臺灣博碩士論文加值系統

摘要

本論文針對溶藻弧菌 （Vibrio alginolyticus） 及相近之病原弧菌Vibrio parahaemolyticus、Vibrio harveyi、Vibrio carchariae 等菌株設計專一性引子，以聚合酶連鎖反應 （PCR） 技術，作親源關係及致病機制之研究探討。各弧菌被比較鑑定的對象為 23S rRNA 及菌體之熱休克蛋白（heat shock proteins, HSPs）。
以 23S rRNA 相關之 23S-based 引子進行 PCR 反應，所得到的產物大小約450 bp，之後利用定序載體構築並進行選殖，選取具有插入片段的殖株進行定序工作。將所得到 443 bp 大小的 23S rRNA 基因序列跟 NCBI 資料庫已知之弧菌 23S rRNA 序列進行比對分析。並利用 GCG 的 GrowTree Program 作出演化樹型圖，得知 Vibrio alginolyticus 其品系（strain）間親源關係上大致相近； V. parahaemolyticus 則和 V. alginolyticus 的關係最為相近； 而 V. harveyi 和 V. carchariea 的親源相近。將 23S rRNA 進行比對後均有88％以上之相似性，說明弧菌屬間 23S rRNA 的相似性極高。
再以熱休克蛋白相關 HSP60 F1/HSP60 R2 引子進行 PCR 反應，所得到的產物大小約450 bp，定序後得到 485 bp 大小的 hsp60 核苷酸基因序列，跟 NCBI 資料庫已知之弧菌 hsp60 核苷酸序列進行比對分析。並利用 GCG的 GrowTree Program 作出演化樹型圖，得知 V. alginolyticus 品系間關係相近，但致病性強之品系 E：6-2 及 A1I1 互相相近，但與其他種關係較獨立；而利用 23S rRNA 基因序列較不易鑑定出種間差異性之 V. parahaemolyticus、V. harveyi及V. carchariae，從hsp60核苷酸基因序列則可容易鑑定其差別。
將所得 hsp60 之核苷酸轉譯成胺基酸序列後，經 NCBI 資料庫比對搜尋後，與其他物種之 GroEL 蛋白質有極高之相似性，這種蛋白質普遍存在於多種細菌中。Heat shock proteins (HSPs) 已知在許多的病原體的致病過程中會引起寄主強烈免疫反應。之前的研究中發現經過熱休克後，菌體會產生熱休克蛋白質同時致病力也有上升的趨勢，為瞭解是何種熱休克蛋白質具有這樣的功能，故利用分子生物的方法分析其胺基酸基因序列，以便進一步瞭解其蛋白質胺基酸序列與其在致病力之相關性，往後可更近一步將其表現繼續研究探討。

Abstract

The thesis investigated the relatedness of phylogeny and pathogenicity among pathogenic Vibrio species including V. alginolyticus, V. parahaemolyticus, V. harveyi, V. carchariae by using PCR technology. Various specific primers were designed for the target-genes amplification of 23S rRNA and heat shock proteins for comparison.
A 450 bp product was obtained after PCR reaction using 23S-based primer for gene targeting. Then, the product was vector constructed and cloned in E. coli DH5α. A 443 bp inserted fragment of 23S rRNA was selected and sequenced. The gene sequence was compared with known sequences in NCBI GeneBank. The GCG GrowTree Program was employed to construct phylogenetic trees. The majority of the V. alginolyticus strains were closely genetic related. Strains of V. parahaemolyticus and V. alginolyticus, and strains of V. harveyi and V. carchariae are closely related. The DNA sequences and alignment of all the Vibrio species had at least 88% relatedness.
In addition, a 450 bp product was obtained after PCR reaction using HSP60 F1/HSP60 R2 primer for gene targeting. Then, the product was also vector constructed and cloned in E. coli DH5α. A 485 bp inserted fragment of hsp60 nucleotide was selected and sequenced. The gene sequence was compared with known sequences in NCBI GeneBank. The GCG GrowTree Program was used to construct phylogenetic trees. The majority of V. alginolyticus strains were approximately genetic related except the two more virulent strains, E：6-2 and A1I1, both were closely related and independent from other Vibrio species. The results revealed that using hsp60 nucleotide sequences could easily identify the species of V. parahaemolyticus and V. alginolyticus, and species of V. harveyi and V. carchariae.
The obtainded hsp60 nucleotide sequences were translated into amino acid sequences, then serched with NCBI Blast. The sequences were highly related to GroEL of other Vibrio species. This protein was generally present in various bacteria. Heat shock proteins of many pathogens may cause overreaction of the host immune system. Some reference indicated that the virulene of pathogenic bacteria can be induced after heat shock. It is necessary to clone and express the protein in a further future study.

目錄

u 中文摘要 ---------------------------------------------------------------------- I
u 英文摘要 -------------------------------------------------------------------- III
u 表目錄 --------------------------------------------------------------------- VIII
u 圖目錄 ----------------------------------------------------------------------- IX
u 一、前言 ----------------------------------------------------------------------- 1
u 二、文獻整理 ----------------------------------------------------------------- 6
u 三、材料與方法 ------------------------------------------------------------- 21
Ø 菌種來源及保存 ----------------------------------------------------- 21
¬ 菌株來源 -------------------------------------------------------- 21
¬ 菌株保存 -------------------------------------------------------- 21
¬ 菌株確認 -------------------------------------------------------- 22
Ø 細菌細胞外產物活性之分析 -------------------------------------- 23
¬ 細菌細胞外產物之備製 -------------------------------------- 23
¬ 蛋白分解酵素活性測定 -------------------------------------- 23
Ø Vibrio alginolyticus sp.對石斑魚之毒性攻擊試驗 ------------- 23
¬ 細菌對石斑魚之毒性試驗 ----------------------------------- 23
Ø Vibrio spp. 23S rRNA、熱休克蛋白基因序列分析 ------------- 24
¬ 染色體去氧核醣核酸之萃取 -------------------------------- 24
¬ 引子之設計 ----------------------------------------------------- 25
¬ 聚合酶連鎖反應（polymerase chainreaction, PCR）---- 26
¬ 洋菜膠體電泳 -------------------------------------------------- 27
¬ 膠體萃取 -------------------------------------------------------- 27
¬ 備製勝任細胞 -------------------------------------------------- 28
¬ 定序載體的構築（Construction）與選殖（Selection）- 29
¬ 接合作用（Ligation）----------------------------------------- 29
¬ 轉形作用（Transformation）--------------------------------- 30
¬ 篩選（Selection）---------------------------------------------- 30
¬ 重組質體DNA之備製（Plasmid Miniprep）------------- 30
¬ 目標片段之定序 ----------------------------------------------- 31
u 四、結果 ---------------------------------------------------------------------- 32
Ø 細菌之生化特性 ---------------------------------------------------- 32
Ø Vibrio alginolyticus 菌株對石斑魚之毒性攻擊試驗 --------- 32
Ø Vibrio spp.之親源性研究分析 ------------------------------------ 33
¬ 16-based 引子PCR結果 ------------------------------------- 33
¬ 23-based 引子PCR結果 ------------------------------------- 33
Ø Vibrio spp. 之 heat shock protein 60（HSP 60）基因序列分
析 ---------------------------------------------------------------------- 36
¬ 利用 PCR 擴增 Vibrio alginolyticus 部分hsp 60核苷酸
基因序列結果 ------------------------------------------------- 36
¬ Vibrio spp. HSP60胺基酸序列與已知弧菌之胺基酸序列
列排列比較 ----------------------------------------------------- 37
u 五、討論 ---------------------------------------------------------------------- 42
u 六、參考文獻 ---------------------------------------------------------------- 48
u 表 ----------------------------------------------------------------------------- 59
u 圖 ----------------------------------------------------------------------------- 71

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