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Studies on the Diagnosis of Schistosoma japonicum Infection Utilizing Real-time PCR and ELISA

DANG TRINH MINH ANH 岐阜大学

2020.03.13

概要

Schistosoma japonicum（日本住血吸虫）の感染による日本住血吸虫症は，中国，フィリピン及びインドネシアの一部において，依然として公衆衛生上の問題となっている人獣共通寄生虫病である。患者は，虫卵が肝臓や脾臓に蓄積することで，肝脾腫や肝硬変といった慢性症状を呈し，重症化すると死に至る。ワクチンによる予防ができないことから，プラジカンテル単剤による集団投薬が主要な予防対策となっている。しかしながら，この薬剤は患者体内に寄生する幼若虫を殺滅することができず，再感染を予防することもできない。また，化学療法剤による感染症対策は一般に薬剤耐性株出現との競争であり，更に効果的な創薬が待たれている。日本住血吸虫症では，病理組織標本を観察して虫卵の蓄積を評価する古典的は手法が依然として用いられていて，その煩雑さや時間を要することが問題になっている。一方，集団投薬の効果は，有病率の変化を指標に評価できるが，日本住血吸虫症ではこの有病率の調査に，Kato-Katz 法と呼ばれる古典的な検便による虫卵検査法が用いられ，その感度の低さが問題になっている。そこで本研究では，日本住血吸虫症対策の更なる推進を図るため，マウス実験感染における各臓器の虫卵蓄積の簡便評価法及び高感度・特異的な診断法を開発した。

【第一章】日本住血吸虫症に実験感染したマウスにおいて，各種臓器での寄生虫卵の蓄積の程度を評価する定量リアルタイム PCR 法（qPCR）を開発した。この qPCR では，実験感染マウスの各種臓器由来の DNA を材料として， 寄生虫のミトコンドリア由来 NADH dehydrogenase I 遺伝子を標的とした Threshold Cycle（Ct 値）を算定し，虫卵蓄積の程度とした。この数値を，組織標本の単位面積当たりの虫卵数と比較した結果，重度の虫卵蓄積が観察される肝臓と中度の虫卵蓄積が観察される脾臓で，虫卵数と Ct 値との間に有意な相関が認められた。また，顕微鏡観察で虫卵を検出出来なかった脳においても，qPCRで寄生虫 DNA を検出した。これらの成績から，日本住血吸虫症の実験感染において，各種臓器の感染病理を評価する手法として qPCR が有用であることが示唆された。

【第二章】ペルオキシレドキシンは，宿主に由来する活性酸素種から寄生虫を保護する一群のチオレドキシンペルオキシダーゼとして知られていて，数種の寄生虫病の血清診断用抗原としても有用であることが報告されている。本章では，日本住血吸虫のペルオキシレドキシン-4（SjPrx-4）の組換え蛋白質を作製して，その性状を解析するとともに，血清診断用抗原としての性能を評価した。データベース上の遺伝子配列から推定された SjPrx-4 のアミノ酸配列は，酵素抗体法（ELISA）抗原として有用であることが既に報告されている同寄生虫のペルオキシレドキシン-1（SjTPx-1）の配列と 75％の相同性を示した。 SjPrx-4 の組換え蛋白質（rSjPrx-4）は，Mixed Function Oxidation 試験において，二重鎖プラスミド DNA を酸化的損傷から保護した。また，rSjPrx-4 に対するマウスポリクローナル抗体は，酵素抗体染色法において，寄生虫卵の内容，成虫の外皮および柔組織を染色した。rSjPrx-4 抗原は，患者血清を対象にした ELISA において，それぞれ，83％及び 88％の感度及び特異性を示した。更に，rSjPrx-4 と組換え体 SjTPx-1（rSjTPx-1）を混合したカクテル抗原を使用した ELISA では，感度及び特異性が，それぞれ，90％及び 93％に向上した。これらの成績から，SjPrx-4 が日本住血吸虫を酸化ストレスから保護する抗酸化蛋白質であること，また，rSjPrx-4 の ELISA 抗原としての性能が SjTPx-1 との組み合わせによって向上することが示唆された。

【第三章】日本住血吸虫蛋白質のアミノ酸配列から設計した複数の B 細胞エピトープ配列を組み合わせた組換え蛋白質（multi-epitope recombinant proteins：MERP）を作製して，日本住血吸虫症の ELISA 抗原に応用した。これまでに日本住血吸虫症の ELISA 抗原として有用であることが報告されている 8 種類の蛋白質のアミノ酸配列から 3 種類の MERP（SjChimeric 1，SjChimeric 2 及び SjChimeric 3）を作製した。これら MERP と 8 種類抗原の組換え蛋白質について，それぞれの ELISA 抗原としての感度及び特異性を評価した結果，SjChimeric 2 及び SjChimeric 3 で，全ての単独抗原の成績を上回る良好な成績が得られた。それぞれの感度及び特異性は，SjChimeric 2 で 93％及び 9７％，SjChimeric 3で 90％及び 93％であった。これらの成績から，MERP を応用することで，日本住血吸虫症を診断する ELISA の性能が向上することが示唆された。

以上の成績から，日本住血吸虫症の創薬研究において，実験動物での感染病理を評価する手法として qPCR が有用であること，また日本住血吸虫症の有病率調査において，MERPを応用した ELISA 法が有用であることが示唆された。これら手法が各国での公衆衛生事業に実装されることで，この寄生虫病が早期にアジアから排除されることが期待される。

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