针对包装信号起始区的硫代反义核苷酸抗HBV的研究.doc

???? 针对包装信号起始区的硫代反义?核苷酸抗HBV的研究 ???? ????【 摘要 】　目的　筛选高效特异的抗HBV反义核酸药物。 方法　针对HBV包装信号ε起始区设计并合成硫代反义寡聚核苷酸(s-asODN)片段，通过ELISA检测法、MTT法、电子显微镜等观察，研究此s-asODN对HBsAg、HBeAg和HBcAg表达，以及对性，细胞形态的影响。 结果　针对ε起始区的s-asODN显著抑制HBsAg、HBeAg和HBcAg的表达，其中，对HBeAg和HBcAg的抑制率分别为38.1%和58.7%高于S基因起始区(32.1%和37.2%,P＜0.01),对HBsAg抑制率为82%，低于S基因起始区(93.41%)，在实验浓度下s-asODN对细胞无毒性，对细胞形态无影响。 结论　HBV包装信号区是反义核酸抗HBV复制研究的重要的靶序列选择区域。　　【 主题词 】　肝炎病毒，乙型　　包装信号　　核苷酸类，反义 Study on the anti-HBV activity of antisense phosphorothioate oligodeoxynucleotide of gene ε initiator　Han Jinxiang,Wang Yulin,Zhang Cui,et al.Shandong Medical Biotechnology Centre,Ji?nan 250062 　　【 Abstract 】　Objective　To explore a new specific anti-HBV agent. Methods　According to the sequence of HBV genome,an antisense phosphorothioate oligodeoxynucleotide (s-asODN) of gene ε initiator was designed and synthesized.The inhibition of HBsAg,HBeAg and HBcAg expression and cytotoxicity and cellular morphology also were studied with ELISA,MTT colorimetric assay and scanning electron microscopy. Results　s-asODN of gene ε could inhibit expression of HBsAg,HBeAg and HBcAg.Both the inhibition rates (38.1% and 58.7%) of HBeAg and HBcAg were higher than gene S initiator (32.1% and 37.2%,P＜0.01) and the inhibition rate (82.0%) of HBsAg was lower than gene S initiator (93,4%),s-asODN was apparently not cytotoxic and induced no effect of morphology on HepG2 2215 cells. Conclusion　The gene ε is important target gene for the antisense oligonucleotide anti-HBV replication.　　【 Subject words 】　Gene ε　　Oligodeoxynucleotides,antisense 　　Hepatitis B virus 　　HBV包装是HBV复制过程中的重要环节，起始于C基因两个起始密码ATG间的3.5kb转录体能被病毒核心颗粒所包装，是前基因组RNA(PgRNA)这一高度特异的包装过程，它主要取决于PgRNA近5?端的一段顺式序列，即包装信号ε 。研究发现 ε不仅在PgRNA选择性包装过程中起决定性作用，并且与基因组逆转录有密切关系〔1〕。因此该区是理想的基因水平治疗HBV的靶序列，通过设计合成针对ε起始区的硫代反义寡聚核苷酸，我们首次研究了其对HBV细胞的HBsAg、HBeAg和HBcAg表达的影响，并对其细胞毒性及形态进行了研究，以期筛选高效特异的抗HBV反义核酸药物。 材料与方法　　1.细胞株和培养基：2.2.15细胞为HBV-DNA转染的HepG2细胞，由中国医科院医药生物技术研究所提供，Eagle?s MEM培养基、胎牛血清、G418为美国GIBCO公司产品。　　2.s-asODN的设计合成及纯化：参照Ono Y等〔2〕克隆的HBV