小反刍兽疫病毒Nigeria75／1株F基因的克隆与原核表达

Cloning of F gene of peste des petits ruminants virus Nigeria75/1 strain and its expression in Escherichia coli

提取小反刍兽疫病毒(PPRV)疫苗株Nigeria 75/1的总RNA,用RT-PCR方法扩增F基因并克隆到pMD18-T载体中,以鉴定正确的质粒为模板克隆去掉N端信号肽和跨膜区的F基因(F-1)并亚克隆于原核表达载体pET-30a(+),得到重组质粒pET30-F-1,转化大肠杆菌BL21(DE3)进行诱导表达,表达产物进行SDS-PAGE、Western-blot分析,同时将重组蛋白免疫SPF家兔制备抗体,进行间接免疫荧光检测并用ELISA测定抗体效价。结果显示,目的基因正确插入了表达载体,在大肠杆菌中主要以包涵体形式表达,用重组蛋白免疫3次后,兔血清抗体达到较高水平,Western-blot分析说明表达产物能够与兔血清抗体发生特异性反应,间接免疫荧光试验显示重组蛋白免疫兔阳性血清能够识别PPRV Nigeria 75/1株全病毒抗原。表明,重组蛋白在原核表达系统内得到了高水平的表达,并且具有较好的反应原性。

Total RNA was extracted from peste des petits ruminants virus（PPRV） vaccine strain Nigeria 75/1. An F gene fragment was amplified by RT-PCR and then cloned into pMD18-T vector. The F-1 gene fragment without N-terminal signal peptide and transmembrane domain was amplified by PCR using plasmid pMD18-F as template and was subcloned into pET-30a （＋） vector. The positive recombinant pET30-F-1 was transformed into Escherichia coli BL21 （DE3） subsequently, which were induced by IPTG. The expressed recombinant protein was identified by SDS PAGE and Western-blotting. The positive serum of anti-F was prepared by immunization of SPF rabbits for indirect immunofluorescence assay, and the titers of serum antibody were detected by ELISA. The results showed that the cloned fragment was correctly inserted into the expression vector. The F gene fragment was expressed efficiently in the form of inclusion bodies. The titer of specific antibody against the expressed protein in sera was significantly higher than that of the un-immunized rabbits by ELISA, and possessed immunoreaction by Western-blot. It also showed that the positive sera could recognize the whole virion-antigen of PPRV Nigeria 75/1 strain by indirect immunofluorescence assay. Meanwhile, the F protein could be highly expressed in bacterialexpression system and had good reactogenicity.