Study on relationship between expression level and molecular conformations of gene drugs targeting to hepatoma cells in vitro.
OBJECTIVE
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(+)/P(40) + 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.


CONCLUSIONS
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.
Yang
Dong-Ye
DY
Department of Gastroenterology, Xiangya Second Hospital, Central South University, Changsha 410011, Hunan, China.
Lu
Fang-Gen
FG
Tang
Xi-Xiang
XX
Zhao
Shui-Ping
SP
Ouyang
Chun-Hui
CH
Wu
Xiao-Ping
XP
Liu
Xiao-Wei
XW
Wu
Xiao-Ying
XY
D006528:Carcinoma, Hepatocellular / Q000628:therapy*; D018390:Gene Targeting; D015316:Genetic Therapy; D006801:Humans; D018664:Interleukin-12 / Q000737:chemistry* / Q000235:genetics*; D008113:Liver Neoplasms / Q000628:therapy*; D008968:Molecular Conformation
D018664:Interleukin-12