Construction and biological characterisation of a genetically engineered Plasmodium berghei P.bzoGFP
Objective To construct a genetically engineered Plasmodium berghei strain that expresses green fluorescent protein at the ookinete stage,providing a tool for studies related to this stage.Methods A targeting vector comprising the GFP gene sequence and the recombinant homology arms of the pb25 gene locus was constructed,linearised and then electro-transfected into Plasmodium berghei.Following drug screening,cloning was carried out using limited dilution,and genotype of parasites was detected using PCR.The biological characteristics of the genetically engineered Plasmodium berghei were analyzed through observation of the morphological characteristics of the intra-erythrocytic phase,zygote and ookinete phase and by comparing the parasitemia and survival of mice infected with WT and genetically engineered parasites.Results Genetically engineered Plasmodium berghei P.bzoGFP,genotyped by PCR,exhibited consistent results with the predicted outcomes and demonstrated the capacity to express green fluores-cent protein at the ookinete stage.P.bzoGFP did not display any significant deviations from the wild strain with respect to the morphology of the intra-erythrocytic phase,the zygote and ookinete phase.The survival curves of the two groups did not differ significantly(χ2=0.309,P>0.05).The number of ookinetes obtained from in vitro culture was not sta-tistically significant(t=0.341,P>0.05).Conclusion The genetically engineered Plasmodium berghei strain P.bzoGFP has been successfully constructed,exhibiting biological characteristics that are not significantly different from those of the wild-type parasite.This strain can effectively express GFP after zygote formation,enabling its direct use in studies related to this phase,such as evaluating the efficacy of malaria transmission blockade,without the need for anti-body labeling.
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