本研究首次由台灣山區採集之野生蠶血中分離昆蟲之抗菌蛋白，建立分子檢測方法，快速鑑定並篩選出蟲體內是否具有被誘導出類似之 cecropin；以抗家蠶 cecropin 製作之抗血清作為診斷探針進行酵素連接抗體吸附法分析，可以偵測出 cecropin 對於在兩種來源之野蠶皆顯示經由誘導後具有高含量之抗菌成分，在經由免疫反應 8-10 小時的處理時間，利用 RT-PCR 使用兩組設計的引子對，僅發現對於家蠶可以產生 cecropinD 及 CecropinB，分別產生 250 bp 及 350bp 之產物。而對於野蠶卻無反應，使用鱗翅目序列設計之引子對可以產生 120bp 之 cDNA。利用親和性層析法，可以得到部分純化物質，具有強抗菌反應。其中發現對於格蘭氏陰性菌可產生抗菌反應，對於格蘭氏陽性菌，如金黃色葡萄球菌及枯草桿菌，亦能有效被抑制，但對於酵母菌則無抑菌反應。並嘗試定序，分析其中之差異，並且檢測其專一性與靈敏度。Rapid screenings of cecropin-like substance on the giant wild silkworm Eriogyna pyretorum trapped in mountain regions of Taiwan are rendered for the first time. The enzyme-linked immunosorbent assay (ELISA) using a polyclonal antiserum against cecropin as probe, like Bombyx mori, was adopted. An increasing amount of the inductive cecropin-like proteinous substance was detected from hemolymph of Eriogyna pyretorum, at 8 to 10 hours after E. coli challenged. Subsequently, a RT-PCR was conducted using 2 sets of primers designed from known cecropin B and cecropin D sequences of Bombyx mori, about 200 b.p. and 350 b.p. cDNAs were obtained, respectively, from fatty bodies of bacteria-challenged Bombyx mori. Nevertheless, negative results were gained in all of 2 lineages for Eriogyna pyretorum. Alternatively, with another nested RT-PCR applied using conserved degenerate primers derived from two known lepidopteran cecropins？？cDNA sequences, the 120 b.p. cDNA bands encoding putative cecropin were observed in both species. Therefore, the transcripts encoding putative Eriogyna cecropins were confirmed by this nested RT-PCR. Furthermore, following partially purified method by affinity chromatography, the antibacterial activity against E. coli, Staphylococcus aureus and B. substils were observed through a growth inhibition zone assay in the fractions containing the putative Eriogyna cecropins from hemolymph of these bacteria-challenged larvae.
