[Impact of fluorescent protein tag on gD envelope protein subcellular localization in BHK-21 cells]

The fluorescent protein and gD envelope protein of equine herpes virus type 1 (EHV-1) were used to study the impact of tags on gD protein subcellular localization in BHK-21 cells.

With the EHV-1 genome as a template, the gD complete gene was amplified by PCR technique. The product of PCR was cloned to pAcGFP1-C1 and pDsRed2-N1 plasmids. The recombinant plasmids were designated as pAc-GFP-gD (GFP-gD) and pDs-gD-Red (gD-Red). The GFP gene was inserted into the posterior position of gD gene signal peptide sequence. The modified gD gene signal peptide sequence was cloned to pVAX-1 plasmid, so that pVAX-S-GFP-gD' (S-GFPgD') recombinant plasmid was constructed. Meanwhile, the flag tag was added to N-terminal of gD sequence and they were cloned to pVAX-1 expression vector for constructing pVAX-Flag-gD recombinant plasmid. The BHK-21 cells were transfected with the 4 different recombinant plasmids and the subcellular localizations of fusion proteins were determined by lasar confocal scan microscopy.

Four eukaryotic expression vectors were constructed successfully. In BHK-21 cells, the vast majority of gD envelope proteins was localized in Golgi, and a small amount of gD was localized in the nucleus.

Our finding reveals that the fluorescent protein of different insertion sites has no significant effects on the subcellular localization of gD, and provides a useful reference for other researchers.