TW201829470A - 用於免疫增強的融合胜肽及使用其增強誘發免疫反應的方法 - Google Patents

用於免疫增強的融合胜肽及使用其增強誘發免疫反應的方法 Download PDF

Info

Publication number
TW201829470A
TW201829470A TW106136884A TW106136884A TW201829470A TW 201829470 A TW201829470 A TW 201829470A TW 106136884 A TW106136884 A TW 106136884A TW 106136884 A TW106136884 A TW 106136884A TW 201829470 A TW201829470 A TW 201829470A
Authority
TW
Taiwan
Prior art keywords
leu
ala
glu
gly
ser
Prior art date
Application number
TW106136884A
Other languages
English (en)
Inventor
廖朝暐
廖亭凱
Original Assignee
廖朝暐
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 廖朝暐 filed Critical 廖朝暐
Publication of TW201829470A publication Critical patent/TW201829470A/zh

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/00113—Growth factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/21—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pseudomonadaceae (F)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1077—Pentosyltransferases (2.4.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/02—Pentosyltransferases (2.4.2)
    • C12Y204/02036—NAD(+)--diphthamide ADP-ribosyltransferase (2.4.2.36)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/542—Mucosal route oral/gastrointestinal
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55544—Bacterial toxins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/70—Multivalent vaccine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20071—Demonstrated in vivo effect
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/10011—Arteriviridae
    • C12N2770/10034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/10011—Arteriviridae
    • C12N2770/10071—Demonstrated in vivo effect

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Zoology (AREA)
  • Virology (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Engineering & Computer Science (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Immunology (AREA)
  • General Engineering & Computer Science (AREA)
  • Oncology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Toxicology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Communicable Diseases (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

本揭露揭示一種融合胜肽，包含：(a)一黏膜標靶多肽；(b)一第一轉位肽，其係用於轉位；以及(c)一表位。此外，本揭露亦揭示一種利用前述融合胜肽以增強誘發免疫反應之方法。

Description

用於免疫增強的融合胜肽及使用其增強誘發免疫反應的方法
本申請案主張在2016年10月26日根據35 USC § 119(e)(1)申請之美國臨時申請案第62/412,896號之優先權。
本揭露係關於一種融合胜肽、一種包含該融合胜肽之組成物、及一種使用該融合胜肽以增強誘發免疫反應的方法。
三十五年來，抗原肽是動員免疫系統對抗外來入侵者和癌症的關鍵因素。而大部分非常成功的治療方法，包括疫苗，都是憑經驗製成的，幾乎沒有經由洞悉免疫學方面知識而製成。直到1986年為止，從來沒有人認為胜肽可能在控制免疫反應的特異性方面起重要作用，所述之免疫反應與MHC I類和II類分子有關，而該類分子因參與移植排斥反應而最為人所知。然而，關於抗原肽的化學和藥理學性質以及抗原加工、呈現和免疫細胞識別的分子生物學方面的新知識，使得現今疫苗和免疫增強劑的設計更加合理。若更理解抗原肽在免疫中的起源和作用，將有助於利用免疫系統治療疾病或癌症治療。
抗原肽在先天性和適應性免疫系統中的作用，是這些關鍵參與者可用於治療傳染性疾病、癌症和自身免疫疾病。先天性免疫為防禦感染的第一道防線，並且以抗微生物肽(antimicrobial peptide)為例，其也被稱為宿主防禦肽(host defense peptide，HDP)。適應性免疫靠著免疫細胞的能力，區分自身抗原和不想要的侵入者和腫瘤細胞。屬於適應性免疫類型的抗原肽，存在一分子途徑，該途徑係從最初通過蛋白水解加工生成至藉由免疫系統中的主要組織相容性複合體(major histocompatibilty complex，MHC)分子呈現給免疫細胞。因此，與抗原肽形成非共價複合物，並且在抗原呈現細胞呈現給免疫系統的T細胞的情況下呈現這些胜肽的MHC-I和MHC-II為關鍵參與者。
雖然已經知道抗原肽是誘發免疫反應的有效方式，但許多研究和研究仍集中在尋找具有改善效果的新型抗原肽。因此，本揭露的目的在於尋找一種佐劑，能在適應性免疫途徑中有效傳遞至免疫標靶器官，達到增強誘發免疫反應的目的。
本揭露提供一種融合胜肽，其可作為誘發免疫反應之一增強劑。
本揭露亦提供一種用於增強誘發免疫反應之組成物，包括前述之融合胜肽。此外，本揭露更提供一種前述融合胜肽之方法。
本揭露之融合胜肽包含：(a)一黏膜標靶多肽；(b)一第一轉位肽，其係用於轉位；以及(c)一第一表位。
該黏膜標靶多肽係促進融合胜肽在有需要的主體中與受體結合的一結合表位。用於轉位的該第一轉位肽被用以作為能夠促進細胞溶質定位和抗原呈現的載體。該第一表位可以增強免疫調節活性。當本揭露的融合胜肽包含上述三種成分時，該融合胜肽可有效地送至有需要的主體中，並增強對內源性處理標靶抗原的免疫反應。
此外，本揭露之另一融合胜肽，包含：(b)一第一轉位肽，其係用於轉位；以及(c)一第一表位，其係選自SEQ ID NO: 5和6。(E622、E713)。
用於轉位的該第一轉位肽被用作為能夠促進細胞溶質定位和抗原呈現的載體。該第一表位係經修飾的表位，其能增強免疫調節活性。特別地，SEQ ID NO: 5和6所示的序列(即E622和E713)係經修飾的，不同於已知的E6和E7的病毒蛋白質的多肽，其能更進一步增強免疫調節活性。當本揭露的融合胜肽包含上述兩種成分時，可以將融合胜肽有效地送至有需要的主體中，並增強對內源性處理標靶抗原的免疫反應。
此外，本揭露還提供了用於增強誘發免疫反應的一組成物，包含：一疫苗和上述任一融合胜肽。本文中，該疫苗可包含：用於轉位的一第二轉位肽和一第二表位。該第二轉位肽與第一轉位肽相似，以及該第二表位與第一表位相似。因此，對於第二轉位肽和第二表位的描述不再重複。此外，在本揭露中，第一表位和第二表位必須彼此相容。舉例而言，第一表位和第二表位皆為HPV表位；但是第一表位和第二表位不一定相同。或者，第一表位和第二表位皆為肌肉生長抑制素表位；但是第一表位和第二表位不一定相同。或者，第一表位和第二表位皆為PRRSV表位；但是第一表位和第二表位不一定相同。
另外，本揭露還提供了增強誘發免疫反應的方法，包括：將上述組成物投予有需要的主體。在本文中，該主體可為哺乳動物，例如人、豬等。
在本揭露的一個實施例中，表位可為HPV表位、肌肉生長抑制素表位或PRRSV表位。在本揭露的另一個實施例中，將組合物口服投予需要的主體。
藉由以下詳細描述可使本揭露之其它目的、優點、及新穎特徵更加清楚明白。
以下揭示本揭露實施例的示範性儀器、裝置、方法及其相關結果，其非用以限制本揭露。其中，實施例中的標題或次標題，僅為了方便閱讀者，而並非用於限制本揭露的範圍。此外，本揭露更揭露特定理論；然而，不論 此些理論對或錯，並非用以限制本揭露，本揭露並非根據特定理論來實施。
本揭露關於一種於黏膜系統中產生特定免疫-調節嵌合多肽，包含第一及二型的黏膜免疫系統，其包括可為M細胞或上皮細胞標靶功能區的黏膜標靶多肽、可來自綠膿桿菌外毒素的轉位用轉位肽、及可為Th1表位的表位。
我們研究嵌合多肽的表位的免疫反應。特別是，當本揭露的融合胜肽透過口服方式投予時，可誘發免疫反應。本揭露的融合胜肽與腸道的黏膜免疫系統有關。接下來，本揭露使用三種類的PE類融合抗原，以顯示其可增強特異性免疫反應。
1. 黏膜標靶多肽的建造
在鑑定M細胞特異性表面標記方面已經取得了重大進展 (Kim SH et al.,J Immunol . 2010 Nov 15;185(10):5787-95)。在口服途徑中，應包含為黏膜標靶的M細胞或上皮細胞配體 (Azizi等人,PLoS Pathog. 2010 Nov 11;6(11)；K J Syrjänen,J Clin Pathol. 2002 Oct；55(10): 721–728；Roy C. 等人, GI Motility online (2006) doi:10.1038/gimo15)。我們通過利用天然性胜肽作為融合胜肽的一分子，探查了幾種生物多肽的有效重組生產。
以下實施例利用三種結合表位的胜肽作為在第一型黏膜免疫系統中的M細胞標靶肽的示範。此外，一種結合表位的胜肽係作為第二型黏膜免疫系統中上皮細胞標靶肽的實例(Azizi等人,PLoS Pathog. 2010 Nov 11;6(11); K J Syrjänen,J Clin Pathol. 2002 Oct; 55(10): 721–728; Roy C. 等人,GI Motility online (2006) doi:10.1038/gimo15; Mulder DJ等人,Dig Dis Sci. 2012 Mar;57(3):630-42)。在以下實施例中所用之M細胞標靶肽列於表1中。上皮細胞標靶肽是HPV L2-200(如表2所示)，用於上皮標靶配體 (Mulder DJ等人,Dig Dis Sci. 2012 Mar;57(3):630-42).
表1：在第一型黏膜免疫系統中的M細胞標靶多肽
表2：腸道上皮細胞標靶肽 *：以斜體標示的VD、EF和LE是為了次轉殖而添加的EcoR1和Xho1限制酶切位點。
1-1. M細胞標靶多肽的建造
根據Kim SH等人,J Immunol . 2010 Nov 15;185(10):5787-95所述，選擇CO-1肽核心序列SFHQLPAR SPAPL Q (SEQ ID NO. 01)。藉由生物資訊學(BLAST)搜尋DQ-2和RV-3胺基酸密碼序列以找到模擬CO-1序列的區域。該程序將蛋白質序列與序列數據庫比較並且計算統計學顯著性。從胺基酸密碼子序列，我們可以發現兩個類似CO-1的胜肽，分別為在櫟迷孔菌(Daedalea quercina Fr.) (類多孔菌)中的DQ-2 (SEQ ID NO. 02)：SFHLFHHLPARAPLAPL，以及在九帶犰狳(Dasypus novemcinctus )中的RV-3 (SEQ ID NO. 03)：STPFHPLPARKPLP。
為了在沒有DNA模板的情形下設計和製備DNA密碼子序列，在不改變所選胜肽片段的原始胺基酸序列的情況下進行密碼子替換，以避免假性限制位(spurious restriction site)，並在大腸桿菌中理想地表現。在編碼胜肽片段的DNA序列末端，在5’末端和3’末端的EcoR1和Xho1切位點，添加限制位連接序列，以插入或次選殖(sub-cloning)至質體載體中。藉由聚合酶連鎖反應(PCR)和多重引子延伸產生DNA片段。如表3所示，該些經由PCR產生的DNAs (PCR DNAs)含有上述胺基酸序列，並藉由重組DNA技術將該些PCR DNAs分別次轉殖至大腸桿菌質體載體 。
通過限制酶切割和電泳分析所有合成及/或次轉殖的核苷酸片段，以確定它們是否具有如預想一般的大小以及正確的切割位。一旦獲得樣品，DNA序列將會經由機器自動產生並且在電腦上顯示其結果。此外，進行序列分析以鑑定核酸中的核苷酸序列或多肽中的胺基酸。
表3：編碼M細胞標靶胜肽表位的DNA序列密碼子
1-2. 腸道上皮標靶多肽之建造
使用表6中的引子對進行多步驟PCR (multi-step PCR)，以合成編碼HPV16 L12蛋白N端功能區(胺基酸1至200；L2-200)序列的L2-200 DNA片段。引子對1-10產生的DNA大小分別為82 bp、147 bp、219 bp、285 bp、354 bp、420 bp、492 bp、552 bp和613 bp。用限制酶Mun I和Sal I切割最終的DNA產物以分離630bp DNA大片段，然後將其次轉殖到EcoRI和Xho I切割的載體中。
表4 *Fw：順向。 **Rv：反向。
表5：編碼L2-200標靶多肽的DNA序列
如表1和2所示，用於本揭露融合胜肽的黏膜標靶多肽可為一M細胞標靶多肽(例如CO1、DQ2以及RV3)或一腸道上皮標靶多肽(例如L2-200)。該些多肽CO1、DQ2、RV3、及L2-200分別具有如SEQ ID NO.1至4所示之胺基酸序列。
在本揭露中，包含在本揭露中的融合胜肽的表位係Th1表位。舉例而言，Th1表位可為HPV表位、肌肉生長抑制素表位、或PRRSV表位。在下文中，詳細記載HPV表位、肌肉生長抑制素表位和PRRSV表位的合成。
2. E6和E7的病毒蛋白的HPV Th1表位及其經修飾的多肽
在本揭露中，HPV表位可為人類乳突病毒第16型的E7胜肽序列或E6胜肽序列。
Sarkar A.K.等人(2005)提出，針對E6和E7胜肽的細胞免疫反應參與了針對與HPV相關的子宮頸原位癌(CIN)的保護性免疫反應 (Sarkar AK 等人,Gynecol Oncol . 2005 Dec;99 (3 Suppl 1):S251-61)。由於HPV-16 E6的胜肽Q15L (QLLRREVYDFAFRDL)以及V10C (VYDFAFRDLC)具備良好的細胞性免疫反應，引入一種融合胜肽E601，SEQ ID NO. 9：EFVDQLLRREVFCGFRDLVYDFA FSDLKLPQLCTELKLPQLCTELLE。
根據Tindle R.W等人在1995年發表的文獻(Tindle R.W et al.,Clin Exp Immunol .1995; 101:265-271)，合成一種融合胜肽E701，SEQ ID NO. 11：EFVDQAEPDQAEPDRARAHYNIRARAHYNILRAHYNIVIFRAHYNIVIF LE。他們指出，一種具有BT5胜肽的HPV16 E7 (其包含線性B表位44QAEPD48 、Th表位48DRAHYNI 54、以及重疊CTL 表位49RAHYNIVTF 57) 的免疫刺激載體(Immunostimulatory Carrier，ISCAR)具有良好的治療效果。
E622和E713係從E601和E701-多肽序列修飾而得，其係根據蛋白酶體切割位預測軟體(http://www.imtech.res.in/raghava/pcleavage/)：一種基於SVM預測Th1途徑的抗原序列中組成型蛋白酶體以及免疫蛋白酶體切割位的方法。E601、E622、E701、以及E713之多肽序列如表6所示。
表6：E6及E7的病毒蛋白的Th1表位及其經修飾的多肽 *：以斜體標示的EF、VD和LE是為了次轉殖而添加的EcoR1和Xho1限制酶切位點。
在此，設計編碼E601、E622、E701和E713的DNA。在不改變所選胜肽片段的原始胺基酸序列的情況下進行密碼子替換，以避免假性限制位(spurious restriction site)，並在大腸桿菌中理想地表現。在編碼胜肽片段的DNA序列末端，在5’末端和3’末端的EcoR1、Sal1和Xho1切位點，添加限制位連接序列，以插入與次選殖(sub-cloning)至質體載體中。藉由聚合酶連鎖反應(polymerase chain reaction，PCR)和多重引子延伸產生E601、E622、E701和E713的目標DNA序列，該DNA密碼子如表7所示。藉由多重引子進行PCR，以產生編碼目標嵌合多肽的經修飾的核酸序列的DNA片段(US 8,372,407)。進行非DNA模板PCR。第一輪PCR之後，將0.01-1 µl的DNA產物當作第二輪PCR的DNA模板，在其中加入第二引子對與dNTP、試劑、及Pfu聚合酶。其餘的引子對以這種方式在隨後的PCR循環中依序添加，直到編碼目標胜肽的DNA片段各自被合成 (US 8,372,407)。
表7：不同HPV多肽的DNA密碼子序列
3.肌肉生長抑制素表位的多肽
肌肉生長抑制素，也稱為生長分化因子8 (growth differentiation factor 8，GDF-8)，是肌動蛋白，其是由肌細胞產生和釋放的蛋白，其作用於肌細胞的自泌作用以抑制肌肉生成：肌細胞生長和分化。在人類中，其係由MSTN基因編碼。肌肉生長抑制素是分泌型生長分化因子，是TGFβ蛋白家族的成員。動物若缺乏肌肉生長抑制素或對其阻斷肌肉生長抑制素活性，則該動物明顯具有較多的肌肉量。此外，在肌肉生長抑制素基因的雙鏈都具有突變的個體明顯具有更多的肌肉量並且比正常個體更強壯。關於肌肉生長抑制素的研究顯示，肌肉生長抑制素可用於治療肌肉萎縮疾病例如肌肉營養不良症(Hamrick MK, IBMS BoneKEy. 2010 January;7(1):8-17)。
3-1. M14和M27 建造
M14胜肽表位係選自肌肉生長抑制素結合區的部分片段； DFGLDCDEHSTESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGECEFVFLQKYPHTHLVHQA NPRGSAGPCCTPTLMSPINMLYFNGKEQIIYGKIPAMVVDRCGCS，其已在US20020127234中揭示。選擇M14胜肽核心序列FLQKYPHTHLVHQA 。在不改變所選胜肽片段的原始胺基酸序列的情況下進行密碼子替換，以避免假性限制性位，並在大腸桿菌中最佳地表現。在編碼胜肽片段的DNA序列末端，在5’末端和3’末端的EcoR1-Sal1、和Xho1切位點，添加限制位連接序列，以插入和次選殖(sub-cloning)至質體載體中。編碼EFVDFLQKYPHTHLVHQALE 胺基酸序列的DNA序列是5’-GAATTCGTCGAC GTGTTTTTACAAAAATATCCTCATACGCACCTGGTCCATCAGGCG CTCGAC-3’。透過PCR和多重引子延伸產生DNA片段。此外，藉由重覆插入的重組技術產生具有四個重複核心序列的多肽，即EFVD VFLQKYPHTHLVHQA LD VFLQKYPHTHLVHQA LD VFLQKYPHTHLVHQA LD VFLQKYPHTHLVHQA LE (SEQ CO NO. 17),。
M27係為了表現Th1表位特徵而經修飾的多肽，其係根據蛋白酶體切割位預測軟體(http://www.imtech.res.in/raghava/pcleavage/)：一種基於SVM以預測抗原序列中組成型蛋白酶體和免疫蛋白酶體的切割位的方法。融合並且修飾以下兩個多肽片段： HTHLVHQA 胜肽以及E FLQKYPH 胜肽，以產生該M27嵌合多肽(no. 3)。藉由生物資訊學(BLAST)搜尋HTHLVHQA胜肽，以找尋生物序列之間的相似區域。該軟體將蛋白質序列與序列數據庫進行比較，並計算統計學顯著性。其胺基酸密碼子序列可在小球藻(Chlorella variabilis)的CHLNCDRAFT_134428蛋白中找到(序列號：ref XP_005847307)。EFLQKYPH胜肽的胺基酸密碼子序列可在鷹嘴豆(Cicer arietinum)的根源缺陷1 (ROOT PRIMORDIUM DEFECTIVE 1) 的同型異構物X4(序列ID：ref XP_012571622.1)中找到。將兩個融合肽基因分別次轉殖到大腸桿菌質體載體中，然後通過重組技術融合在一起。透過引子延伸以及PCR方法產生編碼LEP-HTHLVHQA-NVLLALQLLLEDREF的經修飾的DNA片段，其在5’端加入一Xho1限制酶切位點以及在3’端加入一具有NVLLALQLLLEDREF胜肽編碼的DNA連接序列或連接橋。藉由基於SVM的蛋白酶體切割位預測產生該具有NVLLALQLL胜肽編碼的DNA連接序列。當胜肽在ER位置附近被分泌時，LEDR信號也被添加到融合肽中，以增強Th1免疫反應。透過PCR技術產生編碼LE PHTHLVHQA NVLLALQLLLEDREF胜肽的DNA片段。將編碼 HTHLVHQA 的DNA片段插入大腸桿菌質體載體的Xho1和EcoR1位點。透過PCR技術產生具有兩個限制酶位點(EcoR1和Sal1)的編碼E FLQKYPHV D的DNA片段。LE PHTHLVHQA NVLLALQLLLEDREFV FLQKYPH VD胜肽的DNA片段係透過重組技術，融合編碼 HTHLVHQA 的DNA片段以及編碼E FLQKYPHV D的DNA片段。此外，製造了具有八次重複插入的DNA片段。
如表8所示，產生了融合的表位: EFVFLQKYPH VE PHTHLVHQAN V的核心，其胺基酸序列與US20020127234中的肌肉生長抑制素結構域的 EFVFLQKYPHTHLVHQAN 相似。最後，M14(SEQ ID NO.17)和M27(SEQ ID NO.18)的多肽序列如表8所示。編碼M14和M27的DNA如表9所示。
表8：Myo14和Myo27的經修飾多肽 *：以斜體顯示的EF 、LE 和VD 是為了次轉殖而添加的EcoR1、Sal1 和Xho1限制酶切位點。
表9：Myo14和Myo27的DNA編碼序列
4. PRRSV表位的融合胜肽
豬生殖與呼吸綜合症病毒(Porcine reproductive and respiratory syndrome virus，PRRSV)對於豬隻會造成慢性與經濟破壞性疾病。在病毒基因中的頻繁突變導致產生免疫逃脫突變型的病毒。不論是定期接種疫苗，控制PRRSV對養豬業者來說都是一個挑戰。目前，增強PRRSV感染豬隻的早期免疫機制可以增進預防性或治療性目的(O. J. Lopez 等人,Clin Vaccine Immunol . 2007 Mar; 14(3): 269–275)。最近，我們試圖開發口服免疫增強劑。選擇來自PRRSV GP3和GP4表位的三種免疫調節多肽(GP317、GP417和GP437)。
4-1. GP317、 GP417 和GP437建造
藉由使用引子對的多步驟PCR合成編碼PRRSV GP3和GP4蛋白的病毒中和區域的GP317、GP417和GP437 DNA片段，如表10所示。在不改變所選胜肽片段的原始胺基酸序列的情況下進行密碼子替換，以避免假性限制切位點，並在大腸桿菌中的最佳地表現。在編碼胜肽片段的DNA序列末端，5'末端和3'末端的EcoR1和Xho1切位點，添加限制性位連接序列，用於插入並次轉殖到質體載體中。如表11所示，用限制性內切酶EcoR1和Xho1切割最終的DNA產物以分離DNA片段，然後將其次轉殖到EcoRI和Xho I切割的載體中。一旦獲得樣品，機器將會自動生成DNA序列，並且結果顯示在電腦上。接著進行序列分析以鑑定核酸中的核苷酸序列或多肽中的胺基酸。
表10：免疫調節多肽增強劑 *：以斜體標示的VD、EF和LE是為了次轉殖而添加的EcoR1和Xho1限制酶切位點。
表11：編碼免疫調節多肽增強劑的DNA序列
如表6、8、和10所示，本揭露之融合胜肽使用的表位可為E622、E713、Myo27、Myo14、GP317、GP417、或GP437，其所含序列分別如SEQ ID NO: 10、12、17、18、21、22或23所示。
在本揭露中，本揭露的融合胜肽包含作為載體的轉位肽。轉位肽可來自綠膿桿菌外毒素。在本揭露的一個態樣中，轉位肽包含僅去除功能區III (domain III)的綠膿桿菌外毒素A片段。以下詳細描述轉位肽的合成。
5. 用於在細胞內系統運送抗原肽之綠膿桿菌外毒素(Pseudomonas exotoxin，PE)類毒素載體建造
綠膿桿菌外毒素A(PE)多肽含有功能區(domain) Ia、II、Ib和III。共具有613個胺基酸，其全長PE-DNA片段已被公開(Liao CW等人,Applied Microbiology and Biotechnology , July 1995, Volume 43, Issue 3, pp 498–507)，並且次轉殖的菌株被儲存在Liao Lab。為了研究PE多肽片段的C-末端缺失是否會影響PE融合的Th1表位的免疫原性，使用表12中列出的引子對：PE-425、PE-407、PE-49，以合成一系列編碼不同長度的綠膿桿菌外毒素A多肽的DNA片段。術語「PE-N」代表由胺基酸(aa) 1至N組成的PE片段，其中N是整數。例如，「PE-425」是由aa 1至425組成的PE片段，「PE-407」是由aa 1至407組成的PE。
5-1. 試驗例1：pPE49質體
在強噬菌體T7轉錄和(選擇性地)轉譯信號的控制下，將PE49目標基因轉殖到pET15b質體中；透過在宿主細胞中提供T7 RNA聚合酶來誘導表現。如以下建造質體pPE49。透過使用如表12所示引子對PE-F1和PE-R1的PCR，合成一編碼PE aa 1-49的核苷酸序列的159 bp(鹼基對) DNA片段。用Xho1 I和Nde I切割165 bp的PCR產物，以分離一159 bp片段。然後將其次轉殖到用Xho1 I和Nde I切割pET15b而得的5.9 kb的大DNA片段中，以產生質體pPE-49 (6079bp)。
5-2. 試驗例2：pPE407質體
在強噬菌體T7轉錄和(選擇性地)轉譯信號的控制下，將PE407目標基因轉殖到pET15b質體中；透過在宿主細胞中提供T7 RNA聚合酶來誘導表現。如以下建構質體pPE407。透過使用如表12所示引子對PE-F1和 PE-R2的PCR，合成一編碼PE aa 1-407的核苷酸序列的一1224bp DNA片段。用Xho1 I和Nde I切割1238bp的PCR產物，以分離一1224bp片段。然後將其次轉殖到用Xho1 I和Nde I切割pET15b而得的5.9 kb的大DNA片段中，以產生質體pPE-407 (7159bp)。
5-3 試驗例3：pPE425 質體
在強噬菌體T7轉錄和(選擇性地)轉譯信號的控制下，將PE425目標基因轉殖到pET15b質體中；透過在宿主細胞中提供T7 RNA聚合酶來誘導表現。如以下建構質體pPE425。透過使用如表12所示引子對PE-F1和PE-R3的PCR，合成一編碼PE aa1-425的核苷酸序列的一1299bp DNA片段。用Xho1 I和Nde I切割1299bp的PCR產物，以分離一1293bp片段。然後將其次轉殖到用Xho1 I和Nde I切割pET15b而得的5.9 kb的大DNA片段中，以產生質體pPE-425 (7213bp)。
上述PE片段的核苷酸和胺基酸序列為多組胺酸(poly-His)表位，在5'-和3'-(或N-和C-)末端分別側接有連接序列MGSSHHHHHH和LEHHHHHHZ。
表12 *Fw：順向。 **Rv：反向。
PE融合表位的目標DNA片段可插入至pPE407和pPE425的Xho1限制酶位點。
6. 用於免疫增強劑的黏膜標靶融胜肽的質體建造
6-1. 用於次轉殖的質體載體建造
PE49-3，用於M細胞標靶肽表位的DNA片段的插入的質體建造。該質體係來自pPE49質體。其透過PCR和重組操作進行了修飾。表13和14中為引子和序列修飾。
表13 *Fw：順向。 **Rv：反向。
表14：pPE49和pPE49-3之間的N端胺基酸序列和DNA序列及其限制位的差異
6-2. M細胞標靶多肽的質體建造
將M細胞配體的三個DNA片段(CO1、DQ2、RV3)各自連接到EcoR1和XhoI限制酶切割的pPE49-3/pET質體中，使得編碼M細胞配體的DNA片段插入到T7啟動子的下游。將含有插入片段的質體分別轉殖到大腸桿菌中，並通過安比西林抗藥性篩選菌株。如表15所示，其為pP49-3-CO1、pPE49-3-DQ2、和pPE49-3-RV3質體的插入部分的部分DNA序列。
表15
6-3. 上皮細胞標靶多肽的質體建造
將標靶配體DNA的L2-200連接到用EcoR1和XhoI限制酶切割的pPE49-3/pET質體中。將含有質體的插入片段分別轉殖到大腸桿菌中，並通過安比西林抗藥性篩選出菌株。表16列出pL2-200質體插入部分的部分DNA序列。
表16
6-4. 表現的黏膜標靶融合胜肽的製備
嵌合多肽在大腸桿菌BL21(DE3)系統中有表現。在不改變所選胜肽片段的原始胺基酸序列的情況下進行密碼子替換，以避免假性限制位(spurious restriction site)，並使其在大腸桿菌中理想地表現。在編碼胜肽片段的DNA序列的末端添加限制性位連接序列以插入表現載體中。
7.重組綠膿桿菌類毒素(PE)類融合抗原和免疫增強劑的質體建造和製備
7-1. PE類嵌合抗原的建造
將HPV Th1表位的兩個DNA片段(E601；SEQ ID NO. 13及E701；SEQ ID NO. 15)分別連接至用EcoR1和XhoI限制酶切割的pPE425質體(PE類毒素載體衍生物質體)中，使得融合蛋白被加在PE(ΔIII)片段C-末端。將含有插入片段的質體分別轉殖到大腸桿菌中，並藉由安比西林抗藥性篩選出菌株。
將肌肉生長抑制素表位的兩個DNA片段(M14；SEQ ID NO. 19及M27；SEQ ID NO. 20)連接到用EcoR1和XhoI限制酶切割的pPE407質體中。
將PRRSV表位的三個DNA片段(GP317；SEQ ID NO. 24、GP417；SEQ ID NO. 25及 GP437；SEQ ID NO. 26)分別連接到用EcoR1和XhoI限制酶切割的pPE425質體(pET和PE 類毒素載體衍生物質體)，使得融合蛋白被加在PE(∆III) 片段C-末端。將含有插入片段的質體分別轉殖到大腸桿菌中，並經由安比西林抗藥性篩選出菌株。
表17：PE類重組嵌合抗原的實例
7-2. PE類HPV融合胜肽
為了發展PE類HPV融合蛋白作為疫苗，採用逆向基因工程方法構建高效率的病毒融合蛋白表現載體。透過注射給藥，PE類HPV疫苗可將病毒多肽送至包括抗原呈現細胞(antigen presenting cells，APC)的細胞中以引起強烈的免疫反應(Liao; Cancer research 2005)。因此，發展PE類融合胜肽的策略亦為融合PE片段的C-末端與病毒蛋白Th1表位，例如來自HPV16型的E601和E701多肽，如表6所示。然而，任何其他型的HPV(例如，18型、35型和任何其他型的HPV)也可用作HPV表位。
將HPV抗原Th1表位的四個DNA片段(E601、E622、E701和E713)分別連接到用EcoR1和XhoI限制酶切割的pPE425質體(pET23a和PE類毒素載體衍生物質體)中，使得融合蛋白被加至PE(∆III)片段的C末端。將含有插入片段的質體分別轉殖到大腸桿菌中，並通過安比西林抗藥性篩選出菌株。
透過限制酶切割和電泳分析所有合成的及/或次轉殖的核苷酸片段，以檢查它們是否具有預期的大小和正確的切割位點。一旦獲得樣品，機器將自動產生DNA序列並且在電腦上顯示其結果。進行序列分析以鑑定核酸中的核苷酸序列或多肽中的胺基酸。
7-3. PE類肌肉生長抑制素表位融合胜肽的建造
發展PE類融合蛋白的策略是融合PE片段的C末端與肌肉生長抑制素蛋白結合表位(M14)，以誘發用於阻斷肌肉生長抑制素活性的抗肌肉生長抑制素抗體。
將肌肉生長抑制素結合表位的兩個DNA片段(M27、M14)分別連接到用EcoR1和XhoI限制酶切割的pPE407質體(pET23a和PE類毒素載體衍生物質體)中，使融合蛋白被加至PE(∆III)片段的C末端。將含有插入片段的質體分別轉殖到大腸桿菌中，並通過安比西林抗藥性篩選出菌株。一旦獲得樣品，機器將自動產生DNA序列並且在電腦上顯示其結果。進行序列分析以鑑定核酸中的核苷酸序列或多肽中的胺基酸。
7-4. PE類GP3和GP4融合胜肽的建造
發展PE類融合蛋白的策略係融合PE片段的C末端與病毒血清中和表位(GP3和GP4)，以誘發針對PRRSV感染的抗體。
將DNA片段(GP317、GP417、及GP437)分別連接到用EcoR1和XhoI限制酶切割的pPE425質體(pET23a和PE類毒素載體衍生物質體)中，使得融合蛋白被加至在PE(∆III)片段的C端。將含有插入片段的質體分別轉殖到大腸桿菌中，並通過安比西林抗藥性篩選出菌株。一旦獲得樣品，機器將自動產生DNA序列並且在電腦上顯示其結果。進行序列分析以鑑定核酸中的核苷酸序列或多肽中的胺基酸。
8. PE類黏膜標靶融合肽的質體建造
如表18所示，所有建造的質體屬於融合生物多肽，其可通過黏膜標靶性和Th1免疫蛋白酶體加工起到免疫增強作用。
用HindIII和Pst1從pP49-3-DQ2質體上切下含有DQ2表位的長DNA片段，然後分別連接到不同DNA片段，其包含PE和Th1表位質體，例如pPE-E713、pPE-E622、pPE-M14、pPE-M37、pPE-GP317、pPE-GP417和pPE-GP437。用Mfe1和HindIII切割那些含有DQ2表位的新質體，然後交換另外兩個DNA片段(P493-CO1和RV3)。用HindIII和Pst1從pP49-3-L2-200質體中切下含有HPV上皮細胞標靶表位的較長DNA片段，然後和用HindIII和Pst1限制酶切割的pPE-E713或pPE-E622質體連接，所述質體含有PE和HPV Th1表位。
表18：用於免疫增強的融合生物多肽的實例
在表18所列的融合肽中，黏膜標靶多肽係位於融合肽的N末端，表位則係位於融合肽的C末端，轉位肽位於黏膜標靶多肽和表位之間。然而，本揭露不限於此。在本揭露的其他實施例中，轉位肽位於融合肽的N末端，表位位於融合肽的C末端，且黏膜標靶多肽位於轉位肽和表位之間。
9. 融合胜肽的表現與純化
將受T7啟動子控制的質體送進大腸桿菌BL21 (DE3)中進行表現。在誘導重組多肽表現後，在裂解液中發現包涵體；藉由差速離心(1,450 x g，10分鐘)從不溶部分收集包涵體顆粒。然後將沉澱物在含有100 mM PMSF和1 mg/ml DOC 的TNE緩衝液中均勻化，並且另外收集1,450 x g上清液。在進行三次收集後，透過離心(27,000 x g，20分鐘)從合在一起的上清液收集沉澱物。透過在含有1M尿素的pH 8.5的0.1M Tris-HCl三次洗滌中再懸浮，然後進行離心(27,000 x g，20分鐘)，以除去尿素可溶性污染物。最後將其溶在20體積的含有8M尿素的TNE中，在室溫下輕輕攪拌過夜。然後在變性以及還原條件(10mM DTT)下使用含有6M尿素的TNE緩衝液，透過S200凝膠過濾層析純化蛋白質。藉由在Pellicon機器(Millipore, Billerica, MA, USA)中使用包含從4M至0M尿素的TNE緩衝液透析，使蛋白質沖洗液片段復性。
將甘油冷凍菌種(2 ml)培養在含有LB (200ml)以及安比西林(500 μg/ml)的500ml燒瓶中。在150 rpm和37°C下搖動燒瓶，直到培養物具有1.0±0.3的OD600 值。接著將其50ml等量培養物分別培養到8個各自含有LB (1250 ml)、安比西林 (500 μg/ml)、及10%葡萄糖(50 ml)的3000ml滅菌燒瓶中，在37℃的旋轉培養箱中培養，並以150rpm搖動2至3小時。然後加入IPTG至最終濃度為50 ppm，再將培養物在37℃下振盪培養2小時以完成蛋白質誘導。透過Coomassie Blue染色的SDS-PAGE的密度測定法定量嵌合肽。在小鼠動物實驗中，0.03±0.003 mg的嵌合多肽被用作為高劑量給藥，0.01±0.0001mg被用作為低劑量給藥。在每10公升的細菌培養物中，可獲得約300-400 mg的多肽，其足以進行3000-9000次的給藥。
10.用於口服給藥的嵌合抗原以及免疫增強劑的微球體的製備
嵌合抗原重組多肽以及免疫增強劑的製備
在100級層流中進行多肽的生產和樣品的製備。每種重組多肽溶液相當於300 mg的蛋白質含量。將其轉移到3L容量瓶中，然後加入70-80ml的8M尿素和粉末載體，如表19所述。
表19列出了微球體製劑。如Liao等人(2001)所述，透過使用Model L-8機器(Ohkawara Kakohki)的共噴霧乾燥程序，製備微球體粉末。噴霧乾燥程序根據若干製備條件設定：噴霧器速度為30,000 rpm，進料速度為1mL/min，熱空氣流入溫度為50℃，排氣溫度為35℃，旋風壓力約為110-120 mmH2 O。微球體儲存於4℃下(Liao等人，2001)。
表19：微球體粉末製備之配方
為了測試微球體製劑的穩定性，在不同溫度下儲存至少3個月後，分析微球體的大小和表面結構以及進行體外釋放蛋白研究。根據USP-XXII A (United States Pharmacopoeia, 1990)的pH-變化方法進行腸溶微球體的溶解試驗。將總重量為3g的微球體置於含有500mL的0.02N HCl溶液(pH1.5)的雙層燒杯中，在37℃下以100 rpm旋轉。加入5mL的0.2M磷酸三鈉緩衝液以調節pH值至7，以進行更進一步的溶解實驗，並且每個測試進行三次。在溶解測試中，每隔30分鐘取出1.5 mL的樣品，且用1.5 mL緩衝溶液代替該1.5 mL樣品，以保持溶離瓶中溶液的體積。將樣品以15,000 g離心5分鐘，將上清液吸出並儲存在-20℃。最後，透過Coomassie蛋白質測定法來測定被釋放的蛋白質量。
11. 實驗：口服投予M細胞配體嵌合多肽，增加小鼠針對特定抗原的抗體力價
11-1. 於小鼠試驗中，藉由口服投予重組蛋白的接種計畫，以證明融合生物多肽的免疫增強作用
取4週齡ICR小鼠，在實驗前將其隔離一週，並且在整個實驗中提供丸狀飼料及水令其可自由採食。從第4週開始分析生長曲線，並且每週測量體重兩次，直到第10週，在4週時分隔雄性小鼠與雌性小鼠，將同性的同窩出生小鼠放在同一個籠子中，以便在剩下的實驗中對於進行不同處理的組別維持相同的環境。將動物隨機分配成組，接受融合肽抗原與生物肽增強劑之口服藥劑組成物，如表20所示。對於每種處理，分析至少4隻小鼠。將重量繪製在生長曲線上，並使用Stat View軟體進行Anova統計分析。
用於口服PE類疫苗免疫接種及/或黏膜標靶生物肽增強劑為兩種不同的給藥劑量(3 mg低劑量以及6 mg高劑量的重組抗原製劑，即等於10 mcg和20 mcg蛋白質濃度)。使用鈍頭餵食針插入小鼠的胃中，口服投予微球體溶液一劑/一次/每週，共4次。正對照組透過皮下注射免疫接種0.25 mL PE類疫苗，其包含20 mch (H dose)蛋白質抗原，每次注射間隔兩週，總共4次免疫接種。
在每次免疫接種後兩週，透過穿刺眼窩後叢收集三隻小鼠的血液。從血清和腸道的結果中檢測特異性抗體針對融合抗原的力價。透過在4℃凝固12小時然後離心獲得血清。透過將1mL洗滌緩衝液(含有100 μg/mL大豆胰蛋白酶抑製劑、50 mM EDTA、1 mM PMSF、0.5％明膠和0.05％ NaN3 的PBS)灌注入腸中來收集屍體剖檢時完成的腸灌洗樣品。收集洗滌液並儲存於-20℃，並且在屍體剖檢後透過0.5mL洗滌緩衝液均質化一半肺臟以收集肺臟樣品。最後，在離心之後收集肺臟均質化樣品的上清液，並在-20℃下保存。採集血液樣品，並且利用連續10倍稀釋在ELISA中檢測抗-E7或抗-E6或抗-M14或抗-GP3或抗-GP4特異性抗體的力價。在第二輪免疫後檢測到特異性IgA或IgG或IgG1或IgG2a抗體力價。利用PE-GP417、437和317進行口服和注射疫苗組的PRRS病毒中和試驗檢測。
在第三輪免疫接種後，極高劑量(VH)(100 mcg蛋白)和高劑量注射(H)(20 mcg蛋白)誘發相似的力價，並在第四輪後達到穩定含量。低劑量的注射誘發較低的力價，但在第四輪免疫接種後仍可以1:3250的稀釋度檢測到。
11-2. 實驗表格
表20：小鼠試驗中的融合生物多肽的疫苗接種計畫及免疫增強效果檢測
表20-1： 實驗No.1 *：PE-E6疫苗公開於Cheng WF等人,PLoS One . 2013 Sep 13;8(9):e71216.
表20-2：實驗No.2
表20-3：實驗No.3
表20-4：實驗No.4
表20-5：實驗No.5
表20-6：實驗No.6
表20-7：實驗No.7
表20-8：實驗No.8
11-3.　豬模式中的口服免疫實驗：口服投予可增強針對特異性抗原的豬抗體力價的M細胞配體嵌合多肽
在豬模式(pig model)中，24頭仔豬中的每頭仔豬係從一健康養豬場的6頭母豬中分別選出。將每隻母豬的三隻仔豬隨機地分配和在耳上標記成免疫接種組和控制組，如表21所列。口服疫苗組總共包含9隻仔豬，在4、6、7、9、12、14、16、18、21、24、26和28日齡的時候接受口服給藥低劑量(3 mg)或高劑量(6 mg)，其係懸浮在2%乙酸溶液中。肌肉給藥組另外包含三隻仔豬，其接受注射極高劑量(每種抗原100 mcg)的三價PE-GP兩次。在14和28日齡時透過肌肉注射1mL包含0.5mL福馬林無活性液(GP417、437和317抗原的300 mcg總蛋白)和0.5mL鋁凝膠的疫苗對其進行免疫接種。剩下的3隻仔豬作為控制組。在完成口服和注射給藥後，將所有29-30日齡的實驗仔豬移動並飼養在十組3.8公尺x 4.5公尺(17平方公尺)的稻草床畜欄中。在整個試驗期間，每天記錄空氣溫度和濕度兩次。餵食動物相同的完全飼料混合物。
表21：仔豬試驗中的黏膜標靶多肽的疫苗接種計畫和免疫增強效果的檢測
在仔豬適應新環境的兩天期間，觀察及監測其生長表現及健康。將數據進行變異數分析(ANOVA)，以獲得斷奶後年齡對體重的增加和線性測量的影響。
完成免疫計畫兩週後，通過穿刺眼窩後叢收集仔豬血液。從血清和腸道特異性抗體的結果中檢測針對融合抗原的力價。透過在4℃凝固12小時然後離心獲得血清。採集血液樣品，並在ELISA中用連續2.5倍稀釋度測定血清中抗-Myo14、抗-GP317、或抗-GP417和437特異性抗體的力價。在第二輪免疫接種後檢測到特異性IgA、IgG、IgG1或IgG2a抗體力價。 PRRS病毒中和試驗檢測了PE-GP417、437和317口服及注射疫苗組。
11-4. 利用ELISA測量抗體反應
用抗原塗覆聚苯乙烯微量稀釋盤(Costar)。以蛋白質濃度為2 μg/mL的抗原的塗覆緩衝液(pH9.6的碳酸鹽-碳酸氫鹽緩衝液)，每孔以100μl塗覆微量稀釋盤。使用ELISA試驗分析結合抗體力價誘發六種蛋白質(E7、E6、Myo14、Myo27、GP3、GP4)。然後在4℃下隔夜吸附每種蛋白質抗原後，進行間接ELISA(Liao 2003)。就在使用前將辣根過氧化物酶(HRP)綴合的二抗(山羊抗-小鼠IgG、IgA、IgG2、IgG1)在終止緩衝液中以最佳濃度(x1000倍)稀釋。必須注意的是，應使用抗體力價高的正參考樣品來確定抗體力價。將該參考樣品分成等量並保存在凍乾狀態下。血清樣品分別被稀釋500倍、2500倍、12500倍和62500倍。例行性作業將腸和肺樣品稀釋10倍。為了準確闡述結果，ELISA(E)值係依照下式而得：E值=D×(S-N)/(P-N)，其中注射疫苗樣品的OD值=正控制血清或灌洗液，N =負對照血清或灌洗液，S=測試樣品，D =樣品稀釋倍數。
11-5. 利用間接免疫螢光法進行PRRSV血清中和試驗
利用間接免疫螢光法檢測PRRS病毒中和試驗。在NA試驗中所使用的所有過濾的口腔液體和血清樣品(0.2-μm過濾器)皆在距離裝有樣品的孔盤2吋處使用UV光(254 nm)處理45分鐘，然後在56℃熱滅活30分鐘。每個測試樣品在無血清DMEM(每孔100μl)中進行2倍連續稀釋(1：2至1：128)，並在37℃下與等體積的PRRSV病毒株(PRRSV TC-01)之一的100 TCID50培養1小時。在培養後，將100μl上清液移到含有舖滿單層的肺泡巨噬細胞3D4/31(ATCC CRL-2844；肺泡巨噬細胞；用SV40大T抗原不死化)的96孔微量滴定盤中；每個樣品重複進行。培養1小時後，加入100μl含有2％馬血清和抗生素-抗黴菌混合物的DMEM，並將孔盤在37℃下放置在CO2 培養箱中培養48小時。使用丙酮/Milli-Q水(8：2)混合物在室溫(約20℃)下固定細胞10分鐘，並且在進行如上所述免疫染色之前將孔盤完全乾燥。用抗-PRRSV核衣蛋白-特異性單株抗體(SDOW17) (Rural Technologies, Inc., SD) (1：5,000)在37℃下處理細胞兩小時，隨後用Alexa Fluor 488綴合的山羊抗-小鼠IgG(H+L)(Invitrogen, CA)二抗(1：3,000)處理。在加入甘油-磷酸鹽緩衝鹽水(PBS)(6：4)後，用螢光顯微鏡檢查孔盤。在PRRSV誘導的免疫螢光中觀察到大於90%的抑制時，樣品的稀釋倍數比例被決定為病毒中和抗體(virus-neutralizing antibody，NA)力價。
12. 實驗結果
12-1. 融合胜肽作為免疫增強劑對於PE類E6口服疫苗的效力
高風險人類乳突病毒E6(hrHPV E6)蛋白由於涉及人類細胞惡性轉化過程而被廣泛研究。HPV 16 E6致癌蛋白可影響人類PBMCs中IL-18誘導的IFN-γ產生，以解釋HPV感染的子宮頸病變(包含子宮頸癌)的可能的免疫逃脫機制。HPV-16和HPV-18的E6致癌蛋白透過與宿主IRF3和TYK2的相互作用來抑制免疫反應(Li S等人,Oncogene . 1999 Oct 14;18(42):5727-37; Masaud Shah 等人,Scientific Reports 5 , Article number: 13446 (2015); Cheng WF等人,PLoS One . 2013 Sep 13;8(9):e71216.; Cho YS 等人,FEBS Lett . 2001 Jul 20;501(2-3):139-45)。在我們先前的研究中，透過注射小鼠模式，PE-E6疫苗產生的E6特異性免疫反應比PE-E7產生的E7特異性免疫反應弱。在本揭露中，我們透過口服給藥的小鼠模式，評估了PE-E6口服疫苗對免疫反應的效力。在實驗中，從以下的數據分析，我們證明了包含DQ2和RV3的黏膜標靶表位具有增強E6特異性免疫反應的活性。
在表22中，根據4和5號組的ELISA試驗所得之小鼠血清抗體活性數據，PE-E6或PE-E601的口服給藥對E6或E601抗原表現出較弱的免疫反應。然而，投予PE-E6疫苗以及額外含有PE-E622或DQ2-PE-E622免疫增強劑的2和3號組，其血清IgA和IgG抗體活性可被增強。從血清稀釋500倍樣品中的E6特異性IgG活性可發現，3號組的數據顯著高於沒有增強劑的其他口服疫苗組(p ＜0.05)。
表22：不同小鼠組別中透過ELISA試驗所得的針對HPV-16 E6的IgG和IgA血清含量
在表23中，在口服投予疫苗和/或額外的免疫增強劑後2至3週，透過ELISA測定E6或E601特異性血清IgG1和IgG2a反應。從3號組數據可知，根據高比值的IgG2a/IgG1，口服額外含有DQ2-PE-E622免疫增強劑的PE-E6疫苗以及可誘發良好的Th1途徑。3號組(額外含有DQ2-PE-E622增強劑的PE-E6口服疫苗)的IgG2a/IgG1比值為1.4至1.5，顯著高於其他沒有增強劑的組別(p ＜0.05)。2號組(額外含有PE-E622增強劑的PE-E6口服疫苗)的E6特異性抗體的IgG2a/IgG1比值為1.13，與沒有增強劑的其他組相比差異不顯著(p ＞ 0.05) 。
表23：抗-E6或抗-E601的IgG血清活性及其IgG2a/IgG1比值
在表24中，五組小鼠分別口服投予額外含有包括PE-E601、L2-L200-PE-E622、CO1-PE-E622、DQ2-PE -E622和RV3-PE-E622增強劑的PE-E601疫苗。
根據小鼠血清IgG1、IgG2a抗體活性，透過ELISA試驗檢測這些小鼠對於E601抗原的免疫反應。通過IgG2a/IgG1比值數據，可以預測Th1或Th2免疫途徑。11號組或12號組(額外含有DQ2-PE-E622或RV3-PE-E622增強劑的PE-E601口服疫苗)中的IgG2a/IgG1比值為1.56或1.46，但8、9或10號組(額外含有PE-E601、L2-200-PE-E622或CO1-PE-E622增強劑的PE-E601口服疫苗)的IgG2a/IgG1比值低於1.05。根據小鼠實驗中E601特異性IgG和IgG2a/IgG1數據的高比值得知，當PE類E601口服疫苗與額外的增強劑投予時，DQ2-PE-E622和RV3-PE-E622增強劑可大幅度地引起良好的Th1免疫增強反應。DQ2-PE-E622和RV3-PE-E622的Th1免疫增強效力與其他組相比有顯著差異(P ＜0.05)。
表24
在表25中，五組小鼠分別口服投予額外含有包括PE-E601、L2-200-PE-E601、CO1-PE-E601、DQ2-PE-E601和RV3-PE-E601各種增強劑的PE-E601疫苗。
根據小鼠血清IgG1、IgG2a抗體活性，透過ELISA試驗檢測這些小鼠對於E601抗原的免疫反應。透過IgG2a/IgG1比值數據，可以預測Th1或Th2途徑。在16或17號組(額外含有DQ2-PE-E622或RV3-PE-E622的PE-E601口服疫苗)，IgG2a/IgG1比值為1.24或1.28，但在14、15或18號組(額外含有PE-E601、CO1-PE-E622或L2-200-PE-E622的PE-E601口服疫苗)的IgG2a/IgG1比值低於1.05。根據小鼠實驗中IgG2a/IgG1數據的高比值得知，當投予額外含有增強劑的PE類E016口服疫苗時，DQ2-PE-E622和RV3-PE-E622增強劑可引發良好的Th1途徑(p ＜0.05) 。
表25
12-2. 融合生物多肽增強劑對PE類E7口服疫苗的功效
我們先前的研究指出，基於E7的融合蛋白疫苗增強了E7的MHC第 I和II型的抗原呈現，使E7特異性CD8+和CD4+ T細胞前體的數量急劇增加，並顯著提高了E7特異性抗體的力價。這些結果表示逆轉錄融合蛋白透過外毒素的傳遞結構域和抗原，極度地增強體內抗原特異性免疫反應，並且代表一個新方法以改善癌症注射疫苗效力(Ebrahimpoor S 等人,Iran J Allergy Asthma Immunol . 2013 Aug 28;12(4):361-7)。在本揭露中，我們嘗試透過口服給藥來評估PE-E7口服疫苗對小鼠免疫反應的效力。我們對PE-E7在小鼠免疫試驗中的口服實驗進行了研究，得到與PE-E6研究中的相似結果。口服PE-E7或PE-E701展現出對於E7或E701抗原的弱免疫反應。在本揭露中，我們從以下的數據分析證實了包括DQ2和RV3的黏膜標靶表位具有某種增強E6特異性抗體的活性的能力。
在表26中，根據利用ELISA試驗而得的22、23號組小鼠血清抗體活性數據，PE-E7或PE-E701的口服給藥證實對於E7或E701抗原的微弱免疫反應。
表26
然而，額外含有PE-E622或DQ2-PE-E622的PE-E7疫苗組，即 20和21號組，其血清IgA及IgG抗體活性可被增強。從血清稀釋200倍樣品中的E7特異性IgG活性可得知，21號組的數據顯著高於其他沒有額外含有增強劑的口服疫苗組(p ＜0.05)。
在表27中，五組小鼠分別口服投予額外含有包括PE-E713、L2-L200-PE-E713、CO1-PE-E713、DQ2-PE-E713和 RV3-PE-E713各種增強劑的PE-E701疫苗。
表27
根據小鼠血清IgG1、IgG2a抗體活性，透過ELISA試驗檢測這些小鼠抗E701抗原的免疫反應。藉由IgG2a/IgG1比值數據，可預測Th1或Th2免疫途徑。在27、28、29和30號組(分別為額外含有L2-200-PE-713、CO1-PE-E713、DQ2-PE-E713、RV3-PE-E713增強劑的PE-E701口服疫苗)中，IgG2a/IgG1比值分別為1.55、1.34、1.43 和1.44。26號組(額外含有PE-E713增強劑的PE-E701口服疫苗)的IgG2a/IgG1比值低於1.05。根據E701特異性IgG2a的高活性和IgG2a/IgG1高比值數據，當在小鼠免疫模型投予額外含有該些增強劑的PE類E713口服疫苗時，L2-200-PE-713、CO1-PE-E713、DQ2-PE-E622和RV3-PE-E622增強劑可強烈誘發良好的Th1免疫反應。E701特異性IgG2a免疫增強效果顯示L2-200-PE-713、CO1-PE-E713、DQ2-PE-E713和RV3-PE-E713組與PE-E713增強劑組具有顯著性差異(P ＜0.05 )。
12-3. 融合胜肽作為免疫增強劑對PE類肌肉生長抑制素口服疫苗的效力
重組肌肉生長抑制素可透過口服途徑誘發對肌肉生長抑制素的免疫反應，導致小鼠體重增加。這是將細胞轉型為食用疫苗以改善農場動物的肉類生產和對抗肌肉萎縮遺傳病的重要步驟(Zhang T 等人,BMC Biotechnol . 2012 Dec 19;12:97；Aravind S等人,J Virol Methods. 2012 Nov;185(2):234-8)。
我們建立了用於動物的PE類肌肉生長抑制素融合口服疫苗。針對PE類肌肉生長抑制素融合口服疫苗製劑，開發了幾種融合生物多肽增強劑。在本揭露中，根據以下數據分析，我們證實了包括CO1、DQ2和RV3的黏膜標靶表位具有肌肉生長抑制素表位特異性抗體的增強活性。
在表28和29中，在不同血清樣品中，透過ELISA試驗在連續血清稀釋(1:200-1:1250)中檢測M14特異性血清IgG 和IgA活性。
表28
表29
根據31號組(PE-M14疫苗組)的連續稀釋ELISA數據，證明了口服施予PE-M14疫苗在1：200至1：1250倍血清稀釋液中引起了良好的M14特異性IgG和IgA血清力價。此外，含有額外的CO1-PE-M14、DQ2-PE-M14和RV3-PE-M14、CO1-PE-M27、DQ2-PE-M27和RV3-PE-M27的PE-M14疫苗組別(即32、33、34、36、37和38號組)的血清IgA和IgG抗體活性可被極大幅度地增強。從血清稀釋500倍樣品中M14特異性IgG活性來看，32、33和34號組的數據顯著高於其他口服疫苗組(p ＜0.05)。
在表30中，數據證實PE類M14融合口服疫苗可被黏膜標靶配體CO1、DQ2和RV3增強。32、33和34號組(其分別額外以CO1-PE-M14、DQ2-PE-M14和RV3-PE-M14增強劑處理)的IgG和IgA血清特異性-M14抗體活性(血清500稀釋度，ELISA)略高於31號組的活性(p＞ 0.1)。此外，36、37、38號組的活性較31或35號組(沒有額外的黏膜標靶配體)高許多。具體而言，37和38號組的IgG和IgA含量顯著高於控制組(例如31或35號組)。
表30
12-4. 融合胜肽作為免疫增強劑對PE類PRRS GP3-GP4表位口服疫苗的效力
豬生殖與呼吸綜合症(PRRS)會導致新生仔豬因晚期生殖衰竭和嚴重肺炎而造成嚴重的經濟損失。PRRS疾病是一種高風險性動物疾病，由於PRRS病毒的高度遺傳變異，現有疫苗對感染提供的保護有限。血清中和抗體(NA)被認為和針對PRRSV的保護性免疫有重要關聯。 Lopez等人(2007)已經證實了NA在防止PRRSV感染方面的作用。他的研究結果確定了血清病毒中和(serum virus neutralization，SVN)力價(≥1:8)的特定閾值，在這個閾值下，幼豬血清中PRRSV的散布將被阻斷，並且更高的閾值(≥1:32)意味著可完全保護動物避免受PRRSV感染。我們開發了一種PE類PRRSV次單位疫苗。然而，誘發針對PRRS疾病控制的該次單位疫苗必須改善SVN力價。在本揭露中，以下的實驗結果展示，作為口服免疫增強劑的融合胜肽引發SVN的效力。
在表31中，數據表示PE類GP融合口服疫苗可經由黏膜標靶配體CO1、DQ2和RV3增強。
根據44和48號組(PE-GP417和PE-GP437疫苗組)的連續稀釋ELISA數據，其顯示口服投予PE-GP417和PE-GP437在1:200到1:1250倍血清稀釋中誘發了良好的GP417和GP437特異性IgG血清力價。額外含有CO1-PE-GP417或CO1-PE-GP437、DQ2-PE-GP417或DQ2-PE-GP437以及RV3-PE-GP417或RV3-PE-GP437免抑增強劑的PE-GP417或PE-GP437疫苗組別(即41或45、42或46以及43或47號組)之血清IgG抗體活性可增加抗體活性。從在血清稀釋500倍樣品中的GP417特異性IgG活性來看，41、42和43號組的數據顯著高於其他口服疫苗組(p＜ 0.02)。從46和47號組的稀釋倍數樣品中的GP-437-特異性IgG活性，顯示出良好的免疫增強作用，但是45號組並沒有。從結果論，包括CO1、DQ2和RV3在內的黏膜標靶表位對GP417 表位-特異性抗體具有良好的增強活性，並且DQ2和RV3對GP437抗體具有良好的增強活性。
表31
表32
從表32中所示的結果，包括CO1、DQ2和RV3的黏膜標靶表位小幅度增強PE-GP417和PE-GP437 疫苗的GP417和GP437-特異性IgA活性(p＞0.02)。
在表33和34中，數據顯示，當PE類GP417&437和GP317融合的口服疫苗含有額外的黏膜標靶配體CO1、DQ2和RV3時，血清病毒中和(virus-neutralization，VN)抗體便會增加。
表33
表34
從表31和表32的結果可以看出，只有42、46、52號組具有最高的VN力價。口服施予沒有免疫增強劑的PE-GP317、PE-GP414和PE-GP437疫苗，例如44、48和54號組，皆出現較低的VN力價，只有8分。然而，提供了良好黏膜標靶配體的DQ2融合胜肽在添加到如42、46和52號組的疫苗組時，表現出良好的免疫增強效力。
13. 豬隻實驗：增強針對特異性抗原的豬抗體力價的M細胞配體嵌合多肽的口服給藥
在表21中，列出了豬隻試驗中的疫苗接種程序和融合生物多肽的免疫增強功效的檢測。在沒有增強劑的PE-M14高劑量組的豬隻口服疫苗實驗中，年齡x飲食計劃沒有相互影響到斷奶至10週齡的生長表現。PE-M14高劑量組不影響斷奶至10週齡的生長表現。對10週齡時的豬隻體重沒有影響(P ＞ 0.05)。然而，出生時體重較重的豬隻的斷奶前平均日增重(ADG)(出生體重 ≤ 0.104 kg之豬隻ADG為0.172 kg/day，出生體重 ≥ 1.99 kg之豬隻ADG為0.27 kg/day)、斷奶時體重(5.26 kg至8.85 kg)、斷奶BCS (2.69至2.93)、和斷奶前死亡率(24.2% to 4.6%)得到改善。
在整個5週的斷奶後階段，額外含有DQ2-PE-M14或DQ2-PE-M27增強劑的PE-M14高劑量口服疫苗組，仔豬增加的體重較安慰劑組更重23-24% (P ＜ 0.05)，並且出現更多的玩耍行為(4.0 ± 0.3 vs. 2.8 ± 0.3 freq/h,P ＜ 0.05)。
表35
根據表33所示的數據，口服疫苗組中的IgG-ELISA和VN力價低於注射組。當用DQ2-PE-GP417補充口服疫苗時，可增加VN力價分數。這顯示DQ2融合胜肽提供了一種良好的黏膜標靶配體，為PE類PRRSV-NT口服疫苗提供了良好的免疫增強劑。
綜上所述，當疫苗與本揭露之融合胜肽一起使用時，口服投予該組成物可達到良好的免疫反應。尤其，當本揭露之融合胜肽包含黏膜標靶多肽時，免疫反應會顯著增加。
雖然本揭露已就特定的具體實施例說明本揭露之實施方式，需要理解的是，本揭露亦可藉由其他不同的具體實施例加以施行或應用，本說明書中的各項細節亦可針對不同觀點與應用，在不悖離本創作之精神下進行各種修飾與變更。
無
無
無
SEQUENCE LISTING ＜110＞ 廖朝暐 ＜120＞ 用於免疫增強的融合胜肽及使用其增強誘發免疫反應的方法 ＜130＞ A0859/2060 ＜150＞ 62/412,896 ＜151＞ 2016-10-26 ＜160＞ 82 ＜170＞ PatentIn version 3.5 ＜210＞ 1 ＜211＞ 14 ＜212＞ PRT ＜213＞ Homo sapiens ＜400＞ 1 Ser Phe His Gln Leu Pro Ala Arg Ser Pro Ala Pro Leu Gln 1 5 10 ＜210＞ 2 ＜211＞ 22 ＜212＞ PRT ＜213＞ Daedalea quercina Fr. ＜400＞ 2 Ser Ser Phe His Leu Phe His His Leu Pro Ala Arg Ala Pro Leu Ala 1 5 10 15 Pro Ser Glu Leu Gln Pro 20 ＜210＞ 3 ＜211＞ 17 ＜212＞ PRT ＜213＞ Dasypus novemcinctus ＜400＞ 3 Ser Thr Pro Phe His Pro Leu Pro Ala Arg Lys Pro Leu Pro Leu Gln 1 5 10 15 Pro ＜210＞ 4 ＜211＞ 212 ＜212＞ PRT ＜213＞ Human papillomavirus ＜400＞ 4 Glu Phe His Met Val Asp Gly Met Ser Ile Arg Ala Lys Arg Arg Lys 1 5 10 15 Arg Ala Ser Ala Thr Gln Leu Tyr Lys Thr Cys Lys Gln Ala Gly Thr 20 25 30 Cys Pro Pro Asp Ile Ile Pro Lys Val Glu Gly Lys Thr Ile Ala Glu 35 40 45 Gln Ile Leu Gln Tyr Gly Ser Met Gly Val Phe Phe Gly Gly Leu Gly 50 55 60 Ile Gly Thr Gly Ser Gly Thr Gly Gly Arg Thr Gly Tyr Ile Pro Leu 65 70 75 80 Gly Thr Arg Pro Pro Thr Ala Thr Asp Thr Leu Ala Pro Val Arg Pro 85 90 95 Pro Leu Thr Val Asp Pro Val Gly Pro Ser Asp Pro Ser Ile Val Ser 100 105 110 Leu Val Glu Glu Thr Ser Phe Ile Asp Ala Gly Ala Pro Thr Ser Val 115 120 125 Pro Ser Ile Pro Pro Asp Val Ser Gly Phe Ser Ile Thr Thr Ser Thr 130 135 140 Asp Thr Thr Pro Ala Ile Leu Asp Ile Asn Asn Asn Thr Val Thr Thr 145 150 155 160 Val Thr Thr His Asn Asn Pro Thr Phe Thr Asp Pro Ser Val Leu Gln 165 170 175 Pro Pro Thr Pro Ala Glu Thr Gly Gly His Phe Thr Leu Ser Ser Ser 180 185 190 Thr Ile Ser Thr His Asn Tyr Glu Glu Ile Pro Met Asp Thr Lys Asp 195 200 205 Glu Leu Leu Glu 210 ＜210＞ 5 ＜211＞ 78 ＜212＞ DNA ＜213＞ Artificial sequence ＜220＞ ＜223＞ CO-1 DNA sequence ＜400＞ 5 gaattcagca gctttcatct gttccaccat ctgccagcgc gtgcgccatt agcgccttct 60 gaattacagc ccctcgag 78 ＜210＞ 6 ＜211＞ 78 ＜212＞ DNA ＜213＞ Artificial sequence ＜220＞ ＜223＞ DQ-2 DNA sequence ＜400＞ 6 gaattcagca gctttcatct gttccaccat ctgccagcgc gtgcgccatt agcgccttct 60 gaattacagc ccctcgag 78 ＜210＞ 7 ＜211＞ 63 ＜212＞ DNA ＜213＞ Artificial sequence ＜220＞ ＜223＞ RV-3 DNA sequence ＜400＞ 7 gaattctcta ctcctttcca cccattgcct gcccgcaaac cattgcctct ggtgcccctc 60 gag 63 ＜210＞ 8 ＜211＞ 636 ＜212＞ DNA ＜213＞ Human papillomavirus ＜400＞ 8 gaattccata tggtcgacgg tatgtccatc cgtgctaaac gtcgtaaacg tgcttccgct 60 acccagctgt acaaaacctg caaacaggct ggtacctgcc cgccggacat catcccgaaa 120 gttgaaggta aaaccatcgc tgaacagatc ctgcaatacg gttctatggg tgttttcttc 180 ggcggtctgg gcatcggtac cggttccggt actggcggtc gtaccggtta catcccgctg 240 ggtacccgtc cgccgaccgc taccgacacc ctggctccgg ttcgtccgcc gctgaccgtt 300 gacccggttg gtccgtccga cccgtccatc gtttccctgg ttgaagaaac ctccttcatc 360 gacgctggtg ctccgacctc cgttccgtcc atcccgccgg acgtttccgg tttctccatc 420 accacctcca ccgacactac cccggctatc ctggacatca acaacaacac cgttactacc 480 gtaaccactc acaacaaccc gaccttcacc gacccgtccg ttctgcaacc gccgaccccg 540 gctgaaaccg gtggtcactt caccctgtcc tcttccacca tctccaccca caactacgaa 600 gaaatcccga tggacaccaa agacgaactg ctcgag 636 ＜210＞ 9 ＜211＞ 47 ＜212＞ PRT ＜213＞ Artificial sequence ＜220＞ ＜223＞ fused peptide ＜400＞ 9 Glu Phe Val Asp Gln Leu Leu Arg Arg Glu Val Phe Cys Gly Phe Arg 1 5 10 15 Asp Leu Val Tyr Asp Phe Ala Phe Ser Asp Leu Lys Leu Pro Gln Leu 20 25 30 Cys Thr Glu Leu Lys Leu Pro Gln Leu Cys Thr Glu Leu Leu Glu 35 40 45 ＜210＞ 10 ＜211＞ 96 ＜212＞ PRT ＜213＞ Artificial sequence ＜220＞ ＜223＞ fused peptide ＜400＞ 10 Glu Phe Val Asp Lys Asp Glu Leu Arg Glu Val Tyr Asn Phe Ala Phe 1 5 10 15 Leu Leu Val Leu Arg Arg Glu Val Tyr Asp Lys Asp Glu Leu Leu Leu 20 25 30 Leu Leu Glu Asp Arg Gln Leu Leu Arg Arg Glu Val Phe Cys Gly Phe 35 40 45 Arg Asp Leu Leu Glu Asp Arg Val Tyr Asp Phe Ala Phe Ser Asp Leu 50 55 60 Lys Leu Pro Gln Leu Cys Thr Glu Leu Lys Leu Pro Gln Leu Cys Thr 65 70 75 80 Glu Leu Lys Asp Glu Leu Lys Asp Glu Leu Val Leu Leu Leu Leu Glu 85 90 95 ＜210＞ 11 ＜211＞ 51 ＜212＞ PRT ＜213＞ Artificial sequence ＜220＞ ＜223＞ fused peptide ＜400＞ 11 Glu Phe Val Asp Gln Ala Glu Pro Asp Gln Ala Glu Pro Asp Arg Ala 1 5 10 15 Arg Ala His Tyr Asn Ile Arg Ala Arg Ala His Tyr Asn Ile Leu Arg 20 25 30 Ala His Tyr Asn Ile Val Ile Phe Arg Ala His Tyr Asn Ile Val Ile 35 40 45 Phe Leu Glu 50 ＜210＞ 12 ＜211＞ 70 ＜212＞ PRT ＜213＞ Artificial sequence ＜220＞ ＜223＞ fused peptide ＜400＞ 12 Glu Phe Val Asp Gln Ala Glu Pro Asp Gln Ala Glu Pro Asp Arg Asp 1 5 10 15 Glu Leu Val Leu Arg Ala Arg Ala His Tyr Asn Ile Arg Ala Arg Ala 20 25 30 His Tyr Asn Ile Leu Glu Asp Arg Leu Leu Val Leu Arg Ala His Tyr 35 40 45 Asn Ile Val Ile Phe Arg Ala His Tyr Asn Ile Val Ile Phe Lys Asp 50 55 60 Glu Leu Leu Val Leu Glu 65 70 ＜210＞ 13 ＜211＞ 153 ＜212＞ DNA ＜213＞ Artificial sequence ＜220＞ ＜223＞ E601 DNA sequence ＜400＞ 13 gaattcgtcg accaactgtt gcgtcgtgaa gttttctgtg gctttcgtga tctggtctat 60 gacttcgcct ttagtgattt gaagctgcca caattgtgta cggaactgaa actgcctcaa 120 ctgtgtacag aactgaagga tgagctgctc gag 153 ＜210＞ 14 ＜211＞ 288 ＜212＞ DNA ＜213＞ Artificial sequence ＜220＞ ＜223＞ E622 DNA sequence ＜400＞ 14 gaattcgtcg acaaagatga actgcgtgag gtgtataact ttgcgttcct gttagtgtta 60 cgccgtgagg tttatgacaa ggacgagttg ttactgctgt tagaagatcg ccaactgttg 120 cgtcgtgaag ttttctgtgg ctttcgtgat ctgttagaag accgcgtcta tgacttcgcc 180 tttagtgatt tgaagctgcc acaattgtgt acggaactga aactgcctca actgtgtaca 240 gaactgaagg atgagctgaa agatgaatta gtgctgttat tgctcgag 288 ＜210＞ 15 ＜211＞ 150 ＜212＞ DNA ＜213＞ Artificial sequence ＜220＞ ＜223＞ E701 DNA sequence ＜400＞ 15 gaattcgtcg accaggcgga accagatcaa gcggaacctg accgtgcccg cgcacattat 60 aacattcgcg cacgtgcaca ctataatctg gaggcgcatt ataacattgt catcttccgc 120 gcacattata acatcgtcat tttcctcgag 150 ＜210＞ 16 ＜211＞ 207 ＜212＞ DNA ＜213＞ Artificial sequence ＜220＞ ＜223＞ E713 DNA sequence ＜400＞ 16 gaattcgtcg accaggcgga accagatcaa gcggaacctg accgtgacga gctggtgtta 60 cgcgcccgcg cacattataa cattcgcgca cgtgcacact ataatctgga ggatcgttta 120 ctggtcttgc gtgcgcatta taacattgtc atcttccgcg cacattataa catcgtcatt 180 ttcaaagatg agttgctggt tctcgag 207 ＜210＞ 17 ＜211＞ 72 ＜212＞ PRT ＜213＞ Artificial sequence ＜220＞ ＜223＞ fused peptide ＜400＞ 17 Glu Phe Val Asp Val Phe Leu Gln Lys Tyr Pro His Thr His Leu Val 1 5 10 15 His Gln Ala Leu Asp Val Phe Leu Gln Lys Tyr Pro His Thr His Leu 20 25 30 Val His Gln Ala Leu Asp Val Phe Leu Gln Lys Tyr Pro His Thr His 35 40 45 Leu Val His Gln Ala Leu Asp Val Phe Leu Gln Lys Tyr Pro His Thr 50 55 60 His Leu Val His Gln Ala Leu Glu 65 70 ＜210＞ 18 ＜211＞ 277 ＜212＞ PRT ＜213＞ Artificial sequence ＜220＞ ＜223＞ fused peptide ＜400＞ 18 Glu Phe Leu Leu Glu Pro His Thr His Leu Val His Gln Ala Asn Val 1 5 10 15 Leu Leu Ala Leu Gln Leu Leu Leu Glu Asp Arg Glu Phe Val Phe Leu 20 25 30 Gln Lys Tyr Pro His Val Glu Pro His Thr His Leu Val His Gln Ala 35 40 45 Asn Val Leu Leu Ala Leu Gln Leu Leu Leu Glu Asp Arg Glu Phe Val 50 55 60 Phe Leu Gln Lys Tyr Pro His Val Glu Pro His Thr His Leu Val His 65 70 75 80 Gln Ala Asn Val Leu Leu Ala Leu Gln Leu Leu Leu Glu Asp Arg Glu 85 90 95 Phe Val Phe Leu Gln Lys Tyr Pro His Val Glu Pro His Thr His Leu 100 105 110 Val His Gln Ala Asn Val Leu Leu Ala Leu Gln Leu Leu Leu Glu Asp 115 120 125 Arg Glu Phe Val Phe Leu Gln Lys Tyr Pro His Val Glu Pro His Thr 130 135 140 His Leu Val His Gln Ala Asn Val Leu Leu Ala Leu Gln Leu Leu Leu 145 150 155 160 Glu Asp Arg Glu Phe Val Phe Leu Gln Lys Tyr Pro His Val Glu Pro 165 170 175 His Thr His Leu Val His Gln Ala Asn Val Leu Leu Ala Leu Gln Leu 180 185 190 Leu Leu Glu Asp Arg Glu Phe Val Phe Leu Gln Lys Tyr Pro His Val 195 200 205 Glu Pro His Thr His Leu Val His Gln Ala Asn Val Leu Leu Ala Leu 210 215 220 Gln Leu Leu Leu Glu Asp Arg Glu Phe Val Phe Leu Gln Lys Tyr Pro 225 230 235 240 His Val Glu Pro His Thr His Leu Val His Gln Ala Asn Val Leu Leu 245 250 255 Ala Leu Gln Leu Leu Leu Glu Asp Arg Glu Phe Val Phe Leu Gln Lys 260 265 270 Tyr Pro His Val Asp 275 ＜210＞ 19 ＜211＞ 216 ＜212＞ DNA ＜213＞ Artificial sequence ＜220＞ ＜223＞ M14 DNA sequence ＜400＞ 19 gaattcgtcg acgtgttttt acaaaaatat cctcatacgc acctggtcca tcaggcgctc 60 gacgtgtttt tacaaaaata tcctcatacg cacctggtcc atcaggcgct cgacgtgttt 120 ttacaaaaat atcctcatac gcacctggtc catcaggcgc tcgacgtgtt tttacaaaaa 180 tatcctcata cgcacctggt ccatcaggcg ctcgag 216 ＜210＞ 20 ＜211＞ 831 ＜212＞ DNA ＜213＞ Artificial sequence ＜220＞ ＜223＞ M27 DNA sequence ＜400＞ 20 gaattcctcc tcgagccaca tacgcactta gtgcatcaag cgaacgtttt gctggcactg 60 caattattat tagaagatcg tgaatttgtc ttcttgcaaa aatatccaca cgtcgagcca 120 catacgcact tagtgcatca agcgaacgtt ttgctggcac tgcaattatt attagaagat 180 cgtgaatttg tcttcttgca aaaatatcca cacgtcgagc cacatacgca cttagtgcat 240 caagcgaacg ttttgctggc actgcaatta ttattagaag atcgtgaatt tgtcttcttg 300 caaaaatatc cacacgtcga gccacatacg cacttagtgc atcaagcgaa cgttttgctg 360 gcactgcaat tattattaga agatcgtgaa tttgtcttct tgcaaaaata tccacacgtc 420 gagccacata cgcacttagt gcatcaagcg aacgttttgc tggcactgca attattatta 480 gaagatcgtg aatttgtctt cttgcaaaaa tatccacacg tcgagccaca tacgcactta 540 gtgcatcaag cgaacgtttt gctggcactg caattattat tagaagatcg tgaatttgtc 600 ttcttgcaaa aatatccaca cgtcgagcca catacgcact tagtgcatca agcgaacgtt 660 ttgctggcac tgcaattatt attagaagat cgtgaatttg tcttcttgca aaaatatcca 720 cacgtcgagc cacatacgca cttagtgcat caagcgaacg ttttgctggc actgcaatta 780 ttattagaag atcgtgaatt tgtcttcttg caaaaatatc cacacgtcga c 831 ＜210＞ 21 ＜211＞ 297 ＜212＞ PRT ＜213＞ Porcine reproductive and respiratory syndrome virus ＜400＞ 21 Glu Phe Val Ser Phe Ser Thr Gly Gly Ser Gln Asn Trp Thr Val Glu 1 5 10 15 Arg Leu Leu Gln Ala Glu Phe Cys Ser Thr Ser Gln Ala Ala Arg Gln 20 25 30 Arg Leu Glu Thr Gly Arg Asn Cys Ser Thr Gly Gln Ala Ala Arg Gln 35 40 45 Arg Leu Glu Pro Gly Arg Asn Leu Val Leu Cys Leu Thr Ser Gln Ala 50 55 60 Ala Gln Gln Arg Leu Glu Pro Gly Gly Asn Cys Gln Thr Ser Gln Ala 65 70 75 80 Ala His Gln Arg Leu Glu Pro Gly Arg Asn Cys Arg Thr Ser Gln Ala 85 90 95 Ala Ser Gln Arg Leu Glu Pro Gly Arg Asn Cys Arg Thr Ser Gln Ala 100 105 110 Ala His Gln Arg Leu Glu Pro Gly Arg Asn Cys Ser Thr Arg Gln Ala 115 120 125 Ala Gln Gln Arg Leu Glu Pro Gly Arg Asn Leu Leu Cys Pro Thr Ser 130 135 140 Gln Ala Ala His Gln Arg Arg Leu Glu Pro Gly Arg Asn Cys Ser Thr 145 150 155 160 Ser Gln Ala Ala Tyr Gln Arg Leu Glu Pro Gly Arg Asn Cys Pro Thr 165 170 175 Ser Arg Ala Ala Arg Gln Arg Leu Glu Pro Gly Arg Asn Leu Leu Cys 180 185 190 Ser Thr Ser Gln Ala Ala Leu Gln Arg Leu Glu Pro Gly Arg Asn Leu 195 200 205 Cys Pro Thr Ser Gln Ala Ala Lys Gln Arg Leu Glu Pro Gly Arg Asn 210 215 220 Leu Val Val Cys Leu Thr Ser Gln Ala Ala Arg Gln Arg Leu Glu Pro 225 230 235 240 Gly Arg Asn Cys Ser Thr Ser Gln Ala Ala Ser Gln Arg Leu Glu Pro 245 250 255 Gly Arg Asn Cys Pro Thr Ser Gln Ala Ala Arg Gln Arg Leu Glu Pro 260 265 270 Gly Arg Asn Val Leu Leu Leu Cys Leu Thr Ser Gln Ala Ala His Gln 275 280 285 Arg Leu Glu Pro Gly Arg Asn Leu Glu 290 295 ＜210＞ 22 ＜211＞ 230 ＜212＞ PRT ＜213＞ Porcine reproductive and respiratory syndrome virus ＜400＞ 22 Glu Phe Gly Val Ser Ala Ala Gln Glu Lys Ile Ser Phe Gly Leu Leu 1 5 10 15 Gly Val Pro Thr Ala Gln Glu Thr Thr Ser Ile Arg Glu Val Leu Glu 20 25 30 Val Ser Thr Ala Gln Glu Asn Ser Pro Phe Met Leu Gly Ala Ser Ala 35 40 45 Thr Glu Glu Lys Thr Ser Leu Arg Leu Gly Ala Ser Thr Thr Gln Glu 50 55 60 Thr Ser Phe Gly Lys Cys Leu Arg Pro His Gly Val Ser Ala Ala Gln 65 70 75 80 Gly Thr Thr Pro Phe Arg Gly Val Ser Thr Thr Gln Glu Asn Thr Ser 85 90 95 Phe Gly Arg Val Pro Thr Ala Gln Glu Asn Val Ser Phe Gly Leu His 100 105 110 Gly Val Pro Ala Ala Gln Lys Thr Asn Ser Phe Gly Gly Val Pro Thr 115 120 125 Ala Gln Glu Asn Ile Ser Phe Lys Glu Val Ser Ala Thr Gln Arg Glu 130 135 140 Ile Pro Phe Arg Cys Leu Arg Pro His Gly Val Ser Thr Ala Gln Glu 145 150 155 160 Thr Pro Phe Arg Gly Val Ser Thr Ala Gln Glu Thr Ile Pro Phe Arg 165 170 175 Gly Val Ser Ala Thr His Glu Asn Ile Ser Phe Gly Cys Leu Arg Pro 180 185 190 His Gly Val Ser Ala Ala Gln Glu Ser Ile Pro Ile Arg Leu Gly Ala 195 200 205 Ser Ala Ala Gln Glu Asn Thr Ser Phe Arg Gly Thr Pro Ala Ala Gln 210 215 220 Glu Lys Ile Pro Leu Glu 225 230 ＜210＞ 23 ＜211＞ 289 ＜212＞ PRT ＜213＞ Porcine reproductive and respiratory syndrome virus ＜400＞ 23 Glu Phe Leu Gly Val Ser Ala Ala Gln Glu Arg Ile Pro Ile Arg Glu 1 5 10 15 Val Ser Ala Asp Lys Glu Val Ser Ala Glu Lys Lys Glu Ile Ser Phe 20 25 30 Gly Val Ser Thr Ala Gln Gly Asn Ile Ser Phe Gly Leu Gly Val Ser 35 40 45 Thr Ala Gln Glu Ala Ile Pro Phe Leu Ala Leu Gly Val Ser Thr Ala 50 55 60 Gln Glu Thr Ile Pro Phe Gly Leu Leu Gly Val Ser Thr Ala Gln Gly 65 70 75 80 Ile Ile Ser Phe Gly Gly Val Ser Thr Ala Gln Glu Asn Ile Ser Phe 85 90 95 Gly Gly Val Ser Thr Ala Gln Glu Thr Ile Ser Phe Gly Leu Leu Gly 100 105 110 Val Ser Thr Ala Gln Glu Asn Ile Ser Phe Gly Cys Leu Arg Thr His 115 120 125 Glu Val Ser Ala Ala Gln Glu Lys Ile Ser Phe Gly Gly Val Ser Glu 130 135 140 Ala Gln Lys Ile Ser Phe Gly Val Ser Ala Ala Gly Val Ser Ala Ala 145 150 155 160 Gln Glu Glu Ile Pro Phe Gly Cys Leu Arg Pro His Gly Leu Pro Ala 165 170 175 Ala Gln Glu Lys Thr Ser Phe Gly Gly Val Ser Ala Ala Gln Glu Lys 180 185 190 Thr Ser Phe Gly Gly Val Ser Ala Ala Gln Glu Glu Phe Ser Phe Gly 195 200 205 Cys Leu Arg Pro His Arg Val Ser Ala Ala Gln Glu Lys Ile Ser Phe 210 215 220 Glu Val Ser Ala Leu Glu Val Ser Ala Ala Gln Glu Lys Ile Ser Phe 225 230 235 240 Gly Val Ser Ala Ala Leu Gly Val Ser Ala Ala Gln Glu Lys Asn Ser 245 250 255 Phe Gly Cys Leu Arg Pro His Gly Val Ser Ala Ala Gln Glu Lys Thr 260 265 270 Ser Phe Gly Gly Val Ser Ala Ala Gln Lys Lys Ile Ser Phe Gly Leu 275 280 285 Glu ＜210＞ 24 ＜211＞ 892 ＜212＞ DNA ＜213＞ Porcine reproductive and respiratory syndrome virus ＜400＞ 24 ggaattcgtg agctttagca cgggtggcag ccagaactgg acggtggaac gtctgctgca 60 agccgagttc tgtagtactt ctcaggcggc gcgccagcgt ctggaaccag ggcgtaattg 120 ttctacaggc caggccgcac gtcaacgttt agagccaggt cgcaatttag ttttgtgtct 180 gacgagccag gccgcacagc agcgcttgga accaggcggt aactgtcaaa cttctcaagc 240 ggcccatcaa cgcctggaac caggtcgcaa ctgtcgcact agccaagccg ccagccaacg 300 tttagagcca ggccgcaact gtcgcacgag tcaggcggcg caccaacgtc tggaaccagg 360 ccgtaattgt agtacgcgcc aagcagccca gcaacgctta gaaccagggc gcaacctgtt 420 atgtccaact tctcaggcgg cccatcaacg ccgcttagaa ccagggcgta attgtagcac 480 gtctcaagca gcatatcaac gtctggaacc aggccgcaac tgtccaactt ctcgtgcggc 540 acgccagcgc ttagaaccag gtcgtaattt attatgttct actagccaag ccgcattaca 600 gcgtttagag ccagggcgta acctgtgtcc aactagccaa gcagcaaaac aacgcctgga 660 gccaggtcgt aatttagtgg tctgtttaac gagccaagcg gcgcgtcaac gcttagaacc 720 aggtcgcaat tgttctacta gccaagcggc cagtcaacgt ttagaaccag ggcgcaactg 780 tccaacgagc caagcggcgc gccaacgttt agagccaggg cgcaacgttt tattgttgtg 840 tctgacgagt caagccgccc atcaacgtct ggaaccaggt cgcaatctcg ag 892 ＜210＞ 25 ＜211＞ 696 ＜212＞ DNA ＜213＞ Porcine reproductive and respiratory syndrome virus ＜400＞ 25 gaattcggcg tgagcgcggc ccaggaaaag atcagtttcg gcctgttagg tgtgccaacg 60 gcccaagaga ctacaagtat tcgcgaggtt ttggaagtca gtactgcaca agaaaacagt 120 ccatttatgt taggcgcgag tgccacggag gaaaaaacgt ctttgcgcct gggggcaagc 180 acaacgcagg agacgagttt tggcaagtgt ttacgtccac atggggtttc tgcagcccaa 240 gggacgactc catttcgcgg tgtcagtaca acgcaagaaa acacgagttt tggtcgtgtc 300 ccaacggcac aagagaacgt gtcttttggc ctgcatggtg ttccagcagc gcaaaagacg 360 aacagcttcg gtggcgttcc aacggcacaa gaaaacatta gttttaagga ggttagtgcc 420 acgcaacgtg aaatcccatt ccgttgttta cgcccacacg gggttagcac agcccaggag 480 actccatttc gcggggtgag tactgcccag gagacgatcc cattccgtgg ggtttctgca 540 acgcatgaaa acatcagttt tgggtgtttg cgtccacatg gtgtcagcgc cgcacaggaa 600 tctattccaa tccgtctggg cgcgagcgca gcccaagaga ataccagttt tcgcgggaca 660 ccagcggcac aggagaaaat cccattggaa ctcgag 696 ＜210＞ 26 ＜211＞ 870 ＜212＞ DNA ＜213＞ Porcine reproductive and respiratory syndrome virus ＜400＞ 26 gaattcctgg gcgtgagcgc agcccaagag cgcatcccaa ttcgcgaggt gagcgccgac 60 aaagaggtga gtgccgagaa gaaagagatc tctttcgggg tgagcaccgc gcagggtaat 120 atcagttttg gtttgggcgt cagcaccgca caggaggcaa ttccattctt ggcactgggg 180 gtcagtaccg cccaggaaac tattccattt ggcttgctgg gggttagcac tgcacaaggt 240 atcattagtt tcggcggggt ctctactgcg caggagaata tcagctttgg cggggttagt 300 actgcgcaag agaccattag ttttggtttg ctgggcgttt ctaccgccca ggagaatatt 360 agctttggtt gtttacgcac tcatgaagtt agtgccgcac aagagaaaat tagcttcggc 420 ggcgttagtg aagcgcaaga gaagattagt ttcggggtct ctgcagcagg cgtcagcgcc 480 gcccaagagg agattccatt tgggtgtctg cgcccacacg gcctgccagc ggcgcaggag 540 aaaaccagct tcggcggcgt tagtgccgcc caggaaaaga cctctttcgg tggtgtcagc 600 gcagcacaag aagagttctc ttttggttgt ttgcgcccac atcgtgttag tgccgcacag 660 gaaaagatca gctttgaagt tagcgcgctg gaagtcagtg ccgcgcaaga gaagattagt 720 tttggcgtta gcgcggcatt gggtgtcagc gcagcacaag aaaagaactc tttcggttgt 780 ttacgcccac acggtgttag cgccgcgcaa gagaaaacca gcttcggggg tgttagtgcc 840 gcacaaaaaa agatcagctt tgggctcgag 870 ＜210＞ 27 ＜211＞ 359 ＜212＞ DNA ＜213＞ Artificial sequence ＜220＞ ＜223＞ partial sequence of plasmid ＜400＞ 27 gtttaacttt aagaaggaga tataccatgg ccgaacaatt ggtggacctc tggaacgaat 60 gcgccaaagc ctgcgtgctc gacctcaagg acggcgtgcg ttccagccgc atgagcgtcg 120 acccggccat cgccgacacc aacggccagg gcgtgctgca ctactgcatg gaattctctt 180 ttcatcagct gccagcgcgt tctccagccc cactgcagct cgagcatatg gccgaagaag 240 ctttcgacct ctggaacgaa tgcgccaaag cctgcgtgct cgacctcaag gacggcgtgc 300 gttccagccg catgagcgtc gagcaccacc accaccacca ctgagatccg gctgctaac 359 ＜210＞ 28 ＜211＞ 383 ＜212＞ DNA ＜213＞ Artificial sequence ＜220＞ ＜223＞ partial sequence of plasmid ＜400＞ 28 gtttaacttt aagaaggaga tataccatgg ccgaacaatt ggtggacctc tggaacgaat 60 gcgccaaagc ctgcgtgctc gacctcaagg acggcgtgcg ttccagccgc atgagcgtcg 120 acccggccat cgccgacacc aacggccagg gcgtgctgca ctactgcatg gaattcagca 180 gctttcatct gttccaccat ctgccagcgc gtgcgccatt agcgccttct gaattacagc 240 ccctcgagca tatggccgaa gaagctttcg acctctggaa cgaatgcgcc aaagcctgcg 300 tgctcgacct caaggacggc gtgcgttcca gccgcatgag cgtcgagcac caccaccacc 360 accactgaga tccggctgct aac 383 ＜210＞ 29 ＜211＞ 368 ＜212＞ DNA ＜213＞ Artificial sequence ＜220＞ ＜223＞ partial sequence of plasmid ＜400＞ 29 gtttaacttt aagaaggaga tataccatgg ccgaacaatt ggtggacctc tggaacgaat 60 gcgccaaagc ctgcgtgctc gacctcaagg acggcgtgcg ttccagccgc atgagcgtcg 120 acccggccat cgccgacacc aacggccagg gcgtgctgca ctactgcatg gaattctcta 180 ctcctttcca cccattgcct gcccgcaaac cattgcctct ggtgcccctc gagcatatgg 240 ccgaagaagc tttcgacctc tggaacgaat gcgccaaagc ctgcgtgctc gacctcaagg 300 acggcgtgcg ttccagccgc atgagcgtcg agcaccacca ccaccaccac tgagatccgg 360 ctgctaac 368 ＜210＞ 30 ＜211＞ 941 ＜212＞ DNA ＜213＞ Artificial sequence ＜220＞ ＜223＞ partial sequence of plasmid ＜400＞ 30 gtttaacttt aagaaggaga tataccatgg ccgaacaatt ggtggacctc tggaacgaat 60 gcgccaaagc ctgcgtgctc gacctcaagg acggcgtgcg ttccagccgc atgagcgtcg 120 acccggccat cgccgacacc aacggccagg gcgtgctgca ctactgcatg gaattccata 180 tggtcgacgg tatgtccatc cgtgctaaac gtcgtaaacg tgcttccgct acccagctgt 240 acaaaacctg caaacaggct ggtacctgcc cgccggacat catcccgaaa gttgaaggta 300 aaaccatcgc tgaacagatc ctgcaatacg gttctatggg tgttttcttc ggcggtctgg 360 gcatcggtac cggttccggt actggcggtc gtaccggtta catcccgctg ggtacccgtc 420 cgccgaccgc taccgacacc ctggctccgg ttcgtccgcc gctgaccgtt gacccggttg 480 gtccgtccga cccgtccatc gtttccctgg ttgaagaaac ctccttcatc gacgctggtg 540 ctccgacctc cgttccgtcc atcccgccgg acgtttccgg tttctccatc accacctcca 600 ccgacactac cccggctatc ctggacatca acaacaacac cgttactacc gtaaccactc 660 acaacaaccc gaccttcacc gacccgtccg ttctgcaacc gccgaccccg gctgaaaccg 720 gtggtcactt caccctgtcc tcttccacca tctccaccca caactacgaa gaaatcccga 780 tggacaccaa agacgaactg ctcgagcata tggccgaaga agctttcgac ctctggaacg 840 aatgcgccaa agcctgcgtg ctcgacctca aggacggcgt gcgttccagc cgcatgagcg 900 tcgagcacca ccaccaccac cactgagatc cggctgctaa c 941 ＜210＞ 31 ＜211＞ 501 ＜212＞ PRT ＜213＞ Artificial sequence ＜220＞ ＜223＞ chimeric antigen ＜400＞ 31 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala Glu Glu Ala Phe Asp Leu Trp Asn Glu Cys 20 25 30 Ala Lys Ala Cys Val Leu Asp Leu Lys Asp Gly Val Arg Ser Ser Arg 35 40 45 Met Ser Val Asp Pro Ala Ile Ala Asp Thr Asn Gly Gln Gly Val Leu 50 55 60 His Tyr Ser Met Val Leu Glu Gly Gly Asn Asp Ala Leu Lys Leu Ala 65 70 75 80 Ile Asp Asn Ala Leu Ser Ile Thr Ser Asp Gly Leu Thr Ile Arg Leu 85 90 95 Glu Gly Gly Val Glu Pro Asn Lys Pro Val Arg Tyr Ser Tyr Thr Arg 100 105 110 Gln Ala Arg Gly Ser Trp Ser Leu Asn Trp Leu Val Pro Ile Gly His 115 120 125 Glu Lys Pro Ser Asn Ile Lys Val Phe Ile His Glu Leu Asn Ala Gly 130 135 140 Asn Gln Leu Ser His Met Ser Pro Ile Tyr Thr Ile Glu Met Gly Asp 145 150 155 160 Glu Leu Leu Ala Lys Leu Ala Arg Asp Ala Thr Phe Phe Val Arg Ala 165 170 175 His Glu Ser Asn Glu Met Gln Pro Thr Leu Ala Ile Ser His Ala Gly 180 185 190 Val Ser Val Val Met Ala Gln Thr Gln Pro Arg Arg Glu Lys Arg Trp 195 200 205 Ser Glu Trp Ala Ser Gly Lys Val Leu Cys Leu Leu Asp Pro Leu Asp 210 215 220 Gly Val Tyr Asn Tyr Leu Ala Gln Gln Arg Cys Asn Leu Asp Asp Thr 225 230 235 240 Trp Glu Gly Lys Ile Tyr Arg Val Leu Ala Gly Asn Pro Ala Lys His 245 250 255 Asp Leu Asp Ile Lys Pro Thr Val Ile Ser His Arg Leu His Phe Pro 260 265 270 Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys His Leu 275 280 285 Pro Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg Gly Trp Glu Gln 290 295 300 Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr Leu 305 310 315 320 Ala Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val Ile Arg Asn Ala 325 330 335 Leu Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu Ala Ile Arg Glu 340 345 350 Gln Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala Ala Ala Glu Ser 355 360 365 Glu Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu Ala Gly Ala Ala 370 375 380 Asn Ala Asp Val Val Ser Leu Thr Cys Pro Val Ala Ala Gly Glu Cys 385 390 395 400 Ala Gly Pro Ala Asp Ser Gly Asp Ala Leu Leu Glu Arg Asn Tyr Pro 405 410 415 Thr Gly Ala Glu Phe Leu Gly Asp Gly Gly Asp Val Ser Phe Ser Thr 420 425 430 Arg Gly Thr Gln Asn Trp Thr Val Glu Arg Leu Leu Gln Ala Glu Phe 435 440 445 Gln Leu Leu Arg Arg Glu Val Phe Cys Gly Phe Arg Asp Leu Val Tyr 450 455 460 Asp Phe Ala Phe Ser Asp Leu Lys Leu Pro Gln Leu Cys Thr Glu Leu 465 470 475 480 Lys Leu Pro Gln Leu Cys Thr Glu Leu Lys Asp Glu Leu Leu Glu His 485 490 495 His His His His His 500 ＜210＞ 32 ＜211＞ 500 ＜212＞ PRT ＜213＞ Artificial sequence ＜220＞ ＜223＞ chimeric antigen ＜400＞ 32 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala Glu Glu Ala Phe Asp Leu Trp Asn Glu Cys 20 25 30 Ala Lys Ala Cys Val Leu Asp Leu Lys Asp Gly Val Arg Ser Ser Arg 35 40 45 Met Ser Val Asp Pro Ala Ile Ala Asp Thr Asn Gly Gln Gly Val Leu 50 55 60 His Tyr Ser Met Val Leu Glu Gly Gly Asn Asp Ala Leu Lys Leu Ala 65 70 75 80 Ile Asp Asn Ala Leu Ser Ile Thr Ser Asp Gly Leu Thr Ile Arg Leu 85 90 95 Glu Gly Gly Val Glu Pro Asn Lys Pro Val Arg Tyr Ser Tyr Thr Arg 100 105 110 Gln Ala Arg Gly Ser Trp Ser Leu Asn Trp Leu Val Pro Ile Gly His 115 120 125 Glu Lys Pro Ser Asn Ile Lys Val Phe Ile His Glu Leu Asn Ala Gly 130 135 140 Asn Gln Leu Ser His Met Ser Pro Ile Tyr Thr Ile Glu Met Gly Asp 145 150 155 160 Glu Leu Leu Ala Lys Leu Ala Arg Asp Ala Thr Phe Phe Val Arg Ala 165 170 175 His Glu Ser Asn Glu Met Gln Pro Thr Leu Ala Ile Ser His Ala Gly 180 185 190 Val Ser Val Val Met Ala Gln Thr Gln Pro Arg Arg Glu Lys Arg Trp 195 200 205 Ser Glu Trp Ala Ser Gly Lys Val Leu Cys Leu Leu Asp Pro Leu Asp 210 215 220 Gly Val Tyr Asn Tyr Leu Ala Gln Gln Arg Cys Asn Leu Asp Asp Thr 225 230 235 240 Trp Glu Gly Lys Ile Tyr Arg Val Leu Ala Gly Asn Pro Ala Lys His 245 250 255 Asp Leu Asp Ile Lys Pro Thr Val Ile Ser His Arg Leu His Phe Pro 260 265 270 Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys His Leu 275 280 285 Pro Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg Gly Trp Glu Gln 290 295 300 Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr Leu 305 310 315 320 Ala Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val Ile Arg Asn Ala 325 330 335 Leu Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu Ala Ile Arg Glu 340 345 350 Gln Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala Ala Ala Glu Ser 355 360 365 Glu Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu Ala Gly Ala Ala 370 375 380 Asn Ala Asp Val Val Ser Leu Thr Cys Pro Val Ala Ala Gly Glu Cys 385 390 395 400 Ala Gly Pro Ala Asp Ser Gly Asp Ala Leu Leu Glu Arg Asn Tyr Pro 405 410 415 Thr Gly Ala Glu Phe Leu Gly Asp Gly Gly Asp Val Ser Phe Ser Thr 420 425 430 Arg Gly Thr Gln Asn Trp Thr Val Glu Arg Leu Leu Gln Ala Glu Phe 435 440 445 Gln Ala Glu Pro Asp Gln Ala Glu Pro Asp Arg Ala Arg Ala His Tyr 450 455 460 Asn Ile Arg Ala Arg Ala His Tyr Asn Leu Glu Ala His Tyr Asn Ile 465 470 475 480 Val Ile Phe Arg Ala His Tyr Asn Ile Val Ile Phe Leu Glu His His 485 490 495 His His His His 500 ＜210＞ 33 ＜211＞ 504 ＜212＞ PRT ＜213＞ Artificial sequence ＜220＞ ＜223＞ chimeric antigen ＜400＞ 33 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala Glu Glu Ala Phe Asp Leu Trp Asn Glu Cys 20 25 30 Ala Lys Ala Cys Val Leu Asp Leu Lys Asp Gly Val Arg Ser Ser Arg 35 40 45 Met Ser Val Asp Pro Ala Ile Ala Asp Thr Asn Gly Gln Gly Val Leu 50 55 60 His Tyr Ser Met Val Leu Glu Gly Gly Asn Asp Ala Leu Lys Leu Ala 65 70 75 80 Ile Asp Asn Ala Leu Ser Ile Thr Ser Asp Gly Leu Thr Ile Arg Leu 85 90 95 Glu Gly Gly Val Glu Pro Asn Lys Pro Val Arg Tyr Ser Tyr Thr Arg 100 105 110 Gln Ala Arg Gly Ser Trp Ser Leu Asn Trp Leu Val Pro Ile Gly His 115 120 125 Glu Lys Pro Ser Asn Ile Lys Val Phe Ile His Glu Leu Asn Ala Gly 130 135 140 Asn Gln Leu Ser His Met Ser Pro Ile Tyr Thr Ile Glu Met Gly Asp 145 150 155 160 Glu Leu Leu Ala Lys Leu Ala Arg Asp Ala Thr Phe Phe Val Arg Ala 165 170 175 His Glu Ser Asn Glu Met Gln Pro Thr Leu Ala Ile Ser His Ala Gly 180 185 190 Val Ser Val Val Met Ala Gln Thr Gln Pro Arg Arg Glu Lys Arg Trp 195 200 205 Ser Glu Trp Ala Ser Gly Lys Val Leu Cys Leu Leu Asp Pro Leu Asp 210 215 220 Gly Val Tyr Asn Tyr Leu Ala Gln Gln Arg Cys Asn Leu Asp Asp Thr 225 230 235 240 Trp Glu Gly Lys Ile Tyr Arg Val Leu Ala Gly Asn Pro Ala Lys His 245 250 255 Asp Leu Asp Ile Lys Pro Thr Val Ile Ser His Arg Leu His Phe Pro 260 265 270 Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys His Leu 275 280 285 Pro Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg Gly Trp Glu Gln 290 295 300 Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr Leu 305 310 315 320 Ala Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val Ile Arg Asn Ala 325 330 335 Leu Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu Ala Ile Arg Glu 340 345 350 Gln Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala Ala Ala Glu Ser 355 360 365 Glu Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu Ala Gly Ala Ala 370 375 380 Asn Ala Asp Val Val Ser Leu Thr Cys Pro Val Ala Ala Gly Glu Cys 385 390 395 400 Ala Gly Pro Ala Asp Ser Gly Asp Ala Leu Leu Glu Arg Asn Tyr Pro 405 410 415 Thr Gly Ala Glu Phe Leu Gly Asp Gly Gly Asp Val Glu Phe Val Phe 420 425 430 Leu Gln Lys Tyr Pro His Thr His Leu Val His Gln Ala Leu Asp Val 435 440 445 Phe Leu Gln Lys Tyr Pro His Thr His Leu Val His Gln Ala Leu Asp 450 455 460 Val Phe Leu Gln Lys Tyr Pro His Thr His Leu Val His Gln Ala Leu 465 470 475 480 Asp Val Phe Leu Gln Lys Tyr Pro His Thr His Leu Val His Gln Ala 485 490 495 Leu Glu His His His His His His 500 ＜210＞ 34 ＜211＞ 711 ＜212＞ PRT ＜213＞ Artificial sequence ＜220＞ ＜223＞ chimeric antigen ＜400＞ 34 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala Glu Glu Ala Phe Asp Leu Trp Asn Glu Cys 20 25 30 Ala Lys Ala Cys Val Leu Asp Leu Lys Asp Gly Val Arg Ser Ser Arg 35 40 45 Met Ser Val Asp Pro Ala Ile Ala Asp Thr Asn Gly Gln Gly Val Leu 50 55 60 His Tyr Ser Met Val Leu Glu Gly Gly Asn Asp Ala Leu Lys Leu Ala 65 70 75 80 Ile Asp Asn Ala Leu Ser Ile Thr Ser Asp Gly Leu Thr Ile Arg Leu 85 90 95 Glu Gly Gly Val Glu Pro Asn Lys Pro Val Arg Tyr Ser Tyr Thr Arg 100 105 110 Gln Ala Arg Gly Ser Trp Ser Leu Asn Trp Leu Val Pro Ile Gly His 115 120 125 Glu Lys Pro Ser Asn Ile Lys Val Phe Ile His Glu Leu Asn Ala Gly 130 135 140 Asn Gln Leu Ser His Met Ser Pro Ile Tyr Thr Ile Glu Met Gly Asp 145 150 155 160 Glu Leu Leu Ala Lys Leu Ala Arg Asp Ala Thr Phe Phe Val Arg Ala 165 170 175 His Glu Ser Asn Glu Met Gln Pro Thr Leu Ala Ile Ser His Ala Gly 180 185 190 Val Ser Val Val Met Ala Gln Thr Gln Pro Arg Arg Glu Lys Arg Trp 195 200 205 Ser Glu Trp Ala Ser Gly Lys Val Leu Cys Leu Leu Asp Pro Leu Asp 210 215 220 Gly Val Tyr Asn Tyr Leu Ala Gln Gln Arg Cys Asn Leu Asp Asp Thr 225 230 235 240 Trp Glu Gly Lys Ile Tyr Arg Val Leu Ala Gly Asn Pro Ala Lys His 245 250 255 Asp Leu Asp Ile Lys Pro Thr Val Ile Ser His Arg Leu His Phe Pro 260 265 270 Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys His Leu 275 280 285 Pro Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg Gly Trp Glu Gln 290 295 300 Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr Leu 305 310 315 320 Ala Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val Ile Arg Asn Ala 325 330 335 Leu Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu Ala Ile Arg Glu 340 345 350 Gln Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala Ala Ala Glu Ser 355 360 365 Glu Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu Ala Gly Ala Ala 370 375 380 Asn Ala Asp Val Val Ser Leu Thr Cys Pro Val Ala Ala Gly Glu Cys 385 390 395 400 Ala Gly Pro Ala Asp Ser Gly Asp Ala Leu Leu Glu Arg Asn Tyr Pro 405 410 415 Thr Gly Ala Glu Phe Leu Gly Asp Gly Gly Asp Val Glu Phe Leu Leu 420 425 430 Glu Pro His Thr His Leu Val His Gln Ala Asn Val Leu Leu Ala Leu 435 440 445 Gln Leu Leu Leu Glu Asp Arg Glu Phe Val Phe Leu Gln Lys Tyr Pro 450 455 460 His Val Glu Pro His Thr His Leu Val His Gln Ala Asn Val Leu Leu 465 470 475 480 Ala Leu Gln Leu Leu Leu Glu Asp Arg Glu Phe Val Phe Leu Gln Lys 485 490 495 Tyr Pro His Val Glu Pro His Thr His Leu Val His Gln Ala Asn Val 500 505 510 Leu Leu Ala Leu Gln Leu Leu Leu Glu Asp Arg Glu Phe Val Phe Leu 515 520 525 Gln Lys Tyr Pro His Val Glu Pro His Thr His Leu Val His Gln Ala 530 535 540 Asn Val Leu Leu Ala Leu Gln Leu Leu Leu Glu Asp Arg Glu Phe Val 545 550 555 560 Phe Leu Gln Lys Tyr Pro His Val Glu Pro His Thr His Leu Val His 565 570 575 Gln Ala Asn Val Leu Leu Ala Leu Gln Leu Leu Leu Glu Asp Arg Glu 580 585 590 Phe Val Phe Leu Gln Lys Tyr Pro His Val Glu Pro His Thr His Leu 595 600 605 Val His Gln Ala Asn Val Leu Leu Ala Leu Gln Leu Leu Leu Glu Asp 610 615 620 Arg Glu Phe Val Phe Leu Gln Lys Tyr Pro His Val Glu Pro His Thr 625 630 635 640 His Leu Val His Gln Ala Asn Val Leu Leu Ala Leu Gln Leu Leu Leu 645 650 655 Glu Asp Arg Glu Phe Val Phe Leu Gln Lys Tyr Pro His Val Glu Pro 660 665 670 His Thr His Leu Val His Gln Ala Asn Val Leu Leu Ala Leu Gln Leu 675 680 685 Leu Leu Glu Asp Arg Glu Phe Val Phe Leu Gln Lys Tyr Pro His Val 690 695 700 Glu His His His His His His 705 710 ＜210＞ 35 ＜211＞ 749 ＜212＞ PRT ＜213＞ Artificial sequence ＜220＞ ＜223＞ chimeric antigen ＜400＞ 35 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala Glu Glu Ala Phe Asp Leu Trp Asn Glu Cys 20 25 30 Ala Lys Ala Cys Val Leu Asp Leu Lys Asp Gly Val Arg Ser Ser Arg 35 40 45 Met Ser Val Asp Pro Ala Ile Ala Asp Thr Asn Gly Gln Gly Val Leu 50 55 60 His Tyr Ser Met Val Leu Glu Gly Gly Asn Asp Ala Leu Lys Leu Ala 65 70 75 80 Ile Asp Asn Ala Leu Ser Ile Thr Ser Asp Gly Leu Thr Ile Arg Leu 85 90 95 Glu Gly Gly Val Glu Pro Asn Lys Pro Val Arg Tyr Ser Tyr Thr Arg 100 105 110 Gln Ala Arg Gly Ser Trp Ser Leu Asn Trp Leu Val Pro Ile Gly His 115 120 125 Glu Lys Pro Ser Asn Ile Lys Val Phe Ile His Glu Leu Asn Ala Gly 130 135 140 Asn Gln Leu Ser His Met Ser Pro Ile Tyr Thr Ile Glu Met Gly Asp 145 150 155 160 Glu Leu Leu Ala Lys Leu Ala Arg Asp Ala Thr Phe Phe Val Arg Ala 165 170 175 His Glu Ser Asn Glu Met Gln Pro Thr Leu Ala Ile Ser His Ala Gly 180 185 190 Val Ser Val Val Met Ala Gln Thr Gln Pro Arg Arg Glu Lys Arg Trp 195 200 205 Ser Glu Trp Ala Ser Gly Lys Val Leu Cys Leu Leu Asp Pro Leu Asp 210 215 220 Gly Val Tyr Asn Tyr Leu Ala Gln Gln Arg Cys Asn Leu Asp Asp Thr 225 230 235 240 Trp Glu Gly Lys Ile Tyr Arg Val Leu Ala Gly Asn Pro Ala Lys His 245 250 255 Asp Leu Asp Ile Lys Pro Thr Val Ile Ser His Arg Leu His Phe Pro 260 265 270 Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys His Leu 275 280 285 Pro Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg Gly Trp Glu Gln 290 295 300 Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr Leu 305 310 315 320 Ala Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val Ile Arg Asn Ala 325 330 335 Leu Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu Ala Ile Arg Glu 340 345 350 Gln Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala Ala Ala Glu Ser 355 360 365 Glu Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu Ala Gly Ala Ala 370 375 380 Asn Ala Asp Val Val Ser Leu Thr Cys Pro Val Ala Ala Gly Glu Cys 385 390 395 400 Ala Gly Pro Ala Asp Ser Gly Asp Ala Leu Leu Glu Arg Asn Tyr Pro 405 410 415 Thr Gly Ala Glu Phe Leu Gly Asp Gly Gly Asp Val Ser Phe Ser Thr 420 425 430 Arg Gly Thr Gln Asn Trp Thr Val Glu Arg Leu Leu Gln Ala Glu Phe 435 440 445 Val Ser Phe Ser Thr Gly Gly Ser Gln Asn Trp Thr Val Glu Arg Leu 450 455 460 Leu Gln Ala Glu Phe Cys Ser Thr Ser Gln Ala Ala Arg Gln Arg Leu 465 470 475 480 Glu Thr Gly Arg Asn Cys Ser Thr Gly Gln Ala Ala Arg Gln Arg Leu 485 490 495 Glu Pro Gly Arg Asn Leu Val Leu Cys Leu Thr Ser Gln Ala Ala Gln 500 505 510 Gln Arg Leu Glu Pro Gly Gly Asn Cys Gln Thr Ser Gln Ala Ala His 515 520 525 Gln Arg Leu Glu Pro Gly Arg Asn Cys Arg Thr Ser Gln Ala Ala Ser 530 535 540 Gln Arg Leu Glu Pro Gly Arg Asn Cys Arg Thr Ser Gln Ala Ala His 545 550 555 560 Gln Arg Leu Glu Pro Gly Arg Asn Cys Ser Thr Arg Gln Ala Ala Gln 565 570 575 Gln Arg Leu Glu Pro Gly Arg Asn Leu Leu Cys Pro Thr Ser Gln Ala 580 585 590 Ala His Gln Arg Arg Leu Glu Pro Gly Arg Asn Cys Ser Thr Ser Gln 595 600 605 Ala Ala Tyr Gln Arg Leu Glu Pro Gly Arg Asn Cys Pro Thr Ser Arg 610 615 620 Ala Ala Arg Gln Arg Leu Glu Pro Gly Arg Asn Leu Leu Cys Ser Thr 625 630 635 640 Ser Gln Ala Ala Leu Gln Arg Leu Glu Pro Gly Arg Asn Leu Cys Pro 645 650 655 Thr Ser Gln Ala Ala Lys Gln Arg Leu Glu Pro Gly Arg Asn Leu Val 660 665 670 Val Cys Leu Thr Ser Gln Ala Ala Arg Gln Arg Leu Glu Pro Gly Arg 675 680 685 Asn Cys Ser Thr Ser Gln Ala Ala Ser Gln Arg Leu Glu Pro Gly Arg 690 695 700 Asn Cys Pro Thr Ser Gln Ala Ala Arg Gln Arg Leu Glu Pro Gly Arg 705 710 715 720 Asn Val Leu Leu Leu Cys Leu Thr Ser Gln Ala Ala His Gln Arg Leu 725 730 735 Glu Pro Gly Arg Asn Leu Glu His His His His His His 740 745 ＜210＞ 36 ＜211＞ 682 ＜212＞ PRT ＜213＞ Artificial sequence ＜220＞ ＜223＞ chimeric antigen ＜400＞ 36 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala Glu Glu Ala Phe Asp Leu Trp Asn Glu Cys 20 25 30 Ala Lys Ala Cys Val Leu Asp Leu Lys Asp Gly Val Arg Ser Ser Arg 35 40 45 Met Ser Val Asp Pro Ala Ile Ala Asp Thr Asn Gly Gln Gly Val Leu 50 55 60 His Tyr Ser Met Val Leu Glu Gly Gly Asn Asp Ala Leu Lys Leu Ala 65 70 75 80 Ile Asp Asn Ala Leu Ser Ile Thr Ser Asp Gly Leu Thr Ile Arg Leu 85 90 95 Glu Gly Gly Val Glu Pro Asn Lys Pro Val Arg Tyr Ser Tyr Thr Arg 100 105 110 Gln Ala Arg Gly Ser Trp Ser Leu Asn Trp Leu Val Pro Ile Gly His 115 120 125 Glu Lys Pro Ser Asn Ile Lys Val Phe Ile His Glu Leu Asn Ala Gly 130 135 140 Asn Gln Leu Ser His Met Ser Pro Ile Tyr Thr Ile Glu Met Gly Asp 145 150 155 160 Glu Leu Leu Ala Lys Leu Ala Arg Asp Ala Thr Phe Phe Val Arg Ala 165 170 175 His Glu Ser Asn Glu Met Gln Pro Thr Leu Ala Ile Ser His Ala Gly 180 185 190 Val Ser Val Val Met Ala Gln Thr Gln Pro Arg Arg Glu Lys Arg Trp 195 200 205 Ser Glu Trp Ala Ser Gly Lys Val Leu Cys Leu Leu Asp Pro Leu Asp 210 215 220 Gly Val Tyr Asn Tyr Leu Ala Gln Gln Arg Cys Asn Leu Asp Asp Thr 225 230 235 240 Trp Glu Gly Lys Ile Tyr Arg Val Leu Ala Gly Asn Pro Ala Lys His 245 250 255 Asp Leu Asp Ile Lys Pro Thr Val Ile Ser His Arg Leu His Phe Pro 260 265 270 Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys His Leu 275 280 285 Pro Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg Gly Trp Glu Gln 290 295 300 Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr Leu 305 310 315 320 Ala Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val Ile Arg Asn Ala 325 330 335 Leu Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu Ala Ile Arg Glu 340 345 350 Gln Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala Ala Ala Glu Ser 355 360 365 Glu Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu Ala Gly Ala Ala 370 375 380 Asn Ala Asp Val Val Ser Leu Thr Cys Pro Val Ala Ala Gly Glu Cys 385 390 395 400 Ala Gly Pro Ala Asp Ser Gly Asp Ala Leu Leu Glu Arg Asn Tyr Pro 405 410 415 Thr Gly Ala Glu Phe Leu Gly Asp Gly Gly Asp Val Ser Phe Ser Thr 420 425 430 Arg Gly Thr Gln Asn Trp Thr Val Glu Arg Leu Leu Gln Ala Glu Phe 435 440 445 Gly Val Ser Ala Ala Gln Glu Lys Ile Ser Phe Gly Leu Leu Gly Val 450 455 460 Pro Thr Ala Gln Glu Thr Thr Ser Ile Arg Glu Val Leu Glu Val Ser 465 470 475 480 Thr Ala Gln Glu Asn Ser Pro Phe Met Leu Gly Ala Ser Ala Thr Glu 485 490 495 Glu Lys Thr Ser Leu Arg Leu Gly Ala Ser Thr Thr Gln Glu Thr Ser 500 505 510 Phe Gly Lys Cys Leu Arg Pro His Gly Val Ser Ala Ala Gln Gly Thr 515 520 525 Thr Pro Phe Arg Gly Val Ser Thr Thr Gln Glu Asn Thr Ser Phe Gly 530 535 540 Arg Val Pro Thr Ala Gln Glu Asn Val Ser Phe Gly Leu His Gly Val 545 550 555 560 Pro Ala Ala Gln Lys Thr Asn Ser Phe Gly Gly Val Pro Thr Ala Gln 565 570 575 Glu Asn Ile Ser Phe Lys Glu Val Ser Ala Thr Gln Arg Glu Ile Pro 580 585 590 Phe Arg Cys Leu Arg Pro His Gly Val Ser Thr Ala Gln Glu Thr Pro 595 600 605 Phe Arg Gly Val Ser Thr Ala Gln Glu Thr Ile Pro Phe Arg Gly Val 610 615 620 Ser Ala Thr His Glu Asn Ile Ser Phe Gly Cys Leu Arg Pro His Gly 625 630 635 640 Val Ser Ala Ala Gln Glu Ser Ile Pro Ile Arg Leu Gly Ala Ser Ala 645 650 655 Ala Gln Glu Asn Thr Ser Phe Arg Gly Thr Pro Ala Ala Gln Glu Lys 660 665 670 Ile Pro Leu Glu His His His His His His 675 680 ＜210＞ 37 ＜211＞ 741 ＜212＞ PRT ＜213＞ Artificial sequence ＜220＞ ＜223＞ chimeric antigen ＜400＞ 37 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala Glu Glu Ala Phe Asp Leu Trp Asn Glu Cys 20 25 30 Ala Lys Ala Cys Val Leu Asp Leu Lys Asp Gly Val Arg Ser Ser Arg 35 40 45 Met Ser Val Asp Pro Ala Ile Ala Asp Thr Asn Gly Gln Gly Val Leu 50 55 60 His Tyr Ser Met Val Leu Glu Gly Gly Asn Asp Ala Leu Lys Leu Ala 65 70 75 80 Ile Asp Asn Ala Leu Ser Ile Thr Ser Asp Gly Leu Thr Ile Arg Leu 85 90 95 Glu Gly Gly Val Glu Pro Asn Lys Pro Val Arg Tyr Ser Tyr Thr Arg 100 105 110 Gln Ala Arg Gly Ser Trp Ser Leu Asn Trp Leu Val Pro Ile Gly His 115 120 125 Glu Lys Pro Ser Asn Ile Lys Val Phe Ile His Glu Leu Asn Ala Gly 130 135 140 Asn Gln Leu Ser His Met Ser Pro Ile Tyr Thr Ile Glu Met Gly Asp 145 150 155 160 Glu Leu Leu Ala Lys Leu Ala Arg Asp Ala Thr Phe Phe Val Arg Ala 165 170 175 His Glu Ser Asn Glu Met Gln Pro Thr Leu Ala Ile Ser His Ala Gly 180 185 190 Val Ser Val Val Met Ala Gln Thr Gln Pro Arg Arg Glu Lys Arg Trp 195 200 205 Ser Glu Trp Ala Ser Gly Lys Val Leu Cys Leu Leu Asp Pro Leu Asp 210 215 220 Gly Val Tyr Asn Tyr Leu Ala Gln Gln Arg Cys Asn Leu Asp Asp Thr 225 230 235 240 Trp Glu Gly Lys Ile Tyr Arg Val Leu Ala Gly Asn Pro Ala Lys His 245 250 255 Asp Leu Asp Ile Lys Pro Thr Val Ile Ser His Arg Leu His Phe Pro 260 265 270 Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys His Leu 275 280 285 Pro Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg Gly Trp Glu Gln 290 295 300 Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr Leu 305 310 315 320 Ala Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val Ile Arg Asn Ala 325 330 335 Leu Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu Ala Ile Arg Glu 340 345 350 Gln Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala Ala Ala Glu Ser 355 360 365 Glu Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu Ala Gly Ala Ala 370 375 380 Asn Ala Asp Val Val Ser Leu Thr Cys Pro Val Ala Ala Gly Glu Cys 385 390 395 400 Ala Gly Pro Ala Asp Ser Gly Asp Ala Leu Leu Glu Arg Asn Tyr Pro 405 410 415 Thr Gly Ala Glu Phe Leu Gly Asp Gly Gly Asp Val Ser Phe Ser Thr 420 425 430 Arg Gly Thr Gln Asn Trp Thr Val Glu Arg Leu Leu Gln Ala Glu Phe 435 440 445 Leu Gly Val Ser Ala Ala Gln Glu Arg Ile Pro Ile Arg Glu Val Ser 450 455 460 Ala Asp Lys Glu Val Ser Ala Glu Lys Lys Glu Ile Ser Phe Gly Val 465 470 475 480 Ser Thr Ala Gln Gly Asn Ile Ser Phe Gly Leu Gly Val Ser Thr Ala 485 490 495 Gln Glu Ala Ile Pro Phe Leu Ala Leu Gly Val Ser Thr Ala Gln Glu 500 505 510 Thr Ile Pro Phe Gly Leu Leu Gly Val Ser Thr Ala Gln Gly Ile Ile 515 520 525 Ser Phe Gly Gly Val Ser Thr Ala Gln Glu Asn Ile Ser Phe Gly Gly 530 535 540 Val Ser Thr Ala Gln Glu Thr Ile Ser Phe Gly Leu Leu Gly Val Ser 545 550 555 560 Thr Ala Gln Glu Asn Ile Ser Phe Gly Cys Leu Arg Thr His Glu Val 565 570 575 Ser Ala Ala Gln Glu Lys Ile Ser Phe Gly Gly Val Ser Glu Ala Gln 580 585 590 Lys Ile Ser Phe Gly Val Ser Ala Ala Gly Val Ser Ala Ala Gln Glu 595 600 605 Glu Ile Pro Phe Gly Cys Leu Arg Pro His Gly Leu Pro Ala Ala Gln 610 615 620 Glu Lys Thr Ser Phe Gly Gly Val Ser Ala Ala Gln Glu Lys Thr Ser 625 630 635 640 Phe Gly Gly Val Ser Ala Ala Gln Glu Glu Phe Ser Phe Gly Cys Leu 645 650 655 Arg Pro His Arg Val Ser Ala Ala Gln Glu Lys Ile Ser Phe Glu Val 660 665 670 Ser Ala Leu Glu Val Ser Ala Ala Gln Glu Lys Ile Ser Phe Gly Val 675 680 685 Ser Ala Ala Leu Gly Val Ser Ala Ala Gln Glu Lys Asn Ser Phe Gly 690 695 700 Cys Leu Arg Pro His Gly Val Ser Ala Ala Gln Glu Lys Thr Ser Phe 705 710 715 720 Gly Gly Val Ser Ala Ala Gln Lys Lys Ile Ser Phe Gly Leu Glu His 725 730 735 His His His His His 740 ＜210＞ 38 ＜211＞ 595 ＜212＞ PRT ＜213＞ Artificial sequence ＜220＞ ＜223＞ fusion biogenic polypeptide ＜400＞ 38 Met Ala Glu Gln Leu Val Asp Leu Trp Asn Glu Cys Ala Lys Ala Cys 1 5 10 15 Val Leu Asp Leu Lys Asp Gly Val Arg Ser Ser Arg Met Ser Val Asp 20 25 30 Pro Ala Ile Ala Asp Thr Asn Gly Gln Gly Val Leu His Tyr Cys Met 35 40 45 Glu Phe Ser Phe His Gln Leu Pro Ala Arg Ser Pro Ala Pro Leu Gln 50 55 60 Leu Glu His Met Ala Glu Glu Ala Phe Asp Leu Trp Asn Glu Cys Ala 65 70 75 80 Lys Ala Cys Val Leu Asp Leu Lys Asp Gly Val Arg Ser Ser Arg Met 85 90 95 Ser Val Asp Pro Ala Ile Ala Asp Thr Asn Gly Gln Gly Val Leu His 100 105 110 Tyr Ser Met Val Leu Glu Gly Gly Asn Asp Ala Leu Lys Leu Ala Ile 115 120 125 Asp Asn Ala Leu Ser Ile Thr Ser Asp Gly Leu Thr Ile Arg Leu Glu 130 135 140 Gly Gly Val Glu Pro Asn Lys Pro Val Arg Tyr Ser Tyr Thr Arg Gln 145 150 155 160 Ala Arg Gly Ser Trp Ser Leu Asn Trp Leu Val Pro Ile Gly His Glu 165 170 175 Lys Pro Ser Asn Ile Lys Val Phe Ile His Glu Leu Asn Ala Gly Asn 180 185 190 Gln Leu Ser His Met Ser Pro Ile Tyr Thr Ile Glu Met Gly Asp Glu 195 200 205 Leu Leu Ala Lys Leu Ala Arg Asp Ala Thr Phe Phe Val Arg Ala His 210 215 220 Glu Ser Asn Glu Met Gln Pro Thr Leu Ala Ile Ser His Ala Gly Val 225 230 235 240 Ser Val Val Met Ala Gln Thr Gln Pro Arg Arg Glu Lys Arg Trp Ser 245 250 255 Glu Trp Ala Ser Gly Lys Val Leu Cys Leu Leu Asp Pro Leu Asp Gly 260 265 270 Val Tyr Asn Tyr Leu Ala Gln Gln Arg Cys Asn Leu Asp Asp Thr Trp 275 280 285 Glu Gly Lys Ile Tyr Arg Val Leu Ala Gly Asn Pro Ala Lys His Asp 290 295 300 Leu Asp Ile Lys Pro Thr Val Ile Ser His Arg Leu His Phe Pro Glu 305 310 315 320 Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys His Leu Pro 325 330 335 Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg Gly Trp Glu Gln Leu 340 345 350 Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr Leu Ala 355 360 365 Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val Ile Arg Asn Ala Leu 370 375 380 Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu Ala Ile Arg Glu Gln 385 390 395 400 Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala Ala Ala Glu Ser Glu 405 410 415 Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu Ala Gly Ala Ala Asn 420 425 430 Ala Asp Val Val Ser Leu Thr Cys Pro Val Ala Ala Gly Glu Cys Ala 435 440 445 Gly Pro Ala Asp Ser Gly Asp Ala Leu Leu Glu Arg Asn Tyr Pro Thr 450 455 460 Gly Ala Glu Phe Leu Gly Asp Gly Gly Asp Val Ser Phe Ser Thr Arg 465 470 475 480 Gly Thr Gln Asn Trp Thr Val Glu Arg Leu Leu Gln Ala Glu Phe Val 485 490 495 Asp Lys Asp Glu Leu Arg Glu Val Tyr Asn Phe Ala Phe Leu Leu Val 500 505 510 Leu Arg Arg Glu Val Tyr Asp Lys Asp Glu Leu Leu Leu Leu Leu Glu 515 520 525 Asp Arg Gln Leu Leu Arg Arg Glu Val Phe Cys Gly Phe Arg Asp Leu 530 535 540 Leu Glu Asp Arg Val Tyr Asp Phe Ala Phe Ser Asp Leu Lys Leu Pro 545 550 555 560 Gln Leu Cys Thr Glu Leu Lys Leu Pro Gln Leu Cys Thr Glu Leu Lys 565 570 575 Asp Glu Leu Lys Asp Glu Leu Val Leu Leu Leu Leu Glu His His His 580 585 590 His His His 595 ＜210＞ 39 ＜211＞ 603 ＜212＞ PRT ＜213＞ Artificial sequence ＜220＞ ＜223＞ fusion biogenic polypeptide ＜400＞ 39 Met Ala Glu Gln Leu Val Asp Leu Trp Asn Glu Cys Ala Lys Ala Cys 1 5 10 15 Val Leu Asp Leu Lys Asp Gly Val Arg Ser Ser Arg Met Ser Val Asp 20 25 30 Pro Ala Ile Ala Asp Thr Asn Gly Gln Gly Val Leu His Tyr Cys Met 35 40 45 Glu Phe Ser Ser Phe His Leu Phe His His Leu Pro Ala Arg Ala Pro 50 55 60 Leu Ala Pro Ser Glu Leu Gln Pro Leu Glu His Met Ala Glu Glu Ala 65 70 75 80 Phe Asp Leu Trp Asn Glu Cys Ala Lys Ala Cys Val Leu Asp Leu Lys 85 90 95 Asp Gly Val Arg Ser Ser Arg Met Ser Val Asp Pro Ala Ile Ala Asp 100 105 110 Thr Asn Gly Gln Gly Val Leu His Tyr Ser Met Val Leu Glu Gly Gly 115 120 125 Asn Asp Ala Leu Lys Leu Ala Ile Asp Asn Ala Leu Ser Ile Thr Ser 130 135 140 Asp Gly Leu Thr Ile Arg Leu Glu Gly Gly Val Glu Pro Asn Lys Pro 145 150 155 160 Val Arg Tyr Ser Tyr Thr Arg Gln Ala Arg Gly Ser Trp Ser Leu Asn 165 170 175 Trp Leu Val Pro Ile Gly His Glu Lys Pro Ser Asn Ile Lys Val Phe 180 185 190 Ile His Glu Leu Asn Ala Gly Asn Gln Leu Ser His Met Ser Pro Ile 195 200 205 Tyr Thr Ile Glu Met Gly Asp Glu Leu Leu Ala Lys Leu Ala Arg Asp 210 215 220 Ala Thr Phe Phe Val Arg Ala His Glu Ser Asn Glu Met Gln Pro Thr 225 230 235 240 Leu Ala Ile Ser His Ala Gly Val Ser Val Val Met Ala Gln Thr Gln 245 250 255 Pro Arg Arg Glu Lys Arg Trp Ser Glu Trp Ala Ser Gly Lys Val Leu 260 265 270 Cys Leu Leu Asp Pro Leu Asp Gly Val Tyr Asn Tyr Leu Ala Gln Gln 275 280 285 Arg Cys Asn Leu Asp Asp Thr Trp Glu Gly Lys Ile Tyr Arg Val Leu 290 295 300 Ala Gly Asn Pro Ala Lys His Asp Leu Asp Ile Lys Pro Thr Val Ile 305 310 315 320 Ser His Arg Leu His Phe Pro Glu Gly Gly Ser Leu Ala Ala Leu Thr 325 330 335 Ala His Gln Ala Cys His Leu Pro Leu Glu Thr Phe Thr Arg His Arg 340 345 350 Gln Pro Arg Gly Trp Glu Gln Leu Glu Gln Cys Gly Tyr Pro Val Gln 355 360 365 Arg Leu Val Ala Leu Tyr Leu Ala Ala Arg Leu Ser Trp Asn Gln Val 370 375 380 Asp Gln Val Ile Arg Asn Ala Leu Ala Ser Pro Gly Ser Gly Gly Asp 385 390 395 400 Leu Gly Glu Ala Ile Arg Glu Gln Pro Glu Gln Ala Arg Leu Ala Leu 405 410 415 Thr Leu Ala Ala Ala Glu Ser Glu Arg Phe Val Arg Gln Gly Thr Gly 420 425 430 Asn Asp Glu Ala Gly Ala Ala Asn Ala Asp Val Val Ser Leu Thr Cys 435 440 445 Pro Val Ala Ala Gly Glu Cys Ala Gly Pro Ala Asp Ser Gly Asp Ala 450 455 460 Leu Leu Glu Arg Asn Tyr Pro Thr Gly Ala Glu Phe Leu Gly Asp Gly 465 470 475 480 Gly Asp Val Ser Phe Ser Thr Arg Gly Thr Gln Asn Trp Thr Val Glu 485 490 495 Arg Leu Leu Gln Ala Glu Phe Val Asp Lys Asp Glu Leu Arg Glu Val 500 505 510 Tyr Asn Phe Ala Phe Leu Leu Val Leu Arg Arg Glu Val Tyr Asp Lys 515 520 525 Asp Glu Leu Leu Leu Leu Leu Glu Asp Arg Gln Leu Leu Arg Arg Glu 530 535 540 Val Phe Cys Gly Phe Arg Asp Leu Leu Glu Asp Arg Val Tyr Asp Phe 545 550 555 560 Ala Phe Ser Asp Leu Lys Leu Pro Gln Leu Cys Thr Glu Leu Lys Leu 565 570 575 Pro Gln Leu Cys Thr Glu Leu Lys Asp Glu Leu Lys Asp Glu Leu Val 580 585 590 Leu Leu Leu Leu Glu His His His His His His 595 600 ＜210＞ 40 ＜211＞ 598 ＜212＞ PRT ＜213＞ Artificial sequence ＜220＞ ＜223＞ fusion biogenic polypeptide ＜400＞ 40 Met Ala Glu Gln Leu Val Asp Leu Trp Asn Glu Cys Ala Lys Ala Cys 1 5 10 15 Val Leu Asp Leu Lys Asp Gly Val Arg Ser Ser Arg Met Ser Val Asp 20 25 30 Pro Ala Ile Ala Asp Thr Asn Gly Gln Gly Val Leu His Tyr Cys Met 35 40 45 Glu Phe Ser Thr Pro Phe His Pro Leu Pro Ala Arg Lys Pro Leu Pro 50 55 60 Leu Val Pro Leu Glu His Met Ala Glu Glu Ala Phe Asp Leu Trp Asn 65 70 75 80 Glu Cys Ala Lys Ala Cys Val Leu Asp Leu Lys Asp Gly Val Arg Ser 85 90 95 Ser Arg Met Ser Val Asp Pro Ala Ile Ala Asp Thr Asn Gly Gln Gly 100 105 110 Val Leu His Tyr Ser Met Val Leu Glu Gly Gly Asn Asp Ala Leu Lys 115 120 125 Leu Ala Ile Asp Asn Ala Leu Ser Ile Thr Ser Asp Gly Leu Thr Ile 130 135 140 Arg Leu Glu Gly Gly Val Glu Pro Asn Lys Pro Val Arg Tyr Ser Tyr 145 150 155 160 Thr Arg Gln Ala Arg Gly Ser Trp Ser Leu Asn Trp Leu Val Pro Ile 165 170 175 Gly His Glu Lys Pro Ser Asn Ile Lys Val Phe Ile His Glu Leu Asn 180 185 190 Ala Gly Asn Gln Leu Ser His Met Ser Pro Ile Tyr Thr Ile Glu Met 195 200 205 Gly Asp Glu Leu Leu Ala Lys Leu Ala Arg Asp Ala Thr Phe Phe Val 210 215 220 Arg Ala His Glu Ser Asn Glu Met Gln Pro Thr Leu Ala Ile Ser His 225 230 235 240 Ala Gly Val Ser Val Val Met Ala Gln Thr Gln Pro Arg Arg Glu Lys 245 250 255 Arg Trp Ser Glu Trp Ala Ser Gly Lys Val Leu Cys Leu Leu Asp Pro 260 265 270 Leu Asp Gly Val Tyr Asn Tyr Leu Ala Gln Gln Arg Cys Asn Leu Asp 275 280 285 Asp Thr Trp Glu Gly Lys Ile Tyr Arg Val Leu Ala Gly Asn Pro Ala 290 295 300 Lys His Asp Leu Asp Ile Lys Pro Thr Val Ile Ser His Arg Leu His 305 310 315 320 Phe Pro Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys 325 330 335 His Leu Pro Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg Gly Trp 340 345 350 Glu Gln Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala Leu 355 360 365 Tyr Leu Ala Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val Ile Arg 370 375 380 Asn Ala Leu Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu Ala Ile 385 390 395 400 Arg Glu Gln Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala Ala Ala 405 410 415 Glu Ser Glu Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu Ala Gly 420 425 430 Ala Ala Asn Ala Asp Val Val Ser Leu Thr Cys Pro Val Ala Ala Gly 435 440 445 Glu Cys Ala Gly Pro Ala Asp Ser Gly Asp Ala Leu Leu Glu Arg Asn 450 455 460 Tyr Pro Thr Gly Ala Glu Phe Leu Gly Asp Gly Gly Asp Val Ser Phe 465 470 475 480 Ser Thr Arg Gly Thr Gln Asn Trp Thr Val Glu Arg Leu Leu Gln Ala 485 490 495 Glu Phe Val Asp Lys Asp Glu Leu Arg Glu Val Tyr Asn Phe Ala Phe 500 505 510 Leu Leu Val Leu Arg Arg Glu Val Tyr Asp Lys Asp Glu Leu Leu Leu 515 520 525 Leu Leu Glu Asp Arg Gln Leu Leu Arg Arg Glu Val Phe Cys Gly Phe 530 535 540 Arg Asp Leu Leu Glu Asp Arg Val Tyr Asp Phe Ala Phe Ser Asp Leu 545 550 555 560 Lys Leu Pro Gln Leu Cys Thr Glu Leu Lys Leu Pro Gln Leu Cys Thr 565 570 575 Glu Leu Lys Asp Glu Leu Lys Asp Glu Leu Val Leu Leu Leu Leu Glu 580 585 590 His His His His His His 595 ＜210＞ 41 ＜211＞ 789 ＜212＞ PRT ＜213＞ Artificial sequence ＜220＞ ＜223＞ fusion biogenic polypeptide ＜400＞ 41 Met Ala Glu Gln Leu Val Asp Leu Trp Asn Glu Cys Ala Lys Ala Cys 1 5 10 15 Val Leu Asp Leu Lys Asp Gly Val Arg Ser Ser Arg Met Ser Val Asp 20 25 30 Pro Ala Ile Ala Asp Thr Asn Gly Gln Gly Val Leu His Tyr Cys Met 35 40 45 Glu Phe His Met Val Asp Gly Met Ser Ile Arg Ala Lys Arg Arg Lys 50 55 60 Arg Ala Ser Ala Thr Gln Leu Tyr Lys Thr Cys Lys Gln Ala Gly Thr 65 70 75 80 Cys Pro Pro Asp Ile Ile Pro Lys Val Glu Gly Lys Thr Ile Ala Glu 85 90 95 Gln Ile Leu Gln Tyr Gly Ser Met Gly Val Phe Phe Gly Gly Leu Gly 100 105 110 Ile Gly Thr Gly Ser Gly Thr Gly Gly Arg Thr Gly Tyr Ile Pro Leu 115 120 125 Gly Thr Arg Pro Pro Thr Ala Thr Asp Thr Leu Ala Pro Val Arg Pro 130 135 140 Pro Leu Thr Val Asp Pro Val Gly Pro Ser Asp Pro Ser Ile Val Ser 145 150 155 160 Leu Val Glu Glu Thr Ser Phe Ile Asp Ala Gly Ala Pro Thr Ser Val 165 170 175 Pro Ser Ile Pro Pro Asp Val Ser Gly Phe Ser Ile Thr Thr Ser Thr 180 185 190 Asp Thr Thr Pro Ala Ile Leu Asp Ile Asn Asn Asn Thr Val Thr Thr 195 200 205 Val Thr Thr His Asn Asn Pro Thr Phe Thr Asp Pro Ser Val Leu Gln 210 215 220 Pro Pro Thr Pro Ala Glu Thr Gly Gly His Phe Thr Leu Ser Ser Ser 225 230 235 240 Thr Ile Ser Thr His Asn Tyr Glu Glu Ile Pro Met Asp Thr Lys Asp 245 250 255 Glu Leu Leu Glu His Met Ala Glu Glu Ala Phe Asp Leu Trp Asn Glu 260 265 270 Cys Ala Lys Ala Cys Val Leu Asp Leu Lys Asp Gly Val Arg Ser Ser 275 280 285 Arg Met Ser Val Asp Pro Ala Ile Ala Asp Thr Asn Gly Gln Gly Val 290 295 300 Leu His Tyr Ser Met Val Leu Glu Gly Gly Asn Asp Ala Leu Lys Leu 305 310 315 320 Ala Ile Asp Asn Ala Leu Ser Ile Thr Ser Asp Gly Leu Thr Ile Arg 325 330 335 Leu Glu Gly Gly Val Glu Pro Asn Lys Pro Val Arg Tyr Ser Tyr Thr 340 345 350 Arg Gln Ala Arg Gly Ser Trp Ser Leu Asn Trp Leu Val Pro Ile Gly 355 360 365 His Glu Lys Pro Ser Asn Ile Lys Val Phe Ile His Glu Leu Asn Ala 370 375 380 Gly Asn Gln Leu Ser His Met Ser Pro Ile Tyr Thr Ile Glu Met Gly 385 390 395 400 Asp Glu Leu Leu Ala Lys Leu Ala Arg Asp Ala Thr Phe Phe Val Arg 405 410 415 Ala His Glu Ser Asn Glu Met Gln Pro Thr Leu Ala Ile Ser His Ala 420 425 430 Gly Val Ser Val Val Met Ala Gln Thr Gln Pro Arg Arg Glu Lys Arg 435 440 445 Trp Ser Glu Trp Ala Ser Gly Lys Val Leu Cys Leu Leu Asp Pro Leu 450 455 460 Asp Gly Val Tyr Asn Tyr Leu Ala Gln Gln Arg Cys Asn Leu Asp Asp 465 470 475 480 Thr Trp Glu Gly Lys Ile Tyr Arg Val Leu Ala Gly Asn Pro Ala Lys 485 490 495 His Asp Leu Asp Ile Lys Pro Thr Val Ile Ser His Arg Leu His Phe 500 505 510 Pro Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys His 515 520 525 Leu Pro Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg Gly Trp Glu 530 535 540 Gln Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr 545 550 555 560 Leu Ala Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val Ile Arg Asn 565 570 575 Ala Leu Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu Ala Ile Arg 580 585 590 Glu Gln Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala Ala Ala Glu 595 600 605 Ser Glu Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu Ala Gly Ala 610 615 620 Ala Asn Ala Asp Val Val Ser Leu Thr Cys Pro Val Ala Ala Gly Glu 625 630 635 640 Cys Ala Gly Pro Ala Asp Ser Gly Asp Ala Leu Leu Glu Arg Asn Tyr 645 650 655 Pro Thr Gly Ala Glu Phe Leu Gly Asp Gly Gly Asp Val Ser Phe Ser 660 665 670 Thr Arg Gly Thr Gln Asn Trp Thr Val Glu Arg Leu Leu Gln Ala Glu 675 680 685 Phe Val Asp Lys Asp Glu Leu Arg Glu Val Tyr Asn Phe Ala Phe Leu 690 695 700 Leu Val Leu Arg Arg Glu Val Tyr Asp Lys Asp Glu Leu Leu Leu Leu 705 710 715 720 Leu Glu Asp Arg Gln Leu Leu Arg Arg Glu Val Phe Cys Gly Phe Arg 725 730 735 Asp Leu Leu Glu Asp Arg Val Tyr Asp Phe Ala Phe Ser Asp Leu Lys 740 745 750 Leu Pro Gln Leu Cys Thr Glu Leu Lys Leu Pro Gln Leu Cys Thr Glu 755 760 765 Leu Lys Asp Glu Leu Lys Asp Glu Leu Val Leu Leu Leu Leu Glu His 770 775 780 His His His His His 785 ＜210＞ 42 ＜211＞ 569 ＜212＞ PRT ＜213＞ Artificial sequence ＜220＞ ＜223＞ fusion biogenic polypeptide ＜400＞ 42 Met Ala Glu Gln Leu Val Asp Leu Trp Asn Glu Cys Ala Lys Ala Cys 1 5 10 15 Val Leu Asp Leu Lys Asp Gly Val Arg Ser Ser Arg Met Ser Val Asp 20 25 30 Pro Ala Ile Ala Asp Thr Asn Gly Gln Gly Val Leu His Tyr Cys Met 35 40 45 Glu Phe Ser Phe His Gln Leu Pro Ala Arg Ser Pro Ala Pro Leu Gln 50 55 60 Leu Glu His Met Ala Glu Glu Ala Phe Asp Leu Trp Asn Glu Cys Ala 65 70 75 80 Lys Ala Cys Val Leu Asp Leu Lys Asp Gly Val Arg Ser Ser Arg Met 85 90 95 Ser Val Asp Pro Ala Ile Ala Asp Thr Asn Gly Gln Gly Val Leu His 100 105 110 Tyr Ser Met Val Leu Glu Gly Gly Asn Asp Ala Leu Lys Leu Ala Ile 115 120 125 Asp Asn Ala Leu Ser Ile Thr Ser Asp Gly Leu Thr Ile Arg Leu Glu 130 135 140 Gly Gly Val Glu Pro Asn Lys Pro Val Arg Tyr Ser Tyr Thr Arg Gln 145 150 155 160 Ala Arg Gly Ser Trp Ser Leu Asn Trp Leu Val Pro Ile Gly His Glu 165 170 175 Lys Pro Ser Asn Ile Lys Val Phe Ile His Glu Leu Asn Ala Gly Asn 180 185 190 Gln Leu Ser His Met Ser Pro Ile Tyr Thr Ile Glu Met Gly Asp Glu 195 200 205 Leu Leu Ala Lys Leu Ala Arg Asp Ala Thr Phe Phe Val Arg Ala His 210 215 220 Glu Ser Asn Glu Met Gln Pro Thr Leu Ala Ile Ser His Ala Gly Val 225 230 235 240 Ser Val Val Met Ala Gln Thr Gln Pro Arg Arg Glu Lys Arg Trp Ser 245 250 255 Glu Trp Ala Ser Gly Lys Val Leu Cys Leu Leu Asp Pro Leu Asp Gly 260 265 270 Val Tyr Asn Tyr Leu Ala Gln Gln Arg Cys Asn Leu Asp Asp Thr Trp 275 280 285 Glu Gly Lys Ile Tyr Arg Val Leu Ala Gly Asn Pro Ala Lys His Asp 290 295 300 Leu Asp Ile Lys Pro Thr Val Ile Ser His Arg Leu His Phe Pro Glu 305 310 315 320 Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys His Leu Pro 325 330 335 Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg Gly Trp Glu Gln Leu 340 345 350 Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr Leu Ala 355 360 365 Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val Ile Arg Asn Ala Leu 370 375 380 Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu Ala Ile Arg Glu Gln 385 390 395 400 Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala Ala Ala Glu Ser Glu 405 410 415 Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu Ala Gly Ala Ala Asn 420 425 430 Ala Asp Val Val Ser Leu Thr Cys Pro Val Ala Ala Gly Glu Cys Ala 435 440 445 Gly Pro Ala Asp Ser Gly Asp Ala Leu Leu Glu Arg Asn Tyr Pro Thr 450 455 460 Gly Ala Glu Phe Leu Gly Asp Gly Gly Asp Val Ser Phe Ser Thr Arg 465 470 475 480 Gly Thr Gln Asn Trp Thr Val Glu Arg Leu Leu Gln Ala Glu Phe Val 485 490 495 Asp Gln Ala Glu Pro Asp Gln Ala Glu Pro Asp Arg Asp Glu Leu Val 500 505 510 Leu Arg Ala Arg Ala His Tyr Asn Ile Arg Ala Arg Ala His Tyr Asn 515 520 525 Ile Leu Glu Asp Arg Leu Leu Val Leu Arg Ala His Tyr Asn Ile Val 530 535 540 Ile Phe Arg Ala His Tyr Asn Ile Val Ile Phe Lys Asp Glu Leu Leu 545 550 555 560 Val Leu Glu His His His His His His 565 ＜210＞ 43 ＜211＞ 577 ＜212＞ PRT ＜213＞ Artificial sequence ＜220＞ ＜223＞ fusion biogenic polypeptide ＜400＞ 43 Met Ala Glu Gln Leu Val Asp Leu Trp Asn Glu Cys Ala Lys Ala Cys 1 5 10 15 Val Leu Asp Leu Lys Asp Gly Val Arg Ser Ser Arg Met Ser Val Asp 20 25 30 Pro Ala Ile Ala Asp Thr Asn Gly Gln Gly Val Leu His Tyr Cys Met 35 40 45 Glu Phe Ser Ser Phe His Leu Phe His His Leu Pro Ala Arg Ala Pro 50 55 60 Leu Ala Pro Ser Glu Leu Gln Pro Leu Glu His Met Ala Glu Glu Ala 65 70 75 80 Phe Asp Leu Trp Asn Glu Cys Ala Lys Ala Cys Val Leu Asp Leu Lys 85 90 95 Asp Gly Val Arg Ser Ser Arg Met Ser Val Asp Pro Ala Ile Ala Asp 100 105 110 Thr Asn Gly Gln Gly Val Leu His Tyr Ser Met Val Leu Glu Gly Gly 115 120 125 Asn Asp Ala Leu Lys Leu Ala Ile Asp Asn Ala Leu Ser Ile Thr Ser 130 135 140 Asp Gly Leu Thr Ile Arg Leu Glu Gly Gly Val Glu Pro Asn Lys Pro 145 150 155 160 Val Arg Tyr Ser Tyr Thr Arg Gln Ala Arg Gly Ser Trp Ser Leu Asn 165 170 175 Trp Leu Val Pro Ile Gly His Glu Lys Pro Ser Asn Ile Lys Val Phe 180 185 190 Ile His Glu Leu Asn Ala Gly Asn Gln Leu Ser His Met Ser Pro Ile 195 200 205 Tyr Thr Ile Glu Met Gly Asp Glu Leu Leu Ala Lys Leu Ala Arg Asp 210 215 220 Ala Thr Phe Phe Val Arg Ala His Glu Ser Asn Glu Met Gln Pro Thr 225 230 235 240 Leu Ala Ile Ser His Ala Gly Val Ser Val Val Met Ala Gln Thr Gln 245 250 255 Pro Arg Arg Glu Lys Arg Trp Ser Glu Trp Ala Ser Gly Lys Val Leu 260 265 270 Cys Leu Leu Asp Pro Leu Asp Gly Val Tyr Asn Tyr Leu Ala Gln Gln 275 280 285 Arg Cys Asn Leu Asp Asp Thr Trp Glu Gly Lys Ile Tyr Arg Val Leu 290 295 300 Ala Gly Asn Pro Ala Lys His Asp Leu Asp Ile Lys Pro Thr Val Ile 305 310 315 320 Ser His Arg Leu His Phe Pro Glu Gly Gly Ser Leu Ala Ala Leu Thr 325 330 335 Ala His Gln Ala Cys His Leu Pro Leu Glu Thr Phe Thr Arg His Arg 340 345 350 Gln Pro Arg Gly Trp Glu Gln Leu Glu Gln Cys Gly Tyr Pro Val Gln 355 360 365 Arg Leu Val Ala Leu Tyr Leu Ala Ala Arg Leu Ser Trp Asn Gln Val 370 375 380 Asp Gln Val Ile Arg Asn Ala Leu Ala Ser Pro Gly Ser Gly Gly Asp 385 390 395 400 Leu Gly Glu Ala Ile Arg Glu Gln Pro Glu Gln Ala Arg Leu Ala Leu 405 410 415 Thr Leu Ala Ala Ala Glu Ser Glu Arg Phe Val Arg Gln Gly Thr Gly 420 425 430 Asn Asp Glu Ala Gly Ala Ala Asn Ala Asp Val Val Ser Leu Thr Cys 435 440 445 Pro Val Ala Ala Gly Glu Cys Ala Gly Pro Ala Asp Ser Gly Asp Ala 450 455 460 Leu Leu Glu Arg Asn Tyr Pro Thr Gly Ala Glu Phe Leu Gly Asp Gly 465 470 475 480 Gly Asp Val Ser Phe Ser Thr Arg Gly Thr Gln Asn Trp Thr Val Glu 485 490 495 Arg Leu Leu Gln Ala Glu Phe Val Asp Gln Ala Glu Pro Asp Gln Ala 500 505 510 Glu Pro Asp Arg Asp Glu Leu Val Leu Arg Ala Arg Ala His Tyr Asn 515 520 525 Ile Arg Ala Arg Ala His Tyr Asn Ile Leu Glu Asp Arg Leu Leu Val 530 535 540 Leu Arg Ala His Tyr Asn Ile Val Ile Phe Arg Ala His Tyr Asn Ile 545 550 555 560 Val Ile Phe Lys Asp Glu Leu Leu Val Leu Glu His His His His His 565 570 575 His ＜210＞ 44 ＜211＞ 572 ＜212＞ PRT ＜213＞ Artificial sequence ＜220＞ ＜223＞ fusion biogenic polypeptide ＜400＞ 44 Met Ala Glu Gln Leu Val Asp Leu Trp Asn Glu Cys Ala Lys Ala Cys 1 5 10 15 Val Leu Asp Leu Lys Asp Gly Val Arg Ser Ser Arg Met Ser Val Asp 20 25 30 Pro Ala Ile Ala Asp Thr Asn Gly Gln Gly Val Leu His Tyr Cys Met 35 40 45 Glu Phe Ser Thr Pro Phe His Pro Leu Pro Ala Arg Lys Pro Leu Pro 50 55 60 Leu Val Pro Leu Glu His Met Ala Glu Glu Ala Phe Asp Leu Trp Asn 65 70 75 80 Glu Cys Ala Lys Ala Cys Val Leu Asp Leu Lys Asp Gly Val Arg Ser 85 90 95 Ser Arg Met Ser Val Asp Pro Ala Ile Ala Asp Thr Asn Gly Gln Gly 100 105 110 Val Leu His Tyr Ser Met Val Leu Glu Gly Gly Asn Asp Ala Leu Lys 115 120 125 Leu Ala Ile Asp Asn Ala Leu Ser Ile Thr Ser Asp Gly Leu Thr Ile 130 135 140 Arg Leu Glu Gly Gly Val Glu Pro Asn Lys Pro Val Arg Tyr Ser Tyr 145 150 155 160 Thr Arg Gln Ala Arg Gly Ser Trp Ser Leu Asn Trp Leu Val Pro Ile 165 170 175 Gly His Glu Lys Pro Ser Asn Ile Lys Val Phe Ile His Glu Leu Asn 180 185 190 Ala Gly Asn Gln Leu Ser His Met Ser Pro Ile Tyr Thr Ile Glu Met 195 200 205 Gly Asp Glu Leu Leu Ala Lys Leu Ala Arg Asp Ala Thr Phe Phe Val 210 215 220 Arg Ala His Glu Ser Asn Glu Met Gln Pro Thr Leu Ala Ile Ser His 225 230 235 240 Ala Gly Val Ser Val Val Met Ala Gln Thr Gln Pro Arg Arg Glu Lys 245 250 255 Arg Trp Ser Glu Trp Ala Ser Gly Lys Val Leu Cys Leu Leu Asp Pro 260 265 270 Leu Asp Gly Val Tyr Asn Tyr Leu Ala Gln Gln Arg Cys Asn Leu Asp 275 280 285 Asp Thr Trp Glu Gly Lys Ile Tyr Arg Val Leu Ala Gly Asn Pro Ala 290 295 300 Lys His Asp Leu Asp Ile Lys Pro Thr Val Ile Ser His Arg Leu His 305 310 315 320 Phe Pro Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys 325 330 335 His Leu Pro Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg Gly Trp 340 345 350 Glu Gln Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala Leu 355 360 365 Tyr Leu Ala Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val Ile Arg 370 375 380 Asn Ala Leu Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu Ala Ile 385 390 395 400 Arg Glu Gln Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala Ala Ala 405 410 415 Glu Ser Glu Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu Ala Gly 420 425 430 Ala Ala Asn Ala Asp Val Val Ser Leu Thr Cys Pro Val Ala Ala Gly 435 440 445 Glu Cys Ala Gly Pro Ala Asp Ser Gly Asp Ala Leu Leu Glu Arg Asn 450 455 460 Tyr Pro Thr Gly Ala Glu Phe Leu Gly Asp Gly Gly Asp Val Ser Phe 465 470 475 480 Ser Thr Arg Gly Thr Gln Asn Trp Thr Val Glu Arg Leu Leu Gln Ala 485 490 495 Glu Phe Val Asp Gln Ala Glu Pro Asp Gln Ala Glu Pro Asp Arg Asp 500 505 510 Glu Leu Val Leu Arg Ala Arg Ala His Tyr Asn Ile Arg Ala Arg Ala 515 520 525 His Tyr Asn Ile Leu Glu Asp Arg Leu Leu Val Leu Arg Ala His Tyr 530 535 540 Asn Ile Val Ile Phe Arg Ala His Tyr Asn Ile Val Ile Phe Lys Asp 545 550 555 560 Glu Leu Leu Val Leu Glu His His His His His His 565 570 ＜210＞ 45 ＜211＞ 763 ＜212＞ PRT ＜213＞ Artificial sequence ＜220＞ ＜223＞ fusion biogenic polypeptide ＜400＞ 45 Met Ala Glu Gln Leu Val Asp Leu Trp Asn Glu Cys Ala Lys Ala Cys 1 5 10 15 Val Leu Asp Leu Lys Asp Gly Val Arg Ser Ser Arg Met Ser Val Asp 20 25 30 Pro Ala Ile Ala Asp Thr Asn Gly Gln Gly Val Leu His Tyr Cys Met 35 40 45 Glu Phe His Met Val Asp Gly Met Ser Ile Arg Ala Lys Arg Arg Lys 50 55 60 Arg Ala Ser Ala Thr Gln Leu Tyr Lys Thr Cys Lys Gln Ala Gly Thr 65 70 75 80 Cys Pro Pro Asp Ile Ile Pro Lys Val Glu Gly Lys Thr Ile Ala Glu 85 90 95 Gln Ile Leu Gln Tyr Gly Ser Met Gly Val Phe Phe Gly Gly Leu Gly 100 105 110 Ile Gly Thr Gly Ser Gly Thr Gly Gly Arg Thr Gly Tyr Ile Pro Leu 115 120 125 Gly Thr Arg Pro Pro Thr Ala Thr Asp Thr Leu Ala Pro Val Arg Pro 130 135 140 Pro Leu Thr Val Asp Pro Val Gly Pro Ser Asp Pro Ser Ile Val Ser 145 150 155 160 Leu Val Glu Glu Thr Ser Phe Ile Asp Ala Gly Ala Pro Thr Ser Val 165 170 175 Pro Ser Ile Pro Pro Asp Val Ser Gly Phe Ser Ile Thr Thr Ser Thr 180 185 190 Asp Thr Thr Pro Ala Ile Leu Asp Ile Asn Asn Asn Thr Val Thr Thr 195 200 205 Val Thr Thr His Asn Asn Pro Thr Phe Thr Asp Pro Ser Val Leu Gln 210 215 220 Pro Pro Thr Pro Ala Glu Thr Gly Gly His Phe Thr Leu Ser Ser Ser 225 230 235 240 Thr Ile Ser Thr His Asn Tyr Glu Glu Ile Pro Met Asp Thr Lys Asp 245 250 255 Glu Leu Leu Glu His Met Ala Glu Glu Ala Phe Asp Leu Trp Asn Glu 260 265 270 Cys Ala Lys Ala Cys Val Leu Asp Leu Lys Asp Gly Val Arg Ser Ser 275 280 285 Arg Met Ser Val Asp Pro Ala Ile Ala Asp Thr Asn Gly Gln Gly Val 290 295 300 Leu His Tyr Ser Met Val Leu Glu Gly Gly Asn Asp Ala Leu Lys Leu 305 310 315 320 Ala Ile Asp Asn Ala Leu Ser Ile Thr Ser Asp Gly Leu Thr Ile Arg 325 330 335 Leu Glu Gly Gly Val Glu Pro Asn Lys Pro Val Arg Tyr Ser Tyr Thr 340 345 350 Arg Gln Ala Arg Gly Ser Trp Ser Leu Asn Trp Leu Val Pro Ile Gly 355 360 365 His Glu Lys Pro Ser Asn Ile Lys Val Phe Ile His Glu Leu Asn Ala 370 375 380 Gly Asn Gln Leu Ser His Met Ser Pro Ile Tyr Thr Ile Glu Met Gly 385 390 395 400 Asp Glu Leu Leu Ala Lys Leu Ala Arg Asp Ala Thr Phe Phe Val Arg 405 410 415 Ala His Glu Ser Asn Glu Met Gln Pro Thr Leu Ala Ile Ser His Ala 420 425 430 Gly Val Ser Val Val Met Ala Gln Thr Gln Pro Arg Arg Glu Lys Arg 435 440 445 Trp Ser Glu Trp Ala Ser Gly Lys Val Leu Cys Leu Leu Asp Pro Leu 450 455 460 Asp Gly Val Tyr Asn Tyr Leu Ala Gln Gln Arg Cys Asn Leu Asp Asp 465 470 475 480 Thr Trp Glu Gly Lys Ile Tyr Arg Val Leu Ala Gly Asn Pro Ala Lys 485 490 495 His Asp Leu Asp Ile Lys Pro Thr Val Ile Ser His Arg Leu His Phe 500 505 510 Pro Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys His 515 520 525 Leu Pro Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg Gly Trp Glu 530 535 540 Gln Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr 545 550 555 560 Leu Ala Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val Ile Arg Asn 565 570 575 Ala Leu Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu Ala Ile Arg 580 585 590 Glu Gln Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala Ala Ala Glu 595 600 605 Ser Glu Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu Ala Gly Ala 610 615 620 Ala Asn Ala Asp Val Val Ser Leu Thr Cys Pro Val Ala Ala Gly Glu 625 630 635 640 Cys Ala Gly Pro Ala Asp Ser Gly Asp Ala Leu Leu Glu Arg Asn Tyr 645 650 655 Pro Thr Gly Ala Glu Phe Leu Gly Asp Gly Gly Asp Val Ser Phe Ser 660 665 670 Thr Arg Gly Thr Gln Asn Trp Thr Val Glu Arg Leu Leu Gln Ala Glu 675 680 685 Phe Val Asp Gln Ala Glu Pro Asp Gln Ala Glu Pro Asp Arg Asp Glu 690 695 700 Leu Val Leu Arg Ala Arg Ala His Tyr Asn Ile Arg Ala Arg Ala His 705 710 715 720 Tyr Asn Ile Leu Glu Asp Arg Leu Leu Val Leu Arg Ala His Tyr Asn 725 730 735 Ile Val Ile Phe Arg Ala His Tyr Asn Ile Val Ile Phe Lys Asp Glu 740 745 750 Leu Leu Val Leu Glu His His His His His His 755 760 ＜210＞ 46 ＜211＞ 553 ＜212＞ PRT ＜213＞ Artificial sequence ＜220＞ ＜223＞ fusion biogenic polypeptide ＜400＞ 46 Met Ala Glu Gln Leu Val Asp Leu Trp Asn Glu Cys Ala Lys Ala Cys 1 5 10 15 Val Leu Asp Leu Lys Asp Gly Val Arg Ser Ser Arg Met Ser Val Asp 20 25 30 Pro Ala Ile Ala Asp Thr Asn Gly Gln Gly Val Leu His Tyr Cys Met 35 40 45 Glu Phe Ser Phe His Gln Leu Pro Ala Arg Ser Pro Ala Pro Leu Gln 50 55 60 Leu Glu His Met Ala Glu Glu Ala Phe Asp Leu Trp Asn Glu Cys Ala 65 70 75 80 Lys Ala Cys Val Leu Asp Leu Lys Asp Gly Val Arg Ser Ser Arg Met 85 90 95 Ser Val Asp Pro Ala Ile Ala Asp Thr Asn Gly Gln Gly Val Leu His 100 105 110 Tyr Ser Met Val Leu Glu Gly Gly Asn Asp Ala Leu Lys Leu Ala Ile 115 120 125 Asp Asn Ala Leu Ser Ile Thr Ser Asp Gly Leu Thr Ile Arg Leu Glu 130 135 140 Gly Gly Val Glu Pro Asn Lys Pro Val Arg Tyr Ser Tyr Thr Arg Gln 145 150 155 160 Ala Arg Gly Ser Trp Ser Leu Asn Trp Leu Val Pro Ile Gly His Glu 165 170 175 Lys Pro Ser Asn Ile Lys Val Phe Ile His Glu Leu Asn Ala Gly Asn 180 185 190 Gln Leu Ser His Met Ser Pro Ile Tyr Thr Ile Glu Met Gly Asp Glu 195 200 205 Leu Leu Ala Lys Leu Ala Arg Asp Ala Thr Phe Phe Val Arg Ala His 210 215 220 Glu Ser Asn Glu Met Gln Pro Thr Leu Ala Ile Ser His Ala Gly Val 225 230 235 240 Ser Val Val Met Ala Gln Thr Gln Pro Arg Arg Glu Lys Arg Trp Ser 245 250 255 Glu Trp Ala Ser Gly Lys Val Leu Cys Leu Leu Asp Pro Leu Asp Gly 260 265 270 Val Tyr Asn Tyr Leu Ala Gln Gln Arg Cys Asn Leu Asp Asp Thr Trp 275 280 285 Glu Gly Lys Ile Tyr Arg Val Leu Ala Gly Asn Pro Ala Lys His Asp 290 295 300 Leu Asp Ile Lys Pro Thr Val Ile Ser His Arg Leu His Phe Pro Glu 305 310 315 320 Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys His Leu Pro 325 330 335 Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg Gly Trp Glu Gln Leu 340 345 350 Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr Leu Ala 355 360 365 Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val Ile Arg Asn Ala Leu 370 375 380 Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu Ala Ile Arg Glu Gln 385 390 395 400 Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala Ala Ala Glu Ser Glu 405 410 415 Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu Ala Gly Ala Ala Asn 420 425 430 Ala Asp Val Val Ser Leu Thr Cys Pro Val Ala Ala Gly Glu Cys Ala 435 440 445 Gly Pro Ala Asp Ser Gly Asp Ala Leu Leu Glu Arg Asn Tyr Pro Thr 450 455 460 Gly Ala Glu Phe Leu Gly Asp Gly Gly Asp Val Glu Phe Val Asp Val 465 470 475 480 Phe Leu Gln Lys Tyr Pro His Thr His Leu Val His Gln Ala Leu Asp 485 490 495 Val Phe Leu Gln Lys Tyr Pro His Thr His Leu Val His Gln Ala Leu 500 505 510 Asp Val Phe Leu Gln Lys Tyr Pro His Thr His Leu Val His Gln Ala 515 520 525 Leu Asp Val Phe Leu Gln Lys Tyr Pro His Thr His Leu Val His Gln 530 535 540 Ala Leu Glu His His His His His His 545 550 ＜210＞ 47 ＜211＞ 561 ＜212＞ PRT ＜213＞ Artificial sequence ＜220＞ ＜223＞ fusion biogenic polypeptide ＜400＞ 47 Met Ala Glu Gln Leu Val Asp Leu Trp Asn Glu Cys Ala Lys Ala Cys 1 5 10 15 Val Leu Asp Leu Lys Asp Gly Val Arg Ser Ser Arg Met Ser Val Asp 20 25 30 Pro Ala Ile Ala Asp Thr Asn Gly Gln Gly Val Leu His Tyr Cys Met 35 40 45 Glu Phe Ser Ser Phe His Leu Phe His His Leu Pro Ala Arg Ala Pro 50 55 60 Leu Ala Pro Ser Glu Leu Gln Pro Leu Glu His Met Ala Glu Glu Ala 65 70 75 80 Phe Asp Leu Trp Asn Glu Cys Ala Lys Ala Cys Val Leu Asp Leu Lys 85 90 95 Asp Gly Val Arg Ser Ser Arg Met Ser Val Asp Pro Ala Ile Ala Asp 100 105 110 Thr Asn Gly Gln Gly Val Leu His Tyr Ser Met Val Leu Glu Gly Gly 115 120 125 Asn Asp Ala Leu Lys Leu Ala Ile Asp Asn Ala Leu Ser Ile Thr Ser 130 135 140 Asp Gly Leu Thr Ile Arg Leu Glu Gly Gly Val Glu Pro Asn Lys Pro 145 150 155 160 Val Arg Tyr Ser Tyr Thr Arg Gln Ala Arg Gly Ser Trp Ser Leu Asn 165 170 175 Trp Leu Val Pro Ile Gly His Glu Lys Pro Ser Asn Ile Lys Val Phe 180 185 190 Ile His Glu Leu Asn Ala Gly Asn Gln Leu Ser His Met Ser Pro Ile 195 200 205 Tyr Thr Ile Glu Met Gly Asp Glu Leu Leu Ala Lys Leu Ala Arg Asp 210 215 220 Ala Thr Phe Phe Val Arg Ala His Glu Ser Asn Glu Met Gln Pro Thr 225 230 235 240 Leu Ala Ile Ser His Ala Gly Val Ser Val Val Met Ala Gln Thr Gln 245 250 255 Pro Arg Arg Glu Lys Arg Trp Ser Glu Trp Ala Ser Gly Lys Val Leu 260 265 270 Cys Leu Leu Asp Pro Leu Asp Gly Val Tyr Asn Tyr Leu Ala Gln Gln 275 280 285 Arg Cys Asn Leu Asp Asp Thr Trp Glu Gly Lys Ile Tyr Arg Val Leu 290 295 300 Ala Gly Asn Pro Ala Lys His Asp Leu Asp Ile Lys Pro Thr Val Ile 305 310 315 320 Ser His Arg Leu His Phe Pro Glu Gly Gly Ser Leu Ala Ala Leu Thr 325 330 335 Ala His Gln Ala Cys His Leu Pro Leu Glu Thr Phe Thr Arg His Arg 340 345 350 Gln Pro Arg Gly Trp Glu Gln Leu Glu Gln Cys Gly Tyr Pro Val Gln 355 360 365 Arg Leu Val Ala Leu Tyr Leu Ala Ala Arg Leu Ser Trp Asn Gln Val 370 375 380 Asp Gln Val Ile Arg Asn Ala Leu Ala Ser Pro Gly Ser Gly Gly Asp 385 390 395 400 Leu Gly Glu Ala Ile Arg Glu Gln Pro Glu Gln Ala Arg Leu Ala Leu 405 410 415 Thr Leu Ala Ala Ala Glu Ser Glu Arg Phe Val Arg Gln Gly Thr Gly 420 425 430 Asn Asp Glu Ala Gly Ala Ala Asn Ala Asp Val Val Ser Leu Thr Cys 435 440 445 Pro Val Ala Ala Gly Glu Cys Ala Gly Pro Ala Asp Ser Gly Asp Ala 450 455 460 Leu Leu Glu Arg Asn Tyr Pro Thr Gly Ala Glu Phe Leu Gly Asp Gly 465 470 475 480 Gly Asp Val Glu Phe Val Asp Val Phe Leu Gln Lys Tyr Pro His Thr 485 490 495 His Leu Val His Gln Ala Leu Asp Val Phe Leu Gln Lys Tyr Pro His 500 505 510 Thr His Leu Val His Gln Ala Leu Asp Val Phe Leu Gln Lys Tyr Pro 515 520 525 His Thr His Leu Val His Gln Ala Leu Asp Val Phe Leu Gln Lys Tyr 530 535 540 Pro His Thr His Leu Val His Gln Ala Leu Glu His His His His His 545 550 555 560 His ＜210＞ 48 ＜211＞ 556 ＜212＞ PRT ＜213＞ Artificial sequence ＜220＞ ＜223＞ fusion biogenic polypeptide ＜400＞ 48 Met Ala Glu Gln Leu Val Asp Leu Trp Asn Glu Cys Ala Lys Ala Cys 1 5 10 15 Val Leu Asp Leu Lys Asp Gly Val Arg Ser Ser Arg Met Ser Val Asp 20 25 30 Pro Ala Ile Ala Asp Thr Asn Gly Gln Gly Val Leu His Tyr Cys Met 35 40 45 Glu Phe Ser Thr Pro Phe His Pro Leu Pro Ala Arg Lys Pro Leu Pro 50 55 60 Leu Val Pro Leu Glu His Met Ala Glu Glu Ala Phe Asp Leu Trp Asn 65 70 75 80 Glu Cys Ala Lys Ala Cys Val Leu Asp Leu Lys Asp Gly Val Arg Ser 85 90 95 Ser Arg Met Ser Val Asp Pro Ala Ile Ala Asp Thr Asn Gly Gln Gly 100 105 110 Val Leu His Tyr Ser Met Val Leu Glu Gly Gly Asn Asp Ala Leu Lys 115 120 125 Leu Ala Ile Asp Asn Ala Leu Ser Ile Thr Ser Asp Gly Leu Thr Ile 130 135 140 Arg Leu Glu Gly Gly Val Glu Pro Asn Lys Pro Val Arg Tyr Ser Tyr 145 150 155 160 Thr Arg Gln Ala Arg Gly Ser Trp Ser Leu Asn Trp Leu Val Pro Ile 165 170 175 Gly His Glu Lys Pro Ser Asn Ile Lys Val Phe Ile His Glu Leu Asn 180 185 190 Ala Gly Asn Gln Leu Ser His Met Ser Pro Ile Tyr Thr Ile Glu Met 195 200 205 Gly Asp Glu Leu Leu Ala Lys Leu Ala Arg Asp Ala Thr Phe Phe Val 210 215 220 Arg Ala His Glu Ser Asn Glu Met Gln Pro Thr Leu Ala Ile Ser His 225 230 235 240 Ala Gly Val Ser Val Val Met Ala Gln Thr Gln Pro Arg Arg Glu Lys 245 250 255 Arg Trp Ser Glu Trp Ala Ser Gly Lys Val Leu Cys Leu Leu Asp Pro 260 265 270 Leu Asp Gly Val Tyr Asn Tyr Leu Ala Gln Gln Arg Cys Asn Leu Asp 275 280 285 Asp Thr Trp Glu Gly Lys Ile Tyr Arg Val Leu Ala Gly Asn Pro Ala 290 295 300 Lys His Asp Leu Asp Ile Lys Pro Thr Val Ile Ser His Arg Leu His 305 310 315 320 Phe Pro Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys 325 330 335 His Leu Pro Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg Gly Trp 340 345 350 Glu Gln Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala Leu 355 360 365 Tyr Leu Ala Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val Ile Arg 370 375 380 Asn Ala Leu Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu Ala Ile 385 390 395 400 Arg Glu Gln Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala Ala Ala 405 410 415 Glu Ser Glu Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu Ala Gly 420 425 430 Ala Ala Asn Ala Asp Val Val Ser Leu Thr Cys Pro Val Ala Ala Gly 435 440 445 Glu Cys Ala Gly Pro Ala Asp Ser Gly Asp Ala Leu Leu Glu Arg Asn 450 455 460 Tyr Pro Thr Gly Ala Glu Phe Leu Gly Asp Gly Gly Asp Val Glu Phe 465 470 475 480 Val Asp Val Phe Leu Gln Lys Tyr Pro His Thr His Leu Val His Gln 485 490 495 Ala Leu Asp Val Phe Leu Gln Lys Tyr Pro His Thr His Leu Val His 500 505 510 Gln Ala Leu Asp Val Phe Leu Gln Lys Tyr Pro His Thr His Leu Val 515 520 525 His Gln Ala Leu Asp Val Phe Leu Gln Lys Tyr Pro His Thr His Leu 530 535 540 Val His Gln Ala Leu Glu His His His His His His 545 550 555 ＜210＞ 49 ＜211＞ 758 ＜212＞ PRT ＜213＞ Artificial sequence ＜220＞ ＜223＞ fusion biogenic polypeptide ＜400＞ 49 Met Ala Glu Gln Leu Val Asp Leu Trp Asn Glu Cys Ala Lys Ala Cys 1 5 10 15 Val Leu Asp Leu Lys Asp Gly Val Arg Ser Ser Arg Met Ser Val Asp 20 25 30 Pro Ala Ile Ala Asp Thr Asn Gly Gln Gly Val Leu His Tyr Cys Met 35 40 45 Glu Phe Ser Phe His Gln Leu Pro Ala Arg Ser Pro Ala Pro Leu Gln 50 55 60 Leu Glu His Met Ala Glu Glu Ala Phe Asp Leu Trp Asn Glu Cys Ala 65 70 75 80 Lys Ala Cys Val Leu Asp Leu Lys Asp Gly Val Arg Ser Ser Arg Met 85 90 95 Ser Val Asp Pro Ala Ile Ala Asp Thr Asn Gly Gln Gly Val Leu His 100 105 110 Tyr Ser Met Val Leu Glu Gly Gly Asn Asp Ala Leu Lys Leu Ala Ile 115 120 125 Asp Asn Ala Leu Ser Ile Thr Ser Asp Gly Leu Thr Ile Arg Leu Glu 130 135 140 Gly Gly Val Glu Pro Asn Lys Pro Val Arg Tyr Ser Tyr Thr Arg Gln 145 150 155 160 Ala Arg Gly Ser Trp Ser Leu Asn Trp Leu Val Pro Ile Gly His Glu 165 170 175 Lys Pro Ser Asn Ile Lys Val Phe Ile His Glu Leu Asn Ala Gly Asn 180 185 190 Gln Leu Ser His Met Ser Pro Ile Tyr Thr Ile Glu Met Gly Asp Glu 195 200 205 Leu Leu Ala Lys Leu Ala Arg Asp Ala Thr Phe Phe Val Arg Ala His 210 215 220 Glu Ser Asn Glu Met Gln Pro Thr Leu Ala Ile Ser His Ala Gly Val 225 230 235 240 Ser Val Val Met Ala Gln Thr Gln Pro Arg Arg Glu Lys Arg Trp Ser 245 250 255 Glu Trp Ala Ser Gly Lys Val Leu Cys Leu Leu Asp Pro Leu Asp Gly 260 265 270 Val Tyr Asn Tyr Leu Ala Gln Gln Arg Cys Asn Leu Asp Asp Thr Trp 275 280 285 Glu Gly Lys Ile Tyr Arg Val Leu Ala Gly Asn Pro Ala Lys His Asp 290 295 300 Leu Asp Ile Lys Pro Thr Val Ile Ser His Arg Leu His Phe Pro Glu 305 310 315 320 Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys His Leu Pro 325 330 335 Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg Gly Trp Glu Gln Leu 340 345 350 Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr Leu Ala 355 360 365 Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val Ile Arg Asn Ala Leu 370 375 380 Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu Ala Ile Arg Glu Gln 385 390 395 400 Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala Ala Ala Glu Ser Glu 405 410 415 Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu Ala Gly Ala Ala Asn 420 425 430 Ala Asp Val Val Ser Leu Thr Cys Pro Val Ala Ala Gly Glu Cys Ala 435 440 445 Gly Pro Ala Asp Ser Gly Asp Ala Leu Leu Glu Arg Asn Tyr Pro Thr 450 455 460 Gly Ala Glu Phe Leu Gly Asp Gly Gly Asp Val Glu Phe Leu Leu Glu 465 470 475 480 Pro His Thr His Leu Val His Gln Ala Asn Val Leu Leu Ala Leu Gln 485 490 495 Leu Leu Leu Glu Asp Arg Glu Phe Val Phe Leu Gln Lys Tyr Pro His 500 505 510 Val Glu Pro His Thr His Leu Val His Gln Ala Asn Val Leu Leu Ala 515 520 525 Leu Gln Leu Leu Leu Glu Asp Arg Glu Phe Val Phe Leu Gln Lys Tyr 530 535 540 Pro His Val Glu Pro His Thr His Leu Val His Gln Ala Asn Val Leu 545 550 555 560 Leu Ala Leu Gln Leu Leu Leu Glu Asp Arg Glu Phe Val Phe Leu Gln 565 570 575 Lys Tyr Pro His Val Glu Pro His Thr His Leu Val His Gln Ala Asn 580 585 590 Val Leu Leu Ala Leu Gln Leu Leu Leu Glu Asp Arg Glu Phe Val Phe 595 600 605 Leu Gln Lys Tyr Pro His Val Glu Pro His Thr His Leu Val His Gln 610 615 620 Ala Asn Val Leu Leu Ala Leu Gln Leu Leu Leu Glu Asp Arg Glu Phe 625 630 635 640 Val Phe Leu Gln Lys Tyr Pro His Val Glu Pro His Thr His Leu Val 645 650 655 His Gln Ala Asn Val Leu Leu Ala Leu Gln Leu Leu Leu Glu Asp Arg 660 665 670 Glu Phe Val Phe Leu Gln Lys Tyr Pro His Val Glu Pro His Thr His 675 680 685 Leu Val His Gln Ala Asn Val Leu Leu Ala Leu Gln Leu Leu Leu Glu 690 695 700 Asp Arg Glu Phe Val Phe Leu Gln Lys Tyr Pro His Val Glu Pro His 705 710 715 720 Thr His Leu Val His Gln Ala Asn Val Leu Leu Ala Leu Gln Leu Leu 725 730 735 Leu Glu Asp Arg Glu Phe Val Phe Leu Gln Lys Tyr Pro His Val Glu 740 745 750 His His His His His His 755 ＜210＞ 50 ＜211＞ 766 ＜212＞ PRT ＜213＞ Artificial sequence ＜220＞ ＜223＞ fusion biogenic polypeptide ＜400＞ 50 Met Ala Glu Gln Leu Val Asp Leu Trp Asn Glu Cys Ala Lys Ala Cys 1 5 10 15 Val Leu Asp Leu Lys Asp Gly Val Arg Ser Ser Arg Met Ser Val Asp 20 25 30 Pro Ala Ile Ala Asp Thr Asn Gly Gln Gly Val Leu His Tyr Cys Met 35 40 45 Glu Phe Ser Ser Phe His Leu Phe His His Leu Pro Ala Arg Ala Pro 50 55 60 Leu Ala Pro Ser Glu Leu Gln Pro Leu Glu His Met Ala Glu Glu Ala 65 70 75 80 Phe Asp Leu Trp Asn Glu Cys Ala Lys Ala Cys Val Leu Asp Leu Lys 85 90 95 Asp Gly Val Arg Ser Ser Arg Met Ser Val Asp Pro Ala Ile Ala Asp 100 105 110 Thr Asn Gly Gln Gly Val Leu His Tyr Ser Met Val Leu Glu Gly Gly 115 120 125 Asn Asp Ala Leu Lys Leu Ala Ile Asp Asn Ala Leu Ser Ile Thr Ser 130 135 140 Asp Gly Leu Thr Ile Arg Leu Glu Gly Gly Val Glu Pro Asn Lys Pro 145 150 155 160 Val Arg Tyr Ser Tyr Thr Arg Gln Ala Arg Gly Ser Trp Ser Leu Asn 165 170 175 Trp Leu Val Pro Ile Gly His Glu Lys Pro Ser Asn Ile Lys Val Phe 180 185 190 Ile His Glu Leu Asn Ala Gly Asn Gln Leu Ser His Met Ser Pro Ile 195 200 205 Tyr Thr Ile Glu Met Gly Asp Glu Leu Leu Ala Lys Leu Ala Arg Asp 210 215 220 Ala Thr Phe Phe Val Arg Ala His Glu Ser Asn Glu Met Gln Pro Thr 225 230 235 240 Leu Ala Ile Ser His Ala Gly Val Ser Val Val Met Ala Gln Thr Gln 245 250 255 Pro Arg Arg Glu Lys Arg Trp Ser Glu Trp Ala Ser Gly Lys Val Leu 260 265 270 Cys Leu Leu Asp Pro Leu Asp Gly Val Tyr Asn Tyr Leu Ala Gln Gln 275 280 285 Arg Cys Asn Leu Asp Asp Thr Trp Glu Gly Lys Ile Tyr Arg Val Leu 290 295 300 Ala Gly Asn Pro Ala Lys His Asp Leu Asp Ile Lys Pro Thr Val Ile 305 310 315 320 Ser His Arg Leu His Phe Pro Glu Gly Gly Ser Leu Ala Ala Leu Thr 325 330 335 Ala His Gln Ala Cys His Leu Pro Leu Glu Thr Phe Thr Arg His Arg 340 345 350 Gln Pro Arg Gly Trp Glu Gln Leu Glu Gln Cys Gly Tyr Pro Val Gln 355 360 365 Arg Leu Val Ala Leu Tyr Leu Ala Ala Arg Leu Ser Trp Asn Gln Val 370 375 380 Asp Gln Val Ile Arg Asn Ala Leu Ala Ser Pro Gly Ser Gly Gly Asp 385 390 395 400 Leu Gly Glu Ala Ile Arg Glu Gln Pro Glu Gln Ala Arg Leu Ala Leu 405 410 415 Thr Leu Ala Ala Ala Glu Ser Glu Arg Phe Val Arg Gln Gly Thr Gly 420 425 430 Asn Asp Glu Ala Gly Ala Ala Asn Ala Asp Val Val Ser Leu Thr Cys 435 440 445 Pro Val Ala Ala Gly Glu Cys Ala Gly Pro Ala Asp Ser Gly Asp Ala 450 455 460 Leu Leu Glu Arg Asn Tyr Pro Thr Gly Ala Glu Phe Leu Gly Asp Gly 465 470 475 480 Gly Asp Val Glu Phe Leu Leu Glu Pro His Thr His Leu Val His Gln 485 490 495 Ala Asn Val Leu Leu Ala Leu Gln Leu Leu Leu Glu Asp Arg Glu Phe 500 505 510 Val Phe Leu Gln Lys Tyr Pro His Val Glu Pro His Thr His Leu Val 515 520 525 His Gln Ala Asn Val Leu Leu Ala Leu Gln Leu Leu Leu Glu Asp Arg 530 535 540 Glu Phe Val Phe Leu Gln Lys Tyr Pro His Val Glu Pro His Thr His 545 550 555 560 Leu Val His Gln Ala Asn Val Leu Leu Ala Leu Gln Leu Leu Leu Glu 565 570 575 Asp Arg Glu Phe Val Phe Leu Gln Lys Tyr Pro His Val Glu Pro His 580 585 590 Thr His Leu Val His Gln Ala Asn Val Leu Leu Ala Leu Gln Leu Leu 595 600 605 Leu Glu Asp Arg Glu Phe Val Phe Leu Gln Lys Tyr Pro His Val Glu 610 615 620 Pro His Thr His Leu Val His Gln Ala Asn Val Leu Leu Ala Leu Gln 625 630 635 640 Leu Leu Leu Glu Asp Arg Glu Phe Val Phe Leu Gln Lys Tyr Pro His 645 650 655 Val Glu Pro His Thr His Leu Val His Gln Ala Asn Val Leu Leu Ala 660 665 670 Leu Gln Leu Leu Leu Glu Asp Arg Glu Phe Val Phe Leu Gln Lys Tyr 675 680 685 Pro His Val Glu Pro His Thr His Leu Val His Gln Ala Asn Val Leu 690 695 700 Leu Ala Leu Gln Leu Leu Leu Glu Asp Arg Glu Phe Val Phe Leu Gln 705 710 715 720 Lys Tyr Pro His Val Glu Pro His Thr His Leu Val His Gln Ala Asn 725 730 735 Val Leu Leu Ala Leu Gln Leu Leu Leu Glu Asp Arg Glu Phe Val Phe 740 745 750 Leu Gln Lys Tyr Pro His Val Glu His His His His His His 755 760 765 ＜210＞ 51 ＜211＞ 761 ＜212＞ PRT ＜213＞ Artificial sequence ＜220＞ ＜223＞ fusion biogenic polypeptide ＜400＞ 51 Met Ala Glu Gln Leu Val Asp Leu Trp Asn Glu Cys Ala Lys Ala Cys 1 5 10 15 Val Leu Asp Leu Lys Asp Gly Val Arg Ser Ser Arg Met Ser Val Asp 20 25 30 Pro Ala Ile Ala Asp Thr Asn Gly Gln Gly Val Leu His Tyr Cys Met 35 40 45 Glu Phe Ser Thr Pro Phe His Pro Leu Pro Ala Arg Lys Pro Leu Pro 50 55 60 Leu Val Pro Leu Glu His Met Ala Glu Glu Ala Phe Asp Leu Trp Asn 65 70 75 80 Glu Cys Ala Lys Ala Cys Val Leu Asp Leu Lys Asp Gly Val Arg Ser 85 90 95 Ser Arg Met Ser Val Asp Pro Ala Ile Ala Asp Thr Asn Gly Gln Gly 100 105 110 Val Leu His Tyr Ser Met Val Leu Glu Gly Gly Asn Asp Ala Leu Lys 115 120 125 Leu Ala Ile Asp Asn Ala Leu Ser Ile Thr Ser Asp Gly Leu Thr Ile 130 135 140 Arg Leu Glu Gly Gly Val Glu Pro Asn Lys Pro Val Arg Tyr Ser Tyr 145 150 155 160 Thr Arg Gln Ala Arg Gly Ser Trp Ser Leu Asn Trp Leu Val Pro Ile 165 170 175 Gly His Glu Lys Pro Ser Asn Ile Lys Val Phe Ile His Glu Leu Asn 180 185 190 Ala Gly Asn Gln Leu Ser His Met Ser Pro Ile Tyr Thr Ile Glu Met 195 200 205 Gly Asp Glu Leu Leu Ala Lys Leu Ala Arg Asp Ala Thr Phe Phe Val 210 215 220 Arg Ala His Glu Ser Asn Glu Met Gln Pro Thr Leu Ala Ile Ser His 225 230 235 240 Ala Gly Val Ser Val Val Met Ala Gln Thr Gln Pro Arg Arg Glu Lys 245 250 255 Arg Trp Ser Glu Trp Ala Ser Gly Lys Val Leu Cys Leu Leu Asp Pro 260 265 270 Leu Asp Gly Val Tyr Asn Tyr Leu Ala Gln Gln Arg Cys Asn Leu Asp 275 280 285 Asp Thr Trp Glu Gly Lys Ile Tyr Arg Val Leu Ala Gly Asn Pro Ala 290 295 300 Lys His Asp Leu Asp Ile Lys Pro Thr Val Ile Ser His Arg Leu His 305 310 315 320 Phe Pro Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys 325 330 335 His Leu Pro Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg Gly Trp 340 345 350 Glu Gln Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala Leu 355 360 365 Tyr Leu Ala Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val Ile Arg 370 375 380 Asn Ala Leu Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu Ala Ile 385 390 395 400 Arg Glu Gln Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala Ala Ala 405 410 415 Glu Ser Glu Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu Ala Gly 420 425 430 Ala Ala Asn Ala Asp Val Val Ser Leu Thr Cys Pro Val Ala Ala Gly 435 440 445 Glu Cys Ala Gly Pro Ala Asp Ser Gly Asp Ala Leu Leu Glu Arg Asn 450 455 460 Tyr Pro Thr Gly Ala Glu Phe Leu Gly Asp Gly Gly Asp Val Glu Phe 465 470 475 480 Leu Leu Glu Pro His Thr His Leu Val His Gln Ala Asn Val Leu Leu 485 490 495 Ala Leu Gln Leu Leu Leu Glu Asp Arg Glu Phe Val Phe Leu Gln Lys 500 505 510 Tyr Pro His Val Glu Pro His Thr His Leu Val His Gln Ala Asn Val 515 520 525 Leu Leu Ala Leu Gln Leu Leu Leu Glu Asp Arg Glu Phe Val Phe Leu 530 535 540 Gln Lys Tyr Pro His Val Glu Pro His Thr His Leu Val His Gln Ala 545 550 555 560 Asn Val Leu Leu Ala Leu Gln Leu Leu Leu Glu Asp Arg Glu Phe Val 565 570 575 Phe Leu Gln Lys Tyr Pro His Val Glu Pro His Thr His Leu Val His 580 585 590 Gln Ala Asn Val Leu Leu Ala Leu Gln Leu Leu Leu Glu Asp Arg Glu 595 600 605 Phe Val Phe Leu Gln Lys Tyr Pro His Val Glu Pro His Thr His Leu 610 615 620 Val His Gln Ala Asn Val Leu Leu Ala Leu Gln Leu Leu Leu Glu Asp 625 630 635 640 Arg Glu Phe Val Phe Leu Gln Lys Tyr Pro His Val Glu Pro His Thr 645 650 655 His Leu Val His Gln Ala Asn Val Leu Leu Ala Leu Gln Leu Leu Leu 660 665 670 Glu Asp Arg Glu Phe Val Phe Leu Gln Lys Tyr Pro His Val Glu Pro 675 680 685 His Thr His Leu Val His Gln Ala Asn Val Leu Leu Ala Leu Gln Leu 690 695 700 Leu Leu Glu Asp Arg Glu Phe Val Phe Leu Gln Lys Tyr Pro His Val 705 710 715 720 Glu Pro His Thr His Leu Val His Gln Ala Asn Val Leu Leu Ala Leu 725 730 735 Gln Leu Leu Leu Glu Asp Arg Glu Phe Val Phe Leu Gln Lys Tyr Pro 740 745 750 His Val Glu His His His His His His 755 760 ＜210＞ 52 ＜211＞ 804 ＜212＞ PRT ＜213＞ Artificial sequence ＜220＞ ＜223＞ fusion biogenic polypeptide ＜400＞ 52 Met Ala Glu Gln Leu Val Asp Leu Trp Asn Glu Cys Ala Lys Ala Cys 1 5 10 15 Val Leu Asp Leu Lys Asp Gly Val Arg Ser Ser Arg Met Ser Val Asp 20 25 30 Pro Ala Ile Ala Asp Thr Asn Gly Gln Gly Val Leu His Tyr Cys Met 35 40 45 Glu Phe Ser Ser Phe His Leu Phe His His Leu Pro Ala Arg Ala Pro 50 55 60 Leu Ala Pro Ser Glu Leu Gln Pro Leu Glu His Met Ala Glu Glu Ala 65 70 75 80 Phe Asp Leu Trp Asn Glu Cys Ala Lys Ala Cys Val Leu Asp Leu Lys 85 90 95 Asp Gly Val Arg Ser Ser Arg Met Ser Val Asp Pro Ala Ile Ala Asp 100 105 110 Thr Asn Gly Gln Gly Val Leu His Tyr Ser Met Val Leu Glu Gly Gly 115 120 125 Asn Asp Ala Leu Lys Leu Ala Ile Asp Asn Ala Leu Ser Ile Thr Ser 130 135 140 Asp Gly Leu Thr Ile Arg Leu Glu Gly Gly Val Glu Pro Asn Lys Pro 145 150 155 160 Val Arg Tyr Ser Tyr Thr Arg Gln Ala Arg Gly Ser Trp Ser Leu Asn 165 170 175 Trp Leu Val Pro Ile Gly His Glu Lys Pro Ser Asn Ile Lys Val Phe 180 185 190 Ile His Glu Leu Asn Ala Gly Asn Gln Leu Ser His Met Ser Pro Ile 195 200 205 Tyr Thr Ile Glu Met Gly Asp Glu Leu Leu Ala Lys Leu Ala Arg Asp 210 215 220 Ala Thr Phe Phe Val Arg Ala His Glu Ser Asn Glu Met Gln Pro Thr 225 230 235 240 Leu Ala Ile Ser His Ala Gly Val Ser Val Val Met Ala Gln Thr Gln 245 250 255 Pro Arg Arg Glu Lys Arg Trp Ser Glu Trp Ala Ser Gly Lys Val Leu 260 265 270 Cys Leu Leu Asp Pro Leu Asp Gly Val Tyr Asn Tyr Leu Ala Gln Gln 275 280 285 Arg Cys Asn Leu Asp Asp Thr Trp Glu Gly Lys Ile Tyr Arg Val Leu 290 295 300 Ala Gly Asn Pro Ala Lys His Asp Leu Asp Ile Lys Pro Thr Val Ile 305 310 315 320 Ser His Arg Leu His Phe Pro Glu Gly Gly Ser Leu Ala Ala Leu Thr 325 330 335 Ala His Gln Ala Cys His Leu Pro Leu Glu Thr Phe Thr Arg His Arg 340 345 350 Gln Pro Arg Gly Trp Glu Gln Leu Glu Gln Cys Gly Tyr Pro Val Gln 355 360 365 Arg Leu Val Ala Leu Tyr Leu Ala Ala Arg Leu Ser Trp Asn Gln Val 370 375 380 Asp Gln Val Ile Arg Asn Ala Leu Ala Ser Pro Gly Ser Gly Gly Asp 385 390 395 400 Leu Gly Glu Ala Ile Arg Glu Gln Pro Glu Gln Ala Arg Leu Ala Leu 405 410 415 Thr Leu Ala Ala Ala Glu Ser Glu Arg Phe Val Arg Gln Gly Thr Gly 420 425 430 Asn Asp Glu Ala Gly Ala Ala Asn Ala Asp Val Val Ser Leu Thr Cys 435 440 445 Pro Val Ala Ala Gly Glu Cys Ala Gly Pro Ala Asp Ser Gly Asp Ala 450 455 460 Leu Leu Glu Arg Asn Tyr Pro Thr Gly Ala Glu Phe Leu Gly Asp Gly 465 470 475 480 Gly Asp Val Ser Phe Ser Thr Arg Gly Thr Gln Asn Trp Thr Val Glu 485 490 495 Arg Leu Leu Gln Ala Glu Phe Val Ser Phe Ser Thr Gly Gly Ser Gln 500 505 510 Asn Trp Thr Val Glu Arg Leu Leu Gln Ala Glu Phe Cys Ser Thr Ser 515 520 525 Gln Ala Ala Arg Gln Arg Leu Glu Thr Gly Arg Asn Cys Ser Thr Gly 530 535 540 Gln Ala Ala Arg Gln Arg Leu Glu Pro Gly Arg Asn Leu Val Leu Cys 545 550 555 560 Leu Thr Ser Gln Ala Ala Gln Gln Arg Leu Glu Pro Gly Gly Asn Cys 565 570 575 Gln Thr Ser Gln Ala Ala His Gln Arg Leu Glu Pro Gly Arg Asn Cys 580 585 590 Arg Thr Ser Gln Ala Ala Ser Gln Arg Leu Glu Pro Gly Arg Asn Cys 595 600 605 Arg Thr Ser Gln Ala Ala His Gln Arg Leu Glu Pro Gly Arg Asn Cys 610 615 620 Ser Thr Arg Gln Ala Ala Gln Gln Arg Leu Glu Pro Gly Arg Asn Leu 625 630 635 640 Leu Cys Pro Thr Ser Gln Ala Ala His Gln Arg Arg Leu Glu Pro Gly 645 650 655 Arg Asn Cys Ser Thr Ser Gln Ala Ala Tyr Gln Arg Leu Glu Pro Gly 660 665 670 Arg Asn Cys Pro Thr Ser Arg Ala Ala Arg Gln Arg Leu Glu Pro Gly 675 680 685 Arg Asn Leu Leu Cys Ser Thr Ser Gln Ala Ala Leu Gln Arg Leu Glu 690 695 700 Pro Gly Arg Asn Leu Cys Pro Thr Ser Gln Ala Ala Lys Gln Arg Leu 705 710 715 720 Glu Pro Gly Arg Asn Leu Val Val Cys Leu Thr Ser Gln Ala Ala Arg 725 730 735 Gln Arg Leu Glu Pro Gly Arg Asn Cys Ser Thr Ser Gln Ala Ala Ser 740 745 750 Gln Arg Leu Glu Pro Gly Arg Asn Cys Pro Thr Ser Gln Ala Ala Arg 755 760 765 Gln Arg Leu Glu Pro Gly Arg Asn Val Leu Leu Leu Cys Leu Thr Ser 770 775 780 Gln Ala Ala His Gln Arg Leu Glu Pro Gly Arg Asn Leu Glu His His 785 790 795 800 His His His His ＜210＞ 53 ＜211＞ 737 ＜212＞ PRT ＜213＞ Artificial sequence ＜220＞ ＜223＞ fusion biogenic polypeptide ＜400＞ 53 Met Ala Glu Gln Leu Val Asp Leu Trp Asn Glu Cys Ala Lys Ala Cys 1 5 10 15 Val Leu Asp Leu Lys Asp Gly Val Arg Ser Ser Arg Met Ser Val Asp 20 25 30 Pro Ala Ile Ala Asp Thr Asn Gly Gln Gly Val Leu His Tyr Cys Met 35 40 45 Glu Phe Ser Ser Phe His Leu Phe His His Leu Pro Ala Arg Ala Pro 50 55 60 Leu Ala Pro Ser Glu Leu Gln Pro Leu Glu His Met Ala Glu Glu Ala 65 70 75 80 Phe Asp Leu Trp Asn Glu Cys Ala Lys Ala Cys Val Leu Asp Leu Lys 85 90 95 Asp Gly Val Arg Ser Ser Arg Met Ser Val Asp Pro Ala Ile Ala Asp 100 105 110 Thr Asn Gly Gln Gly Val Leu His Tyr Ser Met Val Leu Glu Gly Gly 115 120 125 Asn Asp Ala Leu Lys Leu Ala Ile Asp Asn Ala Leu Ser Ile Thr Ser 130 135 140 Asp Gly Leu Thr Ile Arg Leu Glu Gly Gly Val Glu Pro Asn Lys Pro 145 150 155 160 Val Arg Tyr Ser Tyr Thr Arg Gln Ala Arg Gly Ser Trp Ser Leu Asn 165 170 175 Trp Leu Val Pro Ile Gly His Glu Lys Pro Ser Asn Ile Lys Val Phe 180 185 190 Ile His Glu Leu Asn Ala Gly Asn Gln Leu Ser His Met Ser Pro Ile 195 200 205 Tyr Thr Ile Glu Met Gly Asp Glu Leu Leu Ala Lys Leu Ala Arg Asp 210 215 220 Ala Thr Phe Phe Val Arg Ala His Glu Ser Asn Glu Met Gln Pro Thr 225 230 235 240 Leu Ala Ile Ser His Ala Gly Val Ser Val Val Met Ala Gln Thr Gln 245 250 255 Pro Arg Arg Glu Lys Arg Trp Ser Glu Trp Ala Ser Gly Lys Val Leu 260 265 270 Cys Leu Leu Asp Pro Leu Asp Gly Val Tyr Asn Tyr Leu Ala Gln Gln 275 280 285 Arg Cys Asn Leu Asp Asp Thr Trp Glu Gly Lys Ile Tyr Arg Val Leu 290 295 300 Ala Gly Asn Pro Ala Lys His Asp Leu Asp Ile Lys Pro Thr Val Ile 305 310 315 320 Ser His Arg Leu His Phe Pro Glu Gly Gly Ser Leu Ala Ala Leu Thr 325 330 335 Ala His Gln Ala Cys His Leu Pro Leu Glu Thr Phe Thr Arg His Arg 340 345 350 Gln Pro Arg Gly Trp Glu Gln Leu Glu Gln Cys Gly Tyr Pro Val Gln 355 360 365 Arg Leu Val Ala Leu Tyr Leu Ala Ala Arg Leu Ser Trp Asn Gln Val 370 375 380 Asp Gln Val Ile Arg Asn Ala Leu Ala Ser Pro Gly Ser Gly Gly Asp 385 390 395 400 Leu Gly Glu Ala Ile Arg Glu Gln Pro Glu Gln Ala Arg Leu Ala Leu 405 410 415 Thr Leu Ala Ala Ala Glu Ser Glu Arg Phe Val Arg Gln Gly Thr Gly 420 425 430 Asn Asp Glu Ala Gly Ala Ala Asn Ala Asp Val Val Ser Leu Thr Cys 435 440 445 Pro Val Ala Ala Gly Glu Cys Ala Gly Pro Ala Asp Ser Gly Asp Ala 450 455 460 Leu Leu Glu Arg Asn Tyr Pro Thr Gly Ala Glu Phe Leu Gly Asp Gly 465 470 475 480 Gly Asp Val Ser Phe Ser Thr Arg Gly Thr Gln Asn Trp Thr Val Glu 485 490 495 Arg Leu Leu Gln Ala Glu Phe Gly Val Ser Ala Ala Gln Glu Lys Ile 500 505 510 Ser Phe Gly Leu Leu Gly Val Pro Thr Ala Gln Glu Thr Thr Ser Ile 515 520 525 Arg Glu Val Leu Glu Val Ser Thr Ala Gln Glu Asn Ser Pro Phe Met 530 535 540 Leu Gly Ala Ser Ala Thr Glu Glu Lys Thr Ser Leu Arg Leu Gly Ala 545 550 555 560 Ser Thr Thr Gln Glu Thr Ser Phe Gly Lys Cys Leu Arg Pro His Gly 565 570 575 Val Ser Ala Ala Gln Gly Thr Thr Pro Phe Arg Gly Val Ser Thr Thr 580 585 590 Gln Glu Asn Thr Ser Phe Gly Arg Val Pro Thr Ala Gln Glu Asn Val 595 600 605 Ser Phe Gly Leu His Gly Val Pro Ala Ala Gln Lys Thr Asn Ser Phe 610 615 620 Gly Gly Val Pro Thr Ala Gln Glu Asn Ile Ser Phe Lys Glu Val Ser 625 630 635 640 Ala Thr Gln Arg Glu Ile Pro Phe Arg Cys Leu Arg Pro His Gly Val 645 650 655 Ser Thr Ala Gln Glu Thr Pro Phe Arg Gly Val Ser Thr Ala Gln Glu 660 665 670 Thr Ile Pro Phe Arg Gly Val Ser Ala Thr His Glu Asn Ile Ser Phe 675 680 685 Gly Cys Leu Arg Pro His Gly Val Ser Ala Ala Gln Glu Ser Ile Pro 690 695 700 Ile Arg Leu Gly Ala Ser Ala Ala Gln Glu Asn Thr Ser Phe Arg Gly 705 710 715 720 Thr Pro Ala Ala Gln Glu Lys Ile Pro Leu Glu His His His His His 725 730 735 His ＜210＞ 54 ＜211＞ 796 ＜212＞ PRT ＜213＞ Artificial sequence ＜220＞ ＜223＞ fusion biogenic polypeptide ＜400＞ 54 Met Ala Glu Gln Leu Val Asp Leu Trp Asn Glu Cys Ala Lys Ala Cys 1 5 10 15 Val Leu Asp Leu Lys Asp Gly Val Arg Ser Ser Arg Met Ser Val Asp 20 25 30 Pro Ala Ile Ala Asp Thr Asn Gly Gln Gly Val Leu His Tyr Cys Met 35 40 45 Glu Phe Ser Ser Phe His Leu Phe His His Leu Pro Ala Arg Ala Pro 50 55 60 Leu Ala Pro Ser Glu Leu Gln Pro Leu Glu His Met Ala Glu Glu Ala 65 70 75 80 Phe Asp Leu Trp Asn Glu Cys Ala Lys Ala Cys Val Leu Asp Leu Lys 85 90 95 Asp Gly Val Arg Ser Ser Arg Met Ser Val Asp Pro Ala Ile Ala Asp 100 105 110 Thr Asn Gly Gln Gly Val Leu His Tyr Ser Met Val Leu Glu Gly Gly 115 120 125 Asn Asp Ala Leu Lys Leu Ala Ile Asp Asn Ala Leu Ser Ile Thr Ser 130 135 140 Asp Gly Leu Thr Ile Arg Leu Glu Gly Gly Val Glu Pro Asn Lys Pro 145 150 155 160 Val Arg Tyr Ser Tyr Thr Arg Gln Ala Arg Gly Ser Trp Ser Leu Asn 165 170 175 Trp Leu Val Pro Ile Gly His Glu Lys Pro Ser Asn Ile Lys Val Phe 180 185 190 Ile His Glu Leu Asn Ala Gly Asn Gln Leu Ser His Met Ser Pro Ile 195 200 205 Tyr Thr Ile Glu Met Gly Asp Glu Leu Leu Ala Lys Leu Ala Arg Asp 210 215 220 Ala Thr Phe Phe Val Arg Ala His Glu Ser Asn Glu Met Gln Pro Thr 225 230 235 240 Leu Ala Ile Ser His Ala Gly Val Ser Val Val Met Ala Gln Thr Gln 245 250 255 Pro Arg Arg Glu Lys Arg Trp Ser Glu Trp Ala Ser Gly Lys Val Leu 260 265 270 Cys Leu Leu Asp Pro Leu Asp Gly Val Tyr Asn Tyr Leu Ala Gln Gln 275 280 285 Arg Cys Asn Leu Asp Asp Thr Trp Glu Gly Lys Ile Tyr Arg Val Leu 290 295 300 Ala Gly Asn Pro Ala Lys His Asp Leu Asp Ile Lys Pro Thr Val Ile 305 310 315 320 Ser His Arg Leu His Phe Pro Glu Gly Gly Ser Leu Ala Ala Leu Thr 325 330 335 Ala His Gln Ala Cys His Leu Pro Leu Glu Thr Phe Thr Arg His Arg 340 345 350 Gln Pro Arg Gly Trp Glu Gln Leu Glu Gln Cys Gly Tyr Pro Val Gln 355 360 365 Arg Leu Val Ala Leu Tyr Leu Ala Ala Arg Leu Ser Trp Asn Gln Val 370 375 380 Asp Gln Val Ile Arg Asn Ala Leu Ala Ser Pro Gly Ser Gly Gly Asp 385 390 395 400 Leu Gly Glu Ala Ile Arg Glu Gln Pro Glu Gln Ala Arg Leu Ala Leu 405 410 415 Thr Leu Ala Ala Ala Glu Ser Glu Arg Phe Val Arg Gln Gly Thr Gly 420 425 430 Asn Asp Glu Ala Gly Ala Ala Asn Ala Asp Val Val Ser Leu Thr Cys 435 440 445 Pro Val Ala Ala Gly Glu Cys Ala Gly Pro Ala Asp Ser Gly Asp Ala 450 455 460 Leu Leu Glu Arg Asn Tyr Pro Thr Gly Ala Glu Phe Leu Gly Asp Gly 465 470 475 480 Gly Asp Val Ser Phe Ser Thr Arg Gly Thr Gln Asn Trp Thr Val Glu 485 490 495 Arg Leu Leu Gln Ala Glu Phe Leu Gly Val Ser Ala Ala Gln Glu Arg 500 505 510 Ile Pro Ile Arg Glu Val Ser Ala Asp Lys Glu Val Ser Ala Glu Lys 515 520 525 Lys Glu Ile Ser Phe Gly Val Ser Thr Ala Gln Gly Asn Ile Ser Phe 530 535 540 Gly Leu Gly Val Ser Thr Ala Gln Glu Ala Ile Pro Phe Leu Ala Leu 545 550 555 560 Gly Val Ser Thr Ala Gln Glu Thr Ile Pro Phe Gly Leu Leu Gly Val 565 570 575 Ser Thr Ala Gln Gly Ile Ile Ser Phe Gly Gly Val Ser Thr Ala Gln 580 585 590 Glu Asn Ile Ser Phe Gly Gly Val Ser Thr Ala Gln Glu Thr Ile Ser 595 600 605 Phe Gly Leu Leu Gly Val Ser Thr Ala Gln Glu Asn Ile Ser Phe Gly 610 615 620 Cys Leu Arg Thr His Glu Val Ser Ala Ala Gln Glu Lys Ile Ser Phe 625 630 635 640 Gly Gly Val Ser Glu Ala Gln Lys Ile Ser Phe Gly Val Ser Ala Ala 645 650 655 Gly Val Ser Ala Ala Gln Glu Glu Ile Pro Phe Gly Cys Leu Arg Pro 660 665 670 His Gly Leu Pro Ala Ala Gln Glu Lys Thr Ser Phe Gly Gly Val Ser 675 680 685 Ala Ala Gln Glu Lys Thr Ser Phe Gly Gly Val Ser Ala Ala Gln Glu 690 695 700 Glu Phe Ser Phe Gly Cys Leu Arg Pro His Arg Val Ser Ala Ala Gln 705 710 715 720 Glu Lys Ile Ser Phe Glu Val Ser Ala Leu Glu Val Ser Ala Ala Gln 725 730 735 Glu Lys Ile Ser Phe Gly Val Ser Ala Ala Leu Gly Val Ser Ala Ala 740 745 750 Gln Glu Lys Asn Ser Phe Gly Cys Leu Arg Pro His Gly Val Ser Ala 755 760 765 Ala Gln Glu Lys Thr Ser Phe Gly Gly Val Ser Ala Ala Gln Lys Lys 770 775 780 Ile Ser Phe Gly Leu Glu His His His His His His 785 790 795 ＜210＞ 55 ＜211＞ 48 ＜212＞ DNA ＜213＞ Artificial sequence ＜220＞ ＜223＞ primer ＜400＞ 55 gttgacccgg ttggtccgtc cgacccgtcc atcgtttccc tggttgaa 48 ＜210＞ 56 ＜211＞ 49 ＜212＞ DNA ＜213＞ Artificial sequence ＜220＞ ＜223＞ primer ＜400＞ 56 aggtcggagc accagcgtcg atgaaggagg tttcttcaac cagggaaac 49 ＜210＞ 57 ＜211＞ 48 ＜212＞ DNA ＜213＞ Artificial sequence ＜220＞ ＜223＞ primer ＜400＞ 57 gacaccctgg ctccggttcg tccgccgctg accgttgacc cggttggt 48 ＜210＞ 58 ＜211＞ 48 ＜212＞ DNA ＜213＞ Artificial sequence ＜220＞ ＜223＞ primer ＜400＞ 58 accggaaacg tccggcggga tggacggaac ggaggtcgga gcaccagc 48 ＜210＞ 59 ＜211＞ 51 ＜212＞ DNA ＜213＞ Artificial sequence ＜220＞ ＜223＞ primer ＜400＞ 59 tacatcccgc tgggtacccg tccgccgacc gctaccgaca ccctggctcc g 51 ＜210＞ 60 ＜211＞ 51 ＜212＞ DNA ＜213＞ Artificial sequence ＜220＞ ＜223＞ primer ＜400＞ 60 agccggggta gtgtcggtgg aggtggtgat ggagaaaccg gaaacgtccg g 51 ＜210＞ 61 ＜211＞ 48 ＜212＞ DNA ＜213＞ Artificial sequence ＜220＞ ＜223＞ primer ＜400＞ 61 ggtaccggtt ccggtactgg cggtcgtacc ggttacatcc cgctgggt 48 ＜210＞ 62 ＜211＞ 51 ＜212＞ DNA ＜213＞ Artificial sequence ＜220＞ ＜223＞ primer ＜400＞ 62 tacggtagta acggtgttgt tgttgatgtc caggatagcc ggggtagtgt c 51 ＜210＞ 63 ＜211＞ 51 ＜212＞ DNA ＜213＞ Artificial sequence ＜220＞ ＜223＞ primer ＜400＞ 63 ggttctatgg gtgttttctt cggcggtctg ggcatcggta ccggttccgg t 51 ＜210＞ 64 ＜211＞ 48 ＜212＞ DNA ＜213＞ Artificial sequence ＜220＞ ＜223＞ primer ＜400＞ 64 cgggtcggtg aaggtcgggt tgttgtgagt ggttacggta gtaacggt 48 ＜210＞ 65 ＜211＞ 48 ＜212＞ DNA ＜213＞ Artificial sequence ＜220＞ ＜223＞ primer ＜400＞ 65 ggtaaaacca tcgctgaaca gatcctgcaa tacggttcta tgggtgtt 48 ＜210＞ 66 ＜211＞ 48 ＜212＞ DNA ＜213＞ Artificial sequence ＜220＞ ＜223＞ primer ＜400＞ 66 ggtttcagcc ggggtcggcg gttgcagaac ggacgggtcg gtgaaggt 48 ＜210＞ 67 ＜211＞ 51 ＜212＞ DNA ＜213＞ Artificial sequence ＜220＞ ＜223＞ primer ＜400＞ 67 ggtacctgcc cgccggacat catcccgaaa gttgaaggta aaaccatcgc t 51 ＜210＞ 68 ＜211＞ 51 ＜212＞ DNA ＜213＞ Artificial sequence ＜220＞ ＜223＞ primer ＜400＞ 68 ggagatggtg gaagaggaca gggtgaagtg accaccggtt tcagccgggg t 51 ＜210＞ 69 ＜211＞ 45 ＜212＞ DNA ＜213＞ Artificial sequence ＜220＞ ＜223＞ primer ＜400＞ 69 acccagctgt acaaaacctg caaacaggct ggtacctgcc cgccg 45 ＜210＞ 70 ＜211＞ 45 ＜212＞ DNA ＜213＞ Artificial sequence ＜220＞ ＜223＞ primer ＜400＞ 70 gtccatcggg atttcttcgt agttgtgggt ggagatggtg gaaga 45 ＜210＞ 71 ＜211＞ 47 ＜212＞ DNA ＜213＞ Artificial sequence ＜220＞ ＜223＞ primer ＜400＞ 71 ccgtgctaaa cgtcgtaaac gtgcttccgc tacccagctg tacaaaa 47 ＜210＞ 72 ＜211＞ 48 ＜212＞ DNA ＜213＞ Artificial sequence ＜220＞ ＜223＞ primer ＜400＞ 72 attatttttc tcgagcagtt cgtctttggt gtccatcggg atttcttc 48 ＜210＞ 73 ＜211＞ 48 ＜212＞ DNA ＜213＞ Artificial sequence ＜220＞ ＜223＞ primer ＜400＞ 73 cccgaattcc atatggtcga cggtatgtcc atccgtgcta aacgtcgt 48 ＜210＞ 74 ＜211＞ 48 ＜212＞ DNA ＜213＞ Artificial sequence ＜220＞ ＜223＞ primer ＜400＞ 74 attatttttc tcgagcagtt cgtctttggt gtccatcggg atttcttc 48 ＜210＞ 75 ＜211＞ 21 ＜212＞ DNA ＜213＞ Artificial sequence ＜220＞ ＜223＞ primer ＜400＞ 75 ccccatatgg ccgaagaagc t 21 ＜210＞ 76 ＜211＞ 49 ＜212＞ DNA ＜213＞ Artificial sequence ＜220＞ ＜223＞ primer ＜400＞ 76 tttctcgagt tgaattccat ggagtagttc atcactccct ggccgttgg 49 ＜210＞ 77 ＜211＞ 21 ＜212＞ DNA ＜213＞ Artificial sequence ＜220＞ ＜223＞ primer ＜400＞ 77 ccccatatgg ccgaagaagc t 21 ＜210＞ 78 ＜211＞ 44 ＜212＞ DNA ＜213＞ Artificial sequence ＜220＞ ＜223＞ primer ＜400＞ 78 tttctcgagg aattcgacgt cgccgccgtc gccgaggaac tccg 44 ＜210＞ 79 ＜211＞ 21 ＜212＞ DNA ＜213＞ Artificial sequence ＜220＞ ＜223＞ primer ＜400＞ 79 ccccatatgg ccgaagaagc t 21 ＜210＞ 80 ＜211＞ 38 ＜212＞ DNA ＜213＞ Artificial sequence ＜220＞ ＜223＞ primer ＜400＞ 80 tttctcgagg aattccgcct ggagcagccg ctccaccg 38 ＜210＞ 81 ＜211＞ 30 ＜212＞ DNA ＜213＞ Artificial sequence ＜220＞ ＜223＞ primer ＜400＞ 81 tataccatgg ccgaacaatt ggtggacctc 30 ＜210＞ 82 ＜211＞ 40 ＜212＞ DNA ＜213＞ Artificial sequence ＜220＞ ＜223＞ primer ＜400＞ 82 tttctcgagg aattcttcca tgcagtagtg cagcacgccc 40

Claims (37)

  1. 一種融合胜肽，包含： (a)一黏膜標靶多肽； (b)一第一轉位肽，其係用於轉位；以及 (c)一第一表位。
  2. 如申請專利範圍第1項所述之融合胜肽，其中，該黏膜標靶多肽係位於該融合胜肽的N-末端，該第一表位係位於該融合胜肽的C-末端，並且該第一轉位肽係位於黏膜標靶胜肽和該第一表位之間。
  3. 如申請專利範圍第1項所述之融合胜肽，其中，該黏膜標靶多肽係一M細胞標靶多肽或一腸道上皮標靶多肽。
  4. 如申請專利範圍第1項所述之融合胜肽，其中，該黏膜標靶多肽包含一胺基酸序列，其係選自SEQ ID NO：1至4。
  5. 如申請專利範圍第1項所述之融合胜肽，其中，該第一轉位肽係來自綠膿桿菌外毒素。
  6. 如申請專利範圍第1項所述之融合胜肽，其中，該第一轉位肽包含一僅去除功能區III之綠膿桿菌外毒素A片段。
  7. 如申請專利範圍第1項所述之融合胜肽，其中，該第一表位係一Th1表位。
  8. 如申請專利範圍第1項所述之融合胜肽，其中，該第一表位係一HPV表位、一肌肉生長抑制素表位或一PRRSV表位。
  9. 如申請專利範圍第8項所述之融合胜肽，其中，該HPV表位係人類乳突病毒第16型之一E7胜肽序列或一E6胜肽序列。
  10. 如申請專利範圍第1項所述之融合胜肽，其中，該第一表位係選自SEQ ID NO：10、12、17、18、21、22和23。
  11. 一種增強誘發免疫反應之方法，包括： 向需要的一主體施用一組成物，其中，該組成物包含一疫苗和一融合胜肽，並且該融合胜肽包含： (a)一黏膜標靶多肽； (b)一第一轉位肽，其係用於轉位；以及 (c)一第一表位。
  12. 如申請專利範圍第11項所述之方法，其中，該黏膜標靶多肽係位於該融合胜肽的N-末端，該第一表位係位於該融合胜肽的C-末端，並且該第一轉位肽係位於黏膜標靶胜肽和該第一表位之間。
  13. 如申請專利範圍第11項所述之方法，其中，該黏膜標靶多肽係一M細胞標靶多肽或一腸道上皮標靶多肽。
  14. 如申請專利範圍第11項所述之方法，其中，該黏膜標靶多肽包含一胺基酸序列，其係選自SEQ ID NO：1至4。
  15. 如申請專利範圍第11項所述之方法，其中，該第一轉位肽係來自綠膿桿菌外毒素。
  16. 如申請專利範圍第11項所述之方法，其中，該第一轉位肽包含一僅去除功能區III之綠膿桿菌外毒素A片段。
  17. 如申請專利範圍第11項所述之方法，其中，該第一表位係一Th1表位。
  18. 如申請專利範圍第11項所述之方法，其中，該第一表位係一HPV表位、一肌肉生長抑制素表位或一PRRSV表位。
  19. 如申請專利範圍第18項所述之方法，其中，該HPV表位係人類乳突病毒第16型之一E7胜肽序列或一E6胜肽序列。
  20. 如申請專利範圍第11項所述之方法，其中，該第一表位係選自SEQ ID NO：10、12、17、18、21、22和23。
  21. 如申請專利範圍第11項所述之方法，該組成物係經口服投予需要之主體。
  22. 如申請專利範圍第11項所述之方法，其中，該疫苗包含：一用於轉位之第二轉位肽以及一第二表位。
  23. 如申請專利範圍第22項所述之方法，其中，該第二轉位肽係來自綠膿桿菌外毒素。
  24. 如申請專利範圍第22項所述之方法，其中，該第二轉位肽包含一僅去除功能區III之綠膿桿菌外毒素A片段。
  25. 如申請專利範圍第22項所述之方法，其中，該第二表位係一Th1表位。
  26. 如申請專利範圍第22項所述之方法，其中，該第二表位係一HPV表位、一肌肉生長抑制素表位或一PRRSV表位。
  27. 如申請專利範圍第11項所述之方法，其中，該疫苗係一抗-HPV疫苗、一抗-肌肉生長抑制素疫苗或一抗-PRRSV疫苗。
  28. 一種融合胜肽，包含： (b)一第一轉位肽，其係用於轉位；以及 (c)一第一表位，其係選自SEQ ID NO：10和12。
  29. 如申請專利範圍第28項所述之融合胜肽，其中，該第一轉位肽係來自綠膿桿菌外毒素。
  30. 如申請專利範圍第28項所述之融合胜肽，其中，該第一轉位肽包含一僅去除功能區III之綠膿桿菌外毒素A片段。
  31. 一種治療或預防HPV之方法，包括： 向需要之一主體施用一組成物，其中，該組成物包含一抗-HPV疫苗和一融合胜肽，並且該融合胜肽包含： (b)一第一轉位肽，其係用於轉位；以及 (c)一第一表位，其係選自SEQ ID NO: 10和12。
  32. 如申請專利範圍第31項所述之方法，其中，該第一轉位肽係來自綠膿桿菌外毒素。
  33. 如申請專利範圍第31項所述之方法，其中，該第一轉位肽包含一僅去除功能區III之綠膿桿菌外毒素A片段。
  34. 如申請專利範圍第31項所述之方法，該組成物係經口服投予需要之主體。
  35. 如申請專利範圍第31項所述之方法，其中，該疫苗包含：一用於轉位之第二轉位肽以及一HPV表位。
  36. 如申請專利範圍第35項所述之方法，其中，該第二轉位肽係來自綠膿桿菌外毒素。
  37. 如申請專利範圍第35項所述之方法，其中，該第二轉位肽包含一僅去除功能區III之綠膿桿菌外毒素A片段。
TW106136884A 2016-10-26 2017-10-26 用於免疫增強的融合胜肽及使用其增強誘發免疫反應的方法 TW201829470A (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201662412896P 2016-10-26 2016-10-26
US62/412,896 2016-10-26

Publications (1)

Publication Number Publication Date
TW201829470A true TW201829470A (zh) 2018-08-16

Family

ID=61971220

Family Applications (1)

Application Number Title Priority Date Filing Date
TW106136884A TW201829470A (zh) 2016-10-26 2017-10-26 用於免疫增強的融合胜肽及使用其增強誘發免疫反應的方法

Country Status (3)

Country Link
US (1) US10400013B2 (zh)
TW (1) TW201829470A (zh)
WO (1) WO2018081355A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
TWI839820B (zh) * 2022-08-25 2024-04-21 國立中興大學 表達質體、生產肌肉生長抑制素抗原胜肽的轉形株、重組肌肉生長抑制素抗原胜肽、其製備方法及其用途

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11246915B2 (en) 2010-09-15 2022-02-15 Applied Molecular Transport Inc. Cholix toxin-derived fusion molecules for oral delivery of biologically active cargo
US11129906B1 (en) 2016-12-07 2021-09-28 David Gordon Bermudes Chimeric protein toxins for expression by therapeutic bacteria
EP4316586A3 (en) 2018-03-08 2024-05-08 Applied Molecular Transport Inc. Toxin-derived delivery constructs for oral delivery
CN110881570B (zh) * 2018-09-10 2023-05-02 廖亭彰 饲料添加剂及包含其的饲料
BR112021009001A8 (pt) 2018-11-07 2021-10-26 Applied Molecular Transport Inc Veículos derivados de cholix para administração oral de carga útil heteróloga
WO2023037320A1 (en) * 2021-09-10 2023-03-16 Intron Biotechnology, Inc. Mucosal messenger rna vaccine

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7037501B2 (en) 2001-01-04 2006-05-02 Regents Of The University Of Minnesota Myostatin immnoconjugate
CA2451741C (en) 2001-07-02 2014-02-18 Elan Corporation, Plc Peyers's patch and/or m-celle targeting ligands
US7335361B2 (en) * 2003-06-09 2008-02-26 Animal Technology Institute Taiwan Fusion antigen used as vaccine
US7314630B2 (en) * 2005-01-07 2008-01-01 Yao-Xiong Hu Compounds and methods of early diagnosis of cervical cancer and genital condyloma with HPV, CHSP60 tumor suppressor H-Ras, K-Ras and PTEN derived peptides modified
WO2009073133A1 (en) * 2007-11-29 2009-06-11 Vaxinnate Corporation Compositions of toll-like receptor agonists and papillomavirus antigens and uses thereof
EP2237794B1 (en) 2008-01-31 2013-04-10 TheVax Genetics Vaccine Co., Ltd. Chimeric hiv fusion proteins as vaccines
KR101309109B1 (ko) * 2011-02-22 2013-09-16 전북대학교산학협력단 보체 단백질 Ｃ５ａ 수용체 리간드를 포함하는 점막 면역 보조제 및 이를 이용한 면역 유도 방법.
KR101849831B1 (ko) * 2014-09-05 2018-04-19 서울대학교산학협력단 M 세포 표적 펩타이드-항원 결합체 및 점막점착제를 포함하는 백신 조성물

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
TWI839820B (zh) * 2022-08-25 2024-04-21 國立中興大學 表達質體、生產肌肉生長抑制素抗原胜肽的轉形株、重組肌肉生長抑制素抗原胜肽、其製備方法及其用途

Also Published As

Publication number Publication date
US20180111964A1 (en) 2018-04-26
WO2018081355A1 (en) 2018-05-03
US10400013B2 (en) 2019-09-03

Similar Documents

Publication Publication Date Title
CN111088283B (zh) mVSV病毒载体及其病毒载体疫苗、一种基于mVSV介导的新冠肺炎疫苗
TW201829470A (zh) 用於免疫增強的融合胜肽及使用其增強誘發免疫反應的方法
WO2022262142A1 (zh) 一种产生广谱交叉中和活性的重组新型冠状病毒rbd三聚体蛋白疫苗、其制备方法和应用
CN104039833B (zh) 针对hpv的疫苗
JP7083362B2 (ja) セネカウイルスａ免疫原性組成物およびその方法
WO2022206222A1 (zh) 一种新型冠状病毒s-rbd三聚体蛋白疫苗、其制备方法和应用
CN113454102A (zh) 非洲猪瘟疫苗
CN113666990B (zh) 一种诱导广谱抗冠状病毒的t细胞疫苗免疫原及其应用
TWI621627B (zh) 用於豬生殖與呼吸綜合症及豬環狀病毒關聯疾病之疫苗組成物
WO2023138334A1 (zh) 一种重组新型冠状病毒蛋白疫苗、其制备方法和应用
WO2023023940A1 (zh) 一种诱导广谱抗冠状病毒的t细胞疫苗免疫原及其应用
JP2013537401A (ja) カンピロバクター感染を減少させるワクチン及び方法
CN106632694B (zh) 一种重组蛋白及药物组合物与应用
Lin et al. Avian reovirus sigma C enhances the mucosal and systemic immune responses elicited by antigen-conjugated lactic acid bacteria
JP6808650B2 (ja) 短縮型ロタウイルスｖｐ４タンパク質およびその適用
Gillam et al. Hepatitis B core antigen based novel vaccine against porcine epidemic diarrhea virus
US20160000901A1 (en) Compositions and Methods for the Production of Virus-Like Particles
WO2023138333A1 (zh) 一种重组新型冠状病毒蛋白疫苗、其制备方法和应用
Kovacs-Nolan et al. Tandem copies of a human rotavirus VP8 epitope can induce specific neutralizing antibodies in BALB/c mice
RU2691302C1 (ru) Иммуногенная композиция на основе рекомбинантных псевдоаденовирусных частиц, а также на основе белковых антигенов и способ получения иммуногенной композиции
WO2023184862A1 (zh) Hpv抗原表位及其鉴定方法、应用
TW528760B (en) Peptide repeat immunogens
EP4138901A1 (en) Compositions and methods for inducing immune responses against class i fusion protein viruses
TWI605056B (zh) 用於治療艱難梭狀芽孢桿菌（Clostridium difficile）相關疾病之組成物及方法
KR20120054465A (ko) 소 코로나바이러스 항원 결정기를 포함하는 스파이크 재조합 단백질 및 이에 대한 항체