Study on relationship between expression level and molecular conformations of gene drugs targeting to hepatoma cells in vitro

AIM: To increase exogenous gene expression level bymodulating molecular conformations of targeting gene drugs.METHODS: The full length cDNAs of both P40 and P35subunits of human interleukin 12 were amplified throughpolymerase chain reaction (PCR) and cloned into eukaryoticexpressing vectors pcDNA3.1(±) to construct plasmids of P(+)/IL-12, P(+)/P40 and P(-)/P35. These plasmids werecombined with ASOR-PLL to form two targeting gene drugs[ASOR-PLL-P(+)/IL-12 and ASOR-PLL-P(+)/P40 + ASOR-PLL-P(-)/P35] in optimal ratios. The conformations of these twodrugs at various concentrations adjuvant were examined underelectron microscope (EM) and the drugs were transfected intoHepG2 (ASGr+) cells. Semi-quantitative reverse transcriptionpolymerase chain reaction (RT-PCR) was performed withtotal RNA extracted from the transfected cells to determinethe hiL12 mRNA transcript level. The hiL12 protein in thecultured supernatant was measured with enzyme-linkedimmunosorbent assay (ELISA) 48 hours after transfection.RESULTS: Targeting gene drugs, whose structures weregranular and circle-like and diameters ranged from 25 nmto 150 nm, had the highest hIL-12 expression level. ThehIL-12 expression level in the group co-transfected withASOR-PLL-P(+)/P4o and ASOR-PLL-P(-)/P35 was higher thanthat of ASOR-PLL-P(+)/IL-12 transfected group.CONCLUSION: The molecular conformations of targetinggene drugs play an important role in exogenous geneexpression level, the best structures are granular and circle-like and their diameters range from 25 nm to 150 nm. Thesizes and linking styles of exogenous genes also have someeffects on their expression level.