201023864 . 1 六、發明說明： % 【發明所屬之技術領域】 本發明係與水產疾病之預防和治療有關。 【先前技術】 禽蛋中的免疫球蛋白包含有IgA、IgM及IgY三種，它的 來源是在齡過程中由母禽的血液巾轉移而來，IgA與IgM會 存在蛋白中，而砂則會移行到蛋黃部分，故稱為蛋黃免疫球 ·· 蛋白。相較於其它種類的免疫球蛋白，作為抗體來源IgY有許 多優點，如_乳_轉槪輯差距較大，在免疫分析上 較不易與哺鶴抗财交叉反應，且更料生產械其抗原具 高度專-性的抗體。此外，因為蛋黃中只含有妙一種抗體， 在抗體分離上更為簡單。目前欲由動物獲得特定抗體來源時， 須先免疫實驗鼠或兔子’私抽血的方式獲得其血清以分離抗 • 體。這種侵入性的抗體生產方式，對於動物容易造成痛苦與壓 ' 力。然而，1gY的取得只需於免疫禽類後，每天收取禽蛋，再 、從蛋黃分離出抗體即可。因此，IgY*失為特定抗體來源更好 的選擇。 —在水產動物疫苗的上，目前市面上所使㈣注射疫 田較難處理大I且小體型水產動物(如仔稚魚)之注射。若能 ^用口服疫苗，利用與飼料混合的方式触，將可大幅縮減人 亦不會有佐劑殘留的安全性問題。口服疫苗是利用水產動 201023864 物口服傳遞系統直接將抗體或抗原混入飼料中，經口服進入動 物體，但在製備的過程中蛋白質容易變性，在水中則可能水解. 即使進入魚的體内’也將被胃酸消化掉大部分，而使保護效果 大打折扣。因此’如何將IgY抗體有效地送入動物體内，將是 目前水產業極需解決的一個問題。 【發明内容】 本發義提供-種供水產動物顧的IgY π服製劑，其中 • 包含1gY抗體的蛋粉包埋於水包油包水型三重相之乳化物 中。此口服製劑結構穩定，方便儲存與運送，在水中不易水解， 同時可保護IgY不受胃酸影響，進入水產動物 出娇抗體，提高預防及保護之效。 _釋放 因此，本發明之一方面在提供一種IgY 口服製劑，其包括 治療或預防有效量之包含IgY抗體之蛋粉;約30_70%總重量比 之油相物質，該油相物質於常溫下呈液狀，係用於包埋該蛋粉; • 約0.1-10%總重量比之第-乳化劑，其係加入該油相物質中； 約10-30〇/〇總重量比之第一水相物質，構成一内水相其係被 ' 糾她f所包覆;約2G-4G%總重量比，構成 —外水相，其係用於包覆該油相物質;及約〇1_1〇%總重量比之 第二乳化劑，其係加入該外水相中。 本發明的另一方面在於提供一種製備如上述之IgY 口服 齊丨之方法’其步驟包括：（1)提供一包含IgY抗體之蛋粉;(2) 將該蛋粉與一約3〇_7〇%總重量比之常溫呈液態之油相物質混 201023864 後再…約0.1_10%總重量比之第一乳化劑混合，得到一油 相產物;(3)該油相產物與約则〇%總重量比之第一水相物 質θ構成内水相，以1:1至1:5的範圍内之一重量比混合後， 可付一油包水乳化產物；（4)將約20-40%總重量比之之第二水 3質’構成—外水相，與約0.1··總重量比之第二乳化劑 /吧口後’形成-屍合物；⑶將該混合物與步驟⑶所得之油包水 乳化產物以1.1至1:5的範圍内之一重量比混合得到IgY 口 ^ 服製劑。 』 在本發明另一方面提供一種包含如上述之IgY 口服製劑 之水產用飼料。 本發明之又另方面為提供一種預防或治療水產動物疾病 的方法，其係為口服給予水產動物如上述之水產用飼料。 【實施方式】 本發明係提供-種IgY σ服製劑’其包括治療或預防有效 ❿ 量之包含1gY抗體之蛋粉;約30-70%總重量比之油相物質，該 • 油相物質於常溫下呈液狀，係用於包埋該蛋粉;約〇.1_10%總重 ， 量比之第一乳化劑，其係加入該油相物質中;約1〇_30%總重量 比之第一水相物質，構成一内水相，其係被該油相物質所包覆. 約20·40°/。總重量比之第二水相物質’構成一外水相，其係用 於包覆該油相物質;及約Ο.μΐΟ%總重量比之第二乳化劑，其係 加入該外水相中。 本發明所使用的IgY係包含於一蛋粉中，該蛋粉之來源 201023864 為家禽類如雞、鴨或鶴。本領域之技藝人士，可根據欲預防或 治療的水產性疾病，以自身之知識或根據文獻，將全株不活化 病毒或具抗原決定區位之多肽片段混合佐劑後免疫家禽，並由 被免疫之家禽所生下的蛋中製備包含IgY抗體之蛋粉^艮據本 發明之一實施例，係將不活化之台灣石斑魚虹彩病毒顆粒 CGiOupei· iridovirus of Taiwan，TO/F)與佐劑免疫雞隻數次後，蒐 集該雞隻所產下的蛋並將蛋黃部分冷凍乾燥後，所得到之包含 -❹ IgY-抗台灣石斑魚虹彩病毒之蛋粉。「有效量」乙辭在本發明 - 中意指能達到預防或治療水產動物疾病效果之含量。根據本發 明之一實施例，係使用於油相物質中約5_2〇%重量比之包含 igY抗體之蛋粉。 本發明中所使狀「油相物質」乙辭，係指於食品和藥物 中、曰'遍使用，且於常溫下呈液態狀之酯類或非酯類油性基底。 非酉曰型油性基底之實例包含礦物油，而酯類油性基底之實例包 • 括天然存在的脂肪酸酯，例如液態植物油如花生油、大豆油、 ‘ 向日葵油、玉米油、橄欖油或菜籽油等，及源自動物之液態油 、 如豬油、烏賊油或魚油等。上述油類可視目的單獨或組合使 用。根據本發明之一實施例，該油相物質為魚油。 本發明中所使用之「第一乳化劑」乙辭，係指HLB值小 於8且轉化點(inversion⑽的在10_4(rc之間之非離子性介面 活性劑。「HLB值」乙辭係指某種物質所具有的親水親油平衡 值0^叩11如1丨1)〇1)11丨1_1311從），數值越低，表示親水性越差。 6 201023864 本發明中，驗製備的第-減叙賴包括己贿酐硬脂酸 酯、己糖醇酐油酸酯、脫水山梨醇脂肪酸酯、二辛基十二烷基 -2月桂祕氨酸、單硬月旨酸甘油醋或大豆碟脂。本領域之技 藝人士可根據不同目的’自行決定或組合使用上述之乳化 劑。根據本發明之-個實_，第—乳化劑係使用脫水山梨醇 脂肪酸酯（SPAMs)。 本發明中所使用之「第二乳化劑」乙辭，衡旨職值大 於8且轉化點(inversion point)在l〇_4〇t之間之非離子性介面 活性劑’亦即為親水性乳化劑。本發明中，用於製備的第二乳 化劑之實例包括己糖醇酐月桂酸酯、己糖醇酐棕櫊酸酯、己糖 醇酐硬脂酸酯、己糖醇酐油酸酯、聚氧乙烯脫水山梨醇脂肪酸 酯、單硬脂酸聚甘油酯一10或聚甘油_10硬脂酸酯。本領域 之技藝人士可根據不同目的，自行決定單獨或組合使用上述之 乳化劑。根據本發明之一個實施例，第二乳化劑係使用聚氧乙 烯脫水山梨醇脂肪酸酯（Tween)。 本發明中所使用之「水相物質」乙辭，係指一水溶液，其 可為水或以水為基底的溶液。於本發明中，作為内水相之水相 物質可進一步視情況包含一營養補充劑、一胺基酸、一蛋白質 粉末、一維生素、一免疫賦活劑、一微量礦物質、一海藻多醣 或一疫苗等水溶性物質或上述任兩種以上物質之搭配。該水溶 性物質之目的在於增進IgY 口服製劑之效果，或是作為其它目 的之使用如營養補充。本領域之技藝人士可根據所需，自行挑 201023864 選欲使用之水溶性物質。 於本發明中’作為外水相之水相物質可視情況進一步包含 一水溶性物質，例如用於安定口服製劑之結構，諸如三仙膠、 海蒸酸鈉、k-鹿角菜膠、褐藻酸鈉、大豆卵麟脂、阿拉伯膠或 上述其他物質之搭配。 本發明亦提供一種製備如上述之IgY 口服製劑之方法， 其步驟包括：（1)提供一包含IgY抗體之蛋粉;將該蛋粉與一 參 約3〇-7〇%總重量比之常溫呈液態之油相物質混合後，再與約 、 〇·Μ〇%總重量比之第一乳化劑混合，得到一油相產物；（3)該油 相產物與約1〇_3〇%總重量比之内水相以1:1至1:5的範圍内重 置比混合後，可得一油包水乳化產物；（4)將約2〇_4〇%總重量 比之外水相與約0·Μ0%總重量比之第二乳化劑混合後，形成 —混合物；（5)將該混合物與步驟(3)所得之油包水乳化產物以 1:1至1:5的範圍内重量比混合，得到IgY 口服製劑。 _ 目本發明之IgY 口服製劑具有結構穩定及可抗胃酸之優 點，適合以口服的方式提供水產動物對抗病毒之被動保護，本 發明之IgY 口服製劑可與水產用飼料混合，再將該飼料饒食目 標水產動物。IgY 口服製劑與水產用飼料混合之比例，可由本 發明所屬技術領域中具有通常知識者根據經驗與混合之飼料 種類自行試驗而得知。 根據上述概念’本發财提供—種獅或治療水產動物疾 病的方法，其係為口服給予水產動物如申上述之缺口服製劑 201023864 之水產用铜料。其中，該水產動物為魚類或蝦類，尤以不易注 射之小型魚類為最有價值。 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p艮於該用於展示之實施例。 實施例1:抗台灣石斑虹彩病毒之igY製備 雞隻免疫與蛋粉製作 Φ 選用3-4月齡的母雞，使其自由攝取飲水及飼料。以肌肉 、 注射方式施打弱毒株的台灣石斑虹彩病毒（TGIV)(實驗室自 行由罹病的石斑魚中分離出的病毒），共施打四次。第一次免疫 時加入完全佐劑（sigma，USA)施打，後三次免疫皆使用不完全 佐劑（sigma，USA)施打，每次免疫皆間隔兩週。在每次注射前 收集母雞所生的蛋’並以1 : 4之蛋黃與〇.1倍pBS溶液的比 例在4°C中水化一天後，收集其蛋液。 • IgY力價測試 * 以0.1 M NaHCCb稀釋TGIV抗原並加入在％井的微盤 ^ 上，每個井加入5〇 pL的抗原(濃度為1〇〇 ng/5〇 μ[)，置於操作 台中於室溫吹乾。吹乾後每個井加入50 μι含有1 %即溶奶粉 的PBS溶液，置於37°C作用2小時。甩乾上述液體後，以 PBS/T混衝液(含有0.05% Tween-20之PBS溶液)清洗1次， 再用PBS溶液清洗2次，自然乾燥後將培養盤保存於_2〇。〇 中備用。 201023864 將蛋液以含有1%即溶奶粉的PBS溶液作連續兩倍稀釋 後’在96井的微盤中’每個井加入50 pL待測液，於37°C作 用2小時後，甩乾上述液體，以PBS/Τ緩衝液清洗3次，再 用PBS溶液清洗5次並拍乾。接著將連接辣根過氧化物酶 (Horseradish Peroxidase)之山羊-抗雞lgY抗體(華肝基因公司， 台灣)以1:1000比例用含有1%即溶奶粉的PBS溶液稀釋，並 在每個井中加入50 pL，放置於37°C作用1小時。反應時間結 束後甩乾上述液體，以PBS/T緩衝液清洗3次，再用PBS溶 • 液清洗5次並拍乾。最後每個井中加入50叫的鄰苯二胺 (O-phenylene diamine，OPD) ( SIGMA，USA)作為呈色基質 後，於室溫下避光反應30分鐘。呈色完畢後，每個井加入5〇 pL 的3NHC1終止反應，並偵測490 nm的吸光值(OD)。樣品最大 稀釋倍率之倒數即為其力價。結果顯示，IgY抗體力價約為 1:16000-64000。201023864 . 1 VI. Description of the invention: % [Technical field to which the invention pertains] The present invention relates to the prevention and treatment of aquatic diseases. [Prior Art] The immunoglobulins in poultry eggs contain three kinds of IgA, IgM and IgY. The source is transferred from the blood fodder of the female poultry during the ageing process. IgA and IgM will be present in the protein, while the sand will be Move to the egg yolk part, so it is called egg yolk immune ball · · protein. Compared with other kinds of immunoglobulins, IgY has many advantages as an antibody source. For example, the gap between the milk and the sputum is relatively large, and it is not easy to cross-react with the crane and the anti-finance in the immunoassay, and it is more suitable for the production of the antigen. Highly specific antibody. In addition, because the egg yolk contains only one antibody, it is easier to isolate the antibody. Currently, when a specific antibody source is to be obtained from an animal, the test mouse or rabbit must be immunized to obtain the serum to separate the anti-body. This invasive method of antibody production is prone to pain and stress in animals. However, 1gY can be obtained by collecting the eggs every day after immunizing the birds, and then separating the antibodies from the egg yolk. Therefore, IgY* loss is a better choice for a particular antibody source. - In the case of aquatic animal vaccines, it is currently difficult to treat injections of large I and small-sized aquatic animals (such as larvae) in the market. If you can use an oral vaccine, it can be used to reduce the safety of people without adjuvant residues. The oral vaccine uses the aquatic product 201023864 oral delivery system to directly mix the antibody or antigen into the feed and enter the animal body by oral administration. However, during the preparation process, the protein is easily denatured and may be hydrolyzed in water. Even if it enters the body of the fish It will be digested by most of the stomach acid, and the protection effect will be greatly reduced. Therefore, how to effectively deliver IgY antibodies into animals will be a problem that the aquaculture industry needs to solve. SUMMARY OF THE INVENTION The present invention provides an IgY π preparation for a water supply animal, wherein • the egg powder containing 1 g of the Y antibody is embedded in an emulsion of a water-in-oil-in-water triple phase. The oral preparation has stable structure, convenient storage and transportation, is not easy to be hydrolyzed in water, and can protect IgY from gastric acid, and enters aquatic animals to produce antibodies, thereby improving the effect of prevention and protection. _ Release Therefore, an aspect of the present invention provides an IgY oral preparation comprising a therapeutically or prophylactically effective amount of egg powder comprising an IgY antibody; an oil phase material of about 30-70% by weight, the oil phase substance being present at room temperature Liquid, used to embed the egg powder; • about 0.1-10% of the total weight ratio of the first emulsifier, which is added to the oil phase material; about 10-30 〇 / 〇 total weight ratio of the first water The phase material, which constitutes an internal water phase, is covered by 'correction'; about 2G-4G% of total weight ratio, constitutes the outer aqueous phase, which is used to coat the oil phase material; and about 1_1〇 A total emulsifier ratio of the second emulsifier which is added to the outer aqueous phase. Another aspect of the present invention provides a method for preparing an oral solution of IgY as described above, the steps of which comprise: (1) providing an egg powder comprising an IgY antibody; (2) providing the egg powder with an amount of about 3 〇 7 〇% total weight is mixed with the oil phase material which is liquid at room temperature, 201023864, and then... about 0.1_10% of the total weight ratio of the first emulsifier is mixed to obtain an oil phase product; (3) the oil phase product and about 〇% The total weight ratio of the first aqueous phase material θ constitutes the internal aqueous phase, and after mixing in a weight ratio ranging from 1:1 to 1:5, a water-in-oil emulsion product can be paid; (4) about 20-40 % of the total weight ratio of the second water 3 constitutes the outer aqueous phase, with a total weight ratio of about 0.1% of the total emulsifier / bar after the 'form-corporate; (3) the mixture and step (3) The water-in-oil emulsified product is mixed in a weight ratio of 1.1 to 1:5 to obtain an IgY preparation. In another aspect of the invention, there is provided an aquatic feed comprising the oral preparation of IgY as described above. Still another aspect of the present invention provides a method for preventing or treating a disease in an aquatic animal by orally administering an aquatic animal such as the above-described aquaculture feed. [Embodiment] The present invention provides an IgY σ-based preparation which comprises a therapeutic or prophylactically effective amount of egg powder comprising 1 g of Y antibody; an oil phase material of about 30-70% by weight, the oil phase substance It is liquid at room temperature and is used to embed the egg powder; about 1% to 10% of total weight, the first emulsifier is added to the oil phase material; about 1〇30% of total weight ratio The first aqueous phase material constitutes an internal aqueous phase which is coated with the oil phase material. About 20·40°/. The total weight ratio of the second aqueous phase material 'constitutes an outer aqueous phase for coating the oil phase material; and about Ο.μΐΟ% of the total weight ratio of the second emulsifier, which is added to the outer aqueous phase . The IgY system used in the present invention is contained in an egg powder, and the source of the egg powder 201023864 is a poultry such as a chicken, a duck or a crane. A person skilled in the art can immunize poultry by immunizing poultry with a whole plant inactivated virus or a polypeptide fragment having an antigenic determining region according to their own knowledge or according to the literature according to the aquatic disease to be prevented or treated. Preparation of egg powder containing IgY antibody in eggs produced by poultry. According to an embodiment of the present invention, the inactivated Taiwan grouper iridescent virus particle CGiOupei·iridovirus of Taiwan, TO/F) and adjuvant chicken After only a few times, the egg produced by the chicken was collected and the yolk portion was freeze-dried, and the obtained egg powder containing -❹ IgY-anti-Tiger grouper iridescent virus was obtained. The "effective amount" in the present invention means the content which can achieve the effect of preventing or treating diseases of aquatic animals. According to one embodiment of the invention, the egg powder comprising the igY antibody is used in an amount of about 5-2% by weight of the oil phase material. The term "oil phase substance" as used in the present invention refers to an ester or non-ester oily base which is used in foods and medicines and which is used in a liquid state at room temperature. Examples of non-酉曰 oily substrates include mineral oil, and examples of ester oily substrates include naturally occurring fatty acid esters such as liquid vegetable oils such as peanut oil, soybean oil, 'sunflower oil, corn oil, olive oil or rapeseed Oil, etc., and liquid oil derived from animals, such as lard, squid oil or fish oil. The above oils may be used singly or in combination for visual purposes. According to an embodiment of the invention, the oil phase material is fish oil. The term "first emulsifier" as used in the present invention means that the HLB value is less than 8 and the conversion point (inversion (10) is between 10 and 4 (a nonionic surfactant between rc. "HLB value" refers to a certain The hydrophilic-lipophilic balance value of the substance is 0^叩11 such as 1丨1)〇1)11丨1_1311), and the lower the value, the worse the hydrophilicity. 6 201023864 In the present invention, the first-subsequent preparation of the test includes succinate stearate, hexitol anhydride oleate, sorbitan fatty acid ester, dioctyldodecyl-2 lauric ammonia Acid, single hard acid glycerin or soy sauce. Those skilled in the art can use the above emulsifiers in their own right or in combination for different purposes. According to the present invention, the emulsifier is a sorbitan fatty acid ester (SPAMs). The "second emulsifier" used in the present invention is a hydrophilic non-ionic surfactant which has a value of more than 8 and an inversion point between l〇_4〇t. Emulsifier. In the present invention, examples of the second emulsifier used for the preparation include hexitol anhydride laurate, hexitol anhydride palmitate, hexitol anhydride stearate, hexitol anhydride oleate, poly Oxyethylene sorbitan fatty acid ester, polyglyceryl monostearate-10 or polyglycerol-10 fatty acid ester. Those skilled in the art can use the above-mentioned emulsifier alone or in combination according to different purposes. According to one embodiment of the invention, the second emulsifier is a polyoxyethylene sorbitan fatty acid ester (Tween). The term "aqueous phase substance" as used in the present invention means an aqueous solution which may be water or a water-based solution. In the present invention, the aqueous phase material as the internal aqueous phase may further comprise a nutritional supplement, an amino acid, a protein powder, a vitamin, an immunostimulating agent, a trace mineral, a seaweed polysaccharide or a A water-soluble substance such as a vaccine or a combination of any two or more of the above. The purpose of the water-soluble substance is to enhance the effect of the IgY oral preparation, or for other purposes such as nutritional supplementation. Those skilled in the art can select the water-soluble substance to be used in 201023864 as needed. In the present invention, the aqueous phase material as the external aqueous phase may further comprise a water-soluble substance, for example, for the structure of a stable oral preparation, such as Sanxian gum, sodium oxalate, k-carrageenan, sodium alginate. Soybean egg gum, gum arabic or a combination of the above. The invention also provides a method for preparing an oral preparation of IgY as described above, the method comprising the steps of: (1) providing an egg powder comprising an IgY antibody; and adding the egg powder to a room temperature of about 3〇-7〇% by weight After mixing the liquid phase oil materials, the first emulsifier is mixed with the total weight ratio of about 〇·〇·Μ〇% to obtain an oil phase product; (3) the oil phase product is about 1〇_3〇% total After the water ratio in the weight ratio is mixed in the range of 1:1 to 1:5, a water-in-oil emulsion product is obtained; (4) about 2〇_4〇% total weight ratio is outside the water phase. After mixing with the second emulsifier at a total weight ratio of about 0·Μ0%, forming a mixture; (5) the mixture of the mixture and the water-in-oil emulsion obtained in the step (3) in a range of 1:1 to 1:5 The weight ratio is mixed to obtain an IgY oral preparation. The IgY oral preparation of the present invention has the advantages of structural stability and anti-acidicity, and is suitable for providing passive protection of aquatic animals against viruses by oral administration. The IgY oral preparation of the present invention can be mixed with aquatic feed, and then the feed is mixed. Eat target aquatic animals. The ratio of the IgY oral preparation to the mixed feed for aquatic products can be known by a person having ordinary knowledge in the art to which the present invention pertains, based on experience and mixed feed types. According to the above concept, the present invention provides a lion or a method for treating an aquatic animal disease, which is an oral administration of an aquatic animal such as the aquaculture copper material of the above-mentioned gap service preparation 201023864. Among them, the aquatic animal is a fish or a shrimp, and especially a small fish that is not easily injected is most valuable. The invention will be further illustrated by the following examples, but the actual invention is not to be construed as being limited to the examples. Example 1: Preparation of igY against Taiwanese spotted iridescent virus Chicken immunization and egg powder production Φ Use 3-4 month old hens to freely ingest drinking water and feed. The Taiwanese spotted iridescent virus (TGIV), a virus isolated from the squid grouper, was applied four times in a muscle and injection manner. The first immunization was performed with the addition of a complete adjuvant (sigma, USA), and the subsequent three immunizations were performed with an incomplete adjuvant (sigma, USA), with each immunization separated by two weeks. The egg produced by the hen was collected before each injection and the egg liquid was collected after hydration at 4 ° C for one day at a ratio of 1:4 egg yolk to 〇.1 times pBS solution. • IgY price test* Dilute TGIV antigen with 0.1 M NaHCCb and add it to the microplate of % well, adding 5〇pL of antigen per well (concentration of 1〇〇ng/5〇μ[), placed in operation Taichung was dried at room temperature. After drying, 50 μM of PBS solution containing 1% of instant milk powder was added to each well and allowed to stand at 37 ° C for 2 hours. After the above liquid was dried, it was washed once with PBS/T mixed solution (PBS containing 0.05% Tween-20), washed twice with PBS solution, and naturally dried, and the plate was stored at _2 Torr. 〇 in standby. 201023864 After the egg liquid was diluted twice in PBS solution containing 1% instant milk powder, add 50 pL of the test solution to each well in a microplate of 96 wells, and then dry at 37 ° C for 2 hours. The above liquid was washed 3 times with PBS/Τ buffer, washed 5 times with PBS solution and patted dry. Then, the goat-anti-chicken lgY antibody (Huahe Gene Company, Taiwan) linked to Horseradish Peroxidase was diluted at a ratio of 1:1000 with PBS solution containing 1% instant milk powder, and in each well. 50 pL was added and placed at 37 ° C for 1 hour. After the reaction time was over, the above liquid was dried, washed 3 times with PBS/T buffer, washed 5 times with PBS solution and patted dry. Finally, 50 O-phenylene diamine (OPD) (SIGMA, USA) was added to each well as a coloring substrate, and then reacted at room temperature for 30 minutes in the dark. After the coloration was completed, 5 〇 pL of 3NHC1 was added to each well to terminate the reaction, and the absorbance (OD) at 490 nm was detected. The reciprocal of the maximum dilution ratio of the sample is its price. The results showed that the IgY antibody was about 1:16000-64000.
IgY中和試驗 蛋液先以〇.45μιη濾膜過濾，再以PBS溶液進行從1:20連 續二倍稀釋至1:10240，並分別加入200 TCID5〇/mL的病毒液 於25°C下混合一小時，另外以PBS溶液混合相同濃度之病毒 液為對照組。而後，把該兩種混合液分別加入長有石斑魚泳鰾細 胞株之96井微量培養盤中，吸附二小時後抽掉液體，加入含有 2%胎牛血清(Hyd〇ne,uSA)的L15細胞培養液。若抗體無法有效 中和病毒，會出現如細胞變圓變大，核濃縮邊緣化，最後細胞 破裂死亡的病變症狀，每天在顯微鏡下觀察並記錄細胞病變產 201023864 生的情形。結果發現，在1:8〇, 1:1β〇, 1:32〇以及ι:_的稀釋 倍率下細胞未產生病變的情形，顯示在上述稀釋倍數下，缺 具有最好的中和效果。 實施例2 : IgY 口服製劑之製備 第-階段先S&製水/油乳化物。魏準備商業魚油3〇址， 其係購自台灣_實#有限公司，商品代號為F〇，由多種魚 類所提煉於魚射分別添加不同濃度之蛋粉(含量分別為％ ⑩1〇%，15%) ’並添加0.9 g親油性乳化劑(Span，由日本林純藥工 業株式會_人），齡均自後明_ (SUv_，En細) 8，_ _的速度進行均質，並同時加入内部相水溶液(10 mL)。均質2分鐘後形成水/油乳化物。 第二階段為將第一階段所形成的水/油乳化物40mL，添加 至含有1 g❸親水性乳化额Tween，由日本林純藥工業株式會 • 社構入)之20mL水溶液中，以漏—均質2分鐘，均質後 即可得到水包油包水型IgY 口服製劑。 實施例3: IgY 口服製劑之製備特性 形成率 經過適當稀釋後的1gY 口服製劑，利用攝影系統掏取5 張照片，每張照片最少有5〇顆乳滴，計算三重相乳滴佔總乳 滴的百分比即為其形成率。結果如表丨所示，包含抓及聰 蛋粉的組職形縣有抓社，*包含蛋粉的組別亦 11 201023864 有78%的形成率。 表1 Ig 口服製劑不同蛋粉比例的形成率 組別 5%組 10%組 15%組 形成率 86% 88% 78% 乳化物安定性 各組多重相乳化物分別取出10 mL裝載於15mL離心管 中，並放置4ΐ，定期觀察其變化並紀錄。結果如表2所示， 蛋粉含量越高，安定性則表現較不好，但低劑量組可達45天 以上。 表2 IgY 口服製劑不同蛋粉濃度組之安定性(儲存天數) 組別 5%組 10%組 15%組 安定性(儲存天數） 45天以上 30天 15天 實施例4:抗台灣石斑虹彩病毒(TGIV)之IgY 口服製劑之活體 效果評估 實驗用之點帶石斑魚講自八八六水產動物生物科技股份 有限公司，其點帶石斑魚係來自於高、屏地區，在鹽度3〇〜33 %〇，溫度26〜3(TC的條件下畜養’每曰以商用鰻魚飼料投餵三 次’畜養一週後進行實驗。於實驗開始前隨機選取石斑魚，採 12 201023864 檢體妨TGIV聚合酶賴反餘測之檢測。 每一循環之反應條件為:先以94〇c進行2分鐘，再依次為94。匚40 秒，粘合溫度52°C3〇秒及72°C 1分鐘，共進行35個循環增幅 反應，最後以72¾作用10分鐘將未完成的反應完成。確認無 TGIV感染後進行實驗。用於聚合酶連鎖反應檢測之引子序列 如下：正向引子：5” CCCTT CCTTG TGTCG TGTCT 3"(序列 編號.1);反向引子IGCCACCGTAATCAGTITCGA^^ 列編號：2)。IgY neutralization test egg liquid was first filtered with 〇.45μιη filter, and then serially diluted 1:20 from 1:20 to 1:10240 in PBS solution, and added with 200 TCID5〇/mL virus solution separately at 25 °C. One hour, the same concentration of virus solution was mixed with PBS solution as a control group. Then, the two mixtures were separately added to a 96 well microplate of the grouper cell line, and after two hours of adsorption, the liquid was removed, and L15 cells containing 2% fetal bovine serum (Hyd〇ne, uSA) were added. Culture medium. If the antibody is not effective in neutralizing the virus, there will be symptoms such as rounding of the cells, marginalization of the nuclear nucleus, and finally rupture of the cells. The cytopathic product is observed under a microscope every day. As a result, it was found that the cells did not produce lesions at a dilution ratio of 1:8 〇, 1:1β〇, 1:32〇, and ι:_, indicating that the best neutralization effect was absent under the above dilution factor. Example 2: Preparation of IgY Oral Formulations Stage-First S& Water/Oil Emulsion. Wei prepared commercial fish oil 3 , site, which was purchased from Taiwan _ _ # Co., Ltd., the commodity code is F 〇, refined by a variety of fish in the fish shot separately added different concentrations of egg powder (content is 101%, respectively, 15 %) 'Add 0.9 g of lipophilic emulsifier (Span, by Nippon Pure Chemical Industry Co., Ltd.), the age is averaged from the speed of _ (SUv_, En fine) 8, _ _, and simultaneously Internal phase aqueous solution (10 mL). A water/oil emulsion was formed after 2 minutes of homogenization. In the second stage, 40 mL of the water/oil emulsion formed in the first stage is added to a 20 mL aqueous solution containing 1 g of a hydrophilic emulsified amount of Tween, which is incorporated by the Japanese Pure Chemical Industry Co., Ltd. After homogenization for 2 minutes, the water-in-oil-in-water type IgY oral preparation can be obtained after homogenization. Example 3: Preparation characteristics of IgY oral preparations After a properly diluted 1 gY oral preparation, 5 photographs were taken by a photographic system, and at least 5 drops of milk droplets per photograph were used to calculate the total emulsion droplets of the triple phase emulsion. The percentage is the rate of formation. As a result, as shown in the table, the group with the grasping and Cong egg powder has a grasping society, and the group containing the egg powder also has a formation rate of 78%. Table 1 Ig Oral preparations Different egg powder ratio formation rate group 5% group 10% group 15% group formation rate 86% 88% 78% emulsion stability Each group of multiphase emulsions were taken out 10 mL loaded in 15 mL centrifuge tubes In the middle, and placed 4 ΐ, periodically observe its changes and record. The results are shown in Table 2. The higher the egg powder content, the less stable the stability, but the low dose group can reach more than 45 days. Table 2 Stability of different egg powder concentration groups of IgY oral preparations (storage days) Group 5% group 10% group 15% group stability (storage days) 45 days and 30 days 15 days Example 4: Resistance to Taiwan group iridescence Virus (TGIV) IgY oral preparation for the evaluation of the in vivo effect of the experimental group with the grouper from 868 Aquatic Animal Biotechnology Co., Ltd., the point with the grouper from the high, screen area, in the salinity 3〇~33 %〇, temperature 26~3 (the condition of TC under the condition of TC 'every carp is fed three times with commercial squid feed', the experiment is carried out one week after the animal is raised. The grouper is randomly selected before the start of the experiment, and the TGIV polymerase is used in the 12 201023864 The detection conditions of each cycle are: first, 94 〇c for 2 minutes, then 94. 匚 40 seconds, bonding temperature 52 ° C 3 〇 seconds and 72 ° C 1 minute for a total of 35 cycles The amplification reaction was completed, and the unfinished reaction was finally completed with 723⁄4 for 10 minutes. It was confirmed that the experiment was carried out without TGIV infection. The primer sequence for polymerase chain reaction detection was as follows: forward primer: 5" CCCTT CCTTG TGTCG TGTCT 3"No.1); The primer IGCCACCGTAATCAGTITCGA ^^ column number: 2).
飼料配製分為六組，分別為：A包埋組：以添加1〇%蛋 粉之多重相乳絲和市售驗(纟榮產業股份有限公q，台灣) 以重量1:1混合舰成齡蝴;B未包埋高繼組：將蛋粉緩 粉混合調配舰性飼料，祕加蛋侧量職簡重的3〇%;= 未包埋低継組：將蛋機粉混合調配成祕缩，添加蛋於 劑量約佔就料㈣5%;以及上述未含有抗義空白蛋粉: 陽性對照組，分別為AV，BV * CV。其中未包埋高劑量組 IgY抗體含量約為包埋紐_的6倍。 病毒攻擊試驗的方法為每組取1G尾魚，_分_ 射攻擊組及浸泡攻擊組。注射組選用的病毒劑量分別為納 和10 TCID5〇/0.〇5mL/每條魚，而浸泡組選用的病毒劑量 和104TCID5〇/l 5〇OmL ’浸泡兩小時。攻擊後每日觀 .、、、〇 亡情形，計算累計死亡率。 纪錄死 13 201023864 預防效果實驗 將點帶石斑魚如表3分組，每組4〇隻魚苗，平均長5 5 么分’重3·9公克’並以兩重覆赠動式海摘養於2尺缸中。 ❿ 表3活體實驗分組The feed preparation is divided into six groups, namely: A-embedded group: multi-phase milk with 1%% egg powder and commercial test (Zhenrong Industry Co., Ltd., Taiwan) Ageing butterfly; B is not embedded in Gaoji group: mixing egg powder and powder to mix ship-based feed, secretly adding 3% of egg-side weight; = un-embedded low-lying group: mixing egg machine powder into Secret shrinkage, adding eggs at a dose of about 5% (4); and the above did not contain anti-sense blank egg powder: positive control group, respectively, AV, BV * CV. The IgY antibody content of the unembedded high-dose group was about 6 times that of the embedded New Zealand. The method of virus attack test was to take 1G tail fish, _ minute _ attack group and immersion attack group in each group. The doses of the virus used in the injection group were N and 10 TCID5〇/0.〇5 mL/per fish, respectively, and the virus dose used in the soaking group was soaked for two hours with 104TCID5〇/l 5〇OmL'. After the attack, the daily death rate is calculated, and the cumulative death rate is calculated. Record death 13 201023864 Preventive effect experiment will be grouped with grouper as shown in Table 3, each group of 4 fry, the average length of 5 5 points 'heavily 3 · 9 grams ' and double-grained mobile sea picking at 2 feet In the tank. ❿ Table 3 Live experiment grouping
飼料處理 病毒攻 擊 包埋組 實驗矣且 陽性對照組 陰性對照組 未包埋高劑量組 有 有Feed treatment virus attack embedding group experiment and positive control group negative control group unembedded high dose group
CC
AVAV
BV 未包埋低劑量組 包埋組空白蛋粉 未包埋高劑量空白蛋粉 未包埋低劑量空白蛋粉 15組空白蛋1 ϋ埋高劑量^白蛋^ 有 有 有BV unembedded low-dose group Embedding group blank egg powder Unembedded high-dose blank egg powder Unembedded low-dose blank egg powder 15 groups of blank eggs 1 ϋ buried high dose ^ white eggs ^ Yes Yes
無 CL 未包埋低劑量空白蛋粉 D 一般飼料 無 無 各組飼料每日投餵三餐，各組總體重以上的各組自製 的濕性飼料當日投紋畢。投餒三天後於第—天或第五天，實 驗組和陽性對驗於第卜5、1G、20天各撈出1G隻，進行 TGIV腹腔注射攻擊試驗，選用劑量為1〇3.3 TclD5〇/〇 〇5mL/ 魚，陰性對照組則以L15培養液的腹腔注射取代病毒攻擊，每 曰觀察並紀錄死亡情形，計算相對活存率。結果如圖i和圖2 201023864 所示。 圖1為投餵三天各組飼料後的第一天進行病毒攻擊，並 錄每日死亡冲算相對活存的結果。如圖玉所示，在病毒攻 擊十四天後除了 IgY σ服製劑組⑷和未包埋高劑量級⑼尚 有50〇/〇左右的存活率’其它各組活存率皆為收。此外，圖工 亦顯示本發明之IgY 口服製劑絲包埋高劑量組的效果相當。 圖2為投餘三天各組飼料後的第五天進行病毒攻擊，並 紀錄每日死亡，計算相對活存的結果。如圖2所示，在病毒攻 ❹冑第十四天肖本發明之口服製劑組(A)的存活率幾乎 100% ’未包埋高劑量組⑼有約8〇%的存活率，而除了饒食一 般飼料的D組外’其它組別僅剩10-20%的存活率。 由上述數據可知，本發明之IgY σ服細在活體試驗上， +於抵抗病毒攻擊有十分顯著的效果，可大幅提昇水產動物的 存活率，且該存活率比未包埋的高劑量相等或更高。 本發明所雜_域巾具魏私識者將可在不挣離本 ❿ 發明精神之下’根據實施例進行改變和修改。要注意的是，本 發明並不文限於說明書中實施例所揭露之範圍，而涵蓋於其他 根據申請範圍内揭露之所有變化之形式。 【圖式簡單說明】 刖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 15 201023864 舉；匕要;主意的是，本發明並不受限於列舉於此的說明。 圖1係為點帶石斑魚投傲三天各組飼料後的第一天進行 病毒攻擊，紀錄每日死亡後而換算之相對活存率的結果。 圖2係為點帶石斑魚投餵三天各組飼料後的第五天進行 病毒攻擊，紀錄每日死亡後而換算之相對活存率的結果。 【主要元件符號說明】 無 ❹ 參 16 201023864 T0U0001TW_seq_list _TW 序列表 <110〉周信佑 任讎 朱家瑩 吳彰哲 <120〉 蛋黃免疫球蛋白(IgY) 口服製劑及其製備方法 <130〉 TOU0001TW <160〉 2 <170〉 Patentln version 3.3 <210〉 1 <211> 20 <212〉 DNA <213〉 人工合成 <220〉 <223> 引子 <400〉 1 cccttccttg tgtcgtgtct 20 <210> 2 <211〉 20 <212〉 DNA <213> 人工合成 <220> <223> 引子 <400〉 2 gccaccgtaa tcagttcgat 20 第1頁None CL Unembedded low-dose blank egg powder D General feed None None Each group of feeds was fed three meals a day, and each group of self-made wet feeds with a total weight of more than one group was cast on the same day. Three days after the injection, on the first day or the fifth day, the experimental group and the positive test were taken out on the 5th, 1G, and 20th days, and 1G was taken out for the TGIV intraperitoneal injection test. The dose was 1〇3.3 TclD5〇. /〇〇5mL/fish, the negative control group was treated with intraperitoneal injection of L15 medium instead of virus. The death was observed and recorded, and the relative survival rate was calculated. The results are shown in Figure i and Figure 2 201023864. Figure 1 shows the virus challenge on the first day after feeding the three groups of feeds, and recorded the results of daily deaths. As shown in Fig. j, after 14 days of virus challenge, except for the IgY σ-treatment group (4) and the un-buried high-dose level (9), there was a survival rate of about 50 〇/〇, and the survival rates of the other groups were all received. In addition, the graphic work also shows that the IgY oral preparation of the present invention is highly effective in the high-dose group. Figure 2 shows the virus attack on the fifth day after the three-day feed for each group, and recorded daily deaths to calculate the relative survival results. As shown in Fig. 2, on the fourteenth day of the virus attack, the survival rate of the oral preparation group (A) of the present invention was almost 100%. The unencapsulated high dose group (9) had a survival rate of about 8%, except The survival rate of the other groups in the D group outside the general feed is only 10-20%. It can be seen from the above data that the IgY σ service of the present invention is fine in the in vivo test, and has a very significant effect against the virus attack, and can greatly improve the survival rate of the aquatic animal, and the survival rate is equal to the unembedded high dose or higher. The invention may be modified and modified in accordance with the embodiments without departing from the spirit of the invention. It is to be understood that the invention is not intended to be limited to the scope of the embodiments disclosed herein. [Simple description of the diagram] The description and implementation of the text can be better explained by the additional drawings. In order to enhance the description of the present invention, the drawings of the present invention are set forth in the accompanying drawings. Figure 1 shows the results of a virus attack on the first day after the three-day group feed of the grouper, and recorded the relative survival rate after the daily death. Figure 2 shows the results of a virus attack on the fifth day after the grouper was fed three days of feed, recording the relative survival rate after daily death. [Explanation of main component symbols] None ❹ 16 16 201023864 T0U0001TW_seq_list _TW Sequence table <110> Zhou Xinyou Ren Yi Zhu Jiaying Wu Zhangzhe <120> Egg yolk immunoglobulin (IgY) Oral preparation and preparation method <130> TOU0001TW <160 〉 2 <170〉 Patentln version 3.3 <210〉 1 <211> 20 <212> DNA <213> Synthetic <220> <223> Introduction <400> 1 cccttccttg tgtcgtgtct 20 <210> 2 <211> 20 <212> DNA <213> Synthetic <220><223> Introduction <400> 2 gccaccgtaa tcagttcgat 20 Page 1