出版年:

研究生:

林桂媛

研究生(英文姓名):

論文名稱:

化膿性鏈球菌蛋白分解酵素基因特性之分析

英文論文名稱:

Study Of The SpeB Gene In Streptococcus Pyogenes

指導教授:

吳俊忠

學位類別:

碩士

校院名稱:

國立成功大學　

系所名稱:

微生物及免疫學研究所

學年度:

85

語文別:

中文

論文頁數:

84

關鍵詞:

化膿性鏈球菌 ; 插入法

英文關鍵詞:

Streptococcus pyogenes ; insertinoal inactivation

被引用次數:

0

[ 摘要 ]

　　化膿性鏈球菌（Streptococcus pyogenes）是一種具完全溶血性的革蘭氏陽性球菌。在臨床上此菌所引起的疾病很廣，從輕微的咽喉炎、化膿性皰疹，到危及人類生命安全的疾病，如壞死性肌膜炎、鏈球菌毒性休克症候群等。化膿性鏈球菌的致病因子很多，由本實驗室先前流行病學調查之結果顯示細菌侵入上皮細胞之能力以及此菌對動物之致死率皆與蛋白水解酵素有關。蛋白水解酵素又為speB基因之產物，因此深入研究speB基因將有助於了解化膿性鏈球菌之致病角色。為了證實SpeB之重要性，我們利用插入法（insertional inactivation）的方式，中斷蛋白水解酵素的基因speB，使其無法產生蛋白水解酵素的突變株。由酵素活性分析、南方墨點法及西方墨點法之結果，證實了重組質體可藉以single crossover 方式和染色體上之sbeB基因進行同源性重組作用，進而將speB基因中斷。以突變株與野生株比較，此兩株之生長曲線圖並無差異，表示中斷speB基因並不會影響細菌生長，由此推論SpeB並非細菌生長所必須的。另外，在動物實驗，發現突發株對於老鼠之存活率較野生株來得高；而在體外試驗，其侵入上皮細胞之能力，突變株其侵襲能力較野生株為低。這些實驗結果更可支持SpeB在化膿性鏈球菌致病機轉中確實扮演一個相當重要的角色。由於臨床所收集的72株化膿性鏈球菌皆有sbeB基因，但有4菌株不會分泌蛋白酵素，因此研究此基因的轉錄或轉譯將有助於了解此基因的調節。以隙縫點墨法分析細菌是否有speBmRNA之產生，結果顯示在不具有SpeB蛋白水解酵素的菌種中，皆未發現有speB基因的轉錄。利用核酸引子延伸（primer extension）的實驗得知，speBmRNA的轉錄起始點位在轉譯起使點上游第78個氮鹼基 ''T'' 之位置。此外，以RCR構築一個完整之speB基因，而後送入大腸桿菌複製。由西方墨點法，推測我們所構築之recombinant SpeB蛋白質可在大腸桿菌表現（40-33kDa）。綜合以上這些實驗，本論文提供很多新的資料，這些研究成果將有助於了解蛋白水解酵素及其基因之特性，更進而了解此酵素在化膿性鏈球菌之致病機轉。

[ 英文摘要 ]

　　Streptococcus pyogenes (β-hemolytic group A streptococcus, GAS) is the causative agent of wide spectrum of disease, ranging from streptococcal pharyngitis and impetigo to life-threatening necrotizing fasciitis and streptococcal toxic-shock syndrome. S. pyogenes expresses a variety of cellsurface proteins and secreted extracellular products that canused human diseases In our laboratory, the preliminary data showed that the efficient invasion was correlated with the amount fo proteolytic activity in GAS. To further investigate the potential role of SpeB in the pathogenesis of GAS, the speB isogenic mutant was constructed by insertional inactivation. A single crossover to chromosome was confirmed by Southern hybridization. The protease activities were detected by skim-milk plate and Western blot analysis. The mutant had no detectable proteolytic activity and had a similar growth rate to that of wild-type strain, suggesting that SpeB was not essential for viability. In addition, the mutant not only increased survival rate in the BALB/c mice but also decrease ability of invasion to epithelial cell than that of wild-type strain. These results provide additional evidence that SpeB may play an important role in streptococcal infections.
　　The structure gene of speB is present in all 72 strains of FGAS. However, only 4 strains can not have proteolytic activity. Whether these protease deficient strains were reflected at the transcriptional level. A slot hybridization was performed on purified RNA from protease positive and negative strains. The results demonstrated that speB mRNA was not detected in all four protease negative strains. For further understanding the regulation of speB, primer extension analysis was used to define the transcription initiation site of speB. The start site is 78 bases upstream from the putative translation initiation codon. Expression of recombinant SpeB proteins in E. coli was also constructed. The preliminary data showed that multiple bands ranging from 40 to 33 kDa were detected by Western blot analysis. This study provides novel information regarding the role of SpeB in the pathogenesis of S. pyogenes and the results of this study will lead to a better understanding of the complex system of streptococcal infection.