狂犬病病毒口服疫苗株SRV9全长基因质粒的构建

Structure of Full-length Plasmid of Rabies Virus Oral Vaccine Strain SRV_9

狂犬病病毒(Rabies Virus)是一种致死性的人畜共患病。为了深入研究狂犬病病毒的基因组特性及构建新型病毒载体,研究根据GenBank中已公布的狂犬病病毒口服疫苗株SRV9(登录号:AF499686)基因组序列设计数对引物,以SRV9病毒为材料,通过RT-PCR技术将总RNA中SRV9全长基因组11,928 bp分5段扩增,分别克隆到pMD18-T或pGEM-T载体测序鉴定。测序正确后,再克隆至pBluescript skⅡ(+)载体,利用扩增片段自身内切酶切位点顺次进行全长连接,获得含SRV9全长基因组的质粒pBLSRV9。通过基因组序列同源性比较分析,SRV9株具有稳定的遗传特性,为进一步探索狂犬病病毒的复制、感染、致病及免疫分子机制和研制新型狂犬病病毒疫苗奠定良好的基础。

Rabies Vires is a lethal zoonosis. To deeply study genomic characterization of SRV9 and construct the new vector, several pairs of primers was designed according to the full - length genomic sequence of rabies virus oral vaccine strain SRV9（GenBank Accession No AF499686）. Using the isolated total RNA from rabies virus as a template, five DNA fragments covering the entire genome of SRV9 were amplified by RT- PCR, and inserted into pMDI8 - T or pGEM - T vector separately and sequenced. Fragments were all cloned into pBluescript SK （ ＋ ） vector thereafter sequencing correctly. Five fragments were ligated by using amplified fragment containing themselves＇ restriction enzyme sites to obtain the full - length plasmid clone pBLSRV9 of SRV9. Compared with the genome sequences of homology analysis, SRV9 have the stable hereditary trait. Therefore the construction of full - length plasmid provides foundation to deeply study of rabies virus on the copy, infectious, pathogenicity and the mechanism of molecular immunity and develop the new vaccine.