Abstract
Background: Avian coccidiosis posts a severe threat to poultry production. In addition to commercial attenuated vaccines, other strategies to combat coccidiosis are urgently needed. Lactobacillus plantarum has been frequently used for expression of foreign proteins as an oral vaccine delivery system using traditional erythromycin resistance gene (erm). However, antibiotic selection markers were often used during protein expression and they pose a risk of transferring antibiotic resistance genes to the environment, and significantly restricting the application in field production. Therefore, a food-grade recombinant L. plantarum vaccine candidate would dramatically improve its application potential in the poultry industry. Results: In this study, we firstly replaced the erythromycin resistance gene (erm) of the pLp_1261Inv-derived expression vector with a non-antibiotic, asd-alr fusion gene, yielding a series of non-antibiotic and reliable, food grade expression vectors. In addition, we designed a dual-expression vector that displayed two foreign proteins on the surface of L. plantarum using the anchoring sequences from either a truncated poly-γ-glutamic acid synthetase A (pgsA') from Bacillus subtilis or the L. acidophilus surface layer protein (SlpA). EGFP and mCherry were used as marker proteins to evaluate the surface displayed properties of recombinant L. plantarum strains and were inspected by western blot, flow cytometry and fluorescence microscopy. To further determine its application as oral vaccine candidate, the AMA1 and EtMIC2 genes of E. tenella were anchored on the surface of L. plantarum strain. After oral immunization in chickens, the recombinant L. plantarum strain was able to induce antigen specific humoral, mucosal, and T cell-mediated immune responses, providing efficient protection against coccidiosis challenge. Conclusions: The novel constructed food grade recombinant L. plantarum strain with double surface displayed antigens provides a potential efficient oral vaccine candidate for coccidiosis.