro Ala Gly Ser Glu Val Lys Tyr Asn Ala Trp Ala Arg Asp Ser Ala Gly Arg Val Trp Leu Gln Gln Pro Arg Glu Asn Gly Lys Asn Gly Tyr Leu Val Gly Arg Val Gly Ser Glu Pro Trp Gly Thr Phe Lys
4). 1 above. Or 2. A recombinant plasmid comprising the gene described.
5. 4. The expression vector, which is an expression vector. The recombinant plasmid as described.
6). 4. above. Or 5. A host cell transformed with the recombinant plasmid described in 1.
7). 5. The gram-positive bacterium as described above, A host cell as described.
8). 5. The aforementioned 6, characterized in that it is Escherichia coli, lactic acid bacteria, Bacillus subtilis or staphylococci. A host cell as described.
9. 2. including the following steps 1) and 2); A method for producing the protein according to claim 1;
1) said 6. Or 8. Culturing the host cell according to any one of the methods under conditions capable of producing a protein having antibacterial activity;
2) A step of recovering a protein having antibacterial activity from the culture obtained by the culture in the step 1).
10. 9 above. A protein obtained by the production method described.
11. 3 above. A composition comprising the described protein as an active ingredient.
12 10. A reagent for research, a food additive, an antiseptic, an industrial fungicide, an agricultural fungicide, or a medical antibiotic, The composition as described.

According to the present invention, a novel protein having strong antibacterial activity and a broad antibacterial spectrum is provided. The protein is preferably used as an active ingredient for applications in the fields of biotechnology and medicine, particularly for research reagents, food additives, preservatives, industrial fungicides, agricultural fungicides, medical antibiotics, etc. be able to.
The antibacterial protein (SH3bgaY) of the present invention has no hydrolysis activity, and conversely inhibits the autolysis of Lactobacillus gasseri JCM1131 ^T and the hydrolysis activity of LysgaY.
Therefore, it was revealed that the antibacterial activity of SH3bgaY is completely different from the hydrolysis activity of LysgaY.
The antibacterial activity of SH3bgaY was thought to be due to inhibition of separation of daughter cells, which is the final stage of cell division in the logarithmic growth phase of Lactobacillus gasseri JCM1131 ^T.

The DNA fragment containing the gene of the present invention is subjected to PCR using primers designed to obtain a gene encoding SH3bgaY using, for example, a recombinant plasmid containing a DNA containing a gene part encoding SH3bgaY as a template. It can be obtained by doing. DNA containing the gene portion encoding SH3bgaY can be obtained by cleaving the bacterial cell DNA of Lactobacillus gasseri JCM1131 ^T with a restriction enzyme Sau3AI or the like. Using the obtained DNA, known methods such as the method of Sambrook et al. (Sambrook et al., Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory,
According to Cold Spring Harbor, NY (1989)), a recombinant plasmid incorporating the DNA can be prepared. Primers used for PCR can be designed based on the homology between the C-terminal portion of the gene encoding LysgaY and the gene encoding the cell wall recognition domain (SH3b) of another lytic enzyme.

A recombinant plasmid containing the obtained DNA fragment was prepared, the recombinant plasmid was introduced into a host cell to obtain a transformant, and then a protein was induced from the transformant to induce expression of about 9 kDa. A molecular weight SH3bgaY protein can be obtained.
When the antibacterial activity of the obtained SH3bgaY protein against phytopathogenic bacteria and standard strains was evaluated, it showed strong antibacterial activity against all the strains used.

  Accordingly, one example of the gene of the present invention is DNA consisting of the nucleotide sequence represented by nucleotide numbers 1 to 288 of SEQ ID NO: 1 below corresponding to a region encoding a protein consisting of 95 amino acids. A gene comprising a DNA comprising the nucleotide sequence represented by nucleotide numbers 1 to 288 of SEQ ID NO: 1 below is also included in the present invention as long as it exhibits antibacterial activity.

Another example of the gene of the present invention is a hybrid under stringent conditions with a single-stranded DNA comprising a nucleotide sequence complementary to the nucleotide sequence represented by nucleotide numbers 1 to 288 of SEQ ID NO: 1 described below. And a DNA comprising a region encoding a protein having antibacterial activity, and nucleotide numbers 1 to 28 of SEQ ID NO: 1 described below
And those consisting of DNA comprising a region encoding a protein having 95% or more nucleotide sequence homology with the nucleotide sequence represented by 8 and having antibacterial activity. Such DNA includes mutant DNA found in nature, artificially modified mutant DNA, homologous DNA derived from a different organism, and the like.

  Further examples of the gene of the present invention include a gene encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 2 below corresponding to the 95 amino acids. A gene encoding a protein containing the amino acid sequence represented by SEQ ID NO: 2 below is also encompassed in the present invention. Here, the codon corresponding to each amino acid can be arbitrarily selected, and can be determined according to a conventional method in consideration of the codon usage frequency of the host to be used, for example.

  On the other hand, examples of the protein of the present invention include a protein having an amino acid sequence represented by SEQ ID NO: 2 below. A protein comprising the amino acid sequence represented by SEQ ID NO: 2 below is also encompassed in the present invention.

  Further, in the amino acid sequence represented by SEQ ID NO: 2 below, the present invention can be used even if it is a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted and / or added, as long as it has antibacterial activity. Is included. Here, the term “several” means a number not exceeding 10, preferably 5 or less.

  Here, whether or not a certain DNA hybridizes with a single-stranded DNA comprising a nucleotide sequence complementary to the nucleotide sequence represented by nucleotide numbers 1 to 288 of SEQ ID NO: 1 is determined by the following procedure, for example. Yes: First, the target DNA is labeled with a probe according to the random primer method, the nick translation method or the like. Next, DNA used for hybridization is adsorbed on a known method, for example, a nitrocellulose membrane or a nylon membrane, and solidified by heating or ultraviolet irradiation. The membrane is then immersed in a prehybridization solution containing, for example, 6 × SSC, 5% Denhardt's solution and 0.1% sodium dodecyl sulfate (SDS) and incubated at 55 ° C. for 4 hours or longer. Here, the previously prepared labeled probe is added to the same prehybridization solution so as to have a final specific activity of 1 × 10 6 cpm / mL, and kept at 60 ° C. overnight. The operation of washing the membrane at 57 ° C. for 5 minutes is repeated 5 times. Further, after washing at 57 ° C. for 20 minutes, autoradiography can be performed to determine whether or not the membrane is hybridized.

  Hybridization conditions are not limited to the above conditions, and may be stringent conditions. In the present invention, hybridization under stringent conditions refers to 5 × SSC (0.75 M sodium chloride, 0.075 M sodium citrate) or a hybridization solution having a salt concentration equivalent thereto at 37 to 42 ° C. For about 12 hours under the above temperature conditions, pre-wash with 5 × SSC or a solution having a salt concentration equivalent thereto, if necessary, and then washing in a solution with 1 × SSC or a salt concentration equivalent thereto. Refers to hybridization consisting of. Further, the washing can be performed in a solution of 0.1 × SSC or a salt concentration equivalent to this.

  Whether or not the protein of the present invention has antibacterial activity can be evaluated by a usual method. More specifically, by performing an antibacterial activity test that is the same as or similar to that detailed in any of the following examples on a modified protein produced by amino acid deletion, substitution and / or addition. The presence or absence of antimicrobial activity of the modified protein can be determined.

A recombinant plasmid in which the DNA of the present invention is inserted into a vector is also encompassed by the present invention. As the vector, various commonly known vectors can be used, and examples thereof include, but are not limited to, a vector for prokaryotic cells, a vector for eukaryotic cells, a vector for cells derived from mammals, and the like. Such recombinant plasmids can transform prokaryotic or eukaryotic host cells. Furthermore, an expression vector can be preferably obtained by using a vector having an appropriate promoter sequence and / or a sequence involved in expression, or by introducing such a sequence.

  Host cells obtained by introducing the recombinant plasmid of the present invention into various cells are also encompassed by the present invention. Preferred as the host cell are gram positive bacteria, particularly Escherichia coli, lactic acid bacteria, Bacillus subtilis, staphylococci and the like.

  The protein of the present invention is obtained by culturing the host cell transformed with the recombinant plasmid under conditions capable of producing a protein having antibacterial activity, and recovering the protein having antibacterial activity from the culture obtained by the culture. Can be manufactured. As a method for culturing host cells and a method for recovering proteins, conventionally known methods can be used. For example, the method is the same as or described in detail in any of the following examples. Can do.

  The present invention also relates to a composition comprising the protein of the present invention as an active ingredient. The composition can be applied in various applications where it is necessary to kill bacteria. Specific examples of the use include research reagents, food additives, preservatives, industrial fungicides, agricultural fungicides, and medical antibiotics.

  Hereinafter, the present invention will be described in more detail with specific examples, but the present invention is not intended to be limited to these specific examples.

Isolation of SH3bgaY gene A recombinant plasmid containing the SH3bgaY gene was prepared by using the recombinant plasmid p119gaY3 described in Japanese Patent Application Laid-Open No. 2006-121993, using the method of Sambrook et al. (Sambrook et al., Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989)).
Using the recombinant plasmid p119gaY3 (about 0.01 μg) as a template, GY1SH32
Using R primer (5'-AAAGCTCATATGTCTGTTGCACAATCAGCATCAGAA-3 ') and M13FW primer (5'-GTTTTCCCAGTCACGACGTTGTA-3'), using KOD-Plus-DNA Polymerase (manufactured by Toyobo Co., Ltd.), PCR method, and DNA containing SH3bgaY A fragment (480 bp) was obtained.
The DNA fragment was completely digested with NdeI, and a 473 bp DNA fragment was recovered and purified. Plasmid pET15b (5708 bp E. coli vector, Amp ^r , pT7) was transformed into Nde
After complete degradation with I and BamHI, the BamHI site of the obtained linear DNA was blunt-ended using Klenow fragment (manufactured by NEW ENGLAND BIOLAB). Subsequently, the 473 bp DNA fragment and the linear plasmid pET15b were ligated by a reaction at 16 ° C. for 6 hours using a DNA binding enzyme (manufactured by NEW ENGLAND BIOLAB) to obtain a recombinant plasmid pHSHgaY2 (6170 bp).

Confirmation of the base sequence of the plasmid pHSHgaY2 (6170 bp) The base sequence of the recombinant plasmid pHSHgaY2 (6170 bp) is the Thermo Sequenase
A cycle sequence reaction was performed using Cy5.5 Dye Terminator Cycle Sequencing Kit (Amersham), and electrophoresis and analysis were performed using a Long Read Tower sequencer (Veritas). When the entire nucleotide sequence of the inserted fragment (473 bp) of plasmid pHSHgaY2 was determined by this method, the sequence was identical to the sequence of plasmid p119gaY3 used as a template and the primer of Example 1, and was a sequence containing the target SH3bgaY gene. It was. FIG. 1 shows a schematic diagram showing regions of proteins expressed from plasmids p119gaY3 and pHSHgaY2. It can be seen that the SH3bgaY protein expressed from the plasmid pHSHgaY2 has only the C-terminal part of the lysgaY protein expressed from the plasmid p119gaY3 (Japanese Patent Laid-Open No. 2006-121993).

Preparation and confirmation of SH3bgaY protein Plasmid pHSHgaY2 was introduced into E. coli BL21DE3 (hsdS, gal [λcIts857, ind1, Sam7, nin5, lacUV5-T7 gene1]) by electroporation. Specifically, E. coli BL21DE3 was first cultured at 37 ° C. using LB (Luria-Bertain) medium. After 1 night (12 hours), 2m culture
L was inoculated into 100 mL of fresh LB medium and cultured at 37 ° C. The number of bacteria is 5 × 10 ⁸ / mL
When reaching the above, the cells were collected by the same centrifugation method as described above. The cells were washed 3 times with sterilized water and suspended in 100 μL of distilled water. 1 μL of recombinant plasmid DNA (0.5 μg / μL) and PEG 6000 (10%) were added to the suspension. The recombinant plasmid was introduced into the E. coli body by electroporation using Gene Pulser (registered trademark, manufactured by Bio-Rad Co., Ltd.) using electric pulse conditions of voltage 1.75 kV / cm, capacitance 25 μF, resistance 600Ω. I went by law. After the electric pulse treatment, 4 times the amount of LB medium was added and incubated at 37 ° C. for 30 minutes to obtain a transformant.
The obtained transformant was cultured in 100 mL of LB medium (containing 30 μg / mL of ampicillin). In the middle of the logarithmic phase (turbidity: 0.3), IPTG was added to the culture solution to a final concentration of 0.4 mM, and the cells were further cultured for 4 hours. Thereafter, the cells were collected, washed once with a binding buffer (50 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, pH 7.5), and suspended in 5 mL of the same buffer. This suspension was treated with an ultrasonic crusher (manufactured by Sonics & Materials Co., Ltd.) for 6 times, 30 seconds per time. Insoluble components were removed from the treatment liquid by a centrifugal method (cooled centrifuge, manufactured by Hitachi, Ltd .: 12000 rpm, 4 ° C., 10 minutes).
The cell disruption supernatant was applied to a nickel chelate affinity column (GE Healthcare, column volume: 1 mL). After washing with the above binding buffer, elution buffer (50 mM Tris-HCl, 500 mM NaCl, 600 mM imidazole) was eluted with a linear concentration gradient in the range of 0-100%. Fractions containing SH3bgaY protein were collected and designated as purified SH3bgaY. Subsequently, the purified SH3bgaY was dialyzed against 0.05M acetic acid buffer (PH = 5.0) and stored at 4 ° C.
FIG. 2 shows the results of analyzing the inducible expression of SH3bgaY protein in E. coli and the purification process by SDS electrophoresis (15% gel concentration). Lane 1 is a molecular weight marker, lane 2 is E. coli before induction, lane 3 is E. coli induced by IPTG, lane 4 is E. coli induced protein expression, cell disruption supernatant, and lane 5 is purified SH3bgaY protein. From this result, it is understood that the SH3bgaY protein having a molecular weight of about 9 kDa is induced by the plasmid pHSHgaY2, and the SH3bgaY protein can be purified by a one-step column operation by the nickel chelate affinity chromatography.

Evaluation of antibacterial activity of SH3bgaY protein The antibacterial activity of SH3bgaY protein against phytopathogenic bacteria and standard strains was evaluated. 10 mL of CSM medium (YEAST NITROGEN BASE (DIFCO No.233520) 0.85 g, CSM (BIO 101 No.4500-022) 0.31 g, D-glucose 10 g / 0.1 M phosphate buffer (pH 5.0)) And cultured at 25 ° C. for 3 days to obtain a cultured bacterial solution. After diluting this bacterial solution 100 times with CSM medium,
A 6-well titer plate (manufactured by NUNK) was dispensed at 180 μL / well, and a test sample diluted to a predetermined concentration with 0.05 M acetic acid buffer (PH = 5.0) was dispensed at 20 μL / well. The plate was incubated at 25 ° C., the growth of the test strain after 4 days was visually examined, and the growth inhibition rate was calculated by comparing with the sample-free treatment group.
Ecc: Erwinia carotovora subsp.carotovora (MAFF301052)
Bg: Burkholderia glumae (MAFF301169)
Rs: Ralstonia solanacearum (MAFF301485)
Xc: Xanthomonas campestris pv.citri (MAFF301078)
Bs: Bacillus subtilis (PCI-219)
Sa: Staphylococcus aureus (IFO-12732)
The growth inhibition rate (%) is shown in Table 1.
As is clear from the test results shown in Table 1, SH3bgaY, which is a protein of the present invention, exhibited antibacterial activity against all six strains. In particular, Burkholderia glumae and Ralstonia solanacearum exhibited stronger antibacterial activity than the positive control chloramphenicol. From these results, it was confirmed that SH3bgaY has a broad antibacterial spectrum.
SH3bgaY was completely different from LysgaY and did not show any hydrolytic activity.
And conversely, the autolysis of Lactobacillus gasseri JCM1131 ^T and the hydrolysis activity of LysgaY were inhibited.
Therefore, the antibacterial activity of SH3bgaY was completely different from the hydrolysis activity of LysgaY.
SH3bgaY markedly inhibited the separation of daughter cells, which is the final stage of cell division in the logarithmic growth phase of Lactobacillus gasseri JCM1131 ^T.

FIG. 1 shows a region of a protein expressed from the plasmid prepared in Example 2. FIG. 2 shows a photograph of the electrophoresis results when the protein produced by transformed Escherichia coli in Example 3 was purified. Lane 1 is a molecular weight marker, lane 2 is E. coli before induction, lane 3 is E. coli induced by IPTG, lane 4 is E. coli induced protein expression, cell disruption supernatant, and lane 5 is purified SH3bgaY protein.

Claims (12)

A gene consisting of the DNA of any one of the following a) to d);
a) DNA comprising the nucleotide sequence represented by nucleotide numbers 1 to 288 of SEQ ID NO: 1 below,
b) DNA comprising the nucleotide sequence represented by nucleotide numbers 1 to 288 of SEQ ID NO: 1 below.
c) DNA comprising a region encoding a protein having antibacterial activity, which hybridizes with a single-stranded DNA comprising a nucleotide sequence complementary to the nucleotide sequence of the DNA described in a) above under stringent conditions;
d) DNA comprising a region encoding a protein having 95% nucleotide sequence homology with the DNA described in a) and having antibacterial activity.
atg tct gtt gca caa tca gca tca gaa aaa aca tgg act gat gta caa ggt atg act tgg cat gaa gaa cat ggt act ttc atc act ggt gga gcg att aat ctt cgc tgg ggc gct aat acg caa agc aca accg ctca acc gca ggt tca gaa gtt aaa tac aat gct tgg gct aga gat agt gct ggg cgt gta tgg tta cag caa ccg aga gaa aat ggt aag aat ggc tat tta gtt ggt cgt gtc ggc agt gag ccc tgg gga act ttc aaa A gene encoding any one of the following proteins a) to c);
a) a protein comprising an amino acid sequence represented by SEQ ID NO: 2 below,
b) a protein comprising the amino acid sequence represented by SEQ ID NO: 2 below,
c) A protein having an antibacterial activity, comprising an amino acid sequence in which one or several amino acids are deleted, substituted and / or added in the amino acid sequence represented by SEQ ID NO: 2 below.
Met Ser Val Ala Gln Ser Ala Ser Glu Lys Thr Trp Thr Asp Val Gln Gly Met Thr Trp His Glu Glu His Gly Thr Phe Ile Thr Gly Gly Ala Ile Asn Leu Arg Trp Gly Ala Asn Thr Gln Ser Thr Leu Ile Thr Thr Leu Pro Ala Gly Ser Glu Val Lys Tyr Asn Ala Trp Ala Arg Asp Ser Ala Gly Arg Val Trp Leu Gln Gln Pro Arg Glu Asn Gly Lys Asn Gly Tyr Leu Val Gly Arg Val Gly Ser Glu Pro Trp Gly Thr Phe Lys Any one of the following proteins a) to d);
a) a protein comprising an amino acid sequence represented by SEQ ID NO: 2 below,
b) a protein comprising the amino acid sequence represented by SEQ ID NO: 2 below,
c) a protein having an antibacterial activity, comprising an amino acid sequence in which one or several amino acids are deleted, substituted and / or added in the amino acid sequence represented by SEQ ID NO: 2 below,
d) A protein encoded by the DNA of claim 1 and having antibacterial activity.
Met Ser Val Ala Gln Ser Ala Ser Glu Lys Thr Trp Thr Asp Val Gln Gly Met Thr Trp His Glu Glu His Gly Thr Phe Ile Thr Gly Gly Ala Ile Asn Leu Arg Trp Gly Ala Asn Thr Gln Ser Thr Leu Ile Thr Thr Leu Pro Ala Gly Ser Glu Val Lys Tyr Asn Ala Trp Ala Arg Asp Ser Ala Gly Arg Val Trp Leu Gln Gln Pro Arg Glu Asn Gly Lys Asn Gly Tyr Leu Val Gly Arg Val Gly Ser Glu Pro Trp Gly Thr Phe Lys A recombinant plasmid comprising the gene according to claim 1 or 2. The recombinant plasmid according to claim 4, which is an expression vector. A host cell transformed with the recombinant plasmid according to claim 4 or 5. The host cell according to claim 6, which is a gram positive bacterium. The host cell according to claim 6, which is Escherichia coli, lactic acid bacteria, Bacillus subtilis or staphylococci. The method for producing a protein according to claim 3, comprising the following steps 1) and 2):
1) a step of culturing the host cell according to any one of claims 6 to 8 under conditions capable of producing a protein having antibacterial activity;
2) A step of recovering a protein having antibacterial activity from the culture obtained by the culture in the step 1). A protein obtained by the production method according to claim 9. A composition comprising the protein according to claim 3 as an active ingredient. 12. Composition according to claim 11, characterized in that it is a research reagent, food additive, preservative, industrial fungicide, agricultural fungicide or medical antibiotic.