[Construction and expression of an eukaryote vector of 14-3-3 protein in Toxoplasma gondii].

OBJECTIVE: To construct and express the eukaryotic expression vector of 14-3-3 protein of Toxoplasma gondii RH stain. METHODS: The structure and physicochemical property of 14-3-3 protein were predicted by bioinformatics analysis tools. The desired gene fragment was amplified from total RNA in T. gondii RH strain by RT-PCR, and sub-cloned into pcDNA3.0 to construct recombinant plasmid pcDNA3.0/14-3-3. After PCR confirming, double restriction enzyme digestion and DNA sequencing, the eukaryotic expression vector pcDNA3.0/14-3-3 was transfected into HeLa cells and the target protein was detected by Western blotting. RESULTS: The prediction of its gene sequence and amino acid sequence suggested that the 14-3-3 protein was acid soluble protein with five conserved regions, existing as homo- or hetero-dimers. The amplified gene fragment was about 800 bp, and the inserted fragment in pcDNA3.0/14-3-3 was 801 bp by sequencing, which had 99% homology to the 14-3-3 gene sequence of T. gondii in GenBank (Accession No. AB012775.1). Western blotting showed that there was more 14-3-3 protein expressed in the pcDNA3.0/14-3-3-transfected HeLa cells than not-transfected and mock transfected cells. Its relative molecular mass (Air) was about 30 000. CONCLUSION: The eukaryotic expression vector pcDNA3.0/14-3-3 is constructed and expressed in eukaryotic cells.

[Effect of Escherichia coli heat-labile enterotoxin B subunit on SAG1-ROP2 compound gene vaccine of Toxoplasma gondii tachyzoite].

OBJECTIVE: To investigate the effect of Escherichia coil heat-labile enterotoxin B subunit (LTB) as a genetic adjuvant in enhancing the immune response induced by Toxoplasma gondii tachyzoite compound gene vaccine. METHODS: The eukaryotic expression plasmids of pcDNA3.1-SAG1-ROP2 and pEASY-E1-LTB were constructed. Eighty-eight BALB/c mice were randomly divided into four groups: PBS (group A), pcDNA3.1(B), pcDNA3.1-SAG1-ROP2 (C) and pcDNA3.1-SAG1-ROP2+pEASY-E1-LTB (D). Fifteen mice in each group were randomly selected, and intranasally immunized weekly with 20 microg plasmid or 20 microl PBS, respectively. Each mouse received four immunizations with the same dose of antigen. Two weeks after the final immunization, the antibodies and cytokines were detected, including the specific IgG and IgA antibodies in serum, sIgA in mucosa douche, IFN-gamma and IL-4 in splenocyte culture supernatant. The remaining mice in each group were immunized three times weekly with 20 microg plasmid or 20 microl PBS, respectively, and challenged by T. gondii tachyzoites at four weeks after the final vaccination (1 x 10(3) per mouse). The survival time of mice was recorded. RESULTS: The recombinant plasmids pEASY-E1-LTB were constructed. The specific IgG (0.626/- 0.100) and IgA antibodies (1.086 +/- 0.138) in serum, sIgA (0.886 +/- 0.164) in mucosa douche, cytokines IFN-gamma [(2017 +/- 266) pg/ml] and IL-4 [(203.31) pg/ml] in splenocyte culture supernatant in group D were all higher than those in other groups (P < 0.05). After challenged with T. gondii tachyzoites, the median survival time of mice in groups A, B, C, and D were 3, 4, 6, and 10 d, respectively. The survival time of mice in group D was longest (P < 0.05). CONCLUSION: E. coil heat-labile enterotoxin can enhance the immune response induced by the compound gene vaccine of T. gondii tachyzoites.

[Construction of monovalent and compound nucleic acid vaccines against Toxoplasma gondii with gene encoding p30].

OBJECTIVE: To construct a monovalent gene vaccine pcDNA3.1-p30 and a compound gene vaccine pcDNA3.1-p30-ROP2 and assess the protective effect of the two vaccines against Toxoplasma gondii. METHODS: The sequences encoding p30 and ROP2 were amplified from the genomic DNA of T. gondii RH strain by polymerase chain reaction (PCR) and inserted into eukaryotic vector pcDNA3.1 to construct pcDNA3.1-p30 and pcDNA3.1-p30-ROP2. Mice were injected with the recombinant plasmid to observe the immunoprotectivity of the nucleic acid vaccine by using ELISA for detection of total IgG and observing the survival time after tachyzoites challenge. RESULTS: The recombinant plasmids pcDNA3.1-p30 and pcDNA3.1-p30-ROP2 were constructed. Mice in pcDNA3.1-p30-ROP2 group showed higher IgG (P < 0.05) and survived longer than those in pcDNA3.1-p30 group (P < 0.01) after challenged with T. gondii. CONCLUSION: Compound vaccine of genes from different stages of T. gondii elicits stronger immunoprotectivity in mice than a single gene vaccine.

[Oral mixed DNA vaccine protects mice from infection of Toxoplasma gondii].

OBJECTIVE: To examine the immune response in BALB/c mice induced by oral mixed Toxoplasma gondii DNA vaccine delivered by live attenuated Salmonella typhimurium. METHODS: Gene fragments SAG1 and SAG2 were amplified from the genomic DNA of T. gondii RH strain by PCR and were subcloned into pcDNA3.1(+/-) eukaryotic expression vector. The positive recombinant plasmid was transformed into aroA- and aroD-attenuated Salmonella typhimurium BRD509 (BRD509/pSAG1/SAG2). After screened by PCR, restrictive enzyme digestion and sequencing, recombinant Salmonella strain was used to immunize BALB/c mice by i.g. route, three times at 2-week interval. Humoral and cellular responses were assayed using ELISA for determining antibody, IFN-gamma and IL-4. MTT assay was used to detect the proliferation activity of T lymphocytes and the activity of NK killers. FCM was also used to sort the lymphocyte subsets of spleen cells All mice were then infected with highly virulent RH tachyzoites of T. gondii intraperitoneally. RESULTS: Significant increase of specific IgG level was observed in immunized mice with a titer of 1:100. Proliferation activity of specific NK cells and T lymphocytes was highly enhanced in BRD509/ pSAG1/SAG2 vaccinated mice: the killing activity of NK cells was 85% +/- 7%, the proliferation SI of T lymphocytes was 2.83, which resulted in a 5 days longer survival time than mice in control group after challenge infection. CONCLUSION: The oral mixed DNA vaccine delivered by attenuated Salmonella typhimurium shows an immunoprotection against T. gondii in mice.

Multi-epitope DNA vaccine linked to the A2/B subunit of cholera toxin protect mice against Toxoplasma gondii.

The search for an effective vaccine against toxoplasmosis remains a challenging and elusive goal. Combination of epitopes from different stages of Toxoplasma gondii life cycle is an optimal strategy to overcome the antigen complexity of the parasite. Based on published epitope derived from several promising candidate vaccine antigens, we construct a DNA vaccine encoding multi-epitope of T. gondii and CpG motif, with or without the A2/B subunit of cholera toxin as a genetic adjuvant. The immunity induced by this vaccine in BALB/c mice and the protection afforded against challenge with the highly virulent RH strain of T. gondii is assessed. This vaccine was able to elicit a significant humoral and cellular immune response in vaccinated mice. Furthermore, CTXA2/B as a genetic adjuvant could enhance the magnitude of immune responses as well as increased survival rate in mice infected with the lethal RH tachyzoites. This study is the first report of a multi-epitope DNA construct strategy as a potential DNA vaccine against toxoplasmosis.