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標題: | 小白鼠貯精囊自體抗原基因的初步研究 Preliminary study of the gene structure of mouse seminal vesicle autoantigen |
作者: | 楊允馨 |
出版年 : | 1994 |
學位: | 碩士 |
摘要: | 自小老鼠genomic library篩選出含貯精囊自體抗原(SVA)基因的genomic clone ;我們定序了SVA基因 1016 個核?酸序列:包括第一個exon (122 個核?酸),第一個 intron 的部份序列( 172個核?酸)及 5'端區域( 5'-flanking region)之722個核?酸的序列。由primer extension的結果定出基因的轉錄起始位置在相對於轉譯起始位置5'端上游第27(A)及第28(A)個核?酸。
我們已知雄性素能刺激SVA在小白鼠貯精囊中的表現,由SVA基因之核?酸序列亦發現在SVA基因的5'端區域有兩段和保守的雄性素反應單元(ARE)相似性極高的DNA單元(單元 A, AGAACAaag AGT-GTG,-124?-110;單元B, AGAACAttc TAATCT,-213?-199)。兩單元和保守的雄性素反應單元(consensus ARE sequence: AGAACAnnn TGTTCT)的相似性分別為66.7%(單元A)和83.3%(單元B)。 將合成的雙股DNA(分別含有單元A或單元B)與貯精囊核蛋白質抽取液進行mobility shift DNA bindig assay ,此兩DNA單元皆可和某貯精囊核蛋白質呈現專一性的結合。經由兩單元和此核蛋白質互相競爭結合的實驗,顯示單元B對此核蛋白質的結合強度大於單元A。 為了進一步的檢視SVA基因上的單元A和單元B是否確實具有androgen-dependent transcriptional enhancer 的活性,我們利用帶有CAT基因做為reporter的兩個質體(pCAT-Basic, pCAT-Promoter),將SVA基因5'端區域的不同段落或單獨的單元A或單元B分別鑲入其中,建構了兩組重組的質體,預備進行transcriptional activity assay。 我們選用LNCap細胞做為CAT-fusion plasmid的表現細胞。初步以positive control(pCAT-Control vector)測試的結果,顯示目前所建立的細胞培養,transfection及CAT assay之系統為可行。未來的工作即是要對我們建構的兩組質體進行測試,以期對SVA受雄性素調控的機制有進一步的瞭解。 From the analysis of a genomic clone screened from mouse geomic DNA library, 1016-bp of SVA gene was established. They covered the first exon (122bp), part of the first intron (172bp) and the upstream 722bp from transcriptional start site. Sequence analysis revealed two putative AREs as follows: one between position -124 and -110 (element A, EA) and the other between -213 and -199 (element B, EB)。EA (AGAACAaagAGTGTG) and EB (AGAACAttcTAATCT) have 66.7% and 83.3% sequence homology with the consensus ARE (AGAACAnnnTGTTCT). Nuclear protein extract of seminal vesicle was able to retard the synthetic EA or EB as evident from mobility shift DNA binding assay The interaction between either elements and nuclear protein was specific and EB showed stronger than EA in its affinity. In order to test if EA or EB is able to act as an androgen-dependent transcriptional enhancer, series of SVA 5'-flanking fragments were ligated to pCAT-Basic while EA or EB fragment was inserted into pCAT-Promoter. The constructed CAT-fusion plasmids could be introduced into LNCaP cells to examine the androgenic inducibility of transcriptional activity. The systems of cell culure, transfection and CAT assay have been successfully established. Future work will be the test of these constructed CAT-fusion plasmids to obtain more insight into the mechanism of androgen action on SVA. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76076 |
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顯示於系所單位: | 生化科學研究所 |
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