鄧麗珍Teng, Lee-Jene臺灣大學:醫學檢驗暨生物技術學研究所林郁瑩Lin, Yu-YinYu-YinLin2010-05-112018-07-062010-05-112018-07-062009U0001-2207200917502300http://ntur.lib.ntu.edu.tw//handle/246246/182976G群鏈球菌Streptococcus dysgalactiae subsp. equisimilis平常存在於皮膚、喉嚨以及一些黏膜表面如腸道、陰道等地方，在人體免疫力低下時可能會造成伺機性感染，然而在近年來被報導的感染案例有增加的趨勢，且一些症狀和A群鏈球菌Streptococcus pyogenes很相似，如菌血症、流行性咽喉炎、腹膜炎、蜂窩組織炎、心內膜炎、腎小球腎炎以及鏈球菌休克症候群…等。根據許多研究顯示，此菌和S. pyogenes有許多相似的地方，除了具有β溶血的特性以外，還有一些毒力因子是兩者皆有的，如M protein、SpeG (streptococcal pyrogenic exotoxin G)、ScpG (C5a peptidase)…等。 有學者發現在C群鏈球菌S. dysgalactiae subsp. equisimilis中，存在一個和S. pyogenes中的毒力因子調控基因mga有61％相似的基因，命名為mgc（multigene regulator-like），但其周圍的基因組成結構和mga大不相同，大致可分為large和small type。所以本研究希望了解G群鏈球菌S. dysgalactiae subsp. equisimilis臨床菌株是否也均有mgc，若有的話是否也扮演類似的角色。 先利用PCR偵測1998年至2004年間由臺大醫院分離，並經本實驗室確定為G群鏈球菌S. dysgalactiae subsp. equisimilis的274株臨床菌株，發現mgc locus廣泛分布於不同emm type的細菌中，其中small type的locus佔66％（183/274）。若依據emm type來分析，發現mgc locus的大小和emm type有部分相關，但與檢體來源無明顯相關。 接著挑選兩株臨床菌株，取一株emm type為stG840.0及一株stG6.1（因stG840之菌株皆為large type，stG6.1則皆為small type），進一步分析mgc locus之全長序列，發現mgc的序列相似度為99.61％，但large type的cpdB基因和之前發表的序列長度不同，再加上比對4株large和4株small type的序列，發現small type存有一小段truncated cpdB，由南方墨點法推斷small type的菌株在染色體的其他位置應不具有和cpdB相似的基因，且證實mgc在染色體中為single copy。 為證實Mgc在此菌中也具有功能，於是進一步以EMSA證實Mgc可結合到emm promoter上。進一步以in vitro DNA-protein pull-down的方法，找出一些Mgc可能會結合的基因，初步篩選到約80個基因，未來希望可以用EMSA對這些候選的基因進行確認，並以LA-PCR將未知的片段定序出來。Group G Streptococcus dysgalactiae subsp. equisimilis is an increasing recognized human pathogen. It is commensal in skin, throat, intestine, and vagina. But it may cause severe disease such as bacteremia, purulent pharyngitis, endocarditis, and streptococcal toxic shock syndrome. Many studies indicated that it resembles Group A Streptococcus pyogenes in many respects. They both are β-hemolysis on blood agar, and carry several virulence factors, for example; M protein、SpeG (streptococcal pyrogenic exotoxin G), and ScpG (C5a peptidase) etc. The mgc (multigene regulator-like) , which is 61％ identical to mga of S. pyogenes has been identified in Group C Streptococcus dysgalactiae subsp. equisimilis. But the genetic organization of mga and mgc are different. Since there is no report about mgc in group G streptococcus, thus, we investigate the distribution and characterization of mgc in Group G S. dysgalactiae subsp. equisimilis clinical isolates. First, using PCR, we found mgc locus is present in all strains tested with different emm types, and ~66％ (183/274) were the small type locus. While the size of mgc locus is associated with some emm types, we cannot link small or large type to niche specificities. Furthermore, we sequenced the mgc from two clinical isolates, with emm type stG840.0 and stG6.1, respectively. The mgc sequence from these two isolates were nearly identical (99.61％), but the sequence of cpdB gene in large type is different from the published before. And from 4 strains of large type and 4 strains of small types sequence, we found that there is truncated cpdB in small type mgc locus. The Southern blot results indicated that there is no other genes similar to cpdB in small type strains. We also confirm mgc is single copy in genome by Southern blot. By EMSA analysis, we confirm that His-tagged Mgc can bind emm promoter, suggesting that Mgc in group G Streptococcus is functional. Then we use “in vitro DNA-protein pull-down assay” to identify genes possibly regulated by Mgc.口試委員審定書..........................................i謝....................................................ii文摘要................................................iiibstract................................................v錄....................................................vii一章 緒論..............................................1.1 G群鏈球菌Streptococcus dysgalactiae subsp. equisimilis的介紹.....................................................1.2 mga調控基因和M蛋白對於A群鏈球菌Streptococcus pyogenes的重要性...................................................2.3 已知關於mgc調控基因的研究以及M蛋白對於G群鏈球菌的重要性.......................................................4.4 研究目的.............................................6二章 實驗材料與方法....................................7.1 實驗用菌株...........................................7.2 萃取鏈球菌DNA........................................7.3 聚合酶連鎖反應(PCR)..................................8.4 核酸定序（DNA sequencing）...........................10.5 RFLP（ristriction fragment length polymorphism）.....12.6 南方墨點法（Southern blot）..........................12.7 反轉錄-聚合酶鏈鎖反應 (reverse transcription-PCR)....15.8 北方墨點法（Northern blot）..........................18.9 基因選殖.............................................19.10 蛋白質表現 (protein expression).....................22.11 西方墨點法 (Western blot)...........................25.12 EMSA (Electrophoresis Mobility Shift Assay).........25.13 in vitro DNA-protein pull-down assay................28三章 實驗結果..........................................31.1 mgc locus的分布情形..................................31.2 mgc locus和檢體間的相關性............................31.3 mgc locus之序列分析..................................32.4 比較不同emm type的mgc序列............................33.5 mgc在細菌染色體中為single copy.......................34.6 small type mgc locus的菌株在染色體的其他位置應不具有cpdB基因.....................................................34.7 mgc和emm可能為同一個operon...........................34.8 Mgc可藉由結合啟動子的機制調控下游基因表現............35.9 Mgc可能調控的下游基因................................36四章 討論..............................................37.1 分析mgc locus和emm type之間的相關性..................37.2 mgc locus和mga regulon的比較.........................37.3 cpdB基因可能扮演的角色...............................39.4 以EMSA證實Mgc可以結合到emm promoter區域..............40.5 以in vitro DNA-protein pull-down assay探討Mgc可能調控的基因.....................................................40.6 比較Mga和Mgc所調控的基因.............................41.7 結論與未來方向.......................................45五章 附圖表............................................46一、臨床G群鏈球菌NTUH 5518和NTUH 7688 mgc locus定序結果.......................................................46二、small type mgc locus的菌株之truncated cpdB序列比對.......................................................47三、以RFLP確認經emmF和relR1為引子對所增幅出來的片段....48四、以北方墨點法分析mgc和emm基因的轉錄情形.............49五、以RT-PCR證實mgc和emm可以一起轉錄...................50六、南方墨點法：證明mgc在細菌染色體中為single copy.....51七、南方墨點法：偵測small type mgc locus的菌株中是否有cpdB基因的存在...............................................52八、臨床菌株NTUH 7688之emm promoter序列................53九、將mgc選殖到質體pRSET A.............................54十、以IPTG誘導E.coli BL21中的pRSET A-mgc的表現.........55十一、以西方墨點法確認E.coli BL21所表現的His-tagged Mgc蛋白質.....................................................55十二、以ProBondTM purification system純化His-tagged Mgc蛋白質.....................................................56十三、EMSA (electrophoresis mobility shift assay)......57十四、以Ni-NTA magnetic agarose beads找尋Mgc可能結合的基因.......................................................58十五、以Ni-NTA magnetic agarose beads結合His-tagged Mgc......................................................59十六、以Ni-NTA magnetic agarose beads所pull-down並clone到pUC19的DNA片段...........................................59一、mgc locus在不同emm type的分布情形..................60二、mgc locus在不同檢體的分布情形......................61三、S. dysgalactiae subsp. equisimilis中可能被Mgc所結合的基因.....................................................62考文獻.................................................64application/pdf1617436 bytesapplication/pdfen-USG群鏈球菌調控基因mgcemm typeEMSAin vitro DNA-protein pull-down assaygroup G streptococcusmgc[SDGs]SDG3http://ntur.lib.ntu.edu.tw/bitstream/246246/182976/1/ntu-98-R96424003-1.pdf