Cloning And Prokaryotic Expression Of Actin Gene Of Toxoplasma Gondii And Protective Immunity Of Recombination Protein

ObjectiveThis study was performed to clone and express and purify the actin gene of Toxoplasmagondii, analyzing the immunogenicity of TgACT and its effects intranasally immunized onprotection of mice against Toxoplasma gondii, it may be used as candidate antigen.MethodsThis study included three parts.First part was to construct pET30a TgACT and express the Actin gene of Toxoplasmagondii. Toxoplasma gondii RH strain tachyzoites were harvested and genomic RNA wasprepared. A pair of primers and restriction site EcoR I/Xho I was designed. The coding region ofActin was amplified with RT PCR and cloned into the prokaryotic expression vector pET30a.The recombinants were confirmed by digestion with EcoR I/Xho I, PCR, and DNA sequencing,and then transformed into E.coli BL21induced by IPTG. The proteins analyzed by SDS PAGEwere expressed in inclusion body. rTgACT were treatmented with the dilution refolding processthen purified with Ni NTA maffinity chromatography. The antigenicity of rTgACT was analyzedby Western blotting with rabbit antiserum against Toxoplasma.Second part was to observe the mucosal and systemic immune responses of mice inducedby intranasal immunization with different dose of rTgACT. Fifty BALB/c mice were divided into5groups at random. The mice were intranasally immunized with10g,20g,30g or40grTgACT per mouse at an internal of2weeks and1week, while mice intranasally administratedwith PBS in the same means were the control group. Fourteen days after the last immunization,collect blood from eye vein plexus. The mice were put to death by cervical vertebra dislocation,to collect the washing fluid of nasopharynx, vagina and intestinal. Detect the IgA and IgG withELISA. Spleen cell suspension were collected to be counted and incubated at37, thecell free supernatants were harvested and assayed for interleukin2IL2and interleukin4(IL4)activity at24h, interferon gamma (IFN γ) activity at96h. The concentration of IL2, IL4andIFN γ were determined with ELISA. In vitro lymphocyte proliferation studies were performedand the proliferation response was measured by CCK8kit. Choose the best dose ofimmunifaction.Third part was to study the protection of30g rTgACT against Toxoplasma infection. BALB/c mice were divided into2groups at random.30g rTgACT per mouse at an internal of2weeks and1week, while mice intranasally administrated with PBS in the same means werecontrol. All mice were challenged intragastrically with8×104tachyzoites per mouse on day14after the last immunization. The condition of mice infected about death was observed every day.Mice were killed on the30th day after challenge, the tachyzoites of their lives and brains werecounted.ResultsT. gondii encoding Actin gene with a molecular size of1131bp and the recombinantpET30a TgACT was successfully constructed. The results of SDS PAGE revealed that themolecular weight of recombinant protein was approximately49kDa, more than7kDa thanexpected. rTgACT were treatmented with the dilution refolding process then purified withNi NTA maffinity chromatography. Concentrate the protein by ultrafilter concentration toincrease the concentration. Westen blotting revealed that rTgACT can be recognized by rabbitantiserum of Toxoplasma.Different dose of rTgACT can induce the mucosal and systemic immune responseseffectively. Specific IgG antibody titers in serum:30g and40g are significantly high(P<0.05). The difference of IgA in flushing fluid of nasopharynx, vagina and intestinal in30gimmunized group had statistical significance (P<0.05). There is no significant difference inlymphocyte proliferation result. IL4and IFN γ was significantly high in the immunized group(P<0.05), but no difference in IL2. Compared with the control group, all the immunized groupscan cause immune response in various degrees. Above all, we choose the30g rTgACT permouse as the best immune dose.Mouse was immunized intranasally30g rTgACT per mouse and PBS as control,2weeksafter the last immunization. All mice were challenged intragastrically with8×104tachyzoites permouse on day14after the last immunization.2days after intraperitoneally injected, the micemove less and in bad condition. Ten mice died in the control group and ten mice died in theimmunized group. Mice were killed on the30thday after challenge, The tachyzoite load in theirlives of immunized mice was77.89±20.00核105/g significantly lower than the control group169.77±59.74核105/g (P<0.05), the tachyzoite load decreased by54.12%. The amount in brainsof immunized mice was13.20±2.54核105/g, also lower than the control group22.29±3.35核105/g,the tachyzoite load decreased by40.78%.ConclusionThe recombinant pET30a TgACT was successfully constructed. The recombinant proteinhas been confirmed with immunogenicity, it may be used as candidate antigen. Compaired withother groups,30g rTgACT had induced the mucosal and systemic immune response against Toxoplasma gondii.