不同品种猪感染HP-PRRSV后肺组织间PRRSV受体基因差异表达的研究
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摘要
为进一步了解藏猪、藏梅猪和大约克夏猪对高致病性猪繁殖与呼吸综合征病毒(HP-PRRSV)JXA1分离株具有不同易感性的分子机理,本研究建立实时荧光定量RT-PCR方法比较分析藏猪、藏梅猪和大约克夏猪感染JXA1分离株前、感染后4、7和14d肺脏中HSPG2、SIGLEC1、CD163、VIM和NMMHC-ⅡA5个PRRSV受体基因的差异表达情况。结果显示,藏猪感染JXA1分离株后4和14d肺脏中HSPG2的表达量显著高于感染前(P<0.05),而感染后7dHSPG2的表达量显著低于感染前(P<0.05),藏梅猪在感染后14dHSPG2的表达量显著高于感染后7d(P<0.05);藏猪感染后4和14d肺脏中SIGLEC1的表达量显著高于感染前(P<0.05),大约克夏猪感染后4、7和14dSIGLEC1的表达量均显著低于感染前(P<0.05);藏猪和藏梅猪感染后14d肺脏中CD163的表达量均显著高于感染前(P<0.05),大约克夏猪则感染后7和14d均显著低于感染前(P<0.05);藏猪感染后7d肺脏中VIM的表达量显著高于感染前(P<0.05),大约克夏猪在感染后7d显著低于感染前(P<0.05);藏梅猪感染后4d肺脏中NMMHC-ⅡA的表达量显著高于感染前(P<0.05),大约克夏猪在感染后4和14dNMMHC-ⅡA的表达量显著高于感染前(P<0.05)。结果表明,5个PRRSV受体基因中,SIGLEC1和VIM基因可能是影响藏猪、藏梅猪和大约克夏猪对JXA1分离株易感性存在差异的潜在关键基因。
In order to understand the molecular mechanism of host susceptibility differential to highly pathogenic porcine reproductive and respiratory syndrome(HP-PRRSV)infection among three breeds of pig,Tibetan pig,ZangMei Black and Yorkshire,the mRNA level of five reported PRRSV receptor genes(HSPG2,SIGLEC1,CD163,VIM and NMMHC-ⅡA)expressed in lung were investigated on 0,4,7and 14 days post infection(dpi)with HP-PRRSV JXA1 isolate.The Real-time RT-PCR results showed that the expression of HSPG2 in Tibetan pig increased significantly on 4and 14 dpi and decreased significantly on 7dpi compared with the expression on 0dpi(P<0.05),and its expression in ZangMei Black on 14 dpi was significantly higher than that of on 7dpi(P<0.05);An increased expression of SIGLEC1 in Tibetan pig was observed on 4and14dpi(P<0.05),and a decreased expression of the same gene was detected in Yorkshire on 4,7and 14dpi(P<0.05);The expression of CD163 elevated significantly on 14 dpi in both Tibetan pig and ZangMei Black(P<0.05),however its expression reduced significantly on 7and 14 dpi in Yorkshire(P<0.05);The expression of VIM gene was significantly higher on 7dpi in Tibetan pig and significantly lower on 7dpi in Yorkshire(P<0.05);And the expression of NMMHC-ⅡA was significantly higher on 4dpi in ZangMei Black,which was also significantly higher in Yorkshire on 4and 14dpi(P<0.05).In conclusion,SIGLEC1 and VIM might be important genes to influence the host resistance of Tibetan pig,ZangMei Black and Yorkshire to HP-PRRSV infection.
In order to understand the molecular mechanism of host susceptibility differential to highly pathogenic porcine reproductive and respiratory syndrome(HP-PRRSV)infection among three breeds of pig,Tibetan pig,ZangMei Black and Yorkshire,the mRNA level of five reported PRRSV receptor genes(HSPG2,SIGLEC1,CD163,VIM and NMMHC-ⅡA)expressed in lung were investigated on 0,4,7and 14 days post infection(dpi)with HP-PRRSV JXA1 isolate.The Real-time RT-PCR results showed that the expression of HSPG2 in Tibetan pig increased significantly on 4and 14 dpi and decreased significantly on 7dpi compared with the expression on 0dpi(P<0.05),and its expression in ZangMei Black on 14 dpi was significantly higher than that of on 7dpi(P<0.05);An increased expression of SIGLEC1 in Tibetan pig was observed on 4and14dpi(P<0.05),and a decreased expression of the same gene was detected in Yorkshire on 4,7and 14dpi(P<0.05);The expression of CD163 elevated significantly on 14 dpi in both Tibetan pig and ZangMei Black(P<0.05),however its expression reduced significantly on 7and 14 dpi in Yorkshire(P<0.05);The expression of VIM gene was significantly higher on 7dpi in Tibetan pig and significantly lower on 7dpi in Yorkshire(P<0.05);And the expression of NMMHC-ⅡA was significantly higher on 4dpi in ZangMei Black,which was also significantly higher in Yorkshire on 4and 14dpi(P<0.05).In conclusion,SIGLEC1 and VIM might be important genes to influence the host resistance of Tibetan pig,ZangMei Black and Yorkshire to HP-PRRSV infection.
引文
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[21]Zhang H,Guo X,Ge X,et al.Changes in the cellular proteins of pulmonary alveolar macrophage infected with porcine reproductiveand respiratory syndrome virus by proteomics analysis[J].J Proteome Res,2009,8(6):3091-3097.
[22]Zhou E M,Xiao Y,Shi Y,et al.Generation of internal image monoclonal anti-idiotypic antibodies against idiotypic antibodies to GP5antigen of porcine reproductive and respiratory syndrome virus[J].J Virol Methods,2008,149(2):300-308.
[23]Rey M,Valenzuela-Fernández A,Urzainqui A,et al.MyosinⅡA is involved in the endocytosis of CXCR4induced by SDF-1alpha[J].J Cell Sci,2007,120(6):1126-1133.
[2]Lager K M,Halbur P G.Gross and microscopic lessions in porcine fetuses infected with porine reporductive and respiratory syndrome virus[J].J Vet Diagn Invest,1996,8(3):275-282.
[3]loula T.Mystery pig disease[J].Agri-practice,1991,12(1):23-24.
[4]Cho J C,Dee S A.Porcine reproductive and respiratory syndrome virus[J].Theriogenology,2006,66(3):655-662.
[5]李彬,王豪男,彭忠,等.华中地区猪繁殖与呼吸综合征病毒临床流行毒株Nsp2和GP5氨基酸的遗传变异分析[J].中国畜牧兽医,2015,42(11):3026-3036.
[6]王振华,曹肖涵,宋禾,等.猪繁殖与呼吸综合征病毒的危害及免疫策略[J].中国畜牧兽医,2014,41(2):239-243.
[7]Halbur P G,Rothschild M F,Thacker B J,et al.Differences in susceptibility of Duroc,Hampshire,and Meishan pigs to infection with a high virulence strain(VR2385)of porcine reproductive and respiratory syn-drome virus(PRRSV)[J].Journal of Animal Breeding and Genetics,1998,115(3):181-189.
[8]Jiang C,Xing F,Xing J,et al.Different expression patterns of PRRSV mediator genes in the lung tissues of PRRSV resistant and susceptible pigs[J].Dev Comp Immunol,2013,39(1-2):127-131.
[9]Liang W,Li Z,Wang P,et al.Differences of immune responses between Tongcheng(Chinese local breed)and Large White pigs after artificial infection with highly pathogenic porcine reproductive and respiratory syndrome virus[J].Developmental&Comparative Immunology,2016,215:84-93.
[10]Kang R M,Ji G S,Yang X,et al.Investigation on host susceptibility of Tibetan pig to infection of porcine reproductive and respiratory syndrome virus through viral challenge study[J].Veterinary Microbiology,2016,183:62-68.
[11]Shi C,Liu Y,Ding Y,et al.PRRSV receptors and their roles in virus infection[J].Arch Microbiology,2015,197(4):503-512.
[12]Tian K,Yu X,Zhao T,et al.Emergence of fatal PRRSV variants:Unparalleled outbreaks of atypical PRRS in China and molecular dissection of the unique hallmark[J].PLoS One,2007,2(6):e526.
[13]Zhou Y S,Yang X,Wang H Y,et al.Molecular characterization of a complete genome and 12 Nsp2genes of PRRSV of Southwestern China[J].Food Environ Virol,2012,4(3):102-114.
[14]Uddin M J,Cinar M U,Tesfaye D,et al.Age-related changes in relative expression stability of commonly used housekeeping genes in selected porcine tissues[J].BMC Res Notes,2011,4:441.
[15]Delputte P L,Costers S,Nauwynck H J.Analysis of porcine reproductive and respiratory syndrome virus attachment and internalization:Distinctive roles for heparan sulphate and sialoadhesin[J].J Gen Virol,2005,86(5):1441-1445.
[16]Delputte P L,Vanderheijden N,Nauwynck H J,et al.Involvement of the matrix protein in attachment of porcine reproductive and respiratory syndrome virus to a heparinlike receptor on porcine alveolar macrophages[J].J Virol,2002,76(9):4312-4320.
[17]Breedam W V,Gorp H V,Zhang J,et al.The M/GP(5)glycoprotein complex of porcine reproductive and respiratory syndrome virus binds the sialoadhesin receptor in a sialic acid-dependent manner[J].PLoS Pathog,2010,6(1):el000730.
[18]Ait-Ali T,Wilson A D,CarréW,et al.Host inhibits replication of European porcine reproductive and respiratory syndrome virus in macrophages by altering differential regulation of type-Ⅰinterferon transcriptional response[J].Immunogenetics,2011,63(7):437-448.
[19]Calvert J G,Slade D E,Shields S L,et al.CD163expression confers susceptibility to porcine reproductive and respiratory syndrome viruses[J].J Virol,2007,81(14):7371-7379.
[20]Kim J K,Fahad A M,Shanmukhappa K,et al.Defining the cellular target(s)of porcine reproductive and respiratory syndrome virus blocking monoclonal antibody 7G10[J].J Virol,2006,80(2):689-696.
[21]Zhang H,Guo X,Ge X,et al.Changes in the cellular proteins of pulmonary alveolar macrophage infected with porcine reproductiveand respiratory syndrome virus by proteomics analysis[J].J Proteome Res,2009,8(6):3091-3097.
[22]Zhou E M,Xiao Y,Shi Y,et al.Generation of internal image monoclonal anti-idiotypic antibodies against idiotypic antibodies to GP5antigen of porcine reproductive and respiratory syndrome virus[J].J Virol Methods,2008,149(2):300-308.
[23]Rey M,Valenzuela-Fernández A,Urzainqui A,et al.MyosinⅡA is involved in the endocytosis of CXCR4induced by SDF-1alpha[J].J Cell Sci,2007,120(6):1126-1133.