| Toxoplasma gondii(T.gondii)is an obligate intracellular parasite that infects almost all warm-blooded animals,including humans,and causes zoonotic toxoplasmosis.Individuals with normal immune function usually have no obvious symptoms after infection with Toxoplasma gondii,or only show mild flu-like symptoms.Pregnant women infected with Toxoplasma gondii during pregnancy can have adverse effects on fetal health,causing miscarriage,deformity,stillbirth,etc.Toxoplasmosis is the most common opportunistic infection.Infecting Toxoplasma gondii in immunocompromised populations can cause serious consequences.For example,toxoplasmosis accounts for 10%to 30%of various opportunistic infections of AIDS,which can cause focal space-occupying lesions in the brain.In addition,toxoplasmosis can also cause serious economic losses to the livestock industry.Toxoplasma gondii mainly has three secretory organelles,namely microneme,rhoutry and dense granule.The three organelles produce microneme protein(MIC),rhoptry protein(ROPs)and dense granule proteins(GRAs),respectively.Among them,dense granule proteins play an important role in the modification of the parsitophorous vacuole(PV)and the survival of the parasite in cells.The Toxoplasma gondii GRA12(TgGRA12)protein can be expressed on the membrane of the parasite and is associated with the formation of a membrane-like nanotube network(MNN)located in the PV of T.gondii.Moreover TgGRA12 protein can be expressed in both the tachyzoites and bradyzoites.Macrophage inflammatory protein-1?(MIP-1?),or CCL3,is an inflammatory chemokine that promotes the differentiation of Thl cells,induces the secretion of more cytokines,and produces a good immune response.MIP-la has been reported as a gene adjuvant in various antiviral DNA vaccines,and experiments have shown that MIP-la can enhance the immune effect of the vaccine.DNA vaccines have been recognized as a promising strategy for the prevention of T.gondii infection.In this study,we evaluated the protective efficacy induced by DNA vaccine encoding TgGRA12 under the joint participation of genetic adjuvant MIP-1?.Objective:The excellent target of vaccine needs to be selected among a large number of candidate proteins.Bioinformatics analysis of TgGRA12 protein provides a theoretical basis for the possibility of this protein as a DNA vaccine against T.gondii.The basic characteristics of TgGRA 12 protein were predicted,and the potential Th and B cell epitopes were analyzed and compared with the classical target SAG1 to confirm its advantage as a new target.The eukaryotic expression plasmid was constructed and immunized mice with the genetic adjuvant MIP-la together to evaluate the protective effect of DNA vaccine,which provided a theoretical basis for finding new vaccine targets and more effective adjuvants.Methods:The bio-informatics method was used to predict the secondary structure and potential B cell epitope and Th cell epitope of TgGRA12 protein by DNASTAR software and IEDB online analysis software and compared with the classical candidate protein SAG1.After amplifying the gene fragment of TgGRA12 and MIP-1?,the recombinant DNA vaccine pBudCE4.1-TgGRA12(pBud-TgGRA12)and the recombinant gene adjuvant pBudCE4.1-MIP-1?(pBud-MIP-1?)were constructed and identified by enzyme digestion and sequencing and were used to immunize mice after successful verification.Kunming mice were randomly divided into 5 groups:vaccine group(pBud-TgGRA12 group),adjuvant group(pBud-MIP-1?group),vaccine + adjuvant group(pBud-TgGRA12+pBud-MIP-1? group),empty plasmid group(pBudCE4.1 group)and blank control group(PBS group).Mice were immunized once every two weeks for a total of three immunizations followed by detection of specific IgG and cytokine products.The T.gondii RH strain tachyzoites were challenged and the survival time was recorded to evaluate the immune effect of the DNA vaccine.results:The secondary structure and potential B cell epitopes of TgGRA12 protein.were predicted by DNASTAR software and Th cell epitopes by IEDB online analysis software,which showed significant advantages compared with SAG1 and provide a theoretical basis for subsequent experiments.After successful construction of the DNA vaccine and the gene adjuvant,mice were immunized to evaluate the immunoprotective effects.Compared with the control groups(PBS group and pBudCE4.1 group),mice in vaccine group produced a significantly strong humoral response and a Thl cellular immune response,and the antibody and cytokine titers were significantly increased(P<0.05).DNA vaccine co-injection of MIP-1?significantly enhanced humoral and cellular immune responses compared with the vaccine group(P<0.05).Survival experiments showed that the mice in the pBud-TgGRA12-M group had the longest survival time which was significantly longer than that of the vaccine group or the adjuvant group(P<0.05).Individual adjuvant did not produce effective humoral immune protection,but induced strong Thl cell differentiation.DNA vaccines have a strong ability to induce immune protection and prolong the survival time of immunized mice after infection with tachyzoites of T.gondii RH strain.Conclusion:Bioinformatics analysis predicts that TgGRA12 has good T and B cell epitopes and is theoretically an effective target and a good candidate for the DNA vaccine against T.gondii.It has also been demonstrated by animal experiments that the TgGRA12 can induce a good protective immune response,and the DNA vaccine co-injection of gene adjuvant elicits the highest level of immunoprotective efficacy in all experimental groups.Mice injected with DNA vaccine produce a high degree of effective protection response to T.gondii infection.TgGRA12 is a promising vaccine against acute toxoplasmosis.MIP-1? acts as a genetic adjuvant to enhance the immune response of Toxoplasma gondii DNA vaccine,providing more options for new effective adjuvants. |
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