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研究生:馮任民
研究生(外文):Jeng-Ming Fong
論文名稱:哈威弧菌中致病因子溶血素之分子選殖與表現
論文名稱(外文):Molecular colning and expression of virulent hemolysin from vibrio harveyi
指導教授:吳東昆
指導教授(外文):Tung-Kung Wu
學位類別:碩士
校院名稱:國立交通大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
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
中文關鍵詞:哈威弧菌溶血素
外文關鍵詞:vibrio harveyihemolysin
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弧菌症是由弧菌屬細菌所感染的疾病,會造成海洋生物養殖大量的死亡,在許多由弧菌屬細菌引起的疾病中,哈威弧菌被報導是對虎明蝦養殖最主要的病原菌。迄今對於哈威弧菌的病原檢測還是根據其顯型特徵與特殊篩選培養基來做作為判斷。然而傳統方法有其時效與成本之考量,因此發展一快速檢測弧菌感染病原菌的方法是有必要的。
由於最普遍存在於弧菌屬的致病因子溶血素會造成宿主出血性敗血症和腹瀉,我們希望藉由分子生物技術達成分子選殖與表現哈威弧菌溶血素並分析其特性。溶血基因的開啓閱讀區域含有1254核苷酸與預定轉譯成418胺基酸,並推算有47kDa分子量大小。我們已成功地建立核酸探針檢測方式,並將帶有EcoRI與SalI限制酶的溶血素核苷酸片段分別選殖到pMAL-c2表現系統和轉殖到XLI-blue細胞。溶血素MBP融合重組菌株在TSA含血培養基上表現其溶血活性。

Vibriosis, caused by bacteria of the genus vibrio, is a major cause of death in maricultural operations. Of the several bacterial diseases caused by vibrio spp., vibrio harveyi has been reported as a primary pathogen of cultured penaeid shrimp. To date, identification of V. harveyi has centered on phenotypic methods and specialized growth media. However, the phenotypic approach is inherently slow. Therefore, rapid techniques are necessary to improve the detection and diagnosis of Vibrio spp. infections.
Because of the most common virulent factor hemolysin causing hemorrhagic septicemia and diarrhea in the host, we will try to clone and express hemolysin gene of vibrio harveyi (vhh) by molecular biotechnical methods towards assessing the characterization of vhh. The open reading frame (ORF) of vhh was found to be 1,254 nucleotides and predicted to encode a polypeptide of 418 amino acids with a deduced molecular mass of ~47 kDa. An EcoRI/SalI digested DNA fragment was inserted into pre-digest pMAL-c2 expression vector and subsequently transformed into Escherichia coli XL1-Blue cells. The recombinant E. coli cells showed hemolytic activity on the TSB sheep blood agar plate.

Table of Contents
Page
中文摘要……………………………………………………………………………….I
Abstract ……………………………………………………………………………….II
Acknowledgement……………………………………………………………………III
Table of Contents ……………………………………………………………………IV
Table of figures……………………………………………………………………....VI
Table of tables……………………………………………………………………….VII
Chapter 1. Introduction
1.1 Vibriosis………………………………………..…….……………….………….1
1.1.1 Vibrio anguillarum……………………………………………………...2
1.1.2 Vibrio cholera…………………………………………………………..3
1.1.3 Vibrio parahaemolyticus………………………………………………..4
1.1.4 Vibrio vulnificus………………………………………………………...5
1.2 Vibrio harveyi…………………………………………………………………….6
1.3 Virulence factors………………………………………………………………….8
1.4 Preventing infection……………………………………………………………...14
1.5 Objectives of research……………………………………………………………17
Chapter 2. Materials and Methods
2.1 Materials…………………………………………………………………………18
2.1.1 Strains and vectors………………………………………………………...18
2.1.2 Reagents………………...………………………………………………...18
2.1.3 Buffers and solutions……………………………………………………...18
2.1.4 Equipments………………………………………………………………..20
2.2 Methods…………………………………………………………………………..21
2.2.1 Overview……………………………………………………………….....21
2.2.2 Culture of stock strain………………………………………………….....21
2.2.3 Isolation of genomic DNA………………………………………………..22
2.2.4 Polymerase chain reaction………………………………………………...23
2.2.5 Construction of recombinant DNA……………………………………….25
2.2.6 DNA sequence…………………………………………………………….26
2.2.7 Protein methods…………………………………………………………...27
2.2.8 Hemolysis assay…………………………………………………………..29
Chapter 3. Results and Discussion
3.1 Vibrio harveyi culture…………………………………………………………….30
3.2 Isolation of genomic DNA……………………………………………………….31
3.3 Polymerase chain reaction………………………………………………………..32
3.4 Construction of recombinant DNA……………………………………………....35
3.5 DNA sequence……………………………………………………………………36
3.6 Expression of MBP fusion protein……………………………………………….37
3.7 Hemolytic activity………………………………………………………………..37
Reference………………………………………………………………...38
Appendix
Appendix 1……………………………………………………………………...43
Appendix 2……………………………………………………………………...44
Appendix 3……………………………………………………………………...47
Appendix 4……………………………………………………………………...48
Appendix 5……………………………………………………………………...50
Appendix 6……………………………………………………………………...51
Appendix 7……………………………………………………………………...52
Appendix 8……………………………………………………………………...53
Table of Figures
Figure 1.1 Vibrio harveyi……………………………………………………………..1
Figure 1.2 Red patches around the base of the fins in pathogenic fish……………….3
Figure 1.3 Cholera toxin mechenism…………………………………………………4
Figure 1.4 Shrimp life cycle…………………………………………………………..7
Figure 1.5 Schematic diagram of a lipopolysaccharide molecule…………………….8
Figure 1.6 Biological iron uptake, transport and storage………………………….....10
Figure 1.7 Hemolysin structure and mechanisms……………………………………12
Figure 1.8 Construction of immunochromatography system………………………...16
Figure 1.9 Flow chart of research steps………………………………………………17
Figure 2.1 Bacteria culture…………………………………………………………...22
Figure 2.2 Restriction map and factor Xa cleavage site of the recombinant vector pJMFVHHMAL………………………………………………………..26
Figure 2.3 The procedure of the experiment…………………………………………29
Figure 3.1 Vibrio harveyi culture in TCBS medium…………………………………30
Figure 3.2 Genomic DNA isolation using QIAamp Boold kit and QIAamp tissue kit and traditional methods…………………………………………………31
Figure 3.3 Real-time PCR products………………………………………………….33
Figure 3.4 Amplification of the vhh gene……………………………………………34
Figure 3.5 Mapping of pJMFVHHIISK recombinant DNA…………………………35
Figure 3.6 Mapping of pJMFVHHMAL-c2 recombinant DNA…………………….36
Figure 3.7 Recombinant clones showed the hemolytic activity on blood agar………37
Table of Tables
Table 1.1 Different infections of aquacultural species……………………………….2
Table 1.2 Vibrio spp. each has different virulence factors…………………………..11
Table 2.1 Primers used in these experiments………………………………………..24
Table 2.2 Compositions of SDS-PAGE……………………………………………..28
Table 3.1 Optimal condition of Vibrio strain culture………………………………..31

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