裂谷热假病毒颗粒研制及鉴定

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

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PstI和HindⅢ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。

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. DH5a 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 puffed 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 4.25×10^2 copies. The construction of virus-like particles could provide positive material for RVF molecular detection.