敲减单纯疱疹病毒Ⅱ型（HSV-2）UL30蛋白表达可抑制HSV-2复制

按照shRNA（small hairpin RNA）设计要求，选择编码单纯疱疹病毒Ⅱ型DNA多聚酶催化亚单位的U_L30（unique long 30，U_L30）基因序列保守区域，设计、合成并构建表达U_L30序列特异性siRNA（short interfering RNA）的质粒载体pUL30．通过磷酸钙转染法将其转染入HEK（human embryonic kidney）293细胞中，用蛋白印迹法检测对HSV-2 UL30蛋白表达的影响，观察受染细胞病变效应（cytopathic effect，CPE），终点滴定法测定细胞上清液中病毒感染滴度（50% tissue culture infective dose，TCID₅₀）．结果表明，针对U_L30基因的siRNA能有效抑制UL30蛋白表达，同时显著抑制受染细胞的CPE，降低上清液中病毒感染滴度．提示本研究建立的针对U_L30基因特异性siRNA能有效阻断HSV-2在HEK293细胞内的复制，U_L30基因是一个潜在的抗HSV-2复制的药物靶标．

Knock-down of UL30 Expression Inhibits Replication of Herpes Simplex Virus Type 2 by Plasmid-mediated Short Interfering RNA

Herpes simplex virus type 2 (HSV-2) accounts for about 2/3 of new sexually transmitted genital infections. To date, no specific therapeutic drugs or preventive vaccines are available. In this study, we explored the effect of RNA interference (RNAi) against unique long 30 gene (U_L30, which encodes HSV-2 DNA polymerase catalytic subunit) of HSV-2 in inhibiting viral proliferation. Based on the rule of shRNA design, a 60 bp DNA fragment targeting conserved sequences of U_L30 was synthesized and cloned into a plasmid vector pUL30 which expressed siRNA specifically targeting U_L30 gene. In an in vitro model of infection, human embryonic kidney (HEK) 293 cells were transfected with pUL30，and then challenged with HSV-2 at a multiplicity of infection (MOI) of 1.5. The expression of HSV-2 DNA polymerase UL30 protein were detected with Western blotting, cytopathic effect (CPE) of HSV-2 infected cells were observed, and the TCID₅₀ in supernatants collected from HSV-2 infected cell were determined by end-point assay. We found that pUL30 caused a 50% inhibition of UL30 protein expression, significantly blocked the CPE, and reduced viral titer (TCID₅₀) by 67-7% in HSV-2 infected cells at 24 hours postinfection. The results demonstrated that RNAi could used as a genetic tool for the HSV-2 replication. Our results reveal that UL30 protein plays a critical role in the replication of HSV-2, implying that U_L30 could be a potential target to develop a novel and safe anti-HSV-2 drug. This demonstrates the effective inhibition of HSV-2 replication by targeting HSV-2 U_L30 gene with RNAi.