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Localization, Purification, and Characterization of the Zinc-Binding Protein in the Head Kidney Tissue of Common Carp, Cyprinus carpio
|Authors: ||Po-Chih Kuo|
|Contributors: ||NTOU:Department of Food Science|
common carp;head kidney;zinc;zinc-binding protein;myeloperoxidase
|Issue Date: ||2011-06-30T08:23:52Z
|Abstract: ||鯉魚 (common carp, Cyprius carpio) 是一種原產於亞洲之廣溫型雜食性淡水魚類，無論是在有無污染的水域中所捕獲之鯉魚，其內臟的鋅濃度皆高達174-585μg/g fresh tissue，是其它魚類之數倍以上，但是銅、鉀、鈣及鎂的濃度卻與其它淡水魚相近，似乎是因鯉魚消化道組織中含有與鋅特異性結合之疏水性蛋白質，而在其它魚類中可能含量甚少。鯉魚血液中之鋅主要是存在於紅血球，而紅血球的鋅主要也是分佈於其疏水性之細胞外膜上。將水中氧氣降低至0 ppm並持續5天，發現約有17% 的鯉魚會死亡，死亡之鯉魚消化道組織鋅濃度皆低於活存的鯉魚。頭腎是魚類特有構造且已被確認具有造血、淋巴和內分泌組織在其中，類似於哺乳動物之骨髓。為瞭解鯉魚頭腎是否也具有膜相關性鋅結合蛋白質，乃利用階段萃取 (sequential extraction) 蛋白質的原理依極性高低做蛋白質分群，去除親水性之細胞質蛋白質等物質後，再將所得到的疏水性蛋白質沉澱以含界面活性劑之緩衝液進行抽取，並進一步以固定化金屬親和層析法分離出鋅結合蛋白質。經串聯式質譜法鑑定此鯉魚頭腎「鋅結合蛋白質」為一種鯉魚和鯽魚 (crucian carp, Carassius auratus) 皆具有的造血組織相關之髓過氧化酶 (myeloperoxidase, MPO)。為了解鯉魚髓過氧化酶是否與其它物種有所不同，乃採用cDNA末端快速擴增技術 (rapid-amplification of cDNA ends, RACE) 與簡併性引子PCR (degenerate PCR) 求得鯉魚的髓過氧化酶之核酸與胜肽更完整的序列，以利於比對生物資料庫瞭解更多資訊。另外，多株抗體的製備將可用於蛋白質層級表現量之分析以及檢視此蛋白質在細胞中分佈情況的變化等。|
Common carp Cyprius carpio is a omnivorous fish natively of Asia, that is living in widely temperate freshwater, whether the capture of common carp in a pollution-free water, zinc concentration of the internal organs are as high as 174-585 μg/g fresh tissue, that is several times higher than other fishes, but copper, potassium, calcium and magnesium levels are similar to other freshwater fishes. The digestive tract tissue of the common carp contain specific hydrophobic zinc-binding protein which may be very little in other fishes. Zinc in the blood of common carp is mainly present in red blood cells, and the distribution of zinc is also on the hydrophobic cell membrane. When the oxygen in the water decreased to 0 ppm about 17% of the common carp died. Higher zinc concentration in the digestive tract tissue of the common carps had lower mortality. The head kidney in fish is a unique structure which is similar to the mammalian bone marrow. In order to understand whether the zinc-binding protein also exist in the membrane of the head kidney, sequential extraction was used to extract the zinc-binding protein, followed by extraction with detergent buffer, and purified by immobilized metal affinity chromatography (IMAC). The isolated zinc binding protein of the head kidney was identified by tandem mass spectrometry. It was found that it may be the myeloperoxidase (MPO) that was associated with the hematopoietic tissue of common carp and crucian carp Carassius auratus. In order to understand whether the carp myeloperoxidase is different from other species, rapid amplification of cDNA ends (RACE) and degenerate PCR were used to obtain more complete sequence of nucleic acid and peptide of the carp myeloperoxidas, to facilitate the comparison of biological database for more information. In addition, polyclonal antibodies have been prepared, for the future study of the protein expression levels and its changes in different situation.
|Appears in Collections:||[食品科學系] 博碩士論文|
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