廣東血線蟲之抗原誘發輔助型T細胞免疫反應之研究

Abstract

世界上第一個廣東血線蟲的人體病例是1945年在台灣發現的，目前廣東血線蟲也是在台灣造成嗜酸性腦膜腦炎的主要原因之一。本實驗以受廣東血線蟲感染之雄性紐西蘭白兔血清免疫篩選已建立互補去氧核醣核酸基因庫 (cDNA library)，找出與兔血清內的抗廣東血線蟲抗體有陽性反應之抗原。得到兩個cDNA clones分別送入表現 載體中將蛋白質表現並分別命名為AC 4與AC 5。AC 4之cDNA片段經核苷酸序列分析長度為483 bp，約為160個氨基酸，胺基酸序列經由BLAST軟體搜尋比對後與線蟲Caenorhabditis elegans 之apyrase具有70%相似度；AC 5之cDNA片段經核苷酸序列分析長度為507 bp，約為168個氨基酸，胺基酸序列經由BLAST軟體搜尋比對後與線蟲C. elegans之Ras associated protein (Rab) family中的Rab2具有98%相似度。將廣東血線蟲重組蛋白AC 4及AC 5與正常或是被廣東血線蟲感染之鼷鼠脾細胞共同進行體外培養，再以酵素免疫吸附法偵測細胞激素表現之情形，發現在AC 4及AC 5刺激後，IL-4、IL-5、IL-13等Th2細胞激素與對照組比較後並無顯著差異，但IFN-γ則有顯著增加的情形，故推測AC 4及AC 5在感染廣東血線蟲後， 主要是幫助Th1細胞激素的表現。

The first human case of angiostrongylosis was reported from Taiwan in 1945. At present, Angiostrongylus cantonensis is the main cause of eosinophilic meningoencephalitis in Taiwan. In this study, serum obtained from rabbit hyperinfection with A. cantonensis was used to screen the cDNA library constructed in our laboratory. Two selected clones (AC 4 and AC 5) which showed strong positive reaction with anti-A. cantonensis antibody were selected for further study. Sequenceing analysis revealed that the cDNA fragment for AC 4 is 483bp (160 a.a) length. BLAST search revealed that it exbits 70% homology to apyrase from Caenorhabditis elegans. The other cDNA fragment for AC 5 is 507bp (168 a.a) long. BLAST search revealed that it exbits 98% homology to Rab2 family (Ras associated protein) family from C.elegans. Cytokine production in spleen cell from normal and A. cantonensis- infected mice in culture supernatants with A. cantonensis recombinant protein AC 4 or AC 5 were assessed by the commercial ELISA kit. The results revealed that the amount of cytokines IL-4, IL-5 and IL-13 were not significantly elevated after stimulation with AC 4 or AC 5 . But the production of IFN-γ have significantly increased when compare with control. The increase in IFN-γ suggest that AC 4 and AC 5 may help stimulating Th1 cytokine production.