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.

AIM:To increase exogenous gene expression level by modulating molecular conformations of targeting gene drugs. METHODS:The full length cDNAs of both P_(40)and P_(35) subunits of human interleukin 12 were amplified through polymerase chain reaction(PCR)and cloned into eukaryotic expressing vectors pcDNA3.1(±)to construct plasmids of P (+)/IL-12,P(+)/P_(40)and P(-)/P_(35).These plasmids were combined with ASOR-PLL to form two targeting gene drugs [ASOR-PLL-P(+)/IL-12 and ASOR-PLL-P(+)/P40+ASOR-PLL- P(-)/P_(35)] in optimal ratios.The conformations of these two drugs at various concentrations adjuvant were examined under electron microscope(EM)and the drugs were transfected into HepG2(ASGr+)cells.Semi-quantitative reverse transcription polymerase chain reaction(RT-PCR)was performed with total RNA extracted from the transfected cells to determine the hiL12 mRNA transcript level.The hiL12 protein in the cultured supernatant was measured with enzyme-linked immunosorbent assay(ELISA)48 hours after transfection. RESULTS:Targeting gene drugs,whose structures were granular and circle-like and diameters ranged from 25 nm to 150 nm,had the highest hIL-12 expression level.The hIL-12 expression level in the group co-transfected with ASOR-PLL-P(+)/P_(40)and ASOR-PLL-P(-)/P_(35)was higher than that of ASOR-PLL-P(+)/IL-12 transfected group. CONCLUSION:The molecular conformations of targeting gene drugs play an important role in exogenous gene expression level,the best structures are granular and circle- like and their diameters range from 25 nm to 150 nm.The sizes and linking styles of exogenous genes also have some effects on their expression level.