稳定表达兔ANXA2的RK13细胞系的建立及其对RHDV吸附研究  

Establishment of Oryctolagus cuniculus RK13 Cell Line Stably Expressing ANXA2 and Study on Its Adsorption for RHDV

作　　者：龙翠琴 武小椿 岳亚辉 张宏燕 张立 马海云 邢小勇[1] 温峰琴[1] 包世俊[1] LONG Cui-Qin;WU Xiao-Chun;YUE Ya-Hui;ZHANG Hong-Yan;ZHANG Li;MAHai-Yun;XING Xiao-Yong;WEN Feng-Qin;BAO Shi-Jun(College of Veterinary Medicine,GansuAgricultural University,Lanzhou 730070,China)

机构地区：[1]甘肃农业大学动物医学院,兰州730070

出　　处：《农业生物技术学报》2023年第2期350-360,共11页Journal of Agricultural Biotechnology

基　　金：甘肃省重点研发计划(20YF3NA015)。

摘　　要：兔出血症(Rabbit hemorrhagic disease, RHD)是兔(Oryctolagus cuniculus)的一种高致死性传染病,其由兔出血症病毒(Rabbit hemorrhagic disease virus, RHDV)引起。膜联蛋白A2(annexin A2, ANXA2)与多种病毒的感染过程密切相关。为探究兔ANXA2在RHDV侵染兔肾细胞(rabbit kidney cell, RK13)过程中的作用,本研究利用PCR扩增获得兔ANXA2基因片段,构建真核表达载体pCD513B-ANXA2,并将其与包装质粒pLP1、pLP2和p LP/VSVG转染至293T细胞,包装出含ANXA2基因的慢病毒颗粒并感染RK13,利用嘌呤霉素筛选稳定过表达ANXA2蛋白的细胞株RK13-513B-ANXA2;应用qPCR和Western blot检测ANXA2基因及其蛋白的表达情况;设计合成siRNA并转染RK13,进而分析基因表达情况;以RHDV感染RK13-513B-ANXA2细胞株2 h后,采用qPCR、免疫荧光(immunological fluorescence assay, IFA)和Western blot检测RHDV吸附量的相对变化。结果表明,利用慢病毒载体包装获得滴度较高的病毒颗粒,其滴度达5.0×10^(7)~1.0×10^(8) TU/mL;RK13-513B-ANXA2细胞中ANXA2基因及其蛋白表达量均极显著增加(P<0.01);过表达ANXA2蛋白的RK13对RHDV的吸附作用明显增加(P<0.01),干扰ANXA2蛋白表达可显著下调RHDV对RK13的吸附作用(P<0.05)。本研究结果提示ANXA2可能参与RHDV对宿主细胞的感染,为进一步探究RHDV感染宿主细胞的机制提供了参考。Rabbit hemorrhagic disease(RHD) is a highly lethal infectious disease of rabbits(Oryctolagus cuniculus) caused by Rabbit hemorrhagic disease virus(RHDV). Annexin A2(ANXA2) is closely associated with the infection process of many viruses. In order to investigate the role of rabbit ANXA2 in the process of RHDV infection of host cells, in this study, rabbit ANXA2 gene fragments were amplified by PCR, and the eukaryotic expression vector pCD513B-ANXA2 was constructed and transfected into 293T cells with packaging plasmids pLP1, pLP2, and pLP/VSVG, then packaged lentiviral particles containing ANXA2 gene, infected rabbit kidney cells(RK13), and screened the cell line RK13-513B-ANXA2 for stable overexpression of ANXA2 protein using puromycin, detected the expression of ANXA2 gene and its protein by qPCR and Western blot. siRNA was synthesized and transfected into RK13 to analyze the gene expression. After 2 h, the relative change of RHDV adsorption was detected by q PCR, immunological fluorescence assay(IFA) and Western blot.The results showed that viral particles with high titers of 5.0×10^(7)~1.0×10^(8) TU/m L were obtained using lentiviral vector packaging. The expression of ANXA2 gene and its protein in RK13-513B-ANXA2 cells was significantly up-regulated(P<0.01). The adsorption of RHDV by RK13 overexpressing ANXA2 protein was significantly up-regulated(P<0.01), and interference with ANXA2 expression protein significantly down-regulated the adsorption of RHDV to RK13(P<0.05). The results suggest that ANXA2 may be involved in the infection of host cells by RHDV, which provides a firm basis for further investigation of the mechanism of RHDV infection in host cells.

关 键 词：兔出血症病毒 慢病毒载体 膜联蛋白A2(ANXA2) 兔肾细胞(RK13) 

分 类 号：S855.3[农业科学—临床兽医学]