The Translationally Controlled Tumor Protein Of Toxoplasma Gondii Prokaryotic Expression And Promotes Histamine Release

Toxoplasma gondii is mainly parasitized in the cells,and is widely distributed in the global world,but the probability of infection with Toxoplasma gondii in different regions is different.Th1-type cellular immune response is rapidly induced,during the tachyzoites grow quickly,producing inflammatory cytokines.Histamine is an important regulator of inflammatory response,produced by basophils and mast cells stimulated by translationally controlled tumor protein(TCTP).The TCTP is highly conserved,but the function and characteristics of Toxoplasma gondii TCTP protein have not been reported now.Therefore,the study of histamine release factor-like activity and mediated host inflammatory response contribute to reveal the infection mechanism of Toxoplasma gondii,and it is essential to the prevention and treatment of toxoplasmosis and the control of the spread of the disease.In this study,to study the molecular mechanism of inflammatory reaction after Toxoplasma gondii infection,the extended PCR method was used to amplify TCTP gene and GST gene.The TCTP and GST genes were fused by overlapping extension PCR,and the pET-30a-TCTP-GST prokaryotic expression vector was constructed.That was transformed into E.coli BL21 competent cells,induced by IPTG,and the expression product was analyzed in the way of SDS-PAGE and Western-blot.The existing form of TgTCTP polymer was confirmed by Native-PAGE.to prepare the TgTCTP subunit vaccine by Freund's adjuvant,and inoculating mice to prepare anti-protein serum.Using indirect immunofluorescence assay(IFAT)to identify the site TgTCTP located,and using Competitive ELISA method,based on histamine-releasing activity,to identify the effect of the activity of histamine released from mouse peritoneal cells stimulated by recombinant TgTCTP protein(rTgTCTP).The results showed that the length of TCTP and GST were respectively 510bp and 690bp.While the fusion of TCTP and GST gene was 200bp.The recombinant plasmid pET-30a-TCTP-GST was successfully constructed.The expressed recombinant protein has a molecular weight of 47 ku and has a good reactionogenicity.Anti-TgTCTP mouse serum polyclonal antibody can recognize the identification of 19ku toxoplasma natural protein.TgTCTP protein can secrete into the cytoplasm.Recombinant TgTCTP protein(rTgTCTP)promotes the release of histamine from mouse peritoneal cells.In conclusion,the results showed that TgTCTP could be located in the cytoplasm of Toxoplasma gondii,which is a secretary protein that secrete to the outer side of the body of the parasite.Meanwhile the effect of TgTCTP protein on histamine release was detected by histamine release assay kit;it shows that TgTCTP protein has the effect of promoting histamine release.In summary,this study laid the foundation for further verification of the molecular mechanism of TgTCTP mediated host inflammatory response.