[Expression and immunogenicity evaluation of ectodomain and subdomains of Plasmodium berghei apical membrane antigen 1].
OBJECTIVE
To express and evaluate the immunogenicity of ectodomain and its subdomains of Plasmodium berghei apical membrane antigen I (PbAMA-1).


METHODS
Sequence of PbAMA-I gene was isolated from the genome of P. berghei, and was redesigned and divided into three overlapped fragments according to its subdomain structure. The codon-optimized DNA fragments of PbAMA-1 were synthesized and inserted into vector pET32a for expression in E. coli and the recombinant proteins were purified by Ni-NTA column, followed by refolding in vitro. Mice and rabbits were immunized with the recombinant proteins formulated with Freund adjuvant. Titer of the specific antibodies was detected by ELISA and IFA. The immunized mice were challenged by P. berghei to evaluate protective efficacy in vivo.


RESULTS
The sequence of the PbAMA-1 gene was shown to be identical to that published before. PbAMA-1 sequence was redesigned via codon optimization and synthesized. Both ectodomain and its subdomains of PbAMA-1 were successfully expressed in E. coli after induction. The proteins were isolated with the purity of more than 90% after Ni column purification and refolding in vitro. Immunization of mice with the recombinant proteins induced high level of specific antibodies. The antibody titer to ectodomain E after the 3rd immunization showed a strong immunogenicity at (34.4+/-0.15) x 10(-4). The antibodies interacted with the parasites by indirect fluorescence. The immunized mice were partially protected from the challenge of P. berghei.


CONCLUSIONS
The recombinant PbAMA-1 is highly immunogenic and induces protective immunity against the challenge of P. berghei.
Li
Shu-Ling
SL
Department of Etiological Biology, The Second Military Medical University, Shanghai 200433, China.
Zhang
Dong-Mei
DM
Cao
Yi
Y
Pan
Wei-Qing
WQ
D000818:Animals; D000913:Antibodies, Protozoan / Q000097:blood; D000953:Antigens, Protozoan / Q000235:genetics / Q000276:immunology*; D001483:Base Sequence; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression; D007114:Immunization / Q000379:methods*; D008288:Malaria / Q000097:blood / Q000276:immunology / Q000469:parasitology; D008565:Membrane Proteins / Q000235:genetics / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D010962:Plasmodium berghei / Q000235:genetics / Q000276:immunology*; D015800:Protozoan Proteins / Q000235:genetics / Q000276:immunology*; D011817:Rabbits; D011994:Recombinant Proteins / Q000276:immunology
D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D008565:Membrane Proteins; D015800:Protozoan Proteins; D011994:Recombinant Proteins; C090077:apical membrane antigen I, Plasmodium