O型口蹄疫病毒亚基因组复制子的构建及其鉴定  

Construction of sub-genomic replicon of O serotype foot-and-mouth disease virus

作　　者：孙超[1] 杨德成[1] 高荣远[1] 周国辉[1] 王海伟[1] 于力[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/牛羊传染病研究创新团队,黑龙江哈尔滨150001

出　　处：《中国预防兽医学报》2014年第8期611-614,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：中央级公益性科研院所基本科研业务费专项(0302013012)

摘　　要：为构建含有海肾萤光素酶(Rluc)报告基因的O型口蹄疫病毒(FMDV)亚基因组复制子,本研究在前期构建的O型FMDV cDNA感染性克隆的基础上,利用融合PCR的方法,以Rluc报告基因替换FMDV的前导蛋白Lb基因和结构蛋白P1基因,构建含有Rluc报告基因的FMDV亚基因组复制子FMDVRep。将该复制子质粒线性化后,经体外转录制备复制子全长RNA,转染BHK-21细胞,通过RT-PCR方法和间接免疫荧光试验分别检测该复制子RNA的自主复制能力以及FMDV非结构蛋白3B的表达情况。结果显示,复制子RNA在BHK-21细胞中能够进行自主复制并且能够表达病毒的非结构蛋白。Rluc活性检测结果表明,该复制子RNA在BHK-21细胞中可以高效表达Rluc,在转染后2 h^12 h Rluc活性呈线性递增,进一步反映该复制子具有良好的表达外源基因的能力。O型FMDV亚基因组复制子的构建,为深入研究FMDV的复制和翻译机制奠定了基础。To construct sub-genomic replicon of O serotype foot-and-mouth disease virus （FMDV） using renilla luciferase （Rluc） as the reporter gene, the Lb and P1 gene in a full-length cDNA clone of O serotype FMDV was replaced with the Rluc reporter gene by fusion PCR. Then the transcripted RNA of replicon was prepared from the linearized replicon in vitro and transfected into BHK-21 cells in which the replication of the replicon with Rluc gene was detected by RT-PCR and immunofluorescence assay, respectively. The results showed that the replicon was able to self-replicate and express nonstructural protein of FMDV in BHK-21 cells. In addition, the Rluc signals increased exponentially during 2 to 12 hours post-transfection, which properly represented the replication and translation efficiency of the replicon. The construction of the replicon would facilitate further study of the regulation mechanisms of FMDV for replication.

关 键 词：O型口蹄疫病毒 海肾萤光素酶 复制子 

分 类 号：S852.65[农业科学—基础兽医学]