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Research on Biological Characteristics and Immunogenicity of Toxoplasma Gondii Potential Vaccine Candidate IMP1

Author: YuHaiJie
Tutor: DuAiFang
School: Zhejiang University
Course: Preventive Veterinary Medicine
Keywords: Toxoplasma gondii seroprevalence Real-time PCR IMP1 subccllularlocalization recombinant protein immune sub-unit vaccine DNA vaccine attenuatedSalmonella typhimurium
CLC: S855.9
Type: PhD thesis
Year: 2013
Downloads: 142
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Abstract


Toxoplasma gondii (T. gondii), an opportunistic intracellular protozoan parasite, can infect almost all worm blood animals including humans and exists as a serious threat to public health. In humans, whilst infections often remain asymptomatic in immunocompetent individuals they can lead to mild or severe sequelae in congenitally infected infants, and can cause life-threatening encephalitis in immunocompromised patients (AIDS, bone marrow and organ transplant patients, etc.). In animals, T. gondii infection is a prominent cause of spontaneous abortion, stillbirth, or severe disease and associated economic losses. At present, there is still no effective medicine for Toxoplasmosis and the only method is still prevention. Therefore, vaccine development plays an important role in the disease prevention. IMP1is a newly discovered protein in2011and considered with great importance in the toxoplasmosis vaccine development. In this study, we used the method of molecular biology, gene engineering, immunobiology to do the further research on the distribution and immunogenicity of IMP1, attempting to know better about the function of IMP1and related proteins, so as to provide a novel strategy for Toxoplasmosis prevention.1. Seroprevalence of Toxoplasma gondii infection in animals in Zhejiang provinceInorder to assess the seroprevalence of T. gondii infection in animals in Zhejiang province, a survey was carried out via enzyme linked immunosorbent assay (ELISA) using serum (813of pigs,131of cattle,151of goats213of dogs and60of cats) collected from11regions of Zhejiang province. Anti-Toxoplasma antibodies were found in53.4%for pigs,16.0%for dogs,13.3%for cats and2.7%(4/151) for goats. No cattle were found seropositive for T. gondii infection. For pigs and cats, results were statistically analyzed by chi-square (χ2) test. We found that seroprevalence in pigs increased progressively with age. Differences were observed in feeding environment, animals’species and so on. This is the first detailed study on seroprevalence of T. gondii infection in pigs, cattle, goats, pigs and cats in Zhejiang province. The result revealed that there was a high seroprevalence of T. gondii infection in animals especially pigs in the province of Zhejiang. Since there in no effective vaccine against this parasite, prevention of zoonotic transmission must be considered in Zhejiang province.2. Establish of a real-time PCR assay based on the single-copy SAG1gene for the detection of Toxoplasma gondiiReal-time PCR-based detection of Toxoplasma gondii is very sensitive and convenient for diagnosing toxoplasmosis. However, the performance of the PCR assays could be influenced by the target gene chosen. Here we evaluate a real-time PCR assay using double-stranded DNA dyes (SYBR(?) Green I assay) with a new set of primers targeting the SAG1gene (X14080) for the fast and specific detection of T. gondii. The assay showed higher sensitivity than conventional PCR protocols using T. gondii DNA as template. The detection limit of the developed real-time PCR assay was in the order of1tachyzoite. The assay was also assessed by experimentally infected mice and showed positive results for blood (25%), spleen (50%) and lung (50%) as early as1dpi. The specificity of the assay was confirmed by using DNA from Neospora caninum, Escherichia coli, Babesia bovis, Tiypanosoma brucei, Cryptosporidium parvum, and Toxocara canis. Assay applicability was successfully tested in blood samples collected from slaughtered pigs. These results indicate that, based on SYBR(?) green I, the quantitative SAG1assay may also be useful in the study of the pathogenicity, immunoprophylaxis, and treatment of T. gondii.3. The subcellular localization of Toxoplasma IMP1IMP1is a newly discovered protein and has highly conserved homologues in Eimeria spp. and Neospora caninum. We got the IMP1gene by PCR using the genomic DNA of RH strain. Sequence analysis showed that the gene we cloned was99.8%identical to the published data of ME49strain (XM002370108;2c-36).There arc two mutations, A changed to G in114and T changed to C in342, but they has no effect on the protein expression. The IMP1was expressed in E.coli and anti-IMP1antibody was collected from immunized rabbit. The subcellular localization of IMP1was carried out by indirect immunofluorescence technique, and was anchored on the membrane of the parasite in all the stage (free, absorbing, invading, after invading or in the PV), though some showed strong fluorescence signal on apical. This study is the foundation for deep research of the function of IMP1.4. The immunogenicity of recombinant sub-unit vaccine rSAG1-IMP1of T. gondiiTo evaluate the immunomodulatory potentials of the recombinant sub-unit vaccine rSAG1-IMP1, the BL21-pET30a-IMP1and BL21-pET30a-SAG1-IMP1Prokaryotic expression system were constructed. ICR mice were immunized subcutaneously with PBS,100μg rSAG1,100μg rIMP1,100μg rSAG1-IMP1on day1and immunized subcutaneously with PBS,50μg rSAGl,50μg rIMP1,50μg rSAG1-IMP1on week2and week4after first immune. After immunization, the immune response was evaluated by lymphoproliferative assay, antibody level detection and cytokine measurements. The mice were challenged with500tachyzoites (RH strain) and the survival times of mice were recorded. The results showed that all the recombinant sub-unit vaccines could elicite a high level of specific immune response against T. gondii, and the multiple antigens recombinant sub-unit vaccine rSAGl-IMP1could elicite a higher immune response. Our data demonstrate that the recombinant sub-unit vaccine rSAG1-IMP1can elicit a strong humoral and cellular response, and can effectively help immunized mice against T. gondii infection.5. The immunogenicity of T. gondii IMP1gene carried by attenuated Salmonella typhimuriumTo evaluate the immunomodulatory potentials of IMP1gene carried by attenuated Salmonella typhimurium, the ZJ111/pcDNA3.1-IMP1and ZJ111/pcDNA3.1-SAG1-IMP1DNA vaccine were constructed and immunized to ICR mice. After immunization, we evaluated the immune response by antibody measurements, lymphoproliferative assay, cytokine test and challenge assay in mice. The results showed that DNA vaccine delivered in attenuated Salmonella typhimurium, ZJ111/pcDNA3.1-IMP1、 ZJ111/pcDNA3.1-SAGl-IMPl could elicit a strong humoral and cellular response as well as provide effective protection against T. gondii infection in immunized mice. The multiple antigens vaccine ZJ111/pcDNA3.1-SAG1-IMP1could elicite a higher immune response than ZJ111/pcDNA3.1-IMP1.In summary, Based on the single-copy SAG1gene, a real-time PCR assay was established for the detection of Toxoplasma gondii, the seroprevalence of T. gondii infection in pigs, cattle, goats, pigs and cats in Zhejiang province was carried out via enzyme linked immunosorbent assay (ELISA) using serum collected from11regions of Zhejiang province, The subcellular localization of IMP1was carried out by indirect immunofluorescence technique, The recombinant sub-unit vaccine rSAG1-IMP1and DNA vaccine ZJ111/pcDNA3.1-IMP1、ZJ111/pcDNA3.1-SAGMMP1against T. gondii were constructed. The possibility and the prospect of IMP1as the potent vaccine was evaluated by humoral and cellular response as well as effective protection against T. gondii infection in immunized mice, hoping to provide a new way for prevent Toxoplasmosis.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Veterinary parasites and disease
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