野生型和突变型APP695稳定转染CHO分泌Aβ40和Aβ42的比较  

Comparison of Aβ40 and Aβ42 Secretion between CHO Cells Stably Transfected with Wild-type Human Amyloid Precursor Protein 695 and Swedish Mutation APP695

作　　者：黄巍[1] 周丽荣[1] 李小鸥[2] 孙红霞[1] 周婷[1] 黄晓刚[1] 刘桥[1] 

机构地区：[1]武汉市医学科学研究所基础医学研究室,湖北武汉430014 [2]武汉大学人民医院儿科,湖北武汉430060

出　　处：《生物技术》2013年第6期63-67,共5页Biotechnology

基　　金：国家自然科学基金项目("干预TACE与BACE的平衡以减少APP毒性产物的分子调控途径";30500085);湖北省自然科学基金项目("保护APPβ水解位点阻断Aβ产生并促进α代谢的分子途径";2009CDB336)资助~~

摘　　要：目的:建立野生型和瑞典突变型淀粉样蛋白前体695(APP695)稳定转染CHO细胞株,比较两者分泌Aβ40和Aβ42的能力,为筛选β分泌酶抑制剂奠定基础。方法:以人淀粉样蛋白前体751(APP751)基因序列为模板,通过突变分别获得野生型APP695wt和瑞典突变型APP695sw序列,再连入真核表达载体pcDNA3.1,转染CHO细胞并经G418筛选出稳转细胞株,通过ELISA法检测培养上清中Aβ40和Aβ42的含量,然后检测APPβ位点抗体对Aβ分泌的影响。结果:获得了APP695wt和APP695sw序列,构建载体并稳定转染的CHO细胞株,可检测到两种目的蛋白均有表达,相对分子质量约为98kDa。ELISA法只能检测到APP695sw稳转细胞有高水平Aβ40和Aβ42产生,Aβ40的分泌量约是Aβ42的24.57倍,可达约14000pg/ml。APPβ位点抗体可降低Aβ分泌量,但不影响Aβ40/Aβ42的比值。结论:成功获得APP695wt和APP695sw稳定转染细胞株,其中后者高分泌Aβ40和A42,适用于β分泌酶抑制剂的筛选。Objective： To screen β - secretase inhibiting drug, CHO cell lines stably transfected with wild - type human amyloid precursor protein 695 and Swedish mutation APP695 were established. Method： The wild type APP695 cDNA was generated based on APP751 plas-mid containing total length of human wild type APP751. The Swedish double mutation NL595/596 （ APP695 numbering） was introduced into APP695wt cDNA using site - directed mutagenesis method. The two APP695 cDNA were subsequently cloned into an expression vector pcDNA3. 1 and transfected into CHO cells, the stably transfected cell chines were screened by G418 respectively. The levels of Aβ40 and Aβ42 in conditioned media and the effect of APP 15 -site antibodies on Aβ secretion were assayed by sandwich ELISA. Result：The analy sis of PCR, restriction endonuclease digest and DNA sequencing showed that the recombinant eukaryotic expression plasmids were con- structed suecesfully, and the expressin of APP695wt and APP695 sw were confirmed by western blotting, their relative molecular mass were about 98kDa. Only the level of Aβ in conditioned media of CHO cells stably tranfected APP695sw could be assayed by sandwich ELISA, the concentration of Aβ40 reached about 14 000pg/ml, and the ratio of Aβ40/Aβ42 was about 24. 57. The antibody against human APP β - seeretase site reduced Aβ40 and Aβ42 levels in conditioned media of CHO cells tranfeeted APP695sw, but the ratio of Aβ40/Aβ42 had no change. Conclusion ： The CHO cell stably transfected with wild - type and Swedish mutation type human APP695 were established sue- eesfully, and the latter could secret high level Aβ40 and Aβ42, which provided a good cell model to screen BACE inhibitors.

关 键 词：人淀粉样蛋白前体695 稳定转染 β-淀粉样蛋白β分泌酶 阿尔茨海默病 

分 类 号：R392.11[医药卫生—免疫学]