Cloning And Expression Of Toxoplasma Gondii ROP2, SAG1 Genes, And Construction And Expression Of SAG1/ROP2 Complex Gene

Background: Toxoplasma gondii is an intracellular protozoan parasite and the etiological agent of toxoplasmosis,and able to infect all warmblooded animals.This infection has worldwide distribution ,and it has been estimated that one-third of the human population is infected.Although toxoplasmosis is generally asymptomatic in immunocompetent individuals,!! may cause severe complications in immunocommpromised and pregnant women.If toxoplasmosis is first acquired during pregnancy,transplacental transmission can occur,leading to abortion or neonatal malformations.In immunodeficient or immunosuppressed patients,toxoplasmosis is one of the major opportunistic infections and can become fatal.In addition,toxoplasmosis can cause considerable economic loss in the farming industry. Research about diagnosis and vaccines to toxoplasmosis become very important .Selection and obtainment of toxoplasma antigens is the base of the research.The SAG1 antigen of toxoplasma is stage-specific antigen of tachyzoites,ROP2 antigen is secreted by the rhoptry of tachyzoites andbradyzoites.They are highly conservative and immunogenic,were selected as antigens for diagnosis and vaccination to toxoplasmosis.AimsrThe SAGl gene and ROP2 gene were cloned and expressed respectively to select antigens for diagnosis;SAGl/ROP2 complex gene was constructed for vaccination of toxoplasmosis.Method:!.The ROP2 gene was amplified from the genomic DNA of the toxoplasma gondii RH strain,and was clond into plasmid pGEX-4T-l,the ROP2 protein was expressed in E.coli and analyzed by western-blotting.2.The SAGl gene was cloned into the plasmid pGEX-4T-l and pET32a through PCR,the SAGl protein was expressed in E.coli and analyzed by western-blotting.3.The complex gene SAG1/ROP2 was constructed through enzyme digest and ligation,and was cloned into pET32a.the recombinant protein was expressed in E.coli and analyzed by western-blotting.Results: l.The truncated ROP2 was cloned,and it open read frame is correct by sequencing,the ROP2 was expressed in E.coli as a fusion protein ,and the protein is insoluble in form of inclusion body.The protein is immunogenic by analysis of the western-blotting.2.The truncated SAGl was cloned into two vectors, the SAGl was expressed in E.coli as a soluble or insoluble fusion protein ,the proteins are immunogenic by analysis of the western-blotting.3.The complex gene SAG1/ROP2 was constructed and expressed in E.coli ,the recombinant protein is immunogenic by analysis of the western-blotting .Conclusions: 1 .The truncated ROP2 was cloned and expressed .2.The truncated SAG1 was cloned and expressed as a soluble or insoluble fusion protein.3.The complex gene SAG1/ROP2 was constructed and expressed.