Soluble Expression And Preliminary Application Of A Truncated Version Of Toxoplasma Gondii SAG1, And Development Of Monoclonal Antibodies Anti-recombinant Truncated SAG1

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 and 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. In addition,at the veterinary level, toxoplasmosis is one of the main causes of fetal abortion, stillbirth and neonatal mortality in domestic animals, and can cause considerable economic loss in the farming industry. So diagnosis of infection with T. gondii, especially diagnosis of acute toxoplasmosis is very important.-6-The initial and primary tests presently used for toxoplasmosis diagnosis are serological assays. Most commercial kits use prepared tachyzoites grown in mice or tissue/cell culture as diagnostic antigen to test IgG and IgM antibodies of T.gondii, which inevitably contain extraparasitic material. The production of constant-quality T. gondii tachyzoites antigens is time-consuming and expensive. It is not suprising that some interblock and interassay variability exists because of the lack of a standard method for preparing a purified antigen. Gene clone and expression in prokaryotic systems can provide large amount of purified protein in a short period, and the method is economical and is easy to perform. The SAG1 antigen of toxoplasma is a stage-specific antigen of tachyzoites, and is highly conservative in different srains. Large tests show SAG1 is higly immunogenic and is a candidate antigen for diagnosis and vaccination to toxoplasmosis. Native SAG1 is a highly conformational antigen defined by intramoleculer cysteine bridges that give rise to immunologically relevant conformational epitopes, and is presumed to be post-translationally modified by removeal of the signal sequence and the C terminus. Thus, expression of correctly folded recombinant SAG1 in bacterial expression systems is inherently difficult. In our lab, Chen Xiaoguang et al have obtained truncated SAG1 as inclusion bodies in E.coli, after refolding, the recombinant SAG1 can be recognized by human Toxoplasma antisera.In this present study, we tried to test (1) whether a truncated SAG1 can be expressed as both a soluble and correctly folded protein in E. coli, (2) whether this recombinant protein reacts with antibodies in human patient sera and could be used as a diagnostic reagent. (3) whether monoclonal antibodies that anti-recombinant SAG1 can be developed and can react with native SAG1.Aims: 1. To obtain truncated SAG1 expressed in E. coli in a solubleform, its immunoreactivity was analysed and primarily applied after purification.-7-2. To develop monoclonal antibody of recombinant truncated SAG1 for the purpose of developing Dipstick kit for the diagonosis of toxoplasmosis.Method: 1. Rabbits were immunized with tachyzoite antigen of T. gondii RH strain to produce anti-tachyzoite polyclonal serum.2. The truncated SAG1 gene of the Toxoplasma gondii RH strain was cloned into plasmid pET32a(+), and was expressed in E.coli, the recombinant plamid was identified by restriction enzymes and sequence analysis.3. The recombinant protein was purified by Ni-NTA, and the immunoreactivity of the recombinant protein was identified by Western-blot and some human sera was tested by recombinant SAG1..4. The mice SP2/0 cells were hybridized with speen cells from mice immunized with recombinant SAG1, the positive hybridimas were identified by an indirect ELISA way. Five strains of...