請用此 Handle URI 來引用此文件: http://hdl.handle.net/11455/94742
標題: 鼠抗犬MAGE-A多株抗體之製備
作者: 馮郁青
關鍵字: 黑色素細胞瘤抗原
抗體
melanoma antigen
antibody
出版社: 獸醫學系暨研究所
摘要: Melanoma antigen-A簡稱MAGE-A於1991年首度被發現,其中包含12個基因:MAGE-A1到MAGE-A12。MAGE-A抗原於正常組織中只特定表現於睪丸及胎盤,但是在多種不同的腫瘤中皆可發現此種抗原表現。本研究目的為製備鼠抗犬之MAGE-A多株抗體,並建立此抗體應用於犬組織之免疫組織化學流程。首先,根據犬MAGE-A之保留區域(conserved region)設計引子,並以引子進行聚合酶鏈鎖反應。由於聚合酶鏈鎖反應所得到之產物大小為237 bp,此產物以MAGE-A237稱之。接著構築一個帶有MAGE-A237二重複片段的載體,並於大腸桿菌BL 21中表現此載體,以轉譯重組MAGE-A蛋白質。之後此重組MAGE-A蛋白質經純化及透析,並與佐劑混合以免疫BALB/c小鼠。免疫流程為每兩週肌肉注射一次,並於第11、25及39天收集小鼠血清。之後以西方墨點法測試收集之血清辨識MAGE-A重組蛋白質之能力。結果顯示於所有收集的血清中皆含有可辨識MAGE-A重組蛋白質之抗體,且第11及25天所採取之血清於1:20000時能夠有特異性地辨識MAGE-A重組蛋白質;第39天的血清則是於1:500000倍稀釋即可達此效力。於免疫組織化學染色方面,第25及39天之血清可應用於犬MAGE-A之偵測。其中又以2號BALB/c小鼠在第25天收集之血清,於1:2000倍稀釋、作用10分鐘所得到之染色效果最佳。而以上述條件偵測犬正常組織的MAGE-A表現,發現除睪丸之精原細胞外,所有測試之正常組織皆未見MAGE-A表現。未來犬MAGE-A可望發展成十分具有潛力之腫瘤標記。Melanoma antigen-A (MAGE-A) which comprises 12 members named MAGE-A1 to MAGE-A12 was first discovered in 1991. These antigens are predominantly expressed in a wide variety of tumors but only limited expressed in normal human testes and placentas. The aim of this study was to generate a polyclonal mouse anti-canine MAGE-A antibody and to establish the immunohistochemical (IHC) staining protocol of this in-house antibody against canine tissues. First, a pair of primers was designed based on conserved region of canine MAGE-A. Given the size of this product amplified by polymerase chain reaction (PCR), the amplicon was named MAGE-A237. A plasmid with two repeats of MAGE-A237 sequence was constructed, and then cloned and expressed in bacteria host, E.coli BL21. The purified recombinant MAGE-A protein as the antigen was mixed with adjuvant for immunizing mice by intramuscular injection every other week. To test the performance of serum antibody, sera that collected at day 11, 25 and 39 were served as the first antibody against recombinant MAGE-A protein by western blot analysis, and against normal canine tissues by IHC staining. Results showed that all the generated serum antibodies are able to detect recombinant MAGE-A protein by western blot, while the most suitable dilution ratio were 1:20000 for sera on day 11, 25 and 1:500000 for sera on day 39. The optimization of IHC protocol was: use of serum on day 25 at 1:2000 dilution as the first antibody and incubated for 10 minutes. Among all the tested normal canine tissues, the IHC staining results showed negative immunoreactivity except testicular spermatogonia. MAGE-A may be a promising tumor marker for further clinical application on benign and malignant tumors.
URI: http://hdl.handle.net/11455/94742
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