| Herpes simplex virus (HSV) belongs to a subfamily of Herpes viridae virus, is one ofthe most common human pathogens disease. HSV-1and HSV-2is the two serotypes of HSV,the nucleic acid sequence homology of two types is as high as70%, but the mode of infectionand clinical manifestations are different, and the HSV-2infection is more common thanHSV-1infection. HSV-2infection is a complex process.Because of immune escapemechanism relating to the virus, the virus removal is not clean. Beside virus lurking in theganglion can be activated, thus it becomes a clinical problem. So the research on themechanism of latent and recurrence of HSV-2has important theoretical and practicalsignificance for drug development and for developing of the vaccine to clear the infectionpotential prevention of HSV-2infection to enhance the life quality of patients.ICP22is a kind of phosphorylation protein encoded by HSV IE/α gene, having aplurality of structural motifs, and is also a kind of very important factor regulating expressionof virus gene.a variety of functions in HSV infection:(I) ICP22can be localized in thenucleus and other viral proteins cooperating to shield the host kernel function to enhance virusinfection.(II) ICP22has strong inhibition on a variety of virus and cell promoter and enhancer,such as inhibition of P53-mdm-2transcription activation function.(III) ICP22influencesthe viral late gene activation and expression,through the influence of polymerase II. At thesame time, studies have shown that ICP22can influence the cell cycle protein activity andexpression level, and increase the number of cells in S phase for supporting virus replicationenvironment. Responsible for the activation of CDK2, ICP22decreased cyclin A and B levelsto block the cell in S phase. Therefore, ICP22may be associated with HSV-1reactivationfrom the latent state recurrence and entering the replication closely.Therefore, virus genome used as the template in the HSV-2333to amplify ICP22gene.After double enzyme digestion by HindIII and KpnI, ICP22gene was connected withvector pEGFPN1by T4ligase, identified the positive clones and sequencing by colony PCRand enzyme digestion. the recombinant plasmid was transfected into Vero cells with Xfectregents, Vero cells was collected for isolating total RNA and protein to detect the transcriptand expression of ICP22in Vero cells by RT-PCR and western blot at48h after transfection,respectively. pEGFP-ICP22was expressed abundantly in Vero cells from24h to48h aftertransfectionAfter pEGFP-ICP22transfected into Vero cells, MTT test results show that the activityof the expressed fusion protein had no obvious effect on cell. Cell cycle experiments showed that S cell cycle ratio of the recombinant plasmid group was significantly higher than that ofnormal control group, G2/M cycle ratio were significantly higher than those in normalcontrol group, indicating that HSV-2ICP22promote Vero cells into S phase, and the cell cansmoothly enter the G2/M phase.Fluorescence quantitative PCR results showed that theexpression of HSV-2ICP22can up-regulate the expression of cyclin E, but have no obviouseffect on the expression of cyclin A and cyclin B, at the same time, the WB resultsdemonstrates the differential expression of cyclin E.Based on the above results, it show that HSV-2ICP22can up-regulate the expression ofcyclin E, activating CDK2to promote the activation of cells in S phase, but did not arrest thecell in S phase by decreasing cyclinAand B levels. |
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