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標題: 偵測分化抗原在犬口腔黑色素瘤的表現及架構一個Melan-A表現質體
Detection of differential antigens expression in canine oral melanomas and construction of Melan-A expression plasmid
作者: 熊祐興
Hsiung, Yu-Hsing
關鍵字: dog

oral melanoma
DNA vaccine
DNA vaccine
出版社: 獸醫學系暨研究所
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摘要: 犬黑色素細胞瘤是最常見的口腔腫瘤之一,一旦發生轉移的情形,不管是化學療法或其他現有的治療方式都無法改善其不良的預後。一些可被免疫系統辨認的分化抗原(例如 Melan-A、tyrosinase及gp100)在黑色素細胞瘤當中具有高表現量,這些分化抗原具有使用在免疫療法的潛力。本實驗一開始針對國立中興大學獸醫教學醫院的犬口腔黑色素瘤病例,利用免疫組織化學染色的方式,偵測這些分化抗原表現的情形,這四種免疫組織化學染色所偵測的抗原,皆為100 %的陽性表現,依據免疫組織化學染色的結果,本實驗使用Melan-A基因及一段具有多個抗原決定位(epitope)的序列,來建構一個在獸醫臨床使用的免疫治療物,在本實驗Melan-A成功個從人類黑色素細胞株A375.S2c增幅出,接著分別轉殖到原核及真核表現宰體pET32a及pcDNA3.1當中,且使用Western blot成功偵測到Melan-A的表現,另外一個具有二重複的多抗原決定位序列也藉由PCR增幅出來。
Melanoma is the most common malignant neoplasm of oral cavity in dogs. Metastasis of this cancer is associated with poor durable response to chemotherapy or any other known treatments. Melanoma differential antigens (e.g., Melan-A, tyrosinase and gp100), which are commonly recognized by the immune system, were highly expressed in melanoma lesions. Immunohistochemistry was used to detect the expression of differential antigens of the oral melanomas obtained from the Veterinary Medical Teaching Hospital of National Chung Hsing University. All four immunohistochemistry markers showed 100 % positivity. Based on the results of immunohistochemistry staining, Melan-A was chosen with a multiepitope sequence (gp100 (280V), gp100 (210M), tyrosinase (370D) to generate an immunogenic agent for canine melanoma therapy. In our present study, Melan-A was successfully amplified from human melanoma cell line A375.S2c and was subsequently cloned into pET32a and pcDNA3.1 vector for expression in prokaryotic and mammalian system, respectively. Melan-A expression had also been detected by Western blot. The partial fragment containing two repeats of multiepitope sequence was also obtained by PCR.
其他識別: U0005-2207200911041100
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