锤头状核酶在HepG2.2.15细胞内的抗HBV特性  

Antiviral Activity of a Hammerhead Ribozyme against HBV in HepG2.2.15 Cells

作　　者：冯英[1] 孔玉英[1] 汪垣[1] 祁国荣[1] 

机构地区：[1]中国科学院上海生命科学研究院生物化学与细胞生物学研究所,上海200031

出　　处：《生物化学与生物物理学报》2002年第2期204-208,共5页

基　　金：中国科学院"九五"基础性研究重大项目 (No .KJ95 1 B16 10 );国家重点基础研究发展规划项目 ( 973) (No.G19990 5 410 5 )~~

摘　　要：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 。The carboxy terminal arginine-rich domain of core protein is highly conserved among HBV subtypes, and plays important roles in the packaging of pregenomic RNA and viral replication. Therefore, the 3′highly conserved region of HBV C gene is an attractive target for antiviral therapy mediated by ribozymes. A hammerhead ribozyme RzC, targeting the above site, was designed; meanwhile, as controls, the disabled form of RzC, dRzC, was also designed. In order to investigate its antiviral effects in cultured cells, in vitro-transcribed ribozyme RzC, dRzC and ribozyme expression vectors pCRzC, pCdRzC were delivered, respectively, into HepG2.2.15 cells ( clonal cells derived from HepG2 cells that contain integrated HBVayw DNA). The preliminary results demonstrated that in vitro-transcribed ribozyme RzC had weak inhibition on HBV replication, possibly due to its quick degradation by RNases in cells, while the endogenously expressed ribozyme RzC showed significant inhibitory effect on HBV replication. In conclusion, the results suggested the possibility of the hammerhead ribozyme RzC to be used for the gene therapy of HBV infection.

关 键 词：锤头状核酶 HBVHepG2·2·15细胞 乙型肝炎病毒 

分 类 号：R373[医药卫生—病原生物学]