Construction Of Dendritic Cell-targeted Recombinant Lactobacillus Against H9N2 Avian Influenza Virus And Evaluation Of Immune Efficiency

H9N2 avian influenza virus(H9N2 AIV), a serious threat to the development of China’s poultry industry, is spreading and epidemic in poultry, which can cause egg drop and immunosuppressive diseases, in particular leading to high mortality when mixed with other pathogens. In order to prevent and control the influenza, vaccination is considered to be one of the most effective methods. Since influenza virus mainly infects the mucosal surface of the respiratory and digestive tracts, the study on mucosal immunity is necessary.Oral immunization is the most convenient and safe because the role of the antigen delivered to the mucosa is closer to the natural infection, eliciting cellular and humoral immune responses and inducing an amount of mucosal antigen-specific s Ig A. However, the antigen must depend on the delivery systems to resist to degration during breaking through the gastrointestinal tract barrier and colonization at the effector site, thereby activating the immune system to produce appropriate and effective immune response. Lactic acid bacteria as antigen delivery carrier have outstanding advantages. Lactic acid bacteria are generally regarded as safe during regulating the immune function, especially inducing the secretion of s Ig A. Therefore, this study constructed the recombinant lactobacillus to evaluate the mucosal immune efficiency in mice.We first optimized the codons of HA-GCN4-DCpep, which contained these sequences of the dendritic cell-targeting peptide(DCpep), the isoleucine zipper(GCN4), and the major protective antigen hemagglutinin(HA) gene of H9N2 AIV. The HA-GCN4-DCpep wasinserted into the shuttle vector p SIP409. The recombinant plasmid identified by double digestion method was electric-transformed into Lactobacillus. The result of western blot showed that the HA-GCN4-DCpep protein was expressed correctly by the recombinant lactobacillus after induction by Spp IP and had the reactionogenicity.The residence time of lactobacillus in the intestinal tract of the mice will affect the immunogenicity of the lactobacillus vaccine. Therefore, the mice were orally immunized with the L.p-HA-GCN4-DCpep recombinant lactobacillus according to the immunization procedure to study the adhesion to the intestinal epithelial cells.In order to investigate the mucosal immune efficiency of L.p-HA-GCN4-DCpep recombinant lactobacillus in mice, the mice were vaccinated for three times. The changes of T cells in the spleen, mesenteric lymph nodes and Peyer patch were detected by the flow cytometry. The ELISA was used to detect the specific s Ig A in the lavage, intestinal lavage and stool, the serum Ig G and cytokines. The results showed that the recombinant lactobacillus can induce the proliferation of CD3+CD4+ and CD3+CD8+ T cells and cause the secretion of specific s Ig A, Ig G and cytokines. More significantly, we found that the L.p-HA-GCN4-DCpep recombinant lactobacillus can lower weight loss and improve the antibody secretion levels after challenge compared to other groups.These results suggest that the L.p-HA-GCN4-DCpep recombinant lactobacillus has good immunogenicity to resist the infection of H9N2 avian influenza virus, in which the dendritic cell targeting peptide(DCpep) plays an important role in mucosal immunity response. It provides a basis for further studying the mechanism of dendritic cells in the regulation of mucosal immune responses and the development of the oral vaccine against avian influenza virus.