病毒性出血性败血症病毒N基因的病毒样颗粒制备及定值  

Construction and Valuation of Virus-like Particles Containing N Gene of Viral Hemorrhagic Septicemia of Salmonids Virus

作　　者：王群[1] 姜帆[1] 岳志芹[1] 孙涛[1] 郑小龙[1] 张晓文[1] 房保海[1] Wang Qun;Jiang Fan;Yue Zhiqin;Sun Tao;Zheng Xiaolong;Zhang Xiaowen;Fang Baohai(Technical Center of Shandong Entry-exit Inspection and Quarantine Bureau,Qingdao,Shandong266002,Chin)

机构地区：[1]山东出入境检验检疫局检验检疫技术中心,山东青岛266002

出　　处：《中国动物检疫》2018年第1期100-103,共4页China Animal Health Inspection

基　　金："十二五"国家科技支撑项目(2013BAD12B02-03)

摘　　要：为制备含有病毒性出血性败血症病毒(VHSV)N基因的RNA病毒样颗粒标准样品,通过RT-RCR扩增VHSV的N基因,将其克隆至含有组氨酸标签的pNH-MS_2his载体中,构建重组原核表达载体pNHMS_2his-N;将重组质粒pNH-MS_2his-N转化至表达菌株BL21(DE3),用1 mmol/L IPTG诱导表达、纯化;对病毒样颗粒进行鉴定及稳定性试验,使用数字PCR对病毒样颗粒进行定值。结果显示,该病毒样颗粒含VHSV N基因序列,并且稳定性良好,定值结果为8×10~6 copies/m L。结果表明,构建的病毒样颗粒可以作为RNA病毒检测时的标准品和质控品使用。To construct virus-like particles（VLPs）standard sample containing N gene of Viral hemorrhagic septicemia virus（VHSV）,the N gene of VHSV was ampli？ed by RT-PCR,then the gene was cloned into vector pNH-MS2his contained MS2 phage coat protein,and the recombinant prokaryotic expression vector pNH-MS2his-N was constructed. The recombinant plasmid pNH-MS2his-N transformed into E.coli strain BL21（DE3）was induced to expression and puri？cation with 1 mmol/L IPTG. The VLPs were identi？ed and the stability of VLPs was tested by real-time RT-PCR. The concentration of VLPs was valued by digital PCR. The results showed that the VLPs contained N gene RNA and had good stability. The concentration of VLPs was 8×106 copies/mL. The results showed that the VLPs could be used as a standard and quality control in RNA virus detection.

关 键 词：病毒性出血性败血症病毒 病毒样颗粒 N基因 数字PCR 

分 类 号：S852.65[农业科学—基础兽医学]