| Toxoplasma gondii (T. gondii) is an intracellular protozoan parasite infecting all kinds of eukaryocyte. The parasite has a complex life cycle and distributes worldwide due to the extensive host rang and the various routs of transmission. T. gondii usually causes abortion in pregnant women and congenital diseases in neonates, it also causes life-threatening diseases in immune-compromised individuals. Toxoplasmosis in livestock always results in abortion and mal-development which brings serious economic losses. People become infection with T. gondii by ingesting the undercooked meat contaminated with cysts. Pork is one of main meat sources for human, thus, toxoplasmosis in pigs is thought to be a threat for human health. Understanding the pathogenic mechanism and development of vaccine are important for preventing and controlling T. gondii.In the present study, a cDNA expression library of T. gondii RH strain tachyzoites was constructed. Then in vivo induced antigen technology (TVIAT) was used for screening the constructed library by anti-T. gondii convalescence sera from pigs and yielded14in vivo induced antigens of T. gondii in pigs. The differential expressions of five of the identified antigens between in vivo and in vitro were confirmed using real-time PCR. Prokaryotic expression of the five antigens was also performed. RNA interference (RNAi) was carried out on T. gondii calmodulin (CaM) to evaluate the function in cell events of tachyzoites such as gliding movement, cell attachment, invasion, egress and intracellular replication. Finally, a DNA vaccine against T. gondii encoding micromene protein11(MIC11) was constructed and the protective immunity was detected in BALB/c mice model.(1) Screening and identification of in vivo induced antigens of T. gondii in pigsT. gondii RH strain tachyzoites were collected and purified, and then total RNA was extracted. A cDNA expression library was constructed based on a phage vector using SMARTTM technology. The titers of initial and amplified library were2.1x106pfu/mL and1.6x109pfu/mL, respectively. The recombinant rate was more than98%. Equal amounts of the positive sera from the infected pigs were pooled and successively adsorbed against the in vitro-grown tachyzoites, ultrasonic lysates and heat-denatured lysates of the in vitro-grown tachyzoites.3264clones from the library were screened using the adsorbed sera and yielded14positive clones containing8functional proteins which involved in ion/protein binding, signal transduction, protein folding, cell invasion, biosynthesis and metabolism, and6hypothetical proteins. (2) Differential expression between in vivo and in vitro and prokaryotic expression of the five in vivo induced antigenFive in vivo induced antigens genes (CaM, dense granule protein5(GRA5), MIC11,18kDa cyclophilin (C-18) and serine proteinase inhibitor (PI)) were selected and detected the differential expression under routine in vitro culture conditions or in the host by real-time PCR. Differences in the degree of up-regulation of the five genes were detected between the in vivo tachyzoites collected from BALB/c mice and the tachyzoites grown in vitro in BHK cells (p<0.05). CaM transcripts were detected at approximately15.04-fold higher levels in the in vivo samples than in the in vitro samples, which showed the most significant differences. Five pairs of primers of the genes were designed for prokaryotic expression. The ORFs were amplified and inserted into pGEX-6p-1, resulting in the recombinant plasmids expressing GST-fusion proteins, respectively. All the4recombinant proteins could be expressed efficiently in E. coli BL21, except pGEX-6p-1/GRA5. In the Western-blot analysis, both the positive sera against T. gondii and anti-GST antibodies recognized the recombinant proteins encoding the four in vivo induced antigens, suggesting the good antigenicity. The antigenic responses of CaM and C-18were stronger.(3) Phenotypic consequences of the down-regulated expression of T. gondii CaMThree pairs of siRNA (siRNA900, siRNA865and siRNA707) targeting different sites of CaM gene were used to suppress CaM expression. A certain degree of suppression by each siRNA on CaM expression was detected using real-time PCR. No effect was shown on the other proteins homologous with T. gondii CaM, suggesting the strong specific suppression by the designed siRNA. Most efficient suppression was found in tachyzoites treated with200nmol/L of siRNA900for24h. The phenotypic consequences of the parasite after treatment with siRNA900were investigated in BHK cells, and the treated tachyzoites showed decreased abilities of cell adherence, invasion and egress, but un-correlation with replication.(4) Immunogenicity of in vivo induced antigen MIC11The DNA fragment of T. gondii MIC11a-chain was inserted into pcDNA3.1vector, resulting in the recombinant plasmid pcDNA3.1/MIC11. Expression of MIC11from this vector was confirmed by indirect immune-fluorescence assay following transfection into BHK cells. The BALB/c mice were received the intramuscular injection of100μg pcDNA3.1/MIC11. The mice injected with100μg pcDNA3.1and100μL sterile PBS only were served as the control groups. Antibodies against TLA were first detected in the mice vaccinated with pcDNA3.1/MIC11two weeks after the first immunization. More anti-TLA IgG was accumulated and antibody titers increased dramatically in mice group vaccinated with pcDNA3.1/MIC11after the second and third immunization (p<0.05). In the ELISA analysis for cytokines, mice immunized with pcDNA3.1/MIC11produced highly significant levels of IFN-y, IL-12, IL-2compared with those immunized with PBS or pcDNA3.1(p<0.05). Mice immunized with pcDNA3.1/MIC11also induced higher level of lymphocyte proliferation compared with control groups (p<0.05). Mice were challenged with T. gondii RH strain tachyzoites at day14after the final immunization. All mice in the control groups died within10days post infection, while17%of the mice immunized with pcDNA3.1/MIC11were survived to day15after the parasite challenging. All the results demonstrated that the constructed DNA vaccine efficiently induced high levels of antibodies against T. gondii, Thl immune responses, and mediated good protection in BALB/c mice.The results of the present studies of identification of T. gondii in vivo induced antigen by IVIAT and the functional analysis improve our understanding of T. gondii pathogenesis and provide vaccine candidates against T. gondii infection. |
