新城疫病毒classⅠ和classⅡ微型基因组的构建及其复制效率差异性的比较  

Construction of mini-genome for ClassⅠ and ClassⅡ Newcastle disease virus and comparison of their replication efficiency

作　　者：于洋[1,2] 仇旭升[2] 韦娜娜[2] 詹媛[2] 丁娜[2] 于圣青[2] 秦爱建[1] 丁铲[1,2] 

机构地区：[1]扬州大学兽医学院,江苏扬州225009 [2]中国农业科学院上海兽医研究所国家家禽工程技术研究中心,上海200241

出　　处：《中国预防兽医学报》2012年第5期355-359,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：公益性行业(农业)专项(201003012);863项目(2011AA10A209);国家自然科学基金(31101822)

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。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, ClassⅡ and Class Ⅱ , 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 （Class I ） and La Sota （Class Ⅱ ）, 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 class Ⅱ minigenome was much higher than that of Class Ⅰ , no matter which set of helper plasmids was used in the experiment. This result revealed that the viral polymerase system of Class Ⅱ NDV is more efficient than that of class I NDV.

关 键 词：新城疫病毒 微型基因组 共转染 

分 类 号：S852.65[农业科学—基础兽医学]