- 學位論文
創傷弧菌外毒素RtxA 高量甘氨酸-天門冬氨酸區域人源單鏈抗體之篩選以及此區域的細胞表面結合特性之研究
Isolation of a human single-chain antibody targeting to the Gly-Asp rich region of Vibrio vulnificus RtxA toxin and characterization of the cell surface adherence property of the region
摘要
創傷弧菌會分泌數種致病因子，造成細胞病變。其中一種主要致病因子為RtxA 外毒素，被歸類為MARTX 蛋白質家族成員。在此蛋白質家族中，成員皆具有多樣功能及自我處理的特性。 典型MARTX 蛋白質的C 端含有多次重複的甘氨酸-天門冬氨酸豐富區域。此外，根據推測MARTX毒素的C 端區域會輔助此類毒素進入真核細胞，並且和細胞膜的表面因子產生交互作用。然而，在創傷弧菌RtxA 中，含有甘氨酸-天門冬氨酸豐富區域的C 端是否具有和宿主細胞表面結合的特性，仍須研究證實。 本研究分為兩個部份。第一部分為選殖創傷弧菌RtxA C 端的甘氨酸-天門冬氨酸重複區域，將所得到的純化蛋白作為抗原，用以篩選噬菌體表面展現的人源單鍊抗體庫。第二部份的實驗則著重於驗證RtxA 的C 端區域具有黏附細胞的特性，並且利用結構預測伺服器來預測 此區域的可能分子構造。 第一部分篩選的結果，得到一株可和此重組的甘氨酸-天門冬氨酸豐富區域反應的抗體。後續西方點墨法分析的結果顯示，此抗體不但可專一性辨認甘氨酸-天門冬氨酸豐富區域，並可偵測到創傷弧菌溶解物中RtxA 片段的存在。在第二部份的實驗裡，證實此蛋白具有黏附人類喉癌表皮細胞表面的特性。從結構預測的結果亦可得知，此豐富區域的分子構造類似於它種RTX 蛋白質C 端區域，因此亦可能具有和鈣離子結合的活性，並且負責毒素和宿主細胞之間的交互作用。
並列摘要
Vibrio vulnificus secretes several virulence factors causing cytopathic effects on the host cells. RtxA is a major virulence factor of this bacterium that was categorized into MARTX protein family, in which members of this family are multifunctional and autoprocessing. The MARTX proteins typically contain Gly-Asp rich regions repetitive several times in the C-terminal region. The C-terminal domain had been proposed to be involved in the translocation of the MARTX toxin into eukaryotic cells, and may act as a host cell surface adhesion factor. Nevertheless, whether the Gly-Asp-rich C-terminal region of V. vulnificus RtxA indeed binds to the host cells remains to be determined. This study is divided into two parts. In the first part, we specifically cloned, overexpressed and purified the Gly-Asp rich portion of C-terminal region of V. vulnificus RtxA (GD-repeat). The recombinant protein was then utilized as the target for screening human single-chain variable fragment (scFv) phage display library. The second part of this study aims at characterization of the cell surface adhesion property of recombinant GD-repeat, and a structure modeling web server (SwissModel) was employed to predict the molecular structure of this cloned region. One antibody (scFv-GD23C) showed binding activity to recombinant GD-repeat. In subsequent Western blot analysis, the antibody not only specifically recognized the GD-repeat, but also detected Vibrio vulnificus RtxA fragment in bacterial lysate. In the second part, the surface adhesions on HEp-2 cells of the cloned GD-repeat was demonstrated by immunofluorescence, and the result of modeling had described a special -barrel structure of this region. The structure is similar to the C-terminal regions of other RTX proteins, suggesting this region may possess calcium-binding activity and being responsible for toxin-host cell interaction.
並列關鍵字
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延伸閱讀
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