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BACTERIAL TOXIN VACCINE

Foreign code F110006029
File No. S2008-0581
Posted date Dec 27, 2011
Country WIPO
International application number 2009JP058345
International publication number WO 2009133882
Date of international filing Apr 28, 2009
Date of international publication Nov 5, 2009
Priority data
  • P2008-120573 (May 2, 2008) JP
Title BACTERIAL TOXIN VACCINE
Abstract The object aims to produce a bacterial toxin protein such as Siga-toxin protein with high efficiency by using a plant cell. A bacterial toxin protein such as Siga-toxin protein can be produced in a plant cell by transforming the plant cell with a DNA construct which comprises DNA encoding a hybrid protein composed of bacterial toxin proteins linked in tandem via a peptide having any one of the following characteristic properties (A) and (B): (A) the number of amino acid residues is 12 to 30; and (B) the proline content is 20 to 35%.
Outline of related art and contending technology BACKGROUND ART
Shiga toxin (Stx, verotoxin) is, in the gastrointestinal tract of the pathogenic E. coli enterohemorrhagic E. coli (enterohemorrhagic Escherichia coli) in protein produced by a pyrogenic exotoxin. Shiga toxins, hemorrhagic colitis, hemolytic uremic syndrome, encephalopathy and others. Shiga toxins, divided into Stx2 large and Stx1, each divided into further sub-class. As Stx2, and Stx2e for example swine edema causes soft rot. Pig edema disease that is, 1-2 week after weaning the piglets are high known to be generated. Edema are inoculated with a mortality rate due to infection, and a high 50-90%. In addition, the cholera toxin (CT), protein produced by Vibrio cholerae toxin-in. Is CT, severe diarrhea and emesis are known to cause. In addition, E. coli heat labile toxin (LT) is, enterotoxigenic E. coli (enterotoxigenic Escherichia coli) in toxin-protein produced. LT is, diarrhea and emesis are known to cause. Stx, LT, of any bacterial toxins CT, involved in cell adhesion to the 5 subunit B subunit A toxicity 1 - fold amount by weight of laminates of known amount. In addition, CT and LT is, structurally functionally similar to the known.
These bacterial toxins as a method for preventing disease, a vaccine, or administered by injection or nasal spray, or orally administered in a known method. For example, the detoxified protein Stx2e and produced using recombinant Escherichia coli strain, a technique is adminstered by injection (non-patent document 1) are known. However, by the recombinant E. coli, and production of detoxified protein Stx2e is not sufficient and a problem in that the administration of the vaccine is by injection, such as human labor problem. In addition, orally administered vaccine with respect to the method, from the viewpoint of husbandry and reduce which are of interest in the art. In view of these circumstances, a bacterial toxin protein, to the plant using a transgenic technology for the production of the development of a technique is being performed. For example, LT B subunit protein encoding DNA (LTB) include, for transgenic plants expressing the DNA described in (patent document 1, patent document 2). In addition, CT LT protein or encoding a protein for a transgenic plant expressing the DNA described in (Patent Document 3). However, these techniques, a sufficient amount of protein production was not a problem. In addition, an example in which the lettuce produced LTB is reported (non-patent document 2). In this study, codon modified LT B subunit gene of the protein, the cauliflower mosaic virus promoter in plant 35S RNA to a high expression promoter and (CaMV35S), an enhancer Kozak sequence may be used, are expressed in lettuce. As a result, lettuce LT B subunit protein of about 2.0 mass % of the total soluble protein accumulation have been reported. However, the amount of accumulation of the protein of this size, using transgenic plant bacterial disease which is insufficient for efficiently controlling considered. That is, a bacterial toxin protein of interest in a plant cell with good efficiency, needs to be stored. The inventors of the present invention, the secretion signal peptide at the amino terminal end of vegetable origin added to the Stx2e protein, plant-derived alcohol dehydrogenase gene of the 5 '- untranslated region (ADH5' UTR) can be expressed by using, a lettuce plant of the Stx2e protein with good efficiency and the like, accumulated at high concentrations in the plant can be found, performing a patent application (patent document 4).
Scope of claims (In Japanese)請求の範囲 [請求項1]
 2つ又は3つの、志賀毒素タンパク質、コレラ毒素タンパク質又は大腸菌易熱性毒素タンパク質が、それぞれ、下記の特徴(A)及び(B)を有するペプチドを介してタンデムに連結された、ハイブリッドタンパク質、
 (A)アミノ酸の個数が12~30個;
 (B)プロリンの含有率が20~35%。

[請求項2]
 前記ペプチドが、さらに下記の特徴(C)を有する、請求項1に記載のハイブリッドタンパク質、
 (C)プロリンが、2アミノ酸置き、又は3アミノ酸置きに配置される。

[請求項3]
 前記ペプチドが、配列番号2、82又は84で表されるアミノ酸配列からなる、請求項2に記載のハイブリッドタンパク質。

[請求項4]
 2つの、志賀毒素タンパク質、コレラ毒素タンパク質又は大腸菌易熱性毒素タンパク質が、配列番号2で表されるアミノ酸配列からなるペプチドを介してタンデムに連結された、請求項3に記載のハイブリッドタンパク質。

[請求項5]
 志賀毒素タンパク質が、志賀毒素タンパク質のBサブユニットである、請求項1~4の何れか一項に記載のハイブリッドタンパク質。

[請求項6]
 志賀毒素タンパク質が、Stx2eタンパク質である、請求項1~5の何れか一項に記載のハイブリッドタンパク質。

[請求項7]
 コレラ毒素タンパク質が、コレラ毒素タンパク質のBサブユニットである、請求項1~4の何れか一項に記載のハイブリッドタンパク質。

[請求項8]
 配列番号10、12、14又は16で表されるアミノ酸配列を有する、請求項4に記載のハイブリッドタンパク質。

[請求項9]
 配列番号86、88、90、92、94、96、98又は100で表されるアミノ酸配列を有する、請求項3に記載のハイブリッドタンパク質。

[請求項10]
 アミノ末端に植物由来の分泌シグナルペプチドが付加された、請求項1~9の何れか一項に記載のハイブリッドタンパク質。

[請求項11]
 カルボキシル末端に小胞体残留シグナルペプチドが付加された、請求項10に記載のハイブリッドタンパク質。

[請求項12]
 アミノ末端に葉緑体移行シグナルペプチドが付加された、請求項1~9の何れか一項に記載のハイブリッドタンパク質。

[請求項13]
 請求項1~12の何れか一項に記載のハイブリッドタンパク質をコードするDNAを含むDNA構築物。

[請求項14]
 配列番号9、11、13又は15で表される塩基配列を有するDNAを含む請求項13に記載のDNA構築物。

[請求項15]
 配列番号85、87、89、91、93、95、97又は99で表される塩基配列を有するDNAを含む請求項13に記載のDNA構築物。

[請求項16]
 ハイブリッドタンパク質をコードするDNAが、植物由来のアルコールデヒドロゲナーゼ遺伝子の5’-非翻訳領域に発現可能に連結されている、請求項13~15の何れか一項に記載のDNA構築物。

[請求項17]
 前記植物由来のアルコールデヒドロゲナーゼ遺伝子の5’-非翻訳領域がタバコ由来である、請求項16に記載のDNA構築物。

[請求項18]
 配列番号24~29の何れかで表される塩基配列を有する請求項17に記載のDNA構築物。

[請求項19]
 配列番号101~111の何れかで表される塩基配列を有する請求項17に記載のDNA構築物。

[請求項20]
 請求項13~19の何れか一項に記載のDNA構築物を含む組み換えベクター。

[請求項21]
 請求項20に記載の組み換えベクターで形質転換された形質転換体。

[請求項22]
 形質転換体が形質転換植物細胞又は形質転換植物である、請求項21に記載の形質転換体。

[請求項23]
 請求項21又は22に記載の形質転換体から得られる種子。

[請求項24]
 配列番号2、82又は84で表されるアミノ酸配列からなるペプチド。

  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • IDEMITSU KOSAN CO., LTD.
  • NATIONAL UNIVERSITY CORPORATION NARA INSTITUTE OF SCIENCE AND TECHNOLOGY
  • Inventor
  • SAWADA, Kazutoshi
  • YOSHIDA, Kazuya
  • MATSUI, Takeshi
IPC(International Patent Classification)
Specified countries National States: AE AG AL AM AO AT AU AZ BA BB BG BH BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DO DZ EC EE EG ES FI GB GD GE GH GM GT HN HR HU ID IL IN IS JP KE KG KM KN KP KR KZ LA LC LK LR LS LT LU LY MA MD ME MG MK MN MW MX MY MZ NA NG NI NO NZ OM PG PH PL PT RO RS RU SC SD SE SG SK SL SM ST SV SY TJ TM TN TR TT TZ UA UG US UZ VC VN ZA ZM ZW
ARIPO: BW GH GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW
EAPO: AM AZ BY KG KZ MD RU TJ TM
EPO: AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LT LU LV MC MK MT NL NO PL PT RO SE SI SK TR
OAPI: BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG
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