猪RNase L与PRRSV nsp4缺失突变体的构建及其互作研究  

Construction and interaction of porcine RNase L and PRRSV nsp4 deletion mutants

作　　者：于莹 李均同 丛晓燕[2] 齐静[2] 刘思当[3,4] 郑乾坤 孙文博 王爱国[4] 郑龙祥 吴香菊 杜以军[2] 单虎 YU Ying;LI Juntong;CONG Xiaoyan;QI Jing;LIU Sidang;ZHENG Qiankun;SUN Wenbo;WANG Aiguo;ZHENG Longxiang;WU Xiangju;DU Yijun;SHAN Hu(College of Veterinary Medicine,Qingdao Agricultural University,Qingdao 266000,China;Shandong Province Key Laboratory of Animal Disease Control and Breeding/Institute of Animal Science and Veterinary Medicine,Shandong Academy of Agricultural Sciences,Jinan 250100,China;College of Animal Science and Veterinary Medicine,Shandong Agricultural University,Taiwan 271018,China;Delis Group Co.,Ltd.,Zhucheng 262216,China)

机构地区：[1]青岛农业大学动物医学院,山东青岛266000 [2]山东省畜禽疫病防治与繁育重点实验室/山东省农业科学院畜牧兽医研究所,山东济南250100 [3]山东农业大学动物科技学院,山东泰安271018 [4]得利斯集团有限公司,山东诸城262216

出　　处：《畜牧与兽医》2021年第11期85-92,共8页Animal Husbandry & Veterinary Medicine

基　　金：山东省自然科学基金青年项目(ZR2020QC196);山东省自然科学基金重点项目(ZR2020KC005)。

摘　　要：RNase L是一种独特的核糖核酸内切酶,在机体的抗病毒过程中发挥重要作用。猪繁殖与呼吸综合征病毒(PRRSV)非结构蛋白4(nsp4)具有3C样丝氨酸蛋白酶活性,是参与病毒多聚蛋白前体切割的主要蛋白酶,是影响病毒粒子成熟的重要蛋白。课题组前期研究发现猪RNase L(sRNase L)能够与nsp4互作发挥抗PRRSV作用。为了筛选sRNase L与nsp4互作的区段或位点,本研究分别构建了sRNase L与nsp4的截短体或突变体,通过免疫共沉淀试验进行筛选,结果显示突变体sRNase L R460A、sRNase L R675A、sRNase L Y720A、sRNase L F724A都能够与nsp4发生互作,只有缺失体sRNase L 1~586 aa不与nsp4互作,表明sRNase L第3结构域(587~744 aa)为与nsp4互作区段,但该区段不包括第675位精氨酸、第720位酪氨酸、第724位苯丙氨酸;分别包含nsp4三个结构域的截短体nsp4-D1(1~80 aa)、nsp4-D2(60~156 aa)、nsp4-D3(157~204 aa)中,只有nsp4-D1与sRNase L发生互作,表明nsp4的1~60 aa为与sRNase L互作区段。本研究为进一步探究sRNase L与nsp4互作抗PRRSV机制奠定了基础,为预防和控制PRRSV感染提供了新的思路和方法。Ribonuclease L(RNase L) is a unique ribonucleic acid endonuclease, which plays an important role in the antiviral process of the body. Porcine reproductive and respiratory syndrome virus nonstructural protein 4(nsp4) has 3 C-like serine protease activity, which is the main protease involved in the cleavage of virus polyprotein precursor and an important protein affecting virion maturation. Previously, our group found that the interaction between swine Ribonuclease L(sRNase L) and nsp4 could exert an anti-PRRSV effect. In order to screen the interaction regions or sites between sRNase L and nsp4, truncated or mutant sRNase L and nsp4 were constructed and screened by co-immunoprecipitation assay. The results showed that the mutants sRNase L R460 A, sRNase L R675 A, sRNase L Y720 A and sRNase L F724 A could interact with nsp4, and only the deletion sRNase L 1-586 aa didn’t interact with nsp4, indicating that the third domain of sRNase L(587-744 aa) interacted with nsp4, but this region didn’t include arginine-675, tyrosine-720 and phenylalanine-724. Among the truncated domains of nsp4-D1(1-80 aa), nsp4-D2(60-156 aa) and nsp4-D3(157-204 aa), each containing three domains of nsp4,only nsp4-D1 interacted with sRNase L, indicating that 1-60 aa of nsp4 was the region that interacted with sRNase L. This study laid a foundation for further exploring the anti-PRRSV mechanism of the interaction between sRNase L and nsp4, and provided new ideas and methods for the prevention and control of PRRSV infection.

关 键 词：猪繁殖与呼吸综合征症 猪核糖核酸内切酶L 非结构蛋白4 免疫共沉淀 

分 类 号：S885.3[农业科学—特种经济动物饲养]