CAJ | 학술논문

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Western blot检测单种及2种联合shRNA干扰EV71 mRNA的表达水平及病毒蛋白的表达水平.结果靶向EV71 VP1～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.
목적 검측단충급2충연합파향EV71병독의각단백VP1、VP2、VP3、VP4기인(VP1～ VP4)적shRNA간우병독복제적효과.방법 파향EV71병독적VP1～VP4기인서렬설계급합성shRNA,병분별장기극륭도만병독질립표체재체pLOV.CMV.EGFP중.중조표체질립동psPAX2화pMD2.G공전염293T세포,3d후수집상청중적중조병독과립.용형광정량RT-PCR법(real-timeqRT-PCR)화Western blot검측단충급2충연합shRNA간우EV71 mRNA적표체수평급병독단백적표체수평.결과 파향EV71 VP1～VP4기인적shRNA대본연구적독주(함사망병례、중증병례、보통병례,FY0805)복제간우차이무통계학의의(P＞0.05),대EV71병독적억제솔분별약위: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％),차유제량의뢰성.간우병독효솔최고적위sh-VP1-2연합sh-VP2-2,억제솔가체96.6％,연합조균비단충가배제량적억제솔고.결론 본연구설계적단독급연합파향EV71병독의각단백매개기인구균사선도억제솔균대우50％적shRNA,차연합shRNA가이증강기간우복제적능력.
Objective To evaluate the inhibitory effects of shRNAs 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 EV71 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 expression of recombinant lentiviruses,which were collected on the third day after transfection.The titers of recombined lentiviruses were determined by real-time PCR.The effects of shRNAs used alone or in combination 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 EV71 replication showed no significant differences among various strains (isolated from fatal cases,severe cases,mild cases and FY0805) (P＞0.05).Their inhibition rates were 51.6％ (sh-VP1-1),85.1％ (sh-VP1-2),76.4％ (sh-VP1-3),57.5％ (sh-VP2-1),81.4％ (sh-VP2-2),79.5％ (sh-VP2-3),68.9％ (sh-VP3) and 56.7％ (sh-VP4) respectively,and they were in a dosage dependent manner.sh-VP1-2 in combination with sh-VP2-2 showed the highest 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.