不同生物型BVDV感染对MDBK细胞类泛素基因转录水平的影响  

Effects of Different Biological-types of Bovine Viral Diarrhea Virus Infection on SUMO Gene Transcription Levels in MDBK Cells

作　　者：韩玉霞[1] 孟露萍[1] 孙志华[1] 刘娟[1] 张辉[1] 陈创夫[1] 

机构地区：[1]石河子大学动物科技学院,新疆石河子832000

出　　处：《动物医学进展》2015年第12期11-17,共7页Progress In Veterinary Medicine

基　　金：国际科技合作项目(2013DFR30970);国家自然科学基金项目(U1303283)

摘　　要：探讨牛病毒性腹泻病毒(BVDV)感染对牛胚肾细胞(MDBK)类泛素基因转录水平的影响。对致细胞病变型(cp型)BVDV标准株NADL毒株及非致细胞病变型(ncp型)BVDV分离株shz 132毒株进行增殖,通过实时荧光定量PCR(RT-qPCR)反应测定2种病毒拷贝数,按照Reed-Muench法测定BVDV NADL病毒TCID50值。以100TCID50BVDV NADL病毒和同等拷贝数的BVDV shz 132病毒分别感染MDBK细胞不同时间(4、12、24、48h)后收集细胞,提取细胞总RNA,反转录得到cDNA。以cDNA为模板,采用特异性引物,通过RT-qPCR反应检测细胞类泛素基因SUMO1、SUMO2、SUMO3、Ubc9的转录水平。结果显示,致细胞病变型BVDV NADL感染MDBK细胞48h,引起细胞显著的病变,而非致细胞病变型BVDV shz 132感染不引起细胞病变。BVDV NADL和shz 132的病毒拷贝数分别是1.13×1011 copies/mL和1.77×1011 copies/mL,BVDV NADL的TCID50是10-4.9 TCID50/0.1mL。RT-qPCR分析显示,与对照组相比,2种BVDV病毒感染都能引起MDBK细胞SUMO1、SUMO2、SUMO3、Ubc9基因的相对表达量变化。在BVDV shz 132感染的细胞中,SUMO1、SUMO2、SUMO3的相对表达量在各个感染时间都高于或接近于BVDV NADL感染的细胞,且在感染后24h,以上3个基因的相对表达量下调,差异极显著(P<0.01);而Ubc9基因的相对表达量则低于或接近于BVDV NADL感染的细胞,且在感染后24h,其相对表达量上调,差异极显著(P<0.01)。结果表明,BVDV感染调控了MDBK细胞SUMO系统,提示细胞SUMO系统参与了BVDV在胞内的活动,且这种调控作用与病毒的生物型存在密切联系。This study explored the effects of bovine viral diarrhea virus（BVDV）infection on SUMO gene transcription levels in Madin-Darby Bovine Kidney（MDBK）cells.The standard NADL strain of cytopathogenic（CP）BVDV and the isolation shz 132 strain of non-cytopathogenic（NCP）BVDV were proliferated.Copies of the two viruses were measured by real-time fluorescence quantitative PCR（RT-qPCR）and TCID50 value of NADL strain was measured based on the Reed-Muench method.MDBK cells were infected with 100TCID50 BVDV NADL virus and equal copies of BVDV shz 132 virus respectivly,and collected after different infecting time（4,12,24 hand 48h）.Total RNA of cells were isolated and cDNA were obtained.Transcription levels of SUMO1,SUMO2,SUMO3 and Ubc9genes were measured by RT-qPCR with special primers and cDNA template.Results showed that MDBK cells infected with CP NADL virus showed significant cytopathic effect after 48 h,while MDBK cells infected with NCP type shz 132 virus showed no cytopathic effect.Copies of NADL and shz 132 viruses were 1.13×1011 copies/mL and 1.77×1011 copies/mL respectively,and the TCID50 value of NADL was 10-4.9 TCID50/mL.RT-qPCR analysis showed that compared with control group,relative expression values of SUMO1,SUMO2,SUMO3 and Ubc9genes of MDBK cells were changed by two virus infections.Relative values of SUMO1,SUMO2,SUMO3 in BVDV shz 132 infected cells were higher than or close to that in BVDV NADL infected cells at each infecting time,and were significantly down regulated post infection 24h（P0.01）.However,relative value of Ubc9 gene was significantly up regulated post infection 24h（P0.01）compared with SUMO genes.All of these results indicated that SUMO system of cells was regulated by BVDV infection,meaning that SUMO system is involved in the intracellular acitivity of BVDV,and this regulation is related with biolgolical types of BVDV.

关 键 词：牛病毒性腹泻病毒 牛胚肾细胞 类泛素基因 

分 类 号：S852.65[农业科学—基础兽医学]