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研究生:陳志銘
研究生(外文):Tz-Ming Chen
論文名稱:洛德乳酸桿菌染色體訊號序列的轉載及定序
論文名稱(外文):Cloning and sequencing of chromosomal signal sequences from Lactobacills reuteri
指導教授:張登欽張登欽引用關係
指導教授(外文):Tung-Ching Chung
學位類別:碩士
校院名稱:國立中興大學
系所名稱:獸醫病理學研究所
學門:獸醫學門
學類:獸醫學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:72
中文關鍵詞:乳酸菌洛德乳酸桿菌訊號序列探針載體疫苗攜帶者西方雜合反應Exonuclease Ⅲ 酵素澱粉脢
外文關鍵詞:Lactic acid bacteriaLactobacills reuterisignal sequenceprobe vectorvaccine carrierWestern blotExonuclease Ⅲ enzymeamylase
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乳酸菌( Lactic acid bacteria )長久以來即應用於人類食品及動物飼料中,除具有預防保健功能外,並對於治療腸炎等疾病皆具有一定的療效,因此近年來開發乳酸菌作為疫苗攜帶者(vaccine carrier)的嘗試廣泛受到重視。本研究之目的為藉由實驗室所開發出來的訊號序列探針載體(probe vector),尋找洛德乳酸桿菌(Lactobacillus reuteri)之訊號序列,俾作為本菌未來建造疫苗攜帶者之基礎。
訊號序列之篩選過程,除了利用2種限制酵素(AluⅠ, PvuⅡ)切割染色體DNA進行篩選外,另外則嘗試利用Exonuclease Ⅲ 酵素加強處理,希望能夠提高找尋訊號序列之機會。由結果得知,單以限制酵素處理者,共獲得6株菌株(AluⅠ, 5株 ; PvuⅡ, 1株),而以Exonuclease Ⅲ 酵素加強處理者,則亦獲得6株菌株(AluⅠ, 2株 ; PvuⅡ, 4株)。分析上述所獲得的12株菌株之α-amylase protein活性,結果發現,A1及P315兩株菌不論是在形成澱粉使用環圈 ( zone )的大小、時間,或者是菌株上清液之α-amylase protein活性結果,皆具有強烈反應,推測原因可能是所篩選到的訊號序列片段,含有一段功能強大之promoter sequence所致。應用西方雜合反應(Western blot)及基因序列的判斷,本次實驗共篩選到2株(A7, P402)含有訊號序列之菌株。將所篩選到的訊號序列與已所發表之訊號序列比較,結果發現這2株菌株所篩選到的序列,均屬於非典型之訊號序列。整體而言,雖然實驗結果所找尋到的訊號序列並非經Exonuclease Ⅲ 酵素處理所找尋到,然而Exonuclease Ⅲ 酵素的加強處理,對於訊號序列的尋找,是有增加找尋的機會。

Lactic acid bacteria has long been added in human food and animal fodder and is believed to have efficacies in treating gastroenteritis as well as being beneficial to the health. Consequently, the development of Lactic acid bacteria as a vaccine carrier has also been widely regarded as a very important an potential direction.
The aim of this study was to search signal sequences from the chromosome of Lactobacillus reuteri, with which an expression— secretion vector will be able to be constructed and the final goal of developing Lactobacillus reuteri as a vaccine carrier will be feasible.
Two strategies, including using 2 kinds of restriction enzyme ( i.e., AluⅠor PvuⅡ) alone and an extra-treatment of restriction enzyme-treated chromosome DNA with Exonuclease Ⅲ to enhance the probability of cloning signal sequences, were employed in this study. As a result, a total of 6 clones from restriction-treated DNA alone ( i.e., 5 clones from AluⅠ-digestion and 1 clones from PvuⅡ-degestion ) and 6 clones from an extra-Exonuclease Ⅲ treated DNA ( i.e., 2 clones from AluⅠdigestion and 4 clones from PvuⅡdigestion ) were obtained. Further analysis of α-amylase activity in the supernatant as well as in the agar-solid media of these 12 clones were able to confirm 2 clones ( i.e., A1 and P315 ) with the best expression-secretion capability, suggesting strong promoters were cloned in the probe vector.
Although comparison of the nucleotide sequence between the cloned DNA fragments and the database in GCG bank were not able to identify any standard signal sequences, employment of the extra-Exnuclease Ⅲ digestion seemed to be capable of increasing the probability of cloning expression-secretion functional DNA fragment from the chromosome.

中文摘要---------------------------------------------------- Ⅰ
英文摘要---------------------------------------------------- Ⅱ
目錄 ---------------------------------------------------- Ⅲ
圖次 ---------------------------------------------------- Ⅳ
表次 ---------------------------------------------------- Ⅵ
第一章 前言--------------------------------------------- 1
第二章 文獻探討----------------------------------------- 2
第一節 乳酸菌簡介------------------------------------ 3
第二節 乳酸菌在動物體中扮演之角色-------------------- 3
第三節 洛德乳酸桿菌 ( Lactobacillus reuteri )之特性-- 5
第四節 訊號序列 ( signal sequence )------------------ 8
第五節 訊號序列之分子特性---------------------------- 9
第六節 訊號序列之分類-------------------------------- 11
第七節 訊號序列之選殖-------------------------------- 12
第三章 材料與方法
第一節 菌株及訊號序列探針載體------------------------ 17
第二節 菌株培養基------------------------------------ 17
第三節 菌株培養-------------------------------------- 17
第四節 洛德乳酸桿菌Chromosome DNA之抽取-------------- 17
第五節 E. coli質體之抽取----------------------------- 18
第六節 限制酵素的選擇-------------------------------- 20
第七節 Chromosome DNA與載體之接合反應---------------- 20
第八節 chromosome DNA之縮減反應---------------------- 21
第九節 Signal sequence之初步轉載--------------------- 23
第十節 電孔 ( Electroporation ) 轉形質體試驗--------- 25
第十一節 轉形菌株之α-amylase活性分析------------------ 26
第十二節 各轉形菌株分泌α-amylase蛋白之確認------------ 27
第四章 實驗結果
第一節 限制酵素之選擇結果---------------------------- 34
第二節 ExonucleaseⅢ酵素之效率測定------------------- 34
第三節 訊號序列菌株之初步篩選結果-------------------- 35
第四節 各轉形菌株之Insert DNA片段大小分析------------ 37
第五節 α-Amylase活性分析----------------------------- 37
第六節 血清學分析------------------------------------ 38
第五章 討論 ------------------------------------------ 61
文獻參考 ------------------------------------------------- 66

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