文档介绍：β分泌酶原核表达载体的构建、表达及融合蛋白纯化
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作者:朱爱华李敬陆军钱进郑元林
【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28a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pET28a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。
【关键词】β分泌酶;大肠杆菌;表达;融合蛋白
【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 Special β secretase gene segment amplified from RNA of mice brain tissue was 1 506 bp. After identified and sequenced, binant 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.
【Key words】βsecretase; E. coli; Expression; Fusion protein