JP7185036B2 - アッカーマンシアムシニフィラ菌株およびその用途 - Google Patents
アッカーマンシアムシニフィラ菌株およびその用途 Download PDFInfo
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- JP7185036B2 JP7185036B2 JP2021519759A JP2021519759A JP7185036B2 JP 7185036 B2 JP7185036 B2 JP 7185036B2 JP 2021519759 A JP2021519759 A JP 2021519759A JP 2021519759 A JP2021519759 A JP 2021519759A JP 7185036 B2 JP7185036 B2 JP 7185036B2
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- akkermansia muciniphila
- snug
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Description
本出願は2018年10月11日付韓国特許出願第10-2018-0121137号および2019年10月10日付韓国特許出願第10-2019-0125670号に基づいた優先権の利益を主張し、当該韓国特許出願の文献に開示された全ての内容は本明細書の一部として含まれる。
寄託機関名:韓国生命工学研究院
受託番号:KCTC 13530BP
受託日付:20180525
Gene:Amuc_1631
UniProtKB-B2UM07
Protein name-Carboxyl-terminal protease
Organism:Akkermansia muciniphila
以下、本発明の実施形態を示す。
(1)アッカーマンシアムシニフィラ(Akkermansia muciniphila)SNUG-61027菌株(受託番号KCTC 13530BP)。
(2)前記菌株は、配列目録1の塩基配列からなる16S rDNAを含むことを特徴とする、(1)に記載のアッカーマンシアムシニフィラSNUG-61027菌株(受託番号KCTC 13530BP)。
(3)(1)のアッカーマンシアムシニフィラSNUG-61027菌株、またはその培養液、上澄液、抽出物または分画物を有効性分として含む、食欲抑制または代謝性疾患の予防、改善または治療用薬学的組成物。
(4)前記代謝性疾患は、耐糖能障害、糖尿、動脈硬化、高脂血症、高コレステロール症、脂肪肝、心血管疾患または肥満であることを特徴とする、(3)に記載の食欲抑制または代謝性疾患の予防、改善または治療用薬学的組成物。
(5)前記アッカーマンシアムシニフィラSNUG-61027菌株、またはその培養液、上澄液、抽出物または分画物は、IL-6増加、GLP-1発現増加、および褐色脂肪の活性増加のうちのいずれか一つ以上を誘導することを特徴とする、(3)に記載の食欲抑制または代謝性疾患の予防、改善または治療用薬学的組成物。
(6)配列番号2のアミノ酸配列からなるB2UM07タンパク質を有効性分として含む、食欲抑制または代謝性疾患の予防、改善または治療用薬学的組成物。
(7)前記B2UM07タンパク質は、アッカーマンシアムシニフィラ菌株に由来したことを特徴とする、(6)に記載の食欲抑制または代謝性疾患の予防、改善または治療用薬学的組成物。
(8)前記アッカーマンシアムシニフィラ菌株は、SNUG-61027菌株(受託番号KCTC 13530BP)であることを特徴とする、(7)に記載の食欲抑制または代謝性疾患の予防、改善または治療用薬学的組成物。
(9)前記代謝性疾患は、耐糖能障害、糖尿、動脈硬化、高脂血症、高コレステロール症、脂肪肝、心血管疾患または肥満であることを特徴とする、(6)に記載の食欲抑制または代謝性疾患の予防、改善または治療用薬学的組成物。
(10)(1)のアッカーマンシアムシニフィラSNUG-61027菌株、またはその培養液、上澄液、抽出物または分画物を有効性分として含む、食欲抑制または代謝性疾患の改善または緩和用健康機能性食品。
(11)前記代謝性疾患は、耐糖能障害、糖尿、動脈硬化、高脂血症、高コレステロール症、脂肪肝、心血管疾患または肥満であることを特徴とする、(10)に記載の食欲抑制または代謝性疾患の改善または緩和用健康機能性食品。
(12)前記アッカーマンシアムシニフィラSNUG-61027菌株、またはその培養液、上澄液、抽出物または分画物は、IL-6増加、GLP-1発現増加、および褐色脂肪の活性増加のうちのいずれか一つ以上を誘導することを特徴とする、(10)に記載の食欲抑制または代謝性疾患の改善または緩和用健康機能性食品。
(13)配列番号2のアミノ酸配列からなるB2UM07タンパク質を有効性分として含む、食欲抑制または代謝性疾患の改善または緩和用健康機能性食品。
(14)前記B2UM07タンパク質は、アッカーマンシアムシニフィラ菌株に由来したことを特徴とする、(13)に記載の食欲抑制または代謝性疾患の改善または緩和用健康機能性食品。
(15)前記アッカーマンシアムシニフィラ菌株は、SNUG-61027菌株(受託番号KCTC 13530BP)であることを特徴とする、(14)に記載の食欲抑制または代謝性疾患の改善または緩和用健康機能性食品。
(16)前記代謝性疾患は、耐糖能障害、糖尿、動脈硬化、高脂血症、高コレステロール症、脂肪肝、心血管疾患または肥満であることを特徴とする、(13)に記載の食欲抑制または代謝性疾患の改善または緩和用健康機能性食品。
(17)アッカーマンシアムシニフィラ(Akkermansia muciniphila)SNUG-61027菌株(受託番号KCTC 13530BP)、またはその培養液、上澄液、抽出物または分画物の食欲抑制または代謝性疾患の予防、治療、改善または緩和のための用途。
(18)前記アッカーマンシアムシニフィラ(Akkermansia muciniphila)SNUG-61027菌株(受託番号KCTC 13530BP)は、配列目録1の塩基配列からなる16S rDNAを含むことを特徴とする、(17)に記載の食欲抑制または代謝性疾患の予防、治療、改善または緩和のための用途。
(19)前記代謝性疾患は、耐糖能障害、糖尿、動脈硬化、高脂血症、高コレステロール症、脂肪肝、心血管疾患または肥満であることを特徴とする、(17)に記載の食欲抑制または代謝性疾患の予防、治療、改善または緩和のための用途。
(20)配列番号2のアミノ酸配列からなるB2UM07タンパク質の食欲抑制または代謝性疾患の予防、治療、改善または緩和のための用途。
(21)前記B2UM07タンパク質は、アッカーマンシアムシニフィラ菌株に由来したことを特徴とする、(20)に記載の食欲抑制または代謝性疾患の予防、治療、改善または緩和のための用途。
(22)前記アッカーマンシアムシニフィラ菌株は、SNUG-61027菌株(受託番号KCTC 13530BP)であることを特徴とする、(21)に記載の食欲抑制または代謝性疾患の予防、治療、改善または緩和のための用途。
(23)アッカーマンシアムシニフィラ(Akkermansia muciniphila)SNUG-61027菌株(受託番号KCTC 13530BP)、またはその培養液、上澄液、抽出物または分画物を処理する段階を含む、食欲抑制または代謝性疾患の予防、治療、改善または緩和方法。
(24)前記アッカーマンシアムシニフィラ(Akkermansia muciniphila)SNUG-61027菌株(受託番号KCTC 13530BP)は、配列目録1の塩基配列からなる16S rDNAを含むことを特徴とする、(23)に記載の食欲抑制または代謝性疾患の予防、治療、改善または緩和方法。
(25)前記代謝性疾患は、耐糖能障害、糖尿、動脈硬化、高脂血症、高コレステロール症、脂肪肝、心血管疾患または肥満であることを特徴とする、(23)に記載の食欲抑制または代謝性疾患の予防、治療、改善または緩和方法。
(26)配列番号2のアミノ酸配列からなるB2UM07タンパク質を処理する段階を含む、食欲抑制または代謝性疾患の予防、治療、改善または緩和方法。
(27)前記B2UM07タンパク質は、アッカーマンシアムシニフィラ菌株に由来したことを特徴とする、(26)に記載の食欲抑制または代謝性疾患の予防、治療、改善または緩和方法。
(28)前記アッカーマンシアムシニフィラ菌株は、SNUG-61027菌株(受託番号KCTC 13530BP)であることを特徴とする、(27)に記載の食欲抑制または代謝性疾患の予防、治療、改善または緩和方法。
アッカーマンシアムシニフィラ(ATCC BAA-835、Akk)菌株を0.5%ムチン(mucin)添加brain heart infusion(BHI)固体培地で72時間嫌気培養した後、スタックを確保した。6週齢雄C57BL/6マウスに高脂肪食餌(60%脂肪)飼料摂取と同時に菌株を4×108CFU/200μl/mouse濃度で毎日経口投与した(HF+Akk、n=8/グループ)。低脂肪食餌(10%脂肪)飼料(LF)と高脂肪食餌飼料(HF)のみ摂取したグループを対照群として使用した。14週後、対照群と菌株投与グループを比較した。16時間絶食後、脂肪組織と肝組織を採取、組織重量を測定した(図1A)。
褐色脂肪組織(iBAT)で、褐色脂肪活性化マーカーであるuncoupling protein(UCP-1)をimmuno-histochemistry(IHC)染色をしてグループ別に比較した(図2A)。組織のRNAを抽出、cDNA合成後、UCP-1の遺伝子発現をqPCRで確認し、褐色脂肪分化関連マーカー(CIDEA、PRDM16、PPARGC1α、Apelin)も確認した(図2B)。
小腸(ileum)組織と大腸(colon)組織のRNAを抽出、cDNAを合成後、免疫サイトカインマーカー(TNF-α、IL-1β、IL-18、IL-6、IL-10)の発現程度をグループ別に比較した(図3A、B)。
アッカーマンシアムシニフィラの褐色脂肪活性化効能がIL-6サイトカイン依存的なのかどうかを確認した。
Akk菌株(アッカーマンシアムシニフィラATCC BAA-835)またはアッカーマンシアムシニフィラSNUG-61027菌株を0.5%ムチン(mucin)培地に培養後、液体培養のために0.1%または5% Fetal bovine serum(FBS)添加BHI培地に36時間培養した。
アッカーマンシアムシニフィラ分泌短鎖脂肪酸(short chain fatty acid、SCFA)分析のためにGC-MSを用いて代表的な短鎖脂肪酸、アセテート(acetate)、プロピオネート(propionate)、ブチレート(butyrate)の生産を確認した(図6A)。アセテートとプロピオネート(1mM、10mM)と菌株培養上清液(100%v/v)を処理後2時間後、GLP-1分泌程度を確認した(図6B)。
培養液内活性物質を分離するために大きさ別フィルターを用いて各分画を確保した。その後、これを濃縮した後、GLP-1誘導能を確認した時、100kDa~300kDaの間の分画で高い水準のGLP-1が分泌されるのを確認した。また、効能分画(100K~300K、30K~100K)内タンパク質を除去するために、タンパク質分解酵素(proteinase K;PK)を100μg/ml濃度で55℃で1時間処理後、90℃で10分間非活性化後、GLP-1分泌を測定した。その結果、タンパク質が除去された分画ではGLP-1分泌が誘導されないのを確認し、これを通じて分画内タンパク質によってGLP-1が誘導されるのを確認した。アッカーマンシアムシニフィラ上清液の100kDa~300kDa(100K)を再び分画化するためにMonoQ陰イオン交換カラム(MomoQ 5/50、GE Healthcare)およびAKTAexplorer system(GE Healthcare)を用いてFPLC(fast protein liquid chromatography)を行った。80μg/mlの100K分画が注入され、1ml/minの速度でサンプルを分画化した。その後、各分画をL細胞に処理した後、GLP-1分泌程度を測定した。実験結果、m2-m4分画で高い含量のGLP-1が分泌されるのを確認した(図8A)。
アッカーマンシアムシニフィラ上清液から確保された、Sample1)100K concentrate、Sample2)MonoQ concentrate、Sample3)GPC concentrateをLC/MS-MSを通じて定性分析した。上清液の基本培地で発見できるbovine関連タンパク質は除き、各分画で確認されるタンパク質個数を確認した。
アッカーマンシアムシニフィラ濃縮分画に由来したタンパク質10種をクローニング後、E.coli BL21細胞で発現してタンパク質を純粋分離した。タンパク質10種のうちの1種(Beta-galactosidase)はクローニングで有効な発現ベクターを得られなくて以下の段階で除いた。その後、発現後分離したタンパク質9種をSDS-PAGEを通じて純粋分離されたのを確認した。Amuc1100はアッカーマンシアムシニフィラ由来抗肥満機能が知られたタンパク質であって(Plovier H. et al., A purified membrane protein from Akkermansia muciniphila or the pasteurized bacterium improves metabolism in obese and diabetic mice. Nat Med.(2017)23:107-113)陽性対照群として使用された。分離された各タンパク質をL細胞に処理してGLP-1分泌を確認した。
発掘されたターゲットタンパク質によって体内グルコース恒常性能力が向上されるか確認するために、P1(B2UKW8)、P5(B2UKW8)、P9(B2UM07)タンパク質を正常食餌マウスに腹腔で100μg/mouse濃度で一週間投与後、耐糖性テストを行った。
発掘されたターゲットタンパク質によって体内グルコース恒常性能力が向上されるか確認するために、P9(B2UM07)タンパク質を高脂肪食餌マウスに口腔で100μg/mouse濃度で8週間投与後、耐糖性テストを行った。グルコースを口腔投与(2g/kg)後、15分~120分間血糖を測定した。
Claims (22)
- 受託番号KCTC 13530BPで受託されたアッカーマンシアムシニフィラ(Akkermansia muciniphila)SNUG-61027菌株。
- 前記菌株は、配列番号1の塩基配列からなる16S rDNAを含むことを特徴とする、請求項1に記載のアッカーマンシアムシニフィラSNUG-61027菌株。
- 請求項1のアッカーマンシアムシニフィラSNUG-61027菌株、またはその培養液、上澄液、抽出物または分画物を有効性分として含む、食欲抑制または代謝性疾患の予防、改善または治療用薬学的組成物。
- 前記代謝性疾患は、耐糖能障害、糖尿、動脈硬化、高脂血症、高コレステロール症、脂肪肝、心血管疾患または肥満であることを特徴とする、請求項3に記載の食欲抑制または代謝性疾患の予防、改善または治療用薬学的組成物。
- 前記アッカーマンシアムシニフィラSNUG-61027菌株、またはその培養液、上澄液、抽出物または分画物は、IL-6増加、GLP-1分泌増加、および褐色脂肪の活性増加のうちのいずれか一つ以上を誘導することを特徴とする、請求項3に記載の食欲抑制または代謝性疾患の予防、改善または治療用薬学的組成物。
- 配列番号2のアミノ酸配列からなるB2UM07タンパク質を有効性分として含む、食欲抑制または代謝性疾患の予防、改善または治療用薬学的組成物。
- 前記B2UM07タンパク質は、アッカーマンシアムシニフィラ菌株に由来したことを特徴とする、請求項6に記載の食欲抑制または代謝性疾患の予防、改善または治療用薬学的組成物。
- 前記アッカーマンシアムシニフィラ菌株は、受託番号KCTC 13530BPで受託されたSNUG-61027菌株であることを特徴とする、請求項7に記載の食欲抑制または代謝性疾患の予防、改善または治療用薬学的組成物。
- 前記代謝性疾患は、耐糖能障害、糖尿、動脈硬化、高脂血症、高コレステロール症、脂肪肝、心血管疾患または肥満であることを特徴とする、請求項6に記載の食欲抑制または代謝性疾患の予防、改善または治療用薬学的組成物。
- 請求項1のアッカーマンシアムシニフィラSNUG-61027菌株、またはその培養液、上澄液、抽出物または分画物を有効性分として含む、食欲抑制または代謝性疾患の改善または緩和用健康機能性食品。
- 前記代謝性疾患は、耐糖能障害、糖尿、動脈硬化、高脂血症、高コレステロール症、脂肪肝、心血管疾患または肥満であることを特徴とする、請求項10に記載の食欲抑制または代謝性疾患の改善または緩和用健康機能性食品。
- 前記アッカーマンシアムシニフィラSNUG-61027菌株、またはその培養液、上澄液、抽出物または分画物は、IL-6増加、GLP-1分泌増加、および褐色脂肪の活性増加のうちのいずれか一つ以上を誘導することを特徴とする、請求項10に記載の食欲抑制または代謝性疾患の改善または緩和用健康機能性食品。
- 配列番号2のアミノ酸配列からなるB2UM07タンパク質を有効性分として含む、食欲抑制または代謝性疾患の改善または緩和用健康機能性食品。
- 前記B2UM07タンパク質は、アッカーマンシアムシニフィラ菌株に由来したことを特徴とする、請求項13に記載の食欲抑制または代謝性疾患の改善または緩和用健康機能性食品。
- 前記アッカーマンシアムシニフィラ菌株は、受託番号KCTC 13530BPで受託されたSNUG-61027菌株であることを特徴とする、請求項14に記載の食欲抑制または代謝性疾患の改善または緩和用健康機能性食品。
- 前記代謝性疾患は、耐糖能障害、糖尿、動脈硬化、高脂血症、高コレステロール症、脂肪肝、心血管疾患または肥満であることを特徴とする、請求項13に記載の食欲抑制または代謝性疾患の改善または緩和用健康機能性食品。
- 受託番号KCTC 13530BPで受託されたアッカーマンシアムシニフィラ(Akkermansia muciniphila)SNUG-61027菌株、またはその培養液、上澄液、抽出物または分画物の食欲抑制または代謝性疾患の予防、治療または改善用薬学的組成物の製造のための使用。
- 前記アッカーマンシアムシニフィラ(Akkermansia muciniphila)SNUG-61027菌株は、配列番号1の塩基配列からなる16S rDNAを含むことを特徴とする、請求項17に記載の食欲抑制または代謝性疾患の予防、治療または改善用薬学的組成物の製造のための使用。
- 前記代謝性疾患は、耐糖能障害、糖尿、動脈硬化、高脂血症、高コレステロール症、脂肪肝、心血管疾患または肥満であることを特徴とする、請求項17に記載の食欲抑制または代謝性疾患の予防、治療または改善用薬学的組成物の製造のための使用。
- 配列番号2のアミノ酸配列からなるB2UM07タンパク質の食欲抑制または代謝性疾患の予防、治療または改善用薬学的組成物の製造のための使用。
- 前記B2UM07タンパク質は、アッカーマンシアムシニフィラ菌株に由来したことを特徴とする、請求項20に記載の食欲抑制または代謝性疾患の予防、治療または改善用薬学的組成物の製造のための使用。
- 前記アッカーマンシアムシニフィラ菌株は、受託番号KCTC 13530BPで受託されたSNUG-61027菌株であることを特徴とする、請求項21に記載の食欲抑制または代謝性疾患の予防、治療または改善用薬学的組成物の製造のための使用。
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AU2019356410B2 (en) | 2023-07-06 |
KR102197180B1 (ko) | 2020-12-31 |
RU2021135116A3 (ja) | 2022-04-29 |
AU2019356410A1 (en) | 2021-06-03 |
MX2021004056A (es) | 2021-06-04 |
EP3865568A4 (en) | 2022-08-17 |
CA3115196A1 (en) | 2020-04-16 |
RU2021135116A (ru) | 2021-12-06 |
BR112021006855A2 (pt) | 2021-08-10 |
CN113330109A (zh) | 2021-08-31 |
JP2022502067A (ja) | 2022-01-11 |
EP3865568A2 (en) | 2021-08-18 |
KR20200041280A (ko) | 2020-04-21 |
US20220002665A1 (en) | 2022-01-06 |
CN113330109B (zh) | 2024-02-09 |
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