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標題: | 鯉魚視紫質蛋白的表現 Expression of Common Carp(Cyprinus carpio) Rhodopsin |
作者: | 鄭俊和 |
出版年 : | 1997 |
學位: | 碩士 |
摘要: | 鯉魚(Cyprinus carpio)是日常生活中常見可以生活在渾濁水池的魚,為了瞭解其視覺生理,將已被篩選到的鯉魚視紫質(rhodopsin) cDNA以PCR轉錄區接入大腸桿菌表現載體,構築成pET32-Rho。這個重組視紫質的胺端與thioredoxin接成融合蛋白,銜接處並有6個組胺酸(histidine)。以大腸桿菌BL21(DE3)為寄主細胞,嗜菌體T7的促進子驅動並用IPTG誘發表現視紫質。細胞抽出液經過鎳離子管柱層析(Ni2+ affinity column chromatography)後,進行蛋白質電泳分析,發現在分子量約55kD的地方有一條明顯的band,用抗牛的視紫質的多株抗體(CERN886)進行Western blot分析,並沒有反應。分子量正確及親和性管柱層析的高專一性應可認定其為正確的融合蛋白,亦構築好的表現載體可以在大腸桿菌中成功的表現。另外以真核系統方面:所得的轉錄區接入轉型桿狀病毒載體,再與線狀的病毒DNA共同感染Sf9昆蟲細胞,篩選重組病毒及放大濃度及2次純化感染Sf9昆蟲細胞(MOI=5),72小時後,收集細胞並處理,然後進行蛋白質電泳分析,結果在58kD、40kD和36kD有3條明顯的band,以相同的多株抗體進行western blot分析,都有正反應,其中40kD的蛋白可能是重組視紫質。表示視紫質的cDNA可以被病毒大量表現,得到重組的視紫質蛋白。綜合以上的實驗,無論大腸桿菌或是真核系統都可以得到重組視紫質這種蛋白,這樣的方法建立後,將有利於視紫質的生物活性的測試,進而瞭解鯉魚受光蛋白在視覺生理上的意義。 In order to understand the physiology of the fish vision, we constructed a plasmid, pET32-Rho, which encoded the coding region of rhodopsin cDNA and was driven by T7 promoter. The recombinant rhodopsin is a fusion protein with thioredoxin and 6 histidine residues at the amino terminus. After IPTG induction, the cell extracts of Escherichia coli BL21(DE3) containing pET32-Rho was collected and run through the Ni2+ affinity column chromatography, and then their proteins were analyzed by SDS-polyacrylamide gel electrophoresis . Results demonstrated that a band with molecular mass of 55 kD was shown on the gel, but no positive reaction for western blot analysis by using anti-bovine rhodopsin polyclonal antibody(CERN886). The coding region of rhodospin cDNA was inserted into the baculovirus transfer vector, pBAC-Rho, under the control of the polyhedrin promoter. Linearlized virus DNA and the construct plasmid were co-infected Sf9 cells. Cells infected by recombinant virus were screened, purified and amplified. Extracts of insect cells infected with the recombinant virus at MOI of 5 after 72 hours. postinfection we recollected and treated with histidine-bind column.. Three major bands of 58 kD, 40 kD, and 36 kD were shown on the gel. However, they were all immunologically positive with the polyclonal antibody (CERN886) in which the 40 kD band was excepted as recombinant rhodopsin. Further confirmation study remains to be investigated. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/75011 |
全文授權: | 未授權 |
顯示於系所單位: | 漁業科學研究所 |
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