Yang Dong-Ye, Lu Fang-Gen, Tang Xi-Xiang, Zhao Shui-Ping, Ouyang Chun-Hui, Wu Xiao-Ping, Liu Xiao-Wei, Wu Xiao-Ying
Department of Gastroenterology, Xiangya Second Hospital, Central South University, Changsha 410011, Hunan, China.
World J Gastroenterol. 2003 Sep;9(9):1954-8. doi: 10.3748/wjg.v9.i9.1954.
To increase exogenous gene expression level by modulating molecular conformations of targeting gene drugs.
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.
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.
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.
通过调节靶向基因药物的分子构象来提高外源基因表达水平。
通过聚合酶链反应（PCR）扩增人白细胞介素12的P(40)和P(35)亚基的全长cDNA，并克隆到真核表达载体pcDNA3.1(+/-)中，构建P(+)/IL-12、P(+)/P(40)和P(-)/P(35)质粒。这些质粒与ASOR-PLL以最佳比例组合形成两种靶向基因药物[ASOR-PLL-P(+)/IL-12和ASOR-PLL-P(+)/P(40)+ASOR-PLL-P(-)/P(35)]。在电子显微镜（EM）下检查这两种药物在不同浓度佐剂下的构象，并将药物转染到HepG2（ASGr+）细胞中。用从转染细胞中提取的总RNA进行半定量逆转录聚合酶链反应（RT-PCR），以确定hIL12 mRNA转录水平。转染后48小时，用酶联免疫吸附测定（ELISA）测量培养上清液中的hIL12蛋白。
结构为颗粒状和环状、直径范围为25nm至150nm的靶向基因药物具有最高的hIL-12表达水平。共转染ASOR-PLL-P(+)/P(40)和ASOR-PLL-P(-)/P(35)的组中的hIL-12表达水平高于ASOR-PLL-P(+)/IL-12转染组。
靶向基因药物的分子构象在外源基因表达水平中起重要作用，最佳结构为颗粒状和环状，其直径范围为25nm至150nm。外源基因的大小和连接方式对其表达水平也有一定影响。
