TWI756948B - Manufacturing method and use of peptide nanotube and immunogenic composition including the same - Google Patents

Manufacturing method and use of peptide nanotube and immunogenic composition including the same Download PDF

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TWI756948B
TWI756948B TW109142028A TW109142028A TWI756948B TW I756948 B TWI756948 B TW I756948B TW 109142028 A TW109142028 A TW 109142028A TW 109142028 A TW109142028 A TW 109142028A TW I756948 B TWI756948 B TW I756948B
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TW202222340A (en
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王祥宇
朱純燕
蕭璦莉
廖嘉鴻
李嘉偉
吳敏嘉
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國立屏東科技大學
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Abstract

The present invention is related to a manufacturing method and use of peptide nanotube as a DNA vaccine adjuvant and the immunogenic composition including the same for improving mucosal immune response. The peptide nanotube is long peptide nanotube and/or short peptide nanotube. The manufacturing method of the peptide nanotube comprises: mixing the cyclo-(D-Trp-Tyr) powder with water or alcohol to form a solution, well mixing the solution by a vibrator and withdrawing the liquid in the solution to obtain the nanotube.

Description

胜肽奈米管之製備方法、用途及其免疫組成物Preparation method, use and immune composition of peptide nanotube

本發明係關於一種胜肽奈米管之製備方法、用途及其免疫組成物，特別是關於促進黏膜免疫之胜肽奈米管之製備方法、用途及其免疫組成物。The present invention relates to a preparation method, application and immune composition of a peptide nanotube, in particular to the preparation method, application and immune composition of a peptide nanotube for promoting mucosal immunity.

黏膜免疫反應是人體或動物的第一道免疫防線，以新冠病毒為例(COVID-19)，其感染途徑跟一般的呼吸道疾病很相似，主要經由飛沫傳染、接觸傳染，而病毒會透過黏膜入侵身體，包含眼結膜、鼻黏膜、口腔黏膜等。許多動物傳染病病毒經常經口腔由腸胃道感染，例如豬流行下痢病毒，若能促進黏膜(例如，口腔黏膜、腸黏膜)免疫反應，則可以在第一時間擋下大部分病毒的攻擊，提高疫苗的有效性。因此，疫苗學家亦致力於提高疫苗的黏膜免疫反應效果。The mucosal immune response is the first line of immune defense of the human body or animal. Taking the new coronavirus (COVID-19) as an example, its infection route is very similar to that of general respiratory diseases. It is mainly transmitted through droplets and contact, and the virus will invade through the mucosa. The body, including the conjunctiva, nasal mucosa, oral mucosa, etc. Many animal infectious disease viruses are often infected by the gastrointestinal tract through the oral cavity, such as swine epidemic diarrhea virus, if it can promote the immune response of the mucosa (for example, oral mucosa, intestinal mucosa), it can block most of the virus attacks in the first time, improve Vaccine effectiveness. Therefore, vaccinologists are also working to improve the mucosal immune response of vaccines.

在各種疫苗當中， DNA疫苗因為純化技術簡便，品質易於控制和評價，能免除污染，而且成本較低，容易進行大規模的生產，也方便運送與儲存，成為傳染病疫苗研究的新寵。特別是DNA疫苗較能克服腸胃道嚴酷的環境，避免抗原蛋白降解、免疫耐受性誘導並延長滯留時間，其安全性也漸被信任，非常適合用來開發口服疫苗。口服疫苗在使用上相較於肌肉注射疫苗更為簡單方便，用於動物及小孩時可以減少採用較為費力和耗時的肌肉注射方式進行免疫，避免注射過程中因壓迫而受傷或是造成注射部位組織損傷等不良影響。Among various vaccines, DNA vaccines have become a new favorite in infectious disease vaccine research because of their simple purification technology, easy quality control and evaluation, free from contamination, low cost, easy large-scale production, and convenient transportation and storage. In particular, DNA vaccines can overcome the harsh environment of the gastrointestinal tract, avoid the degradation of antigenic proteins, induce immune tolerance and prolong the residence time. Oral vaccines are simpler and more convenient to use than intramuscular injections. When used for animals and children, the more laborious and time-consuming intramuscular injections can be used for immunization, and the injection process can be avoided due to compression or injury to the injection site. adverse effects such as tissue damage.

然而，縱使有上述諸多好處，DNA疫苗的免疫原性較差，若想要開發各種DNA疫苗，亟需研發一些佐劑來改善、促進、誘發免疫反應，特別是能安全使用於人體及動物中，且能幫助提高黏膜免疫反應的佐劑。However, despite the above-mentioned benefits, DNA vaccines have poor immunogenicity. If you want to develop various DNA vaccines, it is urgent to develop some adjuvants to improve, promote, and induce immune responses, especially those that can be safely used in humans and animals. And can help improve the adjuvant of mucosal immune response.

於一方面，本發明涉及一種胜肽奈米管做為增加黏膜免疫反應之DNA疫苗佐劑之用途，其中該胜肽奈米管為長胜肽奈米管及/或短胜肽奈米管，該胜肽奈米管之製備方法包含：混合一環-(D-色胺酸-酪胺酸)粉末與一水或酒精形成一溶液；震盪該溶液至混合均勻；以及移除該溶液中之液體以形成該胜肽奈米管。In one aspect, the present invention relates to the use of a peptide nanotube as a DNA vaccine adjuvant for increasing mucosal immune response, wherein the peptide nanotube is a long peptide nanotube and/or a short peptide nanotube , the preparation method of the peptide nanotubes comprises: mixing a cyclo-(D-tryptophan-tyrosine) powder and a water or alcohol to form a solution; shaking the solution until the mixture is uniform; and removing the liquid in the solution liquid to form the peptide nanotubes.

於另一方面，本發明涉及一種免疫組成物，包含：一質體，以及一胜肽奈米管，其中該質體上含有一編碼一抗原蛋白質片段的DNA序列，其中該胜肽奈米管為長胜肽奈米管及/或短胜肽奈米管，該胜肽奈米管之製備方法包含：混合一環-(D-色胺酸-酪胺酸)粉末與一水或酒精形成一溶液；震盪該溶液至混合均勻；以及移除該溶液中之液體以形成該胜肽奈米管。In another aspect, the present invention relates to an immune composition comprising: a plastid, and a peptide nanotube, wherein the plastid contains a DNA sequence encoding an antigenic protein fragment, wherein the peptide nanotube For long peptide nanotubes and/or short peptide nanotubes, the preparation method of the peptide nanotubes comprises: mixing a cyclo-(D-tryptophan-tyrosine) powder with a water or alcohol to form a solution; shake the solution to mix uniformly; and remove the liquid in the solution to form the peptide nanotube.

於另一方面，本發明涉及一種胜肽奈米管之製備方法，包含：混合一環-(D-色胺酸-酪胺酸)粉末與一水或酒精形成一溶液；震盪該溶液至混合均勻；以及移除該溶液中之液體以形成該胜肽奈米管。In another aspect, the present invention relates to a method for preparing peptide nanotubes, comprising: mixing a cyclo-(D-tryptophan-tyrosine) powder with water or alcohol to form a solution; shaking the solution until the solution is uniformly mixed and removing the liquid in the solution to form the peptide nanotube.

本發明製備方法所提供的胜肽奈米管可以做為增加黏膜免疫反應之DNA疫苗佐劑，以其所製成的口服DNA疫苗證實能有效誘發動物的IgA特異性抗體，可以提供一種安全、高局部有效濃度及高體內穿透力之疫苗應用，有利於使用來控制野生動物的人畜共通疾病。使用在動物產業，可以減少抗生素的使用，改善動物福利並提高經濟動物之產量。The peptide nanotube provided by the preparation method of the present invention can be used as a DNA vaccine adjuvant for increasing the mucosal immune response, and the oral DNA vaccine prepared by the preparation method can effectively induce IgA-specific antibodies in animals, and can provide a safe, The application of vaccines with high local effective concentration and high penetration in vivo is beneficial for the control of zoonotic diseases in wild animals. Used in the animal industry, it can reduce the use of antibiotics, improve animal welfare and increase the production of economic animals.

本發明於第一方面提供了一種胜肽奈米管做為增加黏膜免疫反應之DNA疫苗佐劑之用途，其中該胜肽奈米管為長胜肽奈米管及/或短胜肽奈米管，該胜肽奈米管之製備方法包含：混合一環-(D-色胺酸-酪胺酸)粉末與一水或酒精形成一溶液；震盪該溶液至混合均勻；以及移除該溶液中之液體以形成該胜肽奈米管。於某些具體實施例中，其係用於動物口服免疫，包含但不限於實驗動物，例如小鼠、大鼠、倉鼠、天竺鼠，家畜動物，例如豬、牛、山羊、綿羊、兔、馬、驢，家禽動物，例如雞、鴨、鵝、火雞，伴侶動物，例如，狗、貓，動物園動物，例如，長頸鹿、大象、獅、豹、猴。In the first aspect, the present invention provides the use of a peptide nanotube as a DNA vaccine adjuvant for increasing mucosal immune response, wherein the peptide nanotube is a long peptide nanotube and/or a short peptide nanotube Tube, the preparation method of the peptide nanotube comprises: mixing a cyclo-(D-tryptophan-tyrosine) powder with a water or alcohol to form a solution; shaking the solution to mix uniformly; and removing the solution liquid to form the peptide nanotubes. In certain embodiments, it is used for oral immunization of animals, including but not limited to experimental animals such as mice, rats, hamsters, guinea pigs, livestock animals such as pigs, cows, goats, sheep, rabbits, horses, Donkeys, poultry animals such as chickens, ducks, geese, turkeys, companion animals such as dogs, cats, zoo animals such as giraffes, elephants, lions, leopards, monkeys.

於某些具體實施例中，使用的胜肽奈米管包含分別為約0%、2.5%、5%、7.5%、10%、12.5%、15%、17.5%、20%、22.5%、25%、27.5%、30%、32.5%、35%、37.5%、40%、42.5%、45%、47.5%、50%、52.5%、55%、57.5%、60%、62.5%、65%、 67.5%、70%、72.5%、75%、77.5%、80%、82.5%、85%、87.5%、90%、92.5%、95%、97.5%或100%重量的長胜肽奈米管，以及約100%、97.5%、95%、92.5%、90%、87.5%、85%、82.5%、 80%、77.5%、75%、72.5%、70%、67.5%、65%、62.5%、60%、57.5%、55%、52.5%、50%、47.5%、45%、42.5%、40%、37.5%、35%、32.5%、30%、27.5%、25%、22.5%、20%、17.5%、15%、12.5%、10%、7.5%、5%、2.5%或0%重量的短胜肽奈米管。In certain embodiments, the peptide nanotubes used comprise about 0%, 2.5%, 5%, 7.5%, 10%, 12.5%, 15%, 17.5%, 20%, 22.5%, 25%, respectively %, 27.5%, 30%, 32.5%, 35%, 37.5%, 40%, 42.5%, 45%, 47.5%, 50%, 52.5%, 55%, 57.5%, 60%, 62.5%, 65%, 67.5%, 70%, 72.5%, 75%, 77.5%, 80%, 82.5%, 85%, 87.5%, 90%, 92.5%, 95%, 97.5% or 100% by weight of long peptide nanotubes, and about 100%, 97.5%, 95%, 92.5%, 90%, 87.5%, 85%, 82.5%, 80%, 77.5%, 75%, 72.5%, 70%, 67.5%, 65%, 62.5%, 60%, 57.5%, 55%, 52.5%, 50%, 47.5%, 45%, 42.5%, 40%, 37.5%, 35%, 32.5%, 30%, 27.5%, 25%, 22.5%, 20% , 17.5%, 15%, 12.5%, 10%, 7.5%, 5%, 2.5% or 0% by weight of short peptide nanotubes.

本發明於第二方面還提供一種免疫組成物，包含：一質體，以及一胜肽奈米管，其中該質體上含有一編碼一抗原蛋白質片段的DNA序列，其中該胜肽奈米管為長胜肽奈米管及/或短胜肽奈米管，該胜肽奈米管之製備方法包含：混合一環-(D-色胺酸-酪胺酸)粉末與一水或酒精形成一溶液；震盪該溶液至混合均勻；以及移除該溶液中之液體以形成該胜肽奈米管。The present invention also provides an immune composition in a second aspect, comprising: a plastid, and a peptide nanotube, wherein the plastid contains a DNA sequence encoding an antigenic protein fragment, wherein the peptide nanotube For long peptide nanotubes and/or short peptide nanotubes, the preparation method of the peptide nanotubes comprises: mixing a cyclo-(D-tryptophan-tyrosine) powder with a water or alcohol to form a solution; shake the solution to mix uniformly; and remove the liquid in the solution to form the peptide nanotube.

於某些具體實施例中，本發明之免疫組成物包含分別為約2.5%、5%、7.5%、10%、12.5%、15%、17.5%、20%、22.5%、25%、27.5%、30%、32.5%、35%、37.5%、40%、42.5%、45%、47.5%、50%、52.5%、55%、57.5%、60%、62.5%、65%、 67.5%、70%、72.5%、75%、77.5%、80%、82.5%、85%、87.5%或90%重量的質體，以及約97.5%、95%、92.5%、90%、87.5%、85%、82.5%、 80%、77.5%、75%、72.5%、70%、67.5%、65%、62.5%、60%、57.5%、55%、52.5%、50%、47.5%、45%、42.5%、40%、37.5%、35%、32.5%、30%、27.5%、25%、22.5%、20%、17.5%、15%、12.5%或10%重量的胜肽奈米管。 In certain embodiments, the immune composition of the present invention comprises about 2.5%, 5%, 7.5%, 10%, 12.5%, 15%, 17.5%, 20%, 22.5%, 25%, 27.5%, respectively , 30%, 32.5%, 35%, 37.5%, 40%, 42.5%, 45%, 47.5%, 50%, 52.5%, 55%, 57.5%, 60%, 62.5%, 65%, 67.5%, 70 %, 72.5%, 75%, 77.5%, 80%, 82.5%, 85%, 87.5% or 90% by weight of plastids, and about 97.5%, 95%, 92.5%, 90%, 87.5%, 85%, 82.5%, 80%, 77.5%, 75%, 72.5%, 70%, 67.5%, 65%, 62.5%, 60%, 57.5%, 55%, 52.5%, 50%, 47.5%, 45%, 42.5% , 40%, 37.5%, 35%, 32.5%, 30%, 27.5%, 25%, 22.5%, 20%, 17.5%, 15%, 12.5% or 10% by weight of peptide nanotubes.

於某些具體實施例中，本發明之免疫組成物進一步包含一黏性劑，該黏性劑可以包含生物可代謝油，包含但不限於，大豆油、花生油、葵花籽油、玉米油、茶籽油、橄欖油、甜杏仁油、堅果油、亞麻仁油、介花油、棕櫚油、紫蘇油、玄米油、芝麻油、椰子油、豬油或其混合油。生物可代謝油之濃度可以為10~30%體積之黏性劑，更佳為15~25%體積之黏性劑。生物可代謝油可以與乳化劑混合成該黏性劑，乳化劑包含但不限於天然乳化劑、化學乳化劑或其組合。該黏性劑的濃度可以為75~95%體積之免疫組成物，更佳為80~90%體積之免疫組成物。 In certain embodiments, the immune composition of the present invention further comprises a viscous agent, and the viscous agent may comprise biometabolizable oils, including but not limited to soybean oil, peanut oil, sunflower oil, corn oil, tea Seed oil, olive oil, sweet almond oil, nut oil, linseed oil, fenugreek oil, palm oil, perilla oil, brown rice oil, sesame oil, coconut oil, lard oil or a mixture thereof. The concentration of the biometabolizable oil can be 10-30% by volume of viscous agent, more preferably 15-25% by volume of viscous agent. Biometabolizable oils can be mixed with emulsifiers to form the viscous agent, including, but not limited to, natural emulsifiers, chemical emulsifiers, or combinations thereof. The concentration of the viscous agent can be 75-95% by volume of the immune composition, more preferably 80-90% by volume of the immune composition.

本發明於第三方面更提供了一種胜肽奈米管之製備方法，包含：混合一環-(D-色胺酸-酪胺酸)粉末與一水或酒精形成一溶液；震盪該溶液至混合均勻；以及移除該溶液中之液體以形成該胜肽奈米管。 The present invention further provides a method for preparing a peptide nanotube in a third aspect, comprising: mixing a cyclo-(D-tryptophan-tyrosine) powder with a water or alcohol to form a solution; shaking the solution to mix uniform; and removing the liquid in the solution to form the peptide nanotube.

於某些具體實施例中，若混合該環-(D-色胺酸-酪胺酸)粉末與該水時，移除該溶液中之上清液以形成短胜肽奈米管。該環-(D-色胺酸-酪胺酸)粉末與水的重量比例可以為3：2000。 In certain embodiments, when the cyclo-(D-tryptophan-tyrosine) powder is mixed with the water, the supernatant in the solution is removed to form short peptide nanotubes. The weight ratio of the cyclo-(D-tryptophan-tyrosine) powder to water may be 3:2000.

於某些具體實施例中，若混合該環-(D-色胺酸-酪胺酸)粉末與該酒精時，使該溶液中之酒精揮發以形成長胜肽奈米管。其中，使用5毫克之該環-(D-色胺酸-酪胺酸)粉末與15毫升50重量%酒精混合。 In some embodiments, when the cyclo-(D-tryptophan-tyrosine) powder is mixed with the alcohol, the alcohol in the solution is volatilized to form long peptide nanotubes. Therein, 5 mg of the cyclo-(D-tryptophan-tyrosine) powder was mixed with 15 ml of 50% by weight alcohol.

於某些具體實施例中，本發明之製備方法進一步包含保存於冰箱。溫度可以為0℃~10℃，更佳為3℃~6℃，最佳為4℃。 In certain embodiments, the preparation method of the present invention further comprises storing in a refrigerator. The temperature may be 0°C to 10°C, more preferably 3°C to 6°C, and most preferably 4°C.

除非另有定義，本文使用的所有技術和科學術語具有與本發明所屬領域中的技術人員所通常理解相同的含義。 Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

如本文所用，冠詞「一」、「一個」以及「任何」是指一個或多於一個(即至少一個)的物品的文法物品。例如，「一個元件」意指一個元件或多於一個元件。 As used herein, the articles "a," "an," and "any" refer to grammatical items of one or more than one (ie, at least one) item. For example, "an element" means one element or more than one element.

本文所使用的「約」、「大約」或「近乎」一詞實質上代表所述之數值或範圍位於20%以內，較佳為於10%以內，以及更佳者為於5%以內。於本文所提供之數字化的量為近似值，意旨若術語「約」、「大約」或「近乎」沒有被使用時亦可被推得。 As used herein, the terms "about", "approximately" or "approximately" mean substantially that the stated value or range is within 20%, preferably within 10%, and more preferably within 5%. Numerical quantities provided herein are approximations, meaning that they could also be inferred if the terms "about," "approximately," or "approximately" were not used.

如本文所用，術語「免疫組成物(immunogenic composition)」是指一種當投予一宿主時能夠觸發該宿主之免疫系統而誘導任何免疫反應的組成物。免疫組成物可以包括它們的任何藥學上可接受的稀釋劑、佐劑、緩衝劑、賦形劑、載體或組合。在一般情況下，免疫組成物的組成分係基於給藥的方式和途徑，以及標準藥學實行而被選擇的。 As used herein, the term "immunogenic composition" refers to a composition that, when administered to a host, is capable of triggering the host's immune system to induce any immune response. The immunological composition may include any pharmaceutically acceptable diluent, adjuvant, buffer, excipient, carrier or combination thereof. In general, the components of the immune composition are selected based on the mode and route of administration, and standard pharmaceutical practice.

如本文所用，術語「室溫」或「常溫」，若無特別說明，係指18至28℃之間之任意溫度區間。 As used herein, the term "room temperature" or "normal temperature", unless otherwise specified, refers to any temperature range between 18 and 28°C.

如本文所用，術語「疫苗」是指適合施用於動物(包括人類)之成分，其包含一免疫有效量(即，能刺激目標動物之免疫系統)之一或多種抗原(諸如減毒或已被滅殺之微生物及/或其次單位，或任何其他物質，諸如有機體之代謝產物)，此一或多種抗原通常與藥學上可接受之載體(例如：含水之液體)組合，且可選擇地包含免疫刺激劑(佐劑)，當將該疫苗投予一動物時其可誘導足 以治療該動物之疾病或障礙失調(即，至少協助預防、改善或治癒疾病或障礙失調)之免疫反應。 As used herein, the term "vaccine" refers to a composition suitable for administration to animals, including humans, comprising an immunologically effective amount (ie, capable of stimulating the immune system of the target animal) of one or more antigens (such as attenuated or Killed microorganisms and/or their subunits, or any other substance, such as a metabolite of an organism), the one or more antigens are usually combined with a pharmaceutically acceptable carrier (e.g., an aqueous liquid), and optionally an immune Stimulant (adjuvant), which induces stamina when the vaccine is administered to an animal An immune response to treat a disease or disorder in the animal (ie, at least assist in the prevention, amelioration or cure of the disease or disorder).

如本文所用，術語「抗體效價」是指一抗體的物理狀態及其在體內的滯留時間，以其與抗原反應的多少來表示其免疫效果，稱為抗體效價。 As used herein, the term "antibody titer" refers to the physical state of an antibody and its residence time in the body, and its immune effect is expressed by how much it reacts with an antigen, which is called antibody titer.

如本文所用，術語「佐劑」是指任何可增加對一抗原之黏膜、體液或細胞免疫反應的物質，通常係用於達成二種目的：減緩抗原釋出及刺激免疫反應。 As used herein, the term "adjuvant" refers to any substance that increases the mucosal, humoral or cellular immune response to an antigen, usually for two purposes: slowing antigen release and stimulating an immune response.

如本文所用，術語「抗原」或「免疫原」是指任何刺激免疫反應之物質。此名詞包括被殺死的、去活化的、減毒的或經改質的活細菌、病毒或寄生蟲。抗原一詞亦包括個別之多核苷酸、多胜肽、重組蛋白質、合成之胜肽、蛋白質萃取物、細胞(包括腫瘤細胞)、組織、多醣、或脂質、或其片段或彼等之任何組合。抗原一詞亦包括抗體，諸如抗個體遺傳型抗體或其片段，以及可模擬抗原或抗原決定簇(抗原決定部位)之合成胜肽模擬抗原決定部位(mimotopes)。 As used herein, the term "antigen" or "immunogen" refers to any substance that stimulates an immune response. This term includes killed, deactivated, attenuated or modified live bacteria, viruses or parasites. The term antigen also includes individual polynucleotides, polypeptides, recombinant proteins, synthetic peptides, protein extracts, cells (including tumor cells), tissues, polysaccharides, or lipids, or fragments thereof, or any combination thereof . The term antigen also includes antibodies, such as anti-idiotypic antibodies or fragments thereof, as well as synthetic peptide mimotopes (mimotopes) that can mimic antigens or antigenic determinants (epitopes).

如本文所用，術語「乳劑」係指二種不相溶混之液體的組成物，其中一種液體之小滴懸浮於另一種液體之連續相中。 As used herein, the term "emulsion" refers to a composition of two immiscible liquids in which droplets of one liquid are suspended in a continuous phase of the other liquid.

如本文所用，術語「黏膜免疫反應」是指外來物質入侵動物(包括人類)黏膜免疫系統時，黏膜免疫系統被觸發時所產生的反應，主要是透過分泌型免疫球蛋白A(IgA)來反應，其主要存在於唾液、乳液、胃腸液、呼吸道分泌液等，是黏膜部位抵抗病原微生物和有害物質的第一道防線。 As used herein, the term "mucosal immune response" refers to the response produced when the mucosal immune system is triggered when a foreign substance invades the mucosal immune system of animals (including humans), mainly through secretory immunoglobulin A (IgA). , which mainly exists in saliva, emulsion, gastrointestinal fluid, respiratory secretions, etc., and is the first line of defense against pathogenic microorganisms and harmful substances in mucosal sites.

如本文所用，術語「胜肽奈米管」是指使用本文揭露之方法而製成的胜肽奈米管，在本文有兩大態樣，使用酒精作為溶劑而生成的為長胜肽 奈米管，使用水為溶劑而生成的為短胜肽奈米管。命名係為方便理解而定，「長」、「短」是相對於另一態樣而言，平均有此特徵，並非絕對特徵。 As used herein, the term "peptide nanotubes" refers to peptide nanotubes produced by the method disclosed herein. There are two major aspects in this paper. The long-peptide is produced by using alcohol as a solvent. Nanotubes are short peptide nanotubes produced using water as a solvent. The naming is for the convenience of understanding. "Long" and "Short" are relative to another aspect and have this characteristic on average, not an absolute characteristic.

本發明通過下列的實施例進一步說明，其提供了用於示範而非限制的目的。根據本發明公開內容，本領域中的技術人員應當理解，許多變化可以在所公開的特定具體實施例中產生，且仍然獲得相同或類似的結果而不脫離本發明的精神和範圍。 The present invention is further illustrated by the following examples, which are provided for purposes of illustration and not limitation. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments disclosed and still obtain a like or similar result without departing from the spirit and scope of the present invention.

實施例一 胜肽奈米管的製備方法Example 1 Preparation method of peptide nanotubes

秤取5毫克環-(D-色胺酸-酪胺酸)胜肽粉末於500毫升燒杯中，加入15毫升50重量%酒精，以鋁箔紙將燒杯蓋住，再放置於超音波震盪器，常溫震盪5分鐘，使藥品溶於50重量%酒精中並呈現透明清澈。接著放到37℃培養箱中隔夜使酒精揮發或烘乾後，燒杯底部會呈現雪花狀，再使用秤藥紙邊緣將雪花狀奈米載體刮起，放到1.5毫升離心管中於4℃冰箱保存，即為長胜肽奈米管(Long Peptide Nano Tube,LPNT或是L-PNT)。 Weigh 5 mg of cyclo-(D-tryptophan-tyrosine) peptide powder into a 500-ml beaker, add 15 ml of 50 wt% alcohol, cover the beaker with aluminum foil, and place it on an ultrasonic shaker. Shake at room temperature for 5 minutes to dissolve the medicine in 50% by weight of alcohol and be transparent and clear. Then put it in a 37°C incubator overnight to evaporate or dry the alcohol, and the bottom of the beaker will appear snowflake-like. Then use the edge of the weighing paper to scrape up the snowflake-shaped nanocarriers, put them in a 1.5ml centrifuge tube and store in a 4°C refrigerator. Save, it is Long Peptide Nano Tube (LPNT or L-PNT).

秤取1.5毫克環-(D-色胺酸-酪胺酸)胜肽粉末於褐色玻璃瓶(2毫升小玻璃瓶)中，加入1毫升滅菌後二次蒸餾水(double distilled water,DDW)，放置於超音波震盪器，常溫震盪10分鐘，使藥品完全震散，震盪分散後，溶液會轉為乳白色。接著靜置後抽掉上清液再保存於4℃冰箱，即為短胜肽奈米管(Short Peptide Nano Tube,SPNT或是S-PNT)。短胜肽奈米管的製備非常簡單快速，震盪完1-2小時即可抽掉上清液使用，若不馬上使用可置入冰箱保存。 Weigh 1.5 mg of cyclo-(D-tryptophan-tyrosine) peptide powder into a brown glass bottle (2 ml small glass bottle), add 1 ml of sterilized double distilled water (DDW), and set it aside In an ultrasonic shaker, shake at room temperature for 10 minutes to completely disperse the medicine. After shaking and dispersing, the solution will turn milky white. Then, after standing, the supernatant was removed and stored in a refrigerator at 4°C, which was called Short Peptide Nano Tube (SPNT or S-PNT). The preparation of short peptide nanotubes is very simple and fast. After 1-2 hours of shaking, the supernatant can be removed and used. If it is not used immediately, it can be stored in the refrigerator.

如圖1A-1H所示，為了了解二種胜肽奈米管於細胞培養環境中的特性，在六孔培養皿中分別將200微克與400微克的兩種不同胜肽奈米管加入非洲綠猴腎細胞(Vero細胞)進行培養，並於初始日及次日以兩百放大倍率在倒立 式顯微鏡進行觀察，可清楚辨識二種胜肽奈米管的形態及其對細胞的影響。第1A-1D圖可以看到，LPNT呈現長晶體狀，長度可達100~200微米。第1E-1G圖可以看到，SPNT則小於1微米而呈短簇狀叢聚的型態。 As shown in Figures 1A-1H, in order to understand the characteristics of the two peptide nanotubes in the cell culture environment, 200 μg and 400 μg of the two different peptide nanotubes were added to African green in a six-well petri dish. Monkey kidney cells (Vero cells) were cultured and incubated at 200 magnification on the first day and the next day The morphology of the two peptide nanotubes and their effects on cells can be clearly identified by observation under a microscope. As can be seen in Figures 1A-1D, the LPNTs appear as long crystals, and the length can reach 100-200 μm. It can be seen from Figures 1E-1G that the SPNTs are smaller than 1 micron and are clustered in short clusters.

圖1A-1B為200微克長胜肽奈米管在起始日與次日的培養狀況。圖1C-1D為400微克長胜肽奈米管在起始日與次日的培養狀況。圖1E-1F為200微克短胜肽奈米管在起始日與次日的培養狀況。圖1G-1H為400微克短胜肽奈米管在起始日與次日的培養狀況。無論SPNT或LPNT添加的量多寡，在細胞培養基環境中1天後，細胞形態並無明顯改變，可以得知Vero細胞對於兩種胜肽奈米管的添加皆不會產生任何急毒性的現象，細胞皆可正常生長。因此，兩種胜肽奈米管做為藥劑的奈米載體或佐劑對於細胞來說是安全的。 Figures 1A-1B show the culture conditions of 200 micrograms of long peptide nanotubes on the first day and the next day. Figures 1C-1D show the culture conditions of 400 μg long peptide nanotubes on the first day and the next day. Figures 1E-1F show the culture conditions of 200 μg short peptide nanotubes on the first day and the next day. Figures 1G-1H show the culture conditions of 400 μg short peptide nanotubes on the first day and the next day. Regardless of the amount of SPNT or LPNT added, the cell morphology did not change significantly after 1 day in the cell culture medium. It can be seen that Vero cells did not produce any acute toxicity to the addition of the two peptide nanotubes. Cells can grow normally. Therefore, the two peptide nanotubes are safe for cells as nanocarriers or adjuvants for pharmaceuticals.

不同大小、種類、長寬比的奈米載體會有不同乘載體積，也會有不同的內化細胞速率與體內藥物動力學停留時間，因此有不同的功效。例如管狀的載體比球形載體有較快與較多內化細胞速率，也有較長血液停留時間，較大的奈米載體承載量較高，較小的奈米載體較容易進入細胞。為了更佳了解兩種胜肽奈米管的形態及性質，在此使用穿透式電子顯微鏡(Transmission Electron microscopy,TEM)及光散射(Dynamic Light Scattering,DLS)技術分別確認上述兩種胜肽奈米管，並將尺寸資料與介面電位(ζ-potential,zeta potential)結果記錄於表1，經t檢定顯示長胜肽奈米管的尺寸、長寬比相較短胜肽奈米管的尺寸有顯著差異。 Nanocarriers of different sizes, types, and aspect ratios will have different loading volumes, as well as different internalization cell rates and in vivo pharmacokinetic residence times, and thus have different efficacy. For example, tubular carriers have faster and more internalized cell rates than spherical carriers, and also have longer blood residence time, larger nanocarriers have higher loading capacity, and smaller nanocarriers can enter cells more easily. In order to better understand the morphology and properties of the two peptide nanotubes, transmission electron microscopy (TEM) and light scattering (Dynamic Light Scattering, DLS) techniques were used to confirm the above two peptide nanotubes. The size data and zeta potential (ζ-potential, zeta potential) results are recorded in Table 1. The t test shows that the size and aspect ratio of the long peptide nanotubes are higher than those of the shorter peptide nanotubes. There are significant differences.

Figure 109142028-A0305-02-0011-1
Figure 109142028-A0305-02-0011-1

*：代表長胜肽奈米管與短胜肽奈米管間有差異，p<0.05 *: represents the difference between long peptide nanotubes and short peptide nanotubes, p<0.05

如圖2所示，使用x光粉末繞射分析長胜肽奈米管、短胜肽奈米管與原始材料：環-(D-色胺酸-酪胺酸)胜肽粉末。分析顯示長、短胜肽奈米管的晶型形成在x光粉末繞射結果上極為類似，惟有長胜肽奈米管在第1波峰峰長的強度大於短胜肽奈米管。長、短胜肽奈米管的x光粉末繞射結果則與非晶型的原始材料粉末有明顯差異。可見經由本實施例一之製備方法，長、短胜肽奈米管已成為與原始材料不同晶型的奈米材料。 As shown in Figure 2, the long peptide nanotubes, the short peptide nanotubes and the original material: cyclo-(D-tryptophan-tyrosine) peptide powder were analyzed using x-ray powder diffraction. The analysis shows that the crystal forms of long and short peptide nanotubes are very similar in X-ray powder diffraction results, only the intensity of the long peptide nanotubes at the peak length of the first wave is greater than that of the short peptide nanotubes. The X-ray powder diffraction results of long and short peptide nanotubes are significantly different from those of the amorphous raw material powder. It can be seen that through the preparation method of the first embodiment, the long and short peptide nanotubes have become nanomaterials with different crystal forms from the original materials.

實施例二 質體去氧核醣核酸(DNA)製備Example 2 Preparation of plastid deoxyribonucleic acid (DNA)

使用pTCY載體攜帶不同抗原基因之大腸桿菌表現系統，培養放大於1公升LB(Lysogeny broth)培養基，再抽取質體。上述pTCY載體係衍生自pEGFP-N1載體(Clontech公司，Mount View，加州，美國)，其中pEGFP-N1載體上的CMV立即早期啟動子(CMV immediate-early promoter)以一大鼠beta-肌動蛋白啟動子替換，且去除該增強的綠螢光蛋白的編碼區域，使其他目標基因能被***並在beta-肌動蛋白啟動子的控制下表現。pTCY載體詳細的構築方式請參閱H.-C.Wu等人(2019)A DNA priming and protein boosting immunization scheme to augment immune responses against parvovirus in ducks.J.Appl.Microbiol.126(1)：49-57.。 Using the E. coli expression system carrying different antigen genes in the pTCY vector, the culture was amplified in 1 liter of LB (Lysogeny broth) medium, and the plastids were extracted. The above-mentioned pTCY vector system is derived from the pEGFP-N1 vector (Clontech Company, Mount View, California, USA), wherein the CMV immediate-early promoter on the pEGFP-N1 vector is derived from a rat beta-actin protein. The promoter was replaced, and the coding region for this enhanced green fluorescent protein was removed, allowing other genes of interest to be inserted and expressed under the control of the beta-actin promoter. For the detailed construction of pTCY vector, please refer to H.-C.Wu et al. (2019) A DNA priming and protein boosting immunization scheme to augment immune responses against parvovirus in ducks.J.Appl.Microbiol.126(1):49-57 ..

在本實施例中，用到兩種病毒抗原基因進行製備。一種是豬流行性下痢病毒(Porcine Epidemic Diarrhea Virus,PEDV)的棘蛋白(spike protein)S1區域，其胺基酸序列為SEQ ID NO：1，編碼該S1區域的胺基酸序列之示例性DNA序列為SEQ ID NO：2，但不限於此序列，可使用任何可以編碼SEQ ID NO：1之DNA序列。一種是鵝源水禽小病毒(Goose Parvo Virus,GPV)之第2病毒蛋白 (VP2)，其胺基酸序列為SEQ ID NO：3，編碼該VP2蛋白之示例性DNA序列為SEQ ID NO：4，但不限於此序列，可使用任何可以編碼SEQ ID NO：3之DNA序列。最後分別獲得pTCY攜帶PEDV-S1序列的質體(在此稱做「PED質體」)及pTCY攜帶VP2序列的質體(在此稱做「GPV質體」)。 In this example, two viral antigen genes were used for preparation. One is the spike protein (spike protein) S1 region of Porcine Epidemic Diarrhea Virus (PEDV), the amino acid sequence of which is SEQ ID NO: 1, and an exemplary DNA encoding the amino acid sequence of the S1 region The sequence is SEQ ID NO: 2, but it is not limited to this sequence, and any DNA sequence that can encode SEQ ID NO: 1 can be used. One is the second viral protein of Goose Parvo Virus (GPV). (VP2), its amino acid sequence is SEQ ID NO: 3, an exemplary DNA sequence encoding the VP2 protein is SEQ ID NO: 4, but not limited to this sequence, any DNA that can encode SEQ ID NO: 3 can be used sequence. Finally, pTCY plastids carrying PEDV-S1 sequence (herein referred to as "PED plastids") and pTCY plastids carrying VP2 sequences (herein referred to as "GPV plastids") were obtained respectively.

實施例三 口服DNA疫苗製備Example 3 Oral DNA vaccine preparation

使用實施例一所獲得的胜肽奈米管與實施例二所獲得的質體DNA製備口服DNA疫苗。製備流程僅需將質體DNA與胜肽奈米管結晶於褐色玻璃管中均勻混合，最後固定於震盪器，使其混合到隔天，即可以將製成之口服DNA疫苗進行後續試驗。例如要製造PED口服DNA疫苗，可以依不同佐劑需求，選擇先秤取1.5毫克之LPNT或SPNT結晶放置褐色玻璃管中，再與1毫升之260微克/毫升PED質體均勻混合。或者，選擇秤取共1.5毫克之SPNT和LPNT結晶，示例性的比例可以為1：1，但不限於該比例，放入褐色玻璃管中，並加入1毫升之260微克/毫升PED質體均勻混合。如果需要製造GPV口服DNA疫苗，將上述PED質體替換為GPV質體即可。 Oral DNA vaccines were prepared using the peptide nanotubes obtained in Example 1 and the plastid DNA obtained in Example 2. The preparation process only requires uniform mixing of plastid DNA and peptide nanotube crystals in a brown glass tube, and finally fixed in a shaker to allow mixing until the next day, and the prepared oral DNA vaccine can be used for subsequent experiments. For example, to manufacture a PED oral DNA vaccine, according to different adjuvant requirements, you can choose to weigh 1.5 mg of LPNT or SPNT crystals and place them in a brown glass tube, and then evenly mix with 1 ml of 260 μg/ml PED plastid. Alternatively, choose to weigh a total of 1.5 mg of SPNT and LPNT crystals, an exemplary ratio can be 1:1, but not limited to this ratio, put it into a brown glass tube, and add 1 ml of 260 μg/ml PED mass to homogenize mix. If a GPV oral DNA vaccine needs to be produced, the above-mentioned PED plastid can be replaced with a GPV plastid.

為了初步確認兩種胜肽奈米管與質體結合後的狀態，採用代表性質體pCMV-bcl-xL-eGFP，其以巨細胞病毒啟動子做為DNA疫苗啟動，帶有編碼抗凋亡基因(bcl-xL)與增強綠色螢光蛋白基因(eGFP)，以大腸桿菌DH5a菌株放大。以市售Cy5螢光標劑分別在兩種胜肽奈米管進行標記，以TM-rhodamine螢光標劑在代表性質體DNA進行標記，分別於37℃下再與緩衝劑混和4個小時後，離心去除上清液，並利用酒精沉澱純化。將已標記長胜肽奈米管與短胜肽奈米管分別與代表性質體結合後，在雷射螢光共軛焦顯微鏡下，以光學顯微鏡以白光觀察(BF)胜肽奈米管與質體結合的狀況，以螢光顯微鏡在 Cy5通道之激發波長633奈米，發射波長645-740奈米觀察胜肽奈米管(紅色)，且在Rhodamine通道以激發波長為514奈米，發射波長570-620奈米觀察質體(綠色)，最後合併圖像為Cy5及Rhodamine通道疊圖的影像(黃色)，以觀察胜肽奈米管與代表性質體是否共定位。質體建構方式及螢光標記方式請參閱Feichin Hsiao等人之論文：In vitro and in vivo assessment of delivery of hydrophobic molecules and plasmid DNAs with PEO-PPO-PEO polymeric micelles on cornea.Journal of food and drug analysis 26(2018)869-878. In order to preliminarily confirm the state of the two peptide nanotubes combined with the plastid, a representative plastid pCMV-bcl-xL-eGFP was used, which was initiated with the cytomegalovirus promoter as a DNA vaccine, with a gene encoding anti-apoptosis. (bcl-xL) and enhanced green fluorescent protein gene (eGFP), amplified with E. coli DH5a strain. The two peptide nanotubes were labeled with commercially available Cy5 fluorescent reagent, and the representative plastid DNA was labeled with TM-rhodamine fluorescent reagent. After mixing with buffer for 4 hours at 37°C, centrifugation was performed. The supernatant was removed and purified by alcohol precipitation. After the labeled long peptide nanotubes and short peptide nanotubes were combined with representative plasmoids, the peptide nanotubes were observed with white light under a laser fluorescence conjugate focus microscope (BF) with white light. The state of plastid binding was measured by fluorescence microscopy in The excitation wavelength of Cy5 channel is 633 nm, and the emission wavelength is 645-740 nm to observe peptide nanotubes (red), and the Rhodamine channel is used to observe plastids (green) with excitation wavelength of 514 nm and emission wavelength of 570-620 nm. ), and the final merged image is the image of the Cy5 and Rhodamine channel overlays (yellow) to observe whether the peptide nanotubes co-localize with the representative plastids. For plastid construction and fluorescent labeling, please refer to Feichin Hsiao et al.'s paper: In vitro and in vivo assessment of delivery of hydrophobic molecules and plasmid DNAs with PEO-PPO-PEO polymeric micelles on cornea. Journal of food and drug analysis 26 (2018) 869-878.

如圖3A-3D所示，顯示長胜肽奈米管與代表性質體結合時用螢光共軛焦顯微鏡進行觀察的照片，圖3A顯示長胜肽奈米管與代表性質體結合之光學顯微觀察圖，圖3B顯示出被標記Cy5螢光的長胜肽奈米管的螢光顯微圖。圖3C顯示出被標記TM-rhodamine螢光的代表性質體的螢光顯微圖。在圖3D則是圖3B與圖3C的疊圖，在同一個位置顯示結合在一起的代表性質體與長胜肽奈米管共定位。圖3E-3H為短胜肽奈米管與代表性質體結合時用螢光共軛焦顯微鏡進行觀察的照片，圖3E顯示短胜肽奈米管與代表性質體結合之光學顯微觀察圖，圖3F顯示出被標記Cy5螢光的短胜肽奈米管的螢光顯微圖。圖3G顯示出被標記TM-rhodamine螢光的代表性質體的螢光顯微圖。在圖3H則是圖3F與圖3G的疊圖，在同一個位置顯示結合在一起的代表性質體與短胜肽奈米管共定位。由上述結果可知，本發明之兩種胜肽奈米管皆能與質體DNA形成共定位。 As shown in Figures 3A-3D, the photos showing the binding of the long peptide nanotubes to the representative plastids were observed with a fluorescent confocal microscope. Figure 3A shows the optical visualization of the long peptide nanotubes binding to the representative plastids. Microscopic view, Figure 3B shows the fluorescence micrograph of Cy5-labeled long peptide nanotubes. Figure 3C shows a fluorescence micrograph of a representative plastid of labeled TM-rhodamine fluorescence. In Figure 3D, which is an overlay of Figure 3B and Figure 3C, representative plastids bound together are shown co-localized with long peptide nanotubes at the same position. Figures 3E-3H are photos of short peptide nanotubes combined with representative plastids observed with a fluorescent conjugation microscope, and Figure 3E shows optical microscopy images of short peptide nanotubes combined with representative plastids, Figure 3F shows a fluorescence micrograph of Cy5-labeled short peptide nanotubes. Figure 3G shows a fluorescence micrograph of a representative plastid of labeled TM-rhodamine fluorescence. In Fig. 3H is an overlay of Fig. 3F and Fig. 3G, showing the co-localization of representative plastids bound together with short peptide nanotubes in the same position. From the above results, it can be seen that both of the two peptide nanotubes of the present invention can co-localize with plastid DNA.

另外，使用穿透式電子顯微鏡(Transmission Electron microscopy,TEM)及光散射(Dynamic Light Scattering,DLS)技術分別確認兩種胜肽奈米管與代表性質體DNA結合後的形態與性質，包括長、寬及預測長寬比(expected ratio)、尺寸和介面電位，並將結果記綠於下表2。 In addition, transmission electron microscopy (TEM) and light scattering (Dynamic Light Scattering, DLS) techniques were used to confirm the morphology and properties of the two peptide nanotubes combined with representative plastid DNA, including long, Width and expected aspect ratio (expected ratio), size and interface potential, and the results are recorded in Table 2 below.

使用螢光測量，藉由不同DNA濃度的存在下，胜肽奈米管的螢光發射光譜強度變化推估胜肽奈米管與質體DNA的結合常數(Binding constant)。使用小鼠的十二指腸進行體外十二指腸穿透係數分析。將腸組織置於體外垂直擴散裝置，將口服DNA疫苗置於供體室，測量受體室內取出的樣品，以qPCR定量其濃度。腸胃道穿透隙數測試(apparent permeability coefficient,P)=V(dC/dt)/A x C0，其中V(dC/dt)為在初始延遲時間後DNA出現於受體室的穩態速率，C0為在供體室的初始質體濃度，A為十二指腸暴露的面積。 Using fluorescence measurement, the binding constant of peptide nanotubes and plastid DNA was estimated by the change of fluorescence emission spectrum intensity of peptide nanotubes in the presence of different DNA concentrations. In vitro duodenal penetration coefficient analysis was performed using mouse duodenum. The intestinal tissue was placed in an in vitro vertical diffusion device, the oral DNA vaccine was placed in the donor chamber, and the samples taken out of the recipient chamber were measured and their concentrations were quantified by qPCR. Gastrointestinal permeability coefficient test (apparent permeability coefficient, P) = V(dC/dt)/A x C 0 , where V(dC/dt) is the steady-state rate of DNA appearing in the receptor compartment after the initial delay time , C 0 is the initial plastid concentration in the donor compartment, and A is the exposed area of the duodenum.

表2還使用t檢定檢驗兩種胜肽奈米管分別與代表性質體DNA之結合摩爾數(Mole fraction,n)、結合常數(M-1)與腸胃道穿透隙數測試P是否有顯著差異。可以理解的是，短胜肽奈米管做為佐劑時體積較小，可以有較佳的腸胃道穿透能力，因此P顯著較大，而長胜肽奈米管做為佐劑時體積較大，可以與質體DNA有較佳的結合能力，攜帶更多的質體DNA，因此結合常數顯著較大。 Table 2 also uses the t test to test whether the binding molar number (Mole fraction, n), binding constant (M -1 ) and gastrointestinal penetration gap number of the two peptide nanotubes to the representative plastid DNA respectively are significant. difference. It can be understood that the volume of short peptide nanotubes as an adjuvant is smaller and can have better gastrointestinal penetration ability, so P is significantly larger, while the volume of long peptide nanotubes as an adjuvant If it is larger, it can have better binding ability with plastid DNA and carry more plastid DNA, so the binding constant is significantly larger.

Figure 109142028-A0305-02-0015-2
Figure 109142028-A0305-02-0015-2

為了進一步瞭解質體DNA與胜肽奈米管之結合率，採用下列方法進行實驗。胜肽奈米管以吸附的方式結合質體DNA，使用實施例二製備之GPV質體為例，先抽取GPV質體並測其濃度後，調整成260微克/毫升，在連續 十倍稀釋成26微克/毫升、2.6微克/毫升、0.26微克/毫升、0.026微克/毫升、0.0026微克/毫升、0.00026微克/毫升及0.000026微克/毫升，建立即時聚合酶連鎖反應(real-time PCR)定量之標準曲線。 In order to further understand the binding rate of plastid DNA to peptide nanotubes, the following methods were used to conduct experiments. The peptide nanotubes bind the plastid DNA by adsorption. Using the GPV plastid prepared in Example 2 as an example, first extract the GPV plastid and measure its concentration, then adjust it to 260 μg/ml, and continuously. Ten-fold dilution to 26 μg/ml, 2.6 μg/ml, 0.26 μg/ml, 0.026 μg/ml, 0.0026 μg/ml, 0.00026 μg/ml and 0.000026 μg/ml to establish real-time polymerase chain reaction (real-time PCR ) quantitative standard curve.

在GPV質體與胜肽奈米管結合過程中，每2、4、8、12及24小時抽取10微升上清液，使用進行pTCY卡納黴素引子(kanamycin primer)進行即時聚合酶連鎖反應，以前述建立之標準曲線定量殘留於上層水溶液中的DNA含量，用以評估時間的長短是否可能影響DNA的吸附效率。從表3、表4的結果中可以發現長胜肽奈米管及短胜肽奈米管皆可快速吸附質體DNA，並於2小時內達99%以上吸附效率，於室溫(25~28℃)24小時仍不會釋放回水層。 During the binding of GPV plastids to peptide nanotubes, 10 μl of the supernatant was withdrawn every 2, 4, 8, 12 and 24 hours, and the pTCY kanamycin primer was used for real-time polymerase linkage. After the reaction, the DNA content remaining in the upper aqueous solution was quantified according to the standard curve established above to evaluate whether the length of time might affect the DNA adsorption efficiency. From the results in Table 3 and Table 4, it can be found that both long peptide nanotubes and short peptide nanotubes can quickly adsorb plastid DNA, and the adsorption efficiency reaches more than 99% within 2 hours. 28°C) for 24 hours without releasing back to the water layer.

Figure 109142028-A0305-02-0016-3
Figure 109142028-A0305-02-0016-3

Figure 109142028-A0305-02-0016-4
Figure 109142028-A0305-02-0016-4

上述的口服DNA疫苗於管餵時，可以確保餵食的疫苗劑量，適用於體積較小的動物，例如小鼠、鴨隻等。不過，較大的動物不容易進行管餵，若希望能提供嗜口性佳的乳劑型口服DNA疫苗，便於讓動物自行食用，本實施例中的口服DNA疫苗可進一步包含黏性劑，黏性劑可以包含生物可代謝 油。配置方式為：分別事先配置含20體積%生物可代謝油之黏性劑，添加之乳化劑可以為吐溫80(Tween80)、聚山梨醇酯二十(Tween20)等體積比例混合，接著與前項試驗中描述之口服DNA疫苗(質體DNA+胜肽奈米管)混合。以黏性劑：口服DNA疫苗為4：1~9：1的體積比例進行混合後使用，即可成為嗜口性佳的乳劑型口服DNA疫苗 When the above-mentioned oral DNA vaccine is tube-fed, the dose of the vaccine can be ensured, and it is suitable for animals with smaller size, such as mice, ducks, etc. However, tube feeding is not easy for larger animals. If it is desired to provide an oral DNA vaccine in an emulsion form with good palatability, it is convenient for animals to eat by themselves. Agents may contain biometabolizable Oil. The configuration method is as follows: respectively pre-configured a viscous agent containing 20% by volume of bio-metabolizable oil, and the added emulsifier can be Tween 80 (Tween80), polysorbate 20 (Tween20) mixed in an equal volume ratio, and then mixed with the preceding item. Oral DNA vaccines (plastid DNA + peptide nanotubes) as described in the experiments were mixed. Mix the viscous agent:oral DNA vaccine in a volume ratio of 4:1~9:1, and then use it to become an emulsion oral DNA vaccine with good palatability

實施例四 PED口服疫苗小鼠免疫試驗Example 4 PED oral vaccine mouse immunization test

實驗動物為4週大ICR公鼠(來源：樂斯科生物科技股份有限公司)，體重約15~18克左右。採用實施例三之方式以1.5毫克的胜肽奈米管混合260微克/毫升的PED質體製備兩種不同的PED口服疫苗。一種是使用長胜肽奈米管為佐劑的PED口服疫苗(LPNT/PED)，一種則使用短胜肽奈米管為佐劑的PED口服疫苗(SPNT/PED)，以試驗不同佐劑的免疫效果。動物實驗分為四組，每組四隻小鼠，控制組讓小鼠口服生理食鹽水進行免疫試驗，陽性對照組則對小鼠肌肉注射(IM)去活化PEDV蛋白(濃度為105.8TCID50/毫升)進行免疫試驗，第一組試驗組則是對小鼠管餵PED口服疫苗(LPNT/PED)，第二組試驗組則是對小鼠管餵PED口服疫苗(SPNT/PED)進行免疫試驗。 The experimental animals were 4-week-old ICR male mice (source: Lesco Biotechnology Co., Ltd.), weighing about 15-18 grams. Two different PED oral vaccines were prepared by mixing 260 μg/ml PED plastids with 1.5 mg of peptide nanotubes in the manner of Example 3. One is a PED oral vaccine (LPNT/PED) using long peptide nanotubes as an adjuvant, and the other is a PED oral vaccine (SPNT/PED) using short peptide nanotubes as an adjuvant to test the effect of different adjuvants. Immune effect. The animal experiment was divided into four groups with four mice in each group. In the control group, mice were given oral saline for immunization test, while in the positive control group, mice were injected intramuscularly (IM) with deactivated PEDV protein (at a concentration of 10 5.8 TCID 50 ). /ml) for immunization test, the first group of test group was given to mice with PED oral vaccine (LPNT/PED), and the second group of test group was given to mice with tube-fed PED oral vaccine (SPNT/PED) for immunization test.

如圖4所示，第一次實驗僅比較血清中的免疫球蛋白G(Immunoglobulin G,IgG)抗體效價。在0週時，各個組別分別以0.2毫升劑量進行免疫試驗，0及4週時對小鼠採血收集血清，保存於-20℃。使用酵素免疫分析(Enzyme-linked Immunoorbent Assay,ELISA)測試小鼠血清中抗PEDV之IgG抗體效價，使用25微克/毫升的去活化PEDV為ELISA底層抗原，樣品為稀釋16倍樣品血清，加入山羊抗小鼠IgG HRP二級抗體(Goat anti mouse IgG HRP(1：5000))，並測定吸光值，各個組別進行多因子變異數分析(multi-ANOVA)檢 定後相對控制組進行比較(*表示p值<0.05，**表示p值<0.01)。結果顯示，到第四週時可以看出LPNT/PED口服DNA疫苗及SPNT/PED口服DNA疫苗之單次免疫相對於控制組皆有顯著效果。 As shown in Figure 4, the first experiment compared only the immunoglobulin G (Immunoglobulin G, IgG) antibody titers in serum. At week 0, each group was administered an immune test at a dose of 0.2 ml, and blood was collected from mice at weeks 0 and 4, and the serum was stored at -20°C. Enzyme-linked Immunoorbent Assay (ELISA) was used to test the anti-PEDV IgG antibody titer in mouse serum. 25 μg/ml of deactivated PEDV was used as the bottom antigen of ELISA. The sample was diluted 16 times and added to goat. Anti-mouse IgG HRP secondary antibody (Goat anti mouse IgG HRP (1:5000)) was used to measure the absorbance value. Multi-factor analysis of variance (multi-ANOVA) was performed for each group. Compared with the control group after determination (* indicates p value <0.05, ** indicates p value <0.01). The results showed that the single immunization of LPNT/PED oral DNA vaccine and SPNT/PED oral DNA vaccine had significant effects compared to the control group at the fourth week.

第二次實驗為了瞭解二次免疫的效果，在0週時，各個組別分別以0.2毫升劑量進行免疫試驗，第二週再施以0.2毫升劑量進行二次免疫。同時，為了更進一步了解黏膜免疫反應，於第3週全數進行犧牲，犧牲後收集小鼠小腸全段(包含十二指腸，約20公分長)，以5毫升生理食鹽水沖洗小腸，收集之腸液以1,500rpm，離心5分鐘收集上清液，保存於-20℃，用免疫球蛋白A(Immunoglobulin A,IgA)ELISA測驗，使用25微克/毫升的去活化PEDV為ELISA底層抗原，樣品為稀釋16倍樣品小腸沖洗液，加入山羊抗小鼠IgA HRP二級抗體(Goat anti mouse IgA HRP(1：5000))來測試小鼠腸液中抗PEDV之IgA抗體效價。 In the second experiment, in order to understand the effect of the second immunization, at week 0, each group was subjected to the immunization test with a dose of 0.2 ml, and the second immunization was performed with a dose of 0.2 ml in the second week. At the same time, in order to further understand the mucosal immune response, all mice were sacrificed at the third week. After the sacrifice, the entire small intestine of the mice (including the duodenum, about 20 cm long) was collected, and the small intestine was washed with 5 ml of normal saline. rpm, centrifuge for 5 minutes to collect the supernatant, store it at -20°C, and use immunoglobulin A (Immunoglobulin A, IgA) ELISA test, use 25 μg/ml of deactivated PEDV as the bottom antigen of the ELISA, and the sample is a 16-fold dilution. The small intestine was washed with goat anti-mouse IgA HRP secondary antibody (Goat anti mouse IgA HRP (1:5000)) to test the anti-PEDV IgA antibody titer in mouse intestinal fluid.

如圖5所示，使用multi-ANOVA檢定後相對控制組進行比較(**表示p值<0.01)。IgA抗體測試結果，在小鼠有補強免疫的情況下，L-PNT/PED口服DNA疫苗組別和S-PNT/PED口服DNA疫苗組別之腸沖洗液測得之IgA抗體效價相較於控制組有顯著的差異，代表PED口服DNA疫苗(L-PNT/PED和S-PNT/PED)可明顯誘發黏膜免疫反應，可顯著增加小鼠小腸中抗PEDV-IgA抗體效價，而肌肉注射去活化疫苗組則無法測得上升的IgA免疫效果，代表傳統肌肉注射去活化疫苗無明顯誘發黏膜免疫反應。 As shown in Figure 5, comparisons were made to the control group after using the multi-ANOVA test (** indicates p-value < 0.01). The results of the IgA antibody test showed that the IgA antibody titers measured in the intestinal washes of the L-PNT/PED oral DNA vaccine group and the S-PNT/PED oral DNA vaccine group were compared with There were significant differences in the control group, representing that PED oral DNA vaccines (L-PNT/PED and S-PNT/PED) could significantly induce mucosal immune responses and significantly increase the anti-PEDV-IgA antibody titer in the small intestine of mice, while intramuscular injection In the deactivated vaccine group, the increased IgA immune effect could not be measured, which means that the traditional intramuscular injection of the deactivated vaccine did not significantly induce mucosal immune responses.

第三次實驗，同樣使用1.5毫克的胜肽奈米管混合260微克/毫升的PED質體，分別製備成兩種不同的PED口服疫苗(LPNT/PED、SPNT/PED)。另外再以總量為1.5毫克的短胜肽奈米管搭配長胜肽奈米管(短胜肽：長胜肽 =3：1)，製備成3SPNT+1LPNT/PED口服疫苗，以試驗長胜肽奈米管是否可輔助短胜肽奈米管免疫效能。 In the third experiment, 1.5 mg of peptide nanotubes were also used to mix 260 μg/ml of PED plastids to prepare two different PED oral vaccines (LPNT/PED, SPNT/PED). In addition, a total amount of 1.5 mg of short peptide nanotubes was matched with long peptide nanotubes (short peptide: long peptide = 3:1), prepare a 3SPNT+1LPNT/PED oral vaccine to test whether long peptide nanotubes can assist the immune efficacy of short peptide nanotubes.

動物實驗分為四組，每組六隻小鼠。控制組讓小鼠口服生理食鹽水進行免疫試驗，第一組試驗組則是對小鼠管餵PED口服疫苗(SPNT/PED)，第二組試驗組則是對小鼠管餵PED口服疫苗(LPNT/PED)進行免疫試驗，第三組試驗組則是對小鼠管餵PED口服疫苗(3SPNT+1LPNT/PED)進行免疫試驗。 Animal experiments were divided into four groups with six mice in each group. In the control group, mice were given oral saline for immunization test. The first experimental group was administered with PED oral vaccine (SPNT/PED), while the second experimental group was administered with PED oral vaccine (SPNT/PED). LPNT/PED) was used for immune test, and the third group of experimental group was administered with PED oral vaccine (3SPNT+1LPNT/PED) in mice.

於第0週，各個組別初始免疫試驗給予0.2毫升劑量，第2週再以0.2毫升劑量進行二次免疫。於第4週全數動物犧牲，收集小鼠小腸全段(包含十二指腸，約20公分長)，以5毫升生理食鹽水沖洗小腸，收集之腸液以1,500rpm，離心5分鐘收集上清液，保存於-20℃，用免疫球蛋白A(Immunoglobulin A,IgA)ELISA測驗，使用25微克/毫升的去活化PEDV為ELISA底層抗原，樣品為稀釋16倍樣品小腸沖洗液，加入山羊抗小鼠IgA HRP二級抗體(Goat anti mouse IgA HRP(1：5000))來測試小鼠腸液中抗PEDV之IgA抗體效價。 In the 0th week, each group was given a dose of 0.2 ml for the initial immunization test, and the second immunization was performed with a dose of 0.2 ml in the second week. At the 4th week, all animals were sacrificed, and the entire mouse small intestine (including the duodenum, about 20 cm long) was collected, and the small intestine was washed with 5 ml of normal saline. The collected intestinal fluid was centrifuged at 1,500 rpm for 5 minutes to collect the supernatant and stored in -20°C, use Immunoglobulin A (IgA) ELISA test, use 25 μg/ml of deactivated PEDV as the bottom antigen of ELISA, and the sample is the small intestine washing fluid diluted 16 times, and add goat anti-mouse IgA HRP II A grade antibody (Goat anti mouse IgA HRP (1:5000)) was used to test the anti-PEDV IgA antibody titer in mouse intestinal fluid.

如圖6所示，IgA抗體測試結果，免疫SPNT/PED組可顯著誘發黏膜免疫反應，而LPNT/PED組別則不顯著，而結合二種載體的3SPNT+1LPNT組別亦可顯著增加黏膜免疫反應(p值<0.05)。 As shown in Figure 6, the results of the IgA antibody test showed that the immunized SPNT/PED group could significantly induce mucosal immune response, while the LPNT/PED group had no significant effect, and the 3SPNT+1LPNT group combined with the two carriers could also significantly increase mucosal immunity. response (p-value < 0.05).

實施例五 GPV口服疫苗鴨隻免疫試驗Example 5 GPV oral vaccine duck immunization test

第一次實驗之實驗動物為9日齡正番鴨(來源：行政院農業委員會畜產試驗所宜蘭分所)，分成五組進行實驗，每組五隻。採用實施例三之方式以1.5毫克的胜肽奈米管混合50或100微克/毫升的PED質體分別製備兩種不同的GPV口服疫苗。一種是使用長胜肽奈米管為佐劑的GPV口服疫苗(L-PNT/GPV)，一種則使用短胜肽奈米管為佐劑的GPV口服疫苗(S-PNT/GPV)，以 試驗不同佐劑的免疫效果。控制組讓鴨隻口服生理食鹽水進行免疫試驗，第一組及第二組試驗組則是讓鴨隻口服GPV口服疫苗(S-PNT/GPV)，其GPV質體濃度分別為50及100微克/毫升，第三組及第四組試驗組則是讓鴨隻口服GPV口服疫苗(L-PNT/GPV)，其GPV質體濃度分別為50及100微克/毫升。 The experimental animals for the first experiment were 9-day-old Muscovy ducks (source: Yilan Branch, Animal Production Testing Institute, Agricultural Committee, Executive Yuan). They were divided into five groups for the experiment, with five animals in each group. Two different GPV oral vaccines were prepared by mixing 50 or 100 μg/ml PED plastids with 1.5 mg of peptide nanotubes in the manner of Example 3. One is a GPV oral vaccine (L-PNT/GPV) adjuvanted with long peptide nanotubes, and the other is a GPV oral vaccine (S-PNT/GPV) adjuvanted with short peptide nanotubes. The immune effects of different adjuvants were tested. In the control group, ducks were given oral saline for immunization test, while the first and second experimental groups were given oral GPV vaccine (S-PNT/GPV), and the GPV plastid concentration was 50 and 100 micrograms, respectively. The third and fourth experimental groups were given oral GPV vaccine (L-PNT/GPV), and the GPV plastid concentration was 50 and 100 μg/ml, respectively.

如圖7所示，第一次實驗進行口服DNA疫苗劑量測試以及長期抗體效力試驗。於第0週時，給予各組鴨隻1毫升的口服劑量，並於第2週以相同劑量進行第二次免疫。每2週採血收集血清，保存於-20℃，以ELISA檢測鴨隻血清中的抗GPV之IgG抗體，使用10微克/毫升的去活化GPV為ELISA底層抗原，樣品為血清樣本(1：50)，加入山羊抗鴨隻IgG HRP二級抗體(Goat anti dock IgG HRP(1：1000))，測定吸光值，並計算S/P比=(樣本吸光值-陰性對照組吸光值)/(陽性對照組吸光值-陰性對照組吸光值)，陽性對照組為GPV攻毒試驗存活的鴨隻血清，陰性對照組為無一級抗體之空白對照。結果顯示，L-PNT/GPV口服DNA疫苗與S-PNT/GPV口服DNA疫苗皆能引起IgG免疫反應，且能延續長達12週，另外可以看到在ANOVA檢定下(*代表p<0.05，**代表p<0.025)，GPV質體濃度100微克/毫升組別的免疫效果相較於控制組有顯著提升。因此，在鴨隻的動物實驗中，長胜肽奈米管與短胜肽奈米管做為佐劑能促進的IgG免疫效果相近。 As shown in Figure 7, the first experiment performed oral DNA vaccine dose testing as well as long-term antibody efficacy testing. At the 0th week, the ducks in each group were given an oral dose of 1 ml, and the second immunization was carried out with the same dose at the 2nd week. Blood was collected every 2 weeks to collect serum, stored at -20°C, and the anti-GPV IgG antibody in duck serum was detected by ELISA. 10 μg/ml of deactivated GPV was used as the bottom antigen of ELISA, and the sample was serum sample (1:50) , add goat anti-duck IgG HRP secondary antibody (Goat anti dock IgG HRP (1:1000)), measure the absorbance value, and calculate the S/P ratio = (sample absorbance value - negative control group absorbance value)/(positive control Absorbance value of group-absorbance value of negative control group), positive control group was the sera of ducks that survived GPV challenge test, and negative control group was blank control without primary antibody. The results showed that both the L-PNT/GPV oral DNA vaccine and the S-PNT/GPV oral DNA vaccine could induce an IgG immune response, which lasted for up to 12 weeks. In addition, it can be seen that under the ANOVA test (*represents p<0.05, **represents p<0.025), the immune effect of the GPV plastid concentration 100 μg/ml group was significantly improved compared with the control group. Therefore, in the animal experiments of ducks, the long peptide nanotubes and short peptide nanotubes as adjuvants can promote the IgG immune effect similar.

第二次實驗之實驗動物為9日齡正番鴨(來源：行政院農業委員會畜產試驗所宜蘭分所)，這次使用長胜肽奈米管與短胜肽奈米管混合(重量比1：1)做為佐劑，測試混合不同胜肽奈米管的佐劑能否提升免疫效果。採用實施例三之方式以共1.5毫克的長/短胜肽奈米管混合260微克/毫升的GPV質體製備GPV口服疫苗。第0週時，給予試驗組的鴨隻1毫升GPV口服疫苗，給予控制組的鴨隻1毫升生理食鹽水。 The experimental animal for the second experiment was a 9-day-old Muscovy duck (source: Yilan Branch, Animal Production Testing Institute, Agricultural Committee, Executive Yuan). This time, long peptide nanotubes were mixed with short peptide nanotubes (weight ratio 1: 1) As an adjuvant, test whether the adjuvant mixed with different peptide nanotubes can enhance the immune effect. GPV oral vaccine was prepared by mixing 260 μg/ml GPV plastids with a total of 1.5 mg of long/short peptide nanotubes in the manner of Example 3. At the 0th week, the ducks in the experimental group were given 1 ml of GPV oral vaccine, and the ducks in the control group were given 1 ml of normal saline.

如圖8及圖9所示，由於水禽類與哺乳動物不同，水禽類的血清、腸都會產生IgA反應，因此將前述取得的0、2、3周鴨隻血清以及第3週犧牲收集的十二指腸檢體來檢測IgA抗體效價。犧牲後收集鴨隻十二指腸(胃下十二指腸，約10公分長)，以5毫升生理食鹽水沖洗小腸，收集之腸液以4,000rpm，離心30分鐘收集上清液，保存於-20℃。以ELISA檢測血清或腸上清液中IgA抗體效價，使用10微克/毫升的去活化GPV為ELISA底層抗原，樣品為血清或腸上清液樣本(1：200)、加入小鼠抗鴨IgA二級抗體(mouse anti duck IgA(1：2000))，山羊抗小鼠IgG HRP三級抗體(Goat anti mouse IgG HRP(1：2000))，最後測定吸光值。使用ANOVA檢定發現，相較於控制組，不論是接種疫苗後第二週或第三週，試驗組血清中IgA抗體效價都顯著較高(*代表p<0.05，**表示p<0.025)(如圖8所示)；同樣地，相較於控制組，試驗組血清中及腸液中IgA抗體效價都顯著較高(**表示p<0.025)(如圖9所示)，代表混合長/短胜肽奈米管的佐劑可以顯著增加口服疫苗的黏膜免疫反應。 As shown in Figure 8 and Figure 9, since waterfowl is different from mammals, the serum and intestine of waterfowl will produce IgA reaction. Therefore, the 0, 2, and 3 weeks duck serum obtained above and the duodenum collected by sacrifice in the 3rd week were used. Samples to detect IgA antibody titers. After sacrifice, the duck duodenum (lower gastroduodenum, about 10 cm long) was collected, and the small intestine was washed with 5 ml of normal saline. The collected intestinal fluid was centrifuged at 4,000 rpm for 30 minutes to collect the supernatant and stored at -20°C. Detect IgA antibody titer in serum or intestinal supernatant by ELISA, use 10 μg/ml deactivated GPV as the bottom antigen of ELISA, the sample is serum or intestinal supernatant sample (1:200), add mouse anti-duck IgA Secondary antibody (mouse anti duck IgA (1: 2000)), goat anti-mouse IgG HRP tertiary antibody (Goat anti mouse IgG HRP (1: 2000)), and the final absorbance value was measured. Using ANOVA test, it was found that compared with the control group, the IgA antibody titer in the serum of the experimental group was significantly higher in the second or third week after vaccination (*represents p<0.05, **represents p<0.025) (As shown in Figure 8); Similarly, compared with the control group, the IgA antibody titers in the serum and intestinal fluid of the test group were significantly higher (** means p<0.025) (as shown in Figure 9), representing mixed Adjuvant of long/short peptide nanotubes can significantly increase the mucosal immune response of oral vaccines.

圖1A-1H所示為實施例一中胜肽奈米管產物與非洲綠猴腎細胞培養時之倒立式顯微鏡放大圖。圖1A-1B為200微克長胜肽奈米管在起始日與次日的培養狀況。圖1C-1D為400微克長胜肽奈米管在起始日與次日的培養狀況。圖1E-1F為200微克短胜肽奈米管在起始日與次日的培養狀況。圖1G-1H為400微克短胜肽奈米管在起始日與次日的培養狀況。圖中之比例尺標示為200微米。1A-1H are enlarged images of inverted microscope when the peptide nanotube product in Example 1 is cultured with African green monkey kidney cells. Figures 1A-1B show the culture conditions of 200 micrograms of long peptide nanotubes on the first day and the next day. Figures 1C-1D show the culture conditions of 400 μg long peptide nanotubes on the first day and the next day. Figures 1E-1F show the culture conditions of 200 μg short peptide nanotubes on the first day and the next day. Figures 1G-1H show the culture conditions of 400 μg short peptide nanotubes on the first day and the next day. The scale bar in the figure is marked as 200 microns.

圖2所示為實施例一中，長胜肽奈米管、短胜肽奈米管及原始材料之x光粉末繞射譜圖。Figure 2 shows the X-ray powder diffraction spectra of the long peptide nanotubes, the short peptide nanotubes and the original material in Example 1.

圖3A-3H所示為實施例三中長胜肽奈米管與短胜肽奈米管分別與質體DNA結合之光學顯微鏡與螢光顯微鏡觀察圖。圖3A為長胜肽奈米管與質體DNA結合之光學顯微鏡觀察圖。圖3B-圖3C分別為長胜肽奈米管與Cy5螢光標記、質體DNA與TM-rhodamine螢光標記所拍攝的螢光顯微鏡觀察圖。圖3D為圖3B與圖3C之結合圖。圖3E為短胜肽奈米管與質體DNA結合之光學顯微鏡觀察圖。圖3F-圖3G分別為短胜肽奈米管與Cy5螢光標記、質體DNA與TM-rhodamine螢光標記所拍攝的螢光顯微鏡觀察圖。圖3H為圖3F與圖3G之結合圖。圖中之比例尺標示為50微米。Figures 3A-3H show the optical microscope and fluorescence microscope observations of the long peptide nanotubes and the short peptide nanotubes binding to plastid DNA in Example 3, respectively. Figure 3A is an optical microscope observation of the combination of long peptide nanotubes and plastid DNA. Fig. 3B-Fig. 3C are the fluorescence microscope observation pictures taken by long peptide nanotubes and Cy5 fluorescent labeling, plastid DNA and TM-rhodamine fluorescent labeling, respectively. FIG. 3D is a combination diagram of FIG. 3B and FIG. 3C . Figure 3E is an optical microscope observation of the binding of short peptide nanotubes to plastid DNA. FIG. 3F-FIG. 3G are the fluorescence microscope observation pictures taken by short peptide nanotubes and Cy5 fluorescent labeling, plastid DNA and TM-rhodamine fluorescent labeling, respectively. FIG. 3H is a combination diagram of FIG. 3F and FIG. 3G . The scale bar in the figure is marked as 50 microns.

圖4所示為實施例四中，第一次實驗各組於免疫後4週測得的小鼠血清抗PEDV-IgG抗體效價長條圖。Figure 4 shows a bar graph of the anti-PEDV-IgG antibody titers of mouse serum in each group of the first experiment measured 4 weeks after immunization in Example 4.

圖5所示為實施例四中，第二次實驗各組於免疫後第3週測得的小鼠腸液抗PEDV-IgA抗體效價盒鬚圖。Figure 5 shows the box chart of the mouse intestinal fluid anti-PEDV-IgA antibody titers measured at the third week after immunization in each group of the second experiment in Example 4.

圖6所示為實施例四中，第三次實驗各組於免疫後第4週測得的小鼠腸液抗PEDV-IgA抗體效價盒鬚圖。Figure 6 shows the box chart of the mouse intestinal fluid anti-PEDV-IgA antibody titers measured in each group in the third experiment in the fourth week after immunization in Example 4.

圖7所示為實施例五中，第一次實驗各組於免疫後不同週數測得的鴨隻血清抗GPV-IgG抗體S/P值長條圖。Figure 7 is a bar graph showing the S/P values of duck serum anti-GPV-IgG antibodies measured at different weeks after immunization in each group of the first experiment in Example 5.

圖8所示為實施例五中，第二次實驗各組於免疫後不同週數測得的鴨隻血清抗GPV-IgA抗體效價長條圖。Figure 8 is a bar graph showing the anti-GPV-IgA antibody titers of duck serum measured in each group in the second experiment at different weeks after immunization in Example 5.

圖9所示為實施例五中，第二次實驗控制組與試驗組之鴨隻血清與腸液中於免疫後第3週測得的抗GPV-IgA抗體效價長條圖。Figure 9 shows the bar graph of the anti-GPV-IgA antibody titers measured in the serum and intestinal fluid of the ducks in the control group and the test group in the second experiment in the third week after immunization in Example 5. 

                                  序列表
          <![CDATA[<110>  國立屏東科技大學]]>
          <![CDATA[<120>  胜肽奈米管之製備方法、用途及其免疫組成物]]>
          <![CDATA[<130>  P20-0222]]>
          <![CDATA[<160>  4     ]]>
          <![CDATA[<170>  PatentIn version 3.5]]>
          <![CDATA[<210>  1]]>
          <![CDATA[<211>  763]]>
          <![CDATA[<212>  PRT]]>
          <![CDATA[<213>  豬流行性下痢病毒]]>
          <![CDATA[<400>  1]]>
          Pro Thr Leu Ser Leu Pro Gln Asp Val Thr Arg Cys Gln Ser Thr Thr 
          1               5                   10                  15      
          Asn Phe Arg Arg Phe Phe Ser Lys Phe Asn Val Gln Ala Pro Ala Val 
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          Val Val Leu Gly Gly Tyr Leu Pro Ser Met Asn Ser Ser Ser Trp Tyr 
                  35                  40                  45              
          Cys Gly Thr Gly Ile Glu Thr Ala Ser Gly Val His Gly Ile Phe Leu 
              50                  55                  60                  
          Ser Tyr Ile Asp Ser Gly Gln Gly Phe Glu Ile Gly Ile Ser Gln Glu 
          65                  70                  75                  80  
          Pro Phe Asp Pro Ser Gly Tyr Gln Leu Tyr Leu His Lys Ala Thr Asn 
                          85                  90                  95      
          Gly Asn Thr Asn Ala Ile Ala Arg Leu Arg Ile Cys Gln Phe Pro Asp 
                      100                 105                 110         
          Asn Lys Thr Leu Gly Pro Thr Val Asn Asp Val Thr Thr Gly Arg Asn 
                  115                 120                 125             
          Cys Leu Phe Asn Lys Ala Ile Pro Ala Tyr Met Arg Asp Gly Lys Asp 
              130                 135                 140                 
          Ile Val Val Gly Ile Thr Trp Asp Asn Asp Arg Val Thr Val Phe Ala 
          145                 150                 155                 160 
          Asp Lys Ile Tyr His Phe Tyr Leu Lys Asn Asp Trp Ser Arg Val Ala 
                          165                 170                 175     
          Thr Arg Cys Tyr Asn Arg Arg Ser Cys Ala Met Gln Tyr Val Tyr Thr 
                      180                 185                 190         
          Pro Thr Tyr Tyr Met Leu Asn Val Thr Ser Ala Gly Glu Asp Gly Ile 
                  195                 200                 205             
          Tyr Tyr Glu Pro Cys Thr Ala Asn Cys Thr Gly Tyr Ala Ala Asn Val 
              210                 215                 220                 
          Phe Ala Thr Asp Ser Asn Gly His Ile Pro Glu Gly Phe Ser Phe Asn 
          225                 230                 235                 240 
          Asn Trp Phe Leu Leu Ser Asn Asp Ser Thr Leu Leu His Gly Lys Val 
                          245                 250                 255     
          Val Ser Asn Gln Pro Leu Leu Val Asn Cys Leu Leu Ala Ile Pro Lys 
                      260                 265                 270         
          Ile Tyr Gly Leu Gly Gln Phe Phe Ser Phe Asn His Thr Met Asp Gly 
                  275                 280                 285             
          Val Cys Asn Gly Ala Ala Val Asp Arg Ala Pro Glu Ala Leu Arg Phe 
              290                 295                 300                 
          Asn Ile Asn Asp Thr Ser Val Ile Leu Ala Glu Gly Ser Ile Val Leu 
          305                 310                 315                 320 
          His Thr Ala Leu Gly Thr Asn Leu Ser Phe Val Cys Ser Asn Ser Ser 
                          325                 330                 335     
          Asp Pro His Leu Ala Ile Phe Ala Ile Pro Leu Gly Ala Thr Glu Val 
                      340                 345                 350         
          Pro Tyr Tyr Cys Phe Leu Lys Val Asp Thr Tyr Asn Ser Thr Val Tyr 
                  355                 360                 365             
          Lys Phe Leu Ala Val Leu Pro Pro Thr Val Arg Glu Ile Val Ile Thr 
              370                 375                 380                 
          Lys Tyr Gly Asp Val Tyr Val Asn Gly Phe Gly Tyr Leu His Leu Gly 
          385                 390                 395                 400 
          Leu Leu Asp Ala Val Thr Ile Asn Phe Thr Gly His Gly Thr Asp Asp 
                          405                 410                 415     
          Asp Val Ser Gly Phe Trp Thr Ile Ala Ser Thr Asn Phe Val Asp Ala 
                      420                 425                 430         
          Leu Ile Glu Val Gln Gly Thr Ser Ile Gln Arg Ile Leu Tyr Cys Asp 
                  435                 440                 445             
          Asp Pro Val Ser Gln Leu Lys Cys Ser Gln Val Ala Phe Asp Leu Asp 
              450                 455                 460                 
          Asp Gly Phe Tyr Pro Ile Ser Ser Arg Asn Leu Leu Ser His Glu Gln 
          465                 470                 475                 480 
          Pro Ile Ser Phe Val Thr Leu Pro Ser Phe Asn Asp His Ser Phe Val 
                          485                 490                 495     
          Asn Ile Thr Val Ser Ala Ala Phe Gly Gly Leu Ser Ser Ala Asn Leu 
                      500                 505                 510         
          Val Ala Ser Asp Thr Thr Ile Asn Gly Phe Ser Ser Phe Cys Val Asp 
                  515                 520                 525             
          Thr Arg Gln Phe Thr Ile Thr Leu Phe Tyr Asn Val Thr Asn Ser Tyr 
              530                 535                 540                 
          Gly Tyr Val Ser Lys Ser Gln Asp Ser Asn Cys Pro Phe Thr Leu Gln 
          545                 550                 555                 560 
          Ser Val Asn Asp Tyr Leu Ser Phe Ser Lys Phe Cys Val Ser Thr Ser 
                          565                 570                 575     
          Leu Leu Ala Gly Ala Cys Thr Ile Asp Leu Phe Gly Tyr Pro Ala Phe 
                      580                 585                 590         
          Gly Ser Gly Val Lys Leu Thr Ser Leu Tyr Phe Gln Phe Thr Lys Gly 
                  595                 600                 605             
          Glu Leu Ile Thr Gly Thr Pro Lys Pro Leu Glu Gly Ile Thr Asp Val 
              610                 615                 620                 
          Ser Phe Met Thr Leu Asp Val Cys Thr Lys Tyr Thr Ile Tyr Gly Phe 
          625                 630                 635                 640 
          Lys Gly Glu Gly Ile Ile Thr Leu Thr Asn Ser Ser Ile Leu Ala Gly 
                          645                 650                 655     
          Val Tyr Tyr Thr Ser Asp Ser Gly Gln Leu Leu Ala Phe Lys Asn Val 
                      660                 665                 670         
          Thr Ser Gly Ala Val Tyr Ser Val Thr Pro Cys Ser Phe Ser Glu Gln 
                  675                 680                 685             
          Ala Ala Tyr Val Asn Asp Asp Ile Val Gly Val Ile Ser Ser Leu Ser 
              690                 695                 700                 
          Asn Ser Thr Phe Asn Asn Thr Arg Glu Leu Pro Gly Phe Phe Tyr His 
          705                 710                 715                 720 
          Ser Asn Asp Gly Ser Asn Cys Thr Glu Pro Val Leu Val Tyr Ser Asn 
                          725                 730                 735     
          Ile Gly Val Cys Lys Ser Gly Ser Ile Gly Tyr Val Pro Ser Gln Tyr 
                      740                 745                 750         
          Gly Gln Val Lys Ile Ala Pro Thr Val Thr Gly 
                  755                 760             
          <![CDATA[<210>  2]]>
          <![CDATA[<211>  2289]]>
          <![CDATA[<212>  DNA]]>
          <![CDATA[<213>  豬流行性下痢病毒]]>
          <![CDATA[<400>  2]]>
          ccaacactca gcctaccaca agatgtcact aggtgccagt ctactactaa ctttaggcgg       60
          ttcttttcaa aatttaatgt tcaggcacct gccgtcgtcg ttttgggtgg ttacctacct      120
          agtatgaact cttctagctg gtactgtggc acaggcattg aaactgctag tggcgttcat      180
          ggtatttttc tcagctacat cgattctggt cagggctttg agattggcat ttcgcaagag      240
          ccgtttgatc ctagtggtta ccagctttat ttacataagg ccactaatgg taacactaat      300
          gctattgcac gactgcgcat ttgccagttt cccgataata aaacattggg ccctactgtt      360
          aatgatgtta caacaggtcg taactgccta ttcaacaaag ccattccagc ttatatgcgt      420
          gatggaaaag atattgttgt cggcataaca tgggataatg atcgtgtcac tgtttttgct      480
          gacaagatct atcattttta tcttaaaaat gattggtccc gcgttgcgac aagatgttac      540
          aatcgcagaa gttgtgctat gcaatatgtt tatacaccta cctactacat gcttaatgtt      600
          actagtgcag gtgaggatgg catttattat gaaccctgta cagctaattg cactggttac      660
          gctgccaatg tatttgccac tgattccaat ggccatatac cagaaggttt tagttttaat      720
          aattggtttc ttttatccaa tgactccact ttgttgcatg gtaaagtggt ttccaaccaa      780
          cccttgttgg tcaattgtct tttggccatt cctaagattt atggactagg ccaatttttc      840
          tcattcaatc acacgatgga tggcgtttgt aatggagctg ctgtggatcg tgccccagag      900
          gctctgaggt ttaatattaa tgacacctcc gtcattcttg ctgaaggctc aattgtactt      960
          catactgctt taggaacaaa tctttctttt gtttgcagta attcctcaga tcctcattta     1020
          gccatctttg ccatacctct gggtgctact gaagtaccct actattgctt tcttaaagtg     1080
          gatacttaca actccactgt ttataaattc ttggctgttt tacctcctac tgtcagggaa     1140
          attgtcatca ccaagtatgg tgatgtttat gtcaatgggt ttggctattt gcatctcggt     1200
          ttgttggatg ctgtcacaat taatttcact ggtcatggca ctgacgatga cgtttcaggt     1260
          ttctggacca tagcatcgac taattttgtt gatgcactca tcgaggttca aggaacttcc     1320
          attcagcgta ttctttattg tgatgatcct gttagccaac tcaagtgttc tcaggttgct     1380
          tttgaccttg acgatggttt ttaccccatc tcttctagaa accttctgag tcacgaacag     1440
          ccaatttctt ttgttacttt gccatcattt aatgatcatt cttttgttaa tattactgtc     1500
          tctgcggctt ttggtggtct tagtagtgcc aatctcgttg catctgacac tactatcaat     1560
          gggtttagtt ctttctgtgt tgacactaga caatttacca ttacactgtt ttataatgtt     1620
          acaaacagtt atggttatgt gtctaaatca caggatagta attgtccttt caccttgcaa     1680
          tctgttaatg attacctgtc ttttagcaaa ttttgtgttt caaccagcct tttggctggt     1740
          gcttgtacca tagatctttt tggttaccct gcgttcggta gtggtgttaa gttgacgtcc     1800
          ctttattttc aattcacaaa aggtgagttg attactggca cgcctaaacc acttgaaggt     1860
          atcacagacg tttcttttat gactctggat gtgtgtacca agtatactat ctatggcttt     1920
          aaaggtgagg gtattattac ccttacaaat tctagcattt tggcaggtgt ttattataca     1980
          tctgattctg gacagttgtt agcctttaag aatgtcacta gtggtgctgt ttattctgtc     2040
          acgccatgtt ctttttcaga gcaggctgca tatgttaatg atgatatagt gggtgttatt     2100
          tctagtttgt ctaactccac ttttaacaat actagggagt tgcctggttt cttctaccat     2160
          tctaatgacg gctccaattg tacagagcct gtgttggtgt atagtaacat aggtgtttgt     2220
          aaatctggca gtattggcta tgttccatct cagtatggcc aagtcaagat tgcacccacg     2280
          gttactggg                                                             2289
          <![CDATA[<210>  3]]>
          <![CDATA[<211>  587]]>
          <![CDATA[<212>  PRT]]>
          <![CDATA[<213>  鵝源水禽小病毒]]>
          <![CDATA[<400>  3]]>
          Thr Ala Pro Ala Lys Lys Asn Thr Gly Lys Leu Thr Asp His Tyr Pro 
          1               5                   10                  15      
          Val Val Lys Lys Pro Lys Leu Thr Glu Glu Val Ser Ala Gly Gly Gly 
                      20                  25                  30          
          Ser Ser Ala Val Gln Asp Gly Gly Ala Thr Ala Glu Gly Thr Glu Pro 
                  35                  40                  45              
          Val Ala Ala Ser Glu Met Ala Glu Gly Gly Gly Gly Ala Met Gly Asp 
              50                  55                  60                  
          Ser Ser Gly Gly Ala Asp Gly Val Gly Asn Ala Ser Gly Asn Trp His 
          65                  70                  75                  80  
          Cys Asp Ser Gln Trp Met Gly Asn Thr Val Ile Thr Lys Thr Thr Arg 
                          85                  90                  95      
          Thr Trp Val Leu Pro Ser Tyr Asn Asn His Ile Tyr Lys Ala Ile Thr 
                      100                 105                 110         
          Ser Gly Thr Ser Gln Asp Ala Asn Val Gln Tyr Ala Gly Tyr Ser Thr 
                  115                 120                 125             
          Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His Cys His Phe Ser Pro 
              130                 135                 140                 
          Arg Asp Trp Gln Arg Leu Ile Asn Asn His Trp Gly Ile Arg Pro Lys 
          145                 150                 155                 160 
          Ser Leu Lys Phe Lys Ile Phe Asn Val Gln Val Lys Glu Val Thr Thr 
                          165                 170                 175     
          Gln Asp Gln Thr Lys Thr Ile Ala Asn Asn Leu Thr Ser Thr Ile Gln 
                      180                 185                 190         
          Val Phe Thr Asp Asp Glu His Gln Leu Pro Tyr Val Leu Gly Ser Ala 
                  195                 200                 205             
          Thr Glu Gly Thr Met Pro Pro Phe Pro Ser Asp Val Tyr Ala Leu Pro 
              210                 215                 220                 
          Gln Tyr Gly Tyr Cys Thr Met His Thr Asn Gln Asn Gly Ala Arg Phe 
          225                 230                 235                 240 
          Asn Asp Arg Ser Ala Phe Tyr Cys Leu Glu Tyr Phe Pro Ser Gln Met 
                          245                 250                 255     
          Leu Arg Thr Gly Asn Asn Phe Glu Phe Thr Phe Asp Phe Glu Glu Val 
                      260                 265                 270         
          Pro Phe His Ser Met Phe Ala His Ser Gln Asp Leu Asp Arg Leu Met 
                  275                 280                 285             
          Asn Pro Leu Val Asp Gln Tyr Leu Trp Asn Phe Asn Glu Val Asp Ser 
              290                 295                 300                 
          Ser Arg Asn Ala Gln Phe Lys Lys Ala Val Lys Gly Ala Tyr Gly Thr 
          305                 310                 315                 320 
          Met Gly Arg Asn Trp Leu Pro Gly Pro Lys Phe Leu Asp Gln Arg Val 
                          325                 330                 335     
          Arg Ala Tyr Thr Gly Gly Thr Asp Asn Tyr Ala Asn Trp Asn Ile Trp 
                      340                 345                 350         
          Ser Asn Gly Asn Lys Val Asn Leu Lys Asp Arg Gln Tyr Leu Leu Gln 
                  355                 360                 365             
          Pro Gly Pro Val Ser Ala Thr Tyr Thr Glu Gly Glu Ala Ser Ser Leu 
              370                 375                 380                 
          Pro Ala Gln Asn Ile Leu Gly Ile Ala Lys Asp Pro Tyr Arg Ser Gly 
          385                 390                 395                 400 
          Ser Thr Thr Ala Gly Ile Ser Asp Ile Met Val Thr Glu Glu Gln Glu 
                          405                 410                 415     
          Val Ala Pro Thr Asn Gly Val Gly Trp Lys Pro Tyr Gly Arg Thr Val 
                      420                 425                 430         
          Thr Asn Glu Gln Asn Thr Thr Thr Ala Pro Thr Ser Ser Asp Leu Asp 
                  435                 440                 445             
          Val Leu Gly Ala Leu Pro Gly Met Val Trp Gln Asn Arg Asp Ile Tyr 
              450                 455                 460                 
          Leu Gln Gly Pro Ile Gly Ala Lys Ile Pro Lys Thr Asp Gly Lys Phe 
          465                 470                 475                 480 
          His Pro Ser Pro Asn Leu Gly Gly Phe Gly Leu His Asn Pro Pro Pro 
                          485                 490                 495     
          Gln Val Phe Ile Lys Asn Thr Pro Val Pro Ala Asp Pro Pro Val Glu 
                      500                 505                 510         
          Tyr Val His Gln Lys Trp Asn Ser Tyr Ile Thr Gln Tyr Ser Thr Gly 
                  515                 520                 525             
          Gln Cys Thr Val Glu Met Val Trp Glu Leu Arg Lys Glu Asn Ser Lys 
              530                 535                 540                 
          Arg Trp Asn Pro Glu Ile Gln Phe Thr Ser Asn Phe Ser Asn Arg Thr 
          545                 550                 555                 560 
          Ser Ile Met Phe Ala Pro Asn Glu Thr Gly Gly Tyr Val Glu Asp Arg 
                          565                 570                 575     
          Leu Ile Gly Thr Arg Tyr Leu Thr Gln Asn Leu 
                      580                 585         
          <![CDATA[<210>  4]]>
          <![CDATA[<211>  1764]]>
          <![CDATA[<212>  DNA]]>
          <![CDATA[<213>  鵝源水禽小病毒]]>
          <![CDATA[<400>  4]]>
          acggcacctg caaaaaaaaa tacagggaag cttactgacc attacccggt agttaagaag       60
          cctaaactta ccgaggaagt cagtgcggga ggtggtagca gtgccgtaca agacggagga      120
          gccaccgcgg agggcaccga acctgtggca gcatctgaaa tggcagaggg aggaggcgga      180
          gctatgggcg actcttcagg gggtgccgat ggagtgggta atgcctcggg aaattggcat      240
          tgcgattccc aatggatggg aaacacagtc atcacaaaga ccaccagaac ctgggtcctg      300
          ccaagctaca acaaccacat ctacaaagca attaccagcg gaacctctca agatgcaaat      360
          gtccagtatg caggatacag taccccctgg gggtactttg atttcaaccg cttccactgc      420
          cacttctccc ctagagactg gcagagactt atcaacaacc attggggaat cagacccaag      480
          tctcttaaat tcaagatctt caatgtccaa gtcaaagaag tcacaacgca ggatcagaca      540
          aagaccattg caaacaatct cacctcaaca attcaagtct ttacggatga tgagcatcaa      600
          ctcccgtatg tcctgggctc ggctacggaa ggcaccatgc cgccgttccc gtcggatgtc      660
          tatgccctgc cgcagtacgg gtactgcaca atgcacacca accagaatgg agcacggttc      720
          aatgaccgta gtgcattcta ctgcttagag tacttcccta gtcagatgct aagaacaggc      780
          aacaactttg agttcacatt tgactttgaa gaagttcctt tccatagcat gttcgctcat      840
          tcacaggact tagacaggct gatgaacccc ctagtggatc aatacctctg gaatttcaat      900
          gaggtagaca gcagcagaaa tgctcaattt aaaaaggctg tgaaaggggc ttatggcacc      960
          atgggccgca attggctgcc aggacctaaa ttcctggatc aaagagttag ggcctacaca     1020
          ggaggaacag acaactatgc aaactggaac atctggagta atgggaacaa ggtgaatttg     1080
          aaagacagac agtatctcct acaacccgga cctgtgtcag ctacttacac agaaggggag     1140
          gcttccagcc ttccagctca aaatatttta gggatagcta aagatccata cagatcaggc     1200
          agcactacag caggaataag tgacattatg gtcacggaag aacaagaagt agcacctaca     1260
          aatggagtag ggtggaaacc atatggtagg actgtaacga atgaacaaaa cactactaca     1320
          gctcctacaa gttcagatct ggatgttctt ggagctttac caggaatggt ttggcagaac     1380
          agggatatat atctgcaggg acctattggg gcaaaaatac cgaagactga tggtaaattc     1440
          catccttctc cgaatctcgg aggatttggc ctgcacaatc caccaccgca ggtgttcatc     1500
          aagaatacac cagtgcctgc agaccctcca gtagaatacg tgcaccagaa gtggaattcc     1560
          tacataaccc agtactctac gggccagtgt acagtagaga tggtgtggga gctgagaaaa     1620
          gagaattcaa agagatggaa cccagaaatc cagttcacca gtaatttcag taacagaaca     1680
          agcataatgt ttgcacctaa tgaaactggt ggatatgtag aagatagatt gattggaacc     1740
          agatatctaa ctcaaaatct gtaa                                            1764
          Sequence Listing <![CDATA[<110> National Pingtung University of Science and Technology]]> <![CDATA[<120> Preparation method, application and immune composition of peptide nanotube]]> <![CDATA[ <130> P20-0222]]> <![CDATA[<160> 4 ]]> <![CDATA[<170> PatentIn version 3.5]]> <![CDATA[<210> 1]]> <![ CDATA[<211> 763]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Porcine Diarrhea Virus]]> <![CDATA[<400> 1]]> Pro Thr Leu Ser Leu Pro Gln Asp Val Thr Arg Cys Gln Ser Thr Thr 1 5 10 15 Asn Phe Arg Arg Phe Phe Ser Lys Phe Asn Val Gln Ala Pro Ala Val 20 25 30 Val Val Leu Gly Gly Tyr Leu Pro Ser Met Asn Ser Ser Ser Trp Tyr 35 40 45 Cys Gly Thr Gly Ile Glu Thr Ala Ser Gly Val His Gly Ile Phe Leu 50 55 60 Ser Tyr Ile Asp Ser Gly Gln Gly Phe Glu Ile Gly Ile Ser Gln Glu 65 70 75 80 Pro Phe Asp Pro Ser Gly Tyr Gln Leu Tyr Leu His Lys Ala Thr Asn 85 90 95 Gly Asn Thr Asn Ala Ile Ala Arg Leu Arg Ile Cys Gln Phe Pro Asp 100 105 110 Asn Lys Thr Leu Gly Pro Thr Val Asn Asp Val Thr Thr Gly Arg Asn 115 120 125 Cys Leu Phe Asn Lys Ala Ile Pro Ala Tyr Met Arg Asp Gly Lys Asp 130 135 140 Ile Val Val Gly Ile Thr Trp Asp Asn Asp Arg Val Thr Val Phe Ala 145 150 155 160 Asp Lys Ile Tyr His Phe Tyr Leu Lys Asn Asp Trp Ser Arg Val Ala 165 170 175 Thr Arg Cys Tyr Asn Arg Arg Ser Cys Ala Met Gln Tyr Val Tyr Thr 180 185 190 Pro Thr Tyr Tyr Met Leu Asn Val Thr Ser Ala Gly Glu Asp Gly Ile 195 200 205 Tyr Tyr Glu Pro Cys Thr Ala Asn Cys Thr Gly Tyr Ala Ala Asn Val 210 215 220 Phe Ala Thr Asp Ser Asn Gly His Ile Pro Glu Gly Phe Ser Phe Asn 225 230 235 240 Asn Trp Phe Leu Leu Ser Asn Asp Ser Thr Leu Leu His Gly Lys Val 245 250 255 Val Ser Asn Gln Pro Leu Leu Val Asn Cys Leu Leu Ala Ile Pro Lys 260 265 270 Ile Tyr Gly Leu Gly Gln Phe Phe Ser Phe Asn His Thr Met Asp Gly 275 280 285 Val Cys Asn Gly Ala Ala Val Asp Arg Ala Pro Glu Ala Leu Arg Phe 290 295 300 Asn Ile Asn Asp Thr Ser Val Ile Leu Ala Glu Gly Ser Ile Val Leu 305 310 315 320 His Thr Ala Leu Gly Thr Asn Leu Ser Phe Val Cys Ser Asn Ser Ser Ser 325 330 335 Asp Pro His Leu Ala Ile Phe Ala Ile Pro Leu Gly Ala Thr Glu Val 340 345 350 Pro Tyr Tyr Cys Phe Leu Lys Val Asp Thr Tyr Asn Ser Thr Val Tyr 355 360 365 Lys Phe Leu Ala Val Leu Pro Pro Thr Val Arg Glu Ile Val Ile Thr 370 375 380 Lys Tyr Gly Asp Val Tyr Val Asn Gly Phe Gly Tyr Leu His Leu Gly 385 390 395 400 Leu Leu Asp Ala Val Thr Ile Asn Phe Thr Gly His Gly Thr Asp Asp 405 410 415 Asp Val Ser Gly Phe Trp Thr Ile Ala Ser Thr Asn Phe Val Asp Ala 420 425 430 Leu Ile Glu Val Gln Gly Thr Ser Ile Gln Arg Ile Leu Tyr Cys Asp 435 440 445 Asp Pro Val Ser Gln Leu Lys Cys Ser Gln Val Ala Phe Asp Leu Asp 450 455 460 Asp Gly Phe Tyr Pro Ile Ser Ser Arg Asn Leu Leu Ser His Glu Gln 465 470 475 480 Pro Ile Ser Phe Val Thr Leu Pro Ser Phe Asn Asp His Ser Phe Val 485 490 495 Asn Ile Thr Val Ser Ala Ala Phe Gly Gly Leu Ser Ser Ala Asn Leu 500 505 510 Val Ala Ser Asp Thr Thr Ile Asn Gly Phe Ser Ser Phe Cys Val Asp 515 520 525 Thr Arg Gln Phe Thr Ile Thr Leu Phe Tyr Asn Val Thr Asn Ser Tyr 530 535 540 Gly Tyr Val Ser Lys Ser Gln Asp Ser Asn Cys Pro Phe Thr Leu Gln 545 550 555 560 Ser Val Asn Asp Tyr Leu Ser Phe Ser Lys Phe Cys Val Ser Thr Ser 565 570 575 Leu Leu Ala Gly Ala Cys Thr Ile Asp Leu Phe Gly Tyr Pro Ala Phe 580 585 590 Gly Ser Gly Val Lys Leu Thr Ser Leu Tyr Phe Gln Phe Thr Lys Gly 595 600 605 Glu Leu Ile Thr Gly Thr Pro Lys Pro Leu Glu Gly Ile Thr Asp Val 610 615 620 Ser Phe Met Thr Leu Asp Val Cys Thr Lys Tyr Thr Ile Tyr Gly Phe 625 630 635 640 Lys Gly Glu Gly Ile Ile Thr Leu Thr Asn Ser Ser Ile Leu Ala Gly 645 650 655 Val Tyr Tyr Thr Ser Asp Ser Gly Gln Leu Leu Ala Phe Lys Asn Val 660 665 670 Thr Ser Gly Ala Val Tyr Ser Val Thr Pro Cys Ser Phe Ser Glu Gln 675 680 685 Ala Ala Tyr Val Asn Asp Asp Ile Val Gly Val Ile Ser Ser Leu Ser 690 695 700 Asn Ser Thr Phe Asn Asn Thr Arg Glu Leu Pro Gly Phe Phe Tyr His 705 710 715 720 Ser Asn Asp Gly Ser Asn Cys Thr Glu Pro Val Leu Val Tyr Ser Asn 725 730 735 Ile Gly Val Cys Lys Ser Gly Ser Ile Gly Tyr Val Pro Ser Gln Tyr 740 745 750 Gly Gln Val Lys Ile Ala Pro Thr Val Thr Gly 755 760 <![CDATA[<210> 2]]> <![CDATA[<211> 2289]]> <![CDATA[<212> DNA]]> <![CDATA [<213> porcine epidemic diarrhea virus]]> <! [CDATA [<400> 2]]> ccaacactca gcctaccaca agatgtcact aggtgccagt ctactactaa ctttaggcgg 60 ttcttttcaa aatttaatgt tcaggcacct gccgtcgtcg ttttgggtgg ttacctacct 120 agtatgaact cttctagctg gtactgtggc acaggcattg aaactgctag tggcgttcat 180 ggtatttttc tcagctacat cgattctggt cagggctttg agattggcat ttcgcaagag 240 ccgtttgatc ctagtggtta ccagctttat ttacataagg ccactaatgg taacactaat 300 gctattgcac gactgcgcat ttgccagttt cccgataata aaacattggg ccctactgtt 360 aatgatgtta caacaggtcg taactgccta ttcaacaaag ccattccagc ttatataccatagt a tgggataatg atcgtgtcac tgtttttgct 480 gacaagatct atcattttta tcttaaaaat gattggtccc gcgttgcgac aagatgttac 540 aatcgcagaa gttgtgctat gcaatatgtt tatacaccta cctactacat gcttaatgtt 600 actagtgcag gtgaggatgg catttattat gaaccctgta cagctaattg cactggttac 660 gctgccaatg tatttgccac tgattccaat ggccatatac cagaaggttt tagttttaat 720 aattggtttc ttttatccaa tgactccact ttgttgcatg gtaaagtggt ttccaaccaa 780 cccttgttgg tcaattgtct tttggccatt cctaagattt atggactagg ccaatttttc 840 tcattcaatc acacgatgga tggcgtttgt aatggagctg ctgtggatcg tgccccagag 900 gctctgaggt ttaatattaa tgacacctcc gtcattcttg ctgaaggctc aattgtactt 960 catactgctt taggaacaaa tctttctttt gtttgcagta attcctcaga tcctcattta 1020 gccatctttg ccatacctct gggtgctact gaagtaccct actattgctt tcttaaagtg 1080 gatacttaca actccactgt ttataaattc ttggctgttt tacctcctac tgtcagggaa 1140 attgtcatca ccaagtatgg tgatgtttat gtcaatgggt ttggctattt gcatctcggt 1200 ttgttggatg ctgtcacaat taatttcact ggtcatggca ctgacgatga cgtttcaggt 1260 ttctggacca tagcatcgac taattttgtt gatgcactca tc gaggttca aggaacttcc 1320 attcagcgta ttctttattg tgatgatcct gttagccaac tcaagtgttc tcaggttgct 1380 tttgaccttg acgatggttt ttaccccatc tcttctagaa accttctgag tcacgaacag 1440 ccaatttctt ttgttacttt gccatcattt aatgatcatt cttttgttaa tattactgtc 1500 tctgcggctt ttggtggtct tagtagtgcc aatctcgttg catctgacac tactatcaat 1560 gggtttagtt ctttctgtgt tgacactaga caatttacca ttacactgtt ttataatgtt 1620 acaaacagtt atggttatgt gtctaaatca caggatagta attgtccttt caccttgcaa 1680 tctgttaatg attacctgtc ttttagcaaa ttttgtgttt caaccagcct tttggctggt 1740 gcttgtacca tagatctttt tggttaccct gcgttcggta gtggtgttaa gttgacgtcc 1800 ctttattttc aattcacaaa aggtgagttg attactggca cgcctaaacc acttgaaggt 1860 atcacagacg tttcttttat gactctggat gtgtgtacca agtatactat ctatggcttt 1920 tctagtttgt ctaactccac ttttaacaat aaaggtgagg gtattattac ccttacaaat tctagcattt tggcaggtgt ttattataca 1980 tctgattctg gacagttgtt agcctttaag aatgtcacta gtggtgctgt ttattctgtc 2040 acgccatgtt ctttttcaga gcaggctgca tatgttaatg atgatatagt gggtgttatt 2100 actagggagt tgcctggt tt cttctaccat 2160 tctaatgacg gctccaattg tacagagcct gtgttggtgt atagtaacat aggtgtttgt 2220 aaatctggca gtattggcta tgttccatct cagtatggcc aagtcaagat tgcacccacg 2280 gttactggg 2289 <! [CDATA [<210> 3]]> <! [CDATA [<211> 587]]> <! [CDATA [<212 > PRT]]> <![CDATA[<213> Goose Origin Waterfowl Parvovirus]]> <![CDATA[<400> 3]]> Thr Ala Pro Ala Lys Lys Asn Thr Gly Lys Leu Thr Asp His Tyr Pro 1 5 10 15 Val Val Lys Lys Pro Lys Leu Thr Glu Glu Val Ser Ala Gly Gly Gly 20 25 30 Ser Ser Ala Val Gln Asp Gly Gly Ala Thr Ala Glu Gly Thr Glu Pro 35 40 45 Val Ala Ala Ser Glu Met Ala Glu Gly Gly Gly Gly Ala Met Gly Asp 50 55 60 Ser Ser Gly Gly Ala Asp Gly Val Gly Asn Ala Ser Gly Asn Trp His 65 70 75 80 Cys Asp Ser Gln Trp Met Gly Asn Thr Val Ile Thr Lys Thr Thr Arg 85 90 95 Thr Trp Val Leu Pro Ser Tyr Asn Asn His Ile Tyr Lys Ala Ile Thr 100 105 110 Ser Gly Thr Ser Gln Asp Ala Asn Val Gln Tyr Ala Gly Tyr Ser Thr 115 120 125 Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His Cys His Phe Ser Pro 130 135 140 Arg Asp Trp Gln Arg Leu Ile Asn Asn His Trp Gly Ile Arg Pro Lys 145 150 155 160 Ser Leu Lys Phe Lys Ile Phe Asn Val Gln Val Lys Glu Val Thr Thr 165 170 175 Gln Asp Gln Thr Lys Thr Ile Ala Asn Asn Leu Thr Ser Thr Ile Gln 180 185 190 Val Phe Thr Asp Asp Glu His Gln Leu Pro Tyr Val Leu Gly Ser Ala 195 200 205 Thr Glu Gly Thr Met Pro Pro Phe Pro Ser Asp Val Tyr Ala Leu Pro 210 215 220 Gln Tyr Gly Tyr Cys Thr Met His Thr Asn Gln Asn Gly Ala Arg Phe 225 230 235 240 Asn Asp Arg Ser Ala Phe Tyr Cys Leu Glu Tyr Phe Pro Ser Gln Met 245 250 255 Leu Arg Thr Gly Asn Asn Phe Glu Phe Thr Phe Asp Phe Glu Glu Val 260 265 270 Pro Phe His Ser Met Phe Ala His Ser Gln Asp Leu Asp Arg Leu Met 275 280 285 Asn Pro Leu Val Asp Gln Tyr Leu Trp Asn Phe Asn Glu Val Asp Ser 290 295 300 Ser Arg Asn Ala Gln Phe Lys Lys Ala Val Lys Gly Ala Tyr Gly Thr 305 310 315 320 Met Gly Arg Asn Trp Leu Pro Gly Pro Lys Phe Leu Asp Gln Arg Val 325 330 335 Arg Ala Tyr Thr Gly Gly Thr Asp Asn Tyr Ala Asn Trp Asn Ile Trp 340 345 350 Ser Asn Gly Asn Lys Val Asn Leu Lys Asp Arg Gln Tyr Leu Leu Gln 355 360 365 Pro Gly Pro Val Ser Ala Thr Tyr Thr Glu Gly Glu Ala Ser Ser Leu 370 375 380 Pro Ala Gln Asn Ile Leu Gly Ile Ala Lys Asp Pro Tyr Arg Ser Gly 385 390 395 400 Ser Thr Thr Ala Gly Ile Ser Asp Ile Met Val Thr Glu Glu Gln Glu 405 410 415 Val Ala Pro Thr Asn Gly Val Gly Trp Lys Pro Tyr Gly Arg Thr Val 420 425 430 Thr Asn Glu Gln Asn Thr Thr Thr Ala Pro Thr Ser Ser Asp Leu Asp 435 440 445 Val Leu Gly Ala Leu Pro Gly Met Val Trp Gln Asn Arg Asp Ile Tyr 450 455 460 Leu Gln Gly Pro Ile Gly Ala Lys Ile Pro Lys Thr Asp Gly Lys Phe 465 470 475 480 His Pro Ser Pro Asn Leu Gly Gly Phe Gly Leu His Asn Pro Pro Pro 485 490 495 Gln Val Phe Ile Lys Asn Thr Pro Val Pro Ala Asp Pro Pro Val Glu 500 505 510 Tyr Val His Gln Lys Trp Asn Ser Tyr Ile Thr Gln Tyr Ser Thr Gly 515 520 525 Gln Cys Thr Val Glu Met Val Trp Glu Leu Arg Lys Glu Asn Ser Lys 530 535 540 Arg Trp Asn Pro Glu Ile Gln Phe Thr Ser Asn Phe Ser Asn Arg Thr 545 550 555 560 Ser Ile Met Phe Ala Pro Asn Glu Thr Gly Gly Tyr Val Glu Asp Arg 565 570 575 Leu Ile Gly Thr Arg Tyr Leu Thr Gln Asn Leu 580 585 <![CDATA[<210> 4]]> <![CDATA[<211> 1764]] > <![CDATA[<212> DNA]]> <![CDATA[<213> Goose Origin Waterfowl Parvovirus]]> <![CDATA[<400> 4]]> acggcacctg caaaaaaaaa tacagggaag cttactgacc attacccggt agttaagaag 60 cctaaactta ccgaggaagt cagtgcggga ggtggtagca gtgccgtaca agacggagga 120 gccaccgcgg agggcaccga acctgtggca gcatctgaaa tggcagaggg aggaggcgga 180 gctatgggcg actcttcagg gggtgccgat ggagtgggta atgcctcggg aaattggcat 240 tgcgattccc aatggatggg aaacacagtc atcacaaaga ccaccagaac ctgggtcctg 300 ccaagctaca acaaccacat ctacaaagca attaccagcg gaacctctca agatgcaaat 360 gtccagtatg caggatacag taccccctgg gggtactttg atttcaaccg cttccactgc 420 cacttctccc ctagagactg gcagagactt atcaacaacc attggggaat cagacccaag 480 tctcttaaat tcaagatctt caatgtccaa gtcaaagaag tcacaacgca ggatcagaca 540 aagaccattg caaacaatct cacctcaaca attcaagtct ttacggatga tgagcatcaa 600 ctcccgtatg tcctgggctc ggctacggaa g gcaccatgc cgccgttccc gtcggatgtc 660 tatgccctgc cgcagtacgg gtactgcaca atgcacacca accagaatgg agcacggttc 720 aatgaccgta gtgcattcta ctgcttagag tacttcccta gtcagatgct aagaacaggc 780 aacaactttg agttcacatt tgactttgaa gaagttcctt tccatagcat gttcgctcat 840 tcacaggact tagacaggct gatgaacccc ctagtggatc aatacctctg gaatttcaat 900 gaggtagaca gcagcagaaa tgctcaattt aaaaaggctg tgaaaggggc ttatggcacc 960 atgggccgca attggctgcc aggacctaaa ttcctggatc aaagagttag ggcctacaca 1020 ggaggaacag acaactatgc aaactggaac atctggagta atgggaacaa ggtgaatttg 1080 aaagacagac agtatctcct acaacccgga cctgtgtcag ctacttacac agaaggggag 1140 gcttccagcc ttccagctca aaatatttta gggatagcta aagatccata cagatcaggc 1200 agcactacag caggaataag tgacattatg gtcacggaag aacaagaagt agcacctaca 1260 aatggagtag ggtggaaacc atatggtagg actgtaacga atgaacaaaa cactactaca 1320 gctcctacaa gttcagatct ggatgttctt ggagctttac caggaatggt ttggcagaac 1380 agggatatat atctgcaggg acctattggg gcaaaaatac cgaagactga tggtaaattc 1440 catccttctc cgaatctcgg aggatttggc ctgcacaatc ca ccaccgca ggtgttcatc 1500 aagaatacac cagtgcctgc agaccctcca gtagaatacg tgcaccagaa gtggaattcc 1560 tacataaccc agtactctac gggccagtgt acagtagaga tggtgtggga gctgagaaaa 1620 gagaattcaa agagatggaa cccagaaatc cagttcacca gtaatttcag taacagaaca 1680 agcataatgt ttgcacctaa tgaaactggt ggatatgtag aagatagatt gattggaacc 1740 agatatctaa ctcaaaatct gtaa 1764

Claims (10)

一種胜肽奈米管做為增加黏膜免疫反應之DNA疫苗佐劑之用途，該胜肽奈米管之製備方法包含：混合一環-(D-色胺酸-酪胺酸)粉末與一水形成一溶液；震盪該溶液至混合均勻；以及移除該溶液中之上清液以形成該胜肽奈米管。 A use of a peptide nanotube as a DNA vaccine adjuvant for increasing mucosal immune response, the preparation method of the peptide nanotube comprises: mixing a cyclo-(D-tryptophan-tyrosine) powder with a water to form a a solution; shake the solution to mix uniformly; and remove the supernatant in the solution to form the peptide nanotube. 如請求項1之用途，其進一步搭配另一胜肽奈米管做為增加黏膜免疫反應之DNA疫苗佐劑，該另一胜肽奈米管之製備方法包含：混合該環-(D-色胺酸-酪胺酸)粉末與一酒精形成另一溶液；震盪該另一溶液至混合均勻；以及揮發該另一溶液中之酒精以形成該另一胜肽奈米管，其中該另一胜肽奈米管與該胜肽奈米管之重量比之範圍係從5：95至95：5。 According to the use of claim 1, it is further matched with another peptide nanotube as a DNA vaccine adjuvant for increasing mucosal immune response, and the preparation method of the other peptide nanotube comprises: mixing the ring-(D-color) amino acid-tyrosine) powder and an alcohol to form another solution; shake the other solution to mix evenly; and volatilize the alcohol in the other solution to form the other peptide nanotube, wherein the other The weight ratio of peptide nanotubes to the peptide nanotubes ranged from 5:95 to 95:5. 如請求項1之用途，其中該環-(D-色胺酸-酪胺酸)粉末與該水之重量比例為3：2000。 The use according to claim 1, wherein the weight ratio of the cyclo-(D-tryptophan-tyrosine) powder to the water is 3:2000. 如請求項1之用途，其係用於動物口服免疫。 The use according to claim 1 is for oral immunization of animals. 一種免疫組成物，包含：一質體，以及一胜肽奈米管，其中該質體上含有一編碼一抗原蛋白質片段的DNA序列，該胜肽奈米管之製備方法包含：混合一環-(D-色胺酸-酪胺酸)粉末與一水形成一溶液；震盪該溶液至混合均勻；以及移除該溶液中之上清液以形成該胜肽奈米管。 An immune composition, comprising: a plastid, and a peptide nanotube, wherein the plastid contains a DNA sequence encoding an antigenic protein fragment, and the preparation method of the peptide nanotube comprises: mixing a ring-( D-tryptophan-tyrosine) powder and water form a solution; shake the solution to mix well; and remove the supernatant from the solution to form the peptide nanotube. 如請求項5之免疫組成物，其進一步包含另一胜肽奈米管，該另一胜肽奈米管之製備方法包含：混合該環-(D-色胺酸-酪胺酸)粉末與一酒精形成另一溶液；震盪該另一溶液至混合均勻；以及揮發該另一溶液中之酒精以形成該另一胜肽奈米管，其中該另一胜肽奈米管與該胜肽奈米管之重量比之範圍係從5：95至95：5。 The immune composition of claim 5, further comprising another peptide nanotube, and the preparation method of the another peptide nanotube comprises: mixing the cyclo-(D-tryptophan-tyrosine) powder with An alcohol forms another solution; shakes the other solution to mix uniformly; and volatilizes the alcohol in the other solution to form the other peptide nanotube, wherein the other peptide nanotube and the peptide nanotube The weight ratio of the meter tube ranges from 5:95 to 95:5. 如請求項5之免疫組成物，其進一步包含一黏性劑，其佔該免疫組成物75至95體積百分比，該黏劑性包含一生物可代謝油。 The immune composition of claim 5, further comprising a viscous agent, which accounts for 75 to 95 volume percent of the immune composition, and the viscous agent comprises a biometabolizable oil. 如請求項5之免疫組成物，其中該環-(D-色胺酸-酪胺酸)粉末與該水之重量比例為3：2000。 The immune composition of claim 5, wherein the weight ratio of the cyclo-(D-tryptophan-tyrosine) powder to the water is 3:2000. 一種胜肽奈米管之製備方法，包含：混合一環-(D-色胺酸-酪胺酸)粉末與一水形成一溶液；震盪該溶液至混合均勻；以及移除該溶液中之上清液以形成該胜肽奈米管。 A method for preparing a peptide nanotube, comprising: mixing a cyclo-(D-tryptophan-tyrosine) powder and water to form a solution; shaking the solution until the mixture is uniform; and removing the supernatant in the solution solution to form the peptide nanotubes. 如請求項9之製備方法，其中該環-(D-色胺酸-酪胺酸)粉末與該水之重量比例為3：2000。 The preparation method of claim 9, wherein the weight ratio of the cyclo-(D-tryptophan-tyrosine) powder to the water is 3:2000.
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