JP5554414B2 - 植物由来のvlpを調製する方法 - Google Patents
植物由来のvlpを調製する方法 Download PDFInfo
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- JP5554414B2 JP5554414B2 JP2012530059A JP2012530059A JP5554414B2 JP 5554414 B2 JP5554414 B2 JP 5554414B2 JP 2012530059 A JP2012530059 A JP 2012530059A JP 2012530059 A JP2012530059 A JP 2012530059A JP 5554414 B2 JP5554414 B2 JP 5554414B2
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Description
本発明は、植物由来のウイルス様粒子(VLP)を調製する方法に関する。
宿主細胞、例えば、大腸菌、昆虫細胞培養および哺乳動物細胞培養における現行の組換え発現戦略は、培養培地中で非常に高レベルでタンパク質を発現させ、分泌させる。これらの系を使用して、高レベルの発現、適当なタンパク質フォールディングおよびタンパク質の翻訳後修飾をなし遂げる。さらに、細胞内タンパク質が他の成分(DNA、小胞、膜、色素など)から容易に分離され得るため、発現されたタンパク質の精製は簡単にされる。植物または酵母発現系において、細胞壁は、培養培地への発現されたタンパク質の分泌を防止する。
本発明は、植物由来のウイルス様粒子(VLP)を調製する方法に関する。さらに具体的に、本発明は、インフルエンザ抗原を含むVLPを調製する方法に関する。
本発明は、植物由来のウイルス様粒子(VLP)を調製する方法に関する。さらに具体的には、本発明は、インフルエンザの血球凝集素(HA)を含むVLPを調製する方法に関する。
MAKNVAIFGLLFSLLLLVPSQIFAEE;配列番号10
を有するタンパク質ジスルフィドイソメラーゼシグナルペプチド(PDI)、植物病因関連タンパク質(PRP; Szyperskiら PNAS 95:2262-2262)、例えば、タバコ植物病因関連タンパク質2(PRP)、ヒトモノクローナル抗体シグナルペプチド(SP)、またはあらゆる天然血球凝集素シグナルペプチドを含む。
VLPの生産のために使用され得る構築物は、米国仮出願第61/220,161号(2009年6月24日に出願された)、WO2009/009876、WO2009/076778およびWO2010/003225(これら全てを出典明示により本明細書に包含させる)に記載されている。構築物は、また、表2に記載されているものを含み得る。これらの構築物のアセンブリーは、WO2009/009876、WO2009/076778、WO2010/003225およびUS61/220,161に記載されている。しかしながら、既知のHAを含む他の構築物、例えば、限定はしないが、表2に記載されているものおよび同様の、または異なる調節要素およびプロモーターと組み合わせられたものは、また、本明細書に記載されているVLPの生産のために使用され得る。
このカセットのアセンブリーは、WO2009/009876、WO2009/076778およびWO2010/003325(これらを出典明示により本明細書に包含させる)に記載されている。
転写後遺伝子サイレンシング(PTGS)は、植物中の導入遺伝子の発現の制限に関与する場合があり、導入遺伝子mRNAの特異的分解を中和するために、ジャガイモウイルスY(HcPro)由来のサイレンシングのサプレッサーの共発現が使用され得る(Brignetiら 1998)。サイレンシングの代替サプレッサー、例えば、限定はしないが、TEV−p1/HC−Pro(タバコエッチ病ウイルス−p1/HC−Pro)、BYV−p21、トマトブッシースタントウイルスのp19(TBSV p19)、トマトクリンクルウイルスのカプシドタンパク質(TCV−CP)、キュウリモザイクウイルスの2b(CMV−2b)、ジャガイモウイルスXのp25(PVX−p25)、ジャガイモウイルスMのp11(PVM−p11)、ジャガイモウイルスSのp11(PVS−p11)、ブルーベリースコーチウイルスのp16(BScV−p16)、カンキツトリステザウイルスのp23(CTV−p23)、ブドウ葉巻随伴ウイルス−2のp24(GLRaV−2 p24)、ブドウウイルスAのp10(GVA−p10)、ブドウウイルスBのp14(GVB−p14)、ヘラクレウム潜在ウイルスのp10(HLV−p10)、またはニンニク共通潜在ウイルスのp16(GCLV−p16)は、当分野でよく知られており、本明細書に記載されているとおりに使用され得る(Chibaら2006, Virology 346:7-14;これを出典明示により本明細書に包含させる)。したがって、サイレンシングのサプレッサー、例えば、限定はしないが、HcPro、TEV −p1/HC−Pro、BYV−p21、TBSV p19、TCV−CP、CMV−2b、PVX−p25、PVM−p11、PVS−p11、BScV−p16、CTV−p23、GLRaV−2 p24、GBV−p14、HLV−p10、GCLV−p16またはGVA−p10は、興味あるタンパク質をコードする核酸配列と共に共発現され、植物内で高レベルのタンパク質生産をさらに保証し得る。
GTATTAGTAATTAGAATTTGGTGTC(配列番号:11)
およびsupP19−plasto.r
CCTTGTATAGCTCGTTCCATTTTCTCTCAAGATG(配列番号:12)
を使用して、構築物660(WO2010/0003225に記載されている、これを出典明示により本明細書に包含させる)を鋳型として増幅した。並行して、p19のコード配列を含む別のフラグメントを、プライマーsupP19−1c
ATGGAACGAGCTATACAAGG(配列番号:13)
およびSupP19−SacI.r
AGTCGAGCTCTTACTCGCTTTCTTTTTCGAAG(配列番号:14)
でVoinnetら(The Plant Journal 33: 949-956 (2003))に記載されている構築物35S:p19を鋳型として使用して増幅した。次に、増幅産物を混合し、第2の増幅(アセンブリー反応)のための鋳型として、プライマーPlasto−443cおよびSupP19−SacI.rと使用した。得られたフラグメントをBamHI(プラストシアニンプロモーター中)およびSacI(p19コード配列の最後)で消化し、同じ制限酵素で以前に消化された構築物番号660にクローニングし、構築物番号R472を得た。該プラスミドを用いて、エレクトロポレーション(Mattanovichら 1989)により、アグロバクテリウム・ツメファシエンス(AGL1; ATCC, Manassas, VA 20108, USA)を形質転換した。全てのアグロバクテリウム・ツメファシエンス株の完全性を、制限マッピングにより確認した。R472を含むアグロバクテリウム・ツメファシエンス株を「AGL1/R472」と称した。
ベンサミアナタバコ(Nicotiana benthamiana)植物を、市販のピートモス培養器で満たされた平箱中で種子から生長させた。該植物は、16/8光周期および日中25℃/夜間20℃の温度レジメンの下で温室で生長させた。播種の3週間後、個々の小植物を選択し、ポットに移し、同じ環境条件下でさらに3週間温室で生長させておいた。6週間後、植物は80gの平均重量および30cmの高さを有した。
4、5、6、7および8日間のインキュベーション後、植物の地上部を回収し、即座に使用した。3容量の1%のTrition X−100および0.004%のメタ重亜硫酸ナトリウムを含む冷50mMのTris pH8.0、0.15MのNaCl中で植物組織を均質化することにより、全可溶性タンパク質を抽出した。植物組織を、1容量の冷50mMのTris pH8、0.15MのNaCl中で、POLYTRONTMを使用して機械的に均質化し、乳鉢および乳棒またはCOMITROLTMで粉砕した。COMITROLTMで使用されるバッファーは、また、0.04%のメタ重亜硫酸ナトリウムを含んだ。均質化後、粉砕された植物原料のスラリーを5,000gで4℃で5分遠心し、粗抽出物(上清)を分析のために保持した。浄化された粗抽出物の全タンパク質含有量を、参照標準としてウシ血清アルブミンを使用して、Bradfordアッセイ(Bio-Rad, Hercules, CA)により決定した。
葉組織をベンサミアナタバコ植物から回収し、〜1cm2切片に切った。葉切片を、室温(RT)で30分間、500mMのマンニトールに浸した。次に、マンニトール溶液を除去し、プロトプラスト化溶液(500mMのマンニトール、10mMのCaCl2および5mMのMES/KOH(pH5.6))中の酵素混合物(トリコデルマ・ビリデ(Trichoderma viride)由来のセルラーゼの混合物(Onozuka R−10;3%v/v)およびクモノスカビ属由来のペクチナーゼの混合物(MACEROZYMETM;0.75%v/v;両方ともYakult Pharmaceuticalsから)と交換した。使用される比率は、100mLの溶液あたり20gの葉切片であった。この調製物を浅い容器(〜11×18cm)に平坦に広げ、40rpmで26℃で回転震盪器上で16時間インキュベートした。
H5に関する血球凝集アッセイは、Nayak and Reichl (2004)により記載されている方法に基づいた。簡潔には、試験サンプル(100μL)の連続二重希釈を100μLのPBSを含むV字底96ウェルマイクロタイタープレート中で作り、ウェル当たり100μLの希釈されたサンプルを置いた。100マイクロリットルの0.25%のシチメンチョウ赤血球懸濁液(Bio Link Inc., Syracuse, NY)をそれぞれのウェルに加え、プレートを室温で2時間インキュベートした。完全血球凝集を示す最も高い希釈度の逆数を血球凝集活性として記録した。並行して、組換えHA5標準(A/Vietnam/1203/2004 H5N1)(Protein Science Corporation, Meriden, CT)をPBSで希釈し、それぞれのプレート上に対照として使用した。
HA5標準を、1%のTriton X−100での処理、次にTissue LyserTM(Qiagen)中で1分機械的撹拌により破壊された精製されたウイルス様粒子を用いて調製した。U字底96ウェルマイクロタイタープレートを、50mMの炭酸−重炭酸コートバッファー(pH9.6)中の10μg/mLの捕捉抗体(Immune Technology Corporation、#IT-003-005I)で4℃で16−18時間コートした。全ての洗浄を、0.1%のTween−20を含む0.01MのPBS(リン酸緩衝食塩水)pH7.4で行った。インキュベーション後、プレートを3回洗浄し、PBS中の1%のカゼインで37℃で1時間でブロックした。ブロッキング工程後、プレートを3回洗浄した。HA5標準を模造抽出物(AGL1/R472単独で浸潤させた葉組織から調製された)に希釈し、500から50ng/mLの標準曲線を作成した。定量するためのサンプルを、マイクロプレートに負荷する前に、1%のTriton X−100で処理した。プレートを37℃で1時間さらにインキュベートした。洗浄後、1:1000に希釈されたHA5(CBER/FDA)に対するヒツジポリクローナル抗体を加え、プレートを37℃で1時間インキュベートした。洗浄後、1:1000に希釈されたセイヨウワサビペルオキシダーゼ結合ウサギ抗ヒツジ抗体を加え、プレートを37℃で1時間インキュベートした。最終洗浄後、プレートを室温で20分SureBlue TMBペルオキシダーゼ基質(KPL)とインキュベートした。反応を1NのHClの添加により停止させ、A450値をMultiskan Ascent プレートリーダー(Thermo Scientific)を使用して測定した。
本発明の酵素抽出方法から得られたHAの量および相対活性を、一般的な機械的抽出方法で得られたHAのものと比較した。ベンサミアナタバコ植物をAGL1/685で浸潤させ、5から6日のインキュベーション期間後に葉を回収した。葉ホモジネートを次の通りに調製した:2グラムの葉をホモジェナイザーでホモジェナイズし;4gの葉を乳鉢および乳棒で粉砕し;25kgの葉を抽出バッファー(50mMのTris、150mMのNaCl pH8.0、1:1w/v比率)中で、COMITROLTM処理装置(Urschel Laboratories)でホモジェナイズした。酵素抽出を次の通りに行った:20グラムの回収した葉を、上記されているMacerozymeペクチナーゼおよびOnozuka R−10セルラーゼで消化に付した。消化後、葉の破片を濾過(ナイロン濾過、250μmメッシュ)により除去した。懸濁液中のプロトプラストを200×g(15分)で遠心分離により除去し、上清を5000×g(15分)で遠心分離によりさらに浄化した。
表3:植物の葉の機械的および酵素抽出から得られたHAの量および相対活性の比較。全てのデータは、それぞれの抽出方法のために加えられた液体の容量の差を考慮して調整した。ComitrolTM抽出方法を本比較分析の目的のための標準値として選択した。
分画遠心分離およびサイズ排除クロマトグラフィー(SEC)の組合せを、本明細書に記載されている酵素抽出方法により得られたHAがVLPとして組織化されていることを証明するために使用した。ベンサミアナタバコ植物を実施例1に記載されているAGL1/685でアグロ浸潤された。実施例1に記載のように、葉を浸潤から6日後に植物から回収し、〜1cm2切片に切り、次に消化し、粗濾過し、遠心した。
浄化された粗抽出物の全タンパク質含有量を、参照標準としてウシ血清アルブミンを使用するBradford アッセイ(Bio-Rad, Hercules, CA)により決定した。SEC溶出画分に存在するタンパク質をアセトンで沈殿させ(Bollagら 1996)、SDS−PAGE分析または免疫ブロット法分析のぞれぞれのために、0.25容量または0.05容量のいずれかの変性サンプルローディングバッファー(0.1MのTris pH6.8、0.05%のブロモフェノールブルー、12.5%のグリセロール、4%のSDSおよび5%のベータ−メルカプトエタノール)に再懸濁した。SDS−PAGEによる分離を還元条件下で行い、Coomassie Brillant Blue R−250をタンパク質染色のために使用した。
ベンサミアナタバコ植物を、実施例1に記載されているAGL1/685でアグロ浸潤した。実施例1に記載されているように、葉を浸潤から6日後に回収し、〜1cm2切片に切り、消化し、粗濾過し、遠心した。
ベンサミアナタバコ植物を、実施例1に記載されているAGL1/685でアグロインフィルトレーションした。葉を浸潤から6日後に回収し、〜1cm2切片に切り、オービタルシェーカー中で室温で15時間消化した。消化バッファーは、600mMのマンニトール、75mMのクエン酸塩、0.04%の亜硫酸水素ナトリウム pH6.0バッファー溶液中にそれぞれ、1.0%(v/v)のマルチフェクトペクチナーゼFE、1.0%(v/v)のマルチフェクト CX CGおよび/または1.0%(v/v)のマルチフェクト CX B(全てGenencorから)を含み、バイオマス:消化バッファー比率は1:2.5(w/v)を使用した。
ベンサミアナタバコ植物を、実施例1に記載されている興味ある血球凝集素(H1/Cal WT、B/Flo、H5/IndoまたはH1/Cal X179A)を発現する構築物を有するアグロバクテリウムAGL1株でアグロ浸潤した。葉を浸潤から6日後に回収し、〜1cm2切片に切り、以下に記載されていることを除いて実施例4にしたがって消化した。濾過、遠心分離および浄化は、実施例4に記載されているとおりに行った。
表4:HA−VLP回収率に対する消化工程へのNaClの添加の効果(血球凝集活性単位、dil:希釈度の逆数により測定される)
表5:浄化工程後のHA−VLP回収率に対する消化工程へのNaClの添加の効果(血球凝集活性単位により測定される)
ベンサミアナタバコ植物を、実施例1に記載されている興味ある血球凝集素(H5/Indo)を発現する構築物を有するアグロバクテリウムAGL1株でアグロ浸潤した。葉を浸潤から6日後に回収し、〜1cm2切片に切り、500mMのNaClまたは500mMのNaClおよび25mMのEDTAの消化バッファーへの添加で実施例4に記載されているとおりに消化した。濾過、遠心分離および浄化は、実施例4に記載されているとおりに行った。
表6:H5−VLP調製物の緑色着色に対する消化バッファーへの25mMのEDTAの添加の効果
ベンサミアナタバコ植物を、実施例1に記載されている興味ある血球凝集素(H5/Indo)を発現する構築物を有するアグロバクテリウムAGL1株でアグロ浸潤した。葉を浸潤から6日後に回収し、〜1cm2切片に切り、表7−9に記載されているとおり0%、0.25%、0.5%、0.75%または1%v/vのマルチフェクトペクチナーゼFE、マルチフェクトCX−CGセルラーゼおよびマルチフェクトCX Bセルロースを含む消化バッファーの修飾で実施例4にしたがって消化した。濾過、遠心分離および浄化は、実施例4に記載されているとおりに行った。
表7:ベンサミアナタバコの葉の消化によるH5/Indo VLPの放出。全ての条件を反復で試験した(HA−VLPの濃度は、血球凝集活性、dil:希釈度の逆数により測定した)。
消化中のpHの制御は、いくつかのVLPの抽出に重要であり得る。消化工程中に起こる細胞壁の脱重合が、適当なバッファーの存在下で溶液を酸性化することができる(すなわちpH6から5)酸糖を放出することができること、およびいくつかのVLP(例えば、H3/BrisおよびB/Flo HAを含むもの)が穏やかな酸性条件に対する強い感受性をすでに証明していることを考慮して、生産されるVLPの収率に対するかかる起こりうる酸性化の影響を調査した。
表10:B/Flo VLPの抽出収率に対する消化バッファー組成の効果
表11:H5/Indo VLPの抽出収率に対する消化バッファーの最初のpHの効果
表12:B/Flo VLPの抽出収率に対する種々の消化バッファー成分の効果
表13:H3/Bris VLPの抽出収率に対する消化溶液中のマンニトールおよび亜硫酸水素ナトリウムの濃度の効果
表14:異なる濃度のマンニトールを有するバッファー中で実施されたバイオマスの消化からのH5/Indo VLPの放出
22つの試験を、マンニトールなし(試験1)および100mMのマンニトール有り(試験2)対600mMのマンニトールで、VLPの抽出収率を比較するために実施した
本明細書に記載されている植物バイオマスに関する酵素消化方法は、広範な種類のHA−VLPの抽出に適用される可能性がある。上記実施例に示されているH5/Indo、H1/Cal WT VLP、H3/BrisおよびB/Floを含むHA−VLPの抽出に加えて、本明細書に記載されている方法は、また、表15に示されている季節的H1/BrisおよびH1/NC由来のHA−VLPの抽出のために適当であることが示された。
表15:アグロ浸潤されたベンサミアナタバコの葉の消化からの季節的H1/BrisおよびH1/NC VLPの放出(血球凝集活性により測定されるHAの濃度、dil:希釈度の逆数)。
Claims (43)
- 植物由来のウイルス様粒子(VLP)を調製する方法であって、
a.アポプラスト局在VLPを含む植物または植物材料を得ること、
b.1つもしくはそれ以上のセルラーゼを含む細胞壁分解酵素の多成分混合物による植物または植物材料の処理により、プロトプラスト/スフェロプラスト画分およびアポプラスト画分を生産すること;および、
c.アポプラスト画分を回収すること、ここに、該アポプラスト画分は、植物由来のVLPを含む
を含む方法。 - 細胞壁分解酵素の多成分混合物が、1つもしくはそれ以上のペクチナーゼをさらに含む、請求項1に記載の方法。
- 1つもしくはそれ以上のペクチナーゼの濃度が、0.01%(v/v)から2.5%(v/v)である、請求項2に記載の方法。
- 1つもしくはそれ以上のセルラーゼの濃度が、0.1%(w/v)から5%(w/v)である、請求項1に記載の方法。
- 細胞壁分解酵素の多成分混合物が、1つまたはそれ以上のリパーゼ、プロテアーゼまたはペクチナーゼを含まない、請求項1に記載の方法。
- 得る工程(工程a)において、植物が、ウイルスエンベロープタンパク質、ウイルス構造タンパク質、ウイルスカプシドタンパク質、およびウイルスコートタンパク質の群から選択されるタンパク質をコードする核酸配列で形質転換され、該植物または植物材料が回収される、請求項1に記載の方法。
- 核酸が植物に一過性で導入される、請求項6に記載の方法。
- 核酸が植物のゲノム内に安定に組み込まれる、請求項6に記載の方法。
- 得る工程(工程a)において、植物を成長させ、植物または植物材料を回収する、請求項1に記載の方法。
- 核酸がインフルエンザの血球凝集素をコードする、請求項6に記載の方法。
- 植物由来のウイルス様粒子(VLP)がノイラミニダーゼまたはMタンパク質を含まない、請求項1に記載の方法。
- 植物材料が葉および培養された植物細胞の群から選択される、請求項1に記載の方法。
- d)アポプラスト画分から植物由来のウイルス様粒子(VLP)を精製する工程をさらに含む、請求項1に記載の方法。
- 精製する工程が、浄化された抽出物を生産するための深層濾過を使用してアポプラスト画分を濾過すること、次に、陽イオン交換樹脂を使用する浄化された抽出物のクロマトグラフィーを含む、請求項13に記載の方法。
- 植物由来の脂質エンベロープを含む植物由来のウイルス様粒子(VLP)を調製する方法であって、
a.アポプラスト局在VLPを含む植物または植物材料を得ること、
b.植物または植物材料を、1つもしくはそれ以上のセルラーゼを含む酵素の多成分混合物で処理して、プロトプラスト/スフェロプラスト画分およびVLPを含む1つもしくはそれ以上のアポプラストタンパク質複合体を生産すること;
c.プロトプラスト/スフェロプラスト画分から1つもしくはそれ以上のアポプラストタンパク質複合体を分離すること
を含む方法。 - 酵素の多成分混合物が、1つもしくはそれ以上のペクチナーゼをさらに含む、請求項15に記載の方法。
- 1つもしくはそれ以上のペクチナーゼの濃度が、0.01%(v/v)から2.5%(v/v)である、請求項16に記載の方法。
- 1つもしくはそれ以上のセルラーゼの濃度が、0.1%(w/v)から5%(w/v)である、請求項15に記載の方法。
- 酵素の多成分混合物が、1つまたはそれ以上のリパーゼ、プロテアーゼまたはペクチナーゼを含まない、請求項15に記載の方法。
- d)1つもしくはそれ以上のアポプラストタンパク質複合体から植物由来のウイルス様粒子(VLP)を精製する工程をさらに含む、請求項15に記載の方法。
- 精製する工程が、浄化された抽出物を生産するための深層濾過、次に、陽イオン交換樹脂を使用する浄化された抽出物のクロマトグラフィーを含む、請求項20に記載の方法。
- 得る工程(工程a)において、植物が、ウイルスエンベロープタンパク質、ウイルス構造タンパク質、ウイルスカプシドタンパク質、およびウイルスコートタンパク質の群から選択されるタンパク質をコードする核酸配列で形質転換され、該植物または植物材料が回収される、請求項15に記載の方法。
- 核酸が植物に一過性で導入される、請求項22に記載の方法。
- 核酸が植物のゲノム内に安定に組み込まれる、請求項22に記載の方法。
- 得る工程(工程a)において、植物を成長させ、植物または植物材料を回収する、請求項15に記載の方法。
- ウイルス様粒子(VLP)がインフルエンザの血球凝集素を含む、請求項22に記載の方法。
- 植物材料が葉および培養された植物細胞の群から選択される、請求項15に記載の方法。
- ウイルス様粒子(VLP)がノイラミニダーゼまたはMタンパク質を含まない、請求項15に記載の方法。
- a.植物由来のウイルス様粒子(VLP)を含む植物または植物材料を得ること、
b.消化された画分を生産するために、1つもしくはそれ以上のセルラーゼを含む細胞壁分解酵素の多成分混合物を使用して植物または植物材料を消化すること、
c.濾過された画分を生産するために消化された画分を濾過し、濾過された画分から植物由来のVLPを回収すること
を含む、植物由来のVLPを調製する方法。 - 細胞壁分解酵素の多成分混合物が、1つもしくはそれ以上のペクチナーゼをさらに含む、請求項29に記載の方法。
- 1つもしくはそれ以上のペクチナーゼの濃度が、0.01%(v/v)から2.5%(v/v)である、請求項30に記載の方法。
- 1つもしくはそれ以上のセルラーゼの濃度が、0.1%(w/v)から5%(w/v)である、請求項29に記載の方法。
- 細胞壁分解酵素の多成分混合物が、1つまたはそれ以上のリパーゼ、プロテアーゼまたはペクチナーゼを含まない、請求項29に記載の方法。
- 得る工程(工程a)において、植物が、ウイルスエンベロープタンパク質、ウイルス構造タンパク質、ウイルスカプシドタンパク質、およびウイルスコートタンパク質の群から選択されるタンパク質をコードする核酸配列で形質転換され、植物または植物材料が回収される、請求項29に記載の方法。
- 核酸が植物に一過性で導入される、請求項34に記載の方法。
- 核酸が植物のゲノム内に安定に組み込まれる、請求項34に記載の方法。
- 植物由来のウイルス様粒子(VLP)がインフルエンザの血球凝集素を含む、請求項34に記載の方法。
- 植物材料が葉および培養された植物細胞の群から選択される、請求項29に記載の方法。
- d)細胞残屑および不溶性材料から濾過された画分中の該ウイルス様粒子(VLP)を分離する工程をさらに含む、請求項29に記載の方法。
- 分離する工程が遠心分離により行われる、請求項39に記載の方法。
- 分離する工程が深層濾過により行われる、請求項39に記載の方法。
- d)濾過された画分から植物由来のウイルス様粒子(VLP)を精製する工程をさらに含む、請求項29に記載の方法。
- 精製する工程が、浄化された抽出物を生産するための濾過された画分の深層濾過、次に、陽イオン交換樹脂を使用する浄化された抽出物のクロマトグラフィーを含む、請求項42に記載の方法。
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