title: Construction of transgenic Plasmodium berghei as a model for evaluation of blood-stage vaccine candidate of Plasmodium falciparum chimeric protein 2.9.
abstract: BACKGROUND
  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 easily detected in living organisms by fluorescence microscopy and flow cytometry.
  
  
  RESULTS
  In 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 comparison 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.
  
  
  CONCLUSIONS
  We 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.
authors.affiliation: Department of Pathogen Biology, Second Military Medical University, Shanghai, China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Cao
authors.forename: Dongmei
authors.initials: D
authors.lastname: Zhang
authors.forename: Weiqing
authors.initials: W
authors.lastname: Pan
mesh_terms: D000818:Animals; D005434:Flow Cytometry / Q000379:methods; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D005821:Genetic Techniques; D049452:Green Fluorescent Proteins / Q000378:metabolism; D017780:Malaria Vaccines / Q000235:genetics* / Q000378:metabolism; D020066:Merozoite Surface Protein 1 / Q000235:genetics*; D051379:Mice; D008807:Mice, Inbred BALB C; D008856:Microscopy, Fluorescence / Q000379:methods; D010962:Plasmodium berghei / Q000378:metabolism*; D010963:Plasmodium falciparum / Q000378:metabolism*; D011817:Rabbits; D019076:Transgenes; D014614:Vaccines, Synthetic / Q000235:genetics
chemical_list: D017780:Malaria Vaccines; D020066:Merozoite Surface Protein 1; C535092:PfCP2.9 vaccine; D014614:Vaccines, Synthetic; D049452:Green Fluorescent Proteins