In vivo immunoprotective comparison between recombinant protein and DNA vaccine of Eimeria tenella surface antigen 4

摘要

Eimeria tenella, belonging to protozoon, is the causative agent of cecal coccidiosis in chicken and causes enormous impacts for poultry industry. The surface antigens of apicomplexan parasites function as attachment and invasion in host-parasite interaction. Meanwhile, host immune response is triggered as a result of parasitic invasion. Immunogenicity and potency as a vaccinal candidate antigen of E. tenella surface antigen 4 (EtSAG4) have been unknown. Therefore, a gene segment of E. tenella EtSAG4 was amplified and transplanted to pET28a prokaryotic vector for recombinant protein expression. Similarly, pEGFP-N1 eukaryotic vectors with EtSAG4 gene segment (pEGFP-N1-EtSAG4) amplified in 293 T cells as DNA vaccines. Reverse transcription-polymerase chain reaction (RT-PCR) assay and western blot analysis were used to demonstrate successful expressions of EtSAG4 in Escherichia coli or 293 T cells. Subsequently, animal experiments (72 cobb broilers) were performed to evaluate immunoprotective between recombinant protein and DNA vaccine of E. tenella EtSAG4 using different immunizing doses (50 or 100 mu g), respectively. Serum from chickens infected with E. tenella identified recombinant EtSAG4 (rEtSAG4) protein. Chickens vaccinated with either rEtSAG4 protein or pEGFP-N1-EtSAG4 plasmids both shown a significant increase in concentration of IFN-gamma (p < 0.05) compared with control groups indicating production of cell-mediated immunity. Besides, pEGFP-N1-EtSAG4 plasmids motivated more intense immune responses for immunoglobulin Y (IgY) and interleukin 17 (IL-17) (p < 0.05) contrast to control groups. However, there was no increase in concentration of interleukin 10 (IL-10) and interleukin 4 (IL-4) for both rEtSAG4 protein and pEGFP-N1-EtSAG4 plasmids. Chickens vaccinated with rEtSAG4 protein or pEGFP-N1-EtSAG4 plasmids both show higher weight, lower oocyst output and mean lesion scores compared with infection control groups. The highest anticoccidial index (ACI) value of immunized groups was 168.24 from EGFP-N1-EtSAG4 plasmids (100 mu g) group. Generally, EGFP-N1-EtSAG4 plasmids as DNA vaccines provided a more effective immunoprotective for chickens against E. tenalla than that of rEtSAG4 protein as subunit vaccines. EtSAG4 is a promising candidate antigen gene for development of coccidiosis vaccine.