人源蛋白酶体α亚基6的酵母表面展示及表达优化(英文)

YEAST SURFACE DISPLAY OF HUMAN PROTEASOME SUBUNIT ALPHA 6 AND EXPRESSION ENHANCEMENT

为构建人源蛋白酶体α亚基6(hPSA6)的酵母表面展示体系用于抗体表位分析和研究泛素-蛋白酶体途径,并优化hPSA6的展示表达,将基因PSA6_HUMAN克隆于表达载体pICAS-H,该载体带有His.tag标签可检测表达水平.经过重组酵母的培养,用抗His.tag或抗hPSA6的单克隆抗体和荧光二抗进行免疫荧光染色,通过流式细胞仪和荧光显微镜检测到hPSA6已经成功地展示于酿酒酵母表面.通过对培养基中不同初始浓度的葡萄糖和酸水解酪素的诱导培养,发现hPSA6呈现出不同的展示水平,与对照相比,对His.tag进行免疫荧光检测观测到3%酸水解酪素诱导培养可获得超过70%的展示表达率,3%葡萄糖诱导培养可达到50%以上表达率,2%葡萄糖诱导也有接近40%表达率.考虑到葡萄糖效应和灭菌过程中高浓度的葡萄糖易碳化,采用2%葡萄糖进行诱导表达较3%更适合于hPSA6的展示表达.

To construct the yeast display system of the human proteasome subunit alpha 6 （hPSA6） for epitope analysis and mechanism investigation of ubiquitin-proteasome pathway, and enhance the display expression of hPSA6, the gene PSA6_HUMAN coding hPSA6 was cloned into a yeast-displaying expression vector, pICAS-H, which had been inserted a His. tag marker for expression level detection. As probed with a h （MAb） and corresponding MAb, hPSA6 was detected functiona fluorescence microscopy analysis which confirmed that yeast-disp with highly specific affinity was expressed efficiently after 24 h is. tag monoclonal antibody lly by flow cytometry and laying recombinant hPSA6 cultivation of recombinant yeast MTS-1/pICAS-H-PSA6. Induced by different concentration of initial glucose and casein acid in restrictive mediums, the displayed hPSA6 expressions were compared through im- munofluorescence by using anti-His MAbs. Comparing with negative control, more than 70% cells were induced to express hPSA6 in SA with 3% casein acid initially. The 3% glucose could make more than half cells express well and nearly 40% cells were displaying hPSA6 successfully under the 2% initial glucose. Considering the glucose effect and the carbonization in sterilization period, 2% glucose was more appropriate than 3% in hPSA6 displaying.