Expression Of Chicken Eimeria3-1e Gene In Lactococcus Lactis And Its Immune Protection Effect

Coccidiosis is a major economically important enteric disease in poultry industry, which caused by protozoan parasites of the genus Eimeria.The traditional control means based on chemical drugs result in drug residue in animal food, threatening human health. Conventional vaccines including live or attenuated commercial vaccines could cause mild to servere reaction, therefore the new stable and efficient alternatives to control coccidiosis are needed. Sporozoites of Eimeria could recognize, adhere and invade chicken intestinal epithelial cells through its surface protein. The objective protein expressed on the surface of lactic acid bacteria (LAB) is similar to the protein displayed on the surface of sporozoite, andean also stimulate the effective protective immune response. LAB used as antigen delivery vectors had became one of the hot topics in vaccine research.In this study, the application of L.lactis NZ9000using as host strain are reported, E.tenella sporozoite3-1E gene was cloned into E.coli-LAB shuttle expression vector pTX8048to obtain the positive plasmid pTX8048-3-1E. The positive plasmid pTX8048-3-1E was then transformed into L.lactis NZ9000competent cells by electroporation. Western blot was used to detect the expression of3-1E. On days5to7,15to17,25to27, SPF chickens were immunized orally with1×1010CFU L.lactis NZ9000-pTX8048-3-1E, the effect of immune protection after infected with E.tenella was evaluated; T lymphocyte proliferation function in spleen and changes of expression of IL-2, IFN-y mRNA were detected. The method for detecting the expression of several molecules contained in TLR2signaling pathway in spleen was established, and expression of related molecules mRNA in TLR2signaling pathway were detected. The results were as follows:l.The fusion protein with the size of33kDa was successfully detected in the positive recombinant strain of L.lactis NZ9000-pTX8048-3-1E using western blot method.2.Oral administration of L.lactis NZ9000-pTX8048-3-1E can provide partial protective effect against homologous infection compared with the control group, showing with the significant increased body weight gain (P<0.01).the reduced oocyst shedding per grams of fecal and the oocyst decrease rate21.37%, the decreased cecal lesion scores, and ACI index151.26. 3.The proliferation response of T lymphocytes to ConA in spleen in group L.lactis pTX8048-3-1E was significantly higher than that in group L.lactis pTX8048and PBS (P<0.01) after primary immunizaiton. After secondary immunization, T lymphocytes proliferation response in spleen of chickens in group L.lactis pTX8048-3-1E was significantly higher than that in group L.lactis pTX8048(P<0.05), and in group PBS (P<0.01). After third immunization, T lymphocytes proliferation in spleen of chickens in group L.lactis pTX8048-3-1E was significantly higher than that in group L.lactis pTX8048and PBS (P<0.05).4.The results of mRNA level of related molecules in TLR2signal pathway and cytokines IL-2and IFN-γ detected by real-time fluorescence quantitative PCR showed that the expression of TLR2-2and INF-γ mRNA in spleen were respectively upregulated2.53and1.49folds after third immunization.All the above results show that, L.lactis NZ9000-pTX8048-3-1E could induce immune response, provide protective immunity again homologous infection. This study lays a foundation for the study of lactic acid bacteria oral vaccine against coccidiosis.