草蝦細胞凋亡抑制蛋白基因之表現及其功能分析

Abstract

本研究由實驗室建構之草蝦5’EST資料庫和5’RACE選殖出草蝦IAP基因完整序列，稱為草蝦inhibitor of apoptosis protein (IAP)基因，草蝦IAP包含三個桿狀病毒IAP重複區域(BIR)以及一個RING區域，且進行序列上的分析發現草蝦IAP蛋白之BIR1、BIR2、BIR3和RING finger區域與人類cIAP1、cIAP2、XIAP及果蠅DIAP2相似。此基因cDNA全長為4769 bps ，轉錄區域為2097bps ，轉譯長度為698個氨基酸，分子量為76.9 kDa。以RTPCR得知IAP基因在類淋巴器官相對於其他器官如：肝胰腺、胃、血球、鰓、神經組織、心臟和肌肉組織有最高表現量，在感染白點症病毒後也發現在類淋巴器官與胃器官表現量並未隨感染時序而改變，進一步將草蝦IAP基因暫時性轉染進Sf-9昆蟲細胞株，經由免疫螢光染色得知此基因產物主要是位於細胞質。對於草蝦IAP拮抗細胞凋亡測試，以轉染果蠅Reaper使細胞產生細胞凋亡，發現共同轉染草蝦IAP與果蠅Reaper時，草蝦IAP具有抑制細胞凋亡之能力。

We cloned a novel inhibitor of apoptosis protein (IAP) family member, PmIAP, from Penaeus Monodon. PmIAP contains three baculoviral IAP repeat (BIR) domains followed by a RING finger domain. The BIR and RING finger domains of PmIAP share striking amino acid sequence similarity with those of cIAP1, cIAP2, XIAP, and DIAP2, suggesting evolutionary conservation. Pm-IAP was 4.7 kb long, including a coding region of 2097 bp preceded by 633 bp (5' untranslated region) and followed by 2039 bp (3' untranslated region). Pm-IAP encoded a protein (Pm-IAP) of 698 amino acids with a predicted molecular mass of 76.9 kDa. RT-PCR analysis revealed that the steady-state level of PmIAP mRNA is highest in lymphoid organ among examined tissues, including hepatopancrease, stomach, hemocyte, gill, nerve, heart and muscle. Furthermore, the PmIAP RNA level in LO cells was not changed significantly after WSSV infection, although LO cells undergo apoptosis upon viral infection. The immunofluorescence staining of Spodoptera frugiperda Sf-9 cells transiently expressing PmIAP revealed that the PmIAP was mainly localized at cytoplasm of Sf-9 cells. To demonstrate the anti-apoptosis activity of Pm-IAP, we cotransfected the Drosophila reaper and Pm-IAP gene into Sf-9 cells. The result showed that Pm-IAP can block apoptosis in Sf-9 cells induced by Drosophila Reaper.