The Expression And Analysis Of TatD-like DNase Sequence Of Rodent Malaria Parasite

Malaria is an important parasitic disease caused by Plasmodium and epidemicin tropical and subtropical regions. It is known that malaria, HIV/AIDS, andtuberculosis are the most serious infectious diseases to human being. The malariaparasite immune evasion mechanism is very complicated, which is one of thereasons that make the malaria vaccine development difficult. Having studied themain pathological tissues of patients suffering from P.falciparum malaria,researchers didn’t found obvious inflammatory response such as caused by bacteriaor virus where a large numbers of parasites gathered. The gathering of macrophagesand other lymphocytes is similar to normal tissues and few neutrophils wereobserved. This suggests that the parasite may antagonize the host innate immune.Recently, a novel antimicrobial mechanism of neutrophil has been described: duringan active cell death process named NETosis, they release the chromatin andcytosolic lysosomal particles, forming neutrophil extracellular traps (NETs) to trapand kill a broad spectrum of pathogenic microbes, including Gram-positive andGram-negative bacteria, fungi, protozoa and viruses. The pathogen can secret a kindof DNase to degrade the DNA framework of NETs, destruct the NETs effect onpathogen, and escape from the innate immune clearance. DNase is an effectiveweapon for pathogenic microbes to circumvent the NETs attack and associated withits virulence. It has been considered as an important virulence/pathogenicity relatedfactor of pathogens. Several Plasmodium genome analysis results show that theTatD-like DNase sequences encoded in the various Plasmodium genomes are veryconservative, contain a N-terminal signal peptide, and very similar to TatD DNaseof E.coli in the spatial structure. TatD DNase in E.coli is a kind of protein with theactivity of degrading the exogenous DNA. Therefore we predict that Plasmodium can express and secret a kind of protein with DNase activity, TatD-like DNase. Theobjective of this study is the detection of TatD-like DNase expression in the threerodent malaria parasites and its correlation with Plasmodium pathogenicity.First, the recombinant plasmid pUC19-PCH, pUC19-PB, and pUC19-PY(stored in our laboratory) were digested with restriction endonuclease BamHI andXhoI, the target genes PCH, PB, and PY were obtained and cloned into prokaryoticexpression vectors pET-28a and pGEX-4T-1respectively. The recombinantepression plasmids were constructed and transformed into E.coli BL21. The E.coliBL21was induced to express the recombinant protein. Three His-tagged and threeGST-tagged recombinant proteins were purified by affinity chromatograghy. Theresult of Western-blot shows that the molecular weight is consistent with thesoftware predictive value for His-PCH, His-PB, GST-PCH and GST-PBrecombinant proteins, but lower than the predicted value about4kDa for His-PYand GST-PY.The rabbits were immunized with the purified His-tagged recombinant proteinsemulsified with Freund’s adjuvant. When the serum titer was higher than1:16000by ELISA assay, the blood was got from heart and antiserum was collected. Thespecific antibody IgG was purified by affinity chromatography, and applied to theWestern-blot and indirect immunofluoresscence analysis of the TatD-like DNaseexpression in the three rodent malaria parasites.Each female Balb/c mouse was infected with1×105parasitized red blood cells(pRBCs) by intraperitoneal injection. When the percentage of pRBCs was up to50%,the blood was collected by enucleating the mouse eyeball. The pRBCs were lysed byPBS containing0.1%Saponin and the parasites were collected by centrifugalization.The pRBC and parasite samples were analysed by Western-blot with theanti-TatD-like DNase IgG as primary antibody and the alkaline phosphataseconjugated goat anti-rabbit IgG as the second antibody. The specific bands weredetected in the pRBCs and parasites lane, and no band was found in negative control.This indicates that P.chabaudi chabaudi AS、P.bergher NK65and P.yoelii YM express TatD-like DNase, and the prepared polyclonal antibodies can recognize theTatD-like DNase expressed in parasites specifically. When the percentage of pRBCswas up to10%, slide was smeared with the mouse blood from its tail top. The RBCswere fixed by4%paraformaldehyde. The anti-TatD-like DNase IgG was used as theprimary antibody and the Alexa Fluor488goat anti-rabbit IgG as the secondantibody in the indirect immunofluoresscence assay. The specific green fluorescentwas observed in the parasitophorous vacuole and around the pRBCs, and no specificgreen fluorescent was found in negative control. This indicates that the three rodentmalaria parasites express TatD-like DNase and secret it to the extracellular. Thisstudy lays a foundation for further study of the TatD-like DNase functions and itscorrelation with parasite pathogenicity, as well as for novel antimalarials andefficient malaria vaccine research.