慢病毒介导siRNA抑制O型口蹄疫病毒ON株病毒复制

Inhibition of Type O Foot-and-mouth Disease Virus ON Strain Replication by Lentivirus-mediated siRNA

探究RNA干扰(RNA interference,RNAi)技术对口蹄疫病毒复制的抑制作用,本研究选取O型口蹄疫病毒ON株,以ON株口蹄疫病毒3C、3D、VP1蛋白基因作为靶基因。对每一个靶基因设计并合成两对小片段发夹RNA(short hairpin RNA,shRNA)引物,分别命名为VP1-1、VP1-2、3C-1、3C-2、3D-1、3D-2,依据RNAi的原理构建6个shRNA的重组表达质粒,转染BHK21细胞后并接种ON口蹄疫病毒,通过对TCID50的测定和使用实时荧光定量PCR的检测筛选出能高效抑制ON口蹄疫病毒复制的重组质粒;将能够高效抑制ON口蹄疫病毒复制的重组质粒进行慢病毒的制备并筛选稳定的BHK21细胞系,通过对TCID50的测定和使用实时荧光定量PCR的检测慢病毒干扰载体对ON口蹄疫病毒复制的抑制作用。结果显示,构建的6个shRNA重组表达质粒抑制率均在71.5%~93.2%,其中PLKO.1-VP1-2和PLKO.1-3D-1的抑制效果最为明显,分别为93.2%和90.8%;慢病毒干扰载体PLKO.1-VP1-2和PLKO.1-3D-1的抑制效率在88.3%~95.49%。对研究结果表明,在细胞的水平上挑选出有效抑制ON口蹄疫病毒复制的基因干扰片段,并成功制备慢病毒干扰载体,为后续抗口蹄疫病毒的转基因羊生产培育奠定了实验基础。

In order to inhibit replication of foot-and-mouth disease virus（FMDV）by RNAi technology,two pairs of short hairpin RNA（shRNA）targeting 3 D,3 Cand VP1 gene of foot-andmouth disease virus ON strain were designed separately and named as VP1-1,VP1-2,3 C-1,3 C-2,3 D-1 and 3 D-2.According to the principle of RNAi,the six shRNA expression plasmid were constructed and transfected into BHK21 cell that was infected with the FMDV ON strain.TCID50 and real-time fluorescent quantitative PCR were used to analyze inhibition efficiency of shRNA recombinant plasmids on FMDV.ShRNAs with highest inhibition efficiency were chose to produce lentivirus particles and infect BHK21 cell lines,TCID_（50） and real-time fluorescent quantitative PCR were used to analyze lentivirus inference vectors inhibition efficiency on foot-and-mouth dis-ease virus.The results showed that the inhibition rate of shRNAs was between 71.5%-93.2%,and the most obvious inhibitory effect were PLKO.1-VP1-2 as 93.2% and PLKO.1-3 Das 90.8%,respectively;lentivirus inference vectors PLKO.1-VP1-2 and PLKO.1-3 Dinhibition rate of FMDV were between 88.3%-95.49%.Our result screened out shRNAs that can inhibit foot-andmouth disease virus replication at the cellular level.The study laid the foundation for transgenic sheep against FMDV production and cultivation.