【摘要】 目的 体外研究核酶对乙型肝炎病毒(HBV)前S2基因的作用。方法 设计合成一针对HBVayw DNA前S2区第110，122和132位碱基的三靶位串联锤头状核酶基因，体外转录核酶基因和靶基因并进行切割实验；将核酶基因与逆转录病毒重组，转导2.2.15细胞，观察转导细胞聚人血清白蛋白受体(pHSA-R)的表达水平。结果 核酶在体外可有效切割靶基因转录物，在转入2.2.15细胞后4周内对pHSA-R表达抑制率为51.45±4.57%～41.01±4.16%。结论 本文设计合成的针对乙型肝炎病毒前S2基因的核酶在体外具有相应的生物学活性。

    Synthesis and preliminary study on cleavage activity of ribozyme to hepatitis B virus preS2 gene in vitro Xu Dongping,Han Fenglian,Shi Hong,et al.Biological Engineering Laboratory of 302 Hospital,Beijing 100039

    Abstract In this paper,a multi-target hammerhead ribozyme gene was synthesized directed against 110,122 and 132 sites of nucleotide of HBV preS2 gene.The target gene fragment was cut from HBV genome containing plasmid pCP10.Both of the ribozyme and the target gene fragments were cloned into pGEM3Zf(-)plasmid and sequenced by dideoxy chain termination method.The transcription of both gene fragments was performed in vitro utilizing T7 RNA promoter in pGEM3Zf(-)plasmid.The cleavage activity of ribozyme to substrate was confirmed in vitro. For further evaluating intracellular function of ribozyme,two ribozyme-retroviral recombinant plasmids with different promoter type pDOR-ripc and tRNA-ripc were constructed.Pseudo-virus was collected through routine packaging procedure and transduted into 2.2.15 cells.RIA data showed a stable inhibition of pHSA-R antigen expression to the lowest extent of 41.01%±4.16 and the highest extent of 51.45%±4.57 within 4 weeks after transduction.No influence,however,on HBsAg and HBeAg expression was demonstrated after ribozyme gene transfer.

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