小鼠IL-10重组慢病毒载体的构建和病毒包装

Construction of mouse IL-10 recombinanted lentiviral vector and virus packaging

目的:构建小鼠IL-10重组慢病毒表达载体并进行包装,为进一步研究IL-10基因修饰树突细胞在哮喘免疫耐受中的作用提供实验基础。方法:小鼠IL-10基因片段经PCR扩增后,与酶切线性化的慢病毒载体进行定向连接,获得PGC-LV-IL-10重组慢病毒并转化细菌感受态细胞。克隆菌落行PCR鉴定,对阳性的重组质粒进行测序并转入293T细胞,荧光显微镜下观察GFP表达并行Western blot鉴定。将重组质粒与慢病毒包装系统一同转染293T细胞进行病毒包装,Real-time定量PCR法检测病毒滴度。结果:DNA测序结果及Western blot鉴定证实成功构建小鼠IL-10重组慢病毒载体,对其进行包装后测定慢病毒滴度为2×108TU/ml。结论:成功构建并包装小鼠IL-10重组慢病毒表达载体。

Objective：To construct a lentiviral expressing vector carrying mouse interleukln-10 gene and then being packaged, in order to provide an experimental foundation for the immunological tolerance of IL-l0 gent modified dendritic cells on astluna. Methods：The mouse IL-1O gene fragment was amplified through PIER, then was connected with PGC-LV lentiviral vector which had been digested and linearized. The resulting lentiviral vector was named PGC-LV-IL-l0, and it was converted by bacterial competent cells. The cloning bacteria was identified by PCR, then the positive plasmids were detected by DNA sequencing and transfected into 293T cells. The expression of GFP were observed through fluorescence nficmscope and Western blot. 293T cells were contransfected with lentiviral vector PGC-LV-IL-10 and lentiviral packaging systems. The titles of concentrated lentivirus was tested by Real-time q-PCR. Results： DNA sequencing and Western blot results demonstrated that the IL-10 recombinanted lentiviral vector was constructed successfully. The titles of concentrated lentiviras was 2 x l0^8 TU/ml. Conclusion： The mouse IL-10 recombinanted lentiviral vector was successfully constructed and packaged.