| Malaria ramains one of the most devasting infectious diseases in the world. Among four species of human malaria parasite, Plasmodium falciparum is the most important agent which causes severe even fetal malaria. Due to emergence 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 the deisease. Because of complexity of the parasite's life cycle, stage specificity and variation of the antigen and host MHC restriction, an effective malaria vaccine in principle should be composed of multiple antigens derived from different stage of the parasite to generate a multistage, multivalent vaccine . In this study, we aimed at constructing such a vaccine and investigated its immunogenicity in animals. Previous studies in our lab has generated an chimeric antigen named PfCP-2 which is consist of two Plasmodium falciparum erythrocytic stage antigens, AMA-1(III) and MSP1-19. Extremely high level expression of the gene was achieved in Pichia pastoris. It has been showed that PfCP-2 retains some important conformational epitopes and be highly immunogenic in animal. Moreover, immune sera against the antigen could inhibit the parasite growth in vitro. However, problem with the construct is a doublet of the product appeared on SDS-PAGE gel. N-terminal sequencing analysis of the product displayed 9 amino acids deleted in the band with lower Molecular Weight. To solve this problem, we modified the gene to delete sequence encoding the nine residues, generating a new construct named PfCP-2.9. Consequently, expression of the new construct turned out to be a single band on SDS-PAGE. Comparing to the PfCP-2 construct, the PfCP-2.9 has consistency in a level of expression (yield> 1g/L) and in physical and chemical properties as well as in immunogenicity. Moreover, sera from rabbits immunized with PfCP-2.9 inhibited the parasite growth in vitro. It was concluded that the nine residues deleted of the construct doesn't influence its immunogenicity and the parasite inhibition by its immune sera. Comparing to the individual components of the chimeric protein, AMA-1(III) andMSP1-19, the PfCP-2.9 was shown to have following advantages:(1)the yield of the product was increased by about 20 folds.(2) the anti-AMA1(III) antibody titer induced by PfCP -2.9 was about 18 fold higher than that induced by AMA-1(III) and the anti-MSP1-19 antibody titer was 11 fold higher than that induced by MSP1-19 (3)15% immune sera of PfCP-2.9 completely inhibited the parasite growth in vitro whereas no inhibition was observed in immune sera of either AMA-1(III) and MSP1-19. Thus fusion of two malaria vaccine candidates into one molecule extremely increased the product yield, immunogenicity and antibody-mediated inhibition of parasite growth. To develop a multistage, multivalent malaria vaccine, combination of PfCP-2.9 and a pre-erythrocytic antigen was considered. For this purpose, we have constructed three pre-erythrocytic antigens: i.e. (1) PfCP-3tcl : composed of major T and B cell epitopes derived form pre-erythrocytic antigens, CSP,TRAP and LSA-1. (2)PfCSP-1: the entire C-terminus of CSP. (3)PfCSP-2: consisting of region I, (NANP)15 of the repeat region and entire C-terminus of CSP. All these antigens were redesigned using Pichia codon usage,and removed of potential glycosylation sites. These genes were expressed in Pichia pastoris at yield of 1-4g/L and purification of these proteins was carried out by uses of two steps including Cation ion-exchange and Gel filtration chromatography and >95 % purity of the proteins was achieved.Immunization of mice as well as rabbits was carried out in combination of PfCP-2.9 with PfCP-3tcl , PfCSP-1 and PfCSP-2, respectively. Antibody titers to individual components of combination were detected and compared with that induced by individual protein..The result showed that the titers of PfCP-3tcl/PfCSP-2.9 conbination were significantly lower t...
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