Construction And Immunogenicity Of Recombinant Adenovirus Expressing S Gene Fregments Of Porcine Epidemic Diarrhea Disease Virus In Mice

Porcine epidemic diarrhea virus (PEDV) is one of the etiological agents of swine diarrhea. Although the vaccine for PEDV had been commercialized, it still causing epidemic in China, and lead to great economic loses. So, it is very necessary to develop an effective vaccine to prevent the PEDV infection.S protein is one of the main structural protein of PEDV, it can induce neutral antibody in pigs. So, S gene is the main gene for researching the engineer vaccine. According to the sequence of PEDV JS strain(No.AY653204),four PCR primers, P1,P2,P3,P4 that were specific for S gene, were desigened and synthesized. Among them, P1 and P3 had the KpnI enzyme restriction site, and P2, P4 had XhoI enzyme restriction site. Fragments of 426bp (S_498-638), 1917bp (S_1-638), 2661bp (S_639-1384) were amplified by RT-PCR respectively. Then the PCR products were digested with KpnI/XhoI, and recollected with gel purification kit, and cloned into pShuttle-CMV vector. After tranformation to the competent cells DH_5a, the positive colonies were picked from Kan^r plate, cultured at 37℃over night. The plasmids were extracted form the bacteria, and identified with KpnI/XhoI digestion. Then the insert were sequenced and analysed with DNAstar, The results showed that these genes were successfully cloned into pShuttle-CMV in correct open reading frame (ORF). Compared with the PEDV JS strain S gene, these three S gene fragments showed 98.6%, 97.4% and 98.4% homology. These recombinant plasmids were linearized with PmeI and transformed to the competent E·coli BJ5183 containing backbone vector pAdEasy-1 by electroplating. After homologous recombination between linearized recombinant plasmid and pAdEasy-1 vector in E·coli BJ5183, the positive clones were selected by growing the culture on Kan^r plate. The recombinant plasmids were identified and linearized with Pad, transfected into 293A cells with anion lipid transfection reagent. After incubation of the transfected cells for 15 days at 37℃, obvious cell pathological effect (CPE) was observed. The expression of S gene fragments was identified by indirect immunofluorescent assay (IFN) with antibody against PEDV. These results showed that the recombinant adenoviruses could express these three target proteins of PEDV. These recombinant adenoviruses could be stably passaged in HEK-293A cells and TCID₅₀ was 10^-6.29 ,10^-6.5 and 10^-5.75/ml ,respectively.One hundred female mice(weighing 18-20g)were divided into five groups with 20 mice in each group. In group 1, 2 and 3, Mice were inoculated with three recombinant adenovirus rAd-S_498-638, rAd-S_1-638 and rAd-S_639-1384, respectively. In group 4 and 5, mice inoculated with PBS and wtAd were used as blank control and negative control respectively. Mice were immunized twice with interval of 14 days. At 14, 28, 42 and 56 days post primary immunization, five serum samples and lymphocytes were collected from mice in each group. The presence of antibodies against the viral proteins in the sera of mice was analyzed by ELISA and neutralizing antibody assay. And T-cells proliferation assay responses were detected by MTT. The results showed that mice immunized with these recombinant adenoviruses could develop PEDV-specific ELISA antibody and neutralizing antibody, but they couldn't elicit cellular immune response in mice. It identicated that the S protein fragments could induce strong humoral immune responses.These recombinant adenoviruses may be used for further studies to develop the PEDV vaccine.