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乙型肝炎病毒 (HBV)C基因区的 3′端序列在HBV各亚型中是高度保守的 ,它编码的多肽链都富含精氨酸 ,同时与前基因组RNA的包装和病毒DNA的复制有关。因此 ,HBV中C基因的 3′端保守区是核酶介导的抗病毒研究的理想靶位点。针对上述位点设计了锤头状核酶RzC ,同时作为对照 ,设计了相应的突变型核酶dRzC。HepG2 .2 .15细胞系是由头尾相接的HBVayw亚型基因组转染肝癌细胞系HepG2而建立的一株能稳定分泌HBV各种抗原和完整病毒颗粒的细胞系。因而用HepG2 .2 .15细胞进行核酶的抗病毒研究更接近于疾病的治疗过程。为此 ,把体外转录的核酶RzC和dRzC以及核酶表达载体pCRzC和 pCdRzC直接转染HepG2 .2 .15细胞。初步结果表明 ,体外转录的核酶RzC对HBV复制的抑制很弱 ,这可能是由于核酶在细胞内快速被RNA酶降解造成的 ;而通过内源性传递产生的核酶RzC能够明显地抑制HBV的复制。结果说明 ,锤头状核酶RzC在HepG2 .2 .15细胞内显示抗病毒感染的能力 ,有可能作为HBV基因治疗的一种手段
The 3’end of the HBV C gene region is highly conserved among HBV subtypes and encodes a polypeptide chain that is both arginine-rich, with both pre-genomic RNA packaging and viral DNA replication related. Therefore, the 3 ’conserved region of the C gene in HBV is an ideal target site for ribozyme mediated antiviral studies. The hammerhead-like ribozyme RzC was designed for the above sites, and at the same time as a control, the corresponding mutant ribozyme dRzC was designed. The HepG2.2.15 cell line is a cell line that can stably secrete various antigens of HBV and complete virus particles by transfection of Hepatitis A cell line HepG2 with the head-to-tail genome of HBVayw subtype. Therefore, the antiviral study of ribozymes using HepG2.2.15 cells is much closer to the treatment of the disease. To this end, in vitro transcription of ribozymes RzC and dRzC and ribozyme expression vectors pCRzC and pCdRzC HepG2 .15 cells directly transfected. Preliminary results showed that RzC, a transcriptional ribozyme in vitro, showed a weak inhibitory effect on HBV replication, which may be due to the ribozyme being rapidly degraded by RNase in the cell; and the ribozyme RzC produced by endogenous delivery can obviously inhibit HBV replication. The results show that the hammerhead ribozyme RzC in HepG2.2.15 cells showed the ability of antiviral infection may be used as a means of HBV gene therapy