Abstract：
As a series of our previous studies reported, recombinant yeast can be the oral vaccines to deliver designed protein and DNA, as well as functional shRNA, into dendritic cells (DCs) in mice for specific immune regulation. Here, we report the further optimization of oral yeast-based vaccine from two aspects (yeast characteristics and recombinant DNA constitution) to improve the effect of immune regulation. After screening four genes in negative regulation of glucan synthesis in yeast (MNN9, GUP1, PBS2 and EXG1), this research combined HDR-based genome editing technology with Cre-loxP technology to acquire 15 gene-knockout strains without drug resistance-gene to exclude biosafety risks; afterward, oral feeding experiments were performed on the mice using 15 oral recombinant yeast-based vaccines constructed by the gene-knockout strains harboring pCMV-MSTN plasmid to screen the target strain with more effective inducing mstn-specific antibody which in turn increasing weight gain effect. And subsequently based on the selected gene-knockout strain, the recombinant DNA in the oral recombinant yeast-based vaccine is optimized via a combination of protein fusion expression (OVA-MSTN) and interfering RNA technology (shRNA-IL21), comparison in terms of both weight gain effect and antibody titer revealed that the selected gene-knockout strain (GUP1ΔEXG1Δ) combined with specific recombinant DNA (pCMV-OVA-MSTN-shIL2) had a better effect of the vaccine. This study provides a useful reference to the subsequent construction of a more efficient oral recombinant yeast-based vaccine in the food and pharmaceutical industry.
摘要：
正如我们之前的一系列研究报道的那样，重组酵母可以是口服疫苗，以提供设计的蛋白质和DNA，以及功能性shRNA，进入小鼠树突状细胞（DC）进行特异性免疫调节。这里,从酵母特性和重组DNA组成两方面进一步优化口服酵母疫苗,以提高免疫调节效果。筛选出4个对酵母葡聚糖合成负调控的基因(MNN9、GUP1、PBS2和EXG1),本研究将基于HDR的基因组编辑技术与Cre-loxP技术相结合，获得了15个无耐药基因的基因敲除菌株，以排除生物安全风险；使用含有pCMV-MSTN质粒的基因敲除菌株构建的15种基于酵母的口服重组疫苗对小鼠进行了口服喂养实验，以筛选具有更有效诱导mstn特异性抗体的目标菌株，从而提高体重增加效果。随后根据选择的基因敲除菌株，口服重组酵母疫苗中的重组DNA通过蛋白融合表达（OVA-MSTN）和干扰RNA技术（shRNA-IL21）的组合进行优化，增重效果和抗体滴度的比较表明，选定的基因敲除菌株（GUP1ΔEXG1Δ）与特异性重组DNA（pCMV-OVA-MSTN-shIL2）组合具有更好的疫苗效果。该研究为后续在食品和制药行业中构建更有效的基于酵母的口服重组疫苗提供了有用的参考。
