Immunoscreening Of CDNA Expression Library Of Eimeria Tenella YL Strain And Cloning & Expression Of 3-1E Gene

Chicken coccidiosis is an important avian disease which seriously harms the poutry industry. It was caused by various Eimeria spp. parasitized in intestinal mucosal epithelia, which are characterized with enteric pathological lesions. Among various Eimeria spp., E. tenella, which causes caecal coccidiosis, is highly pathogenic. Presengtly, the control of coccidiosis chiefly depends upon prophylactic chemotherapy with anticoccidial drugs. However, the emergence of drug resistance in coccidia is a great problem with most of the drugs, which, in due course, limits their use. Furthermore, drug- or antibiotic-residue in the poultry product is potentially harmful to consumers. These limitations have necessitated the search for alternative Eimeria control measures. Among the several alternative Eimeria control measures only host vaccination against Eimeria appears promising. In this research, our strategy was screening the constructed cDNA expression library of the sporozoites by E. tenella YL strain sporulated oocysts by immunological methods, cloning and expressing the antigen genes, analyzing the protective immunity. Results of studies displayed as following.1. cDNA expression library of the E. tenella YL strain sporozoites was immunoscreened with positive sera of chicken anti-E. tenella and rabbit anti-E. tenella YL strain. Thus 3 positive clones were obtained, one positive clone gene was designated, and the ORF of this gene is consisted of 513 nucleotides encoding 170 amino acids. There were high homologies with published 3-1E gene in genBank. Compared with 3-1E of E. tenella GS strain and US strain, the homology of nucleotides acid sequence of E. tenella YL strain 3-1E were 99.8% and 99.4%, respectively, the homology of amino acid encoded were 98.8% and 98.2%, respectively. All those confirmed that the gene was a new gene of E. tenella. The new gene accession number in GenBank is EF069437, and it was named as Eimeria tenella strain YL 3-1E.2. The interesting gene of 3-1E from library was cloned to expression vector of pGEX-4T-1 with special primer. The fusion expression vector of pGEX-3-1E and was constructed, then transformed into E. coli BL21. The fusion expression of 3-1E was induced with IPTG, the expression product was analyzed by SDS-PAGE. The results showed that the 3-1E fusion protein was expressed successfully in BL21. The fusion protein was about 44.7 ku and had 30.0% of expression level.3. pGEX-3-1E recombinant proteins were injected into chicken muscle, the efficacy against coccidiosis in chickens was observed. The results illustrated that the pGEX-3-1E protein had better anticoccidial efficacy, whereas inclusion body of pGEX-3-1E had poor efficacy. All those would be a good foundation for the development of anti-Eimeria genetic engineering vaccine.