JPS6336786A - Nucleic acid base sequence of antibody light chain manifestation type, plasmid, cell and chimera antibody light chain - Google Patents
Nucleic acid base sequence of antibody light chain manifestation type, plasmid, cell and chimera antibody light chainInfo
- Publication number
- JPS6336786A JPS6336786A JP61178881A JP17888186A JPS6336786A JP S6336786 A JPS6336786 A JP S6336786A JP 61178881 A JP61178881 A JP 61178881A JP 17888186 A JP17888186 A JP 17888186A JP S6336786 A JPS6336786 A JP S6336786A
- Authority
- JP
- Japan
- Prior art keywords
- region
- chain
- sequence
- antibody
- dna
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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- C—CHEMISTRY; METALLURGY
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Abstract
Description
ａ、産業上の利用分野
本発明は、抗体り鎖発現型核酸塩基配列、マウスーヒ）
Ｗのキメラ抗体り鎖発現屋核酸塩晶配列、これら核ｌ！
ｌ塩晶配列が組込まれた組み換えフラスミド、該プラス
ミドが導入されたマウスミエローマ細胞及びマウス−ヒ
ト型のキメラ抗体り鎖に関するものである。
本明細書において、アミノ酸、ポリペプチドはＩＵＰＡ
Ｃ−ＩＵＢ生化学委員会（ｃＢＮ）で採用された方法に
より略記するものとし、たとえば下記の略号を用いる。
ＡｌａＬ−７ラニン
ＡｒｇＬ−フルギニン
Ａｓｎ Ｌ ７スバラギン
Ａｓｐ Ｌ−アスパラギン酸
ｃｙ纂 Ｌ−システィン
Ｇｉｎ Ｌ−グルタミン
Ｇｌｕ Ｌ−グルタミン酸
Ｇ１７ グリシン
Ｈｉｓ Ｌ−ヒスチジン
１１ｅ Ｌ−イン口・ｒシン
Ｌｅｕ Ｌ−ロイシン
Ｌｙｅ Ｌ−リジン
Ｍｅｔ Ｌ−メチオニン
Ｐｈｅ Ｌ−フェニルアラニン
Ｐｒｏ Ｌ−ブーリン
Ｓｅｒ Ｌ−セリン
Ｔｈｒ Ｌ−スレオニン
Ｔｒｐ Ｌ −トリプトファン
Ｔｙｒ Ｌ−チロシン
Ｖａｌ Ｌ−バリン
また、ＤＮＡの配列はそれを構成する各デオキシリポヌ
クレチオドに含まれる塩基の種類で略記するものとし、
たとえば下肥の略号を用いろ。
Ａ アデニン（デオキシアデニル酸を示１゜）Ｃシトシ
ン（デオキシシチジル酸を示す。）Ｇ グアニン（デオ
キシグアニル酸を示す。）Ｔ チミン （デオキシチミ
ジル酸な示す。）ｂ、従来技術
一般に免疫グロブリン遺伝子は、Ｈ鎖（ＨｅａｖｙＣｈ
ａｉｎ）とＬ鎖（Ｌｉｇｈｔ Ｃｈａｉｎ）とから構成
され、Ｈ鎖とＬ鎖のそれぞれは、抗原と特異的に結合す
る機能を有するＶ領域（可変領域）とイフエクター機能
をするＣ領域（定常領域）とを有している。
一万マウスの抗体におけるＨ鎖及びＬ＠の遺伝子に関し
てＶ領域とＣ領域の内領域に含まれる種々の遺伝子単位
をはじめ、プロモーター。
エンハンサ−などの種々の機能な肩する単位の存在が明
らかにされ、その一部はＤＮＡ配列が明らかにされてい
る。
本発明者は、成る株の抗原に対して特異性を有するマウ
ス・・イブリドーマの産生する抗体のＬ鎖について研究
を行った結果、その抗原に対して結合する特異性ｔｔ規
定する■領域の遺伝子並びＫその抗体のＶ領域のタンパ
クを得ることができ、先に提案した。
そこで本発明者は、前記マウス由来のＬ鎖のＶ領域とヒ
ト由来のＬ鎖のＣ領域を結合させた所謂キメラ型のＬ鎖
の発現について＊に研究を進めた。
そこで本発明の目的は、抗体り鎖の高効率の発現製核酸
塩基配列、殊にマウス−ヒト型のキメラ抗体り鎖の発現
型核酸塩晶配列を提供することにある。
本発明の他の目的は、これら核１！！塩基配列が組み込
まれた組み換えプラスミドを提供することＫある。
本発明の更に他の目的は、前記プラスミドが導入された
マウスミエローマ細砲を提供することにある。
本発明の更に他の目的は、マウス−ヒト型のキメラ抗体
り鎖を提供することにある。
本発明の更に他の目的は以下の貌明から明らかとなるで
あろう。
本発明者の研究によれば、前記した本発明の目的は、先
ず
伝） 抗体り鎖におけるＶ領域のＤＮＡ断片（ｂ）
抗体ＬｙａｌｌにおけるＣ領域のＤＮＡ断片及び
（ｅ） ヒト又はマウス抗体Ｈａ由米のエンハンサ−
ＤＮＡ配列
よりなる抗体り鎖発現型核酸塩基配列によって達成され
ることがわかった。
かかる本発明においては、ヒト又はマウス抗体Ｈ鎖由来
のエンハンサ−ＤＮＡ配列を用いることによって、優れ
た発現性を有する抗体り銀核酸塩基配列が得らＴする。
この抗体り銀核酸塩基配列は、■領域及びＣ領域のいず
れもＤＮＡ断片がマウス由来のものであってもよ（ヒト
由来のものであってもよい。また、■領域のＤＮＡ断片
がマウス抗体由来のものであり且つＣ領域のＤＮＡ断片
がヒト抗体由来のものであろキメラ星の核酸塩基配列で
あることができる。
このようなキメラ型の核酸塩基配列は、成る特定の抗原
に対して特異性を有するマウス抗体のＬ鎖のＶ領域の遺
伝子を、■領域のＤＮＡ断片として使用することによっ
て１人類にとって有用なキメラ抗体を創製するために利
用することが期待できる。
本発明の抗体Ｌｌｌｉ１１発税型核散塩基配列は、前述
したようにヒト又はマウス抗体ＨＧ由米のエンハンサ−
Ｌ）ＮＡ配列を含んでいる。このエンハンサ−ＤＮＡ配
列は、ヒト又はマウス抗体のＨａ中に存在するエンハン
サ−機能を有するＤＮＡ配列であればよく、例えばヨー
ロッパ特許＠１４６７４３号明細書に開示さｉたヒト抗
体Ｈ鎖由来のエンハンサ−ＤＮＡ配列を使用することが
できる。殊に添付Ｂｉ図に示されたヒト抗体Ｈ鎖由来の
ＤＮＡ配列及びそれに相補的なＤＮＡ配列な有利に使用
することができる。エンハンサ−ＤＮＡ配列は１本発明
の抗体り鎖発現型核駿塩基配列中に含まれていればよ（
、その位置ｉ工問わない。すなわち、抗体り鎖に遺伝子
においてＶ領域のＤＮＡ断片、Ｃ領域の断片と並ぶＤＮ
Ａ配列上でＶ領域のＤＮＡ断片とＣ領域断片の間に位置
してもよ（、また該Ｖ領域のＤＮＡ断片の５′−側、Ｃ
領域のＤＮＡ断片の３′−側に位置していてもよい。
さらに本発明の抗体ＬｆＪＡ発現型核酸塩基配列には、
プロモーターや他の機能を有するＤＮＡ断片が含まれて
いても差支えがない。
本発明の前記核酸塩基配列において、それがマウス−ヒ
ト型のキメラ抗体り鎖発現型である場合において、その
Ｖ領域のＤＮＡ断片としては、本発明者が見出し先に提
案した抗ヒト急性白血病リンパ脂共通抗原抗体を産生す
るマウスハイプリドーマにおけるＬ＠のＶ領域のアミノ
酸配列を少くともフードする核＃！Ｉ塩基配列であるこ
とができる。このし＠のｖｉａ域のアミノ酸配列を少く
ともコードしている核ｒ１！塩基配列としては、添付第
２図に示したアミノ酸配列において、１番目（Ｇｌ！ｕ
）から９７：Ｉ！−目（Ｌｅｕ）までのアミノ酸配列を
少くとも〕−ドしているものでもよく、また１番目（Ｇ
／ｕ）から１０９番目（Ａｒｇ）までのアミノ酸配列を
少くともコードしているものであってもよい。添付第２
図のアミノ酸配列において１番目（Ｇ／ｕ）から９７７
番目Ｌｅｕ）までの配列は前記ハイプリドーマにおける
抗体のＬ鎖のＶ領域のアミノ酸配列であり、また１番目
（Ｇ／ｕ）から１０９番目（Ａｒｇ）までの配列はＸ’
Ｌ鎖のＶ領域とＪ領域のアミノ酸配列である。
前記Ｖ領域のアミノ酸配列（ｌ番目〜９７番目）ヲ少く
ともフードしている核酸塩基配列としては、＃付第３図
の５７９番目番目から８６９番目（ＱまでのＤＮＡ配列
であることができ、またＶ領域とＪ領域を少くともコー
ドしている核！！塩基配列としては、添付第３図の５７
９番目Ｃ１から第９０５査目σ〕までのＤＮＡ配列であ
ることができる。
さらに前記Ｖ領域の７ミノ酸量列を少くともコードして
いる核酸塩晶配列としては、同第３図において５６２番
目■から９０５査目Ｇ）までのＤＮＡ配列であってもよ
（、また３４０番目囚から９０５査目（ｒ）までのＤＮ
Ａ配列であることができ、また２３０査目のから９０５
査目のまでのＤＮＡ配列であってもよい。
本発明の抗体り鎖発現屋核酸塩晶配列を構成する抗体り
鎖に？けるＣ領域のＤＮＡ断片としては、ヒト抗体に鎖
由来のものであるのが好ましい。またか〜るＣ領域のＤ
ＮＡ断片として、添付第４図のアミノ酸配列を少くとも
コードしているＤＮＡ配列であることができ、特にその
ＤＮＡ配列としては％添付第５図のＤＮＡ配列を一つの
具体的例として挙げることができる。
前述の如（して、（ａ）抗体り鎖におけるＶ領域のＤＮ
Ａ断片、（ｈ）抗体り鎖におゆろＣ領域のＤＮＡ断片及
びヒト又はマウス抗体Ｈａ由来のエンハンサ−ＤＮＡ配
列よりなる抗体Ｌｍ発現型核酸塩基配列が形成される。
このＬ鎖のＤＮＡ配列は、■領域とＣ領域がマウス−マ
ウス屋或いはヒト−ヒト型の如きホモジニアスなＬ鎖で
あってもよくマウス−ヒト型のキメラＬｉ１１であって
もよい。
前記エンハンサ−ＤＮＡ配列は、ヒト又はマウス抗体Ｈ
鎖由来のエンハンサ−を含んでいればよ（、それによっ
て発税可能になるが、そのＤＮＡ配列には、それ以外の
エンハンサ−やマウス或いはヒト由来のイントロンを含
んでいても差支えない。
本発明の抗体り鎖発現型核酸塩基配列は、過当なベクタ
ープラスミド、例えはｐ ｓ ｖ ２ｇｐｔｔＰＳｖ２
ｎａｏ 、ＰＫＳＶ−１０などに組み込むことによって
組み換えプラスミドが調製される。
か（して調製された組み換えプラスミドは、それ自体公
知の例えばプロトプラスト法〔免疫実験操作法Ｘ１ｌ１
４５３３頁、 日本免疫学会編。
（１９８４） ＃照〕又はＤＥＡＥ−テキストラン法〔
セルＣＣｅ１１）第３３巻７２９負（１９８３）＃照〕
などにより宿主動物細胞、例えばマウスミエローマ（例
えばＪ５５８ＬＩＮＳ−１など）に導入し、形質転換細
胞を得る。得られた形質転換細胞を培養することにより
培養物中（目的とする抗体り鎖と蓄積させることができ
、この培養物から抗体り鎖と分離９回収することができ
る。
か（して本発明にＳいては、マウス−ヒト型のキメラ抗
体り鎖発現型核酸塩基配列を組み込んだプラスミドを、
マウスミエローマ細胞中に導入し、この細胞を培養する
ことＫよって、（１）マウス抗体り鎖におけるＶ領域の
アミノ酸配列と
（１）ヒト抗体Ｌ１ａにおけるＣ領域のアミノ酸配列
とからなるマウス−ヒトキメラ抗体Ｌ＠が提供される。
このキメラ抗体り鎖のＶ領域のアミノ酸配列は、例えば
添付第２図の１番目（ＧＪｕ）から９７番目（Ｌｅｕ）
までのアミノ酸配列を少くとも有している。このアミノ
酸配列は同第２図の１番目（ＧＪｕ）から１０９査目（
Ａｒｇ） ｔでのｖｇｔ４域とＪ領域のアミノ酸配列で
あってもよい。
−万前記キメラ抗体り鎖のＣ領域のアミノ酸配列は、例
えば添付＠４図のアミノ酸配列であることができる。
前述の如（して本発明により提供されるキメラ抗体り鎖
は１例えば本発明者の一部が先に提案したマウス−ヒト
抗体Ｈ鎖と組合せて完全な形のマウス−ヒト抗体とする
ことによって、そのま−或いは抗癌剤と結合させてヒト
急性白血病リンパ腫患者に投与することにより急件白血
病すンパ鹿の免疫学的治療に使用することが期待されろ
。
以下実施例を掲げ本発明の核ｒＩＲ塩晶配列その作成及
び抗体り鎖の発現などについて史に詳細に説明する。
実施例１（マウス染色体ＤＮＡの単離）マウスハイプリ
ドーマＮ Ｌ −］ １ ｘ １０！個を１％Ｓ Ｄ
Ｓ （Ｓｏｄｉｕｍ １ａｕｒｙｌ ５ｕｌｆａｔｅ
）存在下プロテアーゼＫ（シグマ社製）で処理した後
、水飽和フェノールを加えＤＮＡｔｔ抽出した。遠心に
より水相な分ｍｌし、１０ ｍＭ Ｔｒｉａ −ＨＣｌ
ｐＨ’７．４ 、０ 、］ｍＭ Ｎａα、０．１ｍＭ
ＥＤＴＡ （ＴＮＥ ）バッファーで透析した。リボヌ
クレアーゼＡ（シグマ）で処理し、再度フェノール抽出
を行なった後、ＴＮＥバッファーで透析しマウス染色体
Ｄ Ｎ Ａ １．２〜を得たＣＮ、ブライン、Ｄ、Ｗ。
スタフオード：ヌクレイツクアシッドリサーチ３巻２３
０３ページ（１９７ｔｉ ） （Ｎ、Ｂｌｔｎ＋ Ｄ−
Ｗ。
５ｔａｆｆｏｒｄ ： Ｎｕｅｌｓｉｃ Ａｃ１ｄｓ
Ｒｅ５−＋ ３ ２３０３（１９７６））ε照〕
実施例２（マウス遺伝子ライブラリーの作成）実施例１
で得られたマウス染色体ＤＮＡ１５０μＩを後述する実
施例４に示した方法に準じて制＠酵素Ｈ１ｎｄｎｌ（宝
酒造）で消化した後、密度勾配遠心〔蔗糖１０〜４０
％ （ｗｔ／ ｖｏｌ ） ＋蔗糖２８０００ｒ、ｐ、
ｍ、Ｘ １５時間＋ ２０℃〕を行ない、４Ｋｂ〜９
Ｋｂに相当するＤＮＡ断片を得た。
次にこのＤ Ｎ Ａ ’ｌＪｒ片０．４５μｐとＣｈａ
ｒｏｎ ２８ベクターＤＮＡ （ベセスダ・リサーチ・
ラボラトリーズ）のＢｉｎｄｌｌアームとの連結を行な
い１次いで７マ一シヤ入社のキットを用（・て、ｊｎ、
ｖｉＬｒ。
パッケージングを行ない、マウスＮＬ−１遺伝子ライブ
ラリー４Ｘ１０’Ｐ；ｉＵ／μＶを得た。
連結はＴ ４ ＤＮＡ Ｕカーゼ（宝酒造）を用い、反
応混液６６ ｍＭＴｒｉｓ Ｈ（Ｊ （ｐＨ７，６）
−６，６ｍＭＭｇｃｉ、 １０ ｍＭジチオスレイト
ール−１ｍＭＡＴＰ水溶液中で４℃１６時間行った。
実施例３（マウス免疫グロブリンに鎖遺伝子のスクリー
ニング）
前記実施例２で得らｎたマウスＮＬ−１由来のＤＮＡ７
！！：含むＣｈａｒｏｎ ２８フアージを大腸菌ＬＥ３
９２株に感染させ、プラークハイブリダイゼーション法
（Ｗ、Ｄ、ベンゼン、）ζ、Ｗ、デービス：サイエンス
１９６巻１８巻尺８０ページ７７）（Ｗ、Ｄ、Ｂｅｎｔ
ａｎ＋ Ｒ，Ｗ、Ｄａｖｉｓ ： ５ｃｊｅｎｅｅ
１ ９６ １８０（１９７７））参照〕を使用して
３２Ｐ標−マウス抗体a. Industrial application field The present invention relates to antibody chain expression type nucleic acid base sequences,
Chimeric antibody chain expression chain nucleic acid crystal arrangement of W, these nuclei l!
The present invention relates to a recombinant plasmid into which the plasmid has been incorporated, a mouse myeloma cell into which the plasmid has been introduced, and a mouse-human chimeric antibody chain. In this specification, amino acids and polypeptides are referred to as IUPA
It shall be abbreviated according to the method adopted by the C-IUB Committee on Biochemistry (cBN), for example, the following abbreviations are used. AlaL-7 Lanine ArgL-Fruginine Asn L 7 Subaragin Asp L-Aspartic acid cystochemistry L-Cystine Gin L-Glutamine Glu L-Glutamic acid G17 Glycine His L-Histidine 11e L-Leu L-Leucine Lye L- Lysine Met L-Methionine Phe L-Phenylalanine Pro L-Borine Ser L-Serine Thr L-Threonine Trp L-Tryptophan Tyr L-Tyrosine Val L-Valine Also, the sequence of DNA is divided into each deoxylipon nucleotide that makes up the DNA. It shall be abbreviated by the type of base contained,
For example, use the abbreviation for manure. A Adenine (1° indicates deoxyadenylic acid) C Cytosine (Deoxycytidylic acid) G Guanine (Deoxyguanylic acid) T Thymine (Deoxythymidylic acid) b, Prior art In general, immunoglobulin genes is H chain (HeavyCh
ain) and an L chain (Light Chain), each of which has a V region (variable region) that has the function of specifically binding to an antigen and a C region (constant region) that has an effector function. It has 10,000 promoters, including various gene units contained in the inner regions of the V region and C region regarding the H chain and L@ genes in the mouse antibody. The existence of various functional units such as enhancers has been revealed, and the DNA sequences of some of them have been revealed. The present inventor conducted research on the L chain of antibodies produced by murine hybridomas that have specificity for the antigen of the strain, and found that the gene in the The protein of the V region of the antibody can be obtained in the same manner as previously proposed. Therefore, the present inventor conducted research on the expression of a so-called chimeric L chain in which the V region of the mouse-derived L chain and the C region of the human-derived L chain are combined. Therefore, an object of the present invention is to provide a nucleic acid base sequence for highly efficient expression of an antibody chain, particularly a nucleic acid base sequence for expression of a mouse-human type chimeric antibody chain. Another object of the present invention is that these nuclei 1! ! It is possible to provide a recombinant plasmid into which the base sequence has been integrated. Still another object of the present invention is to provide a mouse myeloma gun into which the plasmid is introduced. Yet another object of the present invention is to provide a mouse-human chimeric antibody chain. Still other objects of the present invention will become apparent from the following description. According to the research of the present inventors, the above-mentioned object of the present invention is to first obtain a DNA fragment (b) of the V region in an antibody chain.
DNA fragment of C region of antibody Lyall and (e) enhancer of human or mouse antibody Ha
It has been found that this can be achieved using an antibody chain-expressing nucleobase sequence consisting of a DNA sequence. In the present invention, by using an enhancer DNA sequence derived from a human or mouse antibody H chain, an antibody nucleic acid base sequence with excellent expressivity can be obtained. In this antibody silver nucleobase sequence, the DNA fragments of both the region (■) and the C region may be derived from a mouse (or may be derived from a human).Also, the DNA fragment of the region (■) may be derived from a mouse The DNA fragment of the C region can be derived from a human antibody, or it can be a chimeric nucleobase sequence. By using the gene of the V region of the L chain of a mouse antibody having the same sex as the DNA fragment of the region (1), it can be expected to be used to create a chimeric antibody useful for humankind. As mentioned above, the tax-type nuclear scattering sequence is an enhancer of human or mouse antibody HG Yume.
L) Contains an NA sequence. This enhancer DNA sequence may be any DNA sequence having an enhancer function present in the Ha of a human or mouse antibody, for example, the enhancer derived from the human antibody H chain disclosed in European Patent No. 146743. DNA sequences can be used. In particular, the DNA sequence derived from the human antibody heavy chain shown in the attached Figure Bi and the DNA sequence complementary thereto can be advantageously used. The enhancer DNA sequence may be included in the antibody chain expression type nuclear base sequence of the present invention (
, regardless of its location. In other words, in the antibody chain, there is a DNA fragment of the V region and a DNA fragment of the C region in the gene.
It may be located between the V region DNA fragment and the C region fragment on the A sequence (and also on the 5'-side of the V region DNA fragment, the C region
It may be located on the 3' side of the DNA fragment of the region. Furthermore, the antibody LfJA expression type nucleobase sequence of the present invention includes:
There is no problem even if a DNA fragment having a promoter or other functions is included. In the nucleic acid base sequence of the present invention, when it is a mouse-human type chimeric antibody chain expression type, the DNA fragment of the V region is an anti-human acute leukemia lymphoid fragment proposed by the present inventor in the heading. Nucleus # that harbors at least the amino acid sequence of the V region of L@ in a mouse hybridoma that produces fat common antigen antibodies! I base sequence. Nucleus r1 encodes at least the amino acid sequence of the via region of Konoshi@! The base sequence is the first (Gl!u) in the amino acid sequence shown in attached Figure 2.
) to 97:I! The amino acid sequence up to the -th (Leu) may be at least -, or the first (G)
/u) to the 109th (Arg). Attachment 2
From position 1 (G/u) to 977 in the amino acid sequence shown in the figure
The sequence up to position Leu) is the amino acid sequence of the V region of the L chain of the antibody in the hybridoma, and the sequence from position 1 (G/u) to position 109 (Arg) is X'
This is the amino acid sequence of the V region and J region of the L chain. The nucleobase sequence that at least covers the amino acid sequence (1th to 97th) of the V region can be the DNA sequence from the 579th to the 869th (Q) in Figure 3 marked with a number, In addition, the nucleic acid sequence that encodes at least the V region and the J region is 57 in attached Figure 3.
The DNA sequence can be a DNA sequence from the 9th scan C1 to the 905th scan σ]. Furthermore, the nucleic acid crystal sequence encoding at least the 7-mino acid sequence in the V region may be the DNA sequence from the 562nd position (■) to the 905th position (G) in FIG. DN from 340th prisoner to 905th prisoner (r)
A sequence, and 905 from the 230th check
It may be a DNA sequence up to the first scan point. Is the antibody chain expression agent of the present invention applicable to the antibody chain constituting the nucleic acid crystal array? The C region DNA fragment to be used is preferably one derived from a human antibody chain. D of the C area that starts again
The NA fragment can be a DNA sequence that encodes at least the amino acid sequence shown in the attached Figure 4, and in particular, the DNA sequence shown in the attached Figure 5 can be cited as one specific example. can. As described above, (a) the DN of the V region in the antibody chain
An antibody Lm expression type nucleobase sequence is formed consisting of the A fragment, (h) a DNA fragment of the intermediate C region in the antibody chain, and an enhancer DNA sequence derived from human or mouse antibody Ha. The DNA sequence of this L chain may be a homogeneous L chain such as a mouse-mouse type or human-human type in which the ■ region and the C region are, or may be a chimera Li11 of mouse-human type. The enhancer DNA sequence can be used in human or mouse antibody H
As long as it contains an enhancer derived from a chain (this makes it possible to generate tax), the DNA sequence may also contain other enhancers or introns derived from mouse or human.The present invention The antibody chain expression type nucleobase sequence of the antibody chain can be expressed as a suitable vector plasmid, for example, psv2gpttPSv2.
A recombinant plasmid is prepared by integrating into nao, PKSV-10, etc. The recombinant plasmid prepared by
4533 pages, edited by the Japanese Society of Immunology. (1984) #Sho] or DEAE-text run method [
Cell CCe11) Volume 33 729 Negative (1983) #Sho]
The cells are introduced into host animal cells, such as mouse myeloma (eg, J558LINS-1), to obtain transformed cells. By culturing the obtained transformed cells, the desired antibody chains can be accumulated in the culture, and the antibody chains can be separated and recovered from this culture. For S, a plasmid incorporating a chain-expressing nucleobase sequence of a mouse-human chimeric antibody was used.
By introducing the mouse myeloma cells into mouse myeloma cells and culturing the cells, a mouse-human chimeric antibody consisting of (1) the amino acid sequence of the V region of the mouse antibody chain and (1) the amino acid sequence of the C region of the human antibody L1a is produced. L@ is provided. The amino acid sequence of the V region of this chimeric antibody chain is, for example, from position 1 (GJu) to position 97 (Leu) in attached Figure 2.
It has at least the amino acid sequence up to. This amino acid sequence is from position 1 (GJu) to position 109 (GJu) in Figure 2.
Arg) It may be the amino acid sequence of the vgt4 region and J region at t. - The amino acid sequence of the C region of the chimeric antibody chain can be, for example, the amino acid sequence shown in the attached Figure 4. As mentioned above, the chimeric antibody chain provided by the present invention can be combined with, for example, a mouse-human antibody H chain previously proposed by some of the inventors to form a complete mouse-human antibody. It is expected that the rIR of the present invention will be used for immunological treatment of acute leukemia patients by administering it alone or in combination with an anticancer drug to human acute leukemia-lymphoma patients. The preparation of the salt crystal array and the expression of antibody chains will be explained in detail.Example 1 (Isolation of Mouse Chromosomal DNA) Mouse hybridoma NL-] 1 x 10! cells were isolated at 1% SD.
S (Sodium 1auryl 5ulfate
) in the presence of protease K (manufactured by Sigma), water-saturated phenol was added to perform DNA Att extraction. Dilute the aqueous phase by centrifugation and add 10 mM Tria-HCl.
pH'7.4, 0, ]mM Naα, 0.1mM
Dialysis was performed with EDTA (TNE) buffer. After treatment with ribonuclease A (Sigma) and phenol extraction again, CN, brine, D, and W were dialyzed with TNE buffer to obtain mouse chromosomal DNA of 1.2~. Stafford: Nucleitsu Acid Research Volume 3 23
03 page (197ti) (N, Bltn+ D-
W. 5tafford: Nuelsic Ac1ds
Re5-+ 3 2303 (1976)) ε Teru] Example 2 (Creation of mouse gene library) Example 1
150 μl of the mouse chromosomal DNA obtained in 1. was digested with antienzyme H1ndnl (Takara Shuzo) according to the method shown in Example 4 described later, and then subjected to density gradient centrifugation [sucrose 10-40 μl].
% (wt/vol) + sucrose 28000r, p,
m,
A DNA fragment corresponding to Kb was obtained. Next, add this DNA 'lJr piece 0.45μp and Cha
ron 28 vector DNA (Bethesda Research
I connected it with the Bindll arm from Laboratories, and then used a kit from 7 Machinery Co., Ltd. (・te, jn,
viLr. Packaging was performed to obtain a mouse NL-1 gene library 4X10'P; iU/μV. For ligation, T 4 DNA U case (Takara Shuzo) was used, and the reaction mixture was 66 mM Tris H(J (pH 7,6).
-6.6mM Mgci, 10mM dithiothreitol-1mM ATP aqueous solution at 4°C for 16 hours. Example 3 (Screening for mouse immunoglobulin chain genes) DNA 7 derived from mouse NL-1 obtained in Example 2 above
! ! : Containing Charon 28 phages E. coli LE3
Plaque hybridization method (W, D, benzene, ) ζ, W, Davis: Science Vol. 196, 18, p. 80, 77) (W, D, Bent).
an+ R, W, Davis: 5cjenee
1 96 180 (1977)] using the 32P standard-mouse antibody.
【鎖Ｊ遺伝子で選別した。マウス
免疫グロブリンＣ鎖遺伝子を含むＣｈａｒｏｎ ２８フ
アージからのＤ Ｎ Ａ Ｏ）調製はトーツスとデービ
スの方法により行った〔Ｍ、トーツス、 Ｒ，Ｗ、デー
ビス：ジャーナルオズモレキュラーバイオＬ１ジー９１
巻３１５ベージ（］ ９７４ ） （Ｍ、Ｔｈｏｍａｓ
＋ Ｒ，Ｗ、Ｄａｖｉｓ ； Ｊ、Ｍｏｌ。
Ｂｉｏｌ、、９１ ３１５（１９７４））参照〕。
実施例４（マウスに鎖Ｖ領域遺伝子の制限酌累切断地図
の作成）
実施例３で得られたマウスに鎖遺伝子な含むファージＤ
ＮＡ及び後述する実施例５に準じてプラスミドにリクロ
ーニングしたＤＮＡ１ｐＩＩを制限酵素切断用緩衝液（
ＸｂａＩ 、 Ｂｇｉ １１切断でし工５０＋ｙ＋ＭＴ
ｒｉｓＨα（ｐＨ７，４） −１００ｒｒ＋ＭＮａ（Ｊ
−１０ｍＭＭｇ５Ｏ４水溶液を、 ＢａｍＨＩ＋
Ｈｉｎｄｍ ＋ Ｐｓｔｌ＋ＲｓａＩ＋ １ｆｉｎｃ１
１． ＤｒａＩ切断では１０ ｍＭＴｒｉｓ Ｈα（ｐ
Ｈ７，５） ｂ ＯｍＭＮａｃｌ ７ ｍＭ Ｍｇ
Ｃ１ｔ水藩液をそれぞれ用いた。〕２０μｌｉＫ浴解さ
せ、制限＃索（ＲｓａＩはニラポンジーン裂、その他は
宝酒造製を用いた）２ユニツトを添加して３７℃１時間
以上消化を行なった。
制限薄葉による切断後、４μｌの０．２５％ブロモフェ
ノールグルー・５０％グリセロール水浴液を加え、０．
８％〜２．５％アガｐ−スゲル電気泳動を行なった。７
ガロースはシグマ社のタイプ■電気泳動用を使用した。
１！気泳動バツフアーとして、５０ｍＭＴｒｉｓ−酢酸
８０ｍＭ酢酸ナトリウム−７２ｍＭ塩化ナトリウム１ｍ
Ｍ Ｅ Ｄ Ｔ Ａを用い、５朋厚水干ゲルにて２ Ｖ
／ （：ＩＦの電圧で９〜１６時間電気泳動を行なっ
た。この電気泳動の際、ＤＮＡ断片の分子量マーカーと
して、λファージのＤＮＡをＢｉｎｄｌｌｌで消化した
もの（日本ジーン製）を用いた。電気泳動終了後、アガ
ロースゲル中のＤＮＡを２μｇ ／ ｍｌエチジウムブ
ロマイド水浴液で染色し、このゲルに対して長波長紫外
線？：照射して、切断パターンの観察を行なった。各檻
制限ｕＩ、１ｃｊｌｉ独による切断。
及び二種の制＠酵素の組合せによる切断、こ几らの切断
パターンを解析することにより、各制御ａ酵素切断点の
相対位置関係を決定した。
マウス免疫グロブリンに鎖遺伝子断片の制限酵素切断地
図を第６図に示した。Ｄｒａ Ｉ ＋ Ｍｉｎｅ Ｔｌ
ｖＲＩＩＪＬ Ｉ切断点は図示した以外にも存在する。
実ｍ例５（マウス免疫グロ／リンＥ鎖Ｖ−Ｊ領域遺伝子
のサブクｐ−ニング）
マウス免疫グロブリンに鎖Ｖ−Ｊ債域遺伝子を含むＣｈ
ａｒｏｎ ２８フアージＤＮＡを、実施例４０方法に準
じてＨｉｎｄｌ［Ｉで切断し、アガロースゲル電気泳動
を行なった。Ｖｋ −Ｊｋ遺伝子を含む６．５ ｋｂ
１７） ＤＮＡ断片を、エレクトｐ゛エリューション法
を用いて７ガロースゲルエり回収した。
−万、大腸菌用プラスミドｐＨル３２２．１μｇ′４／
実施例４に準じてＨｌｎｄｍで切断したものに対して、
アルカリ性ホスファターゼ（Ｅ、ｃｏｌｉ ７５ ）（
宝Ｗ造％ ） ？：０．５ユニット加えて、６８℃で１
時間反応させた。反応終了後、反応液中のフルカリ性ホ
スファターゼを失活・除去するために、フェノール抽出
を３回繰返した。このよ５にして得られたｐ ＢＨ３２
２のＨｉｎｄ ｔｉｌ ／ ７ ルヵリ性ホスファター
ゼ処理液を、ゲルより回収した６、５ Ｋｂ Ｈｌｎｄ
ｍ断片水溶液と混ぜ、エタノール沈澱の後、連結反応
用バッファー（実施ｆ１１２を参照）１０ｄＫ溶解させ
る。２ユニツトのＴ４−ＤＮＡリガーゼを加ｊえ、１１
℃、１２時間反応させて、ハイブリッドＤＮＡを得る。
大ｉ劇ＤＨ１株の形質転換は、通常のＣａＣｊ、法（Ｍ
、Ｖ、ノーガード、に、キーン、Ｊ、モナハム：ジー７
３巻２７９ ヘ−：） （１９７８） （Ｍ、Ｖ。
Ｎｏｒｇａｒｄ ＋ Ｋ、Ｋｅｅｎ ＋ Ｊ＋Ｍｏｎａ
ｈａｍ： Ｇｅｎｅ ＋ ３ ＋ ２７９（１９７８）
）参照〕の改良法で行なった。すなわチ、５１Ｌｔ（１
）Ｌ培ｆｉ（１，％）！Ｊブト７．０．５９６ｆｌｉ母
エキス、 Ｏ，Ｓ％Ｎａα＋ｐＨ７−５）に大Ｍ ｉｆ
ｆ Ｄ Ｈ−１株の１８時間培養基を接種し、６００
ｎｍにおける光学密度０．３まで生育させる。菌体な冷
たいマグネシウム・バッフ ７− （０，ＩＭ Ｎａｉ
ｌ−５ＷＮａ１１−５Ｗ ５ｍＭ Ｔｒｉｍ
Ｈα（ｐＨ７，６，Ｏ’Ｃ））中で２回洗い、２ＴＩＬ
ｔの冷したカルシウム・バック７−（１００ｒｒＭ Ｃ
ａαｔ２５０ｍＭＫα−５ｍＭＭｇα＠ ５ｍＭ Ｔ
ｒｉｍ−Ｈα（ｐＨ７，６、０”Ｃ） ）中に再懸濁さ
せ、０℃で２５分間放置する。次に菌体なこの容量の１
／Ｉ Ｏ量のカルシウム・バッファーの中に再懸濁し、
ハイブリッドＤＮＡ水浴液と２ ： １ （ｖｏｌ、：
ｖｏｌ、）混合する。この混合物を６０分間、０℃で保
った後、ｌ就のＬＢＧ培地（１％トリプトン、０．５％
酵母エキス、ＩＸ ＮａＣ１，０，０８Ｘグル＝ −ス
＋ ｐＨ７−５）’を添加し、３７℃で１時間培養する
。培ａｍを、選択培地（アンピシリン３０μＩｉ／ｍ
１に含むＬ培地プレート）Ｉｃ１００μｊ／プレートで
接種する。プレートを３７℃で１晩培養して、形質転換
株を生育させる。得ら才したコロニーより、公知の方法
ケ用いてＤＮＡｔ−ＩＭ製し、アガロースゲル電気泳動
により、目的のハイブリッドＤＮＡを確認した。か（し
てｐＢＲ３２２のＨｉｎｄｎｌサイトＫ Ｖｋ −Ｊｋ
ｆｉ伝子を含ｔｒ ６．５ ｋｂ ）ＤＮＡＩＦｒ片
）３’クローニングＬｐＹＮ−ＭＬＶＩ及びｐＹＮ−Ｍ
ＬＶ２を作成した。
第６図のＶに−Ｊに遺伝子を含むＢａｍＨＩ （Ａ −
１） Ｈｉｎｄ［［Ｉ （Ａ−２）断片のＨｉｎｄｌ
ｌＩ （Ａ −２）がｐＢＲ３２２のＥｃｏＲＩサイト
寄りに連結されたものがｐ Ｙ Ｎ −Ｍ Ｌ Ｖ １
、その逆オリエンテーションのものがｐＹＮ−ＭＬＶ
２である。
ｔたｐＹＮ−ＭＬＶＩ Ｙ実施例４１Ｃ準じてＢａｍＨ
１及びＰａｔ Ｉで消化し、０．７％アガロース電気泳
動の後エレクトロエリューションな行う参により第６図
のＢａｍＨＩ （Ａ−１） −Ｐｓｔ Ｉ （Ａ−３）
のＤＮＡ断片を得た。一方ｐｕｃ １８ ベクター（ビ
ーエルバイオケミカルズ製）を同様にＢａｒｎＨＩ及び
Ｐａｔ Ｉで切断し、約２．７ｋｂの線状ベクター断片
を取得した。この二つのＤＮＡ断片を実施例２に準じて
連結し太Ｍ ｌ！ｌ Ｅ、ｅｏｌｉ Ｊ Ｍ２Ｏ３株を
前記ＢＨ１株と同様に処理する事により形質転換株を生
育せしめた。そり後公知の方法により形質転換株よりプ
ラスミドＤＮＡをｖ４類し、アガロース電気泳動により
目的の組み換えプラスミドを確認した。かくして実施例
３でクローニングされた遺伝子の一部、すなわち第６図
に示−ｊＢａｍＨＩ（Ａ １）−ＰｓｔＩ（Ａ−３）
を含むｐＵｃ１８組換えプラスミドｐＹＮ−ＭＬＶＢＰ
を得た。
実施例６（塩基配列の決定）
実施例５記載１）ｐＹＮ−ＭＬＶｌを９１施例４に準じ
てＢａｍＨＩとＰｓｔＩで切断し、第６図のＢａｍＨＩ
（Ａ−１）とＰｓｔＩ（Ａ−３）切断部位で狭まれた
ＤＮＡ断片得た。また同様ＫＢｇｌＩとＰｓｔ Ｉで切
断し第６図のＰａｔ Ｉ （人−３）とＢｇｌ ＩＩで
狭まれた約１．５．ｋｂのＤＮＡ断片を得た。
これらのＤＮＡ断片をＢａｍＨＩ及びＰｓｔ １で切断
したＭ１３ｍｐ１８（ビーエルバイオケミカルズ製）［
クローニングした。塩基配列決定はＭ１３シークエンシ
ングキット（宝酒′！！Ｌ）と〔α−”Ｐ）ｄＣＴＰ（
７−ｑ−−：ｙ−ｗム社１ｍ）を用いジデオキシチェー
ンターミネーション法で行ッた〔高浪満、大井龍夫編ｒ
ＤＮＡシーケ／ス解析マニュアルＪ （１９８３）講談
社参照〕。
ＢａｍＨＩ （Ａ−−１）−Ｐａｔ Ｉ （Ａ ３
）断片についてはＰｓｔＩ＠から５′方向に、Ｐｓｔ
Ｉ （Ａ −３）−ＢｇｌＩＩ断片についてはＰｓｔ
Ｉ側から３′方向に塩基配列決定を行った。更にプラス
ミドｐＹＮ−ＭＬＶＢＰを実施例４に準じてＲａａ Ｉ
とＰ＋ｓｔ工で切断し【約０．６５ ｋｂすＤＮＡ断片
を得、Ｍ１３ｍｐ１９（ビーエルバイオケミカルズ裏）
ファージをＳｍａ Ｉ及びＰａｔ Ｉで切断したものに
クローニングし、Ｒａａ １かも３′方向に塩基配列を
決定した。Ｓｍａ Ｉ消化は１０ ｍＭＴｒｉｍ −Ｈ
α（ｐ）１８．０ ） 、 ７ｍＭ ＭｇＣ１ｇ 、
２０ ｍＭ ＫＣｌ 、７ｍＭ２−メルカプトエタ／−
ル水浴液中で３７℃２時間行い、その後ＮａＣ１５１１
度を５０？ＦＩＭにした上Ｐｓｔ Ｉで消化を行った。
またプラスミドｐＹＮ−ＭＬＶＢＰを実施例４に準じて
Ｒｓａ ＩとＤｒａ Ｉ及びＤｒａ ＩとＰｓｔ Ｉで
切断し、生成したＤＮＡＴｈ面を分画した。分画には２
ＩｏｌＩ厚５９０アクリル７ミド垂直ゲルをｎノいた〔
高木康孝編「遺伝子操作実数マｓ７／しＪ（１９８２）
講映社参照） 、 Ｒｓａ ＩとＤｒａ Ｉ切断では約
０．３ ｋｂ＋ Ｄｒａ ＩとＰｓｔ １切断では約０
．４ｋｂのＤＮＡＶｒ片を得、 Ｍ １３ ｍｐ １８
をＳｍａ １消化し実施例５に準じて脱燐酸処理を行っ
たもの、後者はＭ １３ ｍｐ １９ ’ｔ Ｓｍａ
ＩとＰ＆Ｉｔ Ｉで消化したものに連結反応を行い、第
６図のＲｓａｌ−Ｄｒａ １及びＤｒａ Ｉ Ｐｓｔ
Ｉ （Ａ ３ ）切断部位で囲まれるＤＮＡ断片の
挿入された組換えファージを得、ＩＮ述の如く塩基配列
の決定を行った。
Ｒｓａ Ｉ −Ｄｒａ Ｉ断片ｒｃついてはＤｒａ Ｉ
から５′方向＊ Ｄｒａ Ｉ Ｐｓｔ Ｉ断片につい
てはＤｒａ Ｉから３１方向にそれぞれ行った。
各々の結果を連結してまとめたものを第３図に示した。
実施例７（ヒト染色体ＤＮＡｏ）＊０）ヒト培養細胞Ａ
ＲＨ７７株３ Ｘ １０’個をガラス棒でつぶし、２％
ＳＤＳ存在下、プｐテ７−ゼＫ（シグマ社製）で処理し
た後、］ＯｍＭＴｒｉｓ−ＨＣｌ（ｐＨ８，０） ｌｎ
５Ｍ Ｅ Ｄ Ｔ Ａ水浴液で飽和したフェノールを
加えた。遠心分離によつ水相とフェノール層を分離（フ
ェノール抽出）。
水相を２０ｍＭ Ｔｒｉａ ＨＣＬ （ｐＨ７，５）
−１００ｒｓＭＮａα−５ｍＭ、ＥＤＴＡ水溶液に対
して透析した。
リボヌクレアーゼＡ（シグマ社製）処理し、７エ／−ル
抽出な行なった後、水相な１０ｍＭＴｒｉｍ−Ｈα（ｐ
Ｈ８，０） １ｍＭ ＥＤＴＡ水浴液に対して透析し
、ヒト染色体ＤＮＡ的１．２Ｉｖを取得した〔実施例！
記載のプラインとスタフオードの報告参照〕。
実施例８（ヒト遺伝子ライブラリーの作成）実施例７で
得られたヒト染色体ＤＮＡを後述の実施例１Ｏに示した
方法に準じて制限＃素ＥｅｏＲＩＣ宝酒造】で切断した
後、アガロースゲル電気泳動を行ない％２ ｋｂ〜３
ｋｂに相当するＤ Ｎ Ａ断片をエレクトロ・エリコー
ション法を用いて回収した。次にこのＤＮＡ断片のλｇ
ｔＷＥＳλＢベクター（アマージャム社ｍ）との連結を
実施例２に準じて行ない、λｇｔＷＥｓλＢベクターの
右７−ムと左のアームとのｎＯＫヒト由来のＤＮＡが挿
入されたハイブリッドＤＮＡを得た。得られたハイブリ
ッドＤＮＡについて、１ｎｖｉｔｒｏ パッケージング
を行ない、ヒト遺伝子ライブラリー（アマージャム社キ
ットヶ使用）とした。
実施例９（ヒト免疫グロブリンに釧遺伝子のスクリーニ
ング）
前記実施例２で得られたヒト由来のＤＮＡを含むλｇｔ
ＷＥｓλＢ７７−ジの集合（遺伝子ライブラリー）を大
ＰＩＪＩｌｌｌｌＬＥ３９２株に感染させ、プラークｌ
形放させた。ヒト免疫グロブリンに鎖遺伝子ン含むクロ
ーンは実施ｔｅｌ ３ Ｋ準じて３＊ｐ ｍ ｊｌマウ
スＣＬ遺伝子をクロスハイプリダイゼーションプローズ
として用い選択した。
ヒト免疫グロブリンに鎖遺伝子を含むλＧＴｗｅλＢ７
７−：）からのＤＮＡの、ＳｌｌｊＭは実施？ｌＪ３に
準じて行なった。
実施例ＩＱ（ヒトに鎖Ｃ領域遺伝子の制限酵素地図の作
成）
実施例９で得られたヒトに鎖遺伝子を含むファージＤＮ
Ａ及び後述する実施例１１に準じてプラスミドにリクｐ
−ニングしたＤＮＡ １μｍを制限酵素切断用バッファ
ー（ＥｃｏＲＩ切断ではＴｒｉｓ −ＨＣＬ（ｐＨ７，
５） ５０ｍＭ ｔ ＭｇＣｔ、 ７ｍＭ ＋Ｎａα１
０ＤｍＭ１２−メルカプトエタノール７−。
Ａｃ６Ｉ切断ではＴｒｉｍ −Ｈα（ｐＨ７，５） １
０．ｍＭ 。
ＭｇＣｊＬｔ ７ ｍ Ｍ ＋ Ｎａα６ＯＦＦＩＭ、
２−メルヵプトエタ／−ル２ｙｇＭ ｒ Ｓａｃ切断
ではＴｒｉｓ −ＨＣＪＬ（ｐＨ８，０）１０ ｍＭ
９Ｍｇ０２７ ｍＭ ｍ ２−メルカプトエタノール
７ｍＭ水浴液奢それぞれ用いた〕２０Ａｌ！に溶解させ
、制限酵素（宝酒造製）２ユニツトを株加して３７℃１
時間以上消化を行った。その後実施例４に準じて電気泳
動を行い制＋ａ酵索切断地図を作成した。ＣＰ、Ａ、ヒ
ーター、 Ｅ、Ｅ、マックス、Ｊ、Ｇ、シードマン＋
Ｊ、Ｖ、マイセルＪｒ＋Ｐ。
レーダー：セル２２巻１９７ベージ（１９８０）（Ｐ、
Ａ、 Ｈｉｅｔｅｒ＋ Ｅ、Ｅ、 Ｍａｚ＋ Ｊ、Ｇｅ
Ｓｅｉｄｍａｎ＋Ｊ、Ｖ、Ｍａｉｚｅｌ＋ Ｊｒ−
＊ Ｐ、Ｌｅｄｅｒ ： Ｃｅ１ｌ ２２
１９７（１９８０））＃照 〕
ヒト免疫グロブリンに細のＣ領域を含む遺伝子の制限酊
素切断地図を第７図に示した。
実施例１１（ヒト免疫グロブリンに鎖Ｃ領域遺伝子のサ
ブクローニング）
ヒト免疫グロブリンに頌Ｃ領域を含むλｇｔＷＥＳλＢ
ファージＤＮＡ３μＶを夾り例１０り方法ＫｍじてＥｃ
ｏＲＩで切断し、アガロース電気泳動を行った。Ｃに遺
伝子を含む２．６ ｋｂのＤＮＡ断片をエレクトロエリ
コーション法を用いてアガロースゲルより回収した。
一万、大腸菌用プラスミドｐ Ｂ Ｒ３２２Ｉｎを実施
例１０に準じてＥｃｏＲＩで切断したものに対し、実に
秒り５に準じてアル刀り件フＹスフ７ターゼ処理を行っ
た。このようにして得られたｐＢＲ３２２のＥｃｏＲｒ
切〜ｒ／アルカリ性７オスフアターゼ処Ｎ１）ＮＡをグ
ルより回収した２、６ ｋｂ’ＥｃｏＲ１，Ｄ Ｎ Ａ
断片と混合し、エタノール沈澱の後連結反応用バッファ
ー（実施例２＃照）１０μＵ」屏させ、実施例５に準じ
てＴ４ＤＮＡリガーゼで連結した。
実施例５に準じて大腸菌Ｅ、ｃｏｌｉ ＤＨＩを形質変
換し、プラスミドＤＮＡを調製して目的のハイブリッド
ＤＮＡな確認した。
実施例１２（マウス・ヒト・キメラ抗体遺伝子の作製）
５Ａ施例５で得られた、マウス免疫グ。プリンｔ ｉＱ
Ｖ −Ｊ領域遺伝子１含ｔｒ ｐ Ｙ Ｎ−Ｍ Ｌ
Ｖ １を、実施例４及び】０の方法に準じてＢａｍ１（
Ｉ及びＥｃｏＲＩで切断し、７ガロースゲル蒐気泳動を
行なった。得られた■に−Ｊに遺伝子を含む４．２Ｋｂ
のＥｃｏＲＩ −Ｂａｍ）（Ｉ断片を、＊ｉ例５と同様
な手法によ’）、ｐｓＶ２ｎａｏベクターＣＰ、Ｊ、サ
ザン、Ｐ、バーグ： ジャー力ルオプ七レキュラーアン
ドアプライドンエネテインクス１巻３２７ページ（１９
８２） （Ｐ、Ｊ、５ｏｕｔｈｅｒｎ。
Ｐ、 Ｂｓｒｇ： Ｊ、 Ｍｏｌ 、 Ａｐｐｌ 、
ＧｅｎｅＬ、± ３２７（１９８２））ＪｉＳ照〕のＥ
ｃｏＲＩ −Ｂａｍ Ｈ１間に挿入し、プラスミドｐ
ＳＶ２ ｎｅｏ−ＭＬＶを作成した。これを第８図に示
した。
次に、実施例１１で得られた。ヒト免疫グσプミンに鎖
Ｃ領域遺伝子を含むｐＹＮ−ＨＬＣを実施例１Ｏの方法
１ｃ準じてＥｃｏＲＩで切断し、アガロースゲル電気泳
動を行なった１、得られたＣに遺伝子を含む２．６Ｋｂ
１７）ＤＮＡ断片を６実施例１１と同様な手法を用いて
、上記プラスミドｐ Ｓ Ｖ ２ ｎｅｏ −Ｍ Ｌ
Ｖ Ｏ） ＥｅｏＲＩサイトに挿入した。得られたクロ
ーンのうち、ヒトＣに遺伝子を含むＥｅｏＲＩ （Ｂ−
１） −ＥｃｏＲＩ （Ｂ−２） の断片のＥｃｏＲ
Ｉ （Ｂ−１）がマウスＶに−Ｊに遺伝子側に連結され
たもの（マウスＶに−Ｊに遺伝子とヒトＣに遺伝子の読
み取り方向が一致しているもの）を選択し、このプラス
ミドをｐｓＶ２ｎｅｏ−ＭＨＬと名付けた。これを第８
図に示した。
−万、特開昭６０−１２０９９１公報に記載のヒト免疫
グロブリンｒ＋鎖遺伝子を含むプラスミドｐ’ｒ、ｙａ
を、実施例１０記載の方法に準じてＭｌｕｌ及びＨｐａ
ｌで切断した。但し切断用バッファーとしてはＴｒｉｓ
Ｈ（１（ｐＨ７，５） １０．ｍＭ ＋ Ｍｇα。
７ｍＭ ＋ＮａＣｊ、Ｉ ５０ｒ＋＋Ｍ ＋ ２−）
ルカプトｘ−ｐノール７ｍＭ水溶液を用いた。Ｍｌｕ
Ｚで切断後エタノール沈澱によりＤＮＡを回収した後、
切断用バッファ −Ｔｒｉｓ−ＨＣｉ（ｐＨ７，５）
１０ｍＭ ＋ Ｋα１００ ｍ Ｍ ＋ ＭｇＣ１ｔ
７ ｍ Ｍ 、２−メルヵプトエタ／−ルアｙｎＭ水溶
液を用いてＨｐａｌ切断を行った。７ガロースグル電気
泳動により、約０．９Ｋｂの ヒ） ｒ＋鎖遺伝子エン
ハンサ−領域を含むＭｌｕＩ −Ｈｐａｌ断片を得た。
このＤＮＡ断片をポリメラーゼ川バッファーＣ３７ｍＭ
リン酸カリウム、６．７ｍＭ ＭｇＣＪｓ ＊ １
ｍＭ ２−メルカプトｚ タ／ −ル。
ｐＨ７，４）に溶解し、ｄＮＴＰ存在下でＤＮ人ポリメ
ラーゼ・クンノウ（Ｋｌｅｎｏｗ ）・フラグメント（
二ニー・イングランド バイオラプズ）を加えることに
より、末端の平滑化を行なった。
さらに％Ｔ４−ＤＮＡリガーゼを用いてこの両端にＢａ
ｒ！Ｉ）Ｉ Ｉリンカ−（宝酒造）！連結した後、Ｂａ
ｍＨＩで切断し、アガロースゲル電気泳動により約０．
９ ＫｂのヒトｒＩ＃遺伝子エンハンサー領域を含む両
端がＢａｍＨＩ断片を得た。このＤＮＡ断片を上記プラ
スミドｐ Ｓ Ｖ ２０ｅｏ −Ｍ ＨＬすＢａｍＨＩ
サイト罠挿入し、挿入オリエンテーションのそｎぞれ異
なるプラスミドｐＳＶ２ｎｅｏ−ＭＨＬＥＩ及びｐ Ｓ
Ｖ２ ｎｅｏ−ＭＨＬＥ ２を作製した。これを第８
図に示した。
実に例１３（キメラ抗体り鎖の発現）
マウスミエローマ細胞Ｊ５５８Ｌ及びＸ６３Ａｇ８．６
５３に別に調製したイムノグロブリン１１鎖発現型遺伝
子’ｉｘ７′ｔｙトズラスト融合法により導入し、安定
形質変換様（５ｔａｂｌｅ ｔｒａｎｇｆｏｒｍａｎｔ
）を得た。これらの細胞は導入したＨ鎖遺伝子を発限
している偏胞はそれぞれＪ５５８Ｌ−Ｈ。
Ｘ ６３ Ａｇ ８．６５３ ）１と称する。
Ｊ５５８Ｌ−Ｈ及びＸ ６３ Ａｇ ８．６５３−Ｈに
実施例１２記載のキメラＬＭ遺伝子を含むプラスミドｐ
Ｓ Ｖ２ ｎｅｏ−ＭＨＬＥ−１及びｐｓＶ２ ｎｅ
ｏ−ＭＨＬＥ−２をＤＥＡＥ−デキストラン法で導入し
た（ Ｊ、ペナルジ、Ｌ、オルリン。
Ｗ、汁ワナー：セル３３巻７２９ページ（１９８３）（
Ｊ、Ｂｅｎａｒｊｉ＋ Ｌ−０１ｓＯｎ＋ ｗ、
５ｃｈａｆｆｎｅｒ ：Ｃｅ１１３３ ７２９（１
９８３））参照〕。すなわち１０７個の＃ｌ抱に８０４
のプラスミドＤＮＡとＤＥＡＥ−デキストラン（ファル
マシア社製）混合物を加え、卵 置の後、ＲＰＭ Ｉ
１６４０完全培地（フローラボラトリー社ｇ＞ｃｉｏ％
牛脂児血清含有）Ｋ移した。薬剤耐性株はジェネシチン
（Ｇｏｎｅｔｉｃｉｎ＋ Ｇ ４１８ ＋ギブフ社Ｖ）
ｌ〜１．５１ｋｇ／ｌＬｊを含むＲＰＭ１１６４０完全
培地ノ牛量交換によって行った。１０〜２０日後、生育
してくるｌ１１１１胞を安定形質交換法とした（それぞ
れＪ５５８Ｌ−ＨＬ、Ｘ６３Ａｇ８．６５３−ＨＬと称
する）。
実施例１４（形質転換細胞のｍＲＮＡ分析）実施例１３
によって得た形質転換細胞よりＲＮＡを子山出し、得ら
ｎたＲ Ｎ Ａそハぞれ２゜μｇ’ｔ７ｆＬ気泳動し、
ノーザンハイプリダイゼーンヨンＫよ’） ｍＲＮＡの
分析ｌ行った（ Ｔ−マニ７テイス他著「モレキュラー
クローニンク」コールドスプリングハーパーラボラトリ
ー（１９８２）（ Ｔ． Ｍａｎｉａｔｉｓ ｅｔａｌ
． ：″Ｍｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ”Ｃｏｌ
ｄ Ｓｐｒｉｎｇ Ｌａｂ． （ １ ９ ８ ２ ）
）参照〕。パイプリダイゼーションプロープとしては
Ｖ領域プｐ一プとしてｐＹＮ−ＭＬＶ−１のプラスミド
上の第６図Ｐｓｔ Ｉ （ Ａ − ３ ） − Ｈｉ
ｎｅｌ［に相当するＤＮＡ断片，Ｃ領域プローズとして
ｐＹＮ一ｋＬ Ｌ Ｃ Ｉ） ＳｃａＩ （Ｂ−１ ）
ＳｃａＩ （Ｂ−２）断片を、それぞれ５Ｐにてｍ
ｊｌｉｌＬ−ｔ：用いた。第９図に示す如《いずれのプ
ｐ−ズを用いた場合にも同じ位置にバンドが出現した。
実施例１５（−？メラ抗体Ｌ＠生成物のＳＤＳ−ポリア
クリル７！ドグル電気泳動によ
る分析）
夾施例１３のＸ ６ ３ Ａｇ ８．６５３−ＨＬ １
０’個を燐酸緩衝液で洗浄後メチオニン不含ＲＰＭＩ
一１６４０培地（午胎児血清１０Ｘ含有）に層濁し，３
００μＣｉの（ ：｛ Ｓ Ｓ ］一メチオニンを加え
８時間培養した。かくして生成した蛋白質？ｌ−“Ｓで
内部標識し、培養上清全回収した。上清より生成抗体を
抗ヒ｝ ＩｇＧ抗体による免疫沈降法で回収し、担体よ
つ遊離させた後２メルカプトエタノールで還元し、Ｈ鎖
とＬ鎖の分離をはかった。その後試料をＳＰＳ−ポリア
クリルアミドゲル電気激動で分離し、ゲルを乾燥後オー
トラジオグラフイーをとった。（電気泳動学会編「電気
泳動実験法」文元堂＋１９７６）参照〕Ｈ鎖とともにＫ
鎖の蛋白の生成が認められ、この結果は導入したキメラ
抗体Ｋ鎖が予め導入してあったＨ鎖と結合して分泌され
ている事を示している。オートラジオダラムを第１０図
に示した。[Selected by chain J gene. DNA preparation from Charon 28 phage containing the mouse immunoglobulin C chain gene was performed by the method of Tortz and Davis [M, Tortz, R, W, Davis: Journal Osmolecular Bio L1 G91.
Volume 315 pages (] 974) (M, Thomas
+ R, W, Davis; J, Mol. Biol, 91 315 (1974)]. Example 4 (Creation of restriction truncated map of mouse chain V region genes) Phage D containing mouse chain genes obtained in Example 3
NA and DNA1pII recloned into a plasmid according to Example 5 described later were mixed with restriction enzyme cleavage buffer (
XbaI, Bgi 11 cutting work 50+y+MT
risHα (pH 7,4) −100rr+MNa(J
-10mM Mg5O4 aqueous solution, BamHI+
Hindm + Pstl + RsaI + 1finc1
1. For DraI cleavage, 10 mM Tris Hα (p
H7,5) b OmMNacl 7mM Mg
C1t Mizuhan liquid was used respectively. ] The mixture was dissolved in a 20 µliK bath, and 2 units of restriction #string (RsaI was Nirapongene fission, others were manufactured by Takara Shuzo Co., Ltd.) were added, and digestion was carried out at 37°C for over 1 hour. After cutting with a restrictive thin film, 4 μl of 0.25% bromophenol glue/50% glycerol water bath solution was added.
8% to 2.5% aga p-gel electrophoresis was performed. 7
As the galose, Sigma's type ■ for electrophoresis was used. 1! As an aerophoresis buffer, 50mM Tris-acetic acid 80mM sodium acetate-72mM sodium chloride 1m
Using MEDTA, 2 V with 5 tomo thick water and dried gel.
/ (: Electrophoresis was performed for 9 to 16 hours at an IF voltage. During this electrophoresis, λ phage DNA digested with Bindll (manufactured by Nippon Gene) was used as a molecular weight marker for the DNA fragment. After the electrophoresis was completed, the DNA in the agarose gel was stained with 2 μg/ml ethidium bromide bath solution, and the gel was irradiated with long wavelength ultraviolet rays to observe the cutting pattern. By analyzing the cleavage patterns of cleavage by cleavage and cleavage by a combination of two types of restriction enzymes, we determined the relative positional relationship of the cleavage points of each regulatory a enzyme.Restriction of chain gene fragments in mouse immunoglobulin The enzyme cleavage map is shown in Figure 6.Dra I + Mine Tl
vRIIJL I break points exist other than those shown. Practical example 5 (subcloning of mouse immunoglobulin/rin E chain V-J region gene) Ch containing chain V-J region gene in mouse immunoglobulin
Aron 28 phage DNA was cleaved with Hindl[I and subjected to agarose gel electrophoresis according to the method of Example 40. 6.5 kb containing Vk-Jk gene
17) The DNA fragments were collected on a 7-gallose gel using the electrolysis method. -10,000, plasmid pH for E. coli 322.1μg'4/
For those cut with Hlndm according to Example 4,
Alkaline phosphatase (E, coli 75) (
Takara Wzo%)? : Add 0.5 unit, 1 at 68℃
Allowed time to react. After the reaction was completed, phenol extraction was repeated three times to inactivate and remove the flukaline phosphatase in the reaction solution. pBH32 obtained in this way 5
2 Hind til / 7 The 6,5 Kb Hlnd recovered from the gel
m fragment aqueous solution, and after ethanol precipitation, dissolve in 10 dK of ligation reaction buffer (see run f112). Add 2 units of T4-DNA ligase, 11
C. for 12 hours to obtain hybrid DNA. The transformation of the Daigo DH1 strain was carried out using the usual CaCj, method (M
, V. Norgaard, N., Keene, J., Monaham: G.7.
Volume 3 279 He-:) (1978) (M, V. Norgard + K, Keen + J + Mona
ham: Gene + 3 + 279 (1978)
). In other words, 51Lt (1
) L culture fi (1,%)! JButo7.0.596fli mother extract, O,S%Naα+pH7-5) with large M if
f Inoculated with 18-hour culture medium of DH-1 strain, 600
Grow to an optical density in nm of 0.3. Bacterial cold magnesium buff 7- (0, IM Nai
l-5WNa11-5W 5mM Trim
Wash twice in Hα (pH 7,6, O'C), 2TIL
t of chilled calcium bag 7-(100rrM C
aαt250mMKα-5mMMgα@5mM T
rim-Hα (pH 7.6, 0"C)) and left at 0°C for 25 minutes. Next, 1 volume of the bacterial cells was
/IO volume of calcium buffer,
Hybrid DNA water bath solution and 2:1 (vol,:
vol,) mix. This mixture was kept at 0°C for 60 minutes and then diluted with LBG medium (1% tryptone, 0.5%
Yeast extract, IX NaC1,0,08X glucose + pH7-5)' is added and cultured at 37°C for 1 hour. The culture medium was mixed with selective medium (ampicillin 30μIi/m
1) Inoculate at 100μj/plate of L medium plate. The plates are incubated overnight at 37°C to grow the transformants. DNAt-IM was prepared from the obtained colony using a known method, and the desired hybrid DNA was confirmed by agarose gel electrophoresis. (The Hindnl site of pBR322 K Vk - Jk
tr 6.5 kb) DNA IFr fragment containing fi gene) 3' cloning LpYN-MLVI and pYN-M
Created LV2. BamHI (A-
1) Hind[[I Hindl of (A-2) fragment
lI (A-2) linked to the EcoRI site of pBR322 is p Y N -M L V 1
, the one with the reverse orientation is pYN-MLV
It is 2. tpYN-MLVI Y BamH according to Example 41C
BamHI (A-1) - Pst I (A-3) in Figure 6 was digested with 1 and Pat I, and subjected to electroelution after 0.7% agarose electrophoresis.
A DNA fragment was obtained. On the other hand, puc 18 vector (manufactured by BL Biochemicals) was similarly digested with BarnHI and Pat I to obtain a linear vector fragment of approximately 2.7 kb. These two DNA fragments were ligated according to Example 2 to obtain thick M1! A transformed strain was grown by treating the 1 E, eoli J M2O3 strain in the same manner as the BH1 strain. After washing, plasmid DNA was isolated from the transformed strain by a known method, and the desired recombinant plasmid was confirmed by agarose electrophoresis. Thus, part of the genes cloned in Example 3, namely -jBamHI (A1) -PstI (A-3) shown in FIG.
pUc18 recombinant plasmid pYN-MLVBP containing
I got it. Example 6 (Determination of base sequence) Description of Example 5 1) pYN-MLVl was cleaved with BamHI and PstI according to 91 Example 4, and the BamHI shown in FIG.
(A-1) and a DNA fragment narrowed at the PstI (A-3) cleavage site was obtained. Similarly, approximately 1.5. A DNA fragment of kb was obtained. M13mp18 (manufactured by BL Biochemicals) [manufactured by BL Biochemicals] was obtained by cutting these DNA fragments with BamHI and Pst1.
Cloned. Base sequencing was performed using M13 sequencing kit (Takarashu'!!L) and [α-”P)dCTP (
7-q--: Y-W Co., Ltd. 1m) by the dideoxy chain termination method [edited by Mitsuru Takanami and Tatsuo Oi r
DNA Sequence/Sequence Analysis Manual J (1983) Kodansha]. BamHI (A--1)-Pat I (A 3
) fragment in the 5′ direction from PstI@, Pst
Pst for the I (A-3)-BglII fragment
The base sequence was determined in the 3' direction from the I side. Furthermore, plasmid pYN-MLVBP was converted to Raa I according to Example 4.
and cut with P+st to obtain a DNA fragment of about 0.65 kb, M13mp19 (back of BL Biochemicals)
The phage was cloned into the one cut with Sma I and Pat I, and the base sequence of Raa 1 was also determined in the 3' direction. Sma I digestion was performed using 10 mMTrim-H
α(p)18.0), 7mM MgClg,
20mM KCl, 7mM 2-mercaptoeta/-
NaCl water bath for 2 hours at 37°C, and then
Degree 50? It was made into FIM and then digested with PstI. Further, plasmid pYN-MLVBP was cut with Rsa I and Dra I and Dra I and Pst I according to Example 4, and the resulting DNA Th surface was fractionated. 2 for fractionation
IolI thickness 590 acrylic 7m vertical gel was made.
Yasutaka Takagi (ed.) Genetic manipulation real number math s7/shiJ (1982)
(see Koeisha), approximately 0.3 kb for Rsa I and Dra I cleavage + approximately 0 for Dra I and Pst 1 cleavage.
．． A 4kb DNAVr piece was obtained, M 13 mp 18
was digested with Sma 1 and dephosphorylated according to Example 5, the latter was M 13 mp 19 't Sma
A ligation reaction was performed on the digested product of I and P&It I, resulting in Rsal-Dra 1 and Dra I Pst in Figure 6.
A recombinant phage into which a DNA fragment surrounded by the I (A 3 ) cleavage site was inserted was obtained, and the nucleotide sequence was determined as described above. For Rsa I-Dra I fragment rc, Dra I
*For the Dra I and Pst I fragments, the 31 direction from Dra I was carried out. A concatenation and summary of each result is shown in Figure 3. Example 7 (Human chromosomal DNAo) *0) Human cultured cells A
Crush 3 x 10' pieces of RH77 strain with a glass rod and add 2%
After treatment with Peptase K (manufactured by Sigma) in the presence of SDS, ]OmMTris-HCl (pH 8,0) ln
Phenol saturated with 5M EDTA water bath was added. Separate the aqueous phase and phenol layer by centrifugation (phenol extraction). The aqueous phase was treated with 20mM Tria HCL (pH 7,5).
Dialyzed against -100rsMNaα-5mM, EDTA aqueous solution. After treatment with ribonuclease A (manufactured by Sigma) and extraction with 7 ethanol, 10mM Trim-Hα (p
H8,0) Dialyzed against 1mM EDTA water bath solution to obtain human chromosomal DNA 1.2Iv [Example!
See the report by Prine and Stafford]. Example 8 (Creation of human gene library) The human chromosomal DNA obtained in Example 7 was cut with restriction #EeoRIC Takara Shuzo according to the method shown in Example 1O below, and then subjected to agarose gel electrophoresis. conduct%2 kb~3
A DNA fragment corresponding to kb was recovered using the electroelimination method. Next, λg of this DNA fragment
Ligation with the tWESλB vector (Amarjam Co., Ltd.) was performed according to Example 2 to obtain a hybrid DNA in which nOK human-derived DNA was inserted between the right 7-arm and the left arm of the λgtWEsλB vector. The obtained hybrid DNA was subjected to 1nvitro packaging to form a human gene library (using a kit from Amajam Co., Ltd.). Example 9 (Screening for human immunoglobulin gene) λgt containing the human-derived DNA obtained in Example 2 above
A collection of WEsλB77-di (gene library) was infected with large PIJIllllLE392 strain, and plaque l
I let it go. Clones containing the human immunoglobulin chain gene were selected using the 3*p m jl mouse CL gene as a cross-hybridization probe according to the tel 3 K experiment. λGTweλB7 containing chain gene in human immunoglobulin
Did SlljM carry out the DNA from 7-:)? It was carried out according to lJ3. Example IQ (Creation of restriction enzyme map of human chain C region gene) Phage DNA containing the human chain gene obtained in Example 9
plasmid according to A and Example 11 described below.
1 μm of the nicked DNA was added to a restriction enzyme cleavage buffer (for EcoRI cleavage, Tris-HCL (pH 7,
5) 50mM tMgCt, 7mM +Naα1
0DmM 12-Mercaptoethanol 7-. For Ac6I cleavage, Trim-Hα (pH 7,5) 1
0. mM. MgCjLt 7 m M + Naα6OFFIM,
For 2-mercaptoethyl 2ygM r Sac cleavage, Tris-HCJL (pH 8,0) 10 mM
9Mg027mM m2-mercaptoethanol 7mM water bath solution]20Al! Add 2 units of restriction enzyme (Takara Shuzo) and heat at 37°C.
It took more than an hour to digest. Thereafter, electrophoresis was performed according to Example 4 to create a cleavage map of the +a enzyme. CP, A, Heater, E, E, Max, J, G, Seedman +
J, V, Meisel Jr+P. Radar: Cell 22 volumes 197 pages (1980) (P,
A, Hieter+ E, E, Maz+ J, Ge
Seidman+J, V, Maizel+ Jr-
*P,Leder: Ce1l 22
197 (1980)) #Reference] Figure 7 shows a restriction cleavage map of a gene containing a small C region in human immunoglobulin. Example 11 (Subcloning of chain C region gene into human immunoglobulin) λgtWESλB containing chain C region in human immunoglobulin
Example 10 Method of collecting 3μV of phage DNA and Ec
It was cut with oRI and subjected to agarose electrophoresis. A 2.6 kb DNA fragment containing the gene in C was recovered from an agarose gel using electroelimination. The Escherichia coli plasmid pB R322In was digested with EcoRI according to Example 10, and then treated with Alfa 7tase according to Example 5. EcoRr of pBR322 thus obtained
2,6 kb'EcoR1, DNA recovered from cleavage~r/alkaline 7 osphatase treatment N1) NA from glue
After mixing with the fragments and ethanol precipitation, 10 μU of ligation reaction buffer (see Example 2) was added, and ligation was performed using T4 DNA ligase according to Example 5. Escherichia coli E and coli DHI were transformed according to Example 5, plasmid DNA was prepared, and the desired hybrid DNA was confirmed. Example 12 (Preparation of mouse-human chimeric antibody gene) Mouse immunogen obtained in 5A Example 5. Pudding t iQ
V-J region gene 1 included tr p Y N-M L
Bam1 (
The fragments were cut with I and EcoRI and subjected to 7-garose gel pneumophoresis. The obtained 4.2Kb containing the gene in -J
psV2nao vector CP, J., Southern, P., Berg: Gerriki Luop 7 Recular and Applied Enenetains Volume 1 327 Page (19
82) (P, J, 5outhern. P, Bsrg: J, Mol, Appl,
GeneL, ± 327 (1982)) JiS Teru] E
Insert between coRI-Bam H1 and plasmid p
SV2 neo-MLV was created. This is shown in FIG. Next, Example 11 was obtained. pYN-HLC containing the chain C region gene of human immunogupmin σpmin was cut with EcoRI according to method 1c of Example 1O, and agarose gel electrophoresis was performed.
17) Using the same method as in Example 11, add the DNA fragment to the above plasmid pSV2neo-ML
VO) Inserted into the EeoRI site. Among the clones obtained, EeoRI (B-
1) -EcoRI (B-2) fragment EcoR
Select a plasmid in which I (B-1) is linked to the mouse V-J gene side (the reading direction of the mouse V-J gene and the human C gene are the same), and insert this plasmid. It was named psV2neo-MHL. This is the 8th
Shown in the figure. - Plasmid p'r, ya containing the human immunoglobulin r+ chain gene described in Japanese Patent Application Laid-Open No. 60-120991
Mlul and Hpa according to the method described in Example 10
It was cut at l. However, Tris is used as a cutting buffer.
H (1 (pH 7,5) 10.mM + Mgα. 7mM +NaCj, I 50r++M + 2-)
A 7mM aqueous solution of lucato x-pnol was used. Mlu
After cutting with Z and recovering DNA by ethanol precipitation,
Cutting buffer -Tris-HCi (pH 7,5)
10mM + Kα100mM + MgC1t
Hpal cleavage was performed using a 7 m M, 2-mercaptoeta/-luaynM aqueous solution. By 7 galose gel electrophoresis, an approximately 0.9 Kb MluI-Hpal fragment containing the human r+ chain gene enhancer region was obtained. Add this DNA fragment to polymerase buffer C37mM.
Potassium phosphate, 6.7mM MgCJs*1
mM 2-Mercapto/-Tol. DN polymerase Klenow fragment (pH 7.4) in the presence of dNTPs.
The ends were smoothed by adding Niney England Biolaps). Furthermore, using %T4-DNA ligase, Ba
r! I) I I Linker (Takara Shuzo)! After connecting, Ba
Cut with mHI and perform agarose gel electrophoresis to obtain approximately 0.
A double-ended BamHI fragment containing the 9 Kb human rI# gene enhancer region was obtained. This DNA fragment was added to the above plasmid pSV 20eo-MHL BamHI.
site-trap insertion, and plasmids pSV2neo-MHLEI and pS with different insertion orientations.
V2 neo-MHLE 2 was produced. This is the 8th
Shown in the figure. Indeed Example 13 (Expression of chimeric antibody chain) Mouse myeloma cells J558L and X63Ag8.6
The separately prepared immunoglobulin 11 chain expression gene 'ix7'ty was introduced into 53 by the Tozlast fusion method, and stable transformation-like (5table transformant
) was obtained. These cells are J558L-H, which express the introduced H chain gene. It is called X 63 Ag 8.653 )1. Plasmid p containing the chimeric LM gene described in Example 12 in J558L-H and X 63 Ag 8.653-H
S V2 neo-MHLE-1 and psV2 ne
o-MHLE-2 was introduced by the DEAE-dextran method (J, Penalji, L, Orlin; W, Juice Wanner: Cell Vol. 33, p. 729 (1983) (
J, Benarji+ L-01sOn+ w,
5chaffner: Ce1133 729 (1
983)))]. That is, 804 in 107 #l
plasmid DNA and DEAE-dextran (manufactured by Pharmacia) mixture, and after incubation, RPM I
1640 complete medium (Flow Laboratory G>cio%
(containing beef tallow serum) K was transferred. The drug-resistant strain is Geneticin (Goneticin + G 418 + Gibeuf V)
This was done by replacing the volume of RPM 11640 complete medium containing l~1.51 kg/l Lj. After 10 to 20 days, the growing 11111 cells were subjected to stable transformation (referred to as J558L-HL and X63Ag8.653-HL, respectively). Example 14 (mRNA analysis of transformed cells) Example 13
RNA was extracted from the transformed cells obtained by
Analysis of mRNA was performed (T. Maniatis et al., "Molecular Croninck", Cold Spring Harper Laboratory, 1982) (T. Maniatis et al., 1982).
．． ："Molecular Cloning"Col
d Spring Lab. (1982)
)reference〕. As a pipelidization probe, as a V region probe, as shown in Fig. 6 on the pYN-MLV-1 plasmid, PstI (A-3)-Hi is used.
DNA fragment corresponding to nel[, pYN as C region probe] ScaI (B-1)
The ScaI (B-2) fragments were each m
jlilL-t: used. As shown in FIG. 9, a band appeared at the same position no matter which sample was used. Example 15 (Analysis of −?Mera Antibody L@Product by SDS-Polyacrylic 7!Doggle Electrophoresis) X 6 3 Ag 8.653-HL 1 of Example 13
After washing 0' pieces with phosphate buffer, methionine-free RPMI
- Suspend the layer in 1640 medium (containing 10X fetal serum),
00 μCi of (:{SS] monomethionine was added and cultured for 8 hours. The thus produced protein was internally labeled with ?l-"S, and the entire culture supernatant was collected. The produced antibody was isolated from the supernatant using an anti-human IgG antibody. The carrier was recovered by immunoprecipitation, and after being released, it was reduced with 2-mercaptoethanol to separate the H chain and L chain.The sample was then separated by SPS-polyacrylamide gel electropulsion, and the gel was dried. Autoradiography was performed. (Refer to "Electrophoresis Experimental Methods" edited by the Electrophoresis Society of Japan, Bungendo + 1976)] Along with the H chain, K
Production of chain protein was observed, and this result indicates that the introduced chimeric antibody K chain was secreted in combination with the previously introduced H chain. The autoradio duram is shown in Figure 10.
ａ付ｇ１図は、エンハンサー塩基配列を示したものであ
り，第２図は抗体Ｌ鎖のＶ領域のアミノ酸配列を示した
ものであり、３３図は上記ｖｇＡ域遺伝子の塩基配列を
示したものであり，第４図は抗体Ｌ鎗Ｃ領域のアミノ酸
配列を示したものであり、第５図は上記Ｃ領域遺伝子の
塩基配列を示したものであり ＃！６図は抗体Ｌ鎖Ｖ領
域遺伝子の制限酊素切断地図を示したものであり、第７
図は抗体Ｌ鎖Ｃ領域遺伝子の制限障素切断地図を示した
ものであり，第８図はキメラＬ鎖遺伝子の作成図を示し
たものである。
第９図はキメラ抗体Ｌ鎮転写産物の７−ザンプロット分
析の結果を示したものであり，第１０図は産生されたキ
メラ抗体Ｌ鎖のＳＤＳ−ポリアクリル７ミドゲル電気泳
動の結果を示したものである。Figure G1 with a shows the enhancer base sequence, Figure 2 shows the amino acid sequence of the V region of the antibody L chain, and Figure 33 shows the base sequence of the vgA region gene mentioned above. Figure 4 shows the amino acid sequence of the C region of antibody L, and Figure 5 shows the base sequence of the C region gene. Figure 6 shows a restriction cleavage map of the antibody L chain V region gene.
The figure shows a restriction barrier cleavage map of the antibody L chain C region gene, and FIG. 8 shows a construction diagram of the chimeric L chain gene. Figure 9 shows the results of 7-zanplot analysis of the chimeric antibody L transcript, and Figure 10 shows the results of SDS-polyacryl 7 midgel electrophoresis of the produced chimeric antibody L chain. It is something.
Claims (1)
抗体Ｌ鎖におけるＣ領域のＤＮＡ断片及び （ｃ）ヒト抗体Ｈ鎖由来のエンハンサーＤＮＡ配列 よりなる抗体Ｌ鎖発現型核酸塩基配列。 ２、該抗体Ｌ鎖がヒト及び／又はマウス抗体由来のもの
である第１項の核酸塩基配列。 ３、該Ｖ領域のＤＮＡ断片がマウス抗体由来であり、該
Ｃ領域のＤＮＡ断片がヒト抗体由来のものであるキメラ
型の第１項の核酸塩基配列。 ４、該エンハンサーＤＮＡ配列が添付第１図のＤＮＡ配
列及びそれに相補的なＤＮＡ配列を有する第１項の核酸
塩基配列。 ５、該Ｖ領域のＤＮＡ断片が、添付第２図の１番目（Ｇ
ｌｕ）から９７番目（Ｌｅｕ）までのアミノ酸配列を少
くともコードしている第１項の核酸塩基配列。 ６、該Ｖ領域のＤＮＡ断片が、添付第３図の５７９番目
から８６９番目までの配列を少くとも有する第１項の核
酸塩基配列。 ７、該Ｃ領域のＤＮＡ断片が、ヒト抗体Ｋ鎖由来のもの
である第１項の核酸塩基配列。 ８、該Ｃ領域のＤＮＡ断片が、添付第４図のアミノ酸配
列を少くともコードしている第１項の核酸塩基配列。 ９、該Ｃ領域のＤＮＡ断片が、添付第５図のＤＮＡ配列
を有する第１項の核酸塩基配列。 １０、第１項の抗体Ｌ鎖発現型核酸塩基配列が組込まれ
た組み換えプラスミド。 １１、第１０項のプラスミドが導入されたマウスミエロ
ーマ細胞。 １２、（ｉ）マウス抗体Ｌ鎖におけるＶ領域のアミノ酸
配列と （ｉｉ）ヒト抗体Ｌ鎖におけるＣ領域のアミノ酸配列 とからなるマウス−ヒトキメラ抗体Ｌ鎖。 １３、該Ｖ領域のアミノ酸配列が、添付第２図の１番目
（Ｇｌｕ）から９７番目（Ｌｅｕ）までのアミノ酸配列
を少くとも有している第１２項のキメラ抗体Ｌ鎖。 １４、該Ｃ領域のアミノ酸配列が、添付第４図のアミノ
酸配列を少くとも有している第１２項のキメラ抗体Ｌ鎖
。[Claims] 1. (a) DNA fragment of the V region of an antibody L chain (b)
An antibody L chain expressed nucleobase sequence consisting of a DNA fragment of the C region of an antibody L chain and (c) an enhancer DNA sequence derived from a human antibody H chain. 2. The nucleic acid base sequence of item 1, wherein the antibody L chain is derived from a human and/or mouse antibody. 3. The nucleobase sequence of item 1 of the chimeric type, wherein the DNA fragment of the V region is derived from a mouse antibody, and the DNA fragment of the C region is derived from a human antibody. 4. The nucleobase sequence of item 1, wherein the enhancer DNA sequence has the DNA sequence shown in attached Figure 1 and a DNA sequence complementary thereto. 5. The DNA fragment of the V region is shown in number 1 (G) in attached Figure 2.
The nucleobase sequence of item 1, which encodes at least the amino acid sequence from position lu) to position 97 (Leu). 6. The nucleobase sequence of item 1, in which the V region DNA fragment has at least the sequence from position 579 to position 869 in attached Figure 3. 7. The nucleobase sequence of item 1, wherein the C region DNA fragment is derived from a human antibody K chain. 8. The nucleobase sequence of item 1, wherein the C region DNA fragment encodes at least the amino acid sequence shown in attached FIG. 4. 9. The nucleobase sequence of item 1, in which the DNA fragment of the C region has the DNA sequence shown in attached FIG. 10. A recombinant plasmid into which the antibody L chain expression type nucleic acid base sequence of item 1 has been integrated. 11. Mouse myeloma cells into which the plasmid of item 10 has been introduced. 12. A mouse-human chimeric antibody L chain consisting of (i) the amino acid sequence of the V region in a mouse antibody L chain and (ii) the amino acid sequence of the C region in a human antibody L chain. 13. The chimeric antibody L chain of Item 12, wherein the amino acid sequence of the V region has at least the amino acid sequence from No. 1 (Glu) to No. 97 (Leu) in attached Figure 2. 14. The chimeric antibody L chain of item 12, wherein the amino acid sequence of the C region has at least the amino acid sequence shown in attached Figure 4.
Priority Applications (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP61178881A JPS6336786A (en) | 1986-07-31 | 1986-07-31 | Nucleic acid base sequence of antibody light chain manifestation type, plasmid, cell and chimera antibody light chain |
| NO873164A NO873164L (en) | 1986-07-30 | 1987-07-28 | MUSEUM-HUMAN CHEMICAL ANTIBODIES. |
| EP87110994A EP0255694A1 (en) | 1986-07-30 | 1987-07-29 | Mouse-human chimera antibody and its components and gene therefor |
| DK398887A DK398887A (en) | 1986-07-30 | 1987-07-30 | ANTIBODIES |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP61178881A JPS6336786A (en) | 1986-07-31 | 1986-07-31 | Nucleic acid base sequence of antibody light chain manifestation type, plasmid, cell and chimera antibody light chain |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPS6336786A true JPS6336786A (en) | 1988-02-17 |
Family
ID=16056330
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP61178881A Pending JPS6336786A (en) | 1986-07-30 | 1986-07-31 | Nucleic acid base sequence of antibody light chain manifestation type, plasmid, cell and chimera antibody light chain |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS6336786A (en) |
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| US20160141192A1 (en) | 2013-08-22 | 2016-05-19 | Sakura Color Products Corporation | Indicator used in electronic device manufacturing apparatus and method for designing and/or managing the apparatus |
| US10180392B2 (en) | 2014-05-09 | 2019-01-15 | Sakura Color Products Corporation | Plasma processing detection indicator using inorganic substance as a color-change layer |
| US10180413B2 (en) | 2014-12-02 | 2019-01-15 | Sakura Color Products Corporation | Ink composition for plasma processing detection, and indicator for plasma processing detection using said ink composition |
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|---|---|---|---|---|
| JPS60120991A (en) * | 1983-12-06 | 1985-06-28 | Teijin Ltd | Enhancer base sequence |
| JPS60155132A (en) * | 1983-04-08 | 1985-08-15 | ジエネンテツク・インコ−ポレイテツド | Recombined immunogloblin |
| JPS6147500A (en) * | 1984-08-15 | 1986-03-07 | Res Dev Corp Of Japan | Chimera monoclonal antibody and its preparation |
| JPS61134325A (en) * | 1984-12-04 | 1986-06-21 | Teijin Ltd | Expression of hybrid antibody gene |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS60155132A (en) * | 1983-04-08 | 1985-08-15 | ジエネンテツク・インコ−ポレイテツド | Recombined immunogloblin |
| JPS60120991A (en) * | 1983-12-06 | 1985-06-28 | Teijin Ltd | Enhancer base sequence |
| JPS6147500A (en) * | 1984-08-15 | 1986-03-07 | Res Dev Corp Of Japan | Chimera monoclonal antibody and its preparation |
| JPS61134325A (en) * | 1984-12-04 | 1986-06-21 | Teijin Ltd | Expression of hybrid antibody gene |
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| US7985833B2 (en) | 2000-04-21 | 2011-07-26 | Fuso Pharmaceutical Industries, Ltd. | Collectin |
| US20160141192A1 (en) | 2013-08-22 | 2016-05-19 | Sakura Color Products Corporation | Indicator used in electronic device manufacturing apparatus and method for designing and/or managing the apparatus |
| US10181414B2 (en) | 2013-08-22 | 2019-01-15 | Sakura Color Products Corporation | Indicator used in electronic device manufacturing apparatus and method for designing and/or managing the apparatus |
| US10401338B2 (en) | 2014-02-14 | 2019-09-03 | Sakura Color Products Corporation | Plasma processing detection indicator |
| US10180392B2 (en) | 2014-05-09 | 2019-01-15 | Sakura Color Products Corporation | Plasma processing detection indicator using inorganic substance as a color-change layer |
| US10180413B2 (en) | 2014-12-02 | 2019-01-15 | Sakura Color Products Corporation | Ink composition for plasma processing detection, and indicator for plasma processing detection using said ink composition |
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