Abstract
BackgroundThe function of the 19 kDa C-terminal region of the merozoite surface protein 1 (MSP1-19) expressed by Plasmodium has been demonstrated to be conserved across distantly related Plasmodium species. The green fluorescent protein (GFP) is a reporter protein that has been widely used because it can be easily detected in living organisms by fluorescence microscopy and flow cytometry.Methodology and ResultsIn this study, we used gene targeting to generate transgenic P. berghei (Pb) parasites (designated as PfMSP1-19Pb) that express the MSP1-19 of P. falciparum (Pf) and the GFP reporter protein simultaneously. The replacement of the PbMSP1-19 locus by PfMSP1-19 was verified by PCR and Southern analysis. The expression of the chimeric PbfMSP-1 and the GFP was verified by Western blot and fluorescence microscopy, respectively. Moreover, GFP-expressing transgenic parasites in blood stages can be readily differentiated from other blood cells using flow cytometry. A comparion of growth rates between wild-type and the PfMSP1-19Pb transgenic parasite indicated that the replacement of the MSP1-19 region and the expression of the GFP protein were not deleterious to the transgenic parasites. We used this transgenic mouse parasite as a murine model to evaluate the protective efficacy in vivo of specific IgG elicited by a PfCP-2.9 malaria vaccine that contains the PfMSP1-19. The BALB/c mice passively transferred with purified rabbit IgG to the PfCP-2.9 survived a lethal challenge of the PfMSP1-19Pb transgenic murine parasites, but not the wild-type P. berghei whereas the control mice passively transferred with purified IgG obtained from adjuvant only-immunized rabbits were vulnerable to both transgenic and wild-type infections.ConclusionsWe generated a transgenic P. berghei line that expresses PfMSP1-19 and the GFP reporter gene simultaneously. The availability of this parasite line provides a murine model to evaluate the protective efficacy in vivo of anti-MSP1-19 antibodies, including, potentially, those elicited by the PfCP-2.9 malaria vaccine in human volunteers.
Highlights
There is an urgent need for the development of a malaria vaccine to control the tropical disease because of the emergence and rapid spread of drug-resistant parasites and insecticideresistant mosquitoes [1]
We generated a transgenic P. berghei line that expresses PfMSP1-19 and the green fluorescent protein (GFP) reporter gene simultaneously. The availability of this parasite line provides a murine model to evaluate the protective efficacy in vivo of anti-MSP1-19 antibodies, including, potentially, those elicited by the PfCP-2.9 malaria vaccine in human volunteers
Construction of the transfection vector Since integration into Plasmodium spp. genomes usually occurs via homologous recombination, the two endogenous P. berghei fragments, msp-1 (1.4 kb) and the 39UTR of PbMSP1 (0.5 kb), were amplified from genomic DNA of P. berghei ANKA strain as targeting sequences
Summary
There is an urgent need for the development of a malaria vaccine to control the tropical disease because of the emergence and rapid spread of drug-resistant parasites and insecticideresistant mosquitoes [1]. The 185–215 kDa merozoite surface protein 1 of Plasmodium falciparum (PfMSP1) is a leading anti-blood stage malaria vaccine candidate [2,3]. In addition to PfMSP1, the apical membrane antigen of Plasmodium falciparum (PfAMA-1) is another attractive vaccine candidate against blood-stage parasite [2,10,11,12]. The most C-terminal of the disulphide-bonded domains in AMA-1 (AMA-1(III)) was demonstrated to be the target of inhibitory antibodies isolated from humans in malaria-endemic regions [13].We have constructed a Plasmodium falciparum chimeric protein that consists of AMA-1(III) and MSP1-19 (designated as PfCP-2.9) [14,15]. The function of the 19 kDa C-terminal region of the merozoite surface protein 1 (MSP1-19) expressed by Plasmodium has been demonstrated to be conserved across distantly related Plasmodium species. The green fluorescent protein (GFP) is a reporter protein that has been widely used because it can be detected in living organisms by fluorescence microscopy and flow cytometry
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