【摘要】 为研究新城疫病毒(NDV)微型基因组的复制效率,本实验分别以NDV 9a5b(ClassⅠ)病毒株和La Sota(ClassⅡ)病毒株基因组骨架为平台,插入绿色荧光蛋白(GFP)作为报告基因替代病毒的整个编码区,只保留与病毒复制、转录和病毒包装相关的调控区域Leader和Trailer,构建获得的微型基因组DNA片段反向插入反向遗传载体TVT(0.0)中,从而构建了两株病毒的微型基因组质粒。同时,将相应病毒株的NP、P和L 3个基因分别克隆至pCI-neo载体中,构建3个辅助质粒的真核表达系统。通过将微型基因组和辅助质粒共转染BSR T7/5T7-5细胞,表明在相同转染条件下,无论使用哪一种辅助质粒,ClassⅡ型微型基因组的报告基因的表达效率均高于ClassⅠ型微型基因组。实验结果显示ClassⅠNDV转录复制系统的运行效率明显低于ClassⅡNDV。更多还原

【Abstract】 Newcastle disease(ND) is a highly infectious avian disease in poultry.The causative agent ND virus(NDV) have a broad host range and are known to infect over 200 bird species.Recently,NDV isolates have been classified into two distinct classes,ClassⅠ and ClassⅡ,on the basis of genomic size and sequence analysis of the F and L genes.In this study,we constructed minigenome plasmids and helper plasmids for 9a5b(ClassⅠ) and La Sota(ClassⅡ),respectively.The nucleotide protein(NP),phosphosprotein(P) and large protein(L) gene were cloned from NDV strain 9a5b or La Sota by RT-PCR and subcloned into pCI-neo vector to construct the two sets of helper plasmids.The constructed minigenome consisted of the 3’ leader and 5’ trailer regions of NDV strain 9a5b or La Sota flanking a reporter gene encoding green fluorescent protein(GFP) to examine the efficacy of the three helper plasmids for viral genome replication and transcription.After co-transfecting minigenome and helper plasmids into BSR T7/5 cells,we found that the expression of reporter protein from classⅡ minigenome was much higher than that of ClassⅠ,no matter which set of helper plasmids was used in the experiment.This result revealed that the viral polymerase system of ClassⅡ NDV is more efficient than that of classⅠ NDV.更多还原