Construction And Identification Of Toxoplasma Gondii ROP18in Transgenic Leishmania Tarentolae

Toxoplasma gondii is a kind of protozoon, which can infect both animal and human by chance. The parasites can invasive a variety of human and animal organs, because of toxoplasmosis, which has a high infection rate and caused enormous threat to public health and domestic animanl production. In recent years, the toxoplasmosis has become a worldwide public health problem as the rate of AIDS patients and pregnant women infected with toxoplasmosis are on the rise. Because of the complexity of Toxoplasma gondii’s life and route of transmission of variety, So far, there is no patent medicine against this kind of disease, It is necessary to develop cheap and effective vaccine agaist T. gondii infection in animals.ROP18isa rhoptry kinase controlling the intracellular proliferation of Toxoplasma gondii, and it is involved in the formation of parasitophorous vacuole. It exert a long-lasting effect on intracellular parasite development and virulence.ROP18sequence in the101-113129-142152-163a amino acid residues place contain arginine. It is translocated into the PVM during invasion. Targeted depletion of ROP18using a ribozyme-modified antisense RNA strategy results in disruption of rhoptry biogenesis and affects cytokinesis,association of host cell mitochondria with the PVM,host cell invasion,and virulence in mice. We found that overexpression of ROP18increaseToxoplasma gondii Intracellula, So that the effect of inhibition protein in Toxoplasma gondii proliferation, it has a vaccine antigens of value.Leishmania tarentolae was first isolated from reptiles, which is infect the lizards, also infect mammals but it is not pathogenic. Due to its parasitic lifestyle, Leishmania tarentolae has an analogous way with mammals in post-translational processing of the target protein, such as protein phosphorylation, glycosylation, prenylation and so on. In addition, Leishmania tarentolae has superiorities as a eukaryotic expression system such as easy to culture and broke, and pollution of its expressed protein can be easily avoided. Hence, Leishmania tarentolae as a kind of exogenous expression vector systems receive more attention.Construct the recombinant plasmids pLEXSY-neo2-ROP18The ROP18gene of T. gondii was amplified by PCR method from T. gondii RH strain genome, after that cloned into eukaryotic expression vector (pLEXSY-neo2) to construct the recombinant plasmids pLEXSY-neo2-ROP18. The plasmids pLEXSY-neo2-ROP18was transfected by electroporation into the Leishmania tarentolae genome, and positive clones were screened by G418.Identification of Toxoplasma gondii ROP18in transgenic Leishmania tarentolae Extract negative and positive control parasites’genome, which was used to amplify ROP18fragment using specific primers, the results showed specific target stripe could be detected in the positive parasites. Western-blot analysis suggested the expressed protein can be recognized by anti-histidine antibody, and no target stripe in negative control was detected. So the target protein was expressed successfully in the Leishmania tarentolae.