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National Chung Hsing University Institutional Repository - NCHUIR > 獸醫學院 > 獸醫學系所 > 依資料類型分類 > 碩博士論文 >  貓重組NGAL蛋白的表現純化及腎病貓與NGAL的關聯性

Please use this identifier to cite or link to this item: http://nchuir.lib.nchu.edu.tw/handle/309270000/155291

標題: 貓重組NGAL蛋白的表現純化及腎病貓與NGAL的關聯性
Expression and purification of recombinant feline neutrophil gelatinase-associated lipocalin (NGAL) and correlation of feline renal disease with NGAL
作者: 尹慧瑜
Yin, Hui-Yu
Contributors: 王咸棋
Hsien-Chi Wang
獸醫學系暨研究所
關鍵字: ;腎病
feline;NGAL;renal disease
日期: 2012
Issue Date: 2013-11-21 11:49:45 (UTC+8)
Publisher: 獸醫學系暨研究所
摘要: Neutrohpil gelatinase-associated lipocalin(NGAL)為25 kDa醣蛋白隸屬於lipocalin家族。NGAL為新穎的biomarker,在人醫方面的研究結果顯示當發生急性腎損傷或慢性腎病時血清或尿液中的NGAL濃度的上升都可有效的預測臨床上的進展。然而,貓NGAL的序列或NGAL在腎衰竭貓所扮演的角色尚未明瞭。本實驗的目的具有多個面向,第一個實驗目的為將貓NGAL的部分核苷酸序列進行鑑定再利用此序列製備貓NGAL重組蛋白。第二個實驗目的為利用本實驗室已成功製備出的抗犬NGAL多株抗體與貓NGAL重組蛋白及臨床上收集到的腎衰竭貓尿液中的NGAL是否存在交叉反應,同時會製備抗貓NGAL多株抗體期望能做為臨床上的用途。首先由Crandell Rees feline kidney(CRFK)細胞株中萃取出mRNA後進行RT-PCR得cDNA後不同物種間高度保留區域設計的兩組primers進行Nested PCR得到DNA。將定序完成的DNA序列放入軟體中進行BLAST比對後顯示與犬NGAL序列有74% 的相似度並命名為貓NGAL。再將此序列與pET32b載體進行結合後再轉殖到大腸桿菌系統(E.coli BL21TM),進行重組蛋白的表現。隨後以Ni-NTA chromatography進行純化,得到預期大小為42 kDa的位置有訊號產生。將貓NGAL重組蛋白與佐劑混合後對小鼠進行免疫,製備抗貓NGAL多株抗體。此外,利用immunoblotting測定抗犬NGAL多株抗體與貓NGAL重組蛋白及腎損傷貓尿液之間具有交叉反應。抗貓NGAL多株抗體同樣於預期的位置可辨識到貓NGAL重組蛋白及腎損傷尿液中的NGAL。另外,臨床上收集到20隻患有不同疾病之患貓尿液與抗犬NGAL多株抗體進行偵測,其中有16隻於25 kDa位置有訊號產生。由於抗犬NGAL多株抗體可與貓NGAL產生交叉反應,希望能將此抗體運用到臨床上做為早期診斷及觀察疾病進展的指標。
Neutrophil gelatinase-associated lipocalin (NGAL), a 25 kDa glycoprotein belonging to lipocalin superfamily, is an emerging excellent biomarker in the plasma and urine for predicting clinical outcomes of acute kidney injury (AKI) and chronic kidney disease (CKD) in human. However, neither the sequence of feline NGAL (F-NGAL) nor the role of NGAL in cat with kidney damage have been identified. The aims of this study are several folds: 1. to identify the partial sequence of F-NGAL that would be then used for generation of feline NGAL recombinant protein (rF-NGAL); 2. to evaluate the cross reactivity of polyclonal canine NGAL (C-NGAL) Ab produced in our lab, with rF-NGAL and clinic specimens from cats with kidney diseases. Simultaneously, polyclonal F-NGAL (F-NGAL) Ab will be produced to detect the F-NGAL for clinic purposes. First, cDNA was amplified from Crandell Rees feline kidney (CRFK) by RT-nested PCR. The sequence of F-NGAL, sharing 74% identification with C-NGAL, was cloned into pET 32b expression vector. The rF-NGAL, with expected size of 42 kDa, was produced in E.coli, purified by Ni-NTA chromatography, and used for mouse immunization to produce polyclonal F-NGAL Ab. Moreover, the cross-reactivity of polyclonal C-NGAL Ab with the F-NGAL was analyzed using rF-NGAL protein and urine from kidney-damaged cats by immunoblotting. Results indicated that proteins with expected sizes were recognized by polyclonal F-NGAL Ab. Using polyclonal C-NGAL Ab, among the 20 clinic specimens of feline urine with different diseases from animal hospital, there were 16 specimens showed the expected size of 25 kDa. Since F-NGAL can cross-react with polyclonal C-NGAL Ab, expected it can be used to detect F-NGAL for clinic purposes.
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