Construction And Stable Expression For Eukaryotic Expression Vector Of IL-7 Gene

Interleukin-7 (IL-7) is a kind of cytokines mainly produced by thymic stromal cells, in addition, fibroblasts, fetal liver cells, bone marrow stromal cells and other cells can also produce and secrete it. IL-7 plays an important role in the growth, survival and differentiation of T cells, there is a close relationship between the occurrence, development and treatment of many malignant diseases, such as cancer, HIV, leukemia and so on. It is a new therapeutic agent which has a broad application prospects in vivo. In this study, the eukaryotic cell line producing IL-7 was established, and its secretory expression was realized. It laid the foundation for the development of genetic enginered IL-7 and its related researches.The lentiviral vector pLV-CMV-EF1-GP-IL7 and eukaryotic expression vector pcDNA3.1-IL7 were constructed separately based on the nucleotide sequence of IL-7 in NCBI database. They were successfully expressed in CH0-K1 and HEK293 cells.The lentiviral vector pLV-CMV-EF1-GP-IL7 and packaged plasmids VSVG, PLP, PLP1, PLP2 were co-transfected into HEK-293T cells for the preparation of recombinant human IL-7 lentivirus. The virus titer was 3.0×108 (TU/mL).CHO-K1 and HEK293 cells were infected by the recombinant lentivirus, the infection efficiency was determined by the number of fluorescent cells. It was found that IL-7 protein was secretory expressed. The contents of IL-7 in the supernatant after 48h were 0.6 ng/mL and 0.893 ng/mL.The expressed vector pcDNA3.1-IL7 and control vector pcDNA3.1-EGFP were transfected into CHO-K1 and HEK293 cells, respectively. The efficiency of cell transfection was determined by the number of fluorescent cells. By the cell doning,2 cell lines with high expression were obtained, named CHO-K1-IL7-1E4 and HEK293-IL7-2G3. IL-7 protein was secretory expressed. The contents of IL-7 in the supernatant after 48h were 0.743ng/mL and 3.2ng/mL.In this study, IL-7 was secreted expression, the highest expression level was 3.2 ng/mL. However, the protein expression was relatively low. The experimental methods and cell culture conditions should be further optimized to improve the expression level of IL-7 protein.