All animal experiments were performed according to national and institutional guidelines. One hundred and ninety female BALB/c mice (Vital River Laboratory Animal Technology Co., Beijing, China) were maintained in isolation cages at the Experimental Animal Center of Harbin Veterinary Research Institute (Harbin, China). Mice were divided into three groups: the pVAX1-Ub-M vaccination group (n = 120), the pVAX1 control vaccination group (n = 35) and the PBS control vaccinated group (n = 35). The plasmid DNA used for immunization was purified using the EndoFree Mega plasmid preparation kit (Qiagen). The pVAX1-Ub-M and pVAX1 cohorts were intramuscularly (i.m.) vaccinated with 100 μg pVAX1-Ub-M or pVAX1 plasmid DNA in 100 μl PBS, respectively. The PBS control group received an i.m. injection of 100 μL PBS. Each group was vaccinated four times at 3-week intervals. To enhance the specific CTL responses to M protein, the mice received an intraperitoneal (i.p.) injection containing 0.1 MOI of rWR-PRRSV-M on day 7 following the fourth DNA vaccination. At the same time, five mice from the pVAX1 and PBS vaccination groups were also inoculated intraperitoneally with 0.1 MOI of rWR-PRRSV-M on day 7 following the fourth vaccination in order to compare the specific antibody raised against M protein of different experimental groups following vaccination with rWR-PRRSV-M. All procedures were conducted with the protocols approved by Experimental Animal Center of Harbin Veterinary Research Institute (HVRI) of the Chinese Academy of Agricultural Sciences (CAAS).