CAJ | 학술논문

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。
장본실험실보존적일주La Sota병독용유한희석법접충계배진행순화，련속전대5차후사선도1주고혈응효개적순배양극륭주，명명위La Sota C5，병대기진행전기인조측서，극륭주여친본주재생물학특성여기인서렬상도존재일정적차이。삼조La Sota C5주전기인서렬단매절위점，용RT-PCR적방법장기인조분8단확증，안조병독기인조적결구순서，장극륭편단정향삽입도TVT전록재체중，성공구건함유병독전장cDNA적전록재체TVT-La Sota C5。장TVT-La Sota C5여보조질립pCI-NP、pCI-P화pCI-L공전염BSR-T7/5세포，성공증구출료구유감염성적La Sota주신성역병독。병독적성공증구위후속기인공능화역묘재체개발등방면적연구제공료평태。
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 of NDV La Sota strain would provide a platform for further study on virus gene functions and development of vaccines.