酿酒酵母表面展示鲑鱼降钙素多肽的研究

Displaying of Salmon Calcitonin on the Surface of Saccharomyces Cerevisiae

目的:实现酿酒酵母表面展示鲑鱼降钙素。方法:人工合成鲑鱼降钙素(Salmoncacitonin,sCT)编码基因,克隆到表面展示载体M-pYD1上。用NocI酶切重组质粒M-pYD1-sCT和空白质粒M-pYD1,回收sCT和V5表达框片段后分别以LiAC法转化酿酒酵母EBY100菌株,分别得到重组酵母yAGA2-sCT和yAGA2-V5。两种重组酵母分别经半乳糖诱导表达后,采用FITC荧光标记酵母表面展示的sCT和V5多肽,分别用荧光显微镜和流式细胞仪进行定性和定量分析。结果:诱导12h后,荧光显微镜下清晰观测到了工程菌表面有重组sCT,流式细胞分析结果表明10000个细胞中65.2%的yAGA2-sCT菌株表达了外源sCT,52.4%的yAGA2-V5菌株表达V5。结论:利用酿酒酵母表面展示鲑鱼降钙素多肽获得了成功,为口服型鲑鱼降钙素的研发奠定了基础。

Objective： To display salmon calcitonin on the surface of Saccharomyces cerevisiae. Methods： Codon optimized salmon calcitonin （sCT） gene was synthesized and cloned into the surface displaying vector pYD1. Expression cassettes of sCT and V5 were respectively recovered from Noc I digestion products of plasmid M-pYDI-sCT and M-pYD1, and then were transformed into Saccharomyces cerevisiae EBY100 by LiAC method, resulting in yAGA2-sCT and yAGA2-V5, respectively. The two recombinants were induced by galactose for 12 h, followed with labeling sCT and V5 by FITC, respectively. Results： The expressed products were clearly observed with green fluorescence under fluorescent microscope. By flow cytometry, 65.2% yAGA2-sCT cells and 52.4% yAGA2-V5 cells were found to express foreign protein. Conclusions： Salmon calcitonin was successfully expressed on the surface of S. cerevisiae, which provided base for development of an oral-delivered sCT.