摘要
根据口蹄疫病毒 IRES和 L 串联序列两侧的保守区域设计了 2个引物 ,利用 RT- PCR和 PCR方法扩增出该串联序列 ,并进行了测序。测序结果表明 ,扩增产物与 Gen Bank上相应的序列具有很高的序列同源性 (大于 99% )。在测序的基础上 ,选择了 L 基因上的 1个靶位点 (位于启始密码子下游第 2 2 9nt后 2 1 nt长的序列 ) ,合成了 si RNA表达盒SEC- L2 2 9。细胞单层长成 5 0 %～ 70 %时 ,将纯化的 SEC- L2 2 9转染到 BHK细胞中 ,转染 4 h后用高感染复数 FMDV接种 ,2 4 h后用间接免疫荧光方法对口蹄疫病毒在 BHK细胞中的复制进行检测。研究结果表明 ,SEC- L 2 2 9极大地抑制了口蹄疫病毒在 BHK细胞中的复制 ,且该抑制作用具有序列特异性 ,并降低了 BHK细胞的死亡率。另外 ,2 5 ng和5 0 ng SEC- L2 2 9处理组间对病毒复制的抑制作用差异不明显 ,可能是病毒基因组发生了突变。本试验表明 ,利用 PCR方法合成的 SEC在 BHK细胞中能特异性地抑制 FMDV的复制 。
According to the conservative flanking-sequence,two primers were designed to amplify the tandem sequence,IRES (Internal ribosome entry sites) and L sequence of foot-and-mouth disease virus (FMDV),using RT-PCR and PCR methods.The PCR product was sequenced and sequencing result shows that it has high homology (more than 99%) with its corresponding sequence from GenBank.A target site (21 nt-length) locates on 229 nt downstream from the start codon of gene L was selected on the basis of sequencing result and its siRNA expression cassette (SEC-L229) was generated.The purified SEC was transfected into culture BHK cells when cell monolayer was at about 50%-70% confluency.High multiplicity of infection (MOI) FMDV was added 4 hours after transfection,and inhibition of the virus replication was detected 24 hours after virus inoculation by indirect immunofluorescent assay.The results indicated that the positive SEC had dramatically induced sequence-specific inhibition of the viral replication in BHK cells and decreased the cell death rate.The difference of inhibition effect between 25 ng and 50 ng SEC-L229 treatments was not distinct,the reason probably is mutations on the viral genome have occurred.The study shows that SEC generated through PCR method can dramatically inhibit FMDV replication in culture BHK cells,and RNAi may be a potential therapy pathway for this virus.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2004年第5期453-456,共4页
Chinese Journal of Veterinary Science
