摘要
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.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2007年第8期738-742,共5页
Chinese Journal of Immunology
关键词
人杀菌通透性增强蛋白
酵母表面展示
内毒素
Human bactericidal/permeability increasing protein
Yeast surface display
Lipopolysaccharide