ICP34.5 Protein Enhances The Replication Of Recombinant Type II Herpes Simplex Virus

Objective:To establish the stably transfected cell line and explore the effects of type?(HSV1)and type ?(HSV2)gene products on the replication of recombinant type ? herpes simplex virus(HSV2).Methods: HSV1-ICP34.5 and HSV2-ICP34.5gene sequences were synthesized,connected to Piggy Bac-Dual-promoter(PBDP)eukaryotic expression vector,and confirmed by sequencing.The recombinant eukaryotic expression vector was transfected into Vero cells and BHK cells to construct a stable transfected cell line expressing ICP34.5 gene.Fluorescence microscope and flow cytometry were used to detect the expression of green fluorescent protein(GFP,which is an indicator of stable conversion success),and PCR amplification,real-time fluorescence quantitative PCR and immunoprinting were used to identify the target gene and expression of ICP34.5.The stable transfected cell line was used to produce o HSV2 virus,and the virus titer was detected by CCID50 method to evaluate the effect of ICP34.5 protein on o HSV2 replication.Res ? lts: A total of 4 stable transfected cell lines were obtained,namely VeroHSV1-ICP34.5?VeroHSV2-ICP34.5?BHKHSV1-ICP34.5? BHKHSV2-ICP34.5;all four new stable transfected cell lines were able to function under fluorescence microscope The expression of green fluorescent protein(GFP)was observed,and the proportion of positive cells detected by flow cytometry was more than 90%;all 4 newly stabilized cell lines were able to express ICP34.5 protein by immunostaining,amplified by PCR and fluorescent Quantitative PCR can identify the ICP34.5 gene;virus production and titer res ? lts show that VeroHSV1-ICP34.5,BHKHSV1-ICP34.5,and BHKHSV2-ICP34.5compared with control cells can promote the replication of o HSV2,To increase the virus titer(yield),statistical analysis has a significant difference(P values were 0.0109,0.0484,0.0125).VeroHSV2-ICP34.5has a tendency to promote o HSV2 virus replication compared with control cells,but there is no significant difference in statistical analysis(P value is0.1506).Conclusion: the new cell lines(VeroHSV1-ICP34.5cells,VeroHSV2-ICP34.5cells,BHKHSV1-ICP34.5cells and BHKHSV2-ICP34.5cells)with stable expression of ICP34.5 gene were successfully constructed.ICP34.5 protein can strengthen restructuring ? herpes simplex virus type(o HSV2)to copy,to improve the drop degree o HSV2 virus.