Identification And Quantitative Analysis Of Differential Genes In Porcine Parvovirus Infected ST Cells

Porcine parvovirus(PPV) is one of the most important pathogens responsible for reproductive disorders including stillbirth,mummification,embryonic death and infertility。PPV strains differ in their pathogenicity,some studies reveals that the PPV can cause not only the reproductive failure but also the PMWS and PRDC.It also plays an important role in the co-infection of other pathogens.In order to select the differentially expressed genes in the PPV infected ST cells,two subtractive hybridization cDNA libraries were constructed,forward and reverse.The up-regulated and down-regulated genes were found in the two libraries,respectively.The total RNAs of PPV infected ST cells and control healthy cells were extracted simultaneously as the suggestion of Clontech PCR-Select^TM cDNA Subtraction Kit.The RNAs were transcript into double strain cDNA with the cocktail method.After digestion with Rsa I,a four-base-cutting restriction enzyme, the tester and driver cDNA were cut into 100bp-2000bp length.The tester cDNA was divided to two portions and ligated with a different adaptor at one end.Two hybridizations and nested PCRs were then performed.The differentially expressed genes were enriched.After the gel recovery,the differential genes were cloned into the pMD18-T vector and then transformed into the DH5αE.Coli cells.Two subtractive hybridization libraries with 100 positive clones in each were constructed.The Dot blot assay reveals that the positive ratios of the two libraries are 93%and 90%respectively.Thirty positive clones were selected randomly from the two subtractive hybridization libraries and sequenced.The sequences were aligned with the ESTs database with the Blast software.The results indicated that the forward subtractive hybridization library contained the up-regulated genes such as G protein gene,the heat shock protein gene 70,the aldehyde dehydrogenase family 1 member gene,ferritin gene,polyadenylate-binding protein,ribosomal protein gene and mitochondrion,the reverse subtractive hybridization library contained the down regulated genes such as acryl-coenzyme A binding protein gene, retinol binding protein 4 gene,tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein gene,sus scrofa secreted phosphoprotein 1 gene,heparin binding protein and calcium-binding protein gene and other unknown genes.Four pairs of primers specific for G protein gene,heat shock protein 70 gene,polyadenylate-binding protein,and mitochondrion gene were designed with the Oligo 6 software.The real-time PCR with SYBR Green I were established.The sensitivity tests indicated that the detection limit of these methods were between 10¹-10³ gene copies/reaction.The specific analyses using the nun-target genes were negative.0.4 ml of PPV BQ strain was inoculated onto 10⁶ ST cells at the time of 0 h.The PPV infected cells and the control cells were cultured in CO₂ incubator for 12h,24h,36h,48h,60h,72h,84h,96h and then collected for RNA extraction.The mRNA concentrations of the G protein gene,heat shock protein gene,polyadenylate-binding protein gene,and mitochondrion gene were tested with the real-time PCR. The results indicated that the target genes in PPV infected cells were significantly higher than the control cells at 60 h.From 12 h to 96 h,the concentions of differential genes showed a unique curve.At the time of 12 h.the concentions of the G protein gene.heat shock protein gene.and polyadenylate-binding protein gene were higher then any other times.These four genes experience a silence at 24 h and then went up dramatically from 36h to 72h.The concentrations,of the mitochondrion gene didn't experience this silence at 24 h.It will go up substantially until the 72h.The concentrations of the selected genes were dropping sharply from 72 h because of the apoptosis of the cells.In this study,we constructed two subtractive hybridization libraries with the up- and down- regulated differential expressed genes.The interaction process between the PPV and the ST cells was analyzed which set a base for the study of the pathological mechanism of PPV.