内含埃博拉病毒GP基因序列假病毒粒子的构建及应用

Construction and Application of Pseudovirions Containing Ebola Virus GP Gene Sequence

目的:构建内含埃博拉病毒(EBOV) GP基因序列的假病毒粒子,针对EBOV GP基因建立实时荧光定量PCR检测方法。方法:人工合成GP基因序列,克隆至慢病毒包装载体pGWLV-pseudovirus中,转化DH5α感受态细胞,筛选得到阳性克隆pGWLV-GP质粒。将重组质粒转染293T细胞,培养48 h后进行除菌,纯化后获得高纯度假病毒,通过RT-PCR鉴定该假病毒粒子含有EBOV GP基因。通过荧光定量qPCR对假病毒颗粒进行计数后,利用本研究所获得的假病毒颗粒制备用于EBOV核酸检测的标准品。结果:建立了基于EBOV GP基因的Taqman荧光定量PCR检测方法。结论:该方法所用标准品模拟了真实病毒粒子的结构,无生物传染性,经检测均匀性和稳定性良好,可作为EBOV核酸检测的阳性标准质控品,实现对核酸检测的全程监控。

Objective: To construct pseudovirion containing Ebola virus (EBOV) GP gene sequence and establish EBOV Taqman real-time fluorescence quantitative PCR detection assay. Methods: The GP gene se-quence was synthesized and cloned into lentivirus packaging vector PGWLV-pseudovirus, trans-formed into DH5α competent cells and screened to obtain a positive clone of pGWLV-GP plasmid. The recombinant plasmid was transfected into 293T cells, cultured for 48 h, and purified to obtain a high purity pseudovirus, which was identified by RT-PCR as containing the EBOV GP gene. After counting pseudovirus particles by fluorescence quantitative qPCR, the pseudovirus particles ob-tained in this study were used to prepare standards for EBOV nucleic acid detection. Results: A Taqman real-time PCR method based on EBOV GP gene was established. Conclusion: The standard substance used in this method simulates the structure of real virions, has good uniformity and sta-bility after detection, and can be used as positive standard quality control substance for EBOV nu-cleic acid detection, realizing the whole process monitoring of nucleic acid detection.