Inducing expression of β-neural growth factor and effect on PC12 cells

doi:10.16098/j.issn.0529-1356.2016.04.007

Abstract

Abstract:

Objective To construct recombinant lentiviral vector carrying rat β-neural growth factor (β-NGF) gene by inducing expression of the Tet-on 3G tetracycline，and to observe its expression in HEK293FT cells and effects on PC12 cells. Methods Total RNA was extracted from rat brain as the template. By gene recombination technique，rat β-NGF gene was inserted into lentiviral vector as pLVX-TRE3G-IRES-β-NGF. The supernatant of virus including the recombinant vector of pLVX-TRE3G-IRES-β-NGF and pLVX-Tet3G was collected from packaging cells, respectively, and then cotransfected blank HEK293FT cells. The expression of β-NGF was induced with different doses of doxycycline (Dox) (divided into non-transfection group, 0, 100, 500 and 1000μg/L of Dox inducing group). Cells were collected and analyzed by Western blotting assay, 48 hours after. The supernatant was detected by ELISA assay for the secretion and used as conditional medium for PC12 cells. Results A band of β-NGF gene was about 726bp. After transfection, β-NGF protein was detected to express on 293FT cells and supernatant following the increase of Dox dose. The neuron-specific enolase (NSE) was expressed on PC12 cells after induced by conditional media.
Conclusion That theβ-NGF gene was be expressed successfully on HEK 293FT cells by Tet-on 3G induced expression system, and the protein has effected on inducing PC12 cells differentiation into neuron-like cells.

Key words: β-Nerve growth factor, PC12 cell, Doxycycline, ELISA, Western blotting, Rat

GAO Jie YAN Hui-wen LI Hong HUANG Yue HU Rong SU Min*. Inducing expression of β-neural growth factor and effect on PC12 cells[J]. AAS, 2016, 47(4): 476-481.

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