新城疫病毒La Sota C5株感染性克隆的构建及病毒拯救

CONSTRUCTION AND RESCUE OF INFECTIOUS MOLECULAR CLONE OF NEWCASTLE DISEASE VIRUS STRAIN LA SOTA C5

将本实验室保存的一株La Sota病毒用有限稀释法接种鸡胚进行纯化,连续传代5次后筛选到1株高血凝效价的纯培养克隆株,命名为La Sota C5,并对其进行全基因组测序,克隆株与亲本株在生物学特性与基因序列上都存在一定的差异。参照La Sota C5株全基因序列单酶切位点,用RT-PCR的方法将基因组分8段扩增,按照病毒基因组的结构顺序,将克隆片段定向插入到TVT转录载体中,成功构建含有病毒全长cDNA的转录载体TVT-La Sota C5。将TVT-La Sota C5与辅助质粒pCI-NP、pCI-P和pCI-L共转染BSR-T7/5细胞,成功拯救出了具有感染性的La Sota株新城疫病毒。病毒的成功拯救为后续基因功能和疫苗载体开发等方面的研究提供了平台。

Newcastle virus （NDV） La Sota strain kept in our laboratory was purified by limiting dilution via chicken embryo inoculation. After 5 passages, a high hemagglutinating clone was identified and designated as La Sota C5. The whole genome of La Sota C5 was sequenced. Some differences in biological characteristics and gene sequences were found between La Sota C5 and its parental strain. The La Sota C5 genome was divided into 8 fragments according to its suitable restriction digestion sites. These 8 fragments were then amplified in RT-PCR and cloned into the transcription vector TVT in accordance with the structure of the viral genome sequence. The resulting transcription vector TVT-La Sota C5 contained complete cDNA of La Sota C5. The vector TVT-La Sota C5 together with 3 helper plasmids pCI-NP, pCI-P and pCI-L were transfected into BSR-T7/5 cells. The infectious La Sota strain viruses were successfully rescued. The availability of infectious clone ofNDV La Sota strain would provide a platform for further study on virus gene functions and development of vaccines.