牛乳鐵蛋白對草蝦抗病能力之影響

Abstract

自1987年以來養殖草蝦的產量每下愈況，主要的原因是蝦病的繁生，改善此困境方法之一是從宿主本身加強其抗病能力。乳鐵蛋白(lactoferrin)為可與鐵結合的一種醣蛋白，最早由牛奶中純化出來，具有殺菌活性以及調節免疫反應多項生理功能；N: 11 mer為lactoferrin N端的11個氨基酸片段，亦具殺菌以及免疫刺激的活性；生長激素(Growth hormone, GH)由脊椎動物的腦下垂體前葉所分泌，可刺激動物生長；哺乳動物之Intertukin-1 β(IL–1β)在不同的細胞中有不同的功能，主要當作調控免疫反應的物質。本論文選取前述lactoferrin、N: 11 mer、yellowfin progy (Acanthopagrus latus) recombinant growth hormone (ypGH)及carp(Cyprinus carpio) recombinant intertukin-lβ(cIL-1β)具免疫調節或抗病功能或生長或三者兼備之物質，從離體及活體進行評估，俾提供標的基因(target gene)供抗病基因轉殖蝦的基本資料。 實驗分離體(in vitro)及活體(in vivo）兩部分，前者包含三項：(1) lactoferrin、N: 11 mer、 ypGH以及cIL-1β對蝦血球吞噬作用的影響，利用nitroblue tetrazolium (NBT)染色法測血球內超氧化陰離子(superoxide anion)的產量；(2)測量lactoferrin、N: 11 mer、GH以及IL-1β對蝦的重要病原菌例如Vibrio vulnificus、V.alginolyticus、V.harveyi的最少抑菌濃度(minimum inhibitory concentration, MIC); (3)檢測lactoferrin 及N: 11 mer對蝦血球細胞膜的影響，利用trypan blue染色檢測血球之存活率。 實驗結果顯示不論是lactoferrin、N: 11 mer、ypGH以及cIL-lβ皆可促進蝦血球細胞產生O2-，而後者已被證實為吞噬作用的最初產物。在抑菌方面，不論lactoferrin或是N: 11 mer均可抑制蝦病原弧菌的生長，；至於對血球細胞膜的影響，lactoferrin與血球培養3小時，N: 11 mer與血球培養l時後，其存活率較之對照組顯著的降低。 綜和以上的結果，lactoferrin及N: 11 mer固然對蝦草血球具有免疫刺激以及抑制弧菌生長的功能，但亦會降低血球細胞的存活率。

To investigate the peptide-antimicrobial activity relationship of shrimp, bovine lactoferrin (bLF), the basic amino acid-rich region of lactoferricin B, RRWQWRMKKLG (N: 11 mer), yellowfin progy (Acanthopagrus latus) recombinant growth hormone (ypGH) and carp (Cyprinus carpio) recombinant interlukin-1β(cIL-1β) were selected. Tiger shrimp (Penaeus monodon) hemocytes were incubated with bLF, N: 11 mer, ypGH and cIL-1β, respectively, and quantified the production of superoxide anion (O2-) by nitroblue tetrazolium (NBT) staining. All of the peptides could enhance the production of O2- of shrimp hemocytes, which was the first product of phagocytosis. The antimicrobial activities of the peptides were also tested by determining the minimal inhibitory concentration (MIC) of Vibrio vulnificus, Vibrio alginolyticus and Vibrio harveyi, respectively, in the presence of the peptides. In contrast to ypGH and cIL-1β, bLF and N: 11 mer were successful to inhibit the growth of the pathogen of tiger shrimp. Finally, the membrane disruptions of tiger shrimp hemocytes that caused by bLF and N: 11 mer, respectively, were determined with trypan blue staining. The survival rate of bLF- and N: 11 mer-treated hemocytes was significant differ with untreated hemocytes after 3- or 1-hour (s) incubation, respectively. These results suggest that bLF and N: 11 mer have the ability to stimulate the immune responses of shrimp hemocytes and to inhibit the growth of pathogen, but the two peptides can decrease the survival rate of shrimp hemocytes.