JP6807347B2 - 植物における高品質の組み換えアレルゲンの生産方法 - Google Patents
植物における高品質の組み換えアレルゲンの生産方法 Download PDFInfo
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Description
− 遺伝子からタンパク質への移行のために長い時間を要すること(開発の仕事が数年かかることを意味する)、及び
− 可溶性タンパク質の0.1%〜1%オーダーである、低い収量(ラージスケールでの生産用の植物材料のバイオマスの大容量の処理を意味する)
を伴う植物遺伝子組み換えを採用している。
a)植物を、好ましくは、移動性フロート上、LED照射下で気耕栽培又は水耕栽培し、
b)工程a)で得られた植物を、真空下、組み換えタンパク質をコードするDNAフラグメントを含むアグロバクテリアにより、アグロインフィルトレーションし、次に、
c)植物を、工程b)の後、工程a)についてのものと同一条件下での培養に戻し、次に、
d)組み換えタンパク質を、工程c)で生産された植物の地上部から抽出及び精製する、
を含む方法に関する。
1)真空下、好ましくは、−0.8barで2分間置き、
2)真空を解除し、大気圧に、好ましくは、30秒間戻し、次に、
3)真空下、好ましくは、−0.8barで2分間置き、その後、大気圧に戻す
で行われる。
本明細書に記載の方法を検証するため、ダニ、木、又はカビの複合アレルゲンをコードするcDNAを、pAG01ベクターにクローニングした。次に、これらのベクターを、ニコチアナ・ベンタミアーナにおける一過性発現のために、アグロバクテリア(LBA4404株)に挿入した。
組み換えアレルゲンの成熟化及び均一性をモニターするために、異なるシグナル(R1、R2、R3、及びR4)を、対象のアレルゲンと融合させ、次に融合タンパク質を、pAG01ベクターにクローニングした。次に、これらのベクターを、ニコチアナ・ベンタミアーナでの一過性発現のために、アグロバクテリア(LBA4404株)に挿入した。
この実施例のため、本発明者等は、本発明に記載の方法を用いて結合したpAG01ベクターの使用を、例えば、Medrano et al. (2009)に記載の通常のトランスフェクション方法で結合したバイナリーベクター(−/+サイレンシングインヒビター)の使用と比較した。
ニコチアナ・ベンタミアーナ植物の葉を回収し、タンパク質を、NaCl(0.1M)を添加したリン酸塩バッファー中、pH7.5でこの植物材料を粉砕することにより、抽出した。迅速な濾過後、抽出物を、固定ニッケルカラム上に置いた。クロマトグラフィー基質に対する親和性を有しない抽出物のタンパク質は、カラム上に保持されない。しかしながら、方法により生産された組み換えアレルゲンは、ヘキサ−ヒスチジンタグを有し、この種の基質上に保持される。カラムを洗浄して、混入したタンパク質を除去した後、アレルゲンを、リン酸塩バッファー中の50mMイミダゾールの存在下で特異的に溶出した。
1)固定ニッケル親和性カラム(IMAC)、及び2)分子粉砕。
トラック1:総タンパク質抽出物。
トラック2:固定ニッケルカラム(IMAC)上で精製し、50mM イミダゾールの存在下で溶出したDer p 4。
トラック3:IMAC工程後、分子粉砕により精製したDer p 4。
トラック4:タグをインビトロで切断し、精製したDer p 4。
上部のパネル:SDS−PAGE後、ゲルにおいてクーマシーブルーでタンパク質を染色することによるDer p 4の分析。
下部のパネル:SDS−PAGE後、ウエスタンブロット、及び精製タグの特異的な免疫血清でのプリント上での免疫検出によるDer p 4の分析。
Claims (8)
- タバコ植物における組み換えアレルゲンの生産方法であって、以下の工程:
a)植物を、LED照射下で気耕栽培又は水耕栽培し、次に、
b)工程a)で得られた植物を、真空下、組み換えアレルゲンをコードするDNAフラグメントが挿入された発現ベクターであって配列番号21を含む発現ベクターを含むアグロバクテリアにより、アグロインフィルトレーションし、次に、
c)植物を、工程b)の後、工程a)についてのものと同一条件下での培養に戻し、次に、
d)組み換えアレルゲンを、工程c)で生産された植物の地上部から抽出及び精製する、を含む方法。 - 組み換えアレルゲンが、組み換えダニアレルゲンであることを特徴とする、請求項1記載の方法。
- 組み換えアレルゲンが、デルマトファゴイデス・ファリナエのアレルゲン、デルマトファゴイデス・プテロニッシヌスのアレルゲン、オイログリフス・マネイのアレルゲン、アカルス・シロのアレルゲン、ブロミア・トロピカリスのアレルゲン、ゴキブリアレルゲン、木又は草の花粉アレルゲン、動物のアレルゲン、カビのアレルゲン、ヘベアラテックスのアレルゲン、及び食物アレルギーに関与するアレルゲンから選択されることを特徴とする、請求項1又は2記載の方法。
- 組み換えアレルゲンが、Der f 10、Der f 11、Der f 13、Der f 14、Der f 15、Der f 16、Der f 17、Der f 18、Der f 2、Der f 2.0101、Der f 2.0102、Der f 2.0103、Der f 2.0104、Der f 2.0105、Der f 2.0106、Der f 2.0107、Der f 2.0108、Der f 2.0109、D er f 2.0110、Der f 2.0111、Der f 2.0112、Der f 2.0113、Der f 2.0114、Der f 2.011 5、Der f 2.0116、Der f 2.0117、Der f 20、Der f 3、Der f 4、Der f 5、Der f 6、Der f 7、Der f 8、Der f 9、及びDer f HSP70から選択される、請求項1〜3の1項記載の方法。
- 組み換えアレルゲンが、Der p 10、Der p 11、Der p 14、Der p 15、Der p 18、Der p 2、Der p 2.0101、Der p 2.0102、Der p 2.0103、Der p 2.0104、Der p 2.0105、Der p 2 .0106、Der p 2.0107、Der p 2.0108、Der p 2.0109、Der p 2.0110、Der p 2.0111、Der p 2.0112、Der p 2.0113、Der p 20、Der p 21、Der p 3、Der p 4、Der p 5、Der p 6 、Der p 7、Der p 8、及びDer p 9から選択される、請求項1〜3の1項記載の方法。
- 組み換えアレルゲンが、Blo t 1、Blo t 5、Blo t 9、Blo t 10、Blo t 12、及びBlo t 21から選択されることを特徴とする、請求項1〜3の1項記載の方法。
- アグロインフィルトレーションが、ベンチュリー効果により真空下で行われることを特徴とする、請求項1〜6の1項記載の方法。
- アグロインフィルトレーションが、植物を真空下に2分間置く工程、又は、真空に置き、次に真空を解除して大気圧に戻し、次に真空下置き、最後に大気圧に戻すことにより、行われることを特徴とする、請求項7記載の方法。
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