单种及联合靶向EV71VP1～VP4基因的shRNA干扰病毒复制的研究

Inhibitory effects of shRNAs targeting genes encoding viral capsomeres VP1-VP4 on EV71 replication

目的检测单种及2种联合靶向Ev71病毒衣壳蛋白VP1、VP2、VP3、VP4基因（VP1～VP4）的shRNA干扰病毒复制的效果。方法靶向EV71病毒的VP1～VP4基因序列设计及合成shRNA，并分别将其克隆到慢病毒质粒表达载体pLOV．CMV.EGFP中。重组表达质粒同psPAX2和pMD2．G共转染293T细胞，3d后收集上清中的重组病毒颗粒。用荧光定量RT—PCR法（real—timeqRT—PCR）和Westernblot检测单种及2种联合shRNA干扰EV71mRNA的表达水平及病毒蛋白的表达水平。结果靶向EV71VP1-VP4基因的shRNA对本研究的毒株（含死亡病例、重症病例、普通病例，FY0805）复制干扰差异无统计学意义（P〉0．05），对EV71病毒的抑制率分别约为：sh-VP1—1（51．6％），sh—VP1—2（85．1％），sh—VP1-3（76．4％），sh-VP2—1（57．5％），sh-VP2-2（81．4％），sh—VP2-3（79．5％），sh—VP3（68．9％），sh—VP4（56．7％），且有剂量依赖性。干扰病毒效率最高的为sh—VP1-2联合sh—VP2—2，抑制率可达96．6％，联合组均比单种加倍剂量的抑制率高。结论本研究设计的单独及联合靶向EV71病毒衣壳蛋白每个基因区均筛选到抑制率均大于50％的shRNA，且联合shRNA可以增强其干扰复制的能力。

Objeciive ：Toevaluate ihe inhibitory effects of shR^NAs targeting genes encoding viral capsomeres VP1 - VP4 on enterovirus 71 （ EV71 ） replication when used alone or in combination. Methods Short hairpin RNAs （shRNAs） targeting genes encoding VP1-VP4 protein of EVT1 were designed and then respectively inserted into lentiviral vector pLKD-CMV-GFP-U6 to construct the recombinant plasmids. The expression plasmids together with psPAX2 and pMD2. G were transfected into 293T cells to induce the ex- pression of recombinant lentiviruses, which were collected on the third day after transfection. The titers of re- combined lentiviruses were determined by real-time PCR. The effects of shRNAs used alone or in combina- tion on the expression of EV71 at mRNA and protein levels were respectively detected by real-time PCR and Western blot. Results The inhibitory effects of shRNAs on EVT1 replication showed no significant differ- ences among various strains （isolated from fatal cases, severe cases, mild cases and FY0805） （P〉0.05）. Their inhibition rates were 51.6% （ sh-VPl-1 ）, 85.1% （ sh-VP1-2）, 76.4% （ sh-VPI-3）, 57.5% （ sh- VP2-1）, 81.4% （sh-VP2-2）, 79.5% （sh-VP2-3）, 68.9% （sh-VP3） and 56.7% （sh-VP4） respective- ly, and they were in a dosage dependent manner, sh-VP1-2 in combination with sh-VP2-2 showed the high- est inhibition rate reaching up to 96.6%. Moreover, shRNAs used in combination showed better effects than any one used alone even at double dosage. Conclusion All shRNAs targeting viral capsid VP1-VP4 genes showed inhibitory effects on EV71 replication with inhibition rates over 50% and the effects could be strengthened when using shRNAs in combination.