军事医学2017，Vol.41Issue(3)：194-198,5.DOI:10.7644/j.issn.1674-9960.2017.03.008
携带圣路易斯脑炎病毒特异基因片段的重组假病毒颗粒的构建与鉴定
Construction and characterization of recombinant pseudovirus particles carrying specific genomic RNA of St.Louis encephalitis virus
摘要
Abstract
Objective To prepare quality control samples for St.Louis encephalitis virus(SLEV)molecular detection by constructing pseudovirus containing target sequences of SLEV.Methods According to the principles of armored RNA technique, the prM gene sequence of SLEV was cloned into the prokaryotic expression vector to generate recombinant plasmid pSE380-MS2-SLEV.Then, recombinant E.coli transformed with the corresponding plasmid was induced with IPTG to produce recombinant pseudovirus particles.The particles were purified by chloroform and further characterized by double enzyme digestion and transmission electron microscopy.The temperature sensitivity experiments and quantitative RT-PCR were performed to validate the potential of these pseudovirus particles as quality control samples.Results PCR amplification and sequencing analysis confirmed that the prM gene sequence of SLEV was cloned into vector pSE380-MS2.Transmission electron microscopy showed that homogenous spherical particles with a diameter of about 25 nm were produced upon IPTG induction.The SLEV genomic RNA within the pseudovirus particles was resistant to DNaseⅠand RNase A digestion, and remained stable for 20 days at 37℃.These samples were validated with quantitative RT-PCR for SLEV.Conclusion The RNase-resistant and stable pseudovirus particles containing prM fragment of SLEV are constructed successfully, which can be used as positive quality control samples for RNA extraction and molecular detection.关键词
圣路易斯脑炎病毒/假病毒/阳性质控品Key words
St.Louis encephalitis virus/pseudovirus/positive quality control分类
医药卫生引用本文复制引用
张娜娜,邓永强,年庆功,康晓平,杨银辉,秦成峰..携带圣路易斯脑炎病毒特异基因片段的重组假病毒颗粒的构建与鉴定[J].军事医学,2017,41(3):194-198,5.基金项目
国家科技重大专项资助项目(2013ZX10004805) （2013ZX10004805）
