Expression And Identification Of Toxoplasma Gondii GRA8?GRA10 And OWP2 Proteins

Toxoplasma gondii is an important zoonotic protozoan with a wide host range and it can infect all warm-blooded animals and humans.Appoximately 25%-30% of world population are infected with toxoplasma.Toxoplasma gondii has a complex life cycle and multiple routes of infection,which is an important reason for its high seroprevalance and also brings difficulties for its prevention and control.For any intermediate hosts,it is very important to identify how they get infected for the design of proper prevention strategies.The main infection pathways of Toxoplasma gondii are oocysts and cysts,the key to track the infection of Toxoplasma gondii is to distinguish the infections from oocysts or cysts.This study selected toxoplasma antigens that can potentially distinguish oocysts and cysts derived infections,as ways to inform further design of diagnostic methods to track the origin of infections..We constructed expression plasmids and purified recombinant proteins,which formed the basis for future serological diagnostics to trace the source of T.gondii infection.No matter the infection caused by oocysts or cysts,T.gondii will first transform into tachyzoites and then differentiate into cysts.Therefore,the only way to distinguish the infection routes is oocyst specific antigens.The purpose of this study was to construct three recombinant plasmids expressing: the N terminal part of Toxoplasma oocyst wall protein 2(OWP2)to detect the infection caused by oocysts,and the N terminal part of Toxoplasma granule protein 10(GRA10)as well as the N terminal part of Toxoplasma granule protein 8(GRA8)to detect Toxoplasma gondii infection caused by any routes.The corresponding fragments of GRA10(AA 4-487)and GRA8(AA 25-269)were amplified from the complementary DNA of ME49 tachyzoites,and OWP2(AA 240-442)from the genomic DNA of the ME49 strain.Subsequently they were cloned into the pE-SUMO vector by homologous recombination.The resulting pE-SUMO-GRA10(AA 4-487)?pE-SUMO-GRA8(AA 25-269)and pE-SUMO-OWP2(AA 240-442)plasmids were then sequenced and transformed into E.coli BL21(DE3)competent cells.Recombinant protein expressions were induced with IPTG and subsequently the products were purified by affinity chromatography and then analyzed using SDS-PAGE.In order to evaluate the immuno-reactivity of the recombinant protein,the product was analyzed using Western blotting.The results showed that the recombinant plasmids pE-SUMO-GRA10(AA 4-487)?pE-SUMO-GRA8(AA 25-269)and pE-SUMO-OWP2(AA 240-442)were successfully constructed.SDS-PAGE results indicated that SUMO-GRA10(AA 4-487)fusion protein was efficiently expressed with a molecular weight of about 64 Kd.SDS-PAGE results indicated that the SUMO-GRA8(AA 25-269)fusion protein was efficiently expressed with a molecular weight of about 43 Kd.SDS-PAGE results indicated that the SUMO-OWP2(AA 240-442)fusion protein was efficiently expressed with a molecular weight of about 39 Kd.Western blotting indicated that SUMO-GRA10(AA4-487)and SUMO-GRA8(AA 25-269)fusion proteins were recognized by sera from mice infected with Toxoplasma gondii.And The conclusions are that the GRA10(AA 4-487)?GRA8(AA 25-269)and OWP2(AA 240-442)genes of T.gondii were successfully cloned and expressed in BL21(DE3)cells.The GRA10(AA 4-487)and GRA8(AA 25-269)recombinant proteins have decent immuno-reactivity and purified the OWP2(AA 240-442)recombinant protein to produce polyclonal antibodies.This research established a foundation to further design of diagnostic methods to track the origin of infections.