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
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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.
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.
引文
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[22]张志超.包被叶酸提高围产期荷斯坦牛免疫力的分子基础研究[D].北京:中国农业大学,2016.ZHANG Z C.Study on molecular mechanisms of folic acid to improve the immunity of transition Holstein cow[D].Beijing:China Agricultural University,2016.(in Chinese)
[23]HE Y H,CHU Q,MA P P,et al.Association of bovine CD4and STAT5b single nucleotide polymorphisms with somatic cell scores and milk production traits in Chinese Holsteins[J].J Dairy Res,2011,78(2):242-249.
[24]CARR B V,LEFEVRE E A,WINDSOR M A,et al.CD4+T-cell responses to foot-and-mouth disease virus in vaccinated cattle[J].J Gen Virol,2013,94(Pt 1):97-107.
[25]DUBOVI E J.Laboratory diagnosis of bovine viral diarrhea virus[J].Biologicals,2013,41(1):8-13.
[26]GOYAL S M,RIDPATH J F.Bovine viral diarrhea virus:diagnosis,management and control[M].Oxford:Blackwell Publishing Professional,2005:190.
[27]FALKENBERG S M,JOHNSON C,BAUERMANN F V,et al.Changes observed in the thymus and lymph nodes 14days after exposure to BVDV field strains of enhanced or typical virulence in neonatal calves[J].Vet Immunol Immunopathol,2014,160(1-2):70-80.
[2]BAUERMANN F V,RIDPATH J F,WEIBLEN R,et al.HoBi-like viruses:An emerging group of pestiviruses[J].J Vet Diagn Invest,2013,25(1):6-15.
[3]HOUSE H.Epidemiology of bovine viral diarrhea virus[J].Vet Clin North Am Food Anim Pract,1995,11(3):521-547.
[4]LACKNER T,MLLER A,PANKRAZ A,et al.Temporal modulation of an autoprotease is crucial for replication and pathogenicity of an RNA virus[J].J Virol,2004,78(19):10765-10775.
[5]王炜,武华.牛病毒性腹泻病毒生物学特性、危害及防控[J].中国奶牛,2014(23-24):22-26.WANG W,WU H.Biological characteristics,hazards and control of bovine viral diarrhea virus[J].China Dairy Cattle,2014(23-24):22-26.(in Chinese)
[6]DARWEESH M F,RAJPUT M K S,BRAUN L J,et al.Characterization of the cytopathic BVDV strains isolated from 13mucosal disease cases arising in a cattle herd[J].Virus Res,2015,195:141-147.
[7]李娜,韩猛立,黄新,等.新疆石河子地区牛病毒性腹泻病毒的分子流行病学调查[J].石河子大学学报:自然科学版,2009,27(6):706-711.LI N,HAN M L,HUANG X,et al.Epidemiology investigation of bovine viral diarrhea virus in Shihezi,Xinjiang[J].Journal of Shihezi University:Natural Science,2009,27(6):706-711.(in Chinese)
[8]EVERMANN J F.Pestiviral infection of llamas and alpacas[J].Small Rumin Res,2006,61(2-3):201-206.
[9]HANSEN T R,SMIRNOVA N P,WEBB B T,et al.Innate and adaptive immune responses to in utero infection with bovine viral diarrhea virus[J].Anim Health Res Rev,2015,16(1):15-26.
[10]RICHTER V,LEBL K,BAUMGARTNER W,et al.A systematic worldwide review of the direct monetary losses in cattle due to bovine viral diarrhoea virus infection[J].Vet J,2017,220:80-87.
[11]黄凯,张俊杰,刘英霞,等.北京地区规模化牛场BVDV持续感染牛清除方案的初步实施[J].中国动物检疫,2010,27(5):34-35.HUANG K,ZHANG J J,LIU Y X,et al.Preliminary implemantation of BVDV persistently infected cow eradication program in dairy farms in Beijing area[J].Chinese Journal of Animal Health Inspection,2010,27(5):34-35.(in Chinese)
[12]RIDPATH J.Preventive strategy for BVDV infection in North America[J].Jpn J Vet Res,2012,60(Sl):S41-S49.
[13]韦玮,张浩,毛建平,等.蛋白质SUMO化修饰研究进展[J].中国生物工程杂志,2008,28(7):122-126.WEI W,ZHANG H,MAO J P,et al.Progress on the study of sumoylation[J].China Biotechnology,2008,28(7):122-126.(in Chinese)
[14]汪小杰,毛若雨,张勇,等.SUMO在蛋白表达中的应用[J].生物技术通报,2013(10):28-33.WANG X J,MAO R Y,ZHANG Y,et al.Application of SUMO protein in fusion expression system[J].Biotechnology Bulletin,2013(10):28-33.(in Chinese)
[15]刘慧敏,李文清,苏丽娟,等.SUMO化修饰在流感病毒感染中的作用研究进展[J].病毒学报,2015,31(5):560-564.LIU H M,LI W Q,SU L J,et al.Research progress inthe function of SUMOylation during infection by the influenza virus[J].Chinese Journal of Virology,2015,31(5):560-564.(in Chinese)
[16]PAL S,SANTOS A,ROSAS J M,et al.Influenza a virus interacts extensively with the cellular SUMOylation system during infection[J].Virus Res,2011,158(1-2):12-27.
[17]周鹏,张子平,王艺磊,等.大黄鱼ubc9基因的克隆和组织表达[J].生物技术通报,2009(8):76-82.ZHOU P,ZHANG Z P,WANG Y L,et al.Cloning and characterization of ubc9gene of large yellow croaker larimichthys crocea and its expression pattern[J].Biotechnology Bulletin,2009(8):76-82.(in Chinese)
[18]WU C Y,JENG K S,LAI M M C.The SUMOylation of matrix protein M1modulates the assembly and morphogenesis of influenza a virus[J].J Virol,2011,85(13):6618-6628.
[19]EVERETT R D,BOUTELL C,HALE B G.Interplay between viruses and host sumoylation pathways[J].Nat Rev Microbiol,2013,11(6):400-411.
[20]韩玉霞.BVDV体外感染与细胞SUMO通路关系的初步研究[D].石河子:石河子大学,2015.HAN Y X.Preliminary study of the relationship between bovine viral diarrhea virus infectionin vitro and SUMO pathway of cell[D].Shihezi:Shihezi University,2015.(in Chinese)
[21]CATALFAMO M,WILHELM C,TCHEUNG L,et al.CD4and CD8Tcell immune activation during chronic HIV infection:roles of homeostasis,HIV,type I IFN,and IL-7[J].J Immunol,2011,186(4):2106-2116.
[22]张志超.包被叶酸提高围产期荷斯坦牛免疫力的分子基础研究[D].北京:中国农业大学,2016.ZHANG Z C.Study on molecular mechanisms of folic acid to improve the immunity of transition Holstein cow[D].Beijing:China Agricultural University,2016.(in Chinese)
[23]HE Y H,CHU Q,MA P P,et al.Association of bovine CD4and STAT5b single nucleotide polymorphisms with somatic cell scores and milk production traits in Chinese Holsteins[J].J Dairy Res,2011,78(2):242-249.
[24]CARR B V,LEFEVRE E A,WINDSOR M A,et al.CD4+T-cell responses to foot-and-mouth disease virus in vaccinated cattle[J].J Gen Virol,2013,94(Pt 1):97-107.
[25]DUBOVI E J.Laboratory diagnosis of bovine viral diarrhea virus[J].Biologicals,2013,41(1):8-13.
[26]GOYAL S M,RIDPATH J F.Bovine viral diarrhea virus:diagnosis,management and control[M].Oxford:Blackwell Publishing Professional,2005:190.
[27]FALKENBERG S M,JOHNSON C,BAUERMANN F V,et al.Changes observed in the thymus and lymph nodes 14days after exposure to BVDV field strains of enhanced or typical virulence in neonatal calves[J].Vet Immunol Immunopathol,2014,160(1-2):70-80.