Cloning And Expression Of Bradyzoite-specific Gene Of Toxoplasma Gondii And Preliminary Application Of Recombinant BAG1

Background: The obligate intracellular protozoan parasite Toxoplasma gondii (T. gondii) is worldwide spread, and is responsible for toxoplasmosis and able to infect all species of mammals (include human) and birds. Nearly one-third of the human population is exposed to the threat of this parasite. T. gondii is an opportunistic pathogen that generally forms cysts in an immunocompetent host which is generally asymptomatic. In hosts who are severely immunocompromised ( e.g , neoplastic disease, organ transplatation, and AIDS patients), the tachyzoites of T. gondii may frequently cause a dissemination and subsequent complications, sometimes become fatal. Primary infection during pregnancy may cause abortion, neonatal malformations, and neonatal death or severe congenital defects appearing in later life, such as mental retardation, retinochoroiditis, and blindness. Therefore, the diagnosis of toxoplasmosis and the development of effective vaccine are very crucial to protect susceptible population and promote aristogenesis.The biology basic of opportunistic pathopoiesis of T. gondii is the conversion between the tachyzoite and bradyzoite. Therefore, a better understanding of conversion between the tachyzoite and bradyzoite stages would therefore be a major step in controlling the disease. As this conversion is mainly a differentiation process characterized by the differential expression of stage specific molecules, the identification of these molecules is a prerequisite to study this process. Then the cloning of the stage specifically expressed genes would allow the study of their regulation, and therefore of the differentiation mechanisms.Because the tachyzoite stage is the most readily available for study, many proteins and the corresponding genes have been characterized from tachyzoites. Most of these proteins have been shown to be common to tachyzoites and bradyzoites. Only a few have proven to be specific to the tachyzoite stage. More recently,because of the rising interest in stage conversion, several groups have identified bradyzoite or cyst specific proteins and the genes coding for some of them have been cloned and sequenced.In the present study, the coding sequence of two bradyzoite-specific antigens, BAG1 and SAG4, were cloned and expressed in E.coli. The immunoreactivity of the recombinant proteins was analyzed and the application of recombinant proteins in diagnosis of infection with T. gondii was preliminary investigated. And monoclonal antibodies anti-recombinant BAG1 were developed to further study T. gondii bradyzoites.Objective:1. To clone and express bradyzoite antigen 1(BAG1) gene and bradyzoite surface antigen 4 (SAG4) gene of T.gondii.2. To purify recombinant proteins, analyze the immunoreactivity of the recombinant proteins, and investigate the application of recombinant proteins in the diagnosis of toxoplasmosis.3. To develop and identificate monoclonal antibodies that anti-recombinant BAG1 to further study T. gondii bradyzoites.Methods:1. The conversion of T.gondii RH strain tachyzoites into bradyzoites was induced in vitro, and the coding sequence of BAG1 was amplified from bradyzoites by RT-PCR. The coding sequence of SAG4 was amplified from toxoplasma genomic DNA by PCR.2. BAG1 and SAG4 gene fragments were cloned into pMD18-T vector and pGEX4T-1 respectively to construct recombinant plasmid pMD 18-T-BAG1 and pGEX4T-1-SAG4.3. BAG1 coding sequence was further subcloned into the plasmid pET32a(+) to construct recombinant plasmid pET32a(+)-BAG1. The correction of the inserting were analyzed by double enzyme digestion and sequencing.4. The plasmid pET32a(+)-BAG1 and pGEX4T-1-SAG4 were transformed into BL21(DE3) respectively to express the recombinant proteins after the induction of IPTG.5. Optimize expression conditions and purify expression products.6. Analyze the immunoreactivity of the recombinant proteins by Western blotting and ELISA.7. Detect toxoplasma IgG antibodies of human sera using ELISA based on the recombinant BAG1.8. The mice SP2/0 cells were hybridized with speen cells from mice immunized with recombinant BAG1, the positive hybridomas were identified by an indirect ELISA way. Four strains of monoclonal antibodies were choosed and were confirmed the reactivity with BAG1 by the analysis of the Western-blotting. Results:1. Bradyzoite-specific antigen of T.gondii BAG1 and SAG4 genes were cloned.2. Recombinant cloning plasmid—pMD 18-T-BAG1 and recombinant expression plasmids—pET32a(+)-BAG1 and pGEX4T-1-SAG4 were successfully constructed. After IPTG inducting, BAG1 was expressed in a fusional soluble form and SAG4 in inclusion body form in E.coli. Western blotting showed the recombinant BAG1 and recombinant SAG4 could be specifically recognized by sera from mice which were chronically infected by T.gondii B36 strain.3. The total positive rate of T.gondii IgG antibodies of 350 human sera detected by recombinant BAG1 and recombinant SAG1 is 23.14 %, and the positive rate of BAG1 is higher than that of SAG1.4. Four strains of monoclonal antibodies anti-recombinant BAG1 were developed, which all reacted with BAG1.Conclusion:1. T.gondii BAG 1 and SAG4 genes were cloned and identified.2. BAG 1 and SAG4 were expressed highly in a fusional soluble form and inclusion body form respectively in E.coli, and the recombinant proteins displayed specific immunoreactivity.3. Recombinant BAG1 may be used as a novel diagnostic antigen to detect T.gondii infection.4. Four strains of monoclonal antibodies anti-recombinant BAG1 were developed, which may be applied in study of T. gondii bradyzoites.