Exploration Of GRA15_? Protein Vaccine Against Toxoplasma Gondii Infection In Murine Model

Background:As an obligate intracellular protozoan parasite,Toxoplasma gondii is capable of infecting a wide range of warm-blooded animals including humans.All over the world,toxoplasmosis has done great harm to both human health and animal husbandry due to its zoonotic nature.Most isolates of T.gondii from North America and Europe belong to three types of genetically distinct clonal lineages,the types I,II,and III.In our country,specially,toxoplasmosis is mostly caused by T.gondii which belongs to a unique genotype named Chinese 1?Toxo DB#9?.Essential effector molecules secreted by T.gondii influence on both its own virulence and host cell transcription,and dense granule protein 15(GRA15_?)is one of the most significant effector molecules among them.Interestingly,it has been reported that GRA15 II from T.gondii either belong to Chinese 1 or type II share the same gene sequence,named GRA15 II.For these reasons,exploration of this protein will have a far-reaching influence on toxoplasmosis diagnosis,pathogenic mechanisms,prevention of toxoplasmosis and relative vaccine development.Objective: To explore the polarization of the GRA15 II transfected cells and the possibility of GRA15 II to be used as a kind of candidate molecule for toxoplasmosis protein vaccine.In addition,our study will provide a theoretical and experimental basis for toxoplasmosis prevention,toxoplasmosis diagnosis and protein vaccine against T.gondii.Methods: First,GRA15 II was amplified by PCR and cloned into the pET28 a plasmids to construct prokaryotic expression vectors.The recombinant plasmids were confirmed by PCR,enzyme digestion and sequencing,and then transformed into E.coli BL21?DE3?to induce expression and the recombinant protein was identified by Western-blotting.Bioinformatics were used to predict the physicochemical propertiesand antigen epitope of GRA15 II.GRA15_? expression was confirmed by WB and SDS-PAGE.Purified GRA15 II was used to stimulate RAW264.7,representative cytokines?IL-10,IL12p40,Arg-1andi NOS?were detected by ELISA and q RT-PCR to demonstrate if GRA15 II may induce M1 macrophages.On the other hand,purified protein was also used to immunize mice,the specific antibodies?Ig G?were investigated by ELISA after six weeks post-inoculation.Those mice were challenged with PRU?type II?tachyzoite,cytokines?IL-4,IL-12p40,L-13,TNF-? and TGF-?1?was detected by ELISA and q RT-PCR.The survival rate and the brain cyst number of mice were statistically counted and analyzed to evaluate the protection effect of GRA15 II.Results:?1?GRA15_? was purified and obtained successfully;?2?GRA15_? was predicted by bioinformatics,showing that it may be suitable as a candidate molecule for toxoplasmosis protein vaccine;?3?In vitro,comparison to mock control and negative control,macrophages stimulated by GRA15 II or LPS expressed i NOS and IL-12p40 in high levels,Arg-1and IL-10 shown no significant difference among all the groups?p>0.05?;?4?In vivo,comparison to mock control and negative control,immunized mice expressed Ig G,IL-12p40 and TNF-? in high levels?p<0.05?,as for IL-4,IL-13 and TGF-?1,there is no significant difference among all the mice?p>0.05?.After inoculation of mice with PRU strain tachyzoites,the higher survival rate was noted in immunized mice?p<0.05?.Meanwhile,there was less brain cysts in the vaccinated mice?p<0.05?.Conclusions: According to our study,GRA15 II as a virulence-associated effector of T.gondii could induce M1 phenotypical skewing of mouse macrophages,and this may be responsible for host to resist t T.gondii in the early stage of infection.Dense granule protein 15(GRA15_?)has a high potential in development of Toxoplasma vaccine due to its outstanding inmune protective effect.