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研究生(外文):Yi-Chia Tseng
論文名稱(外文):Immunological characterization of Neisseria gonorrhoeae hypothetical proteins
指導教授(外文):Chiou-Ying Yang
口試委員(外文):Leung-Kei SiuChi-Jan Lin
外文關鍵詞:Neisseria gonorrhoeaehypothetical protein
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淋病 (gonorrhea) 是由淋病雙球菌 (Neisseria gonorrhoeae, Ng) 感染所導致之疾病,主要藉由性行為途徑感染,感染後多數患者無明顯癥兆並且可能經由垂直傳染給下一代。除此之外,康復患者體內不具有免疫記憶性,早期只要使用抗生素治療就能達到有效療效,但 Ng 菌多重抗藥性的問題逐漸增加,使得治療變得棘手,開發有效疫苗是目前最佳解決方式。本研究以反向疫苗學 (Reverse vaccinology) 方法作為基礎,從全序列定序完成的參考菌株 FA 1090 的假設性蛋白質 (hypothetical protein) 中挑選標的,首先藉由電腦軟體 SignalP-5.0 Server 預測蛋白質是否為分泌性蛋白質,再以 Gneg-mPLoc 預測蛋白質在菌體位置並以 Blast 比對,選擇高保守性的目標製備重組蛋白質以進行免疫原性 (immunogenicity) 分析。本研究以台灣分離的臨床菌株為模板共分析14個基因,其中有8個基因透過 PCR 成功被增幅出,分別為 NGO1889、NGO1484、NGO0783、NGO2002、NGO2133、NGO0782、NGO0666和NGO2054 。將上述8個基因分別構築至表現載體,送入 E. coli 表現並純化重組蛋白質,純化後的蛋白質進行小鼠免疫試驗,以獲得之抗血清進行西方墨點分析各蛋白質在 Ng 菌的表現情形,證實有5個蛋白質之抗血清具有認回原菌能力且在不同的臨床菌株都有表現。Whole- cell ELISA與免疫螢光染色法分析結果顯示只有 NGO1889、NGO1484、NGO0783和NGO2002 暴露於菌體表面,繼而分析 這些抗血清的殺菌力,結果只有 NGO0783 抗血清對不同臨床菌株具有殺菌活性。進一步進行in vivo 小鼠保護力分析,證實事先接種NGO0783 可增加小鼠對 Ng 菌感染的抵抗力,因此 NGO0783 具有成為開發Ng疫苗之潛力抗原。
Gonorrhea is a sexually transmitted disease caused by infection with the Neisseria gonorrhoeae (Ng) bacterium. Most patients are asymptomatic or signs and may be transmitted to the next generation through vertical transmission. In addition, recovery patients have no immunological memory in their body, and antibiotic treatment is effective for early stage of infection. However, antibiotic resistance is representing a serious problem for treatment. Vaccine development seem to be the best way for treatment. Based on the reverse vaccinology, we select hypothetical protein from the reference strain FA 1090, complete sequence sequencing. First, use SignalP-5.0 Server to predict whether the protein is a secreted protein, and then use Gneg-mPLoc to predict the location of the protein in the bacterial cell. Select highly conserved targets to prepare recombinant proteins for immunogenicity. In this study, the 14 genes template are from Taiwan clinical strains, which 8 genes are successfully amplified by PCR, named NGO1889, NGO1484, NGO0783, NGO2002, NGO2133, NGO0782, NGO0666 and NGO2054. The 8 genes are constructed into expression vectors. Escherichia coli is used expression host for the production of recombinant proteins. After immunogenicity test, the expression of each protein in Ng was analyzed by western blotting with anti-serum, the result showed that only five protein’s antibodies has the ability to recognize the original bacteria. The results of Whole cell ELISA and immunofluorescence staining test showed that only NGO1889, NGO1484, NGO0783 and NGO2002 were surface-exposed protein. Further, we tested serum bactericidal activity against different Ng strains and it showed only NGO0783 anti-serum effect on Ng. Final, we choose NGO0783 test protection assay and then NGO0783’s anti-serum effect protection mouse prevent infection. To sum up, this study showed that NGO0783 may be a potential vaccine antigen.
摘要 i
Abstract ii
目錄 iii
表目錄 v
圖目錄 vi
縮寫字對照表 (Abbreviation) viii
壹、 前言 1
一、 緒論 1
二、 奈瑟氏淋病雙球菌 1
(一) 奈瑟氏淋病雙球菌歷史 1
(二) 奈瑟氏淋病雙球菌流行病學 1
(三) 奈瑟氏淋病雙球菌之抗藥性 2
三、 奈瑟氏淋病雙球菌之疫苗研發 3
(一) Ng 菌疫苗先前發展 3
(二) Ng疫苗研發近況 4
四、 研究目的 5
貳、 材料與方法 6
一、 材料來源 6
(一) 菌株培養條件 6
(二) 動物實驗 6
二、 實驗方法 6
(一) 基因構築 6
(二) 重組蛋白質之誘導表現與純化 9
(三) 蛋白濃度定量 10
(四) 蛋白質膠體電泳分析 (sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE) 10
(五) 免疫小鼠實驗 12
(六) 酵素結合免疫吸附法 (Whole cell enzyme-linked immunosorbent assay, ELISA) 12
(七) 血清殺菌力分析 (Serum bactericidal assay) 13
(八) 免疫螢光染色法 (Immunostaining assay) 14
(九) 致死劑量 (Lethal dose assay) 14
(十) 保護力分析 (protection assay) 15
參、 結果 16
一、 目標抗原之選擇 16
二、 重組蛋白質之製備 16
(一) 基因選殖與表現載體的構築 16
(二) 重組蛋白質表現與純化 17
三、 目標蛋白質在 Ng 菌之表現情形 18
(一) 目標蛋白質在不同 Ng 分離菌株中廣泛表現 18
(二) 以Whole Cell ELISA 及 immunostain 偵測目標蛋白質在 Ng 菌表面蛋白質表現 19
四、 In vitro 體外試驗-抗血清殺菌力分析 (Serum bactericidal assay; SBA) 20
五、 In vivo 主動保護力分析 21
肆、 討論 23
伍、 參考文獻 28
陸、 圖表 34
柒、 附錄 61
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