Primary Application And Expression Of Toxoplasma Gondii SAG1Protein From A Recombinant Strain

Toxoplasma gondii (TOX) is an intracellular protozoan parasite that infects a large variety of domestic and wild mammals, including humans. Especially pregnant women, as a consequence of infected, abortion, fetal anomaly and even stillbirth can be followed. So from the perspective of eugenics, prenatal diagnosis of TOX is significantly. Enzyme-linked immunosorbent assay (ELISA) is wide used with high sensitivity and specificity, furthermore, the antibody level can be describe as exactly figures. Immune colloidal gold technique is more and more popular because it has several advantages, such as its high specificity, simplicity of procedure, rapid operation, quick results, low cost, and no requirements for special skills or expensive equipment. However, because of these tests based on the antigens extracted from TOX tachyzoites grown in mice or in tissue culture, they lead to the false positive or false negative results sometimes, bring lots of inconveniences in lab-diagnosis. Therefore, use of purified recombinant proteins in serological tests is the only alternative approach to obtain specific and sensitive results.Objective:Prepared recombinant TOX SAG1protein by recombinant DNA technology for immunological diagnosis.Methods:According to the sequence of TOX SAG1gene in GeneBank, a pair of primers was design to amplify the gene by polymerase chain reaction (PCR). Both the PCR product and the pCold-TF vector were digested with NdeI and XhoI, TOX SAG1gene fragment was subcloned into the digested pCold-TF vector to construct a recombinant plasmid (pCold-TF-SAG1). The resultant plasmid was identified by dual-enzyme digestion and sequence analysis, and then transformed into Escherichia coli BL21(DE3) for expression under the induction of isopropyl β-D-1-thiogalactoside (IPTG). The expressed TF-SAG1fragment in the supernatant was detected by SDS-PAGE and purified by His tag affinity using BIO-RAD protein purifier. The activity of the protein was tested by ELISA and immune colloidal gold technique. ELISA based on the TF-SAG1fragment was used for determining IgG or IgM to TOX. The ELISA was also compared with commercial ELISA kits. These results indicated that the TF-SAG1fragment could be used for replacing whole-TOX (tachyzoites) antigen to detect antibody to TOX in human sera. Results:①The SAG1gene was amplified by PCR.②The recombinant plasmid pCold-TF/SAG1was reconstructed successfully.③The recombinant protein TF/SAG1was harvested.④The specificity of recombinant protein TF/SAG1was tested.Conclusion:The recombinant protein can be used for the diagnosis of TOX infection.