猪传染性胃肠炎病毒Nsp2与宿主细胞蛋白PSMD11相互作用的研究  

Study on Interaction Between Transmissible Gastroenteritis Virus Nsp2 and Host Cell Protein PSMD11

作　　者：孙傲颖 王亚楠 姜艳平[1,2] 崔文 乔薪瑗[1,2] 周晗 唐丽杰[1,2] 徐义刚 李一经[1,2] 王丽 SUN Aoying;WANG Yanan;JIANG Yanping;CUI Wen;QIAO Xinyuan;ZHOU Han;TANG Lijie;XU Yigang;LI Yijing;WANG Li(College of Veterinary Medicine,Northeast Agricultural University,Harbin 150030,China;Heilongjiang Key Laboratory for Animal Disease Control and Pharmaceutical Development,Harbin 150030,China)

机构地区：[1]东北农业大学动物医学学院,哈尔滨150030 [2]黑龙江省动物疾病防控技术与制剂创制实验室,哈尔滨150030

出　　处：《中国畜牧兽医》2021年第7期2569-2576,共8页China Animal Husbandry & Veterinary Medicine

基　　金：黑龙江省普通本科高等学校青年创新人才培养计划(UNPYSCT-2018147)。

摘　　要：为确定猪传染性胃肠炎病毒(Transmissible gastroenteritis virus,TGEV)非结构蛋白2(nonstructural protein 2,Nsp2)与宿主细胞蛋白26S蛋白酶体非ATP酶调节亚基11(PSMD11)之间是否存在相互作用,本研究利用RT-PCR方法扩增猪源PSMD11基因,并构建其真核表达载体pCMV-Myc-PSMD11,经测序和双酶切验证正确后,转染猪小肠上皮细胞(IPEC-J2),通过Western blotting和间接免疫荧光试验(IFA)检测真核表达载体pCMV-Myc-PSMD11是否能在IPEC-J2中表达。利用免疫共沉淀(Co-IP)试验检测TGEV Nsp2和宿主细胞PSMD11蛋白之间的相互作用,并通过激光共聚焦显微镜观察TGEV Nsp2与PSMD11在宿主细胞中的共定位情况。结果显示,本研究成功扩增了猪源PSMD11基因,大小约为1474 bp,基因序列经测序比对与标准序列完全一致。构建的真核表达载体pCMV-Myc-PSMD11能在IPEC-J2细胞中成功表达PSMD11蛋白;Co-IP结果表明,PSMD11与Nsp2之间存在相互作用;共定位试验结果显示,PSMD11与Nsp2的相互作用发生在细胞质中,且细胞中PSMD11蛋白的表达位置并未因Nsp2的表达而发生改变。本研究结果为进一步研究TGEV Nsp2在病毒感染过程中所发挥的重要作用提供新的线索。To determine whether there was an interaction between Transmissible gastroenteritis virus(TGEV)nonstructural protein 2(Nsp2)and the host cell protein 26S proteasome non-ATPase regulatory subunit 11(PSMD11),RT-PCR was used to amplify the porcine PSMD11 gene and eukaryotic expression vector pCMV-Myc-PSMD11 was constructed,which was verified by sequencing and double enzyme digestion.And then pCMV-Myc-PSMD11 was transfected into intestinal epithelial cell lines J2(IPEC-J2),Western blotting and indirect immunofluorescence assay(IFA)were used to detect whether pCMV-Myc-PSMD11 could be expressed in IPEC-J2 cells.Then,the co-immunoprecipitation(Co-IP)experiment was used to determine the interaction between TGEV Nsp2 and PSMD11 of the host cell,and the co-localization of TGEV Nsp2 and PSMD11 in the host cell was observed by laser confocal microscope.The results showed that porcine PSMD11 gene was successfully amplified,with a size of 1474 bp,and the gene sequence was completely consistent with the standard sequence.The constructed eukaryotic expression vector pCMV-Myc-PSMD11 could successfully express PSMD11 protein in IPEC-J2 cells.The results of Co-IP indicated that there was an interaction between PSMD11 and Nsp2.The results of the colocalization experiment showed that the interaction between PSMD11 and Nsp2 occured in the cytoplasm,and the expression position of PSMD11 in the cell was not changed due to the expression of Nsp2.The results of this study provided new clues for further study of the important role of TGEV Nsp2 in the process of virus infection.

关 键 词：猪传染性胃肠炎病毒(TGEV) 非结构蛋白2 26S蛋白酶体非ATP酶调节亚基11 蛋白互作 

分 类 号：S852.651[农业科学—基础兽医学]