He Yun-Xia, Hua Rong-Hong, Zhou Yan-Jun, Qiu Hua-Ji, Tong Guang-Zhi
National Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, 427 Maduan Street, Harbin 150001, PR China.
Antiviral Res. 2007 May;74(2):83-91. doi: 10.1016/j.antiviral.2006.04.013. Epub 2006 May 11.
Porcine reproductive and respiratory syndrome (PRRS) is an economically important disease in swine-producing areas of the world. Many vaccine strategies developed to control the disease are not yet completely successful. The objective of this study was to determine if RNA interference (RNAi) could be utilized to inhibit PRRSV replication on MARC-145 cells. Four short interfering RNA (siRNA) sequences (N95, N179, N218 and N294) directed against a well-conserved region of PRRSV genome ORF7 gene were selected. Sense and antisense siRNA encode sequences separated by a hairpin loop sequence were designed as short hairpin RNA (shRNA) expression cassettes driven by mouse U6 promoter. Using a polymerase chain reaction (PCR)-based approach, shRNAs were generated from shRNA expression cassettes. The PCR products were cloned into pEGFP-N1 vector and shRNA expression vectors were constructed. When MARC-145 cells were transfected with shRNA expression vectors and then infected with PRRSV, N179 was found to be the most effective inhibition site in decreasing cytopathic effect (CPE) induced by PRRSV. Western blot, indirect immunofluorescence and fluorescence quantitative PCR (FQ-PCR) confirmed that the expression of ORF7 was reduced both at protein and RNA levels comparing to controls. The results presented here indicated that DNA-based siRNA could effectively inhibit the replication of PRRS virus (approximately 681-fold reduction of viral titers) on MARC-145 cells.
猪繁殖与呼吸综合征（PRRS）是世界养猪地区一种具有重要经济影响的疾病。为控制该疾病而研发的许多疫苗策略尚未完全成功。本研究的目的是确定RNA干扰（RNAi）是否可用于抑制MARC - 145细胞上的PRRSV复制。选择了四个针对PRRSV基因组ORF7基因保守区域的短干扰RNA（siRNA）序列（N95、N179、N218和N294）。将由发夹环序列隔开的正义和反义siRNA编码序列设计为由小鼠U6启动子驱动的短发夹RNA（shRNA）表达盒。使用基于聚合酶链反应（PCR）的方法，从shRNA表达盒中产生shRNAs。将PCR产物克隆到pEGFP - N1载体中，构建shRNA表达载体。当用shRNA表达载体转染MARC - 145细胞，然后感染PRRSV时，发现N179是降低PRRSV诱导的细胞病变效应（CPE）中最有效的抑制位点。蛋白质印迹、间接免疫荧光和荧光定量PCR（FQ - PCR）证实，与对照相比，ORF7在蛋白质和RNA水平上的表达均降低。此处呈现的结果表明，基于DNA的siRNA可有效抑制PRRS病毒在MARC - 145细胞上的复制（病毒滴度降低约681倍）。
