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小反刍兽疫(peste des petits ruminants,PPR)是由副黏病毒科(Paramyxoviridae)麻疹病毒属(Morbillivirus)的小反刍兽疫病毒(Peste des petits ruminants virus,PPRV)引起绵羊(Ovis aries)和山羊(Capra hircus)等小反刍兽的一种急性和高度传染性病毒性疾病,由于其高死亡率常造成绵羊和山羊养殖业的重大经济损失。为研究小反刍兽疫病毒甘肃(GS)株融合蛋白(fusion protein)基因序列特征及其免疫原性,参照Gen Bank中PPRV Nigeria 75/1株F基因序列(Gen Bank登录号:HQ197753)设计引物,应用RT-PCR扩增PPRV GS株F基因,在测序及序列分析的基础上,设计引物扩增去信号肽和跨膜区的F基因片段Fa并将其亚克隆至原核表达载体p ET-32a(+)。重组质粒p ET-PPRV-Fa转化大肠杆菌表达菌(Escherichia coli)Transetta(DE3)后经异丙基-β-D-硫代半乳糖苷(isopropylβ-D-1-thiogalactopyranoside,IPTG)诱导表达,表达产物纯化后免疫新西兰兔(Oryctolagus cuniculus)制备多克隆抗体,进而应用间接酶联免疫吸附实验(enzyme-linked immunosorbent assay,ELISA)和Western blot对重组蛋白免疫原性进行分析。结果表明,PPRV GS株F基因(Gen Bank登录号:KX822738)完整CDS全长1 641 bp,编码546个氨基酸。去信号肽和跨膜区的F基因片段Fa在大肠杆菌中成功获得表达,重组蛋白大小约为59 k D,且主要以包涵体形式存在;间接ELISA和Western blot结果表明,纯化产物免疫原性良好。研究结果为F蛋白的相关功能研究及小反刍兽疫的快速诊断方法的建立提供了理论依据。
The peste des petits ruminants (PPR) are caused by the Pesvis des petits ruminants virus (PPRV) of the Paramyxoviridae genus Ovis aries and Capra hircus) and other small ruminants are acute and highly contagious viral diseases that often result in significant economic losses to sheep and goat farming due to their high mortality rates. In order to study the sequence characteristics and immunogenicity of fusion protein gene of PESV Gansu strain (GS), primers were designed according to the F gene sequence of PPRV Nigeria 75/1 in Gen Bank (GenBank accession number: HQ197753) The F gene of PPRV GS strain was amplified by RT-PCR. Based on the sequencing and sequence analysis, primers F were designed to amplify Fa fragment of signal peptide and transmembrane region and subcloned into prokaryotic expression vector p ET-32a (+). The recombinant plasmid p ET-PPRV-Fa was transformed into Escherichia coli Transetta (DE3) and then induced by IPTG. The expressed product was purified and immunized New Zealand rabbit (Oryctolagus cuniculus) to prepare polyclonal antibody. Then the immunogenicity of the recombinant protein was analyzed by enzyme-linked immunosorbent assay (ELISA) and Western blot. The results showed that the complete CDS of F gene (Gen Bank accession number: KX822738) of PPRV GS strain was 1 641 bp, encoding 546 amino acids. The F gene fragment Fa which passed through the signal peptide and the transmembrane region was successfully expressed in E. coli. The recombinant protein was about 59 kD in size and existed mainly in inclusion bodies. The indirect ELISA and Western blot showed that the immunogenicity of the purified product good. The results provided a theoretical basis for the study of the related functions of F protein and the establishment of a rapid diagnostic method for PRRSV.