小反刍兽疫病毒融合蛋白基因的克隆、序列分析及表达

Cloning,Sequence Analysis and Expression of Fusion Gene of Peste des petits ruminants Virus

根据GenBank报道的小反刍兽疫病毒（PPRV）融合蛋白（F）基因序列,用特异性引物对PPRV疫苗株F蛋白基因进行了RT-PCR扩增,并将其克隆到pGEM-T载体中进行测序。结果表明：F基因ORF全长1641 bp,编码546个氨基酸;推导的氨基酸序列中第1~18位氨基酸构成信号肽序列,第488~510氨基酸为跨膜区。构建原核表达载体pETF1和pETF2,转化E.coliBL21（DE3）,用IPTG诱导表达。SDS-PAGE和Western-blotting的分析结果表明,F1和F2基因在大肠杆菌中均获得了表达,且均具有良好的反应原性。用Ni-NTA试剂盒纯化F1和F2重组蛋白,为研发检测PPRV特异性抗体的诊断试剂奠定了基础。

According to fusion（F） protein gene sequence of Peste des petits ruminants virus strain reported by GenBank,a pair of specific primers was designed and used to amplify F gene of PPRV vaccinal strain.The RT-PCR product was purified and ligatured with pGEM-T for sequencing.The open reading frame length of F gene of PPRV strain was 1641 bp,which encoded 546 amino acids.The front 18 amino acids constitute signal peptide,and 488-510 amino acids form transmembrane.Then F1 and F2 genes were subcloned into pET-28a（＋） to generate prokaryotic expression vector pETF1 and pETF2,respectively.The recombinant vectors were then transformed into E.coli BL21（DE3） for expression under induction of IPTG.SDS-PAGE and Western-blotting showed that F1 and F2 recombinant proteins were successfully expressed and of better reactiongenicity recombinant protein F1 and F2 were successfully purified by the kit of Ni-NTA respectively.The experiment laid a foundation for studying the specific diagnosis kit to detect PPRV infection.