| Eimeria maxima is one of nine species of fowl coccidians which belong to Eimeria genus, Eimeriidae family, Eucoccidiorida order, Sporozoasida class, Apicomplexa phylum. E.maxima exists all over the world and China. Although coccidiosis is mainly controlled by the use of chemotherapeutic agents, alternative control strategies are needed due to the increasing emergence of drug-resistant parasite strains in commercial settings. DNA vaccine which is regarded as the third-generation vaccine is popularly accepetted for strong cellular immunity and long-lasting immunologic memory can be induced and the application of the DNA vaccine has made some progression on infection disease and tumor. With the development of molecular biology technology, DNA vaccine is also gradually applied to the parasited diseases. Many researchers started to study on DNA vaccine of fowl coccidians. The subunit vaccine Cox Abic? with GAM56 as its main component has been commercialized and had a good effect of clinical application. In order to attempt a new method of controlling coccidiosis, the studies had been conducted as the following:1. Construction and identification of nucleic acid vaccine containing GAM56 gene from Eimeria maximaThe ORF fragment of GAM56 gene was amplified from plasmid gene pGEM-T-gam56 DNA which contained GAM56 gene cDNA of E.maxima NT strain. Then GAM56 gene was ligated to pcDNA3.1. Identified by EcoRI and HindⅢdigestion and sequencing, gam56 gene was correctly ligated to the multiple cloning sites of pCDNA3.1 and the gene sequence didn't mutate. The expression of GAM56 was evaluated by IFA, with mouse anti -GAM56 protein serum as the first anti-body and goat-anti-mouse IgG labeled by FITC as the second anti-body. Results showed that GAM56 was expressed in some COS-1 cells at 72 hours post transfection. 2. Study on the immunogenicity of nucleic acid vaccine pcDNA 3.1-gam56In order to determine the immunogenicity of pcDNA3.1-GAM56,7-days-old chickens were immunized by intramuscular injection wtih pcDNA3.1-GAM56 at a dose of 100μg(high dose group), 50μg(middle dose group), 25μg(low dose group) as a DNA vaccine and boosted with the same dosage after two weeks. At the same time,pcDNA3.1 and PBS were injected as negative control. The 2 ml blood of five chickens was randomly collected for lymphocyte transformation test and the detection of specific antibodies at 7, 14, 21, and 28 days, respectively. Results showed that the chickens in middle dose group produced significantly higher lymphocyte proliferation at 7 days after the first immunization, and continously rasied at 14 and 21 days after the first immunization. When serum titers were concerned, chickens of middle dose group also produced higher antibody responses measured by indirect ELISA and continously rasied at 14 and 21 days after the first immunization. DNA vaccines showed good immunogenicity.3. Study on the immune efficacy of nucleic acid vaccine pcDNA 3.1-gam56To determine the protective effects of the pcDNA3.1-GAM56, 7-days-old chickens were immunized by intramuscular injections with pcDNA3.1-GAM56 at a dose of 100μg, 50μg, 25μg as a DNA vaccine and boosted with the same dosage after two weeks. At the same time,pcDNA3.1 and PBS were injected to every group of ten chickens as negative control. One week after the second immunization, each chicken was challenged with 5×104 E.maxima NT sporulated oocysts except the negative group. After 7 days, chickens were weighted and killed,survival rate, weight gain,and oocyst output were then determined and compared with those of the controls. Results demonstrated that the relative weight gain of and reduction of oocyst output in middle dose group were the highest among three dose groups, showed the best immune efficacy.
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