Functional And Immunogenic Analysis Of The TatD-like DNase Of Plasmodium Parasites

Malaria caused by plasmodium spp is an epidemic disease around the world for thousands years.The parasite develops and reproduces in erythrocyte during asexual stage,which causing that the DNA of plasmodium is more suspectiable to intracelluar oxidant pressure than other intacelluar parasites due to lack of necessery organelles in erythrocyte.The repair mechanism of DNA damage in plasmodium parasite is not clear,and relative factors participate in the repair programs are still under discussed.In the present study,we found that PfTatD-like DNase?PF3D7₀112000?contain a conseved TatD domain which participatites in DNA damage repair in Ecoil by bioinformatics analysis.In this study,we wanted to analyse the TatD-founction in plasmodium as well as the immunogenicity of this protein.At first,the conservaiton of the TatD-like DNase gene of Plasmodium falciparum in epidemic districts has been performed by aligment of PCR products amplified by primers designed based on TatD-like DNase gene of Plasmodium falciparum 3D7 strain.The coding regions of the genes are identical,and there are around 5% differences in the non-coding regions among the genes.The conservation of TatD-like DNase provide the basis for new medicine reaearch and vaccine development.Second,we have expressed the recombinant TatD-like DNase by prokaryotic expression system.The polyclone antibodies obtained from immunized mice were used to localize the protein in parasite.The TatD-like DNase has been proven to localized around the parasite surface in sexual stage by IFA.From the data we can infer that the TatD-like DNase is expressed during sexual and asexual stage in plasmodiun falciparum,from which we can conclude that TatD-like DNase can be used as the potencial antigen for transmisson blocking vaccine.Single cell gel eletrophoresisanalysis and protein migration analysis were used to analyse the DNA repair founction under the oxidant pressure.After damage induced,the trancription and expresson level of TatD-like DNase are upgraded and the protein was translocated to nuclei,in mean time,compared with the P.berghei wild type,DNA fragmentation of the P.berghei TatD KO strain was more serioius and the tails were much longer after oxidant conduction.From above,we can conclude that the TatD-like DNase protein participates in the DNA repair progress caused by oxidant pressure.At last,we have proven that anti-TatD-like DNase serum can inhibit the exflagellation of the microgametocyte,formation of gametocyte,ookinete and oocyst.Montanide ISA51,Montanide ISA61,freund?s ajuvant,alhydrogel,and levomisole were used to evaluate the immunogenicity of TatD-like DNase.Depending on the data,Montanide ISA51 adjuvanted with PbTatD-like DNase inhibited the development and proliferation of the parasite more effective and live longer than other grouops,then passive serum tranfer experiment has proved that the specific IgG obtained from the immunized mice is the major factor to inhibit the development of the parasites.In conclusion,Montanide ISA51 adjuvanted with PbTatD-like DNase has the best effect of immune protection in the present experiment,which proved that TatD-like DNase have the potenial to be used as canidate antigen for malaria vaccine.