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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 prepare lentiviral vector containing Marburg virus, Zaire Ebola virus, Sudan Ebola virus, Lassa fever virus ) And yellow fever virus (5) virulent virus nucleic acid fragments, providing a universal positive reference for nucleic acid detection. Methods The target gene fragments of these five viruses were synthesized in vitro. Five fragments were ligated into a single gene by fusion PCR and cloned into a lentiviral vector. The 293T cells were cotransfected with its helper vector. After transfection, After 72 h, the supernatant was harvested, digested with DNase and RNase, purified and concentrated. Finally, nucleic acid was extracted, and PCR and real-time PCR were used to identify whether the pseudovirus was packaged successfully. Results The sequencing results showed that the target gene fragments of these five viruses synthesized in vitro were correctly ligated and inserted into the lentiviral vector. After the 293T cells were transfected with the vector, the pseudovirions in the supernatant were collected and the nucleic acids extracted were identified by the above two PCR methods Specific detection of the target gene indicates that the pseudovirions containing the target genes for the five hemorrhagic fever viruses have been successfully packaged. Conclusions The pseudoviruses obtained in this study can be used as a universal positive reference for the detection of the five haemorrhagic fever-associated virulent nucleic acids.