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遺伝子組換え抗原を用いる牛ヨーネ病の免疫学的診断法に関する研究

永田, 礼子 東京大学 DOI:10.15083/0002006098

2023.03.20

概要





















永田

礼子

ヨーネ病はヨーネ菌(Mycobacterium avium subsp. Paratuberculosis)
により起こるウシなどの反芻動物の慢性消化器感染症である。我が国では,
診断・淘汰による防疫対策を実施しており,2013 年からリアルタイム PCR
による糞便遺伝子検査でより迅速な診断が可能となった。そのため,諸外国
に比べると感染率は極めて低いが,現在でも年間数百頭の発生がある。その
診断法として主に用いられている培養検査や遺伝子検査では,発症前の潜伏
期間における感染牛の摘発は不可能である。一方,ヨーニン皮内反応検査,
インターフェロン・ガンマ(IFN-γ)検査や抗体検査等の免疫学的診断法は
,早期診断や多検体処理が可能であるが,非特異的反応や感度が問題視され
ており,未だに実用化されていない。本研究は,ヨーネ菌抗原遺伝子の解析
と,遺伝子組換え抗原を用いた感染早期に特異性の高い免疫学的診断法を開
発するための基礎的研究を行ったものである。
第1章は,ヨーネ菌遺伝子発現ライブラリーからIFN-γ 誘導能を有する2
つのヨーネ菌抗原遺伝子を特定したものである。これらはいずれも結核菌で知
られている proline-proline-glutamic acid(PPE)family protein に属するタ
ンパク質をコードしており,その組換えタンパク質 Map39(分子量 39,000)
及び Map41(分子量 41,000)を作製している。さらに,Map39 及び Map41
に対するモノクローナル抗体も作製し,ヨーネ菌でのこれらの発現とヨーネ
菌実験感染牛由来末梢血単核球(PBMC)における特異的な IFN-γ 産生を確
認している。この成果は,ヨーネ菌の PPE family proteinが,IFN-γ 誘導能
を有し,さらに,これらがM. avium 種にのみ共通して存在することを初め
て示した価値ある結果である。特に,そのIFN-γ検出は,従来法に比べて特
異性の高い検査法として有用であることも明らかにしており,基礎的研究が

実用化につながることを示したものと評価した。
第章は, Map41がインターロイキン10 (IL-10)を特異的に産生誘導する
ことを示したものである。Map41抗原刺激によるPBMCにおけるIL-10発現誘
導は,MAPKp38経路によるものであり,既報のヨーネ菌感染マクロファージに
おけるIL-10 mRNA発現の細胞内伝達と同様なことを示し,人工的に作製し
た抗原によるサイトカインの産生が自然感染時と同様な経路で起こることを
明らかにしている。さらに,動物実験により,Map41を用いたIL-10検査は
,早期かつ高い特異性で感染を検出できることも明らにしており,学問的だ
けでなく実用的にも価値ある発見と評価した。
第章では,液性免疫を誘導する酵素 Enoyl-CoA hydratase (EchA12_2)
をコードする echA12 遺伝子を発見したものである。その遺伝子組換え抗原
Map-echA を用いたELISA法による抗体検査は,抗酸菌共通抗原を多く含む
現行の ELISA と比較して,感度,特異度共に高く,特に非特異反応が疑わ
れる血清では反応性が低く,排菌量の少ない罹患牛の血清において高い陽性
率を示した。さらに,従来法より 2~7 カ月も早期に抗体上昇を検出してい
る。また,鳥型結核菌にもEchA12_2 と相同なタンパク質が存在するが,in

vivo での遺伝子発現調節やタンパク質の機能がM. avium亜種間で異なる可
能性も見出している。
本研究では,ヨーネ菌感染早期の細胞性免疫応答の指標となるIFN-γ お
よびIL-10産生を誘導するMap41抗原と,早期かつ特異的に抗体応答を誘導す
るMap-echA抗原を同定するという重要な知見を得ている。これらの抗原は
,鳥型結核菌にも非常に高い相同性を示す遺伝子が存在するが,近縁菌種間
で保存された遺伝子でも,宿主体内における発現とその遺伝子産物の免疫原
性は異なるというこれまでにない概念を示しており,今後,病原性や発病機
構を解析する上でも重要な知見となるだけでなく,今後この分野の研究の発
展に大きく寄与するものと思われる。さらに,本研究で発見したMap41およ
びMap-echAを用いる検査は,特異性が高く感染早期にヨーネ病感染個体を
診断する有用な方法にも応用可能な科学的知見と評価した。
このように,これらの研究成果は、学術上応用上寄与するところが少な
くない。よって、審査委員一同は本論文が博士(獣医学)の学位論文として
価値あるものと認めた。

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