JP6871547B2 - ピエリシン1aのadpリボシル化ドメイン遺伝子及びセリシン繭 - Google Patents
ピエリシン1aのadpリボシル化ドメイン遺伝子及びセリシン繭 Download PDFInfo
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Images
Description
項1. 以下の(a)、(b)又は(c)のDNAを含む遺伝子:
(a)配列番号3の塩基配列を含むDNA
(b)配列番号3の塩基配列とストリンジェントな条件下でハイブリダイズし、核酸のグアニン残基のADPリボシル化活性を有するタンパク質をコードするDNA
(c)(a)又は(b)のDNAの相補鎖
項2. 以下の(p)、(q)又は(r)のポリペプチドをコードする遺伝子:
(p)配列番号4のアミノ酸配列を含むポリペプチド
(q)配列番号4のアミノ酸配列において、1もしくは複数のアミノ酸が置換、付加又は欠失されたアミノ酸配列からなり、核酸のグアニン残基のADPリボシル化活性を有するポリペプチド
(r)配列番号4のアミノ酸配列と85%以上の同一性を有し、核酸のグアニン残基のADPリボシル化活性を有するポリペプチド
項3. 項1又は2に記載の遺伝子を含むベクター。
項4. 前記遺伝子が誘導性プロモーター、又は細胞タイプ若しくは組織特異的プロモーターの制御下にある、項3に記載のベクター。
項5. 項3又は4のベクターにより形質転換された複数の組織を有する多細胞生物の細胞。
項6. 項5に記載の細胞を含む標的組織が機能不全化された複数の組織を有する多細胞生物。
項7. 前記多細胞生物がカイコガであり、標的組織が後部絹糸腺である、項6に記載の多細胞生物。
項8. 項7に記載のカイコガを生育し、セリシン繭を分離する工程を含む、セリシン繭の製造方法。
項9. フィブロインを含まないセリシン繭。
項10. 以下の(p)、(q)又は(r)のポリペプチド:
(p)配列番号4のアミノ酸配列を含むポリペプチド
(q)配列番号4のアミノ酸配列において、1もしくは複数のアミノ酸が置換、付加又は欠失されたアミノ酸配列からなり、核酸のグアニン残基のADPリボシル化活性を有するポリペプチド
(r)配列番号4のアミノ酸配列と85%以上の同一性を有し、核酸のグアニン残基のADPリボシル化活性を有するポリペプチド
1.標的とする組織においてピエリシン1AのADPリボシル化ドメインを発現することで、その組織だけを機能不全化する。
2.実施例として、カイコ後部絹糸腺でピエリシン1AのADPリボシル化ドメインを発現させた所、後部絹糸腺が機能しなくなり、フィブロインの産生が行われなくなった。このため、このカイコは中部絹糸腺からのセリシンでできた糸を吐くようになった。
3.化粧品などに使用されているセリシンは生糸からセリシンのみを抽出、精製して使用している。しかし、その抽出操作によってセリシンが変性することが問題であった。しかし、このピエリシン1AのADPリボシル化ドメインを後部絹糸腺で発現することで、フィブロインを全く含まないセリシンのみの糸を得ることができ、そのセリシンは水などで容易に抽出できるようになった。このため、この後部絹糸腺を機能不全化した遺伝子組換えカイコは、化粧品等の材料としてのセリシンの供給材料に最適であると期待される。
実施例1:ピエリシンカイコ作出方法
1.ベクターの構築
遺伝子配列のベクターへのクローニングに用いたプライマーおよびオリゴヌクレオチドは表1にまとめた。
Tamuraらの方法で、産卵後3-8時間のw1-pndの卵の胚発生域に、200ng/μlのドナープラスミドおよびピギーバック発現用ヘルパープラスミドを含むリン酸緩衝液(pH7.0)を、フェムトジェット(エッペンドルフ社製)を用いてマイクロインジェクションした(G0世代;Tamura et al., 2007 JIBS)。G0世代どうしの交配によって得られるG1世代の発生卵を気管形成期に蛍光顕微鏡を用いて観察し、神経でEGFPを発現している個体を選抜した。
選抜したEGFP陽性個体を交配し、得られる子孫の中で特に強いEGFP蛍光の形質を持ち、セリシン繭を作る系統を選抜した(G2世代)。このG2セリシン繭産生系統の絹糸腺からゲノムDNAを抽出し、制限酵素Sau3AIにより一晩処理して得られるDNA断片をリガーゼを用いて自己環状化させた。これを鋳型に、piggyBack right ITR 1: 5’とpiggyBack right ITR 2: 3’の組み合わせ、またはpiggyBack left ITR 1: 5’とpiggyBack left ITR 2: 3’の組み合わせでPCR法を行い、得られた断片の配列を決定することにより、それぞれ転移配列の5’ ITRおよび3’ ITR周辺のカイコ染色体由来配列を決定した(表2)。この結果、第16番染色体に確かに転移配列が挿入されていることを確認した。この系統について、マーカーのEGFP蛍光の強さからホモ系統を選抜して、以後同系交配を繰り返した。
Claims (10)
- 以下の(a)、(b)又は(c)のDNAを含む遺伝子:
(a)配列番号3の塩基配列を含むDNA
(b)配列番号3の塩基配列の相補鎖とストリンジェントな条件下でハイブリダイズし、核酸のグアニン残基のADPリボシル化活性を有するタンパク質をコードするDNA
(c)(a)又は(b)のDNAの相補鎖 - 以下の(p)、(q)又は(r)のポリペプチドをコードする遺伝子:
(p)配列番号4のアミノ酸配列を含むポリペプチド
(q)配列番号4のアミノ酸配列において、1もしくは複数のアミノ酸が置換、付加又は欠失されたアミノ酸配列からなり、核酸のグアニン残基のADPリボシル化活性を有するポリペプチド
(r)配列番号4のアミノ酸配列と90%以上の同一性を有し、核酸のグアニン残基のADPリボシル化活性を有するポリペプチド - 請求項1又は2に記載の遺伝子を含むベクター。
- 前記遺伝子が誘導性プロモーター、又は細胞タイプ若しくは組織特異的プロモーターの制御下にある、請求項3に記載のベクター。
- 請求項3又は4のベクターにより形質転換された複数の組織を有する多細胞生物の細胞。
- 請求項5に記載の細胞を含む標的組織が機能不全化された複数の組織を有する非ヒト多細胞生物。
- 前記多細胞生物がカイコガであり、標的組織が後部絹糸腺である、請求項6に記載の多細胞生物。
- 請求項7に記載のカイコガを生育し、セリシン繭を分離する工程を含む、セリシン繭の製造方法。
- 前記セリシン繭が、フィブロインを含まないセリシン繭である、請求項8に記載の製造方法。
- 以下の(p)、(q)又は(r)のポリペプチド:
(p)配列番号4のアミノ酸配列を含むポリペプチド
(q)配列番号4のアミノ酸配列において、1もしくは複数のアミノ酸が置換、付加又は欠失されたアミノ酸配列からなり、核酸のグアニン残基のADPリボシル化活性を有するポリペプチド
(r)配列番号4のアミノ酸配列と90%以上の同一性を有し、核酸のグアニン残基のADPリボシル化活性を有するポリペプチド
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