Protection of chickens against infectious bronchitis virus with a multivalent DNA vaccine and boosting with an inactivated vaccine

Yan, Fang; Zhao, Yujun; Hu, Yongting; Qiu, Jianyang; Lei, Wenxin; Ji, Wenhui; Li, Xuying; Wu, Qian; Shi, Xiumin; Li, Zhong

A T cell proliferation assay was performed as previously described [1] with some modification. Peripheral blood mononuclear cells (PBMCs) were isolated by Lympholyte-Mammal (Cedarlane, Canada). Briefly, 2 mL freshly heparinized whole blood was diluted with Hanks' balanced salt solution in a ratio of 1：1 and carefully add on top of 2 mL lympholyte-mammal separation medium, centrifuge at 400 × g/min for 20 min, aspirate coat of leukocyte, add 4 mL KPMI 1640 (Invitrogen, USA), centrifuge at 400 × g/min for 20 min, discard supernatant and wash precipitation cell twice by the same method. The cells were resuspended in RPMI 1640 medium (Invitrogen, USA) supplemented with 5% fetal bovine serum (Invitrogen, USA), 100 μg/mL penicillin (Invitrogen, USA), and 100 μg/mL streptomycin (Invitrogen, USA) at a concentration of 1 × 10 7 cells/mL. The freshly isolated PBMCs (10