Construction Of Transgenic Toxoplasma Gondii Expressing Dual-lfuoresecent Stage-speciifc Proteins

Toxoplasma gondii is an ubiquitous, apicomplexan protozan parasite which is capable of infectingalmost all necleated cells and it is a parasitic zoonosis. The spread and prevalence of T. gondii inlivestock and poultry resulted in lower production property, even large amouts of deaths. The mainreason T. gondii can affect widely is it can form cysts in hosts which can reactive and affect other hosts.The mechanism about how T. gondii convert from tachyzoite to bradyzoite is still a challenging project.Identification of bradyzoite is difficult and many investigator tried to mark it. Application oftransgenetics on T. gondii and development of fluorescent technique give rise to fluorescent T. gondii.This experiment aims to construct green fluorescent expressed tachyzoite and red fluorescent expressedbradyzoite which is convenient to distinct the stage conversion and and can facilitate the mechanismresearch of conversion between tachyzoite and bradyzoite.Optimized pDMG vector was introduced to this experiment and genetic engineering was used toconstruct the recombinant plasmid. Promoter and terminator of tachyzoite stage specific protein SAG1gene were fused to enhanced green fluorescent protein (EGFP) gene, while promoter and terminator ofbradyzoite stage specific protein BAG1gene were fused to red fluorescent protein (RFP) gene.Incorporating all these gene segments into pDMG vector and sequencing of all the segments confirmedthe recombinant plasmid was successfully constructed. T. gondii in this lab was inoculated in humanforeskin fibroblast (HFF) and electroproration was introduced to the transformation of plasmid. Greenfluorescent was observed in tachyzoite following by pyrimethimine and low melting point gel screening,then, red fluorescent showed which suggests that tachyzoite specific EGFP gene and bradyzoite specificRFP gene expressed successfully.Western blot was introduced to indentify the green fluorescent expressed parasites and alkalineinduced red fluorescent expressed parasites were in tachyzoite and bradyzoite stage respectively.Biological characteristics and sensitivity to alkaline pH condition of trangenetic parasites werecharacterized by special in vitro and in vivo experiments. Data showed that transgenetic parasites weresensitive to alkaline pH condition like wide type strain did and the mortality rate after inoculation ofKM mice was a little higher than the wide type strain.