小反刍兽疫病毒N基因在大肠杆菌中的表达及鉴定

参照GenBank中小反刍兽疫病毒((Peste des petits ruminants virus，PPRV)N基因序列（Nigeria/75/1）(1 578 bp)设计了1对添加EcoRⅠ和Not Ⅰ酶切位点的引物，对从奥地利引进的pCI-neo-PPRN质粒进行PCR扩增，克隆至pGM-T载体，并转化大肠杆菌Escherichia. coli TOP10感受态细胞，获得重组质粒pGM-T-PPRN。将重组质粒经EcoRⅠ和NotⅠ双酶切的产物插入原核表达载体pET-32a（+），构建原核表达质粒pET-PPRN，将其转化进BL21(DE3)感受态细胞中后用IPTG诱导表达， 收集菌液进行SDS-PAGE电泳和Western blot分析，结果表明小反刍兽疫病毒N基因在BL21(DE3) 成功表达。所表达融合蛋白的相对分子质量约为80 kD，并能被组氨酸单抗、PPRV标准阳性羊血清及PPRV疫苗免疫羊血清所识别。

Expression and Identification of Peste des petits ruminants virus N Gene in Escherichia coli

A pair of primers added EcoRⅠand NotⅠsites was designed and synthesized based on the sequence of the nucleocapsid（N）protein gene of Peste des petits ruminants virus (PPRV), which was for amplifying the full-length N gene fragment of pCI-neo -PPRN plasmid from Austria. The fragment was cloned into pGM-T vector and transformed into Escherichia coli TOP10. Then the cloned fragment was subcloned into prokaryotic expression vector pET-32a(+), and the recombinant expression plasmid pET-PPRN was expressed in E.coli BL21 (DE3) with inducement. The size of the recombinant N fusion protein was 80 kD. The recombinant PPRV N fusion protein could be detected with Histidine monoclonal antibody or goat anti-PPRV sera by Western blot.