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

摘要

目的 制备含有圣路易斯脑炎病毒(SLEV)prM基因片段的假病毒颗粒,旨在提供安全、可靠、稳定的核酸阳性质控品.方法 根据armored RNA技术原理,构建携带含有SLEV prM基因片段的重组质粒pSE380-MS2-SLEV,转化大肠杆菌后利用IPTG进行诱导表达,三氯甲烷法纯化后利用透射电镜观察重组假病毒颗粒形态.通过DNaseⅠ和RNase A双酶消化实验评价重组假病毒颗粒的核酸酶耐受性,并结合温度敏感性实验分析假病毒颗粒中核酸片段的稳定性.利用荧光定量 RT-PCR法对该假病毒颗粒进行验证.结果 PCR扩增和测序分析显示,prM基因片段正确克隆至载体pSE380-MS2.经大肠杆菌表达可形成直径约为25 nm的均一、球形重组假病毒颗粒.核酸酶消化和温度敏感性实验表明假病毒样颗粒包装的病毒核酸能耐受核酶的降解,37℃条件下存放20 d仍保持稳定.荧光定量RT-PCR检测该假病毒颗粒检测限可达1.8×103 拷贝/ml,且与同属的其他病毒无交叉反应.结论 成功构建了含有SLEV prM基因片段的假病毒颗粒,具有良好的核酸酶耐受性和稳定性,可作为核酸阳性质控品对核酸提取与检测过程进行全程监控.%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.