狂犬病病毒SRV_9株修饰全长基因组cDNA克隆的构建

为获得狂犬病病毒(rabies virus,RV)SRV9株的基因组全长cDNA克隆并对其进行基因修饰,深入研究狂犬病病毒的基因组特性及构建新型病毒载体,本研究根据GenBank中公布的SRV9基因组序列设计数对引物,以SRV9病毒为材料,从病毒总RNA中将全长基因组11 928 bp分5段扩增,克隆至载体测序鉴定。测序正确后,设计引物缺失基因组Ψ区并在缺失位置插入人细胞色素C基因,又设计引物删除糖蛋白(G)胞质区(CD区)。利用引物在病毒基因组起始处添加锤头状核酶序列,在病毒序列结尾处添加了丁型肝炎核酶序列。再次测序后克隆至pcDNA3.1(+)载体,利用扩增片段自身内切酶位点顺次进行修饰后全长连接,从而获得含修饰SRV9全长基因组的质粒pcDNA3.1-SRV9,为RV感染性克隆的建立奠定了基础。

Construction of Full-Length Genomes-Modified cDNA Clone for Rabies Virus Strain SRV9

In order to obtain full-length genomes-modified cDNA clone for rabies virus strain SRV9 and to deeply study genomic characteriitics of SRV9 and construct the new virus vector and several pairs of primers were designed according to the full-length genomic sequence of SRV9 published on GenBank by using the total RNA extracted from rabies virus as a template.Five DNA fragments covering the entire genome 11 928 bp were amplified,and inserted into vector and were determined for sequeneing.After identification of the sequence,Ψ region of genome was designed with a lack of primers before the human cytochrome c gene was cloned into such a region,and cytoplasmic region of glycoprotein gene was also deleted by primers designed ahead.Hammerhead ribozyme sequence was inserted into the head region of full-length genomes and hepatitis delta virus ribozyme sequence was added into the end region of full-length genomes with the help of primers.Fragments were all cloned into pcDNA3.1（＋） vector after sequenced correctly.These five fragments were ligated by using amplified fragments that contain their own restriction enzyme sites to obtain the full-length genomes-modified plasmid clone pcDNA3.1-SRV9,which would contribute to the foundation of infectious clone for rabies virus.