TWI417105B - Pharmaceutical composition for preventing or treating mammalian viral disease - Google Patents

Pharmaceutical composition for preventing or treating mammalian viral disease Download PDF

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TWI417105B
TWI417105B TW100119679A TW100119679A TWI417105B TW I417105 B TWI417105 B TW I417105B TW 100119679 A TW100119679 A TW 100119679A TW 100119679 A TW100119679 A TW 100119679A TW I417105 B TWI417105 B TW I417105B
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pharmaceutical composition
virus
antibacterial protein
grouper
japanese encephalitis
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TW201249457A (en
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Jyh Yih Chen
Chang Jer Wu
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Academia Sinica
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Description

用於預防或治療哺乳類病毒性疾病之醫藥組合物Pharmaceutical composition for preventing or treating mammalian viral diseases

本發明係關於一種用於預防或治療哺乳類醫藥組合物，特別是關於一種預防或治療哺乳類的病毒性疾病之醫藥組合物。The present invention relates to a pharmaceutical composition for preventing or treating a mammal, and more particularly to a pharmaceutical composition for preventing or treating a viral disease of a mammal.

黃病毒科(Flaviviridae)包含了至少三個屬，其一為造成畜牧類疾病之瘟病毒屬(Pestivirus )，其二為造成黃熱病或登革熱等疾病之黃病毒屬(Flavivirus )，其三為造成肝的相關疾病之肝病毒屬(Hepacivirus )。這些病毒常造成多數甚至人類發生嚴重疾病甚至死亡。其中，由於黃病毒屬中的日本腦炎病毒會對人類造成嚴重的影響，而特別受到重視。Flaviviridae (Flaviviridae) contains at least three genera, one of which is caused by pestivirus of livestock diseases (Pestivirus), the other for causing flavivirus diseases such as yellow fever or dengue (Flavivirus), which is three for the cause Hepativirus of the liver related disease. These viruses often cause serious illness and even death in most people and even humans. Among them, the Japanese encephalitis virus in the genus Flavivirus has a serious impact on human beings, and is particularly valued.

習知技術中，用於治療黃病毒科疾病的方法係包含合成藥物阻斷黃病毒科在體內的病毒感染途徑、給予一些免疫刺激物(例如干擾素等)，或利用分子生物方法如注射小片段RNA以干擾病毒遺傳物質在動物體內進行複製等方式，然而這些方法在製程上不但需花費高成本更相當耗時。In the prior art, the method for treating a Flaviviridae disease comprises a synthetic drug blocking the viral infection pathway of the Flaviviridae in vivo, administration of some immunostimulating substances (such as interferon, etc.), or using molecular biological methods such as injection. Fragment RNA interferes with viral genetic material in animals, but these methods are not only costly but also time consuming.

透過注射減毒或不活化病毒的方式所製成的疫苗，通常是目前最為有效的預防方法，但疫苗需長時間注射多次且其中的部份成份會造成生物體的不適反應。習知技術中關於疫苗的組成成份，通常包含一特定病毒的主要蛋白質、多醣類、去氧核醣核酸(deoxyribonucleic acid)、合成胜肽及病毒本身等，以作為免疫系統記憶抗原時的標的。然而為引發較佳的免疫反應，通常尚添加幫助引發免疫反應的佐劑(adjuvants)，有些佐劑在含病源菌的疫苗中還扮演了使病源菌失去活性的作用。目前用於疫苗中常見的佐劑主要為含鋁化合物(硫酸鋁、氫氧化鋁或磷酸鋁)或福馬林(formalin)等，然而以鋁化合物作為佐劑時的疫苗，品質易參差不齊；另外像福馬林這種會對生物體造成影響的化學物質，在製程最後需被移除，且若有移除不完全或病源菌去活性不完全都會對生物體造成危害，因此在製程方面較為繁複且易有生物安全性的疑慮。Vaccines made by injecting attenuated or non-activated viruses are usually the most effective preventive methods at present, but the vaccines need to be injected multiple times and some of them may cause uncomfortable reactions in the organism. The composition of the vaccine in the prior art usually includes a major virus, a major protein, a polysaccharide, a deoxyribonucleic acid, a synthetic peptide, and the virus itself, as a target of the immune system memory antigen. However, in order to elicit a better immune response, adjuvants that help elicit an immune response are usually added, and some adjuvants also act to inactivate the pathogenic bacteria in vaccines containing pathogenic bacteria. The adjuvants currently used in vaccines are mainly aluminum-containing compounds (aluminum sulfate, aluminum hydroxide or aluminum phosphate) or formalin, etc. However, when the aluminum compound is used as an adjuvant, the quality is easily uneven; In addition, chemical substances such as fumarin, which affect the organism, need to be removed at the end of the process, and if the removal is incomplete or the pathogen is not fully activated, it will cause harm to the organism, so the process is relatively Complex and easy to have biosafety concerns.

目前已發現許多存在於脊椎動物中的抗菌蛋白(antimicrobial peptide)，其用以保護生物體抵抗外來病源菌。Yin等人於2006年揭露一種源自點帶石斑魚(Epinephelus coioides )的抗菌蛋白並將其命名為石斑魚抗菌蛋白-1(Epinecidin-1)(Yin ZX. et al. Aquaculcure 253:204-11,2006)。當中提到石斑魚抗菌蛋白-1具有抵抗多種病原菌如細菌或真菌等的功效，但目前並沒有任何可作為醫藥組合物的相關報導產出。Many antimicrobial peptides have been found in vertebrates to protect organisms against foreign pathogenic bacteria. In 2006, Yin et al. revealed an antibacterial protein derived from Epinephelus coioides and named it Epinecidin-1 (Yin ZX. et al. Aquaculcure 253:204-11, 2006). ). It is mentioned that the grouper antibacterial protein-1 has the effect of resisting various pathogenic bacteria such as bacteria or fungi, but there is currently no relevant reported output which can be used as a pharmaceutical composition.

對於黃病毒科病毒之防治相關單位仍積極著手並改進疫苗，因此，如何簡便地製造一種具生物安全性的黃病毒科疫苗，一直是目前重要的課題之一。The relevant units for the prevention and treatment of the Flaviviridae virus are still actively pursuing and improving the vaccine. Therefore, how to easily manufacture a biosafety Flaviviridae vaccine has been one of the important issues at present.

有鑑於先前技術之不足，本發明之目的為提供一種用於治療哺乳類病毒性疾病之醫藥組合物，能夠有效刺激免疫能力且具有生物安全性之特性。In view of the deficiencies of the prior art, it is an object of the present invention to provide a pharmaceutical composition for treating a mammalian viral disease which is effective in stimulating immunity and biosafety.

依據本發明之一種用於治療哺乳類的病毒性疾病之醫藥組合物，其包含有效劑量之一石斑魚之抗菌蛋白，以及具感染哺乳類活性之一病毒。A pharmaceutical composition for treating a viral disease of a mammal according to the present invention, which comprises an antibacterial protein of one of an effective dose of grouper, and a virus having an activity of infecting mammals.

為使之後內容能清楚呈現本發明的技術特徵，以下擬先定義特定名詞，爾後進一步說明本發明內容。In order to make the following content clearly show the technical features of the present invention, the following specific terms are first defined, and the present invention will be further described.

本說明書中所使用之「預防」一詞意指將有效劑量之石斑魚抗菌蛋白-1及有效劑量之以哺乳類為宿主之病毒給予尚未出現病癥之宿主，以達到預防、防止、阻止、延緩、抑制病癥之顯現或病癥之蔓延，或達到與上述目的相似之效果。The term "prevention" as used in this specification means the administration of an effective dose of grouper antibacterial protein-1 and an effective amount of a mammalian-hosted virus to a host that has not developed a disease to prevent, prevent, arrest, delay, inhibit The manifestation of a condition or the spread of a condition, or an effect similar to the above purpose.

本說明書中所使用之「治療」一詞意指將有效劑量之石斑魚抗菌蛋白-1及有效劑量之以哺乳類為宿主之病毒給予受病毒感染而出現可觀察到病癥，或受感染有出現病癥傾向之受體。使受體得到適當治療、治癒、改善、緩解、緩和、減輕或改進其病徵之效果。上述所說之「受體」一詞意指受病毒感染，並接受本發明以石斑魚抗菌蛋白-1及以哺乳類為宿主之病毒所構成之醫藥組合物進行治療之細胞、組織、器官或動物。The term "treatment" as used in this specification means the administration of an effective dose of grouper antibacterial protein-1 and an effective amount of a mammalian-hosted virus to a virus-infected patient with an observable condition or a tendency to develop an infection. Receptor. The receptor is properly treated, cured, ameliorated, alleviated, alleviated, reduced or improved. The term "receptor" as used herein refers to a cell, tissue, organ or animal which is infected with a virus and which is treated with a pharmaceutical composition comprising the grouper antibacterial protein-1 and a mammalian host virus.

本說明書中所使用之「抗菌蛋白」一詞意指包含源自生物體中具有抗菌效果的胜肽。需特別說明的是，在本發明之說明書中之抗菌蛋白係包含至少1種長度介於2-200個胺基酸的胜肽所組成，胜肽形狀可為直鏈形、螺旋形、U形或其他可達特定生物功能之構形，甚至可稱此胜肽為蛋白質。在本發明之一實施例中，抗菌蛋白係為石斑魚抗菌蛋白-1。The term "antibacterial protein" as used in the specification means a peptide containing an antibacterial effect derived from an organism. It should be particularly noted that the antibacterial protein in the specification of the present invention comprises at least one peptide having a length of 2 to 200 amino acids, and the peptide shape may be a linear shape, a spiral shape or a U shape. Or other configurations that can reach a specific biological function, and even call this peptide a protein. In one embodiment of the invention, the antimicrobial protein is grouper antibacterial protein-1.

在本發明書中，「病毒」一詞意指感染哺乳類的病毒，特別係屬於黃病毒科(Flaviviridae)之病毒如登革熱出血病毒(dengue hemorrhagic fever，DHF)、黃熱病病毒(yellow fever virus)、日本腦炎病毒(Japanese encephalitis viruses,JEV)、牛病毒腹瀉病毒(bovine viral diarrhea virus)、豬瘟病毒(Classical swine fever virus,CSFV)、C型肝炎病毒(hepatits C virus,HCV)、或其他選自於黃病毒屬(Flavivirus )、瘟病毒屬(Pestivirus )、或肝病毒屬(Hepacivirus )之病毒。在本發明之一實施例中，病毒係為日本腦炎病毒。In the present specification, the term "virus" means a virus that infects a mammal, particularly a virus belonging to the Flaviviridae family, such as dengue hemorrhagic fever (DHF), yellow fever virus, Japanese encephalitis viruses (JEV), bovine viral diarrhea virus, classical swine fever virus (CSFV), hepatitis C virus (HCV), or other options since flavivirus genus (flavivirus), pestivirus (pestivirus), or liver virus genus (Hepacivirus) of the virus. In one embodiment of the invention, the virus is a Japanese encephalitis virus.

本說明書中所說之「有效劑量」一詞意指本發明之醫藥組合物中的石斑魚抗菌蛋白-1及以哺乳類為宿主之病毒用於受病毒感染之受體時，可達到上述預防或治療效果。且有效劑量，可由本領域具有通常知識者，隨受體物種、重量、體型等不同，或所使用之醫藥組合中除石斑魚抗菌蛋白-1及以哺乳類為宿主之病毒外之其他添加成份所佔比例及功能，來進行調配及變更。在本發明一實施例中，石斑魚之抗菌蛋白之有效劑量係介於5-500 μg/ml之間。在本發明之一實施例中，抗菌蛋白之有效劑量較佳地係介於50 μg/ml至200 μg/ml之間，而病毒之有效劑量較佳地係介於40倍至60倍之半致死劑量。The term "effective dose" as used in the specification means that the grouper antibacterial protein-1 and the mammalian host virus in the pharmaceutical composition of the present invention can achieve the above prevention or treatment when used as a virus-infected receptor. effect. And the effective dosage may be determined by those having ordinary knowledge in the art, depending on the species, weight, body type, etc. of the receptor, or other additive components other than the grouper antibacterial protein-1 and the mammalian host virus in the pharmaceutical combination used. Proportion and function to make adjustments and changes. In one embodiment of the invention, the effective dose of the antibacterial protein of the grouper is between 5 and 500 μg/ml. In an embodiment of the invention, the effective dose of the antibacterial protein is preferably between 50 μg/ml and 200 μg/ml, and the effective dose of the virus is preferably between 40 and 60 times Lethal dose.

在本說明書中所使用之「動物」一詞意指受病毒感染之生物體包含人類及陸地和水生動物等之脊椎動物，主要是針對被定義為哺乳類之動物。The term "animal" as used in this specification means that a virus-infected organism comprises humans and vertebrates such as terrestrial and aquatic animals, mainly for animals defined as mammals.

包含本發明中有效劑量之石斑魚抗菌蛋白-1及以哺乳類為宿主之病毒以及包含合適於醫藥上所使用之添加物，其中，石斑魚抗菌蛋白-1之序列係包含序列表中序列編號1所列之胺基酸序列，或是包含與上述序列編號1中，80%以上相似之胺基酸序列片段，而來源可為人工合成或直接自石斑魚(Epinephelus coioides )中萃取而得。An effective dose of the grouper antibacterial protein-1 and a mammalian hosted virus according to the present invention and an additive suitable for use in medicine, wherein the sequence of the grouper antibacterial protein-1 comprises the sequence number 1 in the sequence listing. The amino acid sequence or the amino acid sequence fragment similar to 80% or more of the above SEQ ID NO: 1, and the source may be artificially synthesized or directly extracted from grouper ( Epinephelus coioides ).

在本說明書中所使用之「添加物」一詞意指包括但不限於賦形劑、稀釋液、增溶劑、乳化劑、抗氧化劑、介面活性劑、脂質體或適合受體生理環境所需之各種酸鹼值、離子強度之緩衝溶液或稀釋液等。醫藥組合物的投藥方式包括但不限於口服、肌肉注射、皮下注射、皮內注射、腹腔注射、靜脈注射、或直接於出現病癥處進行注射。The term "additive" as used in this specification is meant to include, but is not limited to, excipients, diluents, solubilizers, emulsifiers, antioxidants, surfactants, liposomes or those suitable for the physiological environment of the recipient. Various pH values, ionic strength buffer solutions or diluents, etc. The mode of administration of the pharmaceutical composition includes, but is not limited to, oral, intramuscular, subcutaneous, intradermal, intraperitoneal, intravenous, or injection directly at the site of the condition.

另外，在本發明之一實施例中，本發明之醫藥組合物係具有免疫刺激之活性。Further, in an embodiment of the present invention, the pharmaceutical composition of the present invention has an immunostimulating activity.

在本發明之一實施例中，本發明之醫藥組合物係為一疫苗。In one embodiment of the invention, the pharmaceutical composition of the invention is a vaccine.

在本發明之一實施例中，醫藥組合物中之石斑魚抗菌蛋白係為一疫苗佐劑。In one embodiment of the invention, the grouper antibacterial protein in the pharmaceutical composition is a vaccine adjuvant.

承上所述，本發明之醫藥組合物，包含有效劑量之一石斑魚之抗菌蛋白，以及具感染哺乳類活性之一病毒。透過免疫途徑，可使哺乳類具有抵抗病毒之能力，以達到治療病毒性疾病的效果。另外，本發明之醫藥組合物可以疫苗之形式有效預防病毒的感染，其中，以石斑魚的抗菌蛋白作為佐劑，利用其抗病毒之特性，在有效劑量下與病毒混合後能使病毒失去活性，以刺激免疫能力並產生高量且持久之專一性抗體。相較於習知技術中使用福馬林作為佐劑的疫苗，本發明以石斑魚的抗菌蛋白作為佐劑之疫苗可誘發較高量的專一性抗體，使哺乳類動物能具有較好的抵抗能力。再者，本發明之醫藥組合物對生物體不具毒性，因此不會有生物安全性的疑慮。總體而言，本發明之醫藥組合物透過刺激哺乳類的免疫能力，達到治療哺乳類病毒性疾病的功效。相較於習知的疫苗，本發明所提供之醫藥組合物更為有效、安全且具有製程簡便及低成本等特色。As described above, the pharmaceutical composition of the present invention comprises an antibacterial protein of one of the effective doses of grouper and a virus having activity of infecting mammals. Through the immunization route, mammals can have the ability to resist viruses to achieve the effect of treating viral diseases. In addition, the pharmaceutical composition of the present invention can effectively prevent viral infection in the form of a vaccine, wherein the anti-viral property of the grouper is used as an adjuvant, and the virus is inactivated by mixing with the virus at an effective dose. To stimulate immunity and produce high and long-lasting specific antibodies. Compared with the vaccine using fumarin as an adjuvant in the prior art, the vaccine of the grouper antibacterial protein as an adjuvant can induce a higher amount of specific antibody, so that the mammal can have better resistance. Furthermore, the pharmaceutical composition of the present invention is not toxic to living organisms, and thus there is no doubt about biosafety. In general, the pharmaceutical composition of the present invention achieves the efficacy of treating mammalian viral diseases by stimulating the immunity of mammals. Compared with the conventional vaccine, the pharmaceutical composition provided by the invention is more effective, safe, and has the characteristics of simple process and low cost.

依據本發明之一種用於治療哺乳類病毒性疾病之醫藥組合物，其包含有效劑量之一源於石斑魚之抗菌蛋白，以及一病毒，且病毒具感染哺乳類之活性。A pharmaceutical composition for treating a mammalian viral disease according to the present invention, which comprises an antibacterial protein derived from grouper, and a virus, wherein the virus has an activity of infecting a mammal.

本發明之醫藥組合物中，石斑魚之抗菌蛋白係包含萃取自石斑魚屬(Epinephelus )的魚種至少其中之一之抗菌蛋白，而感染哺乳類之病毒係選自於黃病毒科(Flaviviridae )的病毒。於此，在本發明中較佳之醫藥組合物係包含萃取自點帶石斑魚體內的石斑魚抗菌蛋白-1(Epinecidin-1,Epi-1)及黃病毒科中的日本腦炎病毒(Japanese encephalitis viruses,JEV)，且將兩者混合於一緩衝溶液而製成。其中，醫藥組合物中的抗菌蛋白不限於直接從生物體中萃取的胜肽，或是經由人工合成與序列表1所列之胺基酸序列相同或至少80%相同之序列之胜肽。本發明經由一實驗證明石斑魚抗菌蛋白-1具抗病毒之功效，係說明於下。透過細胞培養技術對幼倉鼠腎細胞-21(baby hamster kidney cells-21,BHK-21,ATCC CCL10)給予不同量之石斑魚抗菌蛋白-1，以比較石斑魚抗菌蛋白-1及日本腦炎病毒以不同加入順序時對細胞感染影響。第一組細胞進行在加入日本腦炎病毒前1小時先給予石斑魚抗菌蛋白；第二組細胞則在加入日本腦炎病毒後1小時再給予石斑魚抗菌蛋白-1；以及第三組細胞加入事先混合好的石斑魚抗菌蛋白-1及日本腦炎病毒混合液。各組之各個孔洞則分別安排加入6種不同濃度之石斑魚抗菌蛋白-1，包括0,1,0.5,0.25,0.125及0.0625μg/ml，而每個孔洞則均給予5000 pfu的病毒量。各個孔洞於添加石斑魚抗菌蛋白-1及日本腦炎病毒48小時後，取各組細胞利用CellTiter 96 Aqueous One Solution(Promega,Madison,WI,USA)進行細胞增殖分析(cell proliferation assay)，計算各組細胞經過不同處理之後的感染率。In the pharmaceutical composition of the present invention, the antibacterial protein of the grouper comprises an antibacterial protein extracted from at least one of the species of Epinephelus , and the virus infecting the mammal is selected from the group consisting of Flaviviridae . Herein, the preferred pharmaceutical composition of the present invention comprises the grouper antibacterial protein-1 (Epinecidin-1, Epi-1) extracted from the spotted grouper and the Japanese encephalitis virus (Japanese encephalitis viruses). JEV), and the two are mixed in a buffer solution. Among them, the antibacterial protein in the pharmaceutical composition is not limited to a peptide which is directly extracted from an organism, or a peptide which is synthesized by synthesizing a sequence which is identical or at least 80% identical to the amino acid sequence listed in the Sequence Listing 1. The invention proves that the grouper antibacterial protein-1 has an antiviral effect through an experiment, and is described below. Different amounts of grouper antibacterial protein-1 were administered to baby hamster kidney cells-21 (BHK-21, ATCC CCL10) by cell culture technique to compare grouper antibacterial protein-1 and Japanese encephalitis virus. The effect of cell infection on the order of addition. The first group of cells were given the grouper antibacterial protein one hour before the addition of the Japanese encephalitis virus; the second group was given the grouper antibacterial protein-1 one hour after the addition of the Japanese encephalitis virus; and the third group of cells was added before mixing. Good grouper antibacterial protein-1 and Japanese encephalitis virus mixture. Each hole of each group was arranged to add 6 different concentrations of grouper antibacterial protein-1, including 0, 1, 0.5, 0.25, 0.125 and 0.0625 μg/ml, and each hole was given 5000 pfu of virus. After 48 hours of addition of grouper antibacterial protein-1 and Japanese encephalitis virus, each well was subjected to cell proliferation assay using CellTiter 96 Aqueous One Solution (Promega, Madison, WI, USA) to calculate each group. The infection rate of cells after different treatments.

圖1為對BHK-21細胞以日本腦炎病毒感染之前、後以及同時加入石斑魚抗菌蛋白-1之後細胞的感染率。請參考圖1所示，對BHK-21細胞同時加入石斑魚抗菌蛋白-1及日本腦炎病毒，細胞的感染率明顯較另外兩組降低許多，且尤以石斑魚抗菌蛋白-1濃度分別為1μg/ml及0.5μg/ml時與病毒同時加入的細胞，相較於僅加入病毒感染之控制組(如圖1橫座標標示0 μg/ml Epi-1)，感染率分別降至50%及60%。顯示，石斑魚抗菌蛋白-1具有抵抗病毒之特性，且隨石斑魚抗菌蛋白-1之劑量增加而具有更顯著之功效，呈現劑量依賴(dose-dependent)的趨勢。故由實驗結果可知，給予石斑魚抗菌蛋白-1與日本腦炎病毒能夠有效降低細胞受感染的比例，且石斑魚抗菌蛋白-1具有抑制或不活化日本腦炎病毒的功效。Figure 1 shows the infection rate of cells after BHK-21 cells were infected with Japanese encephalitis virus before, after and simultaneously with grouper antibacterial protein-1. Please refer to Figure 1. As shown in Figure 1, the addition of grouper antibacterial protein-1 and Japanese encephalitis virus to BHK-21 cells was significantly lower than that of the other two groups, especially the grouper antibacterial protein-1 concentration was 1μg/ When the ml and 0.5μg/ml were added to the virus at the same time, the infection rate was reduced to 50% and 60%, respectively, compared with the control group in which only the virus infection was added (Fig. 1 horizontal coordinate indicates 0 μg/ml Epi-1). . It has been shown that grouper antibacterial protein-1 is resistant to viruses and has a more pronounced effect with increasing dose of grouper antibacterial protein-1, showing a dose-dependent trend. Therefore, it is known from the experimental results that the grouper antibacterial protein-1 and Japanese encephalitis virus can effectively reduce the proportion of cells infected, and the grouper antibacterial protein-1 has the effect of inhibiting or not activating Japanese encephalitis virus.

於實驗例中，本發明係利用石斑魚抗菌蛋白-1之抗病毒功效，與日本腦炎病毒混合，以將其去活化，製成具治療效用之醫藥組合物。其製法如下：石斑魚抗菌蛋白-1之胺基酸序列係如本發明之序列表所列。經由GL Biochem(Shanghai,China)合成C端醯胺化(amidated)石斑魚抗菌蛋白-1。日本腦炎病毒株，北京-1型(Beijing-1)係藉由感染乳鼠(suckling mouse)腦部，維持病毒活性。將兩者溶於磷酸緩衝溶液(phosphate-buffered saline,PBS)中，配製成三組不同濃度石斑魚抗菌蛋白-1之醫藥組合物。當中，第1組、第2組及第3組醫藥組合物之石斑魚抗菌蛋白-1濃度分別為為200,100及50 μg/ml，病毒濃度則皆為50倍母的C3H/HeN成鼠(購自國家實驗動物繁殖及研究中心，台北，台灣)之半致死劑量，亦即約為5×106 pfu/ml。使用前將各醫藥組合物於室溫下靜置10-30分鐘，以充分將日本腦炎病毒去活化。In the experimental examples, the present invention utilizes the antiviral effect of the grouper antibacterial protein-1, and is mixed with the Japanese encephalitis virus to deactivate it to prepare a pharmaceutical composition having therapeutic effects. The preparation method is as follows: The amino acid sequence of the grouper antibacterial protein-1 is as listed in the sequence listing of the present invention. The C-terminal amidated grouper antibacterial protein-1 was synthesized via GL Biochem (Shanghai, China). The Japanese encephalitis virus strain, Beijing-1 (Beijing-1) maintains viral activity by infecting the brain of a suckling mouse. The two were dissolved in phosphate-buffered saline (PBS) to prepare three groups of pharmaceutical compositions of different concentrations of grouper antibacterial protein-1. Among them, group 1 , group 2 and group 3 pharmaceutical compositions have grouper antibacterial protein-1 concentrations of 200, 100 and 50 μg/ml, respectively, and virus concentrations are 50 times female C3H/HeN adult mice (purchased from The semi-lethal dose of the National Laboratory Animal Breeding and Research Center, Taipei, Taiwan) is approximately 5 × 10 6 pfu/ml. Each of the pharmaceutical compositions was allowed to stand at room temperature for 10 to 30 minutes before use to sufficiently deactivate the Japanese encephalitis virus.

以下，係以複數實驗例來證實本發明之醫藥組合物，能有效刺激生物體免疫能力，並幫助生物體抵抗病毒感染及具有較佳生物安全性之特性。Hereinafter, the pharmaceutical composition of the present invention is confirmed by a plurality of experimental examples, which can effectively stimulate the immunity of the living body and help the organism to resist viral infection and have better biosafety characteristics.

實驗例一：醫藥組合物係具有刺激專一性抗體產生之功效Experimental Example 1: The pharmaceutical composition has the effect of stimulating the production of specific antibodies

取50隻6週大的C3H/HeN成鼠，以10隻為一組，共分成五組。第1至第3組老鼠分別係以腹腔注射途徑注射前述依本發明醫藥組合物所製成的不同濃度之三組醫藥組合物。第4組為對老鼠注射不含石斑魚抗菌蛋白-1，只含50倍半致死劑量的日本腦炎病毒液，及第5組則為對老鼠注射不含病毒之200 μg/ml濃度之石斑魚抗菌蛋白-1。每隻老鼠注射量均為100 μl，並於注射後第14天，每隻老鼠再注射50倍半致死劑量的日本腦炎病毒液進行再感染，並觀察各組老鼠之存活率。Fifty six-week-old C3H/HeN adult rats were divided into five groups and divided into five groups. Groups 1 to 3 were injected with the above three groups of pharmaceutical compositions of different concentrations prepared according to the pharmaceutical composition of the present invention by intraperitoneal injection. The fourth group was to inject the mice with the grouper antibacterial protein-1, which contained only 50 times the lethal dose of Japanese encephalitis virus solution, and the fifth group was to inject the mice with the virus-free grouper 200 μg/ml. Protein-1. Each mouse was injected 100 μl, and on the 14th day after the injection, each mouse was reinjected with a 50-fold LD50 dose of Japanese encephalitis virus solution, and the survival rate of each group was observed.

圖2A為使用不同濃度石斑魚抗菌蛋白-1之醫藥組合物，以幫助老鼠抵抗日本腦炎病毒感染之存活率示意圖。如圖2A所示，在第一次注射後7天內，第4組老鼠全數死亡；其他注射本發明之醫藥組合物的老鼠，第2組及第3組老鼠有部份死亡，存活率分別為90%及60%，而第1組及第5組均維持100%存活率。第一次注射後第14天，以日本腦炎病毒再感染各組老鼠。結果顯示，第1組至第3組老鼠的存活率與未進行再感染前相同，而第5組老鼠於病毒感染後4天全數死亡。此說明了本發明的醫藥組合物中，由於同時含有石斑魚抗菌蛋白-1及日本腦炎病毒的成分，故能使老鼠具有抵抗日本腦炎病毒感染之能力；而僅單純給予石斑魚抗菌蛋白-1或日本腦炎病毒則都不能使老鼠具有抵抗的能力。另外，醫藥組合物中石斑魚抗菌蛋白-1濃度越高，則提升存活率之效果越佳。Figure 2A is a graphical representation of the survival rate of a Japanese encephalitis virus infection using a pharmaceutical composition of different concentrations of grouper antibacterial protein-1. As shown in Fig. 2A, all of the mice in group 4 died within 7 days after the first injection; the mice injected with the pharmaceutical composition of the present invention, the mice in groups 2 and 3 were partially killed, and the survival rates were respectively It was 90% and 60%, while Group 1 and Group 5 maintained 100% survival. On the 14th day after the first injection, each group of mice was re-infected with Japanese encephalitis virus. The results showed that the survival rate of the mice in the first group to the third group was the same as that before the reinfection, and the mice in the fifth group died in all four days after the virus infection. This demonstrates that in the pharmaceutical composition of the present invention, since it contains both the grouper antibacterial protein-1 and the Japanese encephalitis virus, the mouse is capable of resisting the infection of Japanese encephalitis virus; and only the grouper antibacterial protein-1 is simply administered. Or Japanese encephalitis virus can not make mice have the ability to resist. In addition, the higher the concentration of the grouper antibacterial protein-1 in the pharmaceutical composition, the better the effect of improving the survival rate.

於第一次注射後第4天，從每隻老鼠尾部抽取血清，利用酵素連結免疫吸附分析法(Enzyme-linked immunosorbent assay,ELISA)將老鼠體內的抗日本腦炎病毒抗體進行定量，其步驟如下：於96-孔(96-wells)盤底部披覆(coating)日本腦炎病毒，經阻斷試劑(blocking reagent)處理後，配置各種不同濃度之血清作為標準品(standard)，各取50 μl加到孔洞中，共進行三重覆，其他孔洞加入前述抽取自老鼠尾部的血清，37℃下作用1小時。清洗後，加入稀釋1000倍的山羊山葵過氧化氫酶-連接山羊抗-老鼠免疫球蛋白G抗體(goat horseradish peroxidase(HRP)-conjugated goat anti-mouse IgG antibody,ICN/Cappel,Aurora,OH,USA)，37℃下作用2小時。清洗後，各孔洞加入50 μl鄰苯二胺鹽酸鹽(o -phenylenediamine dihydrochloride,Sigma-Aldrich,St. Louis,MO,USA)室溫下避光呈色30分鐘，並測於495 nm波長下之吸光值(OD495 )。各濃度之標準品對應之吸光值製成標準曲線，達到平原曲線的吸光值則定義為最大吸光值(ODmax )，而OD值達ODmax 數值的二分之一時所對應之血清濃度定義為：1單元(unit,U)具中和能力之抗日本腦炎病毒外套膜(anti-JEV-E)。On the 4th day after the first injection, serum was taken from the tail of each mouse, and the anti-Japanese encephalitis virus antibody in the mouse was quantified by Enzyme-linked immunosorbent assay (ELISA). The procedure was as follows: : Coating Japanese encephalitis virus at the bottom of 96-wells, and after treatment with blocking reagent, configure various concentrations of serum as standard, each taking 50 μl It was added to the wells and triple-covered. The other wells were added to the serum extracted from the tail of the mouse and allowed to act at 37 ° C for 1 hour. After washing, add 1000 times dilution of goat wasabi catalase-linked goat anti-mouse IgG antibody (ICP/conjugated goat anti-mouse IgG antibody, ICN/Cappel, Aurora, OH, USA) ), at 37 ° C for 2 hours. After washing, 50 μl of each hole was added o-phenylenediamine hydrochloride (o -phenylenediamine dihydrochloride, Sigma-Aldrich , St. Louis, MO, USA) at room temperature in the dark coloring 30 min, and measured at a wavelength of 495 nm The absorbance value (OD 495 ). The absorbance corresponding to the standard of each concentration is made into a standard curve, and the absorbance value of the plain curve is defined as the maximum absorbance (OD max ), and the serum concentration corresponding to the OD value is one-half of the OD max value. It is: 1 unit (unit, U) with anti-Japanese encephalitis virus mantle film (anti-JEV-E) with neutralizing ability.

圖2B為於第一次注射醫藥組合物後第4天抽取所有老鼠的血清偵測抗日本腦炎病毒外套膜的專一性抗體量。圖2B中，除了第5組(注射僅含200 μg/ml濃度之石斑魚抗菌蛋白-1)以外，其他組老鼠體內均被誘發具有抗日本腦炎病毒外套膜的抗體。Figure 2B is a graph showing the amount of specific antibody against the Japanese encephalitis virus mantle membrane on the 4th day after the first injection of the pharmaceutical composition. In Fig. 2B, except for the fifth group (injection of the grouper antibacterial protein-1 containing only 200 μg/ml), the mice of the other groups were induced to have antibodies against the mantle membrane of the Japanese encephalitis virus.

圖2C為於第二次注射後第7天抽取存活老鼠的血清偵測抗日本腦炎病毒外套膜的專一性抗體量。當第二次注射以病毒進行再感染後第7天，存活下來的老鼠其血清中仍具有抗日本腦炎病毒外套膜的抗體，且較第一次注射後的第4天所測得的含量更高。Figure 2C shows the amount of specific antibody against the Japanese encephalitis virus mantle membrane extracted from the serum of surviving mice on the 7th day after the second injection. On the 7th day after the second injection was reinfected with the virus, the surviving mice still had antibodies against the Japanese encephalitis virus mantle, and the content measured on the 4th day after the first injection. higher.

另外，為評估注射醫藥組合物的老鼠血清中所產生之抗體是否具有中和日本腦炎病毒的能力，於是本發明以BHK-21細胞進行50%病毒斑減少試驗(50% plaque reduction assay)方式進行病毒斑減少中和性試驗(plaque reduction neutralization test,PRNT)，關於此實驗之方法係採用熟習此項技藝者所慣用的技術來進行。最後，病毒斑減少50%的最高稀釋倍數之倒數即可計算出中和性抗體效價(neutralization antibody titer)。In addition, in order to evaluate whether the antibody produced in the serum of the mouse injected with the pharmaceutical composition has the ability to neutralize Japanese encephalitis virus, the present invention uses the BHK-21 cell for 50% plaque reduction assay. The plaque reduction neutralization test (PRNT) was performed, and the method for this experiment was carried out using techniques familiar to those skilled in the art. Finally, the neutralization antibody titer can be calculated by reversing the highest dilution of the plaque by 50%.

如下表1所示，僅注射石斑魚抗菌蛋白-1(0,50,100,200 μg/ml)於老鼠體內，其所測得之中和性抗體效價均為小於1/10，僅注射病毒的老鼠體內所產生之中和性抗體效價為小於1/40，而50,100及200 μg/ml石斑魚抗菌蛋白-1分別與病毒混合後注射到老鼠體內，自老鼠血清中測得之中和性抗體效價分別為大於1/160、大於1/320及大於1/1280。As shown in Table 1 below, only grouper antibacterial protein-1 (0, 50, 100, 200 μg/ml) was injected into mice, and the neutralizing antibody titers measured were less than 1/10, only in mice injected with virus. The neutralizing antibody titer was less than 1/40, and 50, 100 and 200 μg/ml grouper antibacterial protein-1 were mixed with the virus and injected into the mouse. The neutralizing antibody titer was determined from the mouse serum. It is greater than 1/160, greater than 1/320, and greater than 1/1280.

根據上述實驗證實本發明之醫藥組合物可有效誘發老鼠產生專一性抗體，老鼠體內對含有石斑魚抗菌蛋白-1所誘發之抗體量較單純針對活病毒所產生之抗體量高，且醫藥組合物中石斑魚抗菌蛋白-1濃度越高，則能誘發越多有效的專一性抗體，證明本發明之醫藥組合物具有疫苗之功效。According to the above experiment, it was confirmed that the pharmaceutical composition of the present invention can effectively induce specific antibody production in mice, and the amount of antibody induced by the grouper containing the grouper antibacterial protein-1 is higher than that of the live virus alone, and the pharmaceutical composition is high. The higher the concentration of the grouper antibacterial protein-1, the more effective specific antibodies can be induced, demonstrating that the pharmaceutical composition of the present invention has the efficacy of a vaccine.

實驗例二：醫藥組合物係具有刺激體液免疫反應之功效Experimental Example 2: The pharmaceutical composition has the effect of stimulating humoral immune response

同實驗例一所實驗的各個老鼠，在第一次注射後的第4天、第7天及第21天抽取其血清，利用ELISA方法測試其IgG同種型(isotype)表現，其步驟同實驗例一所述。於本實驗例中，在加入血清之後，加生物素-連接大鼠抗小鼠免疫球蛋白G1抗體(biotin-conjugated rat anti-mouse IgG1,San Diego,CA,USA)或生物素-連接大鼠抗-小鼠免疫球蛋白G2a抗體(biotin-conjugated rat anti-mouse IgG2a,Pharmingen,San Diego,CA,USA)，最後加抗生物素蛋白-山葵過氧化氫酶(avidin-HRP)，進行呈色反應並偵測其吸光值。The mice in the same experiment as in the first experiment were taken on the 4th, 7th and 21st day after the first injection, and their IgG isotypes were tested by ELISA. The procedure was the same as the experimental example. One said. In this example, biotin-conjugated rat anti-mouse immunoglobulin G1 antibody (biotin-conjugated rat anti-mouse IgG1, San Diego, CA, USA) or biotin-linked rat was added after serum addition. Anti-mouse immunoglobulin G2a antibody (biotin-conjugated rat anti-mouse IgG2a, Pharmingen, San Diego, CA, USA), and finally avidin-Moss hydrogenate (avidin-HRP) for coloration React and detect its absorbance.

圖3A為經給予疫苗後老鼠血清中的IgG1及IgG2a表現量。如圖3A所示，第1組至第3組老鼠給予不同石斑魚抗菌蛋白-1濃度之醫藥組合物後，三組老鼠體內血清中，可抗日本腦炎病毒外套膜(anti-JEV-E)的IgG1表現量均明顯較IgG2a高許多。Figure 3A shows the amount of IgG1 and IgG2a expressed in the serum of mice after administration of the vaccine. As shown in Fig. 3A, after the first group to the third group of rats were given a pharmaceutical composition of different grouper antibacterial protein-1 concentration, the serum of the three groups of mice was resistant to the Japanese encephalitis virus mantle (anti-JEV-E). The expression levels of IgG1 were significantly higher than those of IgG2a.

另外，使用老鼠細胞激素ELISA套組BD OptEIATM (BD Bioscience,San Diego,CA,USA)偵測T輔助細胞1(T helper 1,Th1 )所活化的細胞激素介白素-12(interleukine-12,IL-2)及干擾素-γ(interferon-γ,IFN-γ)，以及Th2 所活化的細胞激素介白素-4(interleukine-4,IL-4)及介白素-10(interleukine-10,IL-10)。In addition, the cytokine interleukin-12 (interleukine-activated by T helper 1, Th 1 ) was detected using the mouse cytokine ELISA kit BD OptEIA TM (BD Bioscience, San Diego, CA, USA). 12, IL-2) and interferon-γ (IFN-γ), and Th 2 activated cytokine interleukin-4 (IL-4) and interleukin-10 ( Interleukine-10, IL-10).

圖3B為注射醫藥組合物一週後老鼠血清中IL-2、IFN-γ、IL-4及IL-10的表現量。如圖3B所示，經過醫藥組合物處理的老鼠相較僅給予石斑魚抗菌蛋白-1的老鼠血清中，有較高量的IL-4及IL-10表現量。綜合圖3A及圖3B之結果證明本發明之疫苗主要是透過刺激Th2 ，使IgG1大量表現，因此本發明之疫苗能促進體液免疫反應並產生有效專一性抗體對抗病毒之感染。Figure 3B shows the expression levels of IL-2, IFN-γ, IL-4 and IL-10 in rat serum one week after the injection of the pharmaceutical composition. As shown in Fig. 3B, the mice treated with the pharmaceutical composition had higher amounts of IL-4 and IL-10 than the serum of the rats given the grouper antibacterial protein-1 alone. The results of FIGS. 3A and 3B demonstrate that the vaccine of the present invention mainly exerts a large amount of IgG1 by stimulating Th 2 , and thus the vaccine of the present invention can promote a humoral immune response and produce an effective specific antibody against virus infection.

由本實驗例結果可證明，本發明之醫藥組合物具有刺激免疫之功效，隨石斑魚抗菌蛋白-1的劑量提升，而產生越高的專一性抗日本腦炎外套膜抗體效價(antibody titer)。因此，本發明之醫藥組合物實質上具有疫苗之功效。From the results of the present experimental examples, it was confirmed that the pharmaceutical composition of the present invention has an effect of stimulating immunity, and as the dose of the grouper antibacterial protein-1 is increased, a higher specificity is produced against the antibody titer of the Japanese encephalitis mantle. Therefore, the pharmaceutical composition of the present invention has substantially the efficacy of a vaccine.

實驗例三：醫藥組合物的石斑魚抗菌蛋白-1作為佐劑之用途Experimental Example 3: Use of grouper antibacterial protein-1 as an adjuvant for a pharmaceutical composition

取3組4-7隻新生的C3H/HeN幼鼠，第一組老鼠係注射PBS，第二組老鼠係注射福馬林及日本腦炎病毒所組成之疫苗，第三組老鼠係注射本發明200 μg/ml石斑魚抗菌蛋白-1及日本腦炎病毒所組成之疫苗。所使用的日本腦炎病毒劑量為50倍半致死劑量(LD50 )，各組共注射3次，分別於新生幼鼠7天大時給予第一次注射、14天大時給予第二次注射及21天大時給予第三次注射，並偵測注射疫苗三次後老鼠血清中抗日本腦炎病毒外套膜之抗體量。之後，再以等量之日本腦炎病毒對注射三次疫苗後的老鼠進行再感染，並於再感染後第4天再測量老鼠體內的抗體表現量。Three groups of 4-7 newborn C3H/HeN pups were selected. The first group was injected with PBS, the second group was injected with vaccine consisting of formalin and Japanese encephalitis virus, and the third group was injected with the invention 200. A vaccine consisting of μg/ml grouper antibacterial protein-1 and Japanese encephalitis virus. The dose of Japanese encephalitis virus used was 50 times the lethal dose (LD 50 ), and each group was injected a total of 3 times. The newborn rats were given the first injection at 7 days of age and the second injection at 14 days old. The third injection was given at 21 days of age, and the amount of antibody against the Japanese encephalitis virus mantle membrane in the serum of the mice three times after the injection was detected. Thereafter, the mice injected with the three vaccines were re-infected with an equal amount of Japanese encephalitis virus, and the antibody expression in the mice was measured again on the fourth day after the reinfection.

另外，為比較兩種疫苗強化新生幼鼠的免疫能力，另進行一實驗，除上述第一至第三組外，再增加一第四組老鼠係注射日本腦炎病毒以作為對照組，並在各組注射後第35天以日本腦炎病毒進行再感染，之後觀察各組老鼠之存活率。In addition, in order to compare the immunity of the two vaccines to enhance the immunity of newborn rats, another experiment was conducted. In addition to the first to third groups above, a fourth group of rats was injected with Japanese encephalitis virus as a control group, and Each group was reinfected with Japanese encephalitis virus on the 35th day after the injection, and then the survival rate of each group of mice was observed.

圖4A為新生幼鼠分別於第7天、第14天及第21天注射以福馬林為佐劑及以石斑魚抗菌蛋白-1為佐劑之疫苗後的抗日本腦炎病毒外套膜之抗體量。如圖4A顯示，於第二次注射疫苗時，第二組及第三組的老鼠體內抗體量已開始增加，且以石斑魚抗菌蛋白-1為佐劑之疫苗有較高的抗體量。於第三次注射時，所觀察的情況更加明顯，兩組之抗體量均明顯大量提升，且以石斑魚抗菌蛋白-1為佐劑之疫苗，可誘發較高的抗體表現量。Figure 4A shows the amount of antibody against the Japanese encephalitis virus mantle after injection of a vaccine containing fumarin as an adjuvant and grouper antibacterial protein-1 as adjuvants on day 7, day 14, and day 21, respectively. . As shown in Fig. 4A, in the second injection of vaccine, the amount of antibody in the mice of the second group and the third group began to increase, and the vaccine with the grouper antibacterial protein-1 as an adjuvant had a higher antibody amount. At the third injection, the observed situation was more obvious, and the amount of antibody in both groups was significantly increased, and the vaccine with the grouper antibacterial protein-1 as an adjuvant induced higher antibody expression.

圖4B為注射三次疫苗後以病毒感染後老鼠體內抗體表現量。如圖4B所示，第二組及第三組的老鼠體內仍產生較高量的抗體，其中又以石斑魚抗菌蛋白-1為佐劑之疫苗的老鼠具有較高的抗體量。Figure 4B shows the amount of antibody expression in mice after infection with virus after three injections. As shown in Fig. 4B, the mice in the second group and the third group still produced higher amounts of antibodies, and the mice in which the grouper antibacterial protein-1 was adjuvanted had a higher antibody amount.

圖4C為注射三次疫苗後以病毒感染後各組老鼠之存活率。如圖4C所示，老鼠施打以福馬林為佐劑的疫苗經病毒感染後存活率為90%，而施打以石斑魚抗菌蛋白-1為佐劑的疫苗經病毒感染後存活率仍維持100%。顯示以石斑魚抗菌蛋白-1為佐劑的疫苗可使新生幼鼠具有較佳的免疫能力以抵抗病毒之感染。Figure 4C shows the survival rate of mice in each group after virus infection after three injections. As shown in Fig. 4C, the survival rate of the vaccine administered with fumarin as a vaccine was 90%, and the survival rate of the vaccine with the grouper antibacterial protein-1 as a vaccine was maintained at 100. %. Vaccines showing grouper antibacterial protein-1 as an adjuvant can provide neonatal rats with better immunity against viral infection.

根據實驗結果可說明，本發明之石斑魚抗菌蛋白-1可取代福馬林作為疫苗的佐劑，且本發明之疫苗即使用於剛出生的動物，也能有效引發其免疫反應，並產生較高的抗病毒抗體量，來對抗日本腦炎病毒之感染，而不會對其產生不良的副作用造成死亡。According to the experimental results, the grouper antibacterial protein-1 of the present invention can replace fumarin as an adjuvant for the vaccine, and the vaccine of the present invention can effectively induce an immune reaction even when used in a newborn animal, and produces a high The amount of anti-viral antibody is used to combat the infection of the Japanese encephalitis virus without causing death due to adverse side effects.

承上所述，本發明之醫藥組合物包含有效劑量之一石斑魚之抗菌蛋白，以及具感染哺乳類活性之一病毒。透過免疫途徑，可使哺乳類具有抵抗病毒之能力，以達到治療病毒性疾病的效果。另外，本發明之醫藥組合物可以疫苗之形式有效預防病毒的感染，其中，以石斑魚的抗菌蛋白作為佐劑，利用其抗病毒之特性，在有效劑量下與病毒混合後能使病毒失去活性，以刺激的免疫能力並產生高量且持久之專一性抗體。相較於習知技術中使用福馬林作為佐劑的疫苗，本發明以石斑魚的抗菌蛋白作為佐劑之疫苗可誘發較高量的專一性抗體，使哺乳類動物能具有較好的抵抗能力。再者，本發明之醫藥組合物對生物體不具毒性，因此不會有生物安全性的疑慮。總體而言，本發明之醫藥組合物透過刺激哺乳類的免疫能力，達到治療哺乳類病毒性疾病的功效。相較於習知的疫苗，本發明所提供之醫藥組合物更為有效、安全且具有製程簡便及低成本等特色。As described above, the pharmaceutical composition of the present invention comprises an antibacterial protein of one of the effective doses of the grouper, and a virus having an activity of infecting mammals. Through the immunization route, mammals can have the ability to resist viruses to achieve the effect of treating viral diseases. In addition, the pharmaceutical composition of the present invention can effectively prevent viral infection in the form of a vaccine, wherein the anti-viral property of the grouper is used as an adjuvant, and the virus is inactivated by mixing with the virus at an effective dose. It stimulates immunity and produces high and long-lasting specific antibodies. Compared with the vaccine using fumarin as an adjuvant in the prior art, the vaccine of the grouper antibacterial protein as an adjuvant can induce a higher amount of specific antibody, so that the mammal can have better resistance. Furthermore, the pharmaceutical composition of the present invention is not toxic to living organisms, and thus there is no doubt about biosafety. In general, the pharmaceutical composition of the present invention achieves the efficacy of treating mammalian viral diseases by stimulating the immunity of mammals. Compared with the conventional vaccine, the pharmaceutical composition provided by the invention is more effective, safe, and has the characteristics of simple process and low cost.

以上所述僅為舉例性，而非為限制性者。任何未脫離本發明之精神與範疇，而對其進行之等效修改或變更，均應包含於後附之申請專利範圍中。The above is intended to be illustrative only and not limiting. Any equivalent modifications or alterations to the spirit and scope of the invention are intended to be included in the scope of the appended claims.

<110>　中央硏究院<110> Central Research Institute

<120>　用於預防或治療哺乳類病毒性疾病之醫藥組合物<120> Pharmaceutical composition for preventing or treating mammalian viral diseases

<160>　1<160> 1

<170>　PatentIn version 3.5<170> PatentIn version 3.5

<210>　1<210> 1

<211>　26<211> 26

<212>　PRT<212> PRT

<213>　Epinephelus coioides<213> Epinephelus coioides

<400>　1<400> 1

圖1為對BHK-21細胞以日本腦炎病毒感染之前、後以及同時加入石斑魚抗菌蛋白-1之後細胞的感染率；Figure 1 is the infection rate of cells after BHK-21 cells were infected with Japanese encephalitis virus before, after and simultaneously with grouper antibacterial protein-1;

圖2A為給予老鼠本發明之醫藥組合物後經再感染之存活率；Figure 2A is a survival rate of reinfection after administration of a pharmaceutical composition of the present invention to a mouse;

圖2B為老鼠於第一次注射醫藥組合物後第4天的血清中所含抗日本腦炎病毒外套膜的專一性抗體量；Figure 2B is the specific antibody amount of the anti-Japanese encephalitis virus mantle membrane contained in the serum of the mice on the 4th day after the first injection of the pharmaceutical composition;

圖2C為老鼠第二次注射後第7天的血清中所含抗日本腦炎病毒外套膜的專一性抗體量；Figure 2C shows the specific antibody amount of the anti-Japanese encephalitis virus mantle membrane contained in the serum on the 7th day after the second injection of the mouse;

圖3A為經給予疫苗後老鼠血清中抗日本腦炎病毒外套膜的IgG1及IgG2a表現量；Figure 3A shows the expression levels of IgG1 and IgG2a against the Japanese encephalitis virus mantle membrane in the serum of the mice after administration of the vaccine;

圖3B為注射醫藥組合物一週後老鼠血清中IL-2、IFN-γ、IL-4及IL-10的表現量；Figure 3B shows the expression levels of IL-2, IFN-γ, IL-4 and IL-10 in the serum of rats after one week of injection of the pharmaceutical composition;

圖4A為老鼠以福馬林為佐劑及以石斑魚抗菌蛋白-1為佐劑之疫苗個別進行三次注射後所累積之抗日本腦炎病毒外套膜之抗體表現量；Figure 4A shows the antibody expression of the anti-Japanese encephalitis virus mantle accumulated after three injections of fumarin as an adjuvant and grouper antibacterial protein-1 as an adjuvant;

圖4B為連續注射三次疫苗後以病毒挑戰後老鼠體內抗體表現量；以及Figure 4B is the amount of antibody expression in mice after challenge with virus after three consecutive injections;

圖4C為老鼠注射兩種疫苗三次後經病毒再感染後之存活率。Figure 4C shows the survival rate after reinfection by virus after three injections of two vaccines in mice.

Claims (10)

一種用於預防或治療哺乳類病毒性疾病之醫藥組合物，其包含有效劑量之一石斑魚之抗菌蛋白，以及一病毒，且該病毒具感染一哺乳類之活性。A pharmaceutical composition for preventing or treating a mammalian viral disease, comprising an antibacterial protein of one of an effective dose of grouper, and a virus, and the virus is infected with a mammal. 如申請專利範圍第1項所述之醫藥組合物，其中該源於石斑魚之抗菌蛋白係為石斑魚抗菌蛋白-1，其包含序列編號1之胺基酸序列。The pharmaceutical composition according to claim 1, wherein the antibacterial protein derived from grouper is grouper antibacterial protein-1, which comprises the amino acid sequence of SEQ ID NO: 1. 如申請專利範圍第1項所述之醫藥組合物，其中該病毒係選自於黃病毒科之病毒。The pharmaceutical composition according to claim 1, wherein the virus is selected from the group consisting of a virus of the Flaviviridae family. 如申請專利範圍第1項所述之醫藥組合物，其中該病毒係為日本腦炎病毒。The pharmaceutical composition according to claim 1, wherein the virus is a Japanese encephalitis virus. 如申請專利範圍第1項所述之醫藥組合物，其中該病毒性疾病係為日本腦炎。The pharmaceutical composition according to claim 1, wherein the viral disease is Japanese encephalitis. 如申請專利範圍第1項所述之醫藥組合物，其中該石斑魚之抗菌蛋白之有效劑量係介於5 μg/ml至500 μg/ml之間。The pharmaceutical composition according to claim 1, wherein the effective dose of the antibacterial protein of the grouper is between 5 μg/ml and 500 μg/ml. 如申請專利範圍第1項所述之醫藥組合物，其中該病毒之有效劑量係為對於該哺乳類之10-100倍半致死劑量。The pharmaceutical composition according to claim 1, wherein the effective dose of the virus is 10-100 times the semi-lethal dose for the mammal. 如申請專利範圍第1項所述之醫藥組合物，其中該醫藥組合物係具有刺激免疫之活性。The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition has an activity of stimulating immunity. 如申請專利範圍第1項所述之醫藥組合物，其中該醫藥組合物係為一疫苗。The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is a vaccine. 如申請專利範圍第1項所述之醫藥組合物，其中該石斑魚抗菌蛋白係為一疫苗佐劑。The pharmaceutical composition according to claim 1, wherein the grouper antibacterial protein is a vaccine adjuvant.
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