大鼠睪丸專一性RTDPOZ蛋白研究：重組蛋白表現與同源雙體分析

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Title:	大鼠睪丸專一性RTDPOZ蛋白研究：重組蛋白表現與同源雙體分析 Rat testis-specific protein RTDPOZ：recombinant protein expression & homodimerization analysis
Authors:	張元祥
Contributors:	生物科技研究所
Keywords:	腫瘤壞死因子受器結合因子抗血清桿狀病毒表現系統包涵體同分子雙子 TDPOZ yeast two-hybrid system tumor necrosis factor receptor-associated factor domain poxvirus and zinc finger domain baculovirus expression vector system homodimer
Date:	2006
Issue Date:	2014-06-09 10:48:12 (UTC+8)
Abstract:	TDPOZ，為一含腫瘤壞死因子受器結合因子(TD；tumor necrosis factor receptor-associated factor domain)功能區與POZ (poxvirus and zinc finger domain)功能區的新蛋白質家族。TDPOZ蛋白質的功能目前仍未知。不過由TD與POZ功能區於胺基酸序列強烈的保守性顯示出此基因對細胞的功能可能之重要性。本研究目的在於嘗試製備RTDPOZ重組蛋白提供日後研究分析之用，並對於RTDPOZ的作用結構進行初步的分析。首先針對rat的Rtdpoz基因，構築完整的Rtdpoz序列，並找出編譯讀框，再利用大腸桿菌表現系統表現RTDPOZ蛋白（經電泳分析後發現RTDPOZ蛋白主要存在於不溶性層的包涵體中），並製造抗血清。另以昆蟲細胞Sf-21為宿主，配合桿狀病毒表現系統表現RTDPOZ，以克服表現蛋白包涵體的形成，日後可供RTDPOZ之活性與功能性研究之用。同時由觀察RTDPOZ普遍分布於細胞膜上和蛋白聚集(aggresome)的現象推測可能與其功能之間有密切的關聯。另外為了探討RTDPOZ蛋白本身是否會產生同分子雙子，遂以酵母菌雙雜合系統方式，將Rtdpoz基因片段分別選殖至帶有Gal4-AD（activation domain）以及-BD（DNA binding domain）的表現載體中。結果發現於營養限制培養基中並沒有預期的菌落生長；初步推測RTDPOZ並不會形成同分子雙子結構。 TDPOZ, a new protein family member of with contain the tumor necrosis factor receptor-associated factor domain (TD) and the poxvirus and zinc finger domain (POZ). The function of TDPOZ proteins is unknown. However, conservation of the TD and POZ domains strongly suggests that these domains are critical in some cellular functions. This research focused on the rat Rtdpoz gene. The full-length of Rtdpoz DNA sequence was first determine to locate the open reading frame (ORF). The E. coli expression system was then used to express the RTDPOZ protein (existed in inclusion bodies) and to produce anti-serum. We also used the insect cell Sf-21 and a baculovirus expression vector system to express RTDPOZ to overcome the inclusion body problem. Furthermore, RTDPOZ was widespread at cell membrane and the phenomenon of aggregate may indicate a connection with protein functions. We also analyzed RTDPOZ’s self-interaction by yeast two-hybrid system by constructing the Rtdpoz sequence in yeast expression vectors which have either an activation domain (AD) or a binding domain (BD). The results indicate that co-transformed yeast would not growth on nutrition-limited medium plate; initial analysis suggests that RTDPOZ does not form homodimer.
Appears in Collections:	[Graduate Institute of Biotechnology ] thesis

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