Modulation of the molecular conformation of a hepatocyte-targeting gene drug in order to improve its expression efficiency in vitro.

摘要

AIM: To construct different conformations of a plasmid DNA/vector complex (pcDNA3.1/IFN-gamma-ASOR-PLL) and transfect cells of the hepatoma cell line BEL7402 to investigate the optimal conformation of the complex for improved expression efficiency in the target cell. METHODS: Double-distilled water and adjuvant were added to the naked pcDNA3.1/IFN-gamma, target vector ASOR-PLL and the ASOR-PLL-pcDNA3.1/IFN-gamma complex to create different conformations; molecules that were transfected into BEL7402 cells and the expression efficiency was determined by measuring the IFN-g concentration in the culture supernatant by ELISA. RESULTS: Naked pcDNA3.1/IFN-gamma DNA distributed linearly in double-distilled water and condensed into a mica configuration in adjuvant; ASOR-PLL had a net-like distribution without adjuvant and a spider-like form in the adjuvant-treated group; the ASOR-PLL-pcDNA3.1/IFN-g complex had a divaricate form without adjuvant, but a bead-like or granular conformation in 0.1 and 0.2 mol/L of adjuvant, a homogeneous bacilliform or chromatoid-shaped conformation in 0.3 mol/L adjuvant, and varied shapes in 0.4 and 0.5 mol/L adjuvant. The supernatant IFN-gamma expression in the bacilliform/chromatoid conformation complex group was the highest among the different conformation groups and controls. When chloroquine was added the supernatant IFN-gamma concentration increased in the liposome group and decreased in the bacilliform/chromatoid conformation group . CONCLUSIONS: The two structural molecules and their complex, ASOR-PLL-pcDNA3.1/IFN-gamma, were adjustable in the liquid mode. The specific bacilliform/chromatoid conformation of complex was lysosome enzyme-resistant and could play an active role in improving the efficiency of gene expression. The hypothesis that a chromosome-like conformation of the target gene molecule is involved in enhancing exogenous gene expression is proposed.