Construction Of Transgenic Mouse Malaria Parasite Carrying MSP1 Of Plasmodium Falciparum By Gene Targeting Technique And Its Application

Malaria remains one of the most devastating 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 disease.Merozoite surface protein l(MSP-l) of Plasmodium falciparum(Pf) is the major glycoprotein on the surface of merozoite ,the erythrocyte-invasive form of malaria parasite. It can induce protective immunity against the parasite and its 19-kD C-terminal region of this molecule(MSPl-19) has been mapped to be its functional domain for the protective immunity.Antibodies specific for PfMSP1-19 can inhibit erythocyte invasion and parasite growth in vivo and in vitro.This polypeptides is therefore considered to be one of the more promising malaria vaccine candidates.The P.falciparwn blood stage vaccine, PfCP-2.9, which was constructed and expressed in our lab contains this component and now is in the clinic trial stage.Now, the lack of economical and effective animal model is one of the most difficult problems in the malaria vaccine research. Although the Aotus monkeys in the South America can be infected by P.falciparum as a model to evaluate the malaria vaccine, it is expensive and not the natural host of P.falciparum parasites. In the recent years ,the technique of malaria transfection has been developed and applied to many aspects of malaria research, such as the malaria protein structure and founction, the interaction between the host and the parasites, and the control of the gene expression. Through allelic replacement it was showed that despite the highly divergent amino acid sequence aross the distant related Plasmodium species, many important malaria antigens ,for example TRAP and MSP-1, are founctionlly conserved and can be complemented with the corresponding sequence from the otherPlasmodium species. Here we want to use the malaria transfection technique to generate a line of P.berghei that expresses a human malaria(P.falciparum) form of MSP 1-19. In this line of transgenic P.berghei parasites, the endogenous 19KD C-teminal fragment of PbMSP-1 is replaced with PfMSP1-19, the counterpart from P.falciparum. Because it expresses the C-teminal fragment of PfMSP-1, it can be used as a model to value the immunity protection of P.falciparum vaccine containing the component of PfMSP1-19.PCR was performed on P.berghei ANKA genomic DNA using two pairs of oligonucleotides and yielded 1.4Kb and 500bp targeting sequences from P.berghei MSP 1 gene and its 3'untranslated region, respectively. To create chimeric PbfMSPl,the 1.4Kb P.berghei MSP 1 fragment was fused in frame to the 330bp MSP1-19 region amplified by PCR from P.falciparum genome.The chimeric PbfMSPl and 500bp PbMSP-1 3'UTR fragment were cloned into the P.berghei transfection vector pPyrFlu to create the single crossover integration plasmid, PyrFlu/PbfMSP1, and the double crossover integration plasmid, PyrFlu/PbfMSP1/Pb 3'UTR。 The pPyrFlu vector contains anti-drug gene from Pb DHFR-TS and the reporter gene of green fluorescence protein (GFP), which both can be expressed in the P.berghei transformant. The recombinant plasmids identified by endonuclease digestion and DNA sequencing were linearized before electroporation.The infected blood obtained from mice was incubated overnight under standard culture conditions and mature schizonts of P.berghei ANKA cotaining fully developed merozoites were separated from uninfected cells on density gradients, Nycoprep. In preliminary experiment we observed significant differences in the efficiency of transformation with different electroporation volume and concluded that the suspension of 100μl volume can reach the highest efficiency. After electroporation the P.berghei parasites were intravenously inoculated into naive mice and selected by injection of anti-malaria drug (pyrimethamine). The positive transformants were observed after two-cycle drug selection and the expression of GFP were detected with fluorescence microscope. In the parasites transfected with the plasmid, PyrFlu/PbfMSP1/Pb3'UTR , the correct integration into Pb MSP-1 gene andreplacement of endogenous PbMSPl-19 fragment was confirmed by PCR ,which detected not only the plasmid but also the homologous recombination in the genome of transgenic parasites.To evaluate the immunity protection effect of the P.falciparum chimeric antigen, PfCP-2.9, using the transgenic P.berghei parasites, we constructed P.berghei chimeric antigen PbCP-2.9. According to the sequence homology, the coordinate sequences were found in P.berghei in contrast to PfCP-2.9 Redesigned using Pichia coden usage and clned into the expression vector pPIC9K. Then the recombinant plasmid were introduced into the GS115 genome and transfectants were selected with G418 for the clone of higher expression. To verify the protein PbCP-2.9 the anti-parasite serum was prepared and used in the western blotting. The result showed that after induction with methanol a 26KDa protein band could be discerned. Because six tandem histidine was designed and added to the C-terminal of PbCP-2.9, the protein in supernatant was purified by Ni-NAT affinity chromatography and >90% purity of the protein was achieved.The BABL/c mice were immunized subcutaneously 4 times at 3-week interval with chimeric protein PfCP-2.9 and PbCP-2.9 emulsified in the Freund's adjuvant,respectively. The mice immunized with PBS mixed by adjuvant were used as adjuvant control and the unimmunized mice as blank control. Two weeks after the last booster the mice were challenged i.p. with the naive P.berghei and the tansgenic P.berghei expressing PfMSPl-19. The parasitemia level of every mouse was determined and plotted every day to evaluate the immunity protection effect of two chimeric antigens. In the PbCP-2.9-immune group, mice challenged with naive P.berghei can endure longer time than the mice challenged with transgenic P.berghei as well as the mice in adjuvant group and blank group. It showed the chimeric protein PbCP-2.9can induce the immunity to the naive P.berghei. In the PfCP-2.9-immune group, although no difference for survival time, a statistically significant difference was observed for the mean parasitemia between naive P.berghei and transgenic P.berghei. The result that the latter is lower demenstrated that the P. falciparum chimeric protein PfCP-2.9 can induce the immunity to the transgenic P.berghei...