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构建针对乙型肝炎病毒（ＨＢＶ）Ｃ区基因（２０２９－３１及２０６３－６５位点）的双位点核酶的真核表达载体，以观察核酶在细胞内对ＨＢＶ基因表达的抑制作用。利用亚克隆技术，从质粒ｐＧＥＭＲｚ１２３切下ＥｃｏＲⅠ－ＢａｍＨⅠ片段，双粘端克隆于真核质粒ｐＢＢＳ２１２中，将该质粒与ｐ１．２Ⅱ（含ＨＢＶ全序列）共转染人肝癌细胞（ＨＨＣＣ）细胞株。结果：①在ＨＨＣＣ细胞中ｐ１．２Ⅱ可表达出ＨＢｓＡｇ及ＨＢｅＡｇ。②该核酶对ＨＢｅＡｇ的合成抑制率达６５％，而对ＨＢｓＡｇ无抑制作用。结论：该双位点核酶可能通过针对ＨＢＶＣ区基因的剪切作用，阻断Ｃ区基因的表达，抑制ＨＢｅＡｇ的合成。
To construct an eukaryotic expression vector targeting the double-site ribozyme of hepatitis B virus (HBV) C-region gene (2029-31 and 2063-65) to observe the inhibitory effect of ribozyme on HBV gene expression in the cell. The EcoRⅠ-BamHⅠ fragment was excised from the plasmid pGEMRz123 by subcloning technique. The double-stick was cloned into the eukaryotic plasmid pBBS212. The plasmid was co-transfected with the p1.2Ⅱ (complete sequence containing HBV) into the human hepatocellular carcinoma cell line HHCC . Results: ① In HHCC cells, p1.2Ⅱ could express HBsAg and HBeAg. ② The ribozyme on HBeAg synthesis inhibition rate of 65%, while no inhibition of HBsAg. Conclusion: The double-site ribozyme may block the expression of C region gene and inhibit the synthesis of HBeAg by targeting shearing of HBV gene.