JP2019526253A - 皮膚細胞の増殖効果が増大したサソリ毒融合タンパク質及びそれを有効成分として含有するシワ改善、皮膚弾力の増大及びアンチエイジング化粧料組成物 - Google Patents
皮膚細胞の増殖効果が増大したサソリ毒融合タンパク質及びそれを有効成分として含有するシワ改善、皮膚弾力の増大及びアンチエイジング化粧料組成物 Download PDFInfo
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Abstract
Description
ヒトチオレドキシンとサソリ毒を含む融合タンパク質及びヒト上皮細胞成長因子とサソリ毒を含む融合タンパク質をコードする最適化された遺伝子、組換え発現ベクター及び形質転換組換え微生物は、下記の方法で製作した。ヒトチオレドキシンとサソリ毒を含む融合タンパク質及びヒト上皮細胞成長因子とサソリ毒を含む融合タンパク質をコードする遺伝子を鋳型として宿主微生物内で発現されるように最適化された、140個のアミノ酸を暗号化したヒトチオレドキシンとサソリ毒を含む融合タンパク質(配列番号3)をコードする遺伝子(配列番号1)及び89個のアミノ酸を暗号化したヒト上皮細胞成長因子とサソリ毒を含む融合タンパク質(配列番号4)をコードする遺伝子(配列番号2)の断片を製作、合成した。使用したプライマー、下記の通りである。
逆方向プライマー:5’−CGTCACGGACACGGCAGACTAATTCATTAATGGTG−3’(配列番号6)
センスプライマー:5’−CACCATTAATGAATTAGTCATGTGCATGCCGTGCTTC−3’(配列番号7)
アンチセンスプライマー:5’−GAAGCACGGCATGCACATGACTAATTCATTAATGGTG−3’(配列番号8)。
逆方向プライマー:5’−GGTGGTGGTGCTCGAGACGGCACAGGCACTGCGG−3’(配列番号10)
センスプライマー:5’−AAATGGTGGGAGTTGCGCATGTGCATGCCGTGCTTC−3’(配列番号11)
アンチセンスプライマー:5’−GAAGCACGGCATGCACATGCGCAACTCCCACCATTT−3’(配列番号12)。
実施例1で製造したE.coli Rosetta2(DE3)pLysS(Novagen)をそれぞれ1LLBあるいはBSB培地に回分培養するとき、OD600=0.6〜0.8、20L発酵器を使用して連続培養するとき、OD600=15〜20になるまで培養した。次いで、これらをそれぞれ最終1〜5mM IPTGまたは0.2〜2%ラクトースを加えてクローン内の遺伝子の発現を誘導した。その後、さらに4〜5時間培養した後、細胞を遠心分離方法で回収した。この細胞を緩衝溶液(Phosphate buffered saline,NaCl 8g、KCl 0.2g、Na2HPO4 1.44g、KH2PO4 0.24g/L,pH 7.4)に完全に懸濁させた後、超音波破砕機を使用して細胞を破壊し、細胞内のタンパク質含有溶液を分離した。
実施例2において分離、精製されたヒトチオレドキシンとサソリ毒を含む融合タンパク質及びヒト上皮細胞成長因子とサソリ毒を含む融合タンパク質の存在が確認された分画から試料を選んで皮膚線維芽細胞(Human Dermal Fibroblasts adult,HDFa cell)の細胞増殖効果を確認した。
実施例2において分離、精製されたヒトチオレドキシンとサソリ毒を含む融合タンパク質及びヒト上皮細胞成長因子とサソリ毒を含む融合タンパク質の存在が確認された試料を選んでHaCaT細胞(Human,Adult,low Calcium,High Temperature,epithelial keratinocyte)の細胞増殖及び傷治療効果を確認した。図5及び図6は、HaCaT細胞を培養した後、それぞれの濃度(0〜20ppm)のヒトチオレドキシンとサソリ毒を含む融合タンパク質及びヒト上皮細胞成長因子とサソリ毒を含む融合タンパク質を処理した後、24時間顕微鏡(オリンパスCK40)を使用してHaCaT細胞の傷治療効果を24時間観察した。0.02〜20ppm濃度のヒトチオレドキシンとサソリ毒を含む融合タンパク質及びヒト上皮細胞成長因子とサソリ毒を含む融合タンパク質を処理したHaCaT細胞は、対照群に比べて傷治療効果を示した。図5及び図6の結果から、ヒトチオレドキシンとサソリ毒を含む融合タンパク質及びヒト上皮細胞成長因子とサソリ毒を含む融合タンパク質は、低濃度でも傷治療効果を示すことが分かる。
図7は、ヒトチオレドキシンとサソリ毒を含む融合タンパク質及びヒト上皮細胞成長因子とサソリ毒を含む融合タンパク質の抗酸化効能に係わる結果を示す図面である。ヒトチオレドキシンとサソリ毒を含む融合タンパク質及びヒト上皮細胞成長因子とサソリ毒を含む融合タンパク質の抗酸化効能を測定するために、フリーラジカル消去効果測定方法の1つである1,1−ジフェニル−2−ピクリル−ヒドラジル(1,1−Diphenyl−2−pycryl−Hydrazyl;DPPH)方法を使用した。この試薬は、比較的安定したフリーラジカルとして存在し、フリーラジカル消去作用を確認するのに一次的に使用される試験管的な方法の1つである。
紫外線刺激による皮膚老化抑制手段の1つであるコラーゲン纎維(collagen fiber)合成の促進を調べるために、本発明の前記タンパク質を線維芽細胞に処理した後、コラーゲン合成促進如何を評価した。ヒトチオレドキシンとサソリ毒を含む融合タンパク質及びヒト上皮細胞成長因子とサソリ毒を含む融合タンパク質を無血清DMEM培地に0〜20ppmの濃度で線維芽細胞に処理した後、72時間後、細胞抽出物を対象にCollagen−I ELISA kit(Cloud−Clone Corp.,アメリカ)を使用して、第1型プロコラーゲン発現量の変化を測定し、その結果は、図8の通りである。図8から分かるように、第1型プロコラーゲンの発現量は、ヒト上皮細胞成長因子とサソリ毒を含む融合タンパク質によって濃度−依存的に増加した。一方、ヒトチオレドキシンとサソリ毒を含む融合タンパク質の場合、0.2ppm以下の濃度では、濃度−依存的に増加したが、その後、減少パターンを示した。これは、図4に図示されたように、線維芽細胞増殖効果の結果と一致することを確認することができた。したがって、本発明による前記タンパク質は、図8から、コラーゲン消失による皮膚の老化、特に皮膚シワを抑制可能であるということを確認することができた。
Claims (16)
- 配列番号3のアミノ酸配列からなる皮膚細胞の増殖及び抗酸化効果が増大した、ヒトチオレドキシン−サソリ毒融合タンパク質。
- 配列番号4のアミノ酸配列からなる皮膚細胞の増殖効果が増大した、ヒト上皮細胞成長因子−サソリ毒融合タンパク質。
- 請求項1に記載の皮膚細胞の増殖及び抗酸化効果が増大したヒトチオレドキシン−サソリ毒融合タンパク質をコードする、遺伝子。
- 前記遺伝子は、大腸菌コドンに最適化された配列番号1の塩基配列からなることを特徴とする、請求項3に記載の遺伝子。
- 請求項2に記載の皮膚細胞の増殖効果が増大したヒト上皮細胞成長因子−サソリ毒融合タンパク質をコードする、遺伝子。
- 前記遺伝子は、大腸菌コドンに最適化された配列番号2の塩基配列からなることを特徴とする、請求項5に記載の遺伝子。
- 請求項3または5に記載の遺伝子を含む、組換えベクター。
- 請求項7に記載の組換えベクターで形質転換された、宿主細胞。
- 請求項3または4に記載の遺伝子を含む組換えベクターで宿主細胞を形質転換させて、ヒトチオレドキシン−サソリ毒融合タンパク質をコードする遺伝子を過剰発現させる段階を含む、宿主細胞でのヒトチオレドキシン−サソリ毒融合タンパク質の生産方法。
- 前記宿主細胞は、大腸菌であることを特徴とする、請求項9に記載のヒトチオレドキシン−サソリ毒融合タンパク質の生産方法。
- 請求項5または6に記載の遺伝子を含む組換えベクターで宿主細胞を形質転換させて、ヒト上皮細胞成長因子−サソリ毒融合タンパク質をコードする遺伝子を過剰発現させる段階を含む、宿主細胞でのヒト上皮細胞成長因子−サソリ毒融合タンパク質の生産方法。
- 前記宿主細胞は、大腸菌であることを特徴とする、請求項11に記載のヒト上皮細胞成長因子−サソリ毒融合タンパク質の生産方法。
- 請求項9に記載の方法によって生産された、ヒトチオレドキシン−サソリ毒融合タンパク質。
- 請求項11に記載の方法によって生産された、ヒト上皮細胞成長因子−サソリ毒融合タンパク質。
- 配列番号3のアミノ酸配列からなる皮膚細胞の増殖効果と抗酸化機能とが増大したヒトチオレドキシン−サソリ毒融合タンパク質を有効成分として含有する、シワ改善、皮膚弾力の増大及びアンチエイジング機能を有する化粧料組成物。
- 配列番号4のアミノ酸配列からなる皮膚細胞の増殖効果が増大したヒト上皮細胞成長因子−サソリ毒融合タンパク質を有効成分として含有する、シワ改善及び皮膚弾力の増大機能を有する化粧料組成物。
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