内含猪繁殖与呼吸综合征病毒保守基因序列假病毒粒子的构建及应用  

Construction and application of virus-like particles containing conserved gene of porcine reproductive and respiratory syndrome virus

作　　者：乔彩霞[1] 张鹤晓[1] 高志强[1] 尹羿[1] 蒲静[1] 汪琳[1] 刘环[1] 张伟[1] QIAO Cai-xia ZHANG He-xiao GAO Zhi-qiang YIN Yi PU Jing WANG Lin LIU Huan ZHANG Wei(Beijing Entry-Exit Inspection and Quarantine Bureau ,Beijing 101113,China)

机构地区：[1]北京出入境检验检疫局,北京101113

出　　处：《中国兽医学报》2016年第12期2024-2029,共6页Chinese Journal of Veterinary Science

基　　金：国家公益性行业科研专项资助项目(201310253)

摘　　要：为构建内含猪繁殖与呼吸综合征病毒(PRRSV)保守基因序列的假病毒粒子,将MS2噬菌体基因组中5′非编码区、成熟酶蛋白基因、衣壳蛋白基因、包装位点和复制酶基因部分起始位点的cDNA序列克隆于原核表达载体pET32a,在其下游插入PRRSV ORF7和3′非编码区保守序列,获得重组表达载体pET-MS2-PRRSV。将重组质粒pET-MS2-PRRSV转化表达菌株BL21(DE3),经诱导表达、纯化,获得高纯度的病毒样颗粒。病毒样颗粒经RT-PCR和PCR鉴定含有PRRSV ORF7和3′非编码区RNA且没有质粒DNA残留,并能耐受RNase降解。采用荧光定量RT-PCR对病毒样颗粒定值后,制备了用于PRRSV核酸检测的标准品,该标准品模拟了真实病毒粒子的结构,无生物传染性,经检测均匀性和稳定性良好,可作为PRRSV核酸检测的阳性标准质控品,实现对核酸检测的全程监控。The aim of this study is to construct virus-like particles（VLPs） containing conserved gene of porcine reproductive and respiratory syndrome virus（PRRSV）. The eDNA fragment of maturation protein （A-protein）,coat protein,packaging sites of MS2 baeteriophage,ORF7 and 3r untranslated region of PRRSV were cloned into vector pET32a to construct the prokaryotic ex- pression vector pET-MS2-PRRSV. The recombinant plasmid pET-MS2-PRRSV was transformed into E. coli strain BL21 （DE3） and induced to express with 1 mmol/L IPTG. The RNA of VLPs was identified by PCR and RT-PCR. The results showed that the VLPs contain ORF7 and 3＇untranslated region of PRRSV and were resistant to RNase degradation. Based on the quantitation of VLPs using real-time RT-PCR for PRRSV, a standard sample for PRRSV was prepared using VLPs. The standard sample resembled authentic PRRSV, noninfectious, homogenous and stable, which was an ideal reference control for testing of PRRSV nucleic acids and monitoring the entire detection process.

关 键 词：猪繁殖与呼吸综合征病毒 ORF7 3’非编码区 病毒样颗粒 

分 类 号：S852.65[农业科学—基础兽医学]