Expression And Immunoreactivity Analysis Of GRA15 Protein Of Toxoplasma Gondii GT1 Strain

Toxoplasma gondii(T.gondii)is an opportunistic intracellular protozoan parasite that can infect a broad range of mammalian and avian hosts,includ humans.According to epidemiological surveys,there was a wide distribution and high prevalence of T.gondii in many areas of the world.Therefore,the diagnosis of toxoplasmosis is of great significance for the prevention of toxoplasmosis.The dense granule proteins(GRAs)secreted by dense granules have now been identified to about 30 species.Some have been proven to be good diagnostic antigens for toxoplasma,and more importantly,some of these polymorphic proteins can be used for the serotyping of T.gongdii.In this study,the GRA15 gene fragment(encoding a residue from aa52 to aa635)of T.gondii GT1 strain was cloned and expressed,and its immunoreactivity was analyzed.The results showed that GRA15 protein of T.gondii GT1 strain has a good immunoreactivity.The details are as follows:The bioinformatics analysis of GRA15GT1 gene fragment By using bioinformatics softwares including ORF Finder tool,Expasy tool,TMHMM Server v.2.0 tool,Net Phos 3.1 Server tool,Phyre 2.0tool,PREDICTED ANTIGENIC PEPTIDES tool and DNASTAR softwares,the bioinformatics of GRA15 protein of T.gongdii GT1 strain were analyzed.The results showed that the GRA15GT1 gene fragment was1755 bp,having 25 ORF and it encoded 584 amino acids.The theoretical molecular weight of GRA15GT1 protein was 60.835 k Da and the protein was hydrophilic protein,and there were 1 transmembrane regions,97 phosphorylation sites and 18 potential antigenic tables.Therefore,it is predicted that GRA15GT1 protein has a goood immunogenicity.Expression of GRA15GT1 protein A pair of specific primers were designed by using the GRA15 sequence of T.gondii GT1 strain published in Toxo DB and NCBI,and the Bam H? and Eco R? cleavage sites were added to the upstream and downstream primer respectively.The GRA15GT1 gene fragment was amplified by PCR.The GRA15GT1 gene fragment was ligated into p GEX-6P-1 vector,and then identified by enzyme digestion and sequencing.The p GEX-6P-1-GRA15GT1 was transformed into BL21 strain.Expression verification and verification analysis were performed by SDS-PAGE and western blotmethods.The results showed that the enzyme digestion and sequencing were consistent with the expected results.The results showed that the target band,slightly larger than its estimated size,was observed via SDS-PAGE analysis and western blot analysis using anti GST-Tag mouse monoclonal antibody.Therefore,the p GEX-6P-1-GRA15GT1 vector was successfully constructed,and the purified GRA15GT1 fusion protein was obtained.The immunoreactivity analysis of GRA15GT1 protein The immunoreactivity of GRA15GT1 protein was studied by western blot analysis using swine anti-T.gondii GJS strain,swine anti-T.gondii obtained by our laboratory,and rabbit anti-T.gondii RH strain respectively.The results showed that the size of detected band using swine anti-T.gondii GJS strain,swine anti-T.gondii obtained by our laboratory,and rabbit anti-T.gondii RH strain respectively was the same as that revealed by GST-Tag mouse monoclonal antibody.Therefore,the GRA15GT1 protein has a good immunoreactivity with anti-T.gondii antibody.