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標題: 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
Purification of a derivative of a Shigella flexneri antigenic protein, IpaB, and its effects on mammalian cells
作者: 張瑋倫
Chung, Wei-Lun
關鍵字: shigella flexneri
出版社: 分子生物學研究所
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摘要: 福氏志賀菌 (Shigella flexneri) 入侵人類結腸的表皮細胞後,會引發嚴重的發炎反應,大量破壞腸道組織,造成桿菌性痢疾 (Shigellosis) ,於臨床上的症狀有腹瀉、腹痛和血便等。福氏志賀菌帶有一毒性質體,含有許多致病基因,其中的 IpaB 基因所負責產生的 Invasion plasmid antigens B (IpaB) 蛋白能使自腸道 M 細胞侵入的福氏志賀菌,順利進入腸道下層的巨噬細胞中,並活化巨噬細胞中的 caspase-1 ,進而造成巨噬細胞進行細胞凋亡 (apoptosis)。實驗室已構築一個帶有 IpaB 基因前 803 bp 片段 (IpaB803 基因) 的質體,並使蛋白 C 端帶有 6 個組胺酸 (Histidine,His),並利用大腸桿菌大量表現及純化重組蛋白IpaB803-His。本研究主要是將 IpaB803-His 蛋白經過二次純化,探討二次純化的 IpaB803-His 蛋白對於人類不同組織的癌細胞株及非癌細胞株是否會有不同影響。 實驗室曾將 1996 年分離自南投縣臨床菌株 SH2308 中的 IpaB 基因前 803 bp 構築到表現載體 pET-21b (+) ;利用此重組質體 pET-21b-IpaB803-His ,於大腸桿菌中藉 IPTG 誘導大量表現後,經由親和性管柱 (Ni-NTA resin) 進行一次純化,但純化後的 IpaB803-His 蛋白內仍有許多雜質蛋白。為了降低雜質蛋白的存在,一開始藉由重新摺疊 (refolding) 大腸桿菌內的 IpaB803-His 包涵體 (inclusion body) 的方式,希望可得到水溶性的IpaB803-His變性蛋白 (denature protein)。根據 SDS-PAGE 結果發現 : 當利用含有 4 M urea 的 50 mM imidazol lysis buffer 回溶 IpaB803-His包涵體,可擁有最大量的水溶性但可能變性 (denature) 的IpaB803-His包涵體蛋白。將可溶性的 IpaB803 變性體蛋白以 1 μg 或 2 μg的劑量,用 lipofectamine 包裹後轉殖到人類血癌細胞株 U-937 中,均無法讓 U-937 細胞死亡。接著將純化方式改為將原本已一次純化過的水溶性 IpaB803-His 蛋白,多通過一支疏水性管柱 (Butyl-S sepharose 6 FF column);發現一次或是二次純化後的 IpaB803-His 蛋白,均可對人類血癌細胞株 U-937 以及 HL-60 有毒殺能力。故將二次純化後的 IpaB803-His 蛋白分別轉殖到人類的非小細胞肺腺癌細胞株 (CL1-0、CL1-5、A 549),大細胞肺癌細胞株 (H 460),肺非癌細胞株 (BEAS-2B、WI-38),腎臟胚胎細胞株 (HEK-293),肝癌細胞株 (HepG2),結腸癌細胞株 (CaCo-2、COLO 205),血癌細胞株 (HL-60、U-937),周邊血液單核球細胞 (PBMC)。結果發現,IpaB803-His對於 A-549、CL1-0、CL1-5、H 460、BEAS-2B 和 HEK 293,無明顯毒殺細胞作用,甚至A-549有增生現象。但是對於 WI-38、HepG2、CaCo-2、COLO 205、U-937、HL-60和PBMC 而言,則發現都有 20 % 以上毒殺細胞的作用,其中對於 HL-60 和 HepG2 有最大的毒殺效果 (轉殖 24 小時後,毒殺結果大於 50 %)。而將IpaB803-His 蛋白分別被轉殖到 A-549 和HepG2 16 小時後進行免疫螢光染色;結果發現不論是 A-549 或是 HepG2 細胞,可以看得到大部分的 IpaB803-His 蛋白是呈現聚合體 (aggregate) 之型態,並且都黏在細胞的細胞膜上,推測 IpaB803-His 蛋白有可能是和細胞膜上的受體 (receptor) 有交互作用。其中,在 HepG2 細胞中甚至可以看到 IpaB803-His 蛋白與α-tubulin 有重疊之現象。
Shigella flexneri caused diarrhea or dysentery in human (shigellosis). It carries a virulence plasmid on which mant virulence genes reside. One such gene, IpaB, encoded invasion plasmid antigens B (IpaB) had been demonstrated to enable S. flexneri to enter macrophages underneath the M cells of intestine, activate pro-caspase-1 into caspase-1 in macrophages, and cause apopotosis of macrophages. Previously, the 5’ 803 bp of IpaB was cloned in to E. coli expression vector and IpaB803-His protein was over-expressed by IPTG induction. The protein was purified by Ni-NTA affinity chromatography. The purpose of this study was to further purify the protein and investigate the effects of the purified protein on human cancer and non-cancer cells. Initially, the IpaB803-His protein in inclusion body of the over-expressed E. coli was harvested and soluble IpaB803-His protein was obtained by solubilization in 4 M urea in 50 mM imidazol lysis buffer. However, the protein disn’t show ant cytotoxic actibity against hrman leukemia cell U-937. On the other hand, when the soluble IpaB803-His protein was first purified by affinity chromatography through nickel-NTA column followed by hydrophobic interaction chromatography through Butyl-S sepharose 6 FF column, the purified protein did demonstrated cytotoxic activity against human leukemia cells U-937 and HL-60. The purified protein was then tested for its activity against lung cancer cell (CL1-0、CL1-5、A-549、H 460), lung non-cancer cells (BEAS-2B、WI-38), kidney non-cancer cell (HEK 293), liver cancer cell (HepG2), colon cancer cell (CaCo-2、COLO 205), leukemia cell (HL-60、U-937) and peripheral blood mononuclear cell (PBMC). The results showed that the protein didn’t show cytotoxic activity against A-549、CL1-0、CL1-5、H 460、BEAS-2B and HEK 293, but show significant (greater then 20 %) cytotoxic activity against WI-38、HepG2、CaCo-2、COLO 205、U-937、HL-60 and PBMC. Immunostaining of the IpaB803-His protein-containing A-549 and HepG2 indicated that the protein was located the cell membrane of the two cells.
其他識別: U0005-2008201012362200
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