Abstract: Objective To produce an erythrocytic stage chimeric protein of Plasmodium berghei in Pichia pastoris and evaluate its immunogenicity. Methods The DNA sequences of AMA1（Ⅲ） and MSP1-19 from P. berghei homologous to the corresponding sequences of P. falciparum chimeric antigen 2.9 (PfCP-2.9) were fused to generate a chimeric gene，designated as PbCP-2.9. The resulting gene was redesigned using Pichia preferential coden usage and expressed in P. pastoris in the secreted form. The recombinant protein was purified by Ni-NTA affinity chromatography. Three groups each with 10 BALB/c mice were immunized subcutaneously with 20 μg of purified PbCP-2.9 antigen formulated in Freund’s adjuvant，Montanide ISA720 and Montanide IMS 1 312，respectively. Three control groups each with 10 mice received only adjuvants emulsified with PBS. All the mice received three immunizations at 2-week intervals with the same dose of antigen. Serum samples were collected at pre-immunization and one week after each immunization，and were analyzed for specific antibodies by ELISA and reaction with natural P. berghei proteins by IFAT. Results The PbCP-2.9 antigen with Mr 26 400 was successfully expressed in P. pastoris in secreted form. The recombinant protein can be recognized by the serum against blood stage parasites of P. berghei. High antibody responses were detected in all three PbCP-2.9-immune groups of mice by ELISA. However，mice immunized with PbCP-2.9 antigen in Freund’s adjuvant produced higher antibody titers than those with PbCP-2.9 antigen in Montanide ISA 206 and Montanide IMS 1312 adjuvants. The mean antibody titer in Freund’s adjuvant was 6.9-fold higher than in Montanide ISA 206 adjuvant and 5.6-fold higher than in Montanide IMS 1312 adjuvant after the second immunization （F＝81.06，P＜0.01). In addition，after the third immunization the mean antibody titer in Freund’s adjuvant was 3.7-fold higher than in Montanide ISA 206 adjuvant and 3.3-fold higher than in Montanide IMS 1312 adjuvant（F＝13.29，P＜0.01). The results from IFAT assay demonstrated that the immune sera recognized the surface proteins of P. berghei parasites. Conclusion The codenoptimized PbCP-2.9 gene has been constructed and expressed in P. pastoris. The chimeric antigen is highly immunogenic in mice and the immune sera can interact with natural proteins of P. berghei parasite.

Key words: Plasmodium berghei, Erythrocytic stage, Chimeric protein, Gene expreesion, Immunogenicity