JP2016144453A - 免疫調節作用を有する乳酸菌のスクリーニング方法 - Google Patents
免疫調節作用を有する乳酸菌のスクリーニング方法 Download PDFInfo
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- JP2016144453A JP2016144453A JP2016023520A JP2016023520A JP2016144453A JP 2016144453 A JP2016144453 A JP 2016144453A JP 2016023520 A JP2016023520 A JP 2016023520A JP 2016023520 A JP2016023520 A JP 2016023520A JP 2016144453 A JP2016144453 A JP 2016144453A
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- lactic acid
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- umod
- lactobacillus
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Abstract
【解決手段】被検乳酸菌のウロモジュリン(Umod)蛋白質への結合数を測定することを含む、免疫調節作用を有する乳酸菌のスクリーニング方法。
【選択図】なし
Description
[1] 被検乳酸菌のウロモジュリン(Umod)蛋白質への結合数を測定することを含む、免疫調節作用を有する乳酸菌のスクリーニング方法。
[2] 被検乳酸菌のウロモジュリン(Umod) 蛋白質への結合数が、Lactobacillus acidophilus L-92株のウロモジュリン(Umod) 蛋白質への結合数と同等もしくはそれよりも多い場合に、該被検乳酸菌を、免疫調節作用を有する乳酸菌として選択する、[1]に記載のスクリーニング方法。
[3] 下記の工程を含む、免疫調節作用を有する乳酸菌のスクリーニング方法:
(a) 被検乳酸菌とウロモジュリン(Umod) 蛋白質とを接触させる工程、
(b) 上記被検乳酸菌のウロモジュリン(Umod) 蛋白質への結合数を測定する工程、
(c) (b)で測定した結合数を、Lactobacillus acidophilus L-92株のウロモジュリン(Umod) 蛋白質への結合数(対照結合数)と比較する工程、および
(d) (c)の結果に基づいて、被検乳酸菌の中から、対照結合数と同等もしくはそれよりも多い結合数を示す乳酸菌を、免疫調節作用を有する乳酸菌として選択する工程。
[4] 前記結合数を、ウロモジュリン(Umod) 蛋白質に結合した乳酸菌の遺伝子量により測定する、[1]〜[3]のいずれかに記載の方法。
[6] 下記の工程を含む、免疫調節作用を有する乳酸菌の製造方法:
(a) 被検乳酸菌とウロモジュリン(Umod) 蛋白質とを接触させる工程、
(b) 上記被検乳酸菌のウロモジュリン(Umod) 蛋白質への結合数を測定する工程、
(c) (b)で測定した結合数を、Lactobacillus acidophilus L-92株のウロモジュリン(Umod) 蛋白質への結合数(対照結合数)と比較する工程、および
(d) (c)の結果に基づいて、被検乳酸菌の中から、対照結合数と同等もしくはそれよりも多い結合数を示す乳酸菌を、免疫調節作用を有する乳酸菌として取得する工程。
[7] ウロモジュリン(Umod) 蛋白質への結合数が、Lactobacillus acidophilus L-92株のウロモジュリン(Umod) 蛋白質への結合数と同等もしくはそれよりも多く、かつ、免疫調節作用を有する乳酸菌。
[8] [6]に記載の方法により得られる免疫調節作用を有する乳酸菌。
[9] 受託番号NITE BP-1512で特定されるLactobacillus fermentum CP1299株、受託番号NITE BP-1513で特定されるLactobacillus acidophilus CP1613株、受託番号NITE BP-1514で特定されるLactobacillus fermentum CP1753株、又はそれらの類似菌株もしくは変異株である、[7]または[8]のいずれかに記載の乳酸菌。
[11] 前記免疫調節用組成物が医薬である、[10]に記載の免疫調節用組成物。
[12] 前記免疫調節用組成物が飲食品である、[10]に記載の免疫調節用組成物。
[13] ウロモジュリン(Umod)蛋白質への結合数が、Lactobacillus acidophilus L-92株のウロモジュリン(Umod) 蛋白質への結合数と同等もしくはそれよりも多い乳酸菌の、免疫調節用組成物の製造への使用。
[14] ウロモジュリン(Umod)蛋白質を含む、免疫調節作用を有する乳酸菌のスクリーニング用キット。
(a) 被検乳酸菌とウロモジュリン(Umod) 蛋白質とを接触させる工程
(b) 上記被検乳酸菌のウロモジュリン(Umod) 蛋白質への結合数を測定する工程
(c) (b)で測定した結合数を、Lactobacillus acidophilus L-92株のウロモジュリン(Umod) 蛋白質への結合数(対照結合数)と比較する工程
(d) (c)の結果に基づいて、被検乳酸菌の中から、対照結合数と同等もしくはそれよりも多い結合数を示す乳酸菌を、免疫調節作用を有する乳酸菌として選択する工程
Lactobacillus fermentum CP1299株:受託番号NITE BP-1512(識別の表示:CP1299)
Lactobacillus acidophilus CP1613株:受託番号NITE BP-1513(識別の表示:CP1613) Lactobacillus fermentum CP1753株:受託番号NITE BP-1514(識別の表示:CP1753)
(1)被検乳酸菌の調製
実験には、ラクトバチルス属に属する乳酸菌約20株を用いた。また、免疫調節作用を有する基準株としてLactobacillus acidophilus L-92株を用いた。各菌株をMRS培地(Difco)にて37℃、20時間静置培養した後、PBSにて3回洗菌し、PBSに懸濁した。
ヒトIgG1のFcドメインにマウスUmod蛋白質(配列番号5の1−616位)をつなげて発現させた融合蛋白質(Fc-mUmod)を、Hase K. et al., Uptake through glycoprotein 2 of FimH1 bacteria by M cells initiates mucosal immune response, Nature 2009, 462:226-31の記載に従って作製した。mUmod(マウスUmod)配列(配列番号4)を増幅させるためのプライマーとしてForwardプライマー:5’-CGCAGATCTACCATGGGGATCCCTTTGACC-3’(配列番号6)およびReverse プライマー:5’-CGCGTCGACCTTGGACACTGAGGCCTGG-3’(配列番号7)を用い、制限酵素BglIIとSalIを用いてFcドメインを挿入したpcDNA3ベクター(invitrogen)にクローニングした。
Fc-Umod蛋白質およびコントロールFc蛋白質としてhIgGを、5μg/mlになるようにPBSにて希釈したものを96穴プレートに50μlアプライし、4℃にて一晩固相化した。各ウェルを200μlのPBSにて3回洗浄した後、1% BSA/PBS溶液を200μlアプライして室温にて2時間ブロッキングした。ブロッキング溶液を除去した後、106 cells/50μl となるようにPBSにて懸濁した被検乳酸菌の菌体を、50μlずつアプライし、室温にて2時間インキュベートした。各ウェルを200μlのPBSにて5回洗浄した後、PBSを完全に除去した。
基準株を含む被検乳酸菌14株のUmod結合数の算出結果を表1に示す。
Lactobacillus fermentum CP1753株、Lactobacillus fermentum CP1299株、
Lactobacillus johnsonii CP1544株、Lactobacillus helveticus CP2151株、
Lactobacillus delbrueckii subsp. bulgaricus CP2189株、Lactobacillus delbrueckii subsp. bulgaricus CP973株、Lactobacillus acidophilus CP1613株、Lactobacillus brevis CP287
Lactobacillus acidophilus CP734、actobacillus acidophilus CP23、Lactobacillus casei CP2517、Lactobacillus gasseri CP793、Lactobacillus rhamnosus CP1270
(1)被検乳酸菌の調製
実験には、実施例1においてUmod結合数を調べたラクトバチルス属に属する乳酸菌株を用いた。また、抗アレルギー作用を有する基準株としてLactobacillus acidophilus L-92株を用いた。各菌株をMRS培地(Difco)にて37℃、20時間静置培養した後、生理食塩水(0.85%(w/v) NaCl溶液)にて3回洗菌した。生理食塩水に懸濁した後、85℃達温殺菌を行い、菌体は凍結乾燥し、粉末化した。
6週齢の雌性Balb/cマウス(日本チャールス・リバー株式会社)を各群5匹ずつ実験に用いた。実験開始日および3日後に、卵白アルブミン(OVA)をアジュバントである水酸化アルミニウムゲルと共に2回腹腔内投与し、アレルギーモデルマウスを作製した。実験3日後から毎日、約109個/mL水道水に調整した各被検乳酸菌溶液をマウス体重10g当たり0.1mL強制経口投与した。コントロールマウスには水道水を用いた。14日後に腹部下大静脈より採血し、失血死後に腸管膜リンパ節を摘出し、RNAlater(Applied Biosystems社)で一晩冷蔵保存した。
実施例1で測定したUmod結合数を縦軸に、上記(2)で測定したOVA-IgE低下率を横軸にプロットした結果を図2に示す。その結果、Umod結合数とOVA-IgEの間には相関があり、Umod結合数がLactobacillus L-92株と同等またはそれよりも多い乳酸菌株(CP1753, CP1299, CP1613, CP1544, CP287, CP2151, CP2189)はOVA-IgE低下率が大きく、抗アレルギー効果が高いことが示された。
(1)被検乳酸菌の調製
実施例2(1)と同様にして被検乳酸菌の調製を行なった。
実施例2(2)で作製したアレルギーモデルマウスより摘出した腸間膜リンパ節におけるサイトカイン(IL-4, IL-10, IL-12)の発現量を測定した。まず、各個体の腸間膜リンパ節を2-メルカプトエタノールを添加したRNeasy Mini Kit(QIAGEN社)付属のRLT溶液に入れ、ハサミで細切後、ホモジナイザー(ポリトロン社)でホモジナイズした。以降は、RNeasy Mini Kitの説明書に従って全RNAを抽出し、得られた全RNAの濃度及び純度を、Agilent 2100 Bioanalyser(アジレントテクノロジー社)で測定した。
(IL-4増幅用プライマー)
IL-4_F:5’-CCCCAGCTAGTTGTCATCCTG-3’(配列番号10)
IL-4_R:5’-CGCATCCGTGGATATGGCTC-3’ (配列番号11)
(IL-10増幅用プライマー)
IL-10_F:5’-ACAGCCGGGAAGACAATAACT-3’ (配列番号12)
IL-10_R:5’-GCAGCTCTAGGAGCATGTGG-3’ (配列番号13)
(IL-12増幅用プライマー)
IL-12_F:5’-CAATCACGCTACCTCCTCTTTT-3’ (配列番号14)
IL-12_R:5’-CAGCAGTGCAGGAATAATGTTTC-3’ (配列番号15)
(GAPDH増幅用プライマー)
GAPDH_F:5’-AGGTCGGTGTGAACGGATTTG-3’ (配列番号16)
GAPDH_R:5’-GGGGTCGTTGATGGCAACA-3’ (配列番号17)
実施例1で測定したUmod結合数を縦軸に、上記(2)で測定したサイトカイン(IL-4, IL-10, IL-12/IL-4)発現量を横軸にプロットした結果を図3〜5に示す。その結果、Umod結合数とサイトカイン発現量の間には相関があり、Umod結合数がLactobacillus acidophilus L-92株と同等またはそれよりも多い乳酸菌株(CP1753, CP1299, CP1613)は、Th2サイトカイン(IL-4、IL-10)の発現量が低く(図3、4)、Th1サイトカイン(IL-12)の発現量が高かった(図5)。従って、Umod結合数がLactobacillus acidophilus L-92株と同等またはそれよりも多い上記乳酸菌株は、Th1/Th2バランスをTh1優位に調節することから、抗アレルギー効果が高いことが示された。
(1) 腸間膜リンパ節のサイトカイン(TGF-β)発現量測定
実施例2(2)で作製したアレルギーモデルマウスより摘出した腸間膜リンパ節におけるサイトカイン(TGF-β)の発現量を測定した。TGF-β発現量の測定は、実施例3(2)と同様にしてRNAを調製し、PCR増幅することにより行った。TGF-β遺伝子増幅に用いたプライマーの塩基配列を以下に示す。
(TGF-β増幅用プライマー)
TGF-β_F:5’-AGCTGGTGAAACGGAAGCG-3’ (配列番号18)
TGF-β_R:5’-GCGAGCCTTAGTTTGGACAGG-3’ (配列番号19)
実施例1で測定したUmod結合数を縦軸に、上記(1)で測定したTGF-β発現量を横軸にプロットした結果を図6に示す。その結果、Umod結合数とTGF-β発現量の間には相関があり、特にUmod結合数が多い乳酸菌株(CP1753, CP1299, CP1613)は、TGF-βの発現量が高かった(図6)。従って、Umod結合数が多い上記乳酸菌株は、アレルギー性炎症抑制に関与する因子であるTGF-βの産生を促進することから、抗アレルギー効果が高いことが示された。
Claims (6)
- 下記の(i)および(ii)の特徴を有する乳酸菌(ただし、Lactobacillus acidophilus L-92株を除く)。
(i) ラクトバチルス・ファーメンタム、ラクトバチルス・アシドフィルス、ラクトバチルス・ジョンソニー、ラクトバチルス・ヘルベティカス、またはラクトバチルス・デルブリュッキイに属すること;
(ii) 5μg/mlになるようにPBSで希釈したFc-mUmod蛋白質又はコントロールFc蛋白質(hIgG)を、96穴プレートに50μl添加して固相化し、さらに1%BSA/PBS溶液によってブロッキングした後に、106cells/50μlになるようにPBSに懸濁した乳酸菌の菌体50μlを接触させてインキュベートするin vitro結合アッセイにおいて、Fc-mUmod蛋白質に結合した菌数からコントロールFc蛋白質(hIgG)に結合した菌数を引いたUmod結合数(log10)が4.0以上であること。 - 受託番号NITE BP-1512で特定されるLactobacillus fermentum CP1299株もしくは配列番号1に示す塩基配列と98%以上の相同性を有する塩基配列からなる16s rDNAを有し、かつLactobacillus acidophilus L-92株のウロモジュリン(Umod)蛋白質への結合数と同等もしくはそれよりも多いUmod結合数を示す、Lactobacillus fermentum CP1299株の類似菌株、受託番号NITE BP-1513で特定されるLactobacillus acidophilus CP1613株もしくは配列番号2に示す塩基配列と98%以上の相同性を有する塩基配列からなる16s rDNAを有し、かつLactobacillus acidophilus L-92株のウロモジュリン(Umod)蛋白質への結合数と同等もしくはそれよりも多いUmod結合数を示す、Lactobacillus acidophilus CP1613株の類似菌株、または受託番号NITE BP-1514で特定されるLactobacillus fermentum CP1753株もしくは配列番号3に示す塩基配列と98%以上の相同性を有する塩基配列からなる16s rDNAを有し、かつLactobacillus acidophilus L-92株のウロモジュリン(Umod)蛋白質への結合数と同等もしくはそれよりも多いUmod結合数を示す、Lactobacillus fermentum CP1753株の類似菌株。
- 請求項1または2に記載の乳酸菌(ただし、Lactobacillus acidophilus L-92株を除く)を有効成分として含む免疫調節用組成物。
- 前記免疫調節用組成物が医薬である、請求項3に記載の免疫調節用組成物。
- 前記免疫調節用組成物が飲食品である、請求項3に記載の免疫調節用組成物。
- 請求項1または2に記載の乳酸菌(ただし、Lactobacillus acidophilus L-92株を除く)の、免疫調節用組成物の製造への使用。
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