利用噬菌體展示技術製造及分析可辨認金黃色葡萄球菌烯醇化酶
之雞隻單株抗體片段

金黃色葡萄球菌（Staphylococcus aureus, S.aureus）是院內感染及社區感染的主要病原菌之一，其對於人類疾病發生率及致死率之影響長久以來一直是嚴重的問題。在病原菌感染宿主細胞的機制中，病原菌表面的蛋白和宿主細胞胞外基質之間的交互作用扮演了重要的角色。過去的研究指出，alpha-Enolase，一種參與在糖解代謝反應中的酵素，在金黃色葡萄球菌中能夠做為 plasminogen 的結合受體或是 laminin 結合蛋白，進而可能促進病原菌感染及侵入宿主細胞的能力；因此，本篇的研究目標是利用重組表現金黃色葡萄球菌 alpha-Enolase 蛋白來免疫雞隻，並藉由噬菌體展示技術來製作並篩選出具有與金黃色葡萄球菌 alpha-Enolase 特異性結合反應的多株及單株抗體。首先將 1302 bp 長度的 S.aureus alpha-Enolase 基因片段選殖入 pET 101 載體並表現出 His-S.aureus alpha enolase 融合蛋白並且將其純化。之後，為了製造雞隻 IgY 多株抗體，將純化後的 His-S.aureus alpha-Enolase 蛋白與佐劑適當混合均勻後皮下注射入雞隻。利用西方墨點法以及酵素連結免疫分析實驗，證實在免疫後雞隻體內的確誘發出高劑量的抗體反應並可辨認 His-S.aureus alpha-Enolase 融合蛋白。為了製作出可與 His-S.aureus alpha-Enolase 蛋白結合的單株抗體庫，我們將雞隻犧牲後萃取出其脾臟中之 mRNA 並利用聚合酶連鎖反應製造出抗體重鏈和輕鏈基因片段並形成單鏈抗體片段（scFv）。將做出的單鏈抗體片段選殖入噬菌體載體（phagemid）中以製做出單鏈抗體片段基因庫。之後使用四次週期的淘選（panning）方法從所建構的抗體基因庫篩選並富化出具有和 His-S.aureus alpha-Enolase 蛋白結合能力的單株抗體片段。在淘選過後，將噬菌質體之 DNA 轉型進入 TOP10F’大腸桿菌，挑選出了 15 株能夠表現分子量大小約為 28 kDa 單株抗體片段蛋白的菌株。經過序列比對分析後證明在經過淘選後有六個能夠表現抗體片段之菌種族群有成功被篩選並富化出來。之後利用西方墨點法以及酵素連結免疫分析實驗分析這些抗體片段對於 His-S.aureus alpha-Enolase 蛋白之結合能力，結果顯示這些抗體片段能夠專一性辨認 His-S.aureus alpha-Enolase 蛋白。最後利用流式細胞儀分析法及免疫螢光染色法確認雞隻 IgY 多株抗體及兩個單鏈抗體片段對於 S.aureus alpha-Enolase 具有結合能力。未來將進一步分析這些抗體片段之特性，並期望能將其運用到臨床診斷或治療用藥之研究上。

關鍵字

金黃色葡萄球菌；噬菌體展示技術；單株抗體片段

並列摘要

Staphylococcus aureus is a major human pathogen and it has been a serious health problem on human morbidity and mortality. Previous research suggests that cell-surface protein-mediated interactions of bacteria and the extracellular matrix play an important role in the mechanism of pathogen infection. Studies have revealed that a glycolytic enzyme, alpha-enolase (ENO), acts as a enhancer of plasminogen activator or laminin binding protein in Staphylococcus aureus, and may promote pathogen infection and the ability of tissue invasion. In this study, we aimed to generate high affinity of polyclonal and monoclonal chicken antibodies against S.aureus alpha-Enolase protein by using phage display technology. The S.aureus alpha-enolase 1302 bp DNA fragment was cloned into pET-101 vector expressed as His-S.aureus alpha-enolase fusion protein, and purified. These purified His-S.aureus alpha-enolase fusion protein was mixed with adjuvant and the mixture was injected intramuscularly into Leghorn chicken to generate polyclonal IgY antibodies. His-S.aureus alpha-enolase fusion protein can be recognized by these polyclonal antibodies in western blot and ELISA analysis. To construct S.aureus alpha-enolase -specifc antibody libraries, we isolated chicken's spleen mRNA and used overlap PCR to generate DNA fragments containing heavy and light chain variable genes. These scFv DNA fragments were cloned into phagemid vectors. Several antibody libraries against His-S.aureus alpha-enolase fusion protein were obtained. Four rounds of phage panning was performed to enrich antibody fragments against His-S.aureus alpha-enolase protein. After panning, total phagemid DNA was introduced into TOP10F’ E.coli and fifteen clones were selected for antibody expression. Our result indicated that the protein size of E.coli-expressed scFv antibody is about 28 kDa. Sequence analysis of these antibody fragments indicated that six particular clones were enriched through phage panning process. These clones containing scFv antibody fragments were examined for their binding ability to His-S.aureus alpha-enolase protein by ELISA and western blot. Our results showed that several clones of scFv bind to His-S.aureus alpha-enolase protein specifically. Finally, polyclonal IgY antibodies and two scFv antibodies were confirmed their binding ablility to S.aureus alpha-Enolase by flow cytometry analysis and immunofluorescence analysis. Further studies are undergoing to characterize these antibody fragments with specific binding ability in the development of diagnostic or therapeutic agents in the future.

並列關鍵字

Staphylococcus aureus ； phage display ； scFv

參考文獻

Ko-Jiunn Liu and Neng-Yao Shin.,2007. The Role of Enolase in Tissue Invasion and Metastasis ofPathogens and Tumor Cells. Journal of Cancer Molecules 3(2), 45-8

Akita, E.M. and Nakai, S., 1993a. Comparison of four purification methods for the production of immunoglobulins from eggs laid by hens immunized with an enterotoxigenic E. coli strain. J Immunol Methods 160, 207-14.

Akita, E.M. and Nakai, S., 1993b. Production and purification of Fab' fragments from chicken egg yolk immunoglobulin Y (IgY). J Immunol Methods 162, 155-64.

Andris-Widhopf, J., Rader, C., Steinberger, P., Fuller, R. and Barbas, C.F., 3rd, 2000. Methods for the generation of chicken monoclonal antibody fragments by phage display. J Immunol Methods 242, 159-81.

Aström, E., and Ohlson, S., 2006. Detection of weakly interacting anti-carbohydrate scFv phages using surface plasmon resonance, J. Mol. Recognit 19, 282–286

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利用噬菌體展示技術製造及分析可辨認金黃色葡萄球菌烯醇化酶之雞隻單株抗體片段

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