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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/16561

Title: 草蝦抗脂多醣因子之選殖與功能性分析
Molecular Cloning and Functional Analysis of Tiger Shrimp (Penaeus monodon) Anti-lipopolysaccharide Factor
Authors: Wei-Cheng Liu
劉韋誠
Contributors: NTOU:Department of Aquaculture
國立臺灣海洋大學:水產養殖學系
Keywords: 草蝦抗脂多醣因子;基因表現;最小抑制濃度測試;分殖選殖與特性分析
Shrimp anti-lipopolysaccharide factor;gene expression;MIC;molecular cloning and characterization,
Date: 2004
Issue Date: 2011-06-30T08:39:57Z
Abstract: 革蘭氏陰性菌外膜之內毒素是造成敗血症(Sepsis)之主因,敗血症會引發多重器官衰竭,最後可能產生休克甚至是死亡。在水產養殖方面,台灣由於受到草蝦桿狀病(monodon baculovirus; MBV)、弧菌(Vibrio)及白點病毒(white spot baculovirus,WSBV)等病原之感染, 致使草蝦養殖產業瀕臨崩潰之困境。目前所發表之內毒素結和蛋白(Endotoxin-binding Proteins)諸如LALF、LBP及BPI,大多使用價格昂貴之合成胜肽。因此能以低廉的價格來得到大量有內毒素中和效用又具有抗菌活性之蛋白質是當務之急。 本論文中所選殖之SALF經胺基酸序列之比較分析後,發現SALF之第55-76個胺基酸為富含正電胺基酸,兩端帶有可形成雙硫鍵cysteine之區域(Domain)。 化學合成之csSALF51-79經LAL assay證實,它具有中和內毒素之效果。由大腸桿菌所純化之重組SALF在MIC測試結果發現,革蘭氏陽性菌Micrococcus luteus之MIC值為2 μg/μL,Streptococcus bovis之MIC值為1.25 μg/μL、對Staphylococcus aureus之MIC值為1.25 μg/μL及對Streptococcus pyogenes為1.3 μg/μL。在革蘭氏陰性菌方面,SALF對於Escherichia coli之MIC值為1.25 μg/μL、對Klebsiella oxytoca之MIC值為2.5 μg/μL及對Pseudomonas aeruginosa之MIC值為1 μg/μL。在MTT assay中也證實csSALF51-79及lsSALF51-79可抑制人類子宮頸癌細胞(HeLa)、人類乳癌細胞(MCF-7)及人類纖維肉瘤細胞(HT-1080)之增生。 在組織表現量上,草蝦抗脂多醣因子在草蝦之心臟、鰓及腸有微弱表現量,而在血球細胞、肌肉、眼柄及上皮組織表現量較高,其中以血球細胞的表現量最為明顯。以Real-time PCR分析受免疫刺激後草蝦血球細胞中SALF、Peroxinectin、Prophenoloxidase與Transglutaminase基因表現之變化量,除了Prophenoloxidase在第12 hr以後表現量會持續增加外,其他三種基因均是在第72 hr時才會大量表現。
The outer membrane of gram-negative bacteria secrets endotoxin, which is known to cause sepsis leading to multiple organ failure and even death. The tiger shrimp aquaculture industry has once been developed rapidly in Taiwan, and the success of the industry development has been recognized worldwide. However, the shrimp aquaculture industry has unexpectedly suffered from inability of sustained production, primarily due to serious infection of monodon baculovirus (MBV), Vibrio, and white spot syndrome virus (WSSV). Endotoxin binding proteins reported to date include LALF, LBP, and BPI, which are all high-priced synthetic peptides. It is desirable to search for molecular proteins which are capable to neutralize endotoxin and possess antibiotic functions. For this reason, shrimp anti-lipopolysaccharide factor (SALF) was targeted and cloned in this study. SALF is characterized positive charges at 55-76th amino acids and cysteine domains at both ends. Synthesized csSALF (51-79) is shown effectively to neutralize endotoxin , confirmed by LAL assay. MIC values of SALF against gram-positive bacterias , Micrococcus lutus, Streptococccus bovis, Staphylococcus aureus, and Streptococcus pyognes is 2, 1.25, 1.25, and 1.3 mg/ml, respectively, while those against gram-negative bacterias, Escherichia coli, Klebsiella oxytoca, and Pseudomonas aeruginosa is 1.25, 2.5, and 1.0 mg/ml, respectively. In the MTT assay, both csSALF and lsSALF can suppress hyperplasia of HeLa, MCF-7, and HT-1080 cell lines.SALF expresses lesser in the heart, gill and intestine, but higher in the blood cells, muscle, eyestal, and epithelial tissue, the blood cells in particular. The changes in the gene expression of SALF, peroxinectin, prophenoloxidase and transglutaminase extracted from the tiger shrimp blood cells, were further analyzed by real-time PCR. The results indicated that prophenoloxidase expressed continuously after 12-hr post- stimulation, while other three genes started to express in a large quantity after 72-hr post-stimulation.
URI: http://ethesys.lib.ntou.edu.tw/cdrfb3/record/#G0M92330009
http://ntour.ntou.edu.tw/ir/handle/987654321/16561
Appears in Collections:[水產養殖學系] 博碩士論文

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