高致病性PRRSVNsp2基因RNA干扰对病毒复制的影响

Influence of RNA Interference Mediated HP-PRRSV TJ Nsp2 Gene Silencing on Viral Replication

旨在构建针对猪繁殖与呼吸综合征病毒(PRRSV)TJ株Nsp2基因的siRNA的表达载体,研究Nsp2基因的表达抑制对PRRSV复制的影响。设计并合成针对PRRSV TJ株Nsp2基因的3对优势小发卡RNA(shRNA),连接到pRNAT-U6.1/Neo,构建shRNA表达载体,将鉴定正确的载体质粒转染Marc-145细胞,6 h后接毒,每日观察CPE;在接毒PRRSV TJ株后不同时间点取上清样品,测定样品TCID50;并以β-actin为内参,RT-PCR对PRRSV Nsp2 mRNA进行半定量。结果表明,选取的3条Nsp2基因的优势siRNA均能够抑制PRRSV TJ株在Marc-145细胞上增殖,其中shRNA载体S-1和S-2抑制效果明显,与空载体相比较差异极显著。本研究构建的PRRSV Nsp2基因特异性shRNA载体能够有效抑制PRRSV的复制,可使病毒滴度TCID50最多降低103.38。

It was to construct shRNA recombinant expression vector specific to Non-structural protein 2 （ Nsp2 ） gene and to investigated the effect of Nsp2 gene siRNA on porcine reproductive and respiratory syndrome virus replication. Three pairs complementary single strand DNA oligos targeting three various sites of Nsp2 siRNAs were designed, synthesized and cloned to pRNAT-U6.1/Neo expressed vector. The vectors identified by sequencing were transfected into Marc-145 ceils. Then, we infected the Marc-145 ceils with PRRSV TJ strain after 6 h post infection. CPE was observed every day. Sampling at series times was measured by RT-PCR aimed Nsp2 mRNA and TCID50 assay was used to titer. The results showed three siRNAs of Nsp2 all inhibited the replication of PRRSV on Marc-145 cells successfully, among which the effect of S- 1 and S-2 were visible. The specific shRNA vector of PRRSV Nsp2 could effectively inhibit the replication of PRRSV, and decreased the virus titre not less than 10^3.38.