一种高效逆转录病毒载体pSIV-1的构建及其功能的初步鉴定

构建高效的逆转录病毒载体pSIV-1,并对其功能进行初步鉴定。对pEGZ-Term、pLXSN、pGEX-5X-3等各类表达载体的调控元件进行分析后,运用分子生物学手段去除表达载体pEGZ-Term中的部分元件,定向插入启动子SV40、选择性标志G418、多克隆酶切位点等,从而获得一种新型逆转录病毒载体pSIV-1。运用新载体构建含人PD-L1基因片段的重组载体,并用脂质体转染法,将其转染至包装细胞系293T。收集含重组病毒的上清转染L929细胞,用含G418的培养液筛选,成活细胞经流式细胞仪检测到目的蛋白后,扩大培养,从而获得L929/PD-L1基因转染细胞。L929/PD-L1基因转染细胞与活化T细胞体外混合培养,观察PD-L1信号对T细胞活化后表达CD25、CD69的影响。结果,成功构建了pSIV-1逆转录病毒载体;酶切图谱分析与理论值一致;基因转染细胞能持续稳定高表达PD-L1,筛选时间较短,感染效率较高;PD-L1基因转染细胞能显著抑制T细胞的进一步活化。因此构建成一种新型高效逆转录病毒载体pSIV-1,为构建基因转染,并开展相关功能学研究奠定了良好的物质基础。

Construction of a high efficient retrovirus vector pSIV-1 and its preliminary identification of functions

A high efficient retrovirus vector pSIV-1 was constructed and its preliminary identification of functions was performed in the present study. After analyzing the components of several vectors, such as retrovirus vector pGEZ-Term, pLSN and pGEX-5X-3, a new retrovirus vector pSIV-1 was constructed using methods of molecular biology, in which 5 MCS sites were inserted into this new vector and the human PD-L1, PD-L2 and CD40 genes were cloned and inserted to make its further confirmation. The recombinant retrovirus vectors together with their two helper virus vectors were respectively co-transfected into package cells 293T in the context of liposome LipfectAMINE. Then, the supernatant of 293T cell cultures was used to infect L929 cell line, and this cell line stably expressed aimed proteins was selected in the presence of G418(500 μg/ml). The transgenic L929 cells were analyzed by flow cytometry(FCM) to confirm the expression of the target proteins, and CD25 and CD69 molecules on the activated T cells were detected after the activated T cells were incubated with the L929/PD-L1 cells. The experimental results showed that a high efficient retrovirus vector was successfully constructed and was consistent with the theoretical value as demonstrated by the enzymatic restriction map. The transgenic cells could stably express human PD-L1, but it took less time to select the positive cells in comparison with the parental vector pEGZ-Term. The PD-L1 transgenic cells could potently inhibit T cell activation in vitro. From the results of the above experiment, it is evident that the construction of the retrovirus vector pSIV-1 contributes to the further development of transgenic cells applied to the relative biological function study and to the monoclonal antibody preparation.