| Avian coccidiosis is a serve problem for the poultry industry, caused by intracellular protozoa including several species of the coccidia. Coccidia infection results in extensive destruction of the duodenum epithelium accompanied by severe depression in body weight gain, reduced feed efficiency and intestinal shedding of parasite oocysts. 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. Recently, novel vaccinating strategies using recombinant vaccines have been shown to be able to induce protectively cellular and humoral immune responses against coccidia. In order to attempt a new method of controlling coccidiosis in poultry industry, the studies had been conducted as the following:1. Clone and sequence analysis of gene gam56 of Eimeria maximaThe gam56 gene of E.maxima NT strain isolated from NanTong city was amplified by reverse transcription polymerase chain reaction (RT-PCR) from total RNA of macrogamete. The products of RT-PCR were cloned into pGEM-T vector. Two positive clones were identificated with blue/white selection, restriction enzyme digestion, and PCR method. Nucleotide sequence of the two inserted fragments were determined by dideotide chain-termination method, and showed that gam56 gene of E.maxima NT strain included 1 431 bp opening read frame, which code for a polypeptide of 466 amino acids. The ORF sequences of the gene was the same as that cloned from E.maxima Houghton strain. The results of analysis in secondary structure, flexible regions, antigenic index, and surface probability of protein indicated gam56 protein would be a suitable gene for study vaccines against Eimeria infection. 2. Expression of gene gam56 of Eimeria maxima in E.coliThe gam56 gene of E.maxima NT strain without signal peptide encoding region was amplified by polymerase chain reaction (PCR) from plasmid named pGEM-T-gam56 which contained the cDNA of gam56 gene. The fragment of gam56 gene was subcloned into express vector pGEX-6P-1, and positive plasmid named pGEX-6P-1-gam56-1 and pGEX-6P-1-gam56-2 were identified by restriction enzyme digestion and sequencing. After induced by IPTG, insoluble 44 Ku GST-gam56-1 fusion protein and soluble 38Ku GST-gam56-2 fusion protein were expressed, which were confirmed by SDS-PAGE, respectively.3. Protective efficacy of recombinant gam56-2 antigen against Eimeria maxima infectionsThe recombinant gam56-2 protien expressed from recombinant bacteria of pGEX-6p-1-gam56-2/BL21 was emulsified with or without Freund,s adjuvant, which were used as antigen. The group 1 to 6 were served as immunized group, and the chickens at five-day old were subcutaneously immunized with recombinant antigen at the dose of 100, 50, and 25μg per chicken, and boosted with the same dosage at 12, 19 days of age, respectively. The group 7 and 8 were unimmunized, served as positive and negative control, respectively. Chickens in all groups at 26 days of age were orally challenged with 5×104 sporulated oocysts of E.maxima NT strain, and killed at eight days postchallenge. The protective immunities were assessed by the rate of survival and relative weight gain, the reduction in lesion score and oocyst production, and anti-coccidia index (ACI). The results showed that there were partial protective effects in the immunized group according to the weight gains. But for the reduction of lesion score and oocyst production, no significant protective immunity were observed. The ACI of groups given at 50μg and 25μg antigen mixed with FCA, and at 50μg antigen without adjuvanted were between 160～180, which were belonged to in middle valid range against E.maxima infection. The ACI in other immunized groups were below160, and were in low lever. The protective efficacies compared among the immunized groups were more effective in the groups at 50μg and 25μg antigen mixed with FCA, and at 50μg antigen without adjuvanted.
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