Cloning And Prokaryotic Expression And Purification Of Peroxiredoxin Gene Of Toxoplasma Gondii And Immunoprotection

ObjectiveThis study was performed to clone and express and purify the peroxiredoxin gene of Toxoplasma gondii, We analyzed the immunogenicity of TgPrx and its effects intranasally immunized on protection of mice against Toxoplasma gondii, it may be used as candidate antigen.MethodsThis study included three parts. First part was to construct pET30a/TgPrx and express the peroxiredoxin gene of Toxoplasma gondii. RH strain tachyzoites were harvested and genomic RNA was prepared. A pair of primers and restriction site EcoR I/Xho I was designed. The coding region of peroxiredoxin was amplified with RT-PCR and cloned into the prokaryotic expression vector pET30a. The recombinants were confirmed by EcoR I/Xho I, PCR, and DNA sequencing.and then transformed into E.coliBL21 induced by IPTG. The expressed proteins were analyzed by SDS-PAGE.Second part was to study analyze the immunogenicity of rTgPrx and preparation of anti-serum. A lot of soluble rTgPrx were purified with Ni-NTA maffinity chromatography and analyzed by Westen blotting. Rats were immunized by purified rTgPrx through subcutaneous injection, the antibody was detected by ELISA. purified TgPrx were added PMSF to observe protein degradation. Prx was allocated by immunohistochemistryThird part was to study the mucosal and systemic immune responses after intranasal immunization with rTgPrx and protect mices against Toxoplasma gondii . BALB/c mice were intranasally immunized with 10μg, 20μg, 30μg or 40μg rTgPrx per mouse at an internal of 2 weeks, while mice intranasally administrated with PBS in the same means were control. All mice were challenged intragastrically with 1×104 tachyzoites per mouse on day 14 after the last immunization. The condition of mice infected about death was observed every day. Mice were killed on the 30th day after challenge, the tachyzoites of their lives and brains were counted. Serum IgG and IgA in feces were detected by ELISA. Lymphocytes of IEL and spleens were counted.ResultsT. gondii encoding peroxiredoxin gene with a molecular size of 591 bp and the recombinant pET30a/TgPrx was successfully constructed.. The results of SDS-PAGE revealed that the molecular weight of recombinant protein was approximately 32 kDa. Westen blotting revealed that rTgPrx can be recognized by rabbit antiserum of Toxoplasma. Rat immunized with the purified rTgPrx elicited high titer of antibody. rTgPrx began to degrade after two weeks, freeze thawing could accelerate. Immunohistochemistry revealed that Prx can be recognized by Rat antiserum of TgPrx and distribution of Prx was brownIn 10μg group and control group, the amount of mice was 4, 20μg,30μg was 6 and 40μg was 9. Compared with control group, the tachyzoite load in lives and brains in 40μg group was significantly lower (P<0.05). The level of IgG, spleens and IEL in all immunized mice was higher than that of control (P<0.05), IgA was no different.ConclusionThe recombinant pET30a/TgPrx was successfully constructed. A lot of soluble rTgPrx was producted. The recombinant protein has been confirmed with immunogenicity, it may be used as candidate antigen. Compaired with other groups, 40μg rTgPrx had induced the mucosal and systemic immune response against Toxoplasma gondii.