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研究生:周巧巧
研究生(外文):Zhou, Qiao-Qiao
論文名稱:一種新的電位依賴型鉀離子通道的特性
論文名稱(外文):Characterization of a novel voltage-dependent potassium channel
指導教授:曹美玲
指導教授(外文):Tsaur, Meei-Ling
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:神經科學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:1995
畢業學年度:83
語文別:中文
中文關鍵詞:醫學神經科學神經學鉀離子
外文關鍵詞:MEDICINENEUROSCIENCENEUROLOGY
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自1987年Papazian等人篩殖出第一個鉀離子通道後,已有許多電位依賴型的鉀離子通道基因被篩選出來,包括A型和延遲整流型,它們在神經系統中,對訊號的傳導及處理扮演著一個重要的角色.
本研究室使用一段Kv4.2基因上0.6kb大小的DNA片段,於大白鼠海馬迴 cDNA library,中篩選出一個新的電位依賴型鉀離子通道,定名為Kv4.3.透過DNA定序結果,得知Kv4.3基因的整段DNA長度為4﹑275bp,ORF;(open reading frame)有61l個胺基酸,可轉譯成69kDa(69,331 daltons)的蛋白質,與Kv4.x subfamily中其它的基因Kv4.1,Kv4.2作胺基酸相同度比對,分別得到67%,76%.
使用Kv4.33'端未轉錄部份作為特異性的探針,由北方雜化法得知,Kv4.3在大白鼠腦中轉錄的大小是8.2kb.透過生物體外轉錄轉譯法,在35S-methionine存在下,X光片上可看見一條 67kDa的band.而未標識的反應藉由Western blot,在NC試紙上有三條 band(69,59,48 kDa)可以被Kv4.3N抗體偵測到,但只有69kDa的 band可以被抗原性peptid對抗掉.在生物體中,大白鼠腦的細胞膜蛋白質,海瑪迴及小腦的萃取物,以SDS-PAGE分離之後,轉移至NC試紙,用Kv4.3N抗體染色,只有 31kDa的band可以被偵測到,而且可以被抗原性peptide對抗掉.由以上結果,知道 Kv4.3在離體可轉錄轉譯成一條 69kDa大小的蛋白質,在體內則可能有一些未知的作用發生.
由免疫細胞染色法看出,Kv4.3蛋白質在大白鼠腦中retrosplenial cortex,中央革(medial habenula),海馬迴齒狀分子層(hippocampa dentate molecular layer),視丘(anterior ventral thalamus nuclei, dorsal lateral geniculate nuclei),上丘的表層(superficial layer of superior colliculus),及小腦顆粒層中的glomeruli,均有表現.
將免疫細胞化學法與定位雜化法的結果作比較,得知Kv4.3蛋白質在鼠腦中,可能是分佈於神經元的細胞體和樹狀突上.
Multiple voltage-dependent potassium channel genes had been cloned since 1987. Voltage-dependent potassium channels, including delayed rectifier and A-type, are important in the signal transduction, propagation and processing in the nervous system. Our laberatory used a 0.6 kb fragment of Kv4.2 cDNA encoding an A-type K+ channel as probe to screen a cDNA library made from rat hippocampus and cloned a novel gene-Kv4.3. By DNA sequencing, we found that the full length Kv4.3 cDNA is 4,275 base pairs with an ORP of 611 amino acids (m.w. 69,331 daltons). The amino acid identity of Kv4.3 with the Kv4.x subfamily members Kv4.1 and Kv4.2 is 67% and 76%, respectively.
By Northern blot, the size of Kv4.3 mRNA in rat brain is 8.2 kb. Kv4.3 was transcribed and translated in vitro. In the presence of 35S-methionine, a 67 kDa protein could be detected on film. There are three bands (69, 59 and 48 kDa proteins) could be detected by antibody on unlabeled in vitro transcription/translation product through Western blot, but only the 69 kDa protein band could be competed away by antigenic peptide. We prepared membrane proteins from rat whole brain, crude lysates from hippocampus and cerebellum, separated by SDS-PAGE and transfered to NC paper, which was subjected to be detected by antibody. The data revealed a 31 kDa protein band and could be competed away by antigenic peptide. These findings suggest that Kv4.3 encodes a 69 kDa protein in vitro, but some unknown processing may happen in vivo.
By immunocytochemistry of rat brain, we found that Kv4.3 protein is highly expressed in retrosplenial cortex, medial habenula, hippocampal dentate gyrus molecular layer, thalamus (anterioventral thalamus nuclei, dorsal lateral geniculate nuclei), superficial layer of superior colliculus and glomeruli of granular layer in cerebellum.
Comparing with pattern of in situ hybridization,we suggest that Kv4.3 is localized in the dendrite and/or somata of neurons in rat brain.



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