臺灣博碩士論文加值系統

過去實驗室以yeast two- hybrid得到數個可與HBV 核心蛋白有交互作用的細胞蛋白。其中四十六號蛋白為介白素1受體輔助蛋白 (IL-1RAcP)的細胞外側區域。根據 #46 cDNA的核苷酸序列，在EST 資料庫中搜尋到EST06168 clone。此clone的胺端196個胺基酸與已發表的IL-1RAcP (稱IL-1RAcP570) 的253- 448胺基酸序列完全相同，而中間區域的69個胺基酸序列與IL-1RAcP570的第449-567個胺基酸序列具34%相同性及59%的相似性，此外在羧端尚有119個胺基酸為已發表的IL-1RAcP570所不具有。
藉由定序分析包含IL-1RAcP 基因的human BAC clone，發現IL-1RAcP基因由13個exons和12個introns組成，第9個exon有2種，分別稱為9a和9b。其中膜蛋白型IL-1RAcP570由exons 1-12組成，其中第9個exon為9a；分泌型IL-1RAcP356由exons 1-8及9b組成。EST06168 cDNA clone含exons 7-11及13，其中第9個exon為9a，若此 mRNA具有和IL-1RAcP570相同的5' 端，則含exons 1-11和exon 13。即IL-1RAcP570、356和687 mRNAs為IL-1RAcP 基因轉錄後經由不同的替代剪接而產生。IL-1RAcP687 mRNA所表現的蛋白質為一個由687個胺基酸組成的膜蛋白。在功能分析上，發現L-1RAcP687可與IL-1RI結合，因此IL-1RAcP687可能會影響IL-1的訊息傳導。

In our lab, several cellular proteins were found to interact with HBV core protein by yeast two-hybrid system. One of them, #46, is identical to the extracellular region of human interlukin-1 receptor accessory protein (IL-1RAcP). Based on the sequence of #46, an EST clone (EST06168) which contains the #46 sequence was identified. The sequence of N-terminal 196 amino acids of this EST shows 100% identity to that of the 253-448th amino acids of the IL-1RAcP570, while the sequence of middle 69 amino acids of the former shows 34 % identity and 59 % similarity to that of the 449-567th amino acids of the latter. In addition, this EST contains an extra 119 amino acids in its C terminus.
To characterize this EST clone, human BAC clone contains the IL-1RAcP gene was sequenced and analyzed. The result showed that the IL-1RAcP gene is composed of 13 exons and 12 introns. There are two alternative exons 9, 9a and 9b. Three transcripts are generated by alternative splicing. The membrane form IL-1RAcP570 is composed of exons 1-12 and the exon 9 is 9a, the secreted form IL-1RAcP356 is composed of exons 1-8 and 9b, and the membrane form IL-1RAcP687 is composed of exons 1-11, exon 13, and the exon 9 is 9a. On the functional aspect, IL-1RAP687 can interact with the IL-1RI, suggesting that it may influence the IL-1 induced signal transduction.

中文摘要……………………………………………………………….1
英文摘要………………………………………………………………..2
壹、 緒論………………………………………………………………..3
貳、 材料與方法………………………………………………………14
參、 結果………………………………………………………………53
肆、 討論………………………………………………………………61
伍、 參考文獻…………………………………………………………68
陸、 圖表………………………………………………………………74
.表一、受IL-1調控的基因 。
表二、人類IL-1RAcP基因的exon大小及在BAC RPCI-11-211P13與RPCI -11-487B5的相對應位置。
表三、人類IL-1RAcP基因的intron大小及在BAC RPCI-11-211P13與RPCI -11-487B5的相對應位置。
表四、人類IL-1RAcP基因的exons 與introns大小及交界處序列。
表五、人類IL-1RAcP基因三種transcript 之exons大小與對應序列。
圖一、HBV結構。
圖二、HBV基因群構造及四個起動子、兩個加強子。
圖三、IL-1s、IL-1RIs與IL-1RAcP的結合。
圖四、TLR superfamily 之特性與TIR區域。
圖五、三種IL-1RAcP的胺基酸序列比較。
圖六、IL-1 訊息傳導途徑。
圖七、人類IL-1RAcP、#46與EST06168序列的比較。
圖八、人類IL-1RAcP與EST06168 clone 之胺基酸序列比較。
圖九、含有人類IL-1RAcP基因的BAC clones。
圖十、以南方墨點法確認BAC clone含有IL-1RAcP基因。
圖十一、以南方墨點法確認BAC clone含# 46 序列。
圖十二、以南方墨點法確認BAC clone含有EST06168序列。
圖十三、人類IL-1RAcP基因組成。
圖十四、人類IL-1RAcP687 cDNA與胺基酸組成。
圖十五、BAC clone contig 39 與EST特異序列比對結果。
圖十六、人類IL-1RAcP基因exon 13的序列重組現象。
圖十七、偵測人類正常肝細胞與293T 細胞株中IL-1RAcP687的表現。
圖十八、偵測人類各組織中IL-1RAcP687的表現 。
圖十九、IL-1RI與IL-1RAcP的交互作用。
圖二十、CAT assay。
柒、 附錄…………………………………………………………....105
一、 質體構築圖。
二、 圖十六、十七及十八之PCR condition整理。
三、 IL-1RAcP與MyD88之交互作用。
四、 IL-1RAcP與Tollip之交互作用。
五、 以293、293T細胞株探討IL-1RI與IL-1RAcP570對於IL-1誘發NF-B活化之結果比較。
六、 未列入論文之其它質體構築圖。
七、 Slide與照片的製作流程。

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