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Title: 巴斯德桿菌磷脂質分解酵素的分子選殖與表現
Molecular cloning and expression of a phospholipase gene in Photobacterium damselae subsp. piscicida
Authors: Po-Yuan Hsu
Contributors: NTOU:Department of Aquaculture
Keywords: 巴斯德桿菌;磷脂質分解酵素;分子選殖;蛋白質表現
Photobacterium damselae subsp. piscicida;phospholipase;Molecular cloning;protein expression
Date: 2005
Issue Date: 2011-06-30T08:40:57Z
Abstract: 本論文以原核系統來表現巴斯德桿菌 Photobacterium damselae subsp. piscicida 的 phospholipase gene,利用基因重組方式生產 phospholipase,希望能藉由簡單的原核系統生產 phospholipase,進而取代以往較為繁雜的純化步驟。   以 PCR 方式從巴斯德桿菌夾取大小為 1218 bp的片段,將此片段載入 pGEMT-easy Vector中,以EcoRⅠ 和NdeⅠ限制內切酶切下此片段,載入 pET-28b (+) vector 中,完成表現質體之建構。   將建構完成之菌株加入濃度為 100 mM IPTG 進行誘導,將實驗分為控制組(包括:Rosetta TM (DE3)、pET-28b (+)、沒有加入 IPTG 組-0 小時)、實驗組(誘導1~4 小時),將其分為三種不同性質蛋白質(total protein、soluble、insoluble)分別以 SDS-PAGE 電泳分析由 PAGE 圖發現,誘導 1~4 小時組別中有蛋白質被誘導出來,由可溶性蛋白(soluble)、不可溶性蛋白(insoluble)電泳圖中發現,此誘導蛋白質會以包涵體的形式存在。   經由DNA片段大小預測 phospholipase 應可轉譯成 45kDa蛋白質。但由SDS-PAGE 圖發現,誘導蛋白質出現在42 kDa位置。由蛋白質定序及 N 端定序結果發現,1-20 個胺基酸未在被誘導蛋白質中出現,因此推測此 1-20 胺基酸可能為 signal sequence。因此扣除20 個胺基酸(2.2 kDa)後,大小則和結果相似。 用含0.3% egg yolk 膠片覆蓋 Native-PAGE 後發現誘導蛋白質不具有磷脂質分解酵素活性。   誘導蛋白質藉由Amplex Red Phosphatidylcholine-Specific Phospholipase C Assay Kit 後判斷,實驗組皆比負控制組及控制組的吸光值來得高,顯示經由誘導之後 phospholipase C 的濃度有增加的趨勢,但是否為 phospholipase C,則需進一步的實驗來佐證。   由兔子抗 phospholipase C 血清,進行western blotting、ELISA 之分析,結果發現控制組與實驗組皆有反應,以誘導4小時反應最為強烈,推測大腸桿菌和巴斯德桿菌皆具 phospholipase ,皆能和兔子抗 phospholipase C 血清結合,且由吸光值增加的趨勢來判斷,誘導的蛋白質應該為phospholipase C。
This thesis studied the expression of phospholipase gene of Photobacterium damselae subsp. piscicida in prokaryotic system, the utilization of gene recombination to produce phospholipase, and the possibility of producing phospholipase in the simple prokaryotic system to replace the comparatively miscellaneous purification step which employed in the past. Using PCR to fetch an 1218 bp fragment from Photobacterium damselae subsp. piscicida, and then inserted this fragment in a pGEMT-easy Vector. Restriction endonucleases, EcoR Ⅰ and Nde Ⅰ, were used to cut down this fragment, and phospholipase gene was loaded into a pET-28b(+) vector to build and construct an expression plasmid. The constructed bacterial strain induced by the addition of 100 mM IPTG. The tests were divided into control groups(including:Rosetta TM (DE3)、pET-28b (+)、non-IPTG group-induced for 0 hr)、experimental groups(induced for 1∼4 hr). Three kinds of different proteins(total protein、soluble、insoluble)were of tained by using SDS-PAGE electrophoresis analysis. As found on PAGE profile, there were protein induced for 1∼4 hours. As found on electrophoresis profile, there were soluble protein(soluble)and non- soluble protein(insoluble)existed . The induced proteins existed in a form of includion body. The size of DNA of phospholipase might transfer and translate into a 45kDa protein. As found on SDS-PAGE picture, however, a 42kDa protein was visualized. As the protein and N-Terminal sequenced, and an 1-20 amino acid fragment was not induced and was suggested to be a signal peptide. After deletion of 20 amino acid (2.2 kDa), the size of the predicted 45kDa was almost the same as size of 42kDa. By overlaying a 0.3% egg yolk film on Native –PAGE, there was on phospholipase activity detected. The induced ptoteins were assayed by using a Amplex Red Phosphatidylcholine -Specific Phospholipase C Assay Kit. The experimental group exhibited higher absorbance than the control and the constrast groups. The results reveraled that the phospholipase C could be induced, however, the exact induction of phospholipase C needs further experiment to confirm. The used of rabbit anti phospholipase C antiserum in western blotting and ELISA assay resulted in positive reactions suggested that the enzyme may exist in both of control and experiment groups. The strongest activity was induced for 4 hours. Hence, both Escherichia coli and Photobacterium damselae subsp. piscicida may all seret phospholipase and these phospholipase could cross react with the rabbit anti phospholipase C antiserum. From increase of absorbance, the protein that induced should be phospholipase C.
URI: http://ethesys.lib.ntou.edu.tw/cdrfb3/record/#G0M93330017
Appears in Collections:[水產養殖學系] 博碩士論文

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