Prokaryotic Expression Of GRA7Gene Of Toxoplasma Gondii And Establishment Of ELISA Detection T. Gondii Infection In Cattle

Toxoplasmosis is a zoonotic parasitic disease caused by Toxoplasma gondii, which infectshumans and other mammals. Toxoplasmosis is a worldwide important food borne disease, itmakes serious harm to human health and animal husbandry. If people can be infected by eatinguncooked meat which containing raw tissue cysts or the food contaminated with Toxoplasmagondii oocysts.Beef as one of the important food for human, human could be infected with Toxoplasmagondii if the beef containing cysts, so the detection of Toxoplasmosis from cattle has importantpublic health implications. Toxoplasma gondii dense granule proteins (GRAs) are secretoryproteins, which are potentially antigens for detection of Toxoplasmosis. GRA7is a member ofGRAs family identified by cDNA library, its full-length is750bp. GRA7is used as a marker ofserological diagnosis of Toxoplasmosis.Prokaryotic expression and purification of the GRA7gene of Toxoplasma gondiiAccording the sequence of GRA7gene published in GenBank, the signal peptide was removed,a pair of specific primers was designed, the GRA7gene was amplified by RT-PCR. The675bpfragment was gotten and cloned into T vector, the results are that and the sequence homologywas100%compared to the published sequence in GenBank identified by enzyme digestion andsequence analyzed. The prokaryotic expression vector pET-28a-GRA7was constructed, thentransformed into Escherichia coli, the expression was induced by IPTG and analyzed bySDS-PAGE and Western blotting. The molecular mass of the recombinant protein was27kDa.The expression protein was purified by Ni-column, the recombinant GRA7proteinconcentration is about0.6mg/ml.The establishment of ELISA detection method of Toxoplasma gondiiThe purified recombinant GRA7protein as antigen was coated ELISA plate, the ELISA methodto detection of Toxoplasma gondii was established and applied to examine the cattle serum, theoptimal conditions are as followed coating concentration of antigen is5μg/ml, serum dilution is1:100, sealing liquid is5%skim milk powder, the concentration of secondary antibodies is1:20000and the best time to TMB color is20min. It could be detected the Toxoplasma gondiiafter the serum was diluted1:12800, the coefficient of variation of the intra and inter batchrepeatability mean are4.70%and3.51%, they were all less than8%. The method has goodsensitivity and repeatability. There is no cross reactions when using this method to detect the Taenia saginata, Sarcocystis, hydatid and cattle Toxoplasma gondii positive serum, it showed agood specificity. The100cattle sera were detected using this ELISA method, the coincidencerate was up to92%to meet the analysis requirements when compared with Dot-blot detection.The estabilishment of ELISA method lays the foundation of the development of ELISAdetection kit, and providing the supportment of methods to diagnosis and investigation of cattleToxoplasmosis.