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Expression and Identification of Soluble Fusion Protein of Toxoplasma Gondii SAG1 in E.coli, and Preparation of Monoclonal Antibodies Anti-recombinant SAG1

Author: AnLiGang
Tutor: WangQuan
School: Chinese Academy of Agricultural Sciences
Course: Preventive Veterinary Medicine
Keywords: Toxoplasma gondii SAG1 protein expression protein purification monoclonal antibody identification
CLC: S852.7
Type: Master's thesis
Year: 2009
Downloads: 105
Quote: 1
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Abstract

The obligate intracellular protozoan parasite Toxoplasma gondii(T .gondii)is world wide spread, and is responsible for toxoplasmosis and able to infect all species of mammals (include human) and birds. So diagnosis of infection with T. gondii, especially diagnosis of acute toxoplasmosis is very important. The SAG1 antigen of toxoplasma is a stage-specific antigen of tachyzoite,It marks up at about 5% of the total protein of T. gondii tachyzoites. It has been indicated that this protein has a role in host-cell adhesion, invasion and toxicity. Because the host immune responses can be induced with the protein that is protective to the parasite infection, the gene is studied on vaccine development and toxoplasmosis molecular diagnosis.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 difficut. In this present study, we tried to test whether a truncated SAG1 can be expressed as both a soluble and correctly folded protein in E.coli, whether this recombinant protein reacts with antibodies in animal patient Sera and could be used as a diagnostic reagent. Monoclonal antibody against tSAG1 of Toxoplasma gondii was prepared and then can be used for developing the new diagnosis system.Aims:1. To express the Toxoplasma gondii major surface antigen SAG1 truncated fragment in the Escherichia coli and purify the soluble fusion protein which immuoreactivity was analysed2. To prepare monoclonal antibody of recombinant truncated SAG1 for the purpose of developing ELISA kit for the diagonosis of toxoplasmosis.Method: The recombinant of pET-32a-tSAG1 was transformed into a bacterium BL21(DE3) and the recombinant was expressed under the inducement of isopropyl-beta-D-thiogalactosidase(IPTG). The expression product was analyzed by SDS-PAGE. The fusion protein would be induced into supernatant by using the methods of growing the cell in a low speed and temperature, raising pH of the culture of medium. It was purified by His?Bind? Kits.The immunogenicity of recombinant antigen purified was tested with Western blot and ELSA. The truncated fragment of SAG1 was highly expressed in E.coli as fusion protein in 37℃and 1mmol/L of IPTG. The solubility analysis of express product indicated that this recombinant protein expressed in inclusion bodies.In 22℃and pH7.7of the culture medium, the fusion protein of tSAG1 could be induced into supernatant. The recombinant protein purified could be recognized by toxoplasmosis positive serum with Western blot and ELSA. The truncated fragment of SAG1 was highly expressed in supernatant and the fusion protein could be recognized by toxoplasmosis positive serum after purification. The fusion protein can be used for developing the new diagnostic reagent to diagnose the infection of Toxoplasma gondii.Then the His-tSAG1 was quantified with BCA kit and was used to immunize BALB/c mice. According to the hybridoma technique of B lymphocytes, the immunized mouse spleen cells was isolated while attaining required serous antibody titer(1:10000 detected by ELISA)and fused with mouse MM SP2/0 cells by using polyethylene glycol(PEG), then hybridomas were cultured in HAT selection medium. During from the 10th to 20th postfusion days, ELISA method coating with tSAG1 ang His protein was applied to screen for positive clones. Sequentially the positive cell lines were propagated and tested repeatedly for assuring stability of positive clones. Hybridoma cell lines were established finally when subcloning wells were all positive. Using Pristane-primed BALB/c mice, production of the anti- tSAG1 McAbs was made in vivo (ascites), which was induced by intraperitoneal injection of well-grown hybridomas and acquired from the 7th to 10th day. Following precipitaion with Ammonium Sulfate, the McAbs was purified by immunoaffinity chromatography. For McAb-isotyping, fast-strip method analysis was performed. SDS-PAGE was employed for analysis of ascites. Titers of McAbs from ascites and McAbs purified were measured by ELISA. The specificity of McAbs was analyzed by Western-blotand IFA.Results: 1. The truncated SAG1 gene was cloned, and it opened read frame is correct by sequence analysis, the truncated SAG1 gene was expressed in E.coli as a soluble fusion protein.2. The analysis of the Westen-blot and the results of positive sera ELISA showed highly immunoreactivity and good applying performance of recombinant SAG1.3. Four strains of monoclonal antibodies anti-recombinant truncated SAG1 were prepared, which all reacted with native SAGl and tSAG1 by ELISA IFA and Western-blot, and they had two different epitopes.Conclusions: 1. The truncated SAG1 gene was cloned and expressed as a soluble fusion protein in E. coli, and the truncated SAG1 protein showed highly immunoreactivity and applying performance by the analysis of the Western-blot and ELISA.2. Monoclonal antibodies anti-recombinant truncated SAG1 were prepared, which all reacted with native SAG1 and have potential diagnostic value.

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