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

作　　者：倪伟[1,2] 才学鹏[2] 乔军[1] 孟庆玲[1,2] 陈创夫[1] 任艳[1] 

机构地区：[1]石河子大学动物科技学院预防兽医学重点实验室,石河子832003 [2]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室,兰州730046

出　　处：《石河子大学学报（自然科学版）》2011年第1期35-39,共5页Journal of Shihezi University(Natural Science)

基　　金：家畜疫病病原生物学国家重点实验室开放课题(KEYLAB200802)

摘　　要：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.

关 键 词：小反刍兽疫病毒 融合蛋白基因 克隆 原核表达 

分 类 号：S858.2[农业科学—临床兽医学]