臺灣博碩士論文加值系統

對人類所引起急性腸炎的非傷寒沙門氏菌中，最常見的是血清群B（serogroup B），但近年來血清群D（serogroup D）有增多的趨勢，主要為腸炎沙門氏菌（Salmonella enterica serovar Enteritidis），其中又以噬菌體分型第4型（phage type 4, 簡稱PT4）最多，不過第34型（PT34）正逐漸增多，而且在流行病學資料分析顯示第34型較傾向於造成侵襲性感染。在PFGE分型上，第一型大部分為PT4，而第二型大部分為PT34，而且第一型且為PT4與第二型且為PT34之間PFGE分型的比較是非常相似的，我們發現在PFGE分型上，第二型比第一型多出的條帶是來自於質體pSE34，進一步，將pSE34解序，發現此質體與S. Dublin的兩個質體pOU1114與pOU1115相似度非常高，這三個質體在演化上應屬於同源。
pSE34序列全長為32,950 bp，G+C含量佔41.2%，明顯比腸炎沙門氏菌PT4全基因體的52.17%還低，此質體應屬於外來核酸序列。由複製起始位置的序列比對，pSE34和大腸桿菌質體R485和R6K的相似度分別為96%和84%，因此推測也是IncX group。質體pSE34經序列分析，具有53個ORF，不過大部分為未知基因，blastx的結果發現此質體具有三個操縱組（operon），分別為stbDE、pilX和parFG，另外還有DNA distortion peptide 1、2和3，以及taxB、tnpR和hns等基因。其中pilX操縱組的基因表現的蛋白質相似於第四型分泌系統（type IV secretion system），進一步，我們以Tn5 transposon進行隨機突變，並且分析接合（conjugation）能力，結果顯示，類似於第四型系統的pilX操縱組和taxB基因與接合有關，另外突變在DNA distortion peptide 2和tnpR也會影響接合生殖。進一步為了確認PT34是否有異於PT4的侵襲性能力，我們藉由HEp-2、THP-1和MDCK細胞株，分別探討對細胞的侵犯（invasion）能力、巨噬細胞中存活（intracellular survival）能力和對細胞的穿透（penetration）能力。初步結果顯示，PT34與PT4此三項能力並沒有顯著差異。

Salmonella serogrop B is the most common nontyphoidal Salmonella infecting humans worldwide. However, the incidence of serogroup D infections has been increasing in Taiwan over the years. S. Enteritides is among serogroup D the most frequent serotype isolated. In S. Enteritidis, phage type 4 (PT4) was the common phage type that already caused a global pandemic in the past years. Epidemiological analysis from Chang Gung Memorial Hospital and Children’s Hospital showed that S. Enteritidis now surpassed S. Typhimurium as the most common Salmonella serotype that caused extraintestinal infections in 2004. Phage typing further revealed that unusual phage types PT34 and PT21 have been emerging in Taiwan and furthermore expressed a higher predilection to cause extraintestinal infections. In this study, the genomic DNA of various S. Enteritidis strains were digested by XbaI and analyzed by pulsed-field gel electrophoresis (PFGE). We found that different from PT4, PT34 contained an additional 32-kb band. To evaluate the genetic contents of the 32-kb DNA segment of PT34, we cloned it onto a pBluescript II KS (+/-) phagemid and analyzed its sequence. The result showed that the 32-kb DNA segment is the major part of a plasmid (named pSE34). It was found that most of the S. Enteritidis PT34 isolates possessed this plasmid, while PT4 and PT21 did not. The size of pSE34 is 32,950 bp and GC% content is 41.2%, different from 52.17% of the genome sequence of S. Enteritidis PT4 (from Sanger web). The blastn search showed the replication origin of pSE34 is homologous to E. coli plasmid R485 and R6K, so pSE34 could belong to IncX group. The sequence of pSE34 is highly homologous to pOU1114 and pOU1115 of S. Dublin. All of these plasmids contained a complete pilX operon, which encoded a type IV secretion system (T4SS). In annotation, there were 53 orfs, including three operons, stbDE, pilX, and parFG. We used Tn5 transposon to randomly mutate pSE34. Certain genes are involved in the conjugative function, such as pilX operon, taxB and DNA distortion peptide 2. In vitro experiments showed that this plasmid did not enhance the invasion, intracellular survival, and transcytosis properties of PT34.

中文摘要…………………………………………………………………I
英文摘要………………………………………………………………III
目錄………………………………………………………………………V
圖目錄…………………………………………………………………VIII
表目錄……………………………………………………………………X

第一章 緒論……………………………………………………………..1
1.1沙門氏菌屬（Salmonella spp.）的簡介…………………………1
1.1.1沙門氏菌的分類與命名…………………………………….1
1.1.2非傷寒沙門氏菌（non-typhoidal Salmonella）的致病機轉…..................................................2
1.2腸炎沙門氏菌（S. Enteritidis）的簡介………………………3
1.3腸炎沙門氏菌（S. Enteritidis）的次分型（subtyping）…….5
1.3.1噬菌體分型法（phage typing）…………………………….7
1.3.2脈衝式電泳分型法……………….…………………………7
1.4第四型分泌系統（Type IV secretion system）………………8
1.5研究動機及目的…………………………………………..……..11
第二章 實驗材料與方法………………………………………………12
2.1實驗儀器設備和材料列表…………………………..………….12
2.2菌株來源…………………………………………………………12
2.3生長曲線…………………………………………………………12
2.4快速質體分析…………………………………………...……….13
2.5脈衝式電泳法分析………………………………………………13
2.6純化PFGE差異之DNA片段並進行定序……………………..14
2.6.1膠上DNA純化………………………………………….…14
2.6.2黏合作用…………………………………………………...15
2.6.3轉形作用……………………………………………….…..15
2.6.4定序………………………………………………………...16
2.7定序結果整理與分析……………………………………….…..16
2.8質體純化…………………………………………………….…..17
2.8.1小量質體純化…………………………………..………….17
2.8.2大量質體純化…………………………………...…………17
2.9南方墨點法……………………………………………………...18
2.9.1轉印與固定……………………………………….………..18
2.9.2探針的製備…………………………………………….…..19
2.9.3雜合與顯影………………………………………..……….19
2.10基因組微陣列核酸雜合實驗…………………………………..20
2.10.1純化基因體DNA………………………………..………..21
2.10.2基因組微陣列核酸雜合實驗…………………...………..21
2.11雜合扣除法（subtractive hybridization）…………………22
2.11.1基因體DNA的剪切與adaptor的黏合………….………22
2.11.2雜合作用與PCR增幅………………………………….....23
2.11.3 PCR產物的選殖與序列分析………………………….…23
2.12以Tn5 transposon對pSE34進行隨機突變………….………..24
2.13以突變的pSE34取代腸炎沙門氏菌PT34的野生型質體pSE34…24
2.13.1製備勝任細胞…………………......……………………...24
2.13.2轉形作用………………..………………………………...25
2.13.3取代實驗與分析…………………..……………………...25
2.14質體的接合作用（conjugation）…………………………..….26
2.14.1 實驗菌株抗生素耐受性試驗…………………...……….26
2.14.2 接合實驗…………………………………………………26
2.15細胞株的培養…………………………………………………..27
2.16細菌侵犯（invasion）能力分析實驗………..………………..27
2.17細菌於吞噬細胞中存活（survival）能力分析實驗……….28
2.18細菌穿透（penetration）能力分析實驗…………….…………29
第三章 結果與討論………………………………………………….30
3.1腸炎沙門氏菌PFGE分型第一型和第二型的差異………….…30
3.2腸炎沙門氏菌噬菌體分型PT34特異性質體pSE34之分析…..32
3.3質體pSE34的接合作用（conjugation）分析………………....34
3.4致病性探討…………………………………………………...….36
第四章 結論……………………………………………………………40
第五章 參考文獻………………………………………………………88
附錄…………………………………………………………………..…95

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