MOA Key Laboratory of Animal Vaccine Development, Ministry of China, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China.
MOA Key Laboratory of Animal Vaccine Development, Ministry of China, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China.
Infect Genet Evol. 2014 Dec;28:64-70. doi: 10.1016/j.meegid.2014.08.008. Epub 2014 Aug 20.
To screen siRNAs for effectively inhibiting the replication of porcine reproductive and respiratory syndrome virus (PRRSV). Four pairs of siRNA targeting Nsp9 gene of PRRSV and one non-efficient pair used as control were designed, synthesized and cloned into pSilencer4.1-CMV neo, designated as pSi-294, pSi-367, pSi-409, pSi-1488, pSi-Ctr. The recombinant plasmids were transfected into Marc-145 cells and infected with PRRSV 24h post transfection. Subsequently, IFA, real-time PCR, TCID50 and western blot were used for evaluating the inhibitory effect of the siRNA. IFA and western-blot results showed that pSi-294, pSi-1488 can effectively inhibit the expression of Nsp9 and M protein of PRRSV, real-time PCR result showed that the expression of Nsp9 gene were decreased from 86.56% to 93.66% compared to the negative control. siRNAs can be used as candidates for basic research of PRRSV.
为了筛选出有效抑制猪繁殖与呼吸综合征病毒（PRRSV）复制的 siRNA，设计并合成了针对 PRRSV Nsp9 基因的 4 对 siRNA（si-294、si-367、si-409 和 si-1488）和 1 对非有效 siRNA（si-Ctr）作为对照，将其克隆到 pSilencer4.1-CMV neo 载体中，分别命名为 pSi-294、pSi-367、pSi-409、pSi-1488 和 pSi-Ctr。将重组质粒转染 Marc-145 细胞，转染后 24 小时感染 PRRSV。然后通过 IFA、实时 PCR、TCID50 和 Western blot 评估 siRNA 的抑制效果。IFA 和 Western blot 结果表明，pSi-294 和 pSi-1488 可有效抑制 PRRSV Nsp9 和 M 蛋白的表达，实时 PCR 结果显示，与阴性对照相比，Nsp9 基因的表达降低了 86.56%至 93.66%。这些 siRNA 可以作为 PRRSV 基础研究的候选物。
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