Bioinformatics Analysis, Cloning And Expression Of Rhoptry Protein17of Toxoplasma Gondii And Immunoprotection Of Recombination Protein

ObjectiveThe purpose of this study was to analysis and predict the main characteristicsand specific antigen epitopes of rhoptry protein17(TgROP17) from Toxoplasmagondii by bioinformatics. Then we cloned and expressed the rhoptry protein17(ROP17) gene of RH strain of Toxoplasma gondii, purified and analyzed theantigenicity of recombinant protein; And we observed the immune response inducedby different doses of recombinant Toxoplasma gondii rhoptry protein17(rTgROP17)via an intranasal immunization; Then we evaluated the efficacy of intranasalimmunization with recombination Toxoplasma gondii rhoptry protein17(rTgROP17)protects mice against Toxoplasma gondii infection. The feasibility of rTgROP17tobe used as candidate vaccine for toxoplasmosis has been discussed in this study.MethodsThe first part：The physic-chemical properties、transmembrane area、 signalpeptides, solublitys, post-translational modification sites, econdary structure andspecific antigen epitopes of TgROP17were analyzed and predicted by onlinebioinformatics programs, combined with bioinformatics software such as GeneRunner and DNAMAN.The second part：Total RNA was extracted from tachyzoites of RH strain of T.gondii. A pair of specific primers which introduced BamH I and Xho I restrictionsites were designed according to TgROP17gene open reading frame.The target genewas amplified by RT-PCR and connected into the pGEX-6P-1vector after digestingwith double restriction enzyme, the recombinant plasmid was transformed into E. coli (DH5α), positive colonies were selected through the colony-PCR and identifiedby the double restriction enzyme digestion and sequencing. The constructedrecombinant plasmid pGEX-6P-1-TgROP17transformed into E. coli Rosetta (DE3)and induced to express with IPTG. The rTgROP17was purified with GST affinitychromatography.The expression products were analyzed by SDS-PAGE. Westernblotting assay was used to identify the recombinant protein and analyze its antigenicproperties respectively.The third part：Forty BALB/c mice were randomly divided into five groups,Themice were intranasally immunized with20μl of PBS containing15μg,25μg,35μgor45μg rTgROP17on the day0,14and21, respectively, the control mice received20μl of sterile PBS alone. Two weeks after the last immunization, Blood sampleswere collected from the mice in each group via retro-orbital plexus puncture andstored at-20oC for IgG and IgG isotype assays.Mucosal washes, including nasal,vaginal and intestinal washes, were collected and stored at-20oC for secretory IgA(SIgA) assays. The spleens were collected under aseptic conditions to performlymphocyte proliferation assays by CCK-8and calculated stimulation index (SI), andthe levels of IFN-γ, IL-2, IL-4and IL-5of splenic lymphocyte culture supernatantwere detected by ELISA.The fourth part：Forty BALB/c mice were randomly divided into two groups,The mice were intranasally immunized with20μl of PBS containing35μg rTgROP17on the day0,14and21, respectively, the control mice received20μl of sterile PBSalone. Two weeks after the last immunization,12mice from each group werechallenged orally with a lethal dose (4×104tachyzoites per mouse) of T. gondii RHstrain. The symptoms and survival times of the challenged mice were monitored andrecorded for30days, the remaining8mice from each group were orally challengedwith a nonlethal dose (1×104tachyzoites per mouse) of T. gondii strain RH. On the 30th day after being challenged, the mice were killed and the numbers of tachyzoitesin the livers and brains were measured.ResultsThe first part： The TgROP17consists of608amino acids with the listedcharacters: the molecular formula is C3087H4879N889O875S19; the molecular mass is69056.2; the value of isoelectric point is9.55; the half-life is30hr. The proteinpresents a soluble form without transmembrane area and signal peptides.There were8post-translational modification sites,24B cell epitopes,23CTL cell epitopes and24Th cell epitopes.The second part： The product of RT-PCR was1850bp. The recombinantplasmid pGEX-6P-1-TgROP17was constructed successfully. SDS-PAGE revealedthat the recombinant soluble protein was96kDa. Western blotting showed thatTgROP17recombinant protein can be recognized by GST tag antibody and sera ofrabbit anti-Toxoplasma.The third part： Significantly higher titers of SIgA were detected in nasal,vaginal and intestinal washes from the mice immunized with25μg,35μg and45μgrTgROP17compared with the control group (P<0.05or P<0.01). And the highesttiter was generated in the35μg group.Significantly high levels of IgG were observedin the sera from the mice immunized with25μg,35μg and45μg rTgROP17compared with the controls (P <0.05or P <0.01). The levels of IgG2a were higherthan IgG1. The splenocyte stimulation indices (SI) from the25μg,35μg and45μggroups were higher than that of the PBS group (P<0.05or P <0.01). Compared tothe PBS controls, the spleen cells from the mice vaccinated with25μg,35μg and45μg rTgROP17exhibited significantly higher levels of IFN-γ and IL-2(P<0.05or P <0.01). Among the immunized groups, the highest titer was generated in the35μggroup. The secretions of IL-4in the splenocyte supernatants of the35μg and45μgrTgROP17immunized mice were also significantly increased compared to those of the PBS group (P <0.05). However, the production of IL-5did not statistically differamong all groups (P>0.05).The fourth part：The tachyzoite load in lives and brains (49.42±9.15×105/g and9.63±1.64×105/g) of immunity group were significantly lower than that of thecontrol group (120.62±16.7×105/g and18.91±3.24×105/g)(P<0.01). The reductionrates were59.17%and49.08%, respectively, compared to the control group.Thesurvival rates of rTgROP17immunized mice (75%) was significantly increasedcompared to the control group(25%) on day30after challenge (P<0.01).ConclusionThe results of bioinformatics analyze showed that the TgROP17has potentialepitopes, can be used as a candidate antigen for vaccine of toxoplasmosis. Weconstructed recombinant pGEX-6P-1-TgROP17successfully. The recombinantprotein was expressed in E. coli Rosetta （DE3） and showed the specificantigenicity.Intranasal immunisation with35μg of rTgROP17elicited strongmucosal immune responses and systemic immune responses and can elicited partialprotection against acute and chronic T. gondii infection. Therefore, rTgROP17canbe considered a possible candidate antigen for vaccine of toxoplasmosis.