TWI299335B

TWI299335B - Anti-microbal peptides and uses thereof

Info

Publication number: TWI299335B
Application number: TW95145242A
Authority: TW
Inventors: Jyh Yih Chen; Chingt Ming Kuo; Jade Pan
Original assignee: Academia Sinica
Priority date: 2006-12-05
Filing date: 2006-12-05
Publication date: 2008-08-01
Also published as: TW200825098A

Description

1299335 九、發明說明：【發明所屬之技術領域】本發明有關抗微生物蛋白質之胜肽片段及其用途。【先前技術】 ί抗微:生物蛋白皙友极技嫩4 %成；九&丄、、&，_ .1299335 IX. Description of the invention: [Technical field to which the invention pertains] The peptide fragment of the invention relates to an antimicrobial protein and uses thereof. [Prior Art] ί抗微: Bio-protein 皙极极 4 4 ; ; 九九九九九九九九九九九九九九九九九九九

卒取出來，具有抑制一些革蘭氏陽性菌與陰性菌的特性·。 Pleurocidin在1 997年由Cole等人的研究發現（c〇le ΑΜ 過去已知許多抗微生物蛋白質可自脊椎動物中分離出，該性’例如保思定(^16111>〇(：丨(|丨11)，汉切犯术祗禦致病源。魚類交，這些黏液具有殺菌的特最早是由比目魚的體表黏液 et al· J Biol Chem 272: 1 2008-1 3，1 997)，為玉種比目魚中的抗微生物蛋白質，是一種具有25個胺基酸基團的胜肽，存在於體表的黏液中，具有對抗微生物的活性。這種具有對抗微生物的蛋白質，也在不同的生物中被發現’例如鲽魚。在鲽魚中所發現的抗菌蛋白，其具有一段活性區域，胺基酸序列為GWRTLLKAEVKTVGKLALKHYL，此段區域可對抗革蘭氏菌及真菌的感染（patrZykat A. et al.It is taken out and has the characteristics of inhibiting some Gram-positive and Gram-negative bacteria. Pleurocidin was discovered in 1978 by Cole et al. (c〇le ΑΜ in the past many anti-microbial proteins were known to be isolated from vertebrates, such as Paulis (^16111>〇(:丨(|丨11), Han cut is the source of the disease. Fish feed, these mucus have the most bactericidal effect from the surface of the flounder, et al · J Biol Chem 272: 1 2008-1 3,1 997), jade The anti-microbial protein in the flounder is a peptide with 25 amino acid groups, which is present in the mucus of the body surface and has anti-microbial activity. This protein against microorganisms is also in different organisms. It was discovered, for example, squid. The antibacterial protein found in squid has an active region and the amino acid sequence is GWRTLLKAEVKTVGKLALKHYL, which is resistant to infection by Gram and fungi (patrZykat A. et al.

Antimicrob Agents Chemother 47: 2464-70， 2003)。近年來’ Yin等人在石斑魚中發現了一段類似抗菌胜肽的 cDNA 序列，命名為 epinecidin-l(Yin ZX. et al. Aquaculture 253: 204-1 1，2006)。但並沒有在組織中證明 1299335 層脂質體’多層脂質體’或球形體中製成。此組合物可影響生理狀態’溶解度，體内釋放的速率，以及體内清除之速率。組合物之選擇可依據該抗菌蛋白的物理化學特性而決定。有關醫藥組合物的給付式’可由在此領域具通常知識者依據該組合物的物理化學特性而決定，包括但不限於，口服，腹腔注射’皮下注射’靜脈注射，肌肉注射，Antimicrob Agents Chemother 47: 2464-70, 2003). In recent years, Yin et al. found a cDNA sequence similar to the antibacterial peptide in grouper, named epinecidin-l (Yin ZX. et al. Aquaculture 253: 204-1 1, 2006). However, it has not been produced in tissues to demonstrate 1299335 layer liposome 'multilayered liposomes' or spheres. This composition can affect the physiological state 'solubility, the rate of release in vivo, and the rate of clearance in vivo. The choice of composition can be determined based on the physicochemical properties of the antibacterial protein. The dosage form for a pharmaceutical composition can be determined by those of ordinary skill in the art depending on the physicochemical properties of the composition, including, but not limited to, oral, intraperitoneal injection, subcutaneous injection, intravenous injection, intramuscular injection,

在本發明之實例中，該醫藥組合物經由腹腔注射。’ 在本文中，該術語「動物」包括所有動物種類，例如水生動物及哺乳類動物。在-實施例中’該水生動物為魚類。該哺乳類動物係指任何被定義為哺乳類的動物，包括人類。較佳情況下，該哺乳類動物為人類。在本文中’「水生動物的微生物感染」係指任何可導致水生動物活動力下降或死亡的病原’例如_感染。這些主要病原多為水中環境的常在囷’其中最常見的是弧菌。在_實施例中，以死亡率作為判斷感染結果的依據，且造成此感染之細菌為創傷弧菌。在本文中，「感染性疾病」係指與感染相關的疾病，包括由病源感染直接造成的（如細菌感染），或是在疾病進行中所併發的感染症狀，例如免疫功能低下所導致的·，都在本發鶴_範圍巾。在_實施例中，該疾病為綠膿桿菌引發的敗血症。本务明有關於^又胜肽片段及其醫藥組合物，其包含一段序列編號辱或序列編號2之胺基酸序列，及其具活性的變異體。根據本發明之一汽施例，該二胜肽片段命名為石斑魚抗菌蛋白〜3。在此所述的活性為對抗微生物感染的活性，特別是細菌。序列編號丨或序列編號2的胺基 1299335 酸序列係來自本發明中選殖的cDNA。於本發明之一實施例中，以其他水生動物已知的抗菌蛋白密碼設計引子，以石斑魚cDNA資料庫為模板，進行PCR放大後，得到石斑魚抗菌蛋白〜3的cDNA，將此序列以insightII程式的同源模式分析其最小能量（Accelrys，SanDiego，CA，USA)，得到其結構模型，得序列編號1或序列編號2的胺基酸序列，認為是具有活性的片段，即本發明胜肽片段。石斑魚抗菌蛋白-3的結構模型中，石斑魚抗菌蛋白—3被預测為兩性分子的α-螺旋狀結構，與其他抗菌蛋白類似，其功能為在微生物上形成孔洞，造成滲透壓不平衡而導致微生物死亡。在分析多肽的特性之後，推測出石斑魚抗菌蛋白—3應在細胞質内被傳送。此結果暗示石斑魚抗菌蛋白—3應由細胞内傳送至細胞外分泌，以對抗感染。石斑魚抗菌蛋白-3的胺基酸序列具有一個核仁位置訊息（NLS)，NLS可能與先天性免疫的訊息傳導有關，因為NLS不只出現在所有抗菌蛋白中，也在其他具有調節功能的分子中出現 (Schedlich LJ· et al. J Biol Chem 275: 23462-70， 2000)。當考慮本發明的胺基酸序列範圍時，所有本發明所述實施例中包含序列編號：1及序列編號：2的蛋白質或胜肽的胺基酸序列，均包括在本發明範圍中的胺基酸序列中，舉例來說，本發明中提及兩段合成的抗菌蛋白一3胜肽（g-pie 5 —34)與 (g-pie 5-25)，皆在本發明所涵蓋範圍内。進一步包括根據本發明以上所述，併入保留性胺基酸置換之胜肽之所有胺基酸序列。進一步包括更大蛋白質之胺基酸序列，該蛋白質併入本發明中含有序列編號·· 1或序列編號：2的該蛋白質或胜 1299335 狀’包括融合蛋白，以及併入訊息胜肽的未成熟蛋白質。本發明利用其他方式證明該胜肽片段具有對抗微生物的活性’例如RT-PCR或組織染色。分析此可編碼為此胜肽片段 • 的基因或其產生的蛋白質在不同組織的表現量，由組織分佈的情形顯示，此基因或蛋白質主要表現在接觸外來物的器官中，如石斑魚的頭腎，皮膚及小腸中。另外以病源分子刺激後’該基因表現量也上升，更證實了該已分離之蛋白質胜肽片 > 段具有對抗微生物的活性。以藥物動力學分析抗菌蛋白-3胜肽在血清中的濃度，顯示其具有良好的反應速率，不會黏附在水生動物及哺乳類動物鈐血管壁上，具有隨著血液循環全身的特性，符合藥物開發的特色。 _ II本發明胜肽片段具有在水生動物内對抗細菌感染的活 1 性’例如創傷弧菌感染。在一實施例中，以合成並純化的兩段石斑魚抗菌蛋白-3胜肽片段，即具有序列編號：丨與序列編號：2胺基酸序列之本發明胜肽片段，進行抗菌活性測試。該胜肽片段可有效對抗革蘭氏陰 > 性菌及革蘭氏陽性菌，顯示此胜肽片段具有高度的抗菌活性，且以此胜肽片段達到相同抗菌效果，比表現全長蛋白質序列所需成本更低。在另一實施例中，在石斑魚中注射p〇ly( I):p〇ly(C)以觀察抗菌蛋白-3的mRNA表現量。p〇iy(i):poly(c)是類似RM的聚核酸 (Polynucleotide)，由兩條同性聚合物（H〇mo p〇lymers)組成；其中一邊是聚核糖次黃嗓吟核酸（Polyriboinosinic acid)，另一邊是聚核糖細胞嘧啶核酸（Polyribocytidylic acid)。每一邊的鏈狀結構就像自然存 11 (Η ) 1299335 在的RNA -樣。兩條單鏈之間靠氫鏈結合起來。人工合成的核酸 p〇iy(i):p〇iy(c) ’能夠刺激細胞合成如干擾子（Interfer〇n)的蛋白質，保護其他細胞不受病毒的感染。在本發明的實例中，利用p〇ly⑴:吨⑹ . ?丨發抗g蛋白-3的表現’顯示本發明胜肽片段也具有抗病毒之活性。另一方面，本發明有關於一種具有對抗水生動物（例如魚或蝦）賴生物感染活性之醫藥組合物’其包含—段分離之蛋白質及 , 其具相同活性的變異體，其中該分離之蛋白質包含序列編號】或序列編號鲁 2之胺基酸序列。在_實施例中，藉由以創傷弧誠染吳郭魚及石斑魚的實驗本發明所使賴魚類感染模式，為在此領域所熟知的方法，包含只以腹腔注射創傷弧菌的對驗，及以腹腔注射創傷弧菌與本發明胜肽片段的不同實驗組’本發明胜肽片段的劑量為每條魚〇. i料。最後以魚類的死亡率判斷本發明胜肽片段對於細菌感染的保護效果。而該胜狀片段確實可降低水生動物受微生物感染之死亡率。另方面’本發明有關於一種具有預防或治療哺乳類動物的感染 • I疾病活性之醫藥組合物，其包含一段分離之蛋白質及其具相同活性的 - 變異體，其中該分離之蛋白質包含序列編號1或序列編號2之胺基酸序丨根據本發明之具體實施例，湘動物模式，以腹腔注射本發明胜肽的 Ί、、、、且及以腹腔注射綠膿桿菌與本發明胜肽片段的不同實驗組。最後以死亡率判斷本發明胜肽片段對於細菌感染的保護效果。而該蛋白質胜狀片段可在哺乳類動物受細菌侵襲時降低死亡率，因此可作為In an embodiment of the 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 embodiment - the aquatic animal is a fish. The mammal is any animal defined as a mammal, including humans. Preferably, the mammal is a human. As used herein, "microbial infection of aquatic animals" means any pathogen that causes a decrease in the activity or death of aquatic animals, such as infection. Most of these major pathogens are often found in the aquatic environment, the most common of which is Vibrio. In the embodiment, mortality is used as a basis for judging the result of infection, and the bacteria causing the infection is Vibrio vulnificus. As used herein, "infectious disease" refers to an infection-related disease, including a direct infection caused by a source of infection (such as a bacterial infection), or a symptom of an infection that occurs during the course of the disease, such as caused by poor immune function. Both are in the hair of the hair _ range towel. 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: 2 or 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 1299335 acid sequence of SEQ ID NO: or SEQ ID NO: 2 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, and the sequence is obtained by the Insight II program. The homology pattern is analyzed for its minimum energy (Accelrys, SanDiego, CA, USA) to obtain its structural model, and the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 is considered to be an active fragment, ie, the peptide fragment of the present invention. . In the structural model of the grouper antibacterial protein-3, the grouper antibacterial protein-3 is predicted to be an α-helical structure of amphiphiles, similar to other antibacterial proteins, and its function is to form pores in microorganisms, resulting in an imbalance of osmotic pressure. Microbial 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 involved in the transmission of innate immunity because NLS is not only present in all antimicrobial proteins, but also in other molecules with regulatory functions. Appears (Schedlich LJ et al. J Biol Chem 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 acid sequence, for example, the two synthetic antimicrobial proteins 3-g peptide (g-pie 5-34) and (g-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 comprising an amino acid sequence of a larger protein which is incorporated into the present invention comprising the sequence number +1 or SEQ ID NO: 2 or the genus 1299335 </ RTI> including the fusion protein, and the immature incorporated into the message peptide protein. The present invention utilizes other means to demonstrate that the peptide fragment has anti-microbial activity' such as RT-PCR or tissue staining. Analyze the amount of the gene encoding this peptide fragment or the protein produced by it in different tissues. It is shown by the distribution of tissues that the gene or protein is mainly expressed in organs that contact foreign objects, such as the head kidney of grouper. , in the skin and in the small intestine. In addition, the amount of expression of the gene was also increased after stimulation with the pathogenic molecule, and it was confirmed that the isolated protein peptide tablet > segment has antibacterial activity. Pharmacokinetic analysis of the concentration of antibacterial protein-3 peptide in serum, showing that it has a good reaction rate, does not adhere to the blood vessel wall of aquatic animals and mammals, has the characteristics of the whole body with blood circulation, and conforms to the drug. Development features. The peptide fragment of the present invention has a viable activity against bacterial infections in aquatic animals such as V. vulnificus infection. In one embodiment, the antibacterial activity test is carried out by synthesizing and purifying a two-stage grouper antibacterial protein-3 peptide fragment, i.e., a peptide fragment of the invention having the sequence number: 丨 and SEQ ID NO: 2 amino acid sequence. The peptide fragment is effective against Gram-negative bacteria and Gram-positive bacteria, indicating that the peptide fragment has high antibacterial activity, and the peptide fragment achieves the same antibacterial effect, and the full-length protein sequence is expressed. The cost is lower. In another embodiment, p石ly(I): p〇ly(C) is injected into the grouper to observe the mRNA expression level of the antibacterial protein-3. P〇iy(i): poly(c) is an RM-like polynucleotide composed of two homopolymers (H〇mo p〇lymers); one of which is a polyriboinosinic acid (Polyriboinosinic acid) On the other side is Polyribocytidylic acid. The chain structure on each side is like RNA-like in the natural deposit 11 (Η) 1299335. The two single chains are joined by a hydrogen chain. The synthetic nucleic acid p〇iy(i): p〇iy(c) ' can stimulate cells to synthesize proteins such as interferon, and protect other cells from infection by viruses. In the examples of the present invention, the use of p〇ly(1): ton(6). 丨抗抗抗抗抗 ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ 。。。。。。。。。。。。 In another aspect, the present invention relates to a pharmaceutical composition having activity against aquatic organisms (e.g., fish or shrimp), which comprises a segmentally isolated protein and a variant having the same activity, wherein the isolated protein Contains the sequence number] or the amino acid sequence of the sequence number Lu 2 . In an embodiment, the larvae infection model is invented by the invention of the squid and the grouper in a wounded arc. The method known in the art includes a method of intraperitoneal injection of Vibrio vulnificus. And 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. Finally, the protective effect of the peptide fragment of the present invention on bacterial infection was judged by the mortality rate of fish. The winning fragment does reduce the mortality of aquatic animals from microbial infections. In another aspect, the present invention relates to a pharmaceutical composition having an activity for preventing or treating an infection of a mammal, comprising an isolated protein and a variant having the same activity, wherein the isolated protein comprises SEQ ID NO: 1 Or the amino acid sequence of SEQ ID NO: 2, according to a specific embodiment of the present invention, in an animal model of intraperitoneal injection of the peptide of the present invention, sputum, and, and intraperitoneal injection of Pseudomonas aeruginosa with the peptide fragment of the present invention Different experimental groups. Finally, the protective effect of the peptide fragment of the present invention on bacterial infection was judged by the mortality rate. The protein fragment can reduce mortality when the mammal is attacked by bacteria, so it can be used as

12 1299335 下反應兩分鐘，72 °C下反應兩分鐘，共35循環；之後將PCR 反應的結果以2%洋菜膠分析。引子序列如下： P1 ·· 5’ -TCAAGCTTCGATGAGGTGCATCGCCCTCTTTC (序列編號： 3) 以及， P2 : 5’ -CGGGATCCTCAGGCAAAAGCTTTCTCTCGTTC (序列編號： 4) 。引子序列為根據已知的比目魚pleurocidin密碼區域而設計 (登記編號：AY273181 與 AY273180)，美洲擬鲽 ⑽(登記編號：AF301515 與 kY?>Q\b\2)，美 f 綠 Glyptocephalus cynoglossus (登記編號：AY273177)，美洲黃蓋 0$ Umanc/a ferrugjfnea (登記編號：AY2731 75)，以及石斑魚及 (EST clone，登記編號：BQ096584)。經過PCR放大後，將樣品從洋菜膠上萃取出來，並接入載體 pIRES2-EGFP 中（BD Biosciences, San Jose，CA，USA)。隨機選擇菌落並以載體的引子將雙股都進行定序。含有保留性序列的多肽，以psort軟體（可在網站上取得 http ://psort. ims, u-tokvo. ac. jp/form2. html)進行訊息胜肽在細胞内運送的分析。實例一：分離RNA並以RT-PCR定量mRNA的表現量收集五隻石斑魚（體重大約5公斤）的組織，以下列步驟萃取RNA(Chen JY. et al· DNA Cell Biol 17: 359-76，1998)。在組織分佈實驗中，收集血液，鰓，心臟，頭腎，小腸，肝臟，肌肉，以及皮膚，以相同方法得到織 RNA。藉石斑魚抗菌蛋白—3的mRNA表現，以相對即時定| +12 1299335 was reacted for two minutes and reacted 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 was designed according to the known flounder pleurocidin cryptographic region (registration number: AY273181 and AY273180), American cockroach (10) (registration number: AF301515 and kY?>Q\b\2), beauty f green Glyptocephalus cynoglossus (registration No.: AY273177), American yellow cover 0$ Umanc/a ferrugjfnea (registration number: AY2731 75), and grouper and (EST clone, registration number: BQ096584). After PCR amplification, the samples were extracted from the gelatin and incorporated into the vector pIRES2-EGFP (BD Biosciences, San Jose, CA, USA). Colonies were randomly selected and the double strands were sequenced using the primers of the vector. The polypeptide containing the retained sequence is analyzed by the psort software (available on the website http://psort.ims, u-tokvo. ac.jp/form2.html). Example 1: Isolation of RNA and quantification of mRNA expression by RT-PCR Five tissues of grouper (body weight approximately 5 kg) were collected and RNA was extracted by the following procedure (Chen JY. et al. DNA Cell Biol 17: 359-76, 1998) ). In the tissue distribution experiment, blood, clot, heart, head kidney, small intestine, liver, muscle, and skin were collected, and woven RNA was obtained in the same manner. Take the mRNA expression of the grouper antibacterial protein-3, to be relatively instant |

15 1299335 錄聚合酶連鎖反應(RT-PCR)分析在不同組織的分佈情形。在以等量脂質多醣LPS刺激(每隻魚5純魚的平均重量為1〇克），與不同量脂質多酷哪刺激後(每隻魚50，1G，5,與1㈣，分析組織分佈情形。在以不同量 p〇ly(I):P〇ly(C)-刺激的實驗中，濃度為每隻魚〇1，丨，5，以及1〇 #g。在注射LPS或poly(I):p〇ly(C) 360分鐘後萃取總舰。將mRNA轉錄為cDNA，並以cDNA進行RT_PCR分析抗菌蛋白及冷—actin 的表現量。Taqman probe以及引子均根據ABI的步驟設計（AppUed15 1299335 Polymerase chain reaction (RT-PCR) analysis of distribution in different tissues. Stimulated with the same amount of lipopolysaccharide LPS (average weight of 1 pure fish per fish is 1 gram), and after different stimulation of different amounts of lipids (50, 1G, 5, and 1 (four) per fish, analysis of tissue distribution In experiments with different amounts of p〇ly(I):P〇ly(C)-stimulation, the concentration was 1, 丨, 5, and 1〇#g per fish. Injecting LPS or poly(I) :p〇ly(C) After 360 minutes, the total ship was extracted. The mRNA was transcribed into cDNA, and the expression of antibacterial protein and cold-actin was analyzed by RT_PCR. The Taqman probe and the primer were designed according to the steps of ABI (AppUed).

Biosystems，Perkin-Elmer Coid，Boston，ΜΑ 02118, USA)。所有樣品都進行二重複的實驗。PCR反應以AppliedBiosystems系統（7000序列偵測系統）即時PCR儀器及GeneArop序列偵測系統軟體進行。反應中包含5 # g 不同組織的總RNA。相對定量RT-PCR的Mastermix試劑（Applied Biosystems，Perkin-Elmer)用作定量之用。在每個反應盤中均包含cdna模板，Taqman probe，Mastermix試劑，以及反應緩衝液。石斑魚抗菌蛋白-3的即時定量PCR引子序列如下：正向引子catcgccctctttcttgtgttg (序列編號：5)以及反向引子ccctccccgggttcag (序列編號·· 6); 石斑魚beta-act i η基因：正向引子ccagccttccttccttggt (序列編號：7)以及反向引子 gcacttcatgatgctgttgtaggt (序列編號：8); 石斑魚Mx基因：正向引子 tggtcaaggagcagatcaaacag (序列編號：9)以及 16 1299335 反向引子 aacgccttcctaacagtatctccta (序列編號：1 〇)。總反應體積為25 //1，該反應在96孔盤進行，並在96 °C下反應15 秒，60°C下反應60秒，共40循環。以65°C為起始溫度進行分解反應，以分析PCR產物的分解溫度。以平均值±標準差表示n 個實驗的結果，η代表每種RNA來源的實驗次數。以SAS統計軟體分析其組間差異，p (*)<〇· 〇5或；7 (**)< 〇.〇1為具有明顯差異。Biosystems, Perkin-Elmer Coid, Boston, ΜΑ 02118, USA). All samples were subjected to two replicate experiments. The PCR reaction was performed using an Applied Biosystems system (7000 Sequence Detection System) real-time PCR instrument and GeneArop sequence detection system software. The reaction contained 5 #g total RNA from different tissues. Mastermix reagent (Applied Biosystems, Perkin-Elmer) for relative quantitative RT-PCR was used for quantification. A cdna template, Taqman probe, Mastermix reagent, and reaction buffer were included in each reaction tray. The real-time quantitative PCR primer sequence of grouper antibacterial protein-3 is as follows: forward primer catcgccctctttcttgtgttg (sequence number: 5) and reverse primer ccctccccgggttcag (sequence number·6); grouper beta-act i η gene: positive primer ccagccttccttccttggt (sequence No.: 7) and reverse primer gcacttcatgatgctgttgtaggt (sequence number: 8); grouper Mx gene: forward primer tggtcaaggagcagatcaaacag (sequence number: 9) and 16 1299335 reverse primer aacgccttcctaacagtatctccta (sequence number: 1 〇). The total reaction volume was 25 //1. The reaction was carried out on a 96-well plate and reacted at 96 ° C for 15 seconds and at 60 ° C for 60 seconds for a total of 40 cycles. The decomposition reaction was carried out at a temperature of 65 ° C to analyze the decomposition temperature of the PCR product. The results of n experiments are expressed as mean ± standard deviation, and η represents the number of experiments for each RNA source. The SAS statistical software was used to analyze the difference between the groups, p (*) < 〇 · 〇 5 or; 7 (**) < 〇.〇1 was significantly different.

利用相對性即時RT-PCR，可決定抗菌蛋白-3 mRNA在不同組織的表現量，結果如圖1 A所示。每個數據皆以h mRNA的表現量正常化。如圖1A中所顯示，抗菌蛋白〜3在頭腎，小腸，以及皮膚的表現量最多，此結果顯示抗菌蛋白一3 基因的局部作用。圖1B顯示在Lps刺激後，抗菌蛋白—3心難在不同組織的表現量。在以Lps刺激六小時後，在頭腎部位的抗囷蛋白-3以及皮膚中的Mx mRNA最早被偵測到。為了解 LPS及p〇ly I ·· poly c對石斑魚抗菌蛋白-3表現量的調節作用，在石斑魚中注射不同劑量的[以或p〇ly I: p〇iy c ， ▲ wv刀竹七级黑机囷蛋白的表現里如圖2Α所不，相較於對照組（不經處理）魚類，在5〇 # g ml LPS作用下，抗菌蛋白的表現量在穩定狀態下增; 倍。在圖2B中，相认此丄rm , / . 四 …-為口 ϋ的衣巩置隹穩定狀態下增倍。在圖2Β中’相較於對照組（不經處理）魚類，在“g/, 作用下’抗菌蛋白_3的表現量在穩定狀, 旦一、、口果顯不石斑魚抗菌蛋白-3的基因表, 置會在lps及D0】vm ，/ 的處理下增加。The relative amount of real-time RT-PCR was used to determine the amount of antibacterial protein-3 mRNA in different tissues. The results are shown in Figure 1A. Each data was normalized to the amount of h mRNA expression. As shown in Fig. 1A, the antibacterial protein ~3 exhibited the most amount in the head kidney, the small intestine, and the skin, and this result showed a local effect of the antibacterial protein-3 gene. Figure 1B shows the amount of antibacterial protein-3 in different tissues after Lps stimulation. Six hours after stimulation with Lps, anti-prion-3 in the head kidney and Mx mRNA in the skin were first detected. In order to understand the regulation of LPS and p〇ly I ·· poly c on the performance of grouper antibacterial protein-3, different doses were injected into grouper [to or p〇ly I: p〇iy c , ▲ wv The performance of the black cockroach protein is shown in Fig. 2, compared with the control (untreated) fish, under the action of 5 〇 # g ml LPS, the expression of antibacterial protein increased under steady state; In Fig. 2B, it is recognized that the 丄rm, /. four ...- is the 衣 ϋ ϋ 隹。。。。。。。。。。。。。。. In Fig. 2Β, compared with the control group (untreated), the expression of antibacterial protein_3 was stable under the action of “g/, and the fruit of the group was not resistant to the antibacterial protein-3. The gene table will be added under the processing of lps and D0]vm, /.

(S 17 1299335 14些結果可支持石斑魚抗菌蛋白—3表現後往皮膚運送，以對抗微生物的假設，且在先天性免疫的第一線扮演重要的角色。在本發明中也發現，石斑魚抗菌蛋白—3的表現，也會與 1^3及？〇17(1):13〇17(〇刺激的劑量成正比。雙股跗“化肋幻是病毒感染時的分子模式，因為在大多數病毒複製時，會製造雙股RNA。在本文中，也發現石斑魚抗菌蛋白—3可辨認雙股RNA，並在基因活化後進一步引起Μχ基因的活化。此結果表示石斑魚抗菌蛋白-3基因不只受LPS的刺激，同時也避免宿主受到病毒的感染。這些結果顯示抗菌蛋白可對抗細菌與病毒的感染。根據推論的胺基酸序列合成兩種不同長度石斑魚抗菌蛋白 -3的本發明胜肽片段：石斑魚抗菌蛋白-3 ( g-pie 5-34)，序列為 GFIFHIIKGLFHAGKMIHGLVTRRRHGVEE(序列編號：1);以及石斑魚抗菌蛋白-3 (g-ple 5-25)序列為 GFIFHIIKGLFHAGKMIHGLV(序列編號：2)。實例二：石斑魚抗菌蛋白-3於石斑魚體内分佈之分析多株抗體镅借多株抗體委託創生生物科技股份有限公司代為製作（台北，台灣）。簡單來說，為了讓根據本發明合成的抗菌蛋白一3 (g-pie 5-34)胜肽片段與攜帶蛋白質結合，在兩隻兔子中以皮下注射每隻打入i mg胜肽與完全弗氏佐劑（CMWde Freund’ Sadjuvant)1混合的劑量。接下來每隔十四天注射 18 1299335 胜肽與完全弗氏佐劑的混合物，共五次。在第五次注射的一週後，收集兔子的血清抗體並以胜肽親合性方法純化。以西方墨點法測試血清抗體對抗根據本發明胜肽片段之活性。組織免疫染氙方法 • 結織免疫染色實驗中，取得魚肥，小腸，心臟以及頭腎後立即以4/β已緩衝之中性福馬林固定24q分鐘，經過脫 . 水以及石臘包埋。將石斑魚組織連續橫向切片後(5 ”)，在 • 二甲苯中脫臘並再水化(rehydrated)。再水化後，以擰檬酸 • 緩衝液⑽值6.0)在95飞下預先處理切片10分鐘，並在室溫下在含有3%過氧化氫的甲醇中靜置3〇分鐘，以阻斷内生性的過氧化酶活性。將鏈黴和素（strepavidin)阻斷溶液與生物素（biotin)阻斷溶液分別靜置15分鐘，之後以浸潤緩衝液清洗。利用5%BSA阻斷該溶液5分鐘，再以浸潤緩衝液清洗5分鐘。加入以1: 5稀釋的多株抗體或事先採血的血清抗體，與切片在25 下靜置60分鐘，之後將之清洗。將二級抗體（結合生物素的抗兔子IgG)加入樣本中靜置3〇分鐘， • 之後以浸潤緩衝液清洗。在玻片中加入鏈黴和素HRp 3 〇分鐘後’以浸潤緩衝液清洗。加入染色試劑（DAB)丨〇分鐘後顯色，再β洗一遍。加入蘇木精（hemaf〇Xy 1 in) 1 〇分鐘以作為對比染色’且將該玻片以Scot ter’ s溶液清洗以增強顏色。在連縯酒精中將切片脫水，並浸入二甲苯清洗。最後將載玻片固定劑滴在玻片上，蓋上蓋玻片。以附有CCD照相機（DXM —1 200, Nikon)的 Nikon (Tokyo, Japan) E 600 顯微鏡拍攝顯微照片0 19 1299335(S 17 1299335 14 results can support the grouper antibacterial protein-3 after delivery to the skin to counter the hypothesis of microorganisms, and play an important role in the first line of innate immunity. Also found in the present invention, grouper antibacterial protein The performance of -3 will also be proportional to 1^3 and 〇17(1):13〇17 (the dose of sputum stimulation. Double sputum 跗化是 is the molecular pattern of viral infection, because in most viruses When replicating, double-stranded RNA is produced. In this paper, it was also found that the grouper antibacterial protein-3 recognizes double-stranded RNA and further activates the purine gene after gene activation. This result indicates that the grouper antibacterial protein-3 gene is not only affected by LPS. The stimulation also prevents the host from being infected by the virus. These results show that the antibacterial protein is resistant to bacterial and viral infections. The peptide fragments of the invention are synthesized according to the deduced amino acid sequence of two different lengths of grouper antibacterial protein-3: grouper Antibacterial protein-3 (g-pie 5-34), sequence GFIFHIIKGLFHAGKMIHGLVTRRRHGVEE (SEQ ID NO: 1); and grouper antibacterial protein-3 (g-ple 5-25) sequence GFIFHIIKG LFHAGKMIHGLV (SEQ ID NO: 2). Example 2: Analysis of the distribution of grouper antibacterial protein-3 in grouper. Multiple antibodies were produced on behalf of Genesis Biotechnology Co., Ltd. (Taipei, Taiwan). In order to allow the antibacterial protein-3 (g-pie 5-34) peptide fragment synthesized according to the present invention to bind to the carrier protein, each of the two rabbits was subcutaneously injected with i mg peptide and complete Freund's adjuvant. (CMWde Freund' Sadjuvant) 1 mixed dose. Next, every 14 days, a mixture of 18 1299335 peptide and complete Freund's adjuvant was injected five times. After one week of the fifth injection, rabbit serum antibody was collected. Purified by the peptide affinity method. The serum antibody was tested by Western blot method against the activity of the peptide fragment according to the invention. Tissue immunostaining method • In the immature immunostaining experiment, fish fat, small intestine, heart and head kidney were obtained. Immediately after 4/β buffered neutral fumarin for 24 minutes, after dewatering and paraffin embedding. The grouper tissue was continuously sliced horizontally (5 ”) in • xylene Wax and rehydrated. After rehydration, pre-treat the slice with citric acid buffer (10) 6.0) for 95 minutes at 95 °F, and at room temperature in methanol containing 3% hydrogen peroxide. The cells were allowed to stand for 3 minutes to block endogenous peroxidase activity. The strepavidin 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 infiltration buffer for 5 minutes. A plurality of antibodies diluted in 1:5 or serum antibodies previously collected were added, and the sections were allowed to stand at 25 for 60 minutes, and then washed. A secondary antibody (biotin-conjugated anti-rabbit IgG) was added to the sample for 3 minutes, and then washed with an infiltration buffer. After adding streptavidin HRp 3 to the slide for a few minutes, it was washed with an infiltration buffer. After adding the staining reagent (DAB), the color was developed after a minute, and then washed again by β. Hematoxylin (hemaf〇Xy 1 in) was added for 1 minute to serve as a contrast stain' and the slide was washed with a Scot ter's solution to enhance the color. The sections were dehydrated in continuous alcohol and immersed in xylene for cleaning. Finally, the slide fixing agent was dropped on the slide and covered with a cover glass. Photomicrographs taken with a Nikon (Tokyo, Japan) E 600 microscope with a CCD camera (DXM-1200, Nikon) 0 19 1299335

Mi 為决疋石斑魚抗菌蛋白一 3在組織中的表現，以免液染色方法偵測不同器官中的表現量，結果發現石斑魚抗菌蛋白—3在鰓絲表皮細胞與小腸表皮細胞中金清中的多株抗體有強烈的免疫反應（圖3 )，證實石斑魚抗菌蛋白—3存在石斑魚小腸與鲶中。與事先採血的抗血清實驗比較，在鰓與小腸的表皮細胞中並無染色反應。無論心臟或頭腎都沒有任何親和性純化血清多株抗體或事先採血的血清抗體，對抗菌蛋白—3的免疫反應。在石斑魚中，抗菌蛋白一3可能在消化道與氣體交換器官扮演一個重要的角色，用於抵抗外來微生物的侵犯。實例三：抗菌活性分析达l—目魚pleurocidin胜肽輿兩段石斑裊抗链蛋白一 3之合成該等胜肽由Genesis Biotech公司利用Fmoc化學固態流程合成。萃取粗製的胜肽，冷凍乾燥，之後以反向高效液態層析（RP-HPLC)純化。以純化胜肽之分子量及純度分別由質譜儀及HPLC分析。合成的胜肽放置於磷酸鹽緩衝液中（PH值7. 4) 備用。該合成之比目魚 pleurocidin 胜肽序列為 GWGSFFKKAAHVGKHVGKAALTHYL ;而根據本發明之石斑魚抗菌蛋白 -3 (g-pie 5-34) 序列為 GFIFHIIKGLFHAGKMIHGLVTRRRHGVEE(序列編號：1);以及石斑魚抗菌蛋白 -3 (g-pie 5-25)序列為 GFIFHIIKGLFHAGKMIHGLK序列編號：2)。體外抑菌分极· 20 1299335 g-plew5 ··石斑魚抗菌蛋白以（本發明具有序列編號· 2之脞妝Μ ί；Μ wf-ple ··比目魚pleuroddin胜肽 · <肚肌/i仅y ΝΑ:不活化Mi is the expression of antibacterial protein-3 in the tissue of the grouper, and the liquid staining method is used to detect the expression in different organs. The results show that the grouper antibacterial protein-3 is a multi-drug antibody in the epidermal cells of the silkworm and the epidermal cells of the small intestine. There was a strong immune response (Fig. 3), which confirmed that the grouper antibacterial protein-3 was present in the small intestine and sputum of grouper. There was no staining reaction in the epidermal cells of the sputum and the small intestine compared to the antiserum experiment with prior blood collection. Regardless of whether the heart or the head kidney does not have any affinity for purifying serum multiple antibodies or serum antibodies that have been previously collected, the immune response to antibacterial protein-3. In grouper, antibacterial protein-3 may play an important role in the digestive tract and gas exchangers to combat the invasion of foreign microorganisms. Example 3: Antibacterial activity analysis The synthesis of the two-stage grouper of the pleurocidin peptide 袅 ple ci 石该该该该该该该该该该该该该该该该该该该该该该该该该该该该该该该该该该The crude peptide was extracted, lyophilized, and then purified by reverse high performance liquid chromatography (RP-HPLC). The molecular weight and purity of the purified peptide were analyzed by mass spectrometry and HPLC, respectively. The synthesized peptide was placed in a phosphate buffer (pH 7.4) for use. The synthetic flounder pleurocidin peptide sequence is GWGSFFKKAAHVGKHVGKAALTHYL; and the grouper antibacterial protein-3 (g-pie 5-34) sequence according to the invention is GFIFHIIKGLFHAGKMIHGLVTRRRHGVEE (SEQ ID NO: 1); and grouper antibacterial protein-3 (g-pie 5) -25) The sequence is GFIFHIIKGLFHAGKMIHGLK sequence number: 2). In vitro bacteriostasis pole · 20 1299335 g-plew5 · · grouper antibacterial protein to (the invention has the serial number · 2 脞 makeup Μ ί; Μ wf-ple · · flounder pleuroddin peptide · < belly muscle / i only y ΝΑ: not activated

微生物 g-ple 5 革蘭氏陰性菌 pg/ml Enterobacter aerogenes (BCRC10370) 25 Enterobacter cloacae subsp (BCRC10401) 100 Grouper vibrio alginolyticus 3.125 Klebsiella oxytoca (BCRC 13985) 12.5 Salinivibrio costicola subsp (^CRC12910) 3.125 Vibrio vulnificus (YJ016) 6.25 Vibrio harveyi fBCRC13812) 3.125 Vibrio harveyi (^CRC12907) 25 Vibrio vulnificus (204) 6.25 Vibrio alginolyticus fBCRC 12829) 6.25 Pseudomonas aeruginosa fATCC 19660) NA Yersinia enterocolitica subsp (BCRC 13999) 100 g-ple 5-25 MIC wf-ple MIC pg/ml pg/ml 50 50 100 >100 12.5 6.25 100 50 12.5 3.125 50 50 12.5 3.125 12.5 6.25 12.5 6.25 12.5 6.25 60 45 100 50 1之胜肽片段） 2之胜肽片段） g-ple 5-34 :石斑魚抗菌蛋白„4 (本發明具有序 g-plew5 :石斑魚抗菌蛋白纟·2“本發明具有序 wf-ple :比目魚pleur0Cidin胜肽观 NA:不活化在本發:的結果輸’本發明兩财同蝴胜肽片段均蘭氏陰性囷及革蘭氏陽性菌的殺菌活性，平所不，本發明月生肽片段(石斑魚抗菌蛋白—3胜狀心囷治性，如表i 5 25)在體外實驗中具高度活性，顯示其潛在抗菌功敦。5 34 ^ δ-ρΐ6 中，細菌注射前，單敝射—次本發明胜肽片段，可^時切駿内實驗之功放。預先以本發明胜肽片段處理也可以提高魚類存活率、隻房每·Microbial g-ple 5 Gram-negative bacteria pg/ml Enterobacter aerogenes (BCRC10370) 25 Enterobacter cloacae subsp (BCRC10401) 100 Grouper vibrio alginolyticus 3.125 Klebsiella oxytoca (BCRC 13985) 12.5 Salinivibrio costicola subsp (^CRC12910) 3.125 Vibrio vulnificus (YJ016) 6.25 Vibrio harveyi fBCRC13812) 3.125 Vibrio harveyi (^CRC12907) 25 Vibrio vulnificus (204) 6.25 Vibrio alginolyticus fBCRC 12829) 6.25 Pseudomonas aeruginosa fATCC 19660) NA Yersinia enterocolitica subsp (BCRC 13999) 100 g-ple 5-25 MIC wf-ple MIC Pg/ml pg/ml 50 50 100 >100 12.5 6.25 100 50 12.5 3.125 50 50 12.5 3.125 12.5 6.25 12.5 6.25 12.5 6.25 60 45 100 50 1 peptide fragment) 2 peptide fragment) g-ple 5-34 : Grouper antibacterial protein „4 (The present invention has the order g-plew5: Grouper antibacterial protein 纟·2” The present invention has the order wf-ple: flounder pleur0Cidin peptide view NA: not activated in the present: the result of the transmission of the two The bactericidal activity of both the gram-negative sputum and the gram-positive bacterium of the same peptide fragment is not good. The lunar peptide fragment of the present invention (the grouper antibacterial protein - 3 wins heart palpitations, as shown in Table i 5 25) is highly active in vitro, showing its potential antibacterial work. 5 34 ^ δ-ρΐ6, bacterial injection Before, single shot--the sub-peptide fragment of the present invention can be used to cut the power amplifier of the experiment. The treatment of the peptide fragment of the present invention can also improve the survival rate of the fish, only the room.

23 1299335 在Balb/c老鼠（約19〜21克重）腹腔中打入128 χ i〇6 cfu的綠勝於菌，60分鐘後，分別在老鼠腹腔中注射2/干的石斑魚本發明胜肽片段或每隻老鼠1〇〇 Vg萬古霉素。注射後的2小時、 _ 4小時、6小時，犧牲老鼠取一段丨公分的小腸，浸泡在1〇〇w哪中研 .冑，而後稀釋®液並塗在培養皿上，放人37°C培養箱，96G分鐘後計數菌落數目。結果 •如圖4所示，本實驗可驗證本發明胜肽片段在腸内抑制細菌的能力。在以腹腔注射25 的本發明胜肽片段240分鐘後，與注射1〇〇萬古霉素的效能幾乎相近。顯示石斑魚抗菌蛋白-3有可能用來作為取代抗生素的天然物質。實例四：本發明胜肽片段對吳郭魚及石斑魚的保護效果魚類實驗模 .利用一種創傷弧菌（204 ;從死去的吳郭魚中培養）的致死模式來測試本發明合成的石斑魚抗菌蛋白-3 (g~ple 5_25) 势责八 Oreochromis mossambicus)反石抵旁、{E· co/o/i/es)對抗嚴重感染的功效。每組的第一項測試需要50 隻吳郭魚（平均體重〇·2 g，身長3公分）：（a)第一組只注射創傷弧菌（厂· ; (b)在第二組中，同時注射創傷弧菌與石斑魚抗菌蛋白―3 (g-pie 5-25)胜狀的浪合物； (c)第三組以創傷弧菌預處理18 0分鐘後，注射該石班魚抗菌蛋白-3 (g-pie 5-25)胜肽；（d)第四組以石斑魚抗菌蛋白 24 1299335 -3 (g-Ple 5-25)胜肽預處理18〇分料，注射創傷弧第五組只注射PBS;以及⑴第六組只注射石斑魚抗菌蛋二 (g-Ple5-25)胜肽。第二項測試，每組包含3〇隻石斑均體重0.2g，身長3公分赞 L ^ 身长^刀）.（a)第一組只注射創傷弧菌厂叫b)在第二組中，同時注射創傷弧菌與石斑备抗菌蛋白-3 (g-ple 5-25)胜狀的混合物；⑷帛三組㈣傷弧菌預處理60分鐘後，注射石斑魚抗菌蛋白_3 5-25)胜肽；（d)第四組以石斑魚抗菌蛋白_3 (g—pk 胜肽預處理60分鐘後，注射創傷弧菌；（e)第五組只注射 PBS ;以及（f)第六組只注射石斑魚抗菌蛋白_3 (g_pi e卜％胜肽。所有實驗均從肛門以腹腔給予，且石斑魚抗菌蛋白3 (g-ple 5-25)胜肽的劑量為每條魚〇. j # g，在1〇 “ ^中戋單獨^射PBS。創傷弧菌（204)的接種為每條魚注射〗〇#}，丄 X 10 cfu。魚類維持在5-L的水槽中，溫度約為27 γ。每日紀錄其死亡率，且每日兩次以手餵食魚類商品化飼料。死亡率圖示以百分比（％)表示，組間差距以Fisher精確檢定法 (Fisher’ s exact test)分析及測試。强護魚類感染效旲創傷弧菌㈣//7///^幻（2〇4)為陳俊堯助理教授 (慈濟大學，花蓮，台灣）從死亡的吳郭魚中所分離，此吳郭魚生長於南台灣一水產池塘。感染創傷弧菌（2〇4)的吳郭魚及石斑魚所爆發的流行，已在台灣造成大量死亡與嚴重的經濟損失。石斑魚抗菌蛋白-3胜肽的抗菌活性已在上述内容描述，且在體外顯示對抗魚類病源創傷弧菌（2〇4)的優越抗菌活 25 1299335 性。因此，本發明選擇一種石斑魚抗菌蛋白-3(g-ple5-25) 胜肽，來測試其在吳郭魚及石斑魚的保護效果。在第-個實驗中，第五組及第六組以單一劑量（每條魚O.^g，溶於 • I t，或單獨注射PBS)在吳郭魚或石斑魚中注射石斑备抗菌蛋白-3 (g-ple5_25)胜肽。在168小時的試驗中，並益魚類死亡(如圖5Α’5β)’顯示石斑魚抗菌蛋白-3(g-ple5, 胜肽對魚類不具毒性，且在m組中，雖然所使用的魚體型很小，但沒有因為注射技術導致死亡的現象。然而，在⑽ 小時後’只以創傷弧菌注射的吳郭魚累積死亡率已達到 96%，而同時接受創傷弧菌與石斑魚抗菌蛋白-3 (g-ple 5_25) :肽混合注射的組別累積死亡率只有3〇%。接受該胜肽與細囷此合的組別’比只接受細菌注射的組別死亡率明顯較低。另外，在以創傷弧菌（2G4泣射前，先以石斑魚抗菌蛋白_3 (g-ple 5-25)胜肽注射18〇分鐘的吳郭魚，累積死亡率為 :%。然而，在以石斑魚抗菌蛋白_3 ("le 5_25)胜肽注射珂，先以創傷弧菌（204)注射18〇分鐘的吳郭魚，累積死亡率為78%(如圖5A)。在石斑魚的實驗組，168小時後’只注射創傷弧菌(2〇4)的石斑魚存活率A 0%，而注射創傷弧菌(204)與石斑魚抗菌蛋白一3 (g-ple 5-25)胜肽混合物的石斑魚累積死亡率為3〇%。接受胜肽與細菌合併注射的組別，比只接受細菌注射的組別，具有顯著較低的死亡率。另外，在以創傷弧菌（2〇4)注射蝻，先以石斑魚抗菌蛋白_3 (g_ple 5_25)胜肽注射⑽分鐘的組別，石斑魚累積死亡㈣17%。然而，在以石斑魚抗菌23 1299335 In the abdominal cavity of Balb/c mice (about 19 to 21 grams), 128 χ i〇6 cfu of green is better than bacteria. After 60 minutes, 2/dry grouper is injected into the abdominal cavity of mice. Fragment or 1 〇〇Vg vancomycin per mouse. 2 hours, _ 4 hours, 6 hours after the injection, the rats were sacrificed to take a small intestine of the sputum centimeters, soaked in 1 〇〇w, and then immersed in the culture dish and applied to the culture dish, and cultured at 37 ° C. The box counted the number of colonies after 96G minutes. Results • As shown in Fig. 4, this experiment can verify the ability of the peptide fragments of the present invention to inhibit bacteria in the intestine. After 240 minutes of intraperitoneal injection of 25 of the peptide fragments of the present invention, the efficacy of injection of 1 〇〇 vancomycin was almost similar. It is shown that grouper antibacterial protein-3 may be used as a natural substance to replace antibiotics. Example 4: Protective effect of the peptide fragment of the present invention on Wu Guoyu and grouper fish. Experimental model of fish. The grouper antibacterial protein-3 synthesized by the present invention was tested by lethal mode of Vibrio vulnificus (204; cultured from dead Wuguo fish). (g~ple 5_25) The blame of eight Oreochromis mossambicus) against the stone, {E· co/o/i/es) against the effects of serious infections. The first test in each group required 50 Wu Guoyu (average weight 〇 · 2 g, 3 cm in length): (a) The first group was only injected with Vibrio vulnificus (factory; (b) in the second group, Simultaneous injection of Vibrio vulnificus and grouper antibacterial protein-3 (g-pie 5-25) spurs; (c) The third group was pretreated with Vibrio vulnificus for 18 minutes, and injected with the stonefish antibacterial protein - 3 (g-pie 5-25) peptide; (d) The fourth group was pretreated with grouper antibacterial protein 24 1299335 -3 (g-Ple 5-25) peptide 18 〇, injection wound fifth group only PBS was injected; and (1) the sixth group was only injected with the grouper antibacterial egg two (g-Ple5-25) peptide. In the second test, each group contained 3 plaques with a body weight of 0.2 g and a length of 3 cm. L ^ Length ^ Knife). (a) The first group only injected Vibrio vulnificus factory called b) in the second group, simultaneously injected with a mixture of Vibrio vulnificus and gypsum antibacterial protein-3 (g-ple 5-25); (4) 帛 three groups (four) 60 minutes after the pretreatment of Vibrio cholerae, injection of grouper antibacterial protein _3 5-25) peptide; (d) the fourth group of grouper antibacterial protein _3 (g-pk peptide pretreatment 60 minutes later , injection of Vibrio vulnificus; (e) fifth Only PBS was injected; and (f) the sixth group was only injected with the grouper antibacterial protein _3 (g_pi ebu% peptide). All experiments were given intraperitoneally from the anus, and grouper antibacterial protein 3 (g-ple 5-25) peptide The dose is for each rod. j # g, in 1〇" ^ 戋 ^ ^ PBS alone. Inoculation of Vibrio vulnificus (204) for each fish injection 〇 } #}, 丄 X 10 cfu. Fish maintained in In a 5-L sink, the temperature is about 27 γ. The mortality rate is recorded daily, and the commercial feed of fish is fed by hand twice a day. The mortality rate is expressed as a percentage (%), and the gap between the groups is accurate by Fisher. Fisher's exact test analysis and testing. Strong fish protection effect Vibrio vulnificus (4) // 7///^ illusion (2〇4) is Assistant Professor Chen Junxi (Tzu Chi University, Hualien, Taiwan) from Separated from the dead Wu Guoyu, this Wu Guoyu was born in a fish pond in southern Taiwan. The outbreak of Wu Guoyu and grouper infected with Vibrio vulnificus (2〇4) 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 above and displayed in vitro. The superior antibacterial activity against the fish pathogen Vibrio vulnificus (2〇4) is 25 1299335. Therefore, the present invention selects a grouper antibacterial protein-3 (g-ple5-25) peptide to test its in the squid and grouper Protective effect. In the first experiment, the fifth and sixth groups were injected with grouper in Wu Guoyu or grouper in a single dose (O.g. per fish, dissolved in It, or injected with PBS alone). Prepare antibacterial protein-3 (g-ple5_25) peptide. In the 168-hour test, the fish died (see Figure 5Α '5β)', showing grouper antibacterial protein-3 (g-ple5, the peptide is not toxic to fish, and in the m group, although the fish used is very Small, but there is no death caused by injection technology. However, after (10) hours, the cumulative mortality rate of Wu Guoyu, which was only injected with Vibrio vulnificus, reached 96%, while receiving both Vibrio vulnificus and grouper antibacterial protein-3 ( G-ple 5_25): The cumulative mortality rate of the peptide mixed injection group was only 3%. The group receiving the peptide and the fine sputum was significantly lower than the group receiving only the bacterial injection. Vibrio vulnificus (2G4 was first injected with grouper antibacterial protein_3 (g-ple 5-25) peptide for 18 minutes before the cumulative mortality rate was: %. However, in the grouper antibacterial protein _3 ("le 5_25) peptide injection, first injection of Vibrio vulnificus (204) for 18 minutes of Wu Guoyu, cumulative mortality rate of 78% (Figure 5A). In the grouper experimental group, 168 hours After the 'injection of Vibrio vulnificus (2〇4), the survival rate of grouper was A 0%, while the injection of Vibrio vulnificus (204) and The grouper's cumulative mortality rate of grouper antibacterial protein-3 (g-ple 5-25) peptide mixture was 3〇%. The group receiving the combination of peptide and bacteria was significantly more than the group receiving only bacteria. Low mortality. In addition, in the group injected with Vibrio vulnificus (2〇4), the grouper was first injected with the grouper antibacterial protein_3 (g_ple 5_25) peptide for 10 minutes, and the grouper died cumulatively (4) 17%. Grouper antibacterial

26 1299335 蛋白-3 (g-pie 5-25)胜肽注射前，先以創傷弧菌（204)注射 60分鐘的吳郭魚，累積死亡率為97%(如圖5B)。實例五：對感染綠膿桿菌老鼠的保護效果逢膝桿菌感染動物模式實驗共分七組，每組20隻Balb/c老鼠(每隻大約20〜22克）。對照組：每隻老鼠打入1· 28 X 106 cfu綠膿桿菌。12小時後開始記錄死亡的數目。10 :每隻老鼠先在腹腔注射1〇的本發明胜肽片段，540分鐘後，打入1· 28 X 106 cfu綠膿桿菌。12小時後開始記錄死亡的數目。20 :每隻老鼠先在腹腔注射2〇 //g的本發明胜肽片段， 540分鐘後，打入1· 28 X 1〇6 cfu綠膿桿菌。12小時後開始記錄死亡的數目。25 :每隻老鼠先在腹腔注射25 的本發明胜肽片段，540分鐘後，打入1. 28 X 106 cfu綠膿桿菌。12小時後開始記錄死亡的數目。5〇 ••每隻老鼠先在腹腔注射50#g的本發明胜肽片段，54〇分鐘後，打入128χ1〇6 cfu綠膿桿菌。12小時後開始記錄死亡的數目。 100 ·每隻老鼠先在腹腔注射1〇〇 μ的本發明胜肽片段，54〇分鐘後，打26 1299335 Protein-3 (g-pie 5-25) peptides were injected with Vibrio vulnificus (204) for 60 minutes before the cumulative mortality rate was 97% (Fig. 5B). Example 5: Protective effect against Pseudomonas aeruginosa mice The animal model of Klebsiella pneumoniae was divided into seven groups of 20 Balb/c mice (about 20 to 22 grams each). Control group: 1 · 28 X 106 cfu Pseudomonas aeruginosa was injected into each mouse. The number of deaths began to be recorded after 12 hours. 10: Each mouse was intraperitoneally injected with 1 〇 of the peptide fragment of the present invention, and after 540 minutes, 1·28 X 106 cfu of Pseudomonas aeruginosa was injected. The number of deaths began to be recorded after 12 hours. 20: Each mouse was intraperitoneally injected with 2 〇 //g of the peptide fragment of the present invention, and after 540 minutes, 1·28 X 1 〇 6 cfu of Pseudomonas aeruginosa was ingested. The number of deaths began to be recorded after 12 hours. 25: Each mouse was intraperitoneally injected with 25 peptide fragments of the present invention, and after 540 minutes, 1.28 X 106 cfu Pseudomonas aeruginosa was ingested. The number of deaths began to be recorded after 12 hours. 5〇•• Each mouse was intraperitoneally injected with 50#g of the peptide fragment of the present invention, and after 54 minutes, 128χ1〇6 cfu of Pseudomonas aeruginosa was ingested. The number of deaths began to be recorded after 12 hours. 100 · Each mouse was intraperitoneally injected with 1 μ μ of the peptide fragment of the present invention, 54 minutes later,

入1· 28 X 1〇6 Cfu綠膿桿菌。12小時後開始記錄死亡的數目。獅：每隻老敗先在腹腔注射的本發明胜肽片段，54()分鐘後，打人l 28 X 1〇6 cfu綠膿桿菌。12小時後開始記錄死亡的數目。結果如圖6所不，本實驗驗證石斑魚本發明胜肽片段可以當作預防之藥物，防/口老鼠在細囷侵襲時降低壯率，但是必須在—個適當的濃度下，過多 27 1299335 或過y都不適g。最佳劑量為每隻老鼠25 ，而5〇#g/m〇use效率次之，對照組老鼠並無注射本發明胜肽片段，死亡率在24小時内達到最高。實例六·石斑魚抗菌蛋白—3 (g—ple 5—25)胜肽的藥物動力學分析羡物動力學分折方法在雄性吳郭魚（體重約73.7 ± 16.2克）體内進行抗菌蛋白 -3 (g-pie 5-25)胜肽的藥物動力學分析，先在吳郭魚體内以靜脈注射單一劑量（每條魚25 // g)的石斑魚抗菌蛋白一3 (g-ple 5-25)胜肽。在注射2〇, 30, 60, 1 20,以及180分鐘後，測定血清中的抗菌蛋白—3 (g—ple 5一25)胜肽濃度，每個時間點包含兩支吳郭魚的數值。在每個收集樣本的時間點，麻醉吳郭魚並採血，離心血液樣本以獲得血清。抗菌蛋白一3 (g-ple 5-25)胜肽在血清中的濃度以LC-MS/MS決定。取含有〇. U TFA的酸化水950 // 1以及血漿樣品50 # i加入聚丙烯官中，並以混合器混合i 〇秒。固相萃取管柱spE cartridges (Oasis HLB 1 ml/30 mg (Waters, Milford, MA)) 以2 ml甲醇進行前處理，再調整為2 ml水及2 ml酸化水（含有〇· 1% TFA);之後將樣品注入spE管柱中，以2 ml酸化水 (含有0· 1% TFA)及2 ml水清洗管柱。將75 χ 12〇—_的聚丙烯官放在SPE管柱下方，樣品以〇·5 ml ACN沖出，並在 3〇氮氣氣流吹拂下蒸發乾燥。之後將乾燥後的樣品溶解在 1 00 // 1 80%的ACN液體中，並以混合器混合6〇秒，再將樣品溶液放入螺旋瓶中供LC-MS/MS分析。LC-MS/MS儀器包含一個配備有樣品冷卻器（溫度設定為5 。〇的Waters 28 1299335Into 1·28 X 1〇6 Cfu Pseudomonas aeruginosa. The number of deaths began to be recorded after 12 hours. Lion: Each of the old peptides of the present invention injected intraperitoneally, after 54 () minutes, hit humans 28 x 1 6 cfu Pseudomonas aeruginosa. 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 prophylactic drug, and the anti-mouth mice can reduce the growth rate when invasive, but must be at an appropriate concentration, too much 27 1299335 or After y are not g. The optimal dose was 25 per mouse, and the 5 〇#g/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 grouper antibacterial protein-3 (g-ple 5-25) peptide 羡 kinetics method for the determination of antibacterial protein-3 in male squid (body weight about 73.7 ± 16.2 g) G-pie 5-25) Pharmacokinetic analysis of peptides, first in the Wu Guo fish body intravenous injection of a single dose (25 / g per fish) grouper antibacterial protein-3 (g-ple 5-25) Peptide. After 2, 30, 60, 1 20, and 180 minutes of injection, the concentration of the antibacterial protein-3 (g-ple 5-25) peptide in the serum was determined, and each time point contained the value of two Wu Guoyu. 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-ple 5-25) peptide in serum is determined by LC-MS/MS. Acidified water containing 〇. U TFA 950 // 1 and plasma sample 50 # i were added to the polyacrylic acid official and mixed with a mixer for 1 sec. Solid phase extraction column spE cartridges (Oasis HLB 1 ml/30 mg (Waters, Milford, MA)) pretreated with 2 ml of methanol, adjusted to 2 ml water and 2 ml acidified water (containing 〇·1% TFA) The sample was then injected into the spE column and the column was washed with 2 ml of acidified water (containing 0.1% TFA) and 2 ml of water. A 75 χ 12 〇-_ polypropylene was placed under the SPE column, and the sample was punched out with 〇·5 ml ACN and evaporated to dryness under a nitrogen purge of 3 Torr. The dried sample was then dissolved in 1 000 // 1 80% ACN liquid and mixed in a mixer for 6 sec seconds, and the sample solution was placed in a screw bottle for LC-MS/MS analysis. The LC-MS/MS instrument includes a Waters 28 1299335 equipped with a sample cooler (temperature setting of 5. 〇)

Alliance 2795 HPLC。質譜儀為以正向ESI模式操作的Alliance 2795 HPLC. The mass spectrometer is operated in forward ESI mode

Quattro Ultima (Waters/Micromass， Manchester， UK)。所有儀器皆以 MassLynx 4· 0 (Waters/Micromass)軟體控制。液態層析管柱為Luna amino管柱，顆粒大小為5 —_ ，内後為 150 X 2.0-mm (Phenomenex，Torrance，CA)。使用 ACN與水比例為80/20 v/v，並含有1%氨水的混和液為流動相，將樣品沖出，沖出體積為μΐ。The injection volume was 1 〇 μ 1 ·該質譜儀以正向ESI模式運作，其條件如下：毛細管電壓為3·5 kV，錐孔電壓為35V，熱源溫度為8(rc，脫溶劑氣流溫度為320 °C，脫溶劑器流量（N2)為每小時550公升，碰撞能置為60 eV，碰撞氣流為氬氣。數據在SRM模式下利用m/z 779 > 120之轉換獲得。垄iHL魚中的分析钴类以靜脈注射單一劑量的石斑魚抗菌蛋白_3 (g-ple 5-25) 合成胜肽到吳郭魚體内，該本發明石斑魚抗菌蛋白—3 (g—pie 5-25)胜肽顯示一下降的藥物動力學反應曲線（如圖7a)。靜脈注射的平均最高濃度，在2〇分鐘後為1342· 13 ng/mi。注射後半衰期約為60-80分鐘。以體重約251〜300克的大鼠進行實驗，此每個實驗組由3隻大氣進行實驗。分為頸靜脈（IV)(圖7B)、腹腔⑽（圖7C)、皮下（sc)(圖7D)三個不同部位的組別，每隻注射1〇〇〇 Μ的本發明石斑魚抗菌蛋白〜3 (g-Pie 5-25)胜肽，注射後在1〇分鐘，2〇分鐘，3〇分鐘，丨小時， 29 1299335 小時’ 3小時，各採丨ml的血液，再利用&_s測試血清濃度。結果如圖7B ’ 7C，7D所示。本實驗驗證本發明石斑魚抗菌蛋白一3 (g—心 25)胜肽並不會祕在錢血管壁上。符合在哺乳鋪侧發的特色。除非有其他定義，本文中所有技術與科學術語的意義，為 =本I明所屬領域具通常知識者所普遍了解。在此領域具通 =头識者也會判斷本文中所述之任何類似或相等方法及材料’以用於實施或測試本發明。【圖式簡單說明】田結合洋細敘述，所附的申請專利範圍，及圖式閱讀時，將更能了解下列之發明内容，在圖中：圖1為長條圖，係顯示相對性RT—PCR分析特定組織(1A)以及Lps刺激後 ^ 几菌蛋白一3基因表現的結果。圖ία中各組織以字母代表，[Quattro Ultima (Waters/Micromass, Manchester, UK). All instruments are controlled by MassLynx 4· 0 (Waters/Micromass) software. The liquid chromatography column is a Luna amino column with a particle size of 5 - _ and an internal 150 X 2.0-mm (Phenomenex, Torrance, CA). A mixture of ACN and water with a ratio of 80/20 v/v and containing 1% ammonia was used as the mobile phase, and the sample was punched out to a volume of μΐ. The injection volume was 1 〇μ 1 · The mass spectrometer operates in forward ESI mode with the following conditions: capillary voltage is 3.5 volts, cone voltage is 35V, heat source temperature is 8 (rc, desolvation gas flow temperature is 320 °C, the desolventizer flow rate (N2) is 550 liters per hour, the collision energy is set to 60 eV, and the collision gas flow is argon. The data is obtained by conversion of m/z 779 > 120 in SRM mode. Analytical cobalt was synthesized by intravenous injection of a single dose of grouper antibacterial protein_3 (g-ple 5-25) into the body of Wu Guoyu. The grouper antibacterial protein-3 (g-pie 5-25) of the present invention The peptide showed a decreasing pharmacokinetic response curve (Figure 7a). The average highest concentration of intravenous injection was 1342·13 ng/mi after 2 minutes. The half-life after injection was about 60-80 minutes. Experiments were carried out in rats of ~300 g, each of which was tested by 3 atmospheres, divided into jugular vein (IV) (Fig. 7B), abdominal cavity (10) (Fig. 7C), and subcutaneous (sc) (Fig. 7D). Groups of different parts, each injection of 1 〇〇〇Μ of the grouper antibacterial protein ~3 (g-Pie 5-25) peptide After the injection, in 1 minute, 2 minutes, 3 minutes, 丨 hours, 29 1299335 hours '3 hours, each ml of blood was collected, and the serum concentration was measured using &_s. The results are shown in Fig. 7B '7C, 7D This experiment demonstrates that the grouper antibacterial protein-3 (g-heart 25) peptide of the present invention is not secreted on the wall of the blood vessel. It conforms to the characteristics of the side of the breastfeeding shop. Unless otherwise defined, all the techniques and sciences in this article. The meaning of the term is generally understood by those of ordinary skill in the art to which the present invention pertains. In this field, the person skilled in the art will also judge any similar or equivalent methods and materials described herein for use in the implementation or testing of this document. Inventive. [Simplified description of the schema] Tian Tianyang's detailed description, the scope of the attached patent application, and the reading of the schema will better understand the following inventions. In the figure: Figure 1 is a bar graph showing relative RT-PCR analysis of specific tissue (1A) and Lps-stimulated results of the expression of the genomic protein-3 gene. The tissues in Figure ία are represented by letters, [

為肝臟Η為〜臟，κ為頭腎，G為總，M為肌肉，！為小腸，s為皮膚，B 為血液。圖2為長條圖，係顯示相對性RT—pcR分析⑽㈤及邮c (2B)刺激後’對石斑魚抗菌蛋自—3基因表現的影響。九圖3 &組織免疫染色照片’係顯示石斑魚肥及小腸中免疫木色之結果。（3A)右方為靜置於事先採血的血清抗體中的石斑魚腺’㈣照組’而左方為靜置於多株抗體中的石斑魚肥。 (B)右方為靜置於事先採血的血清抗體中的石斑魚小腸，為對…、組，而左方為靜置於多株抗體中的石斑魚小腸。照片中的前號指示出—個典型的染色區域。a為肥；b為小腸。 1299335 圖4為曲線圖，係顯示在以綠膿桿菌感染β—/。老鼠後，本心月胜肽片段與100 萬古霉素具有類似在腸内抑制細菌的能力。 van以腹腔主射1〇〇料萬古霉素的老鼠；咖··以腹腔注射25料本發明胜肽片段的老鼠。圖5為曲線圖，係顯示石斑魚抗菌蛋白_3 (口^ ⑸胜狀保護吳 U5A)或石斑魚(5B)避免受創傷弧賊染致死的能力。在此實驗中共有、、、且（a)第組只注射創傷弧菌厂· ; (b)在第二組中，同時注射創傷弧菌與石斑魚抗菌蛋白—3 (g—ple5 — 25) 胜肽的混合物；（c)第三組以創傷弧菌預處理i8〇分鐘後，注射該石斑魚抗菌蛋白—3 (g—ple 5 —25)胜肽；⑷第四組以石斑魚抗菌蛋白-3 (g-ple 5一25)胜肽預處理18〇分鐘後，注射創傷弧菌；（e)第五組只注射pBS;以及（f)第六組只注射石斑魚抗菌蛋白-3 (g—ple 5 —25)胜肽。在第三組及第四組中，吳郭魚與石斑魚的條件不同，在吳郭魚的感染時間為18〇分鐘，而石斑魚為60分鐘。在i 68小時後決定累積死亡率。 204 pie ·吳郭魚以每條魚1χ1〇3 cfu的劑量腹腔注射創傷弧菌，二小時後，接受每條魚〇· 1 # g抗菌蛋白一3 (g—ple 5 —25) 的腹腔注射劑量；ple_2〇4 :吳郭魚接受每條魚〇·丨# g抗菌蛋白-3 (g-ple 5-25)的腹腔注射劑量，三小時後，接受每條魚lxlO3 cfu創傷弧菌的腹腔注射劑量；2〇4 + ple :吳郭备同日寸接文母條魚〇· 1 # g抗菌蛋白—3 (g—pie 5 —25)與ixl〇3cfu 創傷弧菌的腹腔注射劑量；204 :每條吳郭魚接受1χ1〇3 cfu 創傷弧菌（204)的腹腔注射劑量；ple :每條吳郭魚接受〇.1“忌抗菌蛋白-3 (g-pie 5-25)的腹腔注射劑量。 31For the liver, it is ~ dirty, κ is the head kidney, G is the total, and M is the muscle! For the small intestine, s is the skin and B is the blood. Figure 2 is a bar graph showing the effects of relative RT-pcR analysis (10) (5) and post-c (2B) stimulation on the performance of the anti-strain of the grouper antibacterial egg. Nine Figure 3 & Tissue Immunostaining Photographs show the results of immortalized color in grouper and small intestine. (3A) The right side is the grouper gland 's (4) group in the serum antibody that has been previously collected, and the left side is the grouper fertilizer which is placed in the multi-plant antibody. (B) The right side is the grouper small intestine that is placed in the serum antibody of the pre-collected blood, which is the pair, the group, and the left side is the grouper small intestine that is placed in the multiple antibodies. The top number in the photo indicates a typical stained area. a is fat; b is small intestine. 1299335 Figure 4 is a graph showing the infection of β-/ with Pseudomonas aeruginosa. After the mouse, the heart-to-heart peptide fragment has the ability to inhibit bacteria in the intestine similarly to 100 vancomycin. Van used the abdominal cavity to shoot a rat with vancomycin; the coffee was injected intraperitoneally into the mouse of the peptide fragment of the invention. Figure 5 is a graph showing the ability of grouper antibacterial protein _3 (mouth ^ (5) to protect Wu U5A) or grouper (5B) to avoid death by traumatic arc thieves. In this experiment, there were, and (a) the first group was injected only with the Vibrio Vibrio plant; (b) In the second group, the simultaneous injection of Vibrio vulnificus and grouper antibacterial protein-3 (g-ple5-25) a mixture of peptides; (c) the third group was pretreated with Vibrio vulnificus for 9 minutes, and the grouper antibacterial protein-3 (g-ple 5-25) was injected; (4) the fourth group was grouper antibacterial protein-3 ( G-ple 5-25) Peptide pretreatment for 18 minutes, injection of Vibrio vulnificus; (e) fifth group only injected pBS; and (f) sixth group only injected grouper antibacterial protein-3 (g-ple 5 —25) peptide. In the third and fourth groups, the conditions of the Wu Guoyu and the grouper were different, the infection time of the Wu Guoyu was 18〇 minutes, and the grouper was 60 minutes. The cumulative mortality rate was determined after 68 hours. 204 pie · Wu Guoyu was intraperitoneally injected with Vibrio vulnificus at a dose of 1χ1〇3 cfu per fish. After two hours, each fish was given an intraperitoneal injection of 1 #g antibacterial protein-3 (g-ple 5-25). Dose; ple_2〇4: Wu Guoyu receives an intraperitoneal dose of each fish 〇·丨# g antibacterial protein-3 (g-ple 5-25), and after three hours, receives the peritoneal cavity of each fish lxlO3 cfu V. vulnificus Injection dose; 2〇4 + ple: Wu Guobei same day inch article motherfish rodfish 1 1 g antibacterial protein-3 (g-pie 5-25) and ixl〇3cfu Vibrio vulnificus intraperitoneal injection dose; 204: each Wu Guoyu received an intraperitoneal dose of 1χ1〇3 cfu of Vibrio vulnificus (204); ple: each Wu Guoyu received an intraperitoneal dose of 〇.1 “g-pie 5-25”.

(S 1299335 圖6為曲線圖’係顯示先在老鼠體内以腹腔注射不同劑量的本發明胜肽片段540分鐘後，再以腹腔注射丨· 28 X 1〇6 cfu的綠膿桿菌，並觀察各組的死亡率。對照組：每隻老鼠打入1.找χ cfu綠膿桿菌小時後開始記錄死亡的數目。 1〇 :每隻老鼠先在腹腔注射10 /zg的本發明胜肽片段，54〇分鐘後，打入 1·28 X 1〇6 cfu綠膿桿菌。20 :每隻老鼠先在腹腔注射2() 的本發明胜肽片段，540分鐘後，打入1.28 X 106 cfu綠膿桿菌。25 :每隻老鼠先在腹腔注射25 //g的本發明胜肽片段，540分鐘後，打入1. 28 X 1〇6 Cfu 綠膿桿菌。50 :每隻老鼠先在腹腔注射50 /zg的本發明胜肽片段，540分鐘後，打入1. 28 X 106 cfu綠膿桿菌。100 :每隻老鼠先在腹腔注射ι〇〇料的本發明胜肽片段，540分鐘後，打八1· 28 X 106 cfu綠膿桿菌。2〇〇 :每隻老鼠先在腹腔注射200 /zg的本發明胜肽片段，540分鐘後，打入丨28 X 1〇6 cfu綠膿桿菌。圖7為曲線圖及長條圖，圖7A係顯示在吳郭魚體内注射單一劑量（每條魚25# g)的石斑魚抗菌蛋白-3 (g-ple 5-25)胜肽的藥物動力曲線圖，每個時間點包含兩條吳郭魚的數值。血清濃度以靜脈給予後每個指定的時間點決定。圖7B〜7D係顯示在大鼠體内注射單一劑量（每隻1 000 # g)的石斑魚抗菌蛋白-3 (g-ple 5-25)胜肽的藥物動力曲線圖，每個時間點包含三隻大鼠的數值。血清濃度以靜脈（7B)，腹腔（7C)，及皮下（7D)注射後每個指定的時間點決定。(S 1299335 Figure 6 is a graph 'showing 540 minutes after intraperitoneal injection of different doses of the peptide fragments of the present invention in mice, and then intraperitoneally injected with 丨·28 X 1〇6 cfu of Pseudomonas aeruginosa and observed Mortality of each group. Control group: Each mouse was scored 1. Find the number of deaths after cfu Pseudomonas aeruginosa. 1〇: Each mouse was injected intraperitoneally with 10/zg of the peptide fragment of the invention. After 54 minutes, score 1·28 X 1〇6 cfu Pseudomonas aeruginosa. 20: Each mouse was injected intraperitoneally with 2() of the peptide fragment of the invention, and after 540 minutes, 1.28 X 106 cfu pus. Bacillus. 25: Each mouse was intraperitoneally injected with 25 //g of the peptide fragment of the invention, and after 540 minutes, 1.28 X 1〇6 Cfu Pseudomonas aeruginosa was infused. 50: Each mouse was intraperitoneally injected 50 /zg of the peptide fragment of the present invention, after 540 minutes, scored 1.28 X 106 cfu Pseudomonas aeruginosa. 100: Each mouse was intraperitoneally injected with the peptide fragment of the present invention, 540 minutes later,八1·28 X 106 cfu Pseudomonas aeruginosa. 2〇〇: Each mouse was intraperitoneally injected with 200 / zg of the peptide fragment of the invention, after 540 minutes Into 丨28 X 1〇6 cfu Pseudomonas aeruginosa. Figure 7 is a graph and a bar graph, Figure 7A shows a grouper antibacterial protein-3 injected in a single dose (25# g per fish) in Wu Guoyu. (g-ple 5-25) The pharmacokinetic profile of the peptide, each time point contains the value of two Wu Guoyu. The serum concentration is determined at each specified time point after intravenous administration. Figures 7B to 7D are shown in A pharmacokinetic profile of a single dose (each of 1 000 # g) of grouper antibacterial protein-3 (g-ple 5-25) peptide was injected in rats, and the value of three rats was included at each time point. Concentrations were determined at each specified time point after intravenous (7B), abdominal (7C), and subcutaneous (7D) injections.

3232

Claims

1299335 ---Ί___」,„ Β| …-| 第095145242號專利申請案觀^ 2§修仅)正本 ϋ正申請專利範替換本(97年5月） ^^^ J 1· 一段胜肽片段，其由下列胺基酸序列之一組成： GFIFHIIKGLFHAGKMIHGLnRRRHGVEE (序列編號：1)或 GFIFHIIKGLFHAGKMIHGLV (序列編號：2)。 2·根據申請專利範圍第1項之胜肽片段，其中該胜肽片段具有對抗微生物感染的活性。1299335 ---Ί___", „ Β| ...-| Patent application No. 095145242 ^ 2 § repair only) Sakamoto is applying for a patent replacement (June 1997) ^^^ J 1· A peptide fragment , which consists of one of the following amino acid sequences: GFIFHIIKGLFHAGKMIHGLnRRRHGVEE (SEQ ID NO: 1) or GFIFHIIKGLFHAGKMIHGLV (SEQ ID NO: 2) 2. The peptide fragment according to claim 1 of the patent application, wherein the peptide fragment has an anti-microbial Infection activity.

3·根據申請專利範圍第2項之胜肽片段，其中該微生物為細菌。 4·根據申請專利範圍第3項之胜肽片段，其中該細菌係從以下組成之群中選出：李斯特菌（Listeria /H〇/7〇cy^:oge/]es)，藤黃微球菌 (#icrococcus ，金黃色葡萄球菌 (Staphylococcus aureus)，腹十生鍵球菌 (vSireptococcus pyoge/ies)，無乳鏈球菌（Streptococcus agalactiae)，表皮葡萄球菌（S^ap/iyJococcus epjder/widis)，葡萄球菌（S^ap/jyJococcus sp.)，木糖葡萄球菌（以ap/jyiococms xWosas)，肺炎鏈球菌（Streptococcus pneumoniae)，金黃色葡萄球菌 (Staphylococcus aureus subsp)，腸產氣桿菌 (Enterobacter aerogenes)，陰溝腸桿菌（^terotecter cJoacae subsp.)，石斑魚溶藻弧菌（斤◦叩er Wbrio alginolyticus)，轰酸免雷伯氏 m (Klebsiella oxytoca， fehWWbrio cost/coJa subsp.)，創傷弧菌（Fibrio n/ini/icus)，哈維氏弧菌（Fibrioharv^yi)，溶藻弧菌 1 1299335 ❹7年5·原8曰修(更)正本 (Vibrio ailginolyticus)，綠痕得m(Pseudomonas aer收i/]〇sa)，以及耶耳辛氏腸炎桿菌（}fersi/7ia enterocolitica subsp)。 5. —種醫藥組合物，其包含有效量之根據申請專利範圍第1、2、3或 4項之胜肽片段，及醫藥上可接受載劑。 6. —種具有對抗水生動物的微生物感染活性之醫藥組合物’其包含一段分離之蛋白質，其中該分離之蛋白質為由如申請專利範圍第1項所定義之序列編號1或序列編號2之胺基酸序列組成。 7·根據申請專利範圍第6項之醫藥組合物，其中水生動物為魚或瑕。 8.根據申請專利範圍第6項之醫藥組合物，其中該微生物為細菌。 9 ·根據申睛專利範圍弟8項之醫藥組合物，其中該水生動物以創傷弧菌F· vuhi/icus感染。 10· —種具有預防或治療哺乳類動物的感染性疾病之醫藥組合物，其包含一段分離之蛋白質，其中該分離之蛋白質為由如申凊專利範圍第1項所定義之序列編號1或序列編號2之胺基酸序列組成。 11 ·根據申請專利範圍第10項之醫藥組合物，其中該哺乳動物為人類。 12.根據申請專利範圍第1〇項之醫藥組合物，其中該感染性疾病為細菌感染疾病。 13·根據申請專利範圍第1〇項之醫藥組合物，其中該感染性疾病為敗血症。 23. The peptide fragment according to item 2 of the patent application, wherein the microorganism is a bacterium. 4. The peptide fragment according to item 3 of the patent application scope, wherein the bacteria is selected from the group consisting of: Listeria / H〇 / 7〇cy^: oge /]es, Micrococcus luteus (#icrococcus, Staphylococcus aureus, vSireptococcus pyoge/ies, Streptococcus agalactiae, Staphylococcus epidermidis (S^ap/iyJococcus epjder/widis), Staphylococcus (S^ap/jyJococcus sp.), Staphylococcus aureus (as ap/jyiococms xWosas), Streptococcus pneumoniae, Staphylococcus aureus subsp, Enterobacter aerogenes, culvert Enterobacteriaceae (^terotecter cJoacae subsp.), grouper bream er Wbrio alginolyticus, Klebsiella oxytoca, fehWWbrio cost/coJa subsp., Vibrio n/ Ini/icus), Vibrioharv^yi, Vibrio alginolyticus 1 1299335 ❹ 7 years 5 · original 8 曰 repair (more) original (Vibrio ailginolyticus), green marks m (Pseudomonas aer collection i /] 〇sa), and Fer si 肠 ( } } } fer fer fer fer fer fer fer fer 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. 5. Receiving a carrier. 6. A pharmaceutical composition having antimicrobial activity against aquatic animals' comprising an isolated protein, wherein the isolated protein is SEQ ID NO: 1 or a sequence as defined in claim 1 The pharmaceutical composition of the invention of claim 6, wherein the aquatic animal is a fish or a cockroach. The pharmaceutical composition according to claim 6 wherein the microorganism is a bacterium. 9 · A pharmaceutical composition according to the scope of the patent application, wherein the aquatic animal is infected with Vibrio vulnificus F· vuhi/icus. 10· A pharmaceutical composition having an infectious disease for preventing or treating mammals, It comprises an isolated protein, wherein the isolated protein is an amino acid sequence set of SEQ ID NO: 1 or SEQ ID NO: 2 as defined in claim 1 of the scope of the patent. . 11. The pharmaceutical composition according to claim 10, wherein the mammal is a human. 12. The pharmaceutical composition according to the first aspect of the invention, wherein the infectious disease is a bacterial infection disease. 13. The pharmaceutical composition according to the first aspect of the invention, wherein the infectious disease is sepsis. 2