稳定表达靶向BVDV shRNA细胞系的建立及其对BVDV复制的影响  

Establishment of Stable Cell Line Expressing shRNA Targeting BVDV and Its Effect on the Replication of BVDV

作　　者：范璐[1] 王丽[1] 史西保[1,2] 樊剑鸣[3] 王爱萍[4] 张改平[1,5] 

机构地区：[1]河南省农业科学院农业部动物免疫学重点开放实验室/河南省动物免疫学重点实验室,河南郑州450002 [2]河南师范大学生命科学学院,河南新乡453007 [3]郑州大学公共卫生学院,河南郑州450001 [4]郑州大学生物工程系,河南郑州450001 [5]河南农业大学牧医工程学院,河南郑州450002

出　　处：《河南农业科学》2014年第12期134-139,共6页Journal of Henan Agricultural Sciences

基　　金：国家自然科学基金项目(31302073);河南省农业科学院自主创新专项基金项目(2013zz48)

摘　　要：为研究靶向牛病毒性腹泻病毒(BVDV)的shRNA对BVDV复制的影响,利用脂质体介导法将BVDV shRNA真核表达质粒pSGH1-sh1083和pSGH1-sh8235及对照质粒pSGH1转染MDBK细胞,通过G418抗性以及有限稀释法筛选获得BVDV shRNA单克隆细胞系subclonepSGH1-1083和subclonepSGH1-8235及对照细胞系subclonepshmock。通过观察其细胞病变效应(CPE)、测定TCID50及RT-PCR、流式细胞术检测BVDV shRNA细胞系对BVDV复制的影响。结果表明,与阴性对照组相比,单克隆细胞系subclonepSGH1-1083和subclonepSGH1-8235的细胞在感染BVDV 72h后CPE不明显,其TCID50低于阴性对照组;RT-PCR结果显示,单克隆细胞系subclonepSGH1-1083和subclonepSGH1-8235的细胞在感染BVDV后病毒E2基因的表达减弱;流式细胞术检测不同细胞系接毒24h后的结合率,单克隆细胞系subclonepSGH1-1083和subclonepSGH1-8235与病毒结合率分别为11.37%和11.51%,与对照组(20.25%)相比有所降低。综上,稳定转染BVDV shRNA的单克隆细胞系subclonepSGH1-1083和subclonepSGH1-8235对BVDV的复制具有抑制作用。In order to study the effects of shRNA targeting BVDV on BVDV replication,the pSGH1-sh1083,pSGH1-sh8235 and the control vector were transfected into MDBK cell by lipofectamin.The transfectants,BVDV shRNA monoclonal cell lines subclonepSGH1-1083,subclonepSGH1-8235 and the control monoclonal cell line subclonepshmock,were obtained by the selection of G418 and the limited dilution.CPE,TCID50,RT-PCR and FCM analysis were used to detect the effect of BVDV shRNA cell lines on BVDV replication.The BVDV cytopathic effect of cells expressing pSGH1-sh1083 or pSGH1-sh8235 at 72hpost-infection was less than that of the negative control group,and the expressions of the BVDV E2 gene in monoclonal cell lines subclonepSGH1-1083,subclonepSGH1-8235 were lower than that in the negative control group.The binding rate to BVDV was 11.37% and 11.51% in monoclonal cell lines subclonepSGH1-1083 and subclonepSGH1-8235 respectively,which were lower than the binding rate in the negative control group（20.25%）.In conclusion,this study demonstrated that the shRNAs specific for BVDV could significantly inhibit the replication of the virus.

关 键 词：RNA干扰 牛病毒性腹泻病毒 短发夹RNA 单克隆细胞系 复制 

分 类 号：S855.3[农业科学—临床兽医学]