Title

本土志賀桿菌的ipaB基因及其蛋白產物功能分析

Translated Titles

Characterization of the ipaB gene of Taiwan Shigella flexneri isolates and its protein product

Authors

顏珮詩

Key Words

志賀桿菌 ; ipaB gene

PublicationName

中興大學分子生物學研究所學位論文

Volume or Term/Year and Month of Publication

2006年

Academic Degree Category

碩士

Advisor

陳建華

Content Language

繁體中文

Chinese Abstract

志賀氏桿菌(Shigellae)感染人類會引起桿菌性痢疾(Shigellosis)。志賀氏桿菌帶有毒性質體,毒性質體帶有許多的致病基因,其中 ipaB 基因所轉譯的IpaB 蛋白具有造成巨噬細胞凋亡的能力。本實驗室收集了 1996 年到 2000 年在台灣南投爆發的 Shigellosis 流行中所分離到的菌株,其中在 1996 年台灣爆發的 Shigellosis 流行中分離到的 40 株不同 S. flexneri 菌株以 Pulsed-field gel electrophoresis (PFGE) 方式進行分類,結果發現有 26 株菌具有相同的 PFGE pattern,此些菌稱為流行菌株。在 1996 年實驗室共收集到 26 株流行菌株,其中 7 株 ipaB 基因經 10 天連續培養後仍穩定存在,而 1997、1998、1999、2000 年中實驗室共收集到 47 株流行菌株,其中只有 8 株 ipaB 基因經 10 天連續培養後仍穩定存在。 實驗室已構築一個帶有 ipaB 基因前 803 bp 片段的質體 pET21b-ipaB803,利用此質體將 IpaB803 蛋白誘導純化出來,將 inclusion body 濃縮後打入小鼠製備抗體,製備好的抗體則用來偵測 1996 到 2000 年 ipaB 基因穩定的流行菌株,其中 1996 年的 7 株流行菌株,只有其中一株 SH3160 在連續培養 10 天後,有表現 IpaB 蛋白的能力,其他流行菌株則無此情形。1997 年到 2000 年的 8 株流行菌株,在連續培養 10 天後則皆無法表現 IpaB 蛋白。任選 1996 年一株流行菌株(SH2308)與 檢測 SH3160 的突變率是否較低,但是結果顯示兩菌株的突變率相同。 進一步研究 SH3160 與 SH2308 的 ipaB promoter 之調控,以 β-galactosidase 為報導基因檢測 ipaB promoter 的活性,結果顯示 SH3160 的 ipaB promoter 活性較 SH2308 高約 50﹪。將 virF 基因構築到質體中,送入 SH2308 經 10 天連續培養後 virF 基因缺失的菌株 SH2308-10A 後,Western Blot 檢測顯示送入帶有 virF 基因質體可以使 SH2308-10A 回復產生 IpaB 蛋白的能力。將純化後的 IpaB803 蛋白,感染人類類巨噬細胞 U937,隨著感染的時間增長,U937細胞凋亡的情況也有增加的情況,顯示 IpaB803 蛋白有能力造成 U937 細胞凋亡

English Abstract

Many Shigella flexneri isolates were collected previously by our laboratory during a shigellosis outbreak in Nantou country form 1996 to 2000. These isolates were sub-typed by pulsed-field gel electrophoresis (PFGE), and 26 isolates in 1996 and 47 isolates form 1997 to 2000 were found with the same PFGE types. They belonged to an epidemic strain. In this study, 7 of 26 epidemic isolates in 1996, and 8 of the 47 of the epidemic isolates form 1997 to 2000 were found having the ipaB gene stably maintained in the first- and tenth-day subcultures. Plasmid pET21b-ipaB803 carrying the first 803 bps of the ipaB gene under the T7 promoter was used to over-express the partial IpaB protein. The protein was injected into mice for antibody production. By western hybridization with the IpaB protein stably maintained in the first- and tenth-day subcultures. An epidemic isolate(SH2308) form 1996 was randomly picked and the spontaneous mutation rate was compared with that of SH3160. The result indicates both isolates had the same mutation rate. The promoter activities of the ipaB genes in SH2308 and SH3160 were analyzed by using the β-gala gene as the reporter gene. About 50﹪ higher promoter activity was found in SH3160. A SH2308 mutant called SH2308-10A was previously isolated form the tenth-day subculture of SH2308 and proved to carry a deletion of the virF gene. The virF gene form SH2308 was cloned into a plasmid and transformed into SH2308-10A. Western analysis shows that SH2308-10A could not produce IpaB protein, but the transformant could. The partial IpaB protein was over-produced and purified by induction of BL21(DE3) carrying the plasmid pET21b-ipaB803. The purified protein was used to infect the humand U937 cells for different time spans and the cells were analyzed by flow cytometry. It was found that apoptosis of the U936 cells increased as the infection time increased.

Topic Category 生命科學院 > 分子生物學研究所
生物農學 > 生物科學
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Times Cited
  1. 許哲瑋(2009)。志賀氏桿菌 ipaB 基因及其衍生蛋白 對肺癌細胞的影響。中興大學分子生物學研究所學位論文。2009。1-80。
  2. 張瑋倫(2010)。福氏志賀菌 IpaB 衍生蛋白之純化及其對哺乳動物細胞的影響。中興大學分子生物學研究所學位論文。2010。1-118。
  3. 陳琬蓉(2011)。志賀氏桿菌ipaB基因產物對哺乳動物細胞毒殺作用之探討。中興大學分子生物學研究所學位論文。2011。1-106。
  4. 曾昭傑(2012)。福氏志賀菌IpaB蛋白及其衍生蛋白對大腸癌細胞株HCT116的影響。中興大學分子生物學研究所學位論文。2012。1-63。