裂谷热假病毒颗粒研制及鉴定

生物技术通报 ›› 2013, Vol. 0 ›› Issue (2): 135-139.

裂谷热假病毒颗粒研制及鉴定

邓俊花林祥梅张永宁冯春燕王彩霞吴绍强

（中国检验检疫科学研究院，北京 100029）

收稿日期:2012-06-26 修回日期:2013-02-27 出版日期:2013-02-26 发布日期:2013-02-27
作者简介:邓俊花，女，硕士，助理研究员，研究方向：动物检疫科研工作；E-mail ：dengqifei-198825@163.com
基金资助:
国家质检总局公益项目（200910132），国家质检总局科研专项（2012IK266）

The Development and Identification of Rift Valley Fever Virus-like Particles

Deng Junhua Lin Xiangmei Zhang Yongning Wang Caixia Feng Chunyan Wu Shaoqiang

（Chinese Academy of Inspection and Quarantine，Beijing 100029）

Received:2012-06-26 Revised:2013-02-27 Published:2013-02-26 Online:2013-02-27

摘要/Abstract

摘要： 裂谷热是危害严重的人畜共患病，为了给该病的分子生物学检测提供RNA 阳性质控品，制备了裂谷热假病毒颗粒并进行了检测和验证。将pGEM-T-RVF 质粒与假病毒载体p-MS2 质粒分别进行Pst I 和Hind Ⅲ双酶切，然后连接构建p-MS2-RVF重组质粒。将p-MS2-RVF 重组菌进行表达、酶消化及盐沉淀后纯化假病毒颗粒，在特性检测的基础上，借助荧光RT-PCR 方法进行鉴定。结果表明，裂谷热假病毒颗粒耐核酸酶，室温至少保持1 个月，稳定，易运输；荧光RT-PCR 检测表明最低可达到4.25×102copies 检测限。构建的裂谷热假病毒颗粒将为裂谷热分子生物学检测提供阳性质控物质。

关键词: 裂谷热, 假病毒颗粒, 鉴定

Abstract: It was to develop Rift valley fever（RVF）virus-like particles（VLPs）to effectively offer positive material for molecular biological detection. pGEM-T-RVFV plasmid and p-MS2 were individually digested with both Pst I and Hind Ⅲ , and then purified ligated to generate recombinant plasmid p-MS2-RVFV. The plasmid was cotransformed into E. coli. DH5α cell and induced with IPTG, incubated with RNase A and DNase I, and then subsided with salt to obtain the rough-wrought virus-like particles. The method of gradient centrifugation was used to prepared rarefied virus-like particles which was determined for characteristic property. The puried particles were verified by Real-time RT-PCR. Results showed that the rarefied virus-like particles were able to endure RNase A, saved at any rate one month under subzero climatic condition, which indicated that the virus-like particles were stable and were simple transported；it indicated that the VLPs could attain a minimum detectable limit of 4.25×102 copies. The construction of virus-like particles could provide positive material for RVF molecular detection.

Key words: Rift valley fever virus（RVFV）, Virus-like particles（VLPs）, Identification

邓俊花, 林祥梅, 张永宁, 冯春燕, 王彩霞, 吴绍强. 裂谷热假病毒颗粒研制及鉴定[J]. 生物技术通报, 2013, 0(2): 135-139.

Deng Junhua, Lin Xiangmei, Zhang Yongning, Wang Caixia, Feng Chunyan, Wu Shaoqiang . The Development and Identification of Rift Valley Fever Virus-like Particles[J]. Biotechnology Bulletin, 2013, 0(2): 135-139.

参考文献

［1] Daubney R, Hundson JR, Gamham PC. Enzootic hepatitis or Rift Valley fever. An undescribed virus disease of sheep, cattle and man from East Africa［J]. J Pathol Bacteriol, 1931, 34(4)：545-579.
[2] Peyrefitte CN, Boubis L, Coudrier D, et al. Real-time reversetranscription loop-mediated isothermal amplification for rapid detection of Rift Valley Fever Virus［J]. Journal of Clinical Microbiology, 2008, 46(11)：3653-3659.
[3] Sall AA, Macondo EA, Sene OK, et al. Use of reverse transcriptase PCR in early diagnosis of Rift Valley Fever［J]. Clinical and Diagnostic Laboratory Immunology, 2002, 9(3)：713-715.
[4] Sall AA, Thonnon J, Sene OK, et al. Single-tube and nested reverse transcriptase-polymerase chain reaction for detection of Rift Valley Fever Virus in human and animal sera［J]. Journal of Virological Methods, 2001, 91(1)：85-92.
[5] Drosten C, G?ttig S, et al. Rapid detection and quantification of RNA of Ebola and Marburg viruses, Lassa virus, Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus, dengue virus, and yellow fever virus by real-time reverse transcription-PCR［J]. Journal of Clinical Microbiology, 2002, 40(7)：2323-2330.
[6] Espach A, Romito M, Nel LH, et al. Development of a diagnostic one-tube RT-PCR for the detection of Rift Valley fever virus［J]. American Journal of Veterinary Research, 2002, 69(3)：247-252.
[7] Pasloske BL, Walkerpeach CR, Dawn Obermoeller RD, et al. Armored RNA technology for production of ribonuclease-resistant viral RNA controls and standards［J]. Journal of Clinical Microbiology, 1998, 36(12)：3590-3594.
[8 ] WalkerPeach CR, Winkler M, DuBois DB, et al. Ribonuclease resistant RNA controls(Armored RNA)for reverse transcription- PCR, branched DNA, and genotyping assays for hepatitis C virus［J]. Clinical Chemistry, 1999, 45(12)：2079-2085.
[9] 王彩霞, 林祥梅, 吴绍强, 邓俊花. 用于检测裂谷热病毒S 片段的引物和探针: 中国, 201110415602X［P].
[10] 萨姆布鲁克 J, 拉塞尔 DW, 等. 分子克隆实验指南［M]. 北京：科学出版社, 2002:185-186.
[11] Jorgensen PA, Neuwald PD. Standardized hepatitis C virus RNA panels for nucleic acid testing assays［J]. Journal of Clinical Virology, 2001, 20(1-2)：35-40.
[12] DuBois DB, Wikker MM, Pasloske BL. Ribonuclease resistant RNA viral RNA standards:U.S., 5, 677, 124［P]. 1997-10-124.
[13] Beld M, Minnaar R, Weel J, et al. Highly sensitive assay for detection of enterovirus in clinical specimens by reverse transcription-PCR with an armored RNA internal controls［J]. Journal of Clinical Microbiology, 2004, 42(7)：3059-3064.
[14] Hietala SK, Crossley BM. Armored RNA as virus surrogate in a real-time reverse transcriptase PCR assay proficiency panel［J]. Journal of Clinical Microbiology, 2006, 44(1)：67-70.
[15] Bressler AM, Nolte FS. Preclinical evaluation of two real-time, reverse transcription-PCR assays for detection of the severe acute respiratory syndrome coronavirus［J]. Journal of Clinical Microbiology, 2004, 42(3)：987-991.
[16] 李金明, 王忠芳, 等. T 载体克隆在病毒样颗粒表达载体构建中的应用［J]. 中华检验医学杂志, 2004, 27(11)：786-788.
[17] 李金明, 宋如俊, 等. 耐核糖核酸酶内含HCV RNA 病毒样颗粒的表达［J]. 中华检验医学杂志, 2005, 28(3)：302-304.
[18] Argetsinger JE, Gussin GN. Intact ribonucleic acid from defective particles of bacteriophage R17［J]. Journal of Molecular Biology, 1966, 21(3)：421-434.
[19] Heisenberg M. Formation of defective bacteriophage particles by fr amber mutants. II［J]. Biochem Biophys Res Commun, 1967, 27 (2)：131-137.
[20] Pickett GG, Peabody DS. Encapsidation of heterologous RNAs by bacteriophage MS2 coat protein［J]. Nucleic Acids Research, 1993, 21(19)：4621-4626.
[21] Romaniuk PJ, Lowary P, Wu HN. RNA binding site of R17 coat protein［J]. Biochemistry, 1987, 26(6)：1563-1568.
[22] Shiba T, Suzuki Y. Localization of A protein in the RNA-A protein complex of RNA phage MS2［J]. Biochim Biophys Acta, 1981, 654(2)：249-255.

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