一种新型慢病毒载体制备体系的改进

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。

Improvement of a new poxviral/lentiviral hybrid system for efficient lentiviral vector production

Objective To improve the initially established poxviral/lentiviral hybrid system for efficient lentiviral vector production.Methods D13L-deleted vaccinia recombinant （vTF7-3-ΔD13L）was constructed for preventing the contamination of the infectious vaccinia virus through the homologous recombination plasmid pZIPPY-NEO/GUS.The vTF7-3 D13L ORF was deleted by homologous recombination of the neomycin gene （neo） and the beta-glucuronidase（GUS）.Results Results from RT-PCR showed that the D13L gene was completely knocked out as judged by the results of RT-PCR and the Western blot.Lentiviral vectors were found in the culture supernatant when vTF7-3 was replaced by the vTF7-3-ΔD13L,as judged by immunofluorescence microscopy.LoVo and SW620 cells lines were transfected with the supernatant.Results confirmed the infectivity of the lentiviral vector produced by the help of the vTF7-3-ΔD13L.The characteristics of this system were also evaluated.The best titer of the lentiviral vector stocks was 1.8×108 tu/ml,which is better than the output by the vTF7-3.Conclusion The replication defective vaccinia virus vTF7-3-ΔD13L was successfully established.It may be used for the large scale application of this production system.