半胱胺酸蛋白脢破壞由複合疫苗提供魚體對抗海水白點病的保護力

Abstract

海水白點蟲 (Cryptocaryon irritans) 是一種能在超過95種海水魚身上寄生的纖毛原蟲。箱網中密集養殖的海水魚群容易受到其纖毛幼蟲 (theronts) 感染而導致大量死亡。海水白點病複合疫苗以幼蟲纖毛上的制動蛋白 (immobilization protein) 作為抗原 (iAg)，加上幼蟲熱休克蛋白C-端 (Hsp70C) 作為佐劑，製作去氧核醣核酸疫苗。經注射或灌食，疫苗均能保護石斑 (Epinephelus coioides) 幼魚抗海水白點蟲感染。然而，免疫魚體表上仍殘留17 %的白點狀營養體 (trophonts)，且白點周圍有明顯的組織壞死。由於寄生蟲的半胱胺酸蛋白酶在侵入宿主的過程中有其重要性，因此此酵素經常作為抗寄生蟲疫苗中的重要抗原。另外，寄生蟲半胱胺酸蛋白酶亦有促進Th2型免疫反應活化的特性。本研究選殖蟲源的半胱胺蛋白酶 (cathepsin B cysteine protease)，加入複合疫苗，探討此酵素誘發的免疫反應是否能減少魚體表上白點狀營養體 (trophonts) 以及感染後魚體表組織壞死的情況。 從纖毛幼蟲選殖出兩種半胱胺酸蛋白酶，分別命名為CiCPB 和CiCPL。CiCPB和CiCPL和石斑魚半胱胺酸蛋白酶 (EcCPB、EcCPL) 的序列相似度分別為58% 和39%，在親緣關係上仍分屬不同的群簇。定量PCR檢測發現 CiCPB在纖毛幼蟲時期的表現顯著高於CiCPL的表現，推論纖毛幼蟲附著魚體後再由 CiCPB，而非 CiCPL，承擔入侵的過程。將CiCPB開放閱讀框中10個終止密碼子置換，使其能在魚細胞中表現。將石斑魚免疫球蛋白IgT重鏈的訊號肽連接修飾過的 CiCPB， 插入質體pcDNA3.1中，再將質體轉染至石斑魚鰭細胞 (GF-1 cells)，免疫螢光細胞染色檢測發現GF-1細胞能表現CiCPB重組蛋白。 將iAg、Hsp70C、CiCPB 質體分別以殼聚糖 (chitosan) 包裹成直徑100奈米的奈米微粒，從石斑魚苗的食道灌食(20 μg/g fish)。免疫後至感染前24天的魚隻體重和體長，各組間無顯著差異。觀察免疫後至感染前24天的魚隻胃腸道組織切片，各組皆無發現明顯組織壞死與出血。免疫後第25天，以活纖毛幼蟲50% 致死劑量 (LD50) 浸泡感染魚苗，複合疫苗免疫的魚群有100% 相對存活率 (relative percent survival，RPS)，但是複合疫苗和CiCPB免疫的魚群卻只有8% RPS；各組魚體表營養體負載量，亦無顯著差異。免疫後第31天，再以91% 致死劑量 (LD91) 浸泡感染前次感染中存活魚隻，複合疫苗免疫的魚群有80% RPS，但是複合疫苗加入CiCPB免疫的魚群只有0% RPS。綜合前述結果，在纖毛幼蟲感染後，複合疫苗中的CiCPB引起負面效應，進而破壞由複合疫苗提供石斑魚的抗病力。

Cryptocaryon irritans is a ciliate protozoan parasite which infects no less than 95 species of marine fish. The marine fish cultured densely in cages is vulnerable to C. irritans infection, and it leads to tremendous fish death. The combination vaccine was composed of C. irritans immobilization antigen (iAg) as antigen and the C terminus of C. irritans heat shock protein 70C (Hsp70C) as adjuvant. With both intramuscular injection and oral intubation, the combination vaccine confers high protection against cryptocaryonosis in Epinephelus coioides (grouper) fingerlings. Nevertheless, 17%, white spots (trophonts) remained on immunized fish with the necrotic lesion around the spots. Involved in parasite invasion, parasite cysteine proteases were usually demonstrated as antigen in anti-parasite vaccine. In addition, parasite cysteine proteases were potent triggers of Th2 immune responses. In the present study, the effect induced by C. irritans cathepsin B cysteine protease (CiCPB) as co-antigen in the combination vaccine was analyzed. Two cathepsin cysteine proteases were cloned and characterized as CiCPB and CiCPL, respectively. CiCPB, CiCPL and grouper cysteine proteases (EcCPB and EcCPL) belong to different clusters phylogenetically, although they share 58% and 39% similarity in sequences, respectively. Real-time PCR revealed that CiCPB gene expression was significantly higher than CiCPL in theront stage. It was suggested that CiCPB is important in theront invasion, rather than CiCPL. The open reading frame of CiCPB was computationally reviewed to replace the 10 stop codons with suitable amino acids, and its GC content was intensified. Plasmid pcDNA3.1 was inserted with a signal peptide of grouper IgT heavy chain and CiCPB. The transfected grouper fin cells (GF-1 cells) successfully translated the insert protein. Plasmid with iAg, Hsp70C and CiCPB was separately encapsulated in chitosan nanoparticles and orally intubated to grouper fingerlings with 20 μg/g fish body weight. The growth rate of fish body weight and body length during 24 days post immunization showed no significant difference among each group. The fish stomach and intestine tissue sections during 24 days post immunization showed no tissue lesion and hemorrhage. Fish was then challenged with a 50% lethal dose (LD50) of live theronts at 25 days post immunization. The combination vaccine group and combination vaccine with CiCPB group showed 100% and 8 % relative percent survival (RPS), respectively. Trophont burden on unit fish surface showed no difference among each group. Fish survived from last challenge suffered another 91% lethal dose (LD91) challenge at 31 days post immunization. The combination vaccine group and combination vaccine with CiCPB group showed 80% and 0% RPS, respectively. In conclusion, the effect induced by CiCPB addition almost broke the protection induced by the combination vaccine in grouper fingerlings.