| ObjectiveWe used bioinformatics approaches to analysis the amino acid sequences as well as gene sequences of the gliding associated protein45(TgGAP45). The TgGAP45 gene was cloned and expressed. Then we purified and subsequently analyzed the antigenicity of recombinant protein TgGAP45. To investigate the immune responses induced with different doses of recombinant Toxoplasma gondii gliding associated protein45(rTgGAP45) in BALB/c by intranasal drip, subcutaneous injection respectively, observe the best dose and ways of rTgGAP45and observe the contribution of rTgGAP45against Toxoplasma gondii chronic and lethal infection. Based on the above results, we can expect the probability of rTgGAP45as a kind of candidate antigen against Toxoplasma gondii.MethodsThe second part:Through bioinformatics analysis software such as ProtParam, WoLF PSORT, PSORT Ⅱ, CELLO v2.5, Bcepred, SYFPEITHI, Gener Runner, DNAman, to predict and analyze the open reading frame, the physicochemical characteristics, structural domain, spatial structure and antigenic epitope of gliding associated protein45(GAP45), and so on.The third part:According to GAP45gene sequence published in NCBI, a pair of specific primers were designed and synthesized included a EcoRI and XhoI restriction enzyme site. Toxoplasma gondii tachyzoites of RH strain were harvested and genomic RNA was prepared, which were used to amplify partial fragments of GAP45gene. The PCR product was digested with double restriction enzyme and then inserted into pET30a(+) expression vector. The positive clones were selected through the colony-PCR and confirmed by the double restriction enzyme digestion and sequencing. The recombinant plasmid pET30a(+)-TgGAP45was transfoemed into BL21competent cells and induced with IPTG to express. The recombinant GAP45(r TgGAP45) was purified with Ni-NTA affinity chromatography and analyzed by SDS-PAGE and Western blotting with HIS-tag antibody,anti-sera from rat and human infected with Tgondii RH strain. The fourth part:Divide406-week-old female BALB/c into four groups randomly,10for each.The mice in test groups1were intranasal immunized with20μl of PBS containing30μg rTgGAP45.The mice in test groups2and3were immunized with100μl of PBS containing30μg,60μg rTgGAP45by intranasal drip respectively, and those in control group were immunized with20μl PBS. All groups were vaccinated three times at days0,14and21, respectively. Two weeks after the last immunization, we collect serum through the venous plexus after eyes, then all mice were sacrificed by cervical dislocation and were scraped intestinal mucosa. Serum IgG and IgA in washes of nasopharynx, vagina and intestinal were detected by ELISA. Single-cell suspensions of splenic lymphocyte were prepared and stimulated by rTgGAP45or ConA, then cultured and determine the proliferative immune response with CCK-8kits. Levels of IL-2, IFN-y and IL-4of splenic lymphocyte culture supernatant and intestinal mucosa cell were all detected by ELISA assay.The fifth part:Divide sixty-four6-week-old female BALB/c into four groups randomly,10for each.The mice in test groups1were intranasal immunized with20μl of PBS containing30μg rTgGAP45.The mice in test groups2and3were immunized with100μl of PBS containing30μg,60μg rTgGAP45by intranasal drip respectively, and those in control group were immunized with20μl PBS. All groups were vaccinated three times at days0,14and21, respectively. Two weeks after the last immunization. Firstly, we selected8mice from each group randomly, were used for acute attack experiment. Mices were challenged intragastrically with4×104tachyzoites of the RH strain and the health status and survival time was observed for30days. Secondly, the remaining mice were challenged intragastrically with1×104tachyzoites per mouse.30days after the challenge, we observed the number of tachyzoites in brain and middle hepatic lobule with the microscope. ResultsThe gliding associated protein45is consist of245amino acids, the molecular mass is27317.9, the value of theoretical isoelectric point is5.05.This protein is a soluble protein without signal peptide,and contain23post-translational modification sites. The protein is subcellularly located in the intracellular membranes and have three zones with surface accessibility≥1.9, six zones with flexibility≥1.9, seven zones with hydrophilicity≥1.9, and four potential epitopes.The PCR product of GAP45cDNA was738bp in length and the recombinant plasmid pET30a(+)-TgGAP45was successfully constructed.The recombinant protein GAP45(rTgGAP45) was soluble and expressed efficiently in E.coli BL21(DE3). The molecular weight of fused GAP45was about35kDa by SDS-PAGE. It could be specifically recognized by HIS-tag antibody and T. gondii positive serumThe levels of IgG and IgA in serum,30μg rTgGAP45intranasal immunization group,30μg rTgGAP45subcutaneous injection group and60μg rTgGAP45subcutaneous injection group were significantly higher than that of the control group (P<0.01and P<0.01;P<0.01and P<0.05;P<0.01and P<0.01), while30μg rTgGAP45intranasal immunization group was higher than30μg rTgGAP45subcutaneous injection group (P<0.01). Compared with the control group, stimulated by rTgGAP45, SI were all increased in immune groups, especially in30μg intranasal immunization group and60μg subcutaneous injection group (P<0.01and P<0.05), but there was no statistically difference between the groups,which were stimulated by ConA. Levels of IgA in intestinal and nasopharynx,30μg intranasal immunization group,30μg subcutaneous injection group and60μg subcutaneous injection group were significantly higher than the control group (P<0.01and P<0.01; P<0.05and P<0.01;P<0.01and P<0.01), the levels of IgA in vagina washes of30μg intranasal immunization group,60μg subcutaneous injection group were higher than the level in the control group (P<0.01,P<0.01). While the level IgA in intestinal and nasopharynx were significantly higher than the level in the30μg subcutaneous injection group and60μg subcutaneous injection group (P<0.01,P<0.01). The levels of IL-2, IFN-y of splenic lymphocyte culture supernatant were significantly increased in30μg intranasal immunization group,30μg and60μg subcutaneous injection groups (P<0.01,P<0.05and P<0.01;P<0.01,P<0.05and P<0.01) compared with the control group. The levels of IL-4and IL-5of splenic lymphocyte culture supernatant were significantly increased in30μg intranasal immunization group and60μg subcutaneous injection group (p<0.01and P<0.05;p<0.01and P<0.01) compared with the control group. The levels of IL-2, IFN-y of duodenum, jejunum, ileum mucosa cell were significantly increased in30μg intranasal immunization group (P<0.05,P<0.01and P<0.01;P<0.05,P<0.05and P<0.05) compared with the control group. The levels of IL-2, IFN-y of duodenum, jejunum, ileum mucosa cell were significantly increased in60μg subcutaneous injection group (P<0.01,P<0.05and P<0.05;P<0.05,P<0.05and P<0.05) compared with the control group. The levels of IL-2of jejunum, ileum mucosa cell were significantly increased in30μg subcutaneous injection group (P<0.05and P<0.05) compared with the control group.In chronic infection test, the tachyzoite load in lives and brains of30μg rTgGAP45intranasal immunization group,30μg rTgGAP45subcutaneous injection group and60μg rTgGAP45subcutaneous injection group were significantly lower than the control group (P<0.01and P<0.01;P<0.01and P<0.01;P<0.01and P<0.01). At the same time, the tachyzoite loads of mice in30μg rTgGAP45intranasal immunization group,30μg rTgGAP45subcutaneous injection group and60μg rTgGAP45subcutaneous injection group decreased in the liver and brain by49.55%and61.38%;33.78%and41.94%;43.82%and51.30%, respectively. While the tachyzoite load in lives and brains in30μg rTgGAP45intranasal immunization group were significantly lower than that of the30μg rTgGAP45subcutaneous injection (P<0.05,P<0.01), the tachyzoite load of lives in60μg rTgGAP45subcutaneous injection group were significantly lower than that of the30μg rTgGAP45subcutaneous injection (P<0.01); In lethal infection test, the survival rates of two group (30μg rTgGAP45intranasal immunization group and60μg rTgGAP45subcutaneous injection group) were50.0%%and37.5%, respectively. At the tenth and thirtieth day after challenge,mices in PBS group and30μg rTgGAP45subcutaneous injection group were died.ConclusionThe results of bioinformatics analysis showed that TgGAP45protein can serve as a candidate antigen for toxoplasmosis vaccination because of its four potential epitopes. The recombinant plasmid pET30a(+)-TgGAP45was successfully constructed and rTgGAP45was soluble and expressed efficiently in E.coli BL21(DE3). Through Western bolt assay has confirmed the immunogencitity of rTgGAP45. Subcutaneous injection and intranasal immunization with rTgGAP45can induce the immune response of mice and significantly reduce the tachyzoite load of in murine tissues, provided partial protection against lethal infection. Therefore, rTgGAP45was a promising vaccine candidate against toxoplasmosis, worth further development... |
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