Ma Chun-hong, Sun Wen-sheng, Liu Su-xia, Wang Xiao-yan, Zhang Li-ning, Cao Ying-lin, Han Li-hui
Institute of Immunology, Medical School of Shandong University, Jinan 250012, China.
Zhonghua Gan Zang Bing Za Zhi. 2003 May;11(5):291-4.
To study the specific expression of the antisense RNA against hepatitis B virus X (HBX) gene in hepatoblastoma cell line and its anti -HBV activity.
HBX gene (nt.1370-1827) was amplified by PCR, then cloned into EB virus vector pEBAF which contained human alpha-fetoprotein promoter and enhancer. After transfected into 2.2.15 hepatoma cells and ECV304 human endothelial cells by lipofectin, northern blot, ELISA and real-time qualitative PCR were carried out to assay the expression of HBX mRNA, HBV antigens and HBV DNA level, respectively.
The HBX antisense RNA expression vector pEBAF-as-HBX which could be expressed specifically in 2.2.15 hepatoblastoma cells was successfully constructed. Both HBV DNA level and the expressions of hepatitis B virus surface antigen (HBsAg) and e antigen (HBeAg) in 2.2.15 hepatoblastoma cells were inhibited by pEBAF-as-HBX. Compared with those in sense control (pEBAF-s-HBX), the inhibitory rates of HBsAg, HBeAg, and HBV DNA were 37.9%, 36.8%, and 25%, respectively.
The pEBAF-as-HBX expression vector may lead to targeted-expression of HBX antisense RNA in hepatoma cells and shows great inhibition effect on HBV.
研究针对乙型肝炎病毒X（HBX）基因的反义RNA在肝母细胞瘤细胞系中的特异性表达及其抗HBV活性。
通过PCR扩增HBX基因（核苷酸1370 - 1827），然后克隆到含有人类甲胎蛋白启动子和增强子的EB病毒载体pEBAF中。用脂质体转染2.2.15肝癌细胞和ECV304人内皮细胞后，分别进行Northern印迹、ELISA和实时定量PCR检测HBX mRNA的表达、HBV抗原及HBV DNA水平。
成功构建了能在2.2.15肝母细胞瘤细胞中特异性表达的HBX反义RNA表达载体pEBAF-as-HBX。pEBAF-as-HBX抑制了2.2.15肝母细胞瘤细胞中的HBV DNA水平以及乙型肝炎病毒表面抗原（HBsAg）和e抗原（HBeAg）的表达。与正义对照（pEBAF-s-HBX）相比，HBsAg、HBeAg和HBV DNA的抑制率分别为37.9%、36.8%和25%。
pEBAF-as-HBX表达载体可使HBX反义RNA在肝癌细胞中靶向表达，并对HBV显示出强大的抑制作用。
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