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Title: | 鵝眼球 δ-水晶體蛋白基因之定序分析及其在大腸桿菌表現產物之特性研究 Facile Cloning , Sequencing , and Expression of δ-Crystallin Gene from Goose Lenses |
Authors: | Chih-Wen Yu 尤志文 |
Publication Year : | 1993 |
Degree: | 碩士 |
Abstract: | δ-水晶體蛋白是存在於鳥類和爬蟲類眼球中的主要水晶體蛋白。?了快速選殖定序鵝之δ-水晶體蛋白基因,於是便利用簡易快速的PCR技術從鵝眼球水晶體cDNA混合液中將δ-水晶體蛋白基因增殖。然後將增殖?物接上pUC19載體,殖入大腸桿菌JM109中繁殖。篩選後,取三個含有約1.4kb插入基因的菌落予以核酸定序。結果得到一條1,401bp完整的δ-水晶體蛋白基因,包含了466個胺基酸序列。在與其他鳥類δ-水晶體蛋白胺基酸序列比較後,發現有很大的相似性。其分別與鴨之 δ1 、δ 2 水晶體蛋白有97%.94% 相似性;與雞之δ 1 、δ 2 水晶體蛋白有88% ,90% 相似性;與鴿子之δ 水晶體蛋白有88% 相似性;而與人之精氨琥珀酸分解酵素只有69%相似性。因籍由陰離子交換樹脂純化出來的鵝δ-水晶體蛋白具有與鴨δ-水晶體蛋白相當的精氨琥珀酸分解酵素活性。而有高相似胺基酸序列的雞與鴿子δ-水晶體蛋白卻無比活性。為進一步研究分析,便將得到的鵝δ-水晶體蛋白基因接上pQE31表現載體,與大腸桿菌M15中大量表現,並對表現?物特性予以探討。 δ - Crystallin is a major lens protein in the avian and reptilian lenses. To facilitate the cloning of the δ-crystallin gene, cDNA was constructed from the poly(A)+RNA of goose lenses, amplified by the polymerase chain reation (PCR). The PCR product was then subcloned into pUC19 vector and transformed into E. coli strain JM109. Plasmids purified from the positive clones were prepared for nucleotide sequencing by the dideoxynucleotide chain-termination method. Sequencing three clones, containing 1.4 kb DNA inserts coding for δ-crystallin allowed the construction of a complete, full- length reading frame of 1,417 bp covering a deduced protein sequence of 466 amino acids, including the universal translation- initiating methionine. The goose δ-crystallin shows 97, 94, 88, 90 and 69% sequence identity to duck δ1, δ2, chicken δ1, δ2 crystallins and human argininosuccinate lyase respectively. Purified δ-crystallin has previously been shown to possess the enzymatic activity of argininosuccinate lyase. For further analysis, the expression of the δ-crystallin cDNA was carried out in the bacterial expression plasmid, pQE-3 1 , and the δ-crystallin protein produced in E. coli was purified to homogeneity to determine the enzymatic properties. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76013 |
Fulltext Rights: | 未授權 |
Appears in Collections: | 生化科學研究所 |
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