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作 者:方剑玉[1,2] 朱文豪[1] 郭小参 白献晓[1] 李海利[1] 徐引弟[1] 郎利敏[1] 王治方[1] 王克领[1]
机构地区:[1]河南省农业科学院畜牧兽医研究所,河南郑州450002 [2]南京农业大学动物医学院,江苏南京210095 [3]普莱柯生物工程股份有限公司,河南洛阳471000
出 处:《河南农业科学》2017年第9期126-131,共6页Journal of Henan Agricultural Sciences
基 金:国家自然科学基金项目(31672565)
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。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 p Ad Track-CMV. The recombinant plasmid were identified by enzyme digestion and sequencing analysis. The p Ad Track-s VIP was linearized by Pme Ⅰ and homologous recombined with p Ad Easy-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-293 A cells for packaging whole virus particle r Ad-s VIP. 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.
关 键 词:猪Viperin蛋白 重组腺病毒 抗病毒活性 猪繁殖与呼吸综合征病毒
分 类 号:S855.3[农业科学—临床兽医学]
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