鹅源新城疫病毒ZJ1株微型基因组的构建及其初步应用

Construction of minigenome of Newcastle disease virus of goose origin and its preliminary application

在获得鹅源新城疫病毒ZJ1株全基因组序列的基础上 ,用增强型绿色荧光蛋白 (eGFP)报告基因取代鹅源新城疫病毒ZJ1株整个编码区 ,只保留与病毒复制、转录和病毒粒子包装相关的调控序列 ,将其反向克隆入转录载体TVT7R(0 0 )中 ,构建了该毒株的微型基因组。当转染用辅助病毒ZJ1株感染的HEp_2细胞时报告基因得到表达 ,表明此微型基因组RNA可被辅助病毒提供的NP、P和L蛋白翻译。同时将该病毒NP、P和L蛋白基因分别克隆入真核表达载体pCI_neo中 ,构建了表达该病毒NP、P与L蛋白的辅助质粒 ,用此微型基因组对辅助质粒的表达产物进行了功能鉴定并对该病毒拯救过程中痘苗病毒的最适感染剂量进行了摸索。

On the base of obtaining the full length genome sequence of a Newcastle disease virus (NDV) isolated from goose, the minigenome was constructed by replacing all the encoding region with the reporter gene of enhanced green fluorescent protein (eGFP), except the virus regulating sequences relating to replication, transcription and packing of virus genome. The reporter gene could be expressed after it was transfected into the HEp-2 cells infected with helper virus of NDV. This result indicated that the minigenome could be translated by the NDV NP, P and L proteins provided by helper virus. Furthermore, the support plasmids expressing NDV NP, P and L protein were constructed respectively and the function of these plasmids was identified using the minigenome. Additionally, the virus rescue system was optimized by changing the infection dose of the recombinant vaccinia virus expressing T7 RNA polymerase . The work mentioned above will accelerate greatly the rescue of NDV and other relative research.