CAJ | 학술논문

目的：应用RNA干扰技术，针对单纯疱疹Ⅱ型病毒（HSV-2）UL29基因构建短发夹RNA重组表达载体，观察其对HSV-2的干扰效应。方法：针对HSV-2 UL29基因筛选出4条拟干扰靶位点序列，分别设计、合成4组靶向UL29基因shRNA的基因真核表达载体。通过脂质体转染到HEK293细胞。再将HSV-2接种到HEK293细胞中。采用终点滴定法检测病毒滴度，RT-PCR检测shRNA对转录水平的影响，Western-blot检测shRNA对蛋白质表达水平的影响。结果：终点滴定法结果显示UL29shRNA各组均可不同程度降低病毒感染滴度，与空白组（未转染重组表达载体）比较差异显著（P＜0．01）。RT-PCR结果显示，与空白组比较，4组抑制率分别为28．80％、59．95％、66．08％、36．27％，差异均具有显著性（P＜0．05），shRNANC（不针对任何基因的序列）与空白组相比差异无显著性，其中以UL29shRNA1461组效果为最佳。 Western-blot检测表明UL29shRNA各组ICP8蛋白表达水平比阴性对照组明显降低。结论：UL29shRNA能有效干扰HSV-2UL29基因的表达，抑制HSV-2在HEK293细胞中的复制。
목적：응용RNA간우기술，침대단순포진Ⅱ형병독（HSV-2）UL29기인구건단발협RNA중조표체재체，관찰기대HSV-2적간우효응。방법：침대HSV-2 UL29기인사선출4조의간우파위점서렬，분별설계、합성4조파향UL29기인shRNA적기인진핵표체재체。통과지질체전염도HEK293세포。재장HSV-2접충도HEK293세포중。채용종점적정법검측병독적도，RT-PCR검측shRNA대전록수평적영향，Western-blot검측shRNA대단백질표체수평적영향。결과：종점적정법결과현시UL29shRNA각조균가불동정도강저병독감염적도，여공백조（미전염중조표체재체）비교차이현저（P＜0．01）。RT-PCR결과현시，여공백조비교，4조억제솔분별위28．80％、59．95％、66．08％、36．27％，차이균구유현저성（P＜0．05），shRNANC（불침대임하기인적서렬）여공백조상비차이무현저성，기중이UL29shRNA1461조효과위최가。 Western-blot검측표명UL29shRNA각조ICP8단백표체수평비음성대조조명현강저。결론：UL29shRNA능유효간우HSV-2UL29기인적표체，억제HSV-2재HEK293세포중적복제。
Objective To construct short hairpin RNA (shRNA) recombinant expression vector for herpes simplex virus typeⅡ(HSV-2) UL29 gene and observe its inhibitory effect on HSV-2. Methods Four interference target sites of HSV-2UL29 gene were selected to construct 4 groups of small hairpin RNA respectively,named shRNA recombinant expression vector. The expression vectors were transfected into HEK293 cells with liposome. HEK293 cells were infected with HSV-2 after expression vector being transfected. The viral titer was estimated by end-point titration assay. The level of transcription was estimated by Real-Time PCR method. The expressing effect of protein was detected by Western-blot. Results Recombinant expression vector pGPU6/GFP/Neo-shRNA was constructed successfully. The result of end-point titration assay showed that the viral titer was reduced comparing with blank control (P<0.05). The result of RT-PCR showed that inhibition rates were respectively 28.80%, 59.95%, 66.08%and 36.27% comparing with blank control, and there were significant differences (P < 0.05). The effect of UL29shRNA1461 group was the best one. The result of Western-blot showed that the expressing quantity of ICP8 was reduced. Conclusion Recombinant expression vector pGPU6/GFP/Neo-shRNA can interfere HSV-2 UL29 gene expression from different cell level in vitro, which can inhibit the replication of HSV-2 genome in HEK293 cells. Thus, RNA interference (RNAi) is conducive to the further exploration of viral therapy.