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

目的制备针对人淀粉样蛋白前体(APP)β分泌酶切割位点的特异性单链抗体(ScFv),并进行鉴定。方法设计扩增单克隆抗体细胞株重链和轻链可变区基因片段VH和VL的引物,利用RT-PCR技术,从本室制备的抗人APPβ分泌酶切割位点单克隆抗体细胞株中扩增出VH和VL基因片段,然后通过重叠引物延伸法(SOE)将VH和VL基因拼接成ScFv基因片段,再将其亚克隆入原核表达载体pET-28a中,转化E.coli BL21(DE3)诱导表达,目的蛋白经镍柱纯化和复性后,用SDS-PAGE、ELISA和Western blot等方法对其检测分析。结果成功从一株抗人APPβ位点单克隆抗体细胞株2H10中扩增出VH和VL并拼接成ScFv片段,片段长744 bp,编码248个氨基酸。PCR、酶切和测序表明表达载体构建成功,转化E.coli BL21(DE3),经IPTG诱导可以表达出约29 ku的目的蛋白,主要为包涵体形式,经镍柱纯化和复性后,获得纯度达90%以上的ScFv蛋白,ELISA和Western blot检测表明可溶性ScFv可以与人APPβ分泌酶切割位点序列短肽和全长APP结合。结论成功构建并表达人APPβ分泌酶切割位点的特异性单链抗体,为进一步研究其生物学活性奠定基础。

Aim To prepare a specific single chain Fv antibody against β-secretase cleavage site of amyloid precursor 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） against β-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 BL21（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 β-secretase site were cloned successfully and the prokaryotic expressing vector of APP β-secretase site scFv was constructed.The analysis of DNA sequencing showed 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 β-secretase site analyzed by ELISA and Western blot.Conclusion A single chain Fv antibody against human APP β-secretase site is prepared,which establishes a solid basis of further studying the biological function of ScFv.