 10^6^ cells/well) were cultured in 12-well plates and stimulated with purified-PRRSV antigen (10 µg/mL) for 6 h at 37℃. Brefeldin A (BFA; Becton, Dickinson and Company, USA) was added to block the release of cytokine for the last 2 h. Next, the cells were resuspended for immunofluorescent staining. Specifically, the cells were stained with fluorescein isothiocyanate (FITC)-labeled anti-Mouse-CD4 or allophycocyanin (APC)-labeled anti-Mouse-CD8a monoclonal antibodies in the dark for 30 min at 4℃. After washing with PBS, the cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% saponin at room temperature, then stained with phycoerythrin (PE)-labeled anti-mouse-IFNγ, anti-mouse-IL4 antibody, or Rat IgG1 isotype control (eBioscince, USA) overnight at 4℃. Finally, the cells were washed and analyzed with a FACSCalibur using the Cell Quest Pro Software (BD Bioscience, USA).

Statistical analysis
--------------------

Differences between groups/samples were identified by a Student\'s *t* test, and *p* \< 0.05 was considered statistically significant.

Results
=======

Construction of recombinant Yeast-GP5
-------------------------------------

The pKLAC1 encoding GP5 was constructed and verified by DNA sequencing, after which the vector was digested with *BstX*I to create a linear DNA fragment that was inserted into the native LAC4 promoter by homologous recombination in *K. lactis* GG799 ([Fig. 1A](#F1){ref-type="fig"}). The frequency of correctly targeted vector integration at LAC4 was assessed by whole-cell genomic PCR with Integration Primers. As shown in [Figs. 1B and C](#F1){ref-type="fig"}, recombinant Yeast-GP5 and Yeast-vector yielded 1.9 kb and 2.3 kb fragments when amplified with primers P1/P2 and P2/P3, respectively, indicating that these vectors undergo multicopy integration. Expression of GP5 in recombinant *K. lactis* was confirmed by western blot using PRRSV GP5-specific antibody ([Fig. 1E](#F1){ref-type="fig"}). Yeast-GP5-His, which was detected by anti-His-tag antibody, was used