Oral Immunization Of Chickens With Recombinant Lactococcus Lactis Expressing Protective Antigens CfrA And CjaA Of Campylobacter Jejuni

Campylobacter jejuni(C. jejuni), a microaerophilic gram-negative organism, which colonization and infection in the host intestine can lead to a variety of diseases, such as diarrhea and enteritis, is one of the most important food-borne pathogens. The control of C. jejuni contamination in poultry is the most direct and effective means to reduce human infection with the bacteria, and the present study was to combine specificity and non-specific of immunity by inserting C. jejuni protective antigen genes into food grade lactic acid expression system, constructing the expression of protective antigens recombinant food-grade Lactococcus lactis(L. lactis) and reducing the colonization level of C. jejuni in chicken intestine by oral immunization of recombinant food-grade L. lactis.The purpose of this study is to reduce the colonization level of C. jejuni in chicken intestine by oral immunization of recombinant food-grade L. lactis expressing the ferric enterobactin receptor CfrA of C. jejuni. The whole cfr A gene and its N-terminal fragments were amplified by PCR, inserted into the food-grade expression vector p NZ8149 and transformed into L. lactis NZ3900 respectively. Based on the expression of CfrA in recombinant L. lactis by Western blot, the expression level was optimized by screening nisin concentration, induction temperature and time. Then the recombinant L. lactis strains were used to orally immunized SPF chickens. After oral immunization, the duration of recombinant L. lactis in chickens, as well as the antibody levels of anti-CfrA serum IgG and intestinal mucosal sIgA were measured by PCR and ELISA respectively. Finally, the immunized chickens were orally inoculated C. jejuni to evaluate the inhibitory effect of recombinant L. lactis on colonization of C. jejuni in chickens. Western blot results determined that the whole cfr A gene and its N-terminal fragments were both expressed in recombinant L. lactis in soluble forms while no secreted CfrA protein was detected out of the bacterial cells. The optimal conditions for inducing the expression were grown at 37 ℃ for 1 h with nisin concentration of 25 ng/m L. Detection of chicken cloacal swabs showed that the duration of oral L. lactis was less than 10 days in chicken. The immunized groups produced higher antibody titers of anti-CfrA specific serum IgG and mucosal sIgA than the control groups. Moreover, the colonization rate of C. jejuni in the immunized groups was significantly lower than in the control groups. The recombinant food-grade L. lactis expressing the ferric enterobactin receptor CfrA of C. jejuni was constructed successfully. Oral immunization of chickens with recombinant L. lactis expressing CfrA indicated an inhibitory effect on the colonization of C. jejuni.Based on the expression of CfrA in recombinant L. lactis, we constructed recombinant secretory L. lactis NZ3900 systems expressing CjaA and co-expressing CjaA-LTB. The gene SPusp45 secretion signal peptide coding sequence of unknown Usp45 secretory protein from L. lactis MG1363 was amplified by PCR, inserted into the food-grade expression vector p NZ8149 and transformed into L. lactis NZ3900 respectively. The main protective antigen cja A was selected as the target gene and E.coli heat-labile toxin B subunit protein was inserted. The immunogenicity responses induced by oral mucosal immunizations with recombinant strains were compared. The immunized groups produced higher antibody titers of anti-CjaA specific serum Ig G and mucosal s Ig A than the control groups. Moreover, the colonization rate of C. jejuni in the immunized groups was significantly lower than in the control groups. The recombinant food-grade L. lactis expressing the membrane transporting protein CjaA of C. jejuni was constructed successfully. Oral immunization of chickens with recombinant L. lactis expressing CjaA indicated an inhibitory effect on the colonization of C. jejuni.In summary, the results obtained so far demonstrated that Lactococcus lactis is capable of delivering antigen to the mucosal and systemic immune systems following oral immunization. These results will play an essential role in developing an oral vaccine of recombinant Lactobacillus for control of C. jejuni infection in chicken.