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標題: | 小白鼠子宮分泌液及副睪中乳輸鐵蛋白之生化研究 The biochemical study of lactoferrin in mouse uterine fluid and eqididymis |
作者: | CHEN-KANG CHANG 張振崗 |
出版年 : | 1995 |
學位: | 碩士 |
摘要: | 乳輸鐵蛋白(lactoferrin or lactotransferrin, LF)是一個可與Fe3+結合的醣蛋白,屬於transferrin家族,它可分為兩個約略對稱的半葉:N-半葉和C-半葉,每個半葉可藉著一個HCO3-的參與,和一個Fe3+結合。LF對Fe3+的親和力約比transferrin高300倍。LF廣泛的存在於乳汁、眼淚、唾液、及其他許多外分泌液中,並具有許多的生理功能。LF以數種不同的形態存在;與Fe3+結合後,也會改變它在SDS-PAGE上的移動能力及蛋白表面的各種特性。 在小白鼠的生殖系統中,LF存於子宮分泌液及副睪中,它的表現受到雌性素的調控,因為在它的基因的起始子(promoter)中含有雌性素反應單元(estrogen response element)的存在。子宮液中的LF於1986年被純化出,並由cDNA推定出它的胺基酸序列,LF在生殖系統中所扮演的角色仍是未知。 在本實驗中,我們自小白鼠子宮分泌液中純化出七種LF的異構形態,其中四種已決定出N-端胺基酸序列,都與由cDNA推定出的序列相近似,且都沒有N-block的情形,對LF抗體都有相等的反應強度。LF mRNA在副睪的中段及尾部都有表現,但只有在尾部可偵測到LF蛋白的存在。為瞭解決所面臨的抗體專一性不足的問題,我們以均質化的LF誘發專一性更佳的抗體,以作為往後進一步分析的工具。 Lactoferrin (lactotransferrin, LF), a 70 kDa iron-binding glycoprotein, is composed of two almost symmetrical lobes, N-lobe and C-lobe. Each of them can bind one Fe3+ through the assistance of one bicarbonate ion. Its affinity to Fe3+ is about 300 times larger than that of transferrin. LF is prevalent in milk, tear, saliva, and many other external secretions. LF plays a lot of biological roles in different cells. Previous study suggests the existence of several LF isoforms. Moreover, the mobility of the Fe3+-LF complex differs from that of the apo-protein and the complex formation results in a change in the protein structure. In mouse reproductive system, LF is found in uterine fluid and epididymis. Its expression is controlled by estrogen and an estrogen response element (ERE) has been located in the promoter of LF gene. Despite much effort paid in the past, the physiological role of LF in the reproductive biology remains to be unrevealed. Preparation of antibody specific to LF becomes important in order to perform functional study of LF. With this goal, we purified seven LF isoforms from mouse uterine fluid. Four of them were shown to have very similar N-terminal amino acid sequence to that deduced from the cDNA of mouse uterine LF. Each form of LF could be interacted strongly with anti-LF antibody. One of the seven LFs was used to immunize rabbit intrasplenically. The antibody gave nearly no affinity to serum albumin. Thus, the antibody was used for the specific immunodetection of LF in tissues. LF protein was detectable only in the cauda portion of epididymis but LF mRNA was found in either the corpus portion or the cauda portion. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76102 |
全文授權: | 未授權 |
顯示於系所單位: | 生化科學研究所 |
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