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.