含5种烈性出血热病毒的假病毒阳性参考品的制备

摘要

目的：利用慢病毒载体系统制备含有马尔堡病毒（Marburg virus）、扎伊尔型埃博拉病毒（Zaire Ebola virus）、苏丹型埃博拉病毒（Sudan Ebola virus）、拉沙热病毒（Lassa fever virus）和黄热病毒（Yellow fever virus）5种烈性病毒核酸片段的假病毒，为其核酸检测提供一种通用的阳性参考品。方法体外合成该5种病毒的目的基因片段，通过融合 PCR 技术将5个片段连接成一条基因，然后克隆到慢病毒表达载体上，并与其辅助载体共转染293T细胞；转染后分别于48、72 h 收集培养上清，用 DNase 和 RNase 进行消化处理及纯化浓缩，最后提取核酸，利用普通PCR 和实时荧光定量 PCR 鉴定假病毒是否包装成功。结果测序结果表明，体外合成的该5种病毒的目的基因片段已正确连接并插入慢病毒载体中；载体转染293T 细胞后收集上清中的假病毒，提取核酸经上述两种 PCR 鉴定均可特异性检测到靶基因，表明已成功包装出含该5种出血热病毒靶基因的假病毒颗粒。结论该研究获得的假病毒颗粒可作为上述5种出血热相关烈性病毒核酸检测的通用阳性参考品。%Objective To construct the pseudovirus containing nucleic acid(NA)fragments of Marburg virus,Zaire Ebola virus,Sudan Ebola virus,Lassa fever virus and Yellow fever virus by using a lentiviral vector system in order to provide a reference standard for the detection of the five viruses.Methods The gene fragments of the above five viruses were synthesized in vitro,connected into a single gene by fusion PCR technique,and cloned into lentiviral vectors with its auxiliary vector.After co-transfecting into 293T cells,the supernatants were collected on 48 h and 72 h post transfection. The naked NA was cleaned from the supernatants using DNase and RNase digestion before pseudotype virus was purified and concentrated.After the NA of the pseudotype virus,were extracted normal PCR and real-time PCR were conducted. Results Sequence analysis showed that the five target genes in vitro synthesis were properly connected and inserted into lentivirus vectors.Using the NA of the pseudotype virus as the template,both normal PCR and real-time PCR could sensitively amplify the target gene with the primers and probes of the above five,viruses respectively.The result indicated that the pseudovirus particles containing the five kinds of hemorrhagic fever virus target genes were successfully packaged. Conclusion The pseudovirus particles containing gene fragments of five viruses are constructed,which can be used as a common reference standard for NA detection.