中国药理学通报2012，Vol.28Issue(3)：337-342,6.DOI:10.3969/j.issn.1001-1978.2012.03.010
APPβ分泌酶切割位点特异性单链抗体的制备和鉴定
Preparation and identification of a single chain Fv antibodyagainst human APP β-secretase site
摘要
Abstract
Aim To prepare a specific single chain Fvantibody against β- secretase cleavage site of amyloidprecursor protein( APP ) and identify its biological activity. Methods The VH and VL genes were cloned by RT-PCR from a murine hybridoma cell line 2H10, which produced the monoclonal antibody ( mAb ) a-gainst (3-secretase site of human APP. SOE-PCR was used to splice the VH and VL genes to construct ScFv, which was subcloned into the prokaryotic expressing vector Pet-28a. The positive clone were transformed into E. Coli BL2l( DE3 ) and induced with IPTG, target protein was purified and refolded via Ni-NTA, and was analyzed by SDS-PAGE, ELISA and Western blot. Results The VH and VL genes of mAb against human APP p-secretase site were cloned successfully and the prokaryotic expressing vector of APP p-secretase site scFv was constructed. The analysis of DNA sequencingshowed that the full-length of constructed scFv gene was 744 bp, and code 248 amino acids. ScFv was expressed as inclusion body with IPTG, and showed a molecular 29 ku analyzed by SDS-PAGE and Western blot. About 90% purity of ScFv was obtained following renaturing and purifying via Ni-NTA, and the soluble ScFv exhibited the binding activity to Happ p-secretase site analyzed by ELISA and Western blot. Conclusion A single chain Fv antibody against human APP p-secretase site is prepared, which establishes a solid basis of further studying the biological function of ScFv.关键词
人淀粉样前体蛋白/β分泌酶/β分泌酶切割位点/单链抗体/阿尔采末病/β淀粉样蛋白Key words
human amyloid precursor protein/ BACE/ P-secretase cleavage site/ single-chain Fv ( ScFv )/ Alzheimer' s disease/ amyloid-beta peptide( Ap )分类
医药卫生引用本文复制引用
黄巍,周丽荣,周婷,孙红霞,黄晓刚,刘桥..APPβ分泌酶切割位点特异性单链抗体的制备和鉴定[J].中国药理学通报,2012,28(3):337-342,6.基金项目
国家自然科学基金(No 30500085) （No 30500085）
湖北省自然科学基金(No 2009CDB336) （No 2009CDB336）
武汉市卫生局科研基金(No WX09B13)资助项目 （No WX09B13）
