JP2022542577A - Nt3を融合した絹フィブロイン神経移植片の製造方法 - Google Patents
Nt3を融合した絹フィブロイン神経移植片の製造方法 Download PDFInfo
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Abstract
Description
絹フィブロイン軽鎖及びNT-3を含む遺伝子断片を合成してpET-30発現ベクターに結合し、得られた組換え発現ベクターをBL21大腸菌に形質転換して、融合タンパク質を得るステップと、
融合タンパク質及び絹フィブロイン溶液を混合して、混合タンパク質溶液を得、絹繊維ネットを鋳型内に置き、混合タンパク質溶液を鋳型に注ぎ込んで冷凍乾燥して、神経導管を形成するステップと、変形処理した後、最終的にNT-3活性を有する絹フィブロイン神経移植片を得るステップと、を含む。さらに、前記絹繊維ネットは絹繊維用編機で編まれる。
NT3を融合した絹フィブロイン神経移植片の製造方法であって、以下のステップを含む。
NT3を融合した絹フィブロイン神経移植片の製造方法であって、以下のステップを含む。
FIBL Protein Length=253 MW=26898.8 pI=6.10
MHHHHHHAPSVTINQYSDNEIPRDIDDGKASSVISRAWDYVDDTDKSIAILNVQEILKDMASQGDYASQASAVAQTAGIIAHLSAGIPGDACAAANVINSYTDGVRSGNFAGFRQSLGPFFGHVGQNLNLINQLVINPGQLRYSVGPALGCAGGGRIYDFEAAWDAILASSDSSFLNEEYCIVKRLYNSRNSQSNNIAAYITAHLLPPVAQVFHQSAGSITDLLRGVGNG NDATGLVANAQRYIAQAASQVHV
FIBL-NT3 Protein Length=372 MW=40503.7 pI=7.39
MHHHHHHAPSVTINQYSDNEIPRDIDDGKASSVISRAWDYVDDTDKSIAILNVQEILKDMASQGDYASQASAVAQTAGIIAHLSAGIPGDACAAANVINSYTDGVRSGNFAGFRQSLGPFFGHVGQNLNLINQLVINPGQLRYSVGPALGCAGGGRIYDFEAAWDAILASSDSSFLNEEYCIVKRLYNSRNSQSNNIAAYITAHLLPPVAQVFHQSAGSITDLLRGVGNGNDATGLVANAQRYIAQAASQVHVYAEHKSHRGEYSVCDSESLWVTDKSSAIDIRGHQVTVLGEIKTGNSPVKQYFYETRCKEARPVKNGCRGIDDKHWNSQCKTSQTYVRALTSENNKLVGWRWIRIDTSCVCALSRKIGRT
FIBL-linker-NT3 Protein Length=382 MW=41134.1 pI=7.39
MHHHHHHAPSVTINQYSDNEIPRDIDDGKASSVISRAWDYVDDTDKSIAILNVQEILKDMASQGDYASQASAVAQTAGIIAHLSAGIPGDACAAANVINSYTDGVRSGNFAGFRQSLGPFFGHVGQNLNLINQLVINPGQLRYSVGPALGCAGGGRIYDFEAAWDAILASSDSSFLNEEYCIVKRLYNSRNSQSNNIAAYITAHLLPPVAQVFHQSAGSITDLLRGVGNGNDATGLVANAQRYIAQAASQVHVGGGGSGGGGSYAEHKSHRGEYSVCDSESLWVTDKSSAIDIRGHQVTVLGEIKTGNSPVKQYFYETRCKEARPVKNGCRGIDDKHWNSQCKTSQTYVRALTSENNKLVGWRWIRIDTS CVCALSRKIG RT
FIBL-(NT3)2 Protein Length=501 MW=54739.0 pI=8.28
MHHHHHHAPSVTINQYSDNEIPRDIDDGKASSVISRAWDYVDDTDKSIAILNVQEILKDMASQGDYASQASAVAQTAGIIAHLSAGIPGDACAAANVINSYTDGVRSGNFAGFRQSLGPFFGHVGQNLNLINQLVINPGQLRYSVGPALGCAGGGRIYDFEAAWDAILASSDSSFLNEEYCIVKRLYNSRNSQSNNIAAYITAHLLPPVAQVFHQSAGSITDLLRGVGNGNDATGLVANAQRYIAQAASQVHVYAEHKSHRGEYSVCDSESLWVTDKSSAIDIRGHQVTVLGEIKTGNSPVKQYFYETRCKEARPVKNGCRGIDDKHWNSQCKTSQTYVRALTSENNKLVGWRWIRIDTSCVCALSRKIGRTGGGGSGGGGSYAEHKSHRGEYSVCDSESLWVTDKSSAIDIRGHQVTVLGEIKTGNSPVKQYFYETRCKEARPVKNGCRGIDDKHWNSQCKTSQTYVRA LTSENNKLVGWRWIRIDTSCVCALSRKIGRT
(付記1)
絹フィブロイン軽鎖及びNT-3を含む遺伝子断片を合成してpET-30発現ベクターに結合し、得られた組換え発現ベクターをBL21大腸菌に形質転換して、融合タンパク質を得るステップと、
融合タンパク質及び絹フィブロイン溶液を混合して、混合タンパク質溶液を得、絹繊維ネットを鋳型内に置き、混合タンパク質溶液を鋳型に注ぎ込んで冷凍乾燥して、神経導管を形成するステップと、変形処理した後、最終的にNT-3活性を有する絹フィブロイン神経移植片を得るステップと、を含む、ことを特徴とするNT3を融合した絹フィブロイン神経移植片の製造方法。
前記絹繊維ネットが絹繊維用編機で編まれる、ことを特徴とする付記1に記載のNT3を融合した絹フィブロイン神経移植片の製造方法。
前記絹フィブロイン溶液の調製方法は、絹繊維をチオシアン酸リチウム溶液に入れて溶解させ、溶解後の溶液を透析用袋に入れ、三重蒸留水を透析液として60~80時間透析し、絹フィブロイン溶液を得るものである、ことを特徴とする付記1に記載のNT3を融合した絹フィブロイン神経移植片の製造方法。
前記絹繊維の製造方法は、カイコ糸を炭酸ナトリウム溶液に入れて20分間以上煮沸し、取り出した後、三重蒸留水で十分に洗浄する、という工程を2~4回繰り返し、外部セリシンを除去した絹繊維を得るものである、ことを特徴とする付記2又は3に記載のNT3を融合した絹フィブロイン神経移植片の製造方法。
混合タンパク質溶液の濃度が5%-40%であり、融合タンパク質及び絹フィブロインの質量割合が1:99~50:50である、ことを特徴とする付記1に記載のNT3を融合した絹フィブロイン神経移植片の製造方法。
変形処理は、60%エタノール中の浸漬で10~14h処理するものである、ことを特徴とする付記1に記載のNT3を融合した絹フィブロイン神経移植片の製造方法。
透析用袋の分画分子量が12~16kDaである、ことを特徴とする付記1に記載のNT3を融合した絹フィブロイン神経移植片の製造方法。
冷凍乾燥の温度が-70℃である、ことを特徴とする付記1に記載のNT3を融合した絹フィブロイン神経移植片の製造方法。
チオシアン酸リチウム溶液の濃度が9mol/Lである、ことを特徴とする付記1に記載のNT3を融合した絹フィブロイン神経移植片の製造方法。
付記1から9のいずれか1つに記載の製造方法で製造される、ことを特徴とするNT3を融合した絹フィブロイン神経移植片。
Claims (10)
- 絹フィブロイン軽鎖及びNT-3を含む遺伝子断片を合成してpET-30発現ベクターに結合し、得られた組換え発現ベクターをBL21大腸菌に形質転換して、融合タンパク質を得るステップと、
融合タンパク質及び絹フィブロイン溶液を混合して、混合タンパク質溶液を得、絹繊維ネットを鋳型内に置き、混合タンパク質溶液を鋳型に注ぎ込んで冷凍乾燥して、神経導管を形成するステップと、変形処理した後、最終的にNT-3活性を有する絹フィブロイン神経移植片を得るステップと、を含む、ことを特徴とするNT3を融合した絹フィブロイン神経移植片の製造方法。 - 前記絹繊維ネットが絹繊維用編機で編まれる、ことを特徴とする請求項1に記載のNT3を融合した絹フィブロイン神経移植片の製造方法。
- 前記絹フィブロイン溶液の調製方法は、絹繊維をチオシアン酸リチウム溶液に入れて溶解させ、溶解後の溶液を透析用袋に入れ、三重蒸留水を透析液として60~80時間透析し、絹フィブロイン溶液を得るものである、ことを特徴とする請求項1に記載のNT3を融合した絹フィブロイン神経移植片の製造方法。
- 前記絹繊維の製造方法は、カイコ糸を炭酸ナトリウム溶液に入れて20分間以上煮沸し、取り出した後、三重蒸留水で十分に洗浄する、という工程を2~4回繰り返し、外部セリシンを除去した絹繊維を得るものである、ことを特徴とする請求項2又は3に記載のNT3を融合した絹フィブロイン神経移植片の製造方法。
- 混合タンパク質溶液の濃度が5%-40%であり、融合タンパク質及び絹フィブロインの質量割合が1:99~50:50である、ことを特徴とする請求項1に記載のNT3を融合した絹フィブロイン神経移植片の製造方法。
- 変形処理は、60%エタノール中の浸漬で10~14h処理するものである、ことを特徴とする請求項1に記載のNT3を融合した絹フィブロイン神経移植片の製造方法。
- 透析用袋の分画分子量が12~16kDaである、ことを特徴とする請求項1に記載のNT3を融合した絹フィブロイン神経移植片の製造方法。
- 冷凍乾燥の温度が-70℃である、ことを特徴とする請求項1に記載のNT3を融合した絹フィブロイン神経移植片の製造方法。
- チオシアン酸リチウム溶液の濃度が9mol/Lである、ことを特徴とする請求項1に記載のNT3を融合した絹フィブロイン神経移植片の製造方法。
- 請求項1から9のいずれか1項に記載の製造方法で製造される、ことを特徴とするNT3を融合した絹フィブロイン神経移植片。
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