Toxoplasma Gondii Sag2 Gene Cloning, Expression, And Its Initial Identification

M. S. Candidate:Yong Tao Supervisor:Prof. Moxiang Liu; Vice Prof. HuayanToxoplasma diseases caused by Toxoplasma gondii is a kind of diseases that both human beings and animals sufferred from. It could cause premature delivery, miscarriage, fetal malformation when pregnant women were infected by Toxoplasma gondii. The prenatal diagnosis of pregnant women infected by Toxplasma is of great significance to eugenics.Diagnosis methods of Toxoplasma diseases can be roughly divided into tree major categories:pathogenic examination, immunological methods, molecular biology technology. The traditional method of pathogenic examination is time-consuming, laborious, and its specificity and sensitivity is not high, can't adapt to the current diagnosis of Toxoplasma diseases and the need for prevention and treatment. Although molecular diagnostic techniques with high sensitivity and specificity, but because of its complicated operation or higher experimental conditions needed, it is difficult to promote to use. Immunological method of diagnosis, with high sensitivity, specificity and the advantages of simple, is a common method of surveying Toxoplasma gondii infection, and of the clinical selection and diagnosis of toxoplasmosis, so it is still the the main diagnostic tool of Toxoplasma gondii infection. The most commonly used whole Toxoplasma gondii antigen for serological testing presently, the results sometimes may be false positive, which brings a lot of inconvenience to diagnosis. So the recombinant antigens prepared using gene recombinant technology can greatly improve the diagnosis of toxoplasma diseases.According to the published sequence of Toxoplasma gondii SAG2 gene of GenBank, we designed a pair of primers by software. The primers with the target gene fragments were amplified by RT-PCR. PCR products was purified with purification kit, The pGEX-4T-1 plasmid was extracted from E.coli DH5a with plasmid extraction kit, and then purified PCR products and pGEX-4T-1 plasmid were digested by restriction enzymes EcoR I and Sal I. PCR products and pGEX-4T-1 recycling products Connected by Dalian treasure of reagent to construct pGEX-4T-1/SAG2 recombinant plasmid, which was identified by PCR and double restriction enzyme digestion, confirmed by DNA sequencing. The pGEX-4T-1/SAG2 was transformed into host E. coli BL21 (DE3), which induced by isopropyl thiogalactoside (IPTG) for expression. The expression products were detected and confirmed by SDS-PAGE, dissolved in urea, and then refolded by arginine oxidation/Reduced glutathione renaturation system. The refolded protein was further ultrafiltrated and concentrated. The concentrated protein solution was passed GST affinity chromatography. The specificity of purified recombinant protein GST-SAG2 was identified by enzyme immunoassay (indirect ELISA) and immunogold filtration assay (capture method and double antigen sandwich method. Finally, we acquired recombinant protein with the antigenicity and specificity of natural protein.The recombinant protein GST/SAG2 was successfully acquired, and was used as antigen to detect Toxoplasma gondii antibodies in human serum, which was proved to be of great sensitivity and specificity.