分泌性BACE1蛋白在昆虫细胞的表达  

Eukaryotic Expression of Soluble BACE1 in Insect Cells

作　　者：邢长虹[1] 赖燕来[2] 谢佐平[2] 

机构地区：[1]首都医科大学宣武医院,北京100053 [2]清华大学生物科学与技术系神经生物学实验室,北京100084

出　　处：《科学技术与工程》2005年第17期1252-1255,共4页Science Technology and Engineering

基　　金：北京市自然科学基金(5052015)资助

摘　　要：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。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 pFastBac-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 CellFECTINR 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.

关 键 词：Β-SECRETASE 真核表达 昆虫细胞 Alzheimefs病 

分 类 号：Q349.53[生物学—遗传学]