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目的 :在酵母细胞表面展示登革 2型病毒 4 3株 (D2_4 3)的E基因 ,探索利用酵母表面展示系统建立DNA改组筛选平台的可行性。方法 :通过RT_PCR扩增获得D2_4 3的E基因 ,将该基因亚克隆至T载体后 ,再克隆至酵母表面展示载体pYD1,于酿酒酵母EBY10 0中利用半乳糖进行诱导表达。表达产物采用间接免疫荧光法和FACS进行检测。结果 :酵母表面展示产物可与D2_4 3的腹水抗体特异性地结合 ;在半乳糖诱导后 2 4h ,展示E蛋白的酵母细胞百分数达 2 2 .0 7%。结论 :本研究为建立基于酵母表面展示系统的DNA改组筛选平台奠定了基础。
OBJECTIVE: To display the E gene of Dengue 2 virus (D2_43) on the surface of yeast cells and to explore the feasibility of using yeast surface display system to establish a DNA shuffling screening platform. Methods: E gene of D2_4 3 was amplified by RT_PCR. The gene was subcloned into T vector and cloned into yeast surface display vector pYD1. The recombinant plasmid was induced by galactose in Saccharomyces cerevisiae EBY10 0. The expression products were detected by indirect immunofluorescence and FACS. Results: The yeast surface display products could specifically bind to the ascites antibody of D2_43. At 24 h after galactose induction, the percentage of yeast cells displaying E protein was 22.07%. Conclusion: This study laid the foundation for the establishment of DNA shuffling screening platform based on yeast surface display system.