preS2反义RNA肝癌特异性表达载体的构建及相关特性研究

Construction of hepatocellular specific preS2 antisense RNA expression vector and study of its inhibitory effect

目的 构建针对preS2基因的肝癌特异性反义RNA表达载体 ,并对其特异性和有效性进行研究。方法 PCR扩增preS2 ( 32 0 3～ 340 )基因 ,克隆至含甲胎蛋白启动子的EB病毒表达载体pEBAF ,构建反义RNA表达载体pEBAF as preS2。脂质体转染肝癌细胞和ECV30 4细胞 ,3d后Northernblot检测preS2mRNA的表达 ,ELISA检测HBV抗原 ,荧光定量PCR检测HBVDNA。结果 序列分析表明 ,pEBAF as preS2构建成功 ,Northernblot证实反义RNA仅在AFP阳性的肝癌细胞中表达 ,pEBAF as preS2转染 3d后 ,可显著抑制HepG2 .2 .15细胞HBV复制和抗原表达 ,对HBsAg、HBeAg表达的抑制率分别为 33 .4%和 41.5 %;对HBV复制抑制率为 86 %。结论 反义RNA表达载体pEBAF as preS2仅在肝癌细胞中特异表达、并可有效抑制HBV ,有良好的开发应用前景。

Objective To obtain recombinant preS2 antisense RNA expression vector targeted to hepatocellular carcinoma. Methods preS2 gene (3?203-340) was obtained by PCR and then cloned into EB virus vector pEBAF containing human α-fetoprotein promoter and enhancer. After transfection by lipofectin, RNA dot blot, ELISA and real-time qualitative PCR were used to assay the expression preS2 mRNA, HBV antigens and HBV DNA respectively. Results The recombinant pEBAF-as-preS2 which can be expressed specifically in hepatocellular carcinoma cells was successfully obtained. Both HBV DNA and HBV antigens of hepatocellular cell line HepG2.2.15 were inhibited by transfection by pEBAF-as-preS2. The inhibition rate of HBsAg, HBeAg and HBV DNA were 33.4%, 41.5% and 86% respectively. Conclusion pEBAF-as-preS2 expression vector may lead to targeted-expression of preS2 antisense RNA on hepatoma cells and inhibit HBV multiplication. [