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 throughpo...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.展开更多
基金the National Natural Science Foundation of China, No.39570355Hunan Health Bureau Foundation,No.Y02-038
文摘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.
