Abstract: The specific primers were designed and produced according to E.tenella sporozoite surface antigen 5401 gene sequence. EcoRⅠ and SalⅠ restriction enzyme cut sites and protective bases were added at the two ends of the primer respectively. A fragement of 881bp was cloned by RT-PCR from the E.tenella sporulated oocysts. The recombinant plasmid pGEM-T-5401 was identified with EcoRⅠ and SalⅠ restriction enzyme, the fragements were collected by agarose gel fraction method. After purificaion the fragement was ligated to the pET-30a express vector, the recombinant pET-30a-5401 express plasmid was constructed. After transformation, the engineered strain E.coli BL21/ 6×His-5401 was expressed by the induction of IPTG in a form of dimer protein. The specific approximate 66.2 ku band was obtained by analysis of SDS-PAGE and Western-blotting. The result indicated that E.tenella 5401 protein had been expressed successfully in E.coli expressing system.