摘要
目的 观察病毒基因组 5′端非编码区内与翻译起始有关的位点和结构蛋白编码区的特异性反义核酸对病毒蛋白表达的影响及其量效关系。方法 应用病毒致细胞病变作用保护实验、空斑形成实验和空斑形成减少实验、Westernblot实验等方法 ,观察特定的反义核酸抑制病毒感染的效果。结果 针对IRES位点、AUG区域和VP1区的 3条反义核酸Scb5 6 1、Scb733、Scb2 785 ,对病毒感染有明显的抑制作用。病毒的感染量为 0 0 1MOI时 ,3种反义核酸在 5 μmol L时对病毒的抑制率均在90 %以上 ,当病毒增加到 10MOI时 ,抑制率仍在 5 0 %以上。Scb5 6 1和Scb733可明显的抑制病毒基因表达 ,当Scb5 6 1的浓度在 0 6 2 5 μmol L时感染后的细胞内病毒蛋白量明显减少 ,增加到 2 5 μmol L时病毒蛋白表达几乎看不到。另外Scb5 6 1、Scb733剂量与抗病毒效果呈现正相关关系。随着Scb5 6 1和Scb733浓度的增加 ,其抗病毒活性也随之增加直到抑制率达到 90 %以上 ,有效剂量在 0 6～ 5 μmol L之间 ,且对细胞无毒性作用。非特异寡聚核苷酸对照实验显示 ,5 μmol L浓度时对病毒感染无明显抑制作用。结论 针对核糖体进入位点和翻译起始位点的反义核酸 ,有明显的特异性抑制病毒基因表达的作用。
Objective In this study,the authors investigated inhibition of coxsackievirus B (CVB) gene expression using antisense oligonucleotides complementary to the 5' NCR,translation initiation codon and structural protein coding sequences and also observed the dose-response of the sequence specific inhibition of CVB plaque formation by antisense oligonucleotides. Methods Antiviral activities of these oligonucleotides were evaluated by using plaque reduction assay,yield reduction assay,cytopathic effect (CPE). and Western blot analysis. The cells were treated with random oligonucleotides as a specificity control. Results At a screening concentration of 5 micromole,6 of the phosphorothioate oligonucleotide demonstrated some reduction of virus replication relative to untreated cells. 70%-90% inhibition of virus at 0.1 MOI (multiplicity of infection),50% inhibition of virus infection at 10 MOI. The levels of the VP1 were reduced in CVB-infected cells treated with Scb561 and Scb733. VP1 was significantly reduced after treatment with 0.625 micromole Scb561 and almost undetectable in cells treated with 2.5 micromole Scb561. Dose-response experiments implied that sequence specific oligonucleotide doses were related to effect on inhibintion of CVB3 infection. When oligonucleotide doses were increased from 1.25 to 5 micromole,75% to 90% inhibition were observed with Scb561 and 65% to 80% inhibition with Scb733, whereas random control failed to inhibit CVB replication (<8% inhibition for each). Conclusion The present studies showed that antisense oligonucleotides against internal ribosome entry site (IRES) and translation initiation codon were capable of specifically inhibiting the synthesis of viral protein and subsequent productive CVB replication.The selective inhibition using antisense oligonucleotide might lead to development of an effective antiviral agent for future clinical evaluation.
出处
《中华实验和临床病毒学杂志》
CAS
CSCD
北大核心
2004年第1期62-65,共4页
Chinese Journal of Experimental and Clinical Virology
