TWI394579B - Compositions for treating or preventing infection of riemerella anatipestifer - Google Patents
Compositions for treating or preventing infection of riemerella anatipestifer Download PDFInfo
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Description
本發明係關於一種對抗雷氏桿菌(Riemerella anatipestifer )感染之組合物。The present invention relates to a composition against infection by Riemerella anatipestifer .
雷氏桿菌是禽類的主要感染病原之一,可感染鴨類、火雞、雞、鵪鶉、雉、嘯聲天鵝、鵝等(Avian Dis 2005;49:104-7)。以鴨類而言,雷氏桿菌感染好發於幼鴨,經常伴隨敗血症(septicemia)及多發性漿膜炎(polyserositis),死亡率高,對養鴨產業影響很大(Avian Dis 1985;29: 128-35;及Avian pathol 1997;26:791-802)。然而,目前對於鴨類抵抗病原的防禦系統所知有限,即使已利用基因型變異發展出疫苗,但由於無法在不同血清型的雷氏桿菌產生交互保護(cross-protection),因此限制了該等疫苗在防制雷氏桿菌感染上的運用。也因此,目前大部分的養殖業者主要仍是使用抗生素來防治雷氏桿菌感染。常用的抗生素包括鹽酸頭孢(ceftriofur)、羅弗撒欣(enrofloxacin)、新生霉素(novobiocin)、鹽酸克林霉素(lincomycin)、磺胺類(sulphonamides)等。然而,這些抗生素會產生副作用,且可能導致抗藥性菌株之產生,甚至這些抗藥性菌株可能經由食物鏈進入人體,對人類產生不利影響(J Vet Diagn invest 2003;15:26-9;及Antimicrob Agents Chemother 2005;49:690-8)。Reptilii is one of the major infectious agents of poultry and can infect ducks, turkeys, chickens, crickets, crickets, swan, geese, etc. ( Avian Dis 2005; 49: 104-7). In the case of ducks, the infection with R. cerevisiae is common in young ducks, often accompanied by septicemia and polyserositis, and the mortality rate is high, which has a great impact on the duck industry ( Avian Dis 1985; 29: 128). -35; and Avian pathol 1997; 26:791-802). However, there is currently limited knowledge of defense systems against duck-resistant pathogens, and even if vaccines have been developed using genotypic variation, they are limited in their ability to produce cross-protection in different serotypes of R. falciparum. The use of vaccines to prevent infection with Rep. Therefore, most of the current breeders still use antibiotics to control R. falciparum infections. Commonly used antibiotics include ceftriofur, enrofloxacin, novobiocin, lincomycin, sulphonamides, and the like. However, these antibiotics can cause side effects and can lead to the development of drug-resistant strains, and even these drug-resistant strains may enter the human body via the food chain, adversely affecting humans ( J Vet Diagn invest 2003; 15:26-9; and Antimicrob Agents Chemother 2005; 49: 690-8).
因此,此一領域仍有需要發展可有效對抗雷氏桿菌感染的藥劑,特別是非抗生素類藥劑,用於在禽類養殖產業作為防治雷氏桿菌感染的藥劑。Therefore, there is still a need in this field to develop an agent that is effective against R. falciparum infection, particularly a non-antibiotic agent, for use in the poultry farming industry as a medicament for controlling infection with R. eutropha.
本發明係發現特定片段之抗菌蛋白具有對抗雷氏桿菌感染的功效。本發明突破目前防治禽類養殖的雷氏桿菌感染之疫苗效果有限及抗生素有副作用及抗藥性等問題,提供可有效對抗雷氏桿菌感染的抗菌蛋白,對於雷氏桿菌感染之防治有極大助益The present invention finds that a specific fragment of the antibacterial protein has an effect against infection by a bacterium. The invention breaks through the current limited effect of the vaccine for preventing and treating Atrabacterium infection in poultry breeding, and the antibiotic has side effects and drug resistance, and provides an antibacterial protein which can effectively resist the infection of the bacterium, and is greatly beneficial for the prevention and treatment of the infection of the bacterium
因此,在一方面,本發明提供一種用於治療或預防雷氏桿菌感染之組合物,其包括有效量之胜肽及載劑,該胜肽係選自下列所組成之群:Accordingly, in one aspect, the present invention provides a composition for treating or preventing a Reuters infection comprising an effective amount of a peptide and a carrier selected from the group consisting of:
(1)具有如SEQ ID NO: 1之胺基酸序列之胜肽;(1) a peptide having the amino acid sequence of SEQ ID NO: 1;
(2)具有如SEQ ID NO: 2之胺基酸序列之胜肽;(2) a peptide having the amino acid sequence of SEQ ID NO: 2;
(3)具有如SEQ ID NO: 3之胺基酸序列之胜肽;(3) a peptide having the amino acid sequence of SEQ ID NO: 3;
(4)具有如SEQ ID NO: 4之胺基酸序列之胜肽;及(4) a peptide having the amino acid sequence of SEQ ID NO: 4;
(5)以上(1)至(4)其中一或多項之組合。(5) A combination of one or more of the above (1) to (4).
在另一方面,本發明提供上述胜肽用於製備供治療或預防雷氏桿菌感染之藥物之用途。In another aspect, the invention provides the use of the above-described peptide for the preparation of a medicament for the treatment or prevention of a Re retrovirus infection.
在又一方面,本發明提供一種用於動物體外之殺死雷氏桿菌的方法,其包括以含有效量的上述胜肽與載劑之組合物,施用於可能含有雷氏桿菌之物品或環境。In still another aspect, the present invention provides a method for killing a bacterium of the bacterium in vitro, comprising administering an effective amount of the above-described peptide and a carrier to an article or environment that may contain a bacterium .
下文中將詳細描述本發明的各種具體實施例。本發明的其他特徵將藉由下列有關各種具體實施例的詳細說明以及申請專利範圍而清楚呈現。Various specific embodiments of the invention are described in detail below. Other features of the present invention will be apparent from the following detailed description of various embodiments.
相信在本發明所屬技術領域中具通常知識者在不需進一步說明之情況下可根據此處的描述利用本發明至其最廣範圍。因此,下列描述應被當作例示之目的而非以任何方式作為本發明之範圍的限制。It is believed that those skilled in the art of the invention can <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; Accordingly, the following description is to be considered as illustrative and not restrictive
除非另有說明,否則此處使用之全部技術和科學名詞與本發明所屬技術領域之技藝人士通常所瞭解的意義相同。All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to the invention.
此處所使用的冠詞「一」係指該冠詞的一或一個以上(即,至少一個)的文法受詞。The article "a" as used herein refers to one or more (ie, at least one) grammatical terms of the article.
在一方面,本發明提供一種用於治療或預防雷氏桿菌感染之組合物,其包括有效量之胜肽及載劑,該胜肽係選自下列所組成之群:In one aspect, the invention provides a composition for treating or preventing a Re retrovirus infection comprising an effective amount of a peptide and a carrier selected from the group consisting of:
(1)具有SEQ ID NO: 1之胺基酸序列之胜肽;(1) a peptide having the amino acid sequence of SEQ ID NO: 1;
(2)具有SEQ ID NO: 2之胺基酸序列之胜肽;(2) a peptide having the amino acid sequence of SEQ ID NO: 2;
(3)具有SEQ ID NO: 3之胺基酸序列之胜肽;(3) a peptide having the amino acid sequence of SEQ ID NO: 3;
(4)具有SEQ ID NO: 4之胺基酸序列之胜肽;及(4) a peptide having the amino acid sequence of SEQ ID NO: 4;
(5)以上(1)至(4)其中一或多項之組合。(5) A combination of one or more of the above (1) to (4).
在另一方面,本發明提供上述胜肽用於製備供治療或預防雷氏桿菌感染之組合物之用途。In another aspect, the invention provides the use of the above-described peptide for the preparation of a composition for the treatment or prevention of a Re retrovirus infection.
在又一方面,本發明提供一種用於動物體外之殺死雷氏桿菌之方法,其包括以含有效量的上述胜肽與載劑之組合物,施用於可能含有雷氏桿菌之物品或環境。In still another aspect, the present invention provides a method for killing a bacterium of the bacterium of the bacterium, comprising administering an effective amount of the above-mentioned peptide and a carrier to an article or environment which may contain a bacterium .
本文所使用的「蛋白」、「多肽」或「胜肽」乙詞可交互使用,是指由胺基酸經由肽鍵連結而成的生物性聚合物。可利用化學合成方式或遺傳工程技術製備特定序列之蛋白或多肽。As used herein, the terms "protein", "polypeptide" or "peptide" are used interchangeably and refer to a biological polymer obtained by linking amino acids via peptide bonds. A particular sequence of proteins or polypeptides can be prepared using chemical synthesis or genetic engineering techniques.
根據本發明之胜肽各自具有SEQ ID NO: 1、2、3、或4之胺基酸序列,可由習知的化學合成方式或遺傳工程技術並參酌本文之說明及教示製備而得。相關技術,包括聚合酶連鎖反應(PCR)擴增技術、核酸純化、質體、核酸片段之酵素處理、定序技術及蛋白質表現與合成,可參見Molecular Cloning:A Laboratory Manual,第二版,Cold Spring Harbor Laboratory Press,Sambrook J等人編著,1989,以及Current Protocols in Molecular Biology,Frederick M.A.等人編著,2001,John Wiley & Sons,Inc.。The peptides according to the invention each have the amino acid sequence of SEQ ID NO: 1, 2, 3, or 4 and can be prepared by conventional chemical synthesis or genetic engineering techniques and with reference to the teachings and teachings herein. Related technologies, including polymerase chain reaction (PCR) amplification techniques, nucleic acid purification, plastids, enzyme processing of nucleic acid fragments, sequencing techniques, and protein expression and synthesis, can be found in Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, edited by Sambrook J et al., 1989, and Current Protocols in Molecular Biology, edited by Frederick MA et al., 2001, John Wiley & Sons, Inc.
根據本發明之胜肽亦可自生物體(例如,魚或蝦類)分離而得。舉例而言,可從點帶石斑魚(Epinephelus coioides )分離出石斑魚抗菌蛋白-1(epinecidin-1),其具有SEQ ID NO: 1之胺基酸序列(參見DNA Cell Biol 2007;26: 403-13)。此外,可從草蝦(Penaeus monodon )分離出抗脂多醣分子(anti-lipopolysaccharide factor,ALF),其具有SEQ ID NO: 2之胺基酸序列;該抗脂多醣分子係一種革蘭氏陰性菌的內毒素,包括線性結構或環狀結構,其中該環狀結構係因該抗脂多醣因子之胺基酸序列中C端的胱胺酸與N端胱胺酸產生雙硫鍵而形成(參見Int immunopharmacol 2007;7: 687-700)。在一實施例中,將線性結構之分子置於10%二甲基亞碸(DMSO,Dimethyl sulfoxide)中,在室溫下反應24小時後即可得到該環狀結構之抗脂多醣因子。又,可從從吳郭魚(Oreochromis mossambicus )分離出肝臟抗菌胜肽(hepcidin),包括TH1-5及TH2-3,分別具有SEQ ID NO: 3及4之胺基酸序列(參見Mol immunol 2007;44: 1922-34)。The peptide according to the invention may also be isolated from a living organism (for example, fish or shrimp). For example, grouper antibacterial protein-1 (epinecidin-1) having the amino acid sequence of SEQ ID NO: 1 can be isolated from Epinephelus coioides (see DNA Cell Biol 2007; 26: 403-13) ). Furthermore, an anti-lipopolysaccharide factor (ALF) having the amino acid sequence of SEQ ID NO: 2 can be isolated from Penaeus monodon ; the anti-lipopolysaccharide molecule is a Gram-negative bacterium Endotoxin, including a linear structure or a cyclic structure, wherein the cyclic structure is formed by a disulfide bond between the C-terminal cystine acid and the N-terminal cystic acid in the amino acid sequence of the anti-lipopolysaccharide factor (see Int) Immunopharmacol 2007;7: 687-700). In one embodiment, the linear structure of the molecule is placed in 10% dimethyl sulfoxide (DMSO, Dimethyl sulfoxide), and the cyclic structure of the anti-lipopolysaccharide factor is obtained after reacting at room temperature for 24 hours. Further, hepatidin can be isolated from Oreochromis mossambicus , including TH1-5 and TH2-3, having the amino acid sequences of SEQ ID NOS: 3 and 4, respectively (see Mol immunol 2007). ;44: 1922-34).
根據本發明之胜肽包括變異體,其係指本質上同源於原始胜肽之胜肽,但由於一個或多個刪除、插入或置換,造成具有不同於本發明之胜肽的胺基酸序列,但保有與原始胜肽實質相同的生物活性。該置換可包括保留性置換之序列,意指已知胺基酸基團被一個具有類似生理化學特性之基團取代。在胜肽或蛋白質中,胺基酸之合適保留性置換,為此領域具通常知識者所熟知,且可在不改變結果分子的生物活性而完成。較佳地,此變異體的立體結構與原始蛋白質胜肽相同。A peptide according to the present invention includes a variant which refers to a peptide which is substantially homologous to the original peptide, but which results in an amino acid different from the peptide of the present invention due to one or more deletions, insertions or substitutions. The sequence, but retains substantially the same biological activity as the original peptide. The substitution may include a sequence of retention substitutions, meaning that the amino acid group is known to be substituted with 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 without altering the biological activity of the resulting molecule. Preferably, the variant has the same steric structure as the original protein peptide.
本文中所用「活性」乙詞係指具有對抗雷氏桿菌之效力。在一實施例中,所述活性可根據美國臨床微生物檢驗室標準委員會的操作手冊針對雷氏桿菌進行最低抑菌濃度測定(Minimum Inhibition Concentrations,MIC)而證明。如下述表1的結果顯示針對雷氏桿菌,本發明具有SEQ ID NO: 1之胺基酸序列之胜肽的MIC約為6.25-50 μg/ml;本發明具有SEQ ID NO: 2之胺基酸序列的環狀結構胜肽的MIC約為12.25-25 μg/ml,線性結構胜肽的MIC約為6.25-25 μg/ml;本發明具有SEQ ID NO: 3之胺基酸序列之胜肽的MIC約為25-400 μg/ml;以及本發明具有SEQ ID NO: 4之胺基酸序列之胜肽的MIC約為100-450 μg/ml。As used herein, the term "active" refers to the efficacy against R. vivax. In one embodiment, the activity can be demonstrated in accordance with the Minimization Inhibition Concentrations (MIC) of the R. cerevisiae according to the American National Laboratory for Clinical Microbiological Laboratory Standards. As shown in the following Table 1, the MIC of the peptide having the amino acid sequence of SEQ ID NO: 1 of the present invention is about 6.25-50 μg/ml for the R. cerevisiae; the present invention has the amino group of SEQ ID NO: 2. The cyclic structure of the acid sequence has a MIC of about 12.25-25 μg/ml, and the linear structure of the peptide has a MIC of about 6.25-25 μg/ml; the peptide having the amino acid sequence of SEQ ID NO: 3 of the present invention The MIC is about 25-400 μg/ml; and the MIC of the peptide having the amino acid sequence of SEQ ID NO: 4 of the present invention is about 100-450 μg/ml.
本文所使用的「治療或預防」包括針對疾病的醫療性處理或預防性處理兩者。需要此等處理者包括已罹患該疾病之個體以及需要預防該疾病發生之個體。因此,在一具體實施例中,根據本發明治療或預防疾病係指將本發明之胜肽投予至已感染雷氏桿菌之個體或具有感染雷氏桿菌可能及傾向之個體,或施用於前述個體可能接觸的環境,使該個體達到醫治、治療、減輕、緩和、改變、矯正、改善、促進或影響感染病徵之傾向。As used herein, "treatment or prevention" includes both medical treatment or prophylactic treatment for a disease. Those in need of such treatment include individuals who have developed the disease and individuals who need to prevent the disease from occurring. Therefore, in a specific embodiment, treating or preventing a disease according to the present invention means administering the peptide of the present invention to an individual who has been infected with R. vaginalis or an individual having the possibility and tendency to infect R. vaginalis, or is administered to the aforementioned The environment in which an individual may be exposed to the individual's tendency to heal, treat, alleviate, alleviate, alter, correct, improve, promote, or influence the symptoms of the infection.
本文所使用的「有效量」是指本發明之胜肽在接受其處理的個體中達到上述治療或預防的效果的劑量。本發明組合物的劑量可視各種因素而變動,例如,投藥途徑、接受該藥劑之個體的體重和品種,以及投藥目的。技藝人士可根據此處的揭示及此技藝已建立的方法依經驗決定個案的劑量。As used herein, "effective amount" refers to a dose of the peptide of the present invention that achieves the above-described therapeutic or prophylactic effect in an individual to which it is treated. The dosage of the composition of the present invention may vary depending on various factors such as the route of administration, the weight and variety of the individual receiving the agent, and the purpose of administration. The skilled person can determine the dosage of the case based on experience and methods established by the art.
為進行上述處理,本發明的組合物可進一步與可接受的載劑調配成所需劑型。此處「可接受的載劑」係指該載劑與本發明組合物內所含有效成分相容,其較佳為能穩定該活性成分並且對欲投予的個體或欲施用的環境無害。該載劑可作為活性成分的稀釋劑、載劑、賦形劑或基質。本發明之組合物可利用各種已知的常規方法與適當載劑調配成所需劑型。For carrying out the above treatment, the compositions of the present invention may be further formulated with an acceptable carrier in the desired dosage form. As used herein, "acceptable carrier" means that the carrier is compatible with the active ingredient contained in the compositions of the present invention, preferably to stabilize the active ingredient and not deleterious to the individual to be administered or the environment to be administered. The carrier can act as a diluent, carrier, excipient or base for the active ingredient. The compositions of the present invention can be formulated into the desired dosage forms using a variety of known conventional methods and suitable carriers.
在一具體實施例中,本發明的組合物係與醫藥上可接受的載劑調配成醫藥組合物。醫藥上可接受的載劑之實例包括但不限於賦形劑,例如,乳糖、右旋糖、蔗糖、山梨糖、甘露糖、澱粉、阿拉伯膠、磷酸鈣、褐藻酸鹽、黃蓍樹膠、明膠、矽酸鈣、微晶纖維素、聚乙烯吡咯啶酮、纖維素、滅菌水、糖漿和甲基纖維素;潤滑劑,例如,滑石粉、硬脂酸鎂和礦物油;濕潤劑;乳化和懸浮劑;保存劑,例如,羥基苯甲酸甲酯和丙酯;甜味劑;以及調味劑。In a specific embodiment, the compositions of the present invention are formulated into a pharmaceutical composition with a pharmaceutically acceptable carrier. Examples of pharmaceutically acceptable carriers include, but are not limited to, excipients such as lactose, dextrose, sucrose, sorbose, mannose, starch, gum arabic, calcium phosphate, alginate, tragacanth, gelatin , calcium citrate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, sterilized water, syrup and methylcellulose; lubricants, for example, talc, magnesium stearate and mineral oil; wetting agents; Suspending agents; preservatives, for example, methyl and propyl hydroxybenzoates; sweeteners; and flavoring agents.
醫藥組合物的形式可為錠劑、藥丸、粉末、糖錠、藥包、藥片、酏劑、懸浮液、乳劑、溶劑、糖漿、軟和硬明膠膠囊、栓劑、注射液和包裝粉末。在一特定實施中,本發明的組合物係注射液的形式。The pharmaceutical compositions may be in the form of lozenges, pills, powders, lozenges, sachets, troches, elixirs, suspensions, emulsions, solvents, syrups, soft and hard gelatin capsules, suppositories, injection solutions and packaging powders. In a particular implementation, the compositions of the invention are in the form of an injectable solution.
醫藥組合物可經由任何生理上可接受途徑,例如,口服、腸道外(例如,肌肉、靜脈、皮下、腹腔內)、經皮、直腸、吸入等方式予以輸送。在一特定實施中,本發明的組合物係經注射投與。The pharmaceutical composition can be delivered by any physiologically acceptable route, for example, orally, parenterally (e.g., muscle, intravenous, subcutaneous, intraperitoneal), transdermal, rectal, inhaled, and the like. In a particular implementation, the compositions of the invention are administered by injection.
除上述調配方式外,本發明的組合物亦可調配為飼料添加物,加至動物(例如,禽類)的飼料中,或調配成環境抗菌劑,施用於動物可能接觸的環境中(例如,禽類生長的養殖場或水域中),用以防治雷氏桿菌之感染。In addition to the above formulation, the compositions of the present invention may also be formulated as feed supplements, added to feeds of animals (eg, poultry), or formulated as environmental antibacterial agents, for application to an environment in which the animal may be exposed (eg, poultry Growing farms or waters) to control infection by R. eutropha.
本發明的胜肽對抗雷氏桿菌效果顯著,可在低濃度達抑菌效果濃度,其係直接破壞菌體細胞膜,導致細胞內物質流出細胞,造成細胞死亡,並已在動物活體證實可提高存活率,具極佳治療或預防雷氏桿菌感染的效果。The peptide of the invention has a remarkable effect against R. rees, and can reach a concentration of bacteriostatic effect at a low concentration, which directly destroys the cell membrane of the cell, causes the intracellular substance to flow out of the cell, causes cell death, and has been confirmed to improve survival in the living body of the animal. Rate, with excellent treatment or prevention of infection with Rep.
在一特定實例中,本發明的胜肽對抗雷氏桿菌的最低抑菌濃度範圍為6.25-450 μg/ml。In a specific example, the peptide of the present invention has a minimum inhibitory concentration against Streptococcus in the range of 6.25-450 μg/ml.
在又一特定實例中,針對常見養殖鴨類的測試證實,不論是共同施打本發明的胜肽及細菌,提前施打本發明的胜肽再接受細菌感染,或是先接受細菌感染再施打本發明的胜肽,幼鴨存活率均顯著高於僅施打細菌的對照組。In yet another specific example, testing against common cultured ducks demonstrates that, whether the peptides and bacteria of the present invention are co-administered, the peptide of the present invention is applied in advance to receive a bacterial infection, or a bacterial infection is first applied. With the peptide of the present invention, the survival rate of the young ducks was significantly higher than that of the control group which only applied the bacteria.
本發明另一方面提供一種用於動物體外之殺死雷氏桿菌之方法,其包括以含有效量的上述胜肽與載劑之組合物,施用於可能含有雷氏桿菌之物品或環境。Another aspect of the present invention provides a method for killing a bacterium of the genus of the bacterium, which comprises administering an effective amount of the above-described peptide and a carrier to an article or environment which may contain a bacterium.
在一特定實例中,以本發明的胜肽處理雷氏桿菌,使用穿透式電子顯微鏡清楚觀察到,細菌經處理後,細胞膜呈現不規則形狀且有破損,導致細胞內物質流出細胞,造成細胞死亡。In a specific example, the treatment of the bacterium with the peptide of the present invention, using a transmission electron microscope, clearly observes that after the treatment of the bacteria, the cell membrane exhibits an irregular shape and is broken, causing the intracellular material to flow out of the cell, causing the cell to be caused. death.
在一具體實例中,本發明之組合物係調配成液狀、顆粒狀、或粉末狀。特定而言,本發明之組合物可施用於動物的養殖場或養殖水域中。In one embodiment, the compositions of the present invention are formulated in a liquid, granular, or powder form. In particular, the compositions of the invention can be applied to farms or farmed waters of animals.
本發明將由下列實施例做進一步的說明,但實際發明並不受限於該用於展示之實施例。The invention will be further illustrated by the following examples, but the actual invention is not limited by the examples shown.
本發明使用的抗菌蛋白係由吉爾生化公司(上海,中國)利用Fmoc/tBu化學法的固相流程予以合成。表1列出各個抗菌蛋白的名稱及胺基酸序列。The antibacterial protein used in the present invention was synthesized by Gil Biochemical Corporation (Shanghai, China) using a solid phase procedure of Fmoc/tBu chemistry. Table 1 lists the names of the respective antibacterial proteins and the amino acid sequence.
合成後的多肽經過萃取、凍乾及利用逆相高效液相層析法(reverse-phase high-performance liquid chromatography,RP-HPLC)進行純化,再以質譜及高效液相層析法分析純化後多肽之分子量及純度。純化後的多肽以磷酸鹽緩衝液(pH 7.4)回溶以進行後續實驗。The synthesized polypeptide is extracted, lyophilized and purified by reverse-phase high-performance liquid chromatography (RP-HPLC), and then the purified peptide is analyzed by mass spectrometry and high performance liquid chromatography. Molecular weight and purity. The purified polypeptide was reconstituted with phosphate buffer (pH 7.4) for subsequent experiments.
本實例測試抗菌蛋白對抗數種雷氏桿菌菌株的最低抑菌濃度,包括由國立嘉義大學獸醫學系的余章游教授所提供的A2、B2、T6、RA16、及T10菌株、由國立台灣大學獸醫研究所張照夫教授所提供的CFC27、CFC437、CFC363、RA3及RA9菌株、以及由台灣礁溪私人養鴨場之櫻桃谷鴨(Cherry Valley Duck)所分離的MRS菌株。將含有各菌株的培養液與待測試的抗菌蛋白共同培育,測定最低抑菌濃度,可參照先前發表的微量培養液稀釋法進行(Int Immunopharmacol 2007;7: 687-700)。表2顯示測試結果。This example tests the minimum inhibitory concentration of antibacterial proteins against several strains of R. vaginalis, including the A2, B2, T6, RA16, and T10 strains provided by Professor Yu Zhangyou from the Department of Veterinary Medicine, National Chiayi University, and National Taiwan University. The CFC27, CFC437, CFC363, RA3 and RA9 strains provided by Prof. Zhang Zhaofu from the Veterinary Research Institute and the MRS strain isolated from the Cherry Valley Duck of the private duck farm in Jiaoxi, Taiwan. The culture solution containing each strain is incubated with the antibacterial protein to be tested, and the minimum inhibitory concentration is determined by referring to the previously published microculture dilution method ( Int Immunopharmacol 2007; 7: 687-700). Table 2 shows the test results.
如表2所示,上述抗菌蛋白對於所列雷氏桿菌的各種菌株均有抗菌效果,測得的最低抑菌濃度範圍為6.25-450 μg/ml。As shown in Table 2, the above antibacterial protein has antibacterial effects against various strains of Listeria species, and the minimum inhibitory concentration range is 6.25-450 μg/ml.
穿透式電子顯微鏡的實驗流程與先前發表的文獻之方法相同(Int Immunopharmacol 2007;7: 687-700)。簡言之,將雷氏桿菌菌株T6、MRS及CFC27培養後的菌液經由離心機(Beckman Coulter,Avanti J-20XP,JA-25.50,Palo Alto,CA,USA)以3000 rpm的轉速在4℃下離心5分鐘。將沈澱物以PBS回溶,濃度為OD550nm下測量值1.0。取5 ml的細菌回溶物與各種抗菌蛋白(100 μg/ml)混合,反應16小時。對照組僅含懸浮於PBS或培養液的細菌回溶物(未經抗菌蛋白處理)。經切片後,在75 kV條件下,以穿透式電子顯微鏡(Hitachi,H-7000,東京,日本)進行觀察。The experimental procedure for the transmission electron microscope is the same as that of the previously published literature ( Int Immunopharmacol 2007; 7: 687-700). Briefly, the bacterial cultures of the R. smegmatis strains T6, MRS and CFC27 were passed through a centrifuge (Beckman Coulter, Avanti J-20XP, JA-25.50, Palo Alto, CA, USA) at 3000 rpm at 4 °C. Centrifuge for 5 minutes. The precipitate was reconstituted with PBS at a concentration of 1.0 at OD550 nm. 5 ml of the bacterial reconstituted material was mixed with various antibacterial proteins (100 μg/ml) for 16 hours. The control group contained only bacterial reconstituted material (not treated with antibacterial protein) suspended in PBS or culture medium. After sectioning, observation was performed by a transmission electron microscope (Hitachi, H-7000, Tokyo, Japan) under conditions of 75 kV.
結果顯示,從TEM清楚觀察到,經本發明抗菌蛋白EPI、CA、LA、或TH1-5處理的三種雷氏桿菌菌株MRS(圖1A),CFC27(圖1B)及T6(圖1C)與未經處理的對照組,在細胞型態上有顯著差異;未經處理的對照組,細胞膜完整無破損,而經抗菌蛋白處理的細菌,其細胞膜則呈現不規則形狀且有破損,導致細胞內物質流出細胞,造成細胞死亡。The results showed that the three R. reesei strains MRS (Fig. 1A), CFC27 (Fig. 1B) and T6 (Fig. 1C) treated with the antibacterial protein EPI, CA, LA, or TH1-5 of the present invention were clearly observed from the TEM. The treated control group showed significant differences in cell type; in the untreated control group, the cell membrane was intact without damage, while the bacteria treated with the antibacterial protein showed irregular shape and damage to the cell membrane, resulting in the outflow of intracellular substances. Cells that cause cell death.
本實例使用南瓜鴨(C. moschata duck)及櫻桃谷鴨進行動物感染實驗。動物來源是一般養殖場孵出的四日齡幼鴨,給予一般商業上可獲得的飼料及無菌水餵食,將幼鴨飼養在隔離房間的籠子裏,依照標準作業程序維持隔離房間的溫度。使用注射器(29 G x 1/2,Terumo Medical,Elkton,MD,USA)將雷氏桿菌、抗菌蛋白及/或PBS直接注入至鴨子的腹腔內,每日紀錄死亡率。This example used a pumpkin duck ( C. moschata duck) and a cherry valley duck for animal infection experiments. The source of the animals is a four-day-old duckling hatched from a general farm, fed with generally commercially available feed and sterile water, and the young ducks are kept in cages in an isolated room to maintain the temperature of the isolated room in accordance with standard operating procedures. The bacteria were injected directly into the abdominal cavity of the duck using a syringe (29 G x 1/2, Terumo Medical, Elkton, MD, USA) and the mortality was recorded daily.
在第一組實驗中,每一組有30隻受測幼鴨,對照組接受雷氏桿菌(1×108 菌落形成單位(cfu),每隻鴨)或PBS注射;而實驗組則接受各個抗菌蛋白(100 μg,每隻幼鴨)與雷氏桿菌(1×108 cfu,每隻幼鴨)之混合物的注射。In the first set of experiments, there were 30 tested ducklings in each group, and the control group received R. vaginalis (1 × 10 8 colony forming units (cfu), each duck) or PBS; the experimental group received each Injection of a mixture of antibacterial proteins (100 μg per duckling) and R. brevis (1 x 10 8 cfu per duckling).
第二組實驗是緊接於第一次實驗完成336小時,再追加注射雷氏桿菌(1×106 cfu,每隻幼鴨)。The second set of experiments was followed by 336 hours of the first experiment, followed by additional injections of R. vaginalis (1 × 10 6 cfu, each young duck).
在第三組實驗中,每一組有20隻受測幼鴨,先注射雷氏桿菌(1×108 cfu,每隻幼鴨),再於2或24小時後,注射PBS或各個抗菌蛋白(100 μg,每隻幼鴨)。In the third set of experiments, each group had 20 tested young ducks, first injected with R. rees (1 × 10 8 cfu, each duckling), and then injected with PBS or each antibacterial protein after 2 or 24 hours. (100 μg, each duckling).
在第四組實驗中,每一組有20隻受測幼鴨,先注射PBS或各個抗菌蛋白(100 μg,每隻幼鴨),再於2或24小時後,注射雷氏桿菌(1×108 cfu,每隻幼鴨)。In the fourth set of experiments, each group had 20 tested young ducks, first injected with PBS or each antibacterial protein (100 μg per duckling), and then injected with R. falciparum after 2 or 24 hours (1× 10 8 cfu, each duckling).
以上動物測試,在櫻桃谷鴨中,使用的雷氏桿菌菌株是MRS,受測的抗菌蛋白是EPI、CA、LA、及TH1-5,以及抗菌蛋白與雷氏桿菌的施打間隔為2小時;而在南瓜鴨中,使用的雷氏桿菌菌株是T6,受測的抗菌蛋白是EPI、CA、LA、TH1-5、及TH2-3,以及抗菌蛋白與雷氏桿菌的施打間隔為24小時。The above animal test, in the Cherry Valley duck, the strain of R. serrata is MRS, the tested antibacterial proteins are EPI, CA, LA, and TH1-5, and the interval between the antibacterial protein and the bacterium is 2 hours. In the pumpkin duck, the strain of the bacterium used is T6, the antibacterial proteins tested are EPI, CA, LA, TH1-5, and TH2-3, and the interval between the antibacterial protein and the bacterium is 24 hour.
使用t測試針對每一次注射實驗的數據進行統計分析,以比較兩組間的差異。多組間的比較則是用SPSS軟體之變異分析(ANOVA)進行測試。圖上不同字母顯示在兩組間有顯著差異;而相同字母則顯示在兩組間不具有顯著差異。Statistical analysis was performed on the data for each injection experiment using the t test to compare the differences between the two groups. Comparisons between groups were tested using SPSS software variant analysis (ANOVA). The different letters on the graph show significant differences between the two groups; the same letters show no significant differences between the two groups.
結果顯示,EPI、CA、LA、及TH1-5能顯著減低受MRS感染之櫻桃谷鴨的死亡率(圖2);而EPI、CA、LA、TH1-5、及TH2-3能顯著減低受T6感染之南瓜鴨的死亡率(圖3)。The results showed that EPI, CA, LA, and TH1-5 significantly reduced the mortality of MRS-infected Cherry Valley ducks (Figure 2); while EPI, CA, LA, TH1-5, and TH2-3 significantly reduced the mortality Mortality of T6-infected pumpkin ducks (Figure 3).
如圖2(A)所示,櫻桃谷鴨在MRS菌株(1×108 cfu/鴨)感染後14天內,死亡率極高(10天內30隻動物中有23隻死亡);但若共同施打抗菌蛋白LA或TH1-5(100 μg/ml)及MRS菌株之動物的14天存活率則達93.33%(三天內30隻動物中有2隻死亡),共同施打抗菌蛋白EPI或CA及MRS菌株之動物的14天存活率亦分別達76.66%(九天內30隻動物中有7隻死亡)及73.33%(九天內30隻動物中有8隻死亡)。在以上共同施打抗菌蛋白及細菌的實驗中,幼鴨存活率均顯著高於僅施打細菌的對照組(p<0.05)。As shown in Fig. 2(A), the mortality of Cherry Valley ducks was extremely high within 14 days after infection with MRS strain (1×10 8 cfu/duck) (23 out of 30 animals in 10 days died); The 14-day survival rate of animals with antibacterial protein LA or TH1-5 (100 μg/ml) and MRS strains reached 93.33% (2 out of 30 animals in three days), and the antibacterial protein EPI was applied together. The 14-day survival rates of animals with either CA and MRS strains were also 76.66% (7 out of 30 animals in nine days) and 73.33% (8 out of 30 animals in nine days died). In the above experiments in which antibacterial proteins and bacteria were commonly applied, the survival rate of the young ducks was significantly higher than that of the control group (p<0.05).
如圖2(B)所示,在動物接受上述共同施打抗菌蛋白及細菌之後,於14日後,再次施打MRS菌株(1×108 cfu/鴨),結果顯示,僅接受細菌注射的幼鴨,存活率僅16.66%,而先前有接受共同施打抗菌蛋白EPI、CA、LA、或TH1-5及細菌的幼鴨,存活率則分別提高至47.82%、63.63%、71.42%及75%。As shown in Fig. 2(B), after the animals received the above-mentioned common antibacterial protein and bacteria, the MRS strain (1 × 10 8 cfu/duck) was again applied after 14 days, and the results showed that only the bacteria were injected. The survival rate of ducks was only 16.66%. The survival rate of ducklings that had previously received antibiotics EPI, CA, LA, or TH1-5 and bacteria increased to 47.82%, 63.63%, 71.42% and 75%, respectively. .
又如圖2(C)及2(D)所示,不論是先接受細菌感染再施打抗菌蛋白,或是提前施打抗菌蛋白再接受細菌感染,幼鴨存活率均顯著高於施打細菌及PBS的對照組。As shown in Figures 2(C) and 2(D), the survival rate of young ducks is significantly higher than that of the bacteria, whether it is a bacterial infection or an antibacterial protein, or an antibacterial protein is applied in advance. And PBS control group.
類似地,如圖3(A)所示,南瓜鴨在T6菌株(1×108 cfu/鴨)感染後14天內,存活率僅有30%,僅施打PBS的幼鴨的存活率則達100%,而在共同施打抗菌蛋白EPI、TH1-5、LA、TH2-3、或CA(100 μg/ml)及T6菌株(1×108 cfu/鴨)之動物的14天存活率分別為100%、75%、75%、60%及60%。在以上共同施打抗菌蛋白及細菌的實驗中,幼鴨存活率均顯著高於僅施打細菌的對照組。Similarly, as shown in Fig. 3(A), the survival rate of pumpkin ducks was only 30% within 14 days after infection with T6 strain (1×10 8 cfu/duck), and the survival rate of young ducks administered only with PBS was 14-day survival rate of animals with 100% antibacterial protein EPI, TH1-5, LA, TH2-3, or CA (100 μg/ml) and T6 strain (1×10 8 cfu/duck) They are 100%, 75%, 75%, 60% and 60% respectively. In the above experiments in which antibacterial proteins and bacteria were jointly applied, the survival rate of the young ducks was significantly higher than that of the control group only.
此外,如圖3(B)所示,在南瓜鴨接受上述共同施打抗菌蛋白及細菌之後,於14日後,再次施打T6菌株(1×108 cfu/鴨),結果顯示,僅接受細菌注射的幼鴨,存活率僅40%,而先前有接受共同施打抗菌蛋白TH2-3、EPI、TH1-5、LA、或CA及細菌的幼鴨,存活率較高,分別達91.7%、90%、86.7%、86.7%、及83.3%。Further, as shown in Fig. 3(B), after the pumpkin duck received the above-mentioned common antibacterial protein and bacteria, the T6 strain (1 × 10 8 cfu/duck) was again applied after the 14th day, and the results showed that only the bacteria were received. The survival rate of the injected ducklings was only 40%, while the young ducks who had previously received the antibacterial protein TH2-3, EPI, TH1-5, LA, or CA and bacteria had a higher survival rate of 91.7%. 90%, 86.7%, 86.7%, and 83.3%.
再者,如圖3(C)所示,僅接受T6菌株施打的動物,在14天內會造成高死亡率(4天內20隻動物中20隻死亡);相較之下,若在施打T6菌株後24小時接著施打抗菌蛋白EPI,TH2-3、TH1-5、LA或CA,則動物存活率較高,分別為100%、95%、90%、85%、及80%。Furthermore, as shown in Fig. 3(C), animals that received only T6 strains caused high mortality within 14 days (20 of 20 animals died within 4 days); in contrast, if After 24 hours of T6 strain application, the antibacterial proteins EPI, TH2-3, TH1-5, LA or CA were applied, and the survival rate of the animals was higher, 100%, 95%, 90%, 85%, and 80%, respectively. .
最後,如圖3(D)所示,僅接受T6菌株施打的動物,在14天之死亡率為80%;相較之下,若預先施打抗菌蛋白EPI、CA、LA、TH1-5及TH2-3(100 μg),24小時後再施打T6菌株(1×108 cfu/duck),則動物存活率較高,分別為70%、50%、60%、55%、及65%。Finally, as shown in Fig. 3(D), the animals that received only T6 strains had a mortality rate of 80% in 14 days; in contrast, if the antibacterial proteins EPI, CA, LA, TH1-5 were applied in advance, And TH2-3 (100 μg), T6 strain (1 × 10 8 cfu / duck) was applied after 24 hours, the animal survival rate was higher, 70%, 50%, 60%, 55%, and 65, respectively. %.
幼鴨接受不同方式的抗菌蛋白及/或菌株之施打。在第一組實驗中,動物先施打T6菌株(1×108 cfu/duck),24小時後再施打抗菌蛋白EPI、CA、LA、TH1-5或TH2-3(100 μg/鴨)。在第二組實驗中,動物先施打抗菌蛋白EPI、CA、LA、TH1-5或TH2-3(100 μg/鴨),24小時後再施打T6菌株(1×108 cfu/鴨)。在第三組實驗中,動物共同施打T6菌株(1×108 cfu/鴨)及抗菌蛋白EPI、CA、LA、TH1-5或TH2-3(100 μg/鴨)。在動物接受施打1、3、5、及7天後,分別取肝臟組織進行分析。首先將肝臟組織均質化後放入PBS中,將其塗佈在羊血瓊脂培養盤上,接著將此培養盤放置於37℃培養24小時,最後分別計算細菌菌落的數目。利用SAS分析程式計算顯著差異(p<0.05)。Young ducks receive different ways of applying antibacterial proteins and/or strains. In the first set of experiments, the animals were first beaten with T6 strain (1×10 8 cfu/duck), and after 24 hours, the antibacterial proteins EPI, CA, LA, TH1-5 or TH2-3 (100 μg/duck) were applied. . In the second set of experiments, the animals were first given antibacterial protein EPI, CA, LA, TH1-5 or TH2-3 (100 μg/duck), and T6 strain (1×10 8 cfu/duck) was applied 24 hours later. . In the third set of experiments, animals were co-administered with T6 strain (1 x 10 8 cfu/duck) and antibacterial proteins EPI, CA, LA, TH1-5 or TH2-3 (100 μg/duck). After the animals received 1, 3, 5, and 7 days of application, liver tissues were taken for analysis. The liver tissue was first homogenized, placed in PBS, and spread on a sheep blood agar plate, and then the plate was placed at 37 ° C for 24 hours, and finally the number of bacterial colonies was counted separately. Significant differences were calculated using the SAS analysis program (p < 0.05).
結果顯示,不論抗菌蛋白EPI、CA、LA、TH1-5或TH2-3是與細菌同時施打(圖4A)、感染後施打(圖4B)、或感染前施打(圖4C),注射7天後皆能顯著減低於動物肝臟內之細菌數(p<0.05)。The results showed that whether the antibacterial proteins EPI, CA, LA, TH1-5 or TH2-3 were applied simultaneously with the bacteria (Fig. 4A), after the infection (Fig. 4B), or before the infection (Fig. 4C), the injection After 7 days, the number of bacteria in the liver of the animals was significantly reduced (p<0.05).
總結以上的試驗,本發明證實抗菌蛋白EPI、CA、LA、TH1-5或TH2-3對於雷氏桿菌有殺菌活性,可顯著提高感染動物的存活率。有鑑於抗生素的各種副作用及抗藥性菌株日益增加,本發明證實上述抗菌蛋白有潛力發展成防治雷氏桿菌感染的藥劑或佐劑,特別是在水禽養殖業對抗雷氏桿菌感染有相當貢獻。Summarizing the above test, the present invention confirmed that the antibacterial proteins EPI, CA, LA, TH1-5 or TH2-3 have bactericidal activity against R. vaginalis, and can significantly improve the survival rate of infected animals. In view of the various side effects of antibiotics and the increasing number of resistant strains, the present invention demonstrates that the above-mentioned antibacterial proteins have the potential to develop into agents or adjuvants for the prevention of infection with R. vaginalis, particularly in the waterfowl farming industry.
熟知技藝之人士將可在不背離本發明精神之下,根據實施例進行改變和修改。要注意的是,本發明並不受限於說明書中實施例所揭露之範圍,而涵蓋於其他根據申請範圍內揭露之所有變化之形式。Those skilled in the art will be able to make changes and modifications in accordance with the embodiments without departing from the spirit of the invention. It is to be understood that the invention is not to be limited by the scope of the embodiments disclosed herein,
前文之所述以及實施方式可藉由附加之圖式達到更好的說明效果。為了加強本發明之說明,將適當的實施例之圖式列舉於此。要注意的是,本發明並不受限於列舉於此的說明。The foregoing description and the embodiments may achieve better illustrative effects by the additional drawings. To enhance the description of the invention, the drawings of the appropriate embodiments are set forth herein. It is to be noted that the present invention is not limited to the descriptions set forth herein.
圖1顯示雷氏桿菌接受抗菌蛋白處理後的穿透式電子顯微鏡影像。圖1(A)是針對MAS菌株,(a)至(e)分別是未經處理的MAS菌株、經抗菌蛋白EPI處理的MAS菌株、經抗菌蛋白CA處理的MAS菌株、經抗菌蛋白LA處理的MAS菌株、以及經抗菌蛋白TH1-5處理的MAS菌株,而(f)至(j)是在(a)至(e)的相同條件下之重複試驗結果。圖1(B)是針對CFC27菌株,(a)至(e)分別是未經處理的CFC27菌株、經抗菌蛋白EPI處理的CFC27菌株、經抗菌蛋白CA處理的CFC27菌株、經抗菌蛋白LA處理的CFC27菌株、以及經抗菌蛋白TH1-5處理的CFC27菌株,而(f)至(j)是在(a)至(e)的相同條件下之重複試驗結果。圖1(C)是針對T6菌株,(a)至(e)分別是未經處理的T6菌株、經抗菌蛋白EPI處理的T6菌株、經抗菌蛋白CA處理的T6菌株、經抗菌蛋白LA處理的T6菌株、以及經抗菌蛋白TH1-5處理的T6菌株,而(f)至(j)是在(a)至(e)的相同條件下之重複試驗結果。Figure 1 shows a transmission electron microscope image of R. eutropha after treatment with an antibacterial protein. Figure 1 (A) is for the MAS strain, (a) to (e) are untreated MAS strain, MAS strain treated with antibacterial protein EPI, MAS strain treated with antibacterial protein CA, treated with antibacterial protein LA The MAS strain, and the MAS strain treated with the antibacterial protein TH1-5, and (f) to (j) are the results of repeated experiments under the same conditions of (a) to (e). Figure 1 (B) is for CFC27 strain, (a) to (e) are untreated CFC27 strain, CFC27 strain treated with antibacterial protein EPI, CFC27 strain treated with antibacterial protein CA, treated with antibacterial protein LA The CFC27 strain, and the CFC27 strain treated with the antibacterial protein TH1-5, and (f) to (j) are repeated test results under the same conditions of (a) to (e). Figure 1 (C) is for the T6 strain, (a) to (e) are untreated T6 strain, T6 strain treated with antibacterial protein EPI, T6 strain treated with antibacterial protein CA, treated with antibacterial protein LA The T6 strain, and the T6 strain treated with the antibacterial protein TH1-5, and (f) to (j) are repeated test results under the same conditions of (a) to (e).
圖2顯示本發明之抗菌蛋白可提高受到雷氏桿菌MAS菌株感染之櫻桃谷鴨的存活率。在圖2(A)中,RA表示僅施打MAS菌株;PBS表示僅施打PBS;EPI、CA、LA及TH1-5表示共同施打各個抗菌蛋白與雷氏桿菌。在圖2(B)中,各個線條表示在圖2(A)的相同條件下,再追加注射雷氏桿菌的結果。在圖2(C)中,RA-2hr-PBS是先施打雷氏桿菌,再於2小時後施打PBS;RA-2hr-TH1-5是先施打雷氏桿菌,再於2小時後施打抗菌蛋白TH1-5;RA-2hr-LA是先施打雷氏桿菌,再於2小時後施打抗菌蛋白LA;RA-2hr-CA是先施打雷氏桿菌,再於2小時後施打抗菌蛋白CA;以及RA-2hr-EPI是先施打雷氏桿菌,再於2小時後施打抗菌蛋白EPI。在圖2(D)中,PBS-2hr-RA是先施打PBS,再於2小時後施打雷氏桿菌;TH1-5-2hr-RA是先施打抗菌蛋白TH1-5,再於2小時後施打雷氏桿菌;LA-2hr-RA是先施打抗菌蛋白LA,再於2小時後施打雷氏桿菌;CA-2hr-RA是先施打抗菌蛋白CA,再於2小時後施打雷氏桿菌;以及EPI-2hr-RA是先施打抗菌蛋白EPI,再於2小時後施打雷氏桿菌。Figure 2 shows that the antibacterial protein of the present invention can increase the survival rate of Cherry Valley duck infected by the RES. In Fig. 2(A), RA indicates that only the MAS strain was applied; PBS indicates that only PBS was applied; EPI, CA, LA, and TH1-5 indicated that each of the antibacterial proteins and R. faecalis were co-administered. In Fig. 2(B), each line shows the result of additionally injecting R. eutropha under the same conditions as in Fig. 2(A). In Fig. 2(C), RA-2hr-PBS was first administered with R. vaginalis, and then PBS was administered 2 hours later. RA-2hr-TH1-5 was first administered with R. vaginalis and then administered 2 hours later. Antibacterial protein TH1-5; RA-2hr-LA is first applied to the bacterium, and then the antibacterial protein LA is applied after 2 hours; RA-2hr-CA is first applied to the bacterium, and then the antibacterial protein is applied after 2 hours. CA; and RA-2hr-EPI were first administered with R. rapae, and the antibacterial protein EPI was applied after 2 hours. In Fig. 2(D), PBS-2hr-RA is first administered with PBS, and then 2 days after the application of Rep.; TH1-5-2hr-RA is first applied antibacterial protein TH1-5, and then 2 hours After the application of R. serrata; LA-2hr-RA is first applied antibacterial protein LA, and then 2 days after the application of Rep.; CA-2hr-RA is first applied antibacterial protein CA, and then 2 hours after the application of Rey Bacillus; and EPI-2hr-RA was first applied with the antibacterial protein EPI, and then treated with R. falciparum 2 hours later.
圖3顯示本發明之抗菌蛋白可提高受到雷氏桿菌T6菌株感染之南瓜鴨的存活率。在圖3(A)中,RA表示僅施打雷氏桿菌;PBS表示僅施打PBS;EPI、CA、LA、TH1-5、及TH2-3表示共同施打各個抗菌蛋白與雷氏桿菌。在圖3(B)中,各個線條表示在圖3(A)的相同條件下,再追加注射雷氏桿菌的結果。在圖3(C)中,RA-24hr-PBS是先施打雷氏桿菌,再於24小時後施打PBS;RA-24hr-TH2-3是先施打雷氏桿菌,再於24小時後施打抗菌蛋白TH2-3;RA-24hr-TH1-5是先施打雷氏桿菌,再於24小時後施打抗菌蛋白TH1-5;RA-24hr-LA是先施打雷氏桿菌,再於24小時後施打抗菌蛋白LA;RA-24hr-CA是先施打雷氏桿菌,再於24小時後施打抗菌蛋白CA;以及RA-24hr-EPI是先施打雷氏桿菌,再於24小時後施打抗菌蛋白EPI。在圖3(D)中,PBS-24hr-RA是先施打PBS,再於24小時後施打雷氏桿菌株;TH2-3-24hr-RA是先施打抗菌蛋白TH2-3,再於24小時後施打雷氏桿菌;TH1-5-24hr-RA是先施打抗菌蛋白TH1-5,再於24小時後施打雷氏桿菌;LA-24hr-RA是先施打抗菌蛋白LA,再於24小時後施打雷氏桿菌;CA-24hr-RA是先施打抗菌蛋白CA,再於24小時後施打雷氏桿菌;以及EPI-2hr-RA是先施打抗菌蛋白EPI,再於24小時後施打雷氏桿菌。Figure 3 shows that the antibacterial protein of the present invention can increase the survival rate of pumpkin duck infected by the R. serovar T6 strain. In Fig. 3(A), RA indicates that only R. serrata was administered; PBS indicates that only PBS was applied; EPI, CA, LA, TH1-5, and TH2-3 indicated that each antibacterial protein and R. rees were co-administered. In Fig. 3(B), each line shows the result of additionally injecting R. eutropha under the same conditions as in Fig. 3(A). In Fig. 3(C), RA-24hr-PBS is first administered with R. vaginalis, and then PBS is administered after 24 hours; RA-24hr-TH2-3 is first applied to R. rapae, and then applied after 24 hours. Antibacterial protein TH2-3; RA-24hr-TH1-5 is first applied to the bacterium, and then the antibacterial protein TH1-5 is applied after 24 hours; RA-24hr-LA is first applied to the bacterium, and after 24 hours Apply antibacterial protein LA; RA-24hr-CA is first applied to R. rapae, and then apply antibacterial protein CA after 24 hours; and RA-24hr-EPI is first applied to R. serrata, and then applied antibacterial after 24 hours. Protein EPI. In Fig. 3(D), PBS-24hr-RA was first applied with PBS, and then strained with T. reesei after 24 hours; TH2-3-24hr-RA was first applied with antibacterial protein TH2-3, then 24 After the hour, Streptococcus faecalis was administered; TH1-5-24hr-RA was first applied to the antibacterial protein TH1-5, and then 24 hours later, the bacterium was killed; LA-24hr-RA was first applied antibacterial protein LA, then 24 After 24 hours, the antibiotics CA was applied. CA-24hr-RA was first applied with antibacterial protein CA, and then 24 hours later; and EPI-2hr-RA was first applied with antibacterial protein EPI, and then applied 24 hours later. Hitella bacillus.
圖4顯示抗菌蛋白在動物肝臟內對抗雷氏桿菌T6菌株的殺菌活性測試結果。在圖4(A)中,RA表示僅施打雷氏桿菌;以及EPI+RA、CA+RA、LA+RA、TH1-5+RA、及TH2-3+RA表示共同施打各個抗菌蛋白與雷氏桿菌。在圖4(B)中,RA表示僅施打雷氏桿菌;RA-24hr-TH2-3是先施打雷氏桿菌,再於24小時後施打抗菌蛋白TH2-3;RA-24hr-TH1-5是先施打雷氏桿菌,再於24小時後施打抗菌蛋白TH1-5;RA-24hr-LA是先施打雷氏桿菌,再於24小時後施打抗菌蛋白LA;RA-24hr-CA是先施打雷氏桿菌,再於24小時後施打抗菌蛋白CA;以及RA-24hr-EPI是先施打雷氏桿菌,再於24小時後施打抗菌蛋白EPI。在圖4(C)中,RA表示僅施打雷氏桿菌;TH2-3-24hr-RA是先施打抗菌蛋白TH2-3,再於24小時後施打雷氏桿菌;TH1-5-24hr-RA是先施打抗菌蛋白TH1-5,再於24小時後施打雷氏桿菌;LA-24hr-RA是先施打抗菌蛋白LA,再於24小時後施打雷氏桿菌;CA-24hr-RA是先施打抗菌蛋白CA,再於24小時後施打雷氏桿菌;以及EPI-2hr-RA是先施打抗菌蛋白EPI,再於24小時後施打雷氏桿菌。Figure 4 shows the results of bactericidal activity tests of antibacterial proteins against the R. reuteri T6 strain in the liver of animals. In Fig. 4(A), RA indicates that only R. serrata is administered; and EPI+RA, CA+RA, LA+RA, TH1-5+RA, and TH2-3+RA indicate that each antibacterial protein and thunder are jointly applied. Bacillus. In Fig. 4(B), RA indicates that only R. serrata is administered; RA-24hr-TH2-3 is first applied to R. rapae, and after 24 hours, antibacterial protein TH2-3 is applied; RA-24hr-TH1-5 It is first applied to the bacterium, and then the antibacterial protein TH1-5 is applied after 24 hours; the RA-24hr-LA is first applied to the bacterium, and then the antibacterial protein LA is applied after 24 hours; the RA-24hr-CA is the first The antibacterial protein CA was applied after 24 hours; and the RA-24hr-EPI was first applied to the bacterium, and the antibacterial protein EPI was applied after 24 hours. In Fig. 4(C), RA indicates that only R. serrata was administered; TH2-3-24hr-RA was first applied to the antibacterial protein TH2-3, and then 24 hours after the administration of R. rees; TH1-5-24hr-RA The antibacterial protein TH1-5 was first applied, and then the bacterium was killed by 24 hours; LA-24hr-RA was first applied with the antibacterial protein LA, and then the bacterium was killed after 24 hours; CA-24hr-RA was the first The antibacterial protein CA was applied, and the R. eutropha was administered after 24 hours; and the EPI-2hr-RA was first applied with the antibacterial protein EPI, and then the R. eutropha was administered 24 hours later.
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