【摘要】 目的构建小鼠β分泌酶原核表达载体并诱导其表达和纯化,为进一步研究β分泌酶的作用奠定基础。方法根据基因文库中小鼠β分泌酶基因的cDNA序列设计引物,采用RT-PCR方法扩增到β分泌酶的cDNA片段,克隆进原核表达载体pET-28 a中,并转化大肠杆菌BL21(DE3),测序鉴定后,通过IPTG诱导表达出目的蛋白,经亲合层析纯化。结果从小鼠的脑组织RNA中扩增出特异的β分泌酶基因片段长1 506 bp,经酶切鉴定及DNA序列测定,证实重组载体pET28 a-BACE构建正确,表达融合蛋白分子量约为55 kD,经镍离子亲合柱层析纯化获得电泳级纯度的目的蛋白。结论在大肠杆菌中获得了小鼠β分泌酶的高效表达,为进一步研究其生物学功能和应用奠定了基础。更多还原

【Abstract】 Objective To build mice β secretase prokaryotic expression vector and induce its expression and purification,to afford basis for β secretase research.Methods Primer was designed by mice β secretase gene cDNA in Genbank.cDNA segment of β secretase gene was amplified by RT-PCR,then cloned into prokaryotic expression vector pET-28a and transformed into E.coli BL21(DE3).After sequenced and identified,fusion protein was induced,expressed and purified.Results Special β secretase gene segment amplified from RNA of mice brain tissue was 1 506 bp.After identified and sequenced,recombinant vector was proved correct,its fusion protein had a molecular weight of 55 kD.Electrophoresis purity target protein could be acquired.Conclusions Mice β secretase has a high expression in E.coli,which build a basis for research of β secretase′s biological function and its application.更多还原