合成寡核苷酸对病毒目标序列特异结合反应及其影响因素研究

Specially binding of synthesized oligonucleotide to target viral gene sequences and the influencing factors

目的 观察人工合成的反义寡核苷酸 (ASON)、三螺旋结构寡核苷酸 (TFO)分别对人甲肝病毒 (HAV) 5′非编码区和鸭乙肝病毒 (DHBV) DNAX C区目标基因序列特异结合效应及受影响因素作用的变化。方法 在设定条件下 ,将人工合成的ASON和TFO与目标基因片断混合反应 ,采用电泳转移印迹技术观察特异结合产物及其产量。结果 所设计的ASON与TFO能与目标基因片段直接特异结合 ;在结合缓冲液中 ,ASON与目标片段的结合最佳比例是 2 5～ 5 0∶1,TFO与目标片段的结合最佳比例是 60 0∶1;在最适加入剂量比例下 ,结合反应 60min即有大量TFO的复合物形成 ,复合物产量随作用时间延长而增加 ,180～ 2 40min时结合产物完全稳定形成。但ASON的结合复合物大量形成需要 180～ 2 40min。结论 本试验所设计的ASON和TFO能与HAV和DHBV的目标基因序列发生特异的结合 。

Objective To observe the reaction and effects of artificial synthesized antisense oligonucleotide (ASON), triple helix forming oligonucleotide (TFO) specially binding to the target gene sequences from 5' nontranslated region of human hepatitis A RNA and X/C region of duck hepatitis B DNA. Methods Under designed condition, the artificial synthesized ASON and TFO were mixed with objective gene sequences, and then the outcomes of special linking were detected with gel shift blot assay. Results The designed ASON and TFO combined to the target genes directly. The best proportion of ASON and its target gene was about 25-50:1, and the TFO and its target gene 600:1. On suitable proportion conditions, the complex products were increased with the reaction holding time. After holding time was about 60 min, most of the TFO complex formed, and 180-240 min later the TFO complex formed completely. For ASON,most its complex also needed 180-240 min to forming. Conclusion The designed ASON and TFO could specially combine to objective genes of HAV and DHBV respectively. The added doses of artificial synthesized oligonucleotide, the reaction holding times could affect the production of special complex.