Porcine IL-12 plasmid as an adjuvant improves the cellular and humoral immune responses of DNA vaccine targeting transmissible gastroenteritis virus spike gene in a mouse model

LI, Xunliang; LI, Pengchong; CAO, Liyan; BAI, Yunyun; CHEN, Huijie; LIU, He; REN, Xiaofeng; LI, Guangxing

The full-length S gene of TGEV strain PUR46 (GenBank: M94101.1) was used as a polymerase chain reaction (PCR) template. PCR for TGEV S1 gene amplification was performed using the forward primer (P1) (5′-GGGGAAGCTTATGAAAAAACTATTTGTG-3′) and reverse primer (P2) (5′-CCCCGAATTCTTAGTTAGTTTGTCTAATA-3′) carrying a HindIII (P1) and an EcoRI (P2) restriction enzyme site (underlined), respectively. The PCR parameters for TGEV-S1 gene amplification (primers P1/P2) were as follows: 95°C for 5 min, 30 cycles of 94°C for 1 min, 45.7°C for 1 min, and 72°C for 2 min, followed by a final extension of 72°C for 10 min. Products were purified and subjected to restriction enzyme digestion, followed by ligation into the appropriate pVAX1 (Invitrogen, Carlsbad, CA, U.S.A.) eukaryotic expression vector. A recombinant plasmid expressing the entire pIL-12, comprising p40 and p35 genes, was constructed by overlap extension PCR. The plasmid was previously constructed in our laboratory [13] . Clones were picked and identified by PCR amplification, and then named as pVAX1-(TGEV-S1) and pVAX1-(pIL-12).