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目的完善已初步建立的以痘病毒为辅助病毒的慢病毒载体(LV)瞬时制备体系。方法通过同源重组质粒pZIPPY-NEO/GUS,将vTF7-3痘病毒D13L基因的ORF通过同源重组被新霉素基因(neo)以及可视性筛选基因(GUS)替代,制备D13L缺陷性痘病毒(vTF7-3-ΔD13L)。结果经RT-PCR鉴定,D13L基因被完全敲除。制备体系中将vTF7-3-ΔD13L代替vTF7-3后,制备体系培养上清经RT-PCR、Western blotting分别检测到慢病毒载体特异性RNA序列以及特征性p24蛋白的存在,提示系统可以正常制备出慢病毒载体颗粒,并进一步评价了慢病毒载体的感染性。此外,对此系统的动力学特征也进行了初步分析。结论 vTF7-3-ΔD13L制备成功,通过此系统可制备出没有复制性痘病毒污染的慢病毒载体,本研究为该系统的大规模应用奠定了客观基础。
Objective To perfect the lentiviral vector (LV) transient preparation system that has been initially established and using poxvirus as a helper virus. Methods The homologous recombination plasmid pZIPPY-NEO / GUS was used to replace ORF of vTF7-3 Poxvirus D13L by neo and visual screening (GUS) to prepare D13L-deficient pox Virus (vTF7-3-ΔD13L). Results The D13L gene was completely knocked out by RT-PCR. Preparation system will vTF7-3-ΔD13L instead of vTF7-3, the system culture supernatant by RT-PCR, Western blotting were detected lentiviral vector-specific RNA sequences and characteristic p24 protein, suggesting that the system can be normal preparation The lentiviral vector particles were generated and the infectivity of the lentiviral vector was further evaluated. In addition, the dynamic characteristics of this system have also been initially analyzed. Conclusion The successful preparation of vTF7-3-ΔD13L can be used to prepare lentiviral vector without replication of poxvirus. This study laid an objective foundation for the large-scale application of this system.