KR101016689B1 - Proteins involved in the transmission of peptidoglycan recognition signals, genes encoding them, and bacterial infection detection kits including the same - Google Patents

Abstract

The present invention provides novel proteins involved in peptidoglycan recognition signal transduction and genes encoding them. The present invention also provides a kit for the detection of bacterial infection in a sample, comprising a protein involved in the transmission of the peptidoglycan recognition signal. Proteins involved in the peptidoglycan recognition signal transmission newly discovered by the present invention can be usefully used in kits for detecting bacterial infection in samples such as blood.

Peptidoglycan, pro-phenol oxidase, brown rice wine

Description

Protein involving in peptidoglycan-recognition signal pathway, gene encoding the same, and kit for detecting bacterial infection comprising the same }

The present invention relates to a novel protein involved in the peptidoglycan recognition signal transduction of a brown rice meal ( Tenebrio molitor ), a gene encoding the same, or a kit for detecting a bacterial infection in a sample containing the same.

Through recent genetic engineering studies, Drosophila Drosophila PGRP-SA and Drosophila PGRP-SD, proteins that recognize the peptidoglycan (PG) of melanogaster ), activate the Toll pathway (Michel, T., Reichhart, JM, Hoffmann, JA & Royet, J. (2001) ) Nature 414, 756-759 and Bischoff, V., Vignal, C., Boneca, IG, Michel, T., Hoffmann, JA & Royet, J. (2004) Nat Immunol 5, 1175-1180), Drosophila PGRP-LC and Drosophila It has been reported that PGRP-LE is a receptor for the Imd pathway (Gottar, M., Gobert, V., Michel, T., Belvin, M., Duyk, G., Hoffmann, JA, Ferrandon, D. & Royet , J. (2002) Nature 416, 640-644; Choe, KM, Werner, T., Stoven, S., Hultmark, D. & Anderson, KV (2002) Science 296, 359-362; and Takehana, A. , Katsuyama, T., Yano, T., Oshima, Y., Takada, H., Aigaki, T. & Kurata, S. (2002) Proc Natl Acad Sci USA 99, 13705-13710. Meanwhile, Drosophila Gram-negative bacteria binding protein 1 ( Drosophila GNBP1) is an immune phenotype of loss-of-function mutant Drosophila Indistinguishable from PGRP-SA, this indicates that two proteins are required to activate the Toll pathway in response to infection of Gram-positive bacteria (Gobert, V., Gottar, M., Matskevich, AA, Rutschmann, S Royet, J., Belvin, M., Hoffmann, JA & Ferrandon, D. (2003) Science 302, 2126-2130; Pili-Floury, S., Leulier, F., Takahashi, K., Saigo, K., Samain, E., Ueda, R. & Lemaitre, B. (2004) J Biol Chem 279, 12848-12853; And Wang, L., Weber, AN, Atilano, ML, Filipe, SR, Gay, NJ & Ligoxygakis, P. (2006) EMBO J 25, 5005-5014). However, the mechanism at the molecular level in the upstream portion of the Toll pathway in Gram-positive bacteria recognition is not yet clear.

Pro-phenoloxisase (pro-PO) activating cascades that induce melaninization of invading pathogens are another important inherent immune defense mechanism in invertebrates, which are peptidoglycan (PG). ) And β-1,3-glucan (Cerenius, L. & Soderhall, K. (2004) Immunol Rev 198, 116-126 and Kanost, MR, Jiang, H. & Yu, XQ (2004) Immunol Rev 198, 97-105). Similar to the complement system of vertebrates, the pro-PO cascade is a plasma proteolytic cascade. Therefore, the pro-PO system is a good research model system for biochemical studies of PG and β-1,3-glucan recognition and subsequent signaling in cell-free conditions.

The inventors of the Drosophila Brown gonorrhea with high sequence homology with PGRP-SA ( Tenebrio Molecular Peptidoglycan Recognition Protein (PGRP) has been identified, Tenebrio The PGRP, named PGRP-SA, has been shown to activate the Lys-PG-dependent pro-PO system in Tenebrio insects (Park, JW, Je, BR, Piao, S., Inamura, S., Fujimoto, Y ., Fukase, K., Kusumoto, S., Soderhall, K., Ha, NC & Lee, BL (2006) J Biol Chem 281, 7747-7755). In addition, the inventors have described how Lys-PG recognition signals are transmitted downstream through the in vivo Drosophila Toll pathway, in vitro pro-PO system, and biochemical approaches using recombinant PGRP-SA. Various studies have been conducted to find out the mechanism, and it has been shown that new proteins involved in the pro-PO system can be isolated and used to detect bacterial infections in samples such as blood (Korean Patent Application No. 10-2007 -0095196, Korean Patent Application No. 10-2007-0013231; Park JW, Kim CH, Kim JH, Je BR, Roh KB, Kim SJ, Lee HH, Ryu JH, Lim JH, Oh BH, Lee WJ, Ha NC , Lee BL Proc Natl Acad Sci US A. 104, 6602-6607 (2007).

In addition, the present inventors have peptidoglycan new protein of approximately 41kDa mealworm size of protease derived involved in glycan recognition - were isolated (Tenebrio Tm 41), wherein the Tenebrio Tm - 41 The peptidoglycan of brown modular serine proteases derived from the higher level of geojeori protease that is activated by the recognition signal (Tenebrio It was confirmed that the protein is activated by the modular serine protease ( Tenebrio- MSP), and it has been found that the protein can be usefully used to detect bacterial infection in samples such as blood (Korean Patent Application No. 10-2007-0095195). , Filed Sept. 19, 2007).

The inventors have attempted a variety of studies to elucidate the specific mechanism of how the peptidoglycan recognition signal is transmitted downstream at the molecular biological level using proteins involved in the pro-PO system. As a result, a new protein involved in the peptidoglycan recognition process was isolated and the new protein obtained was activated in the form of Tenebrio. It was found that activated by Tm - 41, the activated protein again degrades the Spatzle protein present in the lower part to activate the Toll pathway. Therefore, it can be usefully used to detect bacterial infection in samples such as blood.

Accordingly, an object of the present invention is to provide a novel protein involved in the Toll activation pathway and pro-phenoloxidase (pro-PO) activation system, which is a biological defense reaction of insects, and a gene encoding the same.

Another object of the present invention is to provide a kit for detecting bacterial infection in a sample using the protein.

According to one aspect of the present invention, there is provided a protein of brown mealworms consisting of the amino acid sequence of SEQ ID NO: 1.

In addition, according to another aspect of the present invention, a gene encoding a protein derived from brown gourd consisting of the amino acid sequence of SEQ ID NO: 1 is provided, and preferably, the gene has a nucleotide sequence of SEQ ID NO: 2.

In addition, according to another aspect of the present invention, there is provided a kit for detecting bacterial infection in a specimen, comprising a protein derived from brown rice wine consisting of the amino acid sequence of SEQ ID NO: 1.

In the kit according to the present invention, the sample may be blood for transfusion, blood of a mammal, food, tap water, groundwater, rainwater, or sterile product, preferably blood for transfusion or blood of a mammal. In addition, the kit may have the form of a solution, lyophilized powder, frozen solution, or strip.

Detection kit according to the invention is Tenebrio PGRP-SA consisting of the amino acid sequence of SEQ ID NO: 3; And Tenebrio GNBP1 consisting of the amino acid sequence of SEQ ID NO: 4, Tenebrio MSP-1 consisting of the amino acid sequence of SEQ ID NO: 5, Tenebrio MSP-2 consisting of the amino acid sequence of SEQ ID NO: 6, and Tenebrio consisting of the amino acid of SEQ ID NO: 7 One or more proteins selected from the group consisting of Tm-41 may be further included, and if necessary, may further include β-lytic protease (β-lytic protease, blp), lysozyme, or blp and lysozyme.

According to the present invention, a factor (ie, a protein consisting of the amino acid sequence of SEQ ID NO: 1) involved in peptidoglycan recognition signal transmission has been identified, and the kit for detecting bacterial infection in a sample such as blood when the protein is used. It can be usefully used for manufacturing. That is, the protein newly revealed by the present invention is the inventors of the present inventors (Korea Patent Application No. 10-2007-0095196, Republic of Korea Patent Application No. 10-2007-0013231; Proc Natl Acad Sci US A. 104, 6602-6607 (2007)) found peptidoglycan recognition proteins involved in the pro-PO system, ie Tenebrio PGRP-SA consisting of the amino acid sequence of SEQ ID NO: 3; And Tenebrio GNBP1 consisting of the amino acid sequence of SEQ ID NO: 4, Tenebrio MSP-1 consisting of the amino acid sequence of SEQ ID NO: 5, Tenebrio MSP-2 consisting of the amino acid sequence of SEQ ID NO: 6, and Tenebrio consisting of the amino acid of SEQ ID NO: 7 It can be made into a kit for detecting bacterial infection with or separately from one or more selected proteins from the group consisting of Tm-41.

The inventors of the present invention found that brown-peptide peptidoglycan recognition proteins, ie, complexes of Tenebrio PGRP-SA and PG, are involved in signaling to downstream stages, thereby reproducing the pro-PO system and Toll. It has been found to activate the pathway. In addition, the protein involved in signaling to the downstream step is Gram-negative bacteria binding protein 1 (GNBP1) -like protein Tenebrio Tenebrio MSP was found to be a T enebrio-multi-domain containing modular SP (MSP) with low density lipid protein-like domain and complement system regulatory protein-like domain at GNBP1 and N-terminus. That is, it has been found that Tenebrio MSP-1 and / or Tenebrio MSP-2 is present (Korean Patent Application No. 10-2007-0013231; Park JW, Kim CH, Kim JH, Je BR, Roh KB, Kim SJ, Lee HH, Ryu JH, Lim JH, Oh BH, Lee WJ, Ha NC, Lee BL Proc Natl Acad Sci US A. 104, 6602-6607 (2007). In addition, a modular serine protease derived from brown rice bran, a protease of higher levels activated by a peptidoglycan recognition signal ( Tenebrio) Tenebrio Tm-41, a protein activated by the modular serine protease ( Tenebrio- MSP), has been identified (Korean Patent Application No. 10-2007-0095196).

Based on the results of the previous studies, the inventors conducted a study on the molecular biological level of recognition mechanisms and surprisingly isolated new proteins involved in the peptidoglycan recognition process. Sequence analysis of the protein revealed that the protein has an amino acid sequence of SEQ ID NO: 1, and the gene encoding it has a nucleotide sequence of SEQ ID NO: 2. Also, the SEQ ID NO: 1. Functional analysis of a protein comprising the amino acid sequence of the result, the protein is a peptidoglycan a serine protease derived from the higher level of the mealworm protease that is activated by the recognition signal (Tenebrio Tm-41) newly revealed that the protein is activated. Therefore, the protein newly revealed by the present invention is a brown worm-derived Gram negative bacteria binding protein ( Tenebrio Gram negative bacteria binding protein 1, Tenebrio GNBP1), brown worm-derived modular serine protease ( Tenebrio modular serine protease, Tenebrio MSP) ( Tenebrio MSP) MSP-1 and / or Tenebrio MSP-2), a serine protease derived from brown skin In a kit for detecting bacterial infections including Tm-41) and the like, it can function as a cleavage enzyme of the final substrate. Therefore, the protein newly discovered by the present invention can be used to detect bacterial infection in samples such as blood.

Accordingly, the present invention provides a protein derived from brown rice wine consisting of the amino acid sequence of SEQ ID NO: 1.

In addition, the present invention provides a gene encoding a protein derived from brown gourd consisting of the amino acid sequence of SEQ ID NO: 1, preferably a gene consisting of the nucleotide sequence of SEQ ID NO: 2.

According to one embodiment of the present invention, there is provided a kit for detecting bacterial infection in a sample, comprising a protein derived from brown gourd consisting of the amino acid sequence of SEQ ID NO: 1.

In the detection kit, the sample is blood for transfusion, blood of mammals including humans, foods such as vegetables, meat, fruits, cooked or uncooked foods, tap water, ground water, rain water, sterile products And all other specimens in need of detection of other microorganisms. Preferably, the detection kit of the present invention may be usefully used for the detection of bacterial infection in blood for transfusion or in the blood of mammals including humans.

The kit for detection of the present invention is a reagent for detecting reactivity, for example, an amino acid or peptide substance combined with para-nitroaniline and a chromophore substrate of other pro-PO activating factor proteins and pro-PO enzymes. And the like. In addition, the detection kit may have the form of a solution, lyophilized powder, frozen solution, or strip, each form can be formulated by conventional methods in the art. For example, a kit for detection in solution form may be formulated separately or by mixing the protein (s) in a buffer such as sodium-phosphate, potassium-phosphate, tris-hydrochloric acid and several other buffers, It may be frozen or lyophilized accordingly.

In addition, the detection kit of the present invention may be prepared as a separate kit for detecting the protein derived from brown gourd consisting of the amino acid sequence of SEQ ID NO: 1, or the peptides found to be involved in the pro-PO system as a result of the previous studies of the present inventors It may also be prepared as a kit for detection included in the glycan recognition protein or in a separate container.

That is, the detection kit of the present invention, in addition to the brown gourd derived protein consisting of the amino acid sequence of SEQ ID NO: 1, the present inventors (Korean Patent Application No. 10-2007-0095196, Korean Patent Application No. 10- 2007-0013231; Park JW, Kim CH, Kim JH, Je BR, Roh KB, Kim SJ, Lee HH, Ryu JH, Lim JH, Oh BH, Lee WJ, Ha NC, Lee BL Proc Natl Acad Sci US A. 104, 6602-6607 (2007)) found peptidoglycan recognition proteins involved in the pro-PO system, ie Tenebrio PGRP-SA consisting of the amino acid sequence of SEQ ID NO: 3; And Tenebrio GNBP1 consisting of the amino acid sequence of SEQ ID NO: 4, Tenebrio MSP-1 consisting of the amino acid sequence of SEQ ID NO: 5, Tenebrio MSP-2 consisting of the amino acid sequence of SEQ ID NO: 6, and Tenebrio consisting of the amino acid sequence of SEQ ID NO: 7 It may further comprise at least one protein selected from the group consisting of Tm-41.

In addition, the kit for detection of the present invention may further comprise β-lytic protease (β-lytic protease, blp) and / or lysozyme. The blp may be derived from various microorganisms including soil microorganisms. For example, the blp is a microorganism of the genus Achromobacter , and preferably, Achromobacter Achromobacter lyticus ), more preferably Achromobacter lyticus ) ATCC 21456 or Achromobacter lyticus ) may be derived from ATCC 21457. In addition, the blp is a known method (Li, S., Norioka, S. & Sakiyama, F. (1998) J Biochem ( Tokyo ) 124, 332-339), purified from commercially available crude Achromopeptidase samples (Wako Pure Chemical Institute, 014-09661) can also be used. In addition, the lysozyme may be used commercially available conventional lysozyme.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the following examples are intended to illustrate the invention, and the invention is not limited by the following examples.

Example

1. Purification of Proteins

(1) Preparation of antibody against protein involved in peptidoglycan recognition process

Proteins involved in the peptidoglycan recognition process found according to the results of our previous studies, ie Tenebrio PG recognition protein (Tm-PGRP-SA) (J Biol Chem. 2006; 281: 7747-55), Tm-GNBP3 ( Tenebrio Gram-negative binding protein 3) (J Biol Chem. 2003; 278: 42072-9), Tm-GNBP1 ( Tenebrio Gram-negative binding protein 1) ( Proc Natl Acad Sci US A. 104, 6602-6607 (2007)), and Tenebrio modular serine protease (Tm-MSP) ( Proc Natl Acad Antibodies against Sci US A. 104, 6602-6607 (2007)) were prepared. Tm-PGRP-SA was used for recombinant protein, Tm-GNBP1, Tm and Tm-GNBP3-MSP was Tm-GNBP1 using the partial amino acid sequence information is Tm as Keyhole limpet hemocyanin (KLH) -cys- LEAYEPKGFRAS-NH 2 -GNBP3 chemically synthesizes a peptide having a partial amino acid sequence with KLH-cys-YFDGKNKLGYPNDDQKF-NH ₂ and Tm-MSP with KLH-cys-VNGKPVKKGDYPWQ-NH ₂ to conjugate KLH, a carrier protein, with adjuvant Injections four times over four weeks subcutaneously in rabbits yielded antibodies to each protein from rabbit serum.

(2) Preparation of the eluate fraction from the body fluids of the brown rice wine ( Tenebrio molitor )

After separating body fluids (4,000 ml, 43.7 g of protein) from the brown edible ( Tenebrio molitor ), non-specifically activated serine protease was inactivated as follows: 0.5 mM diiso, an irreversible serine protease inhibitor Diisopropyl fluorophosphate (DFP) solution was added to the bodily fluid obtained from the brown gourd, Tenebrio molitor , followed by dialysis with 15 liters of Buffer A (50 mM Tris-HCl, 3 mM EDTA, pH 6.0) for 12 hours. It was. After removing the residue by centrifugation, the supernatant was recovered, loaded on a Toyopearl AF-Heparin HC 650M column, and washed with Buffer A sufficiently. Elution was carried out in a linear gradient using a solution containing 1M NaCl in buffer A, and the resulting eluate fractions were divided into three types (hereinafter, referred to as E1, E2, and E3, see FIG. 1).

(3) Immunoblot analysis and in vitro active recombination testing

In order to determine the distribution of proteins involved in the peptidoglycan recognition process present in each of E1, E2, and E3 fractions, immunoblot analysis was performed using the respective antibodies in (1). As a result, it was confirmed that GNBP1 exists in E1, MSP and PGRP-SA exist in E2, and GNBP3 exists in E3. (See Figure 2)

In addition, 5 μL of each of the E1, E2, and E3 fractions was taken and 10 μL of decoagulation buffer (composition: 30 mM trisodium citrate, 26 mM citric acid, 20 mM EDTA, and 15 mM sodium chloride, pH). 4.6), 10 μL of Lys-PGN (Korean Patent Application No. 10-2007-0013231; Proc Natl Acad Sci US A. 104, 6602-6607 (2007)) or β-1,3-glucan solution, 15 μL of buffer A, pre-incubated for 5 minutes, followed by 443 μL of 20 mM Tris-HCl (pH 8.0). ), 5 μL of 1 M CaCl ₂ solution or no addition, 2 μL of 10 mM α-thrombin substrate (Boc-Val-Pro-Arg-MCA), followed by reaction at 30 ° C. for 20 minutes. In vitro active recombination tests were performed by measuring the intensity of methylcoumarin fluorescence. The obtained result is shown in FIG.

The specific activity - as can be seen in Figure 3, E1, E2, and E3 fractions peptidoglycan is free conditions (column 2) and Ca ^{2 +} ion-free conditions (column 3), the peptidoglycan There was also no amidase in the presence of peptidoglycan in combinations lacking individual fractions (columns 4, 5 and 6). In particular, although all of the proteins Tm-PGRP-SA, GNBP1 and MSP, which are involved in the peptidoglycan recognition process, were present in E1 and E2, no peptidoglycan-specific amidase activity was shown (column 6). However, peptidoglycan, Ca ^{+ 2} ion, and E1, E2, peptidoglycan in the presence of all fractions of the E3 - it can be seen that represents the specific amidase activity (column 1).

The results indicate that proteins that recognize the peptidoglycan of bacteria activate the protease (s) in the inactive zymogen form, which is present in the lower stage after the peptidoglycan is recognized, and then alpha Suggests that it has a protease involved in decomposing Boc-Val-Pro-Arg-MCA, a thrombin substrate, to liberate the methylcoumarin, a fluorescent substance. Indicates that a new argument exists. In particular, the E2 fraction suggests the presence of a lower level of inactive zymogen form protease.

(4) Novel Protein Isolation from E2 Fraction

Step 1-Q- Sepharose Fast Flow Column Chromatography : The E2 fraction obtained in (2) (total protein amount was 737 mg) was loaded on a Q-Sepharose Fast Flow column, and the flow-through fractions were serine proteases derived from brown edible meals respectively activated. Tenebrio After mixing with Tm-41 (Korean Patent Application No. 10-2007-0095196), the fractions showing amidase activity were pooled (total protein: 514 mg).

Step 2- CM - Toyopearl 650M Column Chromatography : The pooled elution fraction obtained in step 1 was ultrafiltered with Buffer A, which was loaded onto a CM-Toyopearl 650M column (30 mm x 150 mm) to obtain the protein fraction. Collected and concentrated (total amount of protein: 83 mg).

Step 3- HiTrap Heparin FPLC Column Chromatography : The HiTrap Heparin FPLC column equilibrated with 20 mM Tri-HCl buffer (3 mM EDTA, pH8.0) was loaded with the concentrate obtained in step 2, washed with the same buffer, and then 400 mL of buffer was added per minute. The remaining protein was eluted at a NaCl concentration gradient of 0-1.0 M at a flow rate of ml.

Step 4 - hydroxyl apatite (Hydroxylapatite) was purified by column chromatography: 20 mM sodium phosphate buffer was equilibrated with a (pH 7.0) hydroxyl apatite (hydroxylapatite) FPLC column (5mm x 50mm of the eluate obtained in the third step containing 3 mM EDTA , Bio-Rad). After washing the column with 4 mL of the same buffer, the protein fractions were pooled by elution with a concentration gradient of 25 mL of 20-500 mM sodium phosphate solution (total protein amount: 1.1 mg).

Step 5- TSKgel G2000SW size exclusion (size exclusion ) column chromatography : The pooled fractions obtained in step 4 were loaded onto a TSKgel G2000SW column (4.6 mm x 30 cm), and 50 mM sodium-phosphate buffer (containing 0.3 M NaCl, pH 7.0) was used at a flow rate of 0.3 mL per minute. Eluted using. Fractions containing protein (protein mass: 90 ug) were pooled. The molecular weight of the obtained protein was about 44 kDa and was used as the zymogen form protein in the following characterization test.

Step 6 - benzamidine-Sepharose (Benzamidine-Sepharose) 6B absorption column chromatography to purify the protein in active form, the elution (flow-through) solution (protein 104 mg) obtained from Step 1 Tenebrio Reaction was carried out with Tm-41 (Korean Patent Application No. 10-2007-0095196). The eluate containing the protein in the activated form was loaded onto a benzamidine-cellose 6B (Pharmacia Biotech) column equilibrated with 50 mM Tris-HCl buffer (containing 0.5M NaCl, pH8.0). The column was washed with the same buffer and the adsorbed protein was eluted with 20 mM 4-aminobenzamidine in the same buffer. The eluate containing the activated protein was purified by sequentially performing HiTrap Heparin FPLC column chromatography and TSKgel G2000SW size exclusion column chromatography in the same manner as above. Fractions containing the protein of the active form were pooled to obtain 60 ug (molecular weight of about 44 kDa) of purified protein, which was named Tm-44.

Electrophoresis was performed on the obtained Tm-44, and the result is shown in FIG. 4 (specifically, lane 5 of (A)). Pure protein bands with gel mobility of about 44 kDa on 12% SDS-PAGE can be identified under reducing conditions.

2. Protein and Nucleic acid sequence  analysis

(1) N-terminal sequence and partial amino acid sequence analysis of Tm-44 protein

After reducing and alkylating the Tm-44 protein, the lysylendopeptidase was treated, separated by HPLC, and the N-terminal sequence and partial amino acid sequence were determined by Edman digestion. Same as 1.

peak
number Amino acid sequence One I V G G E K T D L D E F P W M A L V E Y E K P G G S C G F 2 D P C R S P N G Q S G D C K 3 T D L D E F P W M A L V E Y E K P G G S R G F Y C G G V L I S K 4 L F V A G W G K T E N R S E S N I K 5 D I H W Y A A G V V S F G P S P C G M A N W P G V Y T K

(2) cDNA Cloning and Nucleotide Sequence of Tm-44

Reverse transcription-Reverse transcription was performed using total RNA obtained from fat bodies extracted from the larvae of Tenbribrio. Reverse transcriptase was used Superscript II (Invitrogen).

Partial cDNA Sequence Generation -Based on the amino acid sequence information of the Tm-44 protein obtained in (1), the forward primer was designed as 5'-GAYGARTTYCCNTGGATGGC-3 ', and the reverse primer was designed as 5'-CCARTTNGCCATNCCRCANGG-3'. Partial cDNA fragments were obtained by degenerate PCR. Amplified DNA was subjected to agarose gel electrophoresis and nucleic acid sequences were analyzed using pCR2.1-TOPO (Invitrogen) vector.

RACE ( Rapid Amplification of cDNA Ends ) PCR -5'- and 3'-RACE analysis was performed using the SMART RACE method (CLONTECH). Primers used in RACE PCR were designed based on partial cDNA sequence information, the sequence and the position in the nucleic acid sequence is shown in Table 2 below.

name SEQ ID NO: Primer sequence Nucleic Acid Sequence Locations SP 8 5'-GAYGARTTYCCNTGGATGGC-3 ' 384-404 ASP 9 5'-CCARTTNGCCATNCCRCANGG-3 ' 1074-1095 5'GSPa 10 5'-CGTCGCAACCTCAACAGAGCTA-3 ' 695-716 5'GSPb 11 5'-GATGTTGTTGGGTTCGTAGCTCTC-3 ' 665-688 5'GSP1 12 5'-CGTGTCAGTCTCGGTGTTGTACTC-3 ' 553-576 3'GSP1 13 5'-CTCTGACTACATCAAACCCATTTGTC-3 ' 729-754 3'GSP2 14 5'-GAAGAGCTGAGCAAGTCGTACCTC-3 ' 769-792

Full cDNA Sequence Generation—Specific primers were designed for each terminal site to amplify full length cDNA. As forward and reverse primers, full length cDNA was obtained using 5'-ATGTTGGTCCGCTCCTTGTT-3 '(SEQ ID NO: 15) and 5'-CTAGGGCTTCAGCTTGCCG-3' (SEQ ID NO: 16), respectively. The amplified DNA was subjected to electrophoresis to separate the band, and then inserted into a pGEM T-vector (Promega) vector to analyze the entire nucleic acid sequence. The obtained total amino acid sequence is as shown in Figure 5, it was confirmed that the new sequence as a result of sequencing with proteins of other known insects. In addition, the results of BLAST search (www.ncbi.nlm.nih.gov/BLST) of the Tm-44 gene (ie, the gene of SEQ ID NO: 2) confirmed that it is a completely different gene.

(3) Identification of biochemical properties and function of Tm-44 protein

In order to confirm the molecular activation mechanism between the Tm-MSP protein, Tm-41 protein, and Tm-44 protein, cleavage patterns were measured after interacting with the zymogen or active form of each protein. For example, 1 μg of the activated form of Tm-41 protein and 1 μg of Tm-44 in the zymogen form are reacted at 30 ° C. for 1 hour and 30 minutes under the conditions of 20 mM Tris-HCl (pH. 8.0). The solution was subjected to 12% SDS-PGAE under reducing and non-reducing conditions to measure the cleavage pattern, and the results are shown in FIG. 6.

Fig. 6 (A) shows the reactivity of the active Tm-41 protein with Zymogen form Tm-44. Lanes 1 and 4 are active Tm-41 proteins, lanes 2 and 5 are zymogen-type Tm-44 proteins, and lanes 3 and 6 are active Tm-41 proteins and zymogen forms. Is a mixture with Tm-44 protein. Lanes 1-3 and 4-6 show the gel mobility of these proteins under reducing and non-reducing conditions, respectively. Bands (a) and (b) show the catalytic and clip domains derived from Tm-44 after cleavage and band (c) shows the cleaved Tm-44 protein. 6 (B) shows the reactivity of the active Tm-MSP protein with Zymogen form Tm-41. Lanes 1 and 4 are active Tm-MSP proteins, lanes 2 and 5 are zymogen-type Tm-41 proteins, and lanes 3 and 6 are active Tm-MSP proteins and zymogen forms Is a mixture with Tm-41 protein. Lanes 1-3 and 4-6 show the gel mobility of these proteins under reducing and non-reducing conditions, respectively. Bands (d) and (e) show the catalytic and clip domains derived from Tm-41 after cleavage, and band (f) shows the cleaved Tm-41 protein. In FIG. 6 (C), lane 1 is an activated Tm-MSP protein, lane 2 is a Tm-44 protein in a zymogen form, and lane 3 is an activated Tm-MSP protein and a Tm-44 protein in a zymogen form. Represents a mixture of. From the results of FIG. 6C, it can be seen that the Tm-44 protein is not cleaved by the Tm-MSP protein. In FIG. 6 (D), lane 1 is an active Tm-44 protein, lane 2 is a zymogen-type Tm-41 protein, and lane 3 is an active Tm-44 protein and a zymogen form of Tm-. 41 represents a mixture of proteins. From the results in FIG. 6 (D), it can be seen that the active Tm-44 protein does not cleave the Tm-41 protein in the zymogen form.

Spatzle Protein and Tribolium Cleaved by Active Tm-44 Protein Affinity with Toll ectodomain was analyzed by HPLC using a TSK SW3000 size exclusion column. Curves (c) and (b) in FIG. 7 (A) show the cleaved Spatzle protein (8 μg) and purified Tribolium, respectively. The dissolution pattern after injection of toll ectodomain (18 μg) is shown. Curve (a) shows truncated Spatzle protein (8 μg) and purified Tribolium Toll ectodomain (18 μg) truncated Spatzle protein (8 μg) and Tribolium The elution pattern after injecting a mixture with toll ectodomain (18 μg) is shown. Figure 7 (B) shows the results of the analysis of each of the peaks obtained by the HPLC analysis by 12% SDS-PGAE under reducing and non-reducing conditions. Peak a1 is the truncated Spatzle protein and purified Tribolium Toll ectodomain, these two proteins represent a stable complex. Peak a2 represents excess Spatzle protein remaining after complex formation. Peak b and c are each purified Tribolium Toll ectodomain and truncated Spatzle protein.

As can be seen from the results of FIGS. 6 and 7, the activated form of Tm-41 decomposes and activates the zymogen form of Tm-44 protein in the subprocess, and the activated Tm-44 protein is further lowered. The toll pathway is activated by cleaving the existing Spatzle protein and binding it to the Toll-ectodomain. It can be confirmed that the Tm-44 protein is a protease present in the upper level of the Spatzle protein in the peptidoglycan recognition signal path. Therefore, Tm-44 newly isolated by the present invention selectively detects bacterial peptidoglycan when using a fluorescent substrate (eg, methylcoumarin or the like) or a chromophore (p-nitrophenol) capable of cleaving it. As such, it can function as a final substrate cleavage enzyme, and thus can be usefully used in a kit for detecting bacterial infection. A summary of a series of reaction processes of proteins involved in the pro-PO system derived from a series of brown rice bran is as shown in FIG. 8. In FIG. 8, SAE and SPE mean Tm-41 and Tm-44 proteins, respectively.

1 is a chromatogram of a ToyoPal AF-heparin HC 650M column loaded in a brownish bodily fluid.

Figure 2 is a result of performing an immunooblot analysis of each fraction of the brown body fluid fraction separated by chromatogram of TOYOPEL AF-heparin HC 650M column using each antibody.

FIG. 3 shows the results of in vitro activator test for amidase activity on α-thrombin substrate.

4 shows the 12% SDS-PAGE results for reducing and non-reducing conditions, respectively, for Tm-44.

5 shows the amino acid sequence of Tm-44.

FIG. 6 shows the cleavage patterns after interacting the zymogen or active form of each protein to confirm the molecular activation mechanism between the Tm-MSP protein, Tm-41 protein, and Tm-44 protein. The result of the measurement is shown.

Fig. 7 shows Spatzle protein and Tribolium cleaved by active Tm-44 protein. Affinity with Toll ectodomain is analyzed by HPLC using TSK SW3000 size exclusion column.

Figure 8 summarizes the reaction of proteins involved in the pro-PO system found in accordance with the present invention.

<110> YUHAN CORPORATION <120> Protein involving in peptidoglycan-recognition signal pathway,          gene encoding the same, and kit for detecting bacterial infection          configuring the same <130> PN0153 <160> 16 <170> KopatentIn 1.71 <210> 1 <211> 384 <212> PRT <213> Tenebrio molitor <400> 1 Met Leu Val Arg Ser Leu Phe Ile Leu Val Val Thr Ala Gln Val Leu   1 5 10 15 Asn Ala Asp Glu Asn Cys Arg Thr Pro Asp Asn Glu Glu Gly Asp Cys              20 25 30 Lys Pro Ile Asn Gln Cys Arg Pro Leu Tyr Ser Leu Leu Glu Arg Arg          35 40 45 Pro Ile Thr Ala Ser Thr Ala Glu Tyr Leu Arg Arg Ser Asn Cys Gly      50 55 60 Phe Asp Gly Ser Tyr Pro Arg Val Cys Cys Pro Gln Gly Ser Ile Glu  65 70 75 80 Pro Pro Thr Ile Lys Pro Pro Ile Val Asp Gly Pro Thr Glu Ser Asn                  85 90 95 Asn Val Ser Pro Val Thr Ser Asp Leu Leu Pro Asp Gly Ser Ile Cys             100 105 110 Gly Pro Asn Thr Gln Asn Arg Ile Tyr Gly Gly Glu Lys Thr Asp Leu         115 120 125 Asp Glu Phe Pro Trp Met Ala Leu Val Glu Tyr Glu Lys Pro Gly Gly     130 135 140 Ser Arg Gly Phe Tyr Cys Gly Gly Val Leu Ile Ser Lys Arg Tyr Val 145 150 155 160 Leu Thr Ala Ala His Cys Val Lys Gly Lys Asp Leu Pro Lys Thr Trp                 165 170 175 Lys Leu Val Ser Val Arg Leu Gly Glu Tyr Asn Thr Glu Thr Asp Thr             180 185 190 Asp Cys Ile Asn Asn Gly Phe Gly Glu Asp Cys Ala Pro Pro Val         195 200 205 Asn Val Gln Val Glu Ala Arg Ile Ala His Glu Ser Tyr Glu Pro Asn     210 215 220 Asn Ile Asn Gln Tyr His Asp Ile Ala Leu Leu Arg Leu Arg Arg Glu 225 230 235 240 Val Lys Phe Ser Asp Tyr Ile Lys Pro Ile Cys Leu Pro Thr Thr Thr                 245 250 255 Glu Glu Leu Ser Lys Ser Tyr Leu Gly Gln Lys Leu Phe Val Ala Gly             260 265 270 Trp Gly Lys Thr Glu Asn Arg Ser Glu Ser Asn Ile Lys Leu Lys Val         275 280 285 Gln Val Pro Val Lys Gln Met Ser Asp Cys Thr Ala Thr Tyr Ser Ser     290 295 300 Ala Asn Val Arg Leu Gly Ser Gly Gln Leu Cys Ala Gly Gly Glu Ser 305 310 315 320 Gly Lys Asp Ser Cys Arg Gly Asp Ser Gly Gly Pro Leu Met Ile Leu                 325 330 335 Ser Leu Asp Lys Asp Lys Asp Ile His Trp Tyr Ala Ala Gly Val Val             340 345 350 Ser Phe Gly Pro Ser Pro Cys Gly Met Ala Asn Trp Pro Gly Val Tyr         355 360 365 Thr Lys Val Ser Lys Tyr Val Asp Trp Ile Val Gly Lys Leu Lys Pro     370 375 380 <210> 2 <211> 1155 <212> DNA <213> Tenebrio molitor <400> 2 atgttggtcc gctccttgtt catcctggta gtaacagcac aagtgctcaa tgccgacgag 60 aattgtcgta ctcctgataa tgaagaaggt gattgtaagc ctatcaatca atgccgcccc 120 ctctactccc tgttggagcg ccgccccatc accgccagca ccgccgagta tttgcgccga 180 tccaactgcg gcttcgacgg gagctaccct cgcgtctgct gcccccaagg ctcgatcgaa 240 cccccgacca tcaaaccccc aatagtggac gggcccaccg agtccaacaa tgtgtctccc 300 gtgacgagcg acctcctccc agacggctcc atctgcggtc ccaacaccca gaacaggatc 360 tacggcgggg agaaaaccga cctggatgag ttcccctgga tggccctggt ggaatacgag 420 aaacccggag gcagtcgagg gttctactgc ggcggagtgc tgatcagcaa gaggtacgtc 480 ctgacggcgg cgcactgcgt caaagggaag gatctgccca aaacgtggaa actcgtgagc 540 gtgcgtttgg gcgagtacaa caccgagact gacacggact gcatcaacaa cggcttcggg 600 gaggactgcg ccccaccccc cgtcaacgtc caggtggagg ccaggatcgc ccacgagagc 660 tacgaaccca acaacatcaa ccagtaccac gacatagctc tgttgaggtt gcgacgcgaa 720 gtcaaattct ctgactacat caaacccatt tgtctgccga ccaccaccga agagctgagc 780 aagtcgtacc tcggccagaa actcttcgtg gcgggctggg gcaagaccga gaaccggtcc 840 gagagcaaca tcaagctcaa agtgcaagtt cccgtcaagc aaatgtcaga ctgcaccgcc 900 acctacagca gcgccaatgt gaggttaggt tctggtcagc tgtgcgcagg aggcgaatcg 960 gggaaagatt cgtgtcgcgg agacagcgga gggcctttga tgatcctcag tttggacaaa 1020 gacaaggaca tacactggta cgccgcgggg gtggtgtctt tcgggccctc gccctgcggc 1080 atggccaact ggccgggagt ttacaccaaa gtgtccaaat acgtagactg gatcgtcggc 1140 aagctgaagc cctag 1155 <210> 3 <211> 193 <212> PRT <213> Tenebrio molitor <400> 3 Met Leu Leu Ala Thr Ile Ala Arg Gly Val Tyr Gln Ile Ser Ala Leu   1 5 10 15 Ser Gly Ser Thr Ile Pro Arg Ile Cys Pro Glu Ile Ile Ser Arg Thr              20 25 30 Arg Trp Gly Ala Arg Thr Pro Leu Glu Val Asp Tyr Ser Leu Ile Pro          35 40 45 Ile Glu Asn Val Val Val His His Thr Val Thr His Thr Cys Asp Ser      50 55 60 Glu Ser Glu Cys Ala Thr Leu Leu Arg Asn Val Gln Asn Phe His Met  65 70 75 80 Glu Asn Leu Glu Phe His Asp Ile Gly Tyr Asn Phe Leu Val Ala Gly                  85 90 95 Asp Gly Gln Ile Tyr Glu Gly Ala Gly Trp His Lys Val Gly Ala His             100 105 110 Thr Arg Gly Tyr Asn Thr Arg Ser Leu Gly Leu Ala Phe Ile Gly Asn         115 120 125 Phe Thr Ser Gln Leu Pro Val Gln Lys Gln Leu Lys Val Ala Lys Asp     130 135 140 Phe Leu Gln Cys Gly Val Glu Leu Gly Glu Leu Ser Lys Asn Tyr Lys 145 150 155 160 Leu Phe Gly Ala Arg Gln Val Ser Ser Thr Ser Ser Pro Gly Leu Lys                 165 170 175 Leu Tyr Arg Glu Leu Gln Asp Trp Pro His Phe Thr Arg Ser Pro Pro             180 185 190 Lys     <210> 4 <211> 443 <212> PRT <213> Tenebrio molitor <400> 4 Met Phe Ala Lys Ala Ile Ile Leu Phe Leu Ile Leu Thr Thr Phe Gln   1 5 10 15 Cys His Gly Glu Phe Val Ile Pro Glu Val Thr Leu Glu Ala Tyr Glu              20 25 30 Pro Lys Gly Phe Arg Ala Ser Ile Pro Ala Leu Asn Gly Ile Gln Met          35 40 45 Phe Ala Phe His Gly Asn Ile Asn Lys Pro Ile Ser Gln Val Asp Pro      50 55 60 Gly Glu Tyr Ser Gln Asp Tyr Thr Ser Pro Thr Gly Asn Thr Trp Ser  65 70 75 80 Tyr Phe Asn Lys Asp Leu Lys Leu Lys Ala Gly Asp Val Ile His Tyr                  85 90 95 Trp Val Phe Ile Gln Phe Leu Lys Leu Gly Tyr Arg Lys Asp Asn Gln             100 105 110 Val Trp Asn Val Thr Glu Leu Val Gln Leu Lys Asn Ser Ser Cys Glu         115 120 125 Thr Ser Pro Thr Thr Val Arg Gly Arg Ser Val Ile Cys Lys Asn Ser     130 135 140 Ile Ile Phe Glu Glu Asn Phe Asn Gly Glu Gly Ile Asp Thr Lys Lys 145 150 155 160 Trp Leu Ile Glu Gln Tyr Ile Pro Thr Tyr Thr Ser Leu Asp Tyr Glu                 165 170 175 Phe Val Ser Tyr Gln Asn Asp Pro Thr Val Cys Phe Leu Asn Asp Asn             180 185 190 Lys Leu Phe Ile Lys Pro Lys Tyr Ala Gln Ser Glu Ala Glu Val Asn         195 200 205 Gly Glu Leu Asp Phe Arg Asn Arg Cys Thr Arg Lys Thr Asp Glu Glu     210 215 220 Cys Tyr Lys Lys Arg Glu Ile Tyr Phe Ile Ile Pro Pro Val Thr Ser 225 230 235 240 Gly Arg Leu Val Ser Asp Phe Arg Phe Lys Tyr Gly Lys Val Glu Ile                 245 250 255 Arg Ala Lys Leu Pro Ala Gly Asp Trp Ile Tyr Pro Gln Met Tyr Leu             260 265 270 Glu Gln Val Asn Asp Pro Lys Lys Lys Ile Trp Ile Gly Tyr Ala Arg         275 280 285 Gly Asn Asn Lys Leu Leu Ala Asn Asn Gln Glu Asp Ile Gly Gly Asn     290 295 300 Leu Leu Phe Gly Gly Pro Val Leu Asp Pro Glu Glu Pro His Arg Ser 305 310 315 320 Gln Tyr Leu Lys Ser Thr Arg Asn Ser Lys Pro Phe Thr Ser Gln Met                 325 330 335 His Thr Leu Val Val Leu Trp Asp Glu Asp His Ile Ser Leu Gln Leu             340 345 350 Asn Gly Ile Glu Tyr Gly Lys Ile Asp Lys Arg Thr Met Gln Glu Val         355 360 365 Asn Phe Ala Asp Asn Asp Met Val Arg Leu Val Leu Gly Val Gly Val     370 375 380 Gly Gly Val Asn Asp Phe Pro Asp Asp Phe Arg Ser Gly Thr Asn Val 385 390 395 400 Lys Pro Trp Arg Asn Lys Asp Asn Lys Gln Val Lys Asn Phe Phe Thr                 405 410 415 Ala Arg Ser Glu Trp Gly Lys Thr Trp Ser Gly Asp Asn Cys Ala Leu             420 425 430 Gln Val Asp Tyr Ile Lys Val Trp Ala Leu ***         435 440 <210> 5 <211> 633 <212> PRT <213> Tenebrio molitor <400> 5 Met Cys Asn Val Arg Thr Leu Leu Gln Val Ile Cys Leu Ser Leu Ile   1 5 10 15 Val Ile Gln Thr Val Asp Ser Tyr Ser Phe Ala Leu Ser Lys Phe Thr              20 25 30 Arg Ile Arg Arg Gln Ala Arg Arg Thr Cys Thr Ser Thr Glu Phe Ala          35 40 45 Cys Lys Ser Gly Glu Cys Ile Asp Glu Asp Lys Glu Cys Asp Gly Ile      50 55 60 Val Asp Cys Thr Asp Ala Ser Asp Glu Thr Asn Ala Cys His Arg Ile  65 70 75 80 Lys Cys Pro Asn Tyr Leu Phe Arg Cys Lys Tyr Gly Ala Cys Ile Asn                  85 90 95 Pro Asp Leu Glu Cys Asp Gly Lys Pro Asp Cys Met Asp Gly Ser Asp             100 105 110 Glu Lys Thr Ser Lys Cys Lys Pro Asp Asp Ser Ser Pro Glu Cys Lys         115 120 125 Ala Asn Glu Phe Arg Cys Ser Ser Gly Gln Cys Ile Pro Glu Asp Phe     130 135 140 Lys Cys Asp Gly Lys Ala Glu Cys Lys Asp Asn Ser Asp Glu Ile Arg 145 150 155 160 Ala Thr Cys Trp Asn Val Arg Cys Pro Gly Phe Thr His Lys Cys Lys                 165 170 175 Tyr Gly Ala Cys Val Ser Gly Asn Ala Glu Cys Asn Gly Ile Val Glu             180 185 190 Cys Phe Asp Gly Ser Asp Glu Asp Pro Ala Ile Cys Lys Thr Lys Pro         195 200 205 Thr Pro Arg Pro Thr Pro Thr Pro Gly Thr Pro Gly Pro Gln Pro Thr     210 215 220 Gln Gly Gly Cys Val Leu Pro Asn His Pro Glu Phe Gly Glu Trp Gln 225 230 235 240 Val Tyr Gly Ile Pro Gly Gln Phe Ser Pro Gly Met Val Ile Arg Ala                 245 250 255 Gly Ala Thr Leu Arg Ile Gln Cys Lys Lys Arg Tyr Lys Leu Glu Gly             260 265 270 Lys Asn Ala Ile Phe Cys Glu Asn Gly Lys Trp Ser Asp Ala Val Gly         275 280 285 His Cys Leu Lys Leu Cys Pro Ser Ile Gln Ser Thr Ser Thr Met Arg     290 295 300 Val Thr Cys Ile Tyr Asn Lys His Glu Glu Thr Glu Asn Cys Thr Glu 305 310 315 320 Ala Val Glu Gly Thr Leu Val Arg Phe Asp Cys Ala Pro Phe Tyr Glu                 325 330 335 Asp Leu Gly Leu Ser Arg His Pro Ile His Ile Cys Arg Asp Gly Ser             340 345 350 Trp Asp Gln Arg Arg Pro Glu Cys Thr Pro Val Cys Gly Gln Lys Ser         355 360 365 Val Asn Ala Gln Thr Leu Ile Val Asn Gly Lys Pro Val Lys Lys Gly     370 375 380 Asp Tyr Pro Trp Gln Val Ala Leu Tyr Thr Leu Asn Asp Lys Glu Leu 385 390 395 400 Ile Cys Gly Gly Ser Leu Leu Asn Gln Arg Val Val Leu Thr Ala Ala                 405 410 415 His Cys Ile Thr Asp Asp Lys Gly Lys Leu Leu Ser Lys Glu Asn Tyr             420 425 430 Met Val Ala Val Gly Lys Tyr Tyr Arg Pro Phe Asn Asp Ser Arg Asp         435 440 445 Arg Asn Glu Ala Gln Phe Ser Glu Val Lys His Met Phe Ile Pro Glu     450 455 460 Leu Tyr Lys Gly Ser Thr Gln Asn Tyr Val Gly Asp Ile Ala Ile Leu 465 470 475 480 Val Thr Arg Val Thr Phe Thr Leu Ser Arg Arg Val Gln Pro Val Cys                 485 490 495 Ile Asp Tyr Gly Leu Lys Tyr Thr Ser Tyr Thr Asn Glu Phe Gly Tyr             500 505 510 Val Thr Gly Trp Gly Tyr Thr Leu Gln Asn Asp Lys Pro Ser Asp Val         515 520 525 Leu Lys Glu Leu Lys Val Pro Ala Val Ser Thr Glu Gln Cys Ser Ser     530 535 540 Ala Ile Pro Glu Asp Tyr Asp Ile Tyr Leu Thr His Asp Lys Leu Cys 545 550 555 560 Ala Gly Tyr Leu Asp Asn Gly Thr Ser Val Cys Ser Gly Asp Ser Gly                 565 570 575 Gly Gly Leu Val Phe Lys Phe Asp Gly Arg Tyr Tyr Val Thr Gly Ile             580 585 590 Val Ser Leu Ser Pro Gln Ala Ser Thr Gly Gly Cys Asp Thr Gln Gln         595 600 605 Tyr Gly Leu Tyr Thr Lys Val Gly Thr Tyr Ile Ser Asp Phe Ile Ile     610 615 620 Lys Thr Glu Ser Gln Phe Arg Pro *** 625 630 <210> 6 <211> 633 <212> PRT <213> Tenebrio molitor <400> 6 Met Cys Asn Val Arg Thr Leu Leu Gln Val Ile Cys Leu Ser Leu Ile   1 5 10 15 Val Ile Gln Thr Val Asp Ser Tyr Ser Phe Ala Leu Ser Lys Phe Thr              20 25 30 Arg Ile Arg Arg Pro Ala Arg Arg Thr Cys Thr Ser Thr Glu Phe Ala          35 40 45 Cys Lys Ser Gly Glu Cys Ile Asp Glu Asp Lys Glu Cys Asp Gly Ile      50 55 60 Val Asp Cys Thr Asp Ala Ser Asp Glu Thr Asn Ala Cys His Arg Ile  65 70 75 80 Lys Cys Pro Asn Tyr Leu Phe Arg Cys Lys Tyr Gly Ala Cys Ile Asn                  85 90 95 Pro Asp Leu Glu Cys Asp Gly Lys Pro Asp Cys Met Asp Gly Ser Asp             100 105 110 Glu Lys Ala Ser Lys Cys Lys Pro Asp Asp Ser Ser Pro Glu Cys Lys         115 120 125 Ala Asn Glu Phe Arg Cys Ser Ser Gly Gln Cys Ile Pro Glu Asp Tyr     130 135 140 Lys Cys Asp Gly Lys Ala Glu Cys Lys Asp Asn Ser Asp Glu Ile Arg 145 150 155 160 Ala Thr Cys Trp Asn Val Arg Cys Pro Gly Phe Thr His Lys Cys Lys                 165 170 175 Tyr Gly Ala Cys Val Ser Gly Asn Ala Glu Cys Asn Gly Ile Val Glu             180 185 190 Cys Phe Asp Gly Ser Asp Glu Asp Pro Ala Ile Cys Lys Thr Glu Pro         195 200 205 Thr Pro Lys Pro Thr Pro Thr Pro Gly Thr Pro Gly Pro Gln Pro Thr     210 215 220 Gln Gly Gly Cys Val Leu Pro Asn His Pro Glu Phe Gly Glu Trp Gln 225 230 235 240 Val Tyr Gly Ile Pro Gly Gln Phe Ser Pro Gly Met Ala Ile Arg Ala                 245 250 255 Gly Ala Thr Leu Arg Ile Gln Cys Lys Lys Arg Tyr Lys Leu Glu Gly             260 265 270 Lys Asn Ala Ile Phe Cys Glu Asn Gly Lys Trp Ser Asp Ala Val Gly         275 280 285 His Cys Leu Lys Leu Cys Pro Ser Ile Gln Ser Thr Ser Thr Met Arg     290 295 300 Val Thr Cys Ile Tyr Asn Lys His Glu Glu Thr Glu Asn Cys Thr Glu 305 310 315 320 Ala Val Glu Gly Thr Leu Val Arg Phe Asp Cys Ala Pro Phe Tyr Glu                 325 330 335 Asp Leu Gly Leu Ser Arg His Pro Ile His Ile Cys Arg Asp Gly Ser             340 345 350 Trp Asp Gln Arg Arg Pro Glu Cys Thr Pro Val Cys Gly Gln Lys Ser         355 360 365 Val Asn Ala Gln Thr Leu Ile Val Asn Gly Lys Pro Val Lys Lys Gly     370 375 380 Asp Tyr Pro Trp Gln Val Ala Leu Tyr Thr Leu Asn Asp Lys Glu Leu 385 390 395 400 Ile Cys Gly Gly Ser Leu Leu Asn Gln Arg Val Val Leu Thr Ala Ala                 405 410 415 His Cys Ile Thr Asp Asp Lys Gly Lys Leu Leu Ser Lys Glu Asn Tyr             420 425 430 Met Val Ala Val Gly Lys Tyr Tyr Arg Pro Phe Asn Asp Ser Arg Asp         435 440 445 Arg Asn Glu Ala Gln Phe Ser Glu Val Lys His Met Phe Ile Pro Glu     450 455 460 Leu Tyr Lys Gly Ser Thr Gln Asn Tyr Val Gly Asp Ile Ala Ile Leu 465 470 475 480 Val Thr Arg Val Thr Phe Thr Leu Ser Arg Arg Val Gln Pro Val Cys                 485 490 495 Ile Asp Tyr Gly Leu Lys Tyr Thr Ser Tyr Thr Asn Glu Phe Gly Tyr             500 505 510 Val Thr Gly Trp Gly Tyr Thr Leu Gln Asn Asp Lys Pro Ser Asp Val         515 520 525 Leu Lys Glu Leu Lys Val Pro Ala Val Ser Thr Glu Gln Cys Ser Ser     530 535 540 Ala Ile Pro Glu Asp Tyr Asp Ile Tyr Leu Thr His Asp Lys Leu Cys 545 550 555 560 Ala Gly Tyr Leu Asp Asn Gly Thr Ser Val Cys Ser Gly Asp Ser Gly                 565 570 575 Gly Gly Leu Val Phe Lys Phe Asp Gly Arg Tyr Tyr Val Thr Gly Ile             580 585 590 Val Ser Leu Ser Pro Gln Ala Ser Thr Gly Gly Cys Asp Thr Gln Gln         595 600 605 Tyr Gly Leu Tyr Thr Lys Val Gly Thr Tyr Ile Ser Asp Phe Ile Ile     610 615 620 Lys Thr Glu Ser Gln Phe Arg Pro *** 625 630 <210> 7 <211> 374 <212> PRT <213> Tenebrio molitor <400> 7 Met Leu Asn Leu Asn Tyr Phe Thr Cys Phe Val Ile Val Leu Ile Gln   1 5 10 15 Leu Val Ser Ser Gln Arg Phe Val Gly Asp Leu Cys Thr Leu Glu Ser              20 25 30 Ser Gly Ala Pro Gly Val Cys Glu Leu Phe Lys Glu Cys Lys Gln Ala          35 40 45 Arg Asp Asp Leu Gln Lys His Gln Leu Phe Pro Gln Gln Cys Gly Tyr      50 55 60 Gln Lys Asn Glu Pro Ile Val Cys Cys Leu Lys Lys Ser Lys Arg Lys  65 70 75 80 Pro Gly Glu Ile Ser Leu Lys Lys Cys Gln Glu Tyr Ser Arg Leu Val                  85 90 95 Tyr Glu Val Asn Arg Ala Pro Val Leu Ile Ile Asn Ala Pro Asn Ile             100 105 110 Thr Lys Asn Glu Cys Gly His Lys Ile Ile Lys Leu Ile Val Gly Gly         115 120 125 Thr Asn Ala Thr Arg Lys Glu Phe Pro His Met Ala Val Ile Gly Phe     130 135 140 Glu Pro Gln Pro Gly Asp Ile Lys Trp Leu Cys Gly Gly Thr Val Leu 145 150 155 160 Ser Lys His Tyr Ile Leu Thr Ala Ala His Cys Leu Ser His Gln Glu                 165 170 175 His Gly Arg Ala Arg Tyr Val Arg Ile Gly Val Thr Asp Leu Glu Asp             180 185 190 Thr Asn His Arg Gln Gln Leu Glu Val Glu Glu Leu Ile Pro Tyr Pro         195 200 205 Glu Tyr Lys Ser Ser Ser His Tyr His Asp Ile Gly Leu Leu Arg Leu     210 215 220 Lys Arg Ser Ala Lys Leu Asp Ser Phe Thr Val Pro Ala Cys Leu Tyr 225 230 235 240 Arg Lys His Asp Ile Glu Ala Glu Lys Ala Ile Ala Thr Gly Trp Gly                 245 250 255 His Thr Thr Trp Gly Gly Ser Gly Ser Asn Asn Leu Leu Lys Val Thr             260 265 270 Leu Asp Leu Phe Asp His Ala Ser Cys Asn Arg Ser Tyr Lys Asn Gln         275 280 285 Ile Ser Arg Arg Leu Lys Asp Gly Ile Ile Asp Asp Ile Gln Val Cys     290 295 300 Ala Gly Ser Leu Asp Asp Glu Lys Asp Thr Cys Gln Gly Asp Ser Gly 305 310 315 320 Gly Pro Leu Gln Ile Phe His Glu Ser Lys Asp Ile Lys Cys Met Tyr                 325 330 335 Asp Ile Ile Gly Val Thr Ser Phe Gly Lys Ala Cys Ser Gly Ser Pro             340 345 350 Gly Val Tyr Val Arg Val Ser Gln Tyr Ile Gly Trp Ile Glu Asp Ile         355 360 365 Val Trp Pro Glu Asn Ser     370 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 8 gaygarttyc cntggatggc 20 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 9 ccarttngcc atnccrcang g 21 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 10 cgtcgcaacc tcaacagagc ta 22 <210> 11 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 11 gatgttgttg ggttcgtagc tctc 24 <210> 12 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 12 cgtgtcagtc tcggtgttgt actc 24 <210> 13 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 13 ctctgactac atcaaaccca tttgtc 26 <210> 14 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 14 gaagagctga gcaagtcgta cctc 24 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 15 atgttggtcc gctccttgtt 20 <210> 16 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 16 ctagggcttc agcttgccg 19  

Claims (9)

A protein derived from brown rice wine consisting of the amino acid sequence of SEQ ID NO: 1. A gene encoding a protein derived from brown rice wine consisting of the amino acid sequence of SEQ ID NO: 1. According to claim 2, wherein the gene consisting of the nucleotide sequence of SEQ ID NO: 2. Kit for the detection of bacterial infection in a sample comprising a protein derived from brown rice wine consisting of the amino acid sequence of SEQ ID NO: 1. 5. The kit of claim 4, wherein said sample is blood for transfusion, mammalian blood, food, tap water, groundwater, rainwater, or sterile product. The kit according to claim 4, wherein the sample is blood for transfusion or blood of a mammal. The kit of claim 4, wherein the kit is in the form of a solution, lyophilized powder, frozen solution, or strip. 8. The compound of claim 4, further comprising: Tenebrio PGRP-SA consisting of the amino acid sequence of SEQ ID 3; And Tenebrio GNBP1 consisting of the amino acid sequence of SEQ ID NO: 4, Tenebrio MSP-1 consisting of the amino acid sequence of SEQ ID NO: 5, Tenebrio MSP-2 consisting of the amino acid sequence of SEQ ID NO: 6, and Tenebrio Tm- consisting of the amino acid sequence of SEQ ID NO: 7 The kit further comprises at least one protein selected from the group consisting of 41. The kit of claim 8, further comprising β-lytic protease (blp), lysozyme, or blp and lysozyme.

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