TW200825098A - Anti-microbal peptides and uses thereof - Google Patents

Abstract

The present invention relates to an peptide fragment comprising an amino acid sequence of SEQ No:1 or SEQ NO:2, or the variant thereof. The anti-microbal use of the peptide fragment is also provided.

Description

200825098 IX. DESCRIPTION OF THE INVENTION: TECHNICAL FIELD OF THE INVENTION The present invention relates to peptide fragments of antimicrobial proteins and uses thereof. [Prior Art] Many antimicrobial proteins have been known to be isolated from vertebrates, and these antimicrobial proteins play an important role in resisting microbial infections, and are natural substances in which scorpion replaces antibiotics. Aquatic organisms need to rely on their own innate immune function to resist the pathogens because of the complex environment they encounter. Fish (which itself secretes a lot of mucus on the surface of the scales, these mucus have a bactericidal

Sex, such as pleurocidin, was first extracted from the surface mucus of the flounder and has the property of inhibiting some Gram-positive and Gram-negative bacteria. Pleurocidin was discovered in 1978 by Cole et al. (c〇le AM et al. J Biol Chem 272·12008-13, 1 997), an antimicrobial protein in a flounder, which is a 25-amino group. The peptide of the acid group is present in the mucus of the body surface and has activity against microorganisms. This protein with anti-microbes is also found in different organisms, such as salmon. The antibacterial protein found in squid has a living region, and the amino acid sequence is GWRTLLKAEVKTVGKLALKHYL, which is resistant to infection by Gram and fungi (patrzykat A. et ale

Antimicrob Agents Chemother 47: 2464-70, 2003). In recent years, Yin et al. found a cDNA sequence similar to the antibacterial peptide in the grouper, named epinecidin-l (Yin ZX· et al.

Aquaculture 253: 204-1 1,2006). However, it did not prove in the organization 5 200825098 its gene expression, or its effect on LPS or P〇ly (I): poly (C) stimulation, and no animal experiments to prove the effect of anti-microbial infection. SUMMARY OF THE INVENTION One aspect of the invention relates to a peptide fragment comprising a sequence of amino acid sequence SEQ ID NO: 1 or SEQ ID NO: 2 or a variant thereof having the same functional activity. Another aspect of the invention relates to a pharmaceutical composition comprising a peptide fragment of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a variant thereof having the same activity. In one embodiment, the present invention demonstrates a peptide fragment having one of the following amino acid sequences, which has activity against microbial infections (such as bacteria); GFIFHIIKGLFHAGKMIHGLV (SEQ ID NO: 1) GFIFHIIKGLFHAGKMIHGLVTRRRHGVEE (SEQ ID NO: 2) In one aspect, the invention relates to a pharmaceutical composition for microbial infection against aquatic animals comprising an isolated protein and a variant thereof having the same activity, wherein the isolated protein comprises an amino acid of SEQ ID NO: 1 or SEQ ID NO: sequence. In still another aspect, the present invention relates to a pharmaceutical composition for preventing or treating a mammalian infectious disease comprising an isolated protein and a variant having the same activity, wherein the isolated protein comprises SEQ ID NO: 1 or a SEQ ID NO: Aminoguanidine sequence of 2. The following terms used in the present invention and the terms used herein are defined as the following meanings unless otherwise defined. These explanations are for illustrative purposes only and are not intended to limit the scope of the invention. Of course, these explanations can be used to include any other aspects or examples described herein and claimed herein. The terminology used herein is as follows: 6 200825098 As used herein, the term "isolated" relates to a peptide or protein, meaning that the peptide or protein is not a natural product, but essentially consists of a peptide, protein, nucleic acid or other band. A peptide or protein that is removed or artificially synthesized from a macromolecule of nucleic acid, or a naturally occurring analog thereof. Although the term "separated" does not require a certain degree of purity, typically, the protein has a purity of at least about 75% and a purity of at least about 90%. • As used herein, a protein peptide “variant” refers to a peptide that is essentially homologous to • the original peptide, but has an amino acid sequence that is different from the present invention, <, due to one or more Caused by deletion, insertion or replacement. The substitution may include a sequence of retention substitutions, meaning that the amino acid group is known to be substituted by a group having similar physiochemical properties. Suitable retention of the amino acid in the peptide or protein is well known to those of ordinary skill in the art and can be accomplished generally without altering the biological activity of the resulting molecule. Those of ordinary skill in the art will generally recognize that a single amino acid substitution in a non-essential region of a peptide does not substantially alter biological or functional activity. Preferably, the steric structure of this variant must be identical to the original protein peptide. (: In the present context, the term "microorganism" refers to bacteria, viruses and fungi. In an embodiment of the invention, the isolated peptide fragments of the invention are resistant to bacteria, which may include, but are not limited to, Listeria (Z/sier/3, Micrococcus luteus, ^ ^ ^ ^ {Staphylococcus aureus), pyogenes, Staphylococcus agalactide 'Staphylococcus epidermidi s ), Staphylococcus 7 200825098 sp.), Staphylococcus aureus xylosus, Streptococcus pneumoniae (5Yrepiococc"s·p/7ewzff(9/7』'ae), Staphylococcus aureus (subsp), Intestinal gas rabbit iEnterobacter aerogenes), Enterobacter cloacae subsp., grouper, bristles (Vibrio a gino yticus), acid producing Klebsiella oxytoca, Sal ini vibrio cost i col a subsp·, Vibrio vulnificus (plant/△/·/〇ra7/7///cws〇, Harveyi's 'Vibrio alginolyticus', Pseudomonas aeruginosa, Yersin's sausage Bacilli (enterocolitica subsp). As used herein, the term "pharmaceutical composition" comprises an effective amount of a peptide of the invention or a variant having the same activity in the present invention, comprising a sequence of amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and a suitable pharmaceutical A carrier is acceptable. Such suitable pharmaceutically acceptable carriers include, but are not limited to, diluents, adjuvants, and/or carriers. Dilutions, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers may be added as needed. The composition may be in the form of a liquid or a powder or other desiccant dosage form, and may be added with a diluent comprising various buffer components, pH, ionic strength, additives for preventing surface absorption, and detergents (eg Tween 20, Tween 80). , cholate), solubilizers (eg glycerol), antioxidants (eg vitamin C, sodium bisulfite), preservatives (eg thimerosal, benzyl alcohol, benzoates), covalently attached polymers such as polyethylene glycol 'Metal ion complexes, or contained in microlipids, microemulsions, micelles, single 8 200825098 layer liposomes' multilamellar liposomes, or spheres. This composition can affect the rate of release of the physiological state 'solubility' in vivo, as well as the rate of in vivo clearance. The choice of composition can be determined based on the physicochemical properties of the anti-g egg (iv). The manner of administration of the pharmaceutical composition can be determined by those having ordinary knowledge in the field, depending on the physicochemical properties of the composition, including but not limited to, oral, intraperitoneal injection, subcutaneous injection, intravenous injection, intramuscular injection, nasal cavity, etc. . In the examples of the present invention, the pharmaceutical composition is injected intraperitoneally.

As used herein, the term "animal" includes all animal species, such as aquatic animals and mammals. In the - real off towel 'the aquatic secret domain class. Any of these is defined as mammalian animals, including humans. Preferably, the mammalian shaft is human. As used herein, "microbial infection of aquatic animals" means any pathogen infection that causes a decrease in the activity or death of aquatic animals, such as fine thief dyeing. Most of these major pathogens are common bacteria in the water environment, the most « which is _. In the implementation of the fiscal, (four) death rate as the basis for the results of the infection' and the bacteria causing the infection is Vibrio vulnificus. As used herein, "infectious disease" refers to an infection-related disease, which is caused directly by the infection of the source (such as fine-stained infection), or is caused by symptoms of contagion in the disease, such as low immune function. "All of them are within the scope of application of the present invention. In the embodiment, the disease is sepsis caused by Pseudomonas aeruginosa. The present invention relates to a peptide fragment and a pharmaceutical composition thereof comprising an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and an active variant thereof. According to one embodiment of the invention, the dipeptide fragment is designated as the grouper antibacterial protein ~3. The activity described herein is an activity against microbial infections, particularly bacteria. The amino group of SEQ ID NO: 1 or SEQ ID NO: 2 200825098 The acid sequence is derived from the cDNA cloned in the present invention. In one embodiment of the present invention, the primers of the antibacterial protein code known from other aquatic animals are used, and the cDNA of the grouper cDNA is used as a template to obtain the cDNA of the grouper antibacterial protein-3 by PCR amplification. The homology pattern of the program is analyzed for its minimum energy (Accelrys, SanDiego, CA, USA), and its structural model is obtained as the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, which is considered to be an active fragment, ie, the peptide of the present invention. Fragment. In the structural model of grouper antibacterial protein-3, grouper antibacterial protein-3 is predicted to be an α-helical structure of amphiphiles, similar to other antibacterial proteins, whose function is to form pores in microorganisms, resulting in imbalance of osmotic pressure and microbes. death. After analyzing the properties of the polypeptide, it was speculated that the grouper antibacterial protein-3 should be transported in the cytoplasm. This result suggests that the grouper antibacterial protein-3 should be delivered intracellularly to extracellular secretion to combat infection. The amino acid sequence of the grouper antibacterial protein_3 has a nucleolar positional message (NLS), which may be related to the transmission of innate immunity, because NLS is not only found in all antimicrobial proteins, but also in other molecules with regulatory functions. Appears (Schedlich LJ. et al. J Biol Chera 275: 23462-70, 2000). When considering the range of amino acid sequences of the present invention, all of the amino acid sequences of the protein or peptide comprising SEQ ID NO: 1 and SEQ ID NO: 2 in the examples of the present invention include amines within the scope of the present invention. In the base acid sequence, for example, the two-stage synthetic antibacterial protein-3 peptide (g_ple 5 - 34) and U-Pie 5-25) are mentioned in the present invention, and are within the scope of the present invention. Further included are all amino acid sequences incorporating the peptide of the retained amino acid substitution as described above in accordance with the present invention. Further included is the amino acid sequence of a larger protein which is incorporated into the present invention and which contains the SEQ ID NO: 2 or SEQ ID NO: 2 or Win 200825098. Including fusion proteins, as well as immature proteins that incorporate a message. In other ways, the peptide fragment is proved to have anti-microbial (1) RT PCR or tissue staining. The expression of the gene encoding this peptide fragment or the protein of the product of the peptide fragment and the production of the protein of the peptide fragment in different tissues is analyzed by the distribution of the tissue, and the gene or protein is mainly expressed in contact with foreign substances. Crying officer • Medium ^ grouper's head kidney, skin and small intestine. In addition, stimulation with pathogenic molecules • = ' & $ performance $ also rises' confirms the activity of the isolated peptide peptide C = ', has, ^. (4) kinetic analysis of the anti-white-3 peptide in the blood / month / ritual 33 good reaction rate, not _ in the blood vessel wall of aquatic animals and mammals, and ancient left #, /,虿ik耆 The characteristics of the blood circulation system are in line with the characteristics of drug development. This t-month peptide fragment has an activity against bacterial infection in aquatic animals, such as Vibrio vulnificus infection. In one embodiment, the antibacterial activity test is carried out by synthesizing and purifying two segments of the grouper antibacterial protein 3 peptide fragment, i.e., the fragment having the SEQ ID NO: 2 and the amino acid sequence of the amino acid sequence. The peptide fragment is effective against Gram-negative (sexual bacteria and stagnation _, which has a high antibacterial activity, and the peptide/fragment achieves the same antibacterial effect, which is more than the cost of expressing the full-length protein sequence. In another embodiment, 1) is injected into the grouper; ^(1): 1) 0:^((:) to observe the mRNA expression amount of the antibacterial protein-3. p〇iy(i): P〇iy(c) is an RNA-like polynucleotide ' consisting of two homopolymers; one side is polyriboinosinic acid and the other is polyribose cytosine Polyribocytidylic acid. The chain structure on each side is like the RNA that naturally exists in 200825098. The two single chains are linked by a hydrogen chain. The synthetic nucleic acid poly(I): poly(C) can Stimulating cells to synthesize proteins such as interferon, protecting other cells from infection by viruses. In the examples of the present invention, the expression of antibacterial protein-3 is triggered by poly(I):PQly(e), indicating that the present invention wins Peptide fragments also have antiviral activity. In another aspect, the invention A pharmaceutical composition relating to a microbial infective activity against aquatic animals (e.g., fish or shrimp) comprising an isolated protein and a variant having the same activity, wherein the isolated protein comprises SEQ ID NO: 1 or a sequence Number (amino acid sequence of 2). In one embodiment, the fish infection pattern used by the invention for infecting squid and grouper with Vibrio vulnificus is a method well known in the art, including only The control group for intraperitoneal injection of Vibrio vulnificus and the different experimental groups for intraperitoneal injection of Vibrio vulnificus and the peptide fragment of the present invention, the dosage of the peptide fragment of the present invention is per fish. i Ag. Finally, the mortality rate of the fish is judged. The peptide peptide of the present invention has a protective effect against bacterial infection, and the peptide fragment can indeed reduce the mortality of aquatic animals infected by microorganisms. On the other hand, the invention relates to an activity of preventing or treating Lt disease in mammals. a pharmaceutical composition comprising an isolated protein f and an oligo of the same activity, wherein the isolated protein comprises The amino acid sequence of column number i or SEQ ID NO: 2 according to the specific embodiment of the present invention, the animal model of intraperitoneal injection of the winning form of the present invention, and the different experiments of the test and the testicular peptides Finally, the protective effect of the peptide fragment of the present invention on bacterial infection is judged by the rate of rape. The protein peptide fragment can reduce the mortality when the mammal is invaded by the fine g, so it can be used as a preventive or therapeutic mammalian infection in 12 200825098. An effective drug for a sexually transmitted disease, such as a bacterial infection, is typically a case of sepsis. Other features and advantages of the present invention will be apparent from the following description or through the practice of the invention. It is to be understood that the invention is not intended to be limited by the invention. [Embodiment] The present invention is explained by the following examples. These examples are for illustrative purposes only and are not intended to limit the invention.实施 Example 1 strain and Peixiang strip + Escherichia coli XU-blue MRF, (Stratagene, USA) were cultured in LB medium at 37 °C. Gram-negative bacteria aerofMes) (BCRC 1 0370), Aier Macier subsp. (BCRC 10401), grouper algae Vibrio (from assistant professor of Chen Junyi, Huali University, Chili University), acid production Klebsiella oz/ioca ) (BCRC (; ., ι 1 3985), {Salini vibrio costicola subsp. (BCRC 12910) 5 — Vibrio vulnificus (YJ016); Assistant Professor Chen Junxi, Vibrio vulnificus Factory i·Red (204); from Assistant Professor Chen Junxi), Vibrio harveyi (plant 』·Ατ/ο (BCRC 1 381 2), Vibrio alginolyticus (running r/o) (BCRC 12907), algae-alkali Vibrio-like) (BCRC 1 2829), Pseudomonas aeruginosa (/^ own" and 卯77-like aer//like) (ATCC 1 9660), and Yersin's 13 200825098 Enterobacter subsp.) (BCRC 1 3999); and Gram-positive Listeria monocytogenes 677M) (BCRC 1 4930), Micrococcus luteus ([crococcw /"iews1) (BCRC 1 1 034), Staphylococcus aureus ayrei/s1) ( BCRC 1 0780), Streptococcus pyogenes (5YapA7/oC (9cc"s· ;?/ 叹己/7己5〇 (BCRC 1 0797), Staphylococcus aga/aci/如) (819; from Assistant Professor Chen Junxi) Staphylococcus epidermidis (BCRC 10783) - grape ball ζ') (iSYap/z/Zocc^cws sp.) (BCRC 1 0451), Staphylococcus aureus (•SYap/zy/ococcM) (BCRC 1 2930), Streptococcus pneumoniae (^repiococci/sp/7e"7Z7〇/2/ae) (BCRC 10794), and golden yellow grape g awrews subsp· ) (BCRC 10782) were all cultured at the Food Industry Research Institute (Hsinchu) , Taiwan) (BCRC) - or the US Cell Culture and Storage Center (ATCC, Manassas, VA, USA) - recommended in culture or temperature. Other strains from Assistant Professor Chen Junxi were cultured on tryptic soy broth or acacia at 37 ° C.

C Separation of the cDNA of the grouper antibacterial protein-3 - The cDNA library of the grouper is Xianghuaxing Biotechnology Co., Ltd.

North, Taiwan) to buy. The final volume of the PCR reaction was 50 μΐ. The reaction contained a 5 μΐ grouper phage cDNA library as a reaction template, 5 μΐ of lOx PCR buffer, 200 μΜ for each dNTPs, 1 pg of primer pair P1 and P2, and 2.5 units of Taq DNA polymerization. Enzyme (HT Biotechnology, Cambridge, UK). The reaction mixture was reacted at 94 ° C for one minute, at 55 ° C 14 200825098 for two minutes, and at 72 ° C for two minutes for a total of 35 cycles; the results of the PCR reaction were analyzed by 2% acacia. The primer sequence is as follows: P1: 5' -TCAAGCTTCGATGAGGTGCATCGCCCTCTTTC (SEQ ID NO: 3) and P2: 5' - CGGGATCCTCAGGCAAAAGCTTTCTCTCGTTC (SEQ ID NO: 4). The primer sequence is designed according to the known halibut p 1 euroc id iη cipher region (registration number·· ΑΥ273181 and ΑΥ273180),

Pseudopleuronectes americanus (number I, number: AF301515 and AF301512), US 鲧 (registration number: AY273177), American yellow cover scale (registration number · AY273175) ' and grouper brother coj oii / es1 (EST clone, registration number: BQ096584 ). After PCR amplification, the samples were extracted from the gelatin and incorporated into the vector PIRES2-EGFP (BD Bi0sciences, San Jose, CA, USA). Colonies were randomly selected and the double strands were sequenced using the primers of the vector. For peptides containing a reserving sequence, the pS〇rt software (available on the website V^Psort. ims. u-tokvo. ac. ip/f orm2. html) is used to analyze the intracellular transport of the peptide. Example 1: Separation of RM and collection of five grouper (body weight approximately 5 kg) tissue by RT-PCR quantitative expression, extraction of RNA by the following procedure (Chen jy. et al. DNA Cell Biol 17: 359-76, 1998) . In the tissue distribution experiment, sputum RNA was obtained in the same manner by collecting blood, sputum, heart, head kidney, small intestine, liver, muscle, and skin. Based on the mRM performance of the grouper antibacterial protein-3, the distribution of Linzhizhi was analyzed by the polymerase chain anti-hybrid-(10). In the case of lipid vinegar LPS stimulation (average weight of fish per fish) was 1 gram, and after stimulation with different amounts of lipids (50, 10, 5, and 丨 (4) per fish, the distribution of woven fabrics was analyzed. In experiments with different amounts of P〇ly(I):poly(C)-stimulation, the concentration was * 〇]]^ again..., u·1, i, 5, and 10 “g. In the injection of LPS or Poly(I): p〇ly(C) Total RNA was extracted after 360 minutes. - mRNA was transcribed into cD, and the expression of antibacterial protein and cold-- was analyzed by RT-PCR with cDNA. Taqman probe and primer were based on ABI. Step design (Appiied [Biosystems, Perkin-Elmer Coir, Boston, ΜΑ 02118, USA). All samples were subjected to two replicate experiments. The PCR reaction was performed on an Applied Biosystems system (7000 Sequence Debt System) with real-time PCR instrumentation and GeneAmp sequence detection. The test system software was carried out. The reaction contained total RNA of 5 # g different tissues. Mastermix reagent (Applied Biosystems, Perkin-Elmer) for relative quantitative RT-PCR was used for quantification. cDNA template was included in each reaction plate. Taqman probe, Mastermix reagent, and reaction buffer. Grouper antibacterial protein-3 The real-time PCR primer sequence is as follows: , forward primer catcgccctctttcttgtgttg (sequence number: 5) and . reverse primer ccctccccgggttcag (sequence number: 6); grouper beta-act in gene: positive primer ccagccttccttccttggt (sequence number: 7) and anti To the primer gcacttcatgatgctgttgtaggt (sequence number: 8); grouper Mx gene: forward primer tggtcaaggagcagatcaaacag (sequence number: 9) and 16 200825098 reverse primer aacgccttcctaacagtatctccta (sequence number: 10). The total reaction volume is 25 //1, the reaction is The 96-well plate was carried out and reacted at 96 ° C for 15 seconds and at 60 ° C for 60 seconds for 40 cycles. The decomposition reaction was carried out at a temperature of 65 ° C to analyze the decomposition temperature of the PCR product. ± standard deviation indicates the results of n experiments, η represents the number of experiments for each RNA source. The difference between groups * is analyzed by SAS statistical software, Ρ (*) < 0.05 or ρ (forest) < 0.01 is significantly different • Using relative real-time RT-PCR, the amount of antibacterial protein-3 mRNA in different f tissues can be determined, and the results are shown in Figure 1A. Each data was normalized to the amount of stone in mRNA expression. As shown in Fig. ία, the antibacterial protein-3 exhibited the most amount in the first month, the small intestine, and the skin, and this result showed a local effect of the antibacterial protein-3 gene. Figure 1 shows the amount of antibacterial protein-3 mRNA in different tissues after LPS stimulation. Six hours after stimulation with LPS, antibacterial protein-3 in the head kidney and Mx mRNA in the skin were first detected. To understand the regulation of LPS and poly I: polyC on the performance of grouper antibacterial protein _3, different doses of LPS or p〇ly I: poly c were injected into grouper, and then the grouper antibacterial protein was analyzed by relative quantitative RT-PCR. -3 performance - set. As shown in Fig. 2A, the performance of 'bacteria protein_3' increased by four times in the steady state compared with the control (untreated) fish. In Fig. 2B, the expression of anti-g protein-3 was stable under the action of 5 "g/mi p〇iy(i)(10)y(C) compared to the control (untreated) fish: Plus /, a hundred times. The results showed that the gene expression of the grouper antibacterial protein-3 was increased under the treatment of LPS and poly(I): p〇ly(c). 17 200825098 These results support the hypothesis that grouper antibacterial eggs fight microbes and are delivered to the skin after performance. In the present invention, it has also been found that the expression of the zebra sound and the desired angle iPQ β anti-prion protein-3 is also 盥 LPS and P〇ly(I): poly(c)-stimulated sputum, 曰 主 主 β, Nine Du λα v ratio. Double-stranded A (dsRNA) is a sub-mode when it is sensible to ice, because ▲ Μ A 夕 virus replication, will produce double-stranded RNA. In this paper, it was also found that % x spotted antibacterial protein-3 can recognize scorpion RNA and further activate the MX gene after gene activation. This is not the grouper antibacterial protein-3 gene

/ Again, the host is also infected with the virus. The result is that the main 一 一 颍 颍 颍 颍 颍 颍 可 可 可 可 可 可 可 可 可 可 可 可 可 可A peptide fragment of the invention synthesized according to the deduced amino acid sequence of two different jade grouper antibacterial protein-3: grouper antibacterial protein-3 (g-ple 5-34), sequence GFIFHIIKGLFHAGKMIHGLVTRRRHGVEE (sequence number: 1) And the grouper antibacterial protein-3 (g-ple 5-25) sequence is GFIFHIIKGLFHAGKMIHGLV (sequence number: 2). Example 2: Analysis of the distribution of the grouper antibacterial protein _3 in the grouper. The multi-investment antibody was entrusted to create a biotechnology company (△ North Taiwan). Briefly, in order to allow the antibacterial protein-3 (g-pie 5-34) peptide fragment synthesized according to the present invention to bind to a carrier protein, each of the two rabbits was injected subcutaneously with 1 mg of peptide and complete Compile Freund (s adjuvant) 1 mixed dose. Next, a mixture of 18 200825098 peptide and complete Freund's adjuvant was injected for five times per p + diced four days. One week after the fifth injection, rabbit serum antibodies were collected and purified by peptide affinity method. The activity of the antibody against the peptide according to the present invention was tested by Western blotting. Silk A immunostaining method In the tissue immunostaining experiment, fish gills, small intestine, heart and kidneys were obtained, and immediately fixed in 4% buffered neutral formalin for 240 minutes, dehydrated and embedded in paraffin. The grouper tissue was continuously sliced horizontally (5#..., dewaxed and rehydrated in a benzene). After rehydration, citrate f) buffer (pH 6.0) was at 95. (: The pellet was pretreated for 10 minutes and allowed to stand in methanol containing 3% hydrogen peroxide for 3 minutes at ~HZL to block endogenous bismuth peroxide activity. Streptodin The blocking solution and the biotin blocking solution were allowed to stand for 15 minutes, respectively, and then washed with an infiltration buffer. The solution was blocked with 5% BSA for 5 minutes, and then washed with an infiltration buffer for 5 minutes. 5 diluted multiple antibodies or pre-bleeded serum antibodies, and the sections were allowed to stand at 25 ° C for 60 minutes, and then washed. The secondary antibody (biotin-resistant anti-rabbit I gG) was added to the sample and allowed to stand 3 〇 min, " After washing with infiltration buffer. Add streptavidin HRP to the slide for 30 minutes - then rinse with infiltration buffer. Add staining reagent (DAB) 1 minute later, develop color and wash again. Hematoxy 1 in was added for 10 minutes as a contrast stain' and the slide was washed with a Scot ter's solution to enhance the color. The slices were dehydrated in continuous alcohol and immersed in xylene for cleaning. Slide fixative drops on the slide Cover the coverslip. Take a photomicrograph of a Nikon (Tokyo, Japan) E 600 microscope with a CCD camera (DXM-12, Nikon). 0 19 200825098 The result is to determine the performance of the grouper antibacterial protein-3 in the tissue. The liquid staining method was used to measure the amount of expression in different organs. It was found that the grouper antibacterial protein-3 had a strong immune response in the serum of the epidermal cells of the silkworm and the epithelial cells of the small intestine (Fig. 3). _3 There is a grouper small intestine and • sputum. Compared with the antiserum experiment before blood collection, there is no staining reaction in the epidermal cells of the sputum and the small intestine. No affinity of the heart or the head kidney for purifying serum multiple antibodies or blood sampling beforehand. The serum antibody reacts to the immune f of antibacterial protein-3. In grouper, antibacterial protein-3 may play an important role in the digestive tract and gas exchange organs to resist the invasion of foreign microorganisms. Example 3: Antibacterial activity analysis Synthesis of raw J fish pleurocidin peptide and two-stage grouper antibacterial protein-3 ^1" '-- The peptides by Genesis Bio The company uses Fmoc chemical solid state synthesis to extract the crude peptide, which is then dried by reverse high performance liquid chromatography (RP-HPLC). The molecular weight and purity of the purified peptide are analyzed by mass spectrometry and HPLC, respectively. The synthesized peptide was placed in a phosphate buffer (pH 7.4) (alternate. The synthetic flounder pleurocidin peptide sequence was 'GWGSFFKKAAHVGKHVGKAALTHYL; and the grouper antibacterial protein-3 (g-ple 5-34 according to the present invention) The sequence is GFIFHIIKGLFHAGKMIHGLVTRRRHGVEE (SEQ ID NO: 1); and the grouper antibacterial protein-3 (g-pie 5-25) sequence is GFIFHIIKGLFHAGKMIHGLV (SEQ ID NO: 2). In vitro bacteriostatic analysis 20 200825098 The minimum inhibitory concentration (MIC) of the various microorganisms listed above was measured. The MICs of this peptide were determined by dilution of microbial meat emulsion analysis, modified from the method of PatrZykat et al. (Patrzykat A. et al. Antimicrob Agents Chemother 46: 605-14, 2002). Briefly, the peptides were serially diluted in a 96-well plate (NuncIon Surf ace; Nunc, Roski lde, Denmark) to be 100, 50, 25, 12.5, 6.25, and 3.125 /zg/ml. For each of the cultures, 100 #1 test bacteria (1 X 1〇5 CFU/well), add an equal volume of peptide and mix them and place them at 37. C is 960 minutes. Visually confirm the microbial sedimentation and the absorbance reading at a wavelength of 00 nm to determine the MICs. Results Bactericidal activity is a very important property when identifying antibacterial protein peptides. To determine whether the peptide fragment of the present invention has antibacterial activity, the peptide fragment of the present invention is compared with the synthetic flounder pieur0Cidin peptide according to a known method (Patrzykat A. et al. Antimicfob Agents Chemother 47: 2464-70, 2003). The antibacterial activity of the peptide fragment of the present invention against twelve Gram-negative bacteria: Enterobacter aeruginosa (昃, Enterobacter cloacae (炙c/o learned cae subsp.), Grouper algae Vibrio (plant, calving)夂克读伯氏fel (Ι·αγ/ίοα, 61· subsp·), Vibrio vulnificus (factory F"//7///ci7S), Vibrio harveyi (factory, algae ΐησ· alginolyticus ), Green Win 椁n (p· aeruginosa), to Anti-Yellow's Enteritidis (F·e#eroc〇7/"casubsp·); and against ten Gram-positive bacteria-Listeria (ζ) · _(10) sigh (10) π), M. luteus (#· Staphylococcus aureus (& Can), purulent chain 21 200825098 cocci (51·P7 sigh e/jes), Staphylococcus (S· agalactide), table Staphylococcus aureus (51·, Staphylococcus (SiapAy/ococci/s sp.), Staphylococcus aureus (51·Y/7 still "5〇, Streptococcus pneumoniae (51·/?/7己"卯77 /5 (?), and Staphylococcus aureus (yiwrez/s* subsp.), all tested by MIC test. Table 1 shows the results of the MIC test of the peptide fragments of the present invention and other known antibacterial proteins. Antibacterial In white, the grouper antibacterial protein-3 (g-ple 5-34) of the present invention is against Listeria (Z. (BCRC 14930), Staphylococcus (fork aga/aci/ru) (819), Staphylococcus epidermidis (51) · epidermidis, 昃aerogenes, plaque, algae lysate (V· alginoiyticus), Klebsiella A (Γ· ayybca, and traumatic bacterium (factory) (YJO 16) The group has the highest antibacterial activity. The grouper antibacterial protein-3 (g-ple 5-25) of the present invention also exhibits an antibacterial activity close to the soleus pieurocidin peptide when it is resistant to certain bacteria, and the cost required is low. 1 Microbial g-ple 5-34 MIC g-ple 5.25 MIC wf-ple MIC Gram-positive bacteria pg/ml pg/ml pg/ml Listeria monocytogenes fBCRC 14930) 25 50 50 Micrococcus luteus fBCRCl 1034) 3.125 25 3.125 Staphylococcus aureus fBCRC 10780) 6.25 12.5 6.25 Streptococcus pyogenes (BCRC 10797) 25 >12.5 25 Streptococcus agalactiae (819) 12.5 50 25 Staphylococcus epidermidis (^CRC10783) 12.5 NA NA Staphylococcus sp. (BCRC 10451) 6.25 50 6.25 Staphylococcus xylosus fBCRC 12930) NA 50 50 Streptococcus pneumoniae (BCRC 10794) 12.5 25 6.25 Staphylococcus aureus subsp fBCRC 10782) 6.25 6.25 6.25 g-ple 5-34 · Grouper antibacterial protein 5-34 (The present invention has a sequence number··1 winning fragment 22 200825098 g-ple 5-25 : Grouper antibacterial protein 5-25 (The present invention has a sequence number · 1 wf-ple : flounder pleurocidin peptide wins fragment) NA: non-activated microorganism g'P^j-^MTP pg /ml Gram-negative bacteria - ΙΐΕΐ^5^5 MIC Hg/ml J5i£leMlC Hg/ml

Enterobacter aerogenes (BCRC10370) 25 Enterobacter cloacae subsp fBCRC 10401) 100 Grouper vibrio alginolyticus 3.125 Klebsiella oxytoca fBCRC 13985) 12.5 Salinivibrio costicola subsp (BCRC 12910) 3.125 Vibrio vulnificus (YJ016) 6.25 illusion torve〆 (OCRC13 812) 3.125 Vibrio harveyi fBCRC 12907) 25 Vibrio vulnificus (204) 6.25 Vibrio alginolyticus fBCRC 12829) 6.25 Pseudomonas aeruginosa fATCC 19660) NA Yersinia enterocolitica subsp (BCRC 13999) l〇〇50 100 50 12.5 >10°100 6.25 12.5 50 12.5 12.5 12.5 12.5 50 3.125 50 3.125 6.25 6.25 g-ple 5-34 · Grouper antibacterial protein 5.34 (Inventive order g-ple 5·25: Grouper antibacterial protein 5_25 (Inventive order wf-ple: flounder pleurocidin peptide NA: not activated

In the results of the present invention, the bactericidal activity of the two gram-negative bacteria and the Gram-positive bacteria of the present invention, the peptide fragments of different lengths have the same bactericidal activity and are broadly bactericidal, as shown in Table 1, The invention has won the peptide fragment (the grouper antibacterial protein-3 peptide and g„ple 5-25) in the in vitro test towel with high activity and obvious potential effect. _In vivo experiment,,,,,,,,,,,,,,,, Injection of the peptide fragment of the present invention can produce the effect of protecting the infected fish. The treatment of the fish in the early stage can also improve the survival rate of the fish. The internal bacteria inhibition analysis 23 200825098 - the old gas in Balb / c (about 19 to 21 grams In the peritoneal cavity, 128 χ 〇 咖 咖 咖 咖 绿 , , , , , , , , 128 128 128 〇 〇 128 128 绿 绿 绿 绿 绿 绿 绿 绿 绿 绿 绿 绿 绿 绿 绿 绿 绿 绿 绿 绿 绿 绿 绿 绿 绿 绿 绿 绿 绿2 hours, 4] 卞 6], 衿, sacrifice the mouse to take a 1 cm small intestine, soak in 1 〇〇 in 1 pBs grinding ' and then swivel and spread on the petri dish, put the 37t incubator, (four) minutes later Count the number of colonies. The results are shown in Figure 4. The experiment can verify the ability of the peptide fragment of the present invention to inhibit bacteria in the intestine. After 240 minutes of intraperitoneal injection of 25 //g of the peptide fragment of the present invention, it is similar to the injection of 1 〇〇 古 素 。. Non-grouper antibacterial egg from _3 can be made as a natural substance to replace antibiotics. Example 4: The peptide peptide of the present invention protects the squid and grouper. The experimental model of the fish uses a Vibrio vulnificus (204; from the dead The lethal mode of culture in the squid of the squid to test the synthetic grouper antibacterial protein-3 (tple 5-25) version 1% 亀吴郭条八 Oreochron]is m〇ssambicus) anti-stone micro rabbit gossip · C (1) (1) The efficacy of the infection. The first test in each group requires 50 Wu Guoyu (average weight 〇 · 2 g, length 3 cm): (a) The first group only injected Vibrio vulnificus (Γ· ; (b) in the first In the two groups, a mixture of Vibrio vulnificus and grouper antibacterial protein-3 (g-ple 5-25) peptide was injected simultaneously; (c) The third group was pretreated with Vibrio vulnificus for 18 minutes, and the grouper antibacterial protein was injected. -3 (g-ple 5-25) peptide; (d) Group 4 with grouper Phytoprotein 24 200825098 -3 (g-pie 5-25) After 180 minutes of peptide pretreatment, Vibrio vulnificus was injected. (The fifth group was injected with PBS only; and (1) the sixth group was only injected with grouper antibacterial protein 1 (g- Ple 5-25) peptide. The second test, each group contains 3 grouper (flat F"/7?i/iC仏s; (b) in the second group, simultaneous injection of Vibrio vulnificus and grouper antibacterial a mixture of protein-3 (g-ple 5-25) peptides; (c) a third group of 60-minute pretreatment with Vibrio vulnificus, injection of grouper antibacterial protein_3 (g-pk, 5-25) peptide; (d) The fourth group was treated with grouper antibacterial protein-3 (60 days after pretreatment with gpie peptide, injected with Vibrio vulnificus; (e) the fifth group was injected with only PBS; and (f) the sixth group was only injected with grouper antibacterial protein. —3 (g—the peptide. All experiments were performed intraperitoneally from the anal fistula, and the dose of the grouper antibacterial protein-3 (§116 5-25) peptide was 1/^ per fish, in 1〇#1, or PBS alone. Vibrio vulnificus (204) was inoculated with 1 〇#丨, i X 103 cfu per fish. The fish are maintained in a 5-L water tank at a temperature of approximately 27 1 . The mortality rate is recorded every time, and the commercial feed of fish is fed by hand twice a day. The mortality rate chart is not expressed as a percentage (%), and the gap between groups is analyzed and tested by Fisher's exact test (Fisher, s exact test). Infectious effect Vibrio vulgaris (Bu Hong/〇 (204) is an assistant professor Chen Junyi (Tzu Chi University, Hualien, Taiwan). It is isolated from the dead Wu Guoyu. This Wu Guoyu is grown in a fish pond in southern Taiwan. The outbreak of the squid (204) of the squid and grouper has caused a large number of deaths and serious economic losses in Taiwan. The antibacterial activity of the grouper antibacterial protein-3 peptide has been described in the above description and has been shown to be resistant to fish sources in vitro. Vibrio vulnificus (2〇4) superior antimicrobial activity 25 200825098 sex. Therefore, the present invention selects the species of the grouper antibacterial protein to test its protective effect in the squid and grouper. In the first test, the fifth group And the sixth group was injected with the antibacterial protein-3 (g-ple5-25) peptide in the squid or grouper in a single-dose (each fish 〇·_, dissolved in 10W' or PBS alone). In the 168-hour test, the fish died (Fig. 5A, 5B), indicating that the grouper antibacterial protein 3 (gple525) f peptide was not toxic to fish, and in the PBS group, although the fish used was very small. : But not because of injection technology The phenomenon of death. However, after 168 hours, the cumulative mortality rate of Wu Guoyu, which was only injected with Vibrio vulnificus, reached 96%, and the wound was subjected to wound arc g and grouper anti-g egg (1) (g-ple5_25): peptide mixture. The cumulative mortality rate of the injected group was only 30%. The group receiving the peptide and the fine combination was significantly lower than the group receiving only the bacterial injection. In addition, before the injection of Vibrio vulnificus (204) First, the grouper was injected with the grouper antibacterial protein_3 (g pie 5-25) peptide for 180 minutes, and the cumulative mortality rate was 2% ', and the grouper antibacterial protein _3 5 (5) was injected. Before the 'first injection of wounds arc g (2G4) 180 minutes of Wu Guoyu, the cumulative death was 78% (Figure 5A). 'In the experimental group of grouper, after 168 hours, only injection __ (2 () 4) The grouper has a survival rate of 〇%, while the grouper has a cumulative mortality rate of 3 (10) injecting a mixture of Vibrio vulnificus (204) and grouper antibacterial egg/(g pie 5 25) peptides. Significantly lower mortality than group 2 receiving only bacterial injection. In addition, in the case of injection of Vibrio vulnificus (2〇4), Grouper antibacterial protein-3 (g_ple 5-25) peptide injection 6 min group 'Malgroup fish cumulative mortality is m. However, in grouper antibacterial 26 200825098 protein-3 (g-ple 5-25) peptide injection Before, the cumulative mortality rate was 97% (Fig. 5B) by injecting Vibrio vulnificus (2〇4) for 60 minutes (Fig. 5B). Example 5: Protective effect against Pseudomonas aeruginosa mice Green should be infected with Animal model experiments were divided into seven groups of 20 Balb/c mice (about 20 to 22 grams each). Control group: Each mouse was scored 1.28 X 1〇6 cfu green web rods <3θ/τ/^7/7ίλ5<3). The number of deaths began to be recorded after 12 hours. 10: Each mouse was intraperitoneally injected with 10 peptide fragments of the present invention, and after 540 minutes, 1·28 X 106 Cfu Pseudomonas aeruginosa was injected. The number of deaths began to be recorded after 12 hours. 20: Each mouse was intraperitoneally injected with 20 //g of the peptide fragment of the present invention, and after 540 knives and 4 ft., 1.28 X 1 〇 6 cfu Pseudomonas aeruginosa was inserted. The number of deaths began to be recorded after 12 hours. 25: Each mouse was intraperitoneally injected with 25 //g of the peptide fragment of the present invention, and after 54 minutes, 1,28 X 1〇6 cfu of Pseudomonas aeruginosa was inserted. The number of deaths began to be recorded after 2 hours. : Each mouse was intraperitoneally injected with 50//g of the peptide fragment of the present invention, and after 540 minutes, i.28 x 106 cfu Pseudomonas aeruginosa was infiltrated. The number of deaths began to be recorded after 12 hours. 1〇〇 Each mouse was intraperitoneally injected with 1 〇〇"g of the peptide fragment of the present invention, and after 540 minutes, 1·28 X 1〇6 Cfu Pseudomonas aeruginosa was inserted. The number of deaths began to be recorded after η hours. 2 (10): Each mouse was intraperitoneally injected with 2 〇〇 of the peptide fragment of the present invention, and 54 minutes later, ΐ 28 χ l〇6 cfu Pseudomonas aeruginosa was infiltrated. The number of deaths began to be recorded after 12 hours. The results are shown in Fig. 6. This experiment verifies that the grouper of the present invention can be used as a preventive drug, and the control mice can reduce the mortality when the bacteria invade, but must be at an appropriate concentration, too much 27 200825098 or too little. appropriate. The optimal dose was 25/g per mouse, and the 50/zg/m 〇use efficiency was second. The control mice did not inject the peptide fragments of the present invention, and the mortality rate reached the highest within 24 hours. Example 6: Pharmacokinetic analysis of the grouper antibacterial protein-3 (g-pie 5-25) peptide in accordance with the A-kinetic method of sorghum in male squid (body weight about 73.7 soil 1 6 2 g) Pharmacokinetic analysis of the antibacterial protein-3 (g-ple 5-25) peptide, first in the Wu Guoyu body's intravenous injection of a single dose (25/g per fish) of the grouper antibacterial protein-3 -10565-25) peptide. The serum antibacterial protein-3 (g-ple 5_25) peptide concentration was determined after 2, 3, 6, 12, and 18 minutes of injection, and the value of the positive branch Wu fish was measured at each time point. . At each time point of sample collection, Wu Guoyu was inoculated and blood was collected, and blood samples were centrifuged to obtain serum. The concentration of antibacterial protein-3 (g-pie 5-25) peptide in serum was determined by LC-MS/MS. Acidified water 950 // 1 containing 〇·1% TFA and plasma sample 50 # } were added to the polyacrylic acid official and mixed for a leap second with a mixer. Solid phase extraction column sm cartridges (Oasis HLB 1 ml/3 〇mg (Waters, Milford, MA)), pretreated with 2 ml of methanol, adjusted to 2 ml water and 2 ml acidified water (containing 0.1% TFA); The sample was then injected into the spE column and the column was washed with 2...acidified water (containing 0.1% TFA) and 2 ml of water. A 75 χ 12 〇 —_ polypropylene tube was placed under the SPE column, and the sample was punched out with 〇 5 ml and evaporated to dryness under a 3 〇 2 C nitrogen flow. The dried sample was then dissolved in 100 // 18 G% ACN liquid and mixed in a mixer for 6 G seconds, and the sample solution was placed in a screw bottle for analysis by Lc-MS/MS. The LC —ms/ms instrument contains a sample cooler (temperature set to 5 〇 28 200825098)

Alliance 2795 HPLC. The mass spectrometer is operated in forward ESI mode

Quattro Ultima (Waters/Micromass, Manchester, UK). All instruments are controlled by MassLynx 4. 0 (Waters/Micromass) software. The liquid chromatographic column is a Luna amino column with a particle size of 5 μm and an internal diameter of only 150 X 2.0-mm (Phenomenex, Torrance, CA). Using a mixture of ACN and water with a ratio of 80/20 v/v and containing 1% ammonia water as the mobile phase, the sample was punched out and the volume was 1 μμμ. The injecti〇nv〇lume was 10 μΐ·the mass spectrum The instrument operates in the forward ESI mode with the following conditions: • The capillary voltage is 3.5 kV, the cone voltage is 35 V, and the heat source temperature is 80. (: The desolvation gas flow temperature is 320. (:, the desolventizer flow rate (N2) is 55 liters per hour, the collision energy is $60 eV, and the collision gas flow is argon. The data is used in SRM mode. z 779 > 120 conversion obtained. The bifurcation is a single injection of a single dose of grouper antibacterial protein-3 (g-ple 5-2 synthetic peptide to Wu Guoyu, the grouper antibacterial protein of the present invention) The -3 (me 5 -25) peptide showed a decreasing pharmacokinetic response curve (Figure μ). The average maximum concentration of the main shot in the vein was 1342.13 ng/ml after 20 minutes. The half-life after injection was about 60. -80 minutes. 彳 彳 彳 体重 体重 体重 体重 体重 体重 体重 体重 体重 体重 体重 体重 体重 体重 体重 体重 体重 体重 体重 体重 体重 体重 体重 体重 体重 体重 体重 体重 体重 体重 体重 体重 体重 体重 体重 体重 体重 体重 体重 体重 体重 体重 体重 体重 体重 体重 体重 体重 体重(sc) (Fig. 3 = group of the same site, each injection of 1000 Μ of the grouper antibacterial protein of the invention ("le 5-25) peptide, 1 G minutes after injection, 2 () minutes, 3 ( ) minutes, i hours, 29 200825098 inches, each taking 1 ml of blood, and then using LC_MS/MS to test the serum community as shown in Figure 7, Γ, 7Γίπ-丄 also & This experiment verifies that the grouper antibacterial protein ~3 5 25) peptide of the present invention does not stick to the blood vessel wall of the mouse. It is in line with the characteristics of the development of mammals. Unless otherwise defined, all the techniques in this paper The meaning of the terminology of scientific terminology is generally understood by those who have the usual knowledge in the field of the month. Those who have knowledge of the field in this field are also acquainted with any similar or equal methods and materials described in this article; For the purpose of implementing or testing the present invention. [Simple Description of the Drawings] The following description of the invention will be better understood in the following description: Bar graph, noisy vs. sex just analyzed _ (10) after reading the stimulus

The result of liver flank, sputum, and protein 3 gene expression. The organizations in Figure 1A are represented by letters, L

K is the head kidney, G is total, "muscle, 1 is the small intestine skin, B (2Β^2^ bar shows the relative RT-PCR analysis of LPS (2A) and P〇ly I: poly C (10) stimulation, The effect on the expression of the anti-protein gene of the grouper. The second photo of the immunization wood color picture shows the name of the immunized wood color in the grouper and the small intestine. (3a) Right big grouper legs, for the pair ~, However, in the serum antibody of the blood sample collected in advance, the right side of the stone (10) is static: the grouper that is placed in the multiple antibodies is the winner. It is the grouper in the serum antibody of the day. The small intestine, VN...,,, and 'and the arrow in the left rabbit indicate the grouper's small intestine in the blood type of the antibody. The photograph is a typical stained area. a is 腮; 5 is the small intestine. 30 200825098 4 is a graph ' shows that after the Balb/C mouse was infected with Pseudomonas aeruginosa, the peptide fragment of the present invention has the ability to inhibit bacteria in the intestine similarly to the one-dimensional vancomycin. van : 1 intraperitoneal injection Vg vancomycin mouse; ple: a mouse injected intraperitoneally with the peptide fragment of the invention. Figure 5 is a graph, Show grouper antibacterial protein-3 (g ple 5 25) peptide to protect Wu 'box (5A) or grouper (5B) to avoid the ability to die from traumatic arc money. In this experiment there are - six groups: (a) first The group was injected only with the Vibrio vulnificus plant; (b) In the first group, a mixture of Vibrio vulnificus and grouper antibacterial protein-3 (g-ple 5-25) peptide was injected simultaneously; (c) the third group was wounded After 18 minutes of Vibrio pretreatment, the grouper antibacterial protein-3 (g-ple 5_25) peptide was injected; (the group was pretreated with grouper antibacterial protein-3 (g-pie 5-25) peptide for 180 minutes). After the injection of Vibrio vulnificus; (e) the fifth group only injected PBS; and (f) the sixth group only injected grouper antibacterial protein-3 (g-pie 5-25) wins. In the third and fourth groups t, Wu Guoyu and grouper have different conditions, the infection time of Wu Guoyu is 18〇 minutes' and the grouper is 6〇 minutes. The cumulative mortality rate is determined after 68 hours. (Ple squid for each fish A dose of 1 x 1 〇3 cfu was intraperitoneally injected into the traumatic arc sputum, and after three hours, an intraperitoneal dose of each fish l l//g antibacterial protein-3 (g-ple 5-25) was received; ple — 204: Wu Guoyu receives an intraperitoneal dose of iM antibacterial f-33 (g-Ple5-25) for each fish, three hours I, and receives an intraperitoneal dose of each f 1x10 cfu V. vulnificus; 2〇4 + Pie: Wu Guoyu received the peritrophic dose of each fishfish on the same day. (6) Antibacterial protein-3 (g-pie 5-25) and lxl〇3cfu The intraperitoneal dose of Vibrio vulnificus '· 204: 1 χ1〇 per Wuguo fish, Intraperitoneal dose of Vibrio vulnificus (m); ple •• Each Wu Xiang" received an intraperitoneal dose of anti-g protein-3 (g-ple 5-25). 31

200825098 Figure 6 is a graph showing the plot of the peptide of the present invention injected intraperitoneally with different doses in mice for 540 minutes, followed by intraperitoneal injection of ··· 28 χ 1〇6 cfu of Pseudomonas aeruginosa, and observed each group Mortality. Control group: Each mouse was given 丨28 X 1〇6 cfu Pseudomonas aeruginosa/^ from /〇聊侃5 from /7^/77053. The number of deaths was recorded after 12 hours. 10: Each mouse was intraperitoneally injected with 10 peptide fragments of the present invention, and after 540 minutes, 1.28 X 1G6 cfu green pus i was entered. 2G: Each mouse was intraperitoneally injected with 2Q of the peptide fragment of the present invention, and after 540 minutes, L28 x 1Q6 cfu Pseudomonas aeruginosa was infiltrated. 25: Each mouse was intraperitoneally injected with 25 "g of the peptide fragment of the present invention, and after 54 () minutes, n 1〇6 coffee Pseudomonas aeruginosa was hit. 50: Each mouse was intraperitoneally injected with 5 Mg of the winning fragment of the present invention, and after 54 minutes, 1,28 X H) 6 cfu of Pseudomonas aeruginosa was ingested. Each mouse was intraperitoneally injected with (10) Μ of the peptide fragment of the present invention, and 54 minutes later, it was injected into h 28 χ#. Touch: Each mouse was injected with ία X 106 cfu Pseudomonas aeruginosa just after the intraperitoneal injection of the peptide fragment of the present invention. Figure 7 is a graph and a bar graph, and Figure 7A shows a drug-force graph of the grouper antibacterial protein _3 peptide injected with a single mJ = (25 // g per fish) in Wu Guoyu. Each time point contains the value of two Wu Guoyu. Serum concentrations are determined at each specified time point after intravenous administration. Figures 7B to 7D are graphs showing the pharmacokinetic profiles of single-dose (each of the centromere grouper antibacterial protein-3 (g Ple 5-25) peptides in rats, each time point is large and large The serum concentration is determined at each specified time point after intravenous (7B), intraperitoneal (7C), and subcutaneous (7D) injections. 32 200825098 [Sequence List] <110> Academia Sinica < 12 0> Microbial peptide and its use

<130> 682781.0019TW <160> 10 <170〉 Patentln version 3.3

C <210> 1 <211> 30 <212> Protein <213> Shibanyu <400> 1

Gly Phe lie Phe His lie lie Lys Gly Leu Phe His Ala Gly Lys Met 1 5 10 15 lie His Gly Leu Val Thr Arg Arg Arg His Gly Val Glu Glu 20 25 30 <210> 2 <211> 30 <212&gt ; protein <213> Shiban fish <400> 2

Gly Phe lie Phe His lie lie Lys Gly Leu Phe His Ala Gly Lys Met 33 200825098 15 10 15 lie His Gly Leu Val Thr Arg Arg Arg His Gly Val Glu Glu 20 25 30 <210> 3 <211> 32 < 212> Deoxyribonucleic acid <2 1 3> Artificial <220><223> Introduction <400> 3 32 . ί: force ' tcaagcttcg atgaggtgca tcgccctctt tc <210> 4 <21 1> 32 &lt ;212> Deoxyribonucleic acid <213> Artificial <220><223> Introduction <400> 4 cgggatcctc aggcaaaagc tttctctcgt tc 32 34 200825098

<210> 5 <211> 22 <212> Deoxyribonucleic acid <213> Artificial <220><223> Introduction <400> 5 catcgccctc tttcttgtgt tg <210> 6 <21 1> 16 <212> DNA <213> Artificial <220><223> primer <400> 6 ccctccccgg gttcag <210> 7 <21 1> 19 <212> DNA <;213> Artificial <220> 200825098 <223> Introduction <400> 7 ccagccttcc ttccttggt <210> 8 <21 1> 24 <212> Deoxyribonucleic acid <213> Artificial <220>;223> Introduction <400> 8 gcacttcatg atgctgttgt aggt <210> 9 <21 1> 23 <212> Deoxyribonucleic acid <2 1 3> Artificial <220><223> Introduction <400> 9 tggtcaagga gcagatcaaa cag 200825098 <210> 10 <21 1> 25 <212> Deoxyribonucleic acid <213> Artificial <220><223> Introduction <400> 10 aacgccttcc taacagtatc tecta

Claims (1)

200825098 X. Patent application scope: 1. A segment of a peptide comprising a segment having one of the following amino acid sequences or having the same functional activity: GFIFHIIKGLFHAGKMIHGLV (SEQ ID NO: 1) and • GFIFHIIKGLFHAGKMIHGLVTRRRHGVEE (SEQ ID NO: :2). • 2. According to the scope of the patent application, item 1 wherein the peptide fragment has an activity against microbial infection. 3. According to claim 2, wherein the microorganism is a bacterium. 4. According to the third scope of the patent application, the bacteria are selected from the group consisting of: Listeria, Micrococcus luteus (f/crococcM, Staphylococcus aureus ai/rews), Streptococcus pyogenes) {Staphylococcus agalactide), Table I Grape {Wistaphylococcus epidermidis), SiaMWococci/s sp., Staphylococcus aureus (SiaMy/ococcM • xylosus), miso pearl window { Streptococcus pneumoniae), Staphylococcus aureus (Sbp/zWococci/s aarei/s subsp), enterobacter aerogenes, cloacae suhsp·), grouper, stagnation: Vibrio alginolyticus , Ab acid Mebrisella oxytoca (Salinivibrio costicola subsp.), Vibrio vulnificus (F/Z?r/(9 ra//7///aA?), Harvey's fox fungus (Γ/Ζ) ?γ/(9 harveyi), 容薄孤窗{Vibrio alginolyticus), 绿胜杆菌38 200825098 {Pseudomonas aeruginosa), 反 耳 耳 耳 ' ' Y, Yersinja enterocolitica subsp) 〇 5_ — a pharmaceutical composition , which contains an effective amount based on the application A peptide fragment of the first, second, third or fourth aspect of the patent, and a pharmaceutically acceptable carrier. 6. A pharmaceutical composition having activity against microbial infection of aquatic animals, comprising an isolated protein and variants thereof having the same activity, wherein the protein comprises a sequence as defined in paragraph i of the patent scope of the patent application No. i or the amino acid sequence of SEQ ID NO: 2. 7 · According to the scope of the patent application No. 6 8. According to the scope of the patent application, item 6 wherein the aquatic animal is a fish or shrimp, wherein the microorganism is a bacterium. 〇 9. According to the application Patent scope 8th basin-SH Aquatic animal with trauma 1 pack 0 August! Medicine group for preventing or treating infectious diseases of mammals ^ Contains a separate protein containing temporary substances such as towels, special ^ smoke (four) allogeneic, which is looking for The isolated protein beryllium 3 is the i-th sequence of the patent scope of the invention. The amino acid of SEQ ID NO: 1 or SEQ ID NO: 2 is according to the first aspect of the patent application, 12. According to the scope of the patent application, 1 3 According to the scope of the patent application, wherein the mammal is a human. The infectious genus is a thin infectious disease, wherein the infectious disease is sepsis.