人杀菌通透性增强蛋白功能性N端片段在酵母细胞表面的展示和鉴定

Identification of N-terminal fragment of human bactericidal/permeability increasing protein displayed on the surface of yeast cells

目的:建立BPI23酵母细胞表面展示方法,使之表达具有生物活性的BPI23蛋白。方法:构建pYD1-BPI600重组质粒,转化酵母细胞,经半乳糖诱导使BPI23在酵母细胞表面表达;采用间接免疫荧光法,用荧光显微镜和流式细胞仪检测BPI23在酵母细胞表面的展示情况;采用鲎基质显色法,检测BPI23中和内毒素的生物学活性。结果:酶切和DNA测序鉴定证实,pYD1-BPI600重组质粒含有人BPI600基因片段。荧光显微镜和流式细胞仪检测结果显示,BPI23在pYD1-BPI600重组质粒转化的酵母细胞表面得到展示;在诱导后第24小时展示BPI23的酵母细胞数占总细胞数的39%。酵母细胞表面展示的BPI23具有中和内毒素的活性。结论:成功构建了pYD1-BPI600酵母表达载体,转化后在酵母细胞表面展示了功能性目的蛋白,为BPI23突变体库的建立和功能优化BPI23突变体的筛选奠定了基础。

To display human BPI23 on the surface of yeast cells, and to identify its biological function. Methods： The DNA fragment encoding human BPI23 was amplified by PCR from pBV-BPI600-Fcγ1 plasmid, and cloned into file yeast surface display vector pYD1. The plasmid pYD1 and recombinant plasmid pYD1-BPI600 were transformed into the EBY100 yeast strain respectively. Fluorescence microscopy and flow cytometry were used to monitor the expression of BPI23 on the surface of yeast cells. Results： After the cells were induced with galactose for about 24 h and stained with anti-BPI Ab and FITC labeled IgG, the target protein （ BPI23 ） could be detected on the surface of about 39% yeast cells. The protein displayed on yeast cells could neutralize lipopolysaccharide. Conclusion：Functional BPI23 can be successfully displayed on surface of yeast cells, which provide basis for its directed evolution.