牛病毒性腹泻病毒的RT-PCR检测及感染犊牛相关基因差异表达分析  

RT-PCR Detection of Bovine Viral Diarrhea Virus Subtypes and Analysis of Differentially Expressed Candidate Genes in Infected Calves

作　　者：米思远 师科荣[2] 张瑞强 张海亮[1] 徐庆磊 俞英[1] MI Si-yuan;SHI Ke-rong;ZHANG Hal liang;XU Qiing-lei;YU Ying(College of Animal Science and Technolo Beijing 100193,China;College of Animal Science and Technology,Shandong Agricultural University,Tai＇an 271018,China)

机构地区：[1]中国农业大学动物科技学院,北京100193 [2]山东农业大学动物科技学院,泰安271018

出　　处：《畜牧兽医学报》2018年第7期1432-1439,共8页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家奶牛产业技术体系项目(CARS-36);北京市奶牛产业创新团队项目(BAIC06);长江学者与创新团队发展计划(IRT1191)

摘　　要：本研究选择SUMO类基因(SUMO1、SUMO2、SUMO3、UBC9)与CD4基因作为牛病毒性腹泻病毒(BVDV)侵染机体的相关候选基因,以期筛选出BVDV感染牛与未感染牛间的差异表达基因,为抗BVDV犊牛分子标记的发掘提供依据。采集42头中国荷斯坦犊牛(2月龄以内)的血样,通过特异性巢式PCR及测序方法检测犊牛是否感染BVDV;再通过荧光定量PCR检测相关候选基因在BVDV感染牛与未感染牛之间的表达差异。结果表明:42头犊牛中有13头感染BVDV-1型病毒,阳性感染率为30.9%;运用DNAMAN软件进行比对分析,发现13段由扩增得到的BVDV-1病毒的5′非翻译区(untranslated region,UTR)相似性达到95.80%;候选基因中SUMO1、SUMO2和SUMO3的表达量在BVDV-1感染牛及未感染牛间存在显著差异(P<0.05),感染牛呈显著性下调。但UBC9与CD4基因在两组间并未呈现显著性差异。本次试验从样品中仅检测出了BVDV-1型病毒,而没有检测出BVDV-2型病毒,这符合我国BVDV的流行趋势;BVDV-1侵染机体的过程可能与机体SUMO化存在关系,SUMO1、SUMO2和SUMO3可作为与BVDV感染有关的候选基因。To explore molecular markers of anti bovine viral diarrhea virus（BVDV）infection in dairy calves,bovine SUMOs（SUMO1,SUMO2,SUMO3,UBC9）and CD4 were selected as related candidate genes,and their differential expression status between BVDV infected and uninfected Holstein calves were detected and analyzed.Blood samples of 42 Holstein calves（Within 2 months old）were collected and whether they carried BVDV or not were identified by specific nested PCR and sequencing techniques.According to the analyses of the nested PCR and sequencing,the calves were divided into two groups（BVDV-carried and un-carried groups）to identify differentially expressed candidate genes of resistance BVDV by fluorescence quantitative PCR.The results showed that 13 cattle of the 42 Holstein calves carried BVDV-1（positive infection rate is30.9%）;the homologies of these 5′UTR reach 95.80% by the analysis of DNAMAN software;compared with the un-carried group,there were significant decreases（P〈0.05）in the BVDVcarried calves for the expression of SUMO1,SUMO2 and SUMO3 genes.But there was no significant difference between the two groups for the expression of UBC9 and CD4.Only BVDV-1 was detected in the Holstein calves,which is consistent with the epidemic trend of BVDV in China.The results suggest that there may have a close relationship between BVDV-1 infection and sumoylation,SUMO1,SUMO2 and SUMO3 could be served as candidate genes of bovine BVDV resistance.

关 键 词：犊牛腹泻 牛病毒性腹泻病毒 SUMOs UBC9 CD4 

分 类 号：S852.659.6[农业科学—基础兽医学] S858.235.3[农业科学—兽医学]