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

Abstract:

[Objective] 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. [Methods] 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. [Results] 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. [Conclusion] 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.

Key words:EHV-1;gD envelope protein;subcellular localization;fluorescent protein tag