反义寡核苷酸体外抗丁型肝炎病毒的初步研究

INHIBITION OF HEPATITIS DELTA VIRUS GENOMIC RNA RIBOZYME BY ANTI-SENSE OLIGODEOXYNUCLEOTIDES

以质粒（ｓＳＶＬＤ３）为模板，通过聚合酶链反应（ＰＣＲ）扩增得到一条１３９ｂｐ的片段，它含有丁型肝炎病毒（ＨＤＶ）基因组ＲＮＡ中核酶（ｒｉｂｏｚｙｍｅ）区的ｃＤＮＡ，该核酶具有自身裂解功能，将上述片段插入到ｐＧＥＭ－３Ｚ中，经筛选、鉴定，得到一重组质粒（ｐＨＤＶ１０８），经测序发现有２个碱基变异，以该质粒为模板，通过Ｔ７ＲＮＡ聚合酶，转录出核酶的前体，并观察到其自身裂解产物，自裂率达７１％。针对核酶两个重要的单链区，设计并合成二条反义寡核背酸（ＡＳＯＮ），当在转录反应中同时加入ＡＳＯＮ后，核酶的自裂率下降均较明显，当ＡＳＯＮ浓度为１６ｕｍｏ１／Ｌ时，核酶自裂抑制率均在７０％以上。提示，ＡＳＯＮ可与核酶两个重要的单链区结合，从而抑制核酶的自裂活性。

in this rescarch,with PSVLD3 as template,a 139bp cDNA containing the region related tohepatitis delta virus (HDV)genomic RNA ribozyme,which is able to undergo autocatalytic cleavage,wasamplified by PCR.After PCR amplification,the products were inserted into PGEM-3Z,and a recombinantplasmin(pHDV 108)was obtained. Sequencing of the inserted DNA was carried out by the dideoxy chaintermination method, and two-base-mutation was found. With PHDV 108 as template, the precursor ofHDV genomic RNA ribozyme was obtained by in vitro transcription using T7 RNA polymerase,and itsself-cleavage rate was 71%. Two antisense oligodeoxynucleotides (ASON)targeting at two important sin-gle-stranded regions of the HDV genomic RNA ribozyme were designed and synthesized. The self-cleavagerates of the ribozyme were obviously reduced with the addition of ASON to the transcription reaction ,andthe inhibition rates of self-cleavage were over 70. 0% at a concentration of 16umol/L of ASON. The re-sults showed that ASON could bind to single-stranded region of HDV genomic RNA ribozyme ,resulting ininhibition of self-cleavage activities of the ribozyme.