表达猪Viperin蛋白重组腺病毒的构建及其对PRRSV复制的抑制作用

为研究猪Viperin蛋白对猪繁殖与呼吸综合征病毒(PRRSV)复制的抑制作用,通过RTPCR方法扩增猪Viperin基因,并克隆入腺病毒穿梭载体p Ad Track-CMV,经菌液PCR、酶切鉴定后进行测序。将PmeⅠ内切酶线性化的重组穿梭载体质粒(p Ad Track-s VIP)电转化大肠杆菌BJ5183,与腺病毒骨架载体p Ad Easy-1进行同源重组,提取重组后的腺病毒质粒,经内切酶PacⅠ线性化后转染HEK-293A细胞,装配成完整的病毒粒子r Ad-s VIP。重组腺病毒r Ad-s VIP感染细胞后,可见绿色荧光蛋白的表达。RT-PCR、Western blot检测结果表明,重组腺病毒r Ad-s VIP可正确表达猪Viperin蛋白,且表达的猪Viperin蛋白具有良好的反应原性,对PRRSV的复制具有明显的抑制作用。

Expression of Swine Viperin Protein in Recombinant Adenovirus and Study of Its Effect on PRRSV Replication

To study the inhibitory effect of swine Viperin on PRRSV replication,the swine Viperin gene was amplified by RT-PCR and cloned into the adenovirus shuttle vector pAdTrack-CMV.The recombinant plasmid were identified by enzyme digestion and sequencing analysis.The pAdTrack-sVIP was linearized by Pme Ⅰ and homologous recombined with pAdEasy-1 backbone vector in E.coli BJ5183.The recombinant adenoviral genome DNA was extracted and digested by Pac Ⅰ,then the DNA was transfected into the HEK-293A cells for packaging whole virus particle rAd-sVIP.The CPE and GFP fluorescence of cells were observed,the cells were harvested and adenoviral expressed recombinant swine Viperin protein were identified by RT-PCR and Western blot.The result showed that the swine Viperin was correctly expressed as expected.The expressed swine Viperin protein had good reactivity,and it could inhibit the PRRSV replication.