Plasmids construction
All enzymes used for cloning procedures were purchased from Takara except T4 DNA ligase from New England Biolabsand KOD-plus-Neo DNA polymerase from TOYOBO. UL31 ORF (composed of 813 bp) was amplified via PCR from the genomic DNA of the PRV Becker strain using a genome previously purified from vBecker2-infected PK-15 cells[3, 5],as the template. After the PCR amplified product was validated as the intended product, it was purified using a PCR gel purification kit (Qiagen) according to the manufacturer’s instructions. The product was then digested with EcoRI and BamHI and inserted into the correspondinglydigested green fluorescent protein variant mammalian expression vector pEYFP-N1(Clontech)encoding EYFPat 16oC overnight using T4 DNA ligase, to createthe recombinant plasmid named pUL31-EYFP,as described previously[2-4].After mini-scale isolation of plasmid DNAusing Qiagen plasmid Mini kits (Qiagen), the presence of the appropriate insert in the obtained plasmid was confirmed by PCR, restriction analysisand sequencing.The series of mutant deletions including amino acids substitution were generated by PCR-ligation-PCR mutagenesis [1]using appropriate primers (sequences available upon request)and cloned into pEYFP-N1as described above, all the N terminal deleted fusion proteins were inserted with an artificial ATG start codon starting at internal positions in UL31 of the fusion constructs.Finally, allconstructs were verified byPCR, restriction analysisand sequencing.
Transfection andfluorescence microscopy
To express the proteinsin vitro, COS-7 cells were plated onto six-well plates (Corning)in DMEM with 10%FBS at a density of ~2.5×105 cells per well overnight to 60-80% confluency before transfection. The next day, monolayer of COS-7 cells were transfected with 1.0 to 1.5 ug plasmid DNA mixed with Turbofet transfection reagent (Thermo Scientific) according to the manufacturer’s instructions. The transfection efficiency (~60%)was determined by transfection with EYFP. At 24 h posttransfection, cells were washed with fresh growth medium and processed for fluorescence microscopy. In the same experiment, each transfection was performed for at least three times. Data shown were from one representative experiment.Samples were analyzed using a ZeissAxiovert 200M microscope (Germany). All the photomicrographswere taken under a magnification of 400×. Each photomicrographrepresents a vast majority of the cells with similar subcellular localization.Fluorescent images of EYFP fusion proteins were presented in pseudocolor green, and images were processed using Adobe Photoshop.
References
1.Ali SA, Steinkasserer A (1995) PCR-ligation-PCR mutagenesis: a protocol for creating gene fusions and mutations. Biotechniques 18:746-750
2.Li ML, Wang L, Ren XM, Zheng CF (2011) Host cell targets of tegument protein VP22 of herpes simplex virus 1. Arch Virol 156:1079-1084
3.Li ML, Wang S, Cai MS, Zheng CF (2011) Identification of nuclear and nucleolar localization signals of pseudorabies virus (PRV) early protein UL54 reveals that its nuclear targeting is required for efficient production of PRV. J Virol 85:10239-10251
4.Li ML, Li Z, Li WT, Wang BY, Ma CQ, Chen JH, Cai MS (2012) Preparation and characterization of an antiserum against truncated UL54 protein of pseudorabies virus. Acta Virol 56:315-322
5.Smith GA, Enquist LW (2000) A self-recombining bacterial artificial chromosome and its application for analysis of herpesvirus pathogenesis. Proc Natl Acad Sci U S A 97:4873-4878