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

摘要

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RT-PCR方法扩增猪Viperin基因,并克隆入腺病毒穿梭载体pAdTrack-CMV,经菌液PCR、酶切鉴定后进行测序.将Pme Ⅰ内切酶线性化的重组穿梭载体质粒(pAdTrack-sVIP)电转化大肠杆菌BJ5183,与腺病毒骨架载体pAdEasy-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rAd-sVIP.重组腺病毒rAd-sVIP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rAd-sVIP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.%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.