Preparation Of Toxoplasma ROP17 Antibody And Preliminary Study On The Distribution, Function And Mechanism Of Toxoplasma ROP17

Objective:After purifying the protein of Toxoplasma gondii Rhoptry protein 17(ROP17),a polyclonal antibody was prepared,and its titer and immunogenicity were identified.The eukaryotic expression vector of Toxoplasma dihydrofolate reductase-thymidylate synthetase-ROP17(pIRES/TgDHFR-TS-TgROP17)was constructed and then transfected into human embryonic kidney cells(HEK 293T)to express ROP17 protein.The function and mechanism on autophagy of ROP17 were investigated in pIRES/TgDHFR-TS-Tg ROP17 transfected HEK 293 T cells with serum free condition.Method:The first part: The recombinant plasmid pGEX-6P-1-ROP17 was transformed into Escherichia coli BL21(DE3)and was induced by IPTG to express ROP17.Recombinant protein(GST-ROP17)was purified by affinity chromatography.SD rats were immunized,and polyclonal antibodies were identified by Western blotting.The second part: Total RNA of T.gondii(Tg)was extrcted from RH strain tachyzoites,TgDHFR-TS and TgROP17 were amplified and the recombinant vector pIRES/TgDHFR-TS-Tg ROP17 was constructed.After identification by colony-PCR,double enzyme digestion and sequencing,the pIRES/Tg DHFR-TS-TgROP17 was transfected into 293 T cells and the expression of TgROP17 mRNA and protein were detected by RT-PCR and Western blotting.The third part: The expression of LC3 protein in the brain tissue of BALB/c mice infected with T.gondii was observed by immunohistochemistry assay.To study the function of ROP17,pIRES/TgDHFR-TS-TgROP17 recombinant plasmid was transfected into 293 T cells and pIRES/ TgDHFR-TS-transfected 293 T cells was used as a control group.Transfected cells were treated with serum starvation for 0,6,and12 hours,proteins were extracted and autophagy marker proteins LC3-?,Beclin-1,and P62 were tested by Western blotting.The expression of autophagosomes was detected by monodansylcadaverine(MDC)staining,GFP-LC3 fusion protein expression,and transmission electron microscopy.The expressions of JNK,p-JNK,Bcl-2,and p-Bcl-2 were tested by Western blotting to analyze the signal pathway involved in ROP17.Results:The first part: The ROP17 polyclonal antibodies were prepared successfully with immunizing SD rats with purified GST-ROP17.The antibody can recognize the ROP17 protein which expresses in Toxoplasma gondii tachyzoite.Western blotting results also showed that ROP17 protein was distributed in the liver,kidney and spleen tissues of BALB / c mice infected with Toxoplasma gondii.The second part: RT-PCR results showed that a fragment of about 1809 bp was amplified from the RH strain T.gondii tachyzoites.Colony-PCR,double digestion and sequencing showed that the recombinant pIRES/TgDHFR-TS plasmid was successfully constructed.RT-PCR and Western blot results showed that the expression of TgDHFR-TS was found in pIRES/TgDHFR-TS transfected 293 T cells with the gene size was 1809 bp,and the relative molecular mass(Mr)was about 68 kDa.Meanwhile,RT-PCR showed that a fragment of about 1860 bp was amplified from the RH strain T.gondii tachyzoites.The results of colony-PCR,double digestion and sequencing showed that the recombinant plasmid pIRES/TgDHFR-TS-TgROP17 was successfully constructed.The results of RT-PCR and Western blot showed that TgROP17 was expressed in pIRES/TgDHFR-TS-TgROP17 transfected 293 T cells,the gene size was 1860 bp,and the relative molecular mass(Mr)was about 70 kDa.The transfected with pIRES / TgDHFR-TS plasmid group showed no expression of corresponding molecular weight genes and proteins.The third part: The levels of LC3 in the brain tissue of BALB/c mice infected with T.gondii were increased than those in the normal mouse brain via immunohistochemistry assay.After serum starvation administration,the results of Western blotting showed that the LC3-? and Beclin-1 protein were gradually increased,while the P62 protein was gradually decreased with a time dependant manner.There was statistical significance(P<0.05)compared with the control group.The results of MDC staining,GFP-LC3 fusion protein expression,and transmission electron microscopy showed that the autophagosomes in ROP17-transfected cells were gradually increased than those in the control cells with a time dependant manner in serum stravation,There was statistical significance(P<0.05)compared with the control group.Additionally,Western blotting showed that there were no changes in the levels of JNK and p-JNK proteins,while Bcl-2 protein gradually decreased and p-Bcl-2 protein gradually increased with a time dependant manner.There was a statistically significant difference compared with the control group(P<0.05).Conclusion:1.The polyclonal antibody against Toxoplasma gondii Rhoptry protein 17(ROP17)was prepared successfully.2.The recombinant plasmid pIRES/TgDHFR-TS-TgROP17 were constructed sucessfully.3.ROP17 may promote serum starvation-induced cell autophagy through the ROP17-Bcl-2/Beclin1 signaling pathway.