Identification And Immunological Study Of Toxoplasma Gondii Proteases

Toxoplasma gondii is an apicomplexan and obligate intracellular parasite, it can cause toxoplasmosis which is a kind of widely distributed in warm-blood animals including human. This disease is a serious threat to human health and the development of stockbreeding; especially on people have impaired immune function, including AIDS patients and patients receiving long-term chemotherapy. Due to there are no drug treatments available to against T. gondii infection, so development of an effective vaccine is a vital strategy in the control of toxoplasmosis. In this study, we fistly analyze and predict the basic physical and chemical properties, subcellar localization, modification post translation, B-cell and T-cell antigen epitopes of four toxoplasma gondii protease. We have observed the expression of TgASPl and TgASP3in parasites by indirect immunofluorescence assay using laser scanning confocal microscope, among them, TgASP3was clearly indentified for the first time. Recombinant DNA vaccines were constructed to evaluate the immune protection. This study filled the research gap of TgASP3, enriched the basic theoretical knowledge about toxoplasma gondii proteases, and laid a foundation of the development of safe and effective vaccine against toxoplasma infections.Objective:Identified the toxoplasma gondii proteases TgASPl and TgASP3, observed the distribution of them in toxoplasma gondii parasites, then constructed recombinant gene vaccines to evaluate the immune protection of cysteine proteases TgCPB, TgCPL and aspartic proteases TgASP1, TgASP3of toxoplasma gondii.Methods:Firstly, bioinformatics approaches were used to analyze and predict the physical and chemical properties, subcellar localization, modification post translation, B-cell and T-cell antigen epitopes of TgCPB, TgCPL, TgASP1, and TgASP3. The recombinant prokaryotic expression vectors were constructed and were transfected into E.coli BL21(DE3) to prepare soluble recombinant proteins. Mice were immunized subcutaneously with purified rTgCPB, rTgCPL, rTgASP1and rTgASP3proteins to prepare anti sera. Through immunolocalization experiments, TgASP1and TgASP3were observed in both extracellular parasites and intracellular parasites grown overnight in HeLa cells under a laser scanning confoal microscope. Finally, recombinant eukaryotic expression vectors were inoculated into BALB/c mice to evaluate the immune protection.Results:The physical and chemical properties, subcellar localization, modification post translation of TgCPB. TgCPL, TgASP1, and TgASP3were analyzed using bioinformatics softwares, B-cell and T-cell antigen epitopes of TgCPB. TgCPL. TgASP1, and TgASP3were predicted by many bioinformatics approaches and filtered out a lot of potential antigen epitopes areas with good hydrophilic, strong plasticity, strong accessibility and high antigen index. The results showed that toxoplasma gondii proteases TgCPB, TgCPL, TgASP1and TgASP3are all good protein antigens. TgCPB, TgCPL, TgASP1and TgASP3gene fragments were amplified by PCR, molecular weight size are respectively1710bp,1269bp,1863bp and1932bp. and then they were cloned into pET30a prokaryotic expression vector in order to construct cloned plasmids pET30a-TgCPB. pET30a-TgCPL. pET30a-TgASP1and pBT30a-TgASP3, meanwhile, TgCPB and TgCPL gene fragments were cloned into pBudCE4.1eukaryotic expression vector in order to construct cloned plasmids pBudCE4.1-TgCPB. pBudCE4.1-TgCPL and pBudCH4.1-TgCPB-TgCPL. TgASP1and TgASP3gene fragments were cloned into pEGFP eukaryotic expression vector in order to construct cloned plasmids pEGF’P-TgASP1and pEGFP-TgASP3. E, coli BL21(DE3) cells were transformed by pET30a-TgCPB, pET30a-TgCPL, pET30a-Tg ASP1and pET30a-TgASP3. Recombinant proteins TgCPB, TgCPL, TgASP1and TgASP3were induced by IPTG and purified by NiNTA resin. Mice were immunized subcutaneously with purified rTgCPB, rTgCPL, rTgASP1and rTgASP3proteins to prepare anti sera, specific antibody IgG was obtained by further separation and purification. The expression area and expression level of TgASPl and TgASP3were observed by indirect immunofluorescence assay under a flurescence microscope. Through immunolocalization experiments, both extracellular parasites and intracellular parasites grown overnight in HeLa cells were observed under a laser scanning confocal microscope. BALB/c mice were immunized with eukaryotic expression plasmids and the determination of anti-toxoplasma total IgG, IgGl, IgG2a and cytokines IL-4, IL-10, IFN-y levels between experimental and control groups were used to assess its ability to stimulate cellular and humoral immunity. To evaluate the immunoprotection induced by the DNA vaccines, all the mice were challenged intraperitoneally (i.p.) with T.gondii RH strain.Conclusions:The results of immunolocalization experiments show that TgASP1is a membrane protein and has a punctuate apical localization rather than being cytosolic. In addition, it seems like that TgASP1does not localize in typical regions where the secretory organs, including rhoptries, micronemes or dense granules localized in. Previous studies suggested that TgASP1resides in a novel compartment of the secretory system that potentially serves a link between the Golgi and the inner membrane complex (IMC). The results confirmed that recombinant DNA vaccines can induce strong cellular and humoral immunity, significantly enhance the ability of mice against toxoplasma gondii infection, prolong the survival times and verify the feasibility of prediction of protein antigen epitopes using bioinformatics approaches.