靶向S区和C区基因的M1GS RNA核酶共同抑制HBV的表达

Inhibition of HBV replication by M1GS RNA ribozymes targeting the S and C regions of the HBV genome

目的:研究靶向HBVS区和C区基因的M1GSRNA核酶共同作用对HBV基因表达的影响.方法:选择HBVayw亚型S区基因294nt和C区基因2333nt为切割位点,以含有编码M1RNA的DNA序列的质粒pTK117为模板,通过PCR扩增得到M1GSRNA核酶的DNA模板,并将其克隆至真核表达载体pEGFP-C1得到重组质粒pEGFP-GSS和pEGFP-GSC.将2个重组质粒共转染HepG2.2.15细胞,转染后ELISA法测细胞培养液中的HBsAg和HBeAg,RT-PCR检测HBVmRNA.结果:成功构建了分别靶向HBVS区基因和C区基因的真核表达载体.共转染HepG2.2.15细胞后,HBsAg和HBeAg的表达分别被抑制了33.2%和39.1%,HBVCmRNA和SmRNA分别被抑制了32.5%和29.7%,而HepG2.2.15细胞的增殖无明显变化.结论:靶向HBVS区和C区基因的M1GSRNA核酶共同作用可特异性抑制HBVS区和C区基因的表达.

AIM： To construct two MIGS RNA ribozymes targeting the S region and C region of the HBV genome, and evaluate their inhibitory effect on HBV gene expression. METHODS： A 294-nt in the S region and a 2333-nt in the C region of HBV genome were selected as sites of cleavage. DNA templates for site-specific MIGS RNA ribozymes targeting the HBV genome were constructed by polymerase chain reaction （PCR） using plasmid pTK117 as template. Then, the DNA templates were cloned into the eukaryotic expression vector pEGFP-C1. The recombinant vectors pEGFP-GSS and pEGFP-GSC were co-transfected into HepG2.2.15 cells. HBsAg and HBeAg proteins were detected by enzyme linked immunosorbent assay （ELISA） and their mRNA levels were analyzed by RTPCR. RESULTS： The two ribozymes effectively inhibited the secretion of HBsAg and HBeAg in HepG2.2.15 cells, by 33.2% and 39.1%, respectively. RT-PCR results showed that HBV S and C mRNAs were markedly decreased by 29.7% and 32.5%, respectively. Expression of MIGS RNA ribozyme had no effect on the proliferation of HepG2.2.15 cells. CONCLUSION： These results demonstrate that vectors with site-specific MIGS RNA ribozymes targeting the S and C regions of HBV can inhibit HBV replication specificity in vitro.