内含埃博拉病毒NP基因序列假病毒粒子的构建及应用
Construction and Application of Pseudovirions Containing Ebola Virus NP Gene Sequence

目的：构建内含埃博拉病毒(EBOV) NP基因序列的假病毒粒子，建立EBOV Taqman实时荧光定量PCR检测方法。方法：对埃博拉病毒的NP基因序列进行分析，分析基因是否包含重复序列，复杂二级结构以及高GC含量等；根据序列分析结果，合成NP全基因序列，并将NP基因的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-NP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 NP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 NP基因的Taqman荧光定量PCR检测方法。本研究建立的基于包含NP基因的EBOV假病毒核酸cDNA的Taqman荧光定量PCR检测方法的Ct值与核酸cDNA标准品1 × 109~1 × 101个/μL范围内呈良好的线性关系，相关系数可达到0.99。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) NP gene sequence and establish EBOV Taqman real-time fluorescence quantitative PCR detection assay. Methods: The NP gene sequence of EBOV was analyzed to determine whether the gene contained repeatitive sequence, complex secondary structure and high GC content, based on the sequence analysis results, the NP gene sequence was synthesized and cloned into lentivirus packaging vector PGWLV-pseudovirus, transformed into DH5α competent cells and screened to obtain a positive clone of pGWLV-NP 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 NP gene. After counting pseudovirus particles by fluorescence quantitative qPCR, the pseudovirus particles obtained in this study were used to prepare standards for EBOV nucleic acid detection. Results: A Taqman real-time PCR method based on EBOV NP gene was established. In this study, the Ct values of Taqman fluorescence quantitative PCR method based on cDNA of EBOV pseudovirus containing NP gene showed a good linear relationship with cDNA standard nucleic acid in the range of 1× 109 ~1×101 copies/μL, and the correlation coefficient could reach 0.99. Conclusion The standard substance used in this method simulates the structure of real virions, has good uniformity and stability after detection, and can be used as positive standard quality control substance for EBOV nucleic acid detection, realizing the whole process monitoring of nucleic acid detection.