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
|
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 amplified by RT-PCR,then the gene was cloned into vector pNH-MS_2 his contained MS_2 phage coat protein,and the recombinant prokaryotic expression vector pNH-MS_2 his-N was constructed. The recombinant plasmid pNH-MS_2 his-N transformed into E.coli strain BL21(DE3)was induced to expression and purification with 1 mmol/L IPTG. The VLPs were identified 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 8×10~6 copies/m L. The results showed that the VLPs could be used as a standard and quality control in RNA virus detection.
To construct virus-like particles(VLPs)standard sample containing N gene of Viral hemorrhagic septicemia virus(VHSV),the N gene of VHSV was amplified by RT-PCR,then the gene was cloned into vector pNH-MS_2 his contained MS_2 phage coat protein,and the recombinant prokaryotic expression vector pNH-MS_2 his-N was constructed. The recombinant plasmid pNH-MS_2 his-N transformed into E.coli strain BL21(DE3)was induced to expression and purification with 1 mmol/L IPTG. The VLPs were identified 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 8×10~6 copies/m L. The results showed that the VLPs could be used as a standard and quality control in RNA virus detection.
引文
[1]阮红梅,张奇亚.鱼类弹状病毒分子生物学研究动态[J].中国水产科学,2003,10(6):513-519.
[2]安元龙,吴斌,林长军.荧光环介导逆转录等温扩增(RT-LAMP)技术在病毒性出血性败血症(VHS)诊断中的应用[J].中国动物检疫,2012(12):21-26.
[3]SKALL H F,OLESEN N J,MELLERGAARD S.Viral haemorrhagic septicaemia virus in marine fish and its implications for fish farming-a review[J].Journal of fish diseases,2005,28(9):509-529.
[4]World Organization for Animal Health(OIE).Manual of diagnostic tests for aquatic animals[M].6th.Paris:OIE,2016.
[5]JONSTRUP S P,KAHNS S,SKALL H F,et al.Development and validation of a novel Taqman-based real-time RT-PCR assay suitable for demonstrating freedom from viral haemorrhagic septicaemia virus[J].Journal of fish diseases,2013,36(1):9.
[6]GARVER K A,HAWLEY L M,MCCLURE C A,et al.Development and validation of a reverse transcription quantitative PCR for universal detection of viral hemorrhagic septicemia virus[J].Diseases of aquatic organisms,2011,95(2):97-112.
[7]张晓文,姜帆,孙明君,等.含有H3N8马流感病毒HA基因噬菌体病毒样颗粒的构建与纯化[J].中国预防兽医学报,2015,37(3):194-198.
[8]王宇,张晓文,王群,等.含马动脉炎病毒N基因的病毒样颗粒的制备[J].畜牧与兽医,2014,46(6):8-11.
[9]张括,魏玉香,李金明.RNA噬菌体病毒样颗粒包装机制及其应用研究进展[J].微生物与感染,2008,3(2):111-114.
[10]柳方方,张玲,王晶,等.数字PCR测定DNA含量及测量结果不确定度评定[J].化学分析计量,2013(1):18-22.
[11]冯兆民,赵翔,邹晓辉,等.基于微滴式数字PCR技术的甲型流感病毒绝对定量方法的建立及应用[J].病毒学报,2017(1):1-5.
[12]赵丽青,王静,秦燕,等.PMA结合dd PCR检测食品中金黄色葡萄球菌的研究[J].微生物学杂志,2017(1):105-109.
[2]安元龙,吴斌,林长军.荧光环介导逆转录等温扩增(RT-LAMP)技术在病毒性出血性败血症(VHS)诊断中的应用[J].中国动物检疫,2012(12):21-26.
[3]SKALL H F,OLESEN N J,MELLERGAARD S.Viral haemorrhagic septicaemia virus in marine fish and its implications for fish farming-a review[J].Journal of fish diseases,2005,28(9):509-529.
[4]World Organization for Animal Health(OIE).Manual of diagnostic tests for aquatic animals[M].6th.Paris:OIE,2016.
[5]JONSTRUP S P,KAHNS S,SKALL H F,et al.Development and validation of a novel Taqman-based real-time RT-PCR assay suitable for demonstrating freedom from viral haemorrhagic septicaemia virus[J].Journal of fish diseases,2013,36(1):9.
[6]GARVER K A,HAWLEY L M,MCCLURE C A,et al.Development and validation of a reverse transcription quantitative PCR for universal detection of viral hemorrhagic septicemia virus[J].Diseases of aquatic organisms,2011,95(2):97-112.
[7]张晓文,姜帆,孙明君,等.含有H3N8马流感病毒HA基因噬菌体病毒样颗粒的构建与纯化[J].中国预防兽医学报,2015,37(3):194-198.
[8]王宇,张晓文,王群,等.含马动脉炎病毒N基因的病毒样颗粒的制备[J].畜牧与兽医,2014,46(6):8-11.
[9]张括,魏玉香,李金明.RNA噬菌体病毒样颗粒包装机制及其应用研究进展[J].微生物与感染,2008,3(2):111-114.
[10]柳方方,张玲,王晶,等.数字PCR测定DNA含量及测量结果不确定度评定[J].化学分析计量,2013(1):18-22.
[11]冯兆民,赵翔,邹晓辉,等.基于微滴式数字PCR技术的甲型流感病毒绝对定量方法的建立及应用[J].病毒学报,2017(1):1-5.
[12]赵丽青,王静,秦燕,等.PMA结合dd PCR检测食品中金黄色葡萄球菌的研究[J].微生物学杂志,2017(1):105-109.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |