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目的高效包装不同启动子的重组慢病毒(LV),建立LV的定量方法。方法采用分子克隆方法获得p FUGW、p TY-EF1α-EGFP及p TY-CMV-EGFP 3种转移质粒,然后采用脂质体法与另两种包装质粒p SPAX2及p MD2.G共同转染293T细胞,制备含有不同启动子的LV。设计针对3’-LTR的探针和引物,建立LV的荧光定量PCR定量方法。结果获得了不同启动子的转移质粒,制备了3种不同启动子的LV,应用荧光定量PCR方法测定制备的病毒,载量能达到109copies/ml。结论成功构建了不同启动子的转移质粒,获得了三种LV,建立了针对LV的荧光定量PCR方法,为后续研究不同启动子在多种细胞系中驱动目的蛋白的表达奠定了基础。
Objective To efficiently package recombinant lentivirus (LV) with different promoters and establish a quantitative method of LV. Methods Three transfer plasmids, p FUGW, p TY-EF1α-EGFP and p TY-CMV-EGFP, were obtained by molecular cloning. The recombinant plasmids were co-transfected with 293T Cells, LVs containing different promoters were prepared. Probes and primers targeting 3’-LTR were designed and used to establish quantitative real-time quantitative PCR for LV. Results The transfer plasmids of different promoters were obtained. Three LVs with different promoters were prepared. The prepared virus was quantified by fluorescence quantitative PCR, and the loading capacity could reach 109 copies / ml. CONCLUSION: Transfer plasmids with different promoters were successfully constructed and three kinds of LVs were obtained. Fluorescent quantitative PCR for LV was established, which laid the foundation for subsequent study on the expression of different promoters in various cell lines.