臺灣博碩士論文加值系統

1.下痢仔豬病原大腸桿菌之鑑定
離乳豬在離乳後一至三週內，常發生急性或慢性下痢，並且逐漸脫水，嚴重甚至死亡。而存活下來的離乳猪，雖因其腸道黏膜的免疫系統逐漸建立而不會再下痢，但生長緩慢，其飼料利用率(Gain/Food intake)低，稱為離乳猪下痢症(postweaning diarrhea，PWD)。PWD導致的猪隻死亡、醫藥費用及存活的猪隻生長減緩等隊全球養猪產業重大經濟損失。PWD最主要的病因為動物來源的腸毒性大腸桿菌(ETEC)，ETEC產生的腸毒素有LT (heat labile enterotoxin)、STa (heat stable enterotoxin type A)、STb (heat stable enterotoxin type B)和EAST1(enteroaggregative E. coli heat-stable enterotoxin 1)。少數的PWD是由STEAEC (產生滋賀毒素(Stx)腸聚集大腸桿菌；Shiga Toxin (Stx) producing Enteroaggregative E. coli)所引起的。因此本研究分析從2013年陸續收集，並分離的5次健康和下痢離乳豬糞便檢體中的細菌，並以生化八管鑑定到68株E. coli，後做STI、STII、 LTI、astA和stx2e的PCR，以確定所以分離到的68株E. coli是何種腸病原性大腸桿菌。本實驗發現有20株E. coli有F18纖毛基因，且會分泌LT和ST，並且不攜帶產生EAST1毒素的基因。這20株E. coli應是可能引起該牧場離乳豬下痢症(PWD)的ETEC類的致病菌。

從大腸桿菌對DH5α和pACYC184-imm/DH5α抑菌效果顯示，如果此菌均不會殺DH5α和pACYC184-imm/DH5α，表示此菌不會產生毒性；如果此菌只會殺DH5α，不殺pACYC184-imm/DH5α，表示此菌會產生Col Ib；如果此菌均會殺DH5α和pACYC184-imm/DH5α，表示此菌會產生其他Colicins。顯示分離到的健康離乳豬分離菌株17株和下痢離乳豬分離菌株51株，共68株菌株中有41.2%健康離乳豬分離菌株和29.4%下痢離乳豬分離菌株會產生Col Ib；17.6%健康離乳分離豬菌株和54.9%下痢離乳豬分離菌株會產生其他Colicins或其他毒素。這54.9%會產生其他Colicins的菌株可能是導致離乳豬下痢的原因之一。

從68株E. coli對純化Col Ib的敏感性結果可知，10 μg/mL的Col Ib可以抑制51%的下痢離乳豬分離菌株，只會抑制35.3%的健康離乳豬分離菌株。將來我們將測試是否可以將Col Ib加到離乳豬飼料中以防治台灣豬場的離乳猪下痢。會選用10 μg/mL的濃度，此濃度可以達到抑制下痢菌株生長，同時較不會影響健康菌株生長。

本研究發現攜帶有F18纖毛基因，且會分泌LT和ST，並且不攜帶產生EAST1毒素的基因的ETEC(39.2%)和會產生其他Colicins的菌株(54.9%)是引起台灣牧場離乳豬下痢症(PWD)的主因。

2.大腸桿菌素相對應之免疫蛋白在大腸桿菌中的角色

由於目前已知所有產生大腸桿菌素的菌株，皆還會產生一種與大腸桿菌素相對應稱為免疫蛋白小分子。一般認為免疫蛋白的角色是與菌體內的大腸桿菌素結合，使大腸桿菌素對菌株本身不產生毒害。但是我們實驗室卻構築出只產生大腸桿菌素colicin Ib (簡稱Col Ib)，不產生其免疫蛋白 (簡稱imm)的大腸桿菌，並且該菌株生長正常。因此本研究探討免疫蛋白在菌中所扮演的功能。

具有ColIb-C-his質體(Col Ib的C端帶有 his tag)的BW25113、ColIb-C-his質體的W3110和ColIb-N-his質體(Col Ib的N端帶有 his tag)的BW25113，三菌養在20 mL培養基中，經mitomycin C誘導後和ColIb-C-his-T5質體(Col Ib的C端帶有 his tag)的W3110養在20 mL培養基中，經IPTG誘導後均與未經誘導後的菌生長沒有差別。

ColIb-C-his質體的BW25113養在20 mL培養基中和ColIb-N-his質體的BW25113養在20 mL及250mL培養基中，三菌經mitomycin C誘導後其上清液有少量的ColIb-C-his或ColIb-N-his蛋白存在，但是沒有細胞質蛋白DnaK的存在。顯示ColIb-C-his或ColIb-N-his蛋白二蛋白為菌主動分泌，而非由少數菌因死亡而裂解出的。但是ColIb-C-his質體的BW25113養在250 mL培養基中經誘導後，雖然其上清液也有少量的ColIb-C-his蛋白存在，但是也有DnaK的存在，表示250 mL菌液中因少數菌死亡而使ColIb-C-his蛋白釋出至上清液中。

ColIb-C-his質體或ColIb-N-his質體的BW25113養在20 mL和250mL培養基中，誘導後以Col Ib-C-his質體的BW25113有最佳的his tag 雜配訊號和抑菌活性。ColIb-C-his質體的W3110以mitomycin C誘導後較ColIb-C-his-T5質體的W3110以IPTG誘導後其his tag 雜配訊號和抑菌活性高。因此之後實驗將以ColIb-C-his質體或ColIb-C-his-T5質體的W3110，養在20 mL培養基中以mitomycin C或IPTG誘導來進行。

具有imm-C-His質體 (imm的C端帶有his tag)轉型至BW25113、W3110和DH5α，比較三菌經mitomycin C誘導後的菌數及西方雜配的結果。顯示imm-C-his在W3110和DH5α中會引起菌的quasilysis現象，但是在BW25113中則沒有此現象。西方雜配結果顯示在BW25113和W3110中，imm-C-His蛋白的量皆隨誘導時間增加而增強。

具有ColIb-C-his-T5質體和imm-C-his質體的W3110和具有ColIb質體和imm-C-his質體的W3110，經mitomycin C或(和)IPTG誘導後，二菌均沒有quasilysis現象。

具有ColIb-C-his-T5質體和imm-C-his質體的W3110未經IPTG誘導，以mitomycin C誘導產生imm-C-his蛋白其抑菌活性較未經mitomycin C誘導的W3110高。具有ColIb質體和imm-C-his質體的W3110經mitomycin C誘導，以IPTG誘導產生imm-C-his蛋白其抑菌活性較未經IPTG誘導的W3110高。顯示imm-C-his蛋白會幫助提高菌中Col Ib蛋白或ColIb-C-his蛋白的抑菌活性。

具有Col Ib質體和pACYC184-imm質體的W3110經mitomycin C誘導後，其上清液抑菌活性較具有ColIb質體和pACYC184質體的W3110經誘導後高。顯示imm蛋白會幫助Col Ib釋出。

1.Identification of pathogenic Escherichia coli from diarrheal piglets in Taiwan

Piglets after weaning for 1 to 3 weeks, often acute or chronic diarrhea, and gradually dehydration, serious or even death. The survival of piglets, although the intestinal mucosa because of its immune system gradually established without diarrhea, so this piglet slow growth, its feed utilization (Gain/Food intake) is low, known as the postweaning diarrhea (PWD). PWD induced piglet death, medical expenses and survival of the pig's growth slowdown in the global pig industry, major economic losses. PWD is the most important cause of animal origin of enterotoxigenic E. coli (ETEC), which produce heat labile enterotoxin (LT), heat stable enterotoxin type A (STa), heat stable enterotoxin type B (STb) and enteroaggregative E. coli heat-stable enterotoxin 1 (EAST1). Small number of PWDs were caused by STEAEC (producing Shiga Toxin (Stx) Enteroaggregative E. coli). Therefore, this study analyzes the bacteria collected from the samples of the healthy and diarrhea piglets from 2013, and identified 68 strains of E. coli by biochemical eight tubes. Then use PCR to check this 68 isolates strains have STI, STII, LTI, astA and Stx2e or not, then identified this 68 isolates strains were which pathogen of E. coli. This study found that 20 E. coli has F18 flagellum gene, and also have LT and ST genes, but not have EAST1 gene. The 20 strains of E. coli should be ETECs, which maybe cause PWD.

The 68 isolates strains inhibition activities of DH5α and the plasmid for expression of imm was transformed into DH5α. If isolates strains do not kill two host, is mean this isolates strains do not produce toxin; if it can only kill DH5α host, is mean this isolates strains can produce Col Ib protein; if it can kill two host, is mean this isolates strains can produce other Colicin protein. The result shows, we have 41.2% healthy and 29.4% diarrhea piglets isolates strains can produce Col Ib protein; 17.6% healthy and 54.9% diarrhea piglets isolates strains can produce other Colicin protein. This 54.9% diarrhea piglets isolates strains maybe cause PWD.

From the sensitivity of 68 E. coli to purified Col Ib, show that 10 μg/mL of Col Ib can inhibit 51% diarrhea piglets isolates strains, but just inhibit 35.3% healthy piglets isolates strains. In the future, we will test whether Col Ib can be added to the suckling pig feed to prevent Taiwan pig farms from diarrhea. We will use 10 μg/mL concentration, this concentration can be achieved to inhibit the growth of diarrhea piglets isolates strains, while not affecting the growth of healthy piglets isolates strains.
In this study, we found ETEC (39.2%) of the gene carrying the F18 cilia gene and secreting LT and ST, and not carrying the EAST1 gene, and the strain (54.9%) that produced other Colicins, maybe cause PWD.

2.The role of immunity protein of colicin Ib in Escherichia coli

So far, all E. coli strains in nature that produce colicins also produce a small protein called immune protein specific to the colicin. It is generally believed that the immune protein binds the corresponding colicin in vivo, thus has no toxicity to the bacteria producing the colicin. However, our laboratory has constructed an E. coli strain that produces colicin Ib (Col Ib) but not the corresponding immunity protein (Imm), and the strain grew normally. The purpose of this study is to understand the role of Imm in E. coli.

At first, it was demonstrated that BW25113 or W3110 carrying plasmid with genes for ColIb-C-his (his tag at C terminus of Col Ib) or ColIb-N-his (his tag at N terminus of Col Ib) showed no harm and no difference on bacterial growth after induction of either protein. Induction by mytomycin C or IPTC showed no difference on bacterial growth.

BW25113 carrying plasmid responsible for production of ColIb-N-his through mytomycin C induction was grown in 20 mL or 250 mL medium. After induction, ColIb-N-his could be detected in the cell-free supernatant, but not the cytoplasmic protein DnaK, indicating ColIb-N-his could be secreted into medium by bacteria. So was BW25113 carrying plasmid responsible for production of ColIb-C-his grown in 20 mL medium. But for BW25113 carrying the same plasmid grown in 250 mL medium, both both DnaK and ColIb-C-his could be detected in the supernatant, indicating bacterial lysis occurred under this particular growth condition.

The His-tag hybridization signal and the bacteriocidal activity of the bacterial cell extract was determined with BW25113 carrying ColIb-C-his or ColIb-N-his plasmid and grown in 20 mL or 250 mL medium. After induction by mytomycin C, BW25113 carrying ColIb-C-his plasmid grown in 20 mL medium showed the best activity. The his-tag hybridization signal and the bacteriocidal activity of bacterial cell extract was also checked with W3110 carrying ColIb-C-his plasmid with which ColIb-C-his production was induced by mytomycin C or IPTC. Induction by mytomycin C had higher hybridization signal and the bacteriocidal activity than induction by IPTG.

Quasilysis on bacterial growth was found with W3110 and DH5α, but not BW25113, carrying plasmid with genes for imm-C-his (his tag at C terminus of Imm) after mytomycin C induction. The His tag hybridization signal of the protein extract increased as the induction time increased. However, W3110 carrying the same imm-C-his plasmid but plus Col Ib plasmid (Col Ib without his tag) did not show quasilysis on growth.

By using W3110 carrying both Col Ib-C-his plasmid and Imm-C-his plasmid which either induction by mytomycin C or IPTG, it is demonstrated that the bacteriocidal activity of the cell extract increased by induction of Imm-C-his. Thus, Imm-C-his increased the bacteriocidal activity of Col Ib-C-his in the bacteria.

The bacteriocidal activities of supernatants from W3110 carrying both Col Ib plasmid (Col Ib without his tag) and pACYC184-imm plasmid (Imm without his tag), as well as W3110 carrying both Col Ib plasmid and pACYC184 plasmid, were determined after induction by mytomycin C. The former had higher activity than the later, indicating that Imm could facilitate the secretion of Col Ib from bacteria.

1.目錄
前言 1
一、離乳豬下痢症(piglets with postweaning diarrhea; PWD) 1
二、腸病原性大腸桿菌(enterovirulent E. coli；EEC) 1
三、腸毒素 (Enterotoxin) : 2
四、EMB培養基 (Levine Eosin-Methylene Blue Agar Medium) 3
五、以GNB-14電腦密碼鑑定系統之菌種鑑定與判讀 3
六、大腸桿菌素(Colicins) 7
七、實驗動機目的 7
實驗材料 8
一、菌種、質體、引子及菌的培養 8
二、 儀器 8
三、 藥品 8
實驗方法 10
一、菌種的分離和鑑定 10
二、 E. coli的F18纖毛及腸毒素基因分析 10
三、菌株分泌ST蛋白之檢測 11
四、菌株分泌LT蛋白之檢測 12
五、收取菌體蛋白以供SDS-PAGE分析 13
六、收取蛋白液以供SDS-PAGE分析 13
七、10 % SDS-PAGE 13
八、Coomassie blue 染色 13
九、以 mitomycin C 誘導Col Ib-C-his蛋白表現 14
十、Col Ib-C-his蛋白純化 14
十一、蛋白定量 15
十二、分離菌株對Col Ib-C-his的敏感性測試 15
十三、分離菌株對DH5α和pACYC184-imm/DH5α的抑菌活性測試 16
結果 17
一、健康離乳豬糞便和下痢離乳豬直腸拭子檢體分菌及菌種鑑定 17
二、大腸桿菌的F18纖毛及腸毒素基因分析 17
三、純化Col Ib-C-his蛋白 18
四、純化Col Ib對大腸桿菌的抑菌活性 18
五、大腸桿菌對DH5α和pACYC184-imm/DH5α抑菌效果 18
討論 19
參考文獻 20
表 25
圖 38
附表 39
附圖 41
附錄一：培養基與實驗藥劑 48

2.目錄
前言 1
一、大腸桿菌素 (Colicin) 介紹 1
二、大腸桿菌素的結構 1
三、SOS反應(SOS response) 2
四、免疫蛋白 (immunity protein) 2
五、溶菌蛋白 (lysis protein) 2
六、Colicin Ib 基因及其相對應之免疫蛋白基因 3
七、實驗室之前的研究 3
八、研究動機與目的 3
實驗材料 4
一、菌種、質體、引子及菌的培養 4
二、儀器 4
三、 藥品 4
實驗方法 5
一、抽取質體DNA 5
二、DNA黏接反應 5
三、製備勝任細胞 5
四、轉型作用 5
五、DNA 限制酶反應 5
六、質體 DNA的PCR反應 5
七、DNA純化回收 5
八、pQE70-ColIb-c-his-T5質體構築 6
九、pQE70-ColIb-T5質體構築 6
十、收取菌體蛋白以進行SDS-PAGE 7
十一、10 %或15 % SDS-PAGE 7
十二、Coomassie blue 染色 7
十三、蛋白質轉漬 7
十四、西方雜配 (Western blot) 7
十五、以image J軟體將西方雜配結果量化 8
十六、以 mitomycin C 或IPTG誘導Col Ib或imm蛋白表現 8
十七、定量菌液上清液與破菌液的抑菌活性 8
十八、製備菌液上清液的TCA 沉澱以進行SDS-PAGE 9
結果 10
一、菌中ColIb-C-his蛋白與ColIb-N-his蛋白的比較 10
1. pQE70-ColIb-C-his-ok/BW25113、pQE70-ColIb-N-his-ok/BW25113和pQE70-ColIb-C-his-ok/W3110養在20 mL培養基中，經mitomycin C誘導後和pQE70-ColIb-C-his-T5/W3110養在20 mL培養基中，經IPTG誘導後，菌的生長均與未經誘導後沒有顯著差別。 10
2. 以pQE70-ColIb-C-his-ok/BW25113養在20 mL培養基中和pQE70-ColIb-N-his-ok/BW25113養在20 mL和250 mL培養基中，經誘導後ColIb-C-his蛋白和ColIb-N-his蛋白為主動分泌到菌體外。但以pQE70-ColIb-C-his-ok/BW25113養在250 mL培養基中，經誘導後會有少數的菌死亡導致ColIb-C-his蛋白釋出。 10
3. pQE70-ColIb-C-his-ok/BW25113和pQE70-ColIb-N-his-ok/BW25113養在20 mL和250mL培養基中，誘導後以pQE70-ColIb-C-his/BW25113養在20 m培養基中有最佳的his tag 雜配訊號和抑菌活性。pQE70-ColIb-C-his/W3110以mitomycin C誘導後較pQE70-ColIb-C-his-T5/W3110以IPTG誘導後其his tag 雜配訊號和抑菌活性高。 11
二、Imm-C-his蛋白與ColIb-C-his蛋白或Col Ib蛋白的關係 11
1. pACYC184-imm-His/W3110菌的imm-C-his蛋白可經 mitomycin C誘導產生，並且菌經誘導後，有quasilysis現象。 11
2. 以pQE70-ColIb-C-his-T5/pACYC184-imm-His/W3110和pACYC184-colIb-CT/pT5-imm/W3110經mitomycin C或(和)IPTG誘導後，二菌均沒有quasilysis現象。且菌中的Imm蛋白會提高ColIb-C-his蛋白及Col Ib蛋白的抑菌活性。 12
3. 以pQE70-ColIb-ok/pACYC184-imm-His/W3110、pQE70-ColIb-T5/pACYC184-imm-His/W3110和pACYC184-colIb-CT/pT5-imm/W3110三菌經mitomycin C或(和)IPTG誘導後，會多出65 kDa或130 kDa的his tag雜配訊號。 12
三、Imm蛋白與Col Ib蛋白的關係 13
1. pACYC184-imm/W3110菌經 mitomycin C誘導後，沒有quasilysis現象。 13
2. 以pQE70-ColIb-ok/pACYC184-imm/W3110經mitomycin C誘導後，菌中的Imm蛋白會幫助Col Ib蛋白的釋出。 13
討論 14
參考文獻 16
表 19
附表 52
附圖 57
附錄一：培養基與實驗藥劑 64

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