脊髓灰质炎病毒疫苗株Sabin Ⅰ全基因序列克隆的构建及鉴定

摘要

目的 构建脊髓灰质炎病毒疫苗株Sabin Ⅰ的全基因序列克隆.方法 利用分子生物学技术,通过分段扩增和酶切连接逐步构建插入脊髓灰质炎病毒疫苗株Sabin Ⅰ全基因序列的质粒,采用单引物二次PCR法引入点突变,并通过酶切、测序等方法进行鉴定.结果 在pWSK29载体中成功插入脊髓灰质炎病毒疫苗株Sabin Ⅰ全基因序列,酶切鉴定正确,序列测定显示有9个核苷酸变异.结论 成功构建脊髓灰质炎病毒疫苗株Sabin Ⅰ的全基因序列克隆,为进一步改造并研究其功能特别是3D聚合酶的功能打下了基础.%Objective To develop a vector inserted with complete genome of poliovirus strain Sabin Ⅰ. Methods The 3 fragments of the complete genome of Sabin Ⅰ was amplified and cloned to pEASY-T3 by molecular biological technology. These cloned pEASY-T3 were then digested by Restriction enzymes and ligated to pWSK29 step by step and identified. Results The complete genome of poliovirus strain Sabin Ⅰ was successfully cloned into vector pWSK29 with 9 nucleotide mutations. Conclusion The complete genome plasmid was constructed and it provided a basis for further research of the function of Sabin Ⅰ.