Construction Of Recombinant Eukaryotic Expression Plasmids Of Four Eimeria Tenella Genes And Immunoprotection Of Nucleic Acid Vaccine Of EtAMA1Gene

Coccidiosis is a major worldwide extremely harmful parasites disease that is caused by anintracellular protozoan parasite, Eimeria spp. Coccidiosis causes a reduction in weight gain, poorfeed-conversion, high levels of mortality and often results in considerable economic losses on thechicken industry. More than35billion chickens are threatened by coccidiosis per annum in the worldand the global economic loss probably exceeds￡500million every year. Control of chicken coccidiosisis presently based on the use of prophylactic coccidiostatic drugs. With the emergence of drug resistantEimeria strains and food safty get more and more attention, emphasis has been laid on to develop betterways to control coccidiosis instead of coccidiostatic drugs. The use of vaccines to prevent coccidiosiswill play an increasingly important role in the control of coccidiosis. As the third-generation vaccines,nucleic acid vaccines are safe, stable, efficient and easy to use. DNA vaccine can elicite both cellularand humoral immune response, and at the same time the expression of cytokines, which can be anadjuvant, could enhance the effect of DNA vaccines and induce more extensive and long-lastinghumoral and cellular immunity. So DNA vaccines will be the best choice for the effective control ofcoccidiosis in the future. In our research, the antigen genes of Eimeria tenella were used to construct theDNA vaccine, these genes had high pathogenicity of all the Eimeria speices. The DNA vaccines wereconstructed by cloning the gene into the eukaryotic expression vector pCAGGS. The immune efficacyof the DNA vaccines was studied. This research laid a foundation for the further study of the DNAvaccine of coccidiosis.1. Cloning of Eimeria tenella4genes (EtAMA1, EtSerpin, EtRON2and EtCDPK3) andchicken IFN-γ, IL-2geneTaking E. tenella cDNA and chicken spleen cDNA as the templates, the genes AMA1, Serpin,RON2, CDPK3, ChIFN-γ and ChIL-2were cloned using the primers designed with restriction enzymecutting site in the upstream and downstream of the primers according to the gene sequencs of GenBank.The amplified gene was ligated to the pGEM-Teasy vector, and transformated into E.coli Top10competent cell. Verified by PCR and restriction enzyme digestion, the positive clone was sequenced.The results suggested that less than5bases were mutated of each cloned gene and no frameshiftmutation emerged. The homology of the cloned gene and the gene of the GenBank was more than99%.2. Construction of recombinant eukaryotic plasmids pCAGGS-EtAMA1, pCAGGS-EtSerpin,pCAGGS-EtRON2, pCAGGS-EtCDPK3and pCAGGS-EtAMA1-IFN-γ, pCAGGS-EtRON2-IFN-γThe cloned Eimeria tenella4genes (EtAMA1, EtSerpin, EtRON2, EtCDPK3) were ligated to theeukaryotic expression vector pCAGGS, and then four eukaryotic recombinant plasmids-pCAGGS-EtAMA1, pCAGGS-EtSerpin, pCAGGS-EtRON2, pCAGGS-EtCDPK3were constructed. They wereverified by restriction enzyme digestion. Then the ChIFN-γ were ligated to pCAGGS-EtAMA1andpCAGGS-EtRON2to construct recombinant plasmids pCAGGS-EtAMA1-IFN-γ, pCAGGS-EtRON2- IFN-γ. Sequence result suggests IFN-γ were inserted correctly.3. The expression of four recombinant eukaryotic plasmids pCAGGS-EtAMA1, pCAGGS-EtSerpin, pCAGGS-EtRON2, pCAGGS-EtCDPK3in293T cellsFour recombinant plasmid pCAGGS-EtAMA1, pCAGGS-EtSerpin, pCAGGS-EtRON2,pCAGGS-EtCDPK3were transfected into293T cells to express each protein. The expression of the fourproteins was verified by indirect immunofluorescence assay and western-blot. The results showed thefour proteins were expressed in293T cells.4. Protective effects of DNA vaccine pCAGGS-EtAMA1and pCAGGS-EtAMA1-IFN-γ onchickens against Eimeria tenella80chickens were divided into five groups (n=16): pCAGGS-EtAMA1group, pCAGGS-EtAMA1-IFN-γ group, pCAGGS empty vector group, non-immune challenged group, non-immune unchallengedgroup. The chicken of the experimental group were injected100mg recombinant plasmid in leg muscleon the same position at7-day-old and14-day-old respectively. First, the leg was pre-treated with0.2mLhypertonic sucrose solution，then the DNA vaccine was given to the chicken intramuscularly15-20minlater. The chicken of the control groups were injected with100μL PBS in the leg on the same time andposition. Chicken were challenged with10000sporulated oocysts of E. tenella at21-day-old by oralinfection. All chicken were killed at29-day old and the relative body weight gains, the lesion scores ofcecum, the numbers of oocysts shedding were calculated. The results indicated that the DNA vaccinepCAGGS-EtAMA1and pCAGGS-EtAMA1-IFN-γ have a protective effect. The chickens in that twogroups was not significantly different with chickens in unchallenged control group on body weight gain,however, chickens in challenged control group was significantly different with chickens in unchallengedcontrol group; the lesion score of the chickens in that two groups reduced significantly compared withthe chickens in challenged control group; the oocysts reduction ratio of that two groups were67.43%and72.89%respectively. Owing to carrying ChIFN-γ gene, pCAGGS-EtAMA1-IFN-γ group displays abetter outcome on body weight gain, cecum lesion scores and the oocysts reduction rate thanpCAGGS-EtAMA1group, however, the difference was not significant.