Expression And Functional Identification Of DNase Containing EEP Domain In Plasmodium Falciparum

Worldwide, malaria is still a critical public health problem. More than one million people die from malaria every year, most of which are children under 5 years old. Malaria is caused by Plasmodium which is an obligate intracellular parasite. During the blood-stage, asexual development and multiplication within human erythrocytes is responsible for the physiological and pathological process of malaria.Deoxyribonucleases（DNase） are important proteins involved in lots of cell functional process, such as DNA replication, repair and chromosomal recombination, as well as immune escape. EEP domain which could hydrolyze phosphodiester bonds in DNA is one of the catalytic domains in DNase. PF3D7₁238600, PF3D7₀107200, PF3D7₀305600, PF3D7₀319200 and PF3D7₁238600 protein of plasmodium falciparum 3D7 clone are predicted to contain EEP domain and have not been investigated.First of all, transcription in different blood stage of PF3D7₁238600, PF3D7₀107200, PF3D7₀305600, PF3D7₁363500 and PF3D7₀319200 gene was investigated by q PCR with eryl-t RNA synthetase gene worked as internal control. As the date shown, targeted genes transcribed in whole blood stage and reached a peak at trophozoite and schizont. Among the five genes, PF3D7₁238600 showed the higheset transcriptional level and PF3D7₀107200 showed the lowest transcriptional level.Secondly, targetted segments with higher hydrophilicity was expressed and purified based on the hydrophilic analysis of amino acids sequence. In order to get specific antibodies, recombinant proteins emulsified with Freund’s adjuvant was injected subcutaneously to rabbits. With the specific antibodies worked as primary antibodies, targeted genes expression was detected in parasite lysate with Western blot. Indirect immunofluorescence（IFA） was used to analyze the location of targeted DNase in parasite. PF3D7₀319200 located in the nucleus. PF3D7_<sub>1238600, PF3D7₀305600 and PF3D7₁363500 located around the nucleus. PF3D7₀107200 located in cytoplasm of parasite.Functional domain of targeted DNase was expressed and purified to identify their DNase activity in vitro. As the result shown, PF3D7₁363500 and PF3D7₀107200 was divalent metal ion independent endodeoxyribonuclease. PF3D7₀305600 and PF3D7₀319200 was Mg2+/Mn2+ dependent endodeoxyribonuclease. PF3D7₁238600 was Cu2+ dependent exodeoxyribonuclease.In this study, the transcription, expression, subcellular localization of targetted DNase were investigated and these DNase activities were confirmed by DNA degradation assay in vitro. All of this was basement of functional research and malaria therapy.