小鼠B7-DC胞外段原核表达载体的构建及表达

Construction and expression of prokaryotic vector encoding extracellular region of murine B7-DC

目的:构建小鼠B7-DC胞外段基因的原核表达载体,并在E.coli BL21中诱导表达。方法:从小鼠骨髓来源的未成熟树突状细胞(DC)中提取总RNA,经RT-PCR扩增B7-DC胞外段,并将其克隆至原核表达载体pET32a(+)中,构建重组表达质粒pET32a(+)-B7-DCECD。重组质粒经双酶切鉴定及序列测定后,转化E.coli BL21,经IPTG诱导表达目的蛋白,并用SDS-PAGE和Western blot进行检测。结果:获得全长为582bp的小鼠B7-DC胞外段基因,经测序证实其序列正确。SDS-PAGE和Western blot分析证实重组质粒可表达出Mr约为41000的B7-DC胞外段蛋白。结论:成功地构建了小鼠B7-DC胞外段基因原核表达载体,并在E.coli BL21中进行表达,为进一步研究B7-DC的功能奠定实验基础。

AIM:To construct a prokaryotic expression vector for the extracellular domain of murine B7-DC(B7-DCECD)gene,and to express the gene in E.coli BL21.METHODS:The total RNA was extracted from murine immature bone marrow-derived dendritic cells and the extracellular fragment of B7-DC cDNA was amplified by RT-PCR.The recombinant plasmid pET32a(+)-B7-DCECD was constructed by cloning the extracellular fragment of B7-DC cDNA into the prokaryotic expression vector pET32a(+).After the recombinant plasmid was identifie...