建立高表達抗原之益生菌疫苗平台

Abstract

近年來全球爆發多起由病毒所引起的呼吸道傳染疾病，包含 SARS、MERS 以及現階段的 SARS-CoV-2，造成疫苗需求上升，推動了新型疫苗的發展。益生 菌疫苗是一種口服疫苗，可以引起全身性及黏膜免疫反應，產生特異性抗體 IgG 及 SIgA，在病毒入侵黏膜時即被抗體中和，阻止感染。此外，益生菌疫苗具有 低成本、容易儲存、方便接種等優勢，適合應用於大型流行性疾病的預防。然而， 受到嚴峻腸胃道環境以及口服耐受的挑戰，益生菌疫苗需要提高表達水平以及添 加佐劑來引發有效的保護反應。因此，本研究透過基因工程調整啟動子與抗原表 達方式，並設計 SARS-CoV-2 當作抗原，以乳酸菌作為載體發展高表達抗原之 益生菌疫苗平台。分別使用誘導型及連續表現型啟動子，建造錨定型、分泌型與 分泌錨定型抗原表達模式，後續還比較了質體的不同抗生素抗性基因對抗原表達 的影響。透過流式細胞儀偵側乳酸菌表面抗原量，發現在 ampicillin 抗性基因 (AmpR)篩選下整體表現比 erythromycin(ErmR) 好，且 AmpR 當中的連續型 啟動子(P11)比誘導型(sppA)有更高的表現量，又以錨定型表達模式為最高 者。同時藉由螢光顯微鏡確認抗原確實表現在乳酸菌表面。此外，在不同宿主中， 重 組 菌 株 Lactiplantibacillus plantarum PS128 表 現 量 比 Lactiplantibacillus plantarum K68 及 Latilactobacillus sakei 要來得好，因此，選擇 AmpR 連續型啟 動子的錨定表達質體 pA-P11-ASD，搭配 L.plantarumPS128 可作為高表達抗原 之 COVID-19 益生菌疫苗平台。

In recent years, there are many outbreaks of respiratory infectious diseases caused by viruses around the world, including SARS, MERS and the current SARS-CoV-2, resulting in rising demand for vaccines and thus promoting the development of different vaccines. Probiotic vaccine is an oral vaccine that can induce systemic and mucosal immune responses to produce specific antibodies IgG and SIgA, which neutralize viruses to prevent their mucosa invasion. In addition, probiotic vaccines are good choices for pandemic prevention due to the advantages of low cost, easy storage, and convenient vaccination. However, challenged by the harsh gastrointestinal environment and oral tolerance, probiotic vaccines require increased expression levels and the addition of adjuvants to elicit an effective protective response. Therefore, this study adjusted the promoter and antigen expression pattern through genetic engineering, using SARS-CoV-2 as the antigen and lactic acid bacteria as the carrier to develop a probiotic vaccine platform with high antigen expression. Inducible and constitutive promoters were used to construct anchored, secreted and secreted-anchored antigen expression patterns, and antibiotic resistance gene was subsequently replaced for antigen expression analysis. Flow cytometry was used to detect the surface antigen of lactic acid bacteria, and the results displayed that the overall performance of ampicillin resistance gene (AmpR) was better than that of erythromycin, and the constitutive promoter (P11) in AmpR was higher than the inducible type (sppA), furthermore, the anchored expression pattern exhibited the highest expression level. The similar result was revealed under fluorescence microscope, showing that the antigen was indeed expressed on the surface of lactic acid bacteria. Among different hosts, the antigen expression level of Lactiplantibacillus plantarum PS128 was better than that of Lactiplantibacillus plantarum K68 and Latilactobacillus sakei. Therefore, transforming pA-P11-ASD to L. plantarum PS128 could serve as a probiotic vaccine platform for future development of COVID vaccine.