JP6347838B2 - 遺伝子を発現する能力が改善された発現ベクター - Google Patents
遺伝子を発現する能力が改善された発現ベクター Download PDFInfo
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Description
(1)発現ベクターに発現がプロモーターによって調節される標的遺伝子を挿入する工程と、
(2)標的遺伝子が挿入されている発現ベクターで細胞を形質転換する工程と、
(3)発現ベクターによって形質転換された細胞を培養して標的タンパク質を発現させ、次いで標的タンパク質を回収する工程と
を含むことができる。
1−1:MARエレメント選択
ベクターを改善するために、ヒトβ−グロビン(β−グロビンMAR、3kb、2.2kb)のMAR及びヒトCSP−B遺伝子隣接SAR(1.2kb)を候補として選択した。また、タンパク質発現の間接的な評価を可能にするレポータ遺伝子アッセイを実行するために、ルシフェラーゼアッセイを、評価の利便性を考慮して選んだ。
国際特許公開WO 2009/102085に開示されるLCRエレメントのうち、in vivoで第IX因子発現増加効果を示したAAT 108(α−1−アンチトリプシン前駆体、120bp、配列番号13)及びAlb E6(血清アルブミンプレプロタンパク質、81bp、配列番号14)を選択して、in vitroでの効果を評価した。AAT 108又はAlb E6を、SV40後期poly(A)シグナルの5’領域、3’領域、並びにpGL4.2−CMV対照ベクターの5’及び3’領域の両方に付加して、試験用として様々なベクターを得た(図4を参照のこと)。
ヒト補体因子H(dCFH)の欠失形態の発現によってキメライントロンの効果を評価するために、dCFH遺伝子を最初に調製した。
更なる選択を、MAR及びLCRエレメントの一次選択で選択された4つのエレメントを使用してFIX(第IX因子)タンパク質発現のレベルを比較することによって実施した。上で調製したルシフェラーゼベクターからルシフェラーゼ遺伝子を除去し、FIX遺伝子をそこへ挿入することによってベクターを調製しようと計画した。新規ベクター調製において使用する場合、プライマーが、FIX遺伝子の前部にXhoI、PacI及びApaI制限酵素部位、並びに後部にApaI及びFseI部位を含むようなプライマー組(配列番号29及び30)を調製した。
実施例1において選択したエレメントを使用して新規ベクターを調製した。
上で調製した新規ベクターの遺伝子発現能力を、以下の通りに検証した。
最初に、新規ベクター対I及び新規ベクター対IIを使用するER2抗体発現を、CHO−K1及びCHO−DG44付着細胞株において実施した。発現レベルを、一対の市販の二重ベクター及び表4に示す一対のMARベクターと比較した。
新規ベクター対IIを、CHO−S及びCD DG44−S懸濁液細胞株において評価した。
新規ベクターの構成エレメントの特性評価を、CHO−S及びCD DG44−S細胞株において実施した。下記の表9に示すように、試験に使用したベクターの対は、以下の通りであった:pS(一対のプロモーターベクター)、pSI(一対のイントロンベクター)、pMS(新規ベクター対I)及びpMSI(新規ベクター対II)。
Claims (7)
- 作動的に連結されたシミアンウイルス40(SV40)プロモーター、マトリックス結合領域(MAR)エレメント、及びキメライントロンを含み、
該MARエレメントが、配列番号40又は41によって表され、
該キメライントロンが、配列番号25によって表され、
図19に開示されるpMSI−P−ER2LC又はpMSI−D−ER2HCのベクターマップを有する(ただし、前記ベクターマップに含まれたER2LC及びER2HCは前記プロモーターによって発現が調節される標的遺伝子に置換される)、発現ベクター。 - 前記SV40プロモーターが、配列番号12によって表される、請求項1に記載の発現ベクター。
- 配列番号15又は16によって表される遺伝子座制御領域(LCR)エレメントを更に含む、請求項1に記載の発現ベクター。
- 前記標的遺伝子が、
第VII因子(FVII)遺伝子、第VIII因子(FVIII)遺伝子、第IX因子(FIX)遺伝子、上皮増殖因子受容体ファミリー抗体遺伝子、インスリン遺伝子、インターロイキン遺伝子、腫瘍壊死因子遺伝子、インターフェロン遺伝子、コロニー刺激因子遺伝子、ケモカイン遺伝子、エリスロポイエチン遺伝子、α−フェトプロテイン遺伝子、a−グルコシダーゼ遺伝子、α1−アンチトリプシン遺伝子、アンチトロンビン遺伝子、リポタンパク質遺伝子、セルロプラスミン遺伝子、フィブリノゲン遺伝子、グルコセレブロシダーゼ遺伝子、ハプトグロビン遺伝子、プラスミノゲン遺伝子、プロトロンビン遺伝子、及びトランスフェリン遺伝子からなる群から選択される、請求項1に記載の発現ベクター。 - 請求項1〜4のいずれか一項に記載の発現ベクターによって形質転換された細胞。
- 前記細胞が、CHO−K1細胞、CHO−DG44細胞、CHO−S細胞、CHO DUKX−B11細胞、COS7細胞、COS3細胞、COS1細胞、NSO細胞、HeLa細胞、Vero細胞、PER−C6細胞、HepG2細胞、及びBHK細胞からなる群から選択される、請求項5に記載の細胞。
- 請求項1〜4のいずれか一項に記載の発現ベクターによって形質転換された細胞を培養して標的タンパク質を発現させる工程と、
該標的タンパク質を回収する工程と
を含む、標的タンパク質を産生する方法。
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