Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/13737
標題: 犬重組NGAL蛋白的表現純化與其抗體的製備
Expression and purification of recombinant canine neutrophil gelatinase-associated lipocalin (NGAL) and production of anti-NGAL antibody
作者: 張淳婷
Chang, Chun-Ting
關鍵字: 犬腎病蛋白
NGAL
出版社: 獸醫學系暨研究所
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摘要: Neutrophil gelatinase-associated lipocalin (NGAL) 為25 kDa蛋白質隸屬於lipocalin家族。NGAL與癌症、炎症和腎臟疾病相關並且已有研究證實NGAL可應用於急性或慢性腎臟疾病診斷。然而,NGAL確實的機制仍為未知。依據在急性和慢性腎臟傷害NGAL上升的變化程度觀察, NGAL可應用於在急性腎臟損傷早期尿液的檢測。目前,對於NGAL與犬的腎臟疾病的相關性仍未被探討。因此,以RT-PCR 方式自犬乳腺腫瘤細胞增幅NGAL cDNA,並且經核酸定序確定為犬之NGAL序列後。利用PET系統於大腸桿菌表現犬之重組 NGAL (rNGAL) 蛋白質並純化出25 kD 預期大小的蛋白質。並以 rNGAL用於作為抗原進行小鼠免疫製備抗犬rNGAL之多株抗體,最後並以immunoblotting方式確認抗體的抗原性並偵測臨床尿液樣本。此抗體分別可辨識腎衰竭犬尿液檢體約22 kDa 位置的蛋白質及腎衰竭貓的尿液檢體約25 kDa 位置的蛋白質;而對於正常犬、正常幼貓、正常及腎衰竭人類病患的尿液檢體則無反應。
Neutrophil gelatinase-associated lipocalin (NGAL) is a protein of 25 kDa and belongs to the lipocalin family. Over-expression of NGAL correlates with cancer, inflammation, and kidney disease. The evidence for the role NGAL in acute or chronic kidney injury has been reported. However, the precise signaling remains unknown. Based on the observation that levels of NGAL rise in both acute and chronic kidney damage, detection of NGAL in urine has been successfully applied in the early diagnosis of acute kidney damage in human. At present, the role of NGAL in canine kidney damage has not yet been characterized. In this study, NGAL cDNA was amplified from canine mammary gland tumor cells by RT-PCR and its identity was determined by automated sequencing. Production of the recombinant NGAL (rNGAL) protein was performed with the pET system in E. coli. A protein with expected size of 25 kDa was over-expressed and was purified. The rNGAL was used as antigen for immunization of mice to produce polyclonal antibodies against rNGAL. The antigenicity of the antibody was confirmed by immunoblotting and the antibody was further used to test the samples from canine disease. The urine samples from normal dog, normal kitten, normal human patient and renal failure patient was not recognized by the antibody by Western blotting. However, a protein with size of 22 kDa, 25 kDa in accordance with the expected size of the NGAL was detected from urine of dog and cat suffering from renal failure, respectively.
URI: http://hdl.handle.net/11455/13737
其他識別: U0005-0808200914011500
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-0808200914011500
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