JP5524823B2 - 自己免疫疾患を予防および/または治療するワクチン - Google Patents
自己免疫疾患を予防および/または治療するワクチン Download PDFInfo
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Description
1)I型糖尿病自己タンパク質抗原と、前記I型糖尿病自己タンパク質抗原のコード遺伝子がマルチクローニング部位に挿入された組換え真核細胞発現ベクターとからなる混合物
2)I型糖尿病自己タンパク質抗原エピトープポリペプチドと、前記I型糖尿病自己タンパク質抗原エピトープポリペプチドのコード遺伝子がマルチクローニング部位に挿入された組換え真核細胞発現ベクターとからなる混合物
3)I型糖尿病自己タンパク質抗原と、前記I型糖尿病自己タンパク質抗原エピトープポリペプチドのコード遺伝子がマルチクローニング部位に挿入された組換え真核細胞発現ベクターとからなる混合物
4)I型糖尿病自己タンパク質抗原エピトープポリペプチドと、前記I型糖尿病自己タンパク質抗原のコード遺伝子がマルチクローニング部位に挿入された組換え真核細胞発現ベクターとからなる混合物
2)I型糖尿病自己タンパク質抗原エピトープポリペプチドと、前記I型糖尿病自己タンパク質抗原エピトープポリペプチドのコード遺伝子がマルチクローニング部位に挿入された組換え真核細胞発現ベクターとの質量比は1:5〜5:1とし、1:1〜1:2であることが好ましい。
3)I型糖尿病自己タンパク質抗原と、前記I型糖尿病自己タンパク質抗原エピトープポリペプチドのコード遺伝子がマルチクローニング部位に挿入された組換え真核細胞発現ベクターとの質量比は1:5〜5:1とし、1:1〜1:2であることが好ましい。
4)I型糖尿病自己タンパク質抗原エピトープポリペプチドと、前記I型糖尿病自己タンパク質抗原のコード遺伝子がマルチクローニング部位に挿入された組換え真核細胞発現ベクターとの質量比は1:5〜5:1とし、1:1〜1:2であることが好ましい。
ヒト膵臓組織を抽出し、タカラ製のRNA抽出試薬キットを用いて、TrizoL試薬において充分に研磨し、取扱説明書に基づき全RNAの抽出を行った。発表されている遺伝子配列に基づきプライマーを設計した。プライマーの配列は、下記のとおりである。
ヒトインスリンcDNA遺伝子を増幅するプライマー、
一、Balb/cマウス試験
1、Balb/cマウス上の細胞免疫反応
2、細胞免疫反応の特異性
3、細胞免疫反応の用量関係試験
二、NODマウス上の共免疫予防試験
自己免疫性卵巣疾患(AOD:autoimmune ovarian disease)は、ヒトの早発卵巣不全(POF:premature ovarian failure)の誘引の1つである。AODの自己タンパク質抗原には、卵透明帯タンパク質3(ZP3)がある。
1 材料と方法
1.1 材料と試薬
1.2 mzp3遺伝子のクローン
1.3 真核細胞発現ベクターの構築および組換えプラスミドのBHK21細胞における一過性発現
1.4 原核細胞発現ベクターの構築、タンパク質発現およびタンパク質純化
1.5 卵巣炎モデルの誘導
1.6 共免疫による卵巣炎の治療
SI=(各刺激ウェルのOD値−培地のOD値)/(刺激していないウェルのOD値−培地のOD値)。
2.結果
2.1 遺伝子クローンおよび配列分析
2.2 真核細胞発現ベクターの構築および組換えプラスミドのBHK21細胞における一過性発現
2.3 原核細胞発現ベクターの構築、タンパク質発現およびタンパク質純化
2.4 自己免疫疾患−卵巣炎の誘導
2.5 MZP3 DNAおよびタンパク質の共免疫によりAODの発生の抑制
2.6 定性共免疫誘導の調節性T細胞の特徴
研究
1、MOG35−55 2コピーの遺伝子のクローンと発現
1.1 材料と試薬
1.2 方法
1.2.1 MOG35−55遺伝子のクローン
1.2.1 真核細胞発現ベクターの構築および組換えプラスミドのBHK21細胞における一過性発現
1.3 試験結果
2、EAE動物モデルの誘導および同定
2.1 直接免疫の方法:
評価基準:
0−いかなる臨床症状もない。
1−動物の尾部の脱力、麻痺
2−動物の尾部の脱力、前肢または後肢の中等度の脱力。
3−前肢または後肢の重篤な脱力、人為的に仰臥位にした後に元に戻ることができない
4−肢体の麻痺、人為的に仰臥位にした後に元に戻ることができない
5−瀕死状態。
☆最後に免疫したロット(20匹)は、最初の免疫の後5週間で4まで罹患したものが4匹いた。
2.2 罹患したマウスのT細胞誘導モデルの養子移入
2.3 罹患したマウスの生理指標の測定
3、EAEマウスの併用免疫治療
3.1 起案
1、MOG35−55ペプチドl00μl(フロイント完全アジュバントと1:1で混合し、濃度1μg/μlまで完全に乳化させる)
2、pVAXMOG352cプラスミドDNAl00μl(濃度1μg/μl)
3、pVAXMOG352cMOG35−55ペプチド+pVAXMOG352cそれぞれl00μl(濃度は同上)
4、pVAXMOG352cMOG35−55ペプチド+pVAX−insulinそれぞれl00μl(濃度は同上)
5、不治療群
Claims (11)
- 哺乳類の自己免疫疾患を予防および/または治療するためのワクチンであって、
a)自己免疫疾患をもたらす自己抗原及びそのエピトープポリペプチドから成る群から選択されるタンパク質;及び
b)当該自己抗原又はそのエピトープポリペプチドをコードする挿入断片を含んで成る組換え真核細胞発現ベクター、
を含んで成り、前記自己抗原が、インスリン、グルタミン酸脱炭酸酵素及び卵透明帯タンパク質3から成る群から選択され、当該ワクチンが調節性T細胞を刺激して抗原特異性のT細胞反応を抑制する、自己免疫疾患を予防および/または治療するためのワクチン。 - 1)前記自己免疫疾患がI型糖尿病であり、
2)i)前記タンパク質が前記自己抗原であり、且つ前記挿入断片が前記自己抗原をコードするか;
ii)前記タンパク質が前記自己抗原のエピトープポリペプチドであり、且つ前記挿入断片が前記自己抗原のエピトープポリペプチドをコードするか;
iii)前記タンパク質が前記自己抗原であり、且つ前記挿入断片が前記自己抗原のエピトープポリペプチドをコードするか;あるいは
iv)前記タンパク質が前記自己抗原のエピトープポリペプチドであり、且つ前記挿入断片が前記自己抗原をコードし、そして
3)前記自己抗原がインスリン及びグルタミン酸脱炭酸酵素(GAD)から成る群から選択される、請求項1に記載のワクチン。 - 前記インスリンがヒト、イヌ及びネコから成る群から選択される哺乳類由来のものである、請求項2に記載のワクチン。
- 前記自己抗原がヒトインスリンである、請求項3に記載のワクチン。
- 前記自己抗原のアミノ酸配列が配列番号1の配列を有する、請求項3に記載のワクチン。
- 前記ベクターが哺乳動物の細胞発現ベクターである、請求項2に記載のワクチン。
- 前記哺乳動物の細胞発現ベクターが、pcDNA3.0またはpVAX1及びprovaxから成る群から選択される、請求項6に記載のワクチン。
- 前記タンパク質がヒトインスリンであり、前記挿入断片がインスリンをコードし、前記ベクターがpVAXである、請求項7に記載のワクチン。
- 前記タンパク質がヒトインスリン断片B9−23であり、前記挿入断片がヒトインスリン断片B9-23をコードし、前記ベクターがpcDNA3.0である、請求項7に記載のワクチン。
- 前記タンパク質と前記ベクターとの質量比が5:1〜1:5である、請求項2に記載のワクチン。
- 前記前記タンパク質と前記ベクターとの質量比が1:1〜1:2である、請求項2に記載のワクチン。
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CN200710064769.X | 2007-03-26 | ||
CNB200710064769XA CN100571786C (zh) | 2007-03-26 | 2007-03-26 | 一种预防和/或治疗自身免疫疾病的疫苗 |
PCT/CN2008/000540 WO2008116380A1 (en) | 2007-03-26 | 2008-03-19 | A vaccine preventing and/or treating autoimmune diseases |
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US9314790B1 (en) * | 2001-05-21 | 2016-04-19 | Safety Art, Llc | System for separation of a sample |
US9169579B2 (en) * | 2005-03-11 | 2015-10-27 | New Jersey Institute Of Technology | Carbon nanotube mediated membrane extraction |
CN100571786C (zh) * | 2007-03-26 | 2009-12-23 | 中国农业大学 | 一种预防和/或治疗自身免疫疾病的疫苗 |
JP5592080B2 (ja) * | 2009-05-08 | 2014-09-17 | 株式会社日立製作所 | 細胞培養方法、細胞培養システム |
CN103200959A (zh) | 2010-09-27 | 2013-07-10 | 中国农业大学 | 预防和治疗自身免疫性疾病的联合抗原和dna疫苗 |
CN103031322B (zh) * | 2011-09-30 | 2014-10-22 | 浙江大学宁波理工学院 | 谷氨酸脱羧酶热稳定变体g311p基因及其用途 |
US20150044244A1 (en) * | 2011-09-30 | 2015-02-12 | Beijing Advanccine Biotechnology Co. Ltd. | Combined facilitator, antigen and dna vaccine for preventing and treating autoimmune diseases |
WO2013067652A1 (en) | 2011-11-10 | 2013-05-16 | Beijing Advaccine Biotechnology Co., Ltd. | Facilitator-dna combination vaccine |
WO2014165164A2 (en) * | 2013-03-12 | 2014-10-09 | Albany Medical College | Compositions and methods for treating autoimmune diseases |
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CA2681775C (en) | 2018-04-10 |
CN100571786C (zh) | 2009-12-23 |
JP2010522220A (ja) | 2010-07-01 |
US20100143401A1 (en) | 2010-06-10 |
WO2008116380A1 (en) | 2008-10-02 |
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US20130089566A1 (en) | 2013-04-11 |
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