JP2022180395A - プロモーターポリヌクレオチド、シグナルポリペプチド、及びその用途 - Google Patents
プロモーターポリヌクレオチド、シグナルポリペプチド、及びその用途 Download PDFInfo
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Abstract
Description
ラクトバチルス属(Lactobacillus sp.)またはストレプトコッカス属(Streptococcus sp.)のようなバクテリアは、伝達ビークルとして有用である。さらに、一般的に安全であると見られるGRAS(generally recognized as safe)微生物は、ヒトまたは動物にも投与される。
他の様相は、前記プロモーターを含む組み換えポリヌクレオチドを提供する。
他の様相は、前記組み換えポリヌクレオチドを含む宿主細胞を提供する。
他の様相は、前記宿主細胞を使用して産物を生産する方法を提供する。
他の様相は、単離されたシグナルポリペプチド、及びそれをコーディングするポリヌクレオチドを提供する。
他の様相は、単離されたシグナルポリペプチドをコーディングするポリヌクレオチドを含む組み換えポリヌクレオチドを提供する。
他の様相は、単離されたシグナルポリペプチドをコーディングする組み換えポリヌクレオチドを含む宿主細胞を提供する。
他の様相は、単離されたシグナルポリペプチドをコーディングする組み換えポリヌクレオチドを含む宿主細胞を使用し、タンパク質を生産する方法を提供する。
他の様相は、前記組み換えポリヌクレオチドを含む宿主細胞を提供する。前記宿主細胞は、バクテリア細胞でもある。前記バクテリア細胞は、グラム陽性バクテリアでもある。前記バクテリア細胞は、乳酸菌またはエスケリキア属に属するものでもある。前記乳酸菌は、ラクトバチルス(Lactobacillus)属、ラクトコッカス(Lactococcus)属、ビフィドバクテリア(bifidobacteria)属、ストレプトコッカス(Streptococcus)属、ロイコノストック(Leuconostoc)属、ワイセラ(Weissella)属、ペディオコッカス(Pediococcus)属またはエンテロコッカス(Enterococcus)属の中に属するものでもある。
他の様相による単離されたシグナルポリペプチドをコーディングするポリヌクレオチドを含む組み換えポリヌクレオチドを含む宿主細胞は、外来遺伝子の産物を細胞外に効率的に分泌させることができる。
実施例1:プロモーター及びシグナルポリペプチドのクローニング、及びその効果の確認
1.プロモーター及びシグナルポリペプチドのクローニング
プロモーターと、シグナルポリペプチドをコーディングするヌクレオチド配列は、PCRによって増幅した。具体的に、Lactobacillus paracasei LMT1-21(受託番号KCTC 13422BP)のゲノムをテンプレートにし、プライマーを使用したPCRにおいて、593kbサイズの増幅産物を得た。使用したプライマーは、PS4_F/R(配列番号4及び5)である。
(1)実験群ベクター作製:pMT54-PR4-IL10-SP4ベクター
IL-10遺伝子(配列番号12)は、外部に依頼して合成した(Macrogen Inc.、韓国)。合成された前記遺伝子断片と、前記pMT54-PR4-SP4ベクターとに、制限酵素SalI及びXhoIを処理し、前記ベクターのクローニング部位を切断した。切断された産物を、Gel purification kit(Bioneer社)を使用して精製した後、アルカリホスファターゼを使用し、脱リン酸化させた。そのように準備した前記ベクターDNA 1μl、前記遺伝子(IL-10)3μl、T4DNAリガーゼ(Takara社)0.5μl及び緩衝溶液1μlの混合物に、蒸溜水5.5μlを添加し、総10μl反応混合物を作った。前記反応混合物を、16℃で12時間インキュベーションし、前記遺伝子を、前記ベクターのクローニング部位に連結した。得られた連結産物を、前述のところと同一方法で、大腸菌Top10菌株に形質転換させ、その配列を確認した。その結果、前記遺伝子が導入されたことを確認し、それらを、pMT54-PR4-IL10-SP4ベクターと命名した。図2は、pMT54-PR4-IL10-SP4ベクターの構造を示した図面である。図2において、promoter、signal peptide及びtarget geneは、PR4、SP4及びIL-10をそれぞれ示す。前記ベクターは、大腸菌と乳酸菌とのシャトルベクターであり、大腸菌複製原点(origin)、乳酸菌複製原点(rep gene)及びクロラムフェニコール耐性遺伝子を含む。多重クローニング部位に、プロモーター、シグナルペプチド、標的遺伝子、HAタグ及びHisタグが連結されている。
シグナルポリペプチドをコーディングするポリヌクレオチドSP4を、USP45ポリヌクレオチドに置き換えたことを除き、実験群ベクターと同様の方式でベクターを作製した。それは、同一プロモーターにおいて、他のシグナルポリペプチドがIL-10タンパク質の細胞外分泌に及ぼず影響を確認するためのものである。
プロモーターPR4をP11プロモーターに置き換えたことを除き、実験群ベクターと同様の方式でベクターを作製した。それは、シグナルポリペプチドにおいて、他のプロモーターがIL-10タンパク質の発現に及ぼす影響を確認するためのものである。P11は、ラクトバチルス・プランタルム(Lactobacillus plantarum)において、強力な転写開始活性を有する合成プロモーターである(Lars Axelsson, Microbiology (2006), 152, 1011-019)。
(1)pMT54-PR4-IL10-SP4ベクターのIL-10タンパク質発現の確認
pMT54-PR4-IL10-SP4ベクターと、pMT54-P11-IL10-SP4ベクターとをそれぞれ3種乳酸菌に形質転換させた。3種乳酸菌は、ラクトバチルス・パラカセイ(Lactobacillus paracasei)KCTC 13422BP、ラクトバチルス・プランタルム(Lactobacillus plantarum)KCTC 13421BP及びラクトバチルス・ブレビス(Lactobacillus brevis)KCTC 13423BPとして、いずれもキムチから単離したものである。それら菌株は、それぞれLMT1-21、LMT1-9及びLMT1-46とも言う。
pMT54-PR4-IL10-SP4ベクターと、pMT54-P11-IL10-SP4ベクターとを、(1)と同様の方法により、ラクトバチルス・パラカセイ(Lactobacillus paracasei)KCTC 13422BP(LMT1-21)乳酸菌に形質転換した。
前記pMT54-PR4-IL10-SP4ベクター及びpMT54-PR4-IL10-USP45ベクターを、(1)と同様の方法により、ラクトバチルス・パラカセイ(Lactobacillus paracasei)KCTC 13422BP(LMT1-21)乳酸菌に形質転換した。
実施例1の(2)及び(3)において、IL-10遺伝子の代わりに、アルファ-アミラーゼ遺伝子(配列番号19)及びプライマーF/R(配列番号22及び23)を使用したことを除き、同様の過程を経て、pMT54-PR4-amylase-SP4ベクターを製造し、それを、乳酸菌L.Paracasei LMT1-21に形質転換させ、アルファ-アミラーゼが細胞外に発現される程度を確認した。アルファ-アミラーゼ遺伝子増幅は、 ラクトバチルス・アミロボルス(Lactobacillus amylovorus)(KCTC 3597)のゲノムDNAを使用した。
Claims (17)
- 配列番号1のヌクレオチド配列と85%以上の配列同一性を有するポリヌクレオチドを含む単離されたプロモーター。
- 請求項1に記載のプロモーターを含む組み換えポリヌクレオチド。
- 前記組み換えポリヌクレオチドは、クローニングベクターまたは発現ベクターである、請求項2に記載の組み換えポリヌクレオチド。
- 前記発現ベクターは、前記プロモーターと作動自在に連結されている第1ポリヌクレオチドを含み、第1ポリヌクレオチドは、産物をコーディングする,請求項3に記載の組み換えポリヌクレオチド。
- 前記産物は、ポリペプチドである、請求項4に記載の組み換えポリヌクレオチド。
- シグナルポリペプチドをコーディングする第2ポリヌクレオチドが、前記プロモーターと第1ポリヌクレオチドとの間に作動自在に連結されている、請求項2ないし5のうちいずれか1項に記載の組み換えポリヌクレオチド。
- 請求項2ないし6のうちいずれか1項に記載の組み換えポリヌクレオチドを含む宿主細胞。
- 前記宿主細胞は、乳酸菌またはエスケリキア属に属する、請求項7に記載の宿主細胞。
- 前記乳酸菌は、ラクトバチルス属、ラクトコッカス属、ビフィドバクテリア属、ストレプトコッカス属、ロイコノストック属、ワイセラ属、ペディオコッカス属またはエンテロコッカス属に属する、請求項8に記載の宿主細胞。
- 請求項7に記載の宿主細胞を培地中で培養して産物を生成する段階と、
前記産物またはその代謝産物を培養物から単離する段階と、を含む産物またはその代謝産物を生産する方法。 - 配列番号3のアミノ酸配列と85%以上の配列同一性を有するアミノ酸配列を含む単離されたシグナルポリペプチド。
- 請求項11に記載のシグナルポリペプチドをコーディングするポリヌクレオチド。
- プロモーター、請求項11に記載のシグナルポリペプチドをコーディングする第2ポリヌクレオチド、及びタンパク質をコーディングする第1ポリヌクレオチドを含み、前記第2ポリヌクレオチドは前記プロモーターと作動自在に連結されており、前記第1ポリヌクレオチドは前記第2ポリヌクレオチドとインフレームで融合されているベクター。
- 請求項13に記載のベクターを含む宿主細胞。
- 前記宿主細胞は、乳酸菌またはエスケリキア属に属する、請求項14に記載の宿主細胞。
- 前記乳酸菌は、ラクトバチルス属、ラクトコッカス属、ビフィドバクテリア属、ストレプトコッカス属、ロイコノストック属、ワイセラ属、ペディオコッカス属またはエンテロコッカス属に属する、請求項15に記載の宿主細胞。
- 請求項14ないし16のうちいずれか1項に記載の宿主細胞を培地中で培養してタンパク質を生成する段階と、
前記タンパク質を培養物から単離する段階と、を含むタンパク質を生産する方法。
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KR102238519B1 (ko) * | 2018-12-28 | 2021-04-13 | (주)메디톡스 | 외래 단백질을 발현하는 미생물, 및 그의 용도 |
CN110643571B (zh) * | 2019-10-22 | 2021-07-27 | 康妍葆(北京)干细胞科技有限公司 | 人角蛋白6a在干细胞培养中的应用及产品 |
KR20210129516A (ko) * | 2020-04-20 | 2021-10-28 | 주식회사 리비옴 | 혈관 작동성 장 펩티드를 발현하는 미생물, 및 그의 용도 |
WO2022065643A1 (ko) * | 2020-09-28 | 2022-03-31 | 한국화학연구원 | 분비 신호서열에 융합된 표적산물 결합 단백질을 코딩하는 폴리뉴클레오티드를 포함하는 재조합 미생물, 그를 포함하는 조성물 및 그를 이용하여 표적산물을 생산하는 방법 |
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ES2568938T3 (es) * | 2009-04-28 | 2016-05-05 | Vanderbilt University | Composiciones y procedimientos de tratamiento de trastornos que implican apoptosis de células epiteliales |
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KR101992345B9 (ko) | 2023-09-07 |
EP3732292A4 (en) | 2021-09-22 |
US11661445B2 (en) | 2023-05-30 |
JP7189218B2 (ja) | 2022-12-13 |
JP2022180396A (ja) | 2022-12-06 |
JP2021508464A (ja) | 2021-03-11 |
KR101992345B1 (ko) | 2019-09-27 |
CN111601895B (zh) | 2024-05-10 |
WO2019132231A1 (en) | 2019-07-04 |
US20230295249A1 (en) | 2023-09-21 |
US20200339637A1 (en) | 2020-10-29 |
EP3732292A1 (en) | 2020-11-04 |
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