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National Chung Hsing University Institutional Repository - NCHUIR > 生命科學院 > 分子生物學研究所 > 依資料類型分類 > 碩博士論文 >  (1) 台灣南投地區痢疾桿菌菌株的毒性與流行性之研究 (2) 珍珠內蛋白質之檢測與鑑定

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標題: (1) 台灣南投地區痢疾桿菌菌株的毒性與流行性之研究 (2) 珍珠內蛋白質之檢測與鑑定
(1)The virulence and epidemic study of Shigella flexneri 2a isolates recovered from Nantou County (2)Detection and partial identification of proteins in pearls
作者: 楊世駿
Yang, Shih-Chun
Contributors: 胡小婷;劉世東;胡念台;何漣漪;張邦彥;許萬枝
Shiau-Ting Hu;Shih-Tung Liu;Lien-I Hor;Wan-Jr Syu
陳建華
Jiann-Hwa Chen
中興大學
關鍵字: shigellosis;colicin;Col Ib;lysis protein;pearl protein
桿菌性痢疾;大腸桿菌素;大腸桿菌素釋放因子;珍珠蛋白
日期: 2013
Issue Date: 2012-08-31 12:57:44 (UTC+8)
Publisher: 分子生物學研究所
摘要: 摘要 (題目一)
自 1996 到 2000 年間,台灣南投地區爆發了大規模的桿菌性痢疾流行事件,分離到 180 株 S. flexneri 2a 菌株。Not I-脈衝式電泳分子分型得到分屬 52 型的174株遺傳相近的菌株,6 株遺傳不相近的菌株。在 174株遺傳相近的菌株中,其中74 株為 A型,稱為流行菌株,另外 100 株分屬 51 種的A1~A51型,稱為非流行菌株。本部分的研究為探討 173 株 S. flexneri 2a 的毒性與流行性的關聯 (一株分離株死亡)。

首先隨機挑選 10 株菌的單一菌落培養菌液 (1st-day colony cultures),及其連續培養 10 天的任意一菌株 (10th-day colony cultures),以 MOI 50 的菌數感染 U-937 及 Caco-2 細胞後,檢測菌在胞內複製能力及對 U-937 細胞的傷害程度,及菌中毒性蛋白IpaB 蛋白的表現。結果顯示:可以表現 IpaB 蛋白的 5 株 1st-day colony cultures 及 1 株 10th-day colony cultures,在二細胞內皆有較高複製能力,及對 U-937 細胞產生較強的傷害。另外不能表現 IpaB 蛋白的 5 株 1st-day colony cultures 及 9 株 10th-day colony cultures,在二細胞內的複製能力較低,對 U-937 細胞的傷害亦較低。但是這些現象與菌是否為流行菌株無關,與是否為第十天的菌株有關,大部分的流行或非流行菌株連續培養 10 天的菌,會喪失 IpaB 蛋白的表現能力。

隨機挑選一株能表現 IpaB 蛋白的流行菌株的 1st-day colony culture,感染 Caco-2 細胞進行 plaque assay,發現菌株具有強的細胞間擴散能力,但其 10th-day colony culture 不能表現 IpaB 蛋白,亦不能在細胞間擴散,而由 -80℃ stock 直接培養的菌液 (1st-day stock culture),其連續培養 10 天的菌液 (10th-day stock culture),也發現相同的結果。另外挑選可以表現 IpaB 的 2 株流行菌株及 4 株非流行菌株的 1st-day stock culture,檢測其細胞間擴散的能力,發現一株流行菌株具有強的細胞間擴散能力,另外一株流行菌株其 4 株非流行菌株只具有弱的細胞間擴散能力。

檢查 173 株 1st-day stock culture 及 10th-day stock culture 其 IpaB 蛋白的表現。結果顯示,73 株流行菌株 1st-day stock culture 中,52 株 (71.2%) 有 IpaB 蛋白的表現,但是只有 9 (12.3%) 株 10th-day stock culture 有 IpaB 蛋白的表現。而非流行菌株也有類似的情形,100 株非流行菌株 1st-day stock culture 中,75株 (75%) 有IpaB 蛋白的表現,但是只有 16 (16%) 株10th-day stock culture 有 IpaB 蛋白的表現。

檢測173 株分離株 1st-day stock culture 對 32 株人類腸道常在菌的殺菌能力,發現有 65 株 (89%) 的流行菌株可毒殺一株 E. coli (F-34),其中有 63 株還可毒殺另二株 E. coli (F-21,F-24) 中的一株或兩株。而非流行菌株中,只有 46 株 (46%) 具有殺 F-34,其中有 21 株還可毒殺另二株 F-21、F-24 中的一株或兩株。1996 到 2000 年中,除了 1999 年外,具有殺菌能力的流行菌株百分比逐年升高, 在 1998 到 2000 年,非流行菌株的殺菌能力則是逐年下降。在 1996 到 1999 年的分離株中,每年任選流行及非流行各 6 株分離株,以及 2000 年的全部菌株,檢查其 10th-day stock culture 的殺菌能力穩定性,發現其殺菌能力幾乎與 1st-day stock culture 相同。

將不具殺菌力的菌液 (rifampicin-resistant) 以 0.1%,1%,10% 加入具殺菌力的菌液 (rifampicin-sensitive) 中,連續培養五天,發現 rifampicin-resistant的菌會分別佔到 12.1%,54.3% 及89.4%,其中分別有 25%,3% 及1.67% 具有毒殺 F-34 的能力,顯示不具毒性的族群會侵略具有毒性的族群,而導致整個族群皆不具毒性。因此,我假設有一株具有殺菌能力的 S. flexneri 2a 菌株,引起南投地區的痢疾大流行,這株菌具有高度毒性並可以在流行期間被分離出 (其中一株流行菌株),而在流行期間可感染腸道細胞但不具殺菌能力的突變株會逐漸產生,導致分離出許多不同類型低毒性的菌株 (非流行菌株)。

本部分第二章的研究為選取一具殺菌能力的菌株,分離及定性殺菌基因。我將173 株分離株的 Not I-脈衝式電泳圖譜與分離株對大腸桿菌 F-34 毒殺能力相比對,發現在 Not I-PFGE 圖譜中第 31 個 DNA 片段可能具有殺菌基因。將具殺菌能力的 SH3160 的 Not I-PFGE 圖譜中第 31 個 DNA 片段製作成 fosmid library,找到兩個可毒殺 F-34 的菌落 pBacin1/EPI300 及 pBacin2/EPI300,後者的殺菌能力較前著強。以 Tn5 對殺菌能力較強的clone (pBacin2/EPI300) 進行隨機突變,篩選不具殺菌能力的插壞株,找到殺菌基因為 cib (編碼出的蛋白為大腸桿菌素 Colicin Ib)。南方雜配及 SH3160 全基因定序結果顯示:SH3160 中的 cib 基因位於一個 ColIb-P9-like 的大型質體上。我以接合生殖實驗證實:此 ColIb-P9-like 質體可於自 E. coli 自我轉移於至 S. flexneri 2a 中或反之。將純化的重組 Col Ib 蛋白對 32 株人類腸道常在菌進行殺菌能力測試,發現有 6 株 E. coli (包括 F-34、F-21、F-24) 對 Col Ib 具有敏感性。

本部分第三章的研究為找尋能夠幫助 Col Ib 蛋白分泌至菌體外的基因或蛋白。上述以Tn5對殺菌能力較強的clone (pBacin2/EPI300) 進行隨機突變,得到的不具殺菌能力的插壞株中。除了cib 基因插壞株外,還有ibfA 基因插壞株。然而目前對於 ibfA 基因的了解較少。以 PCR 檢測殺菌能力較弱的clone (pBacin1/EPI300) 發現不具 ibfA 基因。將 ibfA 基因互補至 pBacin1/EPI300 中,可發可提升其抑菌能力。因此 ibfA 基因應可表現 trans-acting factor。大量表現 IbfA 或 Colicin Ib 的免疫蛋白 (Imm 蛋白),會引發菌體發生 quasi-lysis 現象。以細菌雙雜交系統檢測 Colicin Ib 蛋白、Imm 蛋白及 IbfA 蛋白在菌體是否具交互作用,發現三者並無交互作用。


摘要 (題目二)
珍珠及珍珠製品除了美觀具有收藏價值外,在中國亦是一種傳統藥物及保健食品。珍珠與貝類中的珍珠母 (nacre,或稱珍珠層) 有著相似的化學成分,目前在珍珠母中,有超過 20 種蛋白被鑑定出,但卻沒有任何一種蛋白在珍珠中被發現。因此,本實驗室與台灣的美梭科技公司合作,目的為開發一種能夠偵測及鑑定珍珠內的蛋白的萃取技術。兩批珍珠分別購自兩個不同的三角帆蚌 Hyriopsis cumingii (Lea) 養殖場,先將珍珠研磨成 >10,000 mesh 的珍珠粉,再經由 RTSES 系統將珍珠粉中的顆粒震碎並溶於水中,經離心後的上清液即為珍珠水萃取液。將珍珠水萃取液以 121℃ 加熱處理 20 分鐘或不經熱處理,再利用 TCA 沉澱其中的珍珠蛋白,以 tricine SDS-PAGE 分離蛋白片段。膠體經硝酸銀染色後,在經 121℃ 加熱處裡的珍珠水萃取液的樣品中有明顯的蛋白訊號,但沒有經過121℃ 加熱的珍珠水萃取液則沒有任何蛋白訊號。將兩次獨立實驗經 121℃ 加熱處理的珍珠萃取液蛋白,分別自膠體中挖出,經由 tryptic 切割,再利用質譜儀分析蛋白片段,分析結果以 MASCOT 軟體進行比對搜尋。發現其中一個蛋白與貽貝 (Mytilus edulis) 的 putative vitelline envelop receptor for lysine 蛋白有顯著的序列同源性,另一個蛋白與象鼻蟲 (Diaprepes abbreviatus) 的 putative imaginal disc growth factor (IDGF) 蛋白有顯著的序列同源性。
Abstract (first study)
From 1996 to 2000 there was a large-scale outbreak of shigellosis in Nantou County, and 180 S. flexneri 2a isolates were recovered. Based on their Not I pulsed-field-gel-electrophoresis (PFGE) patterns, 174 isolates were assigned to 52 genetically closely-related strains but 6 isolates were not. Of these 174 isolates, 74 had the type-A pattern and belonged to the epidemic strain, whereas the other 100 isolates had other 51 patterns (types A1 to A51) and belonged to 51 non-epidemic strains. The first part of this study was to analyze the virulence of 173 of the 174 isolates (one isolate was lost).

Initially, ten 1996 isolates were randomly chosen, and cultures of a single colony from the -80℃ stock, named 1st-day colony cultures, plus cultures of a single colony from the 10th subcultures of the 1st-day single colony cultures, named 10th-day colony cultures, were collected and used to infect U-937 and Caco-2 cells at MOI of 50. After infection for 4 hours, intracellular bacterial multiplication and cytotoxicity of these 20 colony cultures were determined. Meanwhile, expression of the virulence protein IpaB in these cultures was also analyzed. The results showed that five 1st-day and one 10th-day colony cultures showed IpaB expression and had high levels of intracellular bacterial multiplication and cytotoxicity, whereas the other five 1st-day and nine 10th-day colony cultures did not demonstrate any IpaB expression and had low levels of intracellular bacterial multiplication and cytotoxicity. There was no difference in the IpaB expression between the 1st-day colony cultures of the epidemic and the non-epidemic strain isolates. Upon 10 subculturings of the 1st-day colony cultures, most of the epidemic and the non-epidemic isolates lost IpaB expression.

However, plaque assay on Caco-2 cells indicated that the 1st-day colony culture of one randomly-picked epidemic isolate which was IpaB-plus had high cell-to-cell spreading ability, but its 10th-day colony culture which was IpaB-minus had no spreading activity. The 1st-day culture directly from the -80℃ stock of this isolate, named the 1st-day stock culture, and its 10th subculturing, named the 10th-day stock culture, showed the same results as its 1st-day and 10th-day colony cultures, respectively. The 1st-day stock cultures of 2 other epidemic and 4 non-epidemic isolates that had IpaB expression were further tested for their spreading activities. The results showed that the 1st-day stock culture of one epidemic isolate had high spreading activity, but the 1st-day stock cultures of the other one epidemic isolate and the 4 non-epidemic isolates all showed low spreading activities.

Both the 1st-day and 10th–day stock cultures of the 173 isolates were checked for IpaB expression. The result showed that, of the 73 epidemic isolates, 52 (71.2%) had IpaB expression in their 1st-day stock cultures but only 9 (12.3%) in their 10th-day stock cultures. Similar results were obtained with the 100 non-epidemic isolates. Seventy-five isolates (75%) had IpaB expression in their 1st-day stock cultures but only 16 (16%) in their 10th-day stock cultures.

Bacteriostatic activities of the 1st-day stock cultures of the 173 isolates against 32 human enteric isolates were examined. Sixty-five (89%) of the 73 epidemic isolates showed bacteriostatic activities against one E. coli strain (F-34), of which 63 showed the activities against another E. coli strain F-21, F-24 or both. On the other hand, 46 (46%) of the non-epidemic isolates had the activities against F-34, of which 21 had the activities against F-21, F-24 or both. Moreover, percentages of the epidemic isolates that carried the bacteriostatic activities in their 1st-day stock cultures increased yearly from 1996 to 2000 except 1999. On the contrary, percentages of the non-epidemic isolates that carried the bacteriostatic activities decreased yearly from 1998 to 2000. Six epidemic and 6 non-epidemic isolates in each year from 1996 to 1999 and all epidemic and non-epidemic isolates in 2000 were picked and the bacteriostatic activities of the 10th-day stock cultures were also checked and the results were about the same as their 1st-day stock cultures.

The 1st-day colony culture of one isolate (rifampicin-resistant) that did not carry any bacteriostatic activity was added to that of another isolate (rifampicin-sensitive) that carried the bacteriostatic activity in an ratio of 0.1%, 1% or 10%. The three mix cultures were subcultured daily for 5 days and colonies were checked for their sensitivities to rifampicin as well as their bacteriostatic activities against F-34. The results showed that 12.1%, 54.3%, and 89.4%, respectively, of the colonies from the three mix cultures were rifampicin-resistant, and 25%, 3%, and 1.67%, respectively, carried the bacteriostatic activities. It appeared that the isolate without the bacteriostatic activity could invade the population of the isolate with the bacteriostatic activity. Thus, it is hypothesized that an S. flexneri 2a strain that carried the bacteriostatic activity was the culprint for the shigellosis outbreak in Nantou county. This strain was highly virulent and isolates from this strain could be recovered throughout the outbreak (thus one single epidemic strain). Mutants defective in the bacteriostatic activity were generated continuously and were capable of invading the population of the epidemic strain isolate in patients, leading to many different strains with low virulence (the non-epidemic strains).

By comparing the Not I-PFGE patterns and the bacteriostatic activities of the 173 isolates, PFGE band 31 was suspected to contain the gene for the bacteriostatic activity. This band was recovered from the PFGE gel and a fosmid library was constructed. Two clones, pBacin1/EPI300 and pBacin2/EPI300, were found to carry the bacteriostatic activity against F-34, and the latter had higher activity than the former. In vitro Tn5 mutagenesis technique was used to find two genes, cib and ibfA, for the bacteriostatic activity. The cib gene encodes colicin Ib. Southern blot and whole genome sequencing of one epidemic isolate with the bacteriostatic activity showed that the cib gene is located on a colIb-P9-like large plasmid. Conjugation experiment showed that this plasmid could be mobilized from E. coli to S. flexneri 2a or vice versa. Recombinant colicin Ib protein was prepared and its bacteriostatic activity against the 32 human enteric isolates was checked. Six E. coli stains including F-34, F-21 and F-24 were found sensitive to colicin Ib.

Little is known about the ibfA gene. Clone pBacin1/EPI300 was found lacking ibfA by PCR. The ibfA gene was plasmid-transferred into pBacin1/EPI300 and the transformant showed a higher bacteriostatic ability (complementation test). Thus, ibfA gene encodes a trans-acting factor. Overexpression of ibfA or the gene for the immunity protein of colicin Ib, imm, would lead to bacterial quasi-lysis. Bacterial two-hybrid system was used to detect any protein-protein interaction of cib, imm, and ibfA in E. coli. The results indicated that no interaction between any pair of colicin Ib, the immunity protein and the protein encoded by ibfA, if any.

Abstract (second study)
Pearls and pearl products have been used as a traditional medicine and health food in Chinese. Pearl and nacre (mother of pearl) have similar chemical compositions. More than 20 proteins have been identified in nacre, yet none have been detected in pearl thus far. The purpose of this study was to detect and identify proteins in pearls. In collaboration with MesoPhase Technologies, two batches of pearls formed in Hyriopsis cumingii (Lea) were ground into a powder of >10,000 mesh and extracted in water by the RTSES system. A portion of the pearl powder water extract was heated at 121℃ for 20 min, while apportion was not. TCA precipitation and tricine SDS-PAGE were conducted on both the heated and non-heated extracts. After silver nitrate staining, the heated extract demonstrated a distinct protein signal, but the non-heated extract did not. The protein band from each of the two heated extracts was excised from the gel and subjected to tryptic digestion and mass spectrometer analysis. A MASCOT search of the results indicated that one protein had significant sequence homology to a putative vitelline envelop receptor for lysine in the common marine mussel Mytilus edulis, and the other to the putative imaginal disc growth factor (IDGF) of Diaprepes abbreviatus.
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