Abstract:

In order to perform transcription kinetics,eukaryotic expression and subcellular localization of ORFV113 protein.DNAStar software was used to perform alignment analysis of ORFV113 gene.In the presence or absence of cytarabine,Hela cells were harvested and the total RNA were extracted at various time points(2,4,6,12,24 h)post ORFV infection.RT-PCR was used to amplify ORFV113 gene to determine the dynamic transcription level of ORFV113 gene.ORFV113 gene was amplified by PCR and subcloned into pEGFP-N1 vector to construct pEGFP-ORFV113 recombinant plasmid.After being correctly identified by restriction enzyme digestion and Sanger sequencing,the pEGFP-N1 plasmid and pEGFP-ORFV113 plasmid was transiently transfected into HEK293 cells with Lipofectamine 3000.The expression of ORFV113-EGFP fusion protein in HEK293 cells was detected by Western Blot.Hela cells were transiently transfected with pEGFP-N1 plasmid and pEGFP-ORFV113 recombinant plasmid.After 24 h,the nucleus was stained with Hoechst 33342,and the subcellular localization of ORFV113 protein was observed under an inverted fluorescence microscope.The results revealed that ORFV113 gene was 627 bp in length,which was highly conserved among ORFV strains.It belonged to an early gene of ORFV.The pEGFP-ORFV113 recombinant plasmid was successfully constructed.The ORFV113-EGFP fusion protein with a molecular mass between 60 and 70 ku,approximately 10—20 ku larger than the predicted molecular mass,was successfully expressed in HEK293 cells,indicating that ORFV113 protein had undergone post-translational modification.ORFV113 protein mainly localized to the cytoplasm of Hela cells.In conclusion,the transcription kineics,eukaryotic expression and subcellular localization of ORFV113 protein were successfully performed in the present study.

Key words: ORFV, ORFV113 protein, Transcription kinetics, Eukaryotic expression, Subcellular localization