JP5899109B2 - 高発現組換え細胞系の選択方法 - Google Patents
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Description
dhfr:ジヒドロ葉酸リダクターゼ(dihydrofolate reductase)
CHO:チャイニーズハムスター卵巣細胞(Chinese Hamster Ovary)
poly A:ポリアデニル化シグナル(polyadenylation signal)
p−clone:単一細胞由来クローンではない予備クローン(preliminary clone)
MTX:メトトレキサート(methotrexate)
ELISA:酵素結合免疫吸着測定法(enzyme-linked immunosorbent assay)
本明細書中の用語「選択マーカー遺伝子(selection marker gene)」とは、目的蛋白質の遺伝子と連結されて発現ベクターに挿入されたマーカー遺伝子であり、それによって、目的遺伝子が正常に発現される細胞を確認することができる。また、選択マーカー遺伝子によってコードされる蛋白質の抑制剤を培地に添加すれば、選択マーカー遺伝子のコピー数を増加させたり、あるいは増加した選択マーカー遺伝子に連結されている目的蛋白質の遺伝子のコピー数をともに増加させることができる。選択マーカーとしては、dhfr遺伝子、グルタミンシンテターゼ(glutamine synthetase)遺伝子、ネオマイシンホスホトランスフェラーゼ(neomycinephosphotransferase)遺伝子、ハイグロマイシンBホスホトランスフェラーゼ(hygromycin B phosphotransferase)遺伝子、ピューロマイシン−N−アセチルトランスフェラーゼ(pac、puromycin−N−acetyltransferase)遺伝子またはストレプトアロテイクス・ヒンダスタヌス(Sh、Streptoalloteichus hindustanus)ble遺伝子などがある。
本発明者らは、高生産性クローンを選択する新規な方法について研究し、その結果、poly AがmRNAの転写と安定性に大きく影響を及ぼし、そして、poly Aの長さは時間の経過と共に幾分短くなった場合、mRNAが分解され始め、そしてこの理由のため、poly Aが正常に作動しなければ、mRNAの半減期は正常mRNAに比べてはるかに短い。この知見に基づき、poly Aに注目して新規な選択方法を開発するための研究を行った。
poly Aの有無による遺伝子の発現程度を調べるための最初の段階として、IgG抗体発現ベクターであるpCT107ベクター(図1)内の3つの転写ユニットであるdhfr遺伝子、重鎖遺伝子及び軽鎖遺伝子の3’末端に作動可能に連結されたpoly Aを作動不能な状態にするために、前記pCT107ベクターを、それぞれRsr II、Pme I及びCla I制限酵素を用いて切断した。前記制限酵素Rsr II、Pme I及びCla Iは、それぞれdhfr遺伝子、重鎖遺伝子及び軽鎖遺伝子の3’末端と対応するpoly A間に存在する一定配列を特異的に認識して切断することによって、IgG抗体発現ベクターを線形化(linearization)させる酵素である。
実施例1によって線形化されたベクターを、CHO DG44細胞系に同量で形質移入した。次の表1に示すように対照群と実験群への形質移入を行った。
IgG抗体遺伝子の発現有無を確認するために、形質移入3日後に、ELISAを行った。まず、ヤギの抗ヒト免疫グロブリンG(Fcγ)(109−006−098、Jackson ImmunoReserarch)を、96−ウェル・マイクロタイタープレート(449824、Nunc)上に吸着させた。前記プレートを1%ウシ血清アルブミン(bovine serum albumin、BSA)を含むリン酸緩衝生理食塩水(phospate-buffered saline、PBS)で処理してブロッキングした後、連続希釈した試料を各ウェルに添加した。プレートを室温で2時間放置した後、ペルオキシダーゼ標識ヤギ抗ヒトカッパー抗体(peroxidase-labeled goat anti-human κ antibody)(I1514、Sigma)で検出のため処理した。室温で1時間放置した後、前記試料をテトラメチルベンジジン(tetramethyl benzidine、TMB)と反応させ、1N HClを加えて反応を停止させた。スタンダードとしては、骨髄種プラズマから精製したヒトIgG1 カッパー(human IgG1 kappa purified myeloma plasma)(A7164、Sigma)を250ng/mlの濃度から用いた。プレートリーダー(Spectramax plus 384、Molecular Device)を使用し、450/650nmで吸光度を測定した。
選択マーカー遺伝子であるdhfr遺伝子の3’末端に連結されたpoly Aが作動しないように操作するために、前記実施例1と同じ処理条件下で、pCT107ベクターをRsr II制限酵素(R0501、NEB)で処理し、それにより実験群(E)を得た。得られたベクターをCHO DG44細胞系に形質移入した。対照群としては、制限酵素で処理していないpCT107ベクター(対照群(A))を用いた。前記実施例1と同じ方法で、形質移入を行った。対照群(A)のベクターと実験群(E)のベクターとを細胞系に導入し、3日後に測定したIgG抗体の発現レベルは、それぞれ3.9μg/ml及び3.8μg/mlであった。形質移入に続く培養開始後の3日目、対照群(A)細胞及び実験群(E)細胞をそれぞれの細胞を96−ウェルプレートに移し、続いて、2%ウシ胎児血清(fetal bovine serum、FBS)及び100nM MTX(813630、Bedford Labs)を含んだSFM4CHO(商標)培地(SH30549.02、HyClone)中で培養した。対照群(A)の場合、0.5×106細胞/96−ウェルプレートを接種して培養し、そして、実験群(E)の場合、2×106細胞/96−ウェルプレートを接種して培養した。
さまざまな細胞系で、遺伝子増幅に関与しない一般的な選択マーカー遺伝子の利用性を調べるために、各選択マーカー遺伝子に作用する薬剤を、表3に記述した条件下で各細胞系に適用し、細胞増殖の抑制を測定した。
先に確認したとおり、poly Aが作動不能に連結されたdhfr選択マーカー遺伝子を用いた選択方法が、CHO DG44細胞系での高生産性クローン選択において、費用及び時間を低減する観点から、非常に効率的である。このような選択方法が、dhfr遺伝子以外の選択マーカーとCHO DG44細胞系以外の細胞系でも適用されるか否かを調べるために、本実験において、poly Aが完全に欠損したpac選択マーカー遺伝子を用いた選択方法をCHO K1細胞株に適用して、その効能を検討した。
Claims (1)
- 発現ベクターを用いた高生産性クローンの選択方法であって、前記発現ベクターは、(i)ポリアデニル化シグナル(poly A)が作動不能に連結された選択マーカー遺伝子、又は、poly Aを除去した選択マーカー遺伝子を含む遺伝子発現カセット、及び、(ii)poly Aが作動可能に連結された目的組換え蛋白質をコードする遺伝子発現カセットを含み、
前記方法は、前記発現ベクターで宿主細胞をトランスフェトする工程、前記選択マーカー遺伝子に対する選択的条件下で前記トランスフェクトされた宿主細胞を培養する工程、及び前記選択的条件下で生存している前記トランスフェクトされた宿主細胞を選択する工程を含み、
ここで、前記選択マーカー遺伝子が、ジヒドロ葉酸レダクターゼ(dhfr)遺伝子、又は、ピューロマイシン−N−アセチルトランスフェラーゼ(pac)遺伝子であり、
前記目的組換え蛋白質が、モノクローナル抗体であり、そして、
前記宿主細胞が、CHO細胞である、方法。
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