Expression Of Eimeria Tenella Surface Antigen Gene Sag10 And λMZP5-7 In Pichia Pastoris

Cecal coccidiosis has tremendous impact on poultry throughout the world because of consequent mortality, morbidity and weight loss in affected birds. Control of Eimeria tenella in poultry is presently accomplished by prophylactic chemotherapy, but the rapid emergence of drug resistant parasites, coupled with difficulty and expense of developing new drugs, has led to a search for new approaches to coccidiosis control involving immunological, biotechnological and genetical methods. Of these, immunological approach is assuming more importance. The protective effect Eimeria tenella surface antigen geneλMZP5-7 has been confirmed and sag10 was expressed both in the stages of merozoites and sporozoites which were the most dangerous to the chicken.The total RNA was extracted from the Beijing strain of Eimeria tenella and used as the template for the cloning of theλMZP5-7 and sag10 gene by RT-PCR with specific primers designed according to the reported sequence in Genbank( L08257,AJ586552). The expression plasmids pPIC9K-MZP and pPIC9K-sag10 of Pichia pastoris were constructed and transformed into Pichia pastoris GS115. Multi-copy Pichia pastoris strains were screened by G418. The selected strain was cultured in 100 ml flasks and the supernatant was analyzed by SDS-PAGE and Western Blot. The results indicated that the SAG and MZP5-7 protein were successfully expressed and secreted in a soluble form with a molecular weight of approximately 43 kDa and 36 kDa in recombinant Pichia pastoris , respectively ,when induced by 0.5%methanol. The selected strain S7 was ferment in 5L fermentor and the biomass was up to 354mg/mL when induced for 120h.,and the percentage of the recombinant protein was 34% in the tatol protein.