Studies On Gene Cloning Of GRA6 And SAG1 From Toxoplasma Gondii And The Application Of Their Expression Products

Aim To clone gene fragment of coding GRA6 from the total DNA of Kunshan and RH strains and construct the recombinant plasmid of pGEX-GRA6. BL21-Codon Plus(DE3)-RP strain cells of E.coli were transformed with the recombinant plasmids of pGEX-SAGl( which had been constructed before in our lab) and pGEX-GRA6 and then expression products were purified. To construct new genetic engineering diagnostic kits that used the two recombinant antigens as diagnostic antigen for the detection of toxoplasma gondii infection and differential diagnosis of acute and chronic toxoplasmosis.Methods 1. The total DNA of Toxoplasma gondii was isolated from purified tachyzoites that were obtained by passage through a column of CF-11 cellulose. A pair of primers were designed and synthesized according to sequences reported at GenBand database for GRA6. The DNA fragment encoding GRA6 was amplified by polymerase chain reaction(PCR) from the total DNA of Kunshan and RH strains, respectively. The genes were subcloned into the plasmid pBluescriptIISK (-) and the DNA sequences were determined subsequently. The BamHI/EcoRI digested fragments of pSK-GRA6 were inserted into the same site of expression vector pGEX-5x-3. The recombinant plasmid of pGEX-GRA6 was transformed to a bacterium BL21-Codon Plus(DE3)-RP and the recombinant products were expressed under the inducement of IPTG. Cells were lysed by multiple rounds of sonication.The expression products were analyzed by using SDS-PAGE. The expression products in supernatant were purified by sedimentation of ammonium sulphate, desalting using Sephadex G50 gel-filtration, affinity chromatography on glutathione-sepharose system. The immunogenicity of recombinant protein purified was tested with Western blotting. We developed the method of indirectenzyme-linked immuneonosorbent assay (ELISA) for the serological diagnosis of toxoplasmosis that used GST-GRA6 recombinant protein as diagnostic antigen for the detection of IgM and IgG antibodies to Toxoplasma gondii in human sera. We developed the method of dot immunogold filtration assay(DIFA) for the serological diagnosis of toxoplasmosis that used GST-GRA6 recombinant protein as diagnostic antigen for the detection of IgM and IgG antibodies to Toxoplasma gondii in human sera.2.The recombinant plasmid of pGEX-SAGl was transformed to a bacterium BL21-Codon Plus(DE3)-RP and the recombinant protein was expressed under the inducement of IPTG. Cells were lysed by multiple rounds of sonication.The expression products were analyzed by using SDS-PAGE. The recombinant protein would be induced into supernatant by using the method of growing the cell at a low temperature, raising pH of the medium. The expression products were purified by sedimentation of ammonium sulphate, desalting using Sephadex G50 gel-filtration, affinity chromatography on glutathione-sepharose system and Sephadex G150 gel-filtration chromatography. The immunogenicity of recombinant protein purified was tested with Western blotting. We developed the method of ELISA for the serological diagnosis of toxoplasmosis that used the recombinant protein GST-SAG 1 as diagnostic antigen for the detection of IgM and IgG antibodies to Toxoplasma gondii in human sera.Results l.The aligment chart of the GRA6 DNA sequences between Kunshan strain and RH strain showed that there were almost no difference from each other within 336 bp encoding sequences.The fusion protein with a molecular weight of 49 kDa was detected on SDS-PAGE, the recombinant protein could be recognized by toxoplasmosis-specific IgM and IgG in human sera and its antigenicity was confirmed by Western blotting. GRA6 was highly expressed in E.coli as fusion protein consisting of glutathione S-transferase (GST-GRA6).The solubility analysis of expression products indicated that the recombinant protein could be expressed in both supernatant and inclusion bodies. The soluble protein of GRA6 in supernatantyield the final concentration at greater than 90% after purification. The results of detection toxopla...