大腸桿菌抗病毒食物因子篩選平台建構與植物乳桿菌抗原表現系統優化之研究

Abstract

隨著COVID-19等病毒傳染病威脅日益嚴峻，藥物治療與疫苗防疫策略逐漸式微，促使天然食物因子與次世代黏膜疫苗佐劑成為研究焦點。然而，食物因子之抗病毒活性缺乏高效、快速且安全的驗證方式；乳酸菌作為黏膜疫苗載體雖具潛力，但前人研究因設計與技術層面的限制，始終無法成功實現抗原表現。本研究旨在建構一套能快速驗證抗病毒分子活性的細菌平台，並克服乳酸菌轉形與抗原表現的技術障礙。在篩選平台建立部分，本研究將病毒抗原片段表現於大腸桿菌 (Escherichia coli) 內部，並經外膜處理後與抗病毒候選因子反應。隨後藉由免疫螢光標定及顯微鏡影像分析，驗證候選因子競爭性抑制抗原-抗體結合的潛力，作為活性評估的依據。乳酸菌表現部分，則針對質體轉形失敗與蛋白未表現的瓶頸進行深入分析，逐步釐清原因並透過篩選合適抗生素選殖系統（由ampR改為ermR）、電穿孔緩衝液改良（使用PEG-1500）、DNA來源菌株優化（選用E. coli JM110未甲基化修飾質體）及質體序列修正等方式，大幅提升植物乳桿菌 (L. plantarum K68) 轉形效率。最終，由Western blot確認重組菌株在誘導條件下可穩定表現目標Spike抗原。本研究建立的篩選平台具快速且成本低的優勢，適合作為抗病毒功能性食品開發的前期篩選工具；而優化後之乳酸菌轉形與抗原表現系統，則可作為未來益生菌疫苗研發的重要基礎，具延伸應用潛力。

With the increasing threat of infectious diseases such as COVID-19, the efficacy of conventional therapeutics and vaccines are diminishing, which has led to the focus on natural food factors and next-generation mucosal vaccine adjuvants. However, there is a lack of efficient, rapid and safe ways to validate the antiviral activity of food factors, and although lactic acid bacteria (LAB) have the potential to be used as mucosal vaccine carriers, previous studies have been unable to achieve antigenic performance due to design and technical constraints. This study aimed to establish a bacterial platform for rapid screening of antiviral compounds, and to overcome technical barriers related to plasmid transformation and antigen expression in lactic acid bacteria. For the screening platform, viral antigen fragments were expressed in Escherichia coli and subjected to permeabilization treatment, enabling them to interact with candidate antiviral agents. Immunofluorescence labeling and microscopic image analysis were performed to verify the ability of the candidate factors to competitively inhibit antigen-antibody binding, which was used as the basis for the preliminary evaluation of their activity. For the LAB expression system, we identified key bottlenecks in transformation failure and low expression. Through systematic optimization—including switching the selection marker from ampR to ermR selection, replacing electroporation buffer with PEG-1500, using methylation-deficient E. coli JM110 for plasmid preparation, and correcting sequence design errors—we significantly improved transformation efficiency in L. plantarum K68. Finally, we confirmed stable expression of transformants by Western blot. In summary, this study presents a rapid, cost-effective bacterial screening tool for functional food preliminary evaluation. The optimized transformation and antigen expression system in L. plantarum K68 further lays a solid foundation for future applications in probiotic vaccine development.