产肠毒素大肠杆菌DNA疫苗重组酵母菌体微胶囊开发

Recombinant yeast-cell microcapsules carrying the DNA vaccine against enterotoxigenic Escherichia coli

摘要:

产毒素大肠杆菌(enterotoxigenic Escherichia coli, ETEC)感染是制约畜牧业发展的一大因素。然而，抗生素的滥用势必引发细菌耐药与药物残留问题，且现有疫苗难以有效激发肠道免疫，预防效果不佳。因此需要开发一种安全的、能激活肠道免疫、适宜作为饲料添加剂的新型疫苗。本研究旨在通过基因工程技术，构建携带ETEC DNA疫苗的酵母菌体微胶囊(yeast-cell microcapsules, YCM)，并通过动物实验探究饲喂YCM对动物肠道免疫系统的调节作用及其对肠道菌群的潜在影响。选用酿酒酵母菌体作为DNA疫苗的口服递送载体—“微胶囊”，合成密码子哺乳动物源优化的ETEC主要抗原K88核酸序列，利用重组DNA技术构建含有相应DNA疫苗表达盒的酵母穿梭载体，转化酿酒酵母菌株JMY1制备相应的YCM重组菌株。进一步通过荧光报告实验、胃肠液耐受实验、肠上皮细胞黏附实验、肠道滞留性评估、抗血清检测和肠道菌群检测等一系列的实验检测全面评估了YCM菌株的特性及作为口服疫苗的可行性。实验结果显示，DNA疫苗表达盒能够在哺乳动物中有效表达，YCM重组菌能够耐受长达8 h的胃肠液消化，并对肠上皮细胞具有良好的黏附力。小鼠饲喂实验结果表明，YCM重组菌能够在肠道内滞留至少2周，且其所携带的DNA疫苗表达盒能够进入肠道免疫系统并引发免疫反应，诱导产生特异性抗体。此外，饲喂YCM重组菌还能改善小鼠肠道菌群丰度，在调节肠道菌群方面展现了积极作用。综上所述，本研究制备了携带ETEC DNA疫苗的YCM重组菌株，全面评估了其作为口服疫苗的特性及可行性，确认了饲喂YCM重组菌能够诱导特异性免疫反应并调节肠道菌群，为ETEC相关动物疾病的免疫预防提供了参考和借鉴。

Abstract:

The enterotoxigenic Escherichia coli (ETEC) infection is a major factor restricting the development of animal husbandry. However, the abuse of antibiotics will lead to the antibiotic residues and emergence of antibiotic-resistant bacteria. The existing vaccines face challenges in stimulating intestinal immunity, demonstrating limited prevention effects. Therefore, it is indispensable to develop a new vaccine that is safe and suitable as a feed additive to activate intestinal immunity. This study constructed yeast-cell microcapsules (YCM) carrying the DNA vaccine against ETEC by genetic engineering. Furthermore, animal experiments were carried out to explore the regulatory effects of feeding YCM on the intestinal immune system and intestinal microbiota. Saccharomyces cerevisiae was selected as the oral delivery vehicle (microcapsules) of the DNA vaccine. The codon-optimized nucleic acid sequence of K88, the main antigen of mammal-derived ETEC, was synthesized, and the yeast shuttle vector containing the corresponding DNA vaccine expression cassette was constructed by DNA recombination. The recombinant strain of YCM was prepared by transforming JMY1. Additionally, the characteristics of the YCM strain and its feasibility as an oral vaccine were comprehensively evaluated by the fluorescence reporter assay, gastrointestinal fluid tolerance assay, intestinal epithelial cell adhesion assay, intestinal retention assessment, antiserum detection, and intestinal microbiota detection. The experimental results showed that the DNA vaccine expression cassette was expressed in mammals, and the recombinant strain of YCM could tolerate up to 8 hours of gastrointestinal fluid digestion and had good adhesion to intestinal epithelial cells. The results of mouse feeding experiments indicated that the recombinant strain of YCM could stay in the intestinal tract for at least two weeks, and the DNA vaccine expression cassette carried by YCM entered the intestinal immune system and triggered an immune response to induce the production of specific antibodies. Moreover, feeding YCM recombinant bacteria also improved the abundance of gut microbiota in mice, demonstrating a positive effect in regulating intestinal flora. In summary, we prepared the recombinant strain of YCM carrying the DNA vaccine against ETEC and comprehensively evaluated its characteristics and feasibility as an oral vaccine. Feeding the recombinant YCM could induce specific immune responses and regulate intestinal microbiota. The findings provide a reference for the immunoprevention of ETEC-related animal diseases.