淀粉样前体蛋白β位点单链抗体抑制β淀粉样肽的分泌  

Single chain antibody against β-site of amyloid precursor protein inhibits the generation of β-amyloid peptide

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,湖北武汉430014 [2]武汉大学人民医院儿科,湖北武汉430060

出　　处：《细胞与分子免疫学杂志》2015年第10期1408-1412,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(30500085;81000094);湖北省自然科学基金(2009CDB336);武汉市卫计委课题(WX09B13;WX14C71)

摘　　要：目的观察淀粉样前体蛋白(APP)β位点单链抗体(sc Fv)对APP的α和β代谢的影响,为其作为APPβ分泌酶特异抑制剂奠定基础。方法以APPβ位点sc Fv 2H10基因序列为模板,通过PCR引入Igκ轻链信号序列和myc标签,与真核表达载体pc DNA3.1hyg(^+)相连,转染过表达人瑞典突变型淀粉样前体蛋白695(APP695)的CHO细胞株(APP695sw/CHO),经潮霉素B筛选出稳定转染细胞株,用夹心ELISA检测稳定转染细胞株培养上清中Aβ40和分泌型APPα(s APPα)的含量,并分别比较两者的变化。结果经PCR、酶切和测序鉴定证明,获得引入Igκ分泌信号和myc标签的APPβ位点sc Fv基因片段,且与真核表达载体正确相连。转染APP695sw/CHO细胞后,经潮霉素B筛选得到稳定转染株;Western blot法可检测到目的蛋白表达,ELISA结果显示,与对照相比,稳定转染细胞培养上清Aβ40分泌量约下降30.4%、s APPα水平上升23.1%。结论 APPβ位点sc Fv在抑制Aβ分泌的同时还可能有利于APP的α代谢,可作为β分泌酶高特异性抑制剂。Objective To construct and express a single chain fragment of variety region （scFv） against β-site of amyloid precursor protein （APP）, and evaluate the effect of scFv on α- and IS-processing of APP. Methods The β-site specific scFv 2H10 was amplified and fused with signal sequence IgK and myc tag by PCR to create the expression cassette, which was then subcloned into expression vector pcDNA3, lhyg （ ＋ ）. The plasmid was transferred into CHO cells over-expressing Swedish mutation type human APP695 （APP695sw/CHO）. The positive clones were identified by hygromycin B. The concentrations of Aβ40 and soluble APPα （sAPP（x） in the conditioned media supernatant were measured by sandwich ELISA. Results The recombinant eukaryotic expression plasmids were identified by PCR, restriction endonuclease digestion and DNA sequencing. The expression of scFv was confirmed by Western blotting. When scFv 2H10 was stably expressed in APP695sw/CHO, the extracellular level of Aβ40 decreased by 30.4%, while sAPPa increased by 23.1%. Conclusion The APP β-site scFv 2H10 could inhibit the secretion of Aβ. Meanwhile, it might be favorable for the a-processing of APP. Hence it can be explored as an inhibitor of β-site APP-cleaving enzyme （BACE）.

关 键 词：淀粉样前体蛋白 β位点 单链抗体 Β淀粉样肽 α代谢 阿尔茨海默病 

分 类 号：R341[医药卫生—基础医学] R392.11