靶向HP-PRRSV ORF7基因siRNA体外抑制HP-PRRSV HN1株复制研究

Inhibition of highly pathogenic porcine reproductive and respiratory syndrome virus replication by mediated RNA interference in vitro

为了体外评价靶向高致病性猪繁殖与呼吸综合征病毒(HP-PRRSV) ORF7的siRNA对HP-PRRSV复制的抑制效果。将3μg siRNA重组质粒载体pSilencer-N2转染于Marc-145细胞中,24 h后再分别接种0. 01 MOI PRRSV,感染病毒48 h后,观察其抑制HP-PRRSV HN1株复制效果;待细胞感染病毒72 h后,应用间接免疫荧光试验(IFA)、实时荧光定量PCR及Western Blot检测siRNA重组质粒对HP-PRRSV在细胞中复制的抑制作用。体外结果显示,在最佳接毒量0. 01 MOI和最佳接毒时间24 h时,siRNA重组表达质粒在HP-PRRSV感染后48 h能明显抑制病毒的复制,与对照组相比,病毒滴度降低了2个多数量级,抑制达到100～1 000倍。有效干扰持续时间,检测结果表明,与对照组相比,siRNA重组质粒显著抑制HP-PRRSV的增殖,推迟CPE的发生时间,其抑制作用可以持续到感染后的4天。为高致病性猪繁殖与呼吸综合征防控提供有力的物质支持。

Highly pathogenic porcine reproductive and respiratory syndrome virus（ HP- PRRSV）is the variant of porcine repro- ductive and respiratory syndrome virus （PRRSV）. In recent years, it has caused heavy economic losses in swine -producing areas. Although current vaccines are some prophylactic, but they provide only limited protection. Furthermore, up to now there are no effec- tive antiviral drugs. Thus, it is necessary for us to develop a new antiviral strategy. In this study, siRNA targeting N protein gene of highly pathogenic porcine reproductive and respiratory syndrome virus （HP- PRRSV） was observed to inhibit HP- PRRSV replica- tion in Marc - 145 cells. The results showed that siRNA recombinant plasmid inhibited HP - PRRSV replication 48h post - infection in Marc -145 cells infected with an MOI of 0. 01. The TCIDs0 was decreased by 2 orders of magnitude compared with the control group, and the inhibition was to 100 - 1000 times. The results of duration time of effective interference indicated that the recombinant plasmid of siRNA significantly suppressed HP - PRRSV replication and delay the occurrence time of CPE, and its inhibitory effect lasted up to 4 days after inoculation. These results suggest that siRNA targeting N gene of HP - PRRSV can inhibit virus replication.