靶向S区和C区基因的M1GS RNA核酶共同抑制HBV的表达

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.

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

AIM:To construct two M1GS 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 M1GS RNA ribozymes targeting the HBV genome were constructed by polymerase chain reaction(PCR)using plasmid pTK117 astemplate.Then,the DNA templates were cloned into the eukaryotic expression vector pEGFP-C1.The recombinant vectors pEGFP-GSS and pEG-FP-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 RT-PCR.RESULTS:The two ribozymes effectively in-hibited the secretion of HBsAg and HBeAg in HepG2.2.15 cells,by 33.2% and 39.1%,respec-tively.RT-PCR results showed that HBV S and C mRNAs were markedly decreased by 29.7% and 32.5%,respectively.Expression of M1GS RNA ribozyme had no effect on the proliferation of HepG2.2.15 cells.CONCLUSION:These results demonstrate that vectors with site-specific M1GS RNA ribozymes targeting the S and C regions of HBV can inhibit HBV replication specificity in vitro.