分泌性BACE1蛋白在昆虫细胞的表达

为了获得有活性的重组β-secretase并研究其功能,寻找特异性抑制剂,应用RT-PCR技术,从人胚胎脑组织特异性扩增并克隆了人BACE1编码基因的胞外片断(BACE1-454)。测序后与质粒pFastBac连接,得到含BACE1-454基因的重组质粒pFast-BACE1-454。将其转化到含有杆状病毒穿梭载体Bacmid的感受态细胞DH10Bac中进行转座重组,用琼脂糖凝胶电泳和PCR扩增对重组穿梭载体Bacmid-BACE1-454进行鉴定。将Bacmid-BACE1-454经脂质体介导转染Sf9细胞,收获病毒。用重组杆状病毒颗粒感染昆虫细胞,表达蛋白,免疫印迹证实所获蛋白产品具有特异的免疫反应性。

Eukaryotic Expression of Soluble BACE1 in Insect Cells

To obtain recombinant secretive BACE1 proteins, study its function and further look for its specific inhibitor, the ectodomain fragment of human BACE1 coding region from human fetal hippocampal tissue is amplified using RT-PCR method. Confirmed by sequencing, clone the target gene into a pFastBac donor plasmid. After the recombinant pFastBac-BACE1-454 had been determined to be correct, the pFast-Bac-BACE1-454 was transformed into DH10Bac competent cells for transposition into a baculovirus shuttle vector Bacmid. Using agarose gel electrophoresis and PCR analysis to verify successful transposition. Recombinant Bacmid-BACE1-454 was transfected into Sf9 cells with CellFECTIN(?) Reagent and harvest virus from culture medium. Recombinant baculovirus particles were infected into insect cells and express the BACE1-454 protein. These expressed BACE1-454 proteins were identified by immunoblot with anti-BACE monoclonal antibody.