绵羊ARQ基因型PrP前体蛋白基因的克隆及原核表达

目的构建ARQ基因型PrP前体蛋白基因原核表达重组质粒,为TSE的检测及诊断、PrP的结构与功能研究奠定基础。方法根据GenBank中绵羊PrP基因全序列和表达载体pET28a载体的多克隆位点设计了绵羊PrP前体蛋白基因引物,以绵羊基因组DNA为模板,PCR扩增绵羊PrP前体蛋白基因,通过酶切将些基因克隆到pET28a(+)载体中,构建了重组原核表达质粒pET-OPrP。重组质粒转化E.coliBL-21感受态细胞,用IPTG诱导表达,通过PAGE电泳和Western-blo-ting鉴定表达的重组蛋白。结果在pET28a(+)载体中成功克隆了PrP前体蛋白基因,并在E.coli中成功表达了重组的PrP前体蛋白。结论绵羊PrP前体蛋白基因可以在E.coli中表达。本次克隆得到的小尾寒羊的PrP基因型为ARQ。

Cloning and prokaryotic expression of the ARQ genotype of ovine prion protein precursor gene

The ovine prion protein(PrP) precursor gene from ovine genomic DNA was amplified by PCR and inserted into prokaryotic expression vector pET28a(+) at the multiple cloning sites between BamH I and Xho I.Squencing result of the rcombinant vector indicated that complete length of the ovine PrP precursor gene with 771 bps could be correctly inserted. The ovine PrP precursor protein was found to be expressed in E.coli BL-21cell and demonstrated by SDS-PAGE analysis and Western blotting assay, and the cloned genotype of the small tail Han sheep was ARQ. It is evident that ovine prion protein precursor gene can be expressed in E.coliBL-21 cells with a AQR genotype in small tail Han sheep.