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研究生:李紫晴
研究生(外文):Li, Tzuching
論文名稱:噬菌體 phiNK5 之溶菌素分離及基因選殖 抗生素及金屬離子對 A 型鏈球菌莢膜增生與致病基因表現之影響
論文名稱(外文):I.Isolation And Gene Cloning Of Lysin Protein From Lytic Phage phiNK5II.Effects Of Antibiotics And Metalions On Group A Streptococcus Capsule Increase And Pathogenic Genes Expression
指導教授:洪志勳洪志勳引用關係
指導教授(外文):Hung, Chinhsin
口試委員:鄭名芳王竣令洪志勳
口試委員(外文):Cheng, MingfangWang, JiunlingHung, Chinhsin
口試日期:2012-07-17
學位類別:碩士
校院名稱:義守大學
系所名稱:生物技術與化學工程研究所
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:2012
畢業學年度:100
語文別:中文
論文頁數:89
中文關鍵詞:雷伯氏肺炎桿菌噬菌體溶菌蛋白A 型鏈球菌化膿性鏈球菌莢膜
外文關鍵詞:Klebsiella PneumoniaePhageLysin ProteinGroup A StreptococcusStreptococcus PyogenesCapsule
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克雷伯氏肺炎桿菌 (Klebsiella pneumoniae) 為革蘭氏陰性兼性厭氧菌,並長引發嚴重的院內感染,近年產生嚴重的多重抗藥性問題,使抗生素治療出現瓶頸,噬菌體治療因此又受到學者的注意,用以解決抗藥性菌株感染之問題。
本實驗室已篩出可溶裂 K. pneumoniae NK5 (簡稱 NK5) 之噬菌體 phiNK5 ,在動物實驗中, phiNK5 可使受 K. pneumoniae NK5 感染之小鼠有 100% 存活率,但此噬菌體之溶菌素基因尚未被選殖表現,本論文將選殖NK5 的 lysin 基因並測試蛋白活性。本實驗室已將 phiNK5 基因完全定序,並推測可能的 open reading frame (ORF) 至基因庫進行比對,得知 ORF45 及 ORF46 與 Klebsiella sp. phage KP32 (Accession No.: NC_011043) 及 K11 (Accession No.: NC_013647) 的 endolysin protein 具有 97% 的相似性。利用 ORF45基因序列設計引子 (primer) 並利用 PCR 增幅完整基因片段選殖至 pBluescript SK+,經定序確認 DNA 序列後得到選殖體 pBNK5L。pBNK5L之 insert 片段轉殖至 pET21 ,得到 pENK5L 用以大量表現 lysin 蛋白。使用 IPTG 誘導大量表現 lysin protein,在SDS-PAGE 上顯示表現蛋白為 16 kDa,但經純化測試後,此蛋白卻不具有 lysin protein 活性。另外,利用 20% ~ 40% 硫酸銨粗萃噬菌體與宿主共同培養後上清液之溶菌素,再以 SDS-PAGE 分離,用無菌水漂洗去SDS後,再以混合菌體的soft-agar覆蓋,發現在約 120 kDa 處具有強裂溶菌的透明區。將此處蛋白質片段切下後利用液相分析質譜儀 (LC/MS/MS) 分析蛋白質片段,與 NK5 open reading frame 序列做比對,結果與NK5的 ORF8 (fusion protein) 與 ORF24 (DNA polymerase)有 56% 與 25% 的相似度,但都與溶菌無關,因此需再重新純化分析。

A 型鏈球菌 (Group A Streptococcus) 為革蘭氏陽性的兼性厭氧菌,為小兒科主要的臨床病原菌之ㄧ。 鏈球菌的莢膜與此菌株的致病力有關,本研究將探討在不同濃度的抗生素 (penicillin)、鎂離子與鈣離子的刺激下,玻尿酸 (hyaluronic acid, HA) 表現量是否會受到調控。抽取 A 型鏈球菌莢膜上的玻尿酸,再使用酵素免疫法分析,但十五株臨床菌株未有明顯的改變的趨勢。此外使用Real Time-PCR mRNA 定量分析,探討抗生素與金屬離子對致病有關的基因 SPY170、mac、hasB、prtS 表現的影響。目前從M72 與 M93兩株菌株初步分析結果發現,其的 mac 與 hasB 兩個基因,在抗生素(penicillin)濃度增加的刺激下表現量有持續增加,顯示此兩基因受到抗生素的調控。
The Klebsiella pneumonia is a Gram-negative, facultative anaerobic bacterium and always causes the serial nosocomial infections. Recently years, the derivative multiple -drug resistant problems have made the antibiotic treatments on the choke point. The phage therapy is rekindled to combat the multiple-drug resistant bacterium infections.
In our laboratory, phage phiNK5 has been isolated and specific to lyse Klebsiella pneumoniae NK5 (abbrev. NK5). In mouse animal model, phage phiNK5 could rescuse the NK5 infected mice and remained 100% survival. The purposes of this stusy will clone the lysin gene of phage phiNK5 and analyzed the activity of the expressed lysine protein. The complete genome sequences of phage phiNK5 has been finished before. Genome sequences was analyzed and blast on NCBI database, the results showed that the putative open reading frame (ORF) ORF45 and ORF46 were with 97% similarities to the endolysin protein of Klebsiella sp. phage KP32 (Accession No.: NC_011043) and K11 (Accession No.: NC_013647). ORF45 was used as the reference to design the primers and use PCR (polymerase chain reaction) to ampify the whole gene of ORF45. The PCR fragment was cloned into the EcoRV site of pBluescript SK+ and sequenced to ensure the correction of insert sequences and gernerate the plasmid pBNK5L. The pBNK5L was digested with NdeI/XhoI and the insert was cloned into pET21b NdeI/XhoI sites to generate the lysin expression plasmid pENK5L. Using IPTG to induce lysin protein expressing and found that the expressed protein was 16 kDa shown on SDS-PAGE, but this protein didn’t have lysis activity after purification. Lysin protein was also crude extracted from the supernatant of phage fNK5 and host coincubated culture medium by using the 20%-40% ammonium sulfate. The crude extracted proteins were separated by SDS-PAGE and the lysis activity protein patterns was determined by bacterium overlay assay. One protein patterns at 120 kDa resulted in clearn zone on the detected plate. This protein pattern was collected from PAGE and analyzed by LC/MS/MS to identified the protein by blast with the genome sequences of phage fNK5. The data showed that the protein was predicted as ORF8 (fusion protein) and ORF24 (DNA polymerase) with 56% and 25% similarity. But both of them were not relative to endolysis, so lysine protein was needed to extract and analyze again later.

Group A streptococcus Gram Positive and facultative anaerobes is one of the major clinical pathogenic bacteria in pediatrics. In recent years, the research of Group A streptococcus’ virulence factor, such as hyaluronic acid, M protein, protein F, plasminogen receptor, streptokinase, C5a peptidase, streptokinase O, streptokinase S, streptococcal inhibitor of complement-mediated lysis, streptococcal pyogenic exotoxin B, and IgG-binding proteins. It can discover the bacteria earlier and prevent them start to spreading. Furthermore, it’s helpful to do the research of vaccine by understand the infection of bacteria and the reason of the disease.
This research discuss about hyaluronic acid in different penicillin’s concentration, and the excitation of magnesium and Calcium, whether the capsule of bacteria strain getting thick or not, the capsule of streptococcus can cause the disease by this bacteria (Liu et al., 2011).This research use two methods to prove that the penicillin, magnesium and calcium in different concentration, whether their capsule will getting thick or not under excitation. There are two methods could get the conclusion; One is get the hyaluronic acid on Group A streptococcus’s capsule, then analyze it by Enzyme-linked Immunosorbent Assay. Because we can’t survey the standard one, it can’t survey the correct capacity of hyaluronic acid, only can contrast with the control of penicillin, magnesium and calcium’s excitation; so between the different strains, then fifteen kind of clinical strains have seven different kind of situation. The other method is get the mRNA ,then use the Real Time-PCR mRNA quantitative analyze. So far we can discover that the mac and hasB of M72 and M93, two kind of genes’ will get more expression under the excitation, so we can discuss that these two genes are affected by the control of penicillin, and the drug resistance of microbiology.
中文摘要I
英文摘要III
誌謝V
目錄VI
圖目錄X
表目錄XI
附錄XII
壹、前言1
貳、 文獻回顧2
一、Klebsiella pneumoniae 背景2
二、超級細菌之抗藥基因 NDM-1 (New Delhi metallo-b-lactamase 1)3
三、噬菌體 lysin 蛋白4
四、Phage 與 lysin 治療之優點6
参、實驗目的7
肆、材料與方法8
實驗材料8
一、菌株8
二、噬菌體8
三、基因選殖所使用之引子、宿主、質體及構築質體8
四、培養基與緩衝溶液8
五、實驗儀器8
六、抗生素、藥品及其配製方法8
實驗方法9
一、細菌菌種活化及保存9
二、噬菌體增殖9
A. 噬菌體固態培養9
B. 噬菌體液態培養10
三、噬菌體濃縮及保存10
四、噬菌體 DNA 抽取10
五、質體 (plasmid) 抽取11
六、重組質體之製備11
A. 聚合酶鏈鎖反應 (Polymerase Chain Reaction;PCR)11
B. PCR 產物 Clean-up12
C. 電泳法回收 DNA 片段12
D. DNA 端點之補齊 (Fill-in)13
E. DNA 之接核反應 (Ligation)13
F. 限制梅切割 (Restriction Enzyme Digestion) DNA 之分析14
G. 洋菜膠體電泳分析 (Agarose gel electrophoresis)14
七、大腸桿菌勝任細胞 (Competent cells)14
八、勝任細胞之轉形作用 (Transformation)15
九、快速篩選重組質體法15
十、重組質體之蛋白質表現16
十一、噬菌體溶菌素之萃取16
十二、蛋白質濃度檢量線17
十三、SDS-PAGE 蛋白質膠體電泳17
A. 膠體電泳17
B. 孔雀藍染色 (Coomassie blue)18
C. 銀染 (Silver stain)18
D. Lysin 蛋白活性測試19
伍、結果20
一、依據定序比對結果,選殖 lysin 基因20
二、重組質體之蛋白質誘導表現20
三、噬菌體感染宿主之培養液中溶菌蛋白質之粗萃與溶菌測試21
四、溶菌蛋白片段之 LC/MS/MS 分析22
陸、討論23
柒、參考文獻25
第二部份:抗生素及金屬離子對 A 型鏈球菌莢膜增生之影響41
中文摘要42
英文摘要43
壹、前言44
貳、文獻回顧46
一、Group A Streptococcus 背景46
二、A 型鏈球菌致病因子之探討47
三、玻尿酸簡介48
参、實驗目的49
肆、材料與方法50
實驗材料50
一、菌株50
實驗方法50
一、細菌 HA 抽取50
二、酵素免疫分析法(Enzyme-Linked immunosorbent assay;ELISA)51
三、RNA 抽取52
四、Real Time-PCR mRNA 定量分析53
伍、結果54
一、不同濃度 penicillin 刺激對菌體莢膜的表現量之影響54
二、不同濃度鎂離子刺激對菌體莢膜的表現量之影響55
三、不同濃度鈣離子刺激對菌體莢膜的表現量之影響56
四、不同抗生素刺激對菌體 SPY170、prtS、mac 和 hasB基因的表現之影響58
陸、討論59
柒、參考文獻61
圖目錄
Fig. 1. 質體pBNK5L 之選殖片段確認32
Fig. 2. pENK5L 重組質體選殖33
Fig. 3. 重組質體 pENK5L 之蛋白表現34
Fig. 4. 最小抑制濃度35
Fig. 5. 粗萃之溶菌蛋白活性測試36
Fig. 6. 利用 SDS-PAGE 分析溶菌蛋白片段分析37
Fig. 7. 具有溶菌效果之蛋白片段以 LC/MS/MS 分析後與NK5基因體比對之結果38
第二部份:抗生素及金屬離子對 A 型鏈球菌莢膜增生之影響
Fig. 1. 以酵素免疫 (Enzyme-Linked immunosorbent assay;ELISA) 分析菌對不同濃度抗生素 (penicillin) 莢膜的表現量64
Fig. 2. 以酵素免疫 (Enzyme-Linked immunosorbent assay;ELISA) 分析菌對不同濃度鎂離子莢膜的表現量65
Fig. 3. 以酵素免疫 (Enzyme-Linked immunosorbent assay;ELISA) 分析菌對不同濃度鈣離子莢膜的表現量66
Fig. 4. 以 RT-PCR 分析 M72 與 M93 菌株在不同濃度抗生素 (penicillin) 下,SPY170、prtS、mac、hasB 基因的表現情形67
表目錄
表一 本實驗基因選殖所使用之宿主、質體及構築質體40
表二 本實驗基因選殖所使用之引子40
第二部份:抗生素及金屬離子對 A 型鏈球菌莢膜增生之影響
表一 本實驗基因選殖所使用之引子69
附錄
附錄一 實驗用儀器設備70
附錄二 抗生素及藥品71
附錄三 培養基與緩衝溶液製備73
附錄四 實驗藥品配製方法74
I.
中文部份
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