Evaluation on the Efficacy and Immunogenicity of Recombinant DNA Plasmids Expressing Spike Genes from Porcine Transmissible Gastroenteritis Virus and Porcine Epidemic Diarrhea Virus

Meng, Fandan; Ren, Yudong; Suo, Siqingaowa; Sun, Xuejiao; Li, Xunliang; Li, Pengchong; Yang, Wei; Li, Guangxing; Li, Lu; Schwegmann-Wessels, Christel; Herrler, Georg; Ren, Xiaofeng

Cytotoxicity was analyzed using a lactate dehydrogenase (LDH) release assay kit according to the manufacturer's instructions (Jiancheng, Nanjing, China) using splenic lymphocytes and blood from 42 dpi mice. Lymphocytes, suspended in complete RPMI 1640 medium were used as effector cells. Simultaneously, target ST and Vero cells were infected with TGEV and PEDV, respectively, at a titer of 100TCID 50 for 36 h at 37uC under 5% CO 2 . The effector cells were mixed with the sensitized target cells at 25:1, added to each well of a 96-well round-bottom microplate, and incubated for 6 h at 37uC. After centrifugation at 1500 rpm for 10 min, 100 ml of supernatant was collected and transferred to a fresh 96-well flat-bottomed plate, followed by the addition of 100 ml/well of LDH assay reagent. The mixture was allowed to incubate for 15 min at 37uC after which the OD 490 was measured. Spontaneous release of LDH was determined using samples prepared from target cells cultured in medium alone; maximum LDH release was measured using samples prepared by lysis of target cells in medium containing 1% (v/v) Triton X-100. The CTL was calculated using the following equation: [(experimental release-spontaneous release)/(maximum release-spontaneous release)] x 100. All experiments were performed in triplicate.