| Malaria remains one of the most devasting infectious diseases in the world. Due to emergency of the drug resistance of the parasite and the insecticide resistance of Anopheles mosquito, it becomes more difficult to control malaria. So it is very important to develop effective malaria vaccine to control this disease.The apical membrane antigen 1 of Plasmodium is one of the leading asexual blood stage antigens being considered for inclusion in a malaria vaccine. It is a minor surface antigen existing in all malaria species. Although its molecular function is still not clearly known, this protein is thought to be function in the host-cell invasion processing, and is essential for the parasite growth cycle.The molecular of AMA-1 is highly conserved among species. It is a type I integral membrane protein. The ectoplasmic region of this protein contains 16 invariant Cys residues and forms 8 disulfide bonds. According to the disulfide bonds forming pattern, the ectoplasmic region can be divided into three distinct subdomains, domain I, domain II, and domain III (D I, DII, and D III).Research upon peoples living in the malaria endemic regions and malaria animal models showed that AMA-1 have excellent immunogenicity and protection. But the effects of each subdomains in inducing protective immunoreactions is not clear. In this research, we analyzed the immunity responses directed against each subdomains of AMA-1 using the P. berghei, an important mouse malaria and mouse animal model. The results of this assay will be useful to guide the construction of a malaria vaccine containing AMA-1 domains or epitopes.As researches before have shown that protective immune responses induced by AMA-1 were strain specific, in order to appreciate the protective immune responses induced by AMA-1 correctly, we sequenced the AMA-1 coding sequence of P.berghei ANKA isolate stored in our lab. Then, according to the translated amino acid sequence of sequenced DNA gene, we developed a synthetic gene with adapted codon usage of Pichia pastoris. In addition, potential 7 N-glycosylation sites (Asn-X-Ser/Thr) were changed using Gin to substitute Asn.Each subdomains and the entire ectodomain were induce expressed in Pichia and/or E.coli. All induce expressed protein were affinity purified on a Ni-nitrilotriacetic acid column using the fused 6Xhis tag. The DIIpp expressed in Pichia was further purified by ion-exchange chromatography. The expressed proteins expressed in E.coli were refolded in vitro using GSH/GSSG. The purities of all targeted protein were all higher than 90%.All purified proteins induced high titer antibodies both in rabbits and in mouse. The entire ectodomain E induced antibody titer in mouse was significantly higher than the antibody titers induce by other antigens in mouse (P>0.05). The antisera of immunized rabbits can recognize native P. berghei AMA-1, the response of anti-E sera was stronger than other anti-sera. Each immunized rabbit anti-sera can recognize a 66-kDa line in western blot to PbANKA antigens, corresponding to the native P. berghei AMA-1. Using mouse anti-PbANKA seras to western blot each antigen, only E showed a positive response.All immunized mouse was challenged with lethal PbANKA. All antigens showed partial protection. In high dose malaria challenge experiments, antigen E exhibits obvious excellent immuoprotection than the other antigens. All antigens showed difference from the contol in peak parasitemia or survival days, except the DIIpp. In low dose malaria challenge experiments, all antigen exhibit obvious immuoprotection compared with the contol group in survival days. The exhibited protections in low and high dose challenge trails are different,showed the influence of dose to protectin efficiency.In conclusion, AMA-1 is a highly hopeful malaria vaccine candidate antigen. The...
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