Protective Effects Of Lactococcus Lactis Expressing Chicken Eimeria Tenella AMA1 Protein

Coccidiosis, caused by intracellular protozoan Eimeria, is an enteric parasitic disease that seriously harms the poultry industry. Chicken coccidiosis brought huge economic losses to the poultry industry. Currently, the control of coccidiosis is mainly dependent on the drug prevention and treatment, but it is still imperative to explore the safe, stable and effective alternatives to prevent coccidiosis.becacuse of drug resistance, drug residues and high costs for development of new drugs. Sporozoites of Eimeria recognize, adhere and invade the chicken intestinal epithelial cells by the secretory proteins. The protein expressed on the surface of Lactococcus lactis is similar to the surface protein of sporozoite, and also can stimulate the effective protective immune response. The research about mucosal immunity using Lactococcus lactis as live vector vaccine has already become one of the current hot spot. Apical membrane antigen 1(AMA1) of apicomplexan plays an important role during invasion of parasites into host cells. However, the report about the immune function of E. tenella sporozoites AMA1(Et AMA1) was few. In this study, the protective effects of recombinant positive Lactococcus lactis expressing E. tenella AMA1 protein was explored.In this study, the gene fragment coding the extracellular domain of apical membrane antigen AMA1 from E. tenella sporozoites(Ecto AMA1) was cloned into prokaryotic expression vector p ET-30a(+) to construct recombinant plasmid p ET30(a)-Ecto AMA1. The characterized plasmid were transformed into the E. coli competent cells to screen positive strains. The positive strains were induced by IPTG, and then the expression of objective protein was detected by SDS-PAGE and Western blot. The expressed protein was used to immunize New Zealand white rabbits to produce polyclonal antibody. The antibody titer was detected by indirect Enzyme linked immunosorbent assay(ELISA) method, and the antibody reactivity was confirmed by Western blot. The result showed that Ecto AMA1 gene was 1269 bp in longth. SDS-PAGE and Western blot analysis showed that Ecto AMA1 was expressed in E. coli strain in the form of inclusion bodies and the molecular weightof the fusion protein is 52 Ka. The titer of the prepared polyclonal antibody from rabbit was 218. The prepared antibody was characterized to bind with sporozoite using Western blot method. Then the Ecto AMA1 fragment was cloned into the vector p TX8048 to construct recombinant plasmids, and the positive plasmids p TX8048-Ecto AMA1,p TX8048-SP-Ecto AMA1 and p TX8048-SP-Ecto AMA1-CWA was respectively electrotransformed into the host strains L. lactis NZ9000 to generate the recombinant positive bacteria L. lactis NZ9000/p TX8048-Ecto AMA1, L. lactis NZ9000/p TX8048-SP-Ecto AMA1 and L. lactis NZ9000/p TX8048-SP-Ecto AMA1-CWA. The expression of objective protein was detected by Western blot after the positive bacteria were induced by 5 ng/m L nisin. After the induction, about 5 109 CFU(colony forming unit) recombinant bacteria L. lactis NZ9000/p TX8048-Ecto AMA1, L. lactis NZ9000/p TX8048-SP-Ecto AMA1 and L. lactis NZ9000/p TX8048-SP-Ecto AMA1-CWA was respectively immunized orally the chickens for three times on days 4 to 6, 17 to 19, 31 to 33. In addition to the chickens in PBS group, chickens in other groups was challenged with 4×104 Eimeria tenella sporolated occysts at day 43. The lymphocyte proliferative function, the percentage of CD4+, CD8+ T lymphocyte subpopulation in peripheral blood, Ig G antibody dynamic change, the body weight gain, lesion score in cecum, oocyst per gram(OPG) and anti-coccidia index(ACI) were all evaluated. The results showed that the chickens in the group immunized with three kinds of positive bacteria displayed higher protection level than control groups. The experimental results suggested that L. lactis as a live vaccine vector via oral immunization can stimulate the host to produce an effective immune response against homologous infection. This study provide important references for the preparation of L. lactis oral vaccine against avian coccidiosis.